Sample records for nadph thioredoxin reductase

  1. Structure of Hordeum vulgare NADPH-dependent thioredoxin reductase 2. Unwinding the reaction mechanism

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kirkensgaard, Kristine G.; Enzyme and Protein Chemistry, Department of Systems BioIogy, Technical University of Denmark; Hägglund, Per

    2009-09-01

    The first crystal structure of a cereal NTR, a protein involved in seed development and germination, has been determined. The structure is in a conformation that excludes NADPH binding and indicates that a domain reorientation facilitated by Trx binding precedes NADPH binding in the reaction mechanism. Thioredoxins (Trxs) are protein disulfide reductases that regulate the intracellular redox environment and are important for seed germination in plants. Trxs are in turn regulated by NADPH-dependent thioredoxin reductases (NTRs), which provide reducing equivalents to Trx using NADPH to recycle Trxs to the active form. Here, the first crystal structure of a cereal NTR,more » HvNTR2 from Hordeum vulgare (barley), is presented, which is also the first structure of a monocot plant NTR. The structure was determined at 2.6 Å resolution and refined to an R{sub cryst} of 19.0% and an R{sub free} of 23.8%. The dimeric protein is structurally similar to the structures of AtNTR-B from Arabidopsis thaliana and other known low-molecular-weight NTRs. However, the relative position of the two NTR cofactor-binding domains, the FAD and the NADPH domains, is not the same. The NADPH domain is rotated by 25° and bent by a 38% closure relative to the FAD domain in comparison with AtNTR-B. The structure may represent an intermediate between the two conformations described previously: the flavin-oxidizing (FO) and the flavin-reducing (FR) conformations. Here, analysis of interdomain contacts as well as phylogenetic studies lead to the proposal of a new reaction scheme in which NTR–Trx interactions mediate the FO to FR transformation.« less

  2. Thioredoxin and NADPH-Dependent Thioredoxin Reductase C Regulation of Tetrapyrrole Biosynthesis.

    PubMed

    Da, Qingen; Wang, Peng; Wang, Menglong; Sun, Ting; Jin, Honglei; Liu, Bing; Wang, Jinfa; Grimm, Bernhard; Wang, Hong-Bin

    2017-10-01

    In chloroplasts, thioredoxin (TRX) isoforms and NADPH-dependent thioredoxin reductase C (NTRC) act as redox regulatory factors involved in multiple plastid biogenesis and metabolic processes. To date, less is known about the functional coordination between TRXs and NTRC in chlorophyll biosynthesis. In this study, we aimed to explore the potential functions of TRX m and NTRC in the regulation of the tetrapyrrole biosynthesis (TBS) pathway. Silencing of three genes, TRX m1 , TRX m2 , and TRX m4 ( TRX ms ), led to pale-green leaves, a significantly reduced 5-aminolevulinic acid (ALA)-synthesizing capacity, and reduced accumulation of chlorophyll and its metabolic intermediates in Arabidopsis ( Arabidopsis thaliana ). The contents of ALA dehydratase, protoporphyrinogen IX oxidase, the I subunit of Mg-chelatase, Mg-protoporphyrin IX methyltransferase (CHLM), and NADPH-protochlorophyllide oxidoreductase were decreased in triple TRX m- silenced seedlings compared with the wild type, although the transcript levels of the corresponding genes were not altered significantly. Protein-protein interaction analyses revealed a physical interaction between the TRX m isoforms and CHLM. 4-Acetoamido-4-maleimidylstilbene-2,2-disulfonate labeling showed the regulatory impact of TRX ms on the CHLM redox status. Since CHLM also is regulated by NTRC (Richter et al., 2013), we assessed the concurrent functions of TRX m and NTRC in the control of CHLM. Combined deficiencies of three TRX m isoforms and NTRC led to a cumulative decrease in leaf pigmentation, TBS intermediate contents, ALA synthesis rate, and CHLM activity. We discuss the coordinated roles of TRX m and NTRC in the redox control of CHLM stability with its corollary activity in the TBS pathway. © 2017 American Society of Plant Biologists. All Rights Reserved.

  3. Expression, purification and molecular structure modeling of thioredoxin (Trx) and thioredoxin reductase (TrxR) from Acidithiobacillus ferrooxidans.

    PubMed

    Wang, Yiping; Zhang, Xiaojian; Liu, Qing; Ai, Chenbing; Mo, Hongyu; Zeng, Jia

    2009-07-01

    The thioredoxin system consists of thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH, which plays several key roles in maintaining the redox environment of the cell. In Acidithiobacillus ferrooxidans, thioredoxin system may play important functions in the activity regulation of periplasmic proteins and energy metabolism. Here, we cloned thioredoxin (trx) and thioredoxin reductase (trxR) genes from Acidithiobacillus ferrooxidans, and expressed the genes in Escherichia coli. His-Trx and His-TrxR were purified to homogeneity with one-step Ni-NTA affinity column chromatography. Site-directed mutagenesis results confirmed that Cys33, Cys36 of thioredoxin, and Cys142, Cys145 of thioredoxin reductase were active-site residues.

  4. Thioredoxin and NADPH-Dependent Thioredoxin Reductase C Regulation of Tetrapyrrole Biosynthesis1[OPEN

    PubMed Central

    Sun, Ting; Jin, Honglei; Wang, Jinfa

    2017-01-01

    In chloroplasts, thioredoxin (TRX) isoforms and NADPH-dependent thioredoxin reductase C (NTRC) act as redox regulatory factors involved in multiple plastid biogenesis and metabolic processes. To date, less is known about the functional coordination between TRXs and NTRC in chlorophyll biosynthesis. In this study, we aimed to explore the potential functions of TRX m and NTRC in the regulation of the tetrapyrrole biosynthesis (TBS) pathway. Silencing of three genes, TRX m1, TRX m2, and TRX m4 (TRX ms), led to pale-green leaves, a significantly reduced 5-aminolevulinic acid (ALA)-synthesizing capacity, and reduced accumulation of chlorophyll and its metabolic intermediates in Arabidopsis (Arabidopsis thaliana). The contents of ALA dehydratase, protoporphyrinogen IX oxidase, the I subunit of Mg-chelatase, Mg-protoporphyrin IX methyltransferase (CHLM), and NADPH-protochlorophyllide oxidoreductase were decreased in triple TRX m-silenced seedlings compared with the wild type, although the transcript levels of the corresponding genes were not altered significantly. Protein-protein interaction analyses revealed a physical interaction between the TRX m isoforms and CHLM. 4-Acetoamido-4-maleimidylstilbene-2,2-disulfonate labeling showed the regulatory impact of TRX ms on the CHLM redox status. Since CHLM also is regulated by NTRC (Richter et al., 2013), we assessed the concurrent functions of TRX m and NTRC in the control of CHLM. Combined deficiencies of three TRX m isoforms and NTRC led to a cumulative decrease in leaf pigmentation, TBS intermediate contents, ALA synthesis rate, and CHLM activity. We discuss the coordinated roles of TRX m and NTRC in the redox control of CHLM stability with its corollary activity in the TBS pathway. PMID:28827456

  5. NADPH Thioredoxin Reductase C and Thioredoxins Act Concertedly in Seedling Development.

    PubMed

    Ojeda, Valle; Pérez-Ruiz, Juan Manuel; González, Maricruz; Nájera, Victoria A; Sahrawy, Mariam; Serrato, Antonio J; Geigenberger, Peter; Cejudo, Francisco Javier

    2017-07-01

    Thiol-dependent redox regulation of enzyme activity plays a central role in the rapid acclimation of chloroplast metabolism to ever-fluctuating light availability. This regulatory mechanism relies on ferredoxin reduced by the photosynthetic electron transport chain, which fuels reducing power to thioredoxins (Trxs) via a ferredoxin-dependent Trx reductase. In addition, chloroplasts harbor an NADPH-dependent Trx reductase, which has a joint Trx domain at the carboxyl terminus, termed NTRC. Thus, a relevant issue concerning chloroplast function is to establish the relationship between these two redox systems and its impact on plant development. To address this issue, we generated Arabidopsis ( Arabidopsis thaliana ) mutants combining the deficiency of NTRC with those of Trxs f , which participate in metabolic redox regulation, and that of Trx x , which has antioxidant function. The ntrc-trxf1f2 and, to a lower extent, ntrc-trxx mutants showed severe growth-retarded phenotypes, decreased photosynthesis performance, and almost abolished light-dependent reduction of fructose-1,6-bisphosphatase. Moreover, the combined deficiency of both redox systems provokes aberrant chloroplast ultrastructure. Remarkably, both the ntrc-trxf1f2 and ntrc-trxx mutants showed high mortality at the seedling stage, which was overcome by the addition of an exogenous carbon source. Based on these results, we propose that NTRC plays a pivotal role in chloroplast redox regulation, being necessary for the activity of diverse Trxs with unrelated functions. The interaction between the two thiol redox systems is indispensable to sustain photosynthesis performed by cotyledons chloroplasts, which is essential for early plant development. © 2017 American Society of Plant Biologists. All Rights Reserved.

  6. Thioredoxin/Glutaredoxin System of Chlorella1

    PubMed Central

    Tsang, Monica Lik-Shing

    1981-01-01

    Using the thioredoxin/glutaredoxin-dependent adenosine 3′-phosphate 5′-phosphosulfate reductase coupled assay system, the Chlorella thioredoxin/glutaredoxin system has been partially purified and characterized. A NADPH-thioredoxin reductase and two thioredoxin/glutaredoxin activities, designated as Chlorella thioredoxin/glutaredoxin protein I and II (CPI and CPII), were found in crude extracts of Chlorella. Similar to their counterparts from Escherichia coli, both CPI and CPII are heat-stable low molecular proteins of ≃14,000. While CPI (but not CPII) is a substrate for its homologous NADPH-thioredoxin reductase as well as for E. coli NADPH-thioredoxin reductase, CPII is better than CPI as a substrate for reduction by the glutathione system. Based on these properties, CPI and CPII may be classified as Chlorella thioredoxin and Chlorella glutaredoxin, respectively. The Chlorella NADPH-thioredoxin reductase (Mr = 72,000, with two 36,000-dalton subunits) resembles E. coli-thioredoxin reductase in size. Besides Chlorella thioredoxin, the Chlorella thioredoxin reductase will also use E. coli thioredoxin, but not glutaredoxin, as a substrate. Although a thioredoxin-like protein has been implicated in higher plant light-dependent sulfate reaction, neither Chlorella thioredoxin nor glutaredoxin can stimulate the thiol-dependent adenosine 5′-phosphosulfate-sulfotransferase reaction. Furthermore, Chlorella thioredoxin and glutaredoxin, in conjunction with an appropriate reductase system, cannot replace the thiol requirement of Chlorella adenosine 5′-phosphosulfate-sulfotransferase. The exact physiological roles and subcellular localization of the Chlorella thioredoxin and glutaredoxin systems remain to be determined. Images PMID:16662058

  7. A Novel F420-dependent Thioredoxin Reductase Gated by Low Potential FAD

    PubMed Central

    Susanti, Dwi; Loganathan, Usha; Mukhopadhyay, Biswarup

    2016-01-01

    A recent report suggested that the thioredoxin-dependent metabolic regulation, which is widespread in all domains of life, existed in methanogenic archaea about 3.5 billion years ago. We now show that the respective electron delivery enzyme (thioredoxin reductase, TrxR), although structurally similar to flavin-containing NADPH-dependent TrxRs (NTR), lacked an NADPH-binding site and was dependent on reduced coenzyme F420 (F420H2), a stronger reductant with a mid-point redox potential (E′0) of −360 mV; E′0 of NAD(P)H is −320 mV. Because F420 is a deazaflavin, this enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). It transferred electrons from F420H2 to thioredoxin via protein-bound flavin; Km values for thioredoxin and F420H2 were 6.3 and 28.6 μm, respectively. The E′0 of DFTR-bound flavin was approximately −389 mV, making electron transfer from NAD(P)H or F420H2 to flavin endergonic. However, under high partial pressures of hydrogen prevailing on early Earth and present day deep-sea volcanoes, the potential for the F420/F420H2 pair could be as low as −425 mV, making DFTR efficient. The presence of DFTR exclusively in ancient methanogens and mostly in the early Earth environment of deep-sea volcanoes and DFTR's characteristics suggest that the enzyme developed on early Earth and gave rise to NTR. A phylogenetic analysis revealed six more novel-type TrxR groups and suggested that the broader flavin-containing disulfide oxidoreductase family is more diverse than previously considered. The unprecedented structural similarities between an F420-dependent enzyme (DFTR) and an NADPH-dependent enzyme (NTR) brought new thoughts to investigations on F420 systems involved in microbial pathogenesis and antibiotic production. PMID:27590343

  8. Thioredoxin Reductase and its Inhibitors

    PubMed Central

    Saccoccia, Fulvio; Angelucci, Francesco; Boumis, Giovanna; Carotti, Daniela; Desiato, Gianni; Miele, Adriana E; Bellelli, Andrea

    2014-01-01

    Thioredoxin plays a crucial role in a wide number of physiological processes, which span from reduction of nucleotides to deoxyriboucleotides to the detoxification from xenobiotics, oxidants and radicals. The redox function of Thioredoxin is critically dependent on the enzyme Thioredoxin NADPH Reductase (TrxR). In view of its indirect involvement in the above mentioned physio/pathological processes, inhibition of TrxR is an important clinical goal. As a general rule, the affinities and mechanisms of binding of TrxR inhibitors to the target enzyme are known with scarce precision and conflicting results abound in the literature. A relevant analysis of published results as well as the experimental procedures is therefore needed, also in view of the critical interest of TrxR inhibitors. We review the inhibitors of TrxR and related flavoreductases and the classical treatment of reversible, competitive, non competitive and uncompetitive inhibition with respect to TrxR, and in some cases we are able to reconcile contradictory results generated by oversimplified data analysis. PMID:24875642

  9. Thioredoxin and NADP-thioredoxin reductase from cultured carrot cells

    NASA Technical Reports Server (NTRS)

    Johnson, T. C.; Cao, R. Q.; Kung, J. E.; Buchanan, B. B.

    1987-01-01

    Dark-grown carrot (Daucus carota L.) tissue cultures were found to contain both protein components of the NADP/thioredoxin system--NADP-thioredoxin reductase and the thioredoxin characteristic of heterotrophic systems, thioredoxin h. Thioredoxin h was purified to apparent homogeneity and, like typical bacterial counterparts, was a 12-kdalton (kDa) acidic protein capable of activating chloroplast NADP-malate dehydrogenase (EC 1.1.1.82) more effectively than fructose-1,6-bisphosphatase (EC 3.1.3.11). NADP-thioredoxin reductase (EC 1.6.4.5) was partially purified and found to be an arsenite-sensitive enzyme composed of two 34-kDa subunits. Carrot NADP-thioredoxin reductase resembled more closely its counterpart from bacteria rather than animal cells in acceptor (thioredoxin) specificity. Upon greening of the cells, the content of NADP-thioredoxin-reductase activity, and, to a lesser extent, thioredoxin h decreased. The results confirm the presence of a heterotrophic-type thioredoxin system in plant cells and raise the question of its physiological function.

  10. Ebselen: A thioredoxin reductase-dependent catalyst for {alpha}-tocopherol quinone reduction

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fang Jianguo; Zhong Liangwei; Zhao Rong

    2005-09-01

    The thioredoxin system, composed of thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH, is a powerful protein disulfide reductase system with a broad substrate specificity. Recently the selenazol drug ebselen was shown to be a substrate for both mammalian TrxR and Trx. We examined if {alpha}-tocopherol quinone (TQ), a product of {alpha}-tocopherol oxidation, is reduced by ebselen in the presence of TrxR, since TQ was not a substrate for the enzyme itself. Ebselen reduction of TQ in the presence of TrxR was caused by ebselen selenol, generated from fast reduction of ebselen by the enzyme. TQ has no intrinsic antioxidant activity,more » while the product of reduction of TQ, {alpha}-tocopherolhydroquinone (TQH{sub 2}), is a potent antioxidant. The thioredoxin system dependence of ebselen to catalyze reduction of other oxidized species, such as hydrogen peroxide, dehydroascorbate, and peroxynitrite, is discussed. The ability of ebselen to reduce TQ via the thioredoxin system is a novel mechanism to explain the effects of the drug as an antioxidant in vivo.« less

  11. NADPH-Thioredoxin Reductase C Mediates the Response to Oxidative Stress and Thermotolerance in the Cyanobacterium Anabaena sp. PCC7120.

    PubMed

    Sánchez-Riego, Ana M; Mata-Cabana, Alejandro; Galmozzi, Carla V; Florencio, Francisco J

    2016-01-01

    NADPH-thioredoxin reductase C (NTRC) is a bimodular enzyme composed of an NADPH-thioredoxin reductase and a thiioredoxin domain extension in the same protein. In plants, NTRC has been described to be involved in the protection of the chloroplast against oxidative stress damage through reduction of the 2-Cys peroxiredoxin (2-Cys Prx) as well as through other functions related to redox enzyme regulation. In cyanobacteria, the Anabaena NTRC has been characterized in vitro, however, nothing was known about its in vivo function. In order to study that, we have generated the first knockout mutant strain (ΔntrC), apart from the previously described in Arabidopsis. Detailed characterization of this strain reveals a differential sensitivity to oxidative stress treatments with respect to the wild-type Anabaena strain, including a higher level of ROS (reactive oxygen species) in normal growth conditions. In the mutant strain, different oxidative stress treatments such as hydrogen peroxide, methyl-viologen or high light irradiance provoke an increase in the expression of genes related to ROS detoxification, including AnNTRC and peroxiredoxin genes, with a concomitant increase in the amount of AnNTRC and 2-Cys Prx. Moreover, the role of AnNTRC in the antioxidant response is confirmed by the observation of a pronounced overoxidation of the 2-Cys Prx and a time-delay recovery of the reduced form of this protein upon oxidative stress treatments. Our results suggest the participation of this enzyme in the peroxide detoxification in Anabaena. In addition, we describe the role of Anabaena NTRC in thermotolerance, by the appearance of high molecular mass AnNTRC complexes, showing that the mutant strain is more sensitive to high temperature treatments.

  12. NADPH-dependent coenzyme Q reductase is the main enzyme responsible for the reduction of non-mitochondrial CoQ in cells.

    PubMed

    Takahashi, Takayuki; Okuno, Masaaki; Okamoto, Tadashi; Kishi, Takeo

    2008-01-01

    We purified an NADPH-dependent coenzyme Q reductase (NADPH-CoQ reductase) in rat liver cytosol and compared its enzymatic properties with those of the other CoQ10 reductases such as NADPH: quinone acceptor oxidoreductase 1 (NQO1), lipoamide dehydrogenase, thioredoxine reductase and glutathione reductase. NADPH-CoQ reductase was the only enzyme that preferred NADPH to NADH as an electron donor and was also different from the other CoQ10 reductases in the sensitivities to its inhibitors and stimulators. Especially, Zn2+ was the most powerful inhibitor for NADPH-CoQ reductase, but CoQ10 reduction by the other CoQ10 reductases could not be inhibited by Zn2+. Furthermore, the reduction of the CoQ9 incorporated into HeLa cells was also inhibited by Zn2+ in the presence of pyrithione, a zinc ionophore. Moreover, NQO1 gene silencing in HeLa cells by transfection of a small interfering RNA resulted in lowering of both the NQO1 protein level and the NQO1 activity by about 75%. However, this transfection did not affect the NADPH-CoQ reductase activity and the reduction of CoQ9 incorporated into the cells. These results suggest that the NADPH-CoQ reductase located in cytosol may be the main enzyme responsible for the reduction of non-mitochondrial CoQ in cells.

  13. Cancer cell death induced by phosphine gold(I) compounds targeting thioredoxin reductase.

    PubMed

    Gandin, Valentina; Fernandes, Aristi Potamitou; Rigobello, Maria Pia; Dani, Barbara; Sorrentino, Francesca; Tisato, Francesco; Björnstedt, Mikael; Bindoli, Alberto; Sturaro, Alberto; Rella, Rocco; Marzano, Cristina

    2010-01-15

    The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH (nicotinamide adenine dinucleotide phosphate), plays a central role in regulating cellular redox homeostasis and signaling pathways. TrxR, overexpressed in many tumor cells and contributing to drug resistance, has emerged as a new target for anticancer drugs. Gold complexes have been validated as potent TrxR inhibitors in vitro in the nanomolar range. In order to obtain potent and selective TrxR inhibitors, we have synthesized a series of linear, 'auranofin-like' gold(I) complexes all containing the [Au(PEt(3))](+) synthon and the ligands: Cl(-), Br(-), cyanate, thiocyanate, ethylxanthate, diethyldithiocarbamate and thiourea. Phosphine gold(I) complexes efficiently inhibited cytosolic and mitochondrial TrxR at concentrations that did not affect the two related oxidoreductases glutathione reductase (GR) and glutathione peroxidase (GPx). The inhibitory effect of the redox proteins was also observed intracellularly in cancer cells pretreated with gold(I) complexes. Gold(I) compounds were found to induce antiproliferative effects towards several human cancer cells some of which endowed with cisplatin or multidrug resistance. In addition, they were able to activate caspase-3 and induce apoptosis observed as nucleosome formation and sub-G1 cell accumulation. The complexes with thiocyanate and xanthate ligands were particularly effective in inhibiting thioredoxin reductase and inducing apoptosis. Pharmacodynamic studies in human ovarian cancer cells allowed for the correlation of intracellular drug accumulation with TrxR inhibition that leads to the induction of apoptosis via the mitochondrial pathway.

  14. Thioredoxin f1 and NADPH-Dependent Thioredoxin Reductase C Have Overlapping Functions in Regulating Photosynthetic Metabolism and Plant Growth in Response to Varying Light Conditions.

    PubMed

    Thormählen, Ina; Meitzel, Tobias; Groysman, Julia; Öchsner, Alexandra Bianca; von Roepenack-Lahaye, Edda; Naranjo, Belén; Cejudo, Francisco J; Geigenberger, Peter

    2015-11-01

    Two different thiol redox systems exist in plant chloroplasts, the ferredoxin-thioredoxin (Trx) system, which depends on ferredoxin reduced by the photosynthetic electron transport chain and, thus, on light, and the NADPH-dependent Trx reductase C (NTRC) system, which relies on NADPH and thus may be linked to sugar metabolism in the dark. Previous studies suggested, therefore, that the two different systems may have different functions in plants. We now report that there is a previously unrecognized functional redundancy of Trx f1 and NTRC in regulating photosynthetic metabolism and growth. In Arabidopsis (Arabidopsis thaliana) mutants, combined, but not single, deficiencies of Trx f1 and NTRC led to severe growth inhibition and perturbed light acclimation, accompanied by strong impairments of Calvin-Benson cycle activity and starch accumulation. Light activation of key enzymes of these pathways, fructose-1,6-bisphosphatase and ADP-glucose pyrophosphorylase, was almost completely abolished. The subsequent increase in NADPH-NADP(+) and ATP-ADP ratios led to increased nitrogen assimilation, NADP-malate dehydrogenase activation, and light vulnerability of photosystem I core proteins. In an additional approach, reporter studies show that Trx f1 and NTRC proteins are both colocalized in the same chloroplast substructure. Results provide genetic evidence that light- and NADPH-dependent thiol redox systems interact at the level of Trx f1 and NTRC to coordinately participate in the regulation of the Calvin-Benson cycle, starch metabolism, and growth in response to varying light conditions. © 2015 American Society of Plant Biologists. All Rights Reserved.

  15. Inactivation of thioredoxin reductases reveals a complex interplay between thioredoxin and glutathione pathways in Arabidopsis development.

    PubMed

    Reichheld, Jean-Philippe; Khafif, Mehdi; Riondet, Christophe; Droux, Michel; Bonnard, Géraldine; Meyer, Yves

    2007-06-01

    NADPH-dependent thioredoxin reductases (NTRs) are key regulatory enzymes determining the redox state of the thioredoxin system. The Arabidopsis thaliana genome has two genes coding for NTRs (NTRA and NTRB), both of which encode mitochondrial and cytosolic isoforms. Surprisingly, plants of the ntra ntrb knockout mutant are viable and fertile, although with a wrinkled seed phenotype, slower plant growth, and pollen with reduced fitness. Thus, in contrast with mammals, our data demonstrate that neither cytosolic nor mitochondrial NTRs are essential in plants. Nevertheless, in the double mutant, the cytosolic thioredoxin h3 is only partially oxidized, suggesting an alternative mechanism for thioredoxin reduction. Plant growth in ntra ntrb plants is hypersensitive to buthionine sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis, and thioredoxin h3 is totally oxidized under this treatment. Interestingly, this BSO-mediated growth arrest is fully reversible, suggesting that BSO induces a growth arrest signal but not a toxic accumulation of activated oxygen species. Moreover, crossing ntra ntrb with rootmeristemless1, a mutant blocked in root growth due to strongly reduced glutathione synthesis, led to complete inhibition of both shoot and root growth, indicating that either the NTR or the glutathione pathway is required for postembryonic activity in the apical meristem.

  16. Thioredoxin Reductase From Thermoplasma Acidophilum: a New Twist on Redox Regulation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hernandez, H.H.; Jaquez, O.A.; Hamill, M.J.

    2009-05-18

    Thioredoxin reductases (TrxRs) regulate the intracellular redox environment by using NADPH to provide reducing equivalents for thioredoxins (Trxs). Here we present the cloning and biochemical characterization of a putative TrxR (Ta0984) and a putative Trx (Ta0866) from Thermoplasma acidophilum. Our data identify Ta0866 as a Trx through its capacity to reduce insulin and be reduced by Escherichia coli TrxR in a NADPH-dependent manner. Our data also establish Ta0984 as a TrxR due to its ability to reduce T. acidophilum Trx (taTrx), although not in a NADPH- or NADH-dependent manner. To explore the apparent inability of taTrxR to use NADPH ormore » NADH as a reductant, we carried out a complete electrochemical characterization, which suggests that redox potential is not the source of this nonreactivity [Hamill et al. (2008) Biochemistry 47, 9738-9746]. Turning to crystallographic analysis, a 2.35 {angstrom} resolution structure of taTrxR, also presented here, shows that despite the overall structural similarity to the well-characterized TrxR from E. coli (RMSD 1.30 {angstrom}{sup 2} for chain A), the 'NADPH binding pocket' is not conserved. E. coli TrxR residues implicated in NADPH binding, H175, R176, R177, and R181, have been substituted with E185, Y186, M187, and M191 in the ta protein. Thus, we have identified a Trx and TrxR protein system from T. acidophilum for which the TrxR shares overall structural and redox properties with other TrxRs but lacks the appropriate binding motif to use the standard NADPH reductant. Our discovery of a TrxR that does not use NADPH provides a new twist in redox regulation.« less

  17. Inactivation of Thioredoxin Reductases Reveals a Complex Interplay between Thioredoxin and Glutathione Pathways in Arabidopsis Development[W

    PubMed Central

    Reichheld, Jean-Philippe; Khafif, Mehdi; Riondet, Christophe; Droux, Michel; Bonnard, Géraldine; Meyer, Yves

    2007-01-01

    NADPH-dependent thioredoxin reductases (NTRs) are key regulatory enzymes determining the redox state of the thioredoxin system. The Arabidopsis thaliana genome has two genes coding for NTRs (NTRA and NTRB), both of which encode mitochondrial and cytosolic isoforms. Surprisingly, plants of the ntra ntrb knockout mutant are viable and fertile, although with a wrinkled seed phenotype, slower plant growth, and pollen with reduced fitness. Thus, in contrast with mammals, our data demonstrate that neither cytosolic nor mitochondrial NTRs are essential in plants. Nevertheless, in the double mutant, the cytosolic thioredoxin h3 is only partially oxidized, suggesting an alternative mechanism for thioredoxin reduction. Plant growth in ntra ntrb plants is hypersensitive to buthionine sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis, and thioredoxin h3 is totally oxidized under this treatment. Interestingly, this BSO-mediated growth arrest is fully reversible, suggesting that BSO induces a growth arrest signal but not a toxic accumulation of activated oxygen species. Moreover, crossing ntra ntrb with rootmeristemless1, a mutant blocked in root growth due to strongly reduced glutathione synthesis, led to complete inhibition of both shoot and root growth, indicating that either the NTR or the glutathione pathway is required for postembryonic activity in the apical meristem. PMID:17586656

  18. NADPH thioredoxin reductase C is localized in plastids of photosynthetic and nonphotosynthetic tissues and is involved in lateral root formation in Arabidopsis.

    PubMed

    Kirchsteiger, Kerstin; Ferrández, Julia; Pascual, María Belén; González, Maricruz; Cejudo, Francisco Javier

    2012-04-01

    Plastids are organelles present in photosynthetic and nonphotosynthetic plant tissues. While it is well known that thioredoxin-dependent redox regulation is essential for leaf chloroplast function, little is known of the redox regulation in plastids of nonphotosynthetic tissues, which cannot use light as a direct source of reducing power. Thus, the question remains whether redox regulation operates in nonphotosynthetic plastid function and how it is integrated with chloroplasts for plant growth. Here, we show that NADPH-thioredoxin reductase C (NTRC), previously reported as exclusive to green tissues, is also expressed in nonphotosynthetic tissues of Arabidopsis thaliana, where it is localized to plastids. Moreover, we show that NTRC is involved in maintaining the redox homeostasis of plastids also in nonphotosynthetic organs. To test the relationship between plastids of photosynthetic and nonphotosynthetic tissues, transgenic plants were obtained with redox homeostasis restituted exclusively in leaves or in roots, through the expression of NTRC under the control of organ-specific promoters in the ntrc mutant. Our results show that fully functional root amyloplasts are not sufficient for root, or leaf, growth, but fully functional chloroplasts are necessary and sufficient to support wild-type rates of root growth and lateral root formation.

  19. Thioredoxin is required for filamentous phage assembly.

    PubMed Central

    Russel, M; Model, P

    1985-01-01

    Sequence comparisons show that the fip gene product of Escherichia coli, which is required for filamentous phage assembly, is thioredoxin. Thioredoxin serves as a cofactor for reductive processes in many cell types and is a constituent of phage T7 DNA polymerase. The fip-1 mutation makes filamentous phage and T7 growth temperature sensitive in cells that carry it. The lesion lies within a highly conserved thioredoxin active site. Thioredoxin reductase (NADPH), as well as thioredoxin, is required for efficient filamentous phage production. Mutant phages defective in phage gene I are particularly sensitive to perturbations in the fip-thioredoxin system. A speculative model is presented in which thioredoxin reductase, thioredoxin, and the gene I protein interact to drive an engine for filamentous phage assembly. Images PMID:3881756

  20. The chloroplast NADPH thioredoxin reductase C, NTRC, controls non-photochemical quenching of light energy and photosynthetic electron transport in Arabidopsis.

    PubMed

    Naranjo, Belén; Mignée, Clara; Krieger-Liszkay, Anja; Hornero-Méndez, Dámaso; Gallardo-Guerrero, Lourdes; Cejudo, Francisco Javier; Lindahl, Marika

    2016-04-01

    High irradiances may lead to photooxidative stress in plants, and non-photochemical quenching (NPQ) contributes to protection against excess excitation. One of the NPQ mechanisms, qE, involves thermal dissipation of the light energy captured. Importantly, plants need to tune down qE under light-limiting conditions for efficient utilization of the available quanta. Considering the possible redox control of responses to excess light implying enzymes, such as thioredoxins, we have studied the role of the NADPH thioredoxin reductase C (NTRC). Whereas Arabidopsis thaliana plants lacking NTRC tolerate high light intensities, these plants display drastically elevated qE, have larger trans-thylakoid ΔpH and have 10-fold higher zeaxanthin levels under low and medium light intensities, leading to extremely low linear electron transport rates. To test the impact of the high qE on plant growth, we generated an ntrc-psbs double-knockout mutant, which is devoid of qE. This double mutant grows faster than the ntrc mutant and has a higher chlorophyll content. The photosystem II activity is partially restored in the ntrc-psbs mutant, and linear electron transport rates under low and medium light intensities are twice as high as compared with plants lacking ntrc alone. These data uncover a new role for NTRC in the control of photosynthetic yield. © 2015 John Wiley & Sons Ltd.

  1. An event of alternative splicing affects the expression of the NTRC gene, encoding NADPH-thioredoxin reductase C, in seed plants.

    PubMed

    Nájera, Victoria A; González, María Cruz; Pérez-Ruiz, Juan Manuel; Cejudo, Francisco Javier

    2017-05-01

    The NTRC gene encodes a NADPH-dependent thioredoxin reductase with a joint thioredoxin domain, exclusive of photosynthetic organisms. An updated search shows that although most species harbor a single copy of the NTRC gene, two copies were identified in different species of the genus Solanum, Glycine max and the moss Physcomitrella patens. The phylogenetic analysis of NTRCs from different sources produced a tree with the major groups of photosynthetic organisms: cyanobacteria, algae and land plants, indicating the evolutionary success of the NTRC gene among photosynthetic eukaryotes. An event of alternative splicing affecting the expression of the NTRC gene was identified, which is conserved in seed plants but not in algae, bryophytes and lycophytes. The alternative splicing event results in a transcript with premature stop codon, which would produce a truncated form of the enzyme. The standard splicing/alternative splicing (SS/AS) transcripts ratio was higher in photosynthetic tissues from Arabidopsis, Brachypodium and tomato, in line with the higher content of the NTRC polypeptide in these tissues. Moreover, environmental stresses such as cold or high salt affected the SS/AS ratio of the NTRC gene transcripts in Brachypodium seedlings. These results suggest that the alternative splicing of the NTRC gene might be an additional mechanism for modulating the content of NTRC in photosynthetic and non-photosynthetic tissues of seed plants. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Thioredoxins in evolutionarily primitive organisms

    NASA Technical Reports Server (NTRS)

    Buchanan, B. B.

    1986-01-01

    Thioredoxins are low molecular weight redox proteins, alternating between the S-S (oxidized) and SH (reduced) states, that function in a number of biochemical processes, including DNA synthesis, DNA replication, and enzyme regulation. Until recently, reduced ferredoxin was known to serve as the source of reducing power for the reduction of thioredoxins only in oxygenic photosynthetic cells. In all other organisms, the source of hydrogen (electrons) for thioredoxin reduction was considered to be NADPH. It was found that Clostridium pasteurianum, an anaerobic organism normally living in the soil unexposed to light, resembles photosynthetic cells in using ferredoxin for the reduction of thioredoxin. The results reveal the existence of a pathway in which ferredoxin, provides the reducing power for the reduction of thioredoxin via the flavoprotein enzyme, ferredoxinthioredoxin reductase. In related studies, it was found that Chromatium vinosum, an anaerobic photosynthetic purple sulfur bacterium, resembles evolutionarily more advanced micro-organisms in having an NADP-thioredoxin system composed of a single thioredoxin which is reduced by NADPH via NADP-thioredoxin reductase. The adoption of the NADP-thioredoxin system by Chromatium seems appropriate in view of evidence tha the organi sm utilizes ATP-driven reverse electron transport. Finally, results of research directed towards the identification of target enzymes of the ferredoxin/thioredoxin system in a cyanobacterium (Nostoc muscorum), show that thioredoxin-linked photosynthetic enzymes of cyanobateria are similar to those of chloroplasts. It now seems that the ferredoxin/thioredoxin system functions in regulating CO2 assimilation via the reductive pentose phosphate cycle in oxygenic but not anoxygenic photosynthetic cells.

  3. NADPH Thioredoxin Reductase C Is Localized in Plastids of Photosynthetic and Nonphotosynthetic Tissues and Is Involved in Lateral Root Formation in Arabidopsis[W

    PubMed Central

    Kirchsteiger, Kerstin; Ferrández, Julia; Pascual, María Belén; González, Maricruz; Cejudo, Francisco Javier

    2012-01-01

    Plastids are organelles present in photosynthetic and nonphotosynthetic plant tissues. While it is well known that thioredoxin-dependent redox regulation is essential for leaf chloroplast function, little is known of the redox regulation in plastids of nonphotosynthetic tissues, which cannot use light as a direct source of reducing power. Thus, the question remains whether redox regulation operates in nonphotosynthetic plastid function and how it is integrated with chloroplasts for plant growth. Here, we show that NADPH-thioredoxin reductase C (NTRC), previously reported as exclusive to green tissues, is also expressed in nonphotosynthetic tissues of Arabidopsis thaliana, where it is localized to plastids. Moreover, we show that NTRC is involved in maintaining the redox homeostasis of plastids also in nonphotosynthetic organs. To test the relationship between plastids of photosynthetic and nonphotosynthetic tissues, transgenic plants were obtained with redox homeostasis restituted exclusively in leaves or in roots, through the expression of NTRC under the control of organ-specific promoters in the ntrc mutant. Our results show that fully functional root amyloplasts are not sufficient for root, or leaf, growth, but fully functional chloroplasts are necessary and sufficient to support wild-type rates of root growth and lateral root formation. PMID:22505729

  4. Chloroplast NADPH-Dependent Thioredoxin Reductase from Chlorella vulgaris Alleviates Environmental Stresses in Yeast Together with 2-Cys Peroxiredoxin

    PubMed Central

    Machida, Takeshi; Ishibashi, Akiko; Kirino, Ai; Sato, Jun-ichi; Kawasaki, Shinji; Niimura, Youichi; Honjoh, Ken-ichi; Miyamoto, Takahisa

    2012-01-01

    Chloroplast NADPH-dependent thioredoxin reductase (NTRC) catalyzes the reduction of 2-Cys peroxiredoxin (2-Cys Prx) and, thus, probably functions as an antioxidant system. The functions of the enzyme in oxidative and salt stresses have been reported previously. We have previously identified and characterized NTRC in Chlorella vulgaris. In the present study, we isolated a full-length cDNA clone encoding 2-Cys Prx from C. vulgaris and investigated the involvement of Chlorella NTRC/2-Cys Prx system in several environmental stress tolerances by using yeast as a eukaryotic model. Deduced Chlorella 2-Cys Prx was homologous to those of chloroplast 2-Cys Prxs from plants, and two conserved cysteine residues were found in the deduced sequence. Enzyme assay showed that recombinant mature C. vulgaris NTRC (mCvNTRC) transferred electrons from NADPH to recombinant mature C. vulgaris 2-Cys Prx (mCvPrx), and mCvPrx decomposed hydrogen peroxide, tert-butyl hydroperoxide, and peroxynitrite by cooperating with mCvNTRC. Based on the results, the mCvNTRC/mCvPrx antioxidant system was identified in Chlorella. The antioxidant system genes were expressed in yeast separately or coordinately. Stress tolerances of yeast against freezing, heat, and menadione-induced oxidative stresses were significantly improved by expression of mCvNTRC, and the elevated tolerances were more significant when both mCvNTRC and mCvPrx were co-expressed. Our results reveal a novel feature of NTRC: it functions as an antioxidant system with 2-Cys Prx in freezing and heat stress tolerances. PMID:23029353

  5. Purification and kinetic analysis of cytosolic and mitochondrial thioredoxin glutathione reductase extracted from Taenia solium cysticerci.

    PubMed

    Plancarte, Agustin; Nava, Gabriela

    2015-02-01

    Thioredoxin glutathione reductases (TGRs) (EC 1.8.1.9) were purified to homogeneity from the cytosolic (cTsTGR) and mitochondrial (mTsTGR) fractions of Taenia solium, the agent responsible for neurocysticercosis, one of the major central nervous system parasitic diseases in humans. TsTGRs had a relative molecular weight of 132,000, while the corresponding value per subunit obtained under denaturing conditions, was of 62,000. Specific activities for thioredoxin reductase and glutathione reductase substrates for both TGRs explored were in the range or lower than values obtained for other platyhelminths and mammalian TGRs. cTsTGR and mTsTGR also showed hydroperoxide reductase activity using hydroperoxide as substrate. Km(DTNB) and Kcat(DTNB) values for cTsTGR and mTsTGR (88 µM and 1.9 s(-1); 45 µM and 12.6 s(-1), respectively) and Km(GSSG) and Kcat(GSSG) values for cTsTGR and mTsTGR (6.3 µM and 0.96 s(-1); 4 µM and 1.62 s(-1), respectively) were similar to or lower than those reported for mammalian TGRs. Mass spectrometry analysis showed that 12 peptides from cTsTGR and seven from mTsTGR were a match for gi|29825896 thioredoxin glutathione reductase [Echinococcus granulosus], confirming that both enzymes are TGRs. Both T. solium TGRs were inhibited by the gold compound auranofin, a selective inhibitor of thiol-dependent flavoreductases (I₅₀ = 3.25, 2.29 nM for DTNB and GSSG substrates, respectively for cTsTGR; I₅₀ = 5.6, 25.4 nM for mTsTGR toward the same substrates in the described order). Glutathione reductase activity of cTsTGR and mTsTGR exhibited hysteretic behavior with moderate to high concentrations of GSSG; this result was not observed either with thioredoxin, DTNB or NADPH. However, the observed hysteretic kinetics was suppressed with increasing amounts of both parasitic TGRs. These data suggest the existence of an effective substitute which may account for the lack of the detoxification enzymes glutathione reductase

  6. Ebselen is a dehydroascorbate reductase mimic, facilitating the recycling of ascorbate via mammalian thioredoxin systems.

    PubMed

    Zhao, Rong; Holmgren, Arne

    2004-02-01

    Ebselen is a selanazal drug recently revealed as a highly efficient peroxiredoxin mimic catalyzing the hydroperoxide reduction by the mammalian thioredoxin system [thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH]. The mammalian Trx system is a dehydroascorbic acid reductase recycling ascorbic acid essential for cell functions. Here we report that ebselen strongly facilitated the recycling of ascorbic acid by the TrxR both with and without Trx present. Reduction of dehydroascorbic acid by TrxR has a pH optimum of 6.4, and only approximately 55% of this activity at a physiological pH of 7.4. Ebselen at 6 microM enhances this reaction three-fold and with the same pH optimum of 6.4. The mechanism of the ebselen effect is suggested to involve reduction of dehydroascorbic acid by the ebselen selenol, a highly efficient two-electron reductant. Thus, ebselen acts as an antioxidant to lower the peroxide tone inside cells and to facilitate the recycling of dehydroascorbic acid to ascorbic acid, so as to increase the radical scavenging capacity of ascorbic acid directly or indirectly via vitamin E. The high ascorbic acid recycling efficiency of ebselen at pH 6.4 may play a major role in oxidatively stressed cells, where cytosol acidosis may trigger various responses, including apoptosis.

  7. Increased reactive oxygen species production during reductive stress: The roles of mitochondrial glutathione and thioredoxin reductases.

    PubMed

    Korge, Paavo; Calmettes, Guillaume; Weiss, James N

    2015-01-01

    Both extremes of redox balance are known to cause cardiac injury, with mounting evidence revealing that the injury induced by both oxidative and reductive stress is oxidative in nature. During reductive stress, when electron acceptors are expected to be mostly reduced, some redox proteins can donate electrons to O2 instead, which increases reactive oxygen species (ROS) production. However, the high level of reducing equivalents also concomitantly enhances ROS scavenging systems involving redox couples such as NADPH/NADP+ and GSH/GSSG. Here our objective was to explore how reductive stress paradoxically increases net mitochondrial ROS production despite the concomitant enhancement of ROS scavenging systems. Using recombinant enzymes and isolated permeabilized cardiac mitochondria, we show that two normally antioxidant matrix NADPH reductases, glutathione reductase and thioredoxin reductase, generate H2O2 by leaking electrons from their reduced flavoprotein to O2 when electron flow is impaired by inhibitors or because of limited availability of their natural electron acceptors, GSSG and oxidized thioredoxin. The spillover of H2O2 under these conditions depends on H2O2 reduction by peroxiredoxin activity, which may regulate redox signaling in response to endogenous or exogenous factors. These findings may explain how ROS production during reductive stress overwhelms ROS scavenging capability, generating the net mitochondrial ROS spillover causing oxidative injury. These enzymes could potentially be targeted to increase cancer cell death or modulate H2O2-induced redox signaling to protect the heart against ischemia/reperfusion damage. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Identification of an NADP/thioredoxin system in Chlamydomonas reinhardtii

    NASA Technical Reports Server (NTRS)

    Huppe, H. C.; Picaud, A.; Buchanan, B. B.; Miginiac-Maslow, M.

    1991-01-01

    The protein components of the NADP/thioredoxin system, NADP-thioredoxin reductase (NTR) and thioredoxin h, have been purified and characterized from the green alga, Chlamydomonas reinhardtii. The analysis of this system confirms that photoautotrophic Chlamydomonas cells resemble leaves in having both an NADP- and ferrodoxin-linked thioredoxin redox system. Chlamydomonas thioredoxin h, which is smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than thioredoxin m from the same source, cross-reacted with antisera to thioredoxin h from spinach (Spinacia oleracea L.) and wheat germ (Triticum vulgaris L.) but not with antisera to m or f thioredoxins. In these properties, the thioredoxin h resembled a thioredoxin from Chlamydomonas, designated Ch1, whose sequence was reported recently (P. Decottignies et al., 1991, Eur. J. Biochem. 198, 505-512). The differential reactivity of thioredoxin h with antisera was used to demonstrate that thioredoxin h is enriched outside the chloroplast. The NTR was purified from Chlamydomonas using thioredoxin h from the same source. Similar to its counterpart from other organisms, Chlamydomonas NTR had a subunit size of approx. 36 kDa and was specific for NADPH. Chlamydomonas NTR effectively reduced thioredoxin h from the same source but showed little activity with the other thioredoxins tested, including spinach thioredoxin h and Escherichia coli thioredoxin. Comparison of the reduction of Chlamydomonas thioredoxins m and h by each of the endogenous thioredoxin reductases, NTR and ferredoxin-thioredoxin reductase, revealed a differential specificity of each enzyme for thioredoxin. Thus, NTR showed increased activity with thioredoxin h and ferredoxin-thioredoxin reductase with thioredoxins m and f.

  9. Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation

    PubMed Central

    Metcalfe, Clive; Ramasubramoni, Anjana; Pula, Giordano; Harper, Matthew T.; Mundell, Stuart J.; Coxon, Carmen H.

    2016-01-01

    Thioredoxin (Trx) is an oxidoreductase with important physiological function. Imbalances in the NADPH/thioredoxin reductase/thioredoxin system are associated with a number of pathologies, particularly cancer, and a number of clinical trials for thioredoxin and thioredoxin reductase inhibitors have been carried out or are underway. Due to the emerging role and importance of oxidoreductases for haemostasis and the current interest in developing inhibitors for clinical use, we thought it pertinent to assess whether inhibition of the NADPH/thioredoxin reductase/thioredoxin system affects platelet function and thrombosis. We used small molecule inhibitors of Trx (PMX 464 and PX-12) to determine whether Trx activity influences platelet function, as well as an unbiased proteomics approach to identify potential Trx substrates on the surface of platelets that might contribute to platelet reactivity and function. Using LC-MS/MS we found that PMX 464 and PX-12 affected the oxidation state of thiols in a number of cell surface proteins. Key surface receptors for platelet adhesion and activation were affected, including the collagen receptor GPVI and the von Willebrand factor receptor, GPIb. To experimentally validate these findings we assessed platelet function in the presence of PMX 464, PX-12, and rutin (a selective inhibitor of the related protein disulphide isomerase). In agreement with the proteomics data, small molecule inhibitors of thioredoxin selectively inhibited GPVI-mediated platelet activation, and attenuated ristocetin-induced GPIb-vWF-mediated platelet agglutination, thus validating the findings of the proteomics study. These data reveal a novel role for thioredoxin in regulating platelet reactivity via proteins required for early platelet responses at sites of vessel injury (GPVI and GPIb). This work also highlights a potential opportunity for repurposing of PMX 464 and PX-12 as antiplatelet agents. PMID:27716777

  10. Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation.

    PubMed

    Metcalfe, Clive; Ramasubramoni, Anjana; Pula, Giordano; Harper, Matthew T; Mundell, Stuart J; Coxon, Carmen H

    2016-01-01

    Thioredoxin (Trx) is an oxidoreductase with important physiological function. Imbalances in the NADPH/thioredoxin reductase/thioredoxin system are associated with a number of pathologies, particularly cancer, and a number of clinical trials for thioredoxin and thioredoxin reductase inhibitors have been carried out or are underway. Due to the emerging role and importance of oxidoreductases for haemostasis and the current interest in developing inhibitors for clinical use, we thought it pertinent to assess whether inhibition of the NADPH/thioredoxin reductase/thioredoxin system affects platelet function and thrombosis. We used small molecule inhibitors of Trx (PMX 464 and PX-12) to determine whether Trx activity influences platelet function, as well as an unbiased proteomics approach to identify potential Trx substrates on the surface of platelets that might contribute to platelet reactivity and function. Using LC-MS/MS we found that PMX 464 and PX-12 affected the oxidation state of thiols in a number of cell surface proteins. Key surface receptors for platelet adhesion and activation were affected, including the collagen receptor GPVI and the von Willebrand factor receptor, GPIb. To experimentally validate these findings we assessed platelet function in the presence of PMX 464, PX-12, and rutin (a selective inhibitor of the related protein disulphide isomerase). In agreement with the proteomics data, small molecule inhibitors of thioredoxin selectively inhibited GPVI-mediated platelet activation, and attenuated ristocetin-induced GPIb-vWF-mediated platelet agglutination, thus validating the findings of the proteomics study. These data reveal a novel role for thioredoxin in regulating platelet reactivity via proteins required for early platelet responses at sites of vessel injury (GPVI and GPIb). This work also highlights a potential opportunity for repurposing of PMX 464 and PX-12 as antiplatelet agents.

  11. A Ferredoxin Disulfide Reductase Delivers Electrons to the Methanosarcina barkeri Class III Ribonucleotide Reductase

    DOE PAGES

    Wei, Yifeng; Li, Bin; Prakash, Divya; ...

    2015-11-04

    Two subtypes of class III anaerobic ribonucleotide reductases (RNRs) studied so far couple the reduction of ribonucleotides to the oxidation of formate, or the oxidation of NADPH via thioredoxin and thioredoxin reductase. Certain methanogenic archaea contain a phylogenetically distinct third subtype of class III RNR, with distinct active-site residues. Here we report the cloning and recombinant expression of the Methanosarcina barkeri class III RNR and show that the electrons required for ribonucleotide reduction can be delivered by a [4Fe-4S] protein ferredoxin disulfide reductase, and a conserved thioredoxin-like protein NrdH present in the RNR operon. The diversity of class III RNRsmore » reflects the diversity of electron carriers used in anaerobic metabolism« less

  12. Glutathione and Glutaredoxin Act as a Backup of Human Thioredoxin Reductase 1 to Reduce Thioredoxin 1 Preventing Cell Death by Aurothioglucose*

    PubMed Central

    Du, Yatao; Zhang, Huihui; Lu, Jun; Holmgren, Arne

    2012-01-01

    Thioredoxin reductase 1 (TrxR1) in cytosol is the only known reductant of oxidized thioredoxin 1 (Trx1) in vivo so far. We and others found that aurothioglucose (ATG), a well known active-site inhibitor of TrxR1, inhibited TrxR1 activity in HeLa cell cytosol but had no effect on the viability of the cells. Using a redox Western blot analysis, no change was observed in redox state of Trx1, which was mainly fully reduced with five sulfhydryl groups. In contrast, auranofin killed cells and oxidized Trx1, also targeting mitochondrial TrxR2 and Trx2. Combining ATG with ebselen gave a strong synergistic effect, leading to Trx1 oxidation, reactive oxygen species accumulation, and cell death. We hypothesized that there should exist a backup system to reduce Trx1 when only TrxR1 activity was lost. Our results showed that physiological concentrations of glutathione, NADPH, and glutathione reductase reduced Trx1 in vitro and that the reaction was strongly stimulated by glutaredoxin1. Simultaneous depletion of TrxR activity by ATG and glutathione by buthionine sulfoximine led to overoxidation of Trx1 and loss of HeLa cell viability. In conclusion, the glutaredoxin system and glutathione have a backup role to keep Trx1 reduced in cells with loss of TrxR1 activity. Monitoring the redox state of Trx1 shows that cell death occurs when Trx1 is oxidized, followed by general protein oxidation catalyzed by the disulfide form of thioredoxin. PMID:22977247

  13. Direct cloning of the trxB gene that encodes thioredoxin reductase.

    PubMed Central

    Russel, M; Model, P

    1985-01-01

    A strain was constructed which contains mutations in the genes encoding thioredoxin (trxA) and thioredoxin reductase (trxB) such that filamentous phage f1 cannot grow. The complementation of either mutation with its wild-type allele permits phage growth. We used this strain to select f1 phage which contain a cloned trxB gene. The location of the gene on the cloned fragment was determined, and its protein product was identified. Plasmid subclones that contain this gene overproduce thioredoxin reductase. Images PMID:2989245

  14. Sequence analysis, expression profiles and function of thioredoxin 2 and thioredoxin reductase 1 in resistance to nucleopolyhedrovirus in Helicoverpa armigera

    PubMed Central

    Zhang, Songdou; Li, Zhen; Nian, Xiaoge; Wu, Fengming; Shen, Zhongjian; Zhang, Boyu; Zhang, Qingwen; Liu, Xiaoxia

    2015-01-01

    The thioredoxin system, including NADPH, thioredoxin (Trx), and thioredoxin reductase (TrxR), plays significant roles in maintaining intracellular redox homeostasis and protecting organisms against oxidative damage. In this study, the characteristics and functions of H. armigera HaTrx2 and HaTrxR1 were identified. Sequence analysis showed that HaTrx2 and HaTrxR1 were both highly conserved and shared high sequence identity with other insect counterparts. The mRNA of HaTrx2 was expressed the highest in 5th instar 96 h and was mainly detected in heads and epidermis. The expression of HaTrxR1 was highly concentrated in 5th instar 72 h and 96 h, and higher in malpighian tube, midgut and hemocyte than other examined tissues. HaTrx2 and HaTrxR1 were markedly induced by various types of stress. HaTrx2- or HaTrxR1-knockdown increased ROS production in hemocytes and also increased the lipid damage in NPV infected H. armigera larvae. Furthermore, interference with expression of HaTrx2 or HaTrxR1 transcripts in H. armigera larvae resulted in increased sensitivity to NPV infection and shortened LT50 values. Our findings indicated that HaTrx2 and HaTrxR1 contribute to the susceptibility of H. armigera to NPV and also provided the theoretical basis for the in-depth study of insect thioredoxin system. PMID:26502992

  15. Peroxiredoxins and NADPH-dependent thioredoxin systems in the model legume Lotus japonicus.

    PubMed

    Tovar-Méndez, Alejandro; Matamoros, Manuel A; Bustos-Sanmamed, Pilar; Dietz, Karl-Josef; Cejudo, Francisco Javier; Rouhier, Nicolas; Sato, Shusei; Tabata, Satoshi; Becana, Manuel

    2011-07-01

    Peroxiredoxins (Prxs), thioredoxins (Trxs), and NADPH-thioredoxin reductases (NTRs) constitute central elements of the thiol-disulfide redox regulatory network of plant cells. This study provides a comprehensive survey of this network in the model legume Lotus japonicus. The aims were to identify and characterize these gene families and to assess whether the NTR-Trx systems are operative in nodules. Quantitative reverse transcription-polymerase chain reaction and immunological and proteomic approaches were used for expression profiling. We identified seven Prx, 14 Trx, and three NTR functional genes. The PrxQ1 gene was found to be transcribed in two alternative spliced variants and to be expressed at high levels in leaves, stems, petals, pods, and seeds and at low levels in roots and nodules. The 1CPrx gene showed very high expression in the seed embryos and low expression in vegetative tissues and was induced by nitric oxide and cytokinins. In sharp contrast, cytokinins down-regulated all other Prx genes, except PrxQ1, in roots and nodules, but only 2CPrxA and PrxQ1 in leaves. Gene-specific changes in Prx expression were also observed in response to ethylene, abscisic acid, and auxins. Nodules contain significant mRNA and protein amounts of cytosolic PrxIIB, Trxh1, and NTRA and of plastidic NTRC. Likewise, they express cytosolic Trxh3, Trxh4, Trxh8, and Trxh9, mitochondrial PrxIIF and Trxo, and plastidic Trxm2, Trxm4, and ferredoxin-Trx reductase. These findings reveal a complex regulation of Prxs that is dependent on the isoform, tissue, and signaling molecule and support that redox NTR-Trx systems are functional in the cytosol, mitochondria, and plastids of nodules.

  16. Purification and characterization of Taenia crassiceps cysticerci thioredoxin: insight into thioredoxin-glutathione-reductase (TGR) substrate recognition.

    PubMed

    Martínez-González, J J; Guevara-Flores, A; Rendón, J L; Sosa-Peinado, A; Del Arenal Mena, I P

    2015-04-01

    Thioredoxin (Trx) is an oxidoreductase central to redox homeostasis in cells and is involved in the regulation of protein activity through thiol/disulfide exchanges. Based on these facts, our goal was to purify and characterize cytosolic thioredoxin from Taenia crassiceps cysticerci, as well as to study its behavior as a substrate of thioredoxin-glutathione reductase (TGR). The enzyme was purified >133-fold with a total yield of 9.7%. A molecular mass of 11.7kDa and a pI of 4.84 were measured. Native electrophoresis was used to identify the oxidized and reduced forms of the monomer as well as the presence of a homodimer. In addition to the catalytic site cysteines, cysticerci thioredoxin contains Cys28 and Cys65 residues conserved in previously sequenced cestode thioredoxins. The following kinetic parameters were obtained for the substrate of TGR: a Km of 3.1μM, a kcat of 10s(-1) and a catalytic efficiency of 3.2×10(6)M(-1)s(-1). The negative patch around the α3-helix of Trx is involved in the interaction with TGR and suggests variable specificity and catalytic efficiency of the reductase toward thioredoxins of different origins. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  17. JS-K, a Nitric Oxide Prodrug, Has Enhanced Cytotoxicity in Colon Cancer Cells with Knockdown of Thioredoxin Reductase 1

    PubMed Central

    Edes, Kornelia; Cassidy, Pamela; Shami, Paul J.; Moos, Philip J.

    2010-01-01

    Background The selenoenzyme thioredoxin reductase 1 has a complex role relating to cell growth. It is induced as a component of the cellular response to potentially mutagenic oxidants, but also appears to provide growth advantages to transformed cells by inhibiting apoptosis. In addition, selenocysteine-deficient or alkylated forms of thioredoxin reductase 1 have also demonstrated oxidative, pro-apoptotic activity. Therefore, a greater understanding of the role of thioredoxin reductase in redox initiated apoptotic processes is warranted. Methodology The role of thioredoxin reductase 1 in RKO cells was evaluated by attenuating endogenous thioredoxin reductase 1 expression with siRNA and then either inducing a selenium-deficient thioredoxin reductase or treatment with distinct redox challenges including, hydrogen peroxide, an oxidized lipid, 4-hydroxy-2-nonenol, and a nitric oxide donating prodrug. Thioredoxin redox status, cellular viability, and effector caspase activity were measured. Conclusions/Significance In cells with attenuated endogenous thioredoxin reductase 1, a stably integrated selenocysteine-deficient form of the enzyme was induced but did not alter either the thioredoxin redox status or the cellular growth kinetics. The oxidized lipid and the nitric oxide donor demonstrated enhanced cytotoxicity when thioredoxin reductase 1 was knocked-down; however, the effect was more pronounced with the nitric oxide prodrug. These results are consistent with the hypothesis that attenuation of the thioredoxin-system can promote apoptosis in a nitric oxide-dependent manner. PMID:20098717

  18. Cytosolic thioredoxin reductase 1 is required for correct disulfide formation in the ER.

    PubMed

    Poet, Greg J; Oka, Ojore Bv; van Lith, Marcel; Cao, Zhenbo; Robinson, Philip J; Pringle, Marie Anne; Arnér, Elias Sj; Bulleid, Neil J

    2017-03-01

    Folding of proteins entering the secretory pathway in mammalian cells frequently requires the insertion of disulfide bonds. Disulfide insertion can result in covalent linkages found in the native structure as well as those that are not, so-called non-native disulfides. The pathways for disulfide formation are well characterized, but our understanding of how non-native disulfides are reduced so that the correct or native disulfides can form is poor. Here, we use a novel assay to demonstrate that the reduction in non-native disulfides requires NADPH as the ultimate electron donor, and a robust cytosolic thioredoxin system, driven by thioredoxin reductase 1 (TrxR1 or TXNRD1). Inhibition of this reductive pathway prevents the correct folding and secretion of proteins that are known to form non-native disulfides during their folding. Hence, we have shown for the first time that mammalian cells have a pathway for transferring reducing equivalents from the cytosol to the ER, which is required to ensure correct disulfide formation in proteins entering the secretory pathway. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  19. The C-type Arabidopsis thioredoxin reductase ANTR-C acts as an electron donor to 2-Cys peroxiredoxins in chloroplasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Moon, Jeong Chan; Jang, Ho Hee; Chae, Ho Byoung

    2006-09-22

    2-Cys peroxiredoxins (Prxs) play important roles in the antioxidative defense systems of plant chloroplasts. In order to determine the interaction partner for these proteins in Arabidopsis, we used a yeast two-hybrid screening procedure with a C175S-mutant of Arabidopsis 2-Cys Prx-A as bait. A cDNA encoding an NADPH-dependent thioredoxin reductase (NTR) isotype C was identified and designated ANTR-C. We demonstrated that this protein effected efficient transfer of electrons from NADPH to the 2-Cys Prxs of chloroplasts. Interaction between 2-Cys Prx-A and ANTR-C was confirmed by a pull-down experiment. ANTR-C contained N-terminal TR and C-terminal Trx domains. It exhibited both TR andmore » Trx activities and co-localized with 2-Cys Prx-A in chloroplasts. These results suggest that ANTR-C functions as an electron donor for plastidial 2-Cys Prxs and represents the NADPH-dependent TR/Trx system in chloroplasts.« less

  20. Integration between anticipatory blocking and redox signaling by the peroxiredoxin/thioredoxin/thioredoxin-reductase system.

    PubMed

    Selvaggio, Gianluca; Coelho, Pedro M B M; Salvador, Armindo

    2014-10-01

    Cells are occasionally exposed to high H2O2 concentrations, often preceding exposure to other electrophylic compounds. Both H2O2 and these compounds can irreversibly modify protein thiols, with deleterious consequences. Induction of enzymatic defenses against those agents is too slow to avoid significant damage. Cells may solve this conundrum by reversibly "blocking" the thiols once H2O2 concentrations begin to increase. We term this mechanism "anticipatory blocking" because it acts in anticipation of irreversible damage upon detection of early signs of stress. Here we examine the design requirements for the Peroxiredoxin/Thioredoxin/Thioredoxin-Reductase/Protein-Dithiol System (PTTRDS) to effectively integrate H2O2 signaling and anticipatory blocking of protein dithiols as disulfides, and we compared them to the designs found in cells. To that effect, we developed a minimal model of the PTTRDS, and we defined a set of quantitative performance criteria that embody the requirements for (a) efficient scavenging capacity, (b) low NADPH consumption, (c) effective signal propagation, and (d) effective anticipatory blocking. We then sought the design principles (relationships among rate constants and species concentrations) that warrant fulfillment of all these criteria. Experimental data indicates that the design of the PTTRDS in human erythrocytes fulfills these principles and thus accomplishes effective integration between anticipatory blocking, antioxidant protection and redox signaling. A more general analysis suggests that the same principles hold in a wide variety of cell types and organisms. We acknowledge grants PEst-C/SAU/LA0001/2013-2014, PEst-OE/QUI/UI0612/2013, FCOMP-01-0124-FEDER-020978 (PTDC/QUI-BIQ/119657/2010) financed by FEDER through the "Programa Operacional Factores de Competitividade, COMPETE" and by national funds through "FCT, Fundação para a Ciência e a Tecnologia". Copyright © 2014. Published by Elsevier Inc.

  1. Structure and function of NADPH-cytochrome P450 reductase and nitric oxide synthase reductase domain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Iyanagi, Takashi

    2005-12-09

    NADPH-cytochrome P450 reductase (CPR) and the nitric oxide synthase (NOS) reductase domains are members of the FAD-FMN family of proteins. The FAD accepts two reducing equivalents from NADPH (dehydrogenase flavin) and FMN acts as a one-electron carrier (flavodoxin-type flavin) for the transfer from NADPH to the heme protein, in which the FMNH {sup {center_dot}}/FMNH{sub 2} couple donates electrons to cytochrome P450 at constant oxidation-reduction potential. Although the interflavin electron transfer between FAD and FMN is not strictly regulated in CPR, electron transfer is activated in neuronal NOS reductase domain upon binding calmodulin (CaM), in which the CaM-bound activated form canmore » function by a similar mechanism to that of CPR. The oxygenated form and spin state of substrate-bound cytochrome P450 in perfused rat liver are also discussed in terms of stepwise one-electron transfer from CPR. This review provides a historical perspective of the microsomal mixed-function oxidases including CPR and P450. In addition, a new model for the redox-linked conformational changes during the catalytic cycle for both CPR and NOS reductase domain is also discussed.« less

  2. The ferredoxin-thioredoxin system of a green alga, Chlamydomonas reinhardtii: identification and characterization of thioredoxins and ferredoxin-thioredoxin reductase components

    NASA Technical Reports Server (NTRS)

    Huppe, H. C.; de Lamotte-Guery, F.; Buchanan, B. B.

    1990-01-01

    The components of the ferredoxin-thioredoxin (FT) system of Chlamydomonas reinhardtii have been purified and characterized. The system resembled that of higher plants in consisting of a ferredoxin-thioredoxin reductase (FTR) and two types of thioredoxin, a single f and two m species, m1 and m2. The Chlamydomonas m and f thioredoxins were antigenically similar to their higher-plant counterparts, but not to one another. The m thioredoxins were recognized by antibodies to both higher plant m and bacterial thioredoxins, whereas the thioredoxin f was not. Chlamydomonas thioredoxin f reacted, although weakly, with the antibody to spinach thioredoxin f. The algal thioredoxin f differed from thioredoxins studied previously in behaving as a basic protein on ion-exchange columns. Purification revealed that the algal thioredoxins had molecular masses (Mrs) typical of thioredoxins from other sources, m1 and m2 being 10700 and f 11500. Chlamydomonas FTR had two dissimilar subunits, a feature common to all FTRs studied thus far. One, the 13-kDa ("similar") subunit, resembled its counterpart from other sources in both size and antigenicity. The other, 10-kDa ("variable") subunit was not recognized by antibodies to any FTR tested. When combined with spinach, (Spinacia oleracea L.) thylakoid membranes, the components of the FT system functioned in the light activation of the standard target enzymes from chloroplasts, corn (Zea mays L.) NADP-malate dehydrogenase (EC 1.1.1.82) and spinach fructose 1,6-bisphosphatase (EC 3.1.3.11) as well as the chloroplast-type fructose 1,6-bisphosphatase from Chlamydomonas. Activity was greatest if ferredoxin and other components of the FT system were from Chlamydomonas. The capacity of the Chlamydomonas FT system to activate autologous FBPase indicates that light regulates the photosynthetic carbon metabolism of green algae as in other oxygenic photosynthetic organisms.

  3. Peroxiredoxins and NADPH-Dependent Thioredoxin Systems in the Model Legume Lotus japonicus1[W][OA

    PubMed Central

    Tovar-Méndez, Alejandro; Matamoros, Manuel A.; Bustos-Sanmamed, Pilar; Dietz, Karl-Josef; Cejudo, Francisco Javier; Rouhier, Nicolas; Sato, Shusei; Tabata, Satoshi; Becana, Manuel

    2011-01-01

    Peroxiredoxins (Prxs), thioredoxins (Trxs), and NADPH-thioredoxin reductases (NTRs) constitute central elements of the thiol-disulfide redox regulatory network of plant cells. This study provides a comprehensive survey of this network in the model legume Lotus japonicus. The aims were to identify and characterize these gene families and to assess whether the NTR-Trx systems are operative in nodules. Quantitative reverse transcription-polymerase chain reaction and immunological and proteomic approaches were used for expression profiling. We identified seven Prx, 14 Trx, and three NTR functional genes. The PrxQ1 gene was found to be transcribed in two alternative spliced variants and to be expressed at high levels in leaves, stems, petals, pods, and seeds and at low levels in roots and nodules. The 1CPrx gene showed very high expression in the seed embryos and low expression in vegetative tissues and was induced by nitric oxide and cytokinins. In sharp contrast, cytokinins down-regulated all other Prx genes, except PrxQ1, in roots and nodules, but only 2CPrxA and PrxQ1 in leaves. Gene-specific changes in Prx expression were also observed in response to ethylene, abscisic acid, and auxins. Nodules contain significant mRNA and protein amounts of cytosolic PrxIIB, Trxh1, and NTRA and of plastidic NTRC. Likewise, they express cytosolic Trxh3, Trxh4, Trxh8, and Trxh9, mitochondrial PrxIIF and Trxo, and plastidic Trxm2, Trxm4, and ferredoxin-Trx reductase. These findings reveal a complex regulation of Prxs that is dependent on the isoform, tissue, and signaling molecule and support that redox NTR-Trx systems are functional in the cytosol, mitochondria, and plastids of nodules. PMID:21562331

  4. The logic of kinetic regulation in the thioredoxin system

    PubMed Central

    2011-01-01

    Background The thioredoxin system consisting of NADP(H), thioredoxin reductase and thioredoxin provides reducing equivalents to a large and diverse array of cellular processes. Despite a great deal of information on the kinetics of individual thioredoxin-dependent reactions, the kinetic regulation of this system as an integrated whole is not known. We address this by using kinetic modeling to identify and describe kinetic behavioral motifs found within the system. Results Analysis of a realistic computational model of the Escherichia coli thioredoxin system revealed several modes of kinetic regulation in the system. In keeping with published findings, the model showed that thioredoxin-dependent reactions were adaptable (i.e. changes to the thioredoxin system affected the kinetic profiles of these reactions). Further and in contrast to other systems-level descriptions, analysis of the model showed that apparently unrelated thioredoxin oxidation reactions can affect each other via their combined effects on the thioredoxin redox cycle. However, the scale of these effects depended on the kinetics of the individual thioredoxin oxidation reactions with some reactions more sensitive to changes in the thioredoxin cycle and others, such as the Tpx-dependent reduction of hydrogen peroxide, less sensitive to these changes. The coupling of the thioredoxin and Tpx redox cycles also allowed for ultrasensitive changes in the thioredoxin concentration in response to changes in the thioredoxin reductase concentration. We were able to describe the kinetic mechanisms underlying these behaviors precisely with analytical solutions and core models. Conclusions Using kinetic modeling we have revealed the logic that underlies the functional organization and kinetic behavior of the thioredoxin system. The thioredoxin redox cycle and associated reactions allows for a system that is adaptable, interconnected and able to display differential sensitivities to changes in this redox cycle. This

  5. ARSENICALS INHIBIT THIOREDOXIN REDUCTASE ACTIVITY IN CULTURED RAT HEPATOCYTES

    EPA Science Inventory

    ARSENICALS INHIBIT THIOREDOXIN REDUCTASE ACTIVITY IN CULTURED RAT HEPATOCYTES.

    S. Lin1, L. M. Del Razo1, M. Styblo1, C. Wang2, W. R. Cullen2, and D.J. Thomas3. 1Univ. North Carolina, Chapel Hill, NC; 2Univ. British Columbia, Vancouver, BC, Canada; 3National Health and En...

  6. New insights into the reduction systems of plastidial thioredoxins point out the unique properties of thioredoxin z from Arabidopsis.

    PubMed

    Bohrer, Anne-Sophie; Massot, Vincent; Innocenti, Gilles; Reichheld, Jean-Philippe; Issakidis-Bourguet, Emmanuelle; Vanacker, Hélène

    2012-11-01

    In plants, thioredoxins (TRX) constitute a large protein disulphide oxidoreductase family comprising 10 plastidial members in Arabidopsis thaliana and subdivided in five types. The f- and m-types regulate enzymes involved mainly in carbon metabolism whereas the x, y, and z types have an antioxidant function. The reduction of TRXm and f in chloroplasts is performed in the light by ferredoxin:thioredoxin reductase (FTR) that uses photosynthetically reduced ferredoxin (Fd) as a reductant. The reduction system of Arabidopsis TRXx, y, and z has never been demonstrated. Recently, a gene encoding an atypical plastidial NADPH-dependent TRX reductase (NTRC) was found. In the present study, gene expression analysis revealed that both reductases are expressed in all organs of Arabidopsis and could potentially serve as electron donors to plastidial TRX. This ability was tested in vitro either with purified NTRC in presence of NADPH or with a light-driven reconstituted system comprising thylakoids and purified Fd and FTR. The results demonstrate that FTR reduces the x and y TRX isoforms but not the recently identified TRXz. Moreover, the results show that NTRC cannot be an efficient alternative reducing system, neither for TRXz nor for the other plastidial TRX. The data reveal that TRXf, m, x, and y, known as redox regulators in the chloroplast, have also the ability to reduce TRXz in vitro. Overall, the present study points out the unique properties of TRXz among plastidial TRX.

  7. The contribution of NADPH thioredoxin reductase C (NTRC) and sulfiredoxin to 2-Cys peroxiredoxin overoxidation in Arabidopsis thaliana chloroplasts.

    PubMed

    Puerto-Galán, Leonor; Pérez-Ruiz, Juan M; Guinea, Manuel; Cejudo, Francisco Javier

    2015-05-01

    Hydrogen peroxide is a harmful by-product of photosynthesis, which also has important signalling activity. Therefore, the level of hydrogen peroxide needs to be tightly controlled. Chloroplasts harbour different antioxidant systems including enzymes such as the 2-Cys peroxiredoxins (2-Cys Prxs). Under oxidizing conditions, 2-Cys Prxs are susceptible to inactivation by overoxidation of their peroxidatic cysteine, which is enzymatically reverted by sulfiredoxin (Srx). In chloroplasts, the redox status of 2-Cys Prxs is highly dependent on NADPH-thioredoxin reductase C (NTRC) and Srx; however, the relationship of these activities in determining the level of 2-Cys Prx overoxidation is unknown. Here we have addressed this question by a combination of genetic and biochemical approaches. An Arabidopsis thaliana double knockout mutant lacking NTRC and Srx shows a phenotype similar to the ntrc mutant, while the srx mutant resembles wild-type plants. The deficiency of NTRC causes reduced overoxidation of 2-Cys Prxs, whereas the deficiency of Srx has the opposite effect. Moreover, in vitro analyses show that the disulfide bond linking the resolving and peroxidatic cysteines protects the latter from overoxidation, thus explaining the dominant role of NTRC on the level of 2-Cys Prx overoxidation in vivo. The overoxidation of chloroplast 2-Cys Prxs shows no circadian oscillation, in agreement with the fact that neither the NTRC nor the SRX genes show circadian regulation of expression. Additionally, the low level of 2-Cys Prx overoxidation in the ntrc mutant is light dependent, suggesting that the redox status of 2-Cys Prxs in chloroplasts depends on light rather than the circadian clock. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  8. Nitric oxide is required for the auxin-induced activation of NADPH-dependent thioredoxin reductase and protein denitrosylation during root growth responses in arabidopsis.

    PubMed

    Correa-Aragunde, Natalia; Cejudo, Francisco J; Lamattina, Lorenzo

    2015-09-01

    Auxin is the main phytohormone controlling root development in plants. This study uses pharmacological and genetic approaches to examine the role of auxin and nitric oxide (NO) in the activation of NADPH-dependent thioredoxin reductase (NTR), and the effect that this activity has on root growth responses in Arabidopsis thaliana. Arabidopsis seedlings were treated with auxin with or without the NTR inhibitors auranofin (ANF) and 1-chloro-2, 4-dinitrobenzene (DNCB). NTR activity, lateral root (LR) formation and S-nitrosothiol content were measured in roots. Protein S-nitrosylation was analysed by the biotin switch method in wild-type arabidopsis and in the double mutant ntra ntrb. The auxin-mediated induction of NTR activity is inhibited by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO), suggesting that NO is downstream of auxin in this regulatory pathway. The NTR inhibitors ANF and DNCB prevent auxin-mediated activation of NTR and LR formation. Moreover, ANF and DNCB also inhibit auxin-induced DR5 : : GUS and BA3 : : GUS gene expression, suggesting that the auxin signalling pathway is compromised without full NTR activity. Treatment of roots with ANF and DNCB increases total nitrosothiols (SNO) content and protein S-nitrosylation, suggesting a role of the NTR-thioredoxin (Trx)-redox system in protein denitrosylation. In agreement with these results, the level of S-nitrosylated proteins is increased in the arabidopsis double mutant ntra ntrb as compared with the wild-type. The results support for the idea that NTR is involved in protein denitrosylation during auxin-mediated root development. The fact that a high NO concentration induces NTR activity suggests that a feedback mechanism to control massive and unregulated protein S-nitrosylation could be operating in plant cells. © The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions

  9. Manumycin A Is a Potent Inhibitor of Mammalian Thioredoxin Reductase-1 (TrxR-1).

    PubMed

    Tuladhar, Anupama; Rein, Kathleen S

    2018-04-12

    The anticancer effect of manumycin A (Man A) has been attributed to the inhibition of farnesyl transferase (FTase), an enzyme that is responsible for post-translational modification of Ras proteins. However, we have discovered that Man A inhibits mammalian cytosolic thioredoxin reductase 1 (TrxR-1) in a time-dependent manner, with an IC 50 of 272 nM with preincubation and 1586 nM without preincubation. The inhibition of TrxR-1 by Man A is irreversible and is the result of a covalent interaction between Man A and TrxR-1. Evidence presented herein demonstrates that Man A forms a Michael adduct with the selenocysteine residue, which is located in the C-terminal redox center of TrxR-1. Inhibitors of TrxR-1, which act through this mechanism, convert TrxR-1 into a SecTRAP, which utilizes NADPH to reduce oxygen to superoxide radical anion (O 2 -• ).

  10. Docking into Mycobacterium tuberculosis Thioredoxin Reductase Protein Yields Pyrazolone Lead Molecules for Methicillin-Resistant Staphylococcus aureus

    PubMed Central

    Sweeney, Noreena L.; Lipker, Lauren; Hanson, Alicia M.; Bohl, Chris J.; Engel, Katie E.; Kalous, Kelsey S.; Stemper, Mary E.; Sem, Daniel S.; Schwan, William R.

    2017-01-01

    The thioredoxin/thioredoxin reductase system (Trx/TrxR) is an attractive drug target because of its involvement in a number of important physiological processes, from DNA synthesis to regulating signal transduction. This study describes the finding of pyrazolone compounds that are active against Staphylococcus aureus. Initially, the project was focused on discovering small molecules that may have antibacterial properties targeting the Mycobacterium tuberculosis thioredoxin reductase. This led to the discovery of a pyrazolone scaffold-containing compound series that showed bactericidal capability against S. aureus strains, including drug-resistant clinical isolates. The findings support continued development of the pyrazolone compounds as potential anti-S. aureus antibiotics. PMID:28134858

  11. Brevetoxin-2, is a unique inhibitor of the C-terminal redox center of mammalian thioredoxin reductase-1.

    PubMed

    Chen, Wei; Tuladhar, Anupama; Rolle, Shantelle; Lai, Yanhao; Rodriguez Del Rey, Freddy; Zavala, Cristian E; Liu, Yuan; Rein, Kathleen S

    2017-08-15

    Karenia brevis, the Florida red tide dinoflagellate produces a suite of neurotoxins known as the brevetoxins. The most abundant of the brevetoxins PbTx-2, was found to inhibit the thioredoxin-thioredoxin reductase system, whereas the PbTx-3 has no effect on this system. On the other hand, PbTx-2 activates the reduction of small disulfides such as 5,5'-dithio-bis-(2-nitrobenzoic acid) by thioredoxin reductase. PbTx-2 has an α, β-unsaturated aldehyde moiety which functions as an efficient electrophile and selenocysteine conjugates are readily formed. PbTx-2 blocks the inhibition of TrxR by the inhibitor curcumin, whereas curcumin blocks PbTx-2 activation of TrxR. It is proposed that the mechanism of inhibition of thioredoxin reduction is via the formation of a Michael adduct between selenocysteine and the α, β-unsaturated aldehyde moiety of PbTx-2. PbTx-2 had no effect on the rates of reactions catalyzed by related enzymes such as glutathione reductase, glutathione peroxidase or glutaredoxin. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation.

    PubMed

    Droux, M; Miginiac-Maslow, M; Jacquot, J P; Gadal, P; Crawford, N A; Kosower, N S; Buchanan, B B

    1987-07-01

    The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with [14C]iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.

  13. Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Droux, M.; Miginiac-Maslow, M.; Jacquot, J.P.

    The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with (/sup 14/C)iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn,more » reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.« less

  14. The ferredoxin-thioredoxin reductase variable subunit gene from Anacystis nidulans.

    PubMed

    Szekeres, M; Droux, M; Buchanan, B B

    1991-03-01

    The ferredoxin-thioredoxin reductase variable subunit gene of Anacystis nidulans was cloned, and its nucleotide sequence was determined. A single-copy 219-bp open reading frame encoded a protein of 73 amino acid residues, with a calculated Mr of 8,400. The monocistronic transcripts were represented in a 400-base and a less abundant 300-base mRNA form.

  15. Selenium as an electron acceptor during the catalytic mechanism of thioredoxin reductase.

    PubMed

    Lothrop, Adam P; Snider, Gregg W; Ruggles, Erik L; Patel, Amar S; Lees, Watson J; Hondal, Robert J

    2014-02-04

    Mammalian thioredoxin reductase (TR) is a pyridine nucleotide disulfide oxidoreductase that uses the rare amino acid selenocysteine (Sec) in place of the more commonly used amino acid cysteine (Cys) in the redox-active tetrapeptide Gly-Cys-Sec-Gly motif to catalyze thiol/disulfide exchange reactions. Sec can accelerate the rate of these exchange reactions (i) by being a better nucleophile than Cys, (ii) by being a better electrophile than Cys, (iii) by being a better leaving group than Cys, or (iv) by using a combination of all three of these factors, being more chemically reactive than Cys. The role of the selenolate as a nucleophile in the reaction mechanism was recently demonstrated by creating a mutant of human thioredoxin reductase-1 in which the Cys497-Sec498 dyad of the C-terminal redox center was mutated to either a Ser497-Cys498 dyad or a Cys497-Ser498 dyad. Both mutant enzymes were incubated with human thioredoxin (Trx) to determine which mutant formed a mixed disulfide bond complex. Only the mutant containing the Ser497-Cys498 dyad formed a complex, and this structure has been determined by X-ray crystallography [Fritz-Wolf, K., Kehr, S., Stumpf, M., Rahlfs, S., and Becker, K. (2011) Crystal structure of the human thioredoxin reductase-thioredoxin complex. Nat. Commun. 2, 383]. This experimental observation most likely means that the selenolate is the nucleophile initially attacking the disulfide bond of Trx because a complex resulted only when Cys was present in the second position of the dyad. As a nucleophile, the selenolate of Sec helps to accelerate the rate of this exchange reaction relative to Cys in the Sec → Cys mutant enzyme. Another thiol/disulfide exchange reaction that occurs in the enzymatic cycle of the enzyme is the transfer of electrons from the thiolate of the interchange Cys residue of the N-terminal redox center to the eight-membered selenosulfide ring of the C-terminal redox center. The selenium atom of the selenosulfide could

  16. Nitrate transport is independent of NADH and NAD(P)H nitrate reductases in barley seedlings

    NASA Technical Reports Server (NTRS)

    Warner, R. L.; Huffaker, R. C.

    1989-01-01

    Barley (Hordeum vulgare L.) has NADH-specific and NAD(P)H-bispecific nitrate reductase isozymes. Four isogenic lines with different nitrate reductase isozyme combinations were used to determine the role of NADH and NAD(P)H nitrate reductases on nitrate transport and assimilation in barley seedlings. Both nitrate reductase isozymes were induced by nitrate and were required for maximum nitrate assimilation in barley seedlings. Genotypes lacking the NADH isozyme (Az12) or the NAD(P)H isozyme (Az70) assimilated 65 or 85%, respectively, as much nitrate as the wild type. Nitrate assimilation by genotype (Az12;Az70) which is deficient in both nitrate reductases, was only 13% of the wild type indicating that the NADH and NAD(P)H nitrate reductase isozymes are responsible for most of the nitrate reduction in barley seedlings. For all genotypes, nitrate assimilation rates in the dark were about 55% of the rates in light. Hypotheses that nitrate reductase has direct or indirect roles in nitrate uptake were not supported by this study. Induction of nitrate transporters and the kinetics of net nitrate uptake were the same for all four genotypes indicating that neither nitrate reductase isozyme has a direct role in nitrate uptake in barley seedlings.

  17. The ferredoxin-thioredoxin reductase variable subunit gene from Anacystis nidulans.

    PubMed Central

    Szekeres, M; Droux, M; Buchanan, B B

    1991-01-01

    The ferredoxin-thioredoxin reductase variable subunit gene of Anacystis nidulans was cloned, and its nucleotide sequence was determined. A single-copy 219-bp open reading frame encoded a protein of 73 amino acid residues, with a calculated Mr of 8,400. The monocistronic transcripts were represented in a 400-base and a less abundant 300-base mRNA form. Images PMID:1705544

  18. Thioredoxin System from Deinococcus radiodurans

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Obiero, Josiah; Pittet, Vanessa; Bonderoff, Sara A.

    2010-05-03

    This paper describes the cloning, purification, and characterization of thioredoxin (Trx) and thioredoxin reductase (TrxR) and the structure determination of TrxR from the ionizing radiation-tolerant bacterium Deinococcus radiodurans strain R1. The genes from D. radiodurans encoding Trx and TrxR were amplified by PCR, inserted into a pET expression vector, and overexpressed in Escherichia coli. The overexpressed proteins were purified by metal affinity chromatography, and their activity was demonstrated using well-established assays of insulin precipitation (for Trx), 5,5{prime}-dithiobis(2-nitrobenzoic acid) (DTNB) reduction, and insulin reduction (for TrxR). In addition, the crystal structure of oxidized TrxR was determined at 1.9-{angstrom} resolution. The overallmore » structure was found to be very similar to that of E. coli TrxR and homodimeric with both NADPH- and flavin adenine dinucleotide (FAD)-binding domains containing variants of the canonical nucleotide binding fold, the Rossmann fold. The K{sub m} (5.7 {micro}M) of D. radiodurans TrxR for D. radiodurans Trx was determined and is about twofold higher than that of the E. coli thioredoxin system. However, D. radiodurans TrxR has a much lower affinity for E. coli Trx (K{sub m}, 44.4 {micro}M). Subtle differences in the surface charge and shape of the Trx binding site on TrxR may account for the differences in recognition. Because it has been suggested that TrxR from D. radiodurans may have dual cofactor specificity (can utilize both NADH and NADPH), D. radiodurans TrxR was tested for its ability to utilize NADH as well. Our results show that D. radiodurans TrxR can utilize only NADPH for activity.« less

  19. Fluorescence labelling of NADPH-cytochrome P-450 reductase with the monobromomethyl derivative of syn-9,10-dioxabimane.

    PubMed Central

    Vogel, F; Lumper, L

    1983-01-01

    The kinetics of thiol-group alkylation in NADPH-cytochrome P-450 reductase during its inactivation by monobromobimane has been studied using the fluorimetric determination of S-bimane-L-cysteine by high-performance liquid chromatography. Loss of activity during the reaction of NADPH-cytochrome P-450 reductase with monobromobimane is caused by the alkylation of one single critical cysteine residue, which can be protected against thiol-specific reagents by NADP(H). The chemical stability of the bimane group allows the digestion of bimane-labelled NADPH-cytochrome P-450 reductase by CNBr. The critical cysteine residue could be located in a CNBr-cleaved peptide purified to homogeneity with Mr 10 500 +/- 1 000 and valine as N-terminus. Images Fig. 2. PMID:6414464

  20. Nitrate Transport Is Independent of NADH and NAD(P)H Nitrate Reductases in Barley Seedlings 1

    PubMed Central

    Warner, Robert L.; Huffaker, Ray C.

    1989-01-01

    Barley (Hordeum vulgare L.) has NADH-specific and NAD(P)H-bispecific nitrate reductase isozymes. Four isogenic lines with different nitrate reductase isozyme combinations were used to determine the role of NADH and NAD(P)H nitrate reductases on nitrate transport and assimilation in barley seedlings. Both nitrate reductase isozymes were induced by nitrate and were required for maximum nitrate assimilation in barley seedlings. Genotypes lacking the NADH isozyme (Az12) or the NAD(P)H isozyme (Az70) assimilated 65 or 85%, respectively, as much nitrate as the wild type. Nitrate assimilation by genotype (Az12;Az70) which is deficient in both nitrate reductases, was only 13% of the wild type indicating that the NADH and NAD(P)H nitrate reductase isozymes are responsible for most of the nitrate reduction in barley seedlings. For all genotypes, nitrate assimilation rates in the dark were about 55% of the rates in light. Hypotheses that nitrate reductase has direct or indirect roles in nitrate uptake were not supported by this study. Induction of nitrate transporters and the kinetics of net nitrate uptake were the same for all four genotypes indicating that neither nitrate reductase isozyme has a direct role in nitrate uptake in barley seedlings. PMID:11537465

  1. The Anticancer Agent Chaetocin Is a Competitive Substrate and Inhibitor of Thioredoxin Reductase

    PubMed Central

    Tibodeau, Jennifer D.; Benson, Linda M.; Isham, Crescent R.; Owen, Whyte G.

    2009-01-01

    Abstract We recently reported that the antineoplastic thiodioxopiperazine natural product chaetocin potently induces cellular oxidative stress, thus selectively killing cancer cells. In pursuit of underlying molecular mechanisms, we now report that chaetocin is a competitive and selective substrate for the oxidative stress mitigation enzyme thioredoxin reductase-1 (TrxR1) with lower Km than the TrxR1 native substrate thioredoxin (Trx; chaetocin Km = 4.6 ± 0.6 μM, Trx Km = 104.7 ± 26 μM), thereby attenuating reduction of the critical downstream ROS remediation substrate Trx at achieved intracellular concentrations. Consistent with a role for TrxR1 targeting in the anticancer effects of chaetocin, overexpression of the TrxR1 downstream effector Trx in HeLa cells conferred resistance to chaetocin-induced, but not to doxorubicin-induced, cytotoxicity. As the TrxR/Trx pathway is of central importance in limiting cellular reactive oxygen species (ROS)—and as chaetocin exerts its selective anticancer effects via ROS imposition—the inhibition of TrxR1 by chaetocin has potential to explain its selective anticancer effects. These observations have important implications not just with regard to the mechanism of action and clinical development of chaetocin and related thiodioxopiperazines, but also with regard to the utility of molecular targets within the thioredoxin reductase/thioredoxin pathway in the development of novel candidate antineoplastic agents. Antioxid. Redox Signal. 11, 1097–1106. PMID:18999987

  2. Oligo-carrageenan kappa increases NADPH, ascorbate and glutathione syntheses and TRR/TRX activities enhancing photosynthesis, basal metabolism, and growth in Eucalyptus trees.

    PubMed

    González, Alberto; Moenne, Fabiola; Gómez, Melissa; Sáez, Claudio A; Contreras, Rodrigo A; Moenne, Alejandra

    2014-01-01

    In order to analyze the effect of OC kappa in redox status, photosynthesis, basal metabolism and growth in Eucalyptus globulus, trees were treated with water (control), with OC kappa at 1 mg mL(-1), or treated with inhibitors of NAD(P)H, ascorbate (ASC), and glutathione (GSH) syntheses and thioredoxin reductase (TRR) activity, CHS-828, lycorine, buthionine sulfoximine (BSO), and auranofin, respectively, and with OC kappa, and cultivated for 4 months. Treatment with OC kappa induced an increase in NADPH, ASC, and GSH syntheses, TRR and thioredoxin (TRX) activities, photosynthesis, growth and activities of basal metabolism enzymes such as rubisco, glutamine synthetase (GlnS), adenosine 5'-phosphosulfate reductase (APR), involved in C, N, and S assimilation, respectively, Krebs cycle and purine/pyrimidine synthesis enzymes. Treatment with inhibitors and OC kappa showed that increases in ASC, GSH, and TRR/TRX enhanced NADPH synthesis, increases in NADPH and TRR/TRX enhanced ASC and GSH syntheses, and only the increase in NADPH enhanced TRR/TRX activities. In addition, the increase in NADPH, ASC, GSH, and TRR/TRX enhanced photosynthesis and growth. Moreover, the increase in NADPH, ASC and TRR/TRX enhanced activities of rubisco, Krebs cycle, and purine/pyrimidine synthesis enzymes, the increase in GSH, NADPH, and TRR/TRX enhanced APR activity, and the increase in NADPH and TRR/TRX enhanced GlnS activity. Thus, OC kappa increases NADPH, ASC, and GSH syntheses leading to a more reducing redox status, the increase in NADPH, ASC, GSH syntheses, and TRR/TRX activities are cross-talking events leading to activation of photosynthesis, basal metabolism, and growth in Eucalyptus trees.

  3. Crystal structure of conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 complexed with NADPH.

    PubMed

    Qin, Hui-Min; Yamamura, Akihiro; Miyakawa, Takuya; Kataoka, Michihiko; Maruoka, Shintaro; Ohtsuka, Jun; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2013-11-01

    Conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 is a member of the aldo-keto reductase (AKR) superfamily and reduces ketopantoyl lactone to d-pantoyl lactone in a NADPH-dependent and stereospecific manner. We determined the crystal structure of CPR-C1.NADPH complex at 2.20 Å resolution. CPR-C1 adopted a triose-phosphate isomerase (TIM) barrel fold at the core of the structure in which Thr25 and Lys26 of the GXGTX motif bind uniquely to the adenosine 2'-phosphate group of NADPH. This finding provides a novel structural basis for NADPH binding of the AKR superfamily. Copyright © 2013 Wiley Periodicals, Inc.

  4. Thioredoxin: a key regulator of cardiovascular homeostasis.

    PubMed

    Yamawaki, Hideyuki; Haendeler, Judith; Berk, Bradford C

    2003-11-28

    The thioredoxin (TRX) system (TRX, TRX reductase, and NADPH) is a ubiquitous thiol oxidoreductase system that regulates cellular reduction/oxidation (redox) status. The oxidation mechanism for disease pathogenesis states that an imbalance in cell redox state alters function of multiple cell pathways. In this study, we review the essential role for TRX to limit oxidative stress directly via antioxidant effects and indirectly by protein-protein interaction with key signaling molecules, such as apoptosis signal-regulating kinase 1. We propose that TRX and its endogenous regulators are important future targets to develop clinical therapies for cardiovascular disorders associated with oxidative stress.

  5. Trichomonas vaginalis: metronidazole and other nitroimidazole drugs are reduced by the flavin enzyme thioredoxin reductase and disrupt the cellular redox system. Implications for nitroimidazole toxicity and resistance.

    PubMed

    Leitsch, David; Kolarich, Daniel; Binder, Marina; Stadlmann, Johannes; Altmann, Friedrich; Duchêne, Michael

    2009-04-01

    Infections with the microaerophilic parasite Trichomonas vaginalis are treated with the 5-nitroimidazole drug metronidazole, which is also in use against Entamoeba histolytica, Giardia intestinalis and microaerophilic/anaerobic bacteria. Here we report that in T. vaginalis the flavin enzyme thioredoxin reductase displays nitroreductase activity with nitroimidazoles, including metronidazole, and with the nitrofuran drug furazolidone. Reactive metabolites of metronidazole and other nitroimidazoles form covalent adducts with several proteins that are known or assumed to be associated with thioredoxin-mediated redox regulation, including thioredoxin reductase itself, ribonucleotide reductase, thioredoxin peroxidase and cytosolic malate dehydrogenase. Disulphide reducing activity of thioredoxin reductase was greatly diminished in extracts of metronidazole-treated cells and intracellular non-protein thiol levels were sharply decreased. We generated a highly metronidazole-resistant cell line that displayed only minimal thioredoxin reductase activity, not due to diminished expression of the enzyme but due to the lack of its FAD cofactor. Reduction of free flavins, readily observed in metronidazole-susceptible cells, was also absent in the resistant cells. On the other hand, iron-depleted T. vaginalis cells, expressing only minimal amounts of PFOR and hydrogenosomal malate dehydrogenase, remained fully susceptible to metronidazole. Thus, taken together, our data suggest a flavin-based mechanism of metronidazole activation and thereby challenge the current model of hydrogenosomal activation of nitroimidazole drugs.

