Sample records for nadph-dependent aldehyde reductase

  1. 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

  2. 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.

  3. Resolution and partial characterization of two aldehyde reductases of mammalian liver.

    PubMed

    Tulsiani, D R; Touster

    1977-04-25

    Investigation of NADP-dependent aldehyde reductase activity in mouse liver led to the finding that two distinct reductases are separable by DE52 ion exchange chromatography. Aldehyde reductase I (AR I) appears in the effluent, while aldehyde reductase II (AR II) is eluted with a salt gradient. By several procedures AR II was purified over 1100-fold from liver supernatant fraction, but AR I could be pruified only 107-fold because of its instability. The two enzymes are different in regard to pH optimum, substrate specificity, response to inhibitors, and reactivity with antibody to AR II. While both enzymes utilize aromatic aldehydes well, only AR II ACTS ON D-glucuronate, indicating that it is the aldyhyde reductase recently reported to be identical to NADP-L-gulonate dehydrogenase. The presence of two NADP-linked aldehyde reductases in liver has apparently not heretofore been reported.

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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...

  13. Toward aldehyde and alkane production by removing aldehyde reductase activity in Escherichia coli.

    PubMed

    Rodriguez, Gabriel M; Atsumi, Shota

    2014-09-01

    Advances in synthetic biology and metabolic engineering have enabled the construction of novel biological routes to valuable chemicals using suitable microbial hosts. Aldehydes serve as chemical feedstocks in the synthesis of rubbers, plastics, and other larger molecules. Microbial production of alkanes is dependent on the formation of a fatty aldehyde intermediate which is converted to an alkane by an aldehyde deformylating oxygenase (ADO). However, microbial hosts such as Escherichia coli are plagued by many highly active endogenous aldehyde reductases (ALRs) that convert aldehydes to alcohols, which greatly complicates strain engineering for aldehyde and alkane production. It has been shown that the endogenous ALR activity outcompetes the ADO enzyme for fatty aldehyde substrate. The large degree of ALR redundancy coupled with an incomplete database of ALRs represents a significant obstacle in engineering E. coli for either aldehyde or alkane production. In this study, we identified 44 ALR candidates encoded in the E. coli genome using bioinformatics tools, and undertook a comprehensive screening by measuring the ability of these enzymes to produce isobutanol. From the pool of 44 candidates, we found five new ALRs using this screening method (YahK, DkgA, GldA, YbbO, and YghA). Combined deletions of all 13 known ALRs resulted in a 90-99% reduction in endogenous ALR activity for a wide range of aldehyde substrates (C2-C12). Elucidation of the ALRs found in E. coli could guide one in reducing competing alcohol formation during alkane or aldehyde production. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  14. 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.

  15. YLL056C from Saccharomyces cerevisiae encodes a novel protein with aldehyde reductase activity.

    PubMed

    Wang, Han-Yu; Xiao, Di-Fan; Zhou, Chang; Wang, Lin-Lu; Wu, Lan; Lu, Ya-Ting; Xiang, Quan-Ju; Zhao, Ke; Li, Xi; Ma, Meng -Gen

    2017-06-01

    The short-chain dehydrogenase/reductase (SDR) family, the largest family in dehydrogenase/reductase superfamily, is divided into "classical," "extended," "intermediate," "divergent," "complex," and "atypical" groups. Recently, several open reading frames (ORFs) were characterized as intermediate SDR aldehyde reductase genes in Saccharomyces cerevisiae. However, no functional protein in the atypical group has been characterized in S. cerevisiae till now. Herein, we report that an uncharacterized ORF YLL056C from S. cerevisiae was significantly upregulated under high furfural (2-furaldehyde) or 5-(hydroxymethyl)-2-furaldehyde concentrations, and transcription factors Yap1p, Hsf1p, Pdr1/3p, Yrr1p, and Stb5p likely controlled its upregulated transcription. This ORF indeed encoded a protein (Yll056cp), which was grouped into the atypical subgroup 7 in the SDR family and localized to the cytoplasm. Enzyme activity assays showed that Yll056cp is not a quinone or ketone reductase but an NADH-dependent aldehyde reductase, which can reduce at least seven aldehyde compounds. This enzyme showed the best Vmax, Kcat, and Kcat/Km to glycolaldehyde, but the highest affinity (Km) to formaldehyde. The optimum pH and temperature of this enzyme was pH 6.5 for reduction of glycolaldehyde, furfural, formaldehyde, butyraldehyde, and propylaldehyde, and 30 °C for reduction of formaldehyde or 35 °C for reduction of glycolaldehyde, furfural, butyraldehyde, and propylaldehyde. Temperature and pH affected stability of this enzyme and this influence varied with aldehyde substrate. Metal ions, salts, and chemical protective additives, especially at high concentrations, had different influence on enzyme activities for reduction of different aldehydes. This research provided guidelines for study of more uncharacterized atypical SDR enzymes from S. cerevisiae and other organisms.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. Substrate specificity and catalytic efficiency of aldo-keto reductases with phospholipid aldehydes

    PubMed Central

    Spite, Matthew; Baba, Shahid P.; Ahmed, Yonis; Barski, Oleg A.; Nijhawan, Kanchan; Petrash, J. Mark; Bhatnagar, Aruni; Srivastava, Sanjay

    2007-01-01

    Phospholipid oxidation generates several bioactive aldehydes that remain esterified to the glycerol backbone (‘core’ aldehydes). These aldehydes induce endothelial cells to produce monocyte chemotactic factors and enhance monocyte–endothelium adhesion. They also serve as ligands of scavenger receptors for the uptake of oxidized lipoproteins or apoptotic cells. The biochemical pathways involved in phospholipid aldehyde metabolism, however, remain largely unknown. In the present study, we have examined the efficacy of the three mammalian AKR (aldo-keto reductase) families in catalysing the reduction of phospholipid aldehydes. The model phospholipid aldehyde POVPC [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine] was efficiently reduced by members of the AKR1, but not by the AKR6 or the ARK7 family. In the AKR1 family, POVPC reductase activity was limited to AKR1A and B. No significant activity was observed with AKR1C enzymes. Among the active proteins, human AR (aldose reductase) (AKR1B1) showed the highest catalytic activity. The catalytic efficiency of human small intestinal AR (AKR1B10) was comparable with the murine AKR1B proteins 1B3 and 1B8. Among the murine proteins AKR1A4 and AKR1B7 showed appreciably lower catalytic activity as compared with 1B3 and 1B8. The human AKRs, 1B1 and 1B10, and the murine proteins, 1B3 and 1B8, also reduced C-7 and C-9 sn-2 aldehydes as well as POVPE [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphoethanolamine]. AKR1A4, B1, B7 and B8 catalysed the reduction of aldehydes generated in oxidized C16:0-20:4 phosphatidylcholine with acyl, plasmenyl or alkyl linkage at the sn-1 position or C16:0-20:4 phosphatidylglycerol or phosphatidic acid. AKR1B1 displayed the highest activity with phosphatidic acids; AKR1A4 was more efficient with long-chain aldehydes such as 5-hydroxy-8-oxo-6-octenoyl derivatives, whereas AKR1B8 preferred phosphatidylglycerol. These results suggest that proteins of the AKR1A and B families are

  3. 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.

  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. Heat-stable, FE-dependent alcohol dehydrogenase for aldehyde detoxification

    DOEpatents

    Elkins, James G.; Clarkson, Sonya

    2018-04-24

    The present invention relates to microorganisms and polypeptides for detoxifying aldehydes associated with industrial fermentations. In particular, a heat-stable, NADPH- and iron-dependent alcohol dehydrogenase was cloned from Thermoanaerobacter pseudethanolicus 39E and displayed activity against a number of aldehydes including inhibitory compounds that are produced during the dilute-acid pretreatment process of lignocellulosic biomass before fermentation to biofuels. Methods to use the microorganisms and polypeptides of the invention for improved conversion of bio mass to biofuel are provided as well as use of the enzyme in metabolic engineering strategies for producing longer-chain alcohols from sugars using thermophilic, fermentative microorganisms.

  6. 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

  7. 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

  8. 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.

  9. 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

  10. Guinea-pig liver testosterone 17 beta-dehydrogenase (NADP+) and aldehyde reductase exhibit benzene dihydrodiol dehydrogenase activity.

    PubMed Central

    Hara, A; Hayashibara, M; Nakayama, T; Hasebe, K; Usui, S; Sawada, H

    1985-01-01

    We have kinetically and immunologically demonstrated that testosterone 17 beta-dehydrogenase (NADP+) isoenzymes (EC 1.1.1.64) and aldehyde reductase (EC 1.1.1.2) from guinea-pig liver catalyse the oxidation of benzene dihydrodiol (trans-1,2-dihydroxycyclohexa-3,5-diene) to catechol. One isoenzyme of testosterone 17 beta-dehydrogenase, which has specificity for 5 beta-androstanes, oxidized benzene dihydrodiol at a 3-fold higher rate than 5 beta-dihydrotestosterone, and showed a more than 4-fold higher affinity for benzene dihydrodiol and Vmax. value than did another isoenzyme, which exhibits specificity for 5 alpha-androstanes, and aldehyde reductase. Immunoprecipitation of guinea-pig liver cytosol with antisera against the testosterone 17 beta-dehydrogenase isoenzymes and aldehyde reductase indicated that most of the benzene dihydrodiol dehydrogenase activity in the tissue is due to testosterone 17 beta-dehydrogenase. PMID:2983661

  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. 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

  13. 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.

  14. 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.

  15. 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.

  16. A Novel NADPH-Dependent Aldehyde Reductase Gene from Saccharomyces cerevisiae NRRL Y-12632 Involved in the Detoxification of Aldehyde Inhibitors Derived from Lignocellulosic Biomass Conversion

    USDA-ARS?s Scientific Manuscript database

    Aldehyde inhibitors such as furfural, 5-hydroxymethylfurfural (HMF), anisaldehyde, benzaldehyde, cinnamaldehyde, and phenylaldehyde are commonly generated during lignocellulosic biomass conversion process for low-cost cellulosic ethanol production that interferes with subsequent microbial growth and...

  17. 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.

  18. Kinetic mechanism of an aldehyde reductase of Saccharomyces cerevisiae that relieves toxicity of furfural and 5-hydroxymethylfurfural

    USDA-ARS?s Scientific Manuscript database

    An effective means of relieving the toxicity of furan aldehydes, furfural (FFA) and 5-hydroxymethylfurfural (HMF), on fermenting organisms is essential for achieving efficient fermentation of lignocellulosic biomass to ethanol and other products. Ari1p, an aldehyde reductase from Saccharomyces cerev...

  19. 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.

  20. 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

  1. Steady-state kinetic mechanism of the NADP+- and NAD+-dependent reactions catalysed by betaine aldehyde dehydrogenase from Pseudomonas aeruginosa.

    PubMed Central

    Velasco-García, R; González-Segura, L; Muñoz-Clares, R A

    2000-01-01

    Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible oxidation of betaine aldehyde to glycine betaine with the concomitant reduction of NAD(P)(+) to NADP(H). In Pseudomonas aeruginosa this reaction is a compulsory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. The kinetic mechanisms of the NAD(+)- and NADP(+)-dependent reactions were examined by steady-state kinetic methods and by dinucleotide binding experiments. The double-reciprocal patterns obtained for initial velocity with NAD(P)(+) and for product and dead-end inhibition establish that both mechanisms are steady-state random. However, quantitative analysis of the inhibitions, and comparison with binding data, suggest a preferred route of addition of substrates and release of products in which NAD(P)(+) binds first and NAD(P)H leaves last, particularly in the NADP(+)-dependent reaction. Abortive binding of the dinucleotides, or their analogue ADP, in the betaine aldehyde site was inferred from total substrate inhibition by the dinucleotides, and parabolic inhibition by NADH and ADP. A weak partial uncompetitive substrate inhibition by the aldehyde was observed only in the NADP(+)-dependent reaction. The kinetics of P. aeruginosa BADH is very similar to that of glucose-6-phosphate dehydrogenase, suggesting that both enzymes fulfil a similar amphibolic metabolic role when the bacteria grow in choline and when they grow in glucose. PMID:11104673

  2. 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.

  3. YKL071W from Saccharomyces cerevisiae encodes a novel aldehyde reductase for detoxification of glycolaldehyde and furfural derived from lignocellulose.

    PubMed

    Wang, Hanyu; Ouyang, Yidan; Zhou, Chang; Xiao, Difan; Guo, Yaping; Wu, Lan; Li, Xi; Gu, Yunfu; Xiang, Quanju; Zhao, Ke; Yu, Xiumei; Zou, Likou; Ma, Menggen

    2017-12-01

    Aldehydes generated as by-products during the pretreatment of lignocellulose are the key inhibitors to Saccharomyces cerevisiae, which is considered as the most promising microorganism for industrial production of biofuel, xylitol as well as other special chemicals from lignocellulose. S. cerevisiae has the inherent ability to in situ detoxify aldehydes to corresponding alcohols by multiple aldehyde reductases. Herein, we report that an uncharacterized open reading frame YKL071W from S. cerevisiae encodes a novel "classical" short-chain dehydrogenase/reductase (SDR) protein with NADH-dependent enzymatic activities for reduction of furfural (FF), glycolaldehyde (GA), formaldehyde (FA), and benzaldehyde (BZA). This enzyme showed much better specific activities for reduction of GA and FF than FA and BZA, and displayed much higher Km and Kcat/Km but lower Vmax and Kcat for reduction of GA than FF. For this enzyme, the optimum pH was 5.5 and 6.0 for reduction of GA and FF, and the optimum temperature was 30 °C for reduction of GA and FF. Both pH and temperature affected stability of this enzyme in a similar trend for reduction of GA and FF. Cu 2+ , Zn 2+ , Ni 2+ , and Fe 3+ had severe inhibition effects on enzyme activities of Ykl071wp for reduction of GA and FF. Transcription of YKL071W in S. cerevisiae was significantly upregulated under GA and FF stress conditions, and its transcription is most probably regulated by transcription factor genes of YAP1, CAD1, PDR3, and STB5. This research provides guidelines to identify more uncharacterized genes with reductase activities for detoxification of aldehydes derived from lignocellulose in S. cerevisiae.

  4. 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

  5. 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.

  6. Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae

    USDA-ARS?s Scientific Manuscript database

    Aldehyde reductase gene ARI1 is a recently characterized member of intermediate subfamily under SDR (short-chain dehydrogenase/reductase) superfamily that revealed mechanisms of in situ detoxification of furfural and HMF for tolerance of Saccharomyces cerevisiae. Uncharacterized open reading frames ...

  7. [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.

  8. 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...

  9. 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

  10. The influence of fatty acid supply and aldehyde reductase deletion on cyanobacteria alkane generating pathway in Escherichia coli.

    PubMed

    Wang, Juli; Yu, Haiying; Song, Xuejiao; Zhu, Kun

    2018-05-01

    Cyanobacteria alkane synthetic pathway has been heterologously constructed in many microbial hosts. It is by far the most studied and reliable alkane generating pathway. Aldehyde deformylating oxygenase (i.e., ADO, key enzyme in this pathway) obtained from different cyanobacteria species showed diverse catalytic abilities. This work indicated that single aldehyde reductase deletions were beneficial to Nostoc punctiforme ADO-depended alkane production in Escherichia coli even better than double deletions. Fatty acid metabolism regulator (FadR) overexpression and low temperature increased C18:1 fatty acid supply, and in turn stimulated C18:1-derived heptadecene production, suggesting that supplying ADO with preferred substrate was important to overall alkane yield improvement. Using combinational methods, 1 g/L alkane was obtained in fed-batch fermentation with heptadecene accounting for nearly 84% of total alkane.

  11. 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.

  12. 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.

  13. A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E

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

    Clarkson, Sonya M.; Hamilton-Brehm, Scott D.; Giannone, Richard J.

    Background: Chemical and physical pretreatment of lignocellulosic biomass improves substrate reactivity for increased microbial biofuel production, but also restricts growth via the release of furan aldehydes such as furfural and 5-hydroxymethylfurfural (5-HMF). The physiological effects of these inhibitors on thermophilic, fermentative bacteria is important to understand; especially as cellulolytic strains are being developed for consolidated bioprocessing (CBP) of lignocellulosic feedstocks. Identifying mechanisms for detoxification of aldehydes in naturally resistant strains such as Thermoanaerobacter spp. may also enable improvements in candidate CBP microorganisms. Results: T. pseudethanolicus 39E, an anaerobic, saccharolytic thermophile, was found to grow readily in the presence of 30more » mM furfural and 20 mM 5-HMF and reduce these aldehydes to their respective alcohols in situ. The proteomes of T. pseudethanolicus 39E grown in the presence or absence of 15 mM furfural were compared to identify upregulated enzymes potentially responsible for the observed reduction. A total of 225 proteins were differentially regulated in response to the 15 mM furfural treatment with 152 upregulated vs. 73 downregulated. Only 86 proteins exhibited a 2-fold change in abundance in either direction. Of these, 53 were upregulated in the presence of furfural and 33 were downregulated. Two oxidoreductases were upregulated at least 2-fold by furfural and were targeted for further investigation: Teth39_1597, encodes a predicted butanol dehydrogenase (BdhA) and Teth39_1598, a predicted aldo/keto reductase (AKR). Both genes were cloned from T. pseudethanolicus 39E, with the respective enzymes overexpressed in E. coli and specific activities determined against a variety of aldehydes. BdhA showed significant activity with all aldehydes tested, including furfural and 5-HMF, using NADPH as the cofactor. AKR also showed significant activity with NADPH, but only with four carbon butyr

  14. A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E

    DOE PAGES

    Clarkson, Sonya M.; Hamilton-Brehm, Scott D.; Giannone, Richard J.; ...

    2014-12-03

    Background: Chemical and physical pretreatment of lignocellulosic biomass improves substrate reactivity for increased microbial biofuel production, but also restricts growth via the release of furan aldehydes such as furfural and 5-hydroxymethylfurfural (5-HMF). The physiological effects of these inhibitors on thermophilic, fermentative bacteria is important to understand; especially as cellulolytic strains are being developed for consolidated bioprocessing (CBP) of lignocellulosic feedstocks. Identifying mechanisms for detoxification of aldehydes in naturally resistant strains such as Thermoanaerobacter spp. may also enable improvements in candidate CBP microorganisms. Results: T. pseudethanolicus 39E, an anaerobic, saccharolytic thermophile, was found to grow readily in the presence of 30more » mM furfural and 20 mM 5-HMF and reduce these aldehydes to their respective alcohols in situ. The proteomes of T. pseudethanolicus 39E grown in the presence or absence of 15 mM furfural were compared to identify upregulated enzymes potentially responsible for the observed reduction. A total of 225 proteins were differentially regulated in response to the 15 mM furfural treatment with 152 upregulated vs. 73 downregulated. Only 86 proteins exhibited a 2-fold change in abundance in either direction. Of these, 53 were upregulated in the presence of furfural and 33 were downregulated. Two oxidoreductases were upregulated at least 2-fold by furfural and were targeted for further investigation: Teth39_1597, encodes a predicted butanol dehydrogenase (BdhA) and Teth39_1598, a predicted aldo/keto reductase (AKR). Both genes were cloned from T. pseudethanolicus 39E, with the respective enzymes overexpressed in E. coli and specific activities determined against a variety of aldehydes. BdhA showed significant activity with all aldehydes tested, including furfural and 5-HMF, using NADPH as the cofactor. AKR also showed significant activity with NADPH, but only with four carbon butyr

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  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. 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

  2. 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.

  3. 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

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. Characterization of an allylic/benzyl alcohol dehydrogenase from Yokenella sp. strain WZY002, an organism potentially useful for the synthesis of α,β-unsaturated alcohols from allylic aldehydes and ketones.

    PubMed

    Ying, Xiangxian; Wang, Yifang; Xiong, Bin; Wu, Tingting; Xie, Liping; Yu, Meilan; Wang, Zhao

    2014-04-01

    A novel whole-cell biocatalyst with high allylic alcohol-oxidizing activities was screened and identified as Yokenella sp. WZY002, which chemoselectively reduced the C=O bond of allylic aldehydes/ketones to the corresponding α,β-unsaturated alcohols at 30°C and pH 8.0. The strain also had the capacity of stereoselectively reducing aromatic ketones to (S)-enantioselective alcohols. The enzyme responsible for the predominant allylic/benzyl alcohol dehydrogenase activity was purified to homogeneity and designated YsADH (alcohol dehydrogenase from Yokenella sp.), which had a calculated subunit molecular mass of 36,411 Da. The gene encoding YsADH was subsequently expressed in Escherichia coli, and the purified recombinant YsADH protein was characterized. The enzyme strictly required NADP(H) as a coenzyme and was putatively zinc dependent. The optimal pH and temperature for crotonaldehyde reduction were pH 6.5 and 65°C, whereas those for crotyl alcohol oxidation were pH 8.0 and 55°C. The enzyme showed moderate thermostability, with a half-life of 6.2 h at 55°C. It was robust in the presence of organic solvents and retained 87.5% of the initial activity after 24 h of incubation with 20% (vol/vol) dimethyl sulfoxide. The enzyme preferentially catalyzed allylic/benzyl aldehydes as the substrate in the reduction of aldehydes/ketones and yielded the highest activity of 427 U mg(-1) for benzaldehyde reduction, while the alcohol oxidation reaction demonstrated the maximum activity of 79.9 U mg(-1) using crotyl alcohol as the substrate. Moreover, kinetic parameters of the enzyme showed lower Km values and higher catalytic efficiency for crotonaldehyde/benzaldehyde and NADPH than for crotyl alcohol/benzyl alcohol and NADP(+), suggesting the nature of being an aldehyde reductase.

  15. Characterization of an Allylic/Benzyl Alcohol Dehydrogenase from Yokenella sp. Strain WZY002, an Organism Potentially Useful for the Synthesis of α,β-Unsaturated Alcohols from Allylic Aldehydes and Ketones

    PubMed Central

    Ying, Xiangxian; Wang, Yifang; Xiong, Bin; Wu, Tingting; Xie, Liping; Yu, Meilan

    2014-01-01

    A novel whole-cell biocatalyst with high allylic alcohol-oxidizing activities was screened and identified as Yokenella sp. WZY002, which chemoselectively reduced the C=O bond of allylic aldehydes/ketones to the corresponding α,β-unsaturated alcohols at 30°C and pH 8.0. The strain also had the capacity of stereoselectively reducing aromatic ketones to (S)-enantioselective alcohols. The enzyme responsible for the predominant allylic/benzyl alcohol dehydrogenase activity was purified to homogeneity and designated YsADH (alcohol dehydrogenase from Yokenella sp.), which had a calculated subunit molecular mass of 36,411 Da. The gene encoding YsADH was subsequently expressed in Escherichia coli, and the purified recombinant YsADH protein was characterized. The enzyme strictly required NADP(H) as a coenzyme and was putatively zinc dependent. The optimal pH and temperature for crotonaldehyde reduction were pH 6.5 and 65°C, whereas those for crotyl alcohol oxidation were pH 8.0 and 55°C. The enzyme showed moderate thermostability, with a half-life of 6.2 h at 55°C. It was robust in the presence of organic solvents and retained 87.5% of the initial activity after 24 h of incubation with 20% (vol/vol) dimethyl sulfoxide. The enzyme preferentially catalyzed allylic/benzyl aldehydes as the substrate in the reduction of aldehydes/ketones and yielded the highest activity of 427 U mg−1 for benzaldehyde reduction, while the alcohol oxidation reaction demonstrated the maximum activity of 79.9 U mg−1 using crotyl alcohol as the substrate. Moreover, kinetic parameters of the enzyme showed lower Km values and higher catalytic efficiency for crotonaldehyde/benzaldehyde and NADPH than for crotyl alcohol/benzyl alcohol and NADP+, suggesting the nature of being an aldehyde reductase. PMID:24509923

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. The aldo-keto reductase superfamily homepage.