  6. Dimethoxycurcumin, a metabolically stable analogue of curcumin enhances the radiosensitivity of cancer cells: Possible involvement of ROS and thioredoxin reductase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jayakumar, Sundarraj; Patwardhan, R.S.; Pal, Debojyoti

    Dimethoxycurcumin (DIMC), a structural analogue of curcumin, has been shown to have more stability, bioavailability, and effectiveness than its parent molecule curcumin. In this paper the radiosensitizing effect of DIMC has been investigated in A549 lung cancer cells. As compared to its parent molecule curcumin, DIMC showed a very potent radiosensitizing effect as seen by clonogenic survival assay. DIMC in combination with radiation significantly increased the apoptosis and mitotic death in A549 cells. This combinatorial treatment also lead to effective elimination of cancer stem cells. Further, there was a significant increase in cellular ROS, decrease in GSH to GSSG ratiomore » and also significant slowdown in DNA repair when DIMC was combined with radiation. In silico docking studies and in vitro studies showed inhibition of thioredoxin reductase enzyme by DIMC. Overexpression of thioredoxin lead to the abrogation of radiosensitizing effect of DIMC underscoring the role of thioredoxin reductase in radiosensitization. Our results clearly demonstrate that DIMC can synergistically enhance the cancer cell killing when combined with radiation by targeting thioredoxin system. - Highlights: • DIMC enhances radiosensitivity of cancer cells by inducing cell death. • DIMC with radiation disrupted the cellular redox and targeted cancer stem cells. • DNA repair is hampered when cells are treated with DIMC. • DIMC inhibited thioredoxin reductase in cancer cells.« less

  7. G6pd Deficiency Does Not Affect the Cytosolic Glutathione or Thioredoxin Antioxidant Defense in Mouse Cochlea.

    PubMed

    White, Karessa; Kim, Mi-Jung; Ding, Dalian; Han, Chul; Park, Hyo-Jin; Meneses, Zaimary; Tanokura, Masaru; Linser, Paul; Salvi, Richard; Someya, Shinichi

    2017-06-07

    Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the pentose phosphate pathway; it catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconate and NADP + to NADPH and is thought to be the principal source of NADPH for the cytosolic glutathione and thioredoxin antioxidant defense systems. We investigated the roles of G6PD in the cytosolic antioxidant defense in the cochlea of G6pd hypomorphic mice that were backcrossed onto normal-hearing CBA/CaJ mice. Young G6pd -deficient mice displayed a significant decrease in cytosolic G6PD protein levels and activities in the inner ears. However, G6pd deficiency did not affect the cytosolic NADPH redox state, or glutathione or thioredoxin antioxidant defense in the inner ears. No histological abnormalities or oxidative damage was observed in the cochlea of G6pd hemizygous males or homozygous females. Furthermore, G6pd deficiency did not affect auditory brainstem response hearing thresholds, wave I amplitudes or wave I latencies in young males or females. In contrast, G6pd deficiency resulted in increased activities and protein levels of cytosolic isocitrate dehydrogenase 1, an enzyme that catalyzes the conversion of isocitrate to α-ketoglutarate and NADP + to NADPH, in the inner ear. In a mouse inner ear cell line, knockdown of Idh1 , but not G6pd , decreased cell growth rates, cytosolic NADPH levels, and thioredoxin reductase activities. Therefore, under normal physiological conditions, G6pd deficiency does not affect the cytosolic glutathione or thioredoxin antioxidant defense in mouse cochlea. Under G6pd deficiency conditions, isocitrate dehydrogenase 1 likely functions as the principal source of NADPH for cytosolic antioxidant defense in the cochlea. SIGNIFICANCE STATEMENT Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the pentose phosphate pathway; it catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconate and NADP + to NADPH and

  8. A 1,536-Well-Based Kinetic HTS Assay for Inhibitors of Schistosoma mansoni Thioredoxin Glutathione Reductase

    PubMed Central

    Lea, Wendy A.; Jadhav, Ajit; Rai, Ganesha; Sayed, Ahmed A.; Cass, Cynthia L.; Inglese, James; Williams, David L.; Austin, Christopher P.

    2008-01-01

    Abstract Schistosomiasis is a major neglected tropical disease that currently affects over 200 million people and leads to over 200,000 annual deaths. Schistosoma mansoni parasites survive in humans in part because of a set of antioxidant enzymes that continuously degrade reactive oxygen species produced by the host. A principal component of this defense system has been recently identified as thioredoxin glutathione reductase (TGR), a parasite-specific enzyme that combines the functions of two human counterparts, glutathione reductase and thioredoxin reductase, and as such this enzyme presents an attractive new target for anti-schistosomiasis drug development. Herein, we present the development of a highly miniaturized and robust screening assay for TGR. The 5-μl final volume assay is based on the Ellman reagent [5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)] and utilizes a high-speed absorbance kinetic read to minimize the effect of dust, absorbance interference, and meniscus variation. This assay is further applicable to the testing of other redox enzymes that utilize DTNB as a model substrate. PMID:18665782

  9. Isolation and Characterization of a Soluble NADPH-Dependent Fe(III) Reductase from Geobacter sulfurreducens

    PubMed Central

    Kaufmann, Franz; Lovley, Derek R.

    2001-01-01

    NADPH is an intermediate in the oxidation of organic compounds coupled to Fe(III) reduction in Geobacter species, but Fe(III) reduction with NADPH as the electron donor has not been studied in these organisms. Crude extracts of Geobacter sulfurreducens catalyzed the NADPH-dependent reduction of Fe(III)-nitrilotriacetic acid (NTA). The responsible enzyme, which was recovered in the soluble protein fraction, was purified to apparent homogeneity in a four-step procedure. Its specific activity for Fe(III) reduction was 65 μmol · min−1 · mg−1. The soluble Fe(III) reductase was specific for NADPH and did not utilize NADH as an electron donor. Although the enzyme reduced several forms of Fe(III), Fe(III)-NTA was the preferred electron acceptor. The protein possessed methyl viologen:NADP+ oxidoreductase activity and catalyzed the reduction of NADP+ with reduced methyl viologen as electron donor at a rate of 385 U/mg. The enzyme consisted of two subunits with molecular masses of 87 and 78 kDa and had a native molecular mass of 320 kDa, as determined by gel filtration. The purified enzyme contained 28.9 mol of Fe, 17.4 mol of acid-labile sulfur, and 0.7 mol of flavin adenine dinucleotide per mol of protein. The genes encoding the two subunits were identified in the complete sequence of the G. sulfurreducens genome from the N-terminal amino acid sequences derived from the subunits of the purified protein. The sequences of the two subunits had about 30% amino acid identity to the respective subunits of the formate dehydrogenase from Moorella thermoacetica, but the soluble Fe(III) reductase did not possess formate dehydrogenase activity. This soluble Fe(III) reductase differs significantly from previously characterized dissimilatory and assimilatory Fe(III) reductases in its molecular composition and cofactor content. PMID:11443080

  10. Brevetoxin (PbTx-2) influences the redox status and NPQ of Karenia brevis by way of thioredoxin reductase.

    PubMed

    Chen, Wei; Colon, Ricardo; Louda, J William; Del Rey, Freddy Rodriguez; Durham, Michaella; Rein, Kathleen S

    2018-01-01

    The Florida red tide dinoflagellate, Karenia brevis, is the major harmful algal bloom dinoflagellate of the Gulf of Mexico and plays a destructive role in the region. Blooms of K. brevis can produce brevetoxins: ladder-shaped polyether (LSP) compounds, which can lead to adverse human health effects, such as reduced respiratory function through inhalation exposure, or neurotoxic shellfish poisoning through consumption of contaminated shellfish. The endogenous role of the brevetoxins remains uncertain. Recent work has shown that some forms of NADPH dependent thioredoxin reductase (NTR) are inhibited by brevetoxin-2 (PbTx-2). The study presented herein reveals that high toxin and low toxin K. brevis, which have a ten-fold difference in toxin content, also show a significant difference in their ability, not only to produce brevetoxin, but also in their cellular redox status and distribution of xanthophyll cycle pigments. These differences are likely due to the inhibition of NTR by brevetoxin. The work could shed light on the physiological role that brevetoxin fills for K. brevis. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Curcumin targeting the thioredoxin system elevates oxidative stress in HeLa cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cai, Wenqing; Zhang, Baoxin; Duan, Dongzhu

    2012-08-01

    The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, is ubiquitous in all cells and involved in many redox-dependent signaling pathways. Curcumin, a naturally occurring pigment that gives a specific yellow color in curry food, is consumed in normal diet up to 100 mg per day. This molecule has also been used in traditional medicine for the treatment of a variety of diseases. Curcumin has numerous biological functions, and many of these functions are related to induction of oxidative stress. However, how curcumin elicits oxidative stress in cells is unclear. Our previous work has demonstrated the waymore » by which curcumin interacts with recombinant TrxR1 and alters the antioxidant enzyme into a reactive oxygen species (ROS) generator in vitro. Herein we reported that curcumin can target the cytosolic/nuclear thioredoxin system to eventually elevate oxidative stress in HeLa cells. Curcumin-modified TrxR1 dose-dependently and quantitatively transfers electrons from NADPH to oxygen with the production of ROS. Also, curcumin can drastically down-regulate Trx1 protein level as well as its enzyme activity in HeLa cells, which in turn remarkably decreases intracellular free thiols, shifting the intracellular redox balance to a more oxidative state, and subsequently induces DNA oxidative damage. Furthermore, curcumin-pretreated HeLa cells are more sensitive to oxidative stress. Knockdown of TrxR1 sensitizes HeLa cells to curcumin cytotoxicity, highlighting the physiological significance of targeting TrxR1 by curcumin. Taken together, our data disclose a previously unrecognized prooxidant mechanism of curcumin in cells, and provide a deep insight in understanding how curcumin works in vivo. -- Highlights: ► Curcumin induces oxidative stress by targeting the thioredoxin system. ► Curcumin-modified TrxR quantitatively oxidizes NADPH to generate ROS. ► Knockdown of TrxR1 augments curcumin's cytotoxicity in HeLa cells.

  12. Selenite reduction by the thioredoxin system: kinetics and identification of protein-bound selenide.

    PubMed

    Tamura, Takashi; Sato, Kumi; Komori, Kentaro; Imai, Takeshi; Kuwahara, Mitsuhiko; Okugochi, Takahiro; Mihara, Hisaaki; Esaki, Nobuyoshi; Inagaki, Kenji

    2011-01-01

    Selenite (SeO(3)(2-)) assimilation into a bacterial selenoprotein depends on thioredoxin (trx) reductase in Esherichia coli, but the molecular mechanism has not been elucidated. The mineral-oil overlay method made it possible to carry out anaerobic enzyme assay, which demonstrated an initial lag-phase followed by time-dependent steady NADPH consumption with a positive cooperativity toward selenite and trx. SDS-PAGE/autoradiography using (75)Se-labeled selenite as substrate revealed the formation of trx-bound selenium in the reaction mixture. The protein-bound selenium has metabolic significance in being stabilized in the divalent state, and it also produced the selenopersulfide (-S-SeH) form by the catalysis of E. coli trx reductase (TrxB).

  13. Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding.

    PubMed

    Qin, Hui-Min; Yamamura, Akihiro; Miyakawa, Takuya; Kataoka, Michihiko; Nagai, Takahiro; Kitamura, Nahoko; Urano, Nobuyuki; Maruoka, Shintaro; Ohtsuka, Jun; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2014-01-01

    Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to D-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2'-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.

  14. Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania).

    PubMed Central

    Crankshaw, D L; Hetnarski, K; Wilkinson, C F

    1979-01-01

    1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes. Images Fig. 3. PMID:117798

  15. Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania).

    PubMed

    Crankshaw, D L; Hetnarski, K; Wilkinson, C F

    1979-09-01

    1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes.

  16. The crystal structure of NADPH:ferredoxin reductase from Azotobacter vinelandii.

    PubMed Central

    Sridhar Prasad, G.; Kresge, N.; Muhlberg, A. B.; Shaw, A.; Jung, Y. S.; Burgess, B. K.; Stout, C. D.

    1998-01-01

    NADPH:ferredoxin reductase (AvFPR) is involved in the response to oxidative stress in Azotobacter vinelandii. The crystal structure of AvFPR has been determined at 2.0 A resolution. The polypeptide fold is homologous with six other oxidoreductases whose structures have been solved including Escherichia coli flavodoxin reductase (EcFldR) and spinach, and Anabaena ferredoxin:NADP+ reductases (FNR). AvFPR is overall most homologous to EcFldR. The structure is comprised of a N-terminal six-stranded antiparallel beta-barrel domain, which binds FAD, and a C-terminal five-stranded parallel beta-sheet domain, which binds NADPH/NADP+ and has a classical nucleotide binding fold. The two domains associate to form a deep cleft where the NADPH and FAD binding sites are juxtaposed. The structure displays sequence conserved motifs in the region surrounding the two dinucleotide binding sites, which are characteristic of the homologous enzymes. The folded over conformation of FAD in AvFPR is similar to that in EcFldR due to stacking of Phe255 on the adenine ring of FAD, but it differs from that in the FNR enzymes, which lack a homologous aromatic residue. The structure of AvFPR displays three unique features in the environment of the bound FAD. Two features may affect the rate of reduction of FAD: the absence of an aromatic residue stacked on the isoalloxazine ring in the NADPH binding site; and the interaction of a carbonyl group with N10 of the flavin. Both of these features are due to the substitution of a conserved C-terminal tyrosine residue with alanine (Ala254) in AvFPR. An additional unique feature may affect the interaction of AvFPR with its redox partner ferredoxin I (FdI). This is the extension of the C-terminus by three residues relative to EcFldR and by four residues relative to FNR. The C-terminal residue, Lys258, interacts with the AMP phosphate of FAD. Consequently, both phosphate groups are paired with a basic group due to the simultaneous interaction of the FMN

  17. Expression, purification, crystallization and preliminary X-ray crystallographic studies of Deinococcus radiodurans thioredoxin reductase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Obiero, Josiah; Bonderoff, Sara A.; Goertzen, Meghan M.

    2006-08-01

    Recombinant D. radiodurans TrxR with a His tag at the N-terminus was expressed in Escherichia coli and purified by metal-affinity chromatography. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of 35% PEG 4000, 0.2 M ammonium acetate and citric acid buffer pH 5.1 at 293 K. Deinococcus radiodurans, a Gram-positive bacterium capable of withstanding extreme ionizing radiation, contains two thioredoxins (Trx and Trx1) and a single thioredoxin reductase (TrxR) as part of its response to oxidative stress. Thioredoxin reductase is a member of the family of pyridine nucleotide-disulfide oxidoreductase flavoenzymes. Recombinant D. radiodurans TrxR with amore » His tag at the N-terminus was expressed in Escherichia coli and purified by metal-affinity chromatography. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of 35% PEG 4000, 0.2 M ammonium acetate and citric acid buffer pH 5.1 at 293 K. X-ray diffraction data were collected on a cryocooled crystal to a resolution of 1.9 Å using a synchrotron-radiation source. The space group was determined to be P3{sub 2}21, with unit-cell parameters a = b = 84.33, c = 159.88 Å. The structure of the enzyme has been solved by molecular-replacement methods and structure refinement is in progress.« less

  18. Ferredoxin:thioredoxin reductase (FTR) links the regulation of oxygenic photosynthesis to deeply rooted bacteria.

    PubMed

    Balsera, Monica; Uberegui, Estefania; Susanti, Dwi; Schmitz, Ruth A; Mukhopadhyay, Biswarup; Schürmann, Peter; Buchanan, Bob B

    2013-02-01

    Uncovered in studies on photosynthesis 35 years ago, redox regulation has been extended to all types of living cells. We understand a great deal about the occurrence, function, and mechanism of action of this mode of regulation, but we know little about its origin and its evolution. To help fill this gap, we have taken advantage of available genome sequences that make it possible to trace the phylogenetic roots of members of the system that was originally described for chloroplasts-ferredoxin, ferredoxin:thioredoxin reductase (FTR), and thioredoxin as well as target enzymes. The results suggest that: (1) the catalytic subunit, FTRc, originated in deeply rooted microaerophilic, chemoautotrophic bacteria where it appears to function in regulating CO(2) fixation by the reverse citric acid cycle; (2) FTRc was incorporated into oxygenic photosynthetic organisms without significant structural change except for addition of a variable subunit (FTRv) seemingly to protect the Fe-S cluster against oxygen; (3) new Trxs and target enzymes were systematically added as evolution proceeded from bacteria through the different types of oxygenic photosynthetic organisms; (4) an oxygenic type of regulation preceded classical light-dark regulation in the regulation of enzymes of CO(2) fixation by the Calvin-Benson cycle; (5) FTR is not universally present in oxygenic photosynthetic organisms, and in certain early representatives is seemingly functionally replaced by NADP-thioredoxin reductase; and (6) FTRc underwent structural diversification to meet the ecological needs of a variety of bacteria and archaea.

  19. Positive correlation between decreased cellular uptake, NADPH-glutathione reductase activity and adriamycin resistance in Ehrlich ascites tumor lines.

    PubMed

    Scheulen, M E; Hoensch, H; Kappus, H; Seeber, S; Schmidt, C G

    1987-01-01

    From a wild type strain of Ehrlich ascites tumor (EATWT) sublines resistant to daunorubicin (EATDNM), etoposide (EATETO), and cisplatinum (EATCIS) have been developed in vivo. Increase in survival and cure rate caused by adriamycin (doxorubicin) have been determined in female NMRI mice which were inoculated i.p. with EAT cells. Adriamycin concentrations causing 50% inhibition of 3H-thymidine (ICT) and 3H-uridine incorporation (ICU) and intracellular adriamycin steady-state concentrations (SSC) were measured in vitro. Adriamycin resistance increased and SSC decreased in the following sequence: EATWT - EATCIS - EATDNM - EATETO. When ICT and ICU were corrected for intracellular adriamycin concentrations in consideration of the different SSC (ICTc, ICUc), ICTc and ICUc still varied up to the 3.2 fold in EATCIS, EATDNM and EATETO in comparison to EATWT. Thus, in addition to different SSC other factors must be responsible for adriamycin resistance. Therefore, enzymes which may play a role in the cytotoxicity related to adriamycin metabolism (NADPH-cytochrome P-450 reductase, NADPH-glutathione reductase, NADP-glucose-6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase) were measured. In contrast to the other parameters determined, NADPH-glutathione reductase was significantly (p less than 0.01) increased up to the 3.2 fold parallel to adriamycin resistance as determined by increase in life span, cure rate, ICTc, and ICUc, respectively. It is concluded that high activities of NADPH-glutathione reductase may contribute to an increase in adriamycin resistance of malignant tumors.

  20. Enhanced Purification of Recombinant Rat NADPH-P450 Reductase by Using a Hexahistidine-Tag.

    PubMed

    Park, Hyoung-Goo; Lim, Young-Ran; Han, Songhee; Jeong, Dabin; Kim, Donghak

    2017-05-28

    NADPH-P450 reductase (NPR) transfers electrons from NADPH to cytochrome P450 and heme oxygenase enzymes to support their catalytic activities. This protein is localized within the endoplasmic reticulum membrane and utilizes FMN, FAD, and NADPH as cofactors. Although NPR is essential toward enabling the biochemical and pharmacological analyses of P450 enzymes, its production as a recombinant purified protein requires a series of tedious efforts and a high cost due to the use of NADP + in the affinity chromatography process. In the present study, the rat NPR clone containing a 6× Histidine-tag (NPR-His) was constructed and heterologously expressed. The NPR-His protein was purified using Ni 2+ -affinity chromatography, and its functional features were characterized. A single band at 78 kDa was observed from SDS-PAGE and the purified protein displayed a maximum absorbance at 455 nm, indicating the presence of an oxidized flavin cofactor. Cytochrome c and nitroblue tetrazolium were reduced by purified NPR-His in an NADPH-dependent manner. The purified NPR-His successfully supported the catalytic activities of human P450 1A2 and 2A6 and fungal CYP52A21, yielding results similar to those obtained using conventional purified rat reductase. This study will facilitate the use of recombinant NPR-His protein in the various fields of P450 research.

  1. Thioredoxin Glutathione Reductase as a Novel Drug Target: Evidence from Schistosoma japonicum

    PubMed Central

    Xie, ShuYing; Qian, ChunYan; Wang, Jie; Zhang, Wei; Yin, XuRen; Hua, ZiChun; Yu, ChuanXin

    2012-01-01

    Background Schistosomiasis remains a major public health concern affecting billions of people around the world. Currently, praziquantel is the only drug of choice for treatment of human schistosomiasis. The emergence of drug resistance to praziquantel in schistosomes makes the development of novel drugs an urgent task. Thioredoxin glutathione reductase (TGR) enzymes in Schistosoma mansoni and some other platyhelminths have been identified as alternative targets. The present study was designed to confirm the existense and the potential value of TGR as a target for development of novel antischistosomal agents in Schistosoma japonicum, a platyhelminth endemic in Asia. Methods and Findings After cloning the S. japonicum TGR (SjTGR) gene, the recombinant SjTGR selenoprotein was purified and characterized in enzymatic assays as a multifunctional enzyme with thioredoxin reductase (TrxR), glutathione reductase (GR) and glutaredoxin (Grx) activities. Immunological and bioinformatic analyses confirmed that instead of having separate TrxR and GR proteins in mammalian, S. japonicum only encodes TGR, which performs the functions of both enzymes and plays a critical role in maintaining the redox balance in this parasite. These results were in good agreement with previous findings in Schistosoma mansoni and some other platyhelminths. Auranofin, a known inhibitor against TGR, caused fatal toxicity in S. japonicum adult worms in vitro and reduced worm and egg burdens in S. japonicum infected mice. Conclusions Collectively, our study confirms that a multifunctional enzyme SjTGR selenoprotein, instead of separate TrxR and GR enzymes, exists in S. japonicum. Furthermore, TGR may be a potential target for development of novel agents against schistosomes. This assumption is strengthened by our demonstration that the SjTGR is an essential enzyme for maintaining the thiol-disulfide redox homeostasis of S. japonicum. PMID:22384025

  2. Functional expression and characterization of recombinant NADPH-P450 reductase from Malassezia globosa.

    PubMed

    Lee, Hwayoun; Park, Hyoung-Goo; Lim, Young-Ran; Lee, Im-Soon; Kim, Beom Joon; Seong, Cheul-Hun; Chun, Young-Jin; Kim, Donghak

    2012-01-01

    Malassezia globosa is a common pathogenic fungus that causes skin diseases including dandruff and seborrheic dermatitis in humans. Analysis of its genome identified a gene (MGL_1677) coding for a putative NADPH-P450 reductase (NPR) to support the fungal cytochrome P450 enzymes. The heterologously expressed recombinant M. globosa NPR protein was purified, and its functional features were characterized. The purified protein generated a single band on SDS-PAGE at 80.74 kDa and had an absorption maximum at 452 nm, indicating its possible function as an oxidized flavin cofactor. It evidenced NADPH-dependent reducing activity for cytochrome c or nitroblue tetrazolium. Human P450 1A2 and 2A6 were able to successfully catalyze the O-deethylation of 7- ethoxyresorufin and the 7-hydroxylation of coumarin, respectively, with the support of the purified NPR. These results demonstrate that purified NPR is an orthologous reductase protein that supports cytochrome P450 enzymes in M. globosa.

  3. Molecular cloning and characterization of Fasciola gigantica thioredoxin-glutathione reductase.

    PubMed

    Changklungmoa, Narin; Kueakhai, Pornanan; Sangpairoj, Kant; Chaichanasak, Pannigan; Jaikua, Wipaphorn; Riengrojpitak, Suda; Sobhon, Prasert; Chaithirayanon, Kulathida

    2015-06-01

    The Fasciola gigantica thioredoxin-glutathione reductase (FgTGR) gene is a fusion between thioredoxin reductase (TR) and a glutaredoxin (Grx) gene. FgTGR was cloned by polymerase chain reaction (PCR) from adult complementary DNA (cDNA), and its sequences showed two isoforms, i.e., the cytosolic and mitochondrial FgTGR. Cytosolic FgTGR (cytFgTGR) was composed of 2370 bp, and its peptide had no signal sequence and hence was not a secreted protein. Mitochondrial FgTGR (mitFgTGR) was composed of 2506 bp with a signal peptide of 43 amino acids; therefore, it was a secreted protein. The putative cytFgTGR and mitFgTGR peptides comprised of 598 and 641 amino acids, respectively, with a molecular weight of 65.8 kDa for cytFgTGR and mitFgTGR, with a conserved sequence (CPYC) of TR, and ACUG and CVNVGC of Grx domains. The recombinant FgTGR (rFgTGR) was expressed in Escherichia coli BL21 (DE3) and used for production for a polyclonal antibody in rabbits (anti-rFgTGR). The FgTGR protein expression, estimated by indirect ELISA using the rabbit anti-rFgTGR as probe, showed high levels of expression in eggs, and 2- and 4-week-old juveniles and adults. The rFgTGR exhibited specific activities in the 5,5'-dithiobis (2-nitro-benzoic acid) (DTNB) reductase assay for TR activity and in β-hydroxyethul disulfide (HED) for Grx activity. When analyzed by immunoblotting and immunohistochemistry, rabbit anti-rFgTGR reacted with natural FgTGR at a molecular weight of 66 kDa from eggs, whole body fraction (WB) of metacercariae, NEJ, 2- and 4-week-old juveniles and adults, and the tegumental antigen (TA) of adult. The FgTGR protein was expressed at high levels in the tegument of 2- and 4-week-old juveniles. The FgTGR may be one of the major factors acting against oxidative stresses that can damage the parasite; hence, it could be considered as a novel vaccine or a drug target.

  4. Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1.

    PubMed

    Randall, Matthew J; Spiess, Page C; Hristova, Milena; Hondal, Robert J; van der Vliet, Albert

    2013-01-01

    Cigarette smoking remains a major health concern worldwide, and many of the adverse effects of cigarette smoke (CS) can be attributed to its abundant electrophilic aldehydes, such as acrolein (2-propenal). Previous studies indicate that acrolein readily reacts with thioredoxin reductase 1 (TrxR1), a critical enzyme involved in regulation of thioredoxin (Trx)-mediated redox signaling, by alkylation at its selenocysteine (Sec) residue. Because alkylation of Sec within TrxR1 has significant implications for its enzymatic function, we explored the potential importance of TrxR1 alkylation in acrolein-induced activation or injury of bronchial epithelial cells. Exposure of human bronchial epithelial HBE1 cells to acrolein (1-30 μM) resulted in dose-dependent loss of TrxR thioredoxin reductase activity, which coincided with its alkylation, as determined by biotin hydrazide labeling, and was independent of initial GSH status. To test the involvement of TrxR1 in acrolein responses in HBE1 cells, we suppressed TrxR1 using siRNA silencing or augmented TrxR1 by cell supplementation with sodium selenite. Acrolein exposure of HBE1 cells induced dose-dependent activation of the MAP kinases, extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, and activation of JNK was markedly enhanced after selenite-mediated induction of TrxR1, and was associated with increased alkylation of TrxR1. Conversely, siRNA silencing of TrxR1 significantly suppressed the ability of acrolein to activate JNK, and also appeared to attenuate acrolein-dependent activation of ERK and p38. Alteration of initial TrxR1 levels by siRNA or selenite supplementation also affected initial Trx1 redox status and acrolein-mediated alkylation of Trx1, but did not significantly affect acrolein-mediated activation of HO-1 or cytotoxicity. Collectively, our findings indicate that alkylation of TrxR1 and/or Trx1 may contribute directly to acrolein-mediated activation of MAP kinases such as JNK, and

  5. Role of thioredoxin reductase 1 and thioredoxin interacting protein in prognosis of breast cancer

    PubMed Central

    2010-01-01

    Introduction The purpose of this work was to study the prognostic influence in breast cancer of thioredoxin reductase 1 (TXNRD1) and thioredoxin interacting protein (TXNIP), key players in oxidative stress control that are currently evaluated as possible therapeutic targets. Methods Analysis of the association of TXNRD1 and TXNIP RNA expression with the metastasis-free interval (MFI) was performed in 788 patients with node-negative breast cancer, consisting of three individual cohorts (Mainz, Rotterdam and Transbig). Correlation with metagenes and conventional clinical parameters (age, pT stage, grading, hormone and ERBB2 status) was explored. MCF-7 cells with a doxycycline-inducible expression of an oncogenic ERBB2 were used to investigate the influence of ERBB2 on TXNRD1 and TXNIP transcription. Results TXNRD1 was associated with worse MFI in the combined cohort (hazard ratio = 1.955; P < 0.001) as well as in all three individual cohorts. In contrast, TXNIP was associated with better prognosis (hazard ratio = 0.642; P < 0.001) and similar results were obtained in all three subcohorts. Interestingly, patients with ERBB2-status-positive tumors expressed higher levels of TXNRD1. Induction of ERBB2 in MCF-7 cells caused not only an immediate increase in TXNRD1 but also a strong decrease in TXNIP. A subsequent upregulation of TXNIP as cells undergo senescence was accompanied by a strong increase in levels of reactive oxygen species. Conclusions TXNRD1 and TXNIP are associated with prognosis in breast cancer, and ERBB2 seems to be one of the factors shifting balances of both factors of the redox control system in a prognostic unfavorable manner. PMID:20584310

  6. Lichen acids may be used as a potential drug for cancer therapy; by inhibiting mitochondrial thioredoxin reductase purified from rat lung.

    PubMed

    Ozgencli, Ilknur; Budak, Harun; Ciftci, Mehmet; Anar, Mustafa

    2018-05-24

    Thioredoxin reductase (E.C 1.6.4.5.; TrxR) is a widely distributed flavoprotein that catalyzes the NADPH-dependent reduction of thioredoxin (Trx) in many cellular events such as DNA synthesis, DNA repair, angiogenesis, antioxidative defense, and regulating apoptosis. Although TrxR is indispensible in protecting cells against oxidative stress, the overexpression of TrxR is seen in many aggressive tumors. Therefore, targeted inhibition of TrxR has been accepted as a new approach for chemotherapy. In this study, in vitro inhibition effect of the lichen acids (diffractaic, evernic, lobaric, lecanoric, and vulpinic acid) on mitochondrial TrxR purified from rat lung was investigated. It was the first time the enzyme was purified from rat lungs by using 2', 5'-ADP Sepharose 4B affinity chromatography. The purity of the enzyme was checked with SDS-PAGE. In vitro inhibition effect of the lichen acids was investigated spectrophotometrically. To emphasize the importance of the obtained data, the commercial anticancer drugs cisplatin and doxorubicin were used as positive controls. Molecular mass of the enzyme was calculated as approximately 52.4 kDa. The enzyme was purified with a 63.6% yield, 208.3 fold, and 0.5 EU/mg proteins specific activity. The IC50 values of five lichen acids were significantly lower than IC50 values of anticancer drugs. All of the lichen acids, especially lecanoric and vulpinic acid, exhibited much stronger inhibitory effect on TrxR than the anticancer drugs cisplatin and doxorubicin. These lichen acids have pharmacological potential as effective natural antioxidants, antimicrobials, and anticancer agents. . Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  7. DISRUPTION OF THE SACCHAROMYCES CEREVISIAE GENE FOR NADPH-CYTOCHROME P450-REDUCTASE CAUSES INCREASED SENSITIVITY TO KETOCONAZOLE

    EPA Science Inventory

    Strains of Saccharomyces cerevisiae deleted in the NADPH-cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14-demethylase. Resistance is restored through complementation by the plasmid-born...

  8. Novel thioredoxin-related transmembrane protein TMX4 has reductase activity.

    PubMed

    Sugiura, Yoshimi; Araki, Kazutaka; Iemura, Shun-ichiro; Natsume, Tohru; Hoseki, Jun; Nagata, Kazuhiro

    2010-03-05

    In the endoplasmic reticulum (ER), a number of thioredoxin (Trx) superfamily proteins are present to enable correct disulfide bond formation of secretory and membrane proteins via Trx-like domains. Here, we identified a novel transmembrane Trx-like protein 4 (TMX4), in the ER of mammalian cells. TMX4, a type I transmembrane protein, was localized to the ER and possessed a Trx-like domain that faced the ER lumen. A maleimide alkylation assay showed that a catalytic CXXC motif in the TMX4 Trx-like domain underwent changes in its redox state depending on cellular redox conditions, and, in the normal state, most of the endogenous TMX4 existed in the oxidized form. Using a purified recombinant protein containing the Trx-like domain of TMX4 (TMX4-Trx), we confirmed that this domain had reductase activity in vitro. The redox potential of this domain (-171.5 mV; 30 degrees C at pH 7.0) indicated that TMX4 could work as a reductase in the environment of the ER. TMX4 had no effect on the acceleration of ER-associated degradation. Because TMX4 interacted with calnexin and ERp57 by co-immunoprecipitation assay, the role of TMX4 may be to enable protein folding in cooperation with these proteins consisting of folding complex in the ER.

  9. Induction of Thioredoxin Reductase 1 by Korean Red Ginseng Water Extract Regulates Cytoprotective Effects on Human Endothelial Cells

    PubMed Central

    Park, Hye Rim; Lee, Seung Eun; Yang, Hana; Son, Gun Woo; Jin, Young-Ho

    2015-01-01

    Korean Red Ginseng is a popular herbal medicine and is widely used in many food products. KRG has biological benefits related to vascular diseases including diabetes, hypertension, atherosclerosis, and other cardiac diseases and KRG has antioxidant and anti-hyperlipidemic actions. KRG decreases the level of oxidative stress and suppresses proinflammatory cytokines and cell adhesion molecules, thus protecting endothelial dysfunction. Mammalian Thioredoxin reductase 1 is an NADPH-dependent selenoprotein, essential for antioxidant defense and DNA synthesis and repair, that regulates the redox system by modulating redox-sensitive transcription factors and thiol-containing proteins. Here, we show that KRG water extract increases the expression of TrxR1 in human umbilical vein endothelial cells via the p38 and PKC-δ signaling pathways. The induction of TrxR1 expression by KRG was confirmed by Western blot analysis and reverse transcription polymerase chain reaction. However, the increase in TrxR1 expression was abolished by specific silencing of the p38 and PKC-δ genes. In addition, we demonstrated that auranofin, a TrxR1 inhibitor, weakens the protective effect of KRG against H2O2-induced cell death as measured by the terminal transferase dUTP nick end labeling assay. These results suggest that KRG may have protective effects in vascular diseases by upregulating TrxR1 in endothelial cells, thereby inhibiting the generation of reactive oxygen species and cell death. PMID:26236385

  10. Overexpression of Arabidopsis NADPH-dependent thioredoxin reductase C (AtNTRC) confers freezing and cold shock tolerance to plants

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Moon, Jeong Chan; Lee, Sangmin; Shin, Su Young

    Overexpression of AtNTRC (AtNTRC{sup OE}) in Arabidopsis thaliana led to a freezing and cold stress tolerance, whereas a knockout mutant (atntrc) showed a stress-sensitive phenotype. Biochemical analyses showed that the recombinant AtNTRC proteins exhibited a cryoprotective activity for malate dehydrogenase and lactic dehydrogenase. Furthermore, conclusive evidence of its interaction with nucleic acids in vitro is provided here on the basis of gel shift and electron microscopy analysis. Recombinant AtNTRC efficiently protected RNA and DNA from RNase A and metal catalyzed oxidation damage, respectively. The C-terminal thioredoxin domain is required for the nucleic acid–protein complex formation. From these results, it can bemore » hypothesized that AtNTRC, which is known to be an electron donor of peroxiredoxin, contributes the stability of macromolecules under cold stress. - Highlights: • AtNTRC has a cryoprotective activity in vitro. • Overexpression of AtNTRC increases tolerance to freezing and cold shock stresses. • Thioredoxin domain of AtNTRC protects nucleic acids in vitro. • AtNTRC inhibits protein aggregation under freezing stress in vitro.« less

  11. Crystallization and preliminary X-ray analysis of the NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra

    PubMed Central

    Takeshita, Daijiro; Kataoka, Michihiko; Miyakawa, Takuya; Miyazono, Ken-ichi; Uzura, Atsuko; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2009-01-01

    (R)-3-Quinuclidinol is a useful compound that is applicable to the synthesis of various pharmaceuticals. The NADPH-dependent carbonyl reductase 3-­quinuclidinone reductase from Rhodotorula rubra catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol and is expected to be utilized in industrial production of this alcohol. 3-Quinuclidinone reductase from R. rubra was expressed in Escherichia coli and purified using Ni-affinity and ion-exchange column chromatography. Crystals of the protein were obtained by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant. The crystals belonged to space group P41212, with unit-cell parameters a = b = 91.3, c = 265.4 Å, and diffracted X-rays to 2.2 Å resolution. The asymmetric unit contained four molecules of the protein and the solvent content was 48.4%. PMID:19478454

  12. The role of thioredoxin reductase 1 in melanoma metabolism and metastasis.

    PubMed

    Cassidy, Pamela B; Honeggar, Matthew; Poerschke, Robyn L; White, Karen; Florell, Scott R; Andtbacka, Robert H I; Tross, Joycelyn; Anderson, Madeleine; Leachman, Sancy A; Moos, Philip J

    2015-11-01

    Although significant progress has been made in targeted and immunologic therapeutics for melanoma, many tumors fail to respond, and most eventually progress when treated with the most efficacious targeted combination therapies thus far identified. Therefore, alternative approaches that exploit distinct melanoma phenotypes are necessary to develop new approaches for therapeutic intervention. Tissue microarrays containing human nevi and melanomas were used to evaluate levels of the antioxidant protein thioredoxin reductase 1 (TR1), which was found to increase as a function of disease progression. Melanoma cell lines revealed metabolic differences that correlated with TR1 levels. We used this new insight to design a model treatment strategy that creates a synthetic lethal interaction wherein targeting TR1 sensitizes melanoma to inhibition of glycolytic metabolism, resulting in a decrease in metastases in vivo. This approach holds the promise of a new clinical therapeutic strategy, distinct from oncoprotein inhibition. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Methionine sulfoxide reductase A protects hepatocytes against acetaminophen-induced toxicity via regulation of thioredoxin reductase 1 expression.

    PubMed

    Singh, Mahendra Pratap; Kwak, Geun-Hee; Kim, Ki Young; Kim, Hwa-Young

    2017-06-03

    Thioredoxin reductase 1 (TXNRD1) is associated with susceptibility to acetaminophen (APAP)-induced liver damage. Methionine sulfoxide reductase A (MsrA) is an antioxidant and protein repair enzyme that specifically catalyzes the reduction of methionine S-sulfoxide residues. We have previously shown that MsrA deficiency exacerbates acute liver injury induced by APAP. In this study, we used primary hepatocytes to investigate the underlying mechanism of the protective effect of MsrA against APAP-induced hepatotoxicity. MsrA gene-deleted (MsrA -/- ) hepatocytes showed higher susceptibility to APAP-induced cytotoxicity than wild-type (MsrA +/+ ) cells, consistent with our previous in vivo results. MsrA deficiency increased APAP-induced glutathione depletion and reactive oxygen species production. APAP treatment increased Nrf2 activation more profoundly in MsrA -/- than in MsrA +/+ hepatocytes. Basal TXNRD1 levels were significantly higher in MsrA -/- than in MsrA +/+ hepatocytes, while TXNRD1 depletion in both MsrA -/- and MsrA +/+ cells resulted in increased resistance to APAP-induced cytotoxicity. In addition, APAP treatment significantly increased TXNRD1 expression in MsrA -/- hepatocytes, while no significant change was observed in MsrA +/+ cells. Overexpression of MsrA reduced APAP-induced cytotoxicity and TXNRD1 expression levels in APAP-treated MsrA -/- hepatocytes. Collectively, our results suggest that MsrA protects hepatocytes from APAP-induced cytotoxicity through the modulation of TXNRD1 expression. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Crystal Structure of Perakine Reductase, Founding Member of a Novel Aldo-Keto Reductase (AKR) Subfamily That Undergoes Unique Conformational Changes during NADPH Binding*

    PubMed Central

    Sun, Lianli; Chen, Yixin; Rajendran, Chitra; Mueller, Uwe; Panjikar, Santosh; Wang, Meitian; Mindnich, Rebekka; Rosenthal, Cindy; Penning, Trevor M.; Stöckigt, Joachim

    2012-01-01

    Perakine reductase (PR) catalyzes the NADPH-dependent reduction of the aldehyde perakine to yield the alcohol raucaffrinoline in the biosynthetic pathway of ajmaline in Rauvolfia, a key step in indole alkaloid biosynthesis. Sequence alignment shows that PR is the founder of the new AKR13D subfamily and is designated AKR13D1. The x-ray structure of methylated His6-PR was solved to 2.31 Å. However, the active site of PR was blocked by the connected parts of the neighbor symmetric molecule in the crystal. To break the interactions and obtain the enzyme-ligand complexes, the A213W mutant was generated. The atomic structure of His6-PR-A213W complex with NADPH was determined at 1.77 Å. Overall, PR folds in an unusual α8/β6 barrel that has not been observed in any other AKR protein to date. NADPH binds in an extended pocket, but the nicotinamide riboside moiety is disordered. Upon NADPH binding, dramatic conformational changes and movements were observed: two additional β-strands in the C terminus become ordered to form one α-helix, and a movement of up to 24 Å occurs. This conformational change creates a large space that allows the binding of substrates of variable size for PR and enhances the enzyme activity; as a result cooperative kinetics are observed as NADPH is varied. As the founding member of the new AKR13D subfamily, PR also provides a structural template and model of cofactor binding for the AKR13 family. PMID:22334702

  15. Annatto Constituent Cis-Bixin Has Selective Antimyeloma Effects Mediated by Oxidative Stress and Associated with Inhibition of Thioredoxin and Thioredoxin Reductase

    PubMed Central

    Tibodeau, Jennifer D.; Isham, Crescent R.

    2010-01-01

    Abstract In pursuit of the anticancer effects of seeds of the rain forest plant Bixa orellana (annatto), we found that its constituent cis-bixin induced cytotoxicity in a wide variety of tumor cell lines (IC50 values from 10 to 50 μM, 24-h exposures) and, importantly, also selectively killed freshly collected patient multiple myeloma cells and highly drug-resistant multiple myeloma cell lines. Mechanistic studies indicated that cis-bixin–induced cytotoxicity was greatly attenuated by co-treatment with glutathione or N-acetylcysteine (NAC); whereas fluorescence-activated cell sorting (FACS) assays using the cell-permeable dyes 5-(and-6) chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), or dihydroethidium demonstrated that cis-bixin rapidly induced cellular reactive oxygen species (ROS) in dose- and time-dependent fashions, collectively implicating ROS as contributory to cis-bixin–induced cytotoxicity. In pursuit of potential contributors to ROS imposition by cis-bixin, we found that cis-bixin inhibited both thioredoxin (Trx) and thioredoxin reductase (TrxR1) activities at concentrations comparable to those required for cytotoxicity, implicating the inhibition of these redox enzymes as potentially contributing to its ability to impose cellular ROS and to kill cancer cells. Collectively, our studies indicate that the annatto constituent cis-bixin has intriguing selective antimyeloma activity that appears to be mediated through effects on redox signaling. Antioxid. Redox Signal. 13, 987–997. PMID:20170403

  16. Oligo-carrageenan kappa-induced reducing redox status and activation of TRR/TRX system increase the level of indole-3-acetic acid, gibberellin A3 and trans-zeatin in Eucalyptus globulus trees.

    PubMed

    González, Alberto; Contreras, Rodrigo A; Zúiga, Gustavo; Moenne, Alejandra

    2014-08-20

    Eucalyptus globulus trees treated with oligo-carrageenan (OC) kappa showed an increase in NADPH, ascorbate and glutathione levels and activation of the thioredoxin reductase (TRR)/thioredoxin (TRX) system which enhance photosynthesis, basal metabolism and growth. In order to analyze whether the reducing redox status and the activation of thioredoxin reductase (TRR)/thioredoxin (TRX) increased the level of growth-promoting hormones, trees were treated with water (control), with OC kappa, or with inhibitors of ascorbate synthesis, lycorine, glutathione synthesis, buthionine sulfoximine (BSO), NADPH synthesis, CHS-828, and thioredoxin reductase activity, auranofine, and with OC kappa, and cultivated for four additional months. Eucalyptus trees treated with OC kappa showed an increase in the levels of the auxin indole 3-acetic acid (IAA), gibberellin A3 (GA3) and the cytokinin trans-zeatin (t-Z) as well as a decrease in the level of the brassinosteroid epi-brassinolide (EB). In addition, treatment with lycorine, BSO, CHS-828 and auranofine inhibited the increase in IAA, GA3 and t-Z as well as the decrease in EB levels. Thus, the reducing redox status and the activation of TRR/TRX system induced by OC kappa increased the levels of IAA, GA3 and t-Z levels determining, at least in part, the stimulation of growth in Eucalyptus trees.

  17. Thioredoxin Reductase 2 (TXNRD2) mutation associated with familial glucocorticoid deficiency (FGD).

    PubMed

    Prasad, Rathi; Chan, Li F; Hughes, Claire R; Kaski, Juan P; Kowalczyk, Julia C; Savage, Martin O; Peters, Catherine J; Nathwani, Nisha; Clark, Adrian J L; Storr, Helen L; Metherell, Louise A

    2014-08-01

    Classic ACTH resistance, due to disruption of ACTH signaling, accounts for the majority of cases of familial glucocorticoid deficiency (FGD). Recently FGD cases caused by mutations in the mitochondrial antioxidant, nicotinamide nucleotide transhydrogenase, have highlighted the importance of redox regulation in steroidogenesis. We hypothesized that other components of mitochondrial antioxidant systems would be good candidates in the etiology of FGD. Whole-exome sequencing was performed on three related patients, and segregation of putative causal variants confirmed by Sanger sequencing of all family members. A TXNRD2-knockdown H295R cell line was created to investigate redox homeostasis. The study was conducted on patients from three pediatric centers in the United Kingdom. Seven individuals from a consanguineous Kashmiri kindred, six of whom presented with FGD between 0.1 and 10.8 years, participated in the study. There were no interventions. Identification and functional interrogation of a novel homozygous mutation segregating with the disease trait were measured. A stop gain mutation, p.Y447X in TXNRD2, encoding the mitochondrial selenoprotein thioredoxin reductase 2 (TXNRD2) was identified and segregated with disease in this extended kindred. RT-PCR and Western blotting revealed complete absence of TXNRD2 in patients homozygous for the mutation. TXNRD2 deficiency leads to impaired redox homeostasis in a human adrenocortical cell line. In contrast to the Txnrd2-knockout mouse model, in which embryonic lethality as a consequence of hematopoietic and cardiac defects is described, absence of TXNRD2 in humans leads to glucocorticoid deficiency. This is the first report of a homozygous mutation in any component of the thioredoxin antioxidant system leading to inherited disease in humans.

  18. Thioredoxin system in obligate anaerobe Desulfovibrio desulfuricans: Identification and characterization of a novel thioredoxin 2.

    PubMed

    Sarin, Ritu; Sharma, Yagya D

    2006-07-05

    Metal corroding sulfate reducing bacteria have been poorly characterized at molecular level due to difficulties pertaining to isolation and handling of anaerobes. We report here for the first time the presence and characterization of thioredoxin 2 in an obligate anaerobic dissimilatory sulfate reducing bacterium Desulfovibrio desulfuricans. In silico analysis of the D. desulfuricans genome revealed the presence of thioredoxin 1 (dstrx1), thioredoxin 2 (dstrx2) and thioredoxin reductase (dstrxR) genes. These genes were found to be actively expressed by the bacteria under the anaerobic growth conditions. We have overexpressed the anaerobic thioredoxin genes in E. coli to produce functionally active recombinant proteins. Recombinant DsTrxR recognized both DsTrx1 and DsTrx2 as its substrate. Mutation studies revealed that the activity of DsTrx2 can be completely abolished with a single amino acid mutation (C69A) in the signature motif 'WCGPC'. Furthermore, the N-terminal domain of DsTrx2 containing two extra CXXC motifs was found to have a negative regulation on its biochemical activity. In conclusion, we have shown the presence of thioredoxin 2 for the first time in an obligate anaerobe which in this anaerobe may be required for its survival under either oxidative stress conditions or metal ion hemostasis.

  19. Thioredoxin reductase regulates AP-1 activity as well as thioredoxin nuclear localization via active cysteines in response to ionizing radiation.

    PubMed

    Karimpour, Shervin; Lou, Junyang; Lin, Lilie L; Rene, Luis M; Lagunas, Lucio; Ma, Xinrong; Karra, Sreenivasu; Bradbury, C Matthew; Markovina, Stephanie; Goswami, Prabhat C; Spitz, Douglas R; Hirota, Kiichi; Kalvakolanu, Dhananjaya V; Yodoi, Junji; Gius, David

    2002-09-12

    A recently identified class of signaling factors uses critical cysteine motif(s) that act as redox-sensitive 'sulfhydryl switches' to reversibly modulate specific signal transduction cascades regulating downstream proteins with similar redox-sensitive sites. For example, signaling factors such as redox factor-1 (Ref-1) and transcription factors such as the AP-1 complex both contain redox-sensitive cysteine motifs that regulate activity in response to oxidative stress. The mammalian thioredoxin reductase-1 (TR) is an oxidoreductase selenocysteine-containing flavoprotein that also appears to regulate multiple downstream intracellular redox-sensitive proteins. Since ionizing radiation (IR) induces oxidative stress as well as increases AP-1 DNA-binding activity via the activation of Ref-1, the potential roles of TR and thioredoxin (TRX) in the regulation of AP-1 activity in response to IR were investigated. Permanently transfected cell lines that overexpress wild type TR demonstrated constitutive increases in AP-1 DNA-binding activity as well as AP-1-dependent reporter gene expression, relative to vector control cells. In contrast, permanently transfected cell lines expressing a TR gene with the active site cysteine motif deleted were unable to induce AP-1 activity or reporter gene expression in response to IR. Transient genetic overexpression of either the TR wild type or dominant-negative genes demonstrated similar results using a transient assay system. One mechanism through which TR regulates AP-1 activity appears to involve TRX sub-cellular localization, with no change in the total TRX content of the cell. These results identify a novel function of the TR enzyme as a signaling factor in the regulation of AP-1 activity via a cysteine motif located in the protein.

  20. Overexpressed thioredoxin compensates Fanconi anemia related chromosomal instability.

    PubMed

    Kontou, Maria; Adelfalk, Caroline; Ramirez, Maria Helena; Ruppitsch, Werner; Hirsch-Kauffmann, Monica; Schweiger, Manfred

    2002-04-04

    The cause of the molecular defect of Fanconi anemia (FA) remains unknown. Cells from patients with FA exert an elevated spontaneous chromosomal instability which is further triggered by mitomycin C. The induced lability is reduced by overexpression of thioredoxin which is not the case for spontaneous instability. However, both are eliminated by overexpression of thioredoxin cDNA with an added nuclear localization signal. This implies that thioredoxin is lacking in the nuclei of FA cells. The total thioredoxin content in all FA cells tested is reduced. The resultant lack of nuclear thioredoxin can be the explanation for the major symptomatology in FA. Since thioredoxin is known to be the reactive cofactor of ribonucleotid reductase its shortcoming reduces the supply of deoxyribonucleotides thus hindering the DNA and replication repair with resultant chromosomal breaks. Furthermore, depression of tyrosine hydroxylase, the key enzyme of melanine synthesis, could be the basis for the pathognomotic 'café au lait' spots of FA. The observation of thioredoxin reduction in FA cells permits insight into the molecular phathophysiology of FA.

  1. The Enzymatic and Structural Basis for Inhibition of Echinococcus granulosus Thioredoxin Glutathione Reductase by Gold(I).

    PubMed

    Salinas, Gustavo; Gao, Wei; Wang, Yang; Bonilla, Mariana; Yu, Long; Novikov, Andrey; Virginio, Veridiana G; Ferreira, Henrique B; Vieites, Marisol; Gladyshev, Vadim N; Gambino, Dinorah; Dai, Shaodong

    2017-12-20

    New drugs are needed to treat flatworm infections that cause severe human diseases such as schistosomiasis. The unique flatworm enzyme thioredoxin glutathione reductase (TGR), structurally different from the human enzyme, is a key drug target. Structural studies of the flatworm Echinococcus granulosus TGR, free and complexed with Au I -MPO, a novel gold inhibitor, together with inhibition assays were performed. Au I -MPO is a potent TGR inhibitor that achieves 75% inhibition at a 1:1 TGR:Au ratio and efficiently kills E. granulosus in vitro. The structures revealed salient insights: (i) unique monomer-monomer interactions, (ii) distinct binding sites for thioredoxin and the glutaredoxin (Grx) domain, (iii) a single glutathione disulfide reduction site in the Grx domain, (iv) rotation of the Grx domain toward the Sec-containing redox active site, and (v) a single gold atom bound to Cys 519 and Cys 573 in the Au I -TGR complex. Structural modeling suggests that these residues are involved in the stabilization of the Sec-containing C-terminus. Consistently, Cys→Ser mutations in these residues decreased TGR activities. Mass spectroscopy confirmed these cysteines are the primary binding site. The identification of a primary site for gold binding and the structural model provide a basis for gold compound optimization through scaffold adjustments. The structural study revealed that TGR functions are achieved not only through a mobile Sec-containing redox center but also by rotation of the Grx domain and distinct binding sites for Grx domain and thioredoxin. The conserved Cys 519 and Cys 573 residues targeted by gold assist catalysis through stabilization of the Sec-containing redox center. Antioxid. Redox Signal. 27, 1491-1504.

  2. Baicalein induces cell death in murine T cell lymphoma via inhibition of thioredoxin system.

    PubMed

    Patwardhan, Raghavendra S; Pal, Debojyoti; Checker, Rahul; Sharma, Deepak; Sandur, Santosh K

    2017-10-01

    We have earlier demonstrated the radioprotective potential of baicalein using murine splenic lymphocytes. Here, we have studied the effect of baicalein on murine T cell lymphoma EL4 cells and investigated the underlying mechanism of action. We observed that baicalein induced a dose dependent cell death in EL4 cells in vitro and significantly reduced the frequency of cancer stem cells. Previously, we have reported that murine and human T cell lymphoma cells have increased oxidative stress tolerance capacity due to active thioredoxin system. Hence, we monitored the effect of baicalein on thioredoxin system in EL4 cells. Docking studies revealed that baicalein could bind to the active site of thioredoxin reductase. Baicalein treatment led to significant reduction in the activity of thioredoxin reductase and nuclear levels of thioredoxin-1 thereby increasing ASK1 levels and caspase-3 activity. Interestingly, CRISPR-Cas9 based knock-out of ASK1 or over-expression of thioredoxin-1 abolished anti-tumor effects of baicalein in EL4 cells. Further, baicalein administration significantly reduced intra-peritoneal tumor burden of EL4 cells in C57BL/6 mice. Thus, our study describes anti-tumor effects of baicalein in EL4 cells via inhibition of thioredoxin system. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wei, Yifeng; Li, Bin; Prakash, Divya

    Two subtypes of class III anaerobic ribonucleotide reductases (RNRs) studied so far couple the reduction of ribonucleotides to the oxidation of formate, or the oxidation of NADPH via thioredoxin and thioredoxin reductase. Certain methanogenic archaea contain a phylogenetically distinct third subtype of class III RNR, with distinct active-site residues. Here we report the cloning and recombinant expression of the Methanosarcina barkeri class III RNR and show that the electrons required for ribonucleotide reduction can be delivered by a [4Fe-4S] protein ferredoxin disulfide reductase, and a conserved thioredoxin-like protein NrdH present in the RNR operon. The diversity of class III RNRsmore » reflects the diversity of electron carriers used in anaerobic metabolism« less

  4. Absolute gene expression patterns of thioredoxin and glutaredoxin redox systems in mouse.

    PubMed

    Jurado, Juan; Prieto-Alamo, María-José; Madrid-Rísquez, José; Pueyo, Carmen

    2003-11-14

    This work provides the first absolute expression patterns of genes coding for all known components of both thioredoxin (Trx) and glutaredoxin (Grx) systems in mouse: Trx1, Trx2, Grx1, Grx2, TrxR1, TrxR2, thioredoxin/glutathione reductase, and glutathione reductase. We devised a novel assay that, combining the advantages of multiplex and real-time PCR, streamlines the quantitation of the actual mRNA copy numbers in whole-animal experiments. Quantitations reported establish differences among adult organs and embryonic stages, compare mRNA decay rates, explore the significance of alternative mRNA isoforms derived from TrxR1 and Grx2 genes, and examine the time-course expression upon superoxide stress promoted by paraquat. Collectively, these quantitations show: i) unique expression profiles for each transcript and mouse organ examined, yet with some general trends like the higher amounts of mRNA species coding for thioredoxins than those coding for the reductases that control their redox states and activities; ii) continuous expression during embryogenesis with outstanding up-regulations of Trx1 and TrxR1 mRNAs in specific temporal sequences; iii) drastic differences in mRNA stability, liver decay rates range from 2.8 h (thioredoxin/glutathione reductase) to >/= 35 h (Trx1 and Trx2), and directly correlate with mRNA steady-state values; iv) testis-specific differences in the amounts (relative to total isoforms) of transcripts yielding the mitochondrial Grx2a and 67-kDa TrxR1 variants; and v) coordinated up-regulation of TrxR1 and glutathione reductase mRNAs in response to superoxide stress in an organ-specific manner. Further insights into in vivo roles of these redox systems should be gained from more focused studies of the mechanisms underlying the vast differences reported here at the transcript level.

  5. Thioredoxin-interacting Protein Mediates High Glucose-induced Reactive Oxygen Species Generation by Mitochondria and the NADPH Oxidase, Nox4, in Mesangial Cells*

    PubMed Central

    Shah, Anu; Xia, Ling; Goldberg, Howard; Lee, Ken W.; Quaggin, Susan E.; Fantus, I. George

    2013-01-01

    Thioredoxin-interacting protein (TxNIP) is up-regulated by high glucose and is associated with oxidative stress. It has been implicated in hyperglycemia-induced β-cell dysfunction and apoptosis. As high glucose and oxidative stress mediate diabetic nephropathy (DN), the contribution of TxNIP was investigated in renal mesangial cell reactive oxygen species (ROS) generation and collagen synthesis. To determine the role of TxNIP, mouse mesangial cells (MC) cultured from wild-type C3H and TxNIP-deficient Hcb-19 mice were incubated in HG. Confocal microscopy was used to measure total and mitochondrial ROS production (DCF and MitoSOX) and collagen IV. Trx and NADPH oxidase activities were assayed and NADPH oxidase isoforms, Nox2 and Nox4, and antioxidant enzymes were determined by immunoblotting. C3H MC exposed to HG elicited a significant increase in cellular and mitochondrial ROS as well as Nox4 protein expression and NADPH oxidase activation, whereas Hcb-19 MC showed no response. Trx activity was attenuated by HG only in C3H MC. These defects in Hcb-19 MC were not due to increased antioxidant enzymes or scavenging of ROS, but associated with decreased ROS generation. Adenovirus-mediated overexpression of TxNIP in Hcb-19 MC and TxNIP knockdown with siRNA in C3H confirmed the specific role of TxNIP. Collagen IV accumulation in HG was markedly reduced in Hcb-19 cells. TxNIP is a critical component of the HG-ROS signaling pathway, required for the induction of mitochondrial and total cell ROS and the NADPH oxidase isoform, Nox4. TxNIP is a potential target to prevent DN. PMID:23329835

  6. The Enzymatic and Structural Basis for Inhibition of Echinococcus granulosus Thioredoxin Glutathione Reductase by Gold(I)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Salinas, Gustavo; Gao, Wei; Wang, Yang

    Aims: New drugs are needed to treat flatworm infections that cause severe human diseases such as schistosomiasis. The unique flatworm enzyme thioredoxin glutathione reductase (TGR), structurally different from the human enzyme, is a key drug target. Structural studies of the flatworm Echinococcus granulosus TGR, free and complexed with AuI-MPO, a novel gold inhibitor, together with inhibition assays were performed. Results: AuI-MPO is a potent TGR inhibitor that achieves 75% inhibition at a 1:1 TGR:Au ratio and efficiently kills E. granulosus in vitro. The structures revealed salient insights: (i) unique monomer–monomer interactions, (ii) distinct binding sites for thioredoxin and the glutaredoxinmore » (Grx) domain, (iii) a single glutathione disulfide reduction site in the Grx domain, (iv) rotation of the Grx domain toward the Sec-containing redox active site, and (v) a single gold atom bound to Cys519 and Cys573 in the AuI-TGR complex. Structural modeling suggests that these residues are involved in the stabilization of the Sec-containing C-terminus. Consistently, Cys→Ser mutations in these residues decreased TGR activities. Mass spectroscopy confirmed these cysteines are the primary binding site. Innovation: The identification of a primary site for gold binding and the structural model provide a basis for gold compound optimization through scaffold adjustments. Conclusions: The structural study revealed that TGR functions are achieved not only through a mobile Sec-containing redox center but also by rotation of the Grx domain and distinct binding sites for Grx domain and thioredoxin. The conserved Cys519 and Cys573 residues targeted by gold assist catalysis through stabilization of the Sec-containing redox center. Antioxid. Redox Signal. 27, 1491–1504.« less

  7. Epigallocatechin-3-gallate enhances key enzymatic activities of hepatic thioredoxin and glutathione systems in selenium-optimal mice but activates hepatic Nrf2 responses in selenium-deficient mice.

    PubMed

    Dong, Ruixia; Wang, Dongxu; Wang, Xiaoxiao; Zhang, Ke; Chen, Pingping; Yang, Chung S; Zhang, Jinsong

    2016-12-01

    Selenium participates in the antioxidant defense mainly through a class of selenoproteins, including thioredoxin reductase. Epigallocatechin-3-gallate (EGCG) is the most abundant and biologically active catechin in green tea. Depending upon the dose and biological systems, EGCG may function either as an antioxidant or as an inducer of antioxidant defense via its pro-oxidant action or other unidentified mechanisms. By manipulating the selenium status, the present study investigated the interactions of EGCG with antioxidant defense systems including the thioredoxin system comprising of thioredoxin and thioredoxin reductase, the glutathione system comprising of glutathione and glutathione reductase coupled with glutaredoxin, and the Nrf2 system. In selenium-optimal mice, EGCG increased hepatic activities of thioredoxin reductase, glutathione reductase and glutaredoxin. These effects of EGCG appeared to be not due to overt pro-oxidant action because melatonin, a powerful antioxidant, did not influence the increase. However, in selenium-deficient mice, with low basal levels of thioredoxin reductase 1, the same dose of EGCG did not elevate the above-mentioned enzymes; intriguingly EGCG in turn activated hepatic Nrf2 response, leading to increased heme oxygenase 1 and NAD(P)H:quinone oxidoreductase 1 protein levels and thioredoxin activity. Overall, the present work reveals that EGCG is a robust inducer of the Nrf2 system only in selenium-deficient conditions. Under normal physiological conditions, in selenium-optimal mice, thioredoxin and glutathione systems serve as the first line defense systems against the stress induced by high doses of EGCG, sparing the activation of the Nrf2 system. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  8. Atypical Thioredoxins in Poplar: The Glutathione-Dependent Thioredoxin-Like 2.1 Supports the Activity of Target Enzymes Possessing a Single Redox Active Cysteine1[W

    PubMed Central

    Chibani, Kamel; Tarrago, Lionel; Gualberto, José Manuel; Wingsle, Gunnar; Rey, Pascal; Jacquot, Jean-Pierre; Rouhier, Nicolas

    2012-01-01

    Plant thioredoxins (Trxs) constitute a complex family of thiol oxidoreductases generally sharing a WCGPC active site sequence. Some recently identified plant Trxs (Clot, Trx-like1 and -2, Trx-lilium1, -2, and -3) display atypical active site sequences with altered residues between the two conserved cysteines. The transcript expression patterns, subcellular localizations, and biochemical properties of some representative poplar (Populus spp.) isoforms were investigated. Measurements of transcript levels for the 10 members in poplar organs indicate that most genes are constitutively expressed. Using transient expression of green fluorescent protein fusions, Clot and Trx-like1 were found to be mainly cytosolic, whereas Trx-like2.1 was located in plastids. All soluble recombinant proteins, except Clot, exhibited insulin reductase activity, although with variable efficiencies. Whereas Trx-like2.1 and Trx-lilium2.2 were efficiently regenerated both by NADPH-Trx reductase and glutathione, none of the proteins were reduced by the ferredoxin-Trx reductase. Only Trx-like2.1 supports the activity of plastidial thiol peroxidases and methionine sulfoxide reductases employing a single cysteine residue for catalysis and using a glutathione recycling system. The second active site cysteine of Trx-like2.1 is dispensable for this reaction, indicating that the protein possesses a glutaredoxin-like activity. Interestingly, the Trx-like2.1 active site replacement, from WCRKC to WCGPC, suppresses its capacity to use glutathione as a reductant but is sufficient to allow the regeneration of target proteins employing two cysteines for catalysis, indicating that the nature of the residues composing the active site sequence is crucial for substrate selectivity/recognition. This study provides another example of the cross talk existing between the glutathione/glutaredoxin and Trx-dependent pathways. PMID:22523226

  9. Identification and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase involved in dihydrochalcone formation in Malus×domestica Borkh.

    PubMed

    Ibdah, Mwafaq; Berim, Anna; Martens, Stefan; Valderrama, Andrea Lorena Herrera; Palmieri, Luisa; Lewinsohn, Efraim; Gang, David R

    2014-11-01

    The apple tree (Malus sp.) is an agriculturally and economically important source of food and beverages. Many of the health beneficial properties of apples are due to (poly)phenolic metabolites that they contain, including various dihydrochalcones. Although many of the genes and enzymes involved in polyphenol biosynthesis are known in many plant species, the specific reactions that lead to the biosynthesis of the dihydrochalcone precursor, p-dihydrocoumaroyl-CoA (3), are unknown. To identify genes involved in the synthesis of these metabolites, existing genome databases of the Rosaceae were screened for apple genes with significant sequence similarity to Arabidopsis alkenal double bond reductases. Herein described are the isolation and characterization of a Malus hydroxycinnamoyl-CoA double bond reductase, which catalyzed the NADPH-dependent reduction of p-coumaroyl-CoA and feruloyl-CoA to p-dihydrocoumaroyl-CoA and dihydroferuloyl-CoA, respectively. Its apparent Km values for p-coumaroyl-CoA, feruloyl-CoA and NADPH were 96.6, 92.9 and 101.3μM, respectively. The Malus double bond reductase preferred feruloyl-CoA to p-coumaroyl-CoA as a substrate by a factor of 2.1 when comparing catalytic efficiencies in vitro. Expression analysis of the hydroxycinnamoyl-CoA double bond reductase gene revealed that its transcript levels showed significant variation in tissues of different developmental stages, but was expressed when expected for involvement in dihydrochalcone formation. Thus, the hydroxycinnamoyl-CoA double bond reductase appears to be responsible for the reduction of the α,β-unsaturated double bond of p-coumaroyl-CoA, the first step of dihydrochalcone biosynthesis in apple tissues, and may be involved in the production of these compounds. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. [Induction of NAD(P)H: quinone reductase by anticarcinogenic ingredients of tea].

    PubMed

    Qi, L; Han, C

    1998-09-30

    By assaying the activity of NAD(P)H: quinone reductase (QR) in Hep G2 cells exposed to inducing agents, a variety of ingredients in tea, we compared their abilities on inducing QR and preventing cancer. The results showed that tea polyphenols, tea pigments and mixed tea were all able to induce the activity of QR significantly. The single-component ingredients of tea polyphenols and tea pigments, including thearubigens, EGCG and ECG, also enhanced the activity of QR. But EGC, EC, theaflavins, tea polysaccharide and tea caffeine, showed no apparent induction of QR. We found that among those tea ingredients studied, the multi-component ingredients were more effective than the single-component ones. So we thought that the abilities of antioxidation and cancer prevention of tea depended on the combined effects of several kinds of active ingredients, which mainly include tea polyphenols and tea pigments.

  11. Ketopantoyl lactone reductase is a conjugated polyketone reductase.

    PubMed

    Hata, H; Shimizu, S; Hattori, S; Yamada, H

    1989-03-01

    Ketopantoyl lactone reductase (EC 1.1.1.168) of Saccharomyces cerevisiae was found to catalyze the reduction of a variety of natural and unnatural conjugated polyketone compounds and quinones, such as isatin, ninhydrin, camphorquinone and beta-naphthoquinone in the presence of NADPH. 5-Bromoisatin is the best substrate for the enzyme (Km = 3.1 mM; Vmax = 650 mumol/min/mg). The enzyme is inhibited by quercetin, and several polyketones. These results suggest that ketopantoyl lactone reductase is a carbonyl reductase which specifically catalyzes the reduction of conjugated polyketones.

  12. Structural and functional characterization of the recombinant thioredoxin reductase from Candida albicans as a potential target for vaccine and drug design.

    PubMed

    Godoy, Janine Silva Ribeiro; Kioshima, Érika Seki; Abadio, Ana Karina Rodrigues; Felipe, Maria Sueli Soares; de Freitas, Sonia Maria; Svidzinski, Terezinha Inez Estivalet

    2016-05-01

    The thioredoxin system plays a critical role in maintaining the cytoplasm redox state, participating in functions that are important to the cellular viability of fungi. Although functional and structural information on targets in human pathogenic fungi has been scarcely described in the literature, such studies are essential for in silico drug design and biotechnological applications. Therefore, the aims of the present study were to produce recombinant proteins of the thioredoxin system from Candida albicans and evaluate their possible use as prophylactic or alternative therapies against the most important pathogenic fungus associated with nosocomial infections. We focused on biochemical and structural analyses of recombinant thioredoxin reductase from C. albicans with His-tag (CaTrxR-His) for further biotechnology applications. Heterologous CaTrxR-His was efficiently expressed in the soluble fraction of the Escherichia coli lysate. CaTrxR-His was obtained with a high level of purity and presented specific enzymatic activity. Conformational changes of the protein were observed at different pHs and temperatures, with higher thermal stability at pH 8.0. The CaTrxR-His vaccine was shown to effectively induce high levels of CaTrxR-specific immunoglobulin G antibodies in Balb/c mice and reduce the renal fungal burden of experimental disseminated candidiasis in mice. These data may greatly impact future development strategies for vaccine and drug designs against C. albicans infection.

  13. Influence of acute and chronic administration of methadone hydrochloride on NADPH-cytochrome c reductase and cytochrome P-450 of mouse liver microsomes.

    PubMed

    Datta, R K; Johnson, E A; Bhattacharjee, G; Stenger, R J

    1976-03-01

    Administration of a single acute dose (20 mg/kg body weight) of methadone hydrochloride to both male and female mice increased the specific activity of NADPH-cytochrome c reductase and did not change much the content of cytochrome P-450 of their liver microsomes. Administration of multiple acute doses of methadone in male mice increased the specific activity of cytochrome c reductase and the content of cytochrome P-450 of their liver microsomes. Chronic administration of progressively increasing doses of methadone (up to 40 mg/kg body weight) to male mice increased the specific activity of c reductase. Similar chronic administration of methadone up to 28 mg/kg body weight also increased the microsomal content of P-450, but with higher doses of methadone, the content of P-450 declined and finally dropped slightly below control levels. The levels of c reductase activity and P-450 content returned to normal about two weeks after discontinuation of methadone administration.

  14. Thyroid hormone stimulation of NADPH P450 reductase expression in liver and extrahepatic tissues. Regulation by multiple mechanisms.

    PubMed

    Ram, P A; Waxman, D J

    1992-02-15

    The role of thyroid hormone in regulating the expression of the flavoprotein NADPH cytochrome P450 reductase was studied in adult rats. Depletion of circulating thyroid hormone by hypophysectomy, or more selectively, by treatment with the anti-thyroid drug methimazole led to a 75-85% depletion of hepatic microsomal P450 reductase activity and protein in both male and female rats. Thyroxine substantially restored P450 reductase activity at a dose that rendered the thyroid-depleted rats euthyroid. Microsomal P450 reductase activity in several extrahepatic tissues was also dependent on thyroid hormone, but to a lesser extent than in liver (30-50% decrease in kidney, adrenal, lung, and heart but not in testis from hypothyroid rats). Hepatic P450 reductase mRNA levels were also decreased in the hypothyroid state, indicating that the loss of P450 reductase activity is not a consequence of the associated decreased availability of the FMN and FAD cofactors of P450 reductase. Parallel analysis of S14 mRNA, which has been studied extensively as a model thyroid-regulated liver gene product, indicated that P450 reductase and S14 mRNA respond similarly to these changes in thyroid state. In contrast, while the expression of S14 and several other thyroid hormone-dependent hepatic mRNAs is stimulated by feeding a high carbohydrate, fat-free diet, hepatic P450 reductase expression was not increased by this lipogenic diet. Injection of hypothyroid rats with T3 at a supraphysiologic, receptor-saturating dose stimulated a major induction of hepatic P450 reductase mRNA that was detectable 4 h after the T3 injection, and peaked at approximately 650% of euthyroid levels by 12 h. However, this same treatment stimulated a biphasic increase in P450 reductase protein and activity that required 3 days to reach normal euthyroid levels. T3 treatment of euthyroid rats also stimulated a major induction of P450 reductase mRNA that was maximal (12-fold increase) by 12 h, but in this case no major

  15. Coenzyme preference of Streptococcus pyogenes δ1-pyrroline-5-carboxylate reductase: evidence supporting NADPH as the physiological electron donor.

    PubMed

    Petrollino, Davide; Forlani, Giuseppe

    2012-07-01

    The streptococcal enzyme that catalyzes the last step in proline biosynthesis was heterologously expressed and the recombinant protein was purified to electrophoretic homogeneity and characterized thoroughly. As for δ1-pyrroline-5-carboxylate reductases from other sources, it was able to use either NADH or NADPH as the electron donor in vitro. However, with NADH the activity was markedly inhibited by physiological levels of NADP+. Results also strengthen the possibility that an unusual ordered substrate binding occurs, in which the dinucleotide binds last.

  16. Auranofin inactivates Trichomonas vaginalis thioredoxin reductase and is effective against trichomonads in vitro and in vivo.

    PubMed

    Hopper, Melissa; Yun, Jeong-Fil; Zhou, Bianhua; Le, Christine; Kehoe, Katelin; Le, Ryan; Hill, Ryan; Jongeward, Gregg; Debnath, Anjan; Zhang, Liangfang; Miyamoto, Yukiko; Eckmann, Lars; Land, Kirkwood M; Wrischnik, Lisa A

    2016-12-01

    Trichomoniasis, caused by the protozoan parasite Trichomonas vaginalis, is the most common, non-viral, sexually transmitted infection in the world, but only two closely related nitro drugs are approved for its treatment. New antimicrobials against trichomoniasis remain an urgent need. Several organic gold compounds were tested for activity against T. vaginalis thioredoxin reductase (TrxR) in cell-free systems as well as for activity against different trichomonads in vitro and in a murine infection model. The organic gold(I) compounds auranofin and chloro(diethylphenylphosphine)gold(I) inhibited TrxR in a concentration-dependent manner in assays with recombinant purified reductase and in cytoplasmic extracts of T. vaginalis transfected with a haemagglutinin epitope-tagged form of the reductase. Auranofin potently suppressed the growth of three independent clinical T. vaginalis isolates as well as several strains of another trichomonad (Tritrichomonas foetus) in a 24 h-assay, with 50% inhibitory concentrations of 0.7-2.5 µM and minimum lethal concentrations of 2-6 µM. The drug also compromised the ability of the parasite to overcome oxidant stress, supporting the notion that auranofin acts, in part, by inactivating TrxR-dependent antioxidant defences. Chloro(diethylphenylphosphine)gold(I) was 10-fold less effective against T. vaginalis in vitro than auranofin. Oral administration of auranofin for 4 days cleared the parasites in a murine model of vaginal T. foetus infection without displaying any apparent adverse effects. The approved human drug auranofin may be a promising agent as an alternative treatment of trichomoniasis in cases when standard nitro drug therapies have failed. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

  17. Inhibition of thioredoxin reductase but not of glutathione reductase by the major classes of alkylating and platinum-containing anticancer compounds.

    PubMed

    Witte, Anne-Barbara; Anestål, Karin; Jerremalm, Elin; Ehrsson, Hans; Arnér, Elias S J

    2005-09-01

    Mammalian thioredoxin reductase (TrxR) is important for cell proliferation, antioxidant defense, and redox signaling. Together with glutathione reductase (GR) it is the main enzyme providing reducing equivalents to many cellular processes. GR and TrxR are flavoproteins of the same enzyme family, but only the latter is a selenoprotein. With the active site containing selenocysteine, TrxR may catalyze reduction of a wide range of substrates, but can at the same time easily be targeted by electrophilic compounds due to the extraordinarily high reactivity of a selenolate moiety. Here we addressed the inhibition of the enzyme by major anticancer alkylating agents and platinum-containing compounds and we compared it to that of GR. We confirmed prior studies suggesting that the nitrosourea carmustine can inhibit both GR and TrxR. We next found, however, that nitrogen mustards (chlorambucil and melphalan) and alkyl sulfonates (busulfan) efficiently inhibited TrxR while these compounds, surprisingly, did not inhibit GR. Inhibitions were concentration and time dependent and apparently irreversible. Anticancer anthracyclines (daunorubicin and doxorubicin) were, in contrast to the alkylating agents, not inhibitors but poor substrates of TrxR. We also found that TrxR, but not GR, was efficiently inhibited by both cisplatin, its monohydrated complex, and oxaliplatin. Carboplatin, in contrast, could not inhibit any of the two enzymes. These findings lead us to conclude that representative compounds of the major classes of clinically used anticancer alkylating agents and most platinum compounds may easily target TrxR, but not GR. The TrxR inhibition should thereby be considered as a factor that may contribute to the cytotoxicity seen upon clinical use of these drugs.

  18. Thioredoxin, thioredoxin reductase, and alpha-crystallin revive inactivated glyceraldehyde 3-phosphate dehydrogenase in human aged and cataract lens extracts.

    PubMed

    Yan, Hong; Lou, Marjorie F; Fernando, M Rohan; Harding, John J

    2006-10-02

    To investigate whether mammalian thioredoxin (Trx) and thioredoxin reductase (TrxR), with or without alpha-crystallin can revive inactivated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in both the cortex and nucleus of human aged clear and cataract lenses. The lens cortex (including capsule-epithelium) and the nucleus were separated from human aged clear and cataract lenses (grade II and grade IV) with similar average age. The activity of GAPDH in the water-soluble fraction after incubation with or without Trx or/and TrxR for 60 min at 30 degrees C was measured spectrophotometrically. In addition, the effect of a combination of Trx/TrxR and bovine lens alpha-crystallin was investigated. GAPDH activity was lower in the nucleus of clear lenses than in the cortex, and considerably diminished in the cataractous lenses, particularly in the nucleus of cataract lenses grade IV. Trx and TrxR were able to revive the activity of GAPDH markedly in both the cortex and nucleus of the clear and cataract lenses. The percentage increase of activity in the cortex of the clear lenses was less than that of the nucleus in the presence of Trx and TrxR, whereas it was opposite in the cataract lenses. The revival of activity in both the cortex and nucleus from the cataract lenses grade II was higher than that of the grade IV. Moreover, Trx alone, but not TrxR, efficiently enhanced GAPDH activity. The combination of Trx and TrxR had greater effect than that of either alone. In addition, alpha(L)-crystallin enhanced the activity in the cortex of cataract grade II with Trx and TrxR present. However, it failed to provide a statistically significant increase of activity in the nucleus. This is the first evidence to show that mammalian Trx and TrxR are able to revive inactivated GAPDH in human aged clear and cataract lenses, and alpha-crystallin helped this effect. The inactivation of GAPDH during aging and cataract development must be caused in part by disulphide formation and in part by

  19. Pyridine Nucleotide Complexes with Bacillus anthracis Coenzyme A-Disulfide Reductase: A Structural Analysis of Dual NAD(P)H Specificity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wallen,J.; Paige, C.; Mallett, T.

    2008-01-01

    We have recently reported that CoASH is the major low-molecular weight thiol in Bacillus anthracis, and we have now characterized the kinetic and redox properties of the B. anthracis coenzyme A-disulfide reductase (CoADR, BACoADR) and determined the crystal structure at 2.30 Angstroms resolution. While the Staphylococcus aureus and Borrelia burgdorferi CoADRs exhibit strong preferences for NADPH and NADH, respectively, B. anthracis CoADR can use either pyridine nucleotide equally well. Sequence elements within the respective NAD(P)H-binding motifs correctly reflect the preferences for S. aureus and Bo. burgdorferi CoADRs, but leave questions as to how BACoADR can interact with both pyridine nucleotides.more » The structures of the NADH and NADPH complexes at ca. 2.3 Angstroms resolution reveal that a loop consisting of residues Glu180-Thr187 becomes ordered and changes conformation on NAD(P)H binding. NADH and NADPH interact with nearly identical conformations of this loop; the latter interaction, however, involves a novel binding mode in which the 2'-phosphate of NADPH points out toward solvent. In addition, the NAD(P)H-reduced BACoADR structures provide the first view of the reduced form (Cys42-SH/CoASH) of the Cys42-SSCoA redox center. The Cys42-SH side chain adopts a new conformation in which the conserved Tyr367'-OH and Tyr425'-OH interact with the nascent thiol(ate) on the flavin si-face. Kinetic data with Y367F, Y425F, and Y367, 425F BACoADR mutants indicate that Tyr425' is the primary proton donor in catalysis, with Tyr367' functioning as a cryptic alternate donor in the absence of Tyr425'.« less

  20. The thioredoxin reductase--Thioredoxin redox system cleaves the interchain disulphide bond of botulinum neurotoxins on the cytosolic surface of synaptic vesicles.

    PubMed

    Pirazzini, Marco; Azarnia Tehran, Domenico; Zanetti, Giulia; Lista, Florigio; Binz, Thomas; Shone, Clifford C; Rossetto, Ornella; Montecucco, Cesare

    2015-12-01

    Botulinum neurotoxins (BoNTs) are Janus toxins, as they are at the same time the most deadly substances known and one of the safest drugs used in human therapy. They specifically block neurotransmission at peripheral nerves through the proteolysis of SNARE proteins, i.e. the essential proteins which are the core of the neuroexocytosis machinery. Even if BoNTs are traditionally known as seven main serotypes, their actual number is much higher as each serotype exists in many different subtypes, with individual biological properties and little antigenic relations. Since BoNTs can be used as biological weapons, and the only currently available therapy is based on immunological approaches, the existence of so many different subtypes is a major safety problem. Nevertheless, all BoNT isoforms are structurally similar and intoxicate peripheral nerve endings via a conserved mechanism. They consist of two chains linked by a unique disulphide bond which must be reduced to enable their toxicity. We found that thioredoxin 1 and its reductase compose the cell redox system responsible for this reduction, and its inhibition via specific chemicals significantly reduces BoNTs activity, in vitro as well as in vivo. Such molecules can be considered as lead compounds for the development of pan-inhibitors. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Purification and Characterization of Ferredoxin-Nicotinamide Adenine Dinucleotide Phosphate Reductase from a Nitrogen-Fixing Bacterium

    PubMed Central

    Yoch, Duane C.

    1973-01-01

    Evidence suggesting that Bacillus polymyxa has an active ferredoxin-NADP+ reductase (EC 1.6.99.4) was obtained when NADPH was found to provide reducing power for the nitrogenase of this organism; direct evidence was provided when it was shown that B. polymyxa extracts could substitute for the native ferredoxin-NADP+ reductase in the photochemical reduction of NADP+ by blue-green algal particles. The ferredoxin-NADP+ reductase was purified about 80-fold by a combination of high-speed centrifugation, ammonium sulfate fractionation, and chromatography on Sephadex G-100 and diethylaminoethyl-cellulose. The molecular weight was estimated by gel filtration to be 60,000. A small amount of the enzyme was further purified by polyacrylamide gel electrophoresis and shown to be a flavoprotein. The reductase was specific for NADPH in the ferredoxin-dependent reduction of cytochrome c and methyl viologen diaphorase reactions; furthermore, NADP+ was the acceptor of preference when the electron donor was photoreduced ferredoxin. The reductase also has an irreversible NADPH-NAD+ transhydrogenase (reduced-NADP:NAD oxidoreductase, EC 1.6.1.1) activity, the rate of which was proportional to the concentration of NAD (Km = 5.0 × 10−3M). The reductase catalyzed electron transfer from NADPH not only to B. polymyxa ferredoxin but also to the ferredoxins of Clostridium pasteurianum, Azotobacter vinelandii, and spinach chloroplasts, although less effectively. Rubredoxin from Clostridium acidi-urici and azotoflavin from A. vinelandii also accept electrons from the B. polymyxa reductase. The pH optima for the various reactions catalyzed by the B. polymyxa ferredoxin-NADP reductase are similar to those of the chloroplast reductase. NAD and acetyl-coenzyme A, which obligatorily activate NADPH- and NADH-ferredoxin reductases, respectively, in Clostridium kluyveri, have no effect on B. polymyxa reductase. PMID:4147648

  2. Solution Structures of Mycobacterium tuberculosis Thioredoxin C and Models of the Intact Thioredoxin System Suggest New Approaches to Inhibitor and Drug Design

    PubMed Central

    Olson, Andrew L.; Neumann, Terrence S.; Cai, Sheng; Sem, Daniel S.

    2012-01-01

    Here we report the NMR solution structures of Mycobacterium tuberculosis (M. tuberculosis) thioredoxin C in both oxidized and reduced states, with discussion of structural changes that occur in going between redox states. The NMR solution structure of the oxidized TrxC corresponds closely to that of the crystal structure, except in the C-terminal region. It appears that crystal packing effects have caused an artifactual shift in the α4 helix in the previously reported crystal structure, compared to the solution structure. Based on these TrxC structures, chemical shift mapping, a previously reported crystal structure of the M. tuberculosis thioredoxin reductase (not bound to a Trx) and structures for intermediates in the E. coli thioredoxin catalytic cycle, we have modeled the complete M. tuberculosis thioredoxin system for the various steps in the catalytic cycle. These structures and models reveal pockets at the TrxR/TrxC interface in various steps in the catalytic cycle, which can be targeted in the design of uncompetitive inhibitors as potential anti-mycobacterial agents, or as chemical genetic probes of function. PMID:23229911

  3. Thioredoxin reductase 1 knockdown enhances selenazolidine cytotoxicity in human lung cancer cells via mitochondrial dysfunction

    PubMed Central

    Poerschke, Robyn L.; Moos, Philip J.

    2010-01-01

    Thioredoxin reductase (TR1) is a selenoprotein that is involved in cellular redox status control and deoxyribonucleotide biosynthesis. Many cancers, including lung, overexpress TR1, making it a potential cancer therapy target. Previous work has shown that TR1 knockdown enhances the sensitivity of cancer cells to anticancer treatments, as well as certain selenocompounds. However, it is unknown if TR1 knockdown produces similar effect on the sensitivity of human lung cancer cells. To further elucidate the role of TR1 in the mechanism of selenocompounds in lung cancer, a lentiviral microRNA delivery system to knockdown TR1 expression in A549 human lung adenocarcinoma cells was utilized. Cell viability was assessed after 48 hr treatment with the selenocysteine prodrug selenazolidines 2-butylselenazolidine-4(R)-carboxylic acid (BSCA) and 2-cyclohexylselenazolidine-4-(R)-carboxylic acid (ChSCA), selenocystine (SECY), methylseleninic acid (MSA), 1,4-phenylenebis(methylene)selenocyanate (p-XSC), and selenomethionine (SEM). TR1 knockdown increased the cytotoxicity of BSCA, ChSCA, and SECY but did not sensitize cells to MSA, SEM, or p-XSC. GSH and TR1 depletion together decreased cell viability, while no change was observed with GSH depletion alone. Reactive oxygen species generation was induced only in TR1 knockdown cells treated with the selenazolidines or SECY. These three compounds also decreased total intracellular glutathione levels and oxidized thioredoxin, but in a TR1 independent manner. TR1 knockdown increased selenazolidine and SECY-induced mitochondrial membrane depolarization, as well as DNA strand breaks and AIF translocation from the mitochondria. These results indicate the ability of TR1 to modulate the cytotoxic effects of BSCA, ChSCA and SECY in human lung cancer cells through mitochondrial dysfunction. PMID:20920480

  4. Gold(III) bis(thiosemicarbazonate) compounds in breast cancer cells: Cytotoxicity and thioredoxin reductase targeting.

    PubMed

    Rodríguez-Fanjul, Vanessa; López-Torres, Elena; Mendiola, M Antonia; Pizarro, Ana María

    2018-03-25

    Gold(III) compounds have received increasing attention in cancer research. Three gold complexes of general formula [Au III L]Cl, where L is benzil bis(thiosemicarbazonate), compound 1, benzil bis(4-methyl-3-thiosemicarbazonate), compound 2, or benzil bis(4-cyclohexyl-3-thiosemicarbazonate), compound 3, have been synthesized and fully characterized, including the X-ray crystal structure of compound 3, confirming square-planar geometry around the gold(III) centre. Compound 1 showed moderate cytotoxicity and accumulation in MCF7 breast cancer cells but did not inhibit thioredoxin reductase (TrxR) activity and did not induce reactive oxygen species (ROS) production. Compound 2, the least cytotoxic, was found to be capable of modestly inhibiting TrxR activity and produced low levels of ROS in the MCF7 cell line. The most cytotoxic compound, 3, had the highest cellular accumulation and its distribution pattern showed a clear preference for the cytosol and mitochondria of MCF7 cells. It readily hampered intracellular TrxR activity leading to a dramatic alteration of the cellular redox state and to the induction of cell death. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  5. Deep-Sea Water Containing Selenium Provides Intestinal Protection against Duodenal Ulcers through the Upregulation of Bcl-2 and Thioredoxin Reductase 1

    PubMed Central

    Yang, Chih-Ching; Yao, Chien-An; Lin, Yi-Ruu; Yang, Jyh-Chin; Chien, Chiang-Ting

    2014-01-01

    Deep-sea water (DSW), which is rich in micronutrients and minerals and with antioxidant and anti-inflammatory qualities, may be developed as marine drugs to provide intestinal protection against duodenal ulcers. We determined several characteristics in the modified DSW. We explored duodenal pressure, oxygenation, microvascular blood flow, and changes in pH and oxidative redox potential (ORP) values within the stomach and duodenum in response to tap water (TW, hardness: 2.48 ppm), DSW600 (hardness: 600 ppm), and DSW1200 (hardness: 1200 ppm) in Wistar rats and analyzed oxidative stress and apoptosis gene expressions by cDNA and RNA microarrays in the duodenal epithelium. We compared the effects of drinking DSW, MgCl2, and selenium water on duodenal ulcers using pathologic scoring, immunohistochemical analysis, and Western blotting. Our results showed DSW has a higher pH value, lower ORP value, higher scavenging H2O2 and HOCl activity, higher Mg2+ concentrations, and micronutrients selenium compared with TW samples. Water infusion significantly increased intestinal pressure, O2 levels, and microvascular blood flow in DSW and TW groups. Microarray showed DSW600, DSW1200, selenium water upregulated antioxidant and anti-apoptotic genes and downregulated pro-apoptotic gene expression compared with the TW group. Drinking DSW600, DSW1200, and selenium water but not Mg2+ water significantly enhanced Bcl-2 and thioredoxin reductase 1 expression. Bax/Bcl-2/caspase 3/poly-(ADP-ribose)-polymerase signaling was activated during the pathogenesis of duodenal ulceration. DSW drinking reduced ulcer area as well as apoptotic signaling in acetic acid-induced duodenal ulcers. DSW, which contains selenium, provides intestinal protection against duodenal ulcers through the upregulation of Bcl-2 and thioredoxin reductase 1. PMID:24984066

  6. Thioredoxin reductase activity may be more important than GSH level in protecting human lens epithelial cells against UVA light.

    PubMed

    Padgaonkar, Vanita A; Leverenz, Victor R; Bhat, Aparna V; Pelliccia, Sara E; Giblin, Frank J

    2015-01-01

    This study compares the abilities of the glutathione (GSH) and thioredoxin (Trx) antioxidant systems in defending cultured human lens epithelial cells (LECs) against UVA light. Levels of GSH were depleted with either L-buthionine-(S,R)-sulfoximine (BSO) or 1-chloro-2,4-dinitrobenzene (CDNB). CDNB treatment also inhibited the activity of thioredoxin reductase (TrxR). Two levels of O2 , 3% and 20%, were employed during a 1 h exposure of the cells to 25 J cm(-2) of UVA radiation (338-400 nm wavelength, peak at 365 nm). Inhibition of TrxR activity by CDNB, combined with exposure to UVA light, produced a substantial loss of LECs and cell damage, with the effects being considerably more severe at 20% O2 compared to 3%. In contrast, depletion of GSH by BSO, combined with exposure to UVA light, produced only a slight cell loss, with no apparent morphological effects. Catalase was highly sensitive to UVA-induced inactivation, but was not essential for protection. Although UVA light presented a challenge for the lens epithelium, it was well tolerated under normal conditions. The results demonstrate an important role for TrxR activity in defending the lens epithelium against UVA light, possibly related to the ability of the Trx system to assist DNA synthesis following UVA-induced cell damage. © 2014 The American Society of Photobiology.

  7. Thioredoxin Reductase Activity may be More Important than GSH Level in Protecting Human Lens Epithelial Cells Against UVA Light

    PubMed Central

    Padgaonkar, Vanita A.; Leverenz, Victor R.; Bhat, Aparna V.; Pelliccia, Sara E.; Giblin, Frank J.

    2014-01-01

    This study compares the abilities of the glutathione (GSH) and thioredoxin (Trx) antioxidant systems in defending cultured human lens epithelial cells (LECs) against UVA light. Levels of GSH were depleted with either L-buthionine-(S,R)-sulfoximine (BSO) or 1-chloro-2,4-dinitrobenzene (CDNB). CDNB treatment also inhibited the activity of thioredoxin reductase (TrxR). Two levels of O2, 3% and 20%, were employed during a 1 hr exposure of the cells to 25 J/cm2 of UVA radiation (338-400nm wavelength, peak at 365nm). Inhibition of TrxR activity by CDNB, combined with exposure to UVA light, produced a substantial loss of LECs and cell damage, with the effects being considerably more severe at 20% O2 compared to 3%. In contrast, depletion of GSH by BSO, combined with exposure to UVA light, produced only a slight cell loss, with no apparent morphological effects. Catalase was highly sensitive to UVA-induced inactivation, but was not essential for protection. Although UVA light presented a challenge for the lens epithelium, it was well-tolerated under normal conditions. The results demonstrate an important role for TrxR activity in defending the lens epithelium against UVA light, possibly related to the ability of the Trx system to assist DNA synthesis following UVA-induced cell damage. PMID:25495870

  8. A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

    PubMed

    Petrucco, S; Bolchi, A; Foroni, C; Percudani, R; Rossi, G L; Ottonello, S

    1996-01-01

    we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions.

  9. The PedR transcriptional regulator interacts with thioredoxin to connect photosynthesis with gene expression in cyanobacteria.

    PubMed

    Horiuchi, Mayumi; Nakamura, Kinu; Kojima, Kouji; Nishiyama, Yoshitaka; Hatakeyama, Wakako; Hisabori, Toru; Hihara, Yukako

    2010-10-01

    The redox state of the photosynthetic electron transport chain acts as a critical sensing mechanism by regulating the transcription of key genes involved in the acclimation response to a change in the environment. In the present study we show that the small LuxR-type regulator PedR interacts with Trx (thioredoxin) to achieve photosynthetic electron-transport-dependent transcriptional regulation in the cyanobacterium Synechocystis sp. PCC 6803. TrxM, an isoform of Trx, was isolated as an interacting factor of PedR by pull-down assays. In vitro analysis revealed that the intermolecular disulfide bond formed between Cys80 residues of the PedR homodimer was reduced by both TrxM and TrxX. It has been shown previously that, although PedR is active under low-light conditions, it becomes transiently inactivated following a shift to high-light conditions, with a concomitant conformational change [Nakamura and Hihara (2006) J. Biol. Chem. 281, 36758-36766]. In the present study, we found that the conformational change of PedR and the change in the transcript level of its target gene were minimal when mutants of Synechocystis that lack ferredoxin-Trx reductase or NADPH-Trx reductase were exposed to high levels of light. These results indicate that the reduction of PedR by Trx causes transient inactivation of PedR upon the shift of cyanobacterial cells to high-light conditions.

  10. Structural and biochemical characterization of cinnamoyl-coa reductases

    USDA-ARS?s Scientific Manuscript database

    Cinnamoyl-coenzyme A reductase (CCR) catalyzes the reduction of hydroxycinnamoyl-coenzyme A (CoA) esters using NADPH to produce hydroxycinnamyl aldehyde precursors in lignin synthesis. The catalytic mechanism and substrate specificity of cinnamoyl-CoA reductases from sorghum (Sorghum bicolor), a str...

  11. A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

    PubMed Central

    Petrucco, S; Bolchi, A; Foroni, C; Percudani, R; Rossi, G L; Ottonello, S

    1996-01-01

    we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions. PMID:8597660

  12. Targeting the Thioredoxin System for Cancer Therapy.

    PubMed

    Zhang, Junmin; Li, Xinming; Han, Xiao; Liu, Ruijuan; Fang, Jianguo

    2017-09-01

    Thioredoxin (Trx) and thioredoxin reductase (TrxR) are essential components of the Trx system which plays pivotal roles in regulating multiple cellular redox signaling pathways. In recent years TrxR/Trx have been increasingly recognized as an important modulator of tumor development, and hence targeting TrxR/Trx is a promising strategy for cancer treatment. In this review we first discuss the structural details of TrxR, the functions of the Trx system, and the rational of targeting TrxR/Trx for cancer treatment. We also highlight small-molecule TrxR/Trx inhibitors that have potential anticancer activity and review their mechanisms of action. Finally, we examine the challenges of developing TrxR/Trx inhibitors as anticancer agents and perspectives for selectively targeting TrxR/Trx. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. A cellular stress response (CSR) that interacts with NADPH-P450 reductase (NPR) is a new regulator of hypoxic response.

    PubMed

    Oguro, Ami; Koyama, Chika; Xu, Jing; Imaoka, Susumu

    2014-02-28

    NADPH-P450 reductase (NPR) was previously found to contribute to the hypoxic response of cells, but the mechanism was not clarified. In this study, we identified a cellular stress response (CSR) as a new factor interacting with NPR by a yeast two-hybrid system. Overexpression of CSR enhanced the induction of erythropoietin and hypoxia response element (HRE) activity under hypoxia in human hepatocarcinoma cell lines (Hep3B), while knockdown of CSR suppressed them. This new finding regarding the interaction of NPR with CSR provides insight into the function of NPR in hypoxic response. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Reactivation of oxidized PTP1B and PTEN by Thioredoxin 1

    PubMed Central

    Schwertassek, Ulla; Haque, Aftabul; Krishnan, Navasona; Greiner, Romy; Weingarten, Lars; Dick, Tobias P.; Tonks, Nicholas K.

    2014-01-01

    The transient inactivation of protein phosphatases contributes to the efficiency and temporal control of kinase-dependent signal transduction. In particular, members of the protein tyrosine phosphatase family are known to undergo reversible oxidation of their active site cysteine. The thiol oxidation step requires activation of co-localized NADPH oxidases and is mediated by locally produced ROS, in particular H2O2. How oxidized phosphatases are returned to the reduced active state is less well studied. Both major thiol reductive systems, the thioredoxin and the glutathione systems, have been implicated in the reactivation of phosphatases. Here, we show that the protein tyrosine phosphatase PTP1B and the dual-specificity phosphatase PTEN are preferentially reactivated by the thioredoxin system. We show that inducible depletion of TRX1 slows down PTEN re-activation in intact living cells. Finally, using a mechanism-based trapping approach we demonstrate direct thiol disulfide exchange between the active sites of thioredoxin and either phosphatase. The application of thioredoxin trapping mutants represents a complementary approach to direct assays of PTP oxidation in elucidating the significance of redox regulation of PTP function in the control of cell signaling. PMID:24976139

  15. Altered heme catabolism by heme oxygenase-1 caused by mutations in human NADPH cytochrome P450 reductase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pandey, Amit V., E-mail: amit@pandeylab.org; Flueck, Christa E.; Mullis, Primus E.

    2010-09-24

    Research highlights: {yields} Mutations in POR identified from patients lead to reduced HO-1 activities. {yields} POR mutation Y181D affecting FMN binding results in total loss of HO-1 activity. {yields} POR mutations A287P, C569Y and V608F, lost 50-70% activity. {yields} Mutations in FAD binding domain, R457H, Y459H and V492E lost all HO-1 activity. {yields} POR polymorphisms P228L, R316W, G413S, A503V and G504R have normal activity. -- Abstract: Human heme oxygenase-1 (HO-1) carries out heme catabolism supported by electrons supplied from the NADPH through NADPH P450 reductase (POR, CPR). Previously we have shown that mutations in human POR cause a rare formmore » of congenital adrenal hyperplasia. In this study, we have evaluated the effects of mutations in POR on HO-1 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified HO-1 to measure heme degradation in a coupled assay using biliverdin reductase. Here we show that mutations in POR found in patients may reduce HO-1 activity, potentially influencing heme catabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had total loss of HO-1 activity, while POR mutations A287P, C569Y and V608F lost 50-70% activity. The POR variants P228L, R316W and G413S, A503V and G504R identified as polymorphs had close to WT activity. Loss of HO-1 activity may result in increased oxidative neurotoxicity, anemia, growth retardation and iron deposition. Further examination of patients affected with POR deficiency will be required to assess the metabolic effects of reduced HO-1 activity in affected individuals.« less

  16. Pre-steady-state kinetic studies of redox reactions catalysed by Bacillus subtilis ferredoxin-NADP(+) oxidoreductase with NADP(+)/NADPH and ferredoxin.

    PubMed

    Seo, Daisuke; Soeta, Takahiro; Sakurai, Hidehiro; Sétif, Pierre; Sakurai, Takeshi

    2016-06-01

    Ferredoxin-NADP(+) oxidoreductase ([EC1.18.1.2], FNR) from Bacillus subtilis (BsFNR) is a homodimeric flavoprotein sharing structural homology with bacterial NADPH-thioredoxin reductase. Pre-steady-state kinetics of the reactions of BsFNR with NADP(+), NADPH, NADPD (deuterated form) and B. subtilis ferredoxin (BsFd) using stopped-flow spectrophotometry were studied. Mixing BsFNR with NADP(+) and NADPH yielded two types of charge-transfer (CT) complexes, oxidized FNR (FNR(ox))-NADPH and reduced FNR (FNR(red))-NADP(+), both having CT absorption bands centered at approximately 600n m. After mixing BsFNR(ox) with about a 10-fold molar excess of NADPH (forward reaction), BsFNR was almost completely reduced at equilibrium. When BsFNR(red) was mixed with NADP(+), the amount of BsFNR(ox) increased with increasing NADP(+) concentration, but BsFNR(red) remained as the major species at equilibrium even with about 50-fold molar excess NADP(+). In both directions, the hydride-transfer was the rate-determining step, where the forward direction rate constant (~500 s(-1)) was much higher than the reverse one (<10 s(-1)). Mixing BsFd(red) with BsFNR(ox) induced rapid formation of a neutral semiquinone form. This process was almost completed within 1 ms. Subsequently the neutral semiquinone form was reduced to the hydroquinone form with an apparent rate constant of 50 to 70 s(-1) at 10°C, which increased as BsFd(red) increased from 40 to 120 μM. The reduction rate of BsFNR(ox) by BsFd(red) was markedly decreased by premixing BsFNR(ox) with BsFd(ox), indicating that the dissociation of BsFd(ox) from BsFNR(sq) is rate-limiting in the reaction. The characteristics of the BsFNR reactions with NADP(+)/NADPH were compared with those of other types of FNRs. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Molecular cloning and functional characterization of multiple NADPH-cytochrome P450 reductases from Andrographis paniculata.

    PubMed

    Lin, Huixin; Wang, Jian; Qi, Mengdie; Guo, Juan; Rong, Qixian; Tang, Jinfu; Wu, Yisheng; Ma, Xiaojing; Huang, Luqi

    2017-09-01

    Andrographis paniculata (Burm.f.) Wall. ex Nees is widely used as medicinal herb in Southern and Southeastern Asia and andrographolide is its main medicinal constituent. Based on the structure of andrographolide, it has been proposed that cytochrome P450 enzymes play vital roles on its biosynthesis. NADPH:cytochrome P450 reductase (CPR) is the most important redox partner of multiple P450s. In this study, three CPRs were identified in the genomic data of A. paniculata (namely ApCPR1, ApCPR2, and ApCPR3), and their coding regions were cloned. They varied from 62% to 70% identities to each other at the amino acid sequence level. ApCPR1 belongs to Class I of dicotyledonous CPR while both ApCPR2 and ApCPR3 are grouped to Class II. The recombinant enzymes ApCPR1 and ApCPR2 reduced cytochrome c and ferricyanide in an NADPH-dependent manner. In yeast, they supported the activity of CYP76AH1, a ferruginol-forming enzyme. However, ApCPR3 did not show any enzymatic activities either in vitro or in vivo. Quantitative real-time PCR analysis showed that both ApCPR1 and ApCPR2 expressed in all tissues examined, but ApCPR2 showed higher expression in leaves. Expression of ApCPR2 was inducible by MeJA and its pattern matched with andrographolide accumulation. Present investigation suggested ApCPR2 involves in the biosynthesis of secondary metabolites including andrographolide. Copyright © 2017. Published by Elsevier B.V.

  18. Mutations in the mitochondrial thioredoxin reductase gene TXNRD2 cause dilated cardiomyopathy.

    PubMed

    Sibbing, Dirk; Pfeufer, Arne; Perisic, Tamara; Mannes, Alexander M; Fritz-Wolf, Karin; Unwin, Sarah; Sinner, Moritz F; Gieger, Christian; Gloeckner, Christian Johannes; Wichmann, Heinz-Erich; Kremmer, Elisabeth; Schäfer, Zasie; Walch, Axel; Hinterseer, Martin; Näbauer, Michael; Kääb, Stefan; Kastrati, Adnan; Schömig, Albert; Meitinger, Thomas; Bornkamm, Georg W; Conrad, Marcus; von Beckerath, Nicolas

    2011-05-01

    Cardiac energy requirement is met to a large extent by oxidative phosphorylation in mitochondria that are highly abundant in cardiac myocytes. Human mitochondrial thioredoxin reductase (TXNRD2) is a selenocysteine-containing enzyme essential for mitochondrial oxygen radical scavenging. Cardiac-specific deletion of Txnrd2 in mice results in dilated cardiomyopathy (DCM). The aim of this study was to investigate whether TXNRD2 mutations explain a fraction of monogenic DCM cases. Sequencing and subsequent genotyping of TXNRD2 in patients diagnosed with DCM (n = 227) and in DCM-free (n = 683) individuals from the general population sample KORA S4 was performed. The functional impact of observed mutations on Txnrd2 function was tested in mouse fibroblasts. We identified two novel amino acid residue-altering TXNRD2 mutations [175G > A (Ala59Thr) and 1124G > A (Gly375Arg)] in three heterozygous carriers among 227 patients that were not observed in the 683 DCM-free individuals. Both DCM-associated mutations result in amino acid substitutions of highly conserved residues in helices contributing to the flavin-adenine dinucleotide (FAD)-binding domain of TXNRD2. Functional analysis of both mutations in Txnrd2(-/-) mouse fibroblasts revealed that contrasting to wild-type (wt) Txnrd2, neither mutant did restore Txnrd2 function. Mutants even impaired the survival of Txnrd2 wt cells under oxidative stress by a dominant-negative mechanism. For the first time, we describe mutations in DCM patients in a gene involved in the regulation of cellular redox state. TXNRD2 mutations may explain a fraction of human DCM disease burden.

  19. Insights on the mechanism of thioredoxin reductase inhibition by gold N-heterocyclic carbene compounds using the synthetic linear selenocysteine containing C-terminal peptide hTrxR(488-499): an ESI-MS investigation.

    PubMed

    Pratesi, Alessandro; Gabbiani, Chiara; Michelucci, Elena; Ginanneschi, Mauro; Papini, Anna Maria; Rubbiani, Riccardo; Ott, Ingo; Messori, Luigi

    2014-07-01

    Gold-based drugs typically behave as strong inhibitors of the enzyme thioredoxin reductase (hTrxR), possibly as the consequence of direct Gold(I) coordination to its active site selenocysteine. To gain a deeper insight into the molecular basis of enzyme inhibition and prove gold-selenocysteine coordination, the reactions of three parent Gold(I) NHC compounds with the synthetic C-terminal dodecapeptide of hTrxR containing Selenocysteine at position 498, were investigated by electrospray ionization mass spectrometry (ESI-MS). Formation of 1:1 Gold-peptide adducts, though in highly different amounts, was demonstrated in all cases. In these adducts the same [Au-NHC](+) moiety is always associated to the intact peptide. Afterward, tandem MS experiments, conducted on a specific Gold-peptide complex, pointed out that Gold is coordinated to the selenolate group. The relatively large strength of the Gold-selenolate coordinative bond well accounts for potent enzyme inhibition typically afforded by these Gold(I) compounds. In a selected case, the time course of enzyme inhibition was explored. Interestingly, enzyme inhibition turned out to show up very quickly and reached its maximum just few minutes after mixing. Overall, the present results offer some clear insight into the process of thioredoxin reductase inhibition by Gold-based compounds. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. The expression and activity of thioredoxin reductase 1 splice variants v1 and v2 regulate the expression of genes associated with differentiation and adhesion

    PubMed Central

    Nalvarte, Ivan; Damdimopoulos, Anastasios E.; Rüegg, Joëlle; Spyrou, Giannis

    2015-01-01

    The mammalian redox-active selenoprotein thioredoxin reductase (TrxR1) is a main player in redox homoeostasis. It transfers electrons from NADPH to a large variety of substrates, particularly to those containing redox-active cysteines. Previously, we reported that the classical form of cytosolic TrxR1 (TXNRD1_v1), when overexpressed in human embryonic kidney cells (HEK-293), prompted the cells to undergo differentiation [Nalvarte et al. (2004) J. Biol. Chem. 279, 54510–54517]. In the present study, we show that several genes associated with differentiation and adhesion are differentially expressed in HEK-293 cells stably overexpressing TXNRD1_v1 compared with cells expressing its splice variant TXNRD1_v2. Overexpression of these two splice forms resulted in distinctive effects on various aspects of cellular functions including gene regulation patterns, alteration of growth rate, migration and morphology and susceptibility to selenium-induced toxicity. Furthermore, differentiation of the neuroblastoma cell line SH-SY5Y induced by all-trans retinoic acid (ATRA) increased both TXNRD1_v1 and TXNRD1_v2 expressions along with several of the identified genes associated with differentiation and adhesion. Selenium supplementation in the SH-SY5Y cells also induced a differentiated morphology and changed expression of the adhesion protein fibronectin 1 and the differentiation marker cadherin 11, as well as different temporal expression of the studied TXNRD1 variants. These data suggest that both TXNRD1_v1 and TXNRD1_v2 have distinct roles in differentiation, possibly by altering the expression of the genes associated with differentiation, and further emphasize the importance in distinguishing each unique action of different TrxR1 splice forms, especially when studying the gene silencing or knockout of TrxR1. PMID:26464515

  1. EF24 induces ROS-mediated apoptosis via targeting thioredoxin reductase 1 in gastric cancer cells

    PubMed Central

    Chen, Weiqian; Chen, Xi; Ying, Shilong; Feng, Zhiguo; Chen, Tongke; Ye, Qingqing; Wang, Zhe; Qiu, Chenyu; Yang, Shulin; Liang, Guang

    2016-01-01

    Gastric cancer (GC) is one of the leading causes of cancer mortality in the world, and finding novel agents for the treatment of advanced gastric cancer is of urgent need. Diphenyl difluoroketone (EF24), a molecule having structural similarity to curcumin, exhibits potent anti-tumor activities by arresting cell cycle and inducing apoptosis. Although EF24 demonstrates potent anticancer efficacy in numerous types of human cancer cells, the cellular targets of EF24 have not been fully defined. We report here that EF24 may interact with the thioredoxin reductase 1 (TrxR1), an important selenocysteine (Sec)-containing antioxidant enzyme, to induce reactive oxygen species (ROS)-mediated apoptosis in human gastric cancer cells. By inhibiting TrxR1 activity and increasing intracellular ROS levels, EF24 induces a lethal endoplasmic reticulum stress in human gastric cancer cells. Importantly, knockdown of TrxR1 sensitizes cells to EF24 treatment. In vivo, EF24 treatment markedly reduces the TrxR1 activity and tumor cell burden, and displays synergistic lethality with 5-FU against gastric cancer cells. Targeting TrxR1 with EF24 thus discloses a previously unrecognized mechanism underlying the biological activity of EF24, and reveals that TrxR1 is a good target for gastric cancer therapy. PMID:26919110

  2. Lifespan decrease in a Caenorhabditis elegans mutant lacking TRX-1, a thioredoxin expressed in ASJ sensory neurons.

    PubMed

    Miranda-Vizuete, Antonio; Fierro González, Juan Carlos; Gahmon, Gabriele; Burghoorn, Jan; Navas, Plácido; Swoboda, Peter

    2006-01-23

    Thioredoxins are a class of small proteins that play a key role in regulating many cellular redox processes. We report here the characterization of the first member of the thioredoxin family in metazoans that is mainly associated with neurons. The Caenorhabditis elegans gene B0228.5 encodes a thioredoxin (TRX-1) that is expressed in ASJ ciliated sensory neurons, and to some extent also in the posterior-most intestinal cells. TRX-1 is active at reducing protein disulfides in the presence of a heterologous thioredoxin reductase. A mutant worm strain carrying a null allele of the trx-1 gene displays a reproducible decrease in both mean and maximum lifespan when compared to wild-type. The identification and characterization of TRX-1 paves the way to use C. elegans as an in vivo model to study the role of thioredoxins in lifespan and nervous system physiology and pathology.

  3. Selenocysteine Confers Resistance to Inactivation by Oxidation in Thioredoxin Reductase: Comparison of Selenium and Sulfur Enzymes†

    PubMed Central

    Snider, Gregg W.; Ruggles, Erik; Khan, Nadeem; Hondal, Robert J.

    2013-01-01

    Mammalian thioredoxin reductase (TR) is a selenocysteine (Sec)-containing homodimeric pyridine nucleotide oxidoreductase which catalyzes the reduction of oxidized thioredoxin. We have previously demonstrated the full-length mitochondrial mammalian TR (mTR3) enzyme to be resistant to inactivation from exposure to 50 mM H2O2. Because a Sec residue oxidizes more rapidly than a cysteine (Cys) residue, it has been previously thought that Sec-containing enzymes are “sensitive to oxidation” compared to Cys-orthologs. Here we show for the first time a direct comparison of the abilities of Sec-containing mTR3 and the Cys-ortholog from D. melanogaster (DmTR) to resist inactivation by oxidation from a variety of oxidants including H2O2, hydroxyl radical, peroxynitrite, hypochlorous acid, hypobromous acid, and hypothiocyanous acid. The results show that the Sec-containing TR is far superior to the Cys-ortholog TR in resisting inactivation by oxidation. To further test our hypothesis that the use of Sec confers strong resistance to inactivation by oxidation, we constructed a chimeric enzyme in which we replaced the active site Cys nucleophile of DmTR with a Sec residue using semisynthesis. The chimeric Sec-containing enzyme has similar ability to resist inactivation by oxidation as the wild type Sec-containing TR from mouse mitochondria. The use of Sec in the chimeric enzyme “rescued” the enzyme from oxidant-induced inactivation for all of the oxidants tested in this study, in direct contrast to previous understanding. We discuss two possibilities for this rescue effect from inactivation under identical conditions of oxidative stress: (i) Sec resists over-oxidation and inactivation, whereas a Cys residue can be permanently over-oxidized to the sulfinic acid form, and (ii) Sec protects the body of the enzyme from harmful oxidation by allowing the enzyme to metabolize (turnover) various oxidants much better than a Cys-containing TR. PMID:23865454

  4. Toxicological effects of thiomersal and ethylmercury: Inhibition of the thioredoxin system and NADP{sup +}-dependent dehydrogenases of the pentose phosphate pathway

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rodrigues, Juan, E-mail: juanricardorodrigues@gmail.com; Laboratory of Biochemistry, Faculty of Pharmacy, Central University of Venezuela; Branco, Vasco

    Mercury (Hg) is a strong toxicant affecting mainly the central nervous, renal, cardiovascular and immune systems. Thiomersal (TM) is still in use in medical practice as a topical antiseptic and as a preservative in multiple dose vaccines, routinely given to young children in some developing countries, while other forms of mercury such as methylmercury represent an environmental and food hazard. The aim of the present study was to determine the effects of thiomersal (TM) and its breakdown product ethylmercury (EtHg) on the thioredoxin system and NADP{sup +}-dependent dehydrogenases of the pentose phosphate pathway. Results show that TM and EtHg inhibitedmore » the thioredoxin system enzymes in purified suspensions, being EtHg comparable to methylmercury (MeHg). Also, treatment of neuroblastoma and liver cells with TM or EtHg decreased cell viability (GI{sub 50}: 1.5 to 20 μM) and caused a significant (p < 0.05) decrease in the overall activities of thioredoxin (Trx) and thioredoxin reductase (TrxR) in a concentration- and time-dependent manner in cell lysates. Compared to control, the activities of Trx and TrxR in neuroblastoma cells after EtHg incubation were reduced up to 60% and 80% respectively, whereas in hepatoma cells the reduction was almost 100%. In addition, the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were also significantly inhibited by all mercurials, with inhibition intensity of Hg{sup 2+} > MeHg ≈ EtHg > TM (p < 0.05). Cell incubation with sodium selenite alleviated the inhibitory effects on TrxR and glucose-6-phosphate dehydrogenase. Thus, the molecular mechanism of toxicity of TM and especially of its metabolite EtHg encompasses the blockage of the electrons from NADPH via the thioredoxin system. - Highlights: • TM and EtHg inhibit Trx and TrxR both in purified suspensions and cell lysates. • TM and EtHg also inhibit the activities of G6PDH and 6PGDH in cell lysates, • Co-exposure to selenite

  5. The structure of apo and holo forms of xylose reductase, a dimeric aldo-keto reductase from Candida tenuis.

    PubMed

    Kavanagh, Kathryn L; Klimacek, Mario; Nidetzky, Bernd; Wilson, David K

    2002-07-16

    Xylose reductase is a homodimeric oxidoreductase dependent on NADPH or NADH and belongs to the largely monomeric aldo-keto reductase superfamily of proteins. It catalyzes the first step in the assimilation of xylose, an aldose found to be a major constituent monosaccharide of renewable plant hemicellulosic material, into yeast metabolic pathways. It does this by reducing open chain xylose to xylitol, which is reoxidized to xylulose by xylitol dehydrogenase and metabolically integrated via the pentose phosphate pathway. No structure has yet been determined for a xylose reductase, a dimeric aldo-keto reductase or a family 2 aldo-keto reductase. The structures of the Candida tenuis xylose reductase apo- and holoenzyme, which crystallize in spacegroup C2 with different unit cells, have been determined to 2.2 A resolution and an R-factor of 17.9 and 20.8%, respectively. Residues responsible for mediating the novel dimeric interface include Asp-178, Arg-181, Lys-202, Phe-206, Trp-313, and Pro-319. Alignments with other superfamily members indicate that these interactions are conserved in other dimeric xylose reductases but not throughout the remainder of the oligomeric aldo-keto reductases, predicting alternate modes of oligomerization for other families. An arrangement of side chains in a catalytic triad shows that Tyr-52 has a conserved function as a general acid. The loop that folds over the NAD(P)H cosubstrate is disordered in the apo form but becomes ordered upon cosubstrate binding. A slow conformational isomerization of this loop probably accounts for the observed rate-limiting step involving release of cosubstrate. Xylose binding (K(m) = 87 mM) is mediated by interactions with a binding pocket that is more polar than a typical aldo-keto reductase. Modeling of xylose into the active site of the holoenzyme using ordered waters as a guide for sugar hydroxyls suggests a convincing mode of substrate binding.

  6. Is Oxidized Thioredoxin a Major Trigger for Cysteine Oxidation? Clues from a Redox Proteomics Approach

    PubMed Central

    García-Santamarina, Sarela; Boronat, Susanna; Calvo, Isabel A.; Rodríguez-Gabriel, Miguel; Ayté, José; Molina, Henrik

    2013-01-01

    Abstract Cysteine oxidation mediates oxidative stress toxicity and signaling. It has been long proposed that the thioredoxin (Trx) system, which consists of Trx and thioredoxin reductase (Trr), is not only involved in recycling classical Trx substrates, such as ribonucleotide reductase, but it also regulates general cytoplasmic thiol homeostasis. To investigate such a role, we have performed a proteome-wide analysis of cells expressing or not the two components of the Trx system. We have compared the reversibly oxidized thiol proteomes of wild-type Schizosaccharomyces pombe cells with mutants lacking Trx or Trr. Specific Trx substrates are reversibly-oxidized in both strain backgrounds; however, in the absence of Trr, Trx can weakly recycle its substrates at the expense of an alternative electron donor. A massive thiol oxidation occurs only in cells lacking Trr, with 30% of all cysteine-containing peptides being reversibly oxidized; this oxidized cysteine proteome depends on the presence of Trxs. Our observations lead to the hypothesis that, in the absence of its reductase, the natural electron donor Trx becomes a powerful oxidant and triggers general thiol oxidation. Antioxid. Redox Signal. 18, 1549–1556. PMID:23121505

  7. The Hinge Segment of Human NADPH-Cytochrome P450 Reductase in Conformational Switching: The Critical Role of Ionic Strength

    PubMed Central

    Campelo, Diana; Lautier, Thomas; Urban, Philippe; Esteves, Francisco; Bozonnet, Sophie; Truan, Gilles; Kranendonk, Michel

    2017-01-01

    NADPH-cytochrome P450 reductase (CPR) is a redox partner of microsomal cytochromes P450 and is a prototype of the diflavin reductase family. CPR contains 3 distinct functional domains: a FMN-binding domain (acceptor reduction), a linker (hinge), and a connecting/FAD domain (NADPH oxidation). It has been demonstrated that the mechanism of CPR exhibits an important step in which it switches from a compact, closed conformation (locked state) to an ensemble of open conformations (unlocked state), the latter enabling electron transfer to redox partners. The conformational equilibrium between the locked and unlocked states has been shown to be highly dependent on ionic strength, reinforcing the hypothesis of the presence of critical salt interactions at the interface between the FMN and connecting FAD domains. Here we show that specific residues of the hinge segment are important in the control of the conformational equilibrium of CPR. We constructed six single mutants and two double mutants of the human CPR, targeting residues G240, S243, I245 and R246 of the hinge segment, with the aim of modifying the flexibility or the potential ionic interactions of the hinge segment. We measured the reduction of cytochrome c at various salt concentrations of these 8 mutants, either in the soluble or membrane-bound form of human CPR. All mutants were found capable of reducing cytochrome c yet with different efficiency and their maximal rates of cytochrome c reduction were shifted to lower salt concentration. In particular, residue R246 seems to play a key role in a salt bridge network present at the interface of the hinge and the connecting domain. Interestingly, the effects of mutations, although similar, demonstrated specific differences when present in the soluble or membrane-bound context. Our results demonstrate that the electrostatic and flexibility properties of the hinge segment are critical for electron transfer from CPR to its redox partners. PMID:29163152

  8. Identification of acid-base catalytic residues of high-Mr thioredoxin reductase from Plasmodium falciparum.

    PubMed

    McMillan, Paul J; Arscott, L David; Ballou, David P; Becker, Katja; Williams, Charles H; Müller, Sylke

    2006-11-03

    High-M(r) thioredoxin reductase from the malaria parasite Plasmodium falciparum (PfTrxR) contains three redox active centers (FAD, Cys-88/Cys-93, and Cys-535/Cys-540) that are in redox communication. The catalytic mechanism of PfTrxR, which involves dithiol-disulfide interchanges requiring acid-base catalysis, was studied by steady-state kinetics, spectral analyses of anaerobic static titrations, and rapid kinetics analysis of wild-type enzyme and variants involving the His-509-Glu-514 dyad as the presumed acid-base catalyst. The dyad is conserved in all members of the enzyme family. Substitution of His-509 with glutamine and Glu-514 with alanine led to TrxR with only 0.5 and 7% of wild type activity, respectively, thus demonstrating the crucial roles of these residues for enzymatic activity. The H509Q variant had rate constants in both the reductive and oxidative half-reactions that were dramatically less than those of wild-type enzyme, and no thiolateflavin charge-transfer complex was observed. Glu-514 was shown to be involved in dithiol-disulfide interchange between the Cys-88/Cys-93 and Cys-535/Cys-540 pairs. In addition, Glu-514 appears to greatly enhance the role of His-509 in acid-base catalysis. It can be concluded that the His-509-Glu-514 dyad, in analogy to those in related oxidoreductases, acts as the acid-base catalyst in PfTrxR.

  9. EF24 induces ROS-mediated apoptosis via targeting thioredoxin reductase 1 in gastric cancer cells.

    PubMed

    Zou, Peng; Xia, Yiqun; Chen, Weiqian; Chen, Xi; Ying, Shilong; Feng, Zhiguo; Chen, Tongke; Ye, Qingqing; Wang, Zhe; Qiu, Chenyu; Yang, Shulin; Liang, Guang

    2016-04-05

    Gastric cancer (GC) is one of the leading causes of cancer mortality in the world, and finding novel agents for the treatment of advanced gastric cancer is of urgent need. Diphenyl difluoroketone (EF24), a molecule having structural similarity to curcumin, exhibits potent anti-tumor activities by arresting cell cycle and inducing apoptosis. Although EF24 demonstrates potent anticancer effïcacy in numerous types of human cancer cells, the cellular targets of EF24 have not been fully defined. We report here that EF24 may interact with the thioredoxin reductase 1 (TrxR1), an important selenocysteine (Sec)-containing antioxidant enzyme, to induce reactive oxygen species (ROS)-mediated apoptosis in human gastric cancer cells. By inhibiting TrxR1 activity and increasing intracellular ROS levels, EF24 induces a lethal endoplasmic reticulum stress in human gastric cancer cells. Importantly, knockdown of TrxR1 sensitizes cells to EF24 treatment. In vivo, EF24 treatment markedly reduces the TrxR1 activity and tumor cell burden, and displays synergistic lethality with 5-FU against gastric cancer cells. Targeting TrxR1 with EF24 thus discloses a previously unrecognized mechanism underlying the biological activity of EF24, and reveals that TrxR1 is a good target for gastric cancer therapy.

  10. Glutathione and thioredoxin systems contribute to recombinant monoclonal antibody interchain disulfide bond reduction during bioprocessing.

    PubMed

    Handlogten, Michael W; Zhu, Min; Ahuja, Sanjeev

    2017-07-01

    Antibody interchain disulfide bond reduction during biopharmaceutical manufacturing has received increased attention since it was first reported in 2010. Antibody reduction leads to loss of product and reduced product stability. It is therefore critical to understand the underlying mechanisms of reduction. To date, the thioredoxin system has been reported as the sole contributor to antibody reduction during bioprocessing. In this work, we show that the glutathione system, in addition to the thioredoxin system, is involved in reducing antibody molecules and the contributions of the two systems can vary depending upon the cell culture process. The roles of the glutathione and thioredoxin systems were evaluated for three molecules with different IgG subclass where reduction was observed during manufacturing: mAb A, mAb B, and mAb C representing an IgG 1 , IgG 2 , and IgG 4, respectively. The expression of enzymes for both the thioredoxin and glutathione systems were confirmed in all three cell lines. Inhibitors were evaluated using purified mammalian reductases to evaluate their specificity. The optimized experimental conditions enabled both the determination of reductase activity contributed from as well as the amount of antibody reduced by each enzymatic system. Our results demonstrate that the underlying enzymatic mechanisms are different depending upon the cell culture process; one of the two systems may be the dominant mechanism, or both enzymatic systems may be involved. Specifically, the glutathione system was found to be the major contributor to mAb A reduction while the thioredoxin system was the major contributor to mAb C reduction. Intriguingly, mAb B experienced significant reduction from both enzymatic systems. In summary, we have demonstrated that in addition to the thioredoxin pathway, the glutathione system is a second major pathway contributing to antibody reduction and this knowledge can be leveraged to develop more specific antibody reduction

  11. Molecular cloning and functional characterization of NADPH-dependent cytochrome P450 reductase from the green microalga Botryococcus braunii, B race.

    PubMed

    Tsou, Chung-Yau; Matsunaga, Shigeki; Okada, Shigeru

    2018-01-01

    The green microalga Botryococcus braunii of the B race accumulates various lipophilic compounds containing a 10,11-oxidosqualene epoxide moiety in addition to large amounts of triterpene hydrocarbons. While 2,3-squalene epoxidases have already been isolated and characterized from the alga, the enzyme that catalyzes the 10,11-epoxidation of squalene has remained elusive. In order to obtain a molecular tool to explore a 10,11-squalene epoxidase, cDNA cloning of an NADPH-dependent cytochrome P450 reductase (CPR) that is required by both squalene epoxidases and cytochrome P450 enzymes was carried out. The isolated cDNA contained an open reading frame (1998 bp) that encoded for a protein with 665 amino acid residues with a predicted molecular weight of 71.46 kDa and a theoretical pI of 5.49. Analysis of the deduced amino acid sequence revealed the presence of conserved motifs, including FMN, FAD, and NADPH binding domains, which are typical of other CPRs and necessary for enzyme activity. By truncation of the N-terminal transmembrane anchor and addition of a 6× His-tag, BbCPR was heterologously produced in Escherichia coli and purified by Ni-NTA affinity chromatography. The purified recombinant enzyme showed optimal reducing activity of cytochrome c at around a neutral pH at a temperature range of 30-37°C. For steady state kinetic parameters, the recombinant enzyme had a k m for cytochrome c and NADPH of 11.7±1.6 and 9.4±1.4 μM, and a k cat for cytochrome c and NADPH of 2.78±0.09 and 3.66±0.11 μmol/min/mg protein, respectively. This is the first study to perform the functional characterization of a CPR from eukaryotic microalgae. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  12. Thioredoxin and evolution

    NASA Technical Reports Server (NTRS)

    Buchanan, B. B.

    1991-01-01

    Comparisons of primary structure have revealed significant homology between the m type thioredoxins of chloroplasts and the thioredoxins from a variety of bacteria. Chloroplast thioredoxin f, by comparison, remains an enigma: certain residues are invariant with those of the other thioredoxins, but a phylogenetic relationship to bacterial or m thioredoxins seems distant. Knowledge of the evolutionary history of thioredoxin f is, nevertheless, of interest because of its role in photosynthesis. Therefore, we have attempted to gain information on the evolutionary history of chloroplast thioredoxin f, as well as m. Our goal was first to establish the utility of thioredoxin as a phylogenetic marker, and, if found suitable, to deduce the evolutionary histories of the chloroplast thioredoxins. To this end, we have constructed phylogenetic (minimal replacement) trees using computer analysis. The results show that the thioredoxins of bacteria and animals fall into distinct phylogenetic groups - the bacterial group resembling that derived from earlier 16s RNA analysis and the animal group showing a cluster consistent with known relationships. The chloroplast thioredoxins show a novel type of phylogenetic arrangement: one m type aligns with its counterpart of eukaryotic algae, cyanobacteria and other bacteria, whereas the second type (f type) tracks with animal thioredoxin. The results give new insight into the evolution of photosynthesis.

  13. Time-dependent modulation of thioredoxin reductase activity might contribute to sulforaphane-mediated inhibition of NF-kappaB binding to DNA.

    PubMed

    Heiss, Elke; Gerhäuser, Clarissa

    2005-01-01

    The chemopreventive agent sulforaphane (SFN) exerts anti-inflammatory activity by thiol-dependent inhibition of nuclear factor kappaB (NF-kappaB) DNA binding. To further analyze the underlying mechanisms, we focused on the thioredoxin/thioredoxin reductase (TrxR) system as a key redox mechanism regulating NF-kappaB DNA binding. Using cultured Raw 264.7 mouse macrophages as a model, 1-chloro-2,4-dinitrobenzene (CDNB), a known inhibitor of TrxR, was identified as an inhibitor of lipopolysaccharide (LPS)-mediated nitric oxide (NO) production and of NF-kappaB DNA binding. CDNB and SFN acted synergistically with respect to inhibition of LPS-induced NO release, and we consequently identified SFN as a novel inhibitor of TrxR enzymatic activity in vitro. Short-term treatment of Raw macrophages with SFN or CDNB resulted in the inhibition of TrxR activity in vivo with half-maximal inhibitory concentration of 25.0 +/- 3.5 microM and 9.4 +/- 3.7 microM, respectively, whereas after a 24-h treatment with 25 microM SFN, TrxR activity was >1.5-fold elevated. In additional experiments, we could exclude that inhibition of trans-activating activity of NF-kappaB contributed to the reduced expression of pro-inflammatory proteins by SFN, based on transient transfection experiments with a (kappaB)(2)- chloramphenicol acetyltransferase construct and a lack of inhibition of protein kinase A activity. These findings further emphasize the importance of redox modulation or thiol reactivity for the regulation of NF-kappaB-dependent transcription by SFN. Antioxid. Redox Signal. 7, 1601-1611. Antioxid. Redox Signal. 7, 1601-1611.

  14. Selenium deficiency-induced thioredoxin suppression and thioredoxin knock down disbalanced insulin responsiveness in chicken cardiomyocytes through PI3K/Akt pathway inhibition.

    PubMed

    Yang, Jie; Hamid, Sattar; Cai, Jingzeng; Liu, Qi; Xu, Shiwen; Zhang, Ziwei

    2017-10-01

    Thioredoxin (Txn) system is the most crucial antioxidant defense mechanism in cell consisting of Txn, thioredoxin reductase (TR) and Nicotinamide Adenine Dinucleotide Phosphate (NADPH). Perturbations in Txn system may compromise cell survival through oxidative stress induction. Metabolic activity of insulin plays important roles in fulfilling the stable and persistent demands of heart through glucose metabolism. However, the roles of Txn and Txn system in insulin modulated cardiac energy metabolism have been less reported. Therefore, to investigate the role of Txn in myocardial metabolism, we developed a Se-deficient chicken model (0.033mg/kg) for in-vivo and Txn knock down cardiomyocytes culture model (siRNA) for in-vitro studies. Quantitative real time PCR and western blotting was performed. Se deficiency suppressed Txn and TR in cardiac tissues. Significant increases in ROS (P<0.05) levels signify the onset of oxidative stress and in both models. Se deficiency-induced Txn suppression model and Txn knock down cardiomyocytes models significantly decreased (P<0.05), the mRNA and protein levels of insulin-like growth factors (IGF1, IGF2), IGF-binding proteins (IGFBP2, IGFBP4), insulin receptor (IR), insulin receptor substrates (IRS1, IRS2), and glucose transporters (GLUT1, GLUT3, GLUT8), however, IGFBP3 expression increased in Txn knock down cardiomyocytes. In addition, in contrast to their respective controls, Se deficiency-induced Txn depleted tissues and Txn deleted cardiomyocytes showed suppression in mRNA and protein levels of PI3K, AKT, P-PI3K, and repression in FOX, P-FOX JNK genes. Combing the in vitro and in vivo experiments, we demonstrate that Txn gene suppression can cause dysfunction of insulin-modulated cardiac energy metabolism and increase insulin resistance through PI3K-Akt pathway inhibition. Herein, we conclude that inactivation of Txn system can alter cellular insulin response through IRS/PI3K/Akt pathway repression and JNK and FOX expression

  15. Crystal structures of pinoresinol-lariciresinol and phenylcoumaran benzylic ether reductases and their relationship to isoflavone reductases

    NASA Technical Reports Server (NTRS)

    Min, Tongpil; Kasahara, Hiroyuki; Bedgar, Diana L.; Youn, Buhyun; Lawrence, Paulraj K.; Gang, David R.; Halls, Steven C.; Park, HaJeung; Hilsenbeck, Jacqueline L.; Davin, Laurence B.; hide

    2003-01-01

    Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g. in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun. In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids. Of particular interest was in determining how differing regio- and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates. Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 A resolutions, respectively. Not only do they share high gene sequence similarity, but their structures are similar, having a continuous alpha/beta NADPH-binding domain and a smaller substrate-binding domain. IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure. The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed.

  16. Thioredoxin-interacting protein (Txnip) is a critical regulator of hepatic glucose production.

    PubMed

    Chutkow, William A; Patwari, Parth; Yoshioka, Jun; Lee, Richard T

    2008-01-25

    Thioredoxin-interacting protein (Txnip) has been recently described as a possible link between cellular redox state and metabolism; Txnip binds thioredoxin and inhibits its disulfide reductase activity in vitro, while a naturally occurring strain of Txnip-deficient mice has hyperlipidemia, hypoglycemia, and ketosis exacerbated by fasting. We generated Txnip-null mice to investigate the role of Txnip in glucose homeostasis. Txnip-null mice were hypoglycemic, hypoinsulinemic, and had blunted glucose production following a glucagon challenge, consistent with a central liver glucose-handling defect. Glucose release from isolated Txnip-null hepatocytes was 2-fold lower than wild-type hepatocytes, whereas beta-hydroxybutyrate release was increased 2-fold, supporting an intrinsic defect in hepatocyte glucose metabolism. While hepatocyte-specific gene deletion of Txnip did not alter glucose clearance compared with littermate controls, Txnip expression in the liver was required for maintaining normal fasting glycemia and glucose production. In addition, hepatic overexpression of a Txnip transgene in wild-type mice resulted in elevated serum glucose levels and decreased ketone levels. Liver homogenates from Txnip-null mice had no significant differences in the glutathione oxidation state or in the amount of available thioredoxin. However, overexpression of wild-type Txnip in Txnip-null hepatocytes rescued cellular glucose production, whereas overexpression of a C247S mutant Txnip, which does not bind thioredoxin, had no effect. These data demonstrate that Txnip is required for normal glucose homeostasis in the liver. While available thioredoxin is not changed in Txnip-null mice, the effects of Txnip on glucose homeostasis are abolished by a single cysteine mutation that inhibits binding to thioredoxin.

  17. Functional properties and structural characterization of rice δ 1-pyrroline-5-carboxylate reductase

    DOE PAGES

    Forlani, Giuseppe; Bertazzini, Michele; Zarattini, Marco; ...

    2015-07-28

    The majority of plant species accumulate high intracellular levels of proline to cope with hyperosmotic stress conditions. Proline synthesis from glutamate is tightly regulated at both the transcriptional and the translational levels, yet little is known about the mechanisms for post-translational regulation of the enzymatic activities involved. The gene coding in rice ( Oryza sativa L.) for δ 1-pyrroline-5-carboxylate (P5C) reductase, the enzyme that catalyzes the second and final step in this pathway, was isolated and expressed in Escherichia coli. The structural and functional properties of the affinity-purified protein were characterized. As for most species, rice P5C reductase was ablemore » to use in vitro either NADH or NADPH as the electron donor. However, strikingly different effects of cations and anions were found depending on the pyridine nucleotide used, namely inhibition of NADH-dependent activity and stimulation of NADPH-dependent activity. Moreover, physiological concentrations of proline and NADP + were strongly inhibitory for the NADH-dependent reaction, whereas the NADPH-dependent activity was mildly affected. Our results suggest that only NADPH may be used in vivo and that stress-dependent variations in ion homeostasis and NADPH/NADP + ratio could modulate enzyme activity, being functional in promoting proline accumulation and potentially also adjusting NADPH consumption during the defense against hyperosmotic stress. The apparent molecular weight of the native protein observed in size exclusion chromatography indicated a high oligomerization state. We also report the first crystal structure of a plant P5C reductase at 3.40-Å resolution, showing a decameric quaternary assembly. It was possible to identify dynamic structural differences among rice, human, and bacterial enzymes.« less

  18. Ebselen and analogs as inhibitors of Bacillus anthracis thioredoxin reductase and bactericidal antibacterials targeting Bacillus species, Staphylococcus aureus and Mycobacterium tuberculosis.

    PubMed

    Gustafsson, Tomas N; Osman, Harer; Werngren, Jim; Hoffner, Sven; Engman, Lars; Holmgren, Arne

    2016-06-01

    Bacillus anthracis is the causative agent of anthrax, a disease associated with a very high mortality rate in its invasive forms. We studied a number of ebselen analogs as inhibitors of B. anthracis thioredoxin reductase and their antibacterial activity on Bacillus subtilis, Staphylococcus aureus, Bacillus cereus and Mycobacterium tuberculosis. The most potent compounds in the series gave IC(50) values down to 70 nM for the pure enzyme and minimal inhibitory concentrations (MICs) down to 0.4 μM (0.12 μg/ml) for B. subtilis, 1.5 μM (0.64 μg/ml) for S. aureus, 2 μM (0.86 μg/ml) for B. cereus and 10 μg/ml for M. tuberculosis. Minimal bactericidal concentrations (MBCs) were found at 1-1.5 times the MIC, indicating a general, class-dependent, bactericidal mode of action. The combined bacteriological and enzymological data were used to construct a preliminary structure-activity-relationship for the benzoisoselenazol class of compounds. When S. aureus and B. subtilis were exposed to ebselen, we were unable to isolate resistant mutants on both solid and in liquid medium suggesting a high resistance barrier. These results suggest that ebselen and analogs thereof could be developed into a novel antibiotic class, useful for the treatment of infections caused by B. anthracis, S. aureus, M. tuberculosis and other clinically important bacteria. Furthermore, the high barrier against resistance development is encouraging for further drug development. We have characterized the thioredoxin system from B. anthracis as a novel drug target and ebselen and analogs thereof as a potential new class of antibiotics targeting several important human pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase.

    PubMed

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

    Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases.

  20. The Thioredoxin Domain of Neisseria Gonorrhoeae PilB can use Electrons from DsbD to Reduce Downstream Methionine Sulfoxide Reductases

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Brot,N.; Collet, J.; Johnson, L.

    2006-01-01

    The PilB protein from Neisseria gonorrhoeae is located in the periplasm and made up of three domains. The N-terminal, thioredoxin-like domain (NT domain) is fused to tandem methionine sulfoxide reductase A and B domains (MsrA/B). We show that the {alpha} domain of Escherichia coli DsbD is able to reduce the oxidized NT domain, which suggests that DsbD in Neisseria can transfer electrons from the cytoplasmic thioredoxin to the periplasm for the reduction of the MsrA/B domains. An analysis of the available complete genomes provides further evidence for this proposition in other bacteria where DsbD/CcdA, Trx, MsrA, and MsrB gene homologsmore » are all located in a gene cluster with a common transcriptional direction. An examination of wild-type PilB and a panel of Cys to Ser mutants of the full-length protein and the individually expressed domains have also shown that the NT domain more efficiently reduces the MsrA/B domains when in the polyprotein context. Within this framework there does not appear to be a preference for the NT domain to reduce the proximal MsrA domain over MsrB domain. Finally, we report the 1.6 {angstrom} crystal structure of the NT domain. This structure confirms the presence of a surface loop that makes it different from other membrane-tethered, Trx-like molecules including TlpA, CcmG and ResA. Subtle differences are observed in this loop when compared to the N. meningitidis NT domain structure. The data taken together supports the formation of specific NT domain interactions with the MsrA/B domains and its in vivo recycling partner, DsbD.« less

  1. A mutant of barley lacking NADH-hydroxypyruvate reductase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Blackwell, R.; Lea, P.

    1989-04-01

    A mutant of barley, LaPr 88/29, deficient in peroxisomal NADH-hydroxypyruvate reductase (HPR) activity has been identified. Compared to the wild type the activities of NADH-HPR and NADPH-HPR were severely reduced but the mutant was still capable of fixing CO{sub 2} at rates equivalent to 75% of that of the wild type in air. Although lacking an enzyme in the main photorespiratory pathway, there appeared to be little disruption to photorespiratory metabolism as ammonia release, CO{sub 2} efflux and {sup 14}CO{sub 2} release from L-(U-{sup 14}C) serine were similar in both mutant and wild type. LaPr 88/29 has been used tomore » show that NADH-glyoxylate reductase (GR) and NADH-HPR are probably not catalyzed by the same enzyme in barley and that over 80% of the NADPH-HPR activity is due to the NADH-HPR enzyme. Immunological studies, using antibodies raised against spinach HPR, have shown that the NADH-dependent enzyme protein is absent in LaPr 88/29 but there appears to be enhanced synthesis of the NADPH-dependent enzyme protein.« less

  2. Biotransformation of Hexahydro-1,3,5-trinitro-1,3,5-triazine Catalyzed by a NAD(P)H: Nitrate Oxidoreductase from Aspergillus niger

    DTIC Science & Technology

    2002-01-01

    Biotransformation of Hexahydro-1,3,5-trinitro-1,3,5-triazine Catalyzed by a NAD(P)H: Nitrate Oxidoreductase from Aspergillus niger B H A R A T B H U...reductase from Aspergillus niger catalyzed the biotransformation of RDX most effectively at pH 7.0 and 30 °C under anaerobic conditions using NADPH as...nitroreductase. We selected a nitrate reductase (EC 1.6.6.2) from a fungus Aspergillus niger to transform RDX under anaerobic condi- tions because nitrate

  3. Dual pharmacological inhibition of glutathione and thioredoxin systems synergizes to kill colorectal carcinoma stem cells.

    PubMed

    Tanaka, Genki; Inoue, Ken-Ichi; Shimizu, Takayuki; Akimoto, Kazumi; Kubota, Keiichi

    2016-09-01

    NRF2 stabilizes redox potential through genes for glutathione and thioredoxin antioxidant systems. Whether blockade of glutathione and thioredoxin is useful in eliminating cancer stem cells remain unknown. We used xenografts derived from colorectal carcinoma patients to investigate the pharmacological inhibition of glutathione and thioredoxin systems. Higher expression of five glutathione S-transferase isoforms (GSTA1, A2, M4, O2, and P1) was observed in xenograft-derived spheroids than in fibroblasts. Piperlongumine (2.5-10 μmol/L) and auranofin (0.25-4 μmol/L) were used to inhibit glutathione S-transferase π and thioredoxin reductase, respectively. Piperlongumine or auranofin alone up-regulated the expression of NRF2 target genes, but not TP53 targets. While piperlongumine showed modest cancer-specific cell killing (IC50 difference between cancer spheroids and fibroblasts: P = 0.052), auranofin appeared more toxic to fibroblasts (IC50 difference between cancer spheroids and fibroblasts: P = 0.002). The synergism of dual inhibition was evaluated by determining the Combination Index, based on the number of surviving cells with combination treatments. Molar ratios indicated synergism in cancer spheroids, but not in fibroblasts: (auranofin:piperlongumine) = 2:5, 1:5, 1:10, and 1:20. Cancer-specific cell killing was achieved at the following drug concentrations (auranofin:piperlongumine): 0.25:2.5 μmol/L, 0.5:2.5 μmol/L, or 0.25:5 μmol/L. The dual inhibition successfully decreased CD44v9 surface presentation and delayed tumor emergence in nude mouse. However, a small subpopulation persistently survived and accumulated phosphorylated histone H2A. Such "persisters" still retained lesser but significant tumorigenicity. Thus, dual inhibition of glutathione S-transferase π and thioredoxin reductase could be a feasible option for decreasing the tumor mass and CD44v9-positive fraction by disrupting redox regulation. © 2016 The Authors. Cancer Medicine

  4. Improved expression, purification and crystallization of a putative N-acetyl-γ-glutamyl-phosphate reductase from rice (Oryza sativa)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Miura-Ohnuma, Jun; Nonaka, Tsuyoshi; Katoh, Shizue

    2005-12-01

    Crystals of OsAGPR were obtained using the sitting-drop vapour-diffusion method at 293 K and diffract X-rays to at least 1.8 Å resolution. They belong to the hexagonal space group P6{sub 1}, with unit-cell parameters a = 86.11, c = 316.3 Å. N-Acetyl-γ-glutamyl-phosphate reductase (AGPR) catalyzes the third step in an eight-step arginine-biosynthetic pathway that starts with glutamate. This enzyme converts N-acetyl-γ-glutamyl phosphate to N-acetylglutamate-γ-semialdehyde by an NADPH-dependent reductive dephosphorylation. AGPR from Oryza sativa (OsAGPR) was expressed in Escherichia coli at 291 K as a soluble fusion protein with an upstream thioredoxin-hexahistidine [Trx-(His){sub 6}] extension. OsAGPR(Ala50–Pro366) was purified and crystals weremore » obtained using the sitting-drop vapour-diffusion method at 293 K and diffract X-rays to at least 1.8 Å resolution. They belong to the hexagonal space group P6{sub 1}, with unit-cell parameters a = 86.11, c = 316.3 Å.« less

  5. NADPH-cytochrome P450 reductase-mediated denitration reaction of 2,4,6-trinitrotoluene to yield nitrite in mammals.

    PubMed

    Shinkai, Yasuhiro; Nishihara, Yuya; Amamiya, Masahiro; Wakayama, Toshihiko; Li, Song; Kikuchi, Tomohiro; Nakai, Yumi; Shimojo, Nobuhiro; Kumagai, Yoshito

    2016-02-01

    While the biodegradation of 2,4,6-trinitrotoluene (TNT) via the release of nitrite is well established, mechanistic details of the reaction in mammals are unknown. To address this issue, we attempted to identify the enzyme from rat liver responsible for the production of nitrite from TNT. A NADPH-cytochrome P450 reductase (P450R) was isolated and identified from rat liver microsomes as the enzyme responsible for not only the release of nitrite from TNT but also formation of superoxide and 4-hydroxyamino-2,6-dinitrotoluene (4-HADNT) under aerobic conditions. In this context, reactive oxygen species generated during P450R-catalyzed TNT reduction were found to be, at least in part, a mediator for the production of 4-HADNT from TNT via formation of 4-nitroso-2,6-dinitrotoluene. P450R did not catalyze the formation of the hydride-Meisenheimer complex (H(-)-TNT) that is thought to be an intermediate for nitrite release from TNT. Furthermore, in a time-course experiment, 4-HADNT formation reached a plateau level and then declined during the reaction between TNT and P450R with NADPH, while the release of nitrite was subjected to a lag period. Notably, the produced 4-HADNT can react with the parent compound TNT to produce nitrite and dimerized products via formation of a Janovsky complex. Our results demonstrate for the first time that P450R-mediated release of nitrite from TNT results from the process of chemical interaction of TNT and its 4-electron reduction metabolite 4-HADNT. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Inhibition of the thioredoxin system in the brain and liver of zebra-seabreams exposed to waterborne methylmercury

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Branco, Vasco, E-mail: vbranco@ipimar.pt; Marine Environment and Biodiversity Unit, National Institute for Biological Resources; Canario, Joao, E-mail: jcanario@ipimar.pt

    2011-03-01

    Mercury compounds were recently found to interact in vitro with the thioredoxin system, inhibiting both Thioredoxin (Trx) and Thioredoxin reductase (TrxR). In order to evaluate if Trx and TrxR are affected in vivo by methylmercury (MeHg), we exposed juvenile zebra-seabreams to different concentrations of this toxicant in water for 28 days followed by a 14-day depuration period. Methylmercury accumulated to a larger extent in the kidney and liver of fishes, but decreased significantly during the depuration. During the exposure, MeHg percentage in the liver reached levels above 90% of total mercury (HgT) decreasing to 60% of HgT by the endmore » of the depuration period. In the kidney, MeHg accounted for 50-70% of HgT. In the brain and muscle, mercury accumulated throughout the exposure with all mercury being MeHg. The total mercury kept increasing in these organs during the depuration period. However, in the brain, this increase in HgT was accompanied by a decrease in the MeHg percentage ({approx} 10%). In the liver, both Trx and TrxR activities were significantly reduced (TrxR - 40%; Trx - 70%) by the end of the exposure, but recovered to control levels (100%) during the depuration. In the brain, both enzymes where inhibited during the depuration period (TrxR - 75%; Trx - 70%) when some production of inorganic mercury was detected. Activity of glutathione reductase showed increased levels when TrxR activity was low, suggesting complementarity between both systems. These results indicate that in vivo the thioredoxin system is a toxicological target for MeHg with TrxR being particularly affected.« less

  7. Targeting NADPH oxidases in vascular pharmacology

    PubMed Central

    Schramm, Agata; Matusik, Paweł; Osmenda, Grzegorz; Guzik, Tomasz J

    2012-01-01

    Oxidative stress is a molecular dysregulation in reactive oxygen species (ROS) metabolism, which plays a key role in the pathogenesis of atherosclerosis, vascular inflammation and endothelial dysfunction. It is characterized by a loss of nitric oxide (NO) bioavailability. Large clinical trials such as HOPE and HPS have not shown a clinical benefit of antioxidant vitamin C or vitamin E treatment, putting into question the role of oxidative stress in cardiovascular disease. A change in the understanding of the molecular nature of oxidative stress has been driven by the results of these trials. Oxidative stress is no longer perceived as a simple imbalance between the production and scavenging of ROS, but as a dysfunction of enzymes involved in ROS production. NADPH oxidases are at the center of these events, underlying the dysfunction of other oxidases including eNOS uncoupling, xanthine oxidase and mitochondrial dysfunction. Thus NADPH oxidases are important therapeutic targets. Indeed, HMG-CoA reductase inhibitors (statins) as well as drugs interfering with the renin-angiotensin-aldosterone system inhibit NADPH oxidase activation and expression. Angiotensin-converting enzyme (ACE) inhibitors, AT1 receptor antagonists (sartans) and aliskiren, as well as spironolactone or eplerenone, have been discussed. Molecular aspects of NADPH oxidase regulation must be considered, while thinking about novel pharmacological targeting of this family of enzymes consisting of several homologs Nox1, Nox2, Nox3, Nox4 and Nox5 in humans. In order to properly design trials of antioxidant therapies, we must develop reliable techniques for the assessment of local and systemic oxidative stress. Classical antioxidants could be combined with novel oxidase inhibitors. In this review, we discuss NADPH oxidase inhibitors such as VAS2870, VAS3947, GK-136901, S17834 or plumbagin. Therefore, our efforts must focus on generating small molecular weight inhibitors of NADPH oxidases, allowing the

  8. Cloning, functional characterization, and expression profiles of NADPH-cytochrome P450 reductase gene from the Asiatic rice striped stem borer, Chilo suppressalis (Lepidoptera: Pyralidae).

    PubMed

    Liu, Su; Liang, Qing-Mei; Huang, Yuan-Jie; Yuan, Xin; Zhou, Wen-Wu; Qiao, Fei; Cheng, Jiaan; Gurr, Geoff M; Zhu, Zeng-Rong

    2013-01-01

    NADPH-cytochrome P450 reductase (CPR) is one of the most important components of the cytochrome P450 enzyme system. It catalyzes electron transfer from NADPH to all known P450s, thus plays central roles not only in the metabolism of exogenous xenobiotics but also in the regulation of endogenous hormones in insects. In this study, a full-length cDNA encoding of a CPR (named CsCPR) was isolated from the Asiatic rice striped stem borer, Chilo suppressalis, by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The cDNA contains a 2061 bp open reading frame, which encodes an enzyme of 686 amino acid residues, with a calculated molecular mass of 77.6 kDa. The deduced peptide has hallmarks of typical CPR, including an N-terminal membrane anchor and the FMN, FAD and NADPH binding domains. The N-terminal-truncated protein fused with a 6 × His·tag was heterologously expressed in Escherichia coli Rosetta (DE3) cells and purified, specific activity and the Km values of the recombinant enzyme were determined. Tissue- and developmental stage-dependent expression of CsCPR mRNA was investigated by real-time quantitative PCR. The CsCPR mRNA was noticeably expressed in the digestive, metabolic, and olfactory organs of the larvae and adults of C. suppressalis. Our initial results would provide valuable information for further study on the interactions between CPR and cytochrome P450 enzyme systems. © 2013.

  9. Synthesis and evaluation of 1,4-naphthoquinone ether derivatives as SmTGR inhibitors and new antischistosomal drugs

    PubMed Central

    Johann, Laure; Belorgey, Didier; Huang, Hsin-Hung; Day, Latasha; Chessé, Matthieu; Becker, Katja; Williams, David L.; Davioud-Charvet, Elisabeth

    2016-01-01

    Investigations on the chemistry and mechanism of action of 2-methyl-1,4-naphthoquinone (or menadione) derivatives, revealed 3-phenoxymethyl menadiones as a novel antischistosomal series. These newly synthesized compounds 1–7 and their difluoromethylmenadione counterparts 8–9 were found to be potent and specific inhibitors of Schistosoma mansoni thioredoxin-glutathione reductase (SmTGR) identified as a potential target. The compounds were also tested in enzymic assays using both human flavoenzymes, i.e. the glutathione reductase (hGR) and the selenium-dependent human thioredoxin reductase (hTrxR) to evaluate the specificity of the inhibition. Structure-activity relationships as well as physico- and electro-chemical studies showed a high potential for the 3-phenoxymethyl menadiones to inhibit SmTGR selectively versus hGR and hTrxR enzymes, in particular those bearing α-fluorophenol methyl ether moieties to improve antischistosomal action. In particular, the (substituted phenoxy)methyl menadione derivative 7 displayed time-dependent SmTGR inactivation, correlating with unproductive NADPH-dependent redox-cycling of SmTGR, and potent antischistosomal action in ex vivo worms. In contrast, the difluoromethylmenadione analogue 9, which inactivates SmTGR through an irreversible non-consuming NADPH-dependent process, has little killing effect in cultured ex vivo worms. Because none of the compounds tested in vivo was active, a limited bioavailability might compromise compound activity and future studies will be directed toward improving pharmacokinetics properties. PMID:26111549

  10. The thioredoxin-1 system is essential for fueling DNA synthesis during T-cell metabolic reprogramming and proliferation.

    PubMed

    Muri, Jonathan; Heer, Sebastian; Matsushita, Mai; Pohlmeier, Lea; Tortola, Luigi; Fuhrer, Tobias; Conrad, Marcus; Zamboni, Nicola; Kisielow, Jan; Kopf, Manfred

    2018-05-10

    The thioredoxin-1 (Trx1) system is an important contributor to cellular redox balance and is a sensor of energy and glucose metabolism. Here we show critical c-Myc-dependent activation of the Trx1 system during thymocyte and peripheral T-cell proliferation, but repression during T-cell quiescence. Deletion of thioredoxin reductase-1 (Txnrd1) prevents expansion the CD4 - CD8 - thymocyte population, whereas Txnrd1 deletion in CD4 + CD8 + thymocytes does not affect further maturation and peripheral homeostasis of αβT cells. However, Txnrd1 is critical for expansion of the activated T-cell population during viral and parasite infection. Metabolomics show that TrxR1 is essential for the last step of nucleotide biosynthesis by donating reducing equivalents to ribonucleotide reductase. Impaired availability of 2'-deoxyribonucleotides induces the DNA damage response and cell cycle arrest of Txnrd1-deficient T cells. These results uncover a pivotal function of the Trx1 system in metabolic reprogramming of thymic and peripheral T cells and provide a rationale for targeting Txnrd1 in T-cell leukemia.

  11. Ferredoxin-thioredoxin reductase, an iron-sulfur enzyme linking light to enzyme regulation in oxygenic photosynthesis: purification and properties of the enzyme from C3, C4, and cyanobacterial species.

    PubMed

    Droux, M; Jacquot, J P; Miginac-Maslow, M; Gadal, P; Huet, J C; Crawford, N A; Yee, B C; Buchanan, B B

    1987-02-01

    Ferredoxin-thioredoxin reductase (FTR), an enzyme involved in the light regulation of chloroplast enzymes, was purified to homogeneity from leaves of spinach (a C3 plant) and corn (a C4 plant) and from cells of a cyanobacterium (Nostoc muscorum). The enzyme is a yellowish brown iron-sulfur protein, containing four nonheme iron and labile sulfide groups, that catalyzes the activation of NADP-malate dehydrogenase and fructose 1,6-bisphosphatase in the presence of ferredoxin and of thioredoxin m and f, respectively. FTR is synonymous with the protein earlier called ferralterin. FTR showed an Mr of about 30,000 (determined by sedimentation equilibrium ultracentrifugation, amino acid composition, gel filtration, and gradient gel electrophoresis) and was composed of two dissimilar subunits (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). One of the FTR subunits from each source was similar both in Mr (about 13,000) and immunological properties, while the other subunit (of variable molecular weight) was characteristic of a particular organism. The similar subunit contained a disulfide group that was rapidly reduced by a dithiol (dithiothreitol) but not by monothiols (2-mercaptoethanol or reduced glutathione). Homogeneous FTR formed a tight noncovalent complex with ferredoxin on affinity columns. The basis for the structural variation in the different FTR enzymes remains to be determined.

  12. Crystal Structures of Wild-type and Mutant Methicillin-resistant Staphylococcus aureus Dihydrofolate Reductase Reveal an Alternative Conformation of NADPH that may be Linked to Trimethoprim Resistance

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Frey, K.; Liu, J; Lombardo, M

    2009-01-01

    Both hospital- and community-acquired Staphylococcus aureus infections have become major health concerns in terms of morbidity, suffering and cost. Trimethoprim-sulfamethoxazole (TMP-SMZ) is an alternative treatment for methicillin-resistant S. aureus (MRSA) infections. However, TMP-resistant strains have arisen with point mutations in dihydrofolate reductase (DHFR), the target for TMP. A single point mutation, F98Y, has been shown biochemically to confer the majority of this resistance to TMP. Using a structure-based approach, we have designed a series of novel propargyl-linked DHFR inhibitors that are active against several trimethoprim-resistant enzymes. We screened this series against wild-type and mutant (F98Y) S. aureus DHFR and foundmore » that several are active against both enzymes and specifically that the meta-biphenyl class of these inhibitors is the most potent. In order to understand the structural basis of this potency, we determined eight high-resolution crystal structures: four each of the wild-type and mutant DHFR enzymes bound to various propargyl-linked DHFR inhibitors. In addition to explaining the structure-activity relationships, several of the structures reveal a novel conformation for the cofactor, NADPH. In this new conformation that is predominantly associated with the mutant enzyme, the nicotinamide ring is displaced from its conserved location and three water molecules complete a network of hydrogen bonds between the nicotinamide ring and the protein. In this new position, NADPH has reduced interactions with the inhibitor. An equilibrium between the two conformations of NADPH, implied by their occupancies in the eight crystal structures, is influenced both by the ligand and the F98Y mutation. The mutation induced equilibrium between two NADPH-binding conformations may contribute to decrease TMP binding and thus may be responsible for TMP resistance.« less

  13. Glutaredoxin 2 Reduces Both Thioredoxin 2 and Thioredoxin 1 and Protects Cells from Apoptosis Induced by Auranofin and 4-Hydroxynonenal

    PubMed Central

    Zhang, Huihui; Du, Yatao; Zhang, Xu; Lu, Jun

    2014-01-01

    Abstract Aims: Mitochondrial thioredoxin (Trx) is critical for defense against oxidative stress-induced cell apoptosis. To date, mitochondrial thioredoxin reductase (TrxR) is the only known enzyme catalyzing Trx2 reduction in mitochondria. However, TrxR is sensitive to inactivation by exo/endogenous electrophiles, for example, 4-hydroxynonenal (HNE). In this study, we characterized the mitochondrial glutaredoxin 2 (Grx2) system as a backup for the mitochondrial TrxR. Meanwhile, as Grx2 is also present in the cytosol/nucleus of certain cancer cell lines, the reducing activity of Grx2 on Trx1 was also tested. Results: Glutathione alone could reduce oxidized Trx2, and the presence of physiological concentrations of Grx2 markedly increased the reaction rate. HeLa cells with Grx2 overexpression (particularly in the mitochondria) exhibited higher viabilities than the wild-type cells after treatment with TrxR inhibitors (Auranofin or HNE), whereas knockdown of Grx2 sensitized the cells to TrxR inhibitors. Accordingly, Grx2 overexpression in the mitochondria had protected Trx2 from oxidation by HNE treatment, whereas Grx2 knockdown had sensitized Trx2 to oxidation. On the other hand, Grx2 reduced Trx1 with similar activities as that of Trx2. Overexpression of Grx2 in the cytosol had protected Trx1 from oxidation, indicating a supportive role of Grx2 in the cytosolic redox balance of cancer cells. Innovation: This work explores the reductase activity of Grx2 on Trx2/1, and demonstrates the physiological importance of the activity by using in vivo redox western blot assays. Conclusion: Grx2 system could help to keep Trx2/1 reduced during an oxidative stress, thereby contributing to the anti-apoptotic signaling. Antioxid. Redox Signal. 21, 669–681. PMID:24295294

  14. Microbicidal activity of neutrophils is inhibited by isolates from recurrent vaginal candidiasis (RVVC) caused by Candida albicans through fungal thioredoxin reductase.

    PubMed

    Ratti, Bianca Altrão; Godoy, Janine Silva Ribeiro; de Souza Bonfim Mendonça, Patrícia; Bidóia, Danielle Lazarin; Nakamura, Tânia Ueda; Nakamura, Celso Vataru; Lopes Consolaro, Marcia Edilaine; Estivalet Svidzinski, Terezinha Inez; de Oliveira Silva, Sueli

    2015-01-01

    Vulvovaginal candidiasis (VVC) is characterized by an infection of the vulva and vagina, mainly caused by Candida albicans, a commensal microorganism that inhabits the vaginal, digestive, and respiratory mucosae. Vulvovaginal candidiasis affects approximately 75% of women, and 5% develop the recurrent form (RVVC). The aim of the present study was to evaluate whether neutrophils microbicidal response is triggered when activated with RVVC isolates caused by C. albicans. Our results showed that RVVC isolates induced neutrophil migration but significantly decrease the microbicidal activity of neutrophils, compared with VVC and ASS isolates. The microbicidal activity of neutrophils is highly dependent on the production of reactive oxygen species/reactive nitrogen species (ROS/RNS). However, this isolate induced detoxification of ROS/RNS produced by neutrophils, reflected by the high level of thiol groups and by the oxygen consumption. Therefore, RVVC isolates induced biochemical changes in the inflammatory response triggered by neutrophils, and these effects were mainly related to the detoxification of ROS/RNS through the thioredoxin reductase (TR), a key antioxidant enzyme in fungi. This might be one of the resistance mechanisms triggered by RVVC caused by C. albicans. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Thioredoxin links redox to the regulation of fundamental processes of plant mitochondria

    PubMed Central

    Balmer, Yves; Vensel, William H.; Tanaka, Charlene K.; Hurkman, William J.; Gelhaye, Eric; Rouhier, Nicolas; Jacquot, Jean-Pierre; Manieri, Wanda; Schürmann, Peter; Droux, Michel; Buchanan, Bob B.

    2004-01-01

    Mitochondria contain thioredoxin (Trx), a regulatory disulfide protein, and an associated flavoenzyme, NADP/Trx reductase, which provide a link to NADPH in the organelle. Unlike animal and yeast counterparts, the function of Trx in plant mitochondria is largely unknown. Accordingly, we have applied recently devised proteomic approaches to identify soluble Trx-linked proteins in mitochondria isolated from photosynthetic (pea and spinach leaves) and heterotrophic (potato tubers) sources. Application of the mitochondrial extracts to mutant Trx affinity columns in conjunction with proteomics led to the identification of 50 potential Trx-linked proteins functional in 12 processes: photorespiration, citric acid cycle and associated reactions, lipid metabolism, electron transport, ATP synthesis/transformation, membrane transport, translation, protein assembly/folding, nitrogen metabolism, sulfur metabolism, hormone synthesis, and stress-related reactions. Almost all of these targets were also identified by a fluorescent gel electrophoresis procedure in which reduction by Trx can be observed directly. In some cases, the processes targeted by Trx depended on the source of the mitochondria. The results support the view that Trx acts as a sensor and enables mitochondria to adjust key reactions in accord with prevailing redox state. These and earlier findings further suggest that, by sensing redox in chloroplasts and mitochondria, Trx enables the two organelles of photosynthetic tissues to communicate by means of a network of transportable metabolites such as dihydroxyacetone phosphate, malate, and glycolate. In this way, light absorbed and processed by means of chlorophyll can be perceived and function in regulating fundamental mitochondrial processes akin to its mode of action in chloroplasts. PMID:14983062

  16. Thioredoxin links redox to the regulation of fundamental processes of plant mitochondria.

    PubMed

    Balmer, Yves; Vensel, William H; Tanaka, Charlene K; Hurkman, William J; Gelhaye, Eric; Rouhier, Nicolas; Jacquot, Jean-Pierre; Manieri, Wanda; Schürmann, Peter; Droux, Michel; Buchanan, Bob B

    2004-02-24

    Mitochondria contain thioredoxin (Trx), a regulatory disulfide protein, and an associated flavoenzyme, NADP/Trx reductase, which provide a link to NADPH in the organelle. Unlike animal and yeast counterparts, the function of Trx in plant mitochondria is largely unknown. Accordingly, we have applied recently devised proteomic approaches to identify soluble Trx-linked proteins in mitochondria isolated from photosynthetic (pea and spinach leaves) and heterotrophic (potato tubers) sources. Application of the mitochondrial extracts to mutant Trx affinity columns in conjunction with proteomics led to the identification of 50 potential Trx-linked proteins functional in 12 processes: photorespiration, citric acid cycle and associated reactions, lipid metabolism, electron transport, ATP synthesis/transformation, membrane transport, translation, protein assembly/folding, nitrogen metabolism, sulfur metabolism, hormone synthesis, and stress-related reactions. Almost all of these targets were also identified by a fluorescent gel electrophoresis procedure in which reduction by Trx can be observed directly. In some cases, the processes targeted by Trx depended on the source of the mitochondria. The results support the view that Trx acts as a sensor and enables mitochondria to adjust key reactions in accord with prevailing redox state. These and earlier findings further suggest that, by sensing redox in chloroplasts and mitochondria, Trx enables the two organelles of photosynthetic tissues to communicate by means of a network of transportable metabolites such as dihydroxyacetone phosphate, malate, and glycolate. In this way, light absorbed and processed by means of chlorophyll can be perceived and function in regulating fundamental mitochondrial processes akin to its mode of action in chloroplasts.

  17. X-ray structure of the ternary MTX·NADPH complex of the anthrax dihydrofolate reductase: A pharmacophore for dual-site inhibitor design

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bennett, Brad C.; Wan, Qun; Ahmad, Md Faiz

    2009-11-18

    For reasons of bioterrorism and drug resistance, it is imperative to identify and develop new molecular points of intervention against anthrax. Dihydrofolate reductase (DHFR) is a highly conserved enzyme and an established target in a number of species for a variety of chemotherapeutic programs. Recently, the crystal structure of B. anthracis DHFR (baDHFR) in complex with methotrexate (MTX) was determined and, based on the structure, proposals were made for drug design strategies directed against the substrate binding site. However, little is gleaned about the binding site for NADPH, the cofactor responsible for hydride transfer in the catalytic mechanism. In themore » present study, X-ray crystallography at 100 K was used to determine the structure of baDHFR in complex with MTX and NADPH. Although the NADPH binding mode is nearly identical to that seen in other DHFR ternary complex structures, the adenine moiety adopts an off-plane tilt of nearly 90 deg. and this orientation is stabilized by hydrogen bonds to functionally conserved Arg residues. A comparison of the binding site, focusing on this region, between baDHFR and the human enzyme is discussed, with an aim at designing species-selective therapeutics. Indeed, the ternary model, refined to 2.3{angstrom} resolution, provides an accurate template for testing the feasibility of identifying dual-site inhibitors, compounds that target both the substrate and cofactor binding site. With the ternary model in hand, using in silico methods, several compounds were identified which could potentially form key bonding contacts in the substrate and cofactor binding sites. Ultimately, two structurally distinct compounds were verified that inhibit baDHFR at low {mu}M concentrations. The apparent K{sub d} for one of these, (2-(3-(2-(hydroxyimino)-2-(pyridine-4-yl)-6,7-dimethylquinoxalin-2-yl)-1-(pyridine-4-yl)ethanone oxime), was measured by fluorescence spectroscopy to be 5.3 {mu}M.« less

  18. Attenuation of NADPH oxidase activation and glomerular filtration barrier remodeling with statin treatment.

    PubMed

    Whaley-Connell, Adam; Habibi, Javad; Nistala, Ravi; Cooper, Shawna A; Karuparthi, Poorna R; Hayden, Melvin R; Rehmer, Nathan; DeMarco, Vincent G; Andresen, Bradley T; Wei, Yongzhong; Ferrario, Carlos; Sowers, James R

    2008-02-01

    Activation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase by angiotensin II is integral to the formation of oxidative stress in the vasculature and the kidney. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibition is associated with reductions of oxidative stress in the vasculature and kidney and associated decreases in albuminuria. Effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibition on oxidative stress in the kidney and filtration barrier integrity are poorly understood. To investigate, we used transgenic TG(mRen2)27 (Ren2) rats, which harbor the mouse renin transgene and renin-angiotensin system activation, and an immortalized murine podocyte cell line. We treated young, male Ren2 and Sprague-Dawley rats with rosuvastatin (20 mg/kg IP) or placebo for 21 days. Compared with controls, we observed increases in systolic blood pressure, albuminuria, renal NADPH oxidase activity, and 3-nitrotryosine staining, with reductions in the rosuvastatin-treated Ren2. Structural changes on light and transmission electron microscopy, consistent with periarteriolar fibrosis and podocyte foot-process effacement, were attenuated with statin treatment. Nephrin expression was diminished in the Ren2 kidney and trended to normalize with statin treatment. Angiotensin II-dependent increases in podocyte NADPH oxidase activity and subunit expression (NOX2, NOX4, Rac, and p22(phox)) and reactive oxygen species generation were decreased after in vitro statin treatment. These data support a role for increased NADPH oxidase activity and subunit expression with resultant reactive oxygen species formation in the kidney and podocyte. Furthermore, statin attenuation of NADPH oxidase activation and reactive oxygen species formation in the kidney/podocyte seems to play roles in the abrogation of oxidative stress-induced filtration barrier injury and consequent albuminuria.

  19. [Effect of UV-radiation on the level of ascorbic acid, SH-groups, and activity of glutathione reductase in the eye lens].

    PubMed

    Byshneva, L N; Senchuk, V V

    2002-01-01

    The effect of UV radiation in vitro on the level of ascorbate, SH-groups and glutathione reductase activity in the soluble fraction of bovine eye lens was studied. UV-Irradiation increased NADPH-oxidoreductase activity, the level of ascorbate oxidation and decreased the content of SH-groups and activity of glutathione reductase. Significant activation of the NADPH-oxidoreductase activity in the presence of ascorbate and Cu2+ was observed after UV-irradiation. It is suggested that ascorbate may play an important role in the UV-induced lens pathology.

  20. Improvement of NADPH bioavailability in Escherichia coli through the use of phosphofructokinase deficient strains.

    PubMed

    Wang, Yipeng; San, Ka-Yiu; Bennett, George N

    2013-08-01

    NADPH-dependent reactions play important roles in production of industrially valuable compounds. In this study, we used phosphofructokinase (PFK)-deficient strains to direct fructose-6-phosphate to be oxidized through the pentose phosphate pathway (PPP) to increase NADPH generation. pfkA or pfkB single deletion and double-deletion strains were tested for their ability to produce lycopene. Since lycopene biosynthesis requires many NADPH, levels of lycopene were compared in a set of isogenic strains, with the pfkA single deletion strain showing the highest lycopene yield. Using another NADPH-requiring process, a one-step reduction reaction of 2-chloroacrylate to 2-chloropropionic acid by 2-haloacrylate reductase, the pfkA pfkB double-deletion strain showed the highest yield of 2-chloropropionic acid product. The combined effect of glucose-6-phosphate dehydrogenase overexpression or lactate dehydrogenase deletion with PFK deficiency on NADPH bioavailability was also studied. The results indicated that the flux distribution of fructose-6-phosphate between glycolysis and the pentose phosphate pathway determines the amount of NAPDH available for reductive biosynthesis.

  1. Inhibition of Tapeworm Thioredoxin and Glutathione Pathways by an Oxadiazole N-Oxide Leads to Reduced Mesocestoides vogae Infection Burden in Mice.

    PubMed

    Pasquet, Vivian; Bisio, Hugo; López, Gloria V; Romanelli-Cedrez, Laura; Bonilla, Mariana; Saldaña, Jenny; Salinas, Gustavo

    2015-06-26

    Parasitic flatworms cause serious infectious diseases that affect humans and livestock in vast regions of the world, yet there are few effective drugs to treat them. Thioredoxin glutathione reductase (TGR) is an essential enzyme for redox homeostasis in flatworm parasites and a promising pharmacological target. We purified to homogeneity and characterized the TGR from the tapeworm Mesocestoides vogae (syn. M. corti). This purification revealed absence of conventional TR and GR. The glutathione reductase activity of the purified TGR exhibits a hysteretic behavior typical of flatworm TGRs. Consistently, M. vogae genome analysis revealed the presence of a selenocysteine-containing TGR and absence of conventional TR and GR. M. vogae thioredoxin and glutathione reductase activities were inhibited by 3,4-bis(phenylsulfonyl)-1,2,5-oxadiazole N2-oxide (VL16E), an oxadiazole N-oxide previously identified as an inhibitor of fluke and tapeworm TGRs. Finally, we show that mice experimentally infected with M. vogae tetrathyridia and treated with either praziquantel, the reference drug for flatworm infections, or VL16E exhibited a 28% reduction of intraperitoneal larvae numbers compared to vehicle treated mice. Our results show that oxadiazole N-oxide is a promising chemotype in vivo and highlights the convenience of M. vogae as a model for rapid assessment of tapeworm infections in vivo.

  2. Synthesis and evaluation of 1,4-naphthoquinone ether derivatives as SmTGR inhibitors and new anti-schistosomal drugs.

    PubMed

    Johann, Laure; Belorgey, Didier; Huang, Hsin-Hung; Day, Latasha; Chessé, Matthieu; Becker, Katja; Williams, David L; Davioud-Charvet, Elisabeth

    2015-08-01

    Investigations regarding the chemistry and mechanism of action of 2-methyl-1,4-naphthoquinone (or menadione) derivatives revealed 3-phenoxymethyl menadiones as a novel anti-schistosomal chemical series. These newly synthesized compounds (1-7) and their difluoromethylmenadione counterparts (8, 9) were found to be potent and specific inhibitors of Schistosoma mansoni thioredoxin-glutathione reductase (SmTGR), which has been identified as a potential target for anti-schistosomal drugs. The compounds were also tested in enzymic assays using both human flavoenzymes, i.e. glutathione reductase (hGR) and selenium-dependent human thioredoxin reductase (hTrxR), to evaluate the specificity of the inhibition. Structure-activity relationships as well as physico- and electro-chemical studies showed a high potential for the 3-phenoxymethyl menadiones to inhibit SmTGR selectively compared to hGR and hTrxR enzymes, in particular those bearing an α-fluorophenol methyl ether moiety, which improves anti-schistosomal action. Furthermore, the (substituted phenoxy)methyl menadione derivative (7) displayed time-dependent SmTGR inactivation, correlating with unproductive NADPH-dependent redox cycling of SmTGR, and potent anti-schistosomal action in worms cultured ex vivo. In contrast, the difluoromethylmenadione analog 9, which inactivates SmTGR through an irreversible non-consuming NADPH-dependent process, has little killing effect in worms cultured ex vivo. Despite ex vivo activity, none of the compounds tested was active in vivo, suggesting that the limited bioavailability may compromise compound activity. Therefore, future studies will be directed toward improving pharmacokinetic properties and bioavailability. © 2015 FEBS.

  3. Expression, purification, crystallization and preliminary X-ray crystallographic analysis of a novel plant-type ferredoxin/thioredoxin reductase-like protein from Methanosarcina acetivorans

    PubMed Central

    Kumar, Adepu K.; Yennawar, Neela H.; Yennawar, Hemant P.; Ferry, James G.

    2011-01-01

    The genome of Methanosarcina acetivorans contains a gene (ma1659) that is predicted to encode an uncharacterized chimeric protein containing a plant-type ferredoxin/thioredoxin reductase-like catalytic domain in the N-terminal region and a bacterial-like rubredoxin domain in the C-terminal region. To understand the structural and functional properties of the protein, the ma1659 gene was cloned and overexpressed in Escherichia coli. Crystals of the MA1659 protein were grown by the sitting-drop method using 2 M ammonium sulfate, 0.1 M HEPES buffer pH 7.5 and 0.1 M urea. Diffraction data were collected to 2.8 Å resolution using the remote data-collection feature of the Advanced Light Source, Lawrence Berkeley National Laboratory. The crystal belonged to the primitive cubic space group P23 or P213, with unit-cell parameters a = b = c = 92.72 Å. Assuming the presence of one molecule in the asymmetric unit gave a Matthews coefficient (V M) of 3.55 Å3 Da−1, corresponding to a solvent content of 65%. PMID:21795791

  4. Down-regulation of flavin reductase and alcohol dehydrogenase-1 (ADH1) in metronidazole-resistant isolates of Trichomonas vaginalis

    PubMed Central

    Leitsch, David; Drinić, Mirjana; Kolarich, Daniel; Duchêne, Michael

    2012-01-01

    The microaerophilic parasite Trichomonas vaginalis is a causative agent of painful vaginitis or urethritis, termed trichomoniasis, and can also cause preterm delivery or stillbirth. Treatment of trichomoniasis is almost exclusively based on the nitroimidazole drugs metronidazole and tinidazole. Metronidazole resistance in T. vaginalis does occur and is often associated with treatment failure. In most cases, metronidazole-resistant isolates remain susceptible to tinidazole, but cross resistance between the two closely related drugs can be a problem. In this study we measured activities of thioredoxin reductase and flavin reductase in four metronidazole-susceptible and five metronidazole-resistant isolates. These enzyme activities had been previously found to be downregulated in T. vaginalis with high-level metronidazole resistance induced in the laboratory. Further, we aimed at identifying factors causing metronidazole resistance and compared the protein expression profiles of all nine isolates by application of two-dimensional gel electrophoresis (2DE). Thioredoxin reductase activity was nearly equal in all strains assayed but flavin reductase activity was clearly down-regulated, or even absent, in metronidazole-resistant strains. Since flavin reductase has been shown to reduce oxygen to hydrogen peroxide, its down-regulation could significantly contribute to the impairment of oxygen scavenging as reported by others for metronidazole-resistant strains. Analysis by 2DE revealed down-regulation of alcohol dehydrogenase 1 (ADH1) in strains with reduced sensitivity to metronidazole, an enzyme that could be involved in detoxification of intracellular acetaldehyde. PMID:22449940

  5. Ketopantoyl-lactone reductase from Candida parapsilosis: purification and characterization as a conjugated polyketone reductase.

    PubMed

    Hata, H; Shimizu, S; Hattori, S; Yamada, H

    1989-02-24

    Ketopantoyl-lactone reductase (2-dehydropantoyl-lactone reductase, EC 1.1.1.168) was purified and crystallized from cells of Candida parapsilosis IFO 0708. The enzyme was found to be homogeneous on ultracentrifugation, high-performance gel-permeation liquid chromatography and SDS-polyacrylamide gel electrophoresis. The relative molecular mass of the native and SDS-treated enzyme is approximately 40,000. The isoelectric point of the enzyme is 6.3. The enzyme was found to catalyze specifically the reduction of a variety of natural and unnatural polyketones and quinones other than ketopantoyl lactone in the presence of NADPH. Isatin and 5-methylisatin are rapidly reduced by the enzyme, the Km and Vmax values for isatin being 14 microM and 306 mumol/min per mg protein, respectively. Ketopantoyl lactone is also a good substrate (Km = 333 microM and Vmax = 481 mumol/min per mg protein). Reverse reaction was not detected with pantoyl lactone and NADP+. The enzyme is inhibited by quercetin, several polyketones and SH-reagents. 3,4-Dihydroxy-3-cyclobutene-1,2-dione, cyclohexenediol-1,2,3,4-tetraone and parabanic acid are uncompetitive inhibitors for the enzyme, the Ki values being 1.4, 0.2 and 3140 microM, respectively, with isatin as substrate. Comparison of the enzyme with the conjugated polyketone reductase of Mucor ambiguus (S. Shimizu, H. Hattori, H. Hata and H. Yamada (1988) Eur. J. Biochem. 174, 37-44) and ketopantoyl-lactone reductase of Saccharomyces cerevisiae suggested that ketopantoyl-lactone reductase is a kind of conjugated polyketone reductase.

  6. Characterization and localization of progesterone 5 alpha-reductase from cell cultures of foxglove (Digitalis lanata EHRH).

    PubMed Central

    Wendroth, S; Seitz, H U

    1990-01-01

    Progesterone 5 alpha-reductase, which catalyses the reduction of progesterone to 5 alpha-pregnane-3,20-dione, was isolated and characterized from cell cultures of Digitalis lanata (foxglove). Optimum enzyme activity was observed at pH 7.0, and the enzyme had an apparent Km value of 30 microM for its substrate progesterone. The enzyme needs NADPH as reductant, which could not be replaced by NADH. For NADPH, the apparent Km value is 130 microM. The optimum temperature was 40 degrees C; at temperatures below 45 degrees C, the product 5 alpha-pregnane-3,20-dione was reduced by a second reaction to 5 alpha-pregnan-3 beta-ol-20-one. Progesterone 5 alpha-reductase activity was not dependent on bivalent cations. In the presence of EDTA, 0.1 mM-Mn2+ had no influence on enzyme activity, whereas 0.1 mM-Ca2+, -Co2+ and -Zn2+ decreased progesterone 5 alpha-reductase activity. Only 0.1 mM-Mg2+ was slightly stimulatory. EDTA and thiol reagents such as dithiothreitol stimulate progesterone 5 alpha-reductase activity. By means of linear sucrose gradient fractionation of the cellular membranes, progesterone 5 alpha-reductase was found to be located in the endoplasmic reticulum. PMID:2106876

  7. The enzymes with benzil reductase activity conserved from bacteria to mammals.

    PubMed

    Maruyama, Reiji; Nishizawa, Mikio; Itoi, Yasushi; Ito, Seiji; Inoue, Masami

    2002-03-28

    The diketone compound, benzil is reduced to (S)-benzoin with living Bacillus cereus cells. Recently, we isolated a gene responsible for benzil reduction, and Escherichia coli cells in which this gene was overexpressed transformed benzil to (S)-benzoin. Although this benzil reductase showed high identity to the short-chain dehydrogenase/reductase (SDR) family, enzymological features were unknown. Here, we demonstrated that many B. cereus strains had benzil reductase activity in vivo, and that the benzil reductases shared 94-100% amino acid identities. Recombinant B. cereus benzil reductase produced optically pure (S)-benzoin with NADPH in vitro, and the ketone group distal to a benzene ring was asymmetrically reduced. B. cereus benzil reductase showed 31% amino acid identity to the yeast open reading frame YIR036C protein and 28-30% to mammalian sepiapterin reductases, sharing the seven residues consensus for the SDR family. We isolated the genes encoding yeast YIR036C protein and gerbil sepiapterin reductase, and both recombinant proteins also reduced benzil to (S)-benzoin in vitro. Green fluorescent protein-tagged B. cereus benzil reductase distributed in the bipolar cytoplasm in B. cereus cells. Asymmetric reduction with B. cereus benzil reductase, yeast YIR036C protein and gerbil sepiapterin reductase will be utilized to produce important chiral compounds.

  8. Dual utilization of NADPH and NADH cofactors enhances xylitol production in engineered Saccharomyces cerevisiae.

    PubMed

    Jo, Jung-Hyun; Oh, Sun-Young; Lee, Hyeun-Soo; Park, Yong-Cheol; Seo, Jin-Ho

    2015-12-01

    Xylitol, a natural sweetener, can be produced by hydrogenation of xylose in hemicelluloses. In microbial processes, utilization of only NADPH cofactor limited commercialization of xylitol biosynthesis. To overcome this drawback, Saccharomyces cerevisiae D452-2 was engineered to express two types of xylose reductase (XR) with either NADPH-dependence or NADH-preference. Engineered S. cerevisiae DWM expressing both the XRs exhibited higher xylitol productivity than the yeast strain expressing NADPH-dependent XR only (DWW) in both batch and glucose-limited fed-batch cultures. Furthermore, the coexpression of S. cerevisiae ZWF1 and ACS1 genes in the DWM strain increased intracellular concentrations of NADPH and NADH and improved maximum xylitol productivity by 17%, relative to that for the DWM strain. Finally, the optimized fed-batch fermentation of S. cerevisiae DWM-ZWF1-ACS1 resulted in 196.2 g/L xylitol concentration, 4.27 g/L h productivity and almost the theoretical yield. Expression of the two types of XR utilizing both NADPH and NADH is a promising strategy to meet the industrial demands for microbial xylitol production. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Functional characterization of NADPH-cytochrome P450 reductase from Bactrocera dorsalis: Possible involvement in susceptibility to malathion

    PubMed Central

    Huang, Yong; Lu, Xue-Ping; Wang, Luo-Luo; Wei, Dong; Feng, Zi-Jiao; Zhang, Qi; Xiao, Lin-Fan; Dou, Wei; Wang, Jin-Jun

    2015-01-01

    NADPH cytochrome P450 reductase (CPR) is essential for cytochrome P450 catalysis, which is important in the detoxification and activation of xenobiotics. In this study, two transcripts of Bactrocera dorsalis CPR (BdCPR) were cloned, and the deduced amino-acid sequence had an N-terminus membrane anchor for BdCPR-X1 and three conserved binding domains (FMN, FAD, and NADP), as well as an FAD binding motif and catalytic residues for both BdCPR-X1 and BdCPR-X2. BdCPR-X1 was detected to have the high expression levels in adults and in Malpighian tubules, fat bodies, and midguts of adults, but BdCPR-X2 expressed lowly in B. dorsalis. The levels of BdCPRs were similar in malathion-resistant strain compared to susceptible strain. However, injecting adults with double-stranded RNA against BdCPR significantly reduced the transcript levels of the mRNA, and knockdown of BdCPR increased adult susceptibility to malathion. Expressing complete BdCPR-X1 cDNA in Sf9 cells resulted in high activity determined by cytochrome c reduction and these cells had higher viability after exposure to malathion than control. The results suggest that BdCPR could affect the susceptibility of B. dorsalis to malathion and eukaryotic expression of BdCPR would lay a solid foundation for further investigation of P450 in B. dorsalis. PMID:26681597

  10. Catalase as a sulfide-sulfur oxido-reductase: An ancient (and modern?) regulator of reactive sulfur species (RSS).

    PubMed

    Olson, Kenneth R; Gao, Yan; DeLeon, Eric R; Arif, Maaz; Arif, Faihaan; Arora, Nitin; Straub, Karl D

    2017-08-01

    Catalase is well-known as an antioxidant dismutating H 2 O 2 to O 2 and H 2 O. However, catalases evolved when metabolism was largely sulfur-based, long before O 2 and reactive oxygen species (ROS) became abundant, suggesting catalase metabolizes reactive sulfide species (RSS). Here we examine catalase metabolism of H 2 S n , the sulfur analog of H 2 O 2 , hydrogen sulfide (H 2 S) and other sulfur-bearing molecules using H 2 S-specific amperometric electrodes and fluorophores to measure polysulfides (H 2 S n ; SSP4) and ROS (dichlorofluorescein, DCF). Catalase eliminated H 2 S n , but did not anaerobically generate H 2 S, the expected product of dismutation. Instead, catalase concentration- and oxygen-dependently metabolized H 2 S and in so doing acted as a sulfide oxidase with a P 50 of 20mmHg. H 2 O 2 had little effect on catalase-mediated H 2 S metabolism but in the presence of the catalase inhibitor, sodium azide (Az), H 2 O 2 rapidly and efficiently expedited H 2 S metabolism in both normoxia and hypoxia suggesting H 2 O 2 is an effective electron acceptor in this reaction. Unexpectedly, catalase concentration-dependently generated H 2 S from dithiothreitol (DTT) in both normoxia and hypoxia, concomitantly oxidizing H 2 S in the presence of O 2 . H 2 S production from DTT was inhibited by carbon monoxide and augmented by NADPH suggesting that catalase heme-iron is the catalytic site and that NADPH provides reducing equivalents. Catalase also generated H 2 S from garlic oil, diallyltrisulfide, thioredoxin and sulfur dioxide, but not from sulfite, metabisulfite, carbonyl sulfide, cysteine, cystine, glutathione or oxidized glutathione. Oxidase activity was also present in catalase from Aspergillus niger. These results show that catalase can act as either a sulfide oxidase or sulfur reductase and they suggest that these activities likely played a prominent role in sulfur metabolism during evolution and may continue do so in modern cells as well. This also appears

  11. Inhibitory effects of thalidomide on bleomycin-induced pulmonary fibrosis in rats via regulation of thioredoxin reductase and inflammations.