    PubMed

    Hyndman, David; Bauman, David R; Heredia, Vladi V; Penning, Trevor M

    2003-02-01

    The aldo-keto reductases (AKRs) are one of the three enzyme superfamilies that perform oxidoreduction on a wide variety of natural and foreign substrates. A systematic nomenclature for the AKR superfamily was adopted in 1996 and was updated in September 2000 (visit www.med.upenn.edu/akr). Investigators have been diligent in submitting sequences of functional proteins to the Web site. With the new additions, the superfamily contains 114 proteins expressed in prokaryotes and eukaryotes that are distributed over 14 families (AKR1-AKR14). The AKR1 family contains the aldose reductases, the aldehyde reductases, the hydroxysteroid dehydrogenases and steroid 5beta-reductases, and is the largest. Other families of interest include AKR6, which includes potassium channel beta-subunits, and AKR7 the aflatoxin aldehyde reductases. Two new families include AKR13 (yeast aldose reductase) and AKR14 (Escherichia coli aldehyde reductase). Crystal structures of many AKRs and their complexes with ligands are available in the PDB and accessible through the Web site. Each structure has the characteristic (alpha/beta)(8)-barrel motif of the superfamily, a conserved cofactor binding site and a catalytic tetrad, and variable loop structures that define substrate specificity. Although the majority of AKRs are monomeric proteins of about 320 amino acids in length, the AKR2, AKR6 and AKR7 family may form multimers. To expand the nomenclature to accommodate multimers, we recommend that the composition and stoichiometry be listed. For example, AKR7A1:AKR7A4 (1:3) would designate a tetramer of the composition indicated. The current nomenclature is recognized by the Human Genome Project (HUGO) and the Web site provides a link to genomic information including chromosomal localization, gene boundaries, human ESTs and SNPs and much more.

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. The activity of the artemisinic aldehyde Δ11(13) reductase promoter is important for artemisinin yield in different chemotypes of Artemisia annua L.

    PubMed

    Yang, Ke; Monfared, Sajad Rashidi; Monafared, Rashidi Sajad; Wang, Hongzhen; Lundgren, Anneli; Brodelius, Peter E

    2015-07-01

    The artemisinic aldehyde double bond reductase (DBR2) plays an important role in the biosynthesis of the antimalarial artemisinin in Artemisia annua. Artemisinic aldehyde is reduced into dihydroartemisinic aldehyde by DBR2. Artemisinic aldehyde can also be oxidized by amorpha-4,11-diene 12-hydroxylase and/or aldehyde dehydrogenase 1 to artemisinic acid, a precursor of arteannuin B. In order to better understand the effects of DBR2 expression on the flow of artemisinic aldehyde into either artemisinin or arteannuin B, we determined the content of dihydroartemisinic aldehyde, artemisinin, artemisinic acid and arteannuin B content of A. annua varieties sorted into two chemotypes. The high artemisinin producers (HAPs), which includes the '2/39', 'Chongqing' and 'Anamed' varieties, produce more artemisinin than arteannuin B; the low artemisinin producers (LAPs), which include the 'Meise', 'Iran#8', 'Iran#14', 'Iran#24' and 'Iran#47' varieties, produce more arteannuin B than artemisinin. Quantitative PCR showed that the relative expression of DBR2 was significantly higher in the HAP varieties. We cloned and sequenced the promoter of the DBR2 gene from varieties of both the LAP and the HAP groups. There were deletions/insertions in the region just upstream of the ATG start codon in the LAP varities, which might be the reason for the different promoter activities of the HAP and LAP varieties. The relevance of promoter variation, DBR2 expression levels and artemisinin biosynthesis capabilities are discussed and a selection method for HAP varieties with a DNA marker is suggested. Furthermore, putative cis-acting regulatory elements differ between the HAP and LAP varieties.

  14. A spontaneously immortalized Schwann cell line from aldose reductase-deficient mice as a useful tool for studying polyol pathway and aldehyde metabolism.

    PubMed

    Niimi, Naoko; Yako, Hideji; Takaku, Shizuka; Kato, Hiroshi; Matsumoto, Takafumi; Nishito, Yasumasa; Watabe, Kazuhiko; Ogasawara, Saori; Mizukami, Hiroki; Yagihashi, Soroku; Chung, Sookja K; Sango, Kazunori

    2018-03-01

    The increased glucose flux into the polyol pathway via aldose reductase (AR) is recognized as a major contributing factor for the pathogenesis of diabetic neuropathy, whereas little is known about the functional significance of AR in the peripheral nervous system. Spontaneously immortalized Schwann cell lines established from long-term cultures of AR-deficient and normal C57BL/6 mouse dorsal root ganglia and peripheral nerves can be useful tools for studying the physiological and pathological roles of AR. These cell lines, designated as immortalized knockout AR Schwann cells 1 (IKARS1) and 1970C3, respectively, demonstrated distinctive Schwann cell phenotypes, such as spindle-shaped morphology and immunoreactivity to S100, p75 neurotrophin receptor, and vimentin, and extracellular release of neurotrophic factors. Conditioned media obtained from these cells promoted neuronal survival and neurite outgrowth of cultured adult mouse dorsal root ganglia neurons. Microarray and real-time RT-PCR analyses revealed significantly down-regulated mRNA expression of polyol pathway-related enzymes, sorbitol dehydrogenase and ketohexokinase, in IKARS1 cells compared with those in 1970C3 cells. In contrast, significantly up-regulated mRNA expression of aldo-keto reductases (AKR1B7 and AKR1B8) and aldehyde dehydrogenases (ALDH1L2, ALDH5A1, and ALDH7A1) was detected in IKARS1 cells compared with 1970C3 cells. Exposure to reactive aldehydes (3-deoxyglucosone, methylglyoxal, and 4-hydroxynonenal) significantly up-regulated the mRNA expression of AKR1B7 and AKR1B8 in IKARS1 cells, but not in 1970C3 cells. Because no significant differences in viability between these two cell lines after exposure to these aldehydes were observed, it can be assumed that the aldehyde detoxification is taken over by AKR1B7 and AKR1B8 in the absence of AR. © 2017 International Society for Neurochemistry.

  15. 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.

  16. 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

  17. Replacing Escherichia coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways.

    PubMed

    Martínez, Irene; Zhu, Jiangfeng; Lin, Henry; Bennett, George N; San, Ka-Yiu

    2008-11-01

    Reactions requiring reducing equivalents, NAD(P)H, are of enormous importance for the synthesis of industrially valuable compounds such as carotenoids, polymers, antibiotics and chiral alcohols among others. The use of whole-cell biocatalysis can reduce process cost by acting as catalyst and cofactor regenerator at the same time; however, product yields might be limited by cofactor availability within the cell. Thus, our study focussed on the genetic manipulation of a whole-cell system by modifying metabolic pathways and enzymes to improve the overall production process. In the present work, we genetically engineered an Escherichia coli strain to increase NADPH availability to improve the productivity of products that require NADPH in its biosynthesis. The approach involved an alteration of the glycolysis step where glyceraldehyde-3-phosphate (GAP) is oxidized to 1,3 bisphophoglycerate (1,3-BPG). This reaction is catalyzed by NAD-dependent endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoded by the gapA gene. We constructed a recombinant E. coli strain by replacing the native NAD-dependent gapA gene with a NADP-dependent GAPDH from Clostridium acetobutylicum, encoded by the gene gapC. The beauty of this approach is that the recombinant E. coli strain produces 2 mol of NADPH, instead of NADH, per mole of glucose consumed. Metabolic flux analysis showed that the flux through the pentose phosphate (PP) pathway, one of the main pathways that produce NADPH, was reduced significantly in the recombinant strain when compared to that of the parent strain. The effectiveness of the NADPH enhancing system was tested using the production of lycopene and epsilon-caprolactone as model systems using two different background strains. The recombinant strains, with increased NADPH availability, consistently showed significant higher productivity than the parent strains.

  18. Biochemical characterization of a recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius.

    PubMed

    Pennacchio, Angela; Giordano, Assunta; Pucci, Biagio; Rossi, Mosè; Raia, Carlo A

    2010-03-01

    The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases (SDRs) superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh gene was heterologously overexpressed in Escherichia coli, and the protein (SaADH) was purified to homogeneity and characterized. SaADH is a tetrameric enzyme consisting of identical 28,978-Da subunits, each composed of 264 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 75 degrees C and a 30-min half-inactivation temperature of ~90 degrees C, and shows good tolerance to common organic solvents. SaADH has a strict requirement for NAD(H) as the coenzyme, and displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and alpha-keto esters, but is poorly active on aliphatic, cyclic and aromatic alcohols, and shows no activity on aldehydes. The enzyme catalyses the reduction of alpha-methyl and alpha-ethyl benzoylformate, and methyl o-chlorobenzoylformate with 100% conversion to methyl (S)-mandelate [17% enantiomeric excess (ee)], ethyl (R)-mandelate (50% ee), and methyl (R)-o-chloromandelate (72% ee), respectively, with an efficient in situ NADH-recycling system which involves glucose and a thermophilic glucose dehydrogenase. This study provides further evidence supporting the critical role of the D37 residue in discriminating NAD(H) from NAD(P)H in members of the SDR superfamily.

  19. 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.

  20. 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

  1. General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH

    DOE PAGES

    Brinkmann-Chen, Sabine; Flock, Tilman; Cahn, Jackson K. B.; ...

    2013-06-17

    To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymesmore » having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. As a result, high-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.« less

  2. Constitutive NADPH-dependent electron transferase activity of the Nox4 dehydrogenase domain.

    PubMed

    Nisimoto, Yukio; Jackson, Heather M; Ogawa, Hisamitsu; Kawahara, Tsukasa; Lambeth, J David

    2010-03-23

    NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47(phox) and p67(phox) and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a K(m) for NADPH of 55 +/- 10 microM. The concentration of Nox4 in cell lysates was estimated using Western blotting and allowed calculation of a turnover of approximately 200 mol of H(2)O(2) min(-1) (mol of Nox4)(-1). A chimeric protein (Nox2/4) consisting of the Nox2 transmembrane (TM) domain and the Nox4 dehydrogenase (DH) domain showed H(2)O(2) production in the absence of cytosolic regulatory subunits. In contrast, chimera Nox4/2, consisting of the Nox4 TM and Nox2 DH domains, exhibited PMA-dependent activation that required coexpression of regulatory subunits. Nox DH domains from several Nox isoforms were purified and evaluated for their electron transferase activities. Nox1 DH, Nox2 DH, and Nox5 DH domains exhibited barely detectable activities toward artificial electron acceptors, while the Nox4 DH domain exhibited significant rates of reduction of cytochrome c (160 min(-1), largely superoxide dismutase-independent), ferricyanide (470 min(-1)), and other electron acceptors (artificial dyes and cytochrome b(5)). Rates were similar to those observed for H(2)O(2) production by the Nox4 holoenzyme in cell lysates. The activity required added FAD and was seen with NADPH but not NADH. These results indicate that the Nox4 DH domain exists in an intrinsically activated state and that electron transfer from NADPH to FAD is likely to be rate-limiting in the NADPH-dependent reduction of oxygen by holo-Nox4.

  3. 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

  4. 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

  5. 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

  6. 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

  7. Photometric Characterization of the Reductive Amination Scope of the Imine Reductases from Streptomyces tsukubaensis and Streptomyces ipomoeae.

    PubMed

    Matzel, Philipp; Krautschick, Lukas; Höhne, Matthias

    2017-10-18

    Imine reductases (IREDs) have emerged as promising enzymes for the asymmetric synthesis of secondary and tertiary amines starting from carbonyl substrates. Screening the substrate specificity of the reductive amination reaction is usually performed by time-consuming GC analytics. We found two highly active IREDs in our enzyme collection, IR-20 from Streptomyces tsukubaensis and IR-Sip from Streptomyces ipomoeae, that allowed a comprehensive substrate screening with a photometric NADPH assay. We screened 39 carbonyl substrates combined with 17 amines as nucleophiles. Activity data from 663 combinations provided a clear picture about substrate specificity and capabilities in the reductive amination of these enzymes. Besides aliphatic aldehydes, the IREDs accepted various cyclic (C 4 -C 8 ) and acyclic ketones, preferentially with methylamine. IR-Sip also accepted a range of primary and secondary amines as nucleophiles. In biocatalytic reactions, IR-Sip converted (R)-3-methylcyclohexanone with dimethylamine or pyrrolidine with high diastereoselectivity (>94-96 % de). The nucleophile acceptor spectrum depended on the carbonyl substrate employed. The conversion of well-accepted substrates could also be detected if crude lysates were employed as the enzyme source. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. 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

  9. Intracellular NADPH Levels Affect the Oligomeric State of the Glucose 6-Phosphate Dehydrogenase

    PubMed Central

    Tramonti, Angela; Lanini, Claudio; Cialfi, Samantha; De Biase, Daniela; Falcone, Claudio

    2012-01-01

    In the yeast Kluyveromyces lactis, glucose 6-phosphate dehydrogenase (G6PDH) is detected as two differently migrating forms on native polyacrylamide gels. The pivotal metabolic role of G6PDH in K. lactis led us to investigate the mechanism controlling the two activities in respiratory and fermentative mutant strains. An extensive analysis of these mutants showed that the NAD+(H)/NADP+(H)-dependent cytosolic alcohol (ADH) and aldehyde (ALD) dehydrogenase balance affects the expression of the G6PDH activity pattern. Under fermentative/ethanol growth conditions, the concomitant activation of ADH and ALD activities led to cytosolic accumulation of NADPH, triggering an alteration in the oligomeric state of the G6PDH caused by displacement/release of the structural NADP+ bound to each subunit of the enzyme. The new oligomeric G6PDH form with faster-migrating properties increases as a consequence of intracellular redox unbalance/NADPH accumulation, which inhibits G6PDH activity in vivo. The appearance of a new G6PDH-specific activity band, following incubation of Saccharomyces cerevisiae and human cellular extracts with NADP+, also suggests that a regulatory mechanism of this activity through NADPH accumulation is highly conserved among eukaryotes. PMID:23064253

  10. Cytochrome P450BM-3 reduces aldehydes to alcohols through a direct hydride transfer

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

    Kaspera, Ruediger; Sahele, Tariku; Lakatos, Kyle

    Highlights: Black-Right-Pointing-Pointer Cytochrome P450BM-3 reduced aldehydes to alcohols efficiently (k{sub cat} {approx} 25 min{sup -1}). Black-Right-Pointing-Pointer Reduction is a direct hydride transfer from R-NADP{sup 2}H to the carbonyl moiety. Black-Right-Pointing-Pointer P450 domain variants enhance reduction through potential allosteric/redox interactions. Black-Right-Pointing-Pointer Novel reaction will have implications for metabolism of xenobiotics. -- Abstract: Cytochrome P450BM-3 catalyzed the reduction of lipophilic aldehydes to alcohols efficiently. A k{sub cat} of {approx}25 min{sup -1} was obtained for the reduction of methoxy benzaldehyde with wild type P450BM-3 protein which was higher than in the isolated reductase domain (BMR) alone and increased in specific P450-domain variants. Themore » reduction was caused by a direct hydride transfer from preferentially R-NADP{sup 2}H to the carbonyl moiety of the substrate. Weak substrate-P450-binding of the aldehyde, turnover with the reductase domain alone, a deuterium incorporation in the product from NADP{sup 2}H but not D{sub 2}O, and no inhibition by imidazole suggests the reductase domain of P450BM-3 as the potential catalytic site. However, increased aldehyde reduction by P450 domain variants (P450BM-3 F87A T268A) may involve allosteric or redox mechanistic interactions between heme and reductase domains. This is a novel reduction of aldehydes by P450BM-3 involving a direct hydride transfer and could have implications for the metabolism of endogenous substrates or xenobiotics.« less

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

    Kita, Keiko; Fukura, Takanobu; Nakase, Kohichi

    The authors cloned and sequenced the gene encoding and NADPH-dependent aldehyde reductase (ARII) in Sporobolomyces salmonicolor AKU4429, which reduces ethyl 4-chloro-3-oxobutanoate (4-COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate. The ARII gene is 1,032 bp long, is interrupted by four introns, and encodes a 37,315-Da polypeptide. The deduced amino acid sequence exhibited significant levels of similarity to the amino acid sequences of members of the mammalian 3{Beta}-hydroxysteroid dehydrogenase-plant dihydroglavonol 4-reductase superfamily but not to the amino acid sequences of members of the aldo-keto reductase superfamily or to the amino acid sequence of an aldehyde reductase previously isolated from the same organism. The ARII proteinmore » was overproduced in Escherichia coli about 2,000-fold compared to the production in the original y east cells. The enzyme expressed in E. coli was purified to homogeneity and had the same catalytic properties as ARII purified from S. Salmonicolor. To examine the contribution of the dinucleotide-binding motif G{sub 19}-X-X-G{sub 22}-X-X-A{sub 25}, which is located in the N-terminal region, during ARII catalysis, they replaced three amino acid residues in the motif and purified the resulting mutant enzymes. Substrate inhibition of the G{sub 19}{r{underscore}arrow}A and G{sub 22}{r{underscore}arrow}A mutant enzymes by 4-COBE die not occur. The A{sub 25}{r{underscore}arrow}G mutant enzyme could reduce 4-COBE when NADPH was replaced by an equimolar concentration of NADH.« less

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. Cancer-associated Isocitrate Dehydrogenase Mutations Inactivate NADPH-dependent Reductive Carboxylation*

    PubMed Central

    Leonardi, Roberta; Subramanian, Chitra; Jackowski, Suzanne; Rock, Charles O.

    2012-01-01

    Isocitrate dehydrogenase (IDH) is a reversible enzyme that catalyzes the NADP+-dependent oxidative decarboxylation of isocitrate (ICT) to α-ketoglutarate (αKG) and the NADPH/CO2-dependent reductive carboxylation of αKG to ICT. Reductive carboxylation by IDH1 was potently inhibited by NADP+ and, to a lesser extent, by ICT. IDH1 and IDH2 with cancer-associated mutations at the active site arginines were unable to carry out the reductive carboxylation of αKG. These mutants were also defective in ICT decarboxylation and converted αKG to 2-hydroxyglutarate using NADPH. These mutant proteins were thus defective in both of the normal reactions of IDH. Biochemical analysis of heterodimers between wild-type and mutant IDH1 subunits showed that the mutant subunit did not inactivate reductive carboxylation by the wild-type subunit. Cells expressing the mutant IDH are thus deficient in their capacity for reductive carboxylation and may be compromised in their ability to produce acetyl-CoA under hypoxia or when mitochondrial function is otherwise impaired. PMID:22442146

  20. 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

  1. 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.

  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. 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.

  4. 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

  5. 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

  6. 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.

  7. Carboxylic acid reductase enzymes (CARs).

    PubMed

    Winkler, Margit

    2018-04-01

    Carboxylate reductases (CARs) are emerging as valuable catalysts for the selective one-step reduction of carboxylic acids to their corresponding aldehydes. The substrate scope of CARs is exceptionally broad and offers potential for their application in diverse synthetic processes. Two major fields of application are the preparation of aldehydes as end products for the flavor and fragrance sector and the integration of CARs in cascade reactions with aldehydes as the key intermediates. The latest applications of CARs are dominated by in vivo cascades and chemo-enzymatic reaction sequences. The challenge to fully exploit product selectivity is discussed. Recent developments in the characterization of CARs are summarized, with a focus on aspects related to the domain architecture and protein sequences of CAR enzymes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. 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.

  9. 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

  10. 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

  11. 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 ...

  12. Cloning, functional expression and characterization of a bifunctional 3-hydroxybutanal dehydrogenase /reductase involved in acetone metabolism by Desulfococcus biacutus.

    PubMed

    Frey, Jasmin; Rusche, Hendrik; Schink, Bernhard; Schleheck, David

    2016-11-25

    The strictly anaerobic, sulfate-reducing bacterium Desulfococcus biacutus can utilize acetone as sole carbon and energy source for growth. Whereas in aerobic and nitrate-reducing bacteria acetone is activated by carboxylation with CO 2 to acetoacetate, D. biacutus involves CO as a cosubstrate for acetone activation through a different, so far unknown pathway. Proteomic studies indicated that, among others, a predicted medium-chain dehydrogenase/reductase (MDR) superfamily, zinc-dependent alcohol dehydrogenase (locus tag DebiaDRAFT_04514) is specifically and highly produced during growth with acetone. The MDR gene DebiaDRAFT_04514 was cloned and overexpressed in E. coli. The purified recombinant protein required zinc as cofactor, and accepted NADH/NAD + but not NADPH/NADP + as electron donor/acceptor. The pH optimum was at pH 8, and the temperature optimum at 45 °C. Highest specific activities were observed for reduction of C 3 - C 5 -aldehydes with NADH, such as propanal to propanol (380 ± 15 mU mg -1 protein), butanal to butanol (300 ± 24 mU mg -1 ), and 3-hydroxybutanal to 1,3-butanediol (248 ± 60 mU mg -1 ), however, the enzyme also oxidized 3-hydroxybutanal with NAD + to acetoacetaldehyde (83 ± 18 mU mg -1 ). The enzyme might play a key role in acetone degradation by D. biacutus, for example as a bifunctional 3-hydroxybutanal dehydrogenase/reductase. Its recombinant production may represent an important step in the elucidation of the complete degradation pathway.

  13. 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

  14. Dietary sources of aldose reductase inhibitors: prospects for alleviating diabetic complications.

    PubMed

    Saraswat, Megha; Muthenna, P; Suryanarayana, P; Petrash, J Mark; Reddy, G Bhanuprakash

    2008-01-01

    Activation of polyol pathway due to increased aldose reductase activity is one of the several mechanisms that have been implicated in the development of various secondary complications of diabetes. Though numerous synthetic aldose reductase inhibitors have been tested, these have not been very successful clinically. Therefore, a number of common plant/ natural products used in Indian culinary have been evaluated for their aldose reductase inhibitory potential in the present study. The aqueous extracts of 22 plant-derived materials were prepared and evaluated for the inhibitory property against rat lens and human recombinant aldose reductase. Specificity of these extracts towards aldose reductase was established by testing their ability to inhibit a closely related enzyme viz, aldehyde reductase. The ex vivo incubation of erythrocytes in high glucose containing medium was used to underscore the significance in terms of prevention of intracellular sorbitol accumulation. Among the 22 dietary sources tested, 10 showed considerable inhibitory potential against both rat lens and human recombinant aldose reductase. Prominent inhibitory property was found in spinach, cumin, fennel, lemon, basil and black pepper with an approximate IC50 of 0.2 mg/mL with an excellent selectivity towards aldose reductase. As against this, 10 to 20 times higher concentrations were required for 50% inhibition of aldehyde reductase. Reduction in the accumulation of intracellular sorbitol by the dietary extracts further substantiated their in vivo efficacy. The findings reported here indicate the scope of adapting life-style modifications in the form of inclusion of certain common sources in the diet for the management of diabetic complications.

  15. 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.

  16. 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.

  17. 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

  18. Age-dependent neurodegeneration accompanying memory loss in transgenic mice defective in mitochondrial aldehyde dehydrogenase 2 activity.

    PubMed

    Ohsawa, Ikuroh; Nishimaki, Kiyomi; Murakami, Yayoi; Suzuki, Yuko; Ishikawa, Masahiro; Ohta, Shigeo

    2008-06-11

    Oxidative stress may underlie age-dependent memory loss and cognitive decline. Toxic aldehydes, including 4-hydroxy-2-nonenal (HNE), an end product of lipid peroxides, are known to accumulate in the brain in neurodegenerative disease. We have previously shown that mitochondrial aldehyde dehydrogenase 2 (ALDH2) detoxifies HNE by oxidizing its aldehyde group. To investigate the role of such toxic aldehydes, we produced transgenic mice, which expressed a dominant-negative form of ALDH2 in the brain. The mice had decreased ability to detoxify HNE in their cortical neurons and accelerated accumulation of HNE in the brain. Consequently, their lifespan was shortened and age-dependent neurodegeneration and hyperphosphorylation of tau were observed. Object recognition and Morris water maze tests revealed that the onset of cognitive impairment correlated with the degeneration, which was further accelerated by APOE (apolipoprotein E) knock-out; therefore, the accumulation of toxic aldehydes is by itself critical in the progression of neurodegenerative disease, which could be suppressed by ALDH2.