    PubMed

    Dong, Xiaoying; Li, Xin; Li, Minghui; Chen, Ming; Fan, Qian; Wei, Wei

    2017-01-01

    In this study, the potential clinical effects of thalidomide on bleomycin-induced pulmonary fibrosis were investigated. A Sprague-Dawley rats' model of pulmonary fibrosis induced by an intratracheal instillation of bleomycin was adopted. The rats in thalidomide treated groups were intraperitoneally injected with thalidomide (10, 20, 50 mg/kg) daily for 28 days, while the rats in control and bleomycin treated groups were injected with a saline solution. The effects of thalidomide on pulmonary injury were evaluated by the lung wet/dry weight ratios, cell counts, and histopathological examination. Inflammation of lung tissues was assessed by measuring the levels of interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β in bronchoalveolar lavage fluid (BALF). Oxidative stress was evaluated by detecting the levels of reactive oxygen species (ROS), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) in lung tissue. The results indicated that thalidomide treatment remarkably attenuated bleomycin-induced pulmonary fibrosis, oxidative stress and inflammation in rats' lung. The anti-inflammatory and anti-oxidative effects of thalidomide were also found in human lung fibroblasts. Thalidomide administration significantly stimulated the activity of thioredoxin reductase, while other enzymes or proteins involved in biologic oxidation-reduction equilibrium were not affected. Our findings indicate that thalidomide-mediated suppression of fibro-proliferation may contribute to the anti-fibrotic effect against bleomycin-induced pulmonary fibrosis. The mechanisms are related to the inhibition of oxidative stress and inflammatory response. In summary, these results may provide a rationale to explore clinical application of thalidomide for the prevention of pulmonary fibrosis.

  12. Photosynthetic activity of cotyledons is critical during post-germinative growth and seedling establishment.

    PubMed

    Ojeda, Valle; Nájera, Victoria A; González, Maricruz; Pérez-Ruiz, Juan M; Cejudo, Francisco J

    2017-09-02

    Thioredoxins (Trxs) play a relevant role in thiol-dependent redox regulation, which allows the rapid adaptation of chloroplast metabolism to unpredictable environmental conditions. In chloroplasts, Trxs use reducing equivalents provided by photoreduced ferredoxin (Fdx) via the action of a ferredoxin-thioredoxin reductase (FTR), thus linking redox regulation to light. In addition, these organelles contain an NADPH-thioredoxin reductase, NTRC, with a Trx domain at the C-terminus. NTRC efficiently reduces 2-Cys peroxiredoxins (Prxs), hence having antioxidant function. However, NTRC also participates in the redox regulation of processes, such as starch and chlorophyll biosynthesis, which are known to be regulated by Trxs. Thus, the question arising is whether there is a cross-talk between the 2 redox systems. Arabidopsis mutants simultaneously devoid of NTRC and Trx x or Trxs f show a dramatic growth inhibition phenotype, indicating that NTRC is required for the function of these unrelated Trxs. Remarkably, both the ntrc-trxx double mutant and, to a higher extent, the ntrc-trxf1f2 triple mutant show high mortality at the seedling stage, which is rescued by sucrose. These findings show the relevant role of redox regulation for chloroplast performance and uncover the key function of cotyledons chloroplasts at the transition to autotrophic metabolism during seedling establishment.

  13. Thioredoxin targets fundamental processes in a methane-producing archaeon, Methanocaldococcus jannaschii

    PubMed Central

    Susanti, Dwi; Wong, Joshua H.; Vensel, William H.; Loganathan, Usha; DeSantis, Rebecca; Schmitz, Ruth A.; Balsera, Monica; Buchanan, Bob B.; Mukhopadhyay, Biswarup

    2014-01-01

    Thioredoxin (Trx), a small redox protein, controls multiple processes in eukaryotes and bacteria by changing the thiol redox status of selected proteins. The function of Trx in archaea is, however, unexplored. To help fill this gap, we have investigated this aspect in methanarchaea—strict anaerobes that produce methane, a fuel and greenhouse gas. Bioinformatic analyses suggested that Trx is nearly universal in methanogens. Ancient methanogens that produce methane almost exclusively from H2 plus CO2 carried approximately two Trx homologs, whereas nutritionally versatile members possessed four to eight. Due to its simplicity, we studied the Trx system of Methanocaldococcus jannaschii—a deeply rooted hyperthermophilic methanogen growing only on H2 plus CO2. The organism carried two Trx homologs, canonical Trx1 that reduced insulin and accepted electrons from Escherichia coli thioredoxin reductase and atypical Trx2. Proteomic analyses with air-oxidized extracts treated with reduced Trx1 revealed 152 potential targets representing a range of processes—including methanogenesis, biosynthesis, transcription, translation, and oxidative response. In enzyme assays, Trx1 activated two selected targets following partial deactivation by O2, validating proteomics observations: methylenetetrahydromethanopterin dehydrogenase, a methanogenesis enzyme, and sulfite reductase, a detoxification enzyme. The results suggest that Trx assists methanogens in combating oxidative stress and synchronizing metabolic activities with availability of reductant, making it a critical factor in the global carbon cycle and methane emission. Because methanogenesis developed before the oxygenation of Earth, it seems possible that Trx functioned originally in metabolic regulation independently of O2, thus raising the question whether a complex biological system of this type evolved at least 2.5 billion years ago. PMID:24505058

  14. Mercury-resistance and mercuric reductase activity in Chromobacterium, Erwinia, and Bacillus species

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Trevors, J.T.

    1987-06-01

    Mercury resistant bacteria have been the most extensively studied of all the metal-tolerant bacteria. Mercury resistance is usually mediated by two distinctly different enzymes encoded by plasmids. Mercuric reductase reduces Hg/sup 2 +/ to metallic mercury (Hg/sup 0/). Organomercurial lyases have a molecular weight of 20,000 to 40,000, are composed of 1 or 2 subunits and require the presence of thiol. Plasmic-encoded Hg/sup 2 +/ resistance and mercuric reductase activity have not been detected in many species of bacteria. A Chromobacterium, Erwinia and Bacillus species isolated from environmental samples were capable of growth in the presence of 50 ..mu..M HgCl/submore » 2/. Cell-free extracts of the 3 organisms exhibited mercuric reductase activity that oxidized NADPH in the presence of HgCl/sub 2/. Negligible oxidation of NADPH was observed in the absence of HgCl/sub 2/. The Chromobacterium sp. did not contain any plasmid DNA. This would suggest that Hg/sup 2 +/ resistance was carried on the chromosome in Chromobacterium. A single 3 Mdal plasmid in the Bacillus sp. was refractory to curing. The Erwinia sp. contained 3 plasmids which were also refractory to curing. The location of the resistance genes is unknown in the Bacillus and Erwinia isolates.« less

  15. Lack of a peroxiredoxin suppresses the lethality of cells devoid of electron donors by channelling electrons to oxidized ribonucleotide reductase.

    PubMed

    Boronat, Susanna; Domènech, Alba; Carmona, Mercè; García-Santamarina, Sarela; Bañó, M Carmen; Ayté, José; Hidalgo, Elena

    2017-06-01

    The thioredoxin and glutaredoxin pathways are responsible of recycling several enzymes which undergo intramolecular disulfide bond formation as part of their catalytic cycles such as the peroxide scavengers peroxiredoxins or the enzyme ribonucleotide reductase (RNR). RNR, the rate-limiting enzyme of deoxyribonucleotide synthesis, is an essential enzyme relying on these electron flow cascades for recycling. RNR is tightly regulated in a cell cycle-dependent manner at different levels, but little is known about the participation of electron donors in such regulation. Here, we show that cytosolic thioredoxins Trx1 and Trx3 are the primary electron donors for RNR in fission yeast. Unexpectedly, trx1 transcript and Trx1 protein levels are up-regulated in a G1-to-S phase-dependent manner, indicating that the supply of electron donors is also cell cycle-regulated. Indeed, genetic depletion of thioredoxins triggers a DNA replication checkpoint ruled by Rad3 and Cds1, with the final goal of up-regulating transcription of S phase genes and constitutive RNR synthesis. Regarding the thioredoxin and glutaredoxin cascades, one combination of gene deletions is synthetic lethal in fission yeast: cells lacking both thioredoxin reductase and cytosolic dithiol glutaredoxin. We have isolated a suppressor of this lethal phenotype: a mutation at the Tpx1-coding gene, leading to a frame shift and a loss-of-function of Tpx1, the main client of electron donors. We propose that in a mutant strain compromised in reducing equivalents, the absence of an abundant and competitive substrate such as the peroxiredoxin Tpx1 has been selected as a lethality suppressor to favor RNR function at the expense of the non-essential peroxide scavenging function, to allow DNA synthesis and cell growth.

  16. DNA from uncultured organisms as a source of 2,5-diketo-L-gluconic acid reductases.

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Eschenfeldt, W. H.; Stols, L.; Rosenbaum, H.

    2001-09-01

    Total DNA of a population of uncultured organisms was extracted from soil samples, and by using PCR methods, the genes encoding two different 2,5-diketo-D-gluconic acid reductases (DKGRs) were recovered. Degenerate PCR primers based on published sequence information gave internal gene fragments homologous to known DKGRs. Nested primers specific for the internal fragments were combined with random primers to amplify flanking gene fragments from the environmental DNA, and two hypothetical full-length genes were predicted from the combined sequences. Based on these predictions, specific primers were used to amplify the two complete genes in single PCRs. These genes were cloned and expressedmore » in Escherichia coli. The purified gene products catalyzed the reduction of 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid. Compared to previously described DKGRs isolated from Corynebacterium spp., these environmental reductases possessed some valuable properties. Both exhibited greater than 20-fold-higher k{sub cat}/K{sub m} values than those previously determined, primarily as a result of better binding of substrate. The K{sub m} values for the two new reductases were 57 and 67 {mu}M, versus 2 and 13 mM for the Corynebacterium enzymes. Both environmental DKGRs accepted NADH as well as NADPH as a cosubstrate; other DKGRs and most related aldo-keto reductases use only NADPH. In addition, one of the new reductases was more thermostable than known DKGRs.« less

  17. Regiospecificity determinants of human heme oxygenase: differential NADPH- and ascorbate-dependent heme cleavage by the R183E mutant.

    PubMed

    Wang, Jinling; Lad, Latesh; Poulos, Thomas L; Ortiz de Montellano, Paul R

    2005-01-28

    The ability of the human heme oxygenase-1 (hHO-1) R183E mutant to oxidize heme in reactions supported by either NADPH-cytochrome P450 reductase or ascorbic acid has been compared. The NADPH-dependent reaction, like that of wild-type hHO-1, yields exclusively biliverdin IXalpha. In contrast, the R183E mutant with ascorbic acid as the reductant produces biliverdin IXalpha (79 +/- 4%), IXdelta (19 +/- 3%), and a trace of IXbeta. In the presence of superoxide dismutase and catalase, the yield of biliverdin IXdelta is decreased to 8 +/- 1% with a corresponding increase in biliverdin IXalpha. Spectroscopic analysis of the NADPH-dependent reaction shows that the R183E ferric biliverdin complex accumulates, because reduction of the iron, which is required for sequential iron and biliverdin release, is impaired. Reversal of the charge at position 183 makes reduction of the iron more difficult. The crystal structure of the R183E mutant, determined in the ferric and ferrous-NO bound forms, shows that the heme primarily adopts the same orientation as in wild-type hHO-1. The structure of the Fe(II).NO complex suggests that an altered active site hydrogen bonding network supports catalysis in the R183E mutant. Furthermore, Arg-183 contributes to the regiospecificity of the wild-type enzyme, but its contribution is not critical. The results indicate that the ascorbate-dependent reaction is subject to a lower degree of regiochemical control than the NADPH-dependent reaction. Ascorbate may be able to reduce the R183E ferric and ferrous dioxygen complexes in active site conformations that cannot be reduced by NADPH-cytochrome P450 reductase.

  18. Correlated Protein Motion Measurements of Dihydrofolate Reductase Crystals

    NASA Astrophysics Data System (ADS)

    Xu, Mengyang; Niessen, Katherine; Pace, James; Cody, Vivian; Markelz, Andrea

    2014-03-01

    We report the first direct measurements of the long range structural vibrational modes in dihydrofolate reductase (DHFR). DHFR is a universal housekeeping enzyme that catalyzes the reduction of 7,8-dihydrofolate to 5,6,7,8-tetra-hydrofolate, with the aid of coenzyme nicotinamide adenine dinucleotide phosphate (NADPH). This crucial enzymatic role as the target for anti-cancer [methotrexate (MTX)], and other clinically useful drugs, has made DHFR a long-standing target of enzymological studies. The terahertz (THz) frequency range (5-100 cm-1), corresponds to global correlated protein motions. In our lab we have developed Crystal Anisotropy Terahertz Microscopy (CATM), which directly measures these large scale intra-molecular protein vibrations, by removing the relaxational background of the solvent and residue side chain librational motions. We demonstrate narrowband features in the anisotropic absorbance for mouse DHFR with the ligand binding of NADPH and MTX single crystals as well as Escherichia coli DHFR with the ligand binding of NADPH and MTX single crystals. This work is supported by NSF grant MRI2 grant DBI2959989.

  19. Streptococcus sanguinis Class Ib Ribonucleotide Reductase

    PubMed Central

    Makhlynets, Olga; Boal, Amie K.; Rhodes, DeLacy V.; Kitten, Todd; Rosenzweig, Amy C.; Stubbe, JoAnne

    2014-01-01

    Streptococcus sanguinis is a causative agent of infective endocarditis. Deletion of SsaB, a manganese transporter, drastically reduces S. sanguinis virulence. Many pathogenic organisms require class Ib ribonucleotide reductase (RNR) to catalyze the conversion of nucleotides to deoxynucleotides under aerobic conditions, and recent studies demonstrate that this enzyme uses a dimanganese-tyrosyl radical (MnIII2-Y•) cofactor in vivo. The proteins required for S. sanguinis ribonucleotide reduction (NrdE and NrdF, α and β subunits of RNR; NrdH and TrxR, a glutaredoxin-like thioredoxin and a thioredoxin reductase; and NrdI, a flavodoxin essential for assembly of the RNR metallo-cofactor) have been identified and characterized. Apo-NrdF with FeII and O2 can self-assemble a diferric-tyrosyl radical (FeIII2-Y•) cofactor (1.2 Y•/β2) and with the help of NrdI can assemble a MnIII2-Y• cofactor (0.9 Y•/β2). The activity of RNR with its endogenous reductants, NrdH and TrxR, is 5,000 and 1,500 units/mg for the Mn- and Fe-NrdFs (Fe-loaded NrdF), respectively. X-ray structures of S. sanguinis NrdIox and MnII2-NrdF are reported and provide a possible rationale for the weak affinity (2.9 μm) between them. These streptococcal proteins form a structurally distinct subclass relative to other Ib proteins with unique features likely important in cluster assembly, including a long and negatively charged loop near the NrdI flavin and a bulky residue (Thr) at a constriction in the oxidant channel to the NrdI interface. These studies set the stage for identifying the active form of S. sanguinis class Ib RNR in an animal model for infective endocarditis and establishing whether the manganese requirement for pathogenesis is associated with RNR. PMID:24381172

  20. Cotesia vestalis parasitization suppresses expression of a Plutella xylostella thioredoxin.

    PubMed

    Shi, M; Zhao, S; Wang, Z-H; Stanley, D; Chen, X-X

    2016-12-01

    Thioredoxins (Trxs) are a family of small, highly conserved and ubiquitous proteins involved in protecting organisms against toxic reactive oxygen species. In this study, a typical thioredoxin gene, PxTrx, was isolated from Plutella xylostella. The full-length cDNA sequence is composed of 959 bp containing a 321 bp open reading frame that encodes a predicted protein of 106 amino acids, a predicted molecular weight of 11.7 kDa and an isoelectric point of 5.03. PxTrx was mainly expressed in larval Malpighian tubules and the fat body. An enriched recombinant PxTrx had insulin disulphide reductase activity and stimulated Human Embryonic Kidney 293 (HEK293) cell proliferation. It also protected supercoiled DNA and living HEK293 cells from H 2 O 2 -induced damage. Parasitization by Cotesia vestalis and injections of 0.05 and 0.01 equivalents of C. vestalis Bracovirus (CvBv), the symbiotic virus carried by the parasitoid, led to down-regulation of PxTrx expression in host fat body. Taken together, our results indicate that PxTrx contributes to the maintenance of P. xylostella cellular haemostasis. Host fat body expression of PxTrx is strongly attenuated by parasitization and by injections of CvBv. © 2016 The Royal Entomological Society.

  1. Analysis of the Interactions Between Thioredoxin and 20 Selenoproteins in Chicken.

    PubMed

    Liu, Qi; Yang, Jie; Cai, Jingzeng; Luan, Yilin; Sattar, Hamid; Liu, Man; Xu, Shiwen; Zhang, Ziwei

    2017-10-01

    Thioredoxin (Trx) is a small molecular protein with complicated functions in a number of processes, including inflammation, apoptosis, embryogenesis, cardiovascular disease, and redox regulation. Some selenoproteins, such as glutathione peroxidase (Gpx), iodothyronine deiodinase (Dio), and thioredoxin reductase (TR), are involved in redox regulation. However, whether there are interactions between Trx and selenoproteins is still not known. In the present paper, we used a Modeller, Hex 8.0.0, and the KFC2 Server to predict the interactions between Trx and selenoproteins. We used the Modeller to predict the target protein in objective format and assess the accuracy of the results. Molecular interaction studies with Trx and selenoproteins were performed using the molecular docking tools in Hex 8.0.0. Next, we used the KFC2 Server to further test the protein binding sites. In addition to the selenoprotein physiological functions, we also explored potential relationships between Trx and selenoproteins beyond all the results we got. The results demonstrate that Trx has the potential to interact with 19 selenoproteins, including iodothyronine deiodinase 1 (Dio1), iodothyronine deiodinase 3 (Dio3), glutathione peroxidase 1 (Gpx1), glutathione peroxidase 2 (Gpx2), glutathione peroxidase 3 (Gpx3), glutathione peroxidase 4 (Gpx4), selenoprotein H (SelH), selenoprotein I (SelI), selenoprotein M (SelM), selenoprotein N (SelN), selenoprotein T (SelT), selenoprotein U (SelU), selenoprotein W (SelW), selenoprotein 15 (Sep15), methionine sulfoxide reductase B (Sepx1), selenophosphate synthetase 1 (SPS1), TR1, TR2, and TR3, among which TR1, TR2, TR3, SPS1, Sep15, SelN, SelM, SelI, Gpx2, Gpx3, Gpx4, and Dio3 exhibited intense correlations with Trx. However, additional experiments are needed to verify them.

  2. Two Tropinone Reductases with Distinct Stereospecificities from Cultured Roots of Hyoscyamus niger1

    PubMed Central

    Hashimoto, Takashi; Nakajima, Keiji; Ongena, Godelieve; Yamada, Yasuyuki

    1992-01-01

    Tropinone is an alkamine intermediate at the branch point of biosynthetic pathways leading to various tropane alkaloids. Two stereospecifically distinct NADPH-dependent oxidoreductases, TR-I and TR-II, which, respectively, reduce tropinone to 3α-hydroxytropane (tropine) and 3β-hydroxytropane (ψ-tropine), were detected mainly in the root of tropane alkaloid-producing plants but not in nonproducing cultured root. Both reductases were purified to near homogeneity from cultured root of Hyoscyamus niger and characterized. The TR-I reaction was reversible, whereas the TR-II reaction was essentially irreversible, reduction of the ketone being highly favored over oxidation of the alcohol ψ-tropine. Marked differences were found between the two reductase in their affinities for tropinone substrate and in the effects of amino acid modification reagents. Some differences in substrate specificity were apparent. For example, N-propyl-4-piperidone was reduced by TR-II but not by TR-I. Conversely, 3-quinuclidinone and 8-thiabicyclo[3,2,1]octane-3-one were accepted as substrates by TR-I but hardly at all by TR-II. Both enzymes were shown to be class B oxidoreductases, which transfer the pro-S hydrogen of NAD(P)H to their substrates. Possible roles of these tropinone reductases in alkaloid biosynthesis are discussed. Images Figure 6 PMID:16653065

  3. Structure of a bacterial homologue of vitamin K epoxide reductase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, Weikai; Schulman, Sol; Dutton, Rachel J.

    Vitamin K epoxide reductase (VKOR) generates vitamin K hydroquinone to sustain {gamma}-carboxylation of many blood coagulation factors. Here, we report the 3.6 {angstrom} crystal structure of a bacterial homologue of VKOR from Synechococcus sp. The structure shows VKOR in complex with its naturally fused redox partner, a thioredoxin-like domain, and corresponds to an arrested state of electron transfer. The catalytic core of VKOR is a four transmembrane helix bundle that surrounds a quinone, connected through an additional transmembrane segment with the periplasmic thioredoxin-like domain. We propose a pathway for how VKOR uses electrons from cysteines of newly synthesized proteins tomore » reduce a quinone, a mechanism confirmed by in vitro reconstitution of vitamin K-dependent disulphide bridge formation. Our results have implications for the mechanism of the mammalian VKOR and explain how mutations can cause resistance to the VKOR inhibitor warfarin, the most commonly used oral anticoagulant.« less

  4. Carbonyl reductase of dog liver: purification, properties, and kinetic mechanism.

    PubMed

    Hara, A; Nakayama, T; Deyashiki, Y; Kariya, K; Sawada, H

    1986-01-01

    A carbonyl reductase has been extracted into 0.5 M KCl from dog liver and purified to apparent homogeneity by a three-step procedure consisting of chromatography on CM-Sephadex, Matrex green A, and Sephadex G-100 in high-ionic-strength buffers. The enzyme is a dimer composed of two identical subunits of molecular weight 27,000. The pH optimum is 5.5 and the isoelectric point of the enzyme is 9.3. The enzyme reduces aromatic ketones and aldehydes; the aromatic ketones with adjacent medium alkyl chains are the best substrates. Quinones, ketosteroids, prostaglandins, and aliphatic carbonyl compounds are poor or inactive substrates for the enzyme. As a cofactor the enzyme utilizes NADPH, the pro-S hydrogen atom of which is transferred to the substrate. Two moles of NADPH bind to one mole of the enzyme molecule, causing a blue shift and enhancement of the cofactor fluorescence. The reductase reaction is reversible and the equilibrium constant determined at pH 7.0 is 12.8. Steady-state kinetic measurements in both directions suggest that the reaction proceeds through a di-iso ordered bi-bi mechanism.

  5. Gold(I) NHC Complexes: Antiproliferative Activity, Cellular Uptake, Inhibition of Mammalian and Bacterial Thioredoxin Reductases, and Gram-Positive Directed Antibacterial Effects.

    PubMed

    Schmidt, Claudia; Karge, Bianka; Misgeld, Rainer; Prokop, Aram; Franke, Raimo; Brönstrup, Mark; Ott, Ingo

    2017-02-03

    Gold complexes with N-heterocyclic carbene (NHC) ligands represent a promising class of metallodrugs for the treatment of cancer or infectious diseases. In this report, the synthesis and the biological evaluation of halogen-containing NHC-Au I -Cl complexes are described. The complexes 1 and 5 a-5 f displayed good cytotoxic activity against tumor cells, and cellular uptake studies suggested that an intact Au-NHC fragment is essential for the accumulation of high amounts of both the metal and the NHC ligand. However, the bioavailability was negatively affected by serum components of the cell culture media and was influenced by likely transformations of the complex. One example (5 d) efficiently induced apoptosis in vincristine- and daunorubicin-resistant P-glycoprotein overexpressing Nalm-6 leukemia cells. Cellular uptake studies with this compound showed that both the wild-type and resistant Nalm-6 cells accumulated comparable amounts of gold, indicating that the gold drug was not excreted by P-glycoprotein or other efflux transporters. The effective inhibition of mammalian and bacterial thioredoxin reductases (TrxR) was confirmed for all of the gold complexes. Antibacterial screening of the gold complexes showed a particularly high activity against Gram-positive strains, reflecting their high dependence on an intact Trx/TrxR system. This result is of particular interest as the inhibition of bacterial TrxR represents a relatively little explored mechanism of new anti-infectives. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Chaperone-like properties of tobacco plastid thioredoxins f and m

    PubMed Central

    Sanz-Barrio, Ruth; Fernández-San Millán, Alicia; Carballeda, Jon; Corral-Martínez, Patricia; Seguí-Simarro, José M.; Farran, Inmaculada

    2012-01-01

    Thioredoxins (Trxs) are ubiquitous disulphide reductases that play important roles in the redox regulation of many cellular processes. However, some redox-independent functions, such as chaperone activity, have also been attributed to Trxs in recent years. The focus of our study is on the putative chaperone function of the well-described plastid Trxs f and m. To that end, the cDNA of both Trxs, designated as NtTrxf and NtTrxm, was isolated from Nicotiana tabacum plants. It was found that bacterially expressed tobacco Trx f and Trx m, in addition to their disulphide reductase activity, possessed chaperone-like properties. In vitro, Trx f and Trx m could both facilitate the reactivation of the cysteine-free form of chemically denatured glucose-6 phosphate dehydrogenase (foldase chaperone activity) and prevent heat-induced malate dehydrogenase aggregation (holdase chaperone activity). Our results led us to infer that the disulphide reductase and foldase chaperone functions prevail when the proteins occur as monomers and the well-conserved non-active cysteine present in Trx f is critical for both functions. By contrast, the holdase chaperone activity of both Trxs depended on their oligomeric status: the proteins were functional only when they were associated with high molecular mass protein complexes. Because the oligomeric status of both Trxs was induced by salt and temperature, our data suggest that plastid Trxs could operate as molecular holdase chaperones upon oxidative stress, acting as a type of small stress protein. PMID:21948853

  7. Overexpression of NADH-dependent fumarate reductase improves D-xylose fermentation in recombinant Saccharomyces cerevisiae.

    PubMed

    Salusjärvi, Laura; Kaunisto, Sanna; Holmström, Sami; Vehkomäki, Maija-Leena; Koivuranta, Kari; Pitkänen, Juha-Pekka; Ruohonen, Laura

    2013-12-01

    Deviation from optimal levels and ratios of redox cofactors NAD(H) and NADP(H) is common when microbes are metabolically engineered. The resulting redox imbalance often reduces the rate of substrate utilization as well as biomass and product formation. An example is the metabolism of D-xylose by recombinant Saccharomyces cerevisiae strains expressing xylose reductase and xylitol dehydrogenase encoding genes from Scheffersomyces stipitis. This pathway requires both NADPH and NAD(+). The effect of overexpressing the glycosomal NADH-dependent fumarate reductase (FRD) of Trypanosoma brucei in D-xylose-utilizing S. cerevisiae alone and together with an endogenous, cytosol directed NADH-kinase (POS5Δ17) was studied as one possible solution to overcome this imbalance. Expression of FRD and FRD + POS5Δ17 resulted in 60 and 23 % increase in ethanol yield, respectively, on D-xylose under anaerobic conditions. At the same time, xylitol yield decreased in the FRD strain suggesting an improvement in redox balance. We show that fumarate reductase of T. brucei can provide an important source of NAD(+) in yeast under anaerobic conditions, and can be useful for metabolic engineering strategies where the redox cofactors need to be balanced. The effects of FRD and NADH-kinase on aerobic and anaerobic D-xylose and D-glucose metabolism are discussed.

  8. Light Driven CO2 Fixation by Using Cyanobacterial Photosystem I and NADPH-Dependent Formate Dehydrogenase

    PubMed Central

    Ihara, Masaki; Kawano, Yusuke; Urano, Miho; Okabe, Ayako

    2013-01-01

    The ultimate goal of this research is to construct a new direct CO2 fixation system using photosystems in living algae. Here, we report light-driven formate production from CO2 by using cyanobacterial photosystem I (PS I). Formate, a chemical hydrogen carrier and important industrial material, can be produced from CO2 by using the reducing power and the catalytic function of formate dehydrogenase (FDH). We created a bacterial FDH mutant that experimentally switched the cofactor specificity from NADH to NADPH, and combined it with an in vitro-reconstituted cyanobacterial light-driven NADPH production system consisting of PS I, ferredoxin (Fd), and ferredoxin-NADP+-reductase (FNR). Consequently, light-dependent formate production under a CO2 atmosphere was successfully achieved. In addition, we introduced the NADPH-dependent FDH mutant into heterocysts of the cyanobacterium Anabaena sp. PCC 7120 and demonstrated an increased formate concentration in the cells. These results provide a new possibility for photo-biological CO2 fixation. PMID:23936519

  9. Light driven CO2 fixation by using cyanobacterial photosystem I and NADPH-dependent formate dehydrogenase.

    PubMed

    Ihara, Masaki; Kawano, Yusuke; Urano, Miho; Okabe, Ayako

    2013-01-01

    The ultimate goal of this research is to construct a new direct CO2 fixation system using photosystems in living algae. Here, we report light-driven formate production from CO2 by using cyanobacterial photosystem I (PS I). Formate, a chemical hydrogen carrier and important industrial material, can be produced from CO2 by using the reducing power and the catalytic function of formate dehydrogenase (FDH). We created a bacterial FDH mutant that experimentally switched the cofactor specificity from NADH to NADPH, and combined it with an in vitro-reconstituted cyanobacterial light-driven NADPH production system consisting of PS I, ferredoxin (Fd), and ferredoxin-NADP(+)-reductase (FNR). Consequently, light-dependent formate production under a CO2 atmosphere was successfully achieved. In addition, we introduced the NADPH-dependent FDH mutant into heterocysts of the cyanobacterium Anabaena sp. PCC 7120 and demonstrated an increased formate concentration in the cells. These results provide a new possibility for photo-biological CO2 fixation.

  10. An investigation into the interactions of gold nanoparticles and anti-arthritic drugs with macrophages, and their reactivity towards thioredoxin reductase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    James, Lloyd R.A.; Xu, Zhi-Qiang; Sluyter, Ronald

    Gold(I) complexes are an important tool in the arsenal of established approaches for treating rheumatoid arthritis (RA), while some recent studies have suggested that gold nanoparticles (Au NPs) may also be therapeutically efficacious. These observations prompted the current biological studies involving gold(I) anti-RA agents and Au NPs, which are aimed towards improving our knowledge of how they work. The cytotoxicity of auranofin, aurothiomalate, aurothiosulfate and Au NPs towards RAW264.7 macrophages was evaluated using the MTT assay, with the former compound proving to be the most toxic. The extent of cellular uptake of the various gold agents was determined using graphitemore » furnace atomic absorption spectrometry, while their distribution within macrophages was examined using microprobe synchrotron radiation X-ray fluorescence spectroscopy. The latter technique showed accumulation of gold in discrete regions of the cell, and co-localisation with sulfur in the case of cells treated with aurothiomalate or auranofin. Electrospray ionization mass spectrometry was used to characterize thioredoxin reductase (TrxR) in which the penultimate selenocysteine residue was replaced by cysteine. Mass spectra of solutions of TrxR and aurothiomalate, aurothiosulfate or auranofin showed complexes containing bare gold atoms bound to the protein, or protein adducts containing gold atoms retaining some of their initial ligands. These results support TrxR being an important target of gold(I) drugs used to treat RA, while the finding that Au NPs are incorporated into macrophages, but elicit little toxicity, indicates further exploration of their potential for treatment of RA is warranted.« less

  11. Serum thioredoxin reductase is highly increased in mice with hepatocellular carcinoma and its activity is restrained by several mechanisms.

    PubMed

    Zhang, Le; Cheng, Qing; Zhang, Longjie; Wang, Yijun; Merrill, Gary F; Ilani, Tal; Fass, Deborah; Arnér, Elias S J; Zhang, Jinsong

    2016-10-01

    Increased thioredoxin reductase (TrxR) levels in serum were recently identified as possible prognostic markers for human prostate cancer or hepatocellular carcinoma. We had earlier shown that serum levels of TrxR protein are very low in healthy mice, but can in close correlation to alanine aminotransferase (ALT) increase more than 200-fold upon chemically induced liver damage. We also found that enzymatic TrxR activity in serum is counteracted by a yet unidentified oxidase activity in serum. In the present study we found that mice carrying H22 hepatocellular carcinoma tumors present highly increased levels of TrxR in serum, similarly to that reported in human patients. In this case ALT levels did not parallel those of TrxR. We also discovered here that the TrxR-antagonistic oxidase activity in serum is due to the presence of quiescin Q6 sulfhydryl oxidase 1 (QSOX1). We furthermore found that the chemotherapeutic agents cisplatin or auranofin, when given systemically to H22 tumor bearing mice, can further inhibit TrxR activities in serum. The TrxR serum activity was also inhibited by endogenous electrophilic inhibitors, found to increase in tumor-bearing mice and to include protoporphyrin IX (PpIX) and 4-hydroxynonenal (HNE). Thus, hepatocellular carcinoma triggers high levels of serum TrxR that are not paralleled by ALT, and TrxR enzyme activity in serum is counteracted by several different mechanisms. The physiological role of TrxR in serum, if any, as well as its potential value as a prognostic marker for tumor progression, needs to be studied further. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Atomic Structure of Salutaridine Reductase from the Opium Poppy (Papaver somniferum)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Higashi, Yasuhiro; Kutchan, Toni M.; Smith, Thomas J.

    The opium poppy (Papaver somniferum L.) is one of the oldest known medicinal plants. In the biosynthetic pathway for morphine and codeine, salutaridine is reduced to salutaridinol by salutaridine reductase (SalR; EC 1.1.1.248) using NADPH as coenzyme. Here, we report the atomic structure of SalR to a resolution of {approx}1.9 {angstrom} in the presence of NADPH. The core structure is highly homologous to other members of the short chain dehydrogenase/reductase family. The major difference is that the nicotinamide moiety and the substrate-binding pocket are covered by a loop (residues 265-279), on top of which lies a large 'flap'-like domain (residuesmore » 105-140). This configuration appears to be a combination of the two common structural themes found in other members of the short chain dehydrogenase/reductase family. Previous modeling studies suggested that substrate inhibition is due to mutually exclusive productive and nonproductive modes of substrate binding in the active site. This model was tested via site-directed mutagenesis, and a number of these mutations abrogated substrate inhibition. However, the atomic structure of SalR shows that these mutated residues are instead distributed over a wide area of the enzyme, and many are not in the active site. To explain how residues distal to the active site might affect catalysis, a model is presented whereby SalR may undergo significant conformational changes during catalytic turnover.« less

  13. Aerobic Degradation of 2,4,6-Trinitrotoluene by Enterobacter cloacae PB2 and by Pentaerythritol Tetranitrate Reductase

    PubMed Central

    French, Christopher E.; Nicklin, Stephen; Bruce, Neil C.

    1998-01-01

    Enterobacter cloacae PB2 was originally isolated on the basis of its ability to utilize nitrate esters, such as pentaerythritol tetranitrate (PETN) and glycerol trinitrate, as the sole nitrogen source for growth. The enzyme responsible is an NADPH-dependent reductase designated PETN reductase. E. cloacae PB2 was found to be capable of slow aerobic growth with 2,4,6-trinitrotoluene (TNT) as the sole nitrogen source. Dinitrotoluenes were not produced and could not be used as nitrogen sources. Purified PETN reductase was found to reduce TNT to its hydride-Meisenheimer complex, which was further reduced to the dihydride-Meisenheimer complex. Purified PETN reductase and recombinant Escherichia coli expressing PETN reductase were able to liberate nitrogen as nitrite from TNT. The ability to remove nitrogen from TNT suggests that PB2 or recombinant organisms expressing PETN reductase may be useful for bioremediation of TNT-contaminated soil and water. PMID:9687442

  14. Hexavalent Chromium Causes the Oxidation of Thioredoxin in Human Bronchial Epithelial Cells

    PubMed Central

    Myers, Judith M.; Antholine, William E.; Myers, Charles R.

    2008-01-01

    Hexavalent chromium [Cr(VI)] species such as chromates are cytotoxic. Inhalational exposure is a primary concern in many Cr-related industries and their immediate environments, and bronchial epithelial cells are directly exposed to inhaled Cr(VI). Chromates are readily taken up by cells and are reduced to reactive Cr species which may also result in the generation of reactive oxygen species (ROS). The thioredoxin (Trx) system has a key role in the maintenance of cellular thiol redox balance and is essential for cell survival. Cells normally maintain the cytosolic (Trx1) and mitochondrial (Trx2) thioredoxins largely in the reduced state. Redox western blots were used to assess the redox status of the thioredoxins in normal human bronchial epithelial cells (BEAS-2B) incubated with soluble Na2CrO4 or insoluble ZnCrO4 for different periods of time. Both chromates caused a dose- and time-dependent oxidation of Trx2 and Trx1. Trx2 was more susceptible in that it could all be converted to the oxidized form, whereas a small amount of reduced Trx1 remained even after prolonged treatment with higher Cr concentrations. Only one of the dithiols, presumably the active site, of Trx1 was oxidized by Cr(VI). Cr(VI) did not cause significant GSH depletion or oxidation indicating that Trx oxidation does not result from a general oxidation of cellular thiols. With purified Trx and thioredoxin reductase (TrxR) in vitro, Cr(VI) also resulted in Trx oxidation. It was determined that purified TrxR has pronounced Cr(VI) reducing activity, so competition for electron flow from TrxR might impair its ability to reduce Trx. The in vitro data also suggested some direct redox interaction between Cr(VI) and Trx. The ability of Cr(VI) to cause Trx oxidation in cells could contribute to its cytotoxic effects, and could have important implications for cell survival, redox-sensitive cell signaling, and the cells' tolerance of other oxidant insults. PMID:18328613

  15. Corynebacterium diphtheriae methionine sulfoxide reductase a exploits a unique mycothiol redox relay mechanism.

    PubMed

    Tossounian, Maria-Armineh; Pedre, Brandán; Wahni, Khadija; Erdogan, Huriye; Vertommen, Didier; Van Molle, Inge; Messens, Joris

    2015-05-01

    Methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in proteins and play a pivotal role in cellular redox signaling. We have unraveled the redox relay mechanisms of methionine sulfoxide reductase A of the pathogen Corynebacterium diphtheriae (Cd-MsrA) and shown that this enzyme is coupled to two independent redox relay pathways. Steady-state kinetics combined with mass spectrometry of Cd-MsrA mutants give a view of the essential cysteine residues for catalysis. Cd-MsrA combines a nucleophilic cysteine sulfenylation reaction with an intramolecular disulfide bond cascade linked to the thioredoxin pathway. Within this cascade, the oxidative equivalents are transferred to the surface of the protein while releasing the reduced substrate. Alternatively, MsrA catalyzes methionine sulfoxide reduction linked to the mycothiol/mycoredoxin-1 pathway. After the nucleophilic cysteine sulfenylation reaction, MsrA forms a mixed disulfide with mycothiol, which is transferred via a thiol disulfide relay mechanism to a second cysteine for reduction by mycoredoxin-1. With x-ray crystallography, we visualize two essential intermediates of the thioredoxin relay mechanism and a cacodylate molecule mimicking the substrate interactions in the active site. The interplay of both redox pathways in redox signaling regulation forms the basis for further research into the oxidative stress response of this pathogen. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Identification of oleamide in Guatteria recurvisepala by LC/MS-based Plasmodium falciparum thioredoxin reductase ligand binding method.

    PubMed

    Munigunti, Ranjith; Nelson, Nicholas; Mulabagal, Vanisree; Gupta, Mahabir P; Brun, Reto; Calderón, Angela I

    2011-10-01

    Our current research on applications of mass spectrometry to natural product drug discovery against malaria aims to screen plant extracts for new ligands to Plasmodium falciparum thioredoxin reductase (PfTrxR) followed by their identification and structure elucidation. PfTrxR is involved in the antioxidant defense and redox regulation of the parasite and is validated as a promising target for therapeutic intervention against malaria. In the present study, detannified methanol extracts from Guatteria recurvisepala, Licania kallunkiae, and Topobea watsonii were screened for ligands to PfTrxR using ultrafiltration and liquid chromatography/mass spectrometry-based binding experiments. The PfTrxR ligand identified in the extract of Guatteria recurvisepala displayed a relative binding affinity of 3.5-fold when incubated with 1 μM PfTrxR. The ligand corresponding to the protonated molecule m/z 282.2792 [M+ H]+ was eluted at a retention time of 17.95 min in a 20-min gradient of 95% B consisting of (A) 0.1%formic acid in 95% H₂O-5% ACN, and (B) 0.1% formic acid in 95% ACN-5% H₂O in an LC-QTOF-MS.Tandem MS of the protonated molecule m/z 282.2792 [M + H]+, C₁₈H₃₆NO (DBE: 2; error: 1.13 ppm) resulted in two daughter ions m/z 265.2516[M + H-NH₃]+ (DBE: 3; error: 0.35 ppm) and m/z 247.2405 [M + H-NH₃-H₂O] +, (DBE: 4; error:2.26 ppm). The PfTrxR ligand was identified as oleamide and confirmed by comparison of the retention time, molecular formula, accurate mass,and double bond equivalence with the standard oleamide. This is the first report on the identification of oleamide as a PfTrxR ligand from Guatteria recurvisepala R. E. Fr. and the corresponding in vitro activity against P. falciparum strain K1 (IC₅₀ 4.29 μg/mL). © Georg Thieme Verlag KG Stuttgart · New York.

  17. Glutathione reductase-mediated synthesis of tellurium-containing nanostructures exhibiting antibacterial properties.

    PubMed

    Pugin, Benoit; Cornejo, Fabián A; Muñoz-Díaz, Pablo; Muñoz-Villagrán, Claudia M; Vargas-Pérez, Joaquín I; Arenas, Felipe A; Vásquez, Claudio C

    2014-11-01

    Tellurium, a metalloid belonging to group 16 of the periodic table, displays very interesting physical and chemical properties and lately has attracted significant attention for its use in nanotechnology. In this context, the use of microorganisms for synthesizing nanostructures emerges as an eco-friendly and exciting approach compared to their chemical synthesis. To generate Te-containing nanostructures, bacteria enzymatically reduce tellurite to elemental tellurium. In this work, using a classic biochemical approach, we looked for a novel tellurite reductase from the Antarctic bacterium Pseudomonas sp. strain BNF22 and used it to generate tellurium-containing nanostructures. A new tellurite reductase was identified as glutathione reductase, which was subsequently overproduced in Escherichia coli. The characterization of this enzyme showed that it is an NADPH-dependent tellurite reductase, with optimum reducing activity at 30°C and pH 9.0. Finally, the enzyme was able to generate Te-containing nanostructures, about 68 nm in size, which exhibit interesting antibacterial properties against E. coli, with no apparent cytotoxicity against eukaryotic cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  18. Glutathione Reductase-Mediated Synthesis of Tellurium-Containing Nanostructures Exhibiting Antibacterial Properties

    PubMed Central

    Pugin, Benoit; Cornejo, Fabián A.; Muñoz-Díaz, Pablo; Muñoz-Villagrán, Claudia M.; Vargas-Pérez, Joaquín I.; Arenas, Felipe A.

    2014-01-01

    Tellurium, a metalloid belonging to group 16 of the periodic table, displays very interesting physical and chemical properties and lately has attracted significant attention for its use in nanotechnology. In this context, the use of microorganisms for synthesizing nanostructures emerges as an eco-friendly and exciting approach compared to their chemical synthesis. To generate Te-containing nanostructures, bacteria enzymatically reduce tellurite to elemental tellurium. In this work, using a classic biochemical approach, we looked for a novel tellurite reductase from the Antarctic bacterium Pseudomonas sp. strain BNF22 and used it to generate tellurium-containing nanostructures. A new tellurite reductase was identified as glutathione reductase, which was subsequently overproduced in Escherichia coli. The characterization of this enzyme showed that it is an NADPH-dependent tellurite reductase, with optimum reducing activity at 30°C and pH 9.0. Finally, the enzyme was able to generate Te-containing nanostructures, about 68 nm in size, which exhibit interesting antibacterial properties against E. coli, with no apparent cytotoxicity against eukaryotic cells. PMID:25193000

  19. Tropinone reductases, enzymes at the branch point of tropane alkaloid metabolism.

    PubMed

    Dräger, Birgit

    2006-02-01

    Two stereospecific oxidoreductases constitute a branch point in tropane alkaloid metabolism. Products of tropane metabolism are the alkaloids hyoscyamine, scopolamine, cocaine, and polyhydroxylated nortropane alkaloids, the calystegines. Both tropinone reductases reduce the precursor tropinone to yield either tropine or pseudotropine. In Solanaceae, tropine is incorporated into hyoscyamine and scopolamine; pseudotropine is the first specific metabolite on the way to the calystegines. Isolation, cloning and heterologous expression of both tropinone reductases enabled kinetic characterisation, protein crystallisation, and structure elucidation. Stereospecificity of reduction is achieved by binding tropinone in the respective enzyme active centre in opposite orientation. Immunolocalisation of both enzyme proteins in cultured roots revealed a tissue-specific protein accumulation. Metabolite flux through both arms of the tropane alkaloid pathway appears to be regulated by the activity of both enzymes and by their access to the precursor tropinone. Both tropinone reductases are NADPH-dependent short-chain dehydrogenases with amino acid sequence similarity of more than 50% suggesting their descent from a common ancestor. Putative tropinone reductase sequences annotated in plant genomes other that Solanaceae await functional characterisation.

  20. NAD(P)H-dependent aldose reductase from the xylose-assimilating yeast Candida tenuis. Isolation, characterization and biochemical properties of the enzyme.

    PubMed Central

    Neuhauser, W; Haltrich, D; Kulbe, K D; Nidetzky, B

    1997-01-01

    During growth on d-xylose the yeast Candida tenuis produces one aldose reductase that is active with both NADPH and NADH as coenzyme. This enzyme has been isolated by dye ligand and anion-exchange chromatography in yields of 76%. Aldose reductase consists ofa single 43 kDa polypeptide with an isoelectric point of 4.70. Initial velocity, product inhibition and binding studies are consistent with a compulsory-ordered, ternary-complex mechanism with coenzyme binding first and leaving last. The catalytic efficiency (kcat/Km) in d-xylose reduction at pH 7 is more than 60-fold higher than that in xylitol oxidation and reflects significant differences in the corresponding catalytic centre activities as well as apparent substrate-binding constants. The enzyme prefers NADP(H) approx. 2-fold to NAD(H), which is largely due to better apparent binding of the phosphorylated form of the coenzyme. NADP+ is a potent competitive inhibitor of the NADH-linked aldehyde reduction (Ki 1.5 microM), whereas NAD+ is not. Unlike mammalian aldose reductase, the enzyme from C. tenuis is not subject to oxidation-induced activation. Evidence of an essential lysine residue located in or near the coenzyme binding site has been obtained from chemical modification of aldose reductase with pyridoxal 5'-phosphate. The results are discussed in the context of a comparison of the enzymic properties of yeast and mammalian aldose reductase. PMID:9307017

  1. Crystal structure of isoflavone reductase from alfalfa (Medicago sativa L.).

    PubMed

    Wang, Xiaoqiang; He, Xianzhi; Lin, Jianqiao; Shao, Hui; Chang, Zhenzhan; Dixon, Richard A

    2006-05-19

    Isoflavonoids play important roles in plant defense and exhibit a range of mammalian health-promoting activities. Isoflavone reductase (IFR) specifically recognizes isoflavones and catalyzes a stereospecific NADPH-dependent reduction to (3R)-isoflavanone. The crystal structure of Medicago sativa IFR with deletion of residues 39-47 has been determined at 1.6A resolution. Structural analysis, molecular modeling and docking, and comparison with the structures of other NADPH-dependent enzymes, defined the putative binding sites for co-factor and substrate and potential key residues for enzyme activity and substrate specificity. Further mutagenesis has confirmed the role of Lys144 as a catalytic residue. This study provides a structural basis for understanding the enzymatic mechanism and substrate specificity of IFRs as well as the functions of IFR-like proteins.

  2. Aerobic degradation of 2,4,6-trinitrotoluene by Enterobacter cloacae PB2 and by pentaerythritol tetranitrate reductase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    French, C.E.; Bruce, N.C.; Nicklin, S.

    1998-08-01

    Enterobacter cloacae PB2 was originally isolated on the basis of its ability to utilize nitrate esters, such as pentaerythritol tetranitrate (PETN) and glycerol trinitrate, as the sole nitrogen source for growth. The enzyme responsible is an NADPH-dependent reductase designated PETN reductase. E. cloacae PB2 was found to be capable of slow aerobic growth with 2,4,6-trinitrotoluene (TNT) as the sole nitrogen source. Dinitrotoluenes were not produced and could not be used as nitrogen sources. Purified PETN reductase was found to reduce TNT to its hydride-Meisenheimer complex, which was further reduced to the dihydride-Meisenheimer complex. Purified PETN reductase and recombinant Escherichia colimore » expressing PETN reductase were able to liberate nitrogen as nitrite from TNT. The ability to remove nitrogen from TNT suggests that PB2 or recombinant organisms expressing PETN reductase may be useful for bioremediation of TNT-contaminated soil and water.« less

  3. Evolution of the Ferric Reductase Domain (FRD) Superfamily: Modularity, Functional Diversification, and Signature Motifs

    PubMed Central

    Zhang, Xuezhi; Krause, Karl-Heinz; Xenarios, Ioannis; Soldati, Thierry; Boeckmann, Brigitte

    2013-01-01

    A heme-containing transmembrane ferric reductase domain (FRD) is found in bacterial and eukaryotic protein families, including ferric reductases (FRE), and NADPH oxidases (NOX). The aim of this study was to understand the phylogeny of the FRD superfamily. Bacteria contain FRD proteins consisting only of the ferric reductase domain, such as YedZ and short bFRE proteins. Full length FRE and NOX enzymes are mostly found in eukaryotic cells and all possess a dehydrogenase domain, allowing them to catalyze electron transfer from cytosolic NADPH to extracellular metal ions (FRE) or oxygen (NOX). Metazoa possess YedZ-related STEAP proteins, possibly derived from bacteria through horizontal gene transfer. Phylogenetic analyses suggests that FRE enzymes appeared early in evolution, followed by a transition towards EF-hand containing NOX enzymes (NOX5- and DUOX-like). An ancestral gene of the NOX(1-4) family probably lost the EF-hands and new regulatory mechanisms of increasing complexity evolved in this clade. Two signature motifs were identified: NOX enzymes are distinguished from FRE enzymes through a four amino acid motif spanning from transmembrane domain 3 (TM3) to TM4, and YedZ/STEAP proteins are identified by the replacement of the first canonical heme-spanning histidine by a highly conserved arginine. The FRD superfamily most likely originated in bacteria. PMID:23505460

  4. Evolution of the ferric reductase domain (FRD) superfamily: modularity, functional diversification, and signature motifs.

    PubMed

    Zhang, Xuezhi; Krause, Karl-Heinz; Xenarios, Ioannis; Soldati, Thierry; Boeckmann, Brigitte

    2013-01-01

    A heme-containing transmembrane ferric reductase domain (FRD) is found in bacterial and eukaryotic protein families, including ferric reductases (FRE), and NADPH oxidases (NOX). The aim of this study was to understand the phylogeny of the FRD superfamily. Bacteria contain FRD proteins consisting only of the ferric reductase domain, such as YedZ and short bFRE proteins. Full length FRE and NOX enzymes are mostly found in eukaryotic cells and all possess a dehydrogenase domain, allowing them to catalyze electron transfer from cytosolic NADPH to extracellular metal ions (FRE) or oxygen (NOX). Metazoa possess YedZ-related STEAP proteins, possibly derived from bacteria through horizontal gene transfer. Phylogenetic analyses suggests that FRE enzymes appeared early in evolution, followed by a transition towards EF-hand containing NOX enzymes (NOX5- and DUOX-like). An ancestral gene of the NOX(1-4) family probably lost the EF-hands and new regulatory mechanisms of increasing complexity evolved in this clade. Two signature motifs were identified: NOX enzymes are distinguished from FRE enzymes through a four amino acid motif spanning from transmembrane domain 3 (TM3) to TM4, and YedZ/STEAP proteins are identified by the replacement of the first canonical heme-spanning histidine by a highly conserved arginine. The FRD superfamily most likely originated in bacteria.

  5. A Two-component NADPH Oxidase (NOX)-like System in Bacteria Is Involved in the Electron Transfer Chain to the Methionine Sulfoxide Reductase MsrP*

    PubMed Central

    Juillan-Binard, Céline; Picciocchi, Antoine; Andrieu, Jean-Pierre; Petit-Hartlein, Isabelle; Caux-Thang, Christelle; Vivès, Corinne; Nivière, Vincent

    2017-01-01

    MsrPQ is a newly identified methionine sulfoxide reductase system found in bacteria, which appears to be specifically involved in the repair of periplasmic proteins oxidized by hypochlorous acid. It involves two proteins: a periplasmic one, MsrP, previously named YedY, carrying out the Msr activity, and MsrQ, an integral b-type heme membrane-spanning protein, which acts as the specific electron donor to MsrP. MsrQ, previously named YedZ, was mainly characterized by bioinformatics as a member of the FRD superfamily of heme-containing membrane proteins, which include the NADPH oxidase proteins (NOX/DUOX). Here we report a detailed biochemical characterization of the MsrQ protein from Escherichia coli. We optimized conditions for the overexpression and membrane solubilization of an MsrQ-GFP fusion and set up a purification scheme allowing the production of pure MsrQ. Combining UV-visible spectroscopy, heme quantification, and site-directed mutagenesis of histidine residues, we demonstrated that MsrQ is able to bind two b-type hemes through the histidine residues conserved between the MsrQ and NOX protein families. In addition, we identify the E. coli flavin reductase Fre, which is related to the dehydrogenase domain of eukaryotic NOX enzymes, as an efficient cytosolic electron donor to the MsrQ heme moieties. Cross-linking experiments as well as surface Plasmon resonance showed that Fre interacts with MsrQ to form a specific complex. Taken together, these data support the identification of the first prokaryotic two-component protein system related to the eukaryotic NOX family and involved in the reduction of periplasmic oxidized proteins. PMID:28028176

  6. Immunoproteomics based identification of thioredoxin reductase GliT and novel Aspergillus fumigatus antigens for serologic diagnosis of invasive aspergillosis

    PubMed Central

    2012-01-01

    Background There has been a rising incidence of invasive aspergillosis (IA) in critically ill patients, even in the absence of an apparent predisposing immunodeficiency. The diagnosis of IA is difficult because clinical signs are not sensitive and specific, and serum galactomannan has relatively low sensitivity in this group of patients. Therefore, more prompt and accurate disease markers for early diagnosis are needed. To establish disease markers demands a thorough knowledge of fungal antigens which may be detected in the serum or other body fluids of patients. Herein we report novel immunodominant antigens identified from extracellular proteins of Aspergillus fumigatus. Results Extracellular proteins of A. fumigatus were separated by two-dimensional electrophoresis (2-DE) and probed with the sera from critically ill patients with proven IA. The immunoreactive protein spots were identified by MALDI-TOF mass spectrometry (MALDI-TOF -MS). Forty spots from 2DE gels were detected and 17 different proteins were identified as immunogenic in humans. Function annotation revealed that most of these proteins were metabolic enzymes involved in carbohydrate, fatty acid, amino acid, and energy metabolism. One of the proteins, thioredoxin reductase GliT (TR), which showed the best immunoactivity, was analyzed further for secretory signals, protein localization, and homology. The results indicated that TR is a secretory protein with a signal sequence exhibiting a high probability for secretion. Furthermore, TR did not match any human proteins, and had low homology with most other fungi. The recombinant TR was recognized by the sera of all proven IA patients with different underlying diseases in this study. Conclusions The immunoreactive proteins identified in this study may be helpful for the diagnosis of IA in critically ill patients. Our results indicate that TR and other immunodominant antigens have potential as biomarkers for the serologic diagnosis of invasive

  7. Immunoproteomics based identification of thioredoxin reductase GliT and novel Aspergillus fumigatus antigens for serologic diagnosis of invasive aspergillosis.

    PubMed

    Shi, Li-ning; Li, Fang-qiu; Huang, Mei; Lu, Jing-fen; Kong, Xiao-xiang; Wang, Shi-qin; Shao, Hai-feng

    2012-01-18

    There has been a rising incidence of invasive aspergillosis (IA) in critically ill patients, even in the absence of an apparent predisposing immunodeficiency. The diagnosis of IA is difficult because clinical signs are not sensitive and specific, and serum galactomannan has relatively low sensitivity in this group of patients. Therefore, more prompt and accurate disease markers for early diagnosis are needed. To establish disease markers demands a thorough knowledge of fungal antigens which may be detected in the serum or other body fluids of patients. Herein we report novel immunodominant antigens identified from extracellular proteins of Aspergillus fumigatus. Extracellular proteins of A. fumigatus were separated by two-dimensional electrophoresis (2-DE) and probed with the sera from critically ill patients with proven IA. The immunoreactive protein spots were identified by MALDI-TOF mass spectrometry (MALDI-TOF -MS). Forty spots from 2DE gels were detected and 17 different proteins were identified as immunogenic in humans. Function annotation revealed that most of these proteins were metabolic enzymes involved in carbohydrate, fatty acid, amino acid, and energy metabolism. One of the proteins, thioredoxin reductase GliT (TR), which showed the best immunoactivity, was analyzed further for secretory signals, protein localization, and homology. The results indicated that TR is a secretory protein with a signal sequence exhibiting a high probability for secretion. Furthermore, TR did not match any human proteins, and had low homology with most other fungi. The recombinant TR was recognized by the sera of all proven IA patients with different underlying diseases in this study. The immunoreactive proteins identified in this study may be helpful for the diagnosis of IA in critically ill patients. Our results indicate that TR and other immunodominant antigens have potential as biomarkers for the serologic diagnosis of invasive aspergillosis.

  8. RNA interference of NADPH-cytochrome P450 reductase of the rice brown planthopper, Nilaparvata lugens, increases susceptibility to insecticides.

    PubMed

    Liu, Su; Liang, Qing-Mei; Zhou, Wen-Wu; Jiang, Yan-Dong; Zhu, Qing-Zi; Yu, Hang; Zhang, Chuan-Xi; Gurr, Geoff M; Zhu, Zeng-Rong

    2015-01-01

    NADPH-cytochrome P450 reductase (CPR) is essential for numerous biological reactions catalysed by microsomal cytochrome P450 monooxygenases (P450s). Knockdown of CPR in several insects leads to developmental defects and increased susceptibility to insecticides. However, information about the role of CPR in the brown planthopper, Nilaparvata lugens, is still unavailable. A full-length cDNA encoding CPR was cloned from N. lugens (NlCPR). The deduced amino acid sequence showed marked features of classical CPRs, such as an N-terminus membrane anchor, conserved domains for flavin mononucleotide, flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate binding, as well as an FAD-binding motif and catalytic residues. Phylogenetic analysis revealed that NlCPR was located in a branch along with bed bug and pea aphid hemipteran insects. NlCPR mRNA was detectable in all tissues and developmental stages of N. lugens, as determined by real-time quantitative PCR. NlCPR transcripts were most abundant in the abdomen in adults, and in first-instar nymphs. Injection of N. lugens with double-strand RNA (dsRNA) against NlCPR significantly reduced the transcription level of the mRNA, and silencing of NlCPR resulted in increased susceptibility in N. lugens to beta-cypermethrin and imidacloprid. The results provide first evidence that NlCPR contributes to the susceptibility to beta-cypermethrin and imidacloprid in N. lugens. © 2014 Society of Chemical Industry.

  9. The antibacterial prodrug activator Rv2466c is a mycothiol-dependent reductase in the oxidative stress response of Mycobacterium tuberculosis.

    PubMed

    Rosado, Leonardo Astolfi; Wahni, Khadija; Degiacomi, Giulia; Pedre, Brandán; Young, David; de la Rubia, Alfonso G; Boldrin, Francesca; Martens, Edo; Marcos-Pascual, Laura; Sancho-Vaello, Enea; Albesa-Jové, David; Provvedi, Roberta; Martin, Charlotte; Makarov, Vadim; Versées, Wim; Verniest, Guido; Guerin, Marcelo E; Mateos, Luis M; Manganelli, Riccardo; Messens, Joris

    2017-08-11

    The Mycobacterium tuberculosis rv2466c gene encodes an oxidoreductase enzyme annotated as DsbA. It has a CPWC active-site motif embedded within its thioredoxin fold domain and mediates the activation of the prodrug TP053, a thienopyrimidine derivative that kills both replicating and nonreplicating bacilli. However, its mode of action and actual enzymatic function in M. tuberculosis have remained enigmatic. In this study, we report that Rv2466c is essential for bacterial survival under H 2 O 2 stress. Further, we discovered that Rv2466c lacks oxidase activity; rather, it receives electrons through the mycothiol/mycothione reductase/NADPH pathway to activate TP053, preferentially via a dithiol-disulfide mechanism. We also found that Rv2466c uses a monothiol-disulfide exchange mechanism to reduce S -mycothiolated mixed disulfides and intramolecular disulfides. Genetic, phylogenetic, bioinformatics, structural, and biochemical analyses revealed that Rv2466c is a novel mycothiol-dependent reductase, which represents a mycoredoxin cluster of enzymes within the DsbA family different from the glutaredoxin cluster to which mycoredoxin-1 (Mrx1 or Rv3198A) belongs. To validate this DsbA-mycoredoxin cluster, we also characterized a homologous enzyme of Corynebacterium glutamicum (NCgl2339) and observed that it demycothiolates and reduces a mycothiol arsenate adduct with kinetic properties different from those of Mrx1. In conclusion, our work has uncovered a DsbA-like mycoredoxin that promotes mycobacterial resistance to oxidative stress and reacts with free mycothiol and mycothiolated targets. The characterization of the DsbA-like mycoredoxin cluster reported here now paves the way for correctly classifying similar enzymes from other organisms. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Gene Gun Bombardment with DNA-Coated Golden Particles Enhanced the Protective Effect of a DNA Vaccine Based on Thioredoxin Glutathione Reductase of Schistosoma japonicum

    PubMed Central

    Cao, Yan; Zhao, Bin; Han, Yanhui; Zhang, Juan; Li, Xuezhen; Qiu, Chunhui; Wu, Xiujuan; Hong, Yang; Ai, Dezhou; Lin, Jiaojiao; Fu, Zhiqiang

    2013-01-01

    Schistosomiasis, caused by infection with Schistosoma species, remains an important parasitic zoonosis. Thioredoxin glutathione reductase of Schistosoma japonicum (SjTGR) plays an important role in the development of the parasite and for its survival. Here we present a recombinant plasmid DNA vaccine, pVAX1/SjTGR, to estimate its protection against S. japonicum in BALB/c mice. The DNA vaccine administrated by particle bombardment induced higher protection than by intramuscular injection. All animals vaccinated with pVAX1/SjTGR developed significant specific anti-SjTGR antibodies than control groups. Moreover, animals immunized by gene gun exhibited a splenocyte proliferative response, with an increase in IFN-γ and IL-4. The recombinant plasmid administrated by gene gun achieved a medium protective efficacy of 27.83–38.83% (P < 0.01) of worm reduction and 40.38–44.51% (P < 0.01) of liver egg count reduction. It suggests that different modes of administering a DNA vaccine can influence the protective efficacy induced by the vaccine. Interestingly, from the enzymatic activity results, we found that worms obtained from pVAX1/SjTGR-vaccinated animals expressed lower enzymatic activity than the control group and the antibodies weakened the enzymatic activity of SjTGR in vitro, too. It implies that the high-level antibodies may contribute to the protective effects. PMID:23509820

  11. Expression, purification, crystallization and preliminary X-ray analysis of perakine reductase, a new member of the aldo-keto reductase enzyme superfamily from higher plants

    PubMed Central

    Rosenthal, Cindy; Mueller, Uwe; Panjikar, Santosh; Sun, Lianli; Ruppert, Martin; Zhao, Yu; Stöckigt, Joachim

    2006-01-01

    Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C2221 and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å. PMID:17142919

  12. 1-Ene-steroid reductase of Mycobacterium sp. NRRL B-3805.

    PubMed

    Goren, T; Harnik, M; Rimon, S; Aharonowitz, Y

    1983-12-01

    The microbial enzymatic reduction of 1,4-androstadiene-3,17-dione (ADD) to 4-androstene-3,17-dione (AD), testosterone and 1-dehydrotestosterone (DHT) is described. Two reducing activities observed in washed cell suspensions and cell free extracts of Mycobacterium sp. NRRL B-3805 were found to account for these bioconversions. One was a 1-ene-steroid reductase and the other a 17-keto steroid reductase. The first reducing activity was found to appear in the soluble cell fraction whereas the latter could be precipitated by centrifugation. Maximum 1-ene-steroid reductase specific activity was achieved during the exponential growth phase of the organism and significantly increased upon induction with ADD. The 1-ene-steroid reductase was partially purified (30-fold) by ammonium sulfate fractionation, gel-filtration and ion-exchange chromatography, and was eluted from a Sephacryl S-300 column with an Mr = 115,000. The 1-ene-steroid reductase activity was NADPH-dependent and had specificity towards steroid compounds containing C-1,2 double bond with an apparent Km for ADD of 2.2 X 10(-5) M. The reverse reaction catalyzing C-1,2 dehydrogenation could not be detected in our preparations. The results suggest that in Mycobacterium sp NRRL B-3805 and B-3683 the steroid C-1,2 dehydrogenation and 1-ene reduction are two separable activities.

  13. Two Isoforms of Dihydroxyacetone Phosphate Reductase from the Chloroplasts of Dunaliella tertiolecta.

    PubMed

    Gee, R.; Goyal, A.; Byerrum, R. U.; Tolbert, N. E.

    1993-09-01

    Three isoforms of dihydroxyacetone phosphate reductase in extracts from Dunaliella tertiolecta have been separated by a diethylaminoethyl cellulose column chromatography with a shallow NaCl gradient. The chloroplasts contained the two major isoforms, and the third, minor form was in the cytosol. The isoforms are unstable in the absence of glycerol and they are cold labile, but they may be partially reactivated at 35[deg]C. The first chloroplast form to elute from the DEAE cellulose column was the major form when the cells were grown on high NaCl and it has been referred to as the form for glycerol production for osmoregulation or "osmoregulator form." The second form increased in specific activity when inorganic phosphate was increased in the growth media to stimulate growth, and it has been given the designation for the form for glyceride synthesis, "glyceride form." The osmoregulator form was stimulated by NaCl added to the enzyme assay, but not by reduced Escherichia coli thioredoxin. The glyceride form had properties similar to the enzyme in leaf chloroplast, such as inhibition by NaCl and by fatty acyl-coenzyme A derivatives and some stimulation by dithiothreitol, uridine diphosphate galactose, cyti-dine diphosphate dipalmatoyl diglyceride, and reduced E. coli thioredoxin. Thus, Dunaliella chloroplasts have a salt-stimulated osmoregulatory form of dihydroxyacetone phosphate reductase, which seems to have a role in glycerol production, and an isoform, which may be involved in glyceride synthesis and which has properties similar to the enzyme in chloroplasts of higher plants.

  14. Two Isoforms of Dihydroxyacetone Phosphate Reductase from the Chloroplasts of Dunaliella tertiolecta.

    PubMed Central

    Gee, R.; Goyal, A.; Byerrum, R. U.; Tolbert, N. E.

    1993-01-01

    Three isoforms of dihydroxyacetone phosphate reductase in extracts from Dunaliella tertiolecta have been separated by a diethylaminoethyl cellulose column chromatography with a shallow NaCl gradient. The chloroplasts contained the two major isoforms, and the third, minor form was in the cytosol. The isoforms are unstable in the absence of glycerol and they are cold labile, but they may be partially reactivated at 35[deg]C. The first chloroplast form to elute from the DEAE cellulose column was the major form when the cells were grown on high NaCl and it has been referred to as the form for glycerol production for osmoregulation or "osmoregulator form." The second form increased in specific activity when inorganic phosphate was increased in the growth media to stimulate growth, and it has been given the designation for the form for glyceride synthesis, "glyceride form." The osmoregulator form was stimulated by NaCl added to the enzyme assay, but not by reduced Escherichia coli thioredoxin. The glyceride form had properties similar to the enzyme in leaf chloroplast, such as inhibition by NaCl and by fatty acyl-coenzyme A derivatives and some stimulation by dithiothreitol, uridine diphosphate galactose, cyti-dine diphosphate dipalmatoyl diglyceride, and reduced E. coli thioredoxin. Thus, Dunaliella chloroplasts have a salt-stimulated osmoregulatory form of dihydroxyacetone phosphate reductase, which seems to have a role in glycerol production, and an isoform, which may be involved in glyceride synthesis and which has properties similar to the enzyme in chloroplasts of higher plants. PMID:12231930

  15. Silencing of NADPH-Dependent Oxidoreductase Genes (yqhD and dkgA) in Furfural-Resistant Ethanologenic Escherichia coli▿

    PubMed Central

    Miller, E. N.; Jarboe, L. R.; Yomano, L. P.; York, S. W.; Shanmugam, K. T.; Ingram, L. O.

    2009-01-01

    Low concentrations of furfural are formed as a side product during the dilute acid hydrolysis of hemicellulose. Growth is inhibited by exposure to furfural but resumes after the complete reduction of furfural to the less toxic furfuryl alcohol. Growth-based selection was used to isolate a furfural-resistant mutant of ethanologenic Escherichia coli LY180, designated strain EMFR9. Based on mRNA expression levels in the parent and mutant in response to furfural challenge, genes encoding 12 oxidoreductases were found to vary by more than twofold (eight were higher in EMFR9; four were higher in the parent). All 12 genes were cloned. When expressed from plasmids, none of the eight genes in the first group increased furfural tolerance in the parent (LY180). Expression of three of the silenced genes (yqhD, dkgA, and yqfA) in EMFR9 was found to decrease furfural tolerance compared to that in the parent. Purified enzymes encoded by yqhD and dkgA were shown to have NADPH-dependent furfural reductase activity. Both exhibited low Km values for NADPH (8 μM and 23 μM, respectively), similar to those of biosynthetic reactions. Furfural reductase activity was not associated with yqfA. Deleting yqhD and dkgA in the parent (LY180) increased furfural tolerance, but not to the same extent observed in the mutant EMFR9. Together, these results suggest that the process of reducing furfural by using an enzyme with a low Km for NADPH rather than a direct inhibitory action is the primary cause for growth inhibition by low concentrations of furfural. PMID:19429550

  16. Improved NADPH supply for xylitol production by engineered Escherichia coli with glycolytic mutations.

    PubMed

    Chin, Jonathan W; Cirino, Patrick C

    2011-01-01

    Escherichia coli engineered to uptake xylose while metabolizing glucose was previously shown to produce high levels of xylitol from a mixture of glucose and xylose when expressing NADPH-dependent xylose reductase from Candida boidinii (CbXR) (Cirino et al., Biotechnol Bioeng. 2006;95:1167-1176). We then described the effects of deletions of key metabolic pathways (e.g., Embden-Meyerhof-Parnas and pentose phosphate pathway) and reactions (e.g., transhydrogenase and NADH dehydrogenase) on resting-cell xylitol yield (Y RPG: moles of xylitol produced per mole of glucose consumed) (Chin et al., Biotechnol Bioeng. 2009;102:209-220). These prior results demonstrated the importance of direct NADPH supply by NADP+-utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions. This study describes strain modifications that improve coupling between glucose catabolism (oxidation) and xylose reduction using two fundamentally different strategies. We first examined the effects of deleting the phosphofructokinase (pfk) gene(s) on growth-uncoupled xylitol production and found that deleting both pfkA and sthA (encoding the E. coli-soluble transhydrogenase) improved the xylitol Y RPG from 3.4 ± 0.6 to 5.4 ± 0.4. The second strategy focused on coupling aerobic growth on glucose to xylitol production by deleting pgi (encoding phosphoglucose isomerase) and sthA. Impaired growth due to imbalanced NADPH metabolism (Sauer et al., J Biol Chem. 2004;279:6613-6619) was alleviated upon expressing CbXR, resulting in xylitol production similar to that of the growth-uncoupled precursor strains but with much less acetate secretion and more efficient utilization of glucose. Intracellular nicotinamide cofactor levels were also quantified, and the magnitude of the change in the NADPH/NADP+ ratio measured from cells consuming glucose in the absence vs. presence of xylose showed a strong correlation to the resulting Y RPG. Copyright © 2011 American Institute of Chemical

  17. Purification, Characterization, and Potential Bacterial Wax Production Role of an NADPH-Dependent Fatty Aldehyde Reductase from Marinobacter aquaeolei VT8▿ †

    PubMed Central

    Wahlen, Bradley D.; Oswald, Whitney S.; Seefeldt, Lance C.; Barney, Brett M.

    2009-01-01

    Wax esters, ester-linked fatty acids and long-chain alcohols, are important energy storage compounds in select bacteria. The synthesis of wax esters from fatty acids is proposed to require the action of a four-enzyme pathway. An essential step in the pathway is the reduction of a fatty aldehyde to the corresponding fatty alcohol, although the enzyme responsible for catalyzing this reaction has yet to be identified in bacteria. We report here the purification and characterization of an enzyme from the wax ester-accumulating bacterium Marinobacter aquaeolei VT8, which is a proposed fatty aldehyde reductase in this pathway. The enzyme, a 57-kDa monomer, was expressed in Escherichia coli as a fusion protein with the maltose binding protein on the N terminus and was purified to near homogeneity by using amylose affinity chromatography. The purified enzyme was found to reduce a number of long-chain aldehydes to the corresponding alcohols coupled to the oxidation of NADPH. The highest specific activity was observed for the reduction of decanal (85 nmol decanal reduced/min/mg). Short-chain and aromatic aldehydes were not substrates. The enzyme showed no detectable catalysis of the reverse reaction, the oxidation of decanol by NADP+. The mechanism of the enzyme was probed with several site-specific chemical probes. The possible uses of this enzyme in the production of wax esters are discussed. PMID:19270127

  18. The Nox/Ferric reductase/Ferric reductase-like families of Eumycetes.

    PubMed

    Grissa, Ibtissem; Bidard, Frédérique; Grognet, Pierre; Grossetete, Sandrine; Silar, Philippe

    2010-09-01

    Reactive Oxygen Species (ROS) are involved in plant biomass degradation by fungi and development of fungal structures. While the ROS-generating NADPH oxidases from filamentous fungi are under strong scrutiny, much less is known about the related integral Membrane (or Ferric) Reductases (IMRs). Here, we present a survey of these enzymes in 29 fungal genomes covering the entire available range of fungal diversity. IMRs are present in all fungal genomes. They can be classified into at least 24 families, underscoring the high diversity of these enzymes. Some are differentially regulated during colony or fruiting body development, as well as by the nature of the carbon source of the growth medium. Importantly, functional characterization of IMRs has been made on proteins belonging to only two families, while nothing or very little is known about the proteins of the other 22 families. Copyright © 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  19. Mechanism-based Proteomic Screening Identifies Targets of Thioredoxin-like Proteins*

    PubMed Central

    Nakao, Lia S.; Everley, Robert A.; Marino, Stefano M.; Lo, Sze M.; de Souza, Luiz E.; Gygi, Steven P.; Gladyshev, Vadim N.

    2015-01-01

    Thioredoxin (Trx)-fold proteins are protagonists of numerous cellular pathways that are subject to thiol-based redox control. The best characterized regulator of thiols in proteins is Trx1 itself, which together with thioredoxin reductase 1 (TR1) and peroxiredoxins (Prxs) comprises a key redox regulatory system in mammalian cells. However, there are numerous other Trx-like proteins, whose functions and redox interactors are unknown. It is also unclear if the principles of Trx1-based redox control apply to these proteins. Here, we employed a proteomic strategy to four Trx-like proteins containing CXXC motifs, namely Trx1, Rdx12, Trx-like protein 1 (Txnl1) and nucleoredoxin 1 (Nrx1), whose cellular targets were trapped in vivo using mutant Trx-like proteins, under conditions of low endogenous expression of these proteins. Prxs were detected as key redox targets of Trx1, but this approach also supported the detection of TR1, which is the Trx1 reductant, as well as mitochondrial intermembrane proteins AIF and Mia40. In addition, glutathione peroxidase 4 was found to be a Rdx12 redox target. In contrast, no redox targets of Txnl1 and Nrx1 could be detected, suggesting that their CXXC motifs do not engage in mixed disulfides with cellular proteins. For some Trx-like proteins, the method allowed distinguishing redox and non-redox interactions. Parallel, comparative analyses of multiple thiol oxidoreductases revealed differences in the functions of their CXXC motifs, providing important insights into thiol-based redox control of cellular processes. PMID:25561728

  20. Synthetic and Crystallographic Studies of a New Inhibitor Series Targeting Bacillus anthracis Dihydrofolate Reductase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Beierlein, J.; Frey, K; Bolstad, D

    2008-01-01

    Bacillus anthracis, the causative agent of anthrax, poses a significant biodefense danger. Serious limitations in approved therapeutics and the generation of resistance have produced a compelling need for new therapeutic agents against this organism. Bacillus anthracis is known to be insensitive to the clinically used antifolate, trimethoprim, because of a lack of potency against the dihydrofolate reductase enzyme. Herein, we describe a novel lead series of B. anthracis dihydrofolate reductase inhibitors characterized by an extended trimethoprim-like scaffold. The best lead compound adds only 22 Da to the molecular weight and is 82-fold more potent than trimethoprim. An X-ray crystal structuremore » of this lead compound bound to B. anthracis dihydrofolate reductase in the presence of NADPH was determined to 2.25 A resolution. The structure reveals several features that can be exploited for further development of this lead series.« less

  1. Inhibition of glutathione synthesis eliminates the adaptive response of ascitic hepatoma 22 cells to nedaplatin that targets thioredoxin reductase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Yijun; Lu, Hongjuan; Wang, Dongxu

    2012-12-15

    Thioredoxin reductase (TrxR) is a target for cancer therapy and the anticancer mechanism of cisplatin involves TrxR inhibition. We hypothesize that the anticancer drug nedaplatin (NDP), an analogue of cisplatin and a second-generation platinum complex, also targets TrxR. Furthermore, we investigate whether the therapeutic efficacy of NDP can be enhanced by simultaneous modulation of 1) TrxR, via NDP, and 2) glutathione (GSH), via the GSH synthesis inhibitor buthionine sulfoximine (BSO). Mice bearing ascitic hepatoma 22 (H22) cells were treated with NDP alone or NDP plus BSO. TrxR activity of H22 cells was inhibited by NDP in a dose-dependent manner. Amore » high correlation between the inhibition of TrxR activity at 6 h and the inhibition of ascitic fluid volume at 72 h was established (r = 0.978, p < 0.01). As an adaptive response, the viable ascitic cancer cells after NDP treatment displayed an enlarged cell phenotype, assembled with several-fold more antioxidant enzymes and GSH-predominant non-protein free thiols. This adaptive response was largely eliminated when BSO was co-administered with NDP, leading to the decimation of the H22 cell population without enhancing renal toxicity, since at this dose, NDP did not inhibit renal TrxR activity. In conclusion, the pharmacological effect of NDP involves TrxR inhibition, and the adaptive response of NDP-treated ascitic H22 cells can be efficiently counteracted by BSO. Simultaneous modulation of TrxR and GSH on ascitic H22 cells using NDP plus BSO greatly enhances therapeutic efficacy as compared with the single modulation of TrxR using NDP alone. -- Highlights: ► Nedaplatin at a pharmacological dose inhibits TrxR in cancer cells but not in kidney. ► The nedaplatin-treated cancer cells exhibit adaptive response. ► Buthionine sulfoximine inhibits glutathione in both cancer cells and kidney. ► Buthionine sulfoximine counteracts the adaptive response to the nedaplatin treatment. ► Buthionine sulfoximine does

  2. Thioredoxin and Thioredoxin Target Proteins: From Molecular Mechanisms to Functional Significance

    PubMed Central

    Lee, Samuel; Kim, Soo Min

    2013-01-01

    Abstract The thioredoxin (Trx) system is one of the central antioxidant systems in mammalian cells, maintaining a reducing environment by catalyzing electron flux from nicotinamide adenine dinucleotide phosphate through Trx reductase to Trx, which reduces its target proteins using highly conserved thiol groups. While the importance of protecting cells from the detrimental effects of reactive oxygen species is clear, decades of research in this field revealed that there is a network of redox-sensitive proteins forming redox-dependent signaling pathways that are crucial for fundamental cellular processes, including metabolism, proliferation, differentiation, migration, and apoptosis. Trx participates in signaling pathways interacting with different proteins to control their dynamic regulation of structure and function. In this review, we focus on Trx target proteins that are involved in redox-dependent signaling pathways. Specifically, Trx-dependent reductive enzymes that participate in classical redox reactions and redox-sensitive signaling molecules are discussed in greater detail. The latter are extensively discussed, as ongoing research unveils more and more details about the complex signaling networks of Trx-sensitive signaling molecules such as apoptosis signal-regulating kinase 1, Trx interacting protein, and phosphatase and tensin homolog, thus highlighting the potential direct and indirect impact of their redox-dependent interaction with Trx. Overall, the findings that are described here illustrate the importance and complexity of Trx-dependent, redox-sensitive signaling in the cell. Our increasing understanding of the components and mechanisms of these signaling pathways could lead to the identification of new potential targets for the treatment of diseases, including cancer and diabetes. Antioxid. Redox Signal. 18, 1165–1207. PMID:22607099

  3. sigmaR, an RNA polymerase sigma factor that modulates expression of the thioredoxin system in response to oxidative stress in Streptomyces coelicolor A3(2).

    PubMed Central

    Paget, M S; Kang, J G; Roe, J H; Buttner, M J

    1998-01-01

    We have identified an RNA polymerase sigma factor, sigmaR, that is part of a system that senses and responds to thiol oxidation in the Gram-positive, antibiotic-producing bacterium Streptomyces coelicolor A3(2). Deletion of the gene (sigR) encoding sigmaR caused sensitivity to the thiol-specific oxidant diamide and to the redox cycling compounds menadione and plumbagin. This correlated with reduced levels of disulfide reductase activity and an inability to induce this activity on exposure to diamide. The trxBA operon, encoding thioredoxin reductase and thioredoxin, was found to be under the direct control of sigmaR. trxBA is transcribed from two promoters, trxBp1 and trxBp2, separated by 5-6 bp. trxBp1 is transiently induced at least 50-fold in response to diamide treatment in a sigR-dependent manner. Purified sigmaR directed transcription from trxBp1 in vitro, indicating that trxBp1 is a target for sigmaR. Transcription of sigR itself initiates at two promoters, sigRp1 and sigRp2, which are separated by 173 bp. The sigRp2 transcript was undetectable in a sigR-null mutant, and purified sigmaR could direct transcription from sigRp2 in vitro, indicating that sigR is positively autoregulated. Transcription from sigRp2 was also transiently induced (70-fold) following treatment with diamide. We propose a model in which sigmaR induces expression of the thioredoxin system in response to cytoplasmic disulfide bond formation. Upon reestablishment of normal thiol levels, sigmaR activity is switched off, resulting in down-regulation of trxBA and sigR. We present evidence that the sigmaR system also functions in the actinomycete pathogen Mycobacterium tuberculosis. PMID:9755177

  4. sigmaR, an RNA polymerase sigma factor that modulates expression of the thioredoxin system in response to oxidative stress in Streptomyces coelicolor A3(2).

    PubMed

    Paget, M S; Kang, J G; Roe, J H; Buttner, M J

    1998-10-01

    We have identified an RNA polymerase sigma factor, sigmaR, that is part of a system that senses and responds to thiol oxidation in the Gram-positive, antibiotic-producing bacterium Streptomyces coelicolor A3(2). Deletion of the gene (sigR) encoding sigmaR caused sensitivity to the thiol-specific oxidant diamide and to the redox cycling compounds menadione and plumbagin. This correlated with reduced levels of disulfide reductase activity and an inability to induce this activity on exposure to diamide. The trxBA operon, encoding thioredoxin reductase and thioredoxin, was found to be under the direct control of sigmaR. trxBA is transcribed from two promoters, trxBp1 and trxBp2, separated by 5-6 bp. trxBp1 is transiently induced at least 50-fold in response to diamide treatment in a sigR-dependent manner. Purified sigmaR directed transcription from trxBp1 in vitro, indicating that trxBp1 is a target for sigmaR. Transcription of sigR itself initiates at two promoters, sigRp1 and sigRp2, which are separated by 173 bp. The sigRp2 transcript was undetectable in a sigR-null mutant, and purified sigmaR could direct transcription from sigRp2 in vitro, indicating that sigR is positively autoregulated. Transcription from sigRp2 was also transiently induced (70-fold) following treatment with diamide. We propose a model in which sigmaR induces expression of the thioredoxin system in response to cytoplasmic disulfide bond formation. Upon reestablishment of normal thiol levels, sigmaR activity is switched off, resulting in down-regulation of trxBA and sigR. We present evidence that the sigmaR system also functions in the actinomycete pathogen Mycobacterium tuberculosis.

  5. X-ray crystal structure of GarR-tartronate semialdehyde reductase from Salmonella typhimurium.

    PubMed

    Osipiuk, J; Zhou, M; Moy, S; Collart, F; Joachimiak, A

    2009-09-01

    Tartronate semialdehyde reductases (TSRs), also known as 2-hydroxy-3-oxopropionate reductases, catalyze the reduction of tartronate semialdehyde using NAD as cofactor in the final stage of D-glycerate biosynthesis. These enzymes belong to family of structurally and mechanically related beta-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways. Here, we present the crystal structure of GarR a TSR from Salmonella typhimurium determined by the single-wavelength anomalous diffraction method and refined to 1.65 A resolution. The active site of the enzyme contains L-tartrate which most likely mimics a position of a glycerate which is a product of the enzyme reaction. The analysis of the TSR structure shows also a putative NADPH binding site in the enzyme.

  6. Diphenyl diselenide protects against methylmercury-induced inhibition of thioredoxin reductase and glutathione peroxidase in human neuroblastoma cells: a comparison with ebselen.

    PubMed

    Meinerz, Daiane F; Branco, Vasco; Aschner, Michael; Carvalho, Cristina; Rocha, João Batista T

    2017-09-01

    Exposure to methylmercury (MeHg), an important environmental toxicant, may lead to serious health risks, damaging various organs and predominantly affecting the brain function. The toxicity of MeHg can be related to the inhibition of important selenoenzymes, such as glutathione peroxidase (GPx) and thioredoxin reductase (TrxR). Experimental studies have shown that selenocompounds play an important role as cellular detoxifiers and protective agents against the harmful effects of mercury. The present study investigated the mechanisms by which diphenyl diselenide [(PhSe) 2 ] and ebselen interfered with the interaction of mercury (MeHg) and selenoenzymes (TrxR and GPx) in an in vitro experimental model of cultured human neuroblastoma cells (SH-SY5Y). Our results established that (PhSe) 2 and ebselen increased the activity and expression of TrxR. In contrast, MeHg inhibited TrxR activity even at low doses (0.5 μm). Coexposure to selenocompounds and MeHg showed a protective effect of (PhSe) 2 on both the activity and expression of TrxR. When selenoenzyme GPx was evaluated, selenocompounds did not alter its activity or expression significantly, whereas MeHg inhibited the activity of GPx (from 1 μm). Among the selenocompounds only (PhSe) 2 significantly protected against the effects of MeHg on GPx activity. Taken together, these results indicate a potential use for ebselen and (PhSe) 2 against MeHg toxicity. Furthermore, for the first time, we have demonstrated that (PhSe) 2 caused a more pronounced upregulation of TrxR than ebselen in neuroblastoma cells, likely reflecting an important molecular mechanism involved in the antioxidant properties of this compound. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  7. Thioredoxin-independent regulation of metabolism by the alpha-arrestin proteins.

    PubMed

    Patwari, Parth; Chutkow, William A; Cummings, Kiersten; Verstraeten, Valerie L R M; Lammerding, Jan; Schreiter, Eric R; Lee, Richard T

    2009-09-11

    Thioredoxin-interacting protein (Txnip), originally characterized as an inhibitor of thioredoxin, is now known to be a critical regulator of glucose metabolism in vivo. Txnip is a member of the alpha-arrestin protein family; the alpha-arrestins are related to the classical beta-arrestins and visual arrestins. Txnip is the only alpha-arrestin known to bind thioredoxin, and it is not known whether the metabolic effects of Txnip are related to its ability to bind thioredoxin or related to conserved alpha-arrestin function. Here we show that wild type Txnip and Txnip C247S, a Txnip mutant that does not bind thioredoxin in vitro, both inhibit glucose uptake in mature adipocytes and in primary skin fibroblasts. Furthermore, we show that Txnip C247S does not bind thioredoxin in cells, using thiol alkylation to trap the Txnip-thioredoxin complex. Because Txnip function was independent of thioredoxin binding, we tested whether inhibition of glucose uptake was conserved in the related alpha-arrestins Arrdc4 and Arrdc3. Both Txnip and Arrdc4 inhibited glucose uptake and lactate output, while Arrdc3 had no effect. Structure-function analysis indicated that Txnip and Arrdc4 inhibit glucose uptake independent of the C-terminal WW-domain binding motifs, recently identified as important in yeast alpha-arrestins. Instead, regulation of glucose uptake was intrinsic to the arrestin domains themselves. These data demonstrate that Txnip regulates cellular metabolism independent of its binding to thioredoxin and reveal the arrestin domains as crucial structural elements in metabolic functions of alpha-arrestin proteins.

  8. Conformational and functional similarities between glutaredoxin and thioredoxins.

    PubMed Central

    Eklund, H; Cambillau, C; Sjöberg, B M; Holmgren, A; Jörnvall, H; Höög, J O; Brändén, C I

    1984-01-01

    The tertiary structures of thioredoxin from Escherichia coli and bacteriophage T4 have been compared and aligned giving a common fold of 68 C alpha atoms with a root mean square difference of 2.6 A. The amino acid sequence of glutaredoxin has been aligned to those of the thioredoxins assuming that glutaredoxin has the same common fold. A model of the glutaredoxin molecule was built on a vector display using this alignment and the T4 thioredoxin tertiary structure. By comparison of the model with those of the thioredoxins, we have identified a molecular surface area on one side of the redox-active S-S bridge which we suggest is the binding area of these molecules for redox interactions with other proteins. This area comprises residues 33-34, 75-76 and 91-93 in E. coli thioredoxin; 15-16, 65-66 and 76-78 in T4 thioredoxin and 12-13, 59-60 and 69-71 in glutaredoxin. In all three molecules, this part of the surface is flat and hydrophobic. Charged groups are completely absent. In contrast, there is a cluster of charged groups on the other side of the S-S bridge which we suggest participates in the mechanisms of the redox reactions. In particular, a lysine residue close to an aromatic ring is conserved in all molecules. PMID:6378624

  9. Clustered Genes Encoding 2-Keto-l-Gulonate Reductase and l-Idonate 5-Dehydrogenase in the Novel Fungal d-Glucuronic Acid Pathway

    PubMed Central

    Kuivanen, Joosu; Arvas, Mikko; Richard, Peter

    2017-01-01

    D-Glucuronic acid is a biomass component that occurs in plant cell wall polysaccharides and is catabolized by saprotrophic microorganisms including fungi. A pathway for D-glucuronic acid catabolism in fungal microorganisms is only partly known. In the filamentous fungus Aspergillus niger, the enzymes that are known to be part of the pathway are the NADPH requiring D-glucuronic acid reductase forming L-gulonate and the NADH requiring 2-keto-L-gulonate reductase that forms L-idonate. With the aid of RNA sequencing we identified two more enzymes of the pathway. The first is a NADPH requiring 2-keto-L-gulonate reductase that forms L-idonate, GluD. The second is a NAD+ requiring L-idonate 5-dehydrogenase forming 5-keto-gluconate, GluE. The genes coding for these two enzymes are clustered and share the same bidirectional promoter. The GluD is an enzyme with a strict requirement for NADP+/NADPH as cofactors. The kcat for 2-keto-L-gulonate and L-idonate is 21.4 and 1.1 s-1, and the Km 25.3 and 12.6 mM, respectively, when using the purified protein. In contrast, the GluE has a strict requirement for NAD+/NADH. The kcat for L-idonate and 5-keto-D-gluconate is 5.5 and 7.2 s-1, and the Km 30.9 and 8.4 mM, respectively. These values also refer to the purified protein. The gluD deletion resulted in accumulation of 2-keto-L-gulonate in the liquid cultivation while the gluE deletion resulted in reduced growth and cessation of the D-glucuronic acid catabolism. PMID:28261181

  10. Knockdown of NADPH-cytochrome P450 reductase results in reduced resistance to buprofezin in the small brown planthopper, Laodelphax striatellus (fallén).

    PubMed

    Zhang, Yueliang; Wang, Yaming; Wang, Lihua; Yao, Jing; Guo, Huifang; Fang, Jichao

    2016-02-01

    NADPH-cytochrome P450 reductase (CPR) plays an important role in cytochrome P450 function, and CPR knockdown in several insects leads to increased susceptibility to insecticides. However, a putative CPR gene has not yet been fully characterized in the small brown planthopper Laodelphax striatellus, a notorious agricultural pest in rice that causes serious damage by transmitting rice stripe and rice black-streaked dwarf viruses. The objective of this study was to clone the cDNA and to knock down the expression of the gene that encodes L. striatellus CPR (LsCPR) to further determine whether P450s are involved in the resistance of L. striatellus to buprofezin. First, the full-length cDNA of LsCPR was cloned and found to contain an open reading frame (ORF) encoding a polypeptide of 679 amino acids with a calculated molecular mass and isoelectric point of 76.92kDa and 5.37, respectively. The deduced amino acid sequence shares high identity with the CPRs of other insects (98%, 97%, 75% and 68% for Sogatella furcifera, Nilaparvata lugens, Cimex lectularius and Anopheles gambiae, respectively) and possesses the characteristic features of classical CPRs, such as an N-terminal membrane anchor and conserved domains for flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) binding. Phylogenetic analysis revealed that LsCPR is located in a branch along with the CPRs of other hemipteran insects. LsCPR mRNA was detectable in all examined body parts and developmental stages of L. striatellus, as determined by real-time quantitative PCR (qPCR), and transcripts were most abundant in the adult abdomen and in first-instar nymphs and adults. Ingestion of 200μg/mL of LsCPR double-stranded RNA (dsLsCPR) by the planthopper for 5days significantly reduced the transcription level of LsCPR. Moreover, silencing of LsCPR caused increased susceptibility to buprofezin in a buprofezin-resistant (YN-BPF) strain but not in a

  11. Thioredoxin 1 mediates TGF-β-induced epithelial-mesenchymal transition in salivary adenoid cystic carcinoma.

    PubMed

    Jiang, Yang; Feng, Xin; Zheng, Lei; Li, Sheng-Lin; Ge, Xi-Yuan; Zhang, Jian-Guo

    2015-09-22

    Epithelial-mesenchymal transition (EMT) plays an important role in the invasion and metastasis of salivary adenoid cystic carcinoma (SACC) which is characterized by wide local infiltration, perineural spread, a propensity to local recurrence and late distant metastasis. Our recent studies have disclosed that TGF-β is a crucial factor for EMT in metastatic SACC. In this study, we further uncovered small redox protein thioredoxin 1 (TXN) as a critical mediator of TGF-β induced EMT. Immunohistochemistry analysis revealed significantly higher expressions of TXN, thioredoxin reductase 1 (TXNRD1) and N-cadherin, and lower expression of E-cadherin in human metastatic SACC compared to non-metastatic SACC tissues. Consistently, cultured SACC cells with stable TXN overexpression had decreased E-cadherin and increased N-cadherin as well as Snail and Slug expressions. The enhanced migration and invasion potential of these cells was abrogated by Akt or TXNRD1 inhibitors. Expression of N-cadherin and Akt p-Akt decreased, whereas E-cadherin expression increased in a BBSKE (TXNRD1 inhibitor)-dose-dependent manner. In a xenograft mouse model, TXN overexpression facilitated the metastatic potential of SACC-83 cells to the lung. Our results indicate that TXN plays a key role in SACC invasion and metastasis through the modulation of TGF-β-Akt/GSK-3β on EMT. TXN could be a potential therapeutic target for SACC.

  12. Nicotinamide nucleotide transhydrogenase (Nnt) links the substrate requirement in brain mitochondria for hydrogen peroxide removal to the thioredoxin/peroxiredoxin (Trx/Prx) system.

    PubMed

    Lopert, Pamela; Patel, Manisha

    2014-05-30

    Mitochondrial reactive oxygen species are implicated in the etiology of multiple neurodegenerative diseases, including Parkinson disease. Mitochondria are known to be net producers of ROS, but recently we have shown that brain mitochondria can consume mitochondrial hydrogen peroxide (H2O2) in a respiration-dependent manner predominantly by the thioredoxin/peroxiredoxin system. Here, we sought to determine the mechanism linking mitochondrial respiration with H2O2 catabolism in brain mitochondria and dopaminergic cells. We hypothesized that nicotinamide nucleotide transhydrogenase (Nnt), which utilizes the proton gradient to generate NADPH from NADH and NADP(+), provides the link between mitochondrial respiration and H2O2 detoxification through the thioredoxin/peroxiredoxin system. Pharmacological inhibition of Nnt in isolated brain mitochondria significantly decreased their ability to consume H2O2 in the presence, but not absence, of respiration substrates. Nnt inhibition in liver mitochondria, which do not require substrates to detoxify H2O2, had no effect. Pharmacological inhibition or lentiviral knockdown of Nnt in N27 dopaminergic cells (a) decreased H2O2 catabolism, (b) decreased NADPH and increased NADP(+) levels, and (c) decreased basal, spare, and maximal mitochondrial oxygen consumption rates. Nnt-deficient cells possessed higher levels of oxidized mitochondrial Prx, which rendered them more susceptible to steady-state increases in H2O2 and cell death following exposure to subtoxic levels of paraquat. These data implicate Nnt as the critical link between the metabolic and H2O2 antioxidant function in brain mitochondria and suggests Nnt as a potential therapeutic target to improve the redox balance in conditions of oxidative stress associated with neurodegenerative diseases. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Enzymatic reduction of disulfide bonds in lysosomes: Characterization of a Gamma-interferon-inducible lysosomal thiol reductase (GILT)

    NASA Astrophysics Data System (ADS)

    Arunachalam, Balasubramanian; Phan, Uyen T.; Geuze, Hans J.; Cresswell, Peter

    2000-01-01

    Proteins internalized into the endocytic pathway are usually degraded. Efficient proteolysis requires denaturation, induced by acidic conditions within lysosomes, and reduction of inter- and intrachain disulfide bonds. Cytosolic reduction is mediated enzymatically by thioredoxin, but the mechanism of lysosomal reduction is unknown. We describe here a lysosomal thiol reductase optimally active at low pH and capable of catalyzing disulfide bond reduction both in vivo and in vitro. The active site, determined by mutagenesis, consists of a pair of cysteine residues separated by two amino acids, similar to other enzymes of the thioredoxin family. The enzyme is a soluble glycoprotein that is synthesized as a precursor. After delivery into the endosomal/lysosomal system by the mannose 6-phosphate receptor, N- and C-terminal prosequences are removed. The enzyme is expressed constitutively in antigen-presenting cells and induced by IFN-γ in other cell types, suggesting a potentially important role in antigen processing.

  14. X-Ray crystal structure of GarR—tartronate semialdehyde reductase from Salmonella typhimurium

    PubMed Central

    Osipiuk, J.; Zhou, M.; Moy, S.; Collart, F.

    2009-01-01

    Tartronate semialdehyde reductases (TSRs), also known as 2-hydroxy-3-oxopropionate reductases, catalyze the reduction of tartronate semialdehyde using NAD as cofactor in the final stage of D-glycerate biosynthesis. These enzymes belong to family of structurally and mechanically related β-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways. Here, we present the crystal structure of GarR a TSR from Salmonella typhimurium determined by the single-wavelength anomalous diffraction method and refined to 1.65 Å resolution. The active site of the enzyme contains L-tartrate which most likely mimics a position of a glycerate which is a product of the enzyme reaction. The analysis of the TSR structure shows also a putative NADPH binding site in the enzyme. PMID:19184529

  15. Defects in antioxidant defense and calcium transport in the epidermis of xeroderma pigmentosum patients.

    PubMed

    Schallreuter, K U; Pittelkow, M R; Wood, J M

    1991-01-01

    A comparative study of the antioxidant enzymes superoxide dismutase, catalase, glutathione reductase and thioredoxin reductase was undertaken in two families with xeroderma pigmentosum (XP) and in healthy controls of corresponding skin phototypes. Epidermal blister roofs obtained from the XP patients revealed significant decreases in catalase, thioredoxin reductase, and superoxide dismutase, but glutathione reductase was unaffected. In addition, keratinocytes established from XP patients contained a significantly higher than normal intracellular calcium concentration compared with control cells from a corresponding skin type. Keratinocytes established from an XP obligate heterozygote revealed intermediate levels of calcium between XP homozygotes and controls. Previously high intracellular calcium has been shown to compromise the redox status of keratinocytes by allosteric inhibition of the thioredoxin reductase/thioredoxin electron transfer system. In XP homozygous keratinocytes from sun-exposed epidermis, the intracellular concentration of reduced thioredoxin was decreased to 50% compared with these cells from unexposed skin. Taken together, the results from this study indicate that the epidermis in XP patients lacks effective defense against free radicals and peroxides. In addition to the well-established defect in the normal rates of unscheduled DNA repair, these findings provide an even better explanation for the multiple cutaneous neoplasms in these patients.

  16. RNA Interference of NADPH-Cytochrome P450 Reductase Results in Reduced Insecticide Resistance in the Bed Bug, Cimex lectularius

    PubMed Central

    Zhu, Fang; Sams, Sarah; Moural, Tim; Haynes, Kenneth F.; Potter, Michael F.; Palli, Subba R.

    2012-01-01

    Background NADPH-cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The genes coding for P450s are not yet fully identified in the bed bug, Cimex lectularius. Hence, we decided to clone cDNA and knockdown the expression of the gene coding for CPR which is suggested to be required for the function of all P450s to determine whether or not P450s are involved in resistance of bed bugs to insecticides. Methodology/Principal Findings The full length Cimex lectularius CPR (ClCPR) cDNA was isolated from a deltamethrin resistant bed bug population (CIN-1) using a combined PCR strategy. Bioinformatics and in silico modeling were employed to identify three conserved binding domains (FMN, FAD, NADP), a FAD binding motif, and the catalytic residues. The critical amino acids involved in FMN, FAD, NADP binding and their putative functions were also analyzed. No signal peptide but a membrane anchor domain with 21 amino acids which facilitates the localization of ClCPR on the endoplasmic reticulum was identified in ClCPR protein. Phylogenetic analysis showed that ClCPR is closer to the CPR from the body louse, Pediculus humanus corporis than to the CPRs from the other insect species studied. The ClCPR gene was ubiquitously expressed in all tissues tested but showed an increase in expression as immature stages develop into adults. We exploited the traumatic insemination mechanism of bed bugs to inject dsRNA and successfully knockdown the expression of the gene coding for ClCPR. Suppression of the ClCPR expression increased susceptibility to deltamethrin in resistant populations but not in the susceptible population of bed bugs. Conclusions/Significance These data suggest that P450-mediated metabolic detoxification may serve as one of the resistance mechanisms in bed bugs. PMID:22347424

  17. RNA interference of NADPH-cytochrome P450 reductase results in reduced insecticide resistance in the bed bug, Cimex lectularius.

    PubMed

    Zhu, Fang; Sams, Sarah; Moural, Tim; Haynes, Kenneth F; Potter, Michael F; Palli, Subba R

    2012-01-01

    NADPH-cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The genes coding for P450s are not yet fully identified in the bed bug, Cimex lectularius. Hence, we decided to clone cDNA and knockdown the expression of the gene coding for CPR which is suggested to be required for the function of all P450s to determine whether or not P450s are involved in resistance of bed bugs to insecticides. The full length Cimex lectularius CPR (ClCPR) cDNA was isolated from a deltamethrin resistant bed bug population (CIN-1) using a combined PCR strategy. Bioinformatics and in silico modeling were employed to identify three conserved binding domains (FMN, FAD, NADP), a FAD binding motif, and the catalytic residues. The critical amino acids involved in FMN, FAD, NADP binding and their putative functions were also analyzed. No signal peptide but a membrane anchor domain with 21 amino acids which facilitates the localization of ClCPR on the endoplasmic reticulum was identified in ClCPR protein. Phylogenetic analysis showed that ClCPR is closer to the CPR from the body louse, Pediculus humanus corporis than to the CPRs from the other insect species studied. The ClCPR gene was ubiquitously expressed in all tissues tested but showed an increase in expression as immature stages develop into adults. We exploited the traumatic insemination mechanism of bed bugs to inject dsRNA and successfully knockdown the expression of the gene coding for ClCPR. Suppression of the ClCPR expression increased susceptibility to deltamethrin in resistant populations but not in the susceptible population of bed bugs. These data suggest that P450-mediated metabolic detoxification may serve as one of the resistance mechanisms in bed bugs.

  18. Conformational Changes of NADPH-Cytochrome P450 Oxidoreductase Are Essential for Catalysis and Cofactor Binding*

    PubMed Central

    Xia, Chuanwu; Hamdane, Djemel; Shen, Anna L.; Choi, Vivian; Kasper, Charles B.; Pearl, Naw May; Zhang, Haoming; Im, Sang-Choul; Waskell, Lucy; Kim, Jung-Ja P.

    2011-01-01

    The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp147 and Arg514 in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (≥90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function. The crystal structures of the mutant without and with NADP+ revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ∼20° compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP+ shows movement of the Gly631–Asn635 loop. In the NADP+-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP+ shows movement of the Gly631–Asn635 loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly631–Asn635 loop movement controls NADPH binding and NADP+ release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners. PMID:21345800

  19. Yeast mitochondrial glutathione is an essential antioxidant with mitochondrial thioredoxin providing a back-up system

    PubMed Central

    Gostimskaya, Irina; Grant, Chris M.

    2016-01-01

    Glutathione is an abundant, low-molecular-weight tripeptide whose biological importance is dependent upon its redox-active free sulphydryl moiety. Its role as the main determinant of thiol-redox control has been challenged such that it has been proposed to play a crucial role in iron–sulphur clusters maturation, and only a minor role in thiol redox regulation, predominantly as a back-up system for the cytoplasmic thioredoxin system. Here, we have tested the importance of mitochondrial glutathione in thiol-redox regulation. Glutathione reductase (Glr1) is an oxidoreductase which converts oxidized glutathione to its reduced form. Yeast Glr1 localizes to both the cytosol and mitochondria and we have used a Glr1M1L mutant that is constitutively localized to the cytosol to test the requirement for mitochondrial Glr1. We show that the loss of mitochondrial Glr1 specifically accounts for oxidant sensitivity of a glr1 mutant. Loss of mitochondrial Glr1 does not influence iron–sulphur cluster maturation and we have used targeted roGFP2 fluorescent probes to show that oxidant sensitivity is linked to an altered redox environment. Our data indicate mitochondrial glutathione is crucial for mitochondrial thiol-redox regulation, and the mitochondrial thioredoxin system provides a back-up system, but cannot bear the redox load of the mitochondria on its own. PMID:26898146

  20. Stereochemistry of Furfural Reduction by a Saccharomyces cerevisiae Aldehyde Reductase That Contributes to In Situ Furfural Detoxification▿

    PubMed Central

    Bowman, Michael J.; Jordan, Douglas B.; Vermillion, Karl E.; Braker, Jay D.; Moon, Jaewoong; Liu, Z. Lewis

    2010-01-01

    Ari1p from Saccharomyces cerevisiae, recently identified as an intermediate-subclass short-chain dehydrogenase/reductase, contributes in situ to the detoxification of furfural. Furfural inhibits efficient ethanol production by yeast, particularly when the carbon source is acid-treated lignocellulose, which contains furfural at a relatively high concentration. NADPH is Ari1p's best known hydride donor. Here we report the stereochemistry of the hydride transfer step, determined by using (4R)-[4-2H]NADPD and (4S)-[4-2H]NADPD and unlabeled furfural in Ari1p-catalyzed reactions and following the deuterium atom into products 2-furanmethanol or NADP+. Analysis of the products demonstrates unambiguously that Ari1p directs hydride transfer from the si face of NADPH to the re face of furfural. The singular orientation of substrates enables construction of a model of the Michaelis complex in the Ari1p active site. The model reveals hydrophobic residues near the furfural binding site that, upon mutation, may increase specificity for furfural and enhance enzyme performance. Using (4S)-[4-2H]NADPD and NADPH as substrates, primary deuterium kinetic isotope effects of 2.2 and 2.5 were determined for the steady-state parameters kcatNADPH and kcat/KmNADPH, respectively, indicating that hydride transfer is partially rate limiting to catalysis. PMID:20525870

  1. Structural and functional roles of a conserved proline residue in the alpha2 helix of Escherichia coli thioredoxin.

    PubMed

    de Lamotte-Guéry, F; Pruvost, C; Minard, P; Delsuc, M A; Miginiac-Maslow, M; Schmitter, J M; Stein, M; Decottignies, P

    1997-12-01

    Proline 40 in Escherichia coli thioredoxin is located close to the redox active site (Cys32-Cys35) within the alpha2 helix. The conservation of this residue among most of the thioredoxins suggests that it could play an important role in the structure and/or function of this protein. We have substituted Pro40 for Ala by using site-directed mutagenesis and expressed the mutant P40A in E.coli. The effects of the mutation on the biophysical and biological properties of thioredoxin have been analyzed and compared with molecular dynamics simulations. Modeling predicted that the replacement of Pro40 by Ala induced a displacement of the active site which exposes Trp31 to the solvent and opens a cleft located between helices alpha2 and alpha3. The solvation free energy (SFE) calculation also indicated that P40A became more hydrophobic as W31 became more accessible. These predictions were totally in agreement with the experimental results. The mutant P40A exhibited chromatographic behavior and fluorescence properties very different from those of the wild-type (WT) protein, in relationship with the displacement of W31. The determination of the free energy of unfolding of P40A showed that the mutant was globally destabilized by 2.9 kcal/mol. However, the effect of the mutation on the transition curve was highly unusual as the midpoint of the unfolding transition increased, indicating that some local structures were actually stabilized by the mutation. Despite these structural modifications, neither the ability of the protein to reduce a chloroplastic enzyme nor its reactivity with the bacterial reductase decreased. The only functional difference was the higher stability of P40A in light activation of NADP-malate dehydrogenase under air, which suggests that the mutant was less rapidly re-oxidized than WT. Therefore, it can be concluded that Pro40 is not essential for maintaining the redox function of thioredoxin but rather is required for the stability of the protein.

  2. Three-dimensional structure of NADPH–cytochrome P450 reductase: Prototype for FMN- and FAD-containing enzymes

    PubMed Central

    Wang, Ming; Roberts, David L.; Paschke, Rosemary; Shea, Thomas M.; Masters, Bettie Sue Siler; Kim, Jung-Ja P.

    1997-01-01

    Microsomal NADPH–cytochrome P450 reductase (CPR) is one of only two mammalian enzymes known to contain both FAD and FMN, the other being nitric-oxide synthase. CPR is a membrane-bound protein and catalyzes electron transfer from NADPH to all known microsomal cytochromes P450. The structure of rat liver CPR, expressed in Escherichia coli and solubilized by limited trypsinolysis, has been determined by x-ray crystallography at 2.6 Å resolution. The molecule is composed of four structural domains: (from the N- to C- termini) the FMN-binding domain, the connecting domain, and the FAD- and NADPH-binding domains. The FMN-binding domain is similar to the structure of flavodoxin, whereas the two C-terminal dinucleotide-binding domains are similar to those of ferredoxin–NADP+ reductase (FNR). The connecting domain, situated between the FMN-binding and FNR-like domains, is responsible for the relative orientation of the other domains, ensuring the proper alignment of the two flavins necessary for efficient electron transfer. The two flavin isoalloxazine rings are juxtaposed, with the closest distance between them being about 4 Å. The bowl-shaped surface near the FMN-binding site is likely the docking site of cytochrome c and the physiological redox partners, including cytochromes P450 and b5 and heme oxygenase. PMID:9237990

  3. Fructose increases corticosterone production in association with NADPH metabolism alterations in rat epididymal white adipose tissue.

    PubMed

    Prince, Paula D; Santander, Yanina A; Gerez, Estefania M; Höcht, Christian; Polizio, Ariel H; Mayer, Marcos A; Taira, Carlos A; Fraga, Cesar G; Galleano, Monica; Carranza, Andrea

    2017-08-01

    Metabolic syndrome is an array of closely metabolic disorders that includes glucose intolerance/insulin resistance, central obesity, dyslipidemia, and hypertension. Fructose, a highly lipogenic sugar, has profound metabolic effects in adipose tissue, and has been associated with the etiopathology of many components of the metabolic syndrome. In adipocytes, the enzyme 11 β-HSD1 amplifies local glucocorticoid production, being a key player in the pathogenesis of central obesity and metabolic syndrome. 11 β-HSD1 reductase activity is dependent on NADPH, a cofactor generated by H6PD inside the endoplasmic reticulum. Our focus was to explore the effect of fructose overload on epididymal white adipose tissue (EWAT) machinery involved in glucocorticoid production and NADPH and oxidants metabolism. Male Sprague-Dawley rats fed with a fructose solution (10% (w/v) in tap water) during 9 weeks developed some characteristic features of metabolic syndrome, such as hypertriglyceridemia, and hypertension. In addition, high levels of plasma and EWAT corticosterone were detected. Activities and expressions of H6PD and 11 β-HSD1, NAPDH content, superoxide anion production, expression of NADPH oxidase 2 subunits, and indicators of oxidative metabolism were measured. Fructose overloaded rats showed an increased potential in oxidant production respect to control rats. In parallel, in EWAT from fructose overloaded rats we found higher expression/activity of H6PD and 11 β-HSD1, and NADPH/NADP + ratio. Our in vivo results support that fructose overload installs in EWAT conditions favoring glucocorticoid production through higher H6PD expression/activity supplying NADPH for enhanced 11 β-HSD1 expression/activity, becoming this tissue a potential extra-adrenal source of corticosterone under these experimental conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from S-sulfocysteine increases L-cysteine production in Escherichia coli

    PubMed Central

    2012-01-01

    Background Escherichia coli has two L-cysteine biosynthetic pathways; one is synthesized from O-acetyl L-serine (OAS) and sulfate by L-cysteine synthase (CysK), and another is produced via S-sulfocysteine (SSC) from OAS and thiosulfate by SSC synthase (CysM). SSC is converted into L-cysteine and sulfite by an uncharacterized reaction. As thioredoxins (Trx1 and Trx2) and glutaredoxins (Grx1, Grx2, Grx3, Grx4, and NrdH) are known as reductases of peptidyl disulfides, overexpression of such reductases might be a good way for improving L-cysteine production to accelerate the reduction of SSC in E. coli. Results Because the redox enzymes can reduce the disulfide that forms on proteins, we first tested whether these enzymes catalyze the reduction of SSC to L-cysteine. All His-tagged recombinant enzymes, except for Grx4, efficiently convert SSC into L-cysteine in vitro. Overexpression of Grx1 and NrdH enhanced a 15-40% increase in the E. coliL-cysteine production. On the other hand, disruption of the cysM gene cancelled the effect caused by the overexpression of Grx1 and NrdH, suggesting that its improvement was due to the efficient reduction of SSC under the fermentative conditions. Moreover, L-cysteine production in knockout mutants of the sulfite reductase genes (ΔcysI and ΔcysJ) and the L-cysteine synthase gene (ΔcysK) each decreased to about 50% of that in the wild-type strain. Interestingly, there was no significant difference in L-cysteine production between wild-type strain and gene deletion mutant of the upstream pathway of sulfite (ΔcysC or ΔcysH). These results indicate that sulfite generated from the SSC reduction is available as the sulfur source to produce additional L-cysteine molecule. It was finally found that in the E. coliL-cysteine producer that co-overexpress glutaredoxin (NrdH), sulfite reductase (CysI), and L-cysteine synthase (CysK), there was the highest amount of L-cysteine produced per cell. Conclusions In this work, we showed that Grx1 and

  5. Synergy between sulforaphane and selenium in the up-regulation of thioredoxin reductase and protection against hydrogen peroxide-induced cell death in human hepatocytes.

    PubMed

    Li, Dan; Wang, Wei; Shan, Yujuan; Barrera, Lawrence N; Howie, Alexander F; Beckett, Geoffrey J; Wu, Kun; Bao, Yongping

    2012-07-15

    Dietary isothiocyanates and selenium are chemopreventive agents and potent inducers of antioxidant enzymes. It has been previously shown that sulforaphane and selenium have a synergistic effect on the upregulation of thioredoxin reductase-1 (TrxR-1) in human hepatoma HepG2 cells. In this paper, further evidence is presented to show that sulforaphane and selenium synergistically induce TrxR-1 expression in immortalised human hepatocytes. Sulforaphane was found to be more toxic toward hepatocytes than HepG2 cells with IC50=25.1 and 56.4 μM, respectively. Sulforaphane can protect against hydrogen peroxide-induced cell death and this protection was enhanced by co-treatment with selenium. Using siRNA to knock down TrxR-1 or Nrf2, sulforaphane (5 μM)-protected cell viability was reduced from 73% to 46% and 34%, respectively, suggesting that TrxR-1 is an important enzyme in protection against hydrogen peroxide-induced cell death. Sulforaphane-induced TrxR-1 expression was positively associated with significant levels of Nrf2 translocation into the nucleus, but co-treatment with selenium showed no significant increase in Nrf2 translocation. Moreover, MAPK (ERK, JNK and p38) and PI3K/Akt signalling pathways were found to play no significant role in sulforaphane-induced Nrf2 translocation into the nucleus. However, blocking ERK and JNK signalling pathways decreased sulforaphane-induced TrxR-1 mRNA by about 20%; whereas blocking p38 and PI3K/AKT increased TrxR-1 transcription. In summary, a combination of sulforaphane and selenium resulted in a synergistic upregulation of TrxR-1 that contributed to the enhanced protection against free radical-mediated oxidative damage in human hepatocytes. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Metabolism of d-Glycero-d-Manno-Heptitol, Volemitol, in Polyanthus. Discovery of a Novel Ketose Reductase1

    PubMed Central

    Häfliger, Beat; Kindhauser, Elsbeth; Keller, Felix

    1999-01-01

    Volemitol (d-glycero-d-manno-heptitol, α-sedoheptitol) is an unusual seven-carbon sugar alcohol that fulfills several important physiological functions in certain species of the genus Primula. Using the horticultural hybrid polyanthus (Primula × polyantha) as our model plant, we found that volemitol is the major nonstructural carbohydrate in leaves of all stages of development, with concentrations of up to 50 mg/g fresh weight in source leaves (about 25% of the dry weight), followed by sedoheptulose (d-altro-2-heptulose, 36 mg/g fresh weight), and sucrose (4 mg/g fresh weight). Volemitol was shown by the ethylenediaminetetraacetate-exudation technique to be a prominent phloem-mobile carbohydrate. It accounted for about 24% (mol/mol) of the phloem sap carbohydrates, surpassed only by sucrose (63%). Preliminary 14CO2 pulse-chase radiolabeling experiments showed that volemitol was a major photosynthetic product, preceded by the structurally related ketose sedoheptulose. Finally, we present evidence for a novel NADPH-dependent ketose reductase, tentatively called sedoheptulose reductase, in volemitol-containing Primula species, and propose it as responsible for the biosynthesis of volemitol in planta. Using enzyme extracts from polyanthus leaves, we determined that sedoheptulose reductase has a pH optimum between 7.0 and 8.0, a very high substrate specificity, and displays saturable concentration dependence for both sedoheptulose (apparent Km = 21 mm) and NADPH (apparent Km = 0.4 mm). Our results suggest that volemitol is important in certain Primula species as a photosynthetic product, phloem translocate, and storage carbohydrate. PMID:9880360

  7. Analysis of the complex formation, interaction and electron transfer pathway between the "open" conformation of NADPH-cytochrome P450 reductase and aromatase.

    PubMed

    Dai, Yuejie; Zhen, Jing; Zhang, Xiuli; Zhong, Yonghui; Liu, Shaodan; Sun, Ziyue; Guo, Yue; Wu, Qingli

    2015-09-01

    The complex structure of human aromatase (CYP19) and the open form of ΔTGEE mutant NADPH-cytochrome P450 reductase (mCPR) was constructed using template-based protein alignment method. Dynamic simulation of formed complex was performed on NAMD 2.9, in which CHARMm all 27_prot_lipid_na force field and an explicit TIP3P water solvent model were applied. The result showed mCPR in its open conformation could steadily combine with aromatase from the proximal face. Data analysis indicates hydrogen bonds and four salt bridges on the binding surface enhance the interaction between the two protein molecules. Amino acid, Lys108 plays a key role in aromatase activity through the formation of a salt bridge with Asp147 and two hydrogen bonds with Asp147 and Gln150 in mCPR. The optimal pathway for the first electron transfer from CPR to aromatase was revealed and calculated using HARLEM software. The rates for solvent mediated and non-solvent mediated electron transfer from FMNH2 to heme were determined as 1.04×10(6)s(-)(1) and 4.86×10(5)s(-)(1) respectively, which indicates the solvent water can facilitate the electron transfer from FMNH2 to heme. This study presents a novel strategy for the study of the protein-protein interactions based on the template-based protein alignment, which may help new aromtase development targeting the electron transfer between mCPR and aromatase. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Mitochondrial and nuclear localization of a novel pea thioredoxin: identification of its mitochondrial target proteins.

    PubMed

    Martí, María C; Olmos, Enrique; Calvete, Juan J; Díaz, Isabel; Barranco-Medina, Sergio; Whelan, James; Lázaro, Juan J; Sevilla, Francisca; Jiménez, Ana

    2009-06-01

    Plants contain several genes encoding thioredoxins (Trxs), small proteins involved in the regulation of the activity of many enzymes through dithiol-disulfide exchange. In addition to chloroplastic and cytoplasmic Trx systems, plant mitochondria contain a reduced nicotinamide adenine dinucleotide phosphate-dependent Trx reductase and a specific Trx o, and to date, there have been no reports of a gene encoding a plant nuclear Trx. We report here the presence in pea (Pisum sativum) mitochondria and nuclei of a Trx isoform (PsTrxo1) that seems to belong to the Trx o group, although it differs from this Trx type by its absence of introns in the genomic sequence. Western-blot analysis with isolated mitochondria and nuclei, immunogold labeling, and green fluorescent protein fusion constructs all indicated that PsTrxo1 is present in both cell compartments. Moreover, the identification by tandem mass spectrometry of the native mitochondrial Trx after gel filtration using the fast-protein liquid chromatography system of highly purified mitochondria and the in vitro uptake assay into isolated mitochondria also corroborated a mitochondrial location for this protein. The recombinant PsTrxo1 protein has been shown to be reduced more effectively by the Saccharomyces cerevisiae mitochondrial Trx reductase Trr2 than by the wheat (Triticum aestivum) cytoplasmic reduced nicotinamide adenine dinucleotide phosphate-dependent Trx reductase. PsTrxo1 was able to activate alternative oxidase, and it was shown to interact with a number of mitochondrial proteins, including peroxiredoxin and enzymes mainly involved in the photorespiratory process.

  9. Protective effect of tetraethyl pyrazine against focal cerebral ischemia/reperfusion injury in rats: therapeutic time window and its mechanism.

    PubMed

    Jia, Jie; Zhang, Xi; Hu, Yong-Shan; Wu, Yi; Wang, Qing-Zhi; Li, Na-Na; Wu, Cai-Qin; Yu, Hui-Xian; Guo, Qing-Chuan

    2009-03-01

    Tetramethyl pyrazine has been considered an effective agent in treating neurons ischemia/reperfusion injury, but the mechanism of its therapeutic effect remains unclear. This study was to explore the therapeutic time window and mechanism of tetramethyl pyrazine on temporary focal cerebral ischemia/reperfusion injury. Middle cerebral artery occlusion was conducted in male Sprague-Dawley rats and 20 mg/kg of tetramethyl pyrazine was intraperitoneally injected at different time points. At 72 h after reperfusion, all animals' neurologic deficit scores were evaluated. Cerebrums were removed and cerebral infarction volume was measured. The expression of thioredoxin and thioredoxin reductase mRNA was determined at 6 and 24 h after reperfusion. Cerebral infarction volume and neurological deficit scores were significantly decreased in the group with tetramethyl pyrazine treatment. The expression of thioredoxin-1/thioredoxin-2 and thioredoxin reductase-1/thioredoxin reductase-2 was significantly decreased in rats with ischemia/reperfusion injury, while it was increased by tetramethyl pyrazine administration. Treatment with tetramethyl pyrazine, within 4 h after reperfusion, protects the brain from ischemic reperfusion injury in rats. The neuroprotective mechanism of tetramethyl pyrazine treatment is, in part, mediated through the upregulation of thioredoxin transcription.

  10. NADPH oxidase inhibitors: a patent review.

    PubMed

    Kim, Jung-Ae; Neupane, Ganesh Prasad; Lee, Eung Seok; Jeong, Byeong-Seon; Park, Byung Chul; Thapa, Pritam

    2011-08-01

    NADPH oxidases, a family of multi-subunit enzyme complexes, catalyze the production of reactive oxygen species (ROS), which may contribute to the pathogenesis of a variety of diseases. In addition to the first NADPH oxidase found in phagocytes, four non-phagocytic NADPH oxidase isoforms have been identified, which all differ in their catalytic subunit (Nox1-5) and tissue distribution. This paper provides a comprehensive review of the patent literature on NADPH oxidase inhibitors, small molecule Nox inhibitors, peptides and siRNAs. Since each member of the NADPH oxidase family has great potential as a therapeutic target, several different compounds have been registered as NADPH oxidase inhibitors in the patent literature. As yet, none have gone through clinical trials, and some have not completed preclinical trials, including safety and specificity evaluation. Recently, small molecule pyrazolopyridine and triazolopyrimidine derivatives have been submitted as potent NADPH oxidase inhibitors and reported as first-in-class inhibitors for idiopathic pulmonary fibrosis and acute stroke, respectively. Further clinical efficacy and safety data are warranted to prove their actual clinical utility.

  11. Development of liquid chromatography/mass spectrometry based screening assay for PfTrxR inhibitors using relative quantitation of intact thioredoxin.

    PubMed

    Munigunti, Ranjith; Calderón, Angela I

    2012-09-15

    Plasmodium falciparum (Pf) thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin disulfide (Trx-S(2)) to thioredoxin dithiol (Trx-(SH)(2)) that is essential for antioxidant defense mechanism and DNA synthesis in the parasite and is a validated drug target for new antimalarial agents. In this study, we have developed a liquid chromatography/mass spectrometry (LC/MS)-based functional assay to identify inhibitors of PfTrxR by quantifying the product formed (Trx-(SH)(2)) in the enzymatic reaction. Relative quantitation of the reaction product (intact Trx-(SH)(2)) was carried out using an Agilent 6520 QTOF mass spectrometer equipped with a positive mode electrospray ionization (ESI) source. The calibration curve prepared for Trx-(SH)(2) at concentrations ranging from 1.8 to 116.5 µg/mL was linear (R(2) >0.998). The limit of detection (LOD) and limit of quantification (LOQ) of Trx-(SH)(2) were at 0.45 and 1.8 µg/mL respectively. To validate the developed functional assay we have screened reference compounds 1, 2 and 3 for their PfTrxR inhibitory activity and ten natural compounds (at 10 μM) which were earlier identified as ligands of PfTrxR by a UF-LC/MS based binding assay. The developed LC/MS-based functional assay for identification of inhibitors of PfTrxR is a sensitive and reliable method that is also amendable for high-throughput format. This is the first representation of a relative quantitation of intact Trx-(SH)(2) using LC/MS. Copyright © 2012 John Wiley & Sons, Ltd.

  12. Role of Aldo-Keto Reductase Family 1 (AKR1) Enzymes in Human Steroid Metabolism

    PubMed Central

    Rižner, Tea Lanišnik; Penning, Trevor M.

    2013-01-01

    Human aldo-keto reductases AKR1C1-AKR1C4 and AKR1D1 play essential roles in the metabolism of all steroid hormones, the biosynthesis of neurosteroids and bile acids, the metabolism of conjugated steroids, and synthetic therapeutic steroids. These enzymes catalyze NADPH dependent reductions at the C3, C5, C17 and C20 positions on the steroid nucleus and side-chain. AKR1C1-AKR1C4 act as 3-keto, 17-keto and 20-ketosteroid reductases to varying extents, while AKR1D1 acts as the sole Δ4-3-ketosteroid-5β-reductase (steroid 5β-reductase) in humans. AKR1 enzymes control the concentrations of active ligands for nuclear receptors and control their ligand occupancy and trans-activation, they also regulate the amount of neurosteroids that can modulate the activity of GABAA and NMDA receptors. As such they are involved in the pre-receptor regulation of nuclear and membrane bound receptors. Altered expression of individual AKR1C genes is related to development of prostate, breast, and endometrial cancer. Mutations in AKR1C1 and AKR1C4 are responsible for sexual development dysgenesis and mutations in AKR1D1 are causative in bile-acid deficiency. PMID:24189185

  13. Contrasting Influence of NADPH and a NADPH-Regenerating System on the Metabolism of Carbonyl-Containing Compounds in Hepatic Microsomes

    EPA Science Inventory

    Carbonyl containing xenobiotics may be susceptible to NADPH-dependent cytochrome P450 (P450) and carbonyl-reduction reactions. In vitro hepatic microsome assays are routinely supplied NADPH either by direct addition of NADPH or via an NADPH-regenerating system (NRS). In contrast ...

  14. Defining the Structural Basis for Allosteric Product Release from E. coli Dihydrofolate Reductase Using NMR Relaxation Dispersion.

    PubMed

    Oyen, David; Fenwick, R Bryn; Aoto, Phillip C; Stanfield, Robyn L; Wilson, Ian A; Dyson, H Jane; Wright, Peter E

    2017-08-16

    The rate-determining step in the catalytic cycle of E. coli dihydrofolate reductase is tetrahydrofolate (THF) product release, which can occur via an allosteric or an intrinsic pathway. The allosteric pathway, which becomes accessible when the reduced cofactor NADPH is bound, involves transient sampling of a higher energy conformational state, greatly increasing the product dissociation rate as compared to the intrinsic pathway that obtains when NADPH is absent. Although the kinetics of this process are known, the enzyme structure and the THF product conformation in the transiently formed excited state remain elusive. Here, we use side-chain proton NMR relaxation dispersion measurements, X-ray crystallography, and structure-based chemical shift predictions to explore the structural basis of allosteric product release. In the excited state of the E:THF:NADPH product release complex, the reduced nicotinamide ring of the cofactor transiently enters the active site where it displaces the pterin ring of the THF product. The p-aminobenzoyl-l-glutamate tail of THF remains weakly bound in a widened binding cleft. Thus, through transient entry of the nicotinamide ring into the active site, the NADPH cofactor remodels the enzyme structure and the conformation of the THF to form a weakly populated excited state that is poised for rapid product release.

  15. Trichomonas vaginalis Flavin Reductase 1 and its Role in Metronidazole Resistance

    PubMed Central

    Leitsch, David; Janssen, Brian D.; Kolarich, Daniel; Johnson, Patricia J.; Duchêne, Michael

    2015-01-01

    Summary The enzyme flavin reductase 1 (FR1) from Trichomonas vaginalis, formerly known as NADPH oxidase, was isolated and identified. Flavin reductase is part of the antioxidative defense in T. vaginalis and indirectly reduces molecular oxygen to hydrogen peroxide via free flavins. Importantly, a reduced or absent flavin reductase activity has been reported in metronidazole-resistant T. vaginalis, resulting in elevated intracellular oxygen levels and futile cycling of metronidazole. Interestingly, FR1 has no close homologue in any other sequenced genome, but seven full-length and three truncated isoforms exist in the T. vaginalis genome. However, out of these, only FR1 has an affinity for flavins, i.e. FMN, FAD, and riboflavin, which is high enough to be of physiological relevance. Although there are no relevant changes in the gene sequence or any alterations of the predicted FR1-mRNA structure in any of the strains studied, FR1 is not expressed in highly metronidazole-resistant strains. Transfection of a metronidazole-resistant clinical isolate (B7268), which does not express any detectable amounts of FR, with a plasmid bearing a functional FR1 gene nearly completely restored metronidazole sensitivity. Our results indicate that FR1 has a significant role in the emergence of metronidazole resistance in T. vaginalis. PMID:24256032

  16. Membrane proteins from the cyanobacterium Synechocystis sp. PCC 6803 interacting with thioredoxin.

    PubMed

    Mata-Cabana, Alejandro; Florencio, Francisco J; Lindahl, Marika

    2007-11-01

    Cysteine dithiol/disulphide exchange forms the molecular basis for regulation of a wide variety of enzymatic activities and for transduction of cellular signals. Thus, the search for proteins with reactive, accessible cysteines is expected to contribute to the unravelling of new molecular mechanisms for enzyme regulation and signal transduction. Several methods have been designed for this purpose taking advantage of the interactions between thioredoxins and their protein substrates. Thioredoxins comprise a family of redox-active enzymes, which catalyse reduction of protein disulphides and sulphenic acids. Due to the inherent practical difficulties associated with studies of membrane proteins these have been largely overlooked in the many proteomic studies of thioredoxin-interacting proteins. In the present work, we have developed a procedure to isolate membrane proteins interacting with thioredoxin by binding in situ to a monocysteinic His-tagged thioredoxin added directly to the intact membranes. Following fractionation and solubilisation of the membranes, thioredoxin target proteins were isolated by Ni-affinity chromatography and 2-DE SDS-PAGE under nonreducing/reducing conditions. Applying this method to total membranes, including thylakoid and plasma membranes, from the cyanobacterium Synechocystis sp. PCC 6803 we have identified 50 thioredoxin-interacting proteins. Among the 38 newly identified thioredoxin targets are the ATP-binding subunits of several transporters and members of the AAA-family of ATPases.

  17. The effects of chromium(VI) on the thioredoxin system: Implications for redox regulation

    PubMed Central

    Myers, Charles R.

    2014-01-01

    Hexavalent chromium [Cr(VI)] compounds are highly redox active and have long been recognized as potent cytotoxins and carcinogens. The intracellular reduction of Cr(VI) generates reactive Cr intermediates, which are themselves strong oxidants, as well as superoxide, hydrogen peroxide, and hydroxyl radical. These probably contribute to the oxidative damage and effects on redox-sensitive transcription factors that have been reported. However, the identification of events that initiate these signaling changes has been elusive. More recent studies show that Cr(VI) causes irreversible inhibition of thioredoxin reductase (TrxR) and oxidation of thioredoxin (Trx) and peroxiredoxin (Prx). Mitochondrial Trx2/Prx3 are more sensitive to Cr(VI) treatment than cytosolic Trx1/Prx1, although both compartments show thiol oxidation with higher doses or longer treatments. Thiol redox proteomics demonstrate that Trx2, Prx3, and Trx1 are among the most sensitive proteins in cells to Cr(VI) treatment. Their oxidation could therefore represent initiating events that have widespread implications for protein thiol redox control and for multiple aspects of redox signaling. This review summarizes the effects of Cr(VI) on the TrxR/Trx system and how these events could influence a number of downstream redox signaling systems that are influenced by Cr(VI) exposure. Some of the signaling events discussed include the activation of apoptosis signal regulating kinase and MAP kinases (p38 and JNK) and the modulation of a number of redox-sensitive transcription factors including AP-1, NF-κB, p53, and Nrf2. PMID:22542445

  18. Glutathione reductase gsr-1 is an essential gene required for Caenorhabditis elegans early embryonic development.

    PubMed

    Mora-Lorca, José Antonio; Sáenz-Narciso, Beatriz; Gaffney, Christopher J; Naranjo-Galindo, Francisco José; Pedrajas, José Rafael; Guerrero-Gómez, David; Dobrzynska, Agnieszka; Askjaer, Peter; Szewczyk, Nathaniel J; Cabello, Juan; Miranda-Vizuete, Antonio

    2016-07-01

    Glutathione is the most abundant thiol in the vast majority of organisms and is maintained in its reduced form by the flavoenzyme glutathione reductase. In this work, we describe the genetic and functional analysis of the Caenorhabditis elegans gsr-1 gene that encodes the only glutathione reductase protein in this model organism. By using green fluorescent protein reporters we demonstrate that gsr-1 produces two GSR-1 isoforms, one located in the cytoplasm and one in the mitochondria. gsr-1 loss of function mutants display a fully penetrant embryonic lethal phenotype characterized by a progressive and robust cell division delay accompanied by an aberrant distribution of interphasic chromatin in the periphery of the cell nucleus. Maternally expressed GSR-1 is sufficient to support embryonic development but these animals are short-lived, sensitized to chemical stress, have increased mitochondrial fragmentation and lower mitochondrial DNA content. Furthermore, the embryonic lethality of gsr-1 worms is prevented by restoring GSR-1 activity in the cytoplasm but not in mitochondria. Given the fact that the thioredoxin redox systems are dispensable in C. elegans, our data support a prominent role of the glutathione reductase/glutathione pathway in maintaining redox homeostasis in the nematode. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Functional expression of a putative geraniol 8-hydroxylase by reconstitution of bacterially expressed plant CYP76F45 and NADPH-cytochrome P450 reductase CPR I from Croton stellatopilosus Ohba.

    PubMed

    Sintupachee, Siriluk; Promden, Worrawat; Ngamrojanavanich, Nattaya; Sitthithaworn, Worapan; De-Eknamkul, Wanchai

    2015-10-01

    While attempting to isolate the enzyme geranylgeraniol 18-hydroxylase, which is involved in plaunotol biosynthesis in Croton stellatopilosus (Cs), the cDNAs for a cytochrome P450 monooxygenase(designated as CYP76F45) and an NADPH-cytochrome P450 reductase (designated as CPR I based on its classification) were isolated from the leaf. The CYP76F45 and CsCPR I genes have open reading frames (ORFs) encoding 507- and 711-amino acid proteins with predicted relative molecular weights of 56.7 and 79.0 kDa,respectively. Amino acid sequence comparison showed that both CYP76F45 (63–73%) and CsCPR I (79–83%) share relatively high sequence identities with homologous proteins in other plant species.Phylogenetic tree analysis confirmed that CYP76F45 belongs to the CYP76 family and that CsCPR I belongs to Class I of dicotyledonous CPRs, with both being closely related to Ricinus communis genes. Functional characterization of both enzymes, each expressed separately in Escherichia coli as recombinant proteins,showed that only simultaneous incubation of the membrane bound proteins with the substrate geraniol (GOH) and the coenzyme NADPH could form 8-hydroxygeraniol. The enzyme mixture could also utilize acyclic sesquiterpene farnesol (FOH) with a comparable substrate preference ratio (GOH:FOH) of 54:46. The levelsof the CYP76F45 and CsCPR I transcripts in the shoots, leaves and twigs of C. stellatopilosus were correlated with the levels of a major monoterpenoid indole alkaloid, identified tentatively as 19-Evallesamine,that accumulated in these plant parts. These results suggested that CYP76F45 and CPR I function as the enzyme geraniol-8-hydroxylase (G8H), which is likely to be involved in the biosynthesis of the indole alkaloid in C. stellatopilosus [corrected]. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Comparative Analyses of Cu-Zn Superoxide Dismutase (SOD1) and Thioredoxin Reductase (TrxR) at the mRNA Level between Apis mellifera L. and Apis cerana F. (Hymenoptera: Apidae) Under Stress Conditions.

    PubMed

    Koo, Hyun-Na; Lee, Soon-Gyu; Yun, Seung-Hwan; Kim, Hyun Kyung; Choi, Yong Soo; Kim, Gil-Hah

    2016-01-01

    This study compared stress-induced expression of Cu-Zn superoxide dismutase (SOD1) and thioredoxin reductase (TrxR) genes in the European honeybee Apis mellifera L. and Asian honeybee Apis cerana F. Expression of both SOD1 and TrxR rapidly increased up to 5 h after exposure to cold (4 °C) or heat (37 °C) treatment and then gradually decreased, with a stronger effect induced by cold stress in A. mellifera compared with A. cerana. Injection of stress-inducing substances (methyl viologen, [MV] and H2O2) also increased SOD1 and TrxR expression in both A. mellifera and A. cerana, and this effect was more pronounced with MV than H2O2. Additionally, we heterologously expressed the A. mellifera and A. cerana SOD1 and TrxR proteins in an Escherichia coli expression system, and detection by SDS-PAGE, confirmed by Western blotting using anti-His tag antibodies, revealed bands at 16 and 60 kDa, respectively. Our results show that the expression patterns of SOD1 and TrxR differ between A. mellifera and A. cerana under conditions of low or high temperature as well as oxidative stress. © The Author 2016. Published by Oxford University Press on behalf of the Entomological Society of America.

  1. Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia stipitis.

    PubMed

    Watanabe, Seiya; Abu Saleh, Ahmed; Pack, Seung Pil; Annaluru, Narayana; Kodaki, Tsutomu; Makino, Keisuke

    2007-09-01

    A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis (PsXR and PsXDH, respectively) has the ability to convert xylose to ethanol together with the unfavourable excretion of xylitol, which may be due to intercellular redox imbalance caused by the different coenzyme specificity between NADPH-preferring XR and NAD(+)-dependent XDH. In this study, we focused on the effect(s) of mutated NADH-preferring PsXR in fermentation. The R276H and K270R/N272D mutants were improved 52- and 146-fold, respectively, in the ratio of NADH/NADPH in catalytic efficiency [(k(cat)/K(m) with NADH)/(k(cat)/K(m) with NADPH)] compared with the wild-type (WT), which was due to decrease of k(cat) with NADPH in the R276H mutant and increase of K(m) with NADPH in the K270R/N272D mutant. Furthermore, R276H mutation led to significant thermostabilization in PsXR. The most positive effect on xylose fermentation to ethanol was found by using the Y-R276H strain, expressing PsXR R276H mutant and PsXDH WT: 20 % increase of ethanol production and 52 % decrease of xylitol excretion, compared with the Y-WT strain expressing PsXR WT and PsXDH WT. Measurement of intracellular coenzyme concentrations suggested that maintenance of the of NADPH/NADP(+) and NADH/NAD(+) ratios is important for efficient ethanol fermentation from xylose by recombinant S. cerevisiae.

  2. Biotransformation of hexahydro-1,3,5-trinitro-1,3,5-tiazine catalyzed by a NAD(P)H: nitrate oxidoreductase from Aspergillus niger.

    PubMed

    Bhushan, Bharat; Halasz, Annamaria; Spain, Jim; Thiboutot, Sonia; Ampleman, Guy; Hawari, Jalal

    2002-07-15

    Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) can be efficiently mineralized with anaerobic domestic sludge, but the initial enzymatic processes involved in its transformation are unknown. To test the hypothesis that the initial reaction involves reduction of nitro group(s), we designed experiments to test the ability of a nitrate reductase (EC 1.6.6.2) to catalyze the initial reaction leading to ring cleavage and subsequent decomposition. A nitrate reductase from Aspergillus niger catalyzed the biotransformation of RDX most effectively at pH 7.0 and 30 degrees C under anaerobic conditions using NADPH as electron donor. LC/MS (ES-) chromatograms showed the formation of hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and methylenedinitramine as key initial products of RDX, but neither the dinitroso neither (DNX) nor trinitroso (TNX) derivatives were observed. None of the above detected products persisted, and their disappearance was accompanied by the accumulation of nitrous oxide (N20), formaldehyde (HCHO), and ammonium ion (NH4+). Stoichiometric studies showed that three NADPH molecules were consumed, and one molecule of methylenedinitramine was produced per RDX molecule. The carbon and nitrogen mass balances were 96.14% and 82.10%, respectively. The stoichiometries and mass balance measurements supported a mechanism involving initial transformation of RDX to MNX via a two-electron reduction mechanism. Subsequent reduction of MNX followed by rapid ring cleavage gave methylenedinitramine which in turn decomposed in water to produce quantitatively N2O and HCHO. The results clearly indicate that an initial reduction of a nitro group by nitrate reductase is sufficient for the decomposition of RDX.

  3. Thioredoxin binding protein (TBP)-2/Txnip and α-arrestin proteins in cancer and diabetes mellitus.

    PubMed

    Masutani, Hiroshi; Yoshihara, Eiji; Masaki, So; Chen, Zhe; Yodoi, Junji

    2012-01-01

    Thioredoxin binding protein -2/ thioredoxin interacting protein is an α-arrestin protein that has attracted much attention as a multifunctional regulator. Thioredoxin binding protein -2 expression is downregulated in tumor cells and the level of thioredoxin binding protein is correlated with clinical stage of cancer. Mice with mutations or knockout of the thioredoxin binding protein -2 gene are much more susceptible to carcinogenesis than wild-type mice, indicating a role for thioredoxin binding protein -2 in cancer suppression. Studies have also revealed roles for thioredoxin binding protein -2 in metabolic control. Enhancement of thioredoxin binding protein -2 expression causes impairment of insulin sensitivity and glucose-induced insulin secretion, and β-cell apoptosis. These changes are important characteristics of type 2 diabetes mellitus. Thioredoxin binding protein -2 regulates transcription of metabolic regulating genes. Thioredoxin binding protein -2-like inducible membrane protein/ arrestin domain containing 3 regulates endocytosis of receptors such as the β(2)-adrenergic receptor. The α-arrestin family possesses PPXY motifs and may function as an adaptor/scaffold for NEDD family ubiquitin ligases. Elucidation of the molecular mechanisms of α-arrestin proteins would provide a new pharmacological basis for developing approaches against cancer and type 2 diabetes mellitus.

  4. Method to Detect the Cellular Source of Over-Activated NADPH Oxidases Using NAD(P)H Fluorescence Lifetime Imaging.

    PubMed

    Bremer, Daniel; Leben, Ruth; Mothes, Ronja; Radbruch, Helena; Niesner, Raluca

    2017-04-03

    Fluorescence-lifetime imaging microscopy (FLIM) is a technique to generate images, in which the contrast is obtained by the excited-state lifetime of fluorescent molecules instead of their intensity and emission spectrum. The ubiquitous coenzymes NADH and NADPH, hereafter NAD(P)H, in cells show a short fluorescence lifetime ≈400 psec in the free-state and a longer fluorescence lifetime when bound to enzymes. The fluorescence lifetime of NAD(P)H in this state depends on the binding-site on the specific enzyme. In the case of NADPH bound to members of the NADPH oxidases family we measured a fluorescence lifetime of 3650 psec as compared to enzymes typically active in cells, in which case fluorescence lifetimes of ∼2000 psec are measured. Here we present a robust protocol based on NAD(P)H fluorescence lifetime imaging in isolated cells to distinguish between normally active enzymes and NADPH oxidases, mainly responsible for oxidative stress. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  5. Metabolism of hydroxypyruvate in a mutant of barley lacking NADH-dependent hydroxypyruvate reductase, an important photorespiratory enzyme activity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Murray, A.J.S.; Blackwell, R.D.; Lea, P.J.

    1989-09-01

    A mutant of barley (Hordeum vulgare L.), LaPr 88/29, deficient in NADH-dependent hydroxypyruvate reductase (HPR) activity has been isolated. The activities of both NADH (5%) and NADPH-dependent (19%) HPR were severely reduced in this mutant compared to the wild type. Although lacking an enzyme in the main carbon pathway of photorespiration, this mutant was capable of CO{sub 2} fixation rates equivalent to 75% of that of the wild type, in normal atmospheres and 50% O{sub 2}. There also appeared to be little disruption to the photorespiratory metabolism as ammonia release, CO{sub 2} efflux and {sup 14}CO{sub 2} release from L-(U-{supmore » 14}C)serine feeding were similar in both mutant and wild-type leaves. When leaves of LaPr 88/29 were fed either ({sup 14}C)serine or {sup 14}CO{sub 2}, the accumulation of radioactivity was in serine and not in hydroxypyruvate, although the mutant was still able to metabolize over 25% of the supplied ({sup 14}C)serine into sucrose. After 3 hours in air the soluble amino acid pool was almost totally dominated by serine and glycine. LaPr 88/29 has also been used to show that NADH-glyoxylate reductase and NADH-HPR are probably not catalyzed by the same enzyme in barley and that over 80% of the NADPH-dependent HPR activity is due to the NADH-dependent enzyme. We also suggest that the alternative NADPH activity can metabolize a proportion, but not all, of the hydroxypyruvate produced during photorespiration and may thus form a useful backup to the NADH-dependent enzyme under conditions of maximal photorespiration.« less

  6. Purification and Thermal Dependence of Glutathione Reductase from Two Forage Legume Species 1

    PubMed Central

    Kidambi, Saranga P.; Mahan, James R.; Matches, Arthur G.

    1990-01-01

    Alfalfa (Medicago sativa L.) and sainfoin (Onobrychis viciifolia Scop.) are forage legumes that differ in their responses to high and low temperature stresses. Thermal limitations on the function of glutathione reductase (EC 1.6.4.2) could adversely affect the ability of the plant to cope with adverse temperatures. Our objectives were to (a) purify glutathione reductase from `Cimarron' alfalfa and `PI 212241' sainfoin and (b) investigate the intraspecies variation in the thermal dependency of glutathione reductase from each of three cultivars of alfalfa and two cultivars and an introduction of sainfoin. Glutathione reductase was purified 1222-and 1948-fold to a specific activity of 281 and 273 units per milligram of protein, from one species each of alfalfa and sainfoin, respectively. The relative molecular mass of the protein was approximately 140 kilodaltons with subunits of 57 and 37 kilodaltons under denaturing conditions. The activation energies were approximately 50 kilojoules per mole for both species. Over a 5 to 45°C temperature gradient, large variation among species and genotypes within species was found for: (a) the minimum apparent Michaelis constant (0.6-2.1 micromoles of NADPH), (b) the temperature at which the minimum apparent Michaelis constant was observed (10-25°C), and (c) the thermal kinetic windows (6-19°C width). Future studies will focus on relating the thermal dependence of the Michaelis constant of the glutathione reductases and plant growth rates and forage quality of these species throughout the growing season. PMID:16667283

  7. Thioredoxin binding protein (TBP)-2/Txnip and α-arrestin proteins in cancer and diabetes mellitus

    PubMed Central

    Masutani, Hiroshi; Yoshihara, Eiji; Masaki, So; Chen, Zhe; Yodoi, Junji

    2012-01-01

    Thioredoxin binding protein −2/ thioredoxin interacting protein is an α-arrestin protein that has attracted much attention as a multifunctional regulator. Thioredoxin binding protein −2 expression is downregulated in tumor cells and the level of thioredoxin binding protein is correlated with clinical stage of cancer. Mice with mutations or knockout of the thioredoxin binding protein −2 gene are much more susceptible to carcinogenesis than wild-type mice, indicating a role for thioredoxin binding protein −2 in cancer suppression. Studies have also revealed roles for thioredoxin binding protein −2 in metabolic control. Enhancement of thioredoxin binding protein −2 expression causes impairment of insulin sensitivity and glucose-induced insulin secretion, and β-cell apoptosis. These changes are important characteristics of type 2 diabetes mellitus. Thioredoxin binding protein −2 regulates transcription of metabolic regulating genes. Thioredoxin binding protein −2-like inducible membrane protein/ arrestin domain containing 3 regulates endocytosis of receptors such as the β2-adrenergic receptor. The α-arrestin family possesses PPXY motifs and may function as an adaptor/scaffold for NEDD family ubiquitin ligases. Elucidation of the molecular mechanisms of α-arrestin proteins would provide a new pharmacological basis for developing approaches against cancer and type 2 diabetes mellitus. PMID:22247597

  8. Structural insights into the neuroprotective-acting carbonyl reductase Sniffer of Drosophila melanogaster.

    PubMed

    Sgraja, Tanja; Ulschmid, Julia; Becker, Katja; Schneuwly, Stephan; Klebe, Gerhard; Reuter, Klaus; Heine, Andreas

    2004-10-01

    In vivo studies with the fruit-fly Drosophila melanogaster have shown that the Sniffer protein prevents age-dependent and oxidative stress-induced neurodegenerative processes. Sniffer is a NADPH-dependent carbonyl reductase belonging to the enzyme family of short-chain dehydrogenases/reductases (SDRs). The crystal structure of the homodimeric Sniffer protein from Drosophila melanogaster in complex with NADP+ has been determined by multiple-wavelength anomalous dispersion and refined to a resolution of 1.75 A. The observed fold represents a typical dinucleotide-binding domain as detected for other SDRs. With respect to the cofactor-binding site and the region referred to as substrate-binding loop, the Sniffer protein shows a striking similarity to the porcine carbonyl reductase (PTCR). This loop, in both Sniffer and PTCR, is substantially shortened compared to other SDRs. In most enzymes of the SDR family this loop adopts a well-defined conformation only after substrate binding and remains disordered in the absence of any bound ligands or even if only the dinucleotide cofactor is bound. In the structure of the Sniffer protein, however, the conformation of this loop is well defined, although no substrate is present. Molecular modeling studies provide an idea of how binding of substrate molecules to Sniffer could possibly occur.

  9. Chloroplast redox homeostasis is essential for lateral root formation in Arabidopsis.

    PubMed

    Ferrández, Julia; González, Maricruz; Cejudo, Francisco Javier

    2012-09-01

    Redox regulation based on dithiol-disulphide interchange is an essential component of the control of chloroplast metabolism. In contrast to heterotrophic organisms, and non-photosynthetic plant tissues, chloroplast redox regulation relies on ferredoxin (Fd) reduced by the photosynthetic electron transport chain, thus being highly dependent on light. The finding of the NADPH-dependent thioredoxin reductase C (NTRC), a chloroplast-localized NTR with a joint thioredoxin domain, showed that NADPH is also used as source of reducing power for chloroplast redox homeostasis. Recently we have found that NTRC is also in plastids of non-photosynthetic tissues. Because these non-green plastids lack photochemical reactions, their redox homeostasis depends exclusively on NADPH produced from sugars and, thus, NTRC may play an essential role maintaining the redox homeostasis in these plastids. The fact that redox regulation occurs in any type of plastids raises the possibility that the functions of chloroplasts and non-green plastids, such as amyloplasts, are integrated to harmonize the growth of the different organs of the plant. To address this question, we generated Arabidopsis plants the redox homeostasis of which is recovered exclusively in chloroplasts, by leaf-specific expression of NTRC in the ntrc mutant, or exclusively in amyloplasts, by root-specific expression of NTRC. The analysis of these plants suggests that chloroplasts exert a pivotal role on plant growth, as expected because chloroplasts constitute the major source of nutrients and energy, derived from photosynthesis, for growth of heterotrophic tissues. However, NTRC deficiency causes impairment of auxin synthesis and lateral root formation. Interestingly, recovery of redox homeostasis of chloroplasts, but not of amyloplasts, was sufficient to restore wild type levels of lateral roots, showing the important signaling function of chloroplasts for the development of heterotrophic organs.

  10. Thioredoxin 1-Mediated Post-Translational Modifications: Reduction, Transnitrosylation, Denitrosylation, and Related Proteomics Methodologies

    PubMed Central

    Wu, Changgong; Parrott, Andrew M.; Fu, Cexiong; Liu, Tong; Marino, Stefano M.; Gladyshev, Vadim N.; Jain, Mohit R.; Baykal, Ahmet T.; Li, Qing; Oka, Shinichi; Sadoshima, Junichi; Beuve, Annie; Simmons, William J.

    2011-01-01

    Abstract Despite the significance of redox post-translational modifications (PTMs) in regulating diverse signal transduction pathways, the enzymatic systems that catalyze reversible and specific oxidative or reductive modifications have yet to be firmly established. Thioredoxin 1 (Trx1) is a conserved antioxidant protein that is well known for its disulfide reductase activity. Interestingly, Trx1 is also able to transnitrosylate or denitrosylate (defined as processes to transfer or remove a nitric oxide entity to/from substrates) specific proteins. An intricate redox regulatory mechanism has recently been uncovered that accounts for the ability of Trx1 to catalyze these different redox PTMs. In this review, we will summarize the available evidence in support of Trx1 as a specific disulfide reductase, and denitrosylation and transnitrosylation agent, as well as the biological significance of the diverse array of Trx1-regulated pathways and processes under different physiological contexts. The dramatic progress in redox proteomics techniques has enabled the identification of an increasing number of proteins, including peroxiredoxin 1, whose disulfide bond formation and nitrosylation status are regulated by Trx1. This review will also summarize the advancements of redox proteomics techniques for the identification of the protein targets of Trx1-mediated PTMs. Collectively, these studies have shed light on the mechanisms that regulate Trx1-mediated reduction, transnitrosylation, and denitrosylation of specific target proteins, solidifying the role of Trx1 as a master regulator of redox signal transduction. Antioxid. Redox Signal. 15, 2565–2604. PMID:21453190

  11. Holo Structure and Steady State Kinetics of the Thiazolinyl Imine Reductases for Siderophore Biosynthesis

    PubMed Central

    Meneely, Kathleen M.; Ronnebaum, Trey A.; Riley, Andrew P.; Prisinzano, Thomas E.; Lamb, Audrey L.

    2016-01-01

    Thiazolinyl imine reductases catalyze the NADPH-dependent reduction of a thiazoline to a thiazolidine, a required step in the formation of the siderophores yersiniabactin (Yersinia spp.) and pyochelin (Pseudomonas aeruginosa). These stand-alone nonribosomal peptide tailoring domains are structural homologues of sugar oxidoreductases. Two closed structures of the thiazolinyl imine reductase from Yersinia enterocolitica (Irp3) are presented here: an NADP+-bound structure to 1.45 Å resolution and a holo structure to 1.28 Å resolution with NADP+ and a substrate analogue bound. Michaelis—Menten kinetics were measured using the same substrate analogue and the homologue from P. aeruginosa, PchG. The data presented here support the hypothesis that tyrosine 128 is the likely general acid residue for catalysis and also highlight the phosphopantetheine tunnel for tethering of the substrate to the nonribosomal peptide synthetase module during assembly line biosynthesis of the siderophore. PMID:27601130

  12. Thioredoxin-mimetic peptide CB3 lowers MAPKinase activity in the Zucker rat brain☆

    PubMed Central

    Cohen-Kutner, Moshe; Khomsky, Lena; Trus, Michael; Ben-Yehuda, Hila; Lenhard, James M.; Liang, Yin; Martin, Tonya; Atlas, Daphne

    2014-01-01

    Diabetes is a high risk factor for dementia. High glucose may be a risk factor for dementia even among persons without diabetes, and in transgenic animals it has been shown to cause a potentiation of indices that are pre-symptomatic of Alzheimer's disease. To further elucidate the underlying mechanisms linking inflammatory events elicited in the brain during oxidative stress and diabetes, we monitored the activation of mitogen-activated kinsase (MAPKs), c-jun NH2-terminal kinase (JNK), p38 MAP kinases (p38MAPK), and extracellular activating kinsae1/2 (ERK1/2) and the anti-inflammatory effects of the thioredoxin mimetic (TxM) peptides, Ac-Cys-Pro-Cys-amide (CB3) and Ac-Cys-Gly-Pro-Cys-amide (CB4) in the brain of male leptin-receptor-deficient Zucker diabetic fatty (ZDF) rats and human neuroblastoma SH-SY5Y cells. Daily i.p. injection of CB3 to ZDF rats inhibited the phosphorylation of JNK and p38MAPK, and prevented the expression of thioredoxin-interacting-protein (TXNIP/TBP-2) in ZDF rat brain. Although plasma glucose/insulin remained high, CB3 also increased the phosphorylation of AMP-ribose activating kinase (AMPK) and inhibited p70S6K kinase in the brain. Both CB3 and CB4 reversed apoptosis induced by inhibiting thioredoxin reductase as monitored by decreasing caspase 3 cleavage and PARP dissociation in SH-SY5Y cells. The decrease in JNK and p38MAPK activity in the absence of a change in plasma glucose implies a decrease in oxidative or neuroinflammatory stress in the ZDF rat brain. CB3 not only attenuated MAPK phosphorylation and activated AMPK in the brain, but it also diminished apoptotic markers, most likely acting via the MAPK–AMPK–mTOR pathway. These results were correlated with CB3 and CB4 inhibiting inflammation progression and protection from oxidative stress induced apoptosis in human neuronal cells. We suggest that by attenuating neuro-inflammatory processes in the brain Trx1 mimetic peptides could become beneficial for preventing neurological

  13. Contrasting influence of NADPH and a NADPH-regenerating system on the metabolism of carbonyl-containing compounds in hepatic microsomes.

    PubMed

    Mazur, Christopher S; Kenneke, John F; Goldsmith, Michael-Rock; Brown, Cather

    2009-09-01

    Carbonyl containing xenobiotics may be susceptible to NADPH-dependent cytochrome P450 (P450) and carbonyl-reduction reactions. In vitro hepatic microsome assays are routinely supplied NADPH either by direct addition of NADPH or via an NADPH-regenerating system (NRS). In contrast to oxidative P450 transformations, which occur on the periphery of a microsome vesicle, intraluminal carbonyl reduction depends on transport of cofactors across the endoplasmic reticulum (ER) membrane into the lumen. Glucose 6-phosphate, a natural cofactor and component of the NRS matrix, is readily transported across the ER membrane and facilitates intraluminal NADPH production, whereas direct addition of NADPH has limited access to the lumen. In this study, we compared the effects of direct addition of NADPH and use of an NRS on the P450-mediated transformation of propiconazole and 11 beta-hydroxysteroid dehydrogenase type 1 (HSD1) carbonyl reduction of cortisone and the xenobiotic triadimefon in hepatic microsomes. Our results demonstrate that the use of NADPH rather than NRS can underestimate the kinetic rates of intraluminal carbonyl reduction, whereas P450-mediated transformations were unaffected. Therefore, in vitro depletion rates measured for a carbonyl-containing xenobiotic susceptible to both intraluminal carbonyl reduction and P450 processes may not be properly assessed with direct addition of NADPH. In addition, we used in silico predictions as follows: 1) to show that 11 beta-HSD1 carbonyl reduction was energetically more favorable than oxidative P450 transformation; and 2) to calculate chemical binding score and the distance between the carbonyl group and the hydride to be transferred by NADPH to identify other 11 beta-HSD1 substrates for which reaction kinetics may be underestimated by direct addition of NADPH.

  14. Role of aldo-keto reductase family 1 (AKR1) enzymes in human steroid metabolism.

    PubMed

    Rižner, Tea Lanišnik; Penning, Trevor M

    2014-01-01

    Human aldo-keto reductases AKR1C1-AKR1C4 and AKR1D1 play essential roles in the metabolism of all steroid hormones, the biosynthesis of neurosteroids and bile acids, the metabolism of conjugated steroids, and synthetic therapeutic steroids. These enzymes catalyze NADPH dependent reductions at the C3, C5, C17 and C20 positions on the steroid nucleus and side-chain. AKR1C1-AKR1C4 act as 3-keto, 17-keto and 20-ketosteroid reductases to varying extents, while AKR1D1 acts as the sole Δ(4)-3-ketosteroid-5β-reductase (steroid 5β-reductase) in humans. AKR1 enzymes control the concentrations of active ligands for nuclear receptors and control their ligand occupancy and trans-activation, they also regulate the amount of neurosteroids that can modulate the activity of GABAA and NMDA receptors. As such they are involved in the pre-receptor regulation of nuclear and membrane bound receptors. Altered expression of individual AKR1C genes is related to development of prostate, breast, and endometrial cancer. Mutations in AKR1C1 and AKR1C4 are responsible for sexual development dysgenesis and mutations in AKR1D1 are causative in bile-acid deficiency. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Molecular Diagnosis of 5α-Reductase Type II Deficiency in Brazilian Siblings with 46,XY Disorder of Sex Development

    PubMed Central

    de Calais, Flávia Leme; Soardi, Fernanda Caroline; Petroli, Reginaldo José; Lusa, Ana Letícia Gori; de Paiva e Silva, Roberto Benedito; Maciel-Guerra, Andréa Trevas; Guerra-Júnior, Gil; de Mello, Maricilda Palandi

    2011-01-01

    The steroid 5α-reductase type II enzyme catalyzes the conversion of testosterone (T) to dihydrotestosterone (DHT), and its deficiency leads to undervirilization in 46,XY individuals, due to an impairment of this conversion in genital tissues. Molecular analysis in the steroid 5α-reductase type II gene (SRD5A2) was performed in two 46,XY female siblings. SRD5A2 gene sequencing revealed that the patients were homozygous for p.Gln126Arg missense mutation, which results from the CGA > CAA nucleotide substitution. The molecular result confirmed clinical diagnosis of 46,XY disorder of sex development (DSD) for the older sister and directed the investigation to other family members. Studies on SRD5A2 protein structure showed severe changes at NADPH binding region indicating that structural modeling analysis can be useful to evaluate the deleterious role of a mutation as causing 5α-reductase type II enzyme deficiency. PMID:22272144

  16. Activation of neuronal nitric oxide synthase in cerebellum of chronic hepatic encephalopathy rats is associated with up-regulation of NADPH-producing pathway.

    PubMed

    Singh, Santosh; Trigun, Surendra K

    2010-09-01

    Cerebellum-associated functions get affected during mild hepatic encephalopathy (MHE) in patients with chronic liver failure (CLF). Involvement of nitrosative and antioxidant factors in the pathogenesis of chronic hepatic encephalopathy is an evolving concept and needs to be defined in a true CLF animal model. This article describes profiles of NADPH-dependent neuronal nitric oxide synthase (nNOS) and those of glutathione peroxidase and glutathione reductase (GR) vis-a-vis regulation of NADPH-producing pathway in the cerebellum of CLF rats induced by administration of thioacetamide (100 mg kg⁻¹ b.w., i.p.) up to 10 days and confirming MHE on Morris water maze tests. Significant increases in the expression of nNOS protein and nitric oxide (NOx) level coincided with a similar increment in NADPH-diaphorase activity in the cerebellum of CLF rats. Glutathione peroxidase and GR utilize NADPH to regenerate reduced glutathione (GSH) in the cells. Both these enzymes and GSH level were found to be static and thus suggested efficient turnover of GSH in the cerebellum of MHE rats. Relative levels of glucose-6-phosphate dehydrogenase (G6PD) vs. phosphofructokinase 2 (PFK2) determine the rate of pentose phosphate pathway (PPP) responsible to synthesize NADPH. The cerebellum of CLF rats showed overactivation of G6PD with a significant decline in the expression of PFK2 and thus suggested activation of PPP in the cerebellum during MHE. It is concluded that concordant activations of PPP and nNOS in cerebellum of MHE rats could be associated with the implication of NOx in the pathogenesis of MHE.

  17. Two distinct redox cascades cooperatively regulate chloroplast functions and sustain plant viability.

    PubMed

    Yoshida, Keisuke; Hisabori, Toru

    2016-07-05

    The thiol-based redox regulation system is believed to adjust chloroplast functions in response to changes in light environments. A redox cascade via the ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) pathway has been traditionally considered to serve as a transmitter of light signals to target enzymes. However, emerging data indicate that chloroplasts have a complex redox network composed of diverse redox-mediator proteins and target enzymes. Despite extensive research addressing this system, two fundamental questions are still unresolved: How are redox pathways orchestrated within chloroplasts, and why are chloroplasts endowed with a complicated redox network? In this report, we show that NADPH-Trx reductase C (NTRC) is a key redox-mediator protein responsible for regulatory functions distinct from those of the classically known FTR/Trx system. Target screening and subsequent biochemical assays indicated that NTRC and the Trx family differentially recognize their target proteins. In addition, we found that NTRC is an electron donor to Trx-z, which is a key regulator of gene expression in chloroplasts. We further demonstrate that cooperative control of chloroplast functions via the FTR/Trx and NTRC pathways is essential for plant viability. Arabidopsis double mutants impaired in FTR and NTRC expression displayed lethal phenotypes under autotrophic growth conditions. This severe growth phenotype was related to a drastic loss of photosynthetic performance. These combined results provide an expanded map of the chloroplast redox network and its biological functions.

  18. Two distinct redox cascades cooperatively regulate chloroplast functions and sustain plant viability

    PubMed Central

    Yoshida, Keisuke; Hisabori, Toru

    2016-01-01

    The thiol-based redox regulation system is believed to adjust chloroplast functions in response to changes in light environments. A redox cascade via the ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) pathway has been traditionally considered to serve as a transmitter of light signals to target enzymes. However, emerging data indicate that chloroplasts have a complex redox network composed of diverse redox-mediator proteins and target enzymes. Despite extensive research addressing this system, two fundamental questions are still unresolved: How are redox pathways orchestrated within chloroplasts, and why are chloroplasts endowed with a complicated redox network? In this report, we show that NADPH-Trx reductase C (NTRC) is a key redox-mediator protein responsible for regulatory functions distinct from those of the classically known FTR/Trx system. Target screening and subsequent biochemical assays indicated that NTRC and the Trx family differentially recognize their target proteins. In addition, we found that NTRC is an electron donor to Trx-z, which is a key regulator of gene expression in chloroplasts. We further demonstrate that cooperative control of chloroplast functions via the FTR/Trx and NTRC pathways is essential for plant viability. Arabidopsis double mutants impaired in FTR and NTRC expression displayed lethal phenotypes under autotrophic growth conditions. This severe growth phenotype was related to a drastic loss of photosynthetic performance. These combined results provide an expanded map of the chloroplast redox network and its biological functions. PMID:27335455

  19. NADPH-generating systems in bacteria and archaea

    PubMed Central

    Spaans, Sebastiaan K.; Weusthuis, Ruud A.; van der Oost, John; Kengen, Servé W. M.

    2015-01-01

    Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an essential electron donor in all organisms. It provides the reducing power that drives numerous anabolic reactions, including those responsible for the biosynthesis of all major cell components and many products in biotechnology. The efficient synthesis of many of these products, however, is limited by the rate of NADPH regeneration. Hence, a thorough understanding of the reactions involved in the generation of NADPH is required to increase its turnover through rational strain improvement. Traditionally, the main engineering targets for increasing NADPH availability have included the dehydrogenase reactions of the oxidative pentose phosphate pathway and the isocitrate dehydrogenase step of the tricarboxylic acid (TCA) cycle. However, the importance of alternative NADPH-generating reactions has recently become evident. In the current review, the major canonical and non-canonical reactions involved in the production and regeneration of NADPH in prokaryotes are described, and their key enzymes are discussed. In addition, an overview of how different enzymes have been applied to increase NADPH availability and thereby enhance productivity is provided. PMID:26284036

  20. Identification and characterization of NADPH-dependent cytochrome P450 reductase gene and cytochrome b₅ gene from Plutella xylostella: possible involvement in resistance to beta-cypermethrin.

    PubMed

    Chen, Xi'en; Zhang, Yalin

    2015-03-10

    NADPH-cytochrome P450 reductase (CPR) and cytochrome b5 (b5) are essential for cytochrome P450 mediated biological reactions. CPR and b5 in several insects have been found to be associated with insecticide resistance. However, CPR and b5 in the diamondback moth (DBM), Plutella xylostella, are not characterized and their roles remain undefined. A full-length cDNA of CPR encoding 678 amino acids and a full-length cDNA of b5 encoding 127 amino acids were cloned from DBM. Their deduced amino acid sequences shared high identities with those of other insects and showed characteristics of classical CPRs and b5s, respectively. The mRNAs of both genes were detectable in all developmental stages with the highest expression levels occurring in the 4th instar larvae. Tissue-specific expression analysis showed that their transcripts were most abundant in gut. Transcripts of CPR and b5 in the beta-cypermethrin resistant DBM strain were 13.2- and 2.84-fold higher than those in the beta-cypermethrin susceptible strain, respectively. The expression levels of CPR and b5 were enhanced by beta-cypermethrin at the concentration of 12 mg L(-1) (~LC10). The results indicate that CPR and b5 may play essential roles in the P450 mediated resistance of DBM to beta-cypermethrin or even other insecticides. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. The Roles of Peroxiredoxin and Thioredoxin in Hydrogen Peroxide Sensing and in Signal Transduction.

    PubMed

    Netto, Luis E S; Antunes, Fernando

    2016-01-01

    A challenge in the redox field is the elucidation of the molecular mechanisms, by which H2O2 mediates signal transduction in cells. This is relevant since redox pathways are disturbed in some pathologies. The transcription factor OxyR is the H2O2 sensor in bacteria, whereas Cys-based peroxidases are involved in the perception of this oxidant in eukaryotic cells. Three possible mechanisms may be involved in H2O2 signaling that are not mutually exclusive. In the simplest pathway, H2O2 signals through direct oxidation of the signaling protein, such as a phosphatase or a transcription factor. Although signaling proteins are frequently observed in the oxidized state in biological systems, in most cases their direct oxidation by H2O2 is too slow (10(1) M(-1)s(-1) range) to outcompete Cys-based peroxidases and glutathione. In some particular cellular compartments (such as vicinity of NADPH oxidases), it is possible that a signaling protein faces extremely high H2O2 concentrations, making the direct oxidation feasible. Alternatively, high H2O2 levels can hyperoxidize peroxiredoxins leading to local building up of H2O2 that then could oxidize a signaling protein (floodgate hypothesis). In a second model, H2O2 oxidizes Cys-based peroxidases that then through thiol-disulfide reshuffling would transmit the oxidized equivalents to the signaling protein. The third model of signaling is centered on the reducing substrate of Cys-based peroxidases that in most cases is thioredoxin. Is this model, peroxiredoxins would signal by modulating the thioredoxin redox status. More kinetic data is required to allow the identification of the complex network of thiol switches.

  2. Protective effects of the thioredoxin and glutaredoxin systems in dopamine-induced cell death.

    PubMed

    Arodin, Lisa; Miranda-Vizuete, Antonio; Swoboda, Peter; Fernandes, Aristi P

    2014-08-01

    Although the etiology of sporadic Parkinson disease (PD) is unknown, it is well established that oxidative stress plays an important role in the pathogenic mechanism. The thioredoxin (Trx) and glutaredoxin (Grx) systems are two central systems upholding the sulfhydryl homeostasis by reducing disulfides and mixed disulfides within the cell and thereby protecting against oxidative stress. By examining the expression of redox proteins in human postmortem PD brains, we found the levels of Trx1 and thioredoxin reductase 1 (TrxR1) to be significantly decreased. The human neuroblastoma cell line SH-SY5Y and the nematode Caenorhabditis elegans were used as model systems to explore the potential protective effects of the redox proteins against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity. 6-OHDA is highly prone to oxidation, resulting in the formation of the quinone of 6-OHDA, a highly reactive species and powerful neurotoxin. Treatment of human cells with 6-OHDA resulted in an increased expression of Trx1, TrxR1, Grx1, and Grx2, and small interfering RNA for these genes significantly increased the cytotoxic effects exerted by the 6-OHDA neurotoxin. Evaluation of the dopaminergic neurons in C. elegans revealed that nematodes lacking trxr-1 were significantly more sensitive to 6-OHDA, with significantly increased neuronal degradation. Importantly, both the Trx and the Grx systems were also found to directly mediate reduction of the 6-OHDA-quinone in vitro and thus render its cytotoxic effects. In conclusion, our results suggest that the two redox systems are important for neuronal survival in dopamine-induced cell death. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Quinone Reductase 2 Is a Catechol Quinone Reductase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fu, Yue; Buryanovskyy, Leonid; Zhang, Zhongtao

    2008-09-05

    The functions of quinone reductase 2 have eluded researchers for decades even though a genetic polymorphism is associated with various neurological disorders. Employing enzymatic studies using adrenochrome as a substrate, we show that quinone reductase 2 is specific for the reduction of adrenochrome, whereas quinone reductase 1 shows no activity. We also solved the crystal structure of quinone reductase 2 in complexes with dopamine and adrenochrome, two compounds that are structurally related to catecholamine quinones. Detailed structural analyses delineate the mechanism of quinone reductase 2 specificity toward catechol quinones in comparison with quinone reductase 1; a side-chain rotational difference betweenmore » quinone reductase 1 and quinone reductase 2 of a single residue, phenylalanine 106, determines the specificity of enzymatic activities. These results infer functional differences between two homologous enzymes and indicate that quinone reductase 2 could play important roles in the regulation of catecholamine oxidation processes that may be involved in the etiology of Parkinson disease.« less

  4. Enhanced poly(3-hydroxybutyrate) production in transgenic tobacco BY-2 cells using engineered acetoacetyl-CoA reductase.

    PubMed

    Yokoo, Toshinori; Matsumoto, Ken'ichiro; Ooba, Takashi; Morimoto, Kenjiro; Taguchi, Seiichi

    2015-01-01

    Highly active mutant of NADPH-dependent acetoacetyl-CoA reductase (PhaB) was expressed in Nicotiana tabacum cv. Bright Yellow-2 cultured cells to produce poly(3-hydroxybutyrate) [P(3HB)]. The mutated PhaB increased P(3HB) content by three-fold over the control, indicating that the mutant was a versatile tool for P(3HB) production. Additionally, the PhaB-catalyzed reaction was suggested to be a rate-limiting step of P(3HB) biosynthesis in tobacco BY-2 cells.

  5. Cotesia vestalis parasitization suppresses expression of a Plutella xylostella thioredoxin

    USDA-ARS?s Scientific Manuscript database

    Thioredoxins (Trxs) are a family of small, highly conserved and ubiquitous proteins involved in protecting organisms against toxic reactive oxygen species (ROS). In this study, a typical thioredoxin gene, PxTrx, was isolated from Plutella xylostella. The full-length cDNA sequence is composed of 959 ...

  6. Comparative characterization of novel ene-reductases from cyanobacteria.

    PubMed

    Fu, Yilei; Castiglione, Kathrin; Weuster-Botz, Dirk

    2013-05-01

    The growing importance of biocatalysis in the syntheses of enantiopure molecules results from the benefits of enzymes regarding selectivity and specificity of the reaction and ecological issues of the process. Ene-reductases (ERs) from the old yellow enzyme family have received much attention in the last years. These flavo-enzymes catalyze the trans-specific reduction of activated C=C bonds, which is an important reaction in asymmetric synthesis, because up to two stereogenic centers can be created in one reaction. However, limitations of ERs described in the literature such as their moderate catalytic activity and their strong preference for NADPH promote the search for novel ERs with improved properties. In this study, we characterized nine novel ERs from cyanobacterial strains belonging to different taxonomic orders and habitats. ERs were identified with activities towards a broad spectrum of alkenes. The reduction of maleimide was catalyzed with activities of up to 35.5 U mg(-1) using NADPH. Ketoisophorone and (R)-carvone, which were converted to the highly valuable compounds (R)-levodione and (2R,5R)-dihydrocarvone, were reduced with reaction rates of up to 2.2 U mg(-1) with NADPH. In contrast to other homologous ERs from the literature, NADH was accepted at moderate to high rates as well: Enzyme activities of up to 16.7 U mg(-1) were obtained for maleimide and up to 1.3 U mg(-1) for ketoisophorone and (R)-carvone. Additionally, excellent stereoselectivities were achieved in the reduction of (R)-carvone (97-99% de). In particular, AnabaenaER3 from Anabaena variabilis ATCC 29413 and AcaryoER1 from Acaryochloris marina MBIC 11017 were identified as useful biocatalysts. Therefore, novel ERs from cyanobacteria with high catalytic efficiency were added to the toolbox for the asymmetric reduction of alkenes. Copyright © 2012 Wiley Periodicals, Inc.

  7. A second target of benzamide riboside: dihydrofolate reductase.

    PubMed

    Roussel, Breton; Johnson-Farley, Nadine; Kerrigan, John E; Scotto, Kathleen W; Banerjee, Debabrata; Felczak, Krzysztof; Pankiewicz, Krzysztof W; Gounder, Murugesan; Lin, HongXia; Abali, Emine Ercikan; Bertino, Joseph R

    2012-11-01

    Dihydrofolate reductase (DHFR) is an essential enzyme involved in de novo purine and thymidine biosynthesis. For several decades, selective inhibition of DHFR has proven to be a potent therapeutic approach in the treatment of various cancers including acute lymphoblastic leukemia, non-Hodgkin's lymphoma, osteogenic sarcoma, carcinoma of the breast, and head and neck cancer. Therapeutic success with DHFR inhibitor methotrexate (MTX) has been compromised in the clinic, which limits the success of MTX treatment by both acquired and intrinsic resistance mechanisms. We report that benzamide riboside (BR), via anabolism to benzamide adenine dinucleotide (BAD) known to potently inhibit inosine monophosphate dehydrogenase (IMPDH), also inhibits cell growth through a mechanism involving downregulation of DHFR protein. Evidence to support this second site of action of BR includes the finding that CCRF-CEM/R human T-cell lymphoblasic leukemia cells, resistant to MTX as a consequence of gene amplification and overexpression of DHFR, are more resistant to BR than are parental cells. Studies of the mechanism by which BR lowers DHFR showed that BR, through its metabolite BAD, reduced NADP and NADPH cellular levels by inhibiting nicotinamide adenine dinucleotide kinase (NADK). As consequence of the lack of NADPH, DHFR was shown to be destabilized. We suggest that, inhibition of NADK is a new approach to downregulate DHFR and to inhibit cell growth.

  8. The Drosophila carbonyl reductase sniffer is an efficient 4-oxonon-2-enal (4ONE) reductase.

    PubMed

    Martin, Hans-Jörg; Ziemba, Marta; Kisiela, Michael; Botella, José A; Schneuwly, Stephan; Maser, Edmund

    2011-05-30

    Studies with the fruit-fly Drosophila melanogaster demonstrated that the enzyme sniffer prevented oxidative stress-induced neurodegeneration. Mutant flies overexpressing sniffer had significantly extended life spans in a 99.5% oxygen atmosphere compared to wild-type flies. However, the molecular mechanism of this protection remained unclear. Sequence analysis and database searches identified sniffer as a member of the short-chain dehydrogenase/reductase superfamily with a 27.4% identity to the human enzyme carbonyl reductase type I (CBR1). As CBR1 catalyzes the reduction of the lipid peroxidation products 4HNE and 4ONE, we tested whether sniffer is able to metabolize these lipid derived aldehydes by carbonyl reduction. To produce recombinant enzyme, the coding sequence of sniffer was amplified from a cDNA-library, cloned into a bacterial expression vector and the His-tagged protein was purified by Ni-chelate chromatography. We found that sniffer catalyzed the NADPH-dependent carbonyl reduction of 4ONE (K(m)=24±2 μM, k(cat)=500±10 min(-1), k(cat)/K(m)=350 s(-1) mM(-1)) but not that of 4HNE. The reaction product of 4ONE reduction by sniffer was mainly 4HNE as shown by HPLC- and GC/MS analysis. Since 4HNE, though still a potent electrophile, is less neurotoxic and protein reactive than 4ONE, one mechanism by which sniffer exerts its neuroprotective effects in Drosophila after oxidative stress may be enzymatic reduction of 4ONE. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  9. NADPH: Protochlorophyllide Oxidoreductase-Structure, Catalytic Function, and Role in Prolamellar Body Formation and Morphogenesis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Timko, Michael P

    2013-02-01

    The biosynthesis of chlorophyll is a critical biochemical step in the development of photosynthetic vascular plants and green algae. From photosynthetic bacteria (cyanobacteria) to algae, non-vascular plants, gymnosperms and vascular plants, mechanisms have evolved for protochlorophyllide reduction a key step in chlorophyll synthesis. Protochlorophyllide reduction is carried out by both a light-dependent (POR) and light-independent (LIPOR) mechanisms. NADPH: protochlorophyllide oxidoreductase (EC 1.3.1.33, abbreviated POR) catalyzes the light-dependent reduction of protochlorophyllide (PChlide) to chlorophyllide (Chlide). In contrast, a light-independent protochlorophyllide reductase (LIPOR) involves three plastid gene products (chlL, chlN, and chlB) and several nuclear factors. Our work focused on characterization ofmore » both the POR and LIPOR catalyzed processes.« less

  10. [Establishment of an in vitro screening model for steroid 5 alpha-reductase inhibitors with the microplate reader].

    PubMed

    Wu, Jian-Hui; Sun, Zu-Yue

    2013-06-01

    To establish an in vitro screening model for steroid 5 alpha-reductase inhibitors using the microplate reader. Steroid 5 alpha-reductase was obtained from the liver of female rats, an in vitro screening model for steroid 5 alpha-reductase inhibitors established using the 96-well plate and microplate reader after determination of the enzymatic activity, and the reliability of the model verified with the known 5 alpha-reductase inhibitors epristeride and finasteride. Added to the 96-well plate were the final concentrations of testosterone (0-40 micromol/L), NADPH (22 micromol/L), epristeride (0-60 nmol/L) or finasteride (0-60 nmol/ L) and steroid 5 alpha-reductase (20 microl), the total volume of each well adjusted to 200 microl with Tris-Hcl buffer. The 96-well plate was placed in the microplate reader, mixed and incubated at 37 degrees C, followed by detection of the A340nm value at 0 and 10 min and analysis of the data. The Km value of steroid 5 alpha-reductase was 3.794 micromol/L, with a Vmax of 0.271 micromol/(L. min). The Ki of epristeride was 148.2 nmol/L, with an IC50 of 31.5 nmol/L, and the enzymatic reaction kinetic curve suggested that epristeride was an uncompetitive enzyme inhibitor. The Ki of finasteride was 158. 8 nmol/L, with an IC50 of 13.6 nmol/L. The enzymatic reaction kinetic curve showed that both epristeride and finasteride were competitive enzyme inhibitors, similar to those reported in the published literature. A screening model was successfully established, which could rapidly and effectively screen steroid 5 alpha-reductase inhibitors in vitro.

  11. Structural rearrangements occurring upon cofactor binding in the Mycobacterium smegmatis β-ketoacyl-acyl carrier protein reductase MabA.

    PubMed

    Küssau, Tanja; Flipo, Marion; Van Wyk, Niel; Viljoen, Albertus; Olieric, Vincent; Kremer, Laurent; Blaise, Mickaël

    2018-05-01

    In mycobacteria, the ketoacyl-acyl carrier protein (ACP) reductase MabA (designated FabG in other bacteria) catalyzes the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products. This first reductive step in the fatty-acid biosynthesis elongation cycle is essential for bacteria, which makes MabA/FabG an interesting drug target. To date, however, very few molecules targeting FabG have been discovered and MabA remains the only enzyme of the mycobacterial type II fatty-acid synthase that lacks specific inhibitors. Despite the existence of several MabA/FabG crystal structures, the structural rearrangement that occurs upon cofactor binding is still not fully understood. Therefore, unlocking this knowledge gap could help in the design of new inhibitors. Here, high-resolution crystal structures of MabA from Mycobacterium smegmatis in its apo, NADP + -bound and NADPH-bound forms are reported. Comparison of these crystal structures reveals the structural reorganization of the lid region covering the active site of the enzyme. The crystal structure of the apo form revealed numerous residues that trigger steric hindrance to the binding of NADPH and substrate. Upon NADPH binding, these residues are pushed away from the active site, allowing the enzyme to adopt an open conformation. The transition from an NADPH-bound to an NADP + -bound form is likely to facilitate release of the product. These results may be useful for subsequent rational drug design and/or for in silico drug-screening approaches targeting MabA/FabG.

  12. Fundamental roles of reactive oxygen species and protective mechanisms in the female reproductive system.

    PubMed

    Fujii, Junichi; Iuchi, Yoshihito; Okada, Futoshi

    2005-09-02

    Controlled oxidation, such as disulfide bond formation in sperm nuclei and during ovulation, plays a fundamental role in mammalian reproduction. Excess oxidation, however, causes oxidative stress, resulting in the dysfunction of the reproductive process. Antioxidation reactions that reduce the levels of reactive oxygen species are of prime importance in reproductive systems in maintaining the quality of gametes and support reproduction. While anti-oxidative enzymes, such as superoxide dismutase and peroxidase, play a central role in eliminating oxidative stress, reduction-oxidation (redox) systems, comprised of mainly glutathione and thioredoxin, function to reduce the levels of oxidized molecules. Aldo-keto reductase, using NADPH as an electron donor, detoxifies carbonyl compounds resulting from the oxidation of lipids and proteins. Thus, many antioxidative and redox enzyme genes are expressed and aggressively protect gametes and embryos in reproductive systems.

  13. Crystal Structure of Human Liver delta {4}-3-Ketosteroid 5 beta-Reductase (AKR1D1) and Implications for Substrate Binding and Catalysis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Di Costanzo,L.; Drury, J.; Penning, T.

    2008-01-01

    AKR1D1 (steroid 5{beta}-reductase) reduces all 4-3-ketosteroids to form 5{beta}-dihydrosteroids, a first step in the clearance of steroid hormones and an essential step in the synthesis of all bile acids. The reduction of the carbon-carbon double bond in an a,{beta}-unsaturated ketone by 5{beta}-reductase is a unique reaction in steroid enzymology because hydride transfer from NADPH to the {beta}-face of a 4-3-ketosteroid yields a cis-A/B-ring configuration with an {approx}90 bend in steroid structure. Here, we report the first x-ray crystal structure of a mammalian steroid hormone carbon-carbon double bond reductase, human 4-3-ketosteroid 5{beta}-reductase (AKR1D1), and its complexes with intact substrates. We havemore » determined the structures of AKR1D1 complexes with NADP+ at 1.79- and 1.35- Angstroms resolution (HEPES bound in the active site), NADP+ and cortisone at 1.90- Angstroms resolution, NADP+ and progesterone at 2.03- Angstroms resolution, and NADP+ and testosterone at 1.62- Angstroms resolution. Complexes with cortisone and progesterone reveal productive substrate binding orientations based on the proximity of each steroid carbon-carbon double bond to the re-face of the nicotinamide ring of NADP+. This orientation would permit 4-pro-(R)-hydride transfer from NADPH. Each steroid carbonyl accepts hydrogen bonds from catalytic residues Tyr58 and Glu120. The Y58F and E120A mutants are devoid of activity, supporting a role for this dyad in the catalytic mechanism. Intriguingly, testosterone binds nonproductively, thereby rationalizing the substrate inhibition observed with this particular steroid. The locations of disease-linked mutations thought to be responsible for bile acid deficiency are also revealed.« less

  14. Mutations at Several Loci Cause Increased Expression of Ribonucleotide Reductase in Escherichia coli

    PubMed Central

    Feeney, Morgan Anne; Ke, Na

    2012-01-01

    Production of deoxyribonucleotides for DNA synthesis is an essential and tightly regulated process. The class Ia ribonucleotide reductase (RNR), the product of the nrdAB genes, is required for aerobic growth of Escherichia coli. In catalyzing the reduction of ribonucleotides, two of the cysteines of RNR become oxidized, forming a disulfide bond. To regenerate active RNR, the cell uses thioredoxins and glutaredoxins to reduce the disulfide bond. Strains that lack thioredoxins 1 and 2 and glutaredoxin 1 do not grow because RNR remains in its oxidized, inactive form. However, suppressor mutations that lead to RNR overproduction allow glutaredoxin 3 to reduce sufficient RNR for growth of these mutant strains. We previously described suppressor mutations in the dnaA and dnaN genes that had such effects. Here we report the isolation of new mutations that lead to increased levels of RNR. These include mutations that were not known to influence production of RNR previously, such as a mutation in the hda gene and insertions in the nrdAB promoter region of insertion elements IS1 and IS5. Bioinformatic analysis raises the possibility that IS element insertion in this region represents an adaptive mechanism in nrdAB regulation in E. coli and closely related species. We also characterize mutations altering different amino acids in DnaA and DnaN from those isolated before. PMID:22247510

  15. Correlation of enteric NADPH-d positive cell counts with the duration of incubation period in NADPH-d histochemistry.