  19. Catalases Are NAD(P)H-Dependent Tellurite Reductases

    PubMed Central

    Calderón, Iván L.; Arenas, Felipe A.; Pérez, José Manuel; Fuentes, Derie E.; Araya, Manuel A.; Saavedra, Claudia P.; Tantaleán, Juan C.; Pichuantes, Sergio E.; Youderian, Philip A.; Vásquez, Claudio C.

    2006-01-01

    Reactive oxygen species damage intracellular targets and are implicated in cancer, genetic disease, mutagenesis, and aging. Catalases are among the key enzymatic defenses against one of the most physiologically abundant reactive oxygen species, hydrogen peroxide. The well-studied, heme-dependent catalases accelerate the rate of the dismutation of peroxide to molecular oxygen and water with near kinetic perfection. Many catalases also bind the cofactors NADPH and NADH tenaciously, but, surprisingly, NAD(P)H is not required for their dismutase activity. Although NAD(P)H protects bovine catalase against oxidative damage by its peroxide substrate, the catalytic role of the nicotinamide cofactor in the function of this enzyme has remained a biochemical mystery to date. Anions formed by heavy metal oxides are among the most highly reactive, natural oxidizing agents. Here, we show that a natural isolate of Staphylococcus epidermidis resistant to tellurite detoxifies this anion thanks to a novel activity of its catalase, and that a subset of both bacterial and mammalian catalases carry out the NAD(P)H-dependent reduction of soluble tellurite ion (TeO3 2−) to the less toxic, insoluble metal, tellurium (Te°), in vitro. An Escherichia coli mutant defective in the KatG catalase/peroxidase is sensitive to tellurite, and expression of the S. epidermidis catalase gene in a heterologous E. coli host confers increased resistance to tellurite as well as to hydrogen peroxide in vivo, arguing that S. epidermidis catalase provides a physiological line of defense against both of these strong oxidizing agents. Kinetic studies reveal that bovine catalase reduces tellurite with a low Michaelis-Menten constant, a result suggesting that tellurite is among the natural substrates of this enzyme. The reduction of tellurite by bovine catalase occurs at the expense of producing the highly reactive superoxide radical. PMID:17183702

  20. Catalases are NAD(P)H-dependent tellurite reductases.

    PubMed

    Calderón, Iván L; Arenas, Felipe A; Pérez, José Manuel; Fuentes, Derie E; Araya, Manuel A; Saavedra, Claudia P; Tantaleán, Juan C; Pichuantes, Sergio E; Youderian, Philip A; Vásquez, Claudio C

    2006-12-20

    Reactive oxygen species damage intracellular targets and are implicated in cancer, genetic disease, mutagenesis, and aging. Catalases are among the key enzymatic defenses against one of the most physiologically abundant reactive oxygen species, hydrogen peroxide. The well-studied, heme-dependent catalases accelerate the rate of the dismutation of peroxide to molecular oxygen and water with near kinetic perfection. Many catalases also bind the cofactors NADPH and NADH tenaciously, but, surprisingly, NAD(P)H is not required for their dismutase activity. Although NAD(P)H protects bovine catalase against oxidative damage by its peroxide substrate, the catalytic role of the nicotinamide cofactor in the function of this enzyme has remained a biochemical mystery to date. Anions formed by heavy metal oxides are among the most highly reactive, natural oxidizing agents. Here, we show that a natural isolate of Staphylococcus epidermidis resistant to tellurite detoxifies this anion thanks to a novel activity of its catalase, and that a subset of both bacterial and mammalian catalases carry out the NAD(P)H-dependent reduction of soluble tellurite ion (TeO(3)(2-)) to the less toxic, insoluble metal, tellurium (Te(o)), in vitro. An Escherichia coli mutant defective in the KatG catalase/peroxidase is sensitive to tellurite, and expression of the S. epidermidis catalase gene in a heterologous E. coli host confers increased resistance to tellurite as well as to hydrogen peroxide in vivo, arguing that S. epidermidis catalase provides a physiological line of defense against both of these strong oxidizing agents. Kinetic studies reveal that bovine catalase reduces tellurite with a low Michaelis-Menten constant, a result suggesting that tellurite is among the natural substrates of this enzyme. The reduction of tellurite by bovine catalase occurs at the expense of producing the highly reactive superoxide radical.

  1. Expression of a heat-stable NADPH-dependent alcohol dehydrogenase in Caldicellulosiruptor bescii results in furan aldehyde detoxification

    DOE PAGES

    Chung, Daehwan; Verbeke, Tobin J.; Cross, Karissa L.; ...

    2015-07-22

    Compounds such as furfural and 5-hydroxymethylfurfural (5-HMF) are generated through the dehydration of xylose and glucose, respectively, during dilute-acid pretreatment of lignocellulosic biomass and are also potent microbial growth and fermentation inhibitors. The enzymatic reduction of these furan aldehydes to their corresponding, and less toxic, alcohols is an engineering approach that has been successfully implemented in both Saccharomyces cerevisiae and ethanologenicEscherichia coli, but has not yet been investigated in thermophiles relevant to biofuel production through consolidated bioprocessing (CBP). Developing CBP-relevant biocatalysts that are either naturally resistant to such inhibitors, or are amenable to engineered resistance, is therefore, an important componentmore » in making biofuels production from lignocellulosic biomass feasible.« less

  2. 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

  3. 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

  4. 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.

  5. 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

  6. Synthesis and accumulation of aromatic aldehydes in an engineered strain of Escherichia coli.

    PubMed

    Kunjapur, Aditya M; Tarasova, Yekaterina; Prather, Kristala L J

    2014-08-20

    Aromatic aldehydes are useful in numerous applications, especially as flavors, fragrances, and pharmaceutical precursors. However, microbial synthesis of aldehydes is hindered by rapid, endogenous, and redundant conversion of aldehydes to their corresponding alcohols. We report the construction of an Escherichia coli K-12 MG1655 strain with reduced aromatic aldehyde reduction (RARE) that serves as a platform for aromatic aldehyde biosynthesis. Six genes with reported activity on the model substrate benzaldehyde were rationally targeted for deletion: three genes that encode aldo-keto reductases and three genes that encode alcohol dehydrogenases. Upon expression of a recombinant carboxylic acid reductase in the RARE strain and addition of benzoate during growth, benzaldehyde remained in the culture after 24 h, with less than 12% conversion of benzaldehyde to benzyl alcohol. Although individual overexpression results demonstrated that all six genes could contribute to benzaldehyde reduction in vivo, additional experiments featuring subset deletion strains revealed that two of the gene deletions were dispensable under the conditions tested. The engineered strain was next investigated for the production of vanillin from vanillate and succeeded in preventing formation of the byproduct vanillyl alcohol. A pathway for the biosynthesis of vanillin directly from glucose was introduced and resulted in a 55-fold improvement in vanillin titer when using the RARE strain versus the wild-type strain. Finally, synthesis of the chiral pharmaceutical intermediate L-phenylacetylcarbinol (L-PAC) was demonstrated from benzaldehyde and glucose upon expression of a recombinant mutant pyruvate decarboxylase in the RARE strain. Beyond allowing accumulation of aromatic aldehydes as end products in E. coli, the RARE strain expands the classes of chemicals that can be produced microbially via aldehyde intermediates.

  7. [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.

  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. Biotechnological Production of Methyl-Branched Aldehydes.

    PubMed

    Fraatz, Marco Alexander; Goldmann, Michael; Geissler, Torsten; Gross, Egon; Backes, Michael; Hilmer, Jens-Michael; Ley, Jakob; Rost, Johanna; Francke, Alexander; Zorn, Holger

    2018-03-14

    A number of methyl-branched aldehydes impart interesting flavor impressions, and especially 12-methyltridecanal is a highly sought after flavoring compound for savory foods. Its smell is reminiscent of cooked meat and tallow. For the biotechnological production of 12-methyltridecanal, the literature was screened for fungi forming iso-fatty acids. Suitable organisms were identified and successfully grown in submerged cultures. The culture medium was optimized to increase the yields of branched fatty acids. A recombinant carboxylic acid reductase was used to reduce 12-methyltridecanoic acid to 12-methyltridecanal. The efficiency of whole-cell catalysis was compared to that of the purified enzyme preparation. After lipase-catalyzed hydrolysis of the fungal lipid extracts, the released fatty acids were converted to the corresponding aldehydes, including 12-methyltridecanal and 12-methyltetradecanal.

  10. Bioreduction of α,β-unsaturated ketones and aldehydes by non-conventional yeast (NCY) whole-cells.

    PubMed

    Goretti, Marta; Ponzoni, Chiara; Caselli, Elisa; Marchegiani, Elisabetta; Cramarossa, Maria Rita; Turchetti, Benedetta; Forti, Luca; Buzzini, Pietro

    2011-03-01

    The bioreduction of α,β-unsaturated ketones (ketoisophorone, 2-methyl- and 3-methyl-cyclopentenone) and aldehydes [(S)-(-)-perillaldehyde and α-methyl-cinnamaldehyde] by 23 "non-conventional" yeasts (NCYs) belonging to 21 species of the genera Candida, Cryptococcus, Debaryomyces, Hanseniaspora, Kazachstania, Kluyveromyces, Lindnera, Nakaseomyces, Vanderwaltozyma, and Wickerhamomyces was reported. The results highlight the potential of NCYs as whole-cell biocatalysts for selective biotransformation of electron-poor alkenes. A few NCYs exhibited extremely high (>90%) or even total ketoisophorone and 2-methyl-cyclopentenone bioconversion yields via asymmetric reduction of the conjugated CC bond catalyzed by enoate reductases. Catalytic efficiency declined after switching from ketones to aldehydes. High chemoselectivity due to low competing carbonyl reductases was also sometimes observed. Copyright © 2010 Elsevier Ltd. All rights reserved.

  11. 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.

  12. 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.

  13. Ammonification in Bacillus subtilis Utilizing Dissimilatory Nitrite Reductase Is Dependent on resDE

    PubMed Central

    Hoffmann, Tamara; Frankenberg, Nicole; Marino, Marco; Jahn, Dieter

    1998-01-01

    During anaerobic nitrate respiration Bacillus subtilis reduces nitrate via nitrite to ammonia. No denitrification products were observed. B. subtilis wild-type cells and a nitrate reductase mutant grew anaerobically with nitrite as an electron acceptor. Oxygen-sensitive dissimilatory nitrite reductase activity was demonstrated in cell extracts prepared from both strains with benzyl viologen as an electron donor and nitrite as an electron acceptor. The anaerobic expression of the discovered nitrite reductase activity was dependent on the regulatory system encoded by resDE. Mutation of the gene encoding the regulatory Fnr had no negative effect on dissimilatory nitrite reductase formation. PMID:9422613

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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

  1. GRE2 from Scheffersomyces stipitis as an aldehyde reductase contributes tolerance to aldehyde inhibitors derived from lignocellulosic biomass

    USDA-ARS?s Scientific Manuscript database

    Scheffersomyces (Pichia) stipitis is one of the most promising yeasts for industrial bioethanol production from lignocellulosic biomass. S. stipitis is able to in situ detoxify aldehyde inhibitors [such as furfural and 5-hydroxymethylfurfural (HMF)] to less toxic corresponding alcohols. However, the...

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. Ultraviolet Radiation: Cellular Antioxidant Response and the Role of Ocular Aldehyde Dehydrogenase Enzymes

    PubMed Central

    Marchitti, Satori A.; Chen, Ying; Thompson, David C.; Vasiliou, Vasilis

    2011-01-01

    Solar ultraviolet radiation (UVR) exposes the human eye to near constant oxidative stress. Evidence suggests that UVR is the most important environmental insult leading to the development of a variety of ophthalmoheliosis disorders. UVR-induced reactive oxygen species are highly reactive with DNA, proteins and cellular membranes, resulting in cellular and tissue damage. Antioxidant defense systems present in ocular tissues function to combat reactive oxygen species and protect the eye from oxidative damage. Important enzymatic antioxidants are the superoxide dismutases, catalase, glutathione peroxidases, glutathione reductase and members of the aldehyde dehydrogenase (ALDH) superfamily. Glutathione, ascorbic and uric acids, α-tocopherol, NADPH and ferritin serve as small molecule, nonenzymatic antioxidants. Ocular tissues have high levels of these antioxidants which are essential for the maintenance of redox homeostasis in the eye and protection against oxidative damage. ALDH1A1 and ALDH3A1, present abundantly in the cornea and lens, have been shown to have unique roles in the defense against UVR and the downstream effects of oxidative stress. This review presents the properties and functions of ocular antioxidants that play critical roles in the cellular response to UVR exposure, including a focused discussion of the unique roles that the ALDH1A1 and ALDH3A1 enzymes have as multi-functional ocular antioxidants. PMID:21670692

  10. 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

  11. 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.

  12. Fatty Aldehydes in Cyanobacteria Are a Metabolically Flexible Precursor for a Diversity of Biofuel Products

    PubMed Central

    Kaiser, Brett K.; Carleton, Michael; Hickman, Jason W.; Miller, Cameron; Lawson, David; Budde, Mark; Warrener, Paul; Paredes, Angel; Mullapudi, Srinivas; Navarro, Patricia; Cross, Fred; Roberts, James M.

    2013-01-01

    We describe how pathway engineering can be used to convert a single intermediate derived from lipid biosynthesis, fatty aldehydes, into a variety of biofuel precursors including alkanes, free fatty acids and wax esters. In cyanobacteria, long-chain acyl-ACPs can be reduced to fatty aldehydes, and then decarbonylated to alkanes. We discovered a cyanobacteria class-3 aldehyde-dehydrogenase, AldE, that was necessary and sufficient to instead oxidize fatty aldehyde precursors into fatty acids. Overexpression of enzymes in this pathway resulted in production of 50 to 100 fold more fatty acids than alkanes, and the fatty acids were secreted from the cell. Co-expression of acyl-ACP reductase, an alcohol-dehydrogenase and a wax-ester-synthase resulted in a third fate for fatty aldehydes: conversion to wax esters, which accumulated as intracellular lipid bodies. Conversion of acyl-ACP to fatty acids using endogenous cyanobacterial enzymes may allow biofuel production without transgenesis. PMID:23505484

  13. 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.

  14. 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

  15. Engineering and systems-level analysis of Saccharomyces cerevisiae for production of 3-hydroxypropionic acid via malonyl-CoA reductase-dependent pathway.

    PubMed

    Kildegaard, Kanchana R; Jensen, Niels B; Schneider, Konstantin; Czarnotta, Eik; Özdemir, Emre; Klein, Tobias; Maury, Jérôme; Ebert, Birgitta E; Christensen, Hanne B; Chen, Yun; Kim, Il-Kwon; Herrgård, Markus J; Blank, Lars M; Forster, Jochen; Nielsen, Jens; Borodina, Irina

    2016-03-15

    In the future, oil- and gas-derived polymers may be replaced with bio-based polymers, produced from renewable feedstocks using engineered cell factories. Acrylic acid and acrylic esters with an estimated world annual production of approximately 6 million tons by 2017 can be derived from 3-hydroxypropionic acid (3HP), which can be produced by microbial fermentation. For an economically viable process 3HP must be produced at high titer, rate and yield and preferably at low pH to minimize downstream processing costs. Here we describe the metabolic engineering of baker's yeast Saccharomyces cerevisiae for biosynthesis of 3HP via a malonyl-CoA reductase (MCR)-dependent pathway. Integration of multiple copies of MCR from Chloroflexus aurantiacus and of phosphorylation-deficient acetyl-CoA carboxylase ACC1 genes into the genome of yeast increased 3HP titer fivefold in comparison with single integration. Furthermore we optimized the supply of acetyl-CoA by overexpressing native pyruvate decarboxylase PDC1, aldehyde dehydrogenase ALD6, and acetyl-CoA synthase from Salmonella enterica SEacs (L641P). Finally we engineered the cofactor specificity of the glyceraldehyde-3-phosphate dehydrogenase to increase the intracellular production of NADPH at the expense of NADH and thus improve 3HP production and reduce formation of glycerol as by-product. The final strain produced 9.8 ± 0.4 g L(-1) 3HP with a yield of 13% C-mol C-mol(-1) glucose after 100 h in carbon-limited fed-batch cultivation at pH 5. The 3HP-producing strain was characterized by (13)C metabolic flux analysis and by transcriptome analysis, which revealed some unexpected consequences of the undertaken metabolic engineering strategy, and based on this data, future metabolic engineering directions are proposed. In this study, S. cerevisiae was engineered for high-level production of 3HP by increasing the copy numbers of biosynthetic genes and improving flux towards precursors and redox cofactors. This strain represents

  16. Aldo-Keto Reductases 1B in Adrenal Cortex Physiology

    PubMed Central

    Pastel, Emilie; Pointud, Jean-Christophe; Martinez, Antoine; Lefrançois-Martinez, A. Marie

    2016-01-01

    Aldose reductase (AKR1B) proteins are monomeric enzymes, belonging to the aldo-keto reductase (AKR) superfamily. They perform oxidoreduction of carbonyl groups from a wide variety of substrates, such as aliphatic and aromatic aldehydes or ketones. Due to the involvement of human aldose reductases in pathologies, such as diabetic complications and cancer, AKR1B subgroup enzymatic properties have been extensively characterized. However, the issue of AKR1B function in non-pathologic conditions remains poorly resolved. Adrenal activities generated large amount of harmful aldehydes from lipid peroxidation and steroidogenesis, including 4-hydroxynonenal (4-HNE) and isocaproaldehyde (4-methylpentanal), which can both be reduced by AKR1B proteins. More recently, some AKR1B isoforms have been shown to be endowed with prostaglandin F synthase (PGFS) activity, suggesting that, in addition to possible scavenger function, they could instigate paracrine signals. Interestingly, the adrenal gland is one of the major sites for human and murine AKR1B expression, suggesting that their detoxifying/signaling activity could be specifically required for the correct handling of adrenal function. Moreover, chronic effects of ACTH result in a coordinated regulation of genes encoding the steroidogenic enzymes and some AKR1B isoforms. This review presents the molecular mechanisms accounting for the adrenal-specific expression of some AKR1B genes. Using data from recent mouse genetic models, we will try to connect their enzymatic properties and regulation with adrenal functions. PMID:27499746

  17. 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

  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. 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

  20. 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.

  1. 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.

  2. Identification of new potent inhibitor of aldose reductase from Ocimum basilicum.

    PubMed

    Bhatti, Huma Aslam; Tehseen, Yildiz; Maryam, Kiran; Uroos, Maliha; Siddiqui, Bina S; Hameed, Abdul; Iqbal, Jamshed

    2017-12-01

    Recent efforts to develop cure for chronic diabetic complications have led to the discovery of potent inhibitors against aldose reductase (AKR1B1, EC 1.1.1.21) whose role in diabetes is well-evident. In the present work, two new natural products were isolated from the ariel part of Ocimum basilicum; 7-(3-hydroxypropyl)-3-methyl-8-β-O-d-glucoside-2H-chromen-2-one (1) and E-4-(6'-hydroxyhex-3'-en-1-yl)phenyl propionate (2) and confirmed their structures with different spectroscopic techniques including NMR spectroscopy etc. The isolated compounds (1, 2) were evaluated for in vitro inhibitory activity against aldose reductase (AKR1B1) and aldehyde reductase (AKR1A1). The natural product (1) showed better inhibitory activity for AKR1B1 with IC 50 value of 2.095±0.77µM compare to standard sorbinil (IC 50 =3.14±0.02µM). Moreover, the compound (1) also showed multifolds higher activity (IC 50 =0.783±0.07µM) against AKR1A1 as compared to standard valproic acid (IC 50 =57.4±0.89µM). However, the natural product (2) showed slightly lower activity for AKR1B1 (IC 50 =4.324±1.25µM). Moreover, the molecular docking studies of the potent inhibitors were also performed to identify the putative binding modes within the active site of aldose/aldehyde reductases. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Two Atypical l-Cysteine-regulated NADPH-dependent Oxidoreductases Involved in Redox Maintenance, l-Cystine and Iron Reduction, and Metronidazole Activation in the Enteric Protozoan Entamoeba histolytica*

    PubMed Central

    Jeelani, Ghulam; Husain, Afzal; Sato, Dan; Ali, Vahab; Suematsu, Makoto; Soga, Tomoyoshi; Nozaki, Tomoyoshi

    2010-01-01

    We discovered novel catalytic activities of two atypical NADPH-dependent oxidoreductases (EhNO1/2) from the enteric protozoan parasite Entamoeba histolytica. EhNO1/2 were previously annotated as the small subunit of glutamate synthase (glutamine:2-oxoglutarate amidotransferase) based on similarity to authentic bacterial homologs. As E. histolytica lacks the large subunit of glutamate synthase, EhNO1/2 were presumed to play an unknown role other than glutamine/glutamate conversion. Transcriptomic and quantitative reverse PCR analyses revealed that supplementation or deprivation of extracellular l-cysteine caused dramatic up- or down-regulation, respectively, of EhNO2, but not EhNO1 expression. Biochemical analysis showed that these FAD- and 2[4Fe-4S]-containing enzymes do not act as glutamate synthases, a conclusion which was supported by phylogenetic analyses. Rather, they catalyze the NADPH-dependent reduction of oxygen to hydrogen peroxide and l-cystine to l-cysteine and also function as ferric and ferredoxin-NADP+ reductases. EhNO1/2 showed notable differences in substrate specificity and catalytic efficiency; EhNO1 had lower Km and higher kcat/Km values for ferric ion and ferredoxin than EhNO2, whereas EhNO2 preferred l-cystine as a substrate. In accordance with these properties, only EhNO1 was observed to physically interact with intrinsic ferredoxin. Interestingly, EhNO1/2 also reduced metronidazole, and E. histolytica transformants overexpressing either of these proteins were more sensitive to metronidazole, suggesting that EhNO1/2 are targets of this anti-amebic drug. To date, this is the first report to demonstrate that small subunit-like proteins of glutamate synthase could play an important role in redox maintenance, l-cysteine/l-cystine homeostasis, iron reduction, and the activation of metronidazole. PMID:20592025

  4. Structural and biochemical analyses of YvgN and YtbE from Bacillus subtilis

    PubMed Central

    Lei, Jian; Zhou, Yan-Feng; Li, Lan-Fen; Su, Xiao-Dong

    2009-01-01

    Bacillus subtilis is one of the most studied gram-positive bacteria. In this work, YvgN and YtbE from B. subtilis, assigned as AKR5G1 and AKR5G2 of aldo-keto reductase (AKR) superfamily. AKR catalyzes the NADPH-dependent reduction of aldehyde or aldose substrates to alcohols. YvgN and YtbE were studied by crystallographic and enzymatic analyses. The apo structures of these proteins were determined by molecular replacement, and the structure of holoenzyme YvgN with NADPH was also solved, revealing the conformational changes upon cofactor binding. Our biochemical data suggest both YvgN and YtbE have preferential specificity for derivatives of benzaldehyde, such as nitryl or halogen group substitution at the 2 or 4 positions. These proteins also showed broad catalytic activity on many standard substrates of AKR, such as glyoxal, dihydroxyacetone, and DL-glyceraldehyde, suggesting a possible role in bacterial detoxification. PMID:19585557

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. Aldehyde Oxidase 4 Plays a Critical Role in Delaying Silique Senescence by Catalyzing Aldehyde Detoxification.

    PubMed

    Srivastava, Sudhakar; Brychkova, Galina; Yarmolinsky, Dmitry; Soltabayeva, Aigerim; Samani, Talya; Sagi, Moshe

    2017-04-01

    The Arabidopsis ( Arabidopsis thaliana ) aldehyde oxidases are a multigene family of four oxidases (AAO1-AAO4) that oxidize a variety of aldehydes, among them abscisic aldehyde, which is oxidized to the phytohormone abscisic acid. Toxic aldehydes are generated in plants both under normal conditions and in response to stress. The detoxification of such aldehydes by oxidation is attributed to aldehyde dehydrogenases but never to aldehyde oxidases. The feasibility of the detoxification of aldehydes in siliques via oxidation by AAO4 was demonstrated, first, by its ability to efficiently oxidize an array of aromatic and aliphatic aldehydes, including the reactive carbonyl species (RCS) acrolein, hydroxyl-2-nonenal, and malondialdehyde. Next, exogenous application of several aldehydes to siliques in AAO4 knockout (KO) Arabidopsis plants induced severe tissue damage and enhanced malondialdehyde levels and senescence symptoms, but not in wild-type siliques. Furthermore, abiotic stresses such as dark and ultraviolet C irradiation caused an increase in endogenous RCS and higher expression levels of senescence marker genes, leading to premature senescence of KO siliques, whereas RCS and senescence marker levels in wild-type siliques were hardly affected. Finally, in naturally senesced KO siliques, higher endogenous RCS levels were associated with enhanced senescence molecular markers, chlorophyll degradation, and earlier seed shattering compared with the wild type. The aldehyde-dependent differential generation of superoxide and hydrogen peroxide by AAO4 and the induction of AAO4 expression by hydrogen peroxide shown here suggest a self-amplification mechanism for detoxifying additional reactive aldehydes produced during stress. Taken together, our results indicate that AAO4 plays a critical role in delaying senescence in siliques by catalyzing aldehyde detoxification. © 2017 American Society of Plant Biologists. All Rights Reserved.