    PubMed

    Cserni, Tamas; O' Donnel, Annemarie; Paran, Sri; Puri, Prem

    2009-03-01

    Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) staining can be used in the enteric nervous system to determine nitrergic neuronal counts, critical in motility disorders such as intestinal neuronal dysplasia and hypoganglionosis. The reported incubation periods of specimens with NADPH-d staining solution has varied from 2 to 24 h. The aim of this study is to investigate the impact of the incubation period on the overall NADPH-d positive cell counts in porcine rectal submucosal plexus. The submucosal plexus of rectal specimens from 12-week-old pigs (n = 5) were studied. Conventional frozen sections were used to identify nitrergic neurons while whole-mount preparations were used to quantify the effect of prolonged duration of incubation on positively identified ganglion cells with NADPH-d histochemistry. The same submucosal ganglia on the conventional sections, and a minimum of 12 ganglia per whole-mount preparation specimen were photographed sequentially at 2, 6, and 24 h and used to count the number of nitrergic cells per ganglion. The same staining solution was used throughout the experiment. Results were analysed using a one-way ANOVA test. Prolonged incubation with the staining solution revealed new NADPH-d positive cells in the ganglia on the conventional sections. The total number of neurons counted in the 12 adjacent ganglia in the whole-mount specimens was 180 +/- 55, the mean neuronal cell per ganglion was 15 +/- 8 after 2 h of incubation. This increased to 357 +/- 17, and to 29 +/- 12 after 6 h (p < 0.05). A further increase was observed of 515 +/- 19 and 43 +/- 17 after 24 h (p < 0.05). When the photomicrographs were retrospectively analysed, not even the outline of the neuronal cells that stained with prolonged incubation was evident at the earlier time points. NADPH-d positive cell counts increase in proportion to the duration of incubation in NADPH-d histochemistry. Comparative studies attempting to quantify nitrergic cell counts in

  16. Thioredoxin A Is Essential for Motility and Contributes to Host Infection of Listeria monocytogenes via Redox Interactions.

    PubMed

    Cheng, Changyong; Dong, Zhimei; Han, Xiao; Wang, Hang; Jiang, Li; Sun, Jing; Yang, Yongchun; Ma, Tiantian; Shao, Chunyan; Wang, Xiaodu; Chen, Zhongwei; Fang, Weihuan; Freitag, Nancy E; Huang, Huarong; Song, Houhui

    2017-01-01

    Microbes employ the thioredoxin system to defend against oxidative stress and ensure correct disulfide bonding to maintain protein function. Listeria monocytogenes has been shown to encode a putative thioredoxin, TrxA, but its biological roles and underlying mechanisms remain unknown. Here, we showed that expression of L. monocytogenes TrxA is significantly induced in bacteria treated with the thiol-specific oxidizing agent, diamide. Deletion of trxA markedly compromised tolerance of the pathogen to diamide, and mainly impaired early stages of infection in human intestinal epithelial Caco-2 cells. In addition, most trxA mutant bacteria were not associated with polymerized actin, and the rare bacteria that were associated with polymerized actin displayed very short tails or clouds during infection. Deletion or constitutive overexpression of TrxA, which was regulated by SigH, severely attenuated the virulence of the pathogen. Transcriptome analysis of L. monocytogenes revealed over 270 genes that were differentially transcribed in the Δ trxA mutant compared to the wild-type, especially for the virulence-associated genes plcA, mpl, hly, actA , and plcB . Particularly, deletion of TrxA completely reduced LLO expression, and thereby led to a thoroughly impaired hemolytic activity. Expression of these virulence factors are positively regulated by the master regulator PrfA that was found here to use TrxA to maintain its reduced forms for activation. Interestingly, the trxA deletion mutant completely lacked flagella and was non-motile. We further confirmed that this deficiency is attributable to TrxA in maintaining the reduced intracellular monomer status of MogR, the key regulator for flagellar formation, to ensure correct dimerization. In summary, we demonstrated for the first time that L. monocytogenes thioredoxin A as a vital cellular reductase is essential for maintaining a highly reducing environment in the bacterial cytosol, which provides a favorable condition for

  17. Streptococcus sanguinis class Ib ribonucleotide reductase: high activity with both iron and manganese cofactors and structural insights.

    PubMed

    Makhlynets, Olga; Boal, Amie K; Rhodes, Delacy V; Kitten, Todd; Rosenzweig, Amy C; Stubbe, JoAnne

    2014-02-28

    Streptococcus sanguinis is a causative agent of infective endocarditis. Deletion of SsaB, a manganese transporter, drastically reduces S. sanguinis virulence. Many pathogenic organisms require class Ib ribonucleotide reductase (RNR) to catalyze the conversion of nucleotides to deoxynucleotides under aerobic conditions, and recent studies demonstrate that this enzyme uses a dimanganese-tyrosyl radical (Mn(III)2-Y(•)) cofactor in vivo. The proteins required for S. sanguinis ribonucleotide reduction (NrdE and NrdF, α and β subunits of RNR; NrdH and TrxR, a glutaredoxin-like thioredoxin and a thioredoxin reductase; and NrdI, a flavodoxin essential for assembly of the RNR metallo-cofactor) have been identified and characterized. Apo-NrdF with Fe(II) and O2 can self-assemble a diferric-tyrosyl radical (Fe(III)2-Y(•)) cofactor (1.2 Y(•)/β2) and with the help of NrdI can assemble a Mn(III)2-Y(•) cofactor (0.9 Y(•)/β2). The activity of RNR with its endogenous reductants, NrdH and TrxR, is 5,000 and 1,500 units/mg for the Mn- and Fe-NrdFs (Fe-loaded NrdF), respectively. X-ray structures of S. sanguinis NrdIox and Mn(II)2-NrdF are reported and provide a possible rationale for the weak affinity (2.9 μM) between them. These streptococcal proteins form a structurally distinct subclass relative to other Ib proteins with unique features likely important in cluster assembly, including a long and negatively charged loop near the NrdI flavin and a bulky residue (Thr) at a constriction in the oxidant channel to the NrdI interface. These studies set the stage for identifying the active form of S. sanguinis class Ib RNR in an animal model for infective endocarditis and establishing whether the manganese requirement for pathogenesis is associated with RNR.

  18. Quantitative flux analysis reveals folate-dependent NADPH production

    NASA Astrophysics Data System (ADS)

    Fan, Jing; Ye, Jiangbin; Kamphorst, Jurre J.; Shlomi, Tomer; Thompson, Craig B.; Rabinowitz, Joshua D.

    2014-06-01

    ATP is the dominant energy source in animals for mechanical and electrical work (for example, muscle contraction or neuronal firing). For chemical work, there is an equally important role for NADPH, which powers redox defence and reductive biosynthesis. The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway, with malic enzyme sometimes also important. Although the relative contribution of glycolysis and oxidative phosphorylation to ATP production has been extensively analysed, similar analysis of NADPH metabolism has been lacking. Here we demonstrate the ability to directly track, by liquid chromatography-mass spectrometry, the passage of deuterium from labelled substrates into NADPH, and combine this approach with carbon labelling and mathematical modelling to measure NADPH fluxes. In proliferating cells, the largest contributor to cytosolic NADPH is the oxidative pentose phosphate pathway. Surprisingly, a nearly comparable contribution comes from serine-driven one-carbon metabolism, in which oxidation of methylene tetrahydrofolate to 10-formyl-tetrahydrofolate is coupled to reduction of NADP+ to NADPH. Moreover, tracing of mitochondrial one-carbon metabolism revealed complete oxidation of 10-formyl-tetrahydrofolate to make NADPH. As folate metabolism has not previously been considered an NADPH producer, confirmation of its functional significance was undertaken through knockdown of methylenetetrahydrofolate dehydrogenase (MTHFD) genes. Depletion of either the cytosolic or mitochondrial MTHFD isozyme resulted in decreased cellular NADPH/NADP+ and reduced/oxidized glutathione ratios (GSH/GSSG) and increased cell sensitivity to oxidative stress. Thus, although the importance of folate metabolism for proliferating cells has been long recognized and attributed to its function of producing one-carbon units for nucleic acid synthesis, another crucial function of this pathway is generating reducing power.

  19. Molecular cloning and functional characterization of the anthocyanidin reductase gene from Vitis bellula.

    PubMed

    Zhu, Yue; Peng, Qing-Zhong; Li, Ke-Gang; Xie, De-Yu

    2014-08-01

    Anthocyanidin reductase (ANR) is an NADPH-/NADH-dependent enzyme that transfers two hydrides to anthocyanidins to produce three types of isomeric flavan-3-ols. This reductase forms the ANR pathway toward the biosynthesis of proanthocyanidins (PAs, which are also called condensed tannins). Here, we report cloning and functional characterization of an ANR (called VbANR) homolog from the leaves of Vitis bellula, a newly developed grape crop in southern China. The open reading frame (ORF) of VbANR is 1,017 bp in length and encodes 339 amino acids. A phylogenetic analysis and an alignment using 17 sequences revealed that VbANR is approximately 99.9 % identical to the ANR homolog from Vitis vinifera. The VbANR ORF is fused to the Trx gene containing a His-tag in the pET32a(+) vector to obtain a pET32a(+)-VbANR construct for expressing the recombinant VbANR. In vitro enzyme assays show that VbANR converts cyanidin, delphinidin, and pelargonidin to their corresponding flavan-3-ols. Enzymatic products include 2S,3R-trans- and 2R,3R-cis-flavan-3-ols isomers, such as (-)-catechin and (-)-epicatechin. In addition, the third compound that is observed from the enzymatic products is most likely a 2S,3S-cis-flavan-3-ol. To analyze the kinetics and optimize pH and temperature values, a UV spectrometry method was developed to quantify the concentrations of total enzymatic products. The optimum pH and temperature values are 4.0 and 40 °C, respectively. The K m , K cat, V max, and K cat/K m values for pelargonidin and delphinidin were similar. In comparison, VbANR exhibits a slightly lower affinity to cyanidin. VbANR uses both NADPH and NADH but prefers to employ NADPH. GFP fusion and confocal microscopy analyses revealed the cytosolic localization of VbANR. The overexpression of VbANR in ban mutants reconstructed the biosynthetic pathway of PAs in the seed coat. These data demonstrate that VbANR forms the ANR pathway, leading to the formation of three types of isomeric flavan-3-ols

  20. Multiple ligand-binding modes in bacterial R67 dihydrofolate reductase

    NASA Astrophysics Data System (ADS)

    Alonso, Hernán; Gillies, Malcolm B.; Cummins, Peter L.; Bliznyuk, Andrey A.; Gready, Jill E.

    2005-03-01

    R67 dihydrofolate reductase (DHFR), a bacterial plasmid-encoded enzyme associated with resistance to the drug trimethoprim, shows neither sequence nor structural homology with the chromosomal DHFR. It presents a highly symmetrical toroidal structure, where four identical monomers contribute to the unique central active-site pore. Two reactants (dihydrofolate, DHF), two cofactors (NADPH) or one of each (R67•DHF•NADPH) can be found simultaneously within the active site, the last one being the reactive ternary complex. As the positioning of the ligands has proven elusive to empirical determination, we addressed the problem from a theoretical perspective. Several potential structures of the ternary complex were generated using the docking programs AutoDock and FlexX. The variability among the final poses, many of which conformed to experimental data, prompted us to perform a comparative scoring analysis and molecular dynamics simulations to assess the stability of the complexes. Analysis of ligand-ligand and ligand-protein interactions along the 4 ns trajectories of eight different structures allowed us to identify important inter-ligand contacts and key protein residues. Our results, combined with published empirical data, clearly suggest that multipe binding modes of the ligands are possible within R67 DHFR. While the pterin ring of DHF and the nicotinamide ring of NADPH assume a stacked endo-conformation at the centre of the pore, probably assisted by V66, Q67 and I68, the tails of the molecules extend towards opposite ends of the cavity, adopting multiple configurations in a solvent rich-environment where hydrogen-bond interactions with K32 and Y69 may play important roles.

  1. Steroid 5 alpha-reductase deficiency in a 65-year-old male pseudohermaphrodite: the natural history, ultrastructure of the testes, and evidence for inherited enzyme heterogeneity.

    PubMed

    Imperato-McGinley, J; Peterson, R E; Leshin, M; Griffin, J E; Cooper, G; Draghi, S; Berenyi, M; Wilson, J D

    1980-01-01

    We report a 65-yr-old male pseudohermaphrodite with steroid 5 alpha-reductase deficiency in whom there was no medical intervention before, during, or after puberty, enabling us to observe the natural history of this condition. The affected subject has an android build, with more facial and body hair than in previously described affected adults. Although the subject was raised as a girl, a male gender identity evolved with the events of puberty, but social factors have delayed the complete expression of a male gender role. Plasma levels of dihydrotestosterone and the in vivo conversion of radiolabeled testosterone to dihydrotestosterone were decreased. There was an elevated urinary etiocholanolone to androsterone ratio, typical of the syndrome. Characterization of 5 alpha-reductase enzyme activity in cultured genital skin fibroblasts demonstrated a pattern of enzyme activity distinctly different from three previously described families with this condition. There was decreased enzyme affinity for testosterone and NADPH. Also, the stability of the enzyme to elevated temperature was not protected by NADPH, resulting in rapid disappearance of enzyme activity after inhibition of protein synthesis with cycloheximide. Electron microscopic evaluation of the testes was carried out.

  2. Functional characterization of thioredoxin 3 (TRX-3), a Caenorhabditis elegans intestine-specific thioredoxin

    PubMed Central

    Jiménez-Hidalgo, María; Kurz, Cyril Léopold; Pedrajas, José Rafael; Naranjo-Galindo, Francisco José; González-Barrios, María; Cabello, Juan; Sáez, Alberto G.; Lozano, Encarnación; Button, Emma L.; Veal, Elizabeth A.; Fierro-González, Juan Carlos; Swoboda, Peter; Miranda-Vizuete, Antonio

    2014-01-01

    Thioredoxins are a class of evolutionarily conserved proteins that have been demonstrated to play a key role in many cellular processes involving redox reactions. We report here the genetic and biochemical characterization of Caenorhabditis elegans TRX-3, the first metazoan thioredoxin with an intestine-specific expression pattern. By using green fluorescent protein reporters we have found that TRX-3 is expressed in both the cytoplasm and the nucleus of intestinal cells, with a prominent localization at the apical membrane. Although intestinal function, reproductive capacity, longevity, and resistance of trx-3 loss-of-function mutants to many stresses are indistinguishable from those of wild-type animals, we have observed a slight reduction in size and a minor reduction in the defecation cycle timing of trx-3 mutants. Interestingly, trx-3 is induced upon infection by Photorhabdus luminescens and Candida albicans, and TRX-3 overexpression provides a modest protection against these pathogens. Together, our data indicate that TRX-3 function in the intestine is dispensable for C. elegans development but may be important to fight specific bacterial and fungal infections. PMID:24316195

  3. The structural basis for the negative regulation of thioredoxin by thioredoxin-interacting protein

    PubMed Central

    Hwang, Jungwon; Suh, Hyun-Woo; Jeon, Young Ho; Hwang, Eunha; Nguyen, Loi T.; Yeom, Jeonghun; Lee, Seung-Goo; Lee, Cheolju; Kim, Kyung Jin; Kang, Beom Sik; Jeong, Jin-Ok; Oh, Tae-Kwang; Choi, Inpyo; Lee, Jie-Oh; Kim, Myung Hee

    2014-01-01

    The redox-dependent inhibition of thioredoxin (TRX) by thioredoxin-interacting protein (TXNIP) plays a pivotal role in various cancers and metabolic syndromes. However, the molecular mechanism of this regulation is largely unknown. Here, we present the crystal structure of the TRX–TXNIP complex and demonstrate that the inhibition of TRX by TXNIP is mediated by an intermolecular disulphide interaction resulting from a novel disulphide bond-switching mechanism. Upon binding to TRX, TXNIP undergoes a structural rearrangement that involves switching of a head-to-tail interprotomer Cys63-Cys247 disulphide between TXNIP molecules to an interdomain Cys63-Cys190 disulphide, and the formation of a de novo intermolecular TXNIP Cys247-TRX Cys32 disulphide. This disulphide-switching event unexpectedly results in a domain arrangement of TXNIP that is entirely different from those of other arrestin family proteins. We further show that the intermolecular disulphide bond between TRX and TXNIP dissociates in the presence of high concentrations of reactive oxygen species. This study provides insight into TRX and TXNIP-dependent cellular regulation. PMID:24389582

  4. Crystal Structure of Human Liver [delta][superscript 4]-3-Ketosteroid 5[beta]-Reductase (AKR1D1) and Implications for Substrate Binding and Catalysis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Di Costanzo, Luigi; Drury, Jason E.; Penning, Trevor M.

    2008-07-15

    AKR1D1 (steroid 5{beta}-reductase) reduces all {Delta}{sup 4}-3-ketosteroids to form 5{beta}-dihydrosteroids, a first step in the clearance of steroid hormones and an essential step in the synthesis of all bile acids. The reduction of the carbon-carbon double bond in an {alpha}{beta}-unsaturated ketone by 5{beta}-reductase is a unique reaction in steroid enzymology because hydride transfer from NADPH to the {beta}-face of a {Delta}{sup 4}-3-ketosteroid yields a cis-A/B-ring configuration with an {approx}90{sup o} bend in steroid structure. Here, we report the first x-ray crystal structure of a mammalian steroid hormone carbon-carbon double bond reductase, human {Delta}{sup 4}-3-ketosteroid 5{beta}-reductase (AKR1D1), and its complexes withmore » intact substrates. We have determined the structures of AKR1D1 complexes with NADP{sup +} at 1.79- and 1.35-{angstrom} resolution (HEPES bound in the active site), NADP{sup +} and cortisone at 1.90-{angstrom} resolution, NADP{sup +} and progesterone at 2.03-{angstrom} resolution, and NADP{sup +} and testosterone at 1.62-{angstrom} resolution. Complexes with cortisone and progesterone reveal productive substrate binding orientations based on the proximity of each steroid carbon-carbon double bond to the re-face of the nicotinamide ring of NADP{sup +}. This orientation would permit 4-pro-(R)-hydride transfer from NADPH. Each steroid carbonyl accepts hydrogen bonds from catalytic residues Tyr{sup 58} and Glu{sup 120}. The Y58F and E120A mutants are devoid of activity, supporting a role for this dyad in the catalytic mechanism. Intriguingly, testosterone binds nonproductively, thereby rationalizing the substrate inhibition observed with this particular steroid. The locations of disease-linked mutations thought to be responsible for bile acid deficiency are also revealed.« less

  5. Towards a mechanistic and physiological understanding of a ferredoxin disulfide reductase from the domains Archaea and Bacteria.

    PubMed

    Prakash, Divya; Walters, Karim A; Martinie, Ryan J; McCarver, Addison C; Kumar, Adepu K; Lessner, Daniel J; Krebs, Carsten; Golbeck, John H; Ferry, James G

    2018-05-02

    Disulfide reductases reduce other proteins and are critically important for cellular redox signaling and homeostasis. Methanosarcina acetivorans is a methane-producing microbe from the domain Archaea that produces a ferredoxin:disulfide reductase (FDR) for which the crystal structure has been reported, yet its biochemical mechanism and physiological substrates are unknown. FDR and the extensively characterized plant-type ferredoxin:thioredoxin reductase (FTR) belong to a distinct class of disulfide reductases that contain a unique active-site [4Fe-4S] cluster. The results reported here support a mechanism for FDR similar to that reported for FTR with notable exceptions. Unlike FTR, FDR contains a rubredoxin [1Fe-0S] center postulated to mediate electron transfer from ferredoxin to the active-site [4Fe-4S] cluster.  UV-Vis, EPR and Mӧssbauer spectroscopic data indicated that two-electron reduction of the active-site disulfide in FDR involves a one-electron-reduced [4Fe-4S]1+ intermediate previously hypothesized for FTR. Our results support a role for an active-site tyrosine in FDR that occupies the equivalent position of an essential histidine in the active-site of FTR. Of note, one of seven Trxs encoded in the genome (Trx5) and methanoredoxin, a glutaredoxin-like enzyme from M. acetivorans, were reduced by FDR advancing the physiological understanding of FDRs role in the redox metabolism of methanoarchaea. Finally, bioinformatics analyses show FDR homologs are widespread in diverse microbes from the domain Bacteria. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Functional analysis of thioredoxin from the desert lichen-forming fungus, Endocarpon pusillum Hedwig, reveals its role in stress tolerance

    PubMed Central

    Li, Hui; Wei, Jiang-Chun

    2016-01-01

    Endocarpon pusillum is a lichen-forming fungus with an outstanding stress resistance property closely related to its antioxidant system. In this study, thioredoxin (Trx), one of the main components of antioxidant defense systems in E. pusillum (EpTrx), was characterized and analyzed both in transgenic yeasts and in vitro. Our analyses identified that the heterologous expression of EpTrx in the yeast Pichia pastoris significantly enhanced its resistance to osmotic and oxidative stresses. Assays in vitro showed EpTrx acted as a disulfide reductase as well as a molecular chaperone by assembling into various polymeric structures. Upon exposure to heat-shock stress, EpTrx exhibited weaker disulfide reductase activity but stronger chaperone activity, which coincided with the switching of the protein complexes from low molecular weight forms to high molecular weight complexes. Specifically, we found that Cys31 near but not at the active site was crucial in promoting the structural and functional transitions, most likely by accelerating the formation of intermolecular disulfide bond. Transgenic Saccharomyces cerevisiae harboring the native EpTrx exhibited stronger tolerance to oxidative, osmotic and high temperature stresses than the corresponding yeast strain containing the mutant EpTrx (C31S). Our results provide the first molecular evidence on how Trx influences stress response in lichen-forming fungi. PMID:27251605

  7. Structural and Biochemical Characterization of Cinnamoyl-CoA Reductases1

    PubMed Central

    Walker, Alexander M.

    2017-01-01

    Cinnamoyl-coenzyme A reductase (CCR) catalyzes the reduction of hydroxycinnamoyl-coenzyme A (CoA) esters using NADPH to produce hydroxycinnamyl aldehyde precursors in lignin synthesis. The catalytic mechanism and substrate specificity of cinnamoyl-CoA reductases from sorghum (Sorghum bicolor), a strategic plant for bioenergy production, were deduced from crystal structures, site-directed mutagenesis, and kinetic and thermodynamic analyses. Although SbCCR1 displayed higher affinity for caffeoyl-CoA or p-coumaroyl-CoA than for feruloyl-CoA, the enzyme showed significantly higher activity for the latter substrate. Through molecular docking and comparisons between the crystal structures of the Vitis vinifera dihydroflavonol reductase and SbCCR1, residues threonine-154 and tyrosine-310 were pinpointed as being involved in binding CoA-conjugated phenylpropanoids. Threonine-154 of SbCCR1 and other CCRs likely confers strong substrate specificity for feruloyl-CoA over other cinnamoyl-CoA thioesters, and the T154Y mutation in SbCCR1 led to broader substrate specificity and faster turnover. Through data mining using our structural and biochemical information, four additional putative CCR genes were discovered from sorghum genomic data. One of these, SbCCR2, displayed greater activity toward p-coumaroyl-CoA than did SbCCR1, which could imply a role in the synthesis of defense-related lignin. Taken together, these findings provide knowledge about critical residues and substrate preference among CCRs and provide, to our knowledge, the first three-dimensional structure information for a CCR from a monocot species. PMID:27956488

  8. A newly-detected reductase from Rauvolfia closes a gap in the biosynthesis of the antiarrhythmic alkaloid ajmaline.

    PubMed

    Gao, Shujuan; von Schumann, Gerald; Stöckigt, Joachim

    2002-10-01

    A new enzyme, 1,2-dihydrovomilenine reductase (E.C. 1.3.1), has been detected in Rauvolfia cell suspension cultures. The enzyme specifically converts 2beta( R)-1,2-dihydrovomilenine through an NADPH-dependent reaction into 17-O-acetylnorajmaline, a close biosynthetic precursor of the antiarrhythmic alkaloid ajmaline from Rauvolfia. A five-step purification procedure using SOURCE 30Q chromatography, hydroxyapatite chromatography, 2',5'-ADP Sepharose 4B affinity chromatography and ion exchange chromatography on DEAE Sepharose and Mono Q delivered an approximately 200-fold enriched enzyme in a yield of approximately 6%. SDS-PAGE showed an M r for the enzyme of approximately 48 kDa. Optimum pH and optimum temperature of the reductase were at pH 6.0 and 37 degrees C. The enzyme shows a limited distribution in cell cultures expressing ajmaline biosynthesis, and is obviously highly specific for the ajmaline pathway.

  9. Fine tuning of coenzyme specificity in family 2 aldo-keto reductases revealed by crystal structures of the Lys-274 → Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD + and NADP +

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Leitgeb, Stefan; Petschacher, Barbara; Wilson, David K.

    2005-01-11

    Aldo-keto reductases of family 2 employ single site replacement Lys → Arg to switch their cosubstrate preference from NADPH to NADH. X-ray crystal structures of Lys-274 → Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD + and NADP + were determined at a resolution of 2.4 and 2.3 Å, respectively. Due to steric conflicts in the NADP +-bound form, the arginine side chain must rotate away from the position of the original lysine side chain, thereby disrupting a network of direct and water-mediated interactions between Glu-227, Lys-274 and the cofactor 2'-phosphate and 3'-hydroxy groups. Because anchoring contactsmore » of its Glu-227 are lost, the coenzyme-enfolding loop that becomes ordered upon binding of NAD(P) + in the wild-type remains partly disordered in the NADP +-bound mutant. The results delineate a catalytic reaction profile for the mutant in comparison to wild-type.« less

  10. Ferulic acid attenuates the cerebral ischemic injury-induced decrease in peroxiredoxin-2 and thioredoxin expression.

    PubMed

    Sung, Jin-Hee; Gim, Sang-Ah; Koh, Phil-Ok

    2014-04-30

    Ferulic acid, a phenolic phytochemical compound found in various plants, has a neuroprotective effect through its anti-oxidant and anti-inflammation functions. Peroxiredoxin-2 and thioredoxin play a potent neuroprotective function against oxidative stress. We investigated whether ferulic acid regulates peroxiredoxin-2 and thioredoxin levels in cerebral ischemia. Sprague-Dawley rats (male, 210-230g) were treated with vehicle or ferulic acid (100mg/kg) after middle cerebral artery occlusion (MCAO), and cerebral cortex tissues were collected 24h after MCAO. Decreases in peroxiredoxin-2 and thioredoxin levels were elucidated in MCAO-operated animals using a proteomics approach. We found that ferulic acid treatment prevented the MCAO-induced decrease in the expression of peroxiredoxin-2 and thioredoxin. RT-PCR and Western blot analyses confirmed that ferulic acid treatment attenuated the MCAO-induced decrease in peroxiredoxin-2 and thioredoxin levels. Moreover, immunoprecipitation analysis showed that the interaction between thioredoxin and apoptosis signal-regulating kinase 1 (ASK1) decreased during MCAO, whereas ferulic acid prevented the MCAO-induced decrease in this interaction. Our findings suggest that ferulic acid plays a neuroprotective role by attenuating injury-induced decreases in peroxiredoxin-2 and thioredoxin levels in neuronal cell injury. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  11. The antibacterial efficacy of an aceraceous plant [Shantung maple (Acer truncatum Bunge)] may be related to inhibition of bacterial beta-oxoacyl-acyl carrier protein reductase (FabG).

    PubMed

    Zhang, Feng; Luo, Shi-Yun; Ye, Yan-Bin; Zhao, Wen-Hua; Sun, Xu-Guang; Wang, Zhi-Qun; Li, Ran; Sun, Ying-Hui; Tian, Wei-Xi; Zhang, Ying-Xia

    2008-10-01

    Polyphenols, including flavonoids, are the major components of the extracts from aceraceous plants. They possess remarkable antibacterial and antitumour activity. Our study focused on whether the inhibition of the bacterial type II fatty acid synthesis system is the mechanism for the antibacterial effect of the related plant polyphenols. Extracts obtained from the fallen leaves of the Shantung maple (Acer truncatum Bunge) using different solvents, and the related pure compound PGG (1,2,3,4,6-penta-O-galloyl-beta-D-glucose), potently inhibited the FabG (beta-oxoacyl-ACP reductase) steps in the fatty-acid-elongation cycle with the IC(50) values between 0.9 and 7.2 microg/ml. An ethyl acetate extract appeared to inhibit FabG reductase in a mixed manner with NADPH, as did PGG with NADPH, demonstrating that they interfered with the binding of the cofactor to the enzyme. Gram-positive and Gram-negative bacteria and some fungi were used to evaluate the antibacterial abilities of different extract samples. The experiments showed that a higher polyphenol content of the extracts led to a more potent inhibitory capacity against FabG, thus enhancing the antibacterial efficacy.

  12. Cross Talk between Two Antioxidant Systems, Thioredoxin and DJ-1: Consequences for Cancer

    PubMed Central

    Raninga, Prahlad V.; Trapani, Giovanna Di; Tonissen, Kathryn F.

    2014-01-01

    Oxidative stress, which is associated with an increased concentration of reactive oxygen species (ROS), is involved in the pathogenesis of numerous diseases including cancer. In response to increased ROS levels, cellular antioxidant molecules such as thioredoxin, peroxiredoxins, glutaredoxins, DJ-1, and superoxide dismutases are upregulated to counteract the detrimental effect of ROS. However, cancer cells take advantage of upregulated antioxidant molecules for protection against ROS-induced cell damage. This review focuses on two antioxidant systems, Thioredoxin and DJ-1, which are upregulated in many human cancer types, correlating with tumour proliferation, survival, and chemo-resistance. Thus, both of these antioxidant molecules serve as potential molecular targets to treat cancer. However, targeting one of these antioxidants alone may not be an effective anti-cancer therapy. Both of these antioxidant molecules are interlinked and act on similar downstream targets such as NF-κβ, PTEN, and Nrf2 to exert cytoprotection. Inhibiting either thioredoxin or DJ-1 alone may allow the other antioxidant to activate downstream signalling cascades leading to tumour cell survival and proliferation. Targeting both thioredoxin and DJ-1 in conjunction may completely shut down the antioxidant defence system regulated by these molecules. This review focuses on the cross-talk between thioredoxin and DJ-1 and highlights the importance and consequences of targeting thioredoxin and DJ-1 together to develop an effective anti-cancer therapeutic strategy. PMID:25593990

  13. Functional characterization of thioredoxin 3 (TRX-3), a Caenorhabditis elegans intestine-specific thioredoxin.

    PubMed

    Jiménez-Hidalgo, María; Kurz, Cyril Léopold; Pedrajas, José Rafael; Naranjo-Galindo, Francisco José; González-Barrios, María; Cabello, Juan; Sáez, Alberto G; Lozano, Encarnación; Button, Emma L; Veal, Elizabeth A; Fierro-González, Juan Carlos; Swoboda, Peter; Miranda-Vizuete, Antonio

    2014-03-01

    Thioredoxins are a class of evolutionarily conserved proteins that have been demonstrated to play a key role in many cellular processes involving redox reactions. We report here the genetic and biochemical characterization of Caenorhabditis elegans TRX-3, the first metazoan thioredoxin with an intestine-specific expression pattern. By using green fluorescent protein reporters we have found that TRX-3 is expressed in both the cytoplasm and the nucleus of intestinal cells, with a prominent localization at the apical membrane. Although intestinal function, reproductive capacity, longevity, and resistance of trx-3 loss-of-function mutants to many stresses are indistinguishable from those of wild-type animals, we have observed a slight reduction in size and a minor reduction in the defecation cycle timing of trx-3 mutants. Interestingly, trx-3 is induced upon infection by Photorhabdus luminescens and Candida albicans, and TRX-3 overexpression provides a modest protection against these pathogens. Together, our data indicate that TRX-3 function in the intestine is dispensable for C. elegans development but may be important to fight specific bacterial and fungal infections. © 2013 Elsevier Inc. All rights reserved.

  14. Anti-HMG-CoA Reductase, Antioxidant, and Anti-Inflammatory Activities of Amaranthus viridis Leaf Extract as a Potential Treatment for Hypercholesterolemia

    PubMed Central

    Salvamani, Shamala; Gunasekaran, Baskaran; Shukor, Mohd Yunus; Shaharuddin, Noor Azmi; Sabullah, Mohd Khalizan

    2016-01-01

    Inflammation and oxidative stress are believed to contribute to the pathology of several chronic diseases including hypercholesterolemia (elevated levels of cholesterol in blood) and atherosclerosis. HMG-CoA reductase inhibitors of plant origin are needed as synthetic drugs, such as statins, which are known to cause adverse effects on the liver and muscles. Amaranthus viridis (A. viridis) has been used from ancient times for its supposedly medically beneficial properties. In the current study, different parts of A. viridis (leaf, stem, and seed) were evaluated for potential anti-HMG-CoA reductase, antioxidant, and anti-inflammatory activities. The putative HMG-CoA reductase inhibitory activity of A. viridis extracts at different concentrations was determined spectrophotometrically by NADPH oxidation, using HMG-CoA as substrate. A. viridis leaf extract revealed the highest HMG-CoA reductase inhibitory effect at about 71%, with noncompetitive inhibition in Lineweaver-Burk plot analysis. The leaf extract showed good inhibition of hydroperoxides, 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO), and ferric ion radicals in various concentrations. A. viridis leaf extract was proven to be an effective inhibitor of hyaluronidase, lipoxygenase, and xanthine oxidase enzymes. The experimental data suggest that A. viridis leaf extract is a source of potent antioxidant and anti-inflammatory agent and may modulate cholesterol metabolism by inhibition of HMG-CoA reductase. PMID:27051453

  15. A Novel Carbonyl Reductase with Anti-Prelog Stereospecificity from Acetobacter sp. CCTCC M209061: Purification and Characterization

    PubMed Central

    Wang, Xiao-Ting; Zong, Min-Hua; Lou, Wen-Yong

    2014-01-01

    A novel carbonyl reductase (AcCR) catalyzing the asymmetric reduction of ketones to enantiopure alcohols with anti-Prelog stereoselectivity was found in Acetobacter sp. CCTCC M209061 and enriched 27.5-fold with an overall yield of 0.4% by purification. The enzyme showed a homotetrameric structure with an apparent molecular mass of 104 kDa and each subunit of 27 kDa. The gene sequence of AcCR was cloned and sequenced, and a 762 bp gene fragment was obtained. Either NAD(H) or NADP(H) can be used as coenzyme. For the reduction of 4′-chloroacetophenone, the Km value for NADH was around 25-fold greater than that for NADPH (0.66 mM vs 0.026 mM), showing that AcCR preferred NADPH over NADH. However, when NADH was used as cofactor, the response of AcCR activity to increasing concentration of 4′-chloroacetophenone was clearly sigmoidal with a Hill coefficient of 3.1, suggesting that the enzyme might possess four substrate-binding sites cooperating with each other The Vmax value for NADH-linked reduction was higher than that for NADPH-linked reduction (0.21 mM/min vs 0.17 mM/min). For the oxidation of isopropanol, the similar enzymological properties of AcCR were found using NAD+ or NADP+ as cofactor. Furthermore, a broad range of ketones such as aryl ketones, α-ketoesters and aliphatic ketones could be enantioselectively reduced into the corresponding chiral alcohols by this enzyme with high activity. PMID:24740089

  16. Potency of a novel saw palmetto ethanol extract, SPET-085, for inhibition of 5alpha-reductase II.

    PubMed

    Pais, Pilar

    2010-08-01

    The nicotinamide adenine dinucleotide phosphate (NADPH)-dependent membrane protein 5alpha-reductase irreversibly catalyses the conversion of testosterone to the most potent androgen, 5alpha-dihydrotestosterone (DHT). In humans, two 5alpha-reductase isoenyzmes are expressed: type I and type II. Type II is found primarily in prostate tissue. Saw palmetto extract (SPE) has been widely used for the treatment of lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH). The mechanisms of the pharmacological effects of SPE include the inhibition of 5alpha-reductase, among other actions. Clinical studies of SPE have been equivocal, with some showing significant results and others not. These inconsistent results may be due, in part, to varying bioactivities of the SPE used in the studies. The aim of the present study was to determine the in vitro potency of a novel saw palmetto ethanol extract (SPET-085), an inhibitor of the 5alpha-reductase isoenzyme type II, in a cell-free test system. On the basis of the enzymatic conversion of the substrate androstenedione to the 5alpha-reduced product 5alpha-androstanedione, the inhibitory potency was measured and compared to those of finasteride, an approved 5alpha-reductase inhibitor. SPET-085 concentration-dependently inhibited 5alpha-reductase type II in vitro (IC(50)=2.88+/-0.45 microg/mL). The approved 5alpha-reductase inhibitor, finasteride, tested as positive control, led to 61% inhibition of 5alpha-reductase type II. SPET-085 effectively inhibits the enzyme that has been linked to BPH, and the amount of extract required for activity is very low compared to data reported for other extracts. It can be concluded from data in the literature that SPET-085 is as effective as a hexane extract of saw palmetto that exhibited the highest levels of bioactivity, and is more effective than other SPEs tested. This study confirmed that SPET-085 has prostate health-promoting bioactivity that also corresponds favorably to

  17. Human NRDRB1, an alternatively spliced isoform of NADP(H)-dependent retinol dehydrogenase/reductase enhanced enzymatic activity of benzil.

    PubMed

    Yan, Yinxia; Song, Xuhong; Liu, Gefei; Su, Zhongjing; Du, Yongming; Sui, Xuxia; Chang, Xiaolan; Huang, Dongyang

    2012-01-01

    Human NRDRB1, a 226 amino acid alternatively spliced isoform of the NADP(H)- dependent retinol dehydrogenase/reductase (NRDR), lacks the complete coding region of exon 3, but preserves all the important functional motifs for NRDR catalytic activity. Nevertheless, its tissue distribution and physiological function remain to be elucidated. Expression of NRDRB1 and NRDR in cells and tissues was analyzed by semi-quantitative polymerase chain reaction (PCR) and western blot. NRDRB1 was expressed as a His(6) fusion protein and subjected to kinetics assays. Recombinant NRDRB1 had 1.2 to 8.6 fold higher k(cat)/K(m) values than recombinant NRDR, depending on the substrate. NRDRB1 catalyzed the NADPH-dependent reduction of α-dicarbonyl compounds, such as isatin, 9,10-phenanthrenequinone, and especially benzil. The significantly high catalytic activity and the relatively high expression in human liver of NRDRB1 conferred cellular resistance to benzil-induced cell toxicity and over-expression of NRDRB1 in low expressing Ec109 cells significantly enhanced cell tolerance toward benzil. Based on its substrate specificity, catalytic activity and relatively high expression in human liver tissue, our results suggest that NRDRB1, an alternatively spliced isoform of NRDR in vivo functions better than NRDR as a dicarbonyl reductase for xenobiotics containing reactive carbonyls. Our study is the first reporting this phenomenon of the enzymes involved in biochemical reactions. Copyright © 2012 S. Karger AG, Basel.

  18. Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase.

    PubMed

    Trigoso, Yvonne D; Evans, Russell C; Karsten, William E; Chooback, Lilian

    2016-01-01

    The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5'and 3' terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40-50 mgs of protein, an improvement on the previous protein expression and multistep purification.

  19. Expression, purification, crystallization and preliminary X-ray analysis of conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708.

    PubMed

    Yamamura, Akihiro; Maruoka, Shintaro; Ohtsuka, Jun; Miyakawa, Takuya; Nagata, Koji; Kataoka, Michihiko; Kitamura, Nahoko; Shimizu, Sakayu; Tanokura, Masaru

    2009-11-01

    Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708 is a member of the NADPH-dependent aldo-keto reductase (AKR) superfamily and catalyzes the stereospecific reduction of ketopantoyl lactone to d-pantoyl lactone. A diffraction-quality crystal of recombinant CPR-C2 was obtained by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystal diffracted X-rays to 1.7 angstrom resolution on beamline NW12A of the Photon Factory-Advanced Ring (Tsukuba, Japan). The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 55.02, b = 68.30, c = 68.93 angstrom. The Matthews coefficient (V(M) = 1.76 angstrom(3) Da(-1)) indicated that the crystal contained one CPR-C2 molecule per asymmetric unit.

  20. Vitamin C. Biosynthesis, recycling and degradation in mammals.

    PubMed

    Linster, Carole L; Van Schaftingen, Emile

    2007-01-01

    Vitamin C, a reducing agent and antioxidant, is a cofactor in reactions catalyzed by Cu(+)-dependent monooxygenases and Fe(2+)-dependent dioxygenases. It is synthesized, in vertebrates having this capacity, from d-glucuronate. The latter is formed through direct hydrolysis of uridine diphosphate (UDP)-glucuronate by enzyme(s) bound to the endoplasmic reticulum membrane, sharing many properties with, and most likely identical to, UDP-glucuronosyltransferases. Non-glucuronidable xenobiotics (aminopyrine, metyrapone, chloretone and others) stimulate the enzymatic hydrolysis of UDP-glucuronate, accounting for their effect to increase vitamin C formation in vivo. Glucuronate is converted to l-gulonate by aldehyde reductase, an enzyme of the aldo-keto reductase superfamily. l-Gulonate is converted to l-gulonolactone by a lactonase identified as SMP30 or regucalcin, whose absence in mice leads to vitamin C deficiency. The last step in the pathway of vitamin C synthesis is the oxidation of l-gulonolactone to l-ascorbic acid by l-gulonolactone oxidase, an enzyme associated with the endoplasmic reticulum membrane and deficient in man, guinea pig and other species due to mutations in its gene. Another fate of glucuronate is its conversion to d-xylulose in a five-step pathway, the pentose pathway, involving identified oxidoreductases and an unknown decarboxylase. Semidehydroascorbate, a major oxidation product of vitamin C, is reconverted to ascorbate in the cytosol by cytochrome b(5) reductase and thioredoxin reductase in reactions involving NADH and NADPH, respectively. Transmembrane electron transfer systems using ascorbate or NADH as electron donors serve to reduce semidehydroascorbate present in neuroendocrine secretory vesicles and in the extracellular medium. Dehydroascorbate, the fully oxidized form of vitamin C, is reduced spontaneously by glutathione, as well as enzymatically in reactions using glutathione or NADPH. The degradation of vitamin C in mammals is

  1. Evolution of plant δ 1-pyrroline-5-carboxylate reductases from phylogenetic and structural perspectives

    DOE PAGES

    Forlani, Giuseppe; Makarova, Kira S.; Ruszkowski, Milosz; ...

    2015-08-03

    Proline plays a crucial role in cell growth and stress responses, and its accumulation is essential for the tolerance of adverse environmental conditions in plants. Two routes are used to biosynthesize proline in plants. The main route uses glutamate as a precursor, while in the other route proline is derived from ornithine. The terminal step of both pathways, the conversion of δ 1-pyrroline-5-carboxylate (P5C) to L-proline, is catalyzed by P5C reductase (P5CR) using NADH or NADPH as a cofactor. Since P5CRs are important housekeeping enzymes, they are conserved across all domains of life and appear to be relatively unaffected throughoutmore » evolution. However, global analysis of these enzymes unveiled significant functional diversity in the preference for cofactors (NADPH vs. NADH), variation in metal dependence and the differences in the oligomeric state. In our study we investigated evolutionary patterns through phylogenetic and structural analysis of P5CR representatives from all kingdoms of life, with emphasis on the plant species. We attempted to correlate local sequence/structure variation among the functionally and structurally characterized members of the family.« less

  2. NADPH Oxidases in Vascular Pathology

    PubMed Central

    Konior, Anna; Schramm, Agata; Czesnikiewicz-Guzik, Marta

    2014-01-01

    Abstract Significance: Reactive oxygen species (ROS) play a critical role in vascular disease. While there are many possible sources of ROS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases play a central role. They are a source of “kindling radicals,” which affect other enzymes, such as nitric oxide synthase endothelial nitric oxide synthase or xanthine oxidase. This is important, as risk factors for atherosclerosis (hypertension, diabetes, hypercholesterolemia, and smoking) regulate the expression and activity of NADPH oxidases in the vessel wall. Recent Advances: There are seven isoforms in mammals: Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2. Nox1, Nox2, Nox4, and Nox5 are expressed in endothelium, vascular smooth muscle cells, fibroblasts, or perivascular adipocytes. Other homologues have not been found or are expressed at very low levels; their roles have not been established. Nox1/Nox2 promote the development of endothelial dysfunction, hypertension, and inflammation. Nox4 may have a role in protecting the vasculature during stress; however, when its activity is increased, it may be detrimental. Calcium-dependent Nox5 has been implicated in oxidative damage in human atherosclerosis. Critical Issues: NADPH oxidase-derived ROS play a role in vascular pathology as well as in the maintenance of normal physiological vascular function. We also discuss recently elucidated mechanisms such as the role of NADPH oxidases in vascular protection, vascular inflammation, pulmonary hypertension, tumor angiogenesis, and central nervous system regulation of vascular function and hypertension. Future Directions: Understanding the role of individual oxidases and interactions between homologues in vascular disease is critical for efficient pharmacological regulation of vascular NADPH oxidases in both the laboratory and clinical practice. Antioxid. Redox Signal. 20, 2794–2814. PMID:24180474

  3. The endogenous adrenodoxin reductase-like flavoprotein arh1 supports heterologous cytochrome P450-dependent substrate conversions in Schizosaccharomyces pombe.

    PubMed

    Ewen, Kerstin M; Schiffler, Burkhard; Uhlmann-Schiffler, Heike; Bernhardt, Rita; Hannemann, Frank

    2008-05-01

    Mitochondrial cytochromes P450 are essential for biosynthesis of steroid hormones, vitamin D and bile acids. In mammals, the electrons needed for these reactions are provided via adrenodoxin and adrenodoxin reductase (AdR). Recently, Schizosaccharomyces pombe was introduced as a new host for the functional expression of human mitochondrial steroid hydroxylases without the coexpression of their natural redox partners. This fact qualifies S. pombe for the biotechnological production of steroids and for application as inhibitor test organism of heterologously expressed cytochromes P450. In this paper, we present evidence that the S. pombe ferredoxin reductase, arh1, and ferredoxin, etp1fd provide mammalian class I cytochromes P450 with reduction equivalents. The recombinant reductase showed an unusual weak binding of flavin adenine dinucleotide (FAD), which was mastered by modifying the FAD-binding region by site-directed mutagenesis yielding a stable holoprotein. The modified reductase arh1_A18G displayed spectroscopic characteristics similar to AdR and was shown to be capable of accepting electrons with no evident preference for NADH or NADPH, respectively. Arh1_A18G can substitute for AdR by interacting not only with its natural redox partner etp1fd but also with the mammalian homolog adrenodoxin. Cytochrome P450-dependent substrate conversion with all combinations of the mammalian and yeast redox proteins was evaluated in a reconstituted system.

  4. Investigation of interaction between Pax-5 isoforms and thioredoxin using de novo modelling methods.

    PubMed

    Cuperlovic-Culf, Miroslava; Robichaud, Gilles A; Nardini, Michel; Ouellette, Rodney J

    2003-01-01

    Pax-5 transcription factor plays a crucial role in B-cell development, activation and differentiation. In murine B-cells four different isoforms of Pax-5 have been identified, and their role in the regulation of the activity of the wild-type protein was revealed although still not fully understood. Using theoretical methods, we investigated the properties of one region of the Pax-5e and Pax-5d isoforms (named UDE domain) and we present a possible theoretical model for the interaction of this domain with thioredoxin that have been previously postulated based on the experimental results. Domain UDE (MW 4.8 kDa) is characterised by an extremely high ratio of positively charged residues (8) in comparisons to negatively charged amino acids (3), as well as unusually large concentrations of prolines (11.6%) and cysteines (4.7%). This is indicative of its role in protein-protein interaction. The experimental 3D structure for either UDE domain or for any analogous sequence is not yet available, and therefore we resorted to various bioinformatics methods in order to predict the secondary and 3D structure from the primary sequence of UDE. Physicochemical properties of the predicted UDE structure gave more indication about possibilities for UDE-thioredoxin binding. In addition, UDE domain was shown to have both sequence and structure analogous to a segment of NAD-reducing hydrogenase HOXS a subunit which is believed to interact with thioredoxin. These studies showed that the UDE domain in Pax-5d and Pax-5e represents an ideal binding site for thioredoxin and we developed a model of UDE-TRX complex with two disulphide bridges. The active site of thioredoxin remained exposed after binding to UDE in this model and therefore binding of thioredoxin to Pax-5d could explain the unexpectedly high resistance of this isoform to oxidation. The complex between thioredoxin and Pax-5e can be a method for transportation of thioredoxin into the nucleus and also into the the vicinity of Pax-5a

  5. Biallelic mutations in the ferredoxin reductase gene cause novel mitochondriopathy with optic atrophy

    PubMed Central

    Peng, Yanyan; Shinde, Deepali N; Valencia, C Alexander; Mo, Jun-Song; Rosenfeld, Jill; Truitt Cho, Megan; Chamberlin, Adam; Li, Zhuo; Liu, Jie; Gui, Baoheng; Brockhage, Rachel; Basinger, Alice; Alvarez-Leon, Brenda; Heydemann, Peter; Magoulas, Pilar L; Lewis, Andrea M; Scaglia, Fernando; Gril, Solange; Chong, Shuk Ching; Bower, Matthew; Monaghan, Kristin G; Willaert, Rebecca; Plona, Maria-Renee; Dineen, Rich; Milan, Francisca; Hoganson, George; Helbig, Katherine L; Keller-Ramey, Jennifer; Harris, Belinda; Anderson, Laura C; Green, Torrian; Sukoff Rizzo, Stacey J; Kaylor, Julie; Chen, Jiani; Guan, Min-Xin; Sellars, Elizabeth; Sparagana, Steven P; Gibson, James B; Reinholdt, Laura G; Tang, Sha; Huang, Taosheng

    2017-01-01

    Abstract Iron–sulfur (Fe-S) clusters are ubiquitous cofactors essential to various cellular processes, including mitochondrial respiration, DNA repair, and iron homeostasis. A steadily increasing number of disorders are being associated with disrupted biogenesis of Fe–S clusters. Here, we conducted whole-exome sequencing of patients with optic atrophy and other neurological signs of mitochondriopathy and identified 17 individuals from 13 unrelated families with recessive mutations in FDXR, encoding the mitochondrial membrane-associated flavoprotein ferrodoxin reductase required for electron transport from NADPH to cytochrome P450. In vitro enzymatic assays in patient fibroblast cells showed deficient ferredoxin NADP reductase activity and mitochondrial dysfunction evidenced by low oxygen consumption rates (OCRs), complex activities, ATP production and increased reactive oxygen species (ROS). Such defects were rescued by overexpression of wild-type FDXR. Moreover, we found that mice carrying a spontaneous mutation allelic to the most common mutation found in patients displayed progressive gait abnormalities and vision loss, in addition to biochemical defects consistent with the major clinical features of the disease. Taken together, these data provide the first demonstration that germline, hypomorphic mutations in FDXR cause a novel mitochondriopathy and optic atrophy in humans. PMID:29040572

  6. Sulforaphane protects cortical neurons against 5-S-cysteinyl-dopamine-induced toxicity through the activation of ERK1/2, Nrf-2 and the upregulation of detoxification enzymes.

    PubMed

    Vauzour, David; Buonfiglio, Maria; Corona, Giulia; Chirafisi, Joselita; Vafeiadou, Katerina; Angeloni, Cristina; Hrelia, Silvana; Hrelia, Patrizia; Spencer, Jeremy P E

    2010-04-01

    The degeneration of dopaminergic neurons in the substantia nigra has been linked to the formation of the endogenous neurotoxin 5-S-cysteinyl-dopamine. Sulforaphane (SFN), an isothiocyanate derived from the corresponding precursor glucosinolate found in cruciferous vegetables has been observed to exert a range of biological activities in various cell populations. In this study, we show that SFN protects primary cortical neurons against 5-S-cysteinyl-dopamine induced neuronal injury. Pre-treatment of cortical neurons with SFN (0.01-1 microM) resulted in protection against 5-S-cysteinyl-dopamine-induced neurotoxicity, which peaked at 100 nM. This protection was observed to be mediated by the ability of SFN to modulate the extracellular signal-regulated kinase 1 and 2 and the activation of Kelch-like ECH-associated protein 1/NF-E2-related factor-2 leading to the increased expression and activity of glutathione-S-transferase (M1, M3 and M5), glutathione reductase, thioredoxin reductase and NAD(P)H oxidoreductase 1. These data suggest that SFN stimulates the NF-E2-related factor-2 pathway of antioxidant gene expression in neurons and may protect against neuronal injury relevant to the aetiology of Parkinson's disease.

  7. NADPH oxidases of the brain: distribution, regulation, and function.

    PubMed

    Infanger, David W; Sharma, Ram V; Davisson, Robin L

    2006-01-01

    The NADPH oxidase is a multi-subunit enzyme that catalyzes the reduction of molecular oxygen to form superoxide (O(2)(-)). While classically linked to the respiratory burst in neutrophils, recent evidence now shows that O(2)(-) (and associated reactive oxygen species, ROS) generated by NADPH oxidase in nonphagocytic cells serves myriad functions in health and disease. An entire new family of NADPH Oxidase (Nox) homologues has emerged, which vary widely in cell and tissue distribution, as well as in function and regulation. A major concept in redox signaling is that while NADPH oxidase-derived ROS are necessary for normal cellular function, excessive oxidative stress can contribute to pathological disease. This certainly is true in the central nervous system (CNS), where normal NADPH oxidase function appears to be required for processes such as neuronal signaling, memory, and central cardiovascular homeostasis, but overproduction of ROS contributes to neurotoxicity, neurodegeneration, and cardiovascular diseases. Despite implications of NADPH oxidase in normal and pathological CNS processes, still relatively little is known about the mechanisms involved. This paper summarizes the evidence for NADPH oxidase distribution, regulation, and function in the CNS, emphasizing the diversity of Nox isoforms and their new and emerging role in neuro-cardiovascular function. In addition, perspectives for future research and novel therapeutic targets are offered.

  8. The role of thioredoxin h in protein metabolism during wheat (Triticum aestivum L.) seed germination.

    PubMed

    Guo, Hongxiang; Wang, Shaoxin; Xu, Fangfang; Li, Yongchun; Ren, Jiangping; Wang, Xiang; Niu, Hongbin; Yin, Jun

    2013-06-01

    Thioredoxin h can regulate the redox environment in the cell and play an important role in the germination of cereals. In the present study, the thioredoxin s antisense transgenic wheat with down-regulation of thioredoxin h was used to study the role of thioredoxin h in protein metabolism during germination of wheat seeds, and to explore the mechanism of the thioredoxin s antisense transgenic wheat seeds having high resistance to pre-harvest sprouting. The qRT-PCR results showed that the expression of protein disulfide isomerase in the thioredoxin s antisense transgenic wheat was up-regulated, which induced easily forming glutenin macropolymers and the resistance of storage proteins to degradation. The expression of serine protease inhibitor was also up-regulated in transgenic wheat, which might be responsible for the decreased activity of thiocalsin during the germination. The expression of WRKY6 in transgenic wheat was down-regulated, which was consistent with the decreased activity of glutamine oxoglutarate aminotransferase. In transgenic wheat, the activities of glutamate dehydrogenase, glutamic pyruvic transaminase and glutamic oxaloacetic transaminase were down-regulated, indicating that the metabolism of amino acid was lower than that in wild-type wheat during seed germination. A putative model for the role of thioredoxin h in protein metabolism during wheat seed germination was proposed and discussed. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  9. The oxygen evolving enhancer protein 1 (OEE) of photosystem II in green algae exhibits thioredoxin activity.

    PubMed

    Heide, Heinrich; Kalisz, Henryk M; Follmann, Hartmut

    2004-02-01

    A thioredoxin-like chloroplast protein of the fructosebisphosphatase-stimulating f-type, but with an unusually high molecular mass of 28 kDa has previously been identified and purified to homogeneity in a fractionation scheme for resolution of the acid- and heat-stable, regular-size (12kDa) thioredoxins of the unicellular green algae, Scenedesmus obliquus. An apparently analogous protein of 26 kDa was described in a cyanobacterium, Anabaena sp., but no such large thioredoxin species f exists in the thioredoxin profiles of higher plants. The structure of the 28 kDa protein, which had been envisaged to represent a precursor, or fusion product of the two more specialized, common chloroplast thioredoxins f and m has now been determined by amino acid sequencing. Although it exhibits virtually all the properties and enzyme-modulating activities of a thioredoxin proper this algal protein, surprisingly, does not belong to the thioredoxin family of small redox proteins but is identical with OEE (oxygen evolving enhancer) protein 1, an auxiliary component of the photosystem II manganese cluster. Extracts of Chlorella vulgaris and Chlamydomonas reinhardtii also contain heat-stable protein fractions of 23-26 kDa capable of specifically stimulating chloroplast fructosebisphosphatase in vitro. In contrast, OEE protein 1 from spinach is not able to modulate FbPase or NADP malate dehydrogenase from spinach chloroplasts. A dual function of the OEE protein in algal photosynthesis is envisaged.

  10. Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway.

    PubMed

    Tsuchiya, Ayako; Kaku, Yoshiko; Nakano, Takashi; Nishizaki, Tomoyuki

    2015-11-01

    1,2-Diarachidonoyl-sn-glycero-3-phosphoethanolamine (DAPE) induces both necrosis/necroptosis and apoptosis of NCI-H28 malignant pleural mesothelioma (MPM) cells. The present study was conducted to understand the mechanism for DAPE-induced apoptosis of NCI-H28 cells. DAPE induced caspase-independent apoptosis of NCI-H28 malignant pleural mesothelioma (MPM) cells, and the effect of DAPE was prevented by antioxidants or an inhibitor of NADPH oxidase (NOX). DAPE generated reactive oxygen species (ROS) and inhibited activity of thioredoxin (Trx) reductase (TrxR). DAPE decreased an association of apoptosis signal-regulating kinase 1 (ASK1) with thioredoxin (Trx), thereby releasing ASK1. DAPE activated p38 mitogen-activated protein kinase (MAPK), which was inhibited by an antioxidant or knocking-down ASK1. In addition, DAPE-induced NCI-H28 cell death was also prevented by knocking-down ASK1. Taken together, the results of the present study indicate that DAPE stimulates NOX-mediated ROS production and suppresses TrxR activity, resulting in the decrease of reduced Trx and the dissociation of ASK1 from a complex with Trx, allowing sequential activation of ASK1 and p38 MAPK, to induce apoptosis of NCI-H28 MPM cells. Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  11. Activation of Nrf2 target enzymes conferring protection against oxidative stress in PC12 cells by ginger principal constituent 6-shogaol.

    PubMed

    Peng, Shoujiao; Yao, Juan; Liu, Yaping; Duan, Dongzhu; Zhang, Xiaolong; Fang, Jianguo

    2015-08-01

    Natural compounds containing phenoxyl groups and/or Michael acceptor units appear to possess antioxidant and cytoprotective properties. The ginger principal constituent 6-shogaol (6-S) represents one of such compounds. In this study, we reported that 6-S efficiently scavenges various free radicals in vitro, and displays remarkable cytoprotection against oxidative stress-induced cell damage in the neuron-like rat pheochromocytoma cell line, PC12 cells. Pretreatment of PC12 cells with 6-S significantly upregulates a series of phase II antioxidant molecules, such as glutathione, heme oxygenase 1, NAD(P)H: quinone oxidoreductase 1, thioredoxin reductase 1, and thioredoxin 1. A mechanistic study revealed that 6-S enhanced the translocation of Nrf2 from the cytosol to the nucleus and knockdown of Nrf2 abolished such protection, indicating that this cytoprotection is mediated by the activation of the transcription factor Nrf2. Another ginger constituent 6-gingerol (6-G), having a similar structure of 6-S but lacking the alpha,beta-unsaturated ketone structure (Michael acceptor moiety), failed to shelter PC12 cells from oxidative stress. Our results demonstrate that 6-S is a novel small molecule activator of Nrf2 in PC12 cells, and suggest that 6-S might be a potential candidate for the prevention of oxidative stress-mediated neurodegenerative disorders.

  12. In vitro susceptibility of thioredoxins and glutathione to redox modification and aging-related changes in skeletal muscle

    PubMed Central

    Dimauro, Ivan; Pearson, Timothy; Caporossi, Daniela; Jackson, Malcolm J.

    2012-01-01

    Thioredoxins (Trx's) regulate redox signaling and are localized to various cellular compartments. Specific redox-regulated pathways for adaptation of skeletal muscle to contractions are attenuated during aging, but little is known about the roles of Trx's in regulating these pathways. This study investigated the susceptibility of Trx1 and Trx2 in skeletal muscle to oxidation and reduction in vitro and the effects of aging and contractions on Trx1, Trx2, and thioredoxin reductase (TrxR) 1 and 2 contents and nuclear and cytosolic Trx1 and mitochondrial Trx2 redox potentials in vivo. The proportions of cytosolic and nuclear Trx1 and mitochondrial Trx2 in the oxidized or reduced forms were analyzed using redox Western blotting. In myotubes, the mean redox potentials were nuclear Trx1, −251 mV; cytosolic Trx1, −242 mV; mitochondrial Trx2, −346 mV, data supporting the occurrence of differing redox potentials between cell compartments. Exogenous treatment of myoblasts and myotubes with hydrogen peroxide or dithiothreitol modified glutathione redox status and nuclear and cytosolic Trx1, but mitochondrial Trx2 was unchanged. Tibialis anterior muscles from young and old mice were exposed to isometric muscle contractions in vivo. Aging increased muscle contents of Trx1, Trx2, and TrxR2, but neither aging nor endogenous ROS generated during contractions modified Trx redox potentials, although oxidation of glutathione and other thiols occurred. We conclude that glutathione redox couples in skeletal muscle are more susceptible to oxidation than Trx and that Trx proteins are upregulated during aging, but do not appear to modulate redox-regulated adaptations to contractions that fail during aging. PMID:23022873

  13. Expression, purification, crystallization and preliminary X-ray analysis of conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708

    PubMed Central

    Yamamura, Akihiro; Maruoka, Shintaro; Ohtsuka, Jun; Miyakawa, Takuya; Nagata, Koji; Kataoka, Michihiko; Kitamura, Nahoko; Shimizu, Sakayu; Tanokura, Masaru

    2009-01-01

    Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708 is a member of the NADPH-dependent aldo-keto reductase (AKR) superfamily and catalyzes the stereospecific reduction of ketopantoyl lactone to d-pantoyl lactone. A diffraction-quality crystal of recombinant CPR-C2 was obtained by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystal diffracted X-rays to 1.7 Å resolution on beamline NW12A of the Photon Factory-Advanced Ring (Tsukuba, Japan). The crystal belonged to space group P212121, with unit-cell parameters a = 55.02, b = 68.30, c = 68.93 Å. The Matthews coefficient (V M = 1.76 Å3 Da−1) indicated that the crystal contained one CPR-C2 molecule per asymmetric unit. PMID:19923737

  14. Identification of 4-Deoxy-L-Etychro-Hexoseulose Uronic Acid Reductases in an Alginolytic Bacterium Vibrio splendidus and their Uses for L-Lactate Production in an Escherichia coli Cell-Free System.

    PubMed

    Lee, Eun Jeong; Lee, Ok Kyung; Lee, Eun Yeol

    2018-06-01

    4-Deoxy-L-erythro-hexoseulose uronic acid (DEH) reductase is a key enzyme in alginate utilizing metabolism, but the number of characterized DEH reductase is quite limited. In this study, novel two DEH reductases, VsRed-1 and VsRed-2, were identified in marine bacterium Vibrio splendidus, and the recombinant enzymes were expressed in an Escherichia coli system and purified by Ni-NTA chromatography. The optimal pH and temperature of the recombinant VsRed-1 and VsRed-2 were pH 7.5, 30 °C, and pH 7.0, 35 °C, respectively. The specific activities of VsRed-1 (776 U/mg for NADH) and VsRed-2 (176 U/mg for NADPH) were the highest among the DEH reductases reported so far. We also demonstrated that DEH could be converted to L-lactate with a yield of 76.7 and 81.9% in E. coli cell-free system containing VsRed-1 and VsRed-2 enzymes, respectively, indicating that two DEH reductases can be employed for production of biofuels and bio-chemicals from brown macroalgae biomass.

  15. Purification, Characterization, and Overexpression of Flavin Reductase Involved in Dibenzothiophene Desulfurization by Rhodococcus erythropolis D-1

    PubMed Central

    Matsubara, Toshiyuki; Ohshiro, Takashi; Nishina, Yoshihiro; Izumi, Yoshikazu

    2001-01-01

    The dibenzothiophene (DBT)-desulfurizing bacterium, Rhodococcus erythropolis D-1, removes sulfur from DBT to form 2-hydroxybiphenyl using four enzymes, DszC, DszA, DszB, and flavin reductase. In this study, we purified and characterized the flavin reductase from R. erythropolis D-1 grown in a medium containing DBT as the sole source of sulfur. It is conceivable that the enzyme is essential for two monooxygenase (DszC and DszA) reactions in vivo. The purified flavin reductase contains no chromogenic cofactors and was found to have a molecular mass of 86 kDa and four identical 22-kDa subunits. The enzyme catalyzed NADH-dependent reduction of flavin mononucleotide (FMN), and the Km values for NADH and FMN were 208 and 10.8 μM, respectively. Flavin adenine dinucleotide was a poor substrate, and NADPH was inert. The enzyme did not catalyze reduction of any nitroaromatic compound. The optimal temperature and optimal pH for enzyme activity were 35°C and 6.0, respectively, and the enzyme retained 30% of its activity after heat treatment at 80°C for 30 min. The N-terminal amino acid sequence of the purified flavin reductase was identical to that of DszD of R. erythropolis IGTS8 (K. A. Gray, O. S. Pogrebinsky, G. T. Mrachko, L. Xi, D. J. Monticello, and C. H. Squires, Nat. Biotechnol. 14:1705–1709, 1996). The flavin reductase gene was amplified with primers designed by using dszD of R. erythropolis IGTS8, and the enzyme was overexpressed in Escherichia coli. The specific activity in crude extracts of the overexpressed strain was about 275-fold that of the wild-type strain. PMID:11229908

  16. Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase

    PubMed Central

    Trigoso, Yvonne D.; Evans, Russell C.; Karsten, William E.; Chooback, Lilian

    2016-01-01

    The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5’and 3’ terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40–50 mgs of protein, an improvement on the previous protein expression and multistep purification. PMID:26815040

  17. Maneb and Paraquat-Mediated Neurotoxicity: Involvement of Peroxiredoxin/Thioredoxin System

    PubMed Central

    Roede, James R.; Hansen, Jason M.; Go, Young-Mi; Jones, Dean P.

    2011-01-01

    Epidemiological and in vivo studies have demonstrated that exposure to the pesticides paraquat (PQ) and maneb (MB) increase the risk of developing Parkinson’s disease (PD) and cause dopaminergic cell loss, respectively. PQ is a well-recognized cause of oxidative toxicity; therefore, the purpose of this study was to determine if MB potentiates oxidative stress caused by PQ, thus providing a mechanism for enhanced neurotoxicity by the combination. The results show that PQ alone at a moderately toxic dose (20–30% cell death in 24 h) caused increased reactive oxygen species (ROS) generation, oxidation of mitochondrial thioredoxin-2 and peroxiredoxin-3, lesser oxidation of cytoplasmic thioredoxin-1 and peroxiredoxin-1, and no oxidation of cellular GSH/GSSG. In contrast, MB alone at a similar toxic dose resulted in no ROS generation, no oxidation of thioredoxin and peroxiredoxin, and an increase in cellular GSH after 24 h. Together, MB increased GSH and inhibited ROS production and thioredoxin/peroxiredoxin oxidation observed with PQ alone, yet resulted in more extensive (> 50%) cell death. MB treatment resulted in increased abundance of nuclear Nrf2 and mRNA for phase II enzymes under the control of Nrf2, indicating activation of cell protective responses. The results show that MB potentiation of PQ neurotoxicity does not occur by enhancing oxidative stress and suggests that increased toxicity occurs by a combination of divergent mechanisms, perhaps involving alkylation by MB and oxidation by PQ. PMID:21402726

  18. A gold-containing drug against parasitic polyamine metabolism: the X-ray structure of trypanothione reductase from Leishmania infantum in complex with auranofin reveals a dual mechanism of enzyme inhibition.

    PubMed

    Ilari, Andrea; Baiocco, Paola; Messori, Luigi; Fiorillo, Annarita; Boffi, Alberto; Gramiccia, Marina; Di Muccio, Trentina; Colotti, Gianni

    2012-02-01

    Auranofin is a gold(I)-containing drug in clinical use as an antiarthritic agent. Recent studies showed that auranofin manifests interesting antiparasitic actions very likely arising from inhibition of parasitic enzymes involved in the control of the redox metabolism. Trypanothione reductase is a key enzyme of Leishmania infantum polyamine-dependent redox metabolism, and a validated target for antileishmanial drugs. As trypanothione reductase contains a dithiol motif at its active site and gold(I) compounds are known to be highly thiophilic, we explored whether auranofin might behave as an effective enzyme inhibitor and as a potential antileishmanial agent. Notably, enzymatic assays revealed that auranofin causes indeed a pronounced enzyme inhibition. To gain a deeper insight into the molecular basis of enzyme inhibition, crystals of the auranofin-bound enzyme, in the presence of NADPH, were prepared, and the X-ray crystal structure of the auranofin-trypanothione reductase-NADPH complex was solved at 3.5 Å resolution. In spite of the rather low resolution, these data were of sufficient quality as to identify the presence of the gold center and of the thiosugar of auranofin, and to locate them within the overall protein structure. Gold binds to the two active site cysteine residues of TR, i.e. Cys52 and Cys57, while the thiosugar moiety of auranofin binds to the trypanothione binding site; thus auranofin appears to inhibit TR through a dual mechanism. Auranofin kills the promastigote stage of L. infantum at micromolar concentration; these findings will contribute to the design of new drugs against leishmaniasis.

  19. Characterization of WY 14,643 and its Complex with Aldose Reductase

    DOE PAGES

    Sawaya, Michael R.; Verma, Malkhey; Balendiran, Vaishnavi; ...

    2016-10-10

    The peroxisome proliferator, WY 14,643 exhibits a pure non-competitive inhibition pattern in the aldehyde reduction and in alcohol oxidation activities of human Aldose reductase (hAR). Fluorescence emission measurements of the equilibrium dissociation constants, Kd, of oxidized (hAR•NADP+) and reduced (hAR•NADPH) holoenzyme complexes display a 2-fold difference between them. Kd values for the dissociation of WY 14,643 from the oxidized (hAR•NADP+•WY 14,643) and reduced (hAR•NADPH•WY 14,643) ternary complexes are comparable to each other. The ternary complex structure of hAR•NADP+•WY 14,643 reveals the first structural evidence of a fibrate class drug binding to hAR. These observations demonstrate how fibrate molecules such asmore » WY 14,643, besides being valued as agonists for PPAR, also inhibit hAR.« less

  20. Characterization of WY 14,643 and its Complex with Aldose Reductase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sawaya, Michael R.; Verma, Malkhey; Balendiran, Vaishnavi

    The peroxisome proliferator, WY 14,643 exhibits a pure non-competitive inhibition pattern in the aldehyde reduction and in alcohol oxidation activities of human Aldose reductase (hAR). Fluorescence emission measurements of the equilibrium dissociation constants, Kd, of oxidized (hAR•NADP+) and reduced (hAR•NADPH) holoenzyme complexes display a 2-fold difference between them. Kd values for the dissociation of WY 14,643 from the oxidized (hAR•NADP+•WY 14,643) and reduced (hAR•NADPH•WY 14,643) ternary complexes are comparable to each other. The ternary complex structure of hAR•NADP+•WY 14,643 reveals the first structural evidence of a fibrate class drug binding to hAR. These observations demonstrate how fibrate molecules such asmore » WY 14,643, besides being valued as agonists for PPAR, also inhibit hAR.« less

  1. Characterization of WY 14,643 and its Complex with Aldose Reductase

    PubMed Central

    Sawaya, Michael R.; Verma, Malkhey; Balendiran, Vaishnavi; Rath, Nigam P.; Cascio, Duilio; Balendiran, Ganesaratnam K.

    2016-01-01

    The peroxisome proliferator, WY 14,643 exhibits a pure non-competitive inhibition pattern in the aldehyde reduction and in alcohol oxidation activities of human Aldose reductase (hAR). Fluorescence emission measurements of the equilibrium dissociation constants, Kd, of oxidized (hAR•NADP+) and reduced (hAR•NADPH) holoenzyme complexes display a 2-fold difference between them. Kd values for the dissociation of WY 14,643 from the oxidized (hAR•NADP+•WY 14,643) and reduced (hAR•NADPH•WY 14,643) ternary complexes are comparable to each other. The ternary complex structure of hAR•NADP+•WY 14,643 reveals the first structural evidence of a fibrate class drug binding to hAR. These observations demonstrate how fibrate molecules such as WY 14,643, besides being valued as agonists for PPAR, also inhibit hAR. PMID:27721416

  2. Engineering Cofactor Preference of Ketone Reducing Biocatalysts: A Mutagenesis Study on a γ-Diketone Reductase from the Yeast Saccharomyces cerevisiae Serving as an Example

    PubMed Central

    Katzberg, Michael; Skorupa-Parachin, Nàdia; Gorwa-Grauslund, Marie-Françoise; Bertau, Martin

    2010-01-01

    The synthesis of pharmaceuticals and catalysts more and more relies on enantiopure chiral building blocks. These can be produced in an environmentally benign and efficient way via bioreduction of prochiral ketones catalyzed by dehydrogenases. A productive source of these biocatalysts is the yeast Saccharomyces cerevisiae, whose genome also encodes a reductase catalyzing the sequential reduction of the γ-diketone 2,5-hexanedione furnishing the diol (2S,5S)-hexanediol and the γ-hydroxyketone (5S)-hydroxy-2-hexanone in high enantio- as well as diastereoselectivity (ee and de >99.5%). This enzyme prefers NADPH as the hydrogen donating cofactor. As NADH is more stable and cheaper than NADPH it would be more effective if NADH could be used in cell-free bioreduction systems. To achieve this, the cofactor binding site of the dehydrogenase was altered by site-directed mutagenesis. The results show that the rational approach based on a homology model of the enzyme allowed us to generate a mutant enzyme having a relaxed cofactor preference and thus is able to use both NADPH and NADH. Results obtained from other mutants are discussed and point towards the limits of rationally designed mutants. PMID:20480039

  3. Current status of NADPH oxidase research in cardiovascular pharmacology.

    PubMed

    Rodiño-Janeiro, Bruno K; Paradela-Dobarro, Beatriz; Castiñeiras-Landeira, María Isabel; Raposeiras-Roubín, Sergio; González-Juanatey, José R; Alvarez, Ezequiel

    2013-01-01

    The implications of reactive oxygen species in cardiovascular disease have been known for some decades. Rationally, therapeutic antioxidant strategies combating oxidative stress have been developed, but the results of clinical trials have not been as good as expected. Therefore, to move forward in the design of new therapeutic strategies for cardiovascular disease based on prevention of production of reactive oxygen species, steps must be taken on two fronts, ie, comprehension of reduction-oxidation signaling pathways and the pathophysiologic roles of reactive oxygen species, and development of new, less toxic, and more selective nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors, to clarify both the role of each NADPH oxidase isoform and their utility in clinical practice. In this review, we analyze the value of NADPH oxidase as a therapeutic target for cardiovascular disease and the old and new pharmacologic agents or strategies to prevent NADPH oxidase activity. Some inhibitors and different direct or indirect approaches are available. Regarding direct NADPH oxidase inhibition, the specificity of NADPH oxidase is the focus of current investigations, whereas the chemical structure-activity relationship studies of known inhibitors have provided pharmacophore models with which to search for new molecules. From a general point of view, small-molecule inhibitors are preferred because of their hydrosolubility and oral bioavailability. However, other possibilities are not closed, with peptide inhibitors or monoclonal antibodies against NADPH oxidase isoforms continuing to be under investigation as well as the ongoing search for naturally occurring compounds. Likewise, some different approaches include inhibition of assembly of the NADPH oxidase complex, subcellular translocation, post-transductional modifications, calcium entry/release, electron transfer, and genetic expression. High-throughput screens for any of these activities could provide new

  4. Current status of NADPH oxidase research in cardiovascular pharmacology

    PubMed Central

    Rodiño-Janeiro, Bruno K; Paradela-Dobarro, Beatriz; Castiñeiras-Landeira, María Isabel; Raposeiras-Roubín, Sergio; González-Juanatey, José R; Álvarez, Ezequiel

    2013-01-01

    The implications of reactive oxygen species in cardiovascular disease have been known for some decades. Rationally, therapeutic antioxidant strategies combating oxidative stress have been developed, but the results of clinical trials have not been as good as expected. Therefore, to move forward in the design of new therapeutic strategies for cardiovascular disease based on prevention of production of reactive oxygen species, steps must be taken on two fronts, ie, comprehension of reduction-oxidation signaling pathways and the pathophysiologic roles of reactive oxygen species, and development of new, less toxic, and more selective nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors, to clarify both the role of each NADPH oxidase isoform and their utility in clinical practice. In this review, we analyze the value of NADPH oxidase as a therapeutic target for cardiovascular disease and the old and new pharmacologic agents or strategies to prevent NADPH oxidase activity. Some inhibitors and different direct or indirect approaches are available. Regarding direct NADPH oxidase inhibition, the specificity of NADPH oxidase is the focus of current investigations, whereas the chemical structure-activity relationship studies of known inhibitors have provided pharmacophore models with which to search for new molecules. From a general point of view, small-molecule inhibitors are preferred because of their hydrosolubility and oral bioavailability. However, other possibilities are not closed, with peptide inhibitors or monoclonal antibodies against NADPH oxidase isoforms continuing to be under investigation as well as the ongoing search for naturally occurring compounds. Likewise, some different approaches include inhibition of assembly of the NADPH oxidase complex, subcellular translocation, post-transductional modifications, calcium entry/release, electron transfer, and genetic expression. High-throughput screens for any of these activities could provide new

  5. Inhibition of Human Vascular NADPH Oxidase by Apocynin Derived Oligophenols

    PubMed Central

    Mora-Pale, Mauricio; Weïwer, Michel; Yu, Jingjing; Linhardt, Robert J.; Dordick, Jonathan S.

    2009-01-01

    Enzymatic oxidation of apocynin, which may mimic in vivo metabolism, affords a large number of oligomers (apocynin oxidation products, AOP) that inhibit vascular NADPH oxidase. In vitro studies of NADPH oxidase activity were performed to identify active inhibitors, resulting in a trimer hydroxylated quinone (IIIHyQ) that inhibited NADPH oxidase with an IC50 = 31 nM. Apocynin itself possessed minimal inhibitory activity. NADPH oxidase is believed to be inhibited through prevention of the interaction between two NADPH oxidase subunits, p47phox and p22phox. To that end, while apocynin was unable to block the interaction of his-tagged p47phox with a surface immobilized biotinalyted p22phox peptide, the IIIHyQ product strongly interfered with this interaction (apparent IC50 = 1.6 μM). These results provide evidence that peroxidase-catalyzed AOP, which consist of oligomeric phenols and quinones, inhibit critical interactions that are involved in the assembly and activation of human vascular NADPH oxidase. PMID:19523836

  6. Impaired cross-talk between the thioredoxin and glutathione systems is related to ASK-1 mediated apoptosis in neuronal cells exposed to mercury.

    PubMed

    Branco, Vasco; Coppo, Lucia; Solá, Susana; Lu, Jun; Rodrigues, Cecília M P; Holmgren, Arne; Carvalho, Cristina

    2017-10-01

    Mercury (Hg) compounds target both cysteine (Cys) and selenocysteine (Sec) residues in peptides and proteins. Thus, the components of the two major cellular antioxidant systems - glutathione (GSH) and thioredoxin (Trx) systems - are likely targets for mercurials. Hg exposure results in GSH depletion and Trx and thioredoxin reductase (TrxR) are prime targets for mercury. These systems have a wide-range of common functions and interaction between their components has been reported. However, toxic effects over both systems are normally treated as isolated events. To study how the interaction between the glutathione and thioredoxin systems is affected by Hg, human neuroblastoma (SH-SY5Y) cells were exposed to 1 and 5μM of inorganic mercury (Hg 2+ ), methylmercury (MeHg) or ethylmercury (EtHg) and examined for TrxR, GSH and Grx levels and activities, as well as for Trx redox state. Phosphorylation of apoptosis signalling kinase 1 (ASK1), caspase-3 activity and the number of apoptotic cells were evaluated to investigate the induction of Trx-mediated apoptotic cell death. Additionally, primary cerebellar neurons from mice depleted of mitochondrial Grx2 (mGrx2D) were used to examine the link between Grx activity and Trx function. Results showed that Trx was affected at higher exposure levels than TrxR, especially for EtHg. GSH levels were only significantly affected by exposure to a high concentration of EtHg. Depletion of GSH with buthionine sulfoximine (BSO) severely increased Trx oxidation by Hg. Notably, EtHg-induced oxidation of Trx was significantly enhanced in primary neurons of mGrx2D mice. Our results suggest that GSH/Grx acts as backups for TrxR in neuronal cells to maintain Trx turnover during Hg exposure, thus linking different mechanisms of molecular and cellular toxicity. Finally, Trx oxidation by Hg compounds was associated to apoptotic hallmarks, including increased ASK-1 phosphorylation, caspase-3 activation and increased number of apoptotic cells

  7. Comparison of in Vitro Bioactivation of Flutamide and Its Cyano Analogue: Evidence for Reductive Activation by Human NADPH:Cytochrome P450 Reductase

    PubMed Central

    Wen, Bo; Coe, Kevin J.; Rademacher, Peter; Fitch, William L.; Monshouwer, Mario; Nelson, Sidney D.

    2009-01-01

    Flutamide (FLU), a nonsteroidal antiandrogen drug widely used in the treatment of prostate cancer, has been associated with idiosyncratic hepatotoxicity in patients. It is proposed that bioactivation of FLU and subsequent binding of reactive metabolite(s) to cellular proteins play a causative role. A toxicogenomic study comparing FLU and its nitro to cyano analogue (CYA) showed that the nitroaromatic group of FLU enhanced cytotoxicity to hepatocytes, indicating that reduction of the nitroaromatic group may represent a potential route of FLU-induced hepatotoxicity [Coe et al. (2007) Chem. Res. Toxicol. 20, 1277–1290]. In the current study, we compared in vitro bioactivation of FLU and CYA in human liver microsomes and cryopreserved human hepatocytes. A nitroreduction metabolite FLU-6 was formed in liver microsomal incubations of FLU under atmospheric oxygen levels and, to a greater extent, under anaerobic conditions. Seven glutathione (GSH) adducts of FLU, FLU-G1–7, were tentatively identified in human liver microsomal incubations using liquid chromatography–tandem mass spectrometry (LC/MS/MS), while CYA formed only four corresponding GSH adducts, CYA-G1–4, under the same conditions. Of particular interest was the formation of FLU-G5–7 from FLU, where the nitroaromatic group of FLU was reduced to an amino group. A tentative pathway is that upon nitroreduction, the para-diamines undergo cytochrome P450 (P450)-catalyzed two-electron oxidations to form corresponding para-diimine intermediates that react with GSH to form GSH adducts FLU-G5–7, respectively. The identities of FLU-G5–7 were further confirmed by LC/MS/MS analyses of microsomal incubations of a synthesized standard FLU-6. In an attempt to identify enzymes involved in the nitroreduction of FLU, NADPH:cytochrome P450 reductase (CPR) was shown to reduce FLU to FLU-6 under both aerobic and anaerobic conditions. Furthermore, the formation of FLU-G5–7 was completely blocked by the addition of a

  8. Comparison of in vitro bioactivation of flutamide and its cyano analogue: evidence for reductive activation by human NADPH:cytochrome P450 reductase.

    PubMed

    Wen, Bo; Coe, Kevin J; Rademacher, Peter; Fitch, William L; Monshouwer, Mario; Nelson, Sidney D

    2008-12-01

    Flutamide (FLU), a nonsteroidal antiandrogen drug widely used in the treatment of prostate cancer, has been associated with idiosyncratic hepatotoxicity in patients. It is proposed that bioactivation of FLU and subsequent binding of reactive metabolite(s) to cellular proteins play a causative role. A toxicogenomic study comparing FLU and its nitro to cyano analogue (CYA) showed that the nitroaromatic group of FLU enhanced cytotoxicity to hepatocytes, indicating that reduction of the nitroaromatic group may represent a potential route of FLU-induced hepatotoxicity [Coe et al. (2007) Chem. Res. Toxicol. 20, 1277-1290]. In the current study, we compared in vitro bioactivation of FLU and CYA in human liver microsomes and cryopreserved human hepatocytes. A nitroreduction metabolite FLU-6 was formed in liver microsomal incubations of FLU under atmospheric oxygen levels and, to a greater extent, under anaerobic conditions. Seven glutathione (GSH) adducts of FLU, FLU-G1-7, were tentatively identified in human liver microsomal incubations using liquid chromatography-tandem mass spectrometry (LC/ MS/MS), while CYA formed only four corresponding GSH adducts, CYA-G1-4, under the same conditions. Of particular interest was the formation of FLU-G5-7 from FLU, where the nitroaromatic group of FLU was reduced to an amino group. A tentative pathway is that upon nitroreduction, the para-diamines undergo cytochrome P450 (P450)-catalyzed two-electron oxidations to form corresponding para-diimine intermediates that react with GSH to form GSH adducts FLU-G5-7, respectively. The identities of FLU-G5-7 were further confirmed by LC/MS/MS analyses of microsomal incubations of a synthesized standard FLU-6. In an attempt to identify enzymes involved in the nitroreduction of FLU, NADPH:cytochrome P450 reductase (CPR) was shown to reduce FLU to FLU-6 under both aerobic and anaerobic conditions. Furthermore, the formation of FLU-G5-7 was completely blocked by the addition of a reversible CPR

  9. ROS mediated selection for increased NADPH availability in Escherichia coli.

    PubMed

    Reynolds, Thomas S; Courtney, Colleen M; Erickson, Keesha E; Wolfe, Lisa M; Chatterjee, Anushree; Nagpal, Prashant; Gill, Ryan T

    2017-11-01

    The economical production of chemicals and fuels by microbial processes remains an intense area of interest in biotechnology. A key limitation in such efforts concerns the availability of key co-factors, in this case NADPH, required for target pathways. Many of the strategies pursued for increasing NADPH availability in Escherichia coli involve manipulations to the central metabolism, which can create redox imbalances and overall growth defects. In this study we used a reactive oxygen species based selection to search for novel methods of increasing NADPH availability. We report a loss of function mutation in the gene hdfR appears to increase NADPH availability in E. coli. Additionally, we show this excess NADPH can be used to improve the production of 3HP in E. coli. © 2017 Wiley Periodicals, Inc.

  10. Biallelic mutations in the ferredoxin reductase gene cause novel mitochondriopathy with optic atrophy.

    PubMed

    Peng, Yanyan; Shinde, Deepali N; Valencia, C Alexander; Mo, Jun-Song; Rosenfeld, Jill; Truitt Cho, Megan; Chamberlin, Adam; Li, Zhuo; Liu, Jie; Gui, Baoheng; Brockhage, Rachel; Basinger, Alice; Alvarez-Leon, Brenda; Heydemann, Peter; Magoulas, Pilar L; Lewis, Andrea M; Scaglia, Fernando; Gril, Solange; Chong, Shuk Ching; Bower, Matthew; Monaghan, Kristin G; Willaert, Rebecca; Plona, Maria-Renee; Dineen, Rich; Milan, Francisca; Hoganson, George; Powis, Zoe; Helbig, Katherine L; Keller-Ramey, Jennifer; Harris, Belinda; Anderson, Laura C; Green, Torrian; Sukoff Rizzo, Stacey J; Kaylor, Julie; Chen, Jiani; Guan, Min-Xin; Sellars, Elizabeth; Sparagana, Steven P; Gibson, James B; Reinholdt, Laura G; Tang, Sha; Huang, Taosheng

    2017-12-15

    Iron-sulfur (Fe-S) clusters are ubiquitous cofactors essential to various cellular processes, including mitochondrial respiration, DNA repair, and iron homeostasis. A steadily increasing number of disorders are being associated with disrupted biogenesis of Fe-S clusters. Here, we conducted whole-exome sequencing of patients with optic atrophy and other neurological signs of mitochondriopathy and identified 17 individuals from 13 unrelated families with recessive mutations in FDXR, encoding the mitochondrial membrane-associated flavoprotein ferrodoxin reductase required for electron transport from NADPH to cytochrome P450. In vitro enzymatic assays in patient fibroblast cells showed deficient ferredoxin NADP reductase activity and mitochondrial dysfunction evidenced by low oxygen consumption rates (OCRs), complex activities, ATP production and increased reactive oxygen species (ROS). Such defects were rescued by overexpression of wild-type FDXR. Moreover, we found that mice carrying a spontaneous mutation allelic to the most common mutation found in patients displayed progressive gait abnormalities and vision loss, in addition to biochemical defects consistent with the major clinical features of the disease. Taken together, these data provide the first demonstration that germline, hypomorphic mutations in FDXR cause a novel mitochondriopathy and optic atrophy in humans. © The Author 2017. Published by Oxford University Press.

  11. H2O2 Production in Species of the Lactobacillus acidophilus Group: a Central Role for a Novel NADH-Dependent Flavin Reductase

    PubMed Central

    Hertzberger, Rosanne; Arents, Jos; Dekker, Henk L.; Pridmore, R. David; Gysler, Christof; Kleerebezem, Michiel

    2014-01-01

    Hydrogen peroxide production is a well-known trait of many bacterial species associated with the human body. In the presence of oxygen, the probiotic lactic acid bacterium Lactobacillus johnsonii NCC 533 excretes up to 1 mM H2O2, inducing growth stagnation and cell death. Disruption of genes commonly assumed to be involved in H2O2 production (e.g., pyruvate oxidase, NADH oxidase, and lactate oxidase) did not affect this. Here we describe the purification of a novel NADH-dependent flavin reductase encoded by two highly similar genes (LJ_0548 and LJ_0549) that are conserved in lactobacilli belonging to the Lactobacillus acidophilus group. The genes are predicted to encode two 20-kDa proteins containing flavin mononucleotide (FMN) reductase conserved domains. Reductase activity requires FMN, flavin adenine dinucleotide (FAD), or riboflavin and is specific for NADH and not NADPH. The Km for FMN is 30 ± 8 μM, in accordance with its proposed in vivo role in H2O2 production. Deletion of the encoding genes in L. johnsonii led to a 40-fold reduction of hydrogen peroxide formation. H2O2 production in this mutant could only be restored by in trans complementation of both genes. Our work identifies a novel, conserved NADH-dependent flavin reductase that is prominently involved in H2O2 production in L. johnsonii. PMID:24487531

  12. Optical observation of correlated motions in dihydrofolate reductase

    NASA Astrophysics Data System (ADS)

    Xu, Mengyang; Niessen, Katherine; Pace, James; Cody, Vivian; Markelz, Andrea

    2015-03-01

    Enzyme function relies on its structural flexibility to make conformational changes for substrate binding and product release. An example of a metabolic enzyme where such structural changes are vital is dihydrofolate reductase (DHFR). DHFR is essential in both prokaryotes and eukaryotes for the nucleotide biosynthesis by catalyzing the reduction of dihydrofolate to tetrahydrofolate. NMR dynamical measurements found large amplitude fast dynamics that could indicate rigid-body, twisting-hinge motion for ecDHFR that may mediate flux. The role of such long-range correlated motions in function was suggested by the observed sharp decrease in enzyme activity for the single point mutation G121V, which is remote from active sites. This decrease in activity may be caused by the mutation interfering with the long-range intramolecular vibrations necessary for rapid access to functional configurations. We use our new technique of crystal anisotropy terahertz microscopy (CATM), to observe correlated motions in ecDHFR crystals with the bonding of NADPH and methotrexate. We compare the measured intramolecular vibrational spectrum with calculations using normal mode analysis.

  13. Exploiting algal NADPH oxidase for biophotovoltaic energy

    DOE PAGES

    Anderson, Alexander; Laohavisit, Anuphon; Blaby, Ian K.; ...

    2015-01-29

    Photosynthetic microbes exhibit light-dependent electron export across the cell membrane, which can generate electricity in biological photovoltaic (BPV) devices. How electrons are exported remains to be determined; the identification of mechanisms would help selection or generation of photosynthetic microbes capable of enhanced electrical output. We show that plasma membrane NADPH oxidase activity is a significant component of light-dependent generation of electricity by the unicellular green alga Chlamydomonas reinhardtii. NADPH oxidases export electrons across the plasma membrane to form superoxide anion from oxygen. The C. reinhardtii mutant lacking the NADPH oxidase encoded by RBO1 is impaired in both extracellular superoxide anionmore » production and current generation in a BPV device. Complementation with the wild-type gene restores both capacities, demonstrating the role of the enzyme in electron export. Monitoring light-dependent extracellular superoxide production with a colorimetric assay is shown to be an effective way of screening for electrogenic potential of candidate algal strains. Furthermore, the results show that algal NADPH oxidases are important for superoxide anion production and open avenues for optimizing the biological component of these devices.« less

  14. Expression, purification and enzymatic characterization of Brugia malayi dihydrofolate reductase.

    PubMed

    Perez-Abraham, Romy; Sanchez, Karla Garabiles; Alfonso, Melany; Gubler, Ueli; Siekierka, John J; Goodey, Nina M

    2016-12-01

    Brugia malayi (B. malayi) is one of the three causative agents of lymphatic filariasis, a neglected parasitic disease. Current literature suggests that dihydrofolate reductase is a potential drug target for the elimination of B. malayi. Here we report the recombinant expression and purification of a ∼20 kDa B. malayi dihydrofolate reductase (BmDHFR). A His6-tagged construct was expressed in E. coli and purified by affinity chromatography to yield active and homogeneous enzyme for steady-state kinetic characterization and inhibition studies. The catalytic activity kcat was found to be 1.4 ± 0.1 s(-1), the Michaelis Menten constant KM for dihydrofolate 14.7 ± 3.6 μM, and the equilibrium dissociation constant KD for NADPH 25 ± 24 nM. For BmDHFR, IC50 values for a six DHFR inhibitors were determined to be 3.1 ± 0.2 nM for methotrexate, 32 ± 22 μM for trimethoprim, 109 ± 34 μM for pyrimethamine, 154 ± 46 μM for 2,4-diaminoquinazoline, 771 ± 44 μM for cycloguanil, and >20,000 μM for 2,4-diaminopyrimidine. Our findings suggest that antifolate compounds can serve as inhibitors of BmDHFR. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. NADPH oxidases: novel therapeutic targets for neurodegenerative diseases.

    PubMed

    Gao, Hui-Ming; Zhou, Hui; Hong, Jau-Shyong

    2012-06-01

    Oxidative stress is a key pathologic factor in neurodegenerative diseases such as Alzheimer and Parkinson diseases (AD, PD). The failure of free-radical-scavenging antioxidants in clinical trials pinpoints an urgent need to identify and to block major sources of oxidative stress in neurodegenerative diseases. As a major superoxide-producing enzyme complex in activated phagocytes, phagocyte NADPH oxidase (PHOX) is essential for host defense. However, recent preclinical evidence has underscored a pivotal role of overactivated PHOX in chronic neuroinflammation and progressive neurodegeneration. Deficiency in PHOX subunits mitigates neuronal damage induced by diverse insults/stresses relevant to neurodegenerative diseases. More importantly, suppression of PHOX activity correlates with reduced neuronal impairment in models of neurodegenerative diseases. The discovery of PHOX and non-phagocyte NADPH oxidases in astroglia and neurons further reinforces the crucial role of NADPH oxidases in oxidative stress-mediated chronic neurodegeneration. Thus, proper modulation of NADPH oxidase activity might hold therapeutic potential for currently incurable neurodegenerative diseases. Published by Elsevier Ltd.

  16. X-ray structural studies of quinone reductase 2 nanomolar range inhibitors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pegan, Scott D.; Sturdy, Megan; Ferry, Gilles

    Quinone reductase 2 (QR2) is one of two members comprising the mammalian quinone reductase family of enzymes responsible for performing FAD mediated reductions of quinone substrates. In contrast to quinone reductase 1 (QR1) which uses NAD(P)H as its co-substrate, QR2 utilizes a rare group of hydride donors, N-methyl or N-ribosyl nicotinamide. Several studies have linked QR2 to the generation of quinone free radicals, several neuronal degenerative diseases, and cancer. QR2 has been also identified as the third melatonin receptor (MT3) through in cellulo and in vitro inhibition of QR2 by traditional MT3 ligands, and through recent X-ray structures of humanmore » QR2 (hQR2) in complex with melatonin and 2-iodomelatonin. Several MT3 specific ligands have been developed that exhibit both potent in cellulo inhibition of hQR2 nanomolar, affinity for MT3. The potency of these ligands suggest their use as molecular probes for hQR2. However, no definitive correlation between traditionally obtained MT3 ligand affinity and hQR2 inhibition exists limiting our understanding of how these ligands are accommodated in the hQR2 active site. To obtain a clearer relationship between the structures of developed MT3 ligands and their inhibitory properties, in cellulo and in vitro IC{sub 50} values were determined for a representative set of MT3 ligands (MCA-NAT, 2-I-MCANAT, prazosin, S26695, S32797, and S29434). Furthermore, X-ray structures for each of these ligands in complex with hQR2 were determined allowing for a structural evaluation of the binding modes of these ligands in relation to the potency of MT3 ligands.« less

  17. Resolving the cofactor-binding site in the proline biosynthetic enzyme human pyrroline-5-carboxylate reductase 1

    PubMed Central

    Christensen, Emily M.; Patel, Sagar M.; Korasick, David A.; Campbell, Ashley C.; Krause, Kurt L.; Becker, Donald F.; Tanner, John J.

    2017-01-01

    Pyrroline-5-carboxylate reductase (PYCR) is the final enzyme in proline biosynthesis, catalyzing the NAD(P)H-dependent reduction of Δ1-pyrroline-5-carboxylate (P5C) to proline. Mutations in the PYCR1 gene alter mitochondrial function and cause the connective tissue disorder cutis laxa. Furthermore, PYCR1 is overexpressed in multiple cancers, and the PYCR1 knock-out suppresses tumorigenic growth, suggesting that PYCR1 is a potential cancer target. However, inhibitor development has been stymied by limited mechanistic details for the enzyme, particularly in light of a previous crystallographic study that placed the cofactor-binding site in the C-terminal domain rather than the anticipated Rossmann fold of the N-terminal domain. To fill this gap, we report crystallographic, sedimentation-velocity, and kinetics data for human PYCR1. Structures of binary complexes of PYCR1 with NADPH or proline determined at 1.9 Å resolution provide insight into cofactor and substrate recognition. We see NADPH bound to the Rossmann fold, over 25 Å from the previously proposed site. The 1.85 Å resolution structure of a ternary complex containing NADPH and a P5C/proline analog provides a model of the Michaelis complex formed during hydride transfer. Sedimentation velocity shows that PYCR1 forms a concentration-dependent decamer in solution, consistent with the pentamer-of-dimers assembly seen crystallographically. Kinetic and mutational analysis confirmed several features seen in the crystal structure, including the importance of a hydrogen bond between Thr-238 and the substrate as well as limited cofactor discrimination. PMID:28258219

  18. Thioredoxins Play a Crucial Role in Dynamic Acclimation of Photosynthesis in Fluctuating Light.

    PubMed

    Thormählen, Ina; Zupok, Arkadiusz; Rescher, Josephin; Leger, Jochen; Weissenberger, Stefan; Groysman, Julia; Orwat, Anne; Chatel-Innocenti, Gilles; Issakidis-Bourguet, Emmanuelle; Armbruster, Ute; Geigenberger, Peter

    2017-01-09

    Sunlight represents the energy source for photosynthesis and plant growth. When growing in the field, plant photosynthesis has to manage strong fluctuations in light intensities. Regulation based on the thioredoxin (Trx) system is believed to ensure light-responsive control of photosynthetic reactions in the chloroplast. However, direct evidence for a role of this system in regulating dynamic acclimation of photosynthesis in fluctuating conditions is largely lacking. In this report we show that the ferredoxin-dependent Trxs m1 and m2 as well as the NADPH-dependent NTRC are both indispensable for photosynthetic acclimation in fluctuating light intensities. Arabidopsis mutants with combined deficiency in Trxs m1 and m2 show wild-type growth and photosynthesis under constant light condition, while photosynthetic parameters are strongly modified in rapidly alternating high and low light. Two independent trxm1m2 mutants show lower photosynthetic efficiency in high light, but surprisingly significantly higher photosynthetic efficiency in low light. Our data suggest that a main target of Trx m1 and m2 is the NADP-malate dehydrogenase involved in export of excess reductive power from the chloroplast. The decreased photosynthetic efficiency in the high-light peaks may thus be explained by a reduced capacity of the trxm1m2 mutants in the rapid light activation of this enzyme. In the ntrc mutant, dynamic responses of non-photochemical quenching of excitation energy and plastoquinone reduction state both were strongly attenuated in fluctuating light intensities, leading to a massive decrease in PSII quantum efficiency and a specific decrease in plant growth under these conditions. This is likely due to the decreased ability of the ntrc mutant to control the stromal NADP(H) redox poise. Taken together, our results indicate that NTRC is indispensable in ensuring the full range of dynamic responses of photosynthesis to optimize photosynthesis and maintain growth in fluctuating

  19. NO nerves in a tapeworm. NADPH-diaphorase histochemistry in adult Hymenolepis diminuta.

    PubMed

    Gustafsson, M K; Lindholm, A M; Terenina, N B; Reuter, M

    1996-12-01

    The free radical nitric oxide (NO), which is synthesized by nitric oxide synthase (NOS), has recently been discovered to function as a neuronal messenger. The presence of NOS was detected in the nervous system of adult Hymenolepis diminuta with NADPH-diaphorase (NADPH-d) histochemistry. The NADPH-d histochemical reaction is regarded as a selective marker for NOS in neuronal tissue. NADPH-d staining was observed in nerve fibres in the main and minor nerve cords and the transverse ring commissures, and in cell bodies in the brain commissure, along the main nerve cords, in the suckers and the rostellar sac. NADPH-d staining was also observed in the wall of the internal seminal vesicle and the genital atrium. The pattern of NADPH-d staining was compared with that of the 5-HT immunoreactive nervous elements. The NADPH-d staining reaction and the 5-HT immunoreactivity occur in separate sets of neurons. This is the first time the NADPH-d reaction has been demonstrated in the nervous system of a flatworm, indicating that NOS is present and that NO can be produced at this level of evolution.

  20. The Escherichia coli thioredoxin homolog YbbN/Trxsc is a chaperone and a weak protein oxidoreductase.

    PubMed

    Caldas, Thérèse; Malki, Abderrahim; Kern, Renée; Abdallah, Jad; Richarme, Gilbert

    2006-05-12

    Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, which presents a strong homology in its N-terminal part with thioredoxin 1 and 2. YbbN, however, does not possess the canonical Cys-x-x-Cys active site of thioredoxins, but instead a Ser-x-x-Cys site. In addition to Cys-38, located in the SxxC site, it contains a second cysteine, Cys-63, close to Cys-38 in the 3D model. Cys-38 and Cys-63 undergo an oxidoreduction process, suggesting that YbbN functions with two redox cysteines. Accordingly, YbbN catalyzes the oxidation of reduced RNase and the isomerization of scrambled RNase. Moreover, upon oxidation, its oligomeric state changes from dimers to tetramers and higher oligomers. YbbN also possesses chaperone properties, promoting protein folding after urea denaturation and forming complexes with unfolded proteins. This is the first biochemical characterization of a member of the YbbN class of bacterial thioredoxin-like proteins, and in vivo experiments will allow to determine the importance of its redox and chaperone properties in the cellular physiology.

  1. Crystal structures and atomic model of NADPH oxidase.

    PubMed

    Magnani, Francesca; Nenci, Simone; Millana Fananas, Elisa; Ceccon, Marta; Romero, Elvira; Fraaije, Marco W; Mattevi, Andrea

    2017-06-27

    NADPH oxidases (NOXs) are the only enzymes exclusively dedicated to reactive oxygen species (ROS) generation. Dysregulation of these polytopic membrane proteins impacts the redox signaling cascades that control cell proliferation and death. We describe the atomic crystal structures of the catalytic flavin adenine dinucleotide (FAD)- and heme-binding domains of Cylindrospermum stagnale NOX5. The two domains form the core subunit that is common to all seven members of the NOX family. The domain structures were then docked in silico to provide a generic model for the NOX family. A linear arrangement of cofactors (NADPH, FAD, and two membrane-embedded heme moieties) injects electrons from the intracellular side across the membrane to a specific oxygen-binding cavity on the extracytoplasmic side. The overall spatial organization of critical interactions is revealed between the intracellular loops on the transmembrane domain and the NADPH-oxidizing dehydrogenase domain. In particular, the C terminus functions as a toggle switch, which affects access of the NADPH substrate to the enzyme. The essence of this mechanistic model is that the regulatory cues conformationally gate NADPH-binding, implicitly providing a handle for activating/deactivating the very first step in the redox chain. Such insight provides a framework to the discovery of much needed drugs that selectively target the distinct members of the NOX family and interfere with ROS signaling.

  2. Fatty acyl-CoA reductases of birds

    PubMed Central

    2011-01-01

    Background Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR) catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters. Results cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba), domestic chicken (Gallus gallus domesticus) and domestic goose (Anser anser domesticus). Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland. Conclusion The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis. PMID:22151413

  3. Co-expression of human cytochrome P4501A1 (CYP1A1) variants and human NADPH-cytochrome P450 reductase in the baculovirus/insect cell system.

    PubMed

    Schwarz, D; Kisselev, P; Honeck, H; Cascorbi, I; Schunck, W H; Roots, I

    2001-06-01

    1. Three human cytochrome P4501A1 (CYP1A1) variants, wild-type (CYP1A1.1), CYP1A1.2 (1462V) and CYP1A1.4 (T461N), were co-expressed with human NADPH-P450 reductase (OR) in Spodoptera frugiperda (Sf9) insect cells by baculovirus co-infection to elaborate a suitable system for studying the role of CYPA1 polymorphism in the metabolism of exogenous and endogenous substrates. 2. A wide range of conditions was examined to optimize co-expression with regard to such parameters as relative multiplicity of infection (MOI), time of harvest, haem precursor supplementation and post-translational stabilization. tinder optimized conditions, almost identical expression levels and molar OR/CYP1A1 ratios (20:1) were attained for all CYP1A1 variants. 3. Microsomes isolated from co-infected cells demonstrated ethoxyresorufin deethlylase activities (nmol/min(-1) nmol(-1) CYP1A1) of 16.0 (CYP1A1.1), 20.5 (CYP1A1.2) and 22.5 (CYP1A1.4). Pentoxyresorufin was dealkylated approximately 10-20 times slower with all enzyme variants. 4. All three CYP1A1 variants were active in metabolizing the precarcinogen benzo[a]pyrene (B[a]P), with wild-type enzyme showing the highest activity, followed by CYP1A1.4 (60%) and CYP1A1.2 (40%). Each variant produced all major metabolites including B[a]P-7,8-dihydrodiol, the precursor of the ultimate carcinogenic species. 5. These studies demonstrate that the baculovirus-mediated co-expression-by-co-infection approach all CYP1A1 variants yields functionally active enzyme systems with similar molar OR/CYP1A1 ratios, thus providing suitable preconditions to examine the metabolism of and environmental chemicals by the different CY1A1 variants.

  4. The structure of Medicago truncatula δ 1-pyrroline-5-carboxylate reductase provides new insights into regulation of proline biosynthesis in plants

    DOE PAGES

    Ruszkowski, Milosz; Nocek, Boguslaw; Forlani, Giuseppe; ...

    2015-10-30

    The two pathways for proline biosynthesis in higher plants share the last step, the conversion of δ 1-pyrroline-5-carboxylate (P5C) to L-proline, which is catalyzed by P5C reductase (P5CR, EC 1.5.1.2) with the use of NAD(P)H as a coenzyme. There is increasing amount of evidence to suggest a complex regulation of P5CR activity at the post-translational level, yet the molecular basis of these mechanisms is unknown. Here we report the three-dimensional structure of the P5CR enzyme from the model legume Medicago truncatula (Mt). The crystal structures of unliganded MtP5CR decamer, and its complexes with the products NAD +, NADP +, andmore » L-proline were refined using x-ray diffraction data (at 1.7, 1.85, 1.95, and 2.1 Å resolution, respectively). Based on the presented structural data, the coenzyme preference for NADPH over NADH was explained, and NADPH is suggested to be the only coenzyme used by MtP5CR in vivo. Moreover, the insensitivity of MtP5CR to feed-back inhibition by proline, revealed by enzymatic analysis, was correlated with structural features. Additionally, a mechanism for the modulation of enzyme activity by chloride anions is discussed, as well as the rationale for the possible development of effective enzyme inhibitors.« less

  5. The role of extended Fe4S4 cluster ligands in mediating sulfite reductase hemoprotein activity.

    PubMed

    Cepeda, Marisa R; McGarry, Lauren; Pennington, Joseph M; Krzystek, J; Elizabeth Stroupe, M

    2018-05-28

    The siroheme-containing subunit from the multimeric hemoflavoprotein NADPH-dependent sulfite reductase (SiR/SiRHP) catalyzes the six electron-reduction of SO 3 2- to S 2- . Siroheme is an iron-containing isobacteriochlorin that is found in sulfite and homologous siroheme-containing nitrite reductases. Siroheme does not work alone but is covalently coupled to a Fe 4 S 4 cluster through one of the cluster's ligands. One long-standing hypothesis predicted from this observation is that the environment of one iron-containing cofactor influences the properties of the other. We tested this hypothesis by identifying three amino acids (F437, M444, and T477) that interact with the Fe 4 S 4 cluster and probing the effect of altering them to alanine on the function and structure of the resulting enzymes by use of activity assays, X-ray crystallographic analysis, and EPR spectroscopy. We showed that F437 and M444 gate access for electron transfer to the siroheme-cluster assembly and the direct hydrogen bond between T477 and one of the cluster sulfides is important for determining the geometry of the siroheme active site. Copyright © 2018. Published by Elsevier B.V.

  6. NADPH OXIDASE: STRUCTURE AND ACTIVATION MECHANISMS (REVIEW). NOTE I.

    PubMed

    Filip-Ciubotaru, Florina; Manciuc, Carmen; Stoleriu, Gabriela; Foia, Liliana

    2016-01-01

    NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase), with its generically termed NOX isoforms, is the major source of ROS (reactive oxigen species) in biological systems. ROS are small oxygen-derived molecules with an important role in various biological processes (physiological or pathological). If under physiological conditions some processes are beneficial and necessary for life, under pathophysiological conditions they are noxious, harmful. NADPH oxidases are present in phagocytes and in a wide variety of nonphagocytic cells. The enzyme generates superoxide by transferring electrons from NADPH inside the cell across the membrane and coupling them to molecular oxygen to produce superoxide anion, a reactive free-radical. Structurally, NADPH oxidase is a multicomponent enzyme which includes two integral membrane proteins, glycoprotein gp9 1 Phox and adaptor protein p22(phox), which together form the heterodimeric flavocytochrome b558 that constitutes the core of the enzyme. During the resting state, the multidomain regulatory subunits p40P(phox), p47(phox), p67(Phox) are located in the cytosol organized as a complex. The activation of phagocytic NADPH oxidase occurs through a complex series of protein interactions.

  7. Sunflower (Helianthus annuus) fatty acid synthase complex: enoyl-[acyl carrier protein]-reductase genes.

    PubMed

    González-Thuillier, Irene; Venegas-Calerón, Mónica; Garcés, Rafael; von Wettstein-Knowles, Penny; Martínez-Force, Enrique

    2015-01-01

    Enoyl-[acyl carrier protein]-reductases from sunflower. A major factor contributing to the amount of fatty acids in plant oils are the first steps of their synthesis. The intraplastidic fatty acid biosynthetic pathway in plants is catalysed by type II fatty acid synthase (FAS). The last step in each elongation cycle is carried out by the enoyl-[ACP]-reductase, which reduces the dehydrated product of β-hydroxyacyl-[ACP] dehydrase using NADPH or NADH. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus) seeds, two enoyl-[ACP]-reductase genes have been identified and cloned from developing seeds with 75 % identity: HaENR1 (GenBank HM021137) and HaENR2 (HM021138). The two genes belong to the ENRA and ENRB families in dicotyledons, respectively. The genetic duplication most likely originated after the separation of di- and monocotyledons. RT-qPCR revealed distinct tissue-specific expression patterns. Highest expression of HaENR1 was in roots, stems and developing cotyledons whereas that of H a ENR2 was in leaves and early stages of seed development. Genomic DNA gel blot analyses suggest that both are single-copy genes. In vivo activity of the ENR enzymes was tested by complementation experiments with the JP1111 fabI(ts) E. coli strain. Both enzymes were functional demonstrating that they interacted with the bacterial FAS components. That different fatty acid profiles resulted infers that the two Helianthus proteins have different structures, substrate specificities and/or reaction rates. The latter possibility was confirmed by in vitro analysis with affinity-purified heterologous-expressed enzymes that reduced the crotonyl-CoA substrate using NADH with different V max.

  8. Protein Chaperones Q8ZP25_SALTY from Salmonella Typhimurium and HYAE_ECOLI from Escherichia coli Exhibit Thioredoxin-like Structures Despite Lack of Canonical Thioredoxin Active Site Sequence Motif

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Parish, D.; Benach, J; Liu, G

    2008-01-01

    The structure of the 142-residue protein Q8ZP25 SALTY encoded in the genome of Salmonella typhimurium LT2 was determined independently by NMR and X-ray crystallography, and the structure of the 140-residue protein HYAE ECOLI encoded in the genome of Escherichia coli was determined by NMR. The two proteins belong to Pfam (Finn et al. 34:D247-D251, 2006) PF07449, which currently comprises 50 members, and belongs itself to the 'thioredoxin-like clan'. However, protein HYAE ECOLI and the other proteins of Pfam PF07449 do not contain the canonical Cys-X-X-Cys active site sequence motif of thioredoxin. Protein HYAE ECOLI was previously classified as a (NiFe)more » hydrogenase-1 specific chaperone interacting with the twin-arginine translocation (Tat) signal peptide. The structures presented here exhibit the expected thioredoxin-like fold and support the view that members of Pfam family PF07449 specifically interact with Tat signal peptides.« less

  9. Oligo-carrageenan kappa-induced reducing redox status and increase in TRR/TRX activities promote activation and reprogramming of terpenoid metabolism in Eucalyptus trees.

    PubMed

    González, Alberto; Gutiérrez-Cutiño, Marlen; Moenne, Alejandra

    2014-06-05

    In order to analyze whether the reducing redox status and activation of thioredoxin reductase (TRR)/thioredoxin(TRX) system induced by oligo-carrageenan (OC) kappa in Eucalyptus globulus activate secondary metabolism increasing terpenoid synthesis, trees were sprayed on the leaves with water, with OC kappa, or with inhibitors of NAD(P)H, ascorbate (ASC) and (GSH) synthesis and TRR activity, CHS-828, lycorine, buthionine sulfoximine (BSO) and auranofine, respectively, and with OC kappa and cultivated for four months. The main terpenoids in control Eucalyptus trees were eucalyptol (76%), α-pinene (7.4%), aromadendrene (3.6%), silvestrene (2.8%), sabinene (2%) and α-terpineol (0.9%). Treated trees showed a 22% increase in total essential oils as well as a decrease in eucalyptol (65%) and sabinene (0.8%) and an increase in aromadendrene (5%), silvestrene (7.8%) and other ten terpenoids. In addition, treated Eucalyptus showed seven de novo synthesized terpenoids corresponding to carene, α-terpinene, α-fenchene, γ-maaliene, spathulenol and α-camphenolic aldehyde. Most increased and de novo synthesized terpenoids have potential insecticidal and antimicrobial activities. Trees treated with CHS-828, lycorine, BSO and auranofine and with OC kappa showed an inhibition of increased and de novo synthesized terpenoids. Thus, OC kappa-induced reducing redox status and activation of TRR/TRX system enhance secondary metabolism increasing the synthesis of terpenoids and reprogramming of terpenoid metabolism in Eucalyptus trees.

  10. Thioredoxin targets in plants: The first 30 years

    USDA-ARS?s Scientific Manuscript database

    The turn of the century welcomed major developments in redox biology. In one development with plants, proteomics made possible the identification of proteins linked to thioredoxin (Trx), initially in chloroplasts and then other cell compartments. Two procedures, one based on thiol specific probes an...

  11. Disulfide proteome yields a detailed understanding of redox regulations: a model study of thioredoxin-linked reactions in seed germination.

    PubMed

    Yano, Hiroyuki; Kuroda, Masaharu

    2006-01-01

    Accumulating evidence suggests that redox regulations play important roles in a broad spectrum of biological processes. Recently, Yano et al. developed a disulfide proteome technique that comprehensively visualizes redox change in proteins. In this paper, using the disulfide proteome, we examined rice bran and identified fragments of embryo-specific protein and dienelactone hydrolase as putative targets of thioredoxin. Also, monitoring of the endogenous and recombinant effects of thioredoxin on rice bran proteins and supporting in vivo observations propose a mechanism of redox regulation in seed germination, in which thioredoxin activates cysteine protease with a concurrent unfolding of its substrate, the embryo-specific protein. Our findings suggest that thioredoxin controls the lifetime of specific proteins effectively by regulating the redox reactions coordinately. The model study demonstrates that the disulfide proteome technique is useful not only for identifying targets of thioredoxin, but also for clarify the detailed mechanism of redox regulation.

  12. Identification and functional evaluation of the reductases and dehydrogenases from Saccharomyces cerevisiae involved in vanillin resistance.

    PubMed

    Wang, Xinning; Liang, Zhenzhen; Hou, Jin; Bao, Xiaoming; Shen, Yu

    2016-04-01

    Vanillin, a type of phenolic released during the pre-treatment of lignocellulosic materials, is toxic to microorganisms and therefore its presence inhibits the fermentation. The vanillin can be reduced to vanillyl alcohol, which is much less toxic, by the ethanol producer Saccharomyces cerevisiae. The reducing capacity of S. cerevisiae and its vanillin resistance are strongly correlated. However, the specific enzymes and their contribution to the vanillin reduction are not extensively studied. In our previous work, an evolved vanillin-resistant strain showed an increased vanillin reduction capacity compared with its parent strain. The transcriptome analysis suggested the reductases and dehydrogenases of this vanillin resistant strain were up-regulated. Using this as a starting point, 11 significantly regulated reductases and dehydrogenases were selected in the present work for further study. The roles of these reductases and dehydrogenases in the vanillin tolerance and detoxification abilities of S. cerevisiae are described. Among the candidate genes, the overexpression of the alcohol dehydrogenase gene ADH6, acetaldehyde dehydrogenase gene ALD6, glucose-6-phosphate 1-dehydrogenase gene ZWF1, NADH-dependent aldehyde reductase gene YNL134C, and aldo-keto reductase gene YJR096W increased 177, 25, 6, 15, and 18 % of the strain μmax in the medium containing 1 g L(-1) vanillin. The in vitro detected vanillin reductase activities of strain overexpressing ADH6, YNL134C and YJR096W were notably higher than control. The vanillin specific reduction rate increased by 8 times in ADH6 overexpressed strain but not in YNL134C and YJR096W overexpressed strain. This suggested that the enzymes encoded by YNL134C and YJR096W might prefer other substrate and/or could not show their effects on vanillin on the high background of Adh6p in vivo. Overexpressing ALD6 and ZWF1 mainly increased the [NADPH]/[NADP(+)] and [GSH]/[GSSG] ratios but not the vanillin reductase activities. Their

  13. YNL134C from Saccharomyces cerevisiae encodes a novel protein with aldehyde reductase activity for detoxification of furfural derived from lignocellulosic biomass.

    PubMed

    Zhao, Xianxian; Tang, Juan; Wang, Xu; Yang, Ruoheng; Zhang, Xiaoping; Gu, Yunfu; Li, Xi; Ma, Menggen

    2015-05-01

    Furfural and 5-hydroxymethylfurfural (HMF) are the two main aldehyde compounds derived from pentoses and hexoses, respectively, during lignocellulosic biomass pretreatment. These two compounds inhibit microbial growth and interfere with subsequent alcohol fermentation. Saccharomyces cerevisiae has the in situ ability to detoxify furfural and HMF to the less toxic 2-furanmethanol (FM) and furan-2,5-dimethanol (FDM), respectively. Herein, we report that an uncharacterized gene, YNL134C, was highly up-regulated under furfural or HMF stress and Yap1p and Msn2/4p transcription factors likely controlled its up-regulated expression. Enzyme activity assays showed that YNL134C is an NADH-dependent aldehyde reductase, which plays a role in detoxification of furfural to FM. However, no NADH- or NADPH-dependent enzyme activity was observed for detoxification of HMF to FDM. This enzyme did not catalyse the reverse reaction of FM to furfural or FDM to HMF. Further studies showed that YNL134C is a broad-substrate aldehyde reductase, which can reduce multiple aldehydes to their corresponding alcohols. Although YNL134C is grouped into the quinone oxidoreductase family, no quinone reductase activity was observed using 1,2-naphthoquinone or 9,10-phenanthrenequinone as a substrate, and phylogenetic analysis indicates that it is genetically distant to quinone reductases. Proteins similar to YNL134C in sequence from S. cerevisiae and other microorganisms were phylogenetically analysed. Copyright © 2015 John Wiley & Sons, Ltd.

  14. A new member of the aldo-keto reductase family from the plant pathogen Xylella fastidiosa.

    PubMed

    Rosselli, Luciana K; Oliveira, Cristiano L P; Azzoni, Adriano R; Tada, Susely F S; Catani, Cleide F; Saraiva, Antonio M; Soares, José Sérgio M; Medrano, Francisco J; Torriani, Iris L; Souza, Anete P

    2006-09-15

    The Xylella fastidiosa genome program generated a large number of gene sequences that belong to pathogenicity, virulence and adaptation categories from this important plant pathogen. One of these genes (XF1729) encodes a protein similar to a superfamily of aldo-keto reductase together with a number of structurally and functionally related NADPH-dependent oxidoreductases. In this work, the similar sequence XF1729 from X. fastidiosa was cloned onto the pET32Xa/LIC vector in order to overexpress a recombinant His-tag fusion protein in Escherichia coli BL21(DE3). The expressed protein in the soluble fraction was purified by immobilized metal affinity chromatography (agarose-IDA-Ni resin). Secondary structure contents were verified by circular dichroism spectroscopy. Small angle X-ray scattering (SAXS) measurements furnish general structural parameters and provide a strong indication that the protein has a monomeric form in solution. Also, ab initio calculations show that the protein has some similarities with a previously crystallized aldo-keto reductase protein. The recombinant XF1729 purified to homogeneity catalyzed the reduction of dl-glyceraldehyde (K(cat) 2.26s(-1), Km 8.20+/-0.98 mM) and 2-nitrobenzaldehyde (K(cat) 11.74 s(-1), Km 0.14+/-0.04 mM) in the presence of NADPH. The amino acid sequence deduced from XF1729 showed the highest identity (40% or higher) with several functional unknown proteins. Among the identified AKRs, we found approximately 29% of identity with YakC (AKR13), 30 and 28% with AKR11A and AKR11B, respectively. The results establish XF1729 as the new member of AKR family, AKR13B1. Finally, the first characterization by gel filtration chromatography assays indicates that the protein has an elongated shape, which generates an apparent higher molecular weight. The study of this protein is an effort to fight X. fastidiosa, which causes tremendous losses in many economically important plants.

  15. Thermodynamic and NMR analyses of NADPH binding to lipocalin-type prostaglandin D synthase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Qin, Shubin; Shimamoto, Shigeru; Maruno, Takahiro

    2015-12-04

    Lipocalin-type prostaglandin D synthase (L-PGDS) is one of the most abundant proteins in human cerebrospinal fluid (CSF) with dual functions as a prostaglandin D{sub 2} (PGD{sub 2}) synthase and a transporter of lipophilic ligands. Recent studies revealed that L-PGDS plays important roles in protecting against various neuronal diseases induced by reactive oxygen species (ROS). However, the molecular mechanisms of such protective actions of L-PGDS remain unknown. In this study, we conducted thermodynamic and nuclear magnetic resonance (NMR) analyses, and demonstrated that L-PGDS binds to nicotinamide coenzymes, including NADPH, NADP{sup +}, and NADH. Although a hydrophilic ligand is not common formore » L-PGDS, these ligands, especially NADPH showed specific interaction with L-PGDS at the upper pocket of its ligand-binding cavity with an unusually bifurcated shape. The binding affinity of L-PGDS for NADPH was comparable to that previously reported for NADPH oxidases and NADPH in vitro. These results suggested that L-PGDS potentially attenuates the activities of NADPH oxidases through interaction with NADPH. Given that NADPH is the substrate for NADPH oxidases that play key roles in neuronal cell death by generating excessive ROS, these results imply a novel linkage between L-PGDS and ROS. - Highlights: • Interactions of L-PGDS with nicotinamide coenzymes were studied by ITC and NMR. • The binding affinity of L-PGDS was strongest to NADPH among nicotinamide coenzymes. • NADPH binds to the upper part of L-PGDS ligand-binding cavity. • L-PGDS binds to both lipophilic and hydrophilic ligands. • This study implies a novel linkage between L-PGDS and reactive oxygen species.« less

  16. Conversion of Human Steroid 5β-Reductase (AKR1D1) into 3β-Hydroxysteroid Dehydrogenase by Single Point Mutation E120H

    PubMed Central

    Chen, Mo; Drury, Jason E.; Christianson, David W.; Penning, Trevor M.

    2012-01-01

    Human aldo-keto reductase 1D1 (AKR1D1) and AKR1C enzymes are essential for bile acid biosynthesis and steroid hormone metabolism. AKR1D1 catalyzes the 5β-reduction of Δ4-3-ketosteroids, whereas AKR1C enzymes are hydroxysteroid dehydrogenases (HSDs). These enzymes share high sequence identity and catalyze 4-pro-(R)-hydride transfer from NADPH to an electrophilic carbon but differ in that one residue in the conserved AKR catalytic tetrad, His120 (AKR1D1 numbering), is substituted by a glutamate in AKR1D1. We find that the AKR1D1 E120H mutant abolishes 5β-reductase activity and introduces HSD activity. However, the E120H mutant unexpectedly favors dihydrosteroids with the 5α-configuration and, unlike most of the AKR1C enzymes, shows a dominant stereochemical preference to act as a 3β-HSD as opposed to a 3α-HSD. The catalytic efficiency achieved for 3β-HSD activity is higher than that observed for any AKR to date. High resolution crystal structures of the E120H mutant in complex with epiandrosterone, 5β-dihydrotestosterone, and Δ4-androstene-3,17-dione elucidated the structural basis for this functional change. The glutamate-histidine substitution prevents a 3-ketosteroid from penetrating the active site so that hydride transfer is directed toward the C3 carbonyl group rather than the Δ4-double bond and confers 3β-HSD activity on the 5β-reductase. Structures indicate that stereospecificity of HSD activity is achieved because the steroid flips over to present its α-face to the A-face of NADPH. This is in contrast to the AKR1C enzymes, which can invert stereochemistry when the steroid swings across the binding pocket. These studies show how a single point mutation in AKR1D1 can introduce HSD activity with unexpected configurational and stereochemical preference. PMID:22437839

  17. Inhibition of botulinum neurotoxins interchain disulfide bond reduction prevents the peripheral neuroparalysis of botulism.

    PubMed

    Zanetti, Giulia; Azarnia Tehran, Domenico; Pirazzini, Marcon; Binz, Thomas; Shone, Clifford C; Fillo, Silvia; Lista, Florigio; Rossetto, Ornella; Montecucco, Cesare

    2015-12-01

    Botulinum neurotoxins (BoNTs) form a growing family of metalloproteases with a unique specificity either for VAMP, SNAP25 or syntaxin. The BoNTs are grouped in seven different serotypes indicated by letters from A to G. These neurotoxins enter the cytosol of nerve terminals via a 100 kDa chain which binds to the presynaptic membrane and assists the translocation of a 50 kDa metalloprotease chain. These two chains are linked by a single disulfide bridge which plays an essential role during the entry of the metalloprotease chain in the cytosol, but thereafter it has to be reduced to free the proteolytic activity. Its reduction is mediated by thioredoxin which is continuously regenerated by its reductase. Here we show that inhibitors of thioredoxin reductase or of thioredoxin prevent the specific proteolysis of VAMP by the four VAMP-specific BoNTs: type B, D, F and G. These compounds are effective not only in primary cultures of neurons, but also in preventing the in vivo mouse limb neuroparalysis. In addition, one of these inhibitors, Ebselen, largely protects mice from the death caused by a systemic injection. Together with recent results obtained with BoNTs specific for SNAP25 and syntaxin, the present data demonstrate the essential role of the thioredoxin-thioredoxin reductase system in reducing the interchain disulfide during the nerve intoxication mechanism of all serotypes. Therefore its inhibitors should be considered for a possible use to prevent botulism and for treating infant botulism. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Structure of the N-terminal domain of human thioredoxin-interacting protein.

    PubMed

    Polekhina, Galina; Ascher, David Benjamin; Kok, Shie Foong; Beckham, Simone; Wilce, Matthew; Waltham, Mark

    2013-03-01

    Thioredoxin-interacting protein (TXNIP) is one of the six known α-arrestins and has recently received considerable attention owing to its involvement in redox signalling and metabolism. Various stress stimuli such as high glucose, heat shock, UV, H2O2 and mechanical stress among others robustly induce the expression of TXNIP, resulting in the sequestration and inactivation of thioredoxin, which in turn leads to cellular oxidative stress. While TXNIP is the only α-arrestin known to bind thioredoxin, TXNIP and two other α-arrestins, Arrdc4 and Arrdc3, have been implicated in metabolism. Furthermore, owing to its roles in the pathologies of diabetes and cardiovascular disease, TXNIP is considered to be a promising drug target. Based on their amino-acid sequences, TXNIP and the other α-arrestins are remotely related to β-arrestins. Here, the crystal structure of the N-terminal domain of TXNIP is reported. It provides the first structural information on any of the α-arrestins and reveals that although TXNIP adopts a β-arrestin fold as predicted, it is structurally more similar to Vps26 proteins than to β-arrestins, while sharing below 15% pairwise sequence identity with either.

  19. Structure of the detoxification catalyst mercuric ion reductase from Bacillus sp. strain RC607

    NASA Astrophysics Data System (ADS)

    Schiering, N.; Kabsch, W.; Moore, M. J.; Distefano, M. D.; Walsh, C. T.; Pai, E. F.

    1991-07-01

    SEVERAL hundred million tons of toxic mercurials are dispersed in the biosphere1. Microbes can detoxify organo-mercurials and mercury salts through sequential action of two enzymes, organomercury lyase2 and mercuric ion reductase (MerA) 3-5. The latter, a homodimer with homology to the FAD-dependent disulphide oxidoreductases6, catalyses the reaction NADPH + Hg(II) --> NADP+ + H+Hg(0), one of the very rare enzymic reactions with metal substrates. Human glutathione reductase7,8 serves as a reference molecule for FAD-dependent disulphide reductases and between its primary structure9 and that of MerA from Tn501 (Pseudomonas), Tn21 (Shigella), pI258 (Staphylococcus) and Bacillus, 25-30% of the residues have been conserved10,11. All MerAs have a C-terminal extension about 15 residues long but have very varied N termini. Although the enzyme from Streptomyces lividans has no addition, from Pseudomonas aeruginosa Tn5Ol and Bacillus sp. strain RC607 it has one and two copies respectively of a domain of 80-85 residues, highly homologous to MerP, the periplasmic component of proteins encoded by the mer operon11. These domains can be proteolytically cleaved off without changing the catalytic efficiency3. We report here the crystal structure of MerA from the Gram-positive bacterium Bacillus sp. strain RC607. Analysis of its complexes with nicotinamide dinucleotide substrates and the inhibitor Cd(II) reveals how limited structural changes enable an enzyme to accept as substrate what used to be a dangerous inhibitor. Knowledge of the mode of mercury ligation is a prerequisite for understanding this unique detoxification mechanism.

  20. A fluorescence resonance energy transfer (FRET)-based redox sensor reveals physiological role of thioredoxin in the yeast Saccharomyces cerevisiae.

    PubMed

    Oku, Masahide; Hoseki, Jun; Ichiki, Yayoi; Sakai, Yasuyoshi

    2013-03-18

    The physiological roles of the thioredoxin isozymes in the yeast Saccharomyces cerevisiae were investigated using a novel FRET-based redox probe, Redoxfluor. After establishing responsiveness of the probe toward thioredoxin, we followed the fluorescence signal of Redoxfluor expressed in the yeast and found that one of the thioredoxin isozymes, Trx2, was required for maintaining the redox status when stationary culture of the organism was exposed to starvation and mild-heat stresses. The failure to maintain redox balance under the tested condition preceded decreased viability of the trx2 mutants, indicating the functional importance of the cytoplasmic thioredoxin in adaptation to environmental changes. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  1. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Brimfield, A.A.; Mancebo, A.M.; Mason, R.P.

    The biochemical sequelae to chloroethyl mustard exposure correspond very well to toxic processes initiated by free radicals. Additionally, mustard solutions contain spontaneously formed cyclic onium ions which produce carbon free radicals when reduced electrochemically. Therefore, we hypothesized that the onium ions of sulfur or nitrogen mustards might produce carbon free radicals upon being reduced enzymatically, and that these radicals might constitute a metabolic activation. We set out to document radical production using an in vitro metabolic system and electron paramagnetic resonance (EPR). Our system consisted of NADPH, one of several pyridine nucleotide-driven flavoprotein reductases, cytochrome c as a terminal electronmore » acceptor, various sulfur or nitrogen mustards and the spin trap {alpha}-[4-pyridyl-1-oxide]-N-tert-butylnitrone in buffer. Reactions were started by adding the reductase to the other materials, vortexing and immediately transferring the mixture to a 10 mm EPR flat cell. Repeated scans on a Bruker ESP 300E EPR spectrometer produced a triplet of doublets with hyperfine splitting constants of a{sub N} = 15.483 G and a{sub H} = 2.512 G. The outcome supported our hypothesis that carbon-centered free radicals are produced when mustard-related onium ions are enzymatically reduced. The EPR results varied little with the chloroethyl compound used or with porcine or human cytochrome P450 reductase, the reductase domain of rat brain neuronal nitric oxide synthase or rat liver thioredoxin reductase. Our results offer new insight into the basis for mustard-induced vesication and the outcome of exposure to different mustards. The free radical model provides an explanation for similarities in the lesions arising from mustard exposure and energy-based lesions such as those from heat, ultraviolet and nuclear radiation as well as damage across tissue types such as skin, eyes or airway epithelium.« less

  2. [Oxygen and the superoxide anion. Modulation of NADPH oxidase?].

    PubMed

    Delbosc, S; Cristol, J P; Descomps, B; Chénard, J; Sirois, P

    2001-01-01

    Oxidative stress which results from an imbalance between oxidant production and antioxidant defense mechanisms can promote modifications of lipids, proteins and nucleic acids. This review focuses on the different pathways leading to Reactive Oxygen Species (ROS) production in particular on NADPH oxidase activation. This enzyme is localized in numerous cells including phagocytes and vascular cells and composed of membrane and cytosolic sub-units. The activation of the NADPH oxidase is largely involved in inflammation associated diseases such as asthma, Systemic Inflammatory Response Syndrome and aging associated diseases such as atherosclerosis and neurodeneratives diseases. The modulation of NADPH oxidase could be a way to limit or prevent the development of these diseases.

  3. NADPH Oxidase Activation Contributes to Heavy Ion Irradiation–Induced Cell Death

    PubMed Central

    Wang, Yupei; Liu, Qing; Zhao, Weiping; Zhou, Xin; Miao, Guoying; Sun, Chao

    2017-01-01

    Increased oxidative stress plays an important role in heavy ion radiation–induced cell death. The mechanism involved in the generation of elevated reactive oxygen species (ROS) is not fully illustrated. Here we show that NADPH oxidase activation is closely related to heavy ion radiation–induced cell death via excessive ROS generation. Cell death and cellular ROS can be greatly reduced in irradiated cancer cells with the preincubation of diphenyleneiodium, an inhibitor of NADPH oxidase. Most of the NADPH oxidase (NOX) family proteins (NOX1, NOX2, NOX3, NOX4, and NOX5) showed increased expression after heavy ion irradiation. Meanwhile, the cytoplasmic subunit p47phox was translocated to the cell membrane and localized with NOX2 to form reactive NADPH oxidase. Our data suggest for the first time that ROS generation, as mediated by NADPH oxidase activation, could be an important contributor to heavy ion irradiation–induced cell death. PMID:28473742

  4. Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase

    PubMed Central

    2015-01-01

    Homotetrameric R67 dihydrofolate reductase possesses 222 symmetry and a single active site pore. This situation results in a promiscuous binding site that accommodates either the substrate, dihydrofolate (DHF), or the cofactor, NADPH. NADPH interacts more directly with the protein as it is larger than the substrate. In contrast, the p-aminobenzoyl-glutamate tail of DHF, as monitored by nuclear magnetic resonance and crystallography, is disordered when bound. To explore whether smaller active site volumes (which should decrease the level of tail disorder by confinement effects) alter steady state rates, asymmetric mutations that decreased the half-pore volume by ∼35% were constructed. Only minor effects on kcat were observed. To continue exploring the role of tail disorder in catalysis, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide-mediated cross-linking between R67 DHFR and folate was performed. A two-folate, one-tetramer complex results in the loss of enzyme activity where two symmetry-related K32 residues in the protein are cross-linked to the carboxylates of two bound folates. The tethered folate could be reduced, although with a ≤30-fold decreased rate, suggesting decreased dynamics and/or suboptimal positioning of the cross-linked folate for catalysis. Computer simulations that restrain the dihydrofolate tail near K32 indicate that cross-linking still allows movement of the p-aminobenzoyl ring, which allows the reaction to occur. Finally, a bis-ethylene-diamine-α,γ-amide folate adduct was synthesized; both negatively charged carboxylates in the glutamate tail were replaced with positively charged amines. The Ki for this adduct was ∼9-fold higher than for folate. These various results indicate a balance between folate tail disorder, which helps the enzyme bind substrate while dynamics facilitates catalysis. PMID:26637016

  5. The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels

    NASA Astrophysics Data System (ADS)

    DeCoursey, Thomas E.; Morgan, Deri; Cherny, Vladimir V.

    2003-04-01

    The enzyme NADPH oxidase in phagocytes is important in the body's defence against microbes: it produces superoxide anions (O2-, precursors to bactericidal reactive oxygen species). Electrons move from intracellular NADPH, across a chain comprising FAD (flavin adenine dinucleotide) and two haems, to reduce extracellular O2 to O2-. NADPH oxidase is electrogenic, generating electron current (Ie) that is measurable under voltage-clamp conditions. Here we report the complete current-voltage relationship of NADPH oxidase, the first such measurement of a plasma membrane electron transporter. We find that Ie is voltage-independent from -100mV to >0mV, but is steeply inhibited by further depolarization, and is abolished at about +190mV. It was proposed that H+ efflux mediated by voltage-gated proton channels compensates Ie, because Zn2+ and Cd2+ inhibit both H+ currents and O2- production. Here we show that COS-7 cells transfected with four NADPH oxidase components, but lacking H+ channels, produce O2- in the presence of Zn2+ concentrations that inhibit O2- production in neutrophils and eosinophils. Zn2+ does not inhibit NADPH oxidase directly, but through effects on H+ channels. H+ channels optimize NADPH oxidase function by preventing membrane depolarization to inhibitory voltages.

  6. Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae.

    PubMed

    Moon, Jaewoong; Liu, Z Lewis

    2015-04-01

    The aldehyde reductase gene ARI1 is a recently characterized member of an intermediate subfamily within the short-chain dehydrogenase/reductase (SDR) superfamily that clarified mechanisms of in situ detoxification of 2-furaldehyde and 5-hydroxymethyl-2-furaldehyde by Saccharomyces cerevisiae. Uncharacterized open reading frames (ORFs) are common among tolerant candidate genes identified for lignocellulose-to-advanced biofuels conversion. This study presents partially purified proteins of two ORFs, YDR541C and YGL039W, and direct enzyme assay evidence against aldehyde-inhibitory compounds commonly encountered during lignocellulosic biomass fermentation processes. Each of the partially purified proteins encoded by these ORFs showed a molecular mass of approximately 38 kDa, similar to Ari1p, a protein encoded by aldehyde reductase gene. Both proteins demonstrated strong aldehyde reduction activities toward 14 aldehyde substrates, with high levels of reduction activity for Ydr541cp toward both aromatic and aliphatic aldehydes. While Ydr541cp was observed to have a significantly higher specific enzyme activity at 20 U/mg using co-factor NADPH, Ygl039wp displayed a NADH preference at 25 U/mg in reduction of butylaldehyde. Amino acid sequence analysis identified a characteristic catalytic triad, Ser, Tyr and Lys; a conserved catalytic motif of Tyr-X-X-X-Lys; and a cofactor-binding sequence motif, Gly-X-X-Gly-X-X-Ala, near the N-terminus that are shared by Ydr541cp, Ygl039wp, Yol151wp/GRE2 and Ari1p. Findings of aldehyde reductase genes contribute to the yeast gene annotation and aids development of the next-generation biocatalyst for advanced biofuels production. Copyright © 2015 John Wiley & Sons, Ltd.

  7. Thioredoxin-1 Negatively Modulates ADAM17 Activity Through Direct Binding and Indirect Reductive Activity.

    PubMed

    Granato, Daniela C; E Costa, Rute A P; Kawahara, Rebeca; Yokoo, Sami; Aragão, Annelize Z; Domingues, Romênia R; Pauletti, Bianca A; Honorato, Rodrigo V; Fattori, Juliana; Figueira, Ana Carolina M; Oliveira, Paulo S L; Consonni, Silvio R; Fernandes, Denise; Laurindo, Francisco; Hansen, Hinrich P; Paes Leme, Adriana F

    2018-02-27

    A disintegrin and metalloprotease 17 (ADAM17) modulates signaling events by releasing surface protein ectodomains such as TNFa and the EGFR-ligands. We have previously characterized cytoplasmic thioredoxin-1 (Trx-1) as a partner of ADAM17 cytoplasmic domain. Still, the mechanism of ADAM17 regulation by Trx-1 is unknown, and it has become of paramount importance to assess the degree of influence that Trx-1 has on metalloproteinase ADAM17. Combining discovery and targeted proteomic approaches, we uncovered that Trx-1 negatively regulates ADAM17 by direct and indirect effect. We performed cell-based assays with synthetic peptides and site-directed mutagenesis, and we demonstrated that the interaction interface of Trx-1 and ADAM17 is important for the negative regulation of ADAM17 activity. However, both Trx-1 K72A and catalytic site mutant Trx-1 C32/35S rescued ADAM17 activity, although the interaction with Trx-1 C32/35S was unaffected, suggesting an indirect effect of Trx-1. We confirmed that the Trx-1 C32/35S mutant showed diminished reductive capacity, explaining this indirect effect on increasing ADAM17 activity through oxidant levels. Interestingly, Trx-1 K72A mutant showed similar oxidant levels to Trx-1 C32/35S , even though its catalytic site was preserved. We further demonstrated that the general reactive oxygen species inhibitor, Nacetylcysteine (NAC), maintained the regulation of ADAM17 dependent of Trx-1 reductase activity levels; whereas the electron transport chain modulator, rotenone, abolished Trx-1 effect on ADAM17 activity. We show for the first time that the mechanism of ADAM17 regulation, Trx-1 dependent, can be by direct interaction and indirect effect, bringing new insights into the cross-talk between isomerases and mammalian metalloproteinases. This unexpected Trx-1 K72A behavior was due to more dimer formation and, consequently, the reduction of its Trx-1 reductase activity, evaluated through dimer verification, by gel filtration and mass

  8. A homogeneous, high-throughput-compatible, fluorescence intensity-based assay for UDP-N-acetylenolpyruvylglucosamine reductase (MurB) with nanomolar product detection.

    PubMed

    Shapiro, Adam B; Livchak, Stephania; Gao, Ning; Whiteaker, James; Thresher, Jason; Jahić, Haris; Huang, Jian; Gu, Rong-Fang

    2012-03-01

    A novel assay for the NADPH-dependent bacterial enzyme UDP-N-acetylenolpyruvylglucosamine reductase (MurB) is described that has nanomolar sensitivity for product formation and is suitable for high-throughput applications. MurB catalyzes an essential cytoplasmic step in the synthesis of peptidoglycan for the bacterial cell wall, reduction of UDP-N-acetylenolpyruvylglucosamine to UDP-N-acetylmuramic acid (UNAM). Interruption of this biosynthetic pathway leads to cell death, making MurB an attractive target for antibacterial drug discovery. In the new assay, the UNAM product of the MurB reaction is ligated to L-alanine by the next enzyme in the peptidoglycan biosynthesis pathway, MurC, resulting in hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). The ADP is detected with nanomolar sensitivity by converting it to oligomeric RNA with polynucleotide phosphorylase and detecting the oligomeric RNA with a fluorescent dye. The product sensitivity of the new assay is 1000-fold greater than that of the standard assay that follows the absorbance decrease resulting from the conversion of NADPH to NADP(+). This sensitivity allows inhibitor screening to be performed at the low substrate concentrations needed to make the assay sensitive to competitive inhibition of MurB.

  9. Acrolein oxidizes the cytosolic and mitochondrial thioredoxins in human endothelial cells.

    PubMed

    Szadkowski, Adam; Myers, Charles R

    2008-01-14

    Acrolein is a reactive aldehyde that is a widespread environmental pollutant and can be generated endogenously from lipid peroxidation. The thioredoxin (Trx) system in endothelial cells plays a major role in the maintenance of cellular thiol redox balance, and is critical for cell survival. Normally, cells maintain the cytosolic (Trx1) and mitochondrial (Trx2) thioredoxins largely in the reduced state. In human microvascular endothelial cells, Trx1 was more sensitive than Trx2 to oxidation by acrolein. A 30-min exposure to 2.5 microM acrolein caused partial oxidation of Trx1 but not Trx2. The active site dithiol of Trx1 was essentially completely oxidized by 5 microM acrolein whereas 12.5 microM was required for complete oxidation of Trx2. Partial recovery of the Trx1 redox status was observed over a 4h acrolein-free recovery period, with increases in the reduced form and decreases in the fully oxidized form. For cells treated with 2.5 or 5 microM acrolein the recovery did not require protein synthesis, whereas protein synthesis was required for the return of reduced Trx1 in cells treated with 12.5 microM acrolein. Pretreatment of cells with N-acetylcysteine (NAC) resulted in partial protection of Trx1 from oxidation by acrolein. In cells treated with acrolein for 30 min, followed by a 14- to 16-h acrolein-free period, small but significant cytotoxic effects were observed with 2.5 microM acrolein whereas all cells were adversely affected by >or= 12.5 microM. NAC pretreatment significantly decreased the percentage of stressed cells subsequently exposed to 5 or 12.5 microM acrolein. Given the critical role of the thioredoxins in cell survival, the ability of acrolein to oxidize both thioredoxins should be taken into account for a thorough understanding of its cytotoxic effects.

  10. [Effects of melaxen and valdoxan on the activity of glutathione antioxidant system and NADPH-producing enzymes in rat heart under experimental hyperthyroidism conditions].

    PubMed

    Gorbenko, M V; Popova, T N; Shul'gin, K K; Popov, S S

    2013-01-01

    The effects of melaxen and valdoxan on the activity of glutathione antioxidant system and some NADPH-producing enzymes have been studied under conditions of experimental hyperthyroidism in rat heart. Under the action of these drugs, reduced glutathione (GSH) content increased as compared to values observed under the conditions of pathology. It has been established that the activities of glutathione reductase (GR), glutathione peroxidase (GP), glucose-6-phosphate dehydrogenase, and NADP isocitrate dehydrogenase (increased under pathological conditions) change toward the intact control values upon the introduction of both drugs. The influence of melaxen and valdoxan, capable of producing antioxidant effect, leads apparently to the inhibition of free-radical oxidation processes and, as a consequence, the reduction of mobilization degree of the glutathione antioxidant system.

  11. Direct electrochemistry of nitrate reductase from the fungus Neurospora crassa.

    PubMed

    Kalimuthu, Palraj; Ringel, Phillip; Kruse, Tobias; Bernhardt, Paul V

    2016-09-01

    We report the first direct (unmediated) catalytic electrochemistry of a eukaryotic nitrate reductase (NR). NR from the filamentous fungus Neurospora crassa, is a member of the mononuclear molybdenum enzyme family and contains a Mo, heme and FAD cofactor which are involved in electron transfer from NAD(P)H to the (Mo) active site where reduction of nitrate to nitrite takes place. NR was adsorbed on an edge plane pyrolytic graphite (EPG) working electrode. Non-turnover redox responses were observed in the absence of nitrate from holo NR and three variants lacking the FAD, heme or Mo cofactor. The FAD response is due to dissociated cofactor in all cases. In the presence of nitrate, NR shows a pronounced cathodic catalytic wave with an apparent Michaelis constant (KM) of 39μM (pH7). The catalytic cathodic current increases with temperature from 5 to 35°C and an activation enthalpy of 26kJmol(-1) was determined. In spite of dissociation of the FAD cofactor, catalytically activity is maintained. Copyright © 2016. Published by Elsevier B.V.

  12. Protein-bound NAD(P)H Lifetime is Sensitive to Multiple Fates of Glucose Carbon.

    PubMed

    Sharick, Joe T; Favreau, Peter F; Gillette, Amani A; Sdao, Sophia M; Merrins, Matthew J; Skala, Melissa C

    2018-04-03

    While NAD(P)H fluorescence lifetime imaging (FLIM) can detect changes in flux through the TCA cycle and electron transport chain (ETC), it remains unclear whether NAD(P)H FLIM is sensitive to other potential fates of glucose. Glucose carbon can be diverted from mitochondria by the pentose phosphate pathway (via glucose 6-phosphate dehydrogenase, G6PDH), lactate production (via lactate dehydrogenase, LDH), and rejection of carbon from the TCA cycle (via pyruvate dehydrogenase kinase, PDK), all of which can be upregulated in cancer cells. Here, we demonstrate that multiphoton NAD(P)H FLIM can be used to quantify the relative concentrations of recombinant LDH and malate dehydrogenase (MDH) in solution. In multiple epithelial cell lines, NAD(P)H FLIM was also sensitive to inhibition of LDH and PDK, as well as the directionality of LDH in cells forced to use pyruvate versus lactate as fuel sources. Among the parameters measurable by FLIM, only the lifetime of protein-bound NAD(P)H (τ 2 ) was sensitive to these changes, in contrast to the optical redox ratio, mean NAD(P)H lifetime, free NAD(P)H lifetime, or the relative amount of free and protein-bound NAD(P)H. NAD(P)H τ 2 offers the ability to non-invasively quantify diversions of carbon away from the TCA cycle/ETC, which may support mechanisms of drug resistance.

  13. Overexpression of plastidial thioredoxins f and m differentially alters photosynthetic activity and response to oxidative stress in tobacco plants

    PubMed Central

    Rey, Pascal; Sanz-Barrio, Ruth; Innocenti, Gilles; Ksas, Brigitte; Courteille, Agathe; Rumeau, Dominique; Issakidis-Bourguet, Emmanuelle; Farran, Inmaculada

    2013-01-01

    Plants display a remarkable diversity of thioredoxins (Trxs), reductases controlling the thiol redox status of proteins. The physiological function of many of them remains elusive, particularly for plastidial Trxs f and m, which are presumed based on biochemical data to regulate photosynthetic reactions and carbon metabolism. Recent reports revealed that Trxs f and m participate in vivo in the control of starch metabolism and cyclic photosynthetic electron transfer around photosystem I, respectively. To further delineate their in planta function, we compared the photosynthetic characteristics, the level and/or activity of various Trx targets and the responses to oxidative stress in transplastomic tobacco plants overexpressing either Trx f or Trx m. We found that plants overexpressing Trx m specifically exhibit altered growth, reduced chlorophyll content, impaired photosynthetic linear electron transfer and decreased pools of glutathione and ascorbate. In both transplastomic lines, activities of two enzymes involved in carbon metabolism, NADP-malate dehydrogenase and NADP-glyceraldehyde-3-phosphate dehydrogenase are markedly and similarly altered. In contrast, plants overexpressing Trx m specifically display increased capacity for methionine sulfoxide reductases, enzymes repairing damaged proteins by regenerating methionine from oxidized methionine. Finally, we also observed that transplastomic plants exhibit distinct responses when exposed to oxidative stress conditions generated by methyl viologen or exposure to high light combined with low temperature, the plants overexpressing Trx m being notably more tolerant than Wt and those overexpressing Trx f. Altogether, these data indicate that Trxs f and m fulfill distinct physiological functions. They prompt us to propose that the m type is involved in key processes linking photosynthetic activity, redox homeostasis and antioxidant mechanisms in the chloroplast. PMID:24137166

  14. Glucose-6-phosphate dehydrogenase and Trypanothione reductase interaction protects Leishmania donovani from metalloid mediated oxidative stress.

    PubMed

    Ghosh, Ayan Kumar; Saini, Savita; Das, Sushmita; Mandal, Abhishek; Sardar, Abul Hasan; Ansari, Md Yousuf; Abhishek, Kumar; Kumar, Ajay; Singh, Ruby; Verma, Sudha; Equbal, Asif; Ali, Vahab; Das, Pradeep

    2017-05-01

    Exploration of metabolons as viable drug target is rare in kinetoplastid biology. Here we present a novel protein-protein interaction among Glucose-6-phosphate dehydrogenase (LdG6PDH) and Trypanothione reductase (LdTryR) of Leishmania donovani displaying interconnection between central glucose metabolism and thiol metabolism of this parasite. Digitonin fractionation patterns observed through immunoblotting indicated localisation of both LdG6PDH and LdTryR in cytosol. In-silico and in-vitro interaction observed by size exclusion chromatography, co-purification, pull-down assay and spectrofluorimetric analysis revealed LdG6PDH and LdTryR physically interact with each other in a NADPH dependent manner. Coupled enzymatic assay displayed that NADPH generation was severely impaired by addition of Sb III , As III and Te IV extraneously, which hint towards metalloid driven structural changes of the interacting proteins. Co-purification patterns and pull-down assays also depicted that metalloids (Sb III , As III and Te IV ) hinder the in-vitro interaction of these two enzymes. Surprisingly, metalloids at sub-lethal concentrations induced the in-vivo interaction of LdG6PDH and LdTryR, as analyzed by pull-down assays and fluorescence microscopy signifying protection against metalloid mediated ROS. Inhibition of LdTryR by thioridazine in LdG6PDH -/- parasites resulted in metalloid induced apoptotic death of the parasites due to abrupt fall in reduced thiol content, disrupted NADPH/NADP + homeostasis and lethal oxidative stress. Interestingly, clinical isolates of L.donovani resistant to SAG exhibited enhanced interaction between LdG6PDH and LdTryR and showed cross resistivity towards As III and Te IV . Thus, our findings propose the metabolon of LdG6PDH and LdTryR as an alternate therapeutic target and provide mechanistic insight about metalloid resistance in Visceral Leishmaniasis. Copyright © 2017. Published by Elsevier Inc.

  15. Enhancement of Chaperone Activity of Plant-Specific Thioredoxin through γ-Ray Mediated Conformational Change.

    PubMed

    Lee, Seung Sik; Jung, Hyun Suk; Park, Soo-Kwon; Lee, Eun Mi; Singh, Sudhir; Lee, Yuno; Lee, Kyun Oh; Lee, Sang Yeol; Chung, Byung Yeoup

    2015-11-13

    AtTDX, a thioredoxin-like plant-specific protein present in Arabidopsis is a thermo-stable and multi-functional enzyme. This enzyme is known to act as a thioredoxin and as a molecular chaperone depending upon its oligomeric status. The present study examines the effects of γ-irradiation on the structural and functional changes of AtTDX. Holdase chaperone activity of AtTDX was increased and reached a maximum at 10 kGy of γ-irradiation and declined subsequently in a dose-dependent manner, together with no effect on foldase chaperone activity. However, thioredoxin activity decreased gradually with increasing irradiation. Electrophoresis and size exclusion chromatography analysis showed that AtTDX had a tendency to form high molecular weight (HMW) complexes after γ-irradiation and γ-ray-induced HMW complexes were tightly associated with a holdase chaperone activity. The hydrophobicity of AtTDX increased with an increase in irradiation dose till 20 kGy and thereafter decreased further. Analysis of the secondary structures of AtTDX using far UV-circular dichroism spectra revealed that the irradiation remarkably increased the exposure of β-sheets and random coils with a dramatic decrease in α-helices and turn elements in a dose-dependent manner. The data of the present study suggest that γ-irradiation may be a useful tool for increasing holdase chaperone activity without adversely affecting foldase chaperone activity of thioredoxin-like proteins.

  16. Expression and characterization of truncated human heme oxygenase (hHO-1) and a fusion protein of hHO-1 with human cytochrome P450 reductase.

    PubMed

    Wilks, A; Black, S M; Miller, W L; Ortiz de Montellano, P R

    1995-04-04

    A human heme oxygenase (hHO-1) gene without the sequence coding for the last 23 amino acids has been expressed in Escherichia coli behind the pho A promoter. The truncated enzyme is obtained in high yields as a soluble, catalytically-active protein, making it available for the first time for detailed mechanistic studies. The purified, truncated hHO-1/heme complex is spectroscopically indistinguishable from that of the rat enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase. A self-sufficient heme oxygenase system has been obtained by fusing the truncated hHO-1 gene to the gene for human cytochrome P450 reductase without the sequence coding for the 20 amino acid membrane binding domain. Expression of the fusion protein in pCWori+ yields a protein that only requires NADPH for catalytic turnover. The failure of exogenous cytochrome P450 reductase to stimulate turnover and the insensitivity of the catalytic rate toward changes in ionic strength establish that electrons are transferred intramolecularly between the reductase and heme oxygenase domains of the fusion protein. The Vmax for the fusion protein is 2.5 times higher than that for the reconstituted system. Therefore, either the covalent tether does not interfere with normal docking and electron transfer between the flavin and heme domains or alternative but equally efficient electron transfer pathways are available that do not require specific docking.

  17. Detoxification of hexavalent chromium by Leucobacter sp. uses a reductase with specificity for dihydrolipoamide.

    PubMed

    Sarangi, Abhipsa; Krishnan, Chandraraj

    2016-02-01

    Leucobacter sp. belongs to the metal stressed community and possesses higher tolerance to metals including chromium and can detoxify toxic hexavalent chromium by reduction to less toxic trivalent chromium. But, the mechanism of reduction of hexavalent chromium by Leucobacter sp. has not been studied. Understanding the enzyme catalyzing reduction of chromium is important to improve the species for application in bioremediation. Hence, a soluble reductase catalyzing the reduction of hexavalent chromium was purified from a Leucobacter sp. and characterized. The pure chromate reductase was obtained from the cell-free extract through hydrophobic interaction and gel filtration column chromatographic methods. It was a monomeric enzyme and showed similar molecular weights in both gel filtration (∼68 KDa) and SDS-PAGE (64 KDa). It reduced Cr(VI) using both NADH and NADPH as the electron donor, but exhibited higher activity with NADH. The optimal activity was found at pH 5.5 and 30 °C. The K(m) and V(max) for Cr(VI) reduction with NADH were 46.57 μM and 0.37 μmol min(-1) (mg protein) (-1), respectively. The activity was inhibited by p-hydroxy mercury benzoate, Ag(2+) and Hg(2+) indicating the role of thiol groups in the catalysis. The spectrophotometric analysis of the purified enzyme showed the absence of bound flavin in the enzyme. The N-terminal amino acid sequence and LC/MS analysis of trypsin digested purified enzyme showed similarity to dihydrolipoyl dehydrogenase. The purified enzyme had dihydrolipoyl dehydrogenase activity with dihydrolipoamide as the substrate, which suggested that Leucobacter sp. uses reductase with multiple substrate specificity for reduction of Cr(VI) detoxification. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. EXPRESSION AND CHARACTERIZATION OF FULL-LENGTH HUMAN HEME OXYGENASE-1: PRESENCE OF INTACT MEMBRANE-BINDING REGION LEADS TO INCREASED BINDING AFFINITY FOR NADPH-CYTOCHROME P450 REDUCTASE

    PubMed Central

    Huber, Warren J.; Backes, Wayne L.

    2009-01-01

    Heme oxygenase (HO) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, this NADPH and cytochrome P450 reductase (CPR)-dependent oxidation of heme also releases free iron and carbon monoxide. Much of the recent research involving heme oxygenase is done using a 30-kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a GST-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30-kDa degradation product that could not be eliminated. Therefore, we attempted to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces lysine with arginine. This mutation allowed the expression and purification of a full length hHO-1 protein. Unlike wild-type HO-1, the K254R mutant could be purified to a single 32-kDa protein capable of degrading heme at the same rate as the wild-type enzyme. The K254R full-length form had a specific activity of ~200–225 nmol bilirubin hr−1nmol−1 HO-1 as compared to ~140–150 nmol bilirubin hr−1nmol−1 for the WT form, which contains the 30-kDa contaminant. This is a 2–3-fold increase from the previously reported soluble 30-kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other ER-resident enzymes. PMID:17915953

  19. Nitric Oxide Synthase and Neuronal NADPH Diaphorase are Identical in Brain and Peripheral Tissues

    NASA Astrophysics Data System (ADS)

    Dawson, Ted M.; Bredt, David S.; Fotuhi, Majid; Hwang, Paul M.; Snyder, Solomon H.

    1991-09-01

    NADPH diaphorase staining neurons, uniquely resistant to toxic insults and neurodegenerative disorders, have been colocalized with neurons in the brain and peripheral tissue containing nitric oxide synthase (EC 1.14.23.-), which generates nitric oxide (NO), a recently identified neuronal messenger molecule. In the corpus striatum and cerebral cortex, NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in medium to large aspiny neurons. These same neurons colocalize with somatostatin and neuropeptide Y immunoreactivity. NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in the pedunculopontine nucleus with choline acetyltransferase-containing cells and are also colocalized in amacrine cells of the inner nuclear layer and ganglion cells of the retina, myenteric plexus neurons of the intestine, and ganglion cells of the adrenal medulla. Transfection of human kidney cells with NO synthase cDNA elicits NADPH diaphorase staining. The ratio of NO synthase to NADPH diaphorase staining in the transfected cells is the same as in neurons, indicating that NO synthase fully accounts for observed NADPH staining. The identity of neuronal NO synthase and NADPH diaphorase suggests a role for NO in modulating neurotoxicity.

  20. NADPH oxidase mediates depressive behavior induced by chronic stress in mice.

    PubMed

    Seo, Ji-Seon; Park, Jin-Young; Choi, Juli; Kim, Tae-Kyung; Shin, Joo-Hyun; Lee, Ja-Kyeong; Han, Pyung-Lim

    2012-07-11

    Stress is a potent risk factor for depression, yet the underlying mechanism is not clearly understood. In the present study, we explored the mechanism of development and maintenance of depression in a stress-induced animal model. Mice restrained for 2 h daily for 14 d showed distinct depressive behavior, and the altered behavior persisted for >3 months in the absence of intervention. Acute restraint induced a surge of oxidative stress in the brain, and stress-induced oxidative stress progressively increased with repetition of stress. In vitro, the stress hormone glucocorticoid generated superoxide via upregulation of NADPH oxidase. Consistently, repeated restraints increased the expression of the key subunits of NADPH oxidase, p47phox and p67phox, in the brain. Moreover, stressed brains markedly upregulated the expression of p47phox to weak restress evoked in the poststress period, and this molecular response was reminiscent of amplified ROS surge to restress. Pharmacological inhibition of NADPH oxidase by the NADPH oxidase inhibitor apocynin during the stress or poststress period completely blocked depressive behavior. Consistently, heterozygous p47phox knock-out mice (p47phox(+/-)) or molecular inhibition of p47phox with Lenti shRNA-p47phox in the hippocampus suppressed depressive behavior. These results suggest that repeated stress promotes depressive behavior through the upregulation of NADPH oxidase and the resultant metabolic oxidative stress, and that the inhibition of NADPH oxidase provides beneficial antidepression effects.

  1. Ultra-fast HPM detectors improve NAD(P)H FLIM

    NASA Astrophysics Data System (ADS)

    Becker, Wolfgang; Wetzker, Cornelia; Benda, Aleš

    2018-02-01

    Metabolic imaging by NAD(P)H FLIM requires the decay functions in the individual pixels to be resolved into the decay components of bound and unbound NAD(P)H. Metabolic information is contained in the lifetime and relative amplitudes of the components. The separation of the decay components and the accuracy of the amplitudes and lifetimes improves substantially by using ultra-fast HPM-100-06 and HPM-100-07 hybrid detectors. The IRF width in combination with the Becker & Hickl SPC-150N and SPC-150NX TCSPC modules is less than 20 ps. An IRF this fast does not interfere with the fluorescence decay. The usual deconvolution process in the data analysis then virtually becomes a simple curve fitting, and the parameters of the NAD(P)H decay components are obtained at unprecedented accuracy.

  2. Redox Signaling Mediated by Thioredoxin and Glutathione Systems in the Central Nervous System.

    PubMed

    Ren, Xiaoyuan; Zou, Lili; Zhang, Xu; Branco, Vasco; Wang, Jun; Carvalho, Cristina; Holmgren, Arne; Lu, Jun

    2017-11-01

    The thioredoxin (Trx) and glutathione (GSH) systems play important roles in maintaining the redox balance in the brain, a tissue that is prone to oxidative stress due to its high-energy demand. These two disulfide reductase systems are active in various areas of the brain and are considered to be critical antioxidant systems in the central nervous system (CNS). Various neuronal disorders have been characterized to have imbalanced redox homeostasis. Recent Advances: In addition to their detrimental effects, recent studies have highlighted that reactive oxygen species/reactive nitrogen species (ROS/RNS) act as critical signaling molecules by modifying thiols in proteins. The Trx and GSH systems, which reversibly regulate thiol modifications, regulate redox signaling involved in various biological events in the CNS. In this review, we focus on the following: (i) how ROS/RNS are produced and mediate signaling in CNS; (ii) how Trx and GSH systems regulate redox signaling by catalyzing reversible thiol modifications; (iii) how dysfunction of the Trx and GSH systems causes alterations of cellular redox signaling in human neuronal diseases; and (iv) the effects of certain small molecules that target thiol-based signaling pathways in the CNS. Further study on the roles of thiol-dependent redox systems in the CNS will improve our understanding of the pathogenesis of many human neuronal disorders and also help to develop novel protective and therapeutic strategies against neuronal diseases. Antioxid. Redox Signal. 27, 989-1010.

  3. Biodegradation of 2,4'-dichlorobiphenyl, a congener of polychlorinated biphenyl, by Pseudomonas isolates GSa and GSb.

    PubMed

    Gayathri, D; Shobha, K J

    2015-08-01

    Bioegradation of 2,4'-dichlorobiphenyl (2,4 CB), by two isolates of Pseudomonas (GSa and GSb) was compared using GC-MS. Transformer oil polluted soil was used for the isolation of 2,4 CB degrading bacteria. GC-MS analysis of the solvent extracts obtained from Pseudomonas sp. GSa spent culture indicated the presence of Phenol 2,6-bis (1,1-dimethyl)-4-methyl (C15H24O). Further, the enzyme analysis of the cell free extracts showed the presence of 2,4'-dichlorobiphenyl dehalogenase (CBD), 2,4'-dichlorobiphenyl-NADPH-oxido-reductase (2,4 CBOR) and 2,3-dihydroxybiphenyl-NADPH-oxido-reductase (2,3 DHOR) with specific activity of 6.00, 0.4 and 0.22 pmol/min/mg of protein, suggesting that dechlorination as an important step during 2,4 CB catabolism. Further, the cell free extract of GSb showed only 2,4'-dichlorobiphenyl-NADPH-oxido-reductase (2,4 CBOR) and 2,3-dihydroxybiphenyl-NADPH-oxido-reductase (2,3 DHOR), with specific activity of 0.3 and 0.213 μmol/min/mg of protein, suggesting attack on non-chlorinated aromatic ring of 2,4 CB, releasing chlorinated intermediates which are toxic to the environment. Although, both the isolated bacteria (GSa and GSb) belong to Pseudomonas spp., they exhibited different metabolic potential.

  4. Novel Aldo-Keto Reductases for the Biocatalytic Conversion of 3-Hydroxybutanal to 1,3-Butanediol: Structural and Biochemical Studies

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kim, Taeho; Flick, Robert; Brunzelle, Joseph

    The nonnatural alcohol 1,3-butanediol (1,3-BDO) is a valuable building block for the synthesis of various polymers. One of the potential pathways for the biosynthesis of 1,3-BDO includes the biotransformation of acetaldehyde to 1,3-BDO via 3-hydroxybutanal (3-HB) using aldolases and aldo-keto reductases (AKRs). This pathway requires an AKR selective for 3-HB, but inactive toward acetaldehyde, so it can be used for one-pot synthesis. In this work, we screened more than 20 purified uncharacterized AKRs for 3-HB reduction and identified 10 enzymes with significant activity and nine proteins with detectable activity. PA1127 fromPseudomonas aeruginosashowed the highest activity and was selected for comparativemore » studies with STM2406 fromSalmonella entericaserovar Typhimurium, for which we have determined the crystal structure. Both AKRs used NADPH as a cofactor, reduced a broad range of aldehydes, and showed low activities toward acetaldehyde. The crystal structures of STM2406 in complex with cacodylate or NADPH revealed the active site with bound molecules of a substrate mimic or cofactor. Site-directed mutagenesis of STM2406 and PA1127 identified the key residues important for the activity against 3-HB and aromatic aldehydes, which include the residues of the substrate-binding pocket and C-terminal loop. Our results revealed that the replacement of the STM2406 Asn65 by Met enhanced the activity and the affinity of this protein toward 3-HB, resulting in a 7-fold increase ink cat/K m. Our work provides further insights into the molecular mechanisms of the substrate selectivity of AKRs and for the rational design of these enzymes toward new substrates. IMPORTANCEIn this study, we identified several aldo-keto reductases with significant activity in reducing 3-hydroxybutanal to 1,3-butanediol (1,3-BDO), an important commodity chemical. Biochemical and structural studies of these enzymes revealed the key catalytic and substrate-binding residues, including the two

  5. Post-translational regulation of mercaptopyruvate sulfurtransferase via a low redox potential cysteine-sulfenate in the maintenance of redox homeostasis.

    PubMed

    Nagahara, Noriyuki; Katayama, Akira

    2005-10-14

    3-Mercaptopyruvate sulfurtransferase (MST) (EC 2.8.1.2), a multifunctional enzyme, catalyzes a transsulfuration from mercaptopyruvate to pyruvate in the degradation process of cysteine. A stoichiometric concentration of hydrogen peroxide and of tetrathionate (S(4)O(6)(2-)) inhibited rat MST (k(i) = 3.3 min(-1), K(i) = 120.5 microM and k(i) = 2.5 min(-1), K(i) = 178.6 microM, respectively). The activity was completely restored by dithiothreitol or thioredoxin with a reducing system containing thioredoxin reductase and NADPH, but glutathione did not restore the activity. On the other hand, an excess molar ratio dose of hydrogen peroxide inactivated MST. Oxidation with a stoichiometric concentration of hydrogen peroxide protected the enzyme against reaction by iodoacetate, which modifies a catalytic Cys(247), suggesting that Cys(247) is a target of the oxidants. A matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric analysis revealed that hydrogen peroxide- and tetrathionate-inhibited MSTs were increased in molecular mass consistent with the addition of atomic oxygen and with a thiosulfate (S(2)O(3)(-)), respectively. Treatment with dithiothreitol restored modified MST to the original mass. These findings suggested that there was no nearby cysteine with which to form a disulfide, and mild oxidation of MST resulted in formation of a sulfenate (SO(-)) at Cys(247), which exhibited exceptional stability and a lower redox potential than that of glutathione. Oxidative stress decreases MST activity so as to increase the amount of cysteine, a precursor of thioredoxin or glutathione, and furthermore, these cellular reductants restore the activity. Thus the redox state regulates MST activity at the enzymatic level, and on the other hand, MST controls redox to maintain cellular redox homeostasis.

  6. Redox regulation of mammalian sperm capacitation

    PubMed Central

    O’Flaherty, Cristian

    2015-01-01

    Capacitation is a series of morphological and metabolic changes necessary for the spermatozoon to achieve fertilizing ability. One of the earlier happenings during mammalian sperm capacitation is the production of reactive oxygen species (ROS) that will trigger and regulate a series of events including protein phosphorylation, in a time-dependent fashion. The identity of the sperm oxidase responsible for the production of ROS involved in capacitation is still elusive, and several candidates are discussed in this review. Interestingly, ROS-induced ROS formation has been described during human sperm capacitation. Redox signaling during capacitation is associated with changes in thiol groups of proteins located on the plasma membrane and subcellular compartments of the spermatozoon. Both, oxidation of thiols forming disulfide bridges and the increase on thiol content are necessary to regulate different sperm proteins associated with capacitation. Reducing equivalents such as NADH and NADPH are necessary to support capacitation in many species including humans. Lactate dehydrogenase, glucose-6-phospohate dehydrogenase, and isocitrate dehydrogenase are responsible in supplying NAD (P) H for sperm capacitation. Peroxiredoxins (PRDXs) are newly described enzymes with antioxidant properties that can protect mammalian spermatozoa; however, they are also candidates for assuring the regulation of redox signaling required for sperm capacitation. The dysregulation of PRDXs and of enzymes needed for their reactivation such as thioredoxin/thioredoxin reductase system and glutathione-S-transferases impairs sperm motility, capacitation, and promotes DNA damage in spermatozoa leading to male infertility. PMID:25926608

  7. NADH/NADPH bi-cofactor-utilizing and thermoactive ketol-acid reductoisomerase from Sulfolobus acidocaldarius.

    PubMed

    Chen, Chin-Yu; Ko, Tzu-Ping; Lin, Kuan-Fu; Lin, Bo-Lin; Huang, Chun-Hsiang; Chiang, Cheng-Hung; Horng, Jia-Cherng

    2018-05-08

    Ketol-acid reductoisomerase (KARI) is a bifunctional enzyme in the second step of branched-chain amino acids biosynthetic pathway. Most KARIs prefer NADPH as a cofactor. However, KARI with a preference for NADH is desirable in industrial applications including anaerobic fermentation for the production of branched-chain amino acids or biofuels. Here, we characterize a thermoacidophilic archaeal Sac-KARI from Sulfolobus acidocaldarius and present its crystal structure at a 1.75-Å resolution. By comparison with other holo-KARI structures, one sulphate ion is observed in each binding site for the 2'-phosphate of NADPH, implicating its NADPH preference. Sac-KARI has very high affinity for NADPH and NADH, with K M values of 0.4 μM for NADPH and 6.0 μM for NADH, suggesting that both are good cofactors at low concentrations although NADPH is favoured over NADH. Furthermore, Sac-KARI can catalyze 2(S)-acetolactate (2S-AL) with either cofactor from 25 to 60 °C, but the enzyme has higher activity by using NADPH. In addition, the catalytic activity of Sac-KARI increases significantly with elevated temperatures and reaches an optimum at 60 °C. Bi-cofactor utilization and the thermoactivity of Sac-KARI make it a potential candidate for use in metabolic engineering or industrial applications under anaerobic or harsh conditions.

  8. NADPH as a potential intrinsic probe for tumour margin estimation

    NASA Astrophysics Data System (ADS)

    Stewart, Hazel; Hupp, Ted R.; Birch, David J. S.

    2018-03-01

    The fluorescent properties of the reduced coenzyme NADH and its phosphorylated derivative (NADPH) have been explored in order to assess their potential as an intrinsic probe for cancer surgery. NADPH production is increased in cancer cells to quench reactive oxygen species and meet higher demands for biosynthesis, and has attractive fluorescent properties such as emission towards the visible part of the spectrum and a relatively long fluorescence lifetime upon binding to enzymes ( 1 - 6.5 ns) that helps discriminate against other endogenous species. Different environmental effects on NAD(P)H fluorescence are reported here, including an increase in lifetime upon oxygen removal, an ability to retain its fluorescent properties in a complex medium (a silica phantom) and its fluorescence lifetime also being distinguishable in a cell environment. In addition, the development of a miniaturized liquid light guide filter-based timecorrelated single photon counting fluorescence lifetime system is reported as a step towards time-resolved visual imaging in cancer surgery. This system has been demonstrated as being capable of accurately measuring NAD(P)H fluorescence lifetimes in both simple solvent and cellular environments.

  9. Nitrate and periplasmic nitrate reductases

    PubMed Central

    Sparacino-Watkins, Courtney; Stolz, John F.; Basu, Partha

    2014-01-01

    The nitrate anion is a simple, abundant and relatively stable species, yet plays a significant role in global cycling of nitrogen, global climate change, and human health. Although it has been known for quite some time that nitrate is an important species environmentally, recent studies have identified potential medical applications. In this respect the nitrate anion remains an enigmatic species that promises to offer exciting science in years to come. Many bacteria readily reduce nitrate to nitrite via nitrate reductases. Classified into three distinct types – periplasmic nitrate reductase (Nap), respiratory nitrate reductase (Nar) and assimilatory nitrate reductase (Nas), they are defined by their cellular location, operon organization and active site structure. Of these, Nap proteins are the focus of this review. Despite similarities in the catalytic and spectroscopic properties Nap from different Proteobacteria are phylogenetically distinct. This review has two major sections: in the first section, nitrate in the nitrogen cycle and human health, taxonomy of nitrate reductases, assimilatory and dissimilatory nitrate reduction, cellular locations of nitrate reductases, structural and redox chemistry are discussed. The second section focuses on the features of periplasmic nitrate reductase where the catalytic subunit of the Nap and its kinetic properties, auxiliary Nap proteins, operon structure and phylogenetic relationships are discussed. PMID:24141308

  10. Drug-enhanced carbon monoxide production from heme by cytochrome P450 reductase.

    PubMed

    Vukomanovic, Dragic; Rahman, Mona N; Jia, Zongchao; Nakatsu, Kanji

    2017-01-01

    Carbon monoxide (CO) formed endogenously is considered to be cytoprotective, and the vast majority of CO formation is attributed to the degradation of heme by heme oxygenases-1 and -2 (HO-1, HO-2). Previously, we observed that brain microsomes containing HO-2 produced many-fold more CO in the presence of menadione and its congeners; herein we explored these observations further. We determined the effects of various drugs on CO production of rat brain microsomes and recombinant human cytochrome P450 reductase (CPR); CO was measured by gas chromatography with reductive detection. Brain microsomes of Sprague-Dawley rats or recombinant human cytochrome P450 reductase (CPR) were incubated with NADPH and various drugs in closed vials in phosphate buffer at pH 7.4 and 37°C. After 15 minutes, the reaction was stopped by cooling in dry ice, and the headspace gas was analyzed for CO production using gas chromatography with reductive (mercuric oxide) detection. We observed drug-enhanced CO production in the presence of both microsomes and recombinant CPR alone; the presence of HO was not required. A range of structurally diverse drugs were capable of amplifying this CO formation; these molecules had structures consistent with redox cycling capability. The addition of catalase to a reaction mixture, that contained activating drugs, inhibited the production of CO. Drug-enhanced CO formation can be catalyzed by CPR. The mechanism of CPR activation was not through classical drug-receptor mediation. Redox cycling may be involved in the drug-induced amplification of CO production by CPR through the production of reactive oxygen species.

  11. Aldehyde-forming fatty acyl-CoA reductase from cyanobacteria: expression, purification and characterization of the recombinant enzyme.

    PubMed

    Lin, Fengming; Das, Debasis; Lin, Xiaoxia N; Marsh, E Neil G

    2013-10-01

    Long-chain acyl-CoA reductases (ACRs) catalyze a key step in the biosynthesis of hydrocarbon waxes. As such they are attractive as components in engineered metabolic pathways for 'drop in' biofuels. Most ACR enzymes are integral membrane proteins, but a cytosolic ACR was recently discovered in cyanobacteria. The ACR from Synechococcus elongatus was overexpressed in Escherichia coli, purified and characterized. The enzyme was specific for NADPH and catalyzed the reduction of fatty acyl-CoA esters to the corresponding aldehydes, rather than alcohols. Stearoyl-CoA was the most effective substrate, being reduced more rapidly than either longer or shorter chain acyl-CoAs. ACR required divalent metal ions, e.g. Mg(2+), for activity and was stimulated ~ 10-fold by K(+). The enzyme was inactivated by iodoacetamide and was acylated on incubation with stearoyl-CoA, suggesting that reduction occurs through an enzyme-thioester intermediate. Consistent with this, steady state kinetic analysis indicates that the enzyme operates by a 'ping-pong' mechanism with kcat = 0.36 ± 0.023 min(-1), K(m)(stearoyl-CoA) = 31.9 ± 4.2 μM and K(m)(NADPH) = 35.6 ± 4.9 μM. The slow turnover number measured for ACR poses a challenge for its use in biofuel applications where highly efficient enzymes are needed. © 2013 FEBS.

  12. NADPH Oxidase as a Therapeutic Target for Oxalate Induced Injury in Kidneys

    PubMed Central

    Peck, Ammon B.; Khan, Saeed R.

    2013-01-01

    A major role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes is to catalyze the production of superoxides and other reactive oxygen species (ROS). These ROS, in turn, play a key role as messengers in cell signal transduction and cell cycling, but when they are produced in excess they can lead to oxidative stress (OS). Oxidative stress in the kidneys is now considered a major cause of renal injury and inflammation, giving rise to a variety of pathological disorders. In this review, we discuss the putative role of oxalate in producing oxidative stress via the production of reactive oxygen species by isoforms of NADPH oxidases expressed in different cellular locations of the kidneys. Most renal cells produce ROS, and recent data indicate a direct correlation between upregulated gene expressions of NADPH oxidase, ROS, and inflammation. Renal tissue expression of multiple NADPH oxidase isoforms most likely will impact the future use of different antioxidants and NADPH oxidase inhibitors to minimize OS and renal tissue injury in hyperoxaluria-induced kidney stone disease. PMID:23840917

  13. The cyanobacterial thioredoxin gene is required for both photoautotrophic and heterotrophic growth.

    PubMed Central

    Navarro, F; Florencio, F J

    1996-01-01

    The gene encoding thioredoxin in the facultative heterotrophic cyanobacterium Synechocytis sp. PCC 6803 (trxA) has been cloned by heterologous hybridization using the corresponding gene trxM from the cyanobacterium Anacystis nidulans as a probe. The deduced amino acid sequence of trxA predicts a protein of relative molecular weight of 11,750 and has strong identity with its cyanobacterial counterparts and other m-type thioredoxins of photo-synthetic eukaryotes. The trxA gene has been expressed Escherichia coli as a functional protein of 12 kD and has been shown by western blot analysis to be the same size as in Synechocystis. The trxA gene is transcribed in Synechocystis as a single transcript of 450 nucleotides and accumulates to the same level under photosynthetic and heterotrophic growth conditions. The trxA gene was inactivated with a kanamycin-resistant cassette, and total segregation of the disrupted trxA gene was obtained only when the trxM gene from A. nidulans (E.D.G. Muller, B.B. Buchanan [1989] J Biol Chem 264: 4008-4014) was simultaneously expressed in Synechocytis. Our results suggest an essential role of thioredoxin not only when cells grow photosynthetically but also under heterotrophic conditions. PMID:8756494

  14. Conversion of human steroid 5β-reductase (AKR1D1) into 3β-hydroxysteroid dehydrogenase by single point mutation E120H: example of perfect enzyme engineering.

    PubMed

    Chen, Mo; Drury, Jason E; Christianson, David W; Penning, Trevor M

    2012-05-11

    Human aldo-keto reductase 1D1 (AKR1D1) and AKR1C enzymes are essential for bile acid biosynthesis and steroid hormone metabolism. AKR1D1 catalyzes the 5β-reduction of Δ(4)-3-ketosteroids, whereas AKR1C enzymes are hydroxysteroid dehydrogenases (HSDs). These enzymes share high sequence identity and catalyze 4-pro-(R)-hydride transfer from NADPH to an electrophilic carbon but differ in that one residue in the conserved AKR catalytic tetrad, His(120) (AKR1D1 numbering), is substituted by a glutamate in AKR1D1. We find that the AKR1D1 E120H mutant abolishes 5β-reductase activity and introduces HSD activity. However, the E120H mutant unexpectedly favors dihydrosteroids with the 5α-configuration and, unlike most of the AKR1C enzymes, shows a dominant stereochemical preference to act as a 3β-HSD as opposed to a 3α-HSD. The catalytic efficiency achieved for 3β-HSD activity is higher than that observed for any AKR to date. High resolution crystal structures of the E120H mutant in complex with epiandrosterone, 5β-dihydrotestosterone, and Δ(4)-androstene-3,17-dione elucidated the structural basis for this functional change. The glutamate-histidine substitution prevents a 3-ketosteroid from penetrating the active site so that hydride transfer is directed toward the C3 carbonyl group rather than the Δ(4)-double bond and confers 3β-HSD activity on the 5β-reductase. Structures indicate that stereospecificity of HSD activity is achieved because the steroid flips over to present its α-face to the A-face of NADPH. This is in contrast to the AKR1C enzymes, which can invert stereochemistry when the steroid swings across the binding pocket. These studies show how a single point mutation in AKR1D1 can introduce HSD activity with unexpected configurational and stereochemical preference.

  15. Conversion of Human Steroid 5[beta]-Reductase (AKR1D1) into 3[beta]-Hydroxysteroid Dehydrogenase by Single Point Mutation E120H: Example of Perfect Enzyme Engineering

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chen, Mo; Drury, Jason E.; Christianson, David W.

    2012-10-10

    Human aldo-keto reductase 1D1 (AKR1D1) and AKR1C enzymes are essential for bile acid biosynthesis and steroid hormone metabolism. AKR1D1 catalyzes the 5{beta}-reduction of {Delta}{sup 4}-3-ketosteroids, whereas AKR1C enzymes are hydroxysteroid dehydrogenases (HSDs). These enzymes share high sequence identity and catalyze 4-pro-(R)-hydride transfer from NADPH to an electrophilic carbon but differ in that one residue in the conserved AKR catalytic tetrad, His120 (AKR1D1 numbering), is substituted by a glutamate in AKR1D1. We find that the AKR1D1 E120H mutant abolishes 5{beta}-reductase activity and introduces HSD activity. However, the E120H mutant unexpectedly favors dihydrosteroids with the 5{alpha}-configuration and, unlike most of the AKR1Cmore » enzymes, shows a dominant stereochemical preference to act as a 3{beta}-HSD as opposed to a 3{alpha}-HSD. The catalytic efficiency achieved for 3{beta}-HSD activity is higher than that observed for any AKR to date. High resolution crystal structures of the E120H mutant in complex with epiandrosterone, 5{beta}-dihydrotestosterone, and {Delta}{sup 4}-androstene-3,17-dione elucidated the structural basis for this functional change. The glutamate-histidine substitution prevents a 3-ketosteroid from penetrating the active site so that hydride transfer is directed toward the C3 carbonyl group rather than the {Delta}{sup 4}-double bond and confers 3{beta}-HSD activity on the 5{beta}-reductase. Structures indicate that stereospecificity of HSD activity is achieved because the steroid flips over to present its {alpha}-face to the A-face of NADPH. This is in contrast to the AKR1C enzymes, which can invert stereochemistry when the steroid swings across the binding pocket. These studies show how a single point mutation in AKR1D1 can introduce HSD activity with unexpected configurational and stereochemical preference.« less

  16. Eugenol and isoeugenol, characteristic aromatic constituents of spices, are biosynthesized via reduction of a coniferyl alcohol ester

    PubMed Central

    Koeduka, Takao; Fridman, Eyal; Gang, David R.; Vassão, Daniel G.; Jackson, Brenda L.; Kish, Christine M.; Orlova, Irina; Spassova, Snejina M.; Lewis, Norman G.; Noel, Joseph P.; Baiga, Thomas J.; Dudareva, Natalia; Pichersky, Eran

    2006-01-01

    Phenylpropenes such as chavicol, t-anol, eugenol, and isoeugenol are produced by plants as defense compounds against animals and microorganisms and as floral attractants of pollinators. Moreover, humans have used phenylpropenes since antiquity for food preservation and flavoring and as medicinal agents. Previous research suggested that the phenylpropenes are synthesized in plants from substituted phenylpropenols, although the identity of the enzymes and the nature of the reaction mechanism involved in this transformation have remained obscure. We show here that glandular trichomes of sweet basil (Ocimum basilicum), which synthesize and accumulate phenylpropenes, possess an enzyme that can use coniferyl acetate and NADPH to form eugenol. Petunia (Petunia hybrida cv. Mitchell) flowers, which emit large amounts of isoeugenol, possess an enzyme homologous to the basil eugenol-forming enzyme that also uses coniferyl acetate and NADPH as substrates but catalyzes the formation of isoeugenol. The basil and petunia phenylpropene-forming enzymes belong to a structural family of NADPH-dependent reductases that also includes pinoresinol–lariciresinol reductase, isoflavone reductase, and phenylcoumaran benzylic ether reductase. PMID:16782809

  17. Crystallization and preliminary X-ray analysis of the N-terminal domain of human thioredoxin-interacting protein.

    PubMed

    Polekhina, Galina; Ascher, David Benjamin; Kok, Shie Foong; Waltham, Mark

    2011-05-01

    Thioredoxin-interacting protein (TXNIP) is a negative regulator of thioredoxin and its roles in the pathologies of diabetes and cardiovascular diseases have marked it out as a potential drug target. Expression of TXNIP is robustly induced under various stress conditions such as high glucose, heat shock, UV, H(2)O(2) and mechanical stress amongst others. Elevated levels of TXNIP result in the sequestration and inactivation of thioredoxin, leading to cellular oxidative stress. For some time, this was the only known function of TXNIP; however, more recently the protein has been shown to play a role in regulation of glucose uptake and activation of the inflammasome. Based on the primary sequence, TXNIP is remotely related to β-arrestins, which include the visual arrestins. TXNIP has thus been classified as a member of the α-arrestin family, which to date includes five other members. None of the other α-arrestins are known to interact with thioredoxin, although curiously one has been implicated in glucose uptake. In order to gain insight into the structure-function relationships of the α-arrestin protein family, and particularly that of TXNIP, the N-terminal domain of TXNIP has been crystallized. The crystals belonged to a monoclinic space group and diffracted to 3 Å resolution using synchrotron radiation.

  18. Antioxidant Protection of NADPH-Depleted Oligodendrocyte Precursor Cells Is Dependent on Supply of Reduced Glutathione.

    PubMed

    Kilanczyk, Ewa; Saraswat Ohri, Sujata; Whittemore, Scott R; Hetman, Michal

    2016-08-01

    The pentose phosphate pathway is the main source of NADPH, which by reducing oxidized glutathione, contributes to antioxidant defenses. Although oxidative stress plays a major role in white matter injury, significance of NADPH for oligodendrocyte survival has not been yet investigated. It is reported here that the NADPH antimetabolite 6-amino-NADP (6AN) was cytotoxic to cultured adult rat spinal cord oligodendrocyte precursor cells (OPCs) as well as OPC-derived oligodendrocytes. The 6AN-induced necrosis was preceded by increased production of superoxide, NADPH depletion, and lower supply of reduced glutathione. Moreover, survival of NADPH-depleted OPCs was improved by the antioxidant drug trolox. Such cells were also protected by physiological concentrations of the neurosteroid dehydroepiandrosterone (10(-8) M). The protection by dehydroepiandrosterone was associated with restoration of reduced glutathione, but not NADPH, and was sensitive to inhibition of glutathione synthesis. A similar protective mechanism was engaged by the cAMP activator forskolin or the G protein-coupled estrogen receptor (GPER/GPR30) ligand G1. Finally, treatment with the glutathione precursor N-acetyl cysteine reduced cytotoxicity of 6AN. Taken together, NADPH is critical for survival of OPCs by supporting their antioxidant defenses. Consequently, injury-associated inhibition of the pentose phosphate pathway may be detrimental for the myelination or remyelination potential of the white matter. Conversely, steroid hormones and cAMP activators may promote survival of NADPH-deprived OPCs by increasing a NADPH-independent supply of reduced glutathione. Therefore, maintenance of glutathione homeostasis appears as a critical effector mechanism for OPC protection against NADPH depletion and preservation of the regenerative potential of the injured white matter. © The Author(s) 2016.