  16. Nitrite reductase activity of rat and human xanthine oxidase, xanthine dehydrogenase, and aldehyde oxidase: evaluation of their contribution to NO formation in vivo.

    PubMed

    Maia, Luisa B; Pereira, Vânia; Mira, Lurdes; Moura, José J G

    2015-01-27

    Nitrite is presently considered a NO "storage form" that can be made available, through its one-electron reduction, to maintain NO formation under hypoxia/anoxia. The molybdoenzymes xanthine oxidase/dehydrogenase (XO/XD) and aldehyde oxidase (AO) are two of the most promising mammalian nitrite reductases, and in this work, we characterized NO formation by rat and human XO/XD and AO. This is the first characterization of human enzymes, and our results support the employment of rat liver enzymes as suitable models of the human counterparts. A comprehensive kinetic characterization of the effect of pH on XO and AO-catalyzed nitrite reduction showed that the enzyme's specificity constant for nitrite increase 8-fold, while the Km(NO2(-)) decrease 6-fold, when the pH decreases from 7.4 to 6.3. These results demonstrate that the ability of XO/AO to trigger NO formation would be greatly enhanced under the acidic conditions characteristic of ischemia. The dioxygen inhibition was quantified, and the Ki(O2) values found (24.3-48.8 μM) suggest that in vivo NO formation would be fine-tuned by dioxygen availability. The potential in vivo relative physiological relevance of XO/XD/AO-dependent pathways of NO formation was evaluated using HepG2 and HMEC cell lines subjected to hypoxia. NO formation by the cells was found to be pH-, nitrite-, and dioxygen-dependent, and the relative contribution of XO/XD plus AO was found to be as high as 50%. Collectively, our results supported the possibility that XO/XD and AO can contribute to NO generation under hypoxia inside a living human cell. Furthermore, the molecular mechanism of XO/AO-catalyzed nitrite reduction was revised.

  17. 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

  18. Molecular modelling and synthesis of spiroimidazolidine-2,4-diones with dual activities as hypoglycemic agents and selective inhibitors of aldose reductase.

    PubMed

    Salem, Manar G; Abdel Aziz, Yasmine M; Elewa, Marwa; Elshihawy, Hosam A; Said, Mohamed M

    2018-05-02

    Novel derivatives of spiroimidazolidinedione were synthesized and evaluated as hypoglycemic agents through binding to sulfonylurea receptor 1 (SUR1) in pancreatic beta-cells. Their selectivity index was calculated against both aldehyde reductase (ALR1) and aldose reductase (ALR2). Aldehyde reductase is a key enzyme in the polyol pathway that is involved in the etiology of the secondary diabetic complications. All structures were confirmed by microanalysis and by IR, 1 H NMR, 13 C NMR and EI-MS spectroscopy. The investigated compounds were subjected to molecular docking and an in silico prediction study to determine their free energy of binding (ΔG) values and predict their physicochemical properties and drug-likeness scores. Compound 1'-(5-chlorothiophene-2-ylsulfonyl)spiro[cyclohexane-1,5'-imidazolidine]-2',4'-dione showed IC 50 0.47 µM and 79% reduction in blood glucose level with a selectivity index 127 for ALR2. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. 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

  20. Structural insights into the cofactor-assisted substrate recognition of yeast methylglyoxal/isovaleraldehyde reductase Gre2.

    PubMed

    Guo, Peng-Chao; Bao, Zhang-Zhi; Ma, Xiao-Xiao; Xia, Qingyou; Li, Wei-Fang

    2014-09-01

    Saccharomyces cerevisiae Gre2 (EC1.1.1.283) serves as a versatile enzyme that catalyzes the stereoselective reduction of a broad range of substrates including aliphatic and aromatic ketones, diketones, as well as aldehydes, using NADPH as the cofactor. Here we present the crystal structures of Gre2 from S. cerevisiae in an apo-form at 2.00Å and NADPH-complexed form at 2.40Å resolution. Gre2 forms a homodimer, each subunit of which contains an N-terminal Rossmann-fold domain and a variable C-terminal domain, which participates in substrate recognition. The induced fit upon binding to the cofactor NADPH makes the two domains shift toward each other, producing an interdomain cleft that better fits the substrate. Computational simulation combined with site-directed mutagenesis and enzymatic activity analysis enabled us to define a potential substrate-binding pocket that determines the stringent substrate stereoselectivity for catalysis. Copyright © 2014 Elsevier B.V. All rights reserved.

  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. Engineering of a functional human NADH-dependent cytochrome P450 system

    PubMed Central

    Döhr, Olaf; Paine, Mark J. I.; Friedberg, Thomas; Roberts, Gordon C. K.; Wolf, C. Roland

    2001-01-01

    A functional human NADH-dependent cytochrome P450 system has been developed by altering the cofactor preference of human NADPH cytochrome P450 reductase (CPR), the redox partner for P450s. This has been achieved by a single amino acid change of the conserved aromatic amino acid Trp-676, which covers the re-side of the FAD isoalloxazine ring in the nicotinamide-binding site. Of the mutations made, the substitution of Trp-676 with alanine (W676A) resulted in a functional NADH-dependent enzyme, which catalyzed the reduction of cytochrome c and ferricyanide as well as facilitated the metabolism of 7-ethoxyresorufin by CYP1A2. Kinetic analysis measuring cytochrome c activity revealed that the NADH-dependent kcat of W676A is equivalent (90%) to the NADPH-dependent kcat of the wild-type enzyme, with W676A having an approximately 1,000-fold higher specificity for NADH. The apparent KMNADPH and KMNADH values of W676A are 80- and 150-fold decreased, respectively. In accordance with structural data, which show a bipartite binding mode of NADPH, substitution of Trp-676 does not affect 2′-AMP binding as seen by the inhibition of both wild-type CPR and the W676A mutant. Furthermore, NADPH was a potent inhibitor of the W676A NADH-dependent cytochrome c reduction and CYP1A2 activity. Overall, the results show that Trp-676 of human CPR plays a major role in cofactor discrimination, and substitution of this conserved aromatic residue with alanine results in an efficient NADH-dependent cytochrome P450 system. PMID:11136248

  3. Aldehyde Oxidase 4 Plays a Critical Role in Delaying Silique Senescence by Catalyzing Aldehyde Detoxification1[OPEN

    PubMed Central

    Yarmolinsky, Dmitry; Soltabayeva, Aigerim; Samani, Talya

    2017-01-01

    The Arabidopsis (Arabidopsis thaliana) aldehyde oxidases are a multigene family of four oxidases (AAO1–AAO4) that oxidize a variety of aldehydes, among them abscisic aldehyde, which is oxidized to the phytohormone abscisic acid. Toxic aldehydes are generated in plants both under normal conditions and in response to stress. The detoxification of such aldehydes by oxidation is attributed to aldehyde dehydrogenases but never to aldehyde oxidases. The feasibility of the detoxification of aldehydes in siliques via oxidation by AAO4 was demonstrated, first, by its ability to efficiently oxidize an array of aromatic and aliphatic aldehydes, including the reactive carbonyl species (RCS) acrolein, hydroxyl-2-nonenal, and malondialdehyde. Next, exogenous application of several aldehydes to siliques in AAO4 knockout (KO) Arabidopsis plants induced severe tissue damage and enhanced malondialdehyde levels and senescence symptoms, but not in wild-type siliques. Furthermore, abiotic stresses such as dark and ultraviolet C irradiation caused an increase in endogenous RCS and higher expression levels of senescence marker genes, leading to premature senescence of KO siliques, whereas RCS and senescence marker levels in wild-type siliques were hardly affected. Finally, in naturally senesced KO siliques, higher endogenous RCS levels were associated with enhanced senescence molecular markers, chlorophyll degradation, and earlier seed shattering compared with the wild type. The aldehyde-dependent differential generation of superoxide and hydrogen peroxide by AAO4 and the induction of AAO4 expression by hydrogen peroxide shown here suggest a self-amplification mechanism for detoxifying additional reactive aldehydes produced during stress. Taken together, our results indicate that AAO4 plays a critical role in delaying senescence in siliques by catalyzing aldehyde detoxification. PMID:28188272

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. Cytochromes P450 Catalyze the Reduction of α,β-Unsaturated Aldehydes

    PubMed Central

    Amunom, Immaculate; Dieter, Laura J.; Tamasi, Viola; Cai, Jan; Conklin, Daniel J.; Srivastava, Sanjay; Martin, Martha V.; Guengerich, F. Peter; Prough, Russell A.

    2011-01-01

    The metabolism of α,β-unsaturated aldehydes, e.g. 4-hydroxynonenal, involves oxidation to carboxylic acids, reduction to alcohols, and glutathionylation to eventually form mercapturide conjugates. Recently we demonstrated that P450s can oxidize aldehydes to carboxylic acids, a reaction previously thought to involve aldehyde dehydrogenase. When recombinant cytochrome P450 3A4 was incubated with 4-hydroxynonenal, O2, and NADPH, several products were produced, including 1,4-dihydroxynonene (DHN), 4-hydroxy-2-nonenoic acid (HNA), and an unknown metabolite. Several P450s catalyzed the reduction reaction in the order (human) P450 2B6 ≅ P450 3A4 > P450 1A2 > P450 2J2 > (mouse) P450 2c29. Other P450s did not catalyze the reduction reaction (human P450 2E1 & rabbit P450 2B4). Metabolism by isolated rat hepatocytes showed that HNA formation was inhibited by cyanamide, while DHN formation was not affected. Troleandomycin increased HNA production 1.6-fold while inhibiting DHN formation, suggesting that P450 3A11 is a major enzyme involved in rat hepatic clearance of 4-HNE. A fluorescent assay was developed using 9-anthracenealdehyde to measure both reactions. Feeding mice diet containing t-butylated hydroxyanisole increased the level of both activities with hepatic microsomal fractions, but not proportionally. Miconazole (0.5 mM) was a potent inhibitor of these microsomal reduction reactions, while phenytoin and α-naphthoflavone (both at 0.5 mM) were partial inhibitors, suggesting the role of multiple P450 enzymes. The oxidative metabolism of these aldehydes was inhibited >90% in an Ar or CO atmosphere, while the reductive reactions were not greatly affected. These results suggest that P450s are significant catalysts of reduction of α,β-unsaturated aldehydes in liver. PMID:21766881

  9. Expression of Cyanobacterial Acyl-ACP Reductase Elevates the Triacylglycerol Level in the Red Alga Cyanidioschyzon merolae.

    PubMed

    Sumiya, Nobuko; Kawase, Yasuko; Hayakawa, Jumpei; Matsuda, Mami; Nakamura, Mami; Era, Atsuko; Tanaka, Kan; Kondo, Akihiko; Hasunuma, Tomohisa; Imamura, Sousuke; Miyagishima, Shin-ya

    2015-10-01

    Nitrogen starvation is known to induce the accumulation of triacylglycerol (TAG) in many microalgae, and potential use of microalgae as a source of biofuel has been explored. However, nitrogen starvation also stops cellular growth. The expression of cyanobacterial acyl-acyl carrier protein (ACP) reductase in the unicellular red alga Cyanidioschyzon merolae chloroplasts resulted in an accumulation of TAG, which led to an increase in the number and size of lipid droplets while maintaining cellular growth. Transcriptome and metabolome analyses showed that the expression of acyl-ACP reductase altered the activities of several metabolic pathways. The activities of enzymes involved in fatty acid synthesis in chloroplasts, such as acetyl-CoA carboxylase and pyruvate dehydrogenase, were up-regulated, while pyruvate decarboxylation in mitochondria and the subsequent consumption of acetyl-CoA by the tricarboxylic acid (TCA) cycle were down-regulated. Aldehyde dehydrogenase, which oxidizes fatty aldehydes to fatty acids, was also up-regulated in the acyl-ACP reductase expresser. This activation was required for the lipid droplet accumulation and metabolic changes observed in the acyl-ACP reductase expresser. Nitrogen starvation also resulted in lipid droplet accumulation in C. merolae, while cell growth ceased as in the case of other algal species. The metabolic changes that occur upon the expression of acyl-ACP reductase are quite different from those caused by nitrogen starvation. Therefore, there should be a method for further increasing the storage lipid level while still maintaining cell growth that is different from the metabolic response to nitrogen starvation. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  10. 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.

  11. Crystallographic analysis and structure-guided engineering of NADPH-dependent Ralstonia sp. alcohol dehydrogenase toward NADH cosubstrate specificity.

    PubMed

    Lerchner, Alexandra; Jarasch, Alexander; Meining, Winfried; Schiefner, André; Skerra, Arne

    2013-11-01

    The NADP⁺-dependent alcohol dehydrogenase from Ralstonia sp. (RasADH) belongs to the protein superfamily of short-chain dehydrogenases/reductases (SDRs). As an enzyme that accepts different types of substrates--including bulky-bulky as well as small-bulky secondary alcohols or ketones--with high stereoselectivity, it offers potential as a biocatalyst for industrial biotechnology. To understand substrate and cosubstrate specificities of RasADH we determined the crystal structure of the apo-enzyme as well as its NADP⁺-bound state with resolutions down to 2.8 Å. RasADH displays a homotetrameric quaternary structure that can be described as a dimer of homodimers while in each subunit a seven-stranded parallel β-sheet, flanked by three α-helices on each side, forms a Rossmann fold-type dinucleotide binding domain. Docking of the well-known substrate (S)-1-phenylethanol clearly revealed the structural determinants of stereospecificity. To favor practical RasADH application in the context of established cofactor recycling systems, for example, those involving an NADH-dependent amino acid dehydrogenase, we attempted to rationally change its cosubstrate specificity from NADP⁺ to NAD⁺ utilizing the structural information that NADP⁺ specificity is largely governed by the residues Asn15, Gly37, Arg38, and Arg39. Furthermore, an extensive sequence alignment with homologous dehydrogenases that have different cosubstrate specificities revealed a modified general SDR motif ASNG (instead of NNAG) at positions 86-89 of RasADH. Consequently, we constructed mutant enzymes with one (G37D), four (N15G/G37D/R38V/R39S), and six (N15G/G37D/R38V/R39S/A86N/S88A) amino acid exchanges. RasADH (N15G/G37D/R38V/R39S) was better able to accept NAD⁺ while showing much reduced catalytic efficiency with NADP⁺, leading to a change in NADH/NADPH specificity by a factor of ∼3.6 million. © 2013 Wiley Periodicals, Inc.

  12. 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

  13. 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.

  14. 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.

  15. Role of Ser-257 in the sliding mechanism of NADP(H) in the reaction catalyzed by the Aspergillus fumigatus flavin-dependent ornithine N5-monooxygenase SidA.

    PubMed

    Shirey, Carolyn; Badieyan, Somayesadat; Sobrado, Pablo

    2013-11-08

    SidA (siderophore A) is a flavin-dependent N-hydroxylating monooxygenase that is essential for virulence in Aspergillus fumigatus. SidA catalyzes the NADPH- and oxygen-dependent formation of N(5)-hydroxyornithine. In this reaction, NADPH reduces the flavin, and the resulting NADP(+) is the last product to be released. The presence of NADP(+) is essential for activity, as it is required for stabilization of the C4a-hydroperoxyflavin, which is the hydroxylating species. As part of our efforts to determine the molecular details of the role of NADP(H) in catalysis, we targeted Ser-257 for site-directed mutagenesis and performed extensive characterization of the S257A enzyme. Using a combination of steady-state and stopped-flow kinetic experiments, substrate analogs, and primary kinetic isotope effects, we show that the interaction between Ser-257 and NADP(H) is essential for stabilization of the C4a-hydroperoxyflavin. Molecular dynamics simulation results suggest that Ser-257 functions as a pivot point, allowing the nicotinamide of NADP(+) to slide into position for stabilization of the C4a-hydroperoxyflavin.

  16. Chlorella Induces Stomatal Closure via NADPH Oxidase-Dependent ROS Production and Its Effects on Instantaneous Water Use Efficiency in Vicia faba

    PubMed Central

    Li, Yan; Xu, Shan-Shan; Gao, Jing; Pan, Sha; Wang, Gen-Xuan

    2014-01-01

    Reactive oxygen species (ROS) have been established to participate in stomatal closure induced by live microbes and microbe-associated molecular patterns (MAMPs). Chlorella as a beneficial microorganism can be expected to trigger stomatal closure via ROS production. Here, we reported that Chlorella induced stomatal closure in a dose-and time-dependent manner in epidermal peels of Vicia faba. Using pharmacological methods in this work, we found that the Chlorella-induced stomatal closure was almost completely abolished by a hydrogen peroxide (H2O2) scavenger, catalase (CAT), significantly suppressed by an NADPH oxidase inhibitor, diphenylene iodonium chloride (DPI), and slightly affected by a peroxidase inhibitor, salicylhydroxamic acid (SHAM), suggesting that ROS production involved in Chlorella-induced stomatal closure is mainly mediated by DPI-sensitive NADPH oxidase. Additionally, Exogenous application of optimal concentrations of Chlorella suspension improved instantaneous water use efficiency (WUEi) in Vicia faba via a reduction in leaf transpiration rate (E) without a parallel reduction in net photosynthetic rate (Pn) assessed by gas-exchange measurements. The chlorophyll fluorescence and content analysis further demonstrated that short-term use of Chlorella did not influence plant photosynthetic reactions center. These results preliminarily reveal that Chlorella can trigger stomatal closure via NADPH oxidase-dependent ROS production in epidermal strips and improve WUEi in leave levels. PMID:24687099

  17. Chlorella induces stomatal closure via NADPH oxidase-dependent ROS production and its effects on instantaneous water use efficiency in Vicia faba.

    PubMed

    Li, Yan; Xu, Shan-Shan; Gao, Jing; Pan, Sha; Wang, Gen-Xuan

    2014-01-01

    Reactive oxygen species (ROS) have been established to participate in stomatal closure induced by live microbes and microbe-associated molecular patterns (MAMPs). Chlorella as a beneficial microorganism can be expected to trigger stomatal closure via ROS production. Here, we reported that Chlorella induced stomatal closure in a dose-and time-dependent manner in epidermal peels of Vicia faba. Using pharmacological methods in this work, we found that the Chlorella-induced stomatal closure was almost completely abolished by a hydrogen peroxide (H2O2) scavenger, catalase (CAT), significantly suppressed by an NADPH oxidase inhibitor, diphenylene iodonium chloride (DPI), and slightly affected by a peroxidase inhibitor, salicylhydroxamic acid (SHAM), suggesting that ROS production involved in Chlorella-induced stomatal closure is mainly mediated by DPI-sensitive NADPH oxidase. Additionally, Exogenous application of optimal concentrations of Chlorella suspension improved instantaneous water use efficiency (WUEi) in Vicia faba via a reduction in leaf transpiration rate (E) without a parallel reduction in net photosynthetic rate (Pn) assessed by gas-exchange measurements. The chlorophyll fluorescence and content analysis further demonstrated that short-term use of Chlorella did not influence plant photosynthetic reactions center. These results preliminarily reveal that Chlorella can trigger stomatal closure via NADPH oxidase-dependent ROS production in epidermal strips and improve WUEi in leave levels.

  18. 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

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

    Forlani, Giuseppe; Bertazzini, Michele; Zarattini, Marco

    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

  20. Dose-dependent DNA adduct formation by cinnamaldehyde and other food-borne α,β-unsaturated aldehydes predicted by physiologically based in silico modelling.

    PubMed

    Kiwamoto, R; Ploeg, D; Rietjens, I M C M; Punt, A

    2016-03-01

    Genotoxicity of α,β-unsaturated aldehydes shown in vitro raises a concern for the use of the aldehydes as food flavourings, while at low dose exposures the formation of DNA adducts may be prevented by detoxification. Unlike many α,β-unsaturated aldehydes for which in vivo data are absent, cinnamaldehyde was shown to be not genotoxic or carcinogenic in vivo. The present study aimed at comparing dose-dependent DNA adduct formation by cinnamaldehyde and 18 acyclic food-borne α,β-unsaturated aldehydes using physiologically based kinetic/dynamic (PBK/D) modelling. In rats, cinnamaldehyde was predicted to induce higher DNA adducts levels than 6 out of the 18 α,β-unsaturated aldehydes, indicating that these 6 aldehydes may also test negative in vivo. At the highest cinnamaldehyde dose that tested negative in vivo, cinnamaldehyde was predicted to form at least three orders of magnitude higher levels of DNA adducts than the 18 aldehydes at their respective estimated daily intake. These results suggest that for all the 18 α,β-unsaturated aldehydes DNA adduct formation at doses relevant for human dietary exposure may not raise a concern. The present study illustrates a possible use of physiologically based in silico modelling to facilitate a science-based comparison and read-across on the possible risks posed by DNA reactive agents. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Biogenic Aldehydes as Therapeutic Targets for Cardiovascular Disease

    PubMed Central

    Nelson, Margaret-Ann M; Baba, Shahid P; Andersonc, Ethan J

    2017-01-01

    Aldehydes are continuously formed in biological systems through enzyme-dependent and spontaneous oxidation of lipids, glucose, and primary amines. These highly reactive, biogenic electrophiles can become toxic via covalent modification of proteins, lipids and DNA. Thus, agents that scavenge aldehydes through conjugation have therapeutic value for a number of major cardiovascular diseases. Several commonly-prescribed drugs (e.g., hydralazine) have been shown to have potent aldehyde-conjugating properties which may contribute to their beneficial effects. Herein, we briefly describe the major sources and toxicities of biogenic aldehydes in cardiovascular system, and provide an overview of drugs that are known to have aldehyde-conjugating effects. Some compounds of phytochemical origin, and histidyl-dipeptides with emerging therapeutic value in this area are also discussed. PMID:28528297

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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

  10. Resistance of Saccharomyces cerevisiae to High Concentrations of Furfural Is Based on NADPH-Dependent Reduction by at Least Two Oxireductases ▿ †

    PubMed Central

    Heer, Dominik; Heine, Daniel; Sauer, Uwe

    2009-01-01

    Biofuels derived from lignocellulosic biomass hold promises for a sustainable fuel economy, but several problems hamper their economical feasibility. One important problem is the presence of toxic compounds in processed lignocellulosic hydrolysates, with furfural as a key toxin. While Saccharomyces cerevisiae has some intrinsic ability to reduce furfural to the less-toxic furfuryl alcohol, higher resistance is necessary for process conditions. By comparing an evolved, furfural-resistant strain and its parent in microaerobic, glucose-limited chemostats at increasing furfural challenge, we elucidate key mechanism and the molecular basis of both natural and high-level furfural resistance. At lower concentrations of furfural, NADH-dependent oxireductases are the main defense mechanism. At furfural concentrations above 15 mM, however, 13C-flux and global array-based transcript analysis demonstrated that the NADPH-generating flux through the pentose phosphate pathway increases and that NADPH-dependent oxireductases become the major resistance mechanism. The transcript analysis further revealed that iron transmembrane transport is upregulated in response to furfural. While these responses occur in both strains, high-level resistance in the evolved strain was based on strong induction of ADH7, the uncharacterized open reading frame (ORF) YKL071W, and four further, likely NADPH-dependent, oxireductases. By overexpressing the ADH7 gene and the ORF YKL071W, we inversely engineered significantly increased furfural resistance in the parent strain, thereby demonstrating that these two enzymes are key elements of the resistance phenotype. PMID:19854918

  11. Biogenic Aldehydes as Therapeutic Targets for Cardiovascular Disease.

    PubMed

    Nelson, Margaret-Ann M; Baba, Shahid P; Anderson, Ethan J

    2017-04-01

    Aldehydes are continuously formed in biological systems through enzyme-dependent and spontaneous oxidation of lipids, glucose, and primary amines. These highly reactive, biogenic electrophiles can become toxic via covalent modification of proteins, lipids and DNA. Thus, agents that scavenge aldehydes through conjugation have therapeutic value for a number of major cardiovascular diseases. Several commonly-prescribed drugs (e.g., hydralazine) have been shown to have potent aldehyde-conjugating properties which may contribute to their beneficial effects. Herein, we briefly describe the major sources and toxicities of biogenic aldehydes in cardiovascular system, and provide an overview of drugs that are known to have aldehyde-conjugating effects. Some compounds of phytochemical origin, and histidyl-dipeptides with emerging therapeutic value in this area are also discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Applications of Carboxylic Acid Reductases in Oleaginous Microbes

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

    Resch, Michael G.; Linger, Jeffrey; McGeehan, John

    2016-05-26

    Carboxylic acid reductases (CARs) are recently emerging reductive enzymes for the direct production of aldehydes from biologically-produced carboxylic acids. Recent work has demonstrated that these powerful enzymes are able to reduce a very broad range of volatile- to long-chain fatty acids as well as aromatic acids. Here, we express four CAR enzymes from different fungal origins to test their activity against fatty acids commonly produced in oleaginous microbes. These in vitro results will inform metabolic engineering strategies to conduct mild biological reduction of carboxylic acids in situ, which is conventionally done via hydrotreating catalysis at high temperatures and hydrogen pressures.

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. Advanced oxidation protein products induce inflammatory response in fibroblast-like synoviocytes through NADPH oxidase -dependent activation of NF-κB.

    PubMed

    Zheng, Shuai; Zhong, Zhao-Ming; Qin, Shuai; Chen, Guo-Xian; Wu, Qian; Zeng, Ji-Huan; Ye, Wen-Bin; Li, Wei; Yuan, Kai; Yao, Ling; Chen, Jian-Ting

    2013-01-01

    Advanced oxidation protein products (AOPPs), a marker of oxidative stress, are prevalent in many kinds of disorders. Rheumatoid arthritis (RA), mainly resulting from the dysfunction of fibroblast-like synoviocytes (FLSs), is related to oxidative stress. Although the increased levels of AOPPs in RA patients were reported, the effect of AOPPs on FLSs function still remains unclear. Therefore, our study aims to investigate whether AOPPs have an effect on the inflammatory response of FLSs in vitro. FLSs obtained from both knees of rats were treated with or without AOPPs-modified rat serum albumin (AOPPs-RSA) in vitro. The mRNA and protein expression of tumor necrosis factor (TNF)-α, interleukin(IL)-1β, matrix metalloproteinases(MMP)-3, MMP-13 and vascular endothelial growth factor (VEGF) were measured by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA), respectively. Reactive oxygen species (ROS) generation was detected by fluorescent microscope and fluorescence microplate reader. Immunoprecipitation, Co-Immunoprecipitation and western blot were performed to examine the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and nuclear factor kappa B (NF-κB). Exposure of FLSs to AOPPs upregulated the mRNA and protein expression of TNF-α, IL-1β, MMP-3, MMP-13 and VEGF in a concentration dependent manner. AOPPs treatment triggered ROS production in FLSs, which was significantly abolished by ROS scavenger N-acetyl-L-cysteine (NAC), superoxide dismutase (SOD), NADPH oxidase inhibitors diphenyleneiodonium (DPI) and apocynin. Challenged AOPPs induced phosphorylation of p47(phox), triggered an interaction between p47(phox), p22(phox) and gp91(phox), and significantly upregulated expression of NADPH oxidase subunits p47(phox), p22(phox) and gp91(phox). IκB degradation and nuclear translocation of NF-κB p65 induced by AOPPs were significantly blocked by SOD, NAC, DPI and apocynin. These data indicate that

  18. Enhanced performance of the methylerythritol phosphate pathway by manipulation of redox reactions relevant to IspC, IspG, and IspH.

    PubMed

    Zhou, Jia; Yang, Liyang; Wang, Chonglong; Choi, Eui-Sung; Kim, Seon-Won

    2017-04-20

    The 2C-methyl-D-erythritol 4-phosphate (MEP) pathway is a carbon-efficient route for synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the building blocks of isoprenoids. However, practical application of a native or recombinant MEP pathway for the mass production of isoprenoids in Escherichia coli has been unsatisfactory. In this study, the entire recombinant MEP pathway was established with plasmids and used for the production of an isoprenoid, protoilludene. E. coli harboring the recombinant MEP pathway plasmid (ME) and a protoilludene synthesis pathway plasmid (AO) produced 10.4mg/L of protoilludene after 48h of culture. To determine the rate-limiting gene on plasmid ME, each constituent gene of the MEP pathway was additionally overexpressed on the plasmid AO. The additional overexpression of IPP isomerase (IDI) enhanced protoilludene production to 67.4mg/L. Overexpression of the Fpr and FldA protein complex, which could mediate electron transfer from NADPH to Fe-S cluster proteins such as IspG and IspH of the MEP pathway, increased protoilludene production to 318.8mg/L. Given that it is required for IspC as well as IspG/H, the MEP pathway has high demand for NADPH. To increase the supply of NADPH, a NADH kinase from Saccharomyces cerevisiae (tPos5p) that converts NADH to NADPH was introduced along with the deletion of a promiscuous NADPH-dependent aldehyde reductase (YjgB) that consumes NADPH. This resulted in a protoilludene production of 512.7mg/L. The results indicate that IDI, Fpr-FldA redox proteins, and NADPH regenerators are key engineering points for boosting the metabolic flux toward a recombinant MEP pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. 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

  20. A novel aldo-keto reductase from Escherichia coli can increase resistance to methylglyoxal toxicity.

    PubMed

    Grant, Anne W; Steel, Gavin; Waugh, Hugh; Ellis, Elizabeth M

    2003-01-21

    A novel aldo-keto reductase (AKR) from Escherichia coli has been cloned, expressed and purified. This protein, YghZ, is distantly related (<40%) to mammalian aflatoxin dialdehyde reductases of the aldo-keto reductase AKR7 family and to potassium channel beta-subunits in the AKR6 family. The enzyme has been placed in a new AKR family (AKR14), with the designation AKR14A1. Sequences encoding putative homologues of this enzyme exist in many other bacteria. The enzyme can reduce several aldehyde and diketone substrates, including the toxic metabolite methylglyoxal. The K(m) for the model substrate 4-nitrobenzaldehyde is 1.06 mM and for the endogenous dicarbonyl methylglyoxal it is 3.4 mM. Overexpression of the recombinant enzyme in E. coli leads to increased resistance to methylglyoxal. It is possible that this enzyme plays a role in the metabolism of methylglyoxal, and can influence its levels in vivo.

  1. The Involvement of Lipid Peroxide-Derived Aldehydes in Aluminum Toxicity of Tobacco Roots1[W][OA

    PubMed Central

    Yin, Lina; Mano, Jun'ichi; Wang, Shiwen; Tsuji, Wataru; Tanaka, Kiyoshi

    2010-01-01

    Oxidative injury of the root elongation zone is a primary event in aluminum (Al) toxicity in plants, but the injuring species remain unidentified. We verified the hypothesis that lipid peroxide-derived aldehydes, especially highly electrophilic α,β-unsaturated aldehydes (2-alkenals), participate in Al toxicity. Transgenic tobacco (Nicotiana tabacum) overexpressing Arabidopsis (Arabidopsis thaliana) 2-alkenal reductase (AER-OE plants), wild-type SR1, and an empty vector-transformed control line (SR-Vec) were exposed to AlCl3 on their roots. Compared with the two controls, AER-OE plants suffered less retardation of root elongation under AlCl3 treatment and showed more rapid regrowth of roots upon Al removal. Under AlCl3 treatment, the roots of AER-OE plants accumulated Al and H2O2 to the same levels as did the sensitive controls, while they accumulated lower levels of aldehydes and suffered less cell death than SR1 and SR-Vec roots. In SR1 roots, AlCl3 treatment markedly increased the contents of the highly reactive 2-alkenals acrolein, 4-hydroxy-(E)-2-hexenal, and 4-hydroxy-(E)-2-nonenal and other aldehydes such as malondialdehyde and formaldehyde. In AER-OE roots, accumulation of these aldehydes was significantly less. Growth of the roots exposed to 4-hydroxy-(E)-2-nonenal and (E)-2-hexenal were retarded more in SR1 than in AER-OE plants. Thus, the lipid peroxide-derived aldehydes, formed downstream of reactive oxygen species, injured root cells directly. Their suppression by AER provides a new defense mechanism against Al toxicity. PMID:20023145

  2. NADPH oxidase/ROS-dependent PYK2 activation is involved in TNF-α-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells

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

    Yang, Chuen-Mao, E-mail: chuenmao@mail.cgu.edu.tw; Heart Failure Center, Division of Cardiology, Department of Internal Medicine, Chang Gung Memorial Hospital at Keelung, Keelung, Taiwan; Lee, I-Ta

    TNF-α plays a mediator role in the pathogenesis of chronic heart failure contributing to cardiac remodeling and peripheral vascular disturbances. The implication of TNF-α in inflammatory responses has been shown to be mediated through up-regulation of matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of TNF-α-induced MMP-9 expression in rat embryonic-heart derived H9c2 cells are largely not defined. We demonstrated that in H9c2 cells, TNF-α induced MMP-9 mRNA and protein expression associated with an increase in the secretion of pro-MMP-9. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of ROS (N-acetyl-L-cysteine, NAC), NADPH oxidase [apocynin (APO) or diphenyleneiodonium chloride (DPI)],more » MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), NF-κB (Bay11-7082), or PYK2 (PF-431396) and transfection with siRNA of TNFR1, p47{sup phox}, p42, p38, JNK1, p65, or PYK2. Moreover, TNF-α markedly induced NADPH oxidase-derived ROS generation in these cells. TNF-α-enhanced p42/p44 MAPK, p38 MAPK, JNK1/2, and NF-κB (p65) phosphorylation and in vivo binding of p65 to the MMP-9 promoter were inhibited by U0126, SB202190, SP600125, NAC, DPI, or APO. In addition, TNF-α-mediated PYK2 phosphorylation was inhibited by NAC, DPI, or APO. PYK2 inhibition could reduce TNF-α-stimulated MAPKs and NF-κB activation. Thus, in H9c2 cells, we are the first to show that TNF-α-induced MMP-9 expression is mediated through a TNFR1/NADPH oxidase/ROS/PYK2/MAPKs/NF-κB cascade. We demonstrated that NADPH oxidase-derived ROS generation is involved in TNF-α-induced PYK2 activation in these cells. Understanding the regulation of MMP-9 expression and NADPH oxidase activation by TNF-α on H9c2 cells may provide potential therapeutic targets of chronic heart failure. - Highlights: • TNF-α induces MMP-9 secretion and expression via a TNFR1-dependent pathway. • TNF-α induces ROS/PYK2-dependent MMP-9 expression in H9c2 cells. • TNF

  3. 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.

  4. 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.

  5. Fatty aldehyde dehydrogenases in Acinetobacter sp. strain HO1-N: role in hexadecanol metabolism.

    PubMed Central

    Singer, M E; Finnerty, W R

    1985-01-01

    The role of fatty aldehyde dehydrogenases (FALDHs) in hexadecane and hexadecanol metabolism was studied in Acinetobacter sp. strain HO1-N. Two distinct FALDHs were demonstrated in Acinetobacter sp. strain HO1-N: a membrane-bound, NADP-dependent FALDH activity induced 5-, 15-, and 9-fold by growth on hexadecanol, dodecyl aldehyde, and hexadecane, respectively, and a constitutive, NAD-dependent, membrane-localized FALDH. The NADP-dependent FALDH exhibited apparent Km and Vmax values for decyl aldehyde of 5.0, 13.0, 18.0, and 18.3 microM and 537.0, 500.0, 25.0, and 38.0 nmol/min in hexadecane-, hexadecanol-, ethanol-, palmitate-grown cells, respectively. FALDH isozymes ald-a, ald-b, and ald-c were demonstrated by gel electrophoresis in extracts of hexadecane- and hexadecanol-grown cells. ald-a, ald-b, and ald-d were present in dodecyl aldehyde-grown cells, while palmitate-grown control cells contained ald-b and ald-d. Dodecyl aldehyde-negative mutants were isolated and grouped into two phenotypic classes based on growth: class 1 mutants were hexadecane and hexadecanol negative and class 2 mutants were hexadecane and hexadecanol positive. Specific activity of NADP-dependent FALDH in Ald21 (class 1 mutant) was 85% lower than that of wild-type FALDH, while the specific activity of Ald24 (class 2 mutant) was 55% greater than that of wild-type FALDH. Ald21R, a dodecyl aldehyde-positive revertant able to grow on hexadecane, hexadecanol, and dodecyl aldehyde, exhibited a 100% increase in the specific activity of the NADP-dependent FALDH. The oxidation of [3H]hexadecane byAld21 yielded the accumulation of 61% more fatty aldehyde than the wild type, while Ald24 accumulated 27% more fatty aldehyde, 95% more fatty alcohol, and 65% more wax ester than the wild type. This study provides genetic and physiological evidence for the role of fatty aldehyde as an essential metabolic intermediate and NADP-dependent FALDH as a key enzyme in the dissimilation of hexadecane, hexadecanol

  6. Biochemical Properties of Purified Human Retinol Dehydrogenase 12 (RDH12): Catalytic Efficiency toward Retinoids and C9 Aldehydes and Effects of Cellular Retinol-Binding Protein Type I (CRBPI) and Cellular Retinaldehyde-Binding Protein (CRALBP) on the Oxidation and Reduction of Retinoids†

    PubMed Central

    Belyaeva, Olga V.; Korkina, Olga V.; Stetsenko, Anton V.; Kim, Tom; Nelson, Peter S.; Kedishvili, Natalia Y.

    2008-01-01

    Retinol dehydrogenase 12 (RDH12) is a novel member of the short-chain dehydrogenase/reductase superfamily of proteins that was recently linked to Leber’s congenital amaurosis 3 (LCA). We report the first biochemical characterization of purified human RDH12 and analysis of its expression in human tissues. RDH12 exhibits ~2000-fold lower Km values for NADP+ and NADPH than for NAD+ and NADH and recognizes both retinoids and lipid peroxidation products (C9 aldehydes) as substrates. The kcat values of RDH12 for retinaldehydes and C9 aldehydes are similar, but the Km values are, in general, lower for retinoids. The enzyme exhibits the highest catalytic efficiency for all-trans-retinal (kcat/Km ~900 min−1 μM−1), followed by 11-cis-retinal (450 min−1 mM−1) and 9-cis-retinal (100 min−1 mM−1). Analysis of RDH12 activity toward retinoids in the presence of cellular retinol-binding protein (CRBP) type I or cellular retinaldehyde-binding protein (CRALBP) suggests that RDH12 utilizes the unbound forms of all-trans- and 11-cis-retinoids. As a result, the widely expressed CRBPI, which binds all-trans-retinol with much higher affinity than all-trans-retinaldehyde, restricts the oxidation of all-trans-retinol by RDH12, but has little effect on the reduction of all-trans-retinaldehyde, and CRALBP inhibits the reduction of 11-cis-retinal stronger than the oxidation of 11-cis-retinol, in accord with its higher affinity for 11-cis-retinal. Together, the tissue distribution of RDH12 and its catalytic properties suggest that, in most tissues, RDH12 primarily contributes to the reduction of all-trans-retinaldehyde; however, at saturating concentrations of peroxidic aldehydes in the cells undergoing oxidative stress, for example, photoreceptors, RDH12 might also play a role in detoxification of lipid peroxidation products. PMID:15865448

  7. 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.

  8. Microbial Engineering for Aldehyde Synthesis

    PubMed Central

    Kunjapur, Aditya M.

    2015-01-01

    Aldehydes are a class of chemicals with many industrial uses. Several aldehydes are responsible for flavors and fragrances present in plants, but aldehydes are not known to accumulate in most natural microorganisms. In many cases, microbial production of aldehydes presents an attractive alternative to extraction from plants or chemical synthesis. During the past 2 decades, a variety of aldehyde biosynthetic enzymes have undergone detailed characterization. Although metabolic pathways that result in alcohol synthesis via aldehyde intermediates were long known, only recent investigations in model microbes such as Escherichia coli have succeeded in minimizing the rapid endogenous conversion of aldehydes into their corresponding alcohols. Such efforts have provided a foundation for microbial aldehyde synthesis and broader utilization of aldehydes as intermediates for other synthetically challenging biochemical classes. However, aldehyde toxicity imposes a practical limit on achievable aldehyde titers and remains an issue of academic and commercial interest. In this minireview, we summarize published efforts of microbial engineering for aldehyde synthesis, with an emphasis on de novo synthesis, engineered aldehyde accumulation in E. coli, and the challenge of aldehyde toxicity. PMID:25576610

  9. Two-carbon homologation of aldehydes and ketones to α,β-unsaturated aldehydes.

    PubMed

    Petroski, Richard J; Vermillion, Karl; Cossé, Allard A

    2011-06-17

    Phosphonate reagents were developed for the two-carbon homologation of aldehydes or ketones to unbranched- or methyl-branched α,β-unsaturated aldehydes. The phosphonate reagents, diethyl methylformyl-2-phosphonate dimethylhydrazone and diethyl ethylformyl-2-phosphonate dimethylhydrazone, contained a protected aldehyde group instead of the usual ester group. A homologation cycle entailed condensation of the reagent with the starting aldehyde, followed by removal of the dimethylhydrazone protective group with a biphasic mixture of 1 M HCl and petroleum ether. This robust two-step process worked with a variety of aldehydes and ketones. Overall isolated yields of unsaturated aldehyde products ranged from 71% to 86% after the condensation and deprotection steps.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. The Contribution of Nicotinamide Nucleotide Transhydrogenase to Peroxide Detoxification Is Dependent on the Respiratory State and Counterbalanced by Other Sources of NADPH in Liver Mitochondria*

    PubMed Central

    Ronchi, Juliana Aparecida; Francisco, Annelise; Passos, Luiz Augusto Correa; Figueira, Tiago Rezende; Castilho, Roger Frigério

    2016-01-01

    The forward reaction of nicotinamide nucleotide transhydrogenase (NNT) reduces NADP+ at the expense of NADH oxidation and H+ movement down the electrochemical potential across the inner mitochondrial membrane, establishing an NADPH/NADP+ ratio severalfold higher than the NADH/NAD+ ratio in the matrix. In turn, NADPH drives processes, such as peroxide detoxification and reductive biosynthesis. In this study, we generated a congenic mouse model carrying a mutated NntC57BL/6J allele from the C57BL/6J substrain. Suspensions of isolated mitochondria from Nnt+/+, Nnt+/−, and Nnt−/− mouse liver were biochemically evaluated and challenged with exogenous peroxide under different respiratory states. The respiratory substrates were also varied, and the participation of concurrent NADPH sources (i.e. isocitrate dehydrogenase-2, malic enzymes, and glutamate dehydrogenase) was assessed. The principal findings include the following: Nnt+/− and Nnt−/− exhibit ∼50% and absent NNT activity, respectively, but the activities of concurrent NADPH sources are unchanged. The lack of NNT activity in Nnt−/− mice impairs peroxide metabolism in intact mitochondria. The contribution of NNT to peroxide metabolism is decreased during ADP phosphorylation compared with the non-phosphorylating state; however, it is accompanied by increased contributions of concurrent NADPH sources, especially glutamate dehydrogenase. NNT makes a major contribution to peroxide metabolism during the blockage of mitochondrial electron transport. Interestingly, peroxide metabolism in the Nnt+/− mitochondria matched that in the Nnt+/+ mitochondria. Overall, this study demonstrates that the respiratory state and/or substrates that sustain energy metabolism markedly influence the relative contribution of NNT (i.e. varies between nearly 0 and 100%) to NADPH-dependent mitochondrial peroxide metabolism. PMID:27474736

  15. The Contribution of Nicotinamide Nucleotide Transhydrogenase to Peroxide Detoxification Is Dependent on the Respiratory State and Counterbalanced by Other Sources of NADPH in Liver Mitochondria.

    PubMed

    Ronchi, Juliana Aparecida; Francisco, Annelise; Passos, Luiz Augusto Correa; Figueira, Tiago Rezende; Castilho, Roger Frigério

    2016-09-16

    The forward reaction of nicotinamide nucleotide transhydrogenase (NNT) reduces NADP(+) at the expense of NADH oxidation and H(+) movement down the electrochemical potential across the inner mitochondrial membrane, establishing an NADPH/NADP(+) ratio severalfold higher than the NADH/NAD(+) ratio in the matrix. In turn, NADPH drives processes, such as peroxide detoxification and reductive biosynthesis. In this study, we generated a congenic mouse model carrying a mutated Nnt(C57BL/6J) allele from the C57BL/6J substrain. Suspensions of isolated mitochondria from Nnt(+/+), Nnt(+/-), and Nnt(-/-) mouse liver were biochemically evaluated and challenged with exogenous peroxide under different respiratory states. The respiratory substrates were also varied, and the participation of concurrent NADPH sources (i.e. isocitrate dehydrogenase-2, malic enzymes, and glutamate dehydrogenase) was assessed. The principal findings include the following: Nnt(+/-) and Nnt(-/-) exhibit ∼50% and absent NNT activity, respectively, but the activities of concurrent NADPH sources are unchanged. The lack of NNT activity in Nnt(-/-) mice impairs peroxide metabolism in intact mitochondria. The contribution of NNT to peroxide metabolism is decreased during ADP phosphorylation compared with the non-phosphorylating state; however, it is accompanied by increased contributions of concurrent NADPH sources, especially glutamate dehydrogenase. NNT makes a major contribution to peroxide metabolism during the blockage of mitochondrial electron transport. Interestingly, peroxide metabolism in the Nnt(+/-) mitochondria matched that in the Nnt(+/+) mitochondria. Overall, this study demonstrates that the respiratory state and/or substrates that sustain energy metabolism markedly influence the relative contribution of NNT (i.e. varies between nearly 0 and 100%) to NADPH-dependent mitochondrial peroxide metabolism. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Pigment epithelium-derived factor stimulates skeletal muscle glycolytic activity through NADPH oxidase-dependent reactive oxygen species production.

    PubMed

    Carnagarin, Revathy; Carlessi, Rodrigo; Newsholme, Philip; Dharmarajan, Arun M; Dass, Crispin R

    2016-09-01

    Pigment epithelium-derived factor is a multifunctional serpin implicated in insulin resistance in metabolic disorders. Recent evidence suggests that exposure of peripheral tissues such as skeletal muscle to PEDF has profound metabolic consequences with predisposition towards chronic conditions such as obesity, type 2 diabetes, metabolic syndrome and polycystic ovarian syndrome. Chronic inflammation shifts muscle metabolism towards increased glycolysis and decreased oxidative metabolism. In the present study, we demonstrate a novel effect of PEDF on cellular metabolism in mouse cell line (C2C12) and human primary skeletal muscle cells. PEDF addition to skeletal muscle cells induced enhanced phospholipase A2 activity. This was accompanied with increased production of reactive oxygen species in a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent manner that triggered a shift towards a more glycolytic phenotype. Extracellular flux analysis and glucose consumption assays demonstrated that PEDF treatment resulted in enhanced glycolysis but did not change mitochondrial respiration. Our results demonstrate that skeletal muscle cells express a PEDF-inducible oxidant generating system that enhances glycolysis but is sensitive to antioxidants and NADPH oxidase inhibition. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. SAXS fingerprints of aldehyde dehydrogenase oligomers.

    PubMed

    Tanner, John J

    2015-12-01

    Enzymes of the aldehyde dehydrogenase (ALDH) superfamily catalyze the nicotinamide adenine dinucleotide-dependent oxidation of aldehydes to carboxylic acids. ALDHs are important in detoxification of aldehydes, amino acid metabolism, embryogenesis and development, neurotransmission, oxidative stress, and cancer. Mutations in genes encoding ALDHs cause metabolic disorders, including alcohol flush reaction (ALDH2), Sjögren-Larsson syndrome (ALDH3A2), hyperprolinemia type II (ALDH4A1), γ-hydroxybutyric aciduria (ALDH5A1), methylmalonic aciduria (ALDH6A1), pyridoxine dependent epilepsy (ALDH7A1), and hyperammonemia (ALDH18A1). We previously reported crystal structures and small-angle X-ray scattering (SAXS) analyses of ALDHs exhibiting dimeric, tetrameric, and hexameric oligomeric states (Luo et al., Biochemistry 54 (2015) 5513-5522; Luo et al., J. Mol. Biol. 425 (2013) 3106-3120). Herein I provide the SAXS curves, radii of gyration, and distance distribution functions for the three types of ALDH oligomer. The SAXS curves and associated analysis provide diagnostic fingerprints that allow rapid identification of the type of ALDH oligomer that is present in solution. The data sets provided here serve as a benchmark for characterizing oligomerization of ALDHs.

  18. 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

  19. Stimulatory effect of insulin on 5alpha-reductase type 1 (SRD5A1) expression through an Akt-dependent pathway in ovarian granulosa cells.

    PubMed

    Kayampilly, Pradeep P; Wanamaker, Brett L; Stewart, James A; Wagner, Carrie L; Menon, K M J

    2010-10-01

    Elevated levels of 5α-reduced androgens have been shown to be associated with hyperandrogenism and hyperinsulinemia, the leading causes of ovulatory dysfunction in women. 5α-Dihydrotestosterone reduces ovarian granulosa cell proliferation by inhibiting FSH-mediated mitogenic signaling pathways. The present study examined the effect of insulin on 5α-reductase, the enzyme that catalyses the conversion of androgens to their 5α-derivatives. Granulosa cells isolated from immature rat ovaries were cultured in serum-free, phenol red-free DMEM-F12 media and treated with different doses of insulin (0, 0.1, 1.0, and 10.0 μg/ml) for different time intervals up to 12 h. The expression of 5α-reductase type 1 mRNA, the predominant isoform found in granulosa cells, showed a significant (P<0.05) increase in response to the insulin treatment up to 12 h compared with control. The catalytic activity of 5α-reductase enzyme was also stimulated in a dose-depended manner (P<0.05). Inhibiting the Akt-dependent signaling pathway abolished the insulin-mediated increase in 5α-reductase mRNA expression, whereas inhibition of the ERK-dependent pathway had no effect. The dose-dependent increase in 5α-reductase mRNA expression as well as catalytic activity seen in response to insulin treatment was also demonstrated in the human granulosa cell line (KGN). In addition to increased mRNA expression, a dose-dependent increase in 5α-reductase protein expression in response to insulin was also seen in KGN cells, which corroborated well with that of mRNA expression. These results suggest that elevated levels of 5α-reduced androgens seen in hyperinsulinemic conditions might be explained on the basis of a stimulatory effect of insulin on 5α-reductase in granulosa cells. The elevated levels of these metabolites, in turn, might adversely affect growth and proliferation of granulosa cells, thereby impairing follicle growth and ovulation.

  20. Stimulatory Effect of Insulin on 5α-Reductase Type 1 (SRD5A1) Expression through an Akt-Dependent Pathway in Ovarian Granulosa Cells

    PubMed Central

    Kayampilly, Pradeep P.; Wanamaker, Brett L.; Stewart, James A.; Wagner, Carrie L.; Menon, K. M. J.

    2010-01-01

    Elevated levels of 5α-reduced androgens have been shown to be associated with hyperandrogenism and hyperinsulinemia, the leading causes of ovulatory dysfunction in women. 5α-Dihydrotestosterone reduces ovarian granulosa cell proliferation by inhibiting FSH-mediated mitogenic signaling pathways. The present study examined the effect of insulin on 5α-reductase, the enzyme that catalyses the conversion of androgens to their 5α-derivatives. Granulosa cells isolated from immature rat ovaries were cultured in serum-free, phenol red-free DMEM-F12 media and treated with different doses of insulin (0, 0.1, 1.0, and 10.0 μg/ml) for different time intervals up to 12 h. The expression of 5α-reductase type 1 mRNA, the predominant isoform found in granulosa cells, showed a significant (P < 0.05) increase in response to the insulin treatment up to 12 h compared with control. The catalytic activity of 5α-reductase enzyme was also stimulated in a dose-depended manner (P < 0.05). Inhibiting the Akt-dependent signaling pathway abolished the insulin-mediated increase in 5α-reductase mRNA expression, whereas inhibition of the ERK-dependent pathway had no effect. The dose-dependent increase in 5α-reductase mRNA expression as well as catalytic activity seen in response to insulin treatment was also demonstrated in the human granulosa cell line (KGN). In addition to increased mRNA expression, a dose-dependent increase in 5α-reductase protein expression in response to insulin was also seen in KGN cells, which corroborated well with that of mRNA expression. These results suggest that elevated levels of 5α-reduced androgens seen in hyperinsulinemic conditions might be explained on the basis of a stimulatory effect of insulin on 5α-reductase in granulosa cells. The elevated levels of these metabolites, in turn, might adversely affect growth and proliferation of granulosa cells, thereby impairing follicle growth and ovulation. PMID:20810561

  1. 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.

  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. Thermal Dependence of the Apparent Km of Glutathione Reductases from Three Plant Species

    PubMed Central

    Mahan, James R.; Burke, John J.; Orzech, Karen A.

    1990-01-01

    The thermal dependencies of the apparent Km of the glutathione reductases from spinach (Spinacia oleracea L.) corn (Zea mays L.), and cucumber (Cucumis sativus L.) were determined. The apparent Km of the enzymes were found to vary up to 9-fold between 12.5 and 45°C. Values of the apparent Km in excess of 200% of the observed minimum are suggested to be detrimental to the normal function of the enzyme. We propose the term “thermal kinetic window” to describe to the range of temperatures over which the apparent Km of the glutathione reductase is within 200% of its minimum and suggest that it may be a useful indicator of the limits of thermal stress for a given species. The thermal kinetic windows determined in this study are: <16°C for spinach, 23 to 32°C for corn, and 35 to 41°C for cucumber. PMID:16667543

  4. 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 -• ).

  5. New Aldehyde Reductase Genes of Saccharomyces cerevisiae Contribute In Situ Detoxification of Lignocellulose-to-Ethanol Conversion Inhibitiors

    USDA-ARS?s Scientific Manuscript database

    Furfural and 5-hydroxymethylfurfural (HMF) are inhibitory compounds commonly encountered during lignocellulose-to-ethanol conversion for cleaner transportation fuels. It is possible to in situ detoxify the aldehyde inhibitors by tolerant ethanologenic yeast strains. Multiple gene-mediated reductio...

  6. Release and Formation of Oxidation-Related Aldehydes during Wine Oxidation.

    PubMed

    Bueno, Mónica; Carrascón, Vanesa; Ferreira, Vicente

    2016-01-27

    Twenty-four Spanish wines were subjected to five consecutive cycles of air saturation at 25 °C. Free and bound forms of carbonyls were measured in the initial samples and after each saturation. Nonoxidized commercial wines contain important and sensory relevant amounts of oxidation-related carbonyls under the form of odorless bound forms. Models relating the contents in total aldehydes to the wine chemical composition suggest that fermentation can be a major origin for Strecker aldehydes: methional, phenylacetaldehyde, isobutyraldehyde, 2-methylbutanal, and isovaleraldehyde. Bound forms are further cleaved, releasing free aldehydes during the first steps of wine oxidation, as a consequence of equilibrium shifts caused by the depletion of SO2. At low levels of free SO2, de novo formation and aldehyde degradation are both observed. The relative importance of these phenomena depends on both the aldehyde and the wine. Models relating aldehyde formation rates to wine chemical composition suggest that amino acids are in most cases the most important precursors for de novo formation.

  7. 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.

  8. 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

  9. A dual substrate kinetic model for cytochrome P450BM3-F87G catalysis: simultaneous binding of long chain aldehydes and 4-fluorophenol.

    PubMed

    Ledford, Chelsea; McMahon, Monica; Whitesell, Ashley; Khan, Ghalib; Kandagatla, Suneel K; Hurst, Dow P; Reggio, Patricia H; Raner, Gregory M

    2017-02-01

    To develop a model for binding and catalysis associated with the stimulation of 4-fluorophenol (4-FP) oxidation in the presence of long chain aldehydes by the enzymatic catalyst, cytochrome P450 BM3 -F87G. A variation of the Michaeli-Menten kinetic model was employed to describe interactions at the active site of the enzyme, along with computer aided modeling approaches. In addition to the hydroquinone product arising from de-fluorination of 4-FP, a second product (p-fluorocatechol) was also observed and, like the hydroquinone, its rate of formation increased in the presence of the aldehyde. When only aldehyde was present with the enzyme, BM3-F87G catalyzed its oxidation to the corresponding carboxylic acid; however, this activity was inhibited when 4-FP was added to the reaction. A 3D computer model of the active site containing both aldehyde and 4-FP was generated, guided by these kinetic observations. Finally, partitioning between the two phenolic products was examined with an emphasis on the conditions directing the initial epoxidation at either the 2,3- or 3,4-positions on the substrate. Temperature, reaction time, substrate concentration, and the structure of the aldehyde had no substantial effect on the overall product ratios, however the NADPH coupling efficiency decreased when unsaturated aldehydes were included, or when the temperature of the reaction was reduced. The unsaturated aldehyde, trans-2-decenal, stimulates BM3-F87G catalyzed oxidation of 4-fluorophenol through a cooperative active site binding mode that doesn't influence product distributions or coupling efficiencies, while 4-fluorophenol acts as a competitive inhibitor of aldehyde oxidation.

  10. Reversible, partial inactivation of plant betaine aldehyde dehydrogenase by betaine aldehyde: mechanism and possible physiological implications.

    PubMed

    Zárate-Romero, Andrés; Murillo-Melo, Darío S; Mújica-Jiménez, Carlos; Montiel, Carmina; Muñoz-Clares, Rosario A

    2016-04-01

    In plants, the last step in the biosynthesis of the osmoprotectant glycine betaine (GB) is the NAD(+)-dependent oxidation of betaine aldehyde (BAL) catalysed by some aldehyde dehydrogenase (ALDH) 10 enzymes that exhibit betaine aldehyde dehydrogenase (BADH) activity. Given the irreversibility of the reaction, the short-term regulation of these enzymes is of great physiological relevance to avoid adverse decreases in the NAD(+):NADH ratio. In the present study, we report that the Spinacia oleracea BADH (SoBADH) is reversibly and partially inactivated by BAL in the absence of NAD(+)in a time- and concentration-dependent mode. Crystallographic evidence indicates that the non-essential Cys(450)(SoBADH numbering) forms a thiohemiacetal with BAL, totally blocking the productive binding of the aldehyde. It is of interest that, in contrast to Cys(450), the catalytic cysteine (Cys(291)) did not react with BAL in the absence of NAD(+) The trimethylammonium group of BAL binds in the same position in the inactivating or productive modes. Accordingly, BAL does not inactivate the C(450)SSoBADH mutant and the degree of inactivation of the A(441)I and A(441)C mutants corresponds to their very different abilities to bind the trimethylammonium group. Cys(450)and the neighbouring residues that participate in stabilizing the thiohemiacetal are strictly conserved in plant ALDH10 enzymes with proven or predicted BADH activity, suggesting that inactivation by BAL is their common feature. Under osmotic stress conditions, this novel partial and reversible covalent regulatory mechanism may contribute to preventing NAD(+)exhaustion, while still permitting the synthesis of high amounts of GB and avoiding the accumulation of the toxic BAL. © 2016 Authors; published by Portland Press Limited.

  11. Function of Coenzyme F420 in Aerobic Catabolism of 2,4,6-Trinitrophenol and 2,4-Dinitrophenol by Nocardioides simplex FJ2-1A

    PubMed Central

    Ebert, Sybille; Rieger, Paul-Gerhard; Knackmuss, Hans-Joachim

    1999-01-01

    2,4,6-Trinitrophenol (picric acid) and 2,4-dinitrophenol were readily biodegraded by the strain Nocardioides simplex FJ2-1A. Aerobic bacterial degradation of these π-electron-deficient aromatic compounds is initiated by hydrogenation at the aromatic ring. A two-component enzyme system was identified which catalyzes hydride transfer to picric acid and 2,4-dinitrophenol. Enzymatic activity was dependent on NADPH and coenzyme F420. The latter could be replaced by an authentic preparation of coenzyme F420 from Methanobacterium thermoautotrophicum. One of the protein components functions as a NADPH-dependent F420 reductase. A second component is a hydride transferase which transfers hydride from reduced coenzyme F420 to the aromatic system of the nitrophenols. The N-terminal sequence of the F420 reductase showed high homology with an F420-dependent NADP reductase found in archaea. In contrast, no N-terminal similarity to any known protein was found for the hydride-transferring enzyme. PMID:10217752

  12. 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

  13. 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

  14. Substrate specificity and subcellular localization of the aldehyde-alcohol redox-coupling reaction in carp cones.

    PubMed

    Sato, Shinya; Fukagawa, Takashi; Tachibanaki, Shuji; Yamano, Yumiko; Wada, Akimori; Kawamura, Satoru

    2013-12-20

    Our previous study suggested the presence of a novel cone-specific redox reaction that generates 11-cis-retinal from 11-cis-retinol in the carp retina. This reaction is unique in that 1) both 11-cis-retinol and all-trans-retinal were required to produce 11-cis-retinal; 2) together with 11-cis-retinal, all-trans-retinol was produced at a 1:1 ratio; and 3) the addition of enzyme cofactors such as NADP(H) was not necessary. This reaction is probably part of the reactions in a cone-specific retinoid cycle required for cone visual pigment regeneration with the use of 11-cis-retinol supplied from Müller cells. In this study, using purified carp cone membrane preparations, we first confirmed that the reaction is a redox-coupling reaction between retinals and retinols. We further examined the substrate specificity, reaction mechanism, and subcellular localization of this reaction. Oxidation was specific for 11-cis-retinol and 9-cis-retinol. In contrast, reduction showed low specificity: many aldehydes, including all-trans-, 9-cis-, 11-cis-, and 13-cis-retinals and even benzaldehyde, supported the reaction. On the basis of kinetic studies of this reaction (aldehyde-alcohol redox-coupling reaction), we found that formation of a ternary complex of a retinol, an aldehyde, and a postulated enzyme seemed to be necessary, which suggested the presence of both the retinol- and aldehyde-binding sites in this enzyme. A subcellular fractionation study showed that the activity is present almost exclusively in the cone inner segment. These results suggest the presence of an effective production mechanism of 11-cis-retinal in the cone inner segment to regenerate visual pigment.

  15. 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

  16. 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

  17. [Aldose reductase gene polymorphism and rate of appearance of retinopathy in non insulin dependent diabetics].

    PubMed

    Olmos, P; Acosta, A M; Schiaffino, R; Díaz, R; Alvarado, D; O'Brien, A; Muñoz, X; Arriagada, P; Claro, J C; Vega, R; Vollrath, V; Velasco, S; Emmerich, M; Maiz, A

    1999-04-01

    Recent studies suggest that polymorphisms associated to the aldose reductase gene could be related to early retinopathy in noninsulin dependent diabetics (NIDDM). There is also new interest on the genetic modulation of coagulation factors in relation to this complication. To look for a possible relationship between the rate of appearance of retinopathy and the genotype of (AC)n polymorphic marker associated to aldose reductase gene. A random sample of 27 NIDDM, aged 68.1 +/- 10.6 years, with a mean diabetes duration of 20.7 +/- 4.8 years and a mean glycosilated hemoglobin of 10.6 +/- 1.6%, was studied. The genotype of the (AC)n, polymorphic marker associated to the 5' end of the aldose reductase (ALR2) gene was determined by 32P-PCR plus sequenciation. Mutations of the factor XIII-A gene were studied by single stranded conformational polymorphism, sequenciation and restriction fragment length polymorphism. Four patients lacked the (AC)24 and had a higher rate of appearance of retinopathy than patients with the (AC)24 allele (0.0167 and 0.0907 score points per year respectively, p = 0.047). Both groups had similar glycosilated hemoglobin (11.7 +/- 0.2 and 10.5 +/- 1.6% respectively). Factor XIII gene mutations were not related to the rate of appearance of retinopathy. Our data suggest that the absence of the (AC)24 allele of the (AC)n polymorphic marker associated to the 5' end of the aldose reductase gene, is associated to a five fold reduction of retinopathy appearance rate.

  18. 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.

  19. Deficiency of aldose reductase exacerbates early pressure overload-induced cardiac dysfunction and autophagy in mice.

    PubMed

    Baba, Shahid P; Zhang, Deqing; Singh, Mahavir; Dassanayaka, Sujith; Xie, Zhengzhi; Jagatheesan, Ganapathy; Zhao, Jingjing; Schmidtke, Virginia K; Brittian, Kenneth R; Merchant, Michael L; Conklin, Daniel J; Jones, Steven P; Bhatnagar, Aruni

    2018-05-01

    Pathological cardiac hypertrophy is associated with the accumulation of lipid peroxidation-derived aldehydes such as 4-hydroxy-trans-2-nonenal (HNE) and acrolein in the heart. These aldehydes are metabolized via several pathways, of which aldose reductase (AR) represents a broad-specificity route for their elimination. We tested the hypothesis that by preventing aldehyde removal, AR deficiency accentuates the pathological effects of transverse aortic constriction (TAC). We found that the levels of AR in the heart were increased in mice subjected to TAC for 2 weeks. In comparison with wild-type (WT), AR-null mice showed lower ejection fraction, which was exacerbated 2 weeks after TAC. Levels of atrial natriuretic peptide and myosin heavy chain were higher in AR-null than in WT TAC hearts. Deficiency of AR decreased urinary levels of the acrolein metabolite, 3-hydroxypropylmercapturic acid. Deletion of AR did not affect the levels of the other aldehyde-metabolizing enzyme - aldehyde dehydrogenase 2 in the heart, or its urinary product - (N-Acetyl-S-(2-carboxyethyl)-l-cystiene). AR-null hearts subjected to TAC showed increased accumulation of HNE- and acrolein-modified proteins, as well as increased AMPK phosphorylation and autophagy. Superfusion with HNE led to a greater increase in p62, LC3II formation, and GFP-LC3-II punctae formation in AR-null than WT cardiac myocytes. Pharmacological inactivation of JNK decreased HNE-induced autophagy in AR-null cardiac myocytes. Collectively, these results suggest that during hypertrophy the accumulation of lipid peroxidation derived aldehydes promotes pathological remodeling via excessive autophagy, and that metabolic detoxification of these aldehydes by AR may be essential for maintaining cardiac function during early stages of pressure overload. Published by Elsevier Ltd.

  20. Structure and biocatalytic scope of thermophilic flavin-dependent halogenase and flavin reductase enzymes.

    PubMed

    Menon, Binuraj R K; Latham, Jonathan; Dunstan, Mark S; Brandenburger, Eileen; Klemstein, Ulrike; Leys, David; Karthikeyan, Chinnan; Greaney, Michael F; Shepherd, Sarah A; Micklefield, Jason

    2016-10-04

    Flavin-dependent halogenase (Fl-Hal) enzymes have been shown to halogenate a range of synthetic as well as natural aromatic compounds. The exquisite regioselectively of Fl-Hal enzymes can provide halogenated building blocks which are inaccessible using standard halogenation chemistries. Consequently, Fl-Hal are potentially useful biocatalysts for the chemoenzymatic synthesis of pharmaceuticals and other valuable products, which are derived from haloaromatic precursors. However, the application of Fl-Hal enzymes, in vitro, has been hampered by their poor catalytic activity and lack of stability. To overcome these issues, we identified a thermophilic tryptophan halogenase (Th-Hal), which has significantly improved catalytic activity and stability, compared with other Fl-Hal characterised to date. When used in combination with a thermostable flavin reductase, Th-Hal can efficiently halogenate a number of aromatic substrates. X-ray crystal structures of Th-Hal, and the reductase partner (Th-Fre), provide insights into the factors that contribute to enzyme stability, which could guide the discovery and engineering of more robust and productive halogenase biocatalysts.

  1. Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis

    PubMed Central

    Khan, Meraj A.; Philip, Lijy M.; Cheung, Guillaume; Vadakepeedika, Shawn; Grasemann, Hartmut; Sweezey, Neil; Palaniyar, Nades

    2018-01-01

    Neutrophils migrating from the blood (pH 7.35–7.45) into the surrounding tissues encounter changes in extracellular pH (pHe) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H+ ions reducing the intracellular pH (pHi). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pHe (ranging from 6.6 to 7.8; every 0.2 units) increased pHi of both activated and resting neutrophils within 10–20 min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H+ ions, pHi is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative)-, and Staphylococcus aureus (Gram-positive)-induced NETosis. Thus, higher pHe promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pHe-mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H+ ions, whereas each bicarbonate HCO3− ion binds 1H+ ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar

  2. Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis.

    PubMed

    Khan, Meraj A; Philip, Lijy M; Cheung, Guillaume; Vadakepeedika, Shawn; Grasemann, Hartmut; Sweezey, Neil; Palaniyar, Nades

    2018-01-01

    Neutrophils migrating from the blood (pH 7.35-7.45) into the surrounding tissues encounter changes in extracellular pH (pH e ) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H + ions reducing the intracellular pH (pH i ). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pH e (ranging from 6.6 to 7.8; every 0.2 units) increased pH i of both activated and resting neutrophils within 10-20 min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H + ions, pH i is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative)-, and Staphylococcus aureus (Gram-positive)-induced NETosis. Thus, higher pH e promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pH e -mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H + ions, whereas each bicarbonate HCO3 - ion binds 1H + ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar

  3. 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.

  4. 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.

  5. A redox-mediated modulation of stem bolting in transgenic Nicotiana sylvestris differentially expressing the external mitochondrial NADPH dehydrogenase.

    PubMed

    Liu, Yun-Jun; Nunes-Nesi, Adriano; Wallström, Sabá V; Lager, Ida; Michalecka, Agnieszka M; Norberg, Fredrik E B; Widell, Susanne; Fredlund, Kenneth M; Fernie, Alisdair R; Rasmusson, Allan G

    2009-07-01

    Cytosolic NADPH can be directly oxidized by a calcium-dependent NADPH dehydrogenase, NDB1, present in the plant mitochondrial electron transport chain. However, little is known regarding the impact of modified cytosolic NADPH reduction levels on growth and metabolism. Nicotiana sylvestris plants overexpressing potato (Solanum tuberosum) NDB1 displayed early bolting, whereas sense suppression of the same gene led to delayed bolting, with consequential changes in flowering time. The phenotype was dependent on light irradiance but not linked to any change in biomass accumulation. Whereas the leaf NADPH/NADP(+) ratio was unaffected, the stem NADPH/NADP(+) ratio was altered following the genetic modification and strongly correlated with the bolting phenotype. Metabolic profiling of the stem showed that the NADP(H) change affected relatively few, albeit central, metabolites, including 2-oxoglutarate, glutamate, ascorbate, sugars, and hexose-phosphates. Consistent with the phenotype, the modified NDB1 level also affected the expression of putative floral meristem identity genes of the SQUAMOSA and LEAFY types. Further evidence for involvement of the NADPH redox in stem development was seen in the distinct decrease in the stem apex NADPH/NADP(+) ratio during bolting. Additionally, the potato NDB1 protein was specifically detected in mitochondria, and a survey of its abundance in major organs revealed that the highest levels are found in green stems. These results thus strongly suggest that NDB1 in the mitochondrial electron transport chain can, by modifying cell redox levels, specifically affect developmental processes.

  6. [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.

  7. Molecular characterization of an aldo-keto reductase from Marivirga tractuosa that converts retinal to retinol.

    PubMed

    Hong, Seung-Hye; Nam, Hyun-Koo; Kim, Kyoung-Rok; Kim, Seon-Won; Oh, Deok-Kun

    2014-01-01

    A recombinant aldo-keto reductase (AKR) from Marivirga tractuosa was purified with a specific activity of 0.32unitml(-1) for all-trans-retinal with a 72kDa dimer. The enzyme had substrate specificity for aldehydes but not for alcohols, carbonyls, or monosaccharides. The enzyme turnover was the highest for benzaldehyde (kcat=446min(-1)), whereas the affinity and catalytic efficiency were the highest for all-trans-retinal (Km=48μM, kcat/Km=427mM(-1)min(-1)) among the tested substrates. The optimal reaction conditions for the production of all-trans-retinol from all-trans-retinal by M. tractuosa AKR were pH 7.5, 30°C, 5% (v/v) methanol, 1% (w/v) hydroquinone, 10mM NADPH, 1710mgl(-1) all-trans-retinal, and 3unitml(-1) enzyme. Under these optimized conditions, the enzyme produced 1090mgml(-1) all-trans-retinol, with a conversion yield of 64% (w/w) and a volumetric productivity of 818mgl(-1)h(-1). AKR from M. tractuosa showed no activity for all-trans-retinol using NADP(+) as a cofactor, whereas human AKR exhibited activity. When the cofactor-binding residues (Ala158, Lys212, and Gln270) of M. tractuosa AKR were changed to the corresponding residues of human AKR (Ser160, Pro212, and Glu272), the A158S and Q270E variants exhibited activity for all-trans-retinol. Thus, amino acids at positions 158 and 270 of M. tractuosa AKR are determinant residues of the activity for all-trans-retinol. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. Anti-neuroinflammatory efficacy of the aldose reductase inhibitor FMHM via phospholipase C/protein kinase C-dependent NF-κB and MAPK pathways

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

    Zeng, Ke-Wu; Li, Jun; Dong, Xin

    2013-11-15

    Aldose reductase (AR) has a key role in several inflammatory diseases: diabetes, cancer and cardiovascular diseases. Therefore, AR inhibition seems to be a useful strategy for anti-inflammation therapy. In the central nervous system (CNS), microglial over-activation is considered to be a central event in neuroinflammation. However, the effects of AR inhibition in CNS inflammation and its underlying mechanism of action remain unknown. In the present study, we found that FMHM (a naturally derived AR inhibitor from the roots of Polygala tricornis Gagnep.) showed potent anti-neuroinflammatory effects in vivo and in vitro by inhibiting microglial activation and expression of inflammatory mediators.more » Mechanistic studies showed that FMHM suppressed the activity of AR-dependent phospholipase C/protein kinase C signaling, which further resulted in downstream inactivation of the IκB kinase/IκB/nuclear factor-kappa B (NF-κB) inflammatory pathway. Therefore, AR inhibition-dependent NF-κB inactivation negatively regulated the transcription and expression of various inflammatory genes. AR inhibition by FMHM exerted neuroprotective effects in lipopolysaccharide-induced neuron–microglia co-cultures. These findings suggested that AR is a potential target for neuroinflammation inhibition and that FMHM could be an effective agent for treating or preventing neuroinflammatory diseases. - Highlights: • FMHM is a natural-derived aldose reductase (AR) inhibitor. • FMHM inhibits various neuroinflammatory mediator productions in vitro and in vivo. • FMHM inhibits neuroinflammation via aldose reductase/PLC/PKC-dependent NF-κB pathway. • FMHM inhibits neuroinflammation via aldose reductase/PLC/PKC-dependent MAPK pathway. • FMHM protects neurons against inflammatory injury in microglia-neuron co-cultures.« less

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

    USDA-ARS?s Scientific Manuscript database

    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 the yeast, particularly when the carbon source is acid-treated lignocell...

  17. 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.

  18. 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

  19. Aldehydic load and aldehyde dehydrogenase 2 profile during the progression of post-myocardial infarction cardiomyopathy: benefits of Alda-1

    PubMed Central

    Gomes, Katia M.S.; Bechara, Luiz R.G.; Lima, Vanessa M.; Ribeiro, Márcio A.C.; Campos, Juliane C.; Dourado, Paulo M.; Kowaltowski, Alicia J.; Mochly-Rosen, Daria; Ferreira, Julio C.B.

    2015-01-01

    Background/Objectives We previously demonstrated that reducing cardiac aldehydic load by aldehyde dehydrogenase 2 (ALDH2), a mitochondrial enzyme responsible for metabolizing the major lipid peroxidation product, protects against acute ischemia/reperfusion injury and chronic heart failure. However, time-dependent changes in ALDH2 profile, aldehydic load and mitochondrial bioenergetics during progression of post-myocardial infarction (post-MI) cardiomyopathy is unknown and should be established to determine the optimal time window for drug treatment. Methods Here we characterized cardiac ALDH2 activity and expression, lipid peroxidation, 4-hydroxy-2-nonenal (4-HNE) adduct formation, glutathione pool and mitochondrial energy metabolism and H2O2 release during the 4 weeks after permanent left anterior descending (LAD) coronary artery occlusion in rats. Results We observed a sustained disruption of cardiac mitochondrial function during the progression of post-MI cardiomyopathy, characterized by >50% reduced mitochondrial respiratory control ratios and up to 2 fold increase in H2O2 release. Mitochondrial dysfunction was accompanied by accumulation of cardiac and circulating lipid peroxides and 4-HNE protein adducts and down-regulation of electron transport chain complexes I and V. Moreover, increased aldehydic load was associated with a 90% reduction in cardiac ALDH2 activity and increased glutathione pool. Further supporting an ALDH2 mechanism, sustained Alda-1 treatment (starting 24hrs after permanent LAD occlusion surgery) prevented aldehydic overload, mitochondrial dysfunction and improved ventricular function in post-MI cardiomyopathy rats. Conclusion Taken together, our findings demonstrate a disrupted mitochondrial metabolism along with an insufficient cardiac ALDH2-mediated aldehyde clearance during the progression of ventricular dysfunction, suggesting a potential therapeutic value of ALDH2 activators during the progression of post-myocardial infarction

  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. Interaction of glutathione reductase with heavy metal: the binding of Hg(II) or Cd(II) to the reduced enzyme affects both the redox dithiol pair and the flavin.

    PubMed

    Picaud, Thierry; Desbois, Alain

    2006-12-26

    To determine the inhibition mechanism of yeast glutathione reductase (GR) by heavy metal, we have compared the electronic absorption and resonance Raman (RR) spectra of the enzyme in its oxidized (Eox) and two-electron reduced (EH2) forms, in the absence and the presence of Hg(II) or Cd(II). The spectral data clearly show a redox dependence of the metal binding. The metal ions do not affect the absorption and RR spectra of Eox. On the contrary, the EH2 spectra, generated by addition of NADPH, are strongly modified by the presence of heavy metal. The absorption changes of EH2 are metal-dependent. On the one hand, the main flavin band observed at 450 nm for EH2 is red-shifted at 455 nm for the EH2-Hg(II) complex and at 451 nm for the EH2-Cd(II) complex. On the other hand, the characteristic charge-transfer (CT) band at 540 nm is quenched upon metal binding to EH2. In NADPH excess, a new CT band is observed at 610 nm for the EH2-Hg(II)-NADPH complex and at 590 nm for EH2-Cd(II)-NADPH. The RR spectra of the EH2-metal complexes are not sensitive to the NADPH concentration. With reference to the RR spectra of EH2 in which the frequencies of bands II and III were observed at 1582 and 1547 cm-1, respectively, those of the EH2-metal complexes are detected at 1577 and 1542 cm-1, indicating an increased flavin bending upon metal coordination to EH2. From the frequency shifts of band III, a concomitant weakening of the H-bonding state of the N5 atom is also deduced. Taking into account the different chemical properties of Hg(II) and Cd(II), the coordination number of the bound metal ion was deduced to be different in GR. A mechanism of the GR inhibition is proposed. It proceeds primarily by a specific binding of the metal to the redox thiol/thiolate pair and the catalytic histidine of EH2. The bound metal ion then acts on the bending of the isoalloxazine ring of FAD as well as on the hydrophobicity of its microenvironment.

  2. Ni-Catalyzed Dehydrogenative Cross-Coupling: Direct Transformation of Aldehydes to Esters and Amides

    PubMed Central

    Whittaker, Aaron M.; Dong, Vy M.

    2015-01-01

    By exploring a new mode of Ni-catalyzed cross-coupling, we have developed a protocol to transform both aromatic and aliphatic aldehydes into either esters or amides directly. The success of this oxidative coupling depends on the appropriate choice of catalyst and organic oxidant, including the use of either α,α,α-trifluoroacetophenone or excess aldehyde. We present mechanistic data that supports a catalytic cycle involving oxidative addition into the aldehyde C–H bond. PMID:25424967

  3. Regulation of NADPH-dependent Nitric Oxide and reactive oxygen species signalling in endothelial and melanoma cells by a photoactive NADPH analogue

    PubMed Central

    Rouaud, Florian; Romero-Perez, Miguel; Wang, Huan; Lobysheva, Irina; Ramassamy, Booma; Henry, Etienne; Tauc, Patrick; Giacchero, Damien; Boucher, Jean-Luc; Deprez, Eric; Rocchi, Stéphane; Slama-Schwok, Anny

    2014-01-01

    Nitric Oxide (NO) and Reactive oxygen species (ROS) are endogenous regulators of angiogenesis-related events as endothelial cell proliferation and survival, but NO/ROS defect or unbalance contribute to cancers. We recently designed a novel photoactive inhibitor of NO-Synthases (NOS) called NS1, which binds their NADPH site in vitro. Here, we show that NS1 inhibited NO formed in aortic rings. NS1-induced NO decrease led to an inhibition of angiogenesis in a model of VEGF-induced endothelial tubes formation. Beside this effect, NS1 reduced ROS levels in endothelial and melanoma A375 cells and in aorta. In metastatic melanoma cells, NS1 first induced a strong decrease of VEGF and blocked melanoma cell cycle at G2/M. NS1 decreased NOX4 and ROS levels that could lead to a specific proliferation arrest and cell death. In contrast, NS1 did not perturb melanocytes growth. Altogether, NS1 revealed a possible cross-talk between eNOS- and NOX4 –associated pathways in melanoma cells via VEGF, Erk and Akt modulation by NS1 that could be targeted to stop proliferation. NS1 thus constitutes a promising tool that modulates NO and redox stresses by targeting and directly inhibiting eNOS and, at least indirectly, NADPH oxidase(s), with great potential to control angiogenesis. PMID:25296975

  4. Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays.

    PubMed

    Aleshin, Vasily A; Artiukhov, Artem V; Oppermann, Henry; Kazantsev, Alexey V; Lukashev, Nikolay V; Bunik, Victoria I

    2015-08-21

    Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay.

  5. Enhanced production of GDP-L-fucose by overexpression of NADPH regenerator in recombinant Escherichia coli.

    PubMed

    Lee, Won-Heong; Chin, Young-Wook; Han, Nam Soo; Kim, Myoung-Dong; Seo, Jin-Ho

    2011-08-01

    Biosynthesis of guanosine 5'-diphosphate-L-fucose (GDP-L-fucose) requires NADPH as a reducing cofactor. In this study, endogenous NADPH regenerating enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (Icd), and NADP(+)-dependent malate dehydrogenase (MaeB) were overexpressed to increase GDP-L-fucose production in recombinant Escherichia coli. The effects of overexpression of each NADPH regenerating enzyme on GDP-L-fucose production were investigated in a series of batch and fed-batch fermentations. Batch fermentations showed that overexpression of G6PDH was the most effective for GDP-L-fucose production. However, GDP-L-fucose production was not enhanced by overexpression of G6PDH in the glucose-limited fed-batch fermentation. Hence, a glucose feeding strategy was optimized to enhance GDP-L-fucose production. Fed-batch fermentation with a pH-stat feeding mode for sufficient supply of glucose significantly enhanced GDP-L-fucose production compared with glucose-limited fed-batch fermentation. A maximum GDP-L-fucose concentration of 235.2 ± 3.3 mg l(-1), corresponding to a 21% enhancement in the GDP-L-fucose production compared with the control strain overexpressing GDP-L-fucose biosynthetic enzymes only, was achieved in the pH-stat fed-batch fermentation of the recombinant E. coli overexpressing G6PDH. It was concluded that sufficient glucose supply and efficient NADPH regeneration are crucial for NADPH-dependent GDP-L-fucose production in recombinant E. coli.

  6. Selective Rac1 inhibition protects renal tubular epithelial cells from oxalate-induced NADPH oxidase-mediated oxidative cell injury

    PubMed Central

    Thamilselvan, Vijayalakshmi; Menon, Mani

    2013-01-01

    Oxalate-induced oxidative cell injury is one of the major mechanisms implicated in calcium oxalate nucleation, aggregation and growth of kidney stones. We previously demonstrated that oxalate-induced NADPH oxidase-derived free radicals play a significant role in renal injury. Since NADPH oxidase activation requires several regulatory proteins, the primary goal of this study was to characterize the role of Rac GTPase in oxalate-induced NADPH oxidase-mediated oxidative injury in renal epithelial cells. Our results show that oxalate significantly increased membrane translocation of Rac1 and NADPH oxidase activity of renal epithelial cells in a time-dependent manner. We found that NSC23766, a selective inhibitor of Rac1, blocked oxalate-induced membrane translocation of Rac1 and NADPH oxidase activity. In the absence of Rac1 inhibitor, oxalate exposure significantly increased hydrogen peroxide formation and LDH release in renal epithelial cells. In contrast, Rac1 inhibitor pretreatment, significantly decreased oxalate-induced hydrogen peroxide production and LDH release. Furthermore, PKC α and δ inhibitor, oxalate exposure did not increase Rac1 protein translocation, suggesting that PKC resides upstream from Rac1 in the pathway that regulates NADPH oxidase. In conclusion, our data demonstrate for the first time that Rac1-dependent activation of NADPH oxidase might be a crucial mechanism responsible for oxalate-induced oxidative renal cell injury. These findings suggest that Rac1 signaling plays a key role in oxalate-induced renal injury, and may serve as a potential therapeutic target to prevent calcium oxalate crystal deposition in stone formers and reduce recurrence. PMID:21814770

  7. 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.

  8. Granzyme B of cytotoxic T cells induces extramitochondrial reactive oxygen species production via caspase-dependent NADPH oxidase activation.

    PubMed

    Aguiló, Juan I; Anel, Alberto; Catalán, Elena; Sebastián, Alvaro; Acín-Pérez, Rebeca; Naval, Javier; Wallich, Reinhard; Simon, Markus M; Pardo, Julián

    2010-07-01

    Induction of reactive oxygen species (ROS) is a hallmark of granzyme B (gzmB)-mediated pro-apoptotic processes and target cell death. However, it is unclear to what extent the generated ROS derive from mitochondrial and/or extra-mitochondrial sources. To clarify this point, we have produced a mutant EL4 cell line, termed EL4-rho(0), which lacks mitochondrial DNA, associated with a decreased mitochondrial membrane potential and a defective ROS production through the electron transport chain of oxidative phosphorylation. When incubated with either recombinant gzmB plus streptolysin or ex vivo gzmB(+) cytotoxic T cells, EL4-rho(0) cells showed phosphatydylserine translocation, caspase 3 activation, Bak conformational change, cytochrome c release and apoptotic morphology comparable to EL4 cells. Moreover, EL4-rho(0) cells produced ROS at levels similar to EL4 under these conditions. GzmB-mediated ROS production was almost totally abolished in both cell lines by the pan-caspase inhibitor, Z-VAD-fmk. However, addition of apocynin, a specific inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, led to a significant reduction of ROS production and cell death only in EL4-rho(0) but not EL4 cells. These data suggest that gzmB-induced cell death is accompanied by a caspase-dependent pathway of extra-mitochondrial ROS production, most probably through activation of NADPH oxidase.

  9. 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

  10. 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.

  11. Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein

    PubMed Central

    Chen, Huizhong; Hopper, Sherryll L.; Cerniglia, Carl E.

    2018-01-01

    Azo dyes are a predominant class of colourants used in tattooing, cosmetics, foods and consumer products. A gene encoding NADPH-flavin azoreductase (Azo1) from the skin bacterium Staphylococcus aureus ATCC 25923 was identified and overexpressed in Escherichia coli. RT-PCR results demonstrated that the azo1 gene was constitutively expressed at the mRNA level in S. aureus. Azo1 was found to be a tetramer with a native molecular mass of 85 kDa containing four non-covalently bound FMN. Azo1 requires NADPH, but not NADH, as an electron donor for its activity. The enzyme was resolved to dimeric apoprotein by removing the flavin prosthetic groups using hydrophobic-interaction chromatography. The dimeric apoprotein was reconstituted on-column and in free stage with FMN, resulting in the formation of a fully functional native-like tetrameric enzyme. The enzyme cleaved the model azo dye 2-[4-(dimethylamino)phenylazo]benzoic acid (Methyl Red) into N,N-dimethyl-p-phenylenediamine and 2-aminobenzoic acid. The apparent Km values for NADPH and Methyl Red substrates were 0·;074 and 0·057 mM, respectively. The apparent Vmax was 0·4 µM min−1 (mg protein)−1. Azo1 was also able to metabolize Orange II, Amaranth, Ponceau BS and Ponceau S azo dyes. Azo1 represents the first azoreductase to be identified and characterized from human skin microflora. PMID:15870453

  12. MASS SPECTROMETRY OF FATTY ALDEHYDES

    PubMed Central

    Berdyshev, Evgeny V.

    2011-01-01

    Fatty aldehydes are important components of the cellular lipidome. Significant interest has been developed towards the analysis of the short chain α,β-unsaturated and hydroxylated aldehydes formed as a result of oxidation of polyunsaturated fatty acids. Multiple gas chromatography-mass spectrometry (GC/MS) and subsequently liquid chromatography-mass spectrometry (LC/MS) approaches have been developed to identify and quantify short-chain as well as long-chain fatty aldehydes. Due to the ability to non-enzymaticaly form Schiff bases with amino groups of proteins, lipids, and with DNA guanidine, free aldehydes are viewed as a marker or metric of fatty acid oxidation and not the part of intracellular signaling pathways which has significantly limited the overall attention this group of molecules have received. This review provides an overview of current GC/MS and LC/MS approaches of fatty aldehyde analysis as well as discusses technical challenges standing in the way of free fatty aldehyde quantitation. PMID:21930240

  13. 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

  14. 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

  15. Two-carbon homologation of aldehydes and ketones to a,ß-unsaturated aldehydes

    USDA-ARS?s Scientific Manuscript database

    Phosphonate reagents were developed for the two-carbon homologation of aldehydes or ketones to unbranched- or methyl-branched a,ß-unsaturated aldehydes. The phosphonate reagents, diethyl methylformyl-2-phosphonate dimethylhydrazone and diethyl ethylformyl-2-phosphonate dimethylhydrazone, contained a...

  16. Structure and Function of the Unusual Tungsten Enzymes Acetylene Hydratase and Class II Benzoyl-Coenzyme A Reductase.

    PubMed

    Boll, Matthias; Einsle, Oliver; Ermler, Ulrich; Kroneck, Peter M H; Ullmann, G Matthias

    2016-01-01

    In biology, tungsten (W) is exclusively found in microbial enzymes bound to a bis-pyranopterin cofactor (bis-WPT). Previously known W enzymes catalyze redox oxo/hydroxyl transfer reactions by directly coordinating their substrates or products to the metal. They comprise the W-containing formate/formylmethanofuran dehydrogenases belonging to the dimethyl sulfoxide reductase (DMSOR) family and the aldehyde:ferredoxin oxidoreductase (AOR) families, which form a separate enzyme family within the Mo/W enzymes. In the last decade, initial insights into the structure and function of two unprecedented W enzymes were obtained: the acetaldehyde forming acetylene hydratase (ACH) belongs to the DMSOR and the class II benzoyl-coenzyme A (CoA) reductase (BCR) to the AOR family. The latter catalyzes the reductive dearomatization of benzoyl-CoA to a cyclic diene. Both are key enzymes in the degradation of acetylene (ACH) or aromatic compounds (BCR) in strictly anaerobic bacteria. They are unusual in either catalyzing a nonredox reaction (ACH) or a redox reaction without coordinating the substrate or product to the metal (BCR). In organic chemical synthesis, analogous reactions require totally nonphysiological conditions depending on Hg2+ (acetylene hydration) or alkali metals (benzene ring reduction). The structural insights obtained pave the way for biological or biomimetic approaches to basic reactions in organic chemistry. © 2016 S. Karger AG, Basel.

  17. 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.

  18. 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

  19. Phosphatidic acid as a second messenger in human polymorphonuclear leukocytes. Effects on activation of NADPH oxidase.

    PubMed Central

    Agwu, D E; McPhail, L C; Sozzani, S; Bass, D A; McCall, C E

    1991-01-01

    Receptor-mediated agonists, such as FMLP, induce an early, phospholipase D (PLD)-mediated accumulation of phosphatidic acid (PA) which may play a role in the activation of NADPH oxidase in human PMN. We have determined the effect of changes in PA production on O2 consumption in intact PMN and the level of NADPH oxidase activity measured in a cell-free assay. Pretreatment of cells with various concentrations of propranolol enhanced (less than or equal to 200 microM) or inhibited (greater than 300 microM) PLD-induced production of PA (mass and radiolabel) in a manner that correlated with enhancement or inhibition of O2 consumption in PMN stimulated with 1 microM FMLP in the absence of cytochalasin B. The concentration-dependent effects of propranolol on FMLP-induced NADPH oxidase activation was confirmed by direct assay of the enzyme in subcellular fractions. In PA extracted from cells pretreated with 200 microM propranolol before stimulation with 1 microM FMLP, phospholipase A1 (PLA1)-digestion for 90 min, followed by quantitation of residual PA, showed that a minimum of 44% of PA in control (undigested) sample was diacyl-PA; alkylacyl-PA remained undigested by PLA1. Propranolol was also observed to have a concentration-dependent enhancement of mass of 1,2-DG formed in PMN stimulated with FMLP. DG levels reached a maximum at 300 microM propranolol and remained unchanged up to 500 microM propranolol. However, in contrast to PA levels, the level of DG produced did not correlate with NADPH oxidase activation. Exogenously added didecanoyl-PA activated NADPH oxidase in a concentration-dependent manner (1-300 microM) in a reconstitution assay using membrane and cytosolic fractions from unstimulated PMN. In addition, PA synergized with SDS for oxidase activation. Taken together, these results indicate that PA plays a second messenger role in the activation of NADPH oxidase in human PMN and that regulation of phospholipase D is a key step in the activation pathway. Images

  20. Deciphering the Origin, Evolution, and Physiological Function of the Subtelomeric Aryl-Alcohol Dehydrogenase Gene Family in the Yeast Saccharomyces cerevisiae.

    PubMed

    Yang, Dong-Dong; de Billerbeck, Gustavo M; Zhang, Jin-Jing; Rosenzweig, Frank; Francois, Jean-Marie

    2018-01-01

    Homology searches indicate that Saccharomyces cerevisiae strain BY4741 contains seven redundant genes that encode putative aryl-alcohol dehydrogenases (AAD). Yeast AAD genes are located in subtelomeric regions of different chromosomes, and their functional role(s) remain enigmatic. Here, we show that two of these genes, AAD4 and AAD14 , encode functional enzymes that reduce aliphatic and aryl-aldehydes concomitant with the oxidation of cofactor NADPH, and that Aad4p and Aad14p exhibit different substrate preference patterns. Other yeast AAD genes are undergoing pseudogenization. The 5' sequence of AAD15 has been deleted from the genome. Repair of an AAD3 missense mutation at the catalytically essential Tyr 73 residue did not result in a functional enzyme. However, ancestral-state reconstruction by fusing Aad6 with Aad16 and by N-terminal repair of Aad10 restores NADPH-dependent aryl-alcohol dehydrogenase activities. Phylogenetic analysis indicates that AAD genes are narrowly distributed in wood-saprophyte fungi and in yeast that occupy lignocellulosic niches. Because yeast AAD genes exhibit activity on veratraldehyde, cinnamaldehyde, and vanillin, they could serve to detoxify aryl-aldehydes released during lignin degradation. However, none of these compounds induce yeast AAD gene expression, and Aad activities do not relieve aryl-aldehyde growth inhibition. Our data suggest an ancestral role for AAD genes in lignin degradation that is degenerating as a result of yeast's domestication and use in brewing, baking, and other industrial applications. IMPORTANCE Functional characterization of hypothetical genes remains one of the chief tasks of the postgenomic era. Although the first Saccharomyces cerevisiae genome sequence was published over 20 years ago, 22% of its estimated 6,603 open reading frames (ORFs) remain unverified. One outstanding example of this category of genes is the enigmatic seven-member AAD family. Here, we demonstrate that proteins encoded by two

  1. Deciphering the Origin, Evolution, and Physiological Function of the Subtelomeric Aryl-Alcohol Dehydrogenase Gene Family in the Yeast Saccharomyces cerevisiae

    PubMed Central

    de Billerbeck, Gustavo M.; Zhang, Jin-jing; Rosenzweig, Frank

    2017-01-01

    ABSTRACT Homology searches indicate that Saccharomyces cerevisiae strain BY4741 contains seven redundant genes that encode putative aryl-alcohol dehydrogenases (AAD). Yeast AAD genes are located in subtelomeric regions of different chromosomes, and their functional role(s) remain enigmatic. Here, we show that two of these genes, AAD4 and AAD14, encode functional enzymes that reduce aliphatic and aryl-aldehydes concomitant with the oxidation of cofactor NADPH, and that Aad4p and Aad14p exhibit different substrate preference patterns. Other yeast AAD genes are undergoing pseudogenization. The 5′ sequence of AAD15 has been deleted from the genome. Repair of an AAD3 missense mutation at the catalytically essential Tyr73 residue did not result in a functional enzyme. However, ancestral-state reconstruction by fusing Aad6 with Aad16 and by N-terminal repair of Aad10 restores NADPH-dependent aryl-alcohol dehydrogenase activities. Phylogenetic analysis indicates that AAD genes are narrowly distributed in wood-saprophyte fungi and in yeast that occupy lignocellulosic niches. Because yeast AAD genes exhibit activity on veratraldehyde, cinnamaldehyde, and vanillin, they could serve to detoxify aryl-aldehydes released during lignin degradation. However, none of these compounds induce yeast AAD gene expression, and Aad activities do not relieve aryl-aldehyde growth inhibition. Our data suggest an ancestral role for AAD genes in lignin degradation that is degenerating as a result of yeast's domestication and use in brewing, baking, and other industrial applications. IMPORTANCE Functional characterization of hypothetical genes remains one of the chief tasks of the postgenomic era. Although the first Saccharomyces cerevisiae genome sequence was published over 20 years ago, 22% of its estimated 6,603 open reading frames (ORFs) remain unverified. One outstanding example of this category of genes is the enigmatic seven-member AAD family. Here, we demonstrate that proteins encoded

  2. 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

  3. Mapping of aldose reductase gene sequences to human chromosomes 1, 3, 7, 9, 11, and 13

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

    Bateman, J.B.; Kojis, T.; Heinzmann, C.

    1993-09-01

    Aldose reductase (alditol:NAD(P)+ 1-oxidoreductase; EC 1.1.1.21) (AR) catalyzes the reduction of several aldehydes, including that of glucose, to the corresponding sugar alcohol. Using a complementary DNA clone encoding human AR, the authors mapped the gene sequences to human chromosomes 1, 3, 7, 9, 11, 13, 14, and 18 by somatic cell hybridization. By in situ hybridization analysis, sequences were localized to human chromosomes 1q32-q43, 3p12, 7q31-q35, 9q22, 11p14-p15, and 13q14-q21. As a putative functional AR gene has been mapped to chromosome 7 and a putative pseudogene to chromosome 3, the sequences on the other seven chromosomes may represent other activemore » genes, non-aldose reductase homologous sequences, or pseudogenes. 24 refs., 3 figs., 2 tabs.« less

  4. Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays

    PubMed Central

    Aleshin, Vasily A.; Artiukhov, Artem V.; Oppermann, Henry; Kazantsev, Alexey V.; Lukashev, Nikolay V.; Bunik, Victoria I.

    2015-01-01

    Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay. PMID:26308058

  5. SIRT1 inhibits NADPH oxidase activation and protects endothelial function in the rat aorta: implications for vascular aging.

    PubMed

    Zarzuelo, María José; López-Sepúlveda, Rocío; Sánchez, Manuel; Romero, Miguel; Gómez-Guzmán, Manuel; Ungvary, Zoltan; Pérez-Vizcaíno, Francisco; Jiménez, Rosario; Duarte, Juan

    2013-05-01

    Vascular aging is characterized by up-regulation of NADPH oxidase, oxidative stress and endothelial dysfunction. Previous studies demonstrate that the activity of the evolutionarily conserved NAD(+)-dependent deacetylase SIRT1 declines with age and that pharmacological activators of SIRT1 confer significant anti-aging cardiovascular effects. To determine whether dysregulation of SIRT1 promotes NADPH oxidase-dependent production of reactive oxygen species (ROS) and impairs endothelial function we assessed the effects of three structurally different inhibitors of SIRT1 (nicotinamide, sirtinol, EX527) in aorta segments isolated from young Wistar rats. Inhibition of SIRT1 induced endothelial dysfunction, as shown by the significantly reduced relaxation to the endothelium-dependent vasodilators acetylcholine and the calcium ionophore A23187. Endothelial dysfunction induced by SIRT1 inhibition was prevented by treatment of the vessels with the NADPH oxidase inhibitor apocynin or superoxide dismutase. Inhibition of SIRT1 significantly increased vascular superoxide production, enhanced NADPH oxidase activity, and mRNA expression of its subunits p22(phox) and NOX4, which were prevented by resveratrol. Peroxisome proliferator-activated receptor-α (PPARα) activation mimicked the effects of resveratrol while PPARα inhibition prevented the effects of this SIRT1 activator. SIRT1 co-precipitated with PPARα and nicotinamide increased the acetylation of the PPARα coactivator PGC-1α, which was suppressed by resveratrol. In conclusion, impaired activity of SIRT1 induces endothelial dysfunction and up-regulates NADPH oxidase-derived ROS production in the vascular wall, mimicking the vascular aging phenotype. Moreover, a new mechanism for controlling endothelial function after SIRT1 activation involves a decreased PGC-1α acetylation and the subsequent PPARα activation, resulting in both decreased NADPH oxidase-driven ROS production and NO inactivation. Copyright © 2013

  6. DIFFERENTIATING THE TOXICITY OF CARCINOGENIC ALDEHYDES FROM NONCARCINOGENIC ALDEHYDES IN THE RAT NOSE USING CDNA ARRAYS

    EPA Science Inventory

    Differentiating the Toxicity of Carcinogenic Aldehydes from Noncarcinogenic Aldehydes in the Rat Nose Using cDNA Arrays.

    Formaldehyde is a widely used aldehyde in many industrial settings, the tanning process, household products, and is a contaminant in cigarette smoke. H...

  7. Progress toward clonable inorganic nanoparticles

    NASA Astrophysics Data System (ADS)

    Ni, Thomas W.; Staicu, Lucian C.; Nemeth, Richard S.; Schwartz, Cindi L.; Crawford, David; Seligman, Jeffrey D.; Hunter, William J.; Pilon-Smits, Elizabeth A. H.; Ackerson, Christopher J.

    2015-10-01

    Pseudomonas moraviensis stanleyae was recently isolated from the roots of the selenium (Se) hyperaccumulator plant Stanleya pinnata. This bacterium tolerates normally lethal concentrations of SeO32- in liquid culture, where it also produces Se nanoparticles. Structure and cellular ultrastructure of the Se nanoparticles as determined by cellular electron tomography shows the nanoparticles as intracellular, of narrow dispersity, symmetrically irregular and without any observable membrane or structured protein shell. Protein mass spectrometry of a fractionated soluble cytosolic material with selenite reducing capability identified nitrite reductase and glutathione reductase homologues as NADPH dependent candidate enzymes for the reduction of selenite to zerovalent Se nanoparticles. In vitro experiments with commercially sourced glutathione reductase revealed that the enzyme can reduce SeO32- (selenite) to Se nanoparticles in an NADPH-dependent process. The disappearance of the enzyme as determined by protein assay during nanoparticle formation suggests that glutathione reductase is associated with or possibly entombed in the nanoparticles whose formation it catalyzes. Chemically dissolving the nanoparticles releases the enzyme. The size of the nanoparticles varies with SeO32- concentration, varying in size form 5 nm diameter when formed at 1.0 μM [SeO32-] to 50 nm maximum diameter when formed at 100 μM [SeO32-]. In aggregate, we suggest that glutathione reductase possesses the key attributes of a clonable nanoparticle system: ion reduction, nanoparticle retention and size control of the nanoparticle at the enzyme site.Pseudomonas moraviensis stanleyae was recently isolated from the roots of the selenium (Se) hyperaccumulator plant Stanleya pinnata. This bacterium tolerates normally lethal concentrations of SeO32- in liquid culture, where it also produces Se nanoparticles. Structure and cellular ultrastructure of the Se nanoparticles as determined by cellular

  8. Production of long chain alcohols and alkanes upon coexpression of an acyl-ACP reductase and aldehyde-deformylating oxgenase with a bacterial type-I fatty acid synthase in E. coli

    DOE PAGES

    Coursolle, Dan; Shanklin, John; Lian, Jiazhang; ...

    2015-06-23

    Microbial long chain alcohols and alkanes are renewable biofuels that could one day replace petroleum-derived fuels. Here we report a novel pathway for high efficiency production of these products in Escherichia coli strain BL21(DE3). We first identified the acyl-ACP reductase/aldehyde deformylase combinations with the highest activity in this strain. Next, we used catalase coexpression to remove toxic byproducts and increase the overall titer. Finally, by introducing the type-I fatty acid synthase from Corynebacterium ammoniagenes, we were able to bypass host regulatory mechanisms of fatty acid synthesis that have thus far hampered efforts to optimize the yield of acyl-ACP-derived products inmore » BL21(DE3). When all these engineering strategies were combined with subsequent optimization of fermentation conditions, we were able to achieve a final titer around 100 mg/L long chain alcohol/alkane products including a 57 mg/L titer of pentadecane, the highest titer reported in E. coli BL21(DE3) to date. The expression of prokaryotic type-I fatty acid synthases offer a unique strategy to produce fatty acid-derived products in E. coli that does not rely exclusively on the endogenous type-II fatty acid synthase system.« less

  9. First general methods toward aldehyde enolphosphates.

    PubMed

    Barthes, Nicolas; Grison, Claude

    2012-02-01

    We herein report two innovative methods toward aldehyde enolphosphates and the first saccharidic aldehyde enolphosphates. Aldehyde enolphosphate function is worthwhile to be considered as a good phosphoenolpyruvate analogue. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. BYK191023 (2-[2-(4-methoxy-pyridin-2-yl)-ethyl]-3h-imidazo[4,5-b]pyridine) is an NADPH- and time-dependent irreversible inhibitor of inducible nitric-oxide synthase.

    PubMed

    Tiso, Mauro; Strub, Andreas; Hesslinger, Christian; Kenney, Claire T; Boer, Rainer; Stuehr, Dennis J

    2008-04-01

    Imidazopyridine derivates were recently shown to be a novel class of selective and arginine-competitive inhibitors of inducible nitric-oxide synthase (iNOS), and 2-[2-(4-methoxypyridin-2-yl)-ethyl]-3H-imidazo[4,5-b]pyridine (BYK191023) was found to have very high selectivity in enzymatic and cellular models ( Mol Pharmacol 69: 328-337, 2006 ). Here, we show that BYK191023 irreversibly inactivates murine iNOS in an NADPH- and time-dependent manner, whereas it acts only as a reversible l-arginine-competitive inhibitor in the absence of NADPH or during anaerobic preincubation. Time-dependent irreversible inhibition by BYK191023 could also be demonstrated in intact cells using the RAW macrophage or iNOS-overexpressing human embryonic kidney 293 cell lines. The mechanism of BYK191023 inhibition in the presence of NADPH was studied using spectral, kinetic, chromatographic, and radioligand binding methods. BYK191023-bound iNOS was spectrally indistinguishable from l-arginine-bound iNOS, pointing to an interaction of BYK191023 with the catalytic center of the enzyme. [(3)H]BYK191023 was recovered quantitatively from irreversibly inactivated iNOS, and no inhibitor metabolite was detected by high-performance liquid chromatography (HPLC). Size exclusion chromatography revealed only about 20% iNOS dissociation into monomers. Furthermore, HPLC and spectrophotometric analysis showed that the irreversible inhibition was associated with loss of heme from iNOS and a reduced ability to form the distinctive ferrous heme-CO complex (cytochrome P450). Thus, enzyme inactivation is mainly caused by heme loss, and it occurs in the inhibitor-bound enzyme in the presence of electron flux from NADPH.

  11. Flavoring Compounds Dominate Toxic Aldehyde Production during E-Cigarette Vaping.

    PubMed

    Khlystov, Andrey; Samburova, Vera

    2016-12-06

    The growing popularity of electronic cigarettes (e-cigarettes) raises concerns about the possibility of adverse health effects to primary users and people exposed to e-cigarette vapors. E-Cigarettes offer a very wide variety of flavors, which is one of the main factors that attract new, especially young, users. How flavoring compounds in e-cigarette liquids affect the chemical composition and toxicity of e-cigarette vapors is practically unknown. Although e-cigarettes are marketed as safer alternatives to traditional cigarettes, several studies have demonstrated formation of toxic aldehydes in e-cigarette vapors during vaping. So far, aldehyde formation has been attributed to thermal decomposition of the main components of e-cigarette e-liquids (propylene glycol and glycerol), while the role of flavoring compounds has been ignored. In this study, we have measured several toxic aldehydes produced by three popular brands of e-cigarettes with flavored and unflavored e-liquids. We show that, within the tested e-cigarette brands, thermal decomposition of flavoring compounds dominates formation of aldehydes during vaping, producing levels that exceed occupational safety standards. Production of aldehydes was found to be exponentially dependent on concentration of flavoring compounds. These findings stress the need for a further, thorough investigation of the effect of flavoring compounds on the toxicity of e-cigarettes.

  12. 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

  13. Aldehyde Detection in Electronic Cigarette Aerosols

    PubMed Central

    2017-01-01

    Acetaldehyde, acrolein, and formaldehyde are the principal toxic aldehydes present in cigarette smoke and contribute to the risk of cardiovascular disease and noncancerous pulmonary disease. The rapid growth of the use of electronic cigarettes (e-cigarettes) has raised concerns over emissions of these harmful aldehydes. This work determines emissions of these aldehydes in both free and bound (aldehyde–hemiacetal) forms and other carbonyls from the use of e-cigarettes. A novel silicon microreactor with a coating phase of 4-(2-aminooxyethyl)-morpholin-4-ium chloride (AMAH) was used to trap carbonyl compounds in the aerosols of e-cigarettes via oximation reactions. AMAH–aldehyde adducts were measured using gas chromatography–mass spectrometry. 1H nuclear magnetic resonance spectroscopy was used to analyze hemiacetals in the aerosols. These aldehydes were detected in the aerosols of all e-cigarettes. Newer-generation e-cigarette devices generated more aldehydes than the first-generation e-cigarettes because of higher battery power output. Formaldehyde–hemiacetal was detected in the aerosols generated from some e-liquids using the newer e-cigarette devices at a battery power output of 11.7 W and above. The emission of these aldehydes from all e-cigarettes, especially higher levels of aldehydes from the newer-generation e-cigarette devices, indicates the risk of using e-cigarettes. PMID:28393137

  14. Alcohol, Aldehydes, Adducts and Airways

    PubMed Central

    Sapkota, Muna; Wyatt, Todd A.

    2015-01-01

    Drinking alcohol and smoking cigarettes results in the formation of reactive aldehydes in the lung, which are capable of forming adducts with several proteins and DNA. Acetaldehyde and malondialdehyde are the major aldehydes generated in high levels in the lung of subjects with alcohol use disorder who smoke cigarettes. In addition to the above aldehydes, several other aldehydes like 4-hydroxynonenal, formaldehyde and acrolein are also detected in the lung due to exposure to toxic gases, vapors and chemicals. These aldehydes react with nucleophilic targets in cells such as DNA, lipids and proteins to form both stable and unstable adducts. This adduction may disturb cellular functions as well as damage proteins, nucleic acids and lipids. Among several adducts formed in the lung, malondialdehyde DNA (MDA-DNA) adduct and hybrid malondialdehyde-acetaldehyde (MAA) protein adducts have been shown to initiate several pathological conditions in the lung. MDA-DNA adducts are pre-mutagenic in mammalian cells and induce frame shift and base-pair substitution mutations, whereas MAA protein adducts have been shown to induce inflammation and inhibit wound healing. This review provides an insight into different reactive aldehyde adducts and their role in the pathogenesis of lung disease. PMID:26556381

  15. Alcohol, Aldehydes, Adducts and Airways.

    PubMed

    Sapkota, Muna; Wyatt, Todd A

    2015-11-05

    Drinking alcohol and smoking cigarettes results in the formation of reactive aldehydes in the lung, which are capable of forming adducts with several proteins and DNA. Acetaldehyde and malondialdehyde are the major aldehydes generated in high levels in the lung of subjects with alcohol use disorder who smoke cigarettes. In addition to the above aldehydes, several other aldehydes like 4-hydroxynonenal, formaldehyde and acrolein are also detected in the lung due to exposure to toxic gases, vapors and chemicals. These aldehydes react with nucleophilic targets in cells such as DNA, lipids and proteins to form both stable and unstable adducts. This adduction may disturb cellular functions as well as damage proteins, nucleic acids and lipids. Among several adducts formed in the lung, malondialdehyde DNA (MDA-DNA) adduct and hybrid malondialdehyde-acetaldehyde (MAA) protein adducts have been shown to initiate several pathological conditions in the lung. MDA-DNA adducts are pre-mutagenic in mammalian cells and induce frame shift and base-pair substitution mutations, whereas MAA protein adducts have been shown to induce inflammation and inhibit wound healing. This review provides an insight into different reactive aldehyde adducts and their role in the pathogenesis of lung disease.

  16. 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.

  17. Toxicity of polyunsaturated aldehydes of diatoms to Indo-Pacific bioindicator organism Echinometra mathaei.

    PubMed

    Sartori, Davide; Gaion, Andrea

    2016-01-01

    Although it is well known suitability of early developmental stages of sea urchin as recommended model for pollutant toxicity testing, little is known about the sensitivity of Indo-Pacific species Echinometra mathaei to polyunsaturated aldehydes. In this study, the effect of three short chain aldehydes, 2,4-decadienal (DD), 2,4-octadienal (OD) and 2,4-heptadienal (HD), normally found in many diatoms, such as Skeletonema costatum, Skeletonema marinoi and Thalassiosira rotula, was evaluated on larval development of E. mathaei embryos. Aldehydes affected larval development in a dose-dependent manner, in particular HD>OD>DD; the results of this study highlighted the higher sensitivity of this species toward aldehydes compared with data registered for other sea urchin species. In comparison with studies reported in the literature, contrasting results were observed during our tests; therefore, an increasing toxic effect was registered with decreasing the chain length of aldehydes. This work could provide new insights in the development of new toxicological assays toward most sensitive species.

  18. Interrupted reperfusion reduces the activation of NADPH oxidase after cerebral I/R injury.

    PubMed

    Shen, Jia; Bai, Xiao-Yin; Qin, Yuan; Jin, Wei-Wei; Zhou, Jing-Yin; Zhou, Ji-Ping; Yan, Ying-Gang; Wang, Qiong; Bruce, Iain C; Chen, Jiang-Hua; Xia, Qiang

    2011-06-15

    Interrupted reperfusion reduces ischemia/reperfusion (I/R) injury. This study was designed to determine whether NADPH oxidase participates in the neural protection against global I/R injury after interrupted reperfusion. Mice were randomly divided into five groups: sham (sham-operated), I/R (20-min global I/R), RR (I/R+interrupted reperfusion), Apo (I/R+apocynin administration), and RR+Apo. Behavioral tests (pole test, beam walking, and Morris water maze) and Nissl staining were undertaken in all five groups; superoxide levels, expression of gp91(phox) and p47(phox), p47(phox) translocation, and Rac1 activation were measured in the sham, I/R, and RR groups. The motor coordination, bradykinesia, and spatial learning and memory, as well as the neuron survival rates, were better in the RR, Apo, and RR+Apo groups than in the I/R group. The NADPH oxidase-dependent superoxide levels, p47(phox) and gp91(phox) expression, p47(phox) translocation, and Rac1 activation were lower in the RR group than in the I/R group. In conclusion, the neural protective effect of interrupted reperfusion is at least partly mediated by decreasing the expression and assembly of NADPH oxidase and the levels of NADPH oxidase-derived superoxide. The most striking reduction Rac1-GTP in the RR group suggests that interrupted reperfusion also acts on the activation of assembled NADPH oxidase by reducing the availability of Rac1-GTP. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. Engineering a cyanobacterium as the catalyst for the photosynthetic conversion of CO2 to 1,2-propanediol.

    PubMed

    Li, Han; Liao, James C

    2013-01-22

    The modern society primarily relies on petroleum and natural gas for the production of fuels and chemicals. One of the major commodity chemicals 1,2-propanediol (1,2-PDO), which has an annual production of more than 0.5 million tons in the United States, is currently produced by chemical processes from petroleum derived propylene oxide, which is energy intensive and not sustainable. In this study, we sought to achieve photosynthetic production of 1,2-PDO from CO2 using a genetically engineered cyanobacterium Synechococcus elongatus PCC 7942. Compared to the previously reported biological 1,2-PDO production processes which used sugar or glycerol as the substrates, direct chemical production from CO2 in photosynthetic organisms recycles the atmospheric CO2 and will not compete with food crops for arable land. In this study, we reported photosynthetic production of 1,2-PDO from CO2 using a genetically engineered cyanobacterium Synechococcus elongatus PCC 7942. Introduction of the genes encoding methylglyoxal synthase (mgsA), glycerol dehydrogenase (gldA), and aldehyde reductase (yqhD) resulted in the production of ~22 mg/L 1,2-PDO from CO2. However, a comparable amount of the pathway intermediate acetol was also produced, especially during the stationary phase. The production of 1,2-PDO requires a robust input of reducing equivalents from cellular metabolism. To take advantage of cyanobacteria's NADPH pool, the synthetic pathway of 1,2-PDO was engineered to be NADPH-dependent by exploiting the NADPH-specific secondary alcohol dehydrogenases which have not been reported for 1,2-PDO production previously. This optimization strategy resulted in the production of ~150 mg/L 1,2-PDO and minimized the accumulation of the incomplete reduction product, acetol. This work demonstrated that cyanobacteria can be engineered as a catalyst for the photosynthetic conversion of CO2 to 1,2-PDO. This work also characterized two NADPH-dependent sADHs for their catalytic capacity in 1

  20. 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.

  1. Catalytic mechanism and substrate specificity of the β-subunit of the voltage-gated potassium (Kv) channel

    PubMed Central

    Tipparaju, Srinivas M.; Barski, Oleg A.; Srivastava, Sanjay; Bhatnagar, Aruni

    2008-01-01

    The β-subunits of voltage-gated potassium (Kv) channels are members of aldo-keto reductase (AKR) superfamily. These proteins regulate inactivation and membrane localization of Kv1 and Kv4 channels. The Kvβ proteins bind to pyridine nucleotides with high affinity; however, their catalytic properties remain unclear. Here we report that recombinant rat Kvβ2 catalyzes the reduction of a wide range of aldehydes and ketones. The rate of catalysis was slower (0.06 to 0.2 min−1) than that of other AKRs, but displayed the expected hyperbolic dependence on substrate concentration, with no evidence of allosteric cooperativity. Catalysis was prevented by site-directed substitution of Tyr-90 with phenylalanine, indicating that the acid-base catalytic residue, identified in other AKRs, has a conserved function in Kvβ2. The protein catalyzed the reduction of a broad range of carbonyls including aromatic carbonyls, electrophilic aldehydes and prostaglandins, phospholipid and sugar aldehydes. Little or no activity was detected with carbonyl steroids. Initial velocity profiles were consistent with an ordered bi