  19. The protein inhibitor of nNOS (PIN/DLC1/LC8) binding does not inhibit the NADPH-dependent heme reduction in nNOS, a key step in NO synthesis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Parhad, Swapnil S.; Jaiswal, Deepa; TIFR Centre for Interdisciplinary Sciences, 21 Brundavan Colony, Narsingi, Hyderabad 500075

    The neuronal nitric oxide synthase (nNOS) is an essential enzyme involved in the synthesis of nitric oxide (NO), a potent neurotransmitter. Although previous studies have indicated that the dynein light chain 1 (DLC1) binding to nNOS could inhibit the NO synthesis, the claim is challenged by contradicting reports. Thus, the mechanism of nNOS regulation remained unclear. nNOS has a heme-bearing, Cytochrome P450 core, and the functional enzyme is a dimer. The electron flow from NADPH to Flavin, and finally to the heme of the paired nNOS subunit within a dimer, is facilitated upon calmodulin (CaM) binding. Here, we show thatmore » DLC1 binding to nNOS-CaM complex does not affect the electron transport from the reductase to the oxygenase domain. Therefore, it cannot inhibit the rate of NADPH-dependent heme reduction in nNOS, which results in L-Arginine oxidation. Also, the NO release activity does not decrease with increasing DLC1 concentration in the reaction mix, which further confirmed that DLC1 does not inhibit nNOS activity. These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell. - Highlights: • The effect of interaction of nNOS with DLC1 has been debatable with contradicting reports in literature. • Purified DLC1 has no effect on electron transport between reductase and oxygenase domain of purified nNOS-CaM. • The NO release activity of nNOS was not altered by DLC1, supporting that DLC1 does not inhibit the enzyme. • These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell.« less

  20. NADPH-Cytochrome P450 Reductase: Molecular Cloning and Functional Characterization of Two Paralogs from Withania somnifera (L.) Dunal

    PubMed Central

    Rana, Satiander; Lattoo, Surrinder K.; Dhar, Niha; Razdan, Sumeer; Bhat, Wajid Waheed; Dhar, Rekha S.; Vishwakarma, Ram

    2013-01-01

    Withania somnifera (L.) Dunal, a highly reputed medicinal plant, synthesizes a large array of steroidal lactone triterpenoids called withanolides. Although its chemical profile and pharmacological activities have been studied extensively during the last two decades, limited attempts have been made to decipher the biosynthetic route and identification of key regulatory genes involved in withanolide biosynthesis. Cytochrome P450 reductase is the most imperative redox partner of multiple P450s involved in primary and secondary metabolite biosynthesis. We describe here the cloning and characterization of two paralogs of cytochrome P450 reductase from W. somnifera. The full length paralogs of WsCPR1 and WsCPR2 have open reading frames of 2058 and 2142 bp encoding 685 and 713 amino acid residues, respectively. Phylogenetic analysis demonstrated that grouping of dual CPRs was in accordance with class I and class II of eudicotyledon CPRs. The corresponding coding sequences were expressed in Escherichia coli as glutathione-S-transferase fusion proteins, purified and characterized. Recombinant proteins of both the paralogs were purified with their intact membrane anchor regions and it is hitherto unreported for other CPRs which have been purified from microsomal fraction. Southern blot analysis suggested that two divergent isoforms of CPR exist independently in Withania genome. Quantitative real-time PCR analysis indicated that both genes were widely expressed in leaves, stalks, roots, flowers and berries with higher expression level of WsCPR2 in comparison to WsCPR1. Similar to CPRs of other plant species, WsCPR1 was un-inducible while WsCPR2 transcript level increased in a time-dependent manner after elicitor treatments. High performance liquid chromatography of withanolides extracted from elicitor-treated samples showed a significant increase in two of the key withanolides, withanolide A and withaferin A, possibly indicating the role of WsCPR2 in withanolide biosynthesis

  1. Exposure of the blue mussel, Mytilus edulis, to gold nanoparticles and the pro-oxidant menadione.

    PubMed

    Tedesco, Sara; Doyle, Hugh; Blasco, Julian; Redmond, Gareth; Sheehan, David

    2010-03-01

    Relatively little is known about how gold nanoparticles (GNP) might interact in vivo with marine organisms. Mytilus edulis was exposed (24h) to approximately 15 nm GNP, menadione and both compounds simultaneously (GNP/menadione). GNP was detected by inductively coupled plasma-optical emission spectroscopy mainly in digestive gland of samples exposed to GNP though not GNP/menadione, perhaps due to impaired feeding. Thioredoxin reductase activity and malondialdehyde levels were determined in all tissues. Thioredoxin reductase inhibition was detected only in digestive gland exposed to menadione whilst malondialdehyde levels did not vary in response to treatment in all tissues. GNP caused a decrease in the reduced/oxidized glutathione ratio in digestive gland, but no difference was found in other tissues or for other treatments. One dimensional electrophoresis of proteins containing thiol groups was performed in all tissues and revealed a reduction in protein thiols for all treatments in digestive gland. Two dimensional electrophoresis of digestive gland extracts, from GNP and control groups, showed decreased levels of thiol proteins in response to GNP which we attribute to oxidation. Our results suggest that GNP causes a modest level of oxidative stress sufficient to oxidize thiols in glutathione and proteins but without causing lipid peroxidation or induction of thioredoxin reductase activity.

  2. Cell-free NADPH oxidase activation assays: "in vitro veritas".

    PubMed

    Pick, Edgar

    2014-01-01

    The superoxide (O2 (∙-))-generating NADPH oxidase complex of phagocytes comprises a membrane-imbedded heterodimeric flavocytochrome, known as cytochrome b 558 (consisting of Nox2 and p22 (phox) ) and four cytosolic regulatory proteins, p47 (phox) , p67 (phox) , p40 (phox) , and the small GTPase Rac. Under physiological conditions, in the resting phagocyte, O2 (∙-) generation is initiated by engagement of membrane receptors by a variety of stimuli, followed by specific signal transduction sequences leading to the translocation of the cytosolic components to the membrane and their association with the cytochrome. A consequent conformational change in Nox2 initiates the electron "flow" along a redox gradient, from NADPH to oxygen, leading to the one-electron reduction of molecular oxygen to O2 (∙-). Methodological difficulties in the dissection of this complex mechanism led to the design "cell-free" systems (also known as "broken cells" or in vitro systems). In these, membrane receptor stimulation and all or part of the signal transduction sequence are missing, the accent being placed on the actual process of "NADPH oxidase assembly," thus on the formation of the complex between cytochrome b 558 and the cytosolic components and the resulting O2 (∙-) generation. Cell-free assays consist of a mixture of the individual components of the NADPH oxidase complex, derived from resting phagocytes or in the form of purified recombinant proteins, exposed in vitro to an activating agent (distinct from and unrelated to whole cell stimulants), in the presence of NADPH and oxygen. Activation is commonly quantified by measuring the primary product of the reaction, O2 (∙-), trapped immediately after its generation by an appropriate acceptor in a kinetic assay, permitting the calculation of the linear rate of O2 (∙-) production, but numerous variations exist, based on the assessment of reaction products or the consumption of substrates. Cell-free assays played a paramount

  3. NADPH oxidases: new kids on the block.

    PubMed

    Geiszt, Miklós

    2006-07-15

    Reactive oxygen species (ROS) play a pivotal role in many physiological processes including host defense, hormone biosynthesis, fertilization and cellular signaling. Altered production of ROS has been implicated in the development of immunodeficiency, hypothyroidism and cardiovascular pathologies. In the last few years, several enzymes were identified at the molecular level, which are now thought to be responsible for ROS production observed in diverse tissues. These enzymes show a high degree of homology to the phagocytic NADPH oxidase and are now designated the Nox family of NADPH oxidases. This review updates our knowledge on six new members of the Nox family: Nox1, Nox3, Nox4, Nox5, Duox1 and Duox2.

  4. Genetic characterization and role in virulence of the ribonucleotide reductases of Streptococcus sanguinis.

    PubMed

    Rhodes, DeLacy V; Crump, Katie E; Makhlynets, Olga; Snyder, Melanie; Ge, Xiuchun; Xu, Ping; Stubbe, JoAnne; Kitten, Todd

    2014-02-28

    Streptococcus sanguinis is a cause of infective endocarditis and has been shown to require a manganese transporter called SsaB for virulence and O2 tolerance. Like certain other pathogens, S. sanguinis possesses aerobic class Ib (NrdEF) and anaerobic class III (NrdDG) ribonucleotide reductases (RNRs) that perform the essential function of reducing ribonucleotides to deoxyribonucleotides. The accompanying paper (Makhlynets, O., Boal, A. K., Rhodes, D. V., Kitten, T., Rosenzweig, A. C., and Stubbe, J. (2014) J. Biol. Chem. 289, 6259-6272) indicates that in the presence of O2, the S. sanguinis class Ib RNR self-assembles an essential diferric-tyrosyl radical (Fe(III)2-Y(•)) in vitro, whereas assembly of a dimanganese-tyrosyl radical (Mn(III)2-Y(•)) cofactor requires NrdI, and Mn(III)2-Y(•) is more active than Fe(III)2-Y(•) with the endogenous reducing system of NrdH and thioredoxin reductase (TrxR1). In this study, we have shown that deletion of either nrdHEKF or nrdI completely abolishes virulence in an animal model of endocarditis, whereas nrdD mutation has no effect. The nrdHEKF, nrdI, and trxR1 mutants fail to grow aerobically, whereas anaerobic growth requires nrdD. The nrdJ gene encoding an O2-independent adenosylcobalamin-cofactored RNR was introduced into the nrdHEKF, nrdI, and trxR1 mutants. Growth of the nrdHEKF and nrdI mutants in the presence of O2 was partially restored. The combined results suggest that Mn(III)2-Y(•)-cofactored NrdF is required for growth under aerobic conditions and in animals. This could explain in part why manganese is necessary for virulence and O2 tolerance in many bacterial pathogens possessing a class Ib RNR and suggests NrdF and NrdI may serve as promising new antimicrobial targets.

  5. Monocyte and macrophage-targeted NADPH oxidase mediates antifungal host defense and regulation of acute inflammation in mice

    PubMed Central

    Grimm, Melissa J.; Vethanayagam, R. Robert; Almyroudis, Nikolaos G.; Dennis, Carly G.; Khan, A. Nazmul H.; D’Auria, Anthony; Singel, Kelly L.; Davidson, Bruce A.; Knight, Paul R.; Blackwell, Timothy S.; Hohl, Tobias M.; Mansour, Michael K.; Vyas, Jatin M.; Röhm, Marc; Urban, Constantin F.; Kelkka, Tiina; Holmdahl, Rikard; Segal, Brahm H.

    2013-01-01

    Chronic granulomatous disease, an inherited disorder of the NADPH oxidase in which phagocytes are defective in the generation of superoxide anion and downstream reactive oxidant species, is characterized by severe bacterial and fungal infections and excessive inflammation. Although NADPH oxidase isoforms exist in several lineages, reactive oxidant generation is greatest in neutrophils, where NADPH oxidase has been deemed vital for pathogen killing. In contrast, the function and importance of NADPH oxidase in macrophages are less clear. Therefore, we evaluated susceptibility to pulmonary aspergillosis in globally NADPH oxidase-deficient mice versus transgenic mice with monocyte/macrophage-targeted NADPH oxidase activity. We found that the lethal inoculum was more than 100-fold greater in transgenic versus globally NADPH oxidase-deficient mice. Consistent with these in vivo results, NADPH oxidase in mouse alveolar macrophages limited germination of phagocytosed Aspergillus fumigatus spores. Finally, globally NADPH oxidase-deficient mice developed exuberant neutrophilic lung inflammation and pro-inflammatory cytokine responses to zymosan, a fungal cell wall-derived product composed principally of particulate beta-glucans, whereas inflammation in transgenic and wildtype mice was mild and transient. Together, our studies identify a central role for monocyte/macrophage NADPH oxidase in controlling fungal infection and in limiting acute lung inflammation. PMID:23509361

  6. Towards a systematic analysis of human short-chain dehydrogenases/reductases (SDR): Ligand identification and structure-activity relationships.

    PubMed

    Bhatia, Chitra; Oerum, Stephanie; Bray, James; Kavanagh, Kathryn L; Shafqat, Naeem; Yue, Wyatt; Oppermann, Udo

    2015-06-05

    Short-chain dehydrogenases/reductases (SDRs) constitute a large, functionally diverse branch of enzymes within the class of NAD(P)(H) dependent oxidoreductases. In humans, over 80 genes have been identified with distinct metabolic roles in carbohydrate, amino acid, lipid, retinoid and steroid hormone metabolism, frequently associated with inherited genetic defects. Besides metabolic functions, a subset of atypical SDR proteins appears to play critical roles in adapting to redox status or RNA processing, and thereby controlling metabolic pathways. Here we present an update on the human SDR superfamily and a ligand identification strategy using differential scanning fluorimetry (DSF) with a focused library of oxidoreductase and metabolic ligands to identify substrate classes and inhibitor chemotypes. This method is applicable to investigate structure-activity relationships of oxidoreductases and ultimately to better understand their physiological roles. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  7. NADPH oxidase activation in neutrophils: Role of the Phosphorylation of its subunits.

    PubMed

    Belambri, Sahra A; Rolas, Loïc; Raad, Houssam; Hurtado-Nedelec, Margarita; Dang, Pham My-Chan; El-Benna, Jamel

    2018-05-14

    Neutrophils are key cells of innate immunity and during inflammation. Upon activation, they produce large amounts of superoxide anion (O 2 -. ) and ensuing reactive oxygen species (ROS) to kill phagocytized microbes. The enzyme responsible for O 2 -. production is called the phagocyte NADPH oxidase. This is a multicomponent enzyme system that becomes active after assembly of four cytosolic proteins (p47 phox , p67 phox , p40 phox and Rac2) with the transmembrane proteins (p22 phox and gp91 phox , which form the cytochrome b 558 ). gp91 phox represents the catalytic subunit of the NADPH oxidase and is also called NOX2. NADPH oxidase-derived ROS are essential for microbial killing and innate immunity; however, excessive ROS production induces tissue injury and prolonged inflammatory reactions that contribute to inflammatory diseases. Thus, NADPH oxidase activation must be tightly regulated in time and space in order to limit ROS production. NADPH oxidase activation is regulated by several processes such as phosphorylation of its components, exchange of GDP/GTP on Rac2 and binding of p47 phox and p40 phox to phospholipids. This review aims to provide new insights into the role of the phosphorylation of the NADPH oxidase components, i.e., gp91 phox , p22 phox , p47 phox , p67 phox and p40 phox , in the activation of this enzyme. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  8. Structural analysis of NADPH depleted bovine liver catalase and its inhibitor complexes

    PubMed Central

    Sugadev, Ragumani; Ponnuswamy, M.N.; Sekar, K.

    2011-01-01

    To study the functional role of NADPH during mammalian catalase inhibition, the X-ray crystal structures of NADPH-depleted bovine liver catalase and its inhibitor complexes, cyanide and azide, determined at 2.8Å resolution. From the complex structures it is observed that subunits with and without an inhibitor/catalytic water molecule are linked by N-terminal domain swapping. Comparing mammalian- and fungal- catalases, we speculate that NADPH-depleted mammalian catalases may function as a domain-swapped dimer of dimers, especially during inactivation by inhibitors like cyanide and azide. We further speculate that in mammalian catalases the N-terminal hinge-loop region and α-helix is the structural element that senses NADPH binding. Although the above arguments are speculative and need further verification, as a whole our studies have opened up a new possibility, viz. that mammalian catalase acts as a domain-swapped dimer of dimers, especially during inhibitor binding. To generalize this concept to the formation of the inactive state in mammalian catalases in the absence of tightly bound NADPH molecules needs further exploration. The present study adds one more intriguing fact to the existing mysteries of mammalian catalases. PMID:21968615

  9. Evidence for two interconverting protein isomers in the methotrexate complex of dihydrofolate reductase from Escherichia coli

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Falzone, C.J.; Benkovic, S.J.; Wright, P.E.

    1991-02-26

    Two-dimensional {sup 1}H NMR methods and a knowledge of the X-ray crystal structure have been used to make resonance assignments for the amino acid side chains of dihydrofolate reductase from Escherichia coli complexed with methotrexate. The H7 proton on the pteridine ring of methotrexate was found to have NOEs to the methyl protons of Leu-28 which were assigned by using the L28F mutant. These NOEs indicated that the orientation of the methotrexate pteridine ring is similar in both solution and crystal structures. During the initial assignment process, it became evident that many of the resonances in this complex, unlike thosemore » of the folate complex, are severally broadened or doubled. The observation of two distinct sets of resonances in a ratio of approximately 2:1 was attributed to the presence of two protein isomers. Many of the side chains with clearly doubled resonances were located in the {beta}-sheet and the active site. Preliminary studies on the apoprotein also revealed doubled resonances in the absence of the inhibitor, indicating the existence of the protein isomers prior to methotrexate binding. In contrast to the methotrexate complex, the binary complex with folate and the ternary MTX-NADPH-DHFR complex presented a single enzyme form. These results are proposed to reflect the ability of folate and NADPH to bind predominantly to one protein isomer.« less

  10. Effects of 3G cell phone exposure on the structure and function of the human cytochrome P450 reductase.

    PubMed

    Tanvir, Shazia; Thuróczy, György; Selmaoui, Brahim; Silva Pires Antonietti, Viviane; Sonnet, Pascal; Arnaud-Cormos, Delia; Lévêque, Philippe; Pulvin, Sylviane; de Seze, René

    2016-10-01

    Cell phones increase exposure to radiofrequency (RF) electromagnetic fields (EMFs). Whether EMFs exert specific effects on biological systems remains debatable. This study investigated the effect of cell phone exposure on the structure and function of human NADPH-cytochrome P450 reductase (CPR). CPR plays a key role in the electron transfer to cytochrome P450, which takes part in a wide range of oxidative metabolic reactions in various organisms from microbes to humans. Human CPR was exposed for 60min to 1966-MHz RF inside a transverse electromagnetic cell (TEM-cell) placed in an incubator. The specific absorption rate (SAR) was 5W·kg(-1). Conformation changes have been detected through fluorescent spectroscopy of flavin and tryptophan residues, and investigated through circular dichroism, dynamic light scattering and microelectrophoresis. These showed that CPR was narrowed. By using cytochrome C reductase activity to assess the electron flux through the CPR, the Michaelis Menten constant (Km) and the maximum initial velocity (Vmax) decreased by 22% as compared with controls. This change was due to small changes in the tertiary and secondary structures of the protein at 37°C. The relevance of these findings to an actual RF exposure scenario demands further biochemical and in-vivo confirmation. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Redox regulation of myocardial ERK 1/2 phosphorylation in experimental hyperthyroidism: role of thioredoxin-peroxiredoxin system.

    PubMed

    Araujo, Alex Sander da Rosa; Fernandes, Tania; Ribeiro, Maria Flavia; Khaper, Neelam; Belló-Klein, Adriane

    2010-11-01

    The present study was conducted to test whether adaptation in the antioxidant system would differentially modulate prosurvival and proapoptotic proteins in hyperthyroidism-induced cardiac hypertrophy. Male Wistar rats were divided into 4 groups: control, vitamin E (20 mg · kg(-1) · d(-1) subcutaneously, 28 days), thyroxine (T4) (12 mg/L in drinking water for 28 days), and T4 + vitamin E. Cardiac mass, redox ratio, glutathione peroxidase (GPx) and glutathione reductase (GR) activities, NF-E2-related factor 2 (Nrf2) thioredoxin-1 (Trx-1), peroxiredoxin-6 (Prx-6), phospho-extracellular-signal-regulated kinases 1/2 (p-ERK 1/2)/extracellular-signal-regulated kinases 1/2 (ERK1/2), and phospho-c-Jun N-terminal kinase (p-JNK)/c-Jun N-terminal kinase (JNK) myocardial protein expression were quantified. Cardiac hypertrophy was attenuated in the T4 + vitamin E group. The redox ratio; GPx and GR; as well as Nrf2, Trx-1, Prx-6, and p-ERK1/2/ERK1/2 immunocontent were elevated in T4 group. All these effects were attenuated by vitamin E administration. p-JNK/JNK remained unchanged in all the groups. The overall results suggest that redox imbalance due to hyperthyroidism induce adaptation of antioxidant systems, favoring ERK1/2 activation and leading to development of cardiac hypertrophy.

  12. Enhancing biomass and ethanol production by increasing NADPH production in Synechocystis sp. PCC 6803.

    PubMed

    Choi, Yun-Nam; Park, Jong Moon

    2016-08-01

    This study demonstrates that increased NADPH production can improve biomass and ethanol production in cyanobacteria. We over-expressed the endogenous zwf gene, which encodes glucose-6-phosphate dehydrogenase of pentose phosphate pathway, in the model cyanobacterium Synechocystis sp. PCC 6803. zwf over-expression resulted in increased NADPH production, and promoted biomass production compared to the wild type in both autotrophic and mixotrophic conditions. Ethanol production pathway including NADPH-dependent alcohol dehydrogenase was also integrated with and without zwf over-expression. Excessive NADPH production by zwf over-expression could improve both biomass and ethanol production in the autotrophic conditions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Purification and characterization of a highly active chromate reductase from endophytic Bacillus sp. DGV19 of Albizzia lebbeck (L.) Benth. actively involved in phytoremediation of tannery effluent-contaminated sites.

    PubMed

    Manikandan, Muthu; Gopal, Judy; Kumaran, Rangarajulu Senthil; Kannan, Vijayaraghavan; Chun, Sechul

    2016-01-01

    Phytoremediation using timber-yielding tree species is considered to be the most efficient method for chromium/tannery effluent-contaminated sites. In this study, we have chosen Albizzia lebbeck, a chromium hyperaccumulator plant, and studied one of its chromium detoxification processes operated by its endophytic bacterial assemblage. Out of the four different groups of endophytic bacteria comprising Pseudomonas, Rhizobium, Bacillus, and Salinicoccus identified from A. lebbeck employed in phytoremediation of tannery effluent-contaminated soil, Bacillus predominated with three species, which exhibited not only remarkable chromium accumulation ability but also high chromium reductase activity. A chromate reductase was purified to homogeneity from the most efficient chromium accumulator, Bacillus sp. DGV 019, and the purified 34.2-kD enzyme was observed to be stable at temperatures from 20°C to 60°C. The enzyme was active over a wide range of pH values (4.0-9.0). Furthermore, the enzyme activity was enhanced with the electron donors NADH, followed by NADPH, not affected by glutathione and ascorbic acid. Cu(2+) enhanced the activity of the purified enzyme but was inhibited by Zn(2+) and etheylenediamine tetraacetic acid (EDTA). In conclusion, due to its versatile adaptability the chromate reductase can be used for chromium remediation.

  14. Cloning and sequence analysis demonstrate the chromate reduction ability of a novel chromate reductase gene from Serratia sp.

    PubMed

    Deng, Peng; Tan, Xiaoqing; Wu, Ying; Bai, Qunhua; Jia, Yan; Xiao, Hong

    2015-03-01

    The ChrT gene encodes a chromate reductase enzyme which catalyzes the reduction of Cr(VI). The chromate reductase is also known as flavin mononucleotide (FMN) reductase (FMN_red). The aim of the present study was to clone the full-length ChrT DNA from Serratia sp. CQMUS2 and analyze the deduced amino acid sequence and three-dimensional structure. The putative ChrT gene fragment of Serratia sp. CQMUS2 was isolated by polymerase chain reaction (PCR), according to the known FMN_red gene sequence from Serratia sp. AS13. The flanking sequences of the ChrT gene were obtained by high efficiency TAIL-PCR, while the full-length gene of ChrT was cloned in Escherichia coli for subsequent sequencing. The nucleotide sequence of ChrT was submitted onto GenBank under the accession number, KF211434. Sequence analysis of the gene and amino acids was conducted using the Basic Local Alignment Search Tool, and open reading frame (ORF) analysis was performed using ORF Finder software. The ChrT gene was found to be an ORF of 567 bp that encodes a 188-amino acid enzyme with a calculated molecular weight of 20.4 kDa. In addition, the ChrT protein was hypothesized to be an NADPH-dependent FMN_red and a member of the flavodoxin-2 superfamily. The amino acid sequence of ChrT showed high sequence similarity to the FMN reductase genes of Klebsiella pneumonia and Raoultella ornithinolytica , which belong to the flavodoxin-2 superfamily. Furthermore, ChrT was shown to have a 85.6% similarity to the three-dimensional structure of Escherichia coli ChrR, sharing four common enzyme active sites for chromate reduction. Therefore, ChrT gene cloning and protein structure determination demonstrated the ability of the gene for chromate reduction. The results of the present study provide a basis for further studies on ChrT gene expression and protein function.

  15. Cloning and sequence analysis demonstrate the chromate reduction ability of a novel chromate reductase gene from Serratia sp

    PubMed Central

    DENG, PENG; TAN, XIAOQING; WU, YING; BAI, QUNHUA; JIA, YAN; XIAO, HONG

    2015-01-01

    The ChrT gene encodes a chromate reductase enzyme which catalyzes the reduction of Cr(VI). The chromate reductase is also known as flavin mononucleotide (FMN) reductase (FMN_red). The aim of the present study was to clone the full-length ChrT DNA from Serratia sp. CQMUS2 and analyze the deduced amino acid sequence and three-dimensional structure. The putative ChrT gene fragment of Serratia sp. CQMUS2 was isolated by polymerase chain reaction (PCR), according to the known FMN_red gene sequence from Serratia sp. AS13. The flanking sequences of the ChrT gene were obtained by high efficiency TAIL-PCR, while the full-length gene of ChrT was cloned in Escherichia coli for subsequent sequencing. The nucleotide sequence of ChrT was submitted onto GenBank under the accession number, KF211434. Sequence analysis of the gene and amino acids was conducted using the Basic Local Alignment Search Tool, and open reading frame (ORF) analysis was performed using ORF Finder software. The ChrT gene was found to be an ORF of 567 bp that encodes a 188-amino acid enzyme with a calculated molecular weight of 20.4 kDa. In addition, the ChrT protein was hypothesized to be an NADPH-dependent FMN_red and a member of the flavodoxin-2 superfamily. The amino acid sequence of ChrT showed high sequence similarity to the FMN reductase genes of Klebsiella pneumonia and Raoultella ornithinolytica, which belong to the flavodoxin-2 superfamily. Furthermore, ChrT was shown to have a 85.6% similarity to the three-dimensional structure of Escherichia coli ChrR, sharing four common enzyme active sites for chromate reduction. Therefore, ChrT gene cloning and protein structure determination demonstrated the ability of the gene for chromate reduction. The results of the present study provide a basis for further studies on ChrT gene expression and protein function. PMID:25667630

  16. Functional Assembly of Soluble and Membrane Recombinant Proteins of Mammalian NADPH Oxidase Complex.

    PubMed

    Souabni, Hajer; Ezzine, Aymen; Bizouarn, Tania; Baciou, Laura

    2017-01-01

    Activation of phagocyte cells from an innate immune system is associated with a massive consumption of molecular oxygen to generate highly reactive oxygen species (ROS) as microbial weapons. This is achieved by a multiprotein complex, the so-called NADPH oxidase. The activity of phagocyte NADPH oxidase relies on an assembly of more than five proteins, among them the membrane heterodimer named flavocytochrome b 558 (Cytb 558 ), constituted by the tight association of the gp91 phox (also named Nox2) and p22 phox proteins. The Cytb 558 is the membrane catalytic core of the NADPH oxidase complex, through which the reducing equivalent provided by NADPH is transferred via the associated prosthetic groups (one flavin and two hemes) to reduce dioxygen into superoxide anion. The other major proteins (p47 phox , p67 phox , p40 phox , Rac) requisite for the complex activity are cytosolic proteins. Thus, the NADPH oxidase functioning relies on a synergic multi-partner assembly that in vivo can be hardly studied at the molecular level due to the cell complexity. Thus, a cell-free assay method has been developed to study the NADPH oxidase activity that allows measuring and eventually quantifying the ROS generation based on optical techniques following reduction of cytochrome c. This setup is a valuable tool for the identification of protein interactions, of crucial components and additives for a functional enzyme. Recently, this method was improved by the engineering and the production of a complete recombinant NADPH oxidase complex using the combination of purified proteins expressed in bacterial and yeast host cells. The reconstitution into artificial membrane leads to a fully controllable system that permits fine functional studies.

  17. Targeting NADPH oxidase decreases oxidative stress in the transgenic sickle cell mouse penis.

    PubMed

    Musicki, Biljana; Liu, Tongyun; Sezen, Sena F; Burnett, Arthur L

    2012-08-01

    Sickle cell disease (SCD) is a state of chronic vasculopathy characterized by endothelial dysfunction and increased oxidative stress, but the sources and mechanisms responsible for reactive oxygen species (ROS) production in the penis are unknown. We evaluated whether SCD activates NADPH oxidase, induces endothelial nitric oxide synthase (eNOS) uncoupling, and decreases antioxidants in the SCD mouse penis. We further tested the hypothesis that targeting NADPH oxidase decreases oxidative stress in the SCD mouse penis. SCD transgenic (sickle) mice were used as an animal model of SCD. Hemizygous (hemi) mice served as controls. Mice received an NADPH oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Penes were excised at baseline for molecular studies. Markers of oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NADPH oxidase subunits p67(phox) , p47(phox) , and gp91(phox) ), and enzymatic antioxidants (superoxide dismutase [SOD]1, SOD2, catalase, and glutathione peroxidase-1 [GPx1]) were measured by Western blot in penes. Sources of ROS, oxidative stress, and enzymatic antioxidants in the SCD penis. Relative to hemi mice, SCD increased (P<0.05) protein expression of NADPH oxidase subunits p67(phox) , p47(phox) , and gp91(phox) , 4-HNE-modified proteins, induced eNOS uncoupling, and reduced Gpx1 expression in the penis. Apocynin treatment of sickle mice reversed (P<0.05) the abnormalities in protein expressions of p47(phox) , gp91(phox) (but not p67(phox) ) and 4-HNE, but only slightly (P>0.05) prevented eNOS uncoupling in the penis. Apocynin treatment of hemi mice did not affect any of these parameters. NADPH oxidase and eNOS uncoupling are sources of oxidative stress in the SCD penis; decreased GPx1 further contributes to oxidative stress. Inhibition of NADPH oxidase upregulation decreases oxidative stress, implying a major role for NADPH oxidase as a ROS source and a potential target for improving vascular function in

  18. Targeting NADPH Oxidase Decreases Oxidative Stress in the Transgenic Sickle Cell Mouse Penis

    PubMed Central

    Musicki, Biljana; Liu, Tongyun; Sezen, Sena F.; Burnett, Arthur L.

    2012-01-01

    Introduction Sickle cell disease (SCD) is a state of chronic vasculopathy characterized by endothelial dysfunction and increased oxidative stress, but the sources and mechanisms responsible for reactive oxygen species (ROS) production in the penis are unknown. Aims We evaluated whether SCD activates NADPH oxidase, induces endothelial nitric oxide synthase (eNOS) uncoupling, and decreases antioxidants in the SCD mouse penis. We further tested the hypothesis that targeting NADPH oxidase decreases oxidative stress in the SCD mouse penis. Methods SCD transgenic (sickle) mice were used as an animal model of SCD. Hemizygous (hemi) mice served as controls. Mice received an NADPH oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Penes were excised at baseline for molecular studies. Markers of oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NADPH oxidase subunits p67phox, p47phox, and gp91phox), and enzymatic antioxidants (superoxide dismutase [SOD]1, SOD2, catalase, and glutathione peroxidase-1 [GPx1]) were measured by Western blot in penes. Main Outcome Measures Sources of ROS, oxidative stress, and enzymatic antioxidants in the SCD penis. Results Relative to hemi mice, SCD increased (P < 0.05) protein expression of NADPH oxidase subunits p67phox, p47phox, and gp91phox, 4-HNE-modified proteins, induced eNOS uncoupling, and reduced Gpx1 expression in the penis. Apocynin treatment of sickle mice reversed (P < 0.05) the abnormalities in protein expressions of p47phox, gp91phox (but not p67phox) and 4-HNE, but only slightly (P > 0.05) prevented eNOS uncoupling in the penis. Apocynin treatment of hemi mice did not affect any of these parameters. Conclusion NADPH oxidase and eNOS uncoupling are sources of oxidative stress in the SCD penis; decreased GPx1 further contributes to oxidative stress. Inhibition of NADPH oxidase upregulation decreases oxidative stress, implying a major role for NADPH oxidase as a ROS source and a

  19. Elemental selenium at nano size possesses lower toxicity without compromising the fundamental effect on selenoenzymes: comparison with selenomethionine in mice.

    PubMed

    Wang, Huali; Zhang, Jinsong; Yu, Hanqing

    2007-05-15

    Glutathione peroxidase and thioredoxin reductase are major selenoenzymes through which selenium exerts powerful antioxidant effects. Selenium also elicits pro-oxidant effects at toxic levels. The antioxidant and pro-oxidant effects, or bioavailability and toxicity, of selenium depend on its chemical form. Selenomethionine is considered to be the most appropriate supplemental form due to its excellent bioavailability and lower toxicity compared to various selenium compounds. The present studies reveal that, compared with selenomethionine, elemental selenium at nano size (Nano-Se) possesses equal efficacy in increasing the activities of glutathione peroxidase and thioredoxin reductase but has much lower toxicity as indicated by median lethal dose, acute liver injury, and short-term toxicity. Our results suggest that Nano-Se can serve as an antioxidant with reduced risk of selenium toxicity.

  20. NADPH-diaphorase activity and neurovascular coupling in the rat cerebral cortex.

    PubMed

    Vlasenko, O V; Maisky, V A; Maznychenko, A V; Pilyavskii, A I

    2008-01-01

    The distribution of NADPH-diaphorase-reactive (NADPH-dr) neurons and neuronal processes in the cerebral cortex and basal forebrain and their association with parenchymal vessels were studied in normal adult rats using NADPH-d histochemical protocol. The intensely stained cortical interneurons and reactive subcortically originating afferents, and stained microvessels were examined through a light microscope at law (x250) and high (x630) magnifications. NADPH-dr interneurons were concentrated in layers 2-6 of the M1 and M2 areas. However, clear predominance in their concentration (14 +/- 0.8 P < 0.05 per section) was found in layer 6. A mean number of labeled neurons in auditory (AuV), granular and agranular (GI, AIP) areas of the insular cortex was calculated to reach 12.3 +/- 0.7, 18.5 +/- 1.0 and 23.3 +/- 1.7 units per section, respectively (P < 0.05). The distinct apposition of labelled neurons to intracortical vessels was found in the M1, M2. The order of frequency of neurovascular coupling in different zones of the cerebral cortex was as following sequence: AuV (31.2%, n = 1040) > GI (18.0%, n = 640) > S1 (13.3%, n = 720) > M1 (6.3%, n = 1360). A large number of structural associations between labeled cells and vessels in the temporal and insular cortex indicate that NADPH-d-reactive interneurons can contribute to regulation of the cerebral regional blood flow in these areas.

  1. Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguinis * ♦

    PubMed Central

    Rhodes, DeLacy V.; Crump, Katie E.; Makhlynets, Olga; Snyder, Melanie; Ge, Xiuchun; Xu, Ping; Stubbe, JoAnne; Kitten, Todd

    2014-01-01

    Streptococcus sanguinis is a cause of infective endocarditis and has been shown to require a manganese transporter called SsaB for virulence and O2 tolerance. Like certain other pathogens, S. sanguinis possesses aerobic class Ib (NrdEF) and anaerobic class III (NrdDG) ribonucleotide reductases (RNRs) that perform the essential function of reducing ribonucleotides to deoxyribonucleotides. The accompanying paper (Makhlynets, O., Boal, A. K., Rhodes, D. V., Kitten, T., Rosenzweig, A. C., and Stubbe, J. (2014) J. Biol. Chem. 289, 6259–6272) indicates that in the presence of O2, the S. sanguinis class Ib RNR self-assembles an essential diferric-tyrosyl radical (FeIII2-Y•) in vitro, whereas assembly of a dimanganese-tyrosyl radical (MnIII2-Y•) cofactor requires NrdI, and MnIII2-Y• is more active than FeIII2-Y• with the endogenous reducing system of NrdH and thioredoxin reductase (TrxR1). In this study, we have shown that deletion of either nrdHEKF or nrdI completely abolishes virulence in an animal model of endocarditis, whereas nrdD mutation has no effect. The nrdHEKF, nrdI, and trxR1 mutants fail to grow aerobically, whereas anaerobic growth requires nrdD. The nrdJ gene encoding an O2-independent adenosylcobalamin-cofactored RNR was introduced into the nrdHEKF, nrdI, and trxR1 mutants. Growth of the nrdHEKF and nrdI mutants in the presence of O2 was partially restored. The combined results suggest that MnIII2-Y•-cofactored NrdF is required for growth under aerobic conditions and in animals. This could explain in part why manganese is necessary for virulence and O2 tolerance in many bacterial pathogens possessing a class Ib RNR and suggests NrdF and NrdI may serve as promising new antimicrobial targets. PMID:24381171

  2. Trimethoprim Resistance of Dihydrofolate Reductase Variants from Clinical Isolates of Pneumocystis jirovecii

    PubMed Central

    Cody, V.; Pace, J.; Torkelson, P.; Gangjee, A.

    2013-01-01

    Pneumocystis jirovecii is an opportunistic pathogen that causes serious pneumonia in immunosuppressed patients. Standard therapy and prophylaxis include trimethoprim (TMP)-sulfamethoxazole; trimethoprim in this combination targets dihydrofolate reductase (DHFR). Fourteen clinically observed variants of P. jirovecii DHFR were produced recombinantly to allow exploration of the causes of clinically observed failure of therapy and prophylaxis that includes trimethoprim. Six DHFR variants (S31F, F36C, L65P, A67V, V79I, and I158V) showed resistance to inhibition by trimethoprim, with Ki values for trimethoprim 4-fold to 100-fold higher than those for the wild-type P. jirovecii DHFR. An experimental antifolate with more conformational flexibility than trimethoprim showed strong activity against one trimethoprim-resistant variant. The two variants that were most resistant to trimethoprim (F36C and L65P) also had increased Km values for dihydrofolic acid (DHFA). The catalytic rate constant (kcat) was unchanged for most variant forms of P. jirovecii DHFR but was significantly lowered in F36C protein; one naturally occurring variant with two amino acid substitutions (S106P and E127G) showed a doubling of kcat, as well as a Km for NADPH half that of the wild type. The strongest resistance to trimethoprim occurred with amino acid changes in the binding pocket for DHFA or trimethoprim, and the strongest effect on binding of NADPH was linked to a mutation involved in binding the phosphate group of the cofactor. This study marks the first confirmation that naturally occurring mutations in the gene for DHFR from P. jirovecii produce variant forms of DHFR that are resistant to trimethoprim and may contribute to clinically observed failures of standard therapy or prophylaxis. PMID:23896474

  3. NADPH oxidases differentially regulate ROS metabolism and nutrient uptake under cadmium toxicity.

    PubMed

    Gupta, D K; Pena, L B; Romero-Puertas, M C; Hernández, A; Inouhe, M; Sandalio, L M

    2017-04-01

    The role of NADPH oxidases under cadmium (Cd) toxicity was studied using Arabidopsis thaliana mutants AtrbohC, AtrbohD and AtrbohF, which were grown under hydroponic conditions with 25 and 100 μM Cd for 1 and 5 days. Cadmium reduced the growth of leaves in WT, AtrbohC and D, but not in AtrbohF. A time-dependent increase in H 2 O 2 and lipid peroxidation was observed in all genotypes, with AtrbohC showing the smallest increase. An opposite behaviour was observed with NO accumulation. Cadmium increased catalase activity in WT plants and decreased it in Atrboh mutants, while glutathione reductase and glycolate oxidase activities increased in Atrboh mutants, and superoxide dismutases were down-regulated in AtrbohC. The GSH/GSSG and ASA/DHA couples were also affected by the treatment, principally in AtrbohC and AtrbohF, respectively. Cadmium translocation to the leaves was severely reduced in Atrboh mutants after 1 day of treatment and even after 5 days in AtrbohF. Similar results were observed for S, P, Ca, Zn and Fe accumulation, while an opposite trend was observed for K accumulation, except in AtrbohF. Thus, under Cd stress, RBOHs differentially regulate ROS metabolism, redox homeostasis and nutrient balance and could be of potential interest in biotechnology for the phytoremediation of polluted soils. © 2016 John Wiley & Sons Ltd.

  4. Sulfurtransferase and thioredoxin specifically interact as demonstrated by bimolecular fluorescence complementation analysis and biochemical tests.

    PubMed

    Henne, Melina; König, Nicolas; Triulzi, Tiziana; Baroni, Sara; Forlani, Fabio; Scheibe, Renate; Papenbrock, Jutta

    2015-01-01

    Sulfurtransferases (Strs) and thioredoxins (Trxs) are members of large protein families. Trxs are disulfide reductases and play an important role in redox-related cellular processes. They interact with a broad range of proteins. Strs catalyze the transfer of a sulfur atom from a suitable sulfur donor to nucleophilic sulfur acceptors in vitro, but the physiological roles of these enzymes are not well defined. Several studies in different organisms demonstrate protein-protein interactions of Strs with members of the Trx family. We are interested in investigating the specificity of the interaction between Str and Trx isoforms. In order to use the bimolecular fluorescence complementation (BiFC), several Str and Trx sequences from Arabidopsis thaliana were cloned into the pUC-SPYNE and pUC-SPYCE split-YFP vectors, respectively. Each couple of plasmids containing the sequences for the putative interaction partners were transformed into Arabidopsis protoplasts and screened using a confocal laser scanning microscope. Compartment- and partner-specific interactions could be observed in transformed protoplasts. Replacement of cysteine residues in the redox-active site of Trxs abolished the interaction signal. Therefore, the redox site is not only involved in the redox reaction but also responsible for the interaction with partner proteins. Biochemical assays support a specific interaction among Strs and certain Trxs. Based on the results obtained, the interaction of Strs and Trxs indicates a role of Strs in the maintenance of the cellular redox homeostasis.

  5. Dynamic single-cell NAD(P)H measurement reveals oscillatory metabolism throughout the E. coli cell division cycle.

    PubMed

    Zhang, Zheng; Milias-Argeitis, Andreas; Heinemann, Matthias

    2018-02-01

    Recent work has shown that metabolism between individual bacterial cells in an otherwise isogenetic population can be different. To investigate such heterogeneity, experimental methods to zoom into the metabolism of individual cells are required. To this end, the autofluoresence of the redox cofactors NADH and NADPH offers great potential for single-cell dynamic NAD(P)H measurements. However, NAD(P)H excitation requires UV light, which can cause cell damage. In this work, we developed a method for time-lapse NAD(P)H imaging in single E. coli cells. Our method combines a setup with reduced background emission, UV-enhanced microscopy equipment and optimized exposure settings, overall generating acceptable NAD(P)H signals from single cells, with minimal negative effect on cell growth. Through different experiments, in which we perturb E. coli's redox metabolism, we demonstrated that the acquired fluorescence signal indeed corresponds to NAD(P)H. Using this new method, for the first time, we report that intracellular NAD(P)H levels oscillate along the bacterial cell division cycle. The developed method for dynamic measurement of NAD(P)H in single bacterial cells will be an important tool to zoom into metabolism of individual cells.

  6. Biochemical and Structural Properties of a Thermostable Mercuric Ion Reductase from Metallosphaera sedula

    PubMed Central

    Artz, Jacob H.; White, Spencer N.; Zadvornyy, Oleg A.; Fugate, Corey J.; Hicks, Danny; Gauss, George H.; Posewitz, Matthew C.; Boyd, Eric S.; Peters, John W.

    2015-01-01

    Mercuric ion reductase (MerA), a mercury detoxification enzyme, has been tuned by evolution to have high specificity for mercuric ions (Hg2+) and to catalyze their reduction to a more volatile, less toxic elemental form. Here, we present a biochemical and structural characterization of MerA from the thermophilic crenarchaeon Metallosphaera sedula. MerA from M. sedula is a thermostable enzyme, and remains active after extended incubation at 97°C. At 37°C, the NADPH oxidation-linked Hg2+ reduction specific activity was found to be 1.9 μmol/min⋅mg, increasing to 3.1 μmol/min⋅mg at 70°C. M. sedula MerA crystals were obtained and the structure was solved to 1.6 Å, representing the first solved crystal structure of a thermophilic MerA. Comparison of both the crystal structure and amino acid sequence of MerA from M. sedula to mesophillic counterparts provides new insights into the structural determinants that underpin the thermal stability of the enzyme. PMID:26217660

  7. Hypoglycemic neuronal death is triggered by glucose reperfusion and activation of neuronal NADPH oxidase

    PubMed Central

    Suh, Sang Won; Gum, Elizabeth T.; Hamby, Aaron M.; Chan, Pak H.; Swanson, Raymond A.

    2007-01-01

    Hypoglycemic coma and brain injury are potential complications of insulin therapy. Certain neurons in the hippocampus and cerebral cortex are uniquely vulnerable to hypoglycemic cell death, and oxidative stress is a key event in this cell death process. Here we show that hypoglycemia-induced oxidative stress and neuronal death are attributable primarily to the activation of neuronal NADPH oxidase during glucose reperfusion. Superoxide production and neuronal death were blocked by the NADPH oxidase inhibitor apocynin in both cell culture and in vivo models of insulin-induced hypoglycemia. Superoxide production and neuronal death were also blocked in studies using mice or cultured neurons deficient in the p47phox subunit of NADPH oxidase. Chelation of zinc with calcium disodium EDTA blocked both the assembly of the neuronal NADPH oxidase complex and superoxide production. Inhibition of the hexose monophosphate shunt, which utilizes glucose to regenerate NADPH, also prevented superoxide formation and neuronal death, suggesting a mechanism linking glucose reperfusion to superoxide formation. Moreover, the degree of superoxide production and neuronal death increased with increasing glucose concentrations during the reperfusion period. These results suggest that high blood glucose concentrations following hypoglycemic coma can initiate neuronal death by a mechanism involving extracellular zinc release and activation of neuronal NADPH oxidase. PMID:17404617

  8. Repurposing Auranofin, Ebselen, and PX-12 as Antimicrobial Agents Targeting the Thioredoxin System

    PubMed Central

    May, Holly C.; Yu, Jieh-Juen; Guentzel, M. N.; Chambers, James P.; Cap, Andrew P.; Arulanandam, Bernard P.

    2018-01-01

    As microbial resistance to drugs continues to rise at an alarming rate, finding new ways to combat pathogens is an issue of utmost importance. Development of novel and specific antimicrobial drugs is a time-consuming and expensive process. However, the re-purposing of previously tested and/or approved drugs could be a feasible way to circumvent this long and costly process. In this review, we evaluate the U.S. Food and Drug Administration tested drugs auranofin, ebselen, and PX-12 as antimicrobial agents targeting the thioredoxin system. These drugs have been shown to act on bacterial, fungal, protozoan, and helminth pathogens without significant toxicity to the host. We propose that the thioredoxin system could serve as a useful therapeutic target with broad spectrum antimicrobial activity. PMID:29556223

  9. Glyphosate-induced oxidative stress in Arabidopsis thaliana affecting peroxisomal metabolism and triggers activity in the oxidative phase of the pentose phosphate pathway (OxPPP) involved in NADPH generation.

    PubMed

    de Freitas-Silva, Larisse; Rodríguez-Ruiz, Marta; Houmani, Hayet; da Silva, Luzimar Campos; Palma, José M; Corpas, Francisco J

    2017-11-01

    Glyphosate is a broad-spectrum systemic herbicide used worldwide. In susceptible plants, glyphosate affects the shikimate pathway and reduces aromatic amino acid synthesis. Using Arabidopsis seedlings grown in the presence of 20μM glyphosate, we analyzed H 2 O 2 , ascorbate, glutathione (GSH) and protein oxidation content as well as antioxidant catalase, superoxide dismutase (SOD) and ascorbate-glutathione cycle enzyme activity. We also examined the principal NADPH-generating system components, including glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-malic enzyme (NADP-ME) and NADP-isocitrate dehydrogenase (NADP-ICDH). Glyphosate caused a drastic reduction in growth parameters and an increase in protein oxidation. The herbicide also resulted in an overall increase in GSH content, antioxidant enzyme activity (catalase and all enzymatic components of the ascorbate-glutathione cycle) in addition to the two oxidative phase enzymes, G6PDH and 6PGDH, in the pentose phosphate pathway involved in NADPH generation. In this study, we provide new evidence on the participation of G6PDH and 6PGDH in the response to oxidative stress induced by glyphosate in Arabidopsis, in which peroxisomal enzymes, such as catalase and glycolate oxidase, are positively affected. We suggest that the NADPH provided by the oxidative phase of the pentose phosphate pathway (OxPPP) should serve to maintain glutathione reductase (GR) activity, thus preserving and regenerating the intracellular GSH pool under glyphosate-induced stress. It is particularly remarkable that the 6PGDH activity was unaffected by pro-oxidant and nitrating molecules such as H 2 0 2 , nitric oxide or peroxynitrite. Copyright © 2017 Elsevier GmbH. All rights reserved.

  10. Evaluation of rice tetraticopeptide domain-containing thioredoxin as a novel solubility-enhancing fusion tag in Escherichia coli.

    PubMed

    Xiao, Wenjun; Jiang, Li; Wang, Weiyu; Wang, Ruyue; Fan, Jun

    2018-02-01

    Fusion of solubility-enhancing tag is frequently used for improving soluble production of target protein in Escherichia coli. The Arabidopsis tetraticopeptide domain-containing thioredoxin (TDX) has been documented to exhibit functions of disulfide reductase, foldase chaperone, and holdase chaperone. Here, we identified that fusion of rice TDX with the smaller size increased soluble expression levels of three fluorescent proteins with different fluorophores in the E. coli strain BL21(DE3) or the Rosetta (DE3) strain with coexpression of six rare tRNAs, but decreased conformational quality of certain fluorescent proteins, as comparison with the His6-tagged ones. Among five maize proteins, the rice TDX fusion carrier displayed higher solubility-enhancing activity than the yeast SUMO3 tag toward three proteins in both E. coli strains. Five fusion constructs were cleaved with the co-expressed TEV protease variant, but the released target proteins were partly insolubly aggregated in vivo. Attachment of the His6-tag to the TDX tagged proteins had little impact on protein solubility. After Ni-NTA purification, five His6-TDX tagged proteins displayed different apparent purities. Taken together, our work presents that rice TDX tag is a novel solubility enhancer. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  11. The Importance of NADPH Oxidases and Redox Signaling in Angiogenesis

    PubMed Central

    Prieto-Bermejo, Rodrigo; Hernández-Hernández, Angel

    2017-01-01

    Eukaryotic cells have to cope with the constant generation of reactive oxygen species (ROS). Although the excessive production of ROS might be deleterious for cell biology, there is a plethora of evidence showing that moderate levels of ROS are important for the control of cell signaling and gene expression. The family of the nicotinamide adenine dinucleotide phosphate oxidases (NADPH oxidases or Nox) has evolved to produce ROS in response to different signals; therefore, they fulfil a central role in the control of redox signaling. The role of NADPH oxidases in vascular physiology has been a field of intense study over the last two decades. In this review we will briefly analyze how ROS can regulate signaling and gene expression. We will address the implication of NADPH oxidases and redox signaling in angiogenesis, and finally, the therapeutic possibilities derived from this knowledge will be discussed. PMID:28505091

  12. Bioreduction with Efficient Recycling of NADPH by Coupled Permeabilized Microorganisms▿

    PubMed Central

    Zhang, Wei; O'Connor, Kevin; Wang, Daniel I. C.; Li, Zhi

    2009-01-01

    The glucose dehydrogenase (GDH) from Bacillus subtilis BGSC 1A1 was cloned and functionally expressed in Escherichia coli BL21(pGDH1) and XL-1 Blue(pGDH1). Controlled permeabilization of recombinant E. coli BL21 and XL-1 Blue with EDTA-toluene under optimized conditions resulted in permeabilized cells with specific activities of 61 and 14 U/g (dry weight) of cells, respectively, for the conversion of NADP+ to NADPH upon oxidation of glucose. The permeabilized recombinant strains were more active than permeabilized B. subtilis BGSC 1A1, did not exhibit NADPH/NADH oxidase activity, and were useful for regeneration of both NADH and NADPH. Coupling of permeabilized cells of Bacillus pumilus Phe-C3 containing an NADPH-dependent ketoreductase and an E. coli recombinant expressing GDH as a novel biocatalytic system allowed enantioselective reduction of ethyl 3-keto-4,4,4-trifluorobutyrate with efficient recycling of NADPH; a total turnover number (TTN) of 4,200 mol/mol was obtained by using E. coli BL21(pGDH1) as the cofactor-regenerating microorganism with initial addition of 0.005 mM NADP+. The high TTN obtained is in the practical range for producing fine chemicals. Long-term stability of the permeabilized cell couple and a higher product concentration were demonstrated by 68 h of bioreduction of ethyl 3-keto-4,4,4-trifluorobutyrate with addition of 0.005 mM NADP+ three times; 50.5 mM (R)-ethyl 3-hydroxy-4,4,4-trifluorobutyrate was obtained with 95% enantiomeric excess, 84% conversion, and an overall TTN of 3,400 mol/mol. Our method results in practical synthesis of (R)-ethyl 3-hydroxy-4,4,4-trifluorobutyrate, and the principle described here is generally applicable to other microbial reductions with cofactor recycling. PMID:19047388

  13. Neovascularization in an arterio-venous loop-containing tissue engineering chamber: role of NADPH oxidase

    PubMed Central

    Jiang, F; Zhang, G; Hashimoto, I; Kumar, B S; Bortolotto, S; Morrison, W A; Dusting, G J

    2008-01-01

    Using an in vivo arterio-venous loop-containing tissue-engineering chamber, we have created a variety of vascularized tissue blocks, including functional myocardium. The viability of the transplanted cells is limited by the rate of neovascularization in the chamber. A Nox2-containing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is thought to have a critical role in ischaemic angiogenesis. In this study we investigated whether NADPH oxidase is involved in the neovascularization process in the tissue-engineering chamber. New blood vessels originating from the venous and the arterial ends of the loop could be identified after 3 days, and the vessel density (by lectin staining) peaked after 7 days and was maintained for at least 14 days. This was accompanied by granulation tissue formation and concomitant increase in the mRNA level of Nox4 NADPH oxidase. Although the total level of Nox2 mRNA in the chamber tissue decreased from day 3 to day 7, immunohistochemistry identified a strong expression of Nox2 in the endothelial cells of the new vessels. In human microvascular endothelial cells, the NADPH oxidase inhibitor apocynin reduced NADPH oxidase activity and inhibited the angiogenic responses in vitro. Local treatment with the NADPH oxidase inhibitors apocynin or gp91ds-tat peptide significantly suppressed the vessel growth in the chamber. In conclusion, NADPH oxidase-dependent redox signalling is important for neovascularization in this novel tissue-engineering chamber in vivo, and boosting this signalling might be a new approach to extending vascularization and tissue growth. PMID:19012731

  14. Structural Studies of Cinnamoyl-CoA Reductase and Cinnamyl-Alcohol Dehydrogenase, Key Enzymes of Monolignol Biosynthesis[C][W

    PubMed Central

    Pan, Haiyun; Zhou, Rui; Louie, Gordon V.; Mühlemann, Joëlle K.; Bomati, Erin K.; Bowman, Marianne E.; Dudareva, Natalia; Dixon, Richard A.; Noel, Joseph P.; Wang, Xiaoqiang

    2014-01-01

    The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4- to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates. PMID:25217505

  15. Time course of hydrogen peroxide-thioredoxin balance and its influence on the intracellular signalling in myocardial infarction.

    PubMed

    Schenkel, Paulo Cavalheiro; Tavares, Angela Maria Vicente; Fernandes, Rafael Oliveira; Diniz, Gabriela Placoná; Ludke, Ana Raquel Lehenbauer; Ribeiro, Maria Flavia Marques; Araujo, Alex Sander da Rosa; Barreto-Chaves, Maria Luiza; Belló-Klein, Adriane

    2012-06-01

    We investigated the myocardial thioredoxin-1 and hydrogen peroxide concentrations and their association with some prosurvival and pro-apoptotic proteins, during the transition from myocardial infarction (MI) to heart failure in rats. Male Wistar rats were divided into the following six groups: three sham-operated groups and three MI groups, each at at 2, 7 and 28 days postsurgery. Cardiac function was analysed by echocardiography; the concentration of H(2)O(2) and the ratio of reduced to oxidized glutathione were measured spectrophotometrically, while the myocardial immunocontent of thioredoxin-1, angiotensin II, angiotensin II type 1 and type 2 receptors, p-JNK/JNK, p-ERK/ERK, p-Akt/Akt, p-mTOR/mTOR and p-GSK3β/GSK3β was evaluated by Western blot. Our results show that thioredoxin-1 appears to make an important contribution to the reduced H(2)O(2) concentration. It was associated with lower JNK expression in the early period post-MI (2 days). However, thioredoxin-1 decreased, while renin-angiotensin system markers and levels of H(2)O(2) increased, over 28 days post-MI, in parallel with some signalling proteins involved in maladaptative cardiac remodelling and ventricular dysfunction. These findings provide insight into the time course profile of endogenous antioxidant adaptation to ischaemic injury, which may be useful for the design of therapeutical strategies targeting oxidative stress post-MI.

  16. NADPH Oxidase-Dependent Signaling in Endothelial Cells: Role in Physiology and Pathophysiology

    PubMed Central

    Ushio-Fukai, Masuko; Malik, Asrar B.

    2009-01-01

    Abstract Reactive oxygen species (ROS) including superoxide (O2·−) and hydrogen peroxide (H2O2) are produced endogenously in response to cytokines, growth factors; G-protein coupled receptors, and shear stress in endothelial cells (ECs). ROS function as signaling molecules to mediate various biological responses such as gene expression, cell proliferation, migration, angiogenesis, apoptosis, and senescence in ECs. Signal transduction activated by ROS, “oxidant signaling,” has received intense investigation. Excess amount of ROS contribute to various pathophysiologies, including endothelial dysfunction, atherosclerosis, hypertension, diabetes, and acute respiratory distress syndrome (ARDS). The major source of ROS in EC is a NADPH oxidase. The prototype phagaocytic NADPH oxidase is composed of membrane-bound gp91phox and p22hox, as well as cytosolic subunits such as p47phox, p67phox and small GTPase Rac. In ECs, in addition to all the components of phagocytic NADPH oxidases, homologues of gp91phox (Nox2) including Nox1, Nox4, and Nox5 are expressed. The aim of this review is to provide an overview of the emerging area of ROS derived from NADPH oxidase and oxidant signaling in ECs linked to physiological and pathophysiological functions. Understanding these mechanisms may provide insight into the NADPH oxidase and oxidant signaling components as potential therapeutic targets. Antioxid. Redox Signal. 11, 791–810. PMID:18783313

  17. Structural studies of cinnamoyl-CoA reductase and cinnamyl-alcohol dehydrogenase, key enzymes of monolignol biosynthesis.

    PubMed

    Pan, Haiyun; Zhou, Rui; Louie, Gordon V; Mühlemann, Joëlle K; Bomati, Erin K; Bowman, Marianne E; Dudareva, Natalia; Dixon, Richard A; Noel, Joseph P; Wang, Xiaoqiang

    2014-09-01

    The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4- to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates. © 2014 American Society of Plant Biologists. All rights reserved.

  18. Thioredoxin-dependent Redox Regulation of Chloroplastic Phosphoglycerate Kinase from Chlamydomonas reinhardtii*

    PubMed Central

    Morisse, Samuel; Michelet, Laure; Bedhomme, Mariette; Marchand, Christophe H.; Calvaresi, Matteo; Trost, Paolo; Fermani, Simona; Zaffagnini, Mirko; Lemaire, Stéphane D.

    2014-01-01

    In photosynthetic organisms, thioredoxin-dependent redox regulation is a well established mechanism involved in the control of a large number of cellular processes, including the Calvin-Benson cycle. Indeed, 4 of 11 enzymes of this cycle are activated in the light through dithiol/disulfide interchanges controlled by chloroplastic thioredoxin. Recently, several proteomics-based approaches suggested that not only four but all enzymes of the Calvin-Benson cycle may withstand redox regulation. Here, we characterized the redox features of the Calvin-Benson enzyme phosphoglycerate kinase (PGK1) from the eukaryotic green alga Chlamydomonas reinhardtii, and we show that C. reinhardtii PGK1 (CrPGK1) activity is inhibited by the formation of a single regulatory disulfide bond with a low midpoint redox potential (−335 mV at pH 7.9). CrPGK1 oxidation was found to affect the turnover number without altering the affinity for substrates, whereas the enzyme activation appeared to be specifically controlled by f-type thioredoxin. Using a combination of site-directed mutagenesis, thiol titration, mass spectrometry analyses, and three-dimensional modeling, the regulatory disulfide bond was shown to involve the not strictly conserved Cys227 and Cys361. Based on molecular mechanics calculation, the formation of the disulfide is proposed to impose structural constraints in the C-terminal domain of the enzyme that may lower its catalytic efficiency. It is therefore concluded that CrPGK1 might constitute an additional light-modulated Calvin-Benson cycle enzyme with a low activity in the dark and a TRX-dependent activation in the light. These results are also discussed from an evolutionary point of view. PMID:25202015

  19. PaTrx1 and PaTrx3, two cytosolic thioredoxins of the filamentous ascomycete Podospora anserina involved in sexual development and cell degeneration.

    PubMed

    Malagnac, Fabienne; Klapholz, Benjamin; Silar, Philippe

    2007-12-01

    In various organisms, thioredoxins are known to be involved in the reduction of protein disulfide bonds and in protecting the cell from oxidative stress. Genes encoding thioredoxins were found by searching the complete genome sequence of the filamentous ascomycete Podospora anserina. Among them, PaTrx1, PaTrx2, and PaTrx3 are predicted to be canonical cytosolic proteins without additional domains. Targeted disruption of PaTrx1, PaTrx2, and PaTrx3 shows that PaTrx1 is the major thioredoxin involved in sulfur metabolism. Deletions have no effect on peroxide resistance; however, data show that either PaTrx1 or PaTrx3 is necessary for sexual reproduction and for the development of the crippled growth cell degeneration (CG), processes that also required the PaMpk1 mitogen-activated protein kinase (MAPK) pathway. Since PaTrx1 PaTrx3 mutants show not an enhancement but rather an impairment in CG, it seems unlikely that PaTrx1 and PaTrx3 thioredoxins participate in the inhibition of this MAPK pathway. Altogether, these results underscore a role for thioredoxins in fungal development.

  20. S-nitrosoglutathione reductase in human lung cancer.

    PubMed

    Marozkina, Nadzeya V; Wei, Christina; Yemen, Sean; Wallrabe, Horst; Nagji, Alykhan S; Liu, Lei; Morozkina, Tatiana; Jones, David R; Gaston, Benjamin

    2012-01-01

    S-Nitrosoglutathione (GSNO) reductase regulates cell signaling pathways relevant to asthma and protects cells from nitrosative stress. Recent evidence suggests that this enzyme may prevent human hepatocellular carcinoma arising in the setting of chronic hepatitis. We hypothesized that GSNO reductase may also protect the lung against potentially carcinogenic reactions associated with nitrosative stress. We report that wild-type Ras is S-nitrosylated and activated by nitrosative stress and that it is denitrosylated by GSNO reductase. In human lung cancer, the activity and expression of GSNO reductase are decreased. Further, the distribution of the enzyme (including its colocalization with wild-type Ras) is abnormal. We conclude that decreased activity of GSNO reductase could leave the human lung vulnerable to the oncogenic effects of nitrosative stress, as is the case in the liver. This potential should be considered when developing therapies that inhibit pulmonary GSNO reductase to treat asthma and other conditions.

  1. The role of electrostatics in TrxR electron transfer mechanism: A computational approach.

    PubMed

    Teixeira, Vitor H; Capacho, Ana Sofia C; Machuqueiro, Miguel

    2016-12-01

    Thioredoxin reductase (TrxR) is an important enzyme in the control of the intracellular reduced redox environment. It transfers electrons from NADPH to several molecules, including its natural partner, thioredoxin. Although there is a generally accepted model describing how the electrons are transferred along TrxR, which involves a flexible arm working as a "shuttle," the molecular details of such mechanism are not completely understood. In this work, we use molecular dynamics simulations with Poisson-Boltzmann/Monte Carlo pKa calculations to investigate the role of electrostatics in the electron transfer mechanism. We observed that the combination of redox/protonation states of the N-terminal (FAD and Cys59/64) and C-terminal (Cys497/Selenocysteine498) redox centers defines the preferred relative positions and allows for the flexible arm to work as the desired "shuttle." Changing the redox/ionization states of those key players, leads to electrostatic triggers pushing the arm into the pocket when oxidized, and pulling it out, once it has been reduced. The calculated pKa values for Cys497 and Selenocysteine498 are 9.7 and 5.8, respectively, confirming that the selenocysteine is indeed deprotonated at physiological pH. This can be an important advantage in terms of reactivity (thiolate/selenolate are more nucleophilic than thiol/selenol) and ability to work as an electrostatic trigger (the "shuttle" mechanism) and may be the reason why TrxR uses selenium instead of sulfur. Proteins 2016; 84:1836-1843. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  2. Reconstituted high-density lipoprotein suppresses leukocyte NADPH oxidase activation by disrupting lipid rafts.

    PubMed

    Peshavariya, Hitesh; Dusting, Gregory J; Di Bartolo, Belinda; Rye, Kerry-Anne; Barter, Philip J; Jiang, Fan

    2009-08-01

    Reconstituted discoidal high-density lipoprotein (rHDL) has potent vascular protective actions. Native HDL suppresses cellular generation of reactive oxygen species, whereas this antioxidant effect of rHDL is less clear. This study examined the effects of rHDL on NADPH oxidase, a major source of cellular superoxide generation, in both leukocytes and human umbilical vein endothelial cells. Superoxide was measured with lucigenin-enhanced chemiluminescence. Expression of NADPH oxidase sub-units was determined by real-time PCR. Pre-treatment of HL-60 cells with rHDL (10 and 25 microM) for 1 h significantly reduced phorbol 12-myristate 13-acetate-stimulated superoxide production. Treatment with rHDL for up to 24 h did not change the mRNA expression of NADPH oxidase sub-units. In HL-60 cells, depletion of cholesterol from the plasma membrane by methyl-beta-cyclodextrin mimicked the effect of rHDL, whereas cholesterol repletion blunted the effects of rHDL. Treatment with rHDL induced disruption of the lipid raft structures and blunted PMA-induced redistribution of p47phox into lipid rafts. In contrast, treatment of endothelial cells with rHDL for up to 18 h had no effect on either basal or tumour necrosis factor-alpha-stimulated NADPH oxidase activity, but markedly suppressed the cytokine-induced expression of proinflammatory adhesion molecules. The results suggest that rHDL inhibits NADPH oxidase activation in leukocytes, probably by interrupting the assembly of NADPH oxidase sub-units at the lipid rafts. This effect may contribute to the vascular protective actions of rHDL against inflammation-mediated oxidative damage.

  3. A Novel Aldo-Keto Reductase, HdRed, from the Pacific Abalone Haliotis discus hannai, Which Reduces Alginate-derived 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid to 2-Keto-3-deoxy-d-gluconate*

    PubMed Central

    Mochizuki, Shogo; Nishiyama, Ryuji; Inoue, Akira; Ojima, Takao

    2015-01-01

    Abalone feeds on brown seaweeds and digests seaweeds' alginate with alginate lyases (EC 4.2.2.3). However, it has been unclear whether the end product of alginate lyases (i.e. unsaturated monouronate-derived 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH)) is assimilated by abalone itself, because DEH cannot be metabolized via the Embden-Meyerhof pathway of animals. Under these circumstances, we recently noticed the occurrence of an NADPH-dependent reductase, which reduced DEH to 2-keto-3-deoxy-d-gluconate, in hepatopancreas extract of the pacific abalone Haliotis discus hannai. In the present study, we characterized this enzyme to some extent. The DEH reductase, named HdRed in the present study, could be purified from the acetone-dried powder of hepatopancreas by ammonium sulfate fractionation followed by conventional column chromatographies. HdRed showed a single band of ∼40 kDa on SDS-PAGE and reduced DEH to 2-keto-3-deoxy-d-gluconate with an optimal temperature and pH at around 50 °C and 7.0, respectively. HdRed exhibited no appreciable activity toward 28 authentic compounds, including aldehyde, aldose, ketose, α-keto-acid, uronic acid, deoxy sugar, sugar alcohol, carboxylic acid, ketone, and ester. The amino acid sequence of 371 residues of HdRed deduced from the cDNA showed 18–60% identities to those of aldo-keto reductase (AKR) superfamily enzymes, such as human aldose reductase, halophilic bacterium reductase, and sea hare norsolorinic acid (a polyketide derivative) reductase-like protein. Catalytic residues and cofactor binding residues known in AKR superfamily enzymes were fairly well conserved in HdRed. Phylogenetic analysis for HdRed and AKR superfamily enzymes indicated that HdRed is an AKR belonging to a novel family. PMID:26555267

  4. A Novel Aldo-Keto Reductase, HdRed, from the Pacific Abalone Haliotis discus hannai, Which Reduces Alginate-derived 4-Deoxy-L-erythro-5-hexoseulose Uronic Acid to 2-Keto-3-deoxy-D-gluconate.

    PubMed

    Mochizuki, Shogo; Nishiyama, Ryuji; Inoue, Akira; Ojima, Takao

    2015-12-25

    Abalone feeds on brown seaweeds and digests seaweeds' alginate with alginate lyases (EC 4.2.2.3). However, it has been unclear whether the end product of alginate lyases (i.e. unsaturated monouronate-derived 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH)) is assimilated by abalone itself, because DEH cannot be metabolized via the Embden-Meyerhof pathway of animals. Under these circumstances, we recently noticed the occurrence of an NADPH-dependent reductase, which reduced DEH to 2-keto-3-deoxy-D-gluconate, in hepatopancreas extract of the pacific abalone Haliotis discus hannai. In the present study, we characterized this enzyme to some extent. The DEH reductase, named HdRed in the present study, could be purified from the acetone-dried powder of hepatopancreas by ammonium sulfate fractionation followed by conventional column chromatographies. HdRed showed a single band of ∼ 40 kDa on SDS-PAGE and reduced DEH to 2-keto-3-deoxy-D-gluconate with an optimal temperature and pH at around 50 °C and 7.0, respectively. HdRed exhibited no appreciable activity toward 28 authentic compounds, including aldehyde, aldose, ketose, α-keto-acid, uronic acid, deoxy sugar, sugar alcohol, carboxylic acid, ketone, and ester. The amino acid sequence of 371 residues of HdRed deduced from the cDNA showed 18-60% identities to those of aldo-keto reductase (AKR) superfamily enzymes, such as human aldose reductase, halophilic bacterium reductase, and sea hare norsolorinic acid (a polyketide derivative) reductase-like protein. Catalytic residues and cofactor binding residues known in AKR superfamily enzymes were fairly well conserved in HdRed. Phylogenetic analysis for HdRed and AKR superfamily enzymes indicated that HdRed is an AKR belonging to a novel family. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Localization of nitric oxide synthase and NADPH-diaphorase in guinea pig and human cochleae.

    PubMed

    Ruan, R S; Leong, S K; Yeoh, K H

    1997-01-01

    The distributions of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) and nitric oxide synthase (NOS) in mammalian cochlea were studied at light and electron microscope levels by NADPH-d histochemistry and brain NOS (bNOS) immunohistochemistry. The cochleae from 15 albino guinea pigs were perilymphatically fixed with 2% periodate-lysine-paraformaldehyde, decalcified in 10% EDTA and processed for light and electron microscopy after NADPH-d or NOS staining in frozen and vibratome sections respectively. One human cochlea was available for light microscope examination of NADPH-d or bNOS stained sections. Light microscope results revealed that type I neurons and nerve fibers of the spiral ganglion cells were labeled by bNOS immunohistochemistry as well as NADPH-d histochemistry in both guinea pig and human cochleae. At subcellular level, NADPH-d reaction product was localized in the mitochondria of the neuronal cytoplasm and axoplasm and in the cytoplasm of the vascular endothelium. The immunoreaction products of bNOS were evenly distributed in the neuronal cytoplasm and axoplasm. Myelinated and unmyelinated fibers in the intraganglionic spiral bundle and the inner spiral and inner radial fibers below the inner hair cells were labeled for bNOS. The nerve endings below the outer hair cells were not stained. NOS immunoreaction product was also found in the outer hair cells, Schwann cells of myelinated nerve fibers, Deiter's cells, pillar cells and the tympanic lamina cells. No difference was found in the staining pattern of both NADPH-d and NOS reaction products between human and guinea pig cochleae at the light microscope level. The results suggest that NO plays an important role in the maintenance of auditory function in the mammal.

  6. Expression and characterization of full-length human heme oxygenase-1: the presence of intact membrane-binding region leads to increased binding affinity for NADPH cytochrome P450 reductase.

    PubMed

    Huber, Warren J; Backes, Wayne L

    2007-10-30

    Heme oxygenase-1 (HO-1) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, HO-1 receives the electrons necessary for catalysis from the flavoprotein NADPH cytochrome P450 reductase (CPR), releasing free iron and carbon monoxide. Much of the recent research involving heme oxygenase has been done using a 30 kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a glutathione-s-transferase (GST)-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30 kDa degradation product that could not be eliminated. Therefore, attempts were made to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces arginine with lysine. This mutation allowed the expression and purification of a full-length hHO-1 protein. Unlike wild type (WT) HO-1, the R254K mutant could be purified to a single 32 kDa protein capable of degrading heme at the same rate as the WT enzyme. The R254K full-length form had a specific activity of approximately 200-225 nmol of bilirubin h-1 nmol-1 HO-1 as compared to approximately 140-150 nmol of bilirubin h-1 nmol-1 for the WT form, which contains the 30 kDa contaminant. This is a 2-3-fold increase from the previously reported soluble 30 kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane-spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other endoplasmic reticulum resident enzymes.

  7. NADPH Oxidase versus Mitochondria-Derived ROS in Glucose-Induced Apoptosis of Pericytes in Early Diabetic Retinopathy

    PubMed Central

    Mustapha, Nik M.; Tarr, Joanna M.; Kohner, Eva M.; Chibber, Rakesh

    2010-01-01

    Objectives. Using apocynin (inhibitor of NADPH oxidase), and Mitoquinol 10 nitrate (MitoQ; mitochondrial-targeted antioxidant), we addressed the importance of mitochondria versus NADPH oxidase-derived ROS in glucose-induced apoptosis of pericytes. Methods. NADPH oxidase was localised using Western blot analysis and cytochrome C reduction assay. Apoptosis was detected by measuring caspase-3 activity. Intracellular glucose concentration, ROS formation and Nε-(carboxymethyl) lysine (CML) content were measured using Amplex Red assay kit, dihydroethidium (DHE), and competitive immunoabsorbant enzyme-linked assay (ELISA), respectively. Results. NADPH oxidase was localised in the cytoplasm of pericytes suggesting ROS production within intracellular compartments. High glucose (25 mM) significantly increased apoptosis, intracellular glucose concentration, and CML content. Apoptosis was associated with increased gp91phox expression, activity of NADPH oxidase, and intracellular ROS production. Apocynin and not MitoQ significantly blunted the generation of ROS, formation of intracellular CML and apoptosis. Conclusions. NADPH oxidase and not mitochondria-derived ROS is responsible for the accelerated apoptosis of pericytes in diabetic retinopathy. PMID:20652059

  8. PKC delta and NADPH oxidase in retinoic acid-induced neuroblastoma cell differentiation.

    PubMed

    Nitti, Mariapaola; Furfaro, Anna Lisa; Cevasco, Claudia; Traverso, Nicola; Marinari, Umberto Maria; Pronzato, Maria Adelaide; Domenicotti, Cinzia

    2010-05-01

    The role of reactive oxygen species (ROS) in the regulation of signal transduction processes has been well established in many cell types and recently the fine tuning of redox signalling in neurons received increasing attention. With regard to this, the involvement of NADPH oxidase (NOX) in neuronal pathophysiology has been proposed but deserves more investigation. In the present study, we used SH-SY5Y neuroblastoma cells to analyse the role of NADPH oxidase in retinoic acid (RA)-induced differentiation, pointing out the involvement of protein kinase C (PKC) delta in the activation of NOX. Retinoic acid induces neuronal differentiation as revealed by the increased expression of MAP2, the decreased cell doubling rate, and the gain in neuronal morphological features and these events are accompanied by the increased expression level of PKC delta and p67(phox), one of the components of NADPH oxidase. Using DPI to inhibit NOX activity we show that retinoic acid acts through this enzyme to induce morphological changes linked to the differentiation. Moreover, using rottlerin to inhibit PKC delta or transfection experiments to overexpress it, we show that retinoic acid acts through this enzyme to induce MAP2 expression and to increase p67(phox) membrane translocation leading to NADPH oxidase activation. These findings identify the activation of PKC delta and NADPH oxidase as crucial steps in RA-induced neuroblastoma cell differentiation. 2010 Elsevier Inc. All rights reserved.

  9. Effect of pregnant mare's serum gonadotrophin on the activities of delta 4-5 alpha-reductase, aromatase, and other enzymes in the ovaries of immature rats.

    PubMed

    Suzuki, K; Kawakura, K; Tamaoki, B I

    1978-05-01

    After incubation of progesterone, 17 alpha-hydroxyprogesterone, androstenedione, and testostrone with an ovarian preparation (supernatant fluid at 10,000 x g) of immature rats (21-23 days of age) in the presence of NADPH, 3 alpha- and 3 beta-hydroxy-5 alpha-reduced steroids were obtained as the major metabolites. Among the enzyme activities relevant to the metabolism, delta 4-5 alpha-reductase and 3 beta-hydroxysteroid dehydrogenase were intracellularly localized to the microsomal fraction (10,000--105,000 x g), and 3 alpha-hydroxysteroid dehydrogenase was detected exclusively in the cytosol fraction (supernatant fluid at 105,000 x g). Within 2 days after a single injection of pregnant mare's serum gonadotrophin (10 IU/rat) to 21-day-old female rats, the following occurred: 1) an enhancement of 17 alpha-hydroxylase and C-17-C-20 lyase activities; 2) a suppression of delta 4-5 alpha-reductase activity; and 3) an increase in aromatizing activity. From the above-mentioned results, it was concluded that the increased secretion of estrogen from ovaries of immature rats stimulated by pregnant mare's serum gonadotrophin administration was caused by a modification of the ovarian enzyme activities relevant to estrogen production.

  10. Purification, cloning, functional expression and characterization of perakine reductase: the first example from the AKR enzyme family, extending the alkaloidal network of the plant Rauvolfia.

    PubMed

    Sun, Lianli; Ruppert, Martin; Sheludko, Yuri; Warzecha, Heribert; Zhao, Yu; Stöckigt, Joachim

    2008-07-01

    Perakine reductase (PR) catalyzes an NADPH-dependent step in a side-branch of the 10-step biosynthetic pathway of the alkaloid ajmaline. The enzyme was cloned by a "reverse-genetic" approach from cell suspension cultures of the plant Rauvolfia serpentina (Apocynaceae) and functionally expressed in Escherichia coli as the N-terminal His(6)-tagged protein. PR displays a broad substrate acceptance, converting 16 out of 28 tested compounds with reducible carbonyl function which belong to three substrate groups: benzaldehyde, cinnamic aldehyde derivatives and monoterpenoid indole alkaloids. The enzyme has an extraordinary selectivity in the group of alkaloids. Sequence alignments define PR as a new member of the aldo-keto reductase (AKR) super family, exhibiting the conserved catalytic tetrad Asp52, Tyr57, Lys84, His126. Site-directed mutagenesis of each of these functional residues to an alanine residue results in >97.8% loss of enzyme activity, in compounds of each substrate group. PR represents the first example of the large AKR-family which is involved in the biosynthesis of plant monoterpenoid indole alkaloids. In addition to a new esterase, PR significantly extends the Rauvolfia alkaloid network to the novel group of peraksine alkaloids.

  11. Nitrosative/Oxidative Stress Conditions Regulate Thioredoxin-Interacting Protein (TXNIP) Expression and Thioredoxin-1 (TRX-1) Nuclear Localization

    PubMed Central

    Ogata, Fernando Toshio; Batista, Wagner Luiz; Sartori, Adriano; Gesteira, Tarsis Ferreira; Masutani, Hiroshi; Arai, Roberto Jun; Yodoi, Junji; Stern, Arnold; Monteiro, Hugo Pequeno

    2013-01-01

    Thioredoxin (TRX-1) is a multifunctional protein that controls the redox status of other proteins. TRX-1 can be found in the extracellular milieu, cytoplasm and nucleus, and it has distinct functions in each environment. Previously, we studied the intracellular localization of TRX-1 and its relationship with the activation of the p21Ras - ERK1/2 MAP Kinases signaling pathway. In situations where this pathway was activated by stress conditions evoked by a nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), TRX-1 accumulated in the nuclear compartment due to nitrosylation of p21Ras and activation of downstream ERK1/2 MAP kinases. Presently, we demonstrate that ERK1/2 MAP Kinases activation and spatial distribution within cells trigger TRX-1 nuclear translocation through down-regulation of the physiological inhibitor of TRX-1, Thioredoxin Interacting Protein (TXNIP). Once activated by the oxidants, SNAP and H2O2, the ERK1/2 MAP kinases migrate to the nucleus. This is correlated with down-regulation of TXNIP. In the presence of the MEK inhibitors (PD98059 or UO126), or in cells transfected with the Protein Enriched in Astrocytes (PEA-15), a cytoplasmic anchor of ERK1/2 MAP kinases, TRX-1 nuclear migration and TXNIP down-regulation are no longer observed in cells exposed to oxidants. On the other hand, over-expression of TXNIP abolishes nuclear migration of TRX-1 under nitrosative/oxidative stress conditions, whereas gene silencing of TXNIP facilitates nuclear migration even in the absence of stress conditions. Studies based on the TXNIP promoter support this regulation. In conclusion, changes in TRX-1 compartmentalization under nitrosative/oxidative stress conditions are dependent on the expression levels of TXNIP, which are regulated by cellular compartmentalization and activation of the ERK1/2 MAP kinases. PMID:24376827

  12. Nitrosative/oxidative stress conditions regulate thioredoxin-interacting protein (TXNIP) expression and thioredoxin-1 (TRX-1) nuclear localization.

    PubMed

    Ogata, Fernando Toshio; Batista, Wagner Luiz; Sartori, Adriano; Gesteira, Tarsis Ferreira; Masutani, Hiroshi; Arai, Roberto Jun; Yodoi, Junji; Stern, Arnold; Monteiro, Hugo Pequeno

    2013-01-01

    Thioredoxin (TRX-1) is a multifunctional protein that controls the redox status of other proteins. TRX-1 can be found in the extracellular milieu, cytoplasm and nucleus, and it has distinct functions in each environment. Previously, we studied the intracellular localization of TRX-1 and its relationship with the activation of the p21Ras-ERK1/2 MAP Kinases signaling pathway. In situations where this pathway was activated by stress conditions evoked by a nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), TRX-1 accumulated in the nuclear compartment due to nitrosylation of p21Ras and activation of downstream ERK1/2 MAP kinases. Presently, we demonstrate that ERK1/2 MAP Kinases activation and spatial distribution within cells trigger TRX-1 nuclear translocation through down-regulation of the physiological inhibitor of TRX-1, Thioredoxin Interacting Protein (TXNIP). Once activated by the oxidants, SNAP and H₂O₂, the ERK1/2 MAP kinases migrate to the nucleus. This is correlated with down-regulation of TXNIP. In the presence of the MEK inhibitors (PD98059 or UO126), or in cells transfected with the Protein Enriched in Astrocytes (PEA-15), a cytoplasmic anchor of ERK1/2 MAP kinases, TRX-1 nuclear migration and TXNIP down-regulation are no longer observed in cells exposed to oxidants. On the other hand, over-expression of TXNIP abolishes nuclear migration of TRX-1 under nitrosative/oxidative stress conditions, whereas gene silencing of TXNIP facilitates nuclear migration even in the absence of stress conditions. Studies based on the TXNIP promoter support this regulation. In conclusion, changes in TRX-1 compartmentalization under nitrosative/oxidative stress conditions are dependent on the expression levels of TXNIP, which are regulated by cellular compartmentalization and activation of the ERK1/2 MAP kinases.

  13. NADPH oxidases in the arbuscular mycorrhizal symbiosis.

    PubMed

    Belmondo, Simone; Calcagno, Cristina; Genre, Andrea; Puppo, Alain; Pauly, Nicolas; Lanfranco, Luisa

    2016-01-01

    Plant NADPH oxidases are the major source of reactive oxygen species (ROS) that plays key roles as both signal and stressor in several plant processes, including defense responses against pathogens. ROS accumulation in root cells during arbuscular mycorrhiza (AM) development has raised the interest in understanding how ROS-mediated defense programs are modulated during the establishment of this mutualistic interaction. We have recently analyzed the expression pattern of 5 NADPH oxidase (also called RBOH) encoding genes in Medicago truncatula, showing that only one of them (MtRbohE) is specifically upregulated in arbuscule-containing cells. In line with this result, RNAi silencing of MtRbohE generated a strong alteration in root colonization, with a significant reduction in the number of arbusculated cells. On this basis, we propose that MtRBOHE-mediated ROS production plays a crucial role in the intracellular accommodation of arbuscules.

  14. NADPH oxidases in the arbuscular mycorrhizal symbiosis

    PubMed Central

    Belmondo, Simone; Calcagno, Cristina; Genre, Andrea; Puppo, Alain; Pauly, Nicolas; Lanfranco, Luisa

    2016-01-01

    ABSTRACT Plant NADPH oxidases are the major source of reactive oxygen species (ROS) that plays key roles as both signal and stressor in several plant processes, including defense responses against pathogens. ROS accumulation in root cells during arbuscular mycorrhiza (AM) development has raised the interest in understanding how ROS-mediated defense programs are modulated during the establishment of this mutualistic interaction. We have recently analyzed the expression pattern of 5 NADPH oxidase (also called RBOH) encoding genes in Medicago truncatula, showing that only one of them (MtRbohE) is specifically upregulated in arbuscule-containing cells. In line with this result, RNAi silencing of MtRbohE generated a strong alteration in root colonization, with a significant reduction in the number of arbusculated cells. On this basis, we propose that MtRBOHE-mediated ROS production plays a crucial role in the intracellular accommodation of arbuscules. PMID:27018627

  15. Overexpression of the FAD-binding domain of the sulphite reductase flavoprotein component from Escherichia coli and its inhibition by iodonium diphenyl chloride.

    PubMed Central

    Covès, J; Lebrun, C; Gervasi, G; Dalbon, P; Fontecave, M

    1999-01-01

    SiR-FP43, the NADPH- and FAD-binding domain of the Escherichia coli sulphite reductase flavoprotein component (SiR-FP), has been overexpressed and characterized. It folds independently, retaining FAD as a cofactor and the catalytic properties associated with the presence of this cofactor. Iodonium diphenyl chloride (IDP) was shown to be a very efficient inhibitor of SiR-FP43 and SiR-FP60, the monomeric form of SiR-FP, containing both FMN and FAD as cofactors (K(i) = 18.5 +/- 5 microM, maximal inactivation rate = 0.053 +/- 0.005 s(-1)). In both cases, inactivation was shown to result from covalent binding of a phenyl group to FAD exclusively, in marked contrast with previous results obtained with cytochrome P450 reductase (CPR), where FMN and a tryptophan were phenylated, but not FAD. However, our kinetic analyses are in agreement with the inhibition mechanism demonstrated with CPR [Tew (1993) Biochemistry 32, 10209-10215]. Nine different FAD phenylated adducts were isolated and, for the first time, two FAD phenylated adducts were identified directly after extraction from a protein. Taken together, our results have shown that flavoprotein inactivation by IDP is not a reliable indicator for a flavin radical intermediate in catalysis. PMID:10455035

  16. Inhibition of the NADPH oxidase regulates HO-1 expression in chronic myeloid leukemia

    PubMed Central

    Singh, Melissa M.; Irwin, Mary E.; Gao, Yin; Ban, Kechen; Shi, Ping; Arlinghaus, Ralph B.; Amin, Hesham M.; Chandra, Joya

    2011-01-01

    Background Patients with blast crisis phase chronic myelogeneous leukemia (CML) have poor response to tyrosine kinase inhibitors designed to inhibit the BCR-ABL1 oncogene. Recent work has shown that heme oxygenase 1 (HO-1) expression is increased in BCR-ABL1 expressing cells and that inhibition of HO-1 in CML leads to reduced cellular growth suggesting HO-1 may be a plausible target for therapy. Here we sought to clarify the mechanism of HO-1 overexpression and the role of the NADPH oxidase as a contributor to this mechanism in CML. Methods HO-1 expression was evaluated in CML bone marrow specimens from patients in various stages of disease, in a transplant based model for CML and in CML cell lines. Chemical and genetic inhibition of the NADPH oxidase was carried out in CML cells. Results Blast crisis CML patient specimens displayed higher levels of HO-1 staining than chronic or accelerated phase. HO-1 upregulation in BCR-ABL1 expressing cells was suppressed by diphenyliodonium (DPI), a chemical inhibitor of the NADPH oxidase. Targeting the NADPH oxidase through RNAi to Rac1, a dominant negative Rac1 construct or an inhibitor of Rac1 activity also blunted HO-1 protein expression. Moreover, inhibition of the NADPH oxidase by RNAi directed towards p47phox similarly abrogated HO-1 levels. Conclusion BCR-ABL1 expression upregulates HO-1, a survival factor for CML cells. This upregulation is more pronounced in blast crisis CML relative to early stage disease and is mediated by the NADPH oxidase components Rac1 and p47phox. Expression of p47phox is increased in BCR-ABL1 expressing cells. PMID:22139798

  17. Studies on the interactions between drugs and estrogen: analytical method for prediction system of gynecomastia induced by drugs on the inhibitory metabolism of estradiol using Escherichia coli coexpressing human CYP3A4 with human NADPH-cytochrome P450 reductase.

    PubMed

    Satoh, T; Fujita, K I; Munakata, H; Itoh, S; Nakamura, K; Kamataki, T; Itoh, S; Yoshizawa, I

    2000-11-15

    To establish a prediction system for drug-induced gynecomastia in clinical fields, a model reaction system was developed to explain numerically this side effect. The principle is based on the assumption that 50% inhibition concentration (IC(50)) of drugs on the in vitro metabolism of estradiol (E2) to its major product 2-hydroxyestradiol (2-OH-E2) can be regarded as the index for achieving this purpose. By using human cytochrome P450s coexpressed with human NADPH-cytochrome P450 reductase in Escherichia coli as the enzyme, the reaction was examined. Among the nine enzymes (CYP1A1, 1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) tested, CYP3A4 having a V(max)/K(m) (ml/min/nmol P450) value of 0.32 for production of 2-OH-E2 was shown to be the most suitable enzyme as the reagent. The inhibitory effects of ketoconazole, cyclosporin A, and cimetidine toward the 2-hydroxylation of E2 catalyzed by CYP3A4 were obtained, and their IC(50) values were 7 nM, 64 nM, and 290 microM, respectively. The present results suggest that IC(50) values thus obtained can be substituted as the prediction index for gynecomastia induced by drugs, considering the patients' individual information. Copyright 2000 Academic Press.

  18. PaTrx1 and PaTrx3, Two Cytosolic Thioredoxins of the Filamentous Ascomycete Podospora anserina Involved in Sexual Development and Cell Degeneration▿ †

    PubMed Central

    Malagnac, Fabienne; Klapholz, Benjamin; Silar, Philippe

    2007-01-01

    In various organisms, thioredoxins are known to be involved in the reduction of protein disulfide bonds and in protecting the cell from oxidative stress. Genes encoding thioredoxins were found by searching the complete genome sequence of the filamentous ascomycete Podospora anserina. Among them, PaTrx1, PaTrx2, and PaTrx3 are predicted to be canonical cytosolic proteins without additional domains. Targeted disruption of PaTrx1, PaTrx2, and PaTrx3 shows that PaTrx1 is the major thioredoxin involved in sulfur metabolism. Deletions have no effect on peroxide resistance; however, data show that either PaTrx1 or PaTrx3 is necessary for sexual reproduction and for the development of the crippled growth cell degeneration (CG), processes that also required the PaMpk1 mitogen-activated protein kinase (MAPK) pathway. Since PaTrx1 PaTrx3 mutants show not an enhancement but rather an impairment in CG, it seems unlikely that PaTrx1 and PaTrx3 thioredoxins participate in the inhibition of this MAPK pathway. Altogether, these results underscore a role for thioredoxins in fungal development. PMID:17933907

  19. Selective Targeting of the Cysteine Proteome by Thioredoxin and Glutathione Redox Systems

    PubMed Central

    Go, Young-Mi; Roede, James R.; Walker, Douglas I.; Duong, Duc M.; Seyfried, Nicholas T.; Orr, Michael; Liang, Yongliang; Pennell, Kurt D.; Jones, Dean P.

    2013-01-01

    Thioredoxin (Trx) and GSH are the major thiol antioxidants protecting cells from oxidative stress-induced cytotoxicity. Redox states of Trx and GSH have been used as indicators of oxidative stress. Accumulating studies suggest that Trx and GSH redox systems regulate cell signaling and metabolic pathways differently and independently during diverse stressful conditions. In the current study, we used a mass spectrometry-based redox proteomics approach to test responses of the cysteine (Cys) proteome to selective disruption of the Trx- and GSH-dependent systems. Auranofin (ARF) was used to inhibit Trx reductase without detectable oxidation of the GSH/GSSG couple, and buthionine sulfoximine (BSO) was used to deplete GSH without detectable oxidation of Trx1. Results for 606 Cys-containing peptides (peptidyl Cys) showed that 36% were oxidized more than 1.3-fold by ARF, whereas BSO-induced oxidation of peptidyl Cys was only 10%. Mean fold oxidation of these peptides was also higher by ARF than BSO treatment. Analysis of potential functional pathways showed that ARF oxidized peptides associated with glycolysis, cytoskeleton remodeling, translation and cell adhesion. Of 60 peptidyl Cys oxidized due to depletion of GSH, 41 were also oxidized by ARF and included proteins of translation and cell adhesion but not glycolysis or cytoskeletal remodeling. Studies to test functional correlates showed that pyruvate kinase activity and lactate levels were decreased with ARF but not BSO, confirming the effects on glycolysis-associated proteins are sensitive to oxidation by ARF. These data show that the Trx system regulates a broader range of proteins than the GSH system, support distinct function of Trx and GSH in cellular redox control, and show for the first time in mammalian cells selective targeting peptidyl Cys and biological pathways due to deficient function of the Trx system. PMID:23946468

  20. Depletion of NADP(H) due to CD38 activation triggers endothelial dysfunction in the postischemic heart.

    PubMed

    Reyes, Levy A; Boslett, James; Varadharaj, Saradhadevi; De Pascali, Francesco; Hemann, Craig; Druhan, Lawrence J; Ambrosio, Giuseppe; El-Mahdy, Mohamed; Zweier, Jay L

    2015-09-15

    In the postischemic heart, coronary vasodilation is impaired due to loss of endothelial nitric oxide synthase (eNOS) function. Although the eNOS cofactor tetrahydrobiopterin (BH4) is depleted, its repletion only partially restores eNOS-mediated coronary vasodilation, indicating that other critical factors trigger endothelial dysfunction. Therefore, studies were performed to characterize the unidentified factor(s) that trigger endothelial dysfunction in the postischemic heart. We observed that depletion of the eNOS substrate NADPH occurs in the postischemic heart with near total depletion from the endothelium, triggering impaired eNOS function and limiting BH4 rescue through NADPH-dependent salvage pathways. In isolated rat hearts subjected to 30 min of ischemia and reperfusion (I/R), depletion of the NADP(H) pool occurred and was most marked in the endothelium, with >85% depletion. Repletion of NADPH after I/R increased NOS-dependent coronary flow well above that with BH4 alone. With combined NADPH and BH4 repletion, full restoration of NOS-dependent coronary flow occurred. Profound endothelial NADPH depletion was identified to be due to marked activation of the NAD(P)ase-activity of CD38 and could be prevented by inhibition or specific knockdown of this protein. Depletion of the NADPH precursor, NADP(+), coincided with formation of 2'-phospho-ADP ribose, a CD38-derived signaling molecule. Inhibition of CD38 prevented NADP(H) depletion and preserved endothelium-dependent relaxation and NO generation with increased recovery of contractile function and decreased infarction in the postischemic heart. Thus, CD38 activation is an important cause of postischemic endothelial dysfunction and presents a novel therapeutic target for prevention of this dysfunction in unstable coronary syndromes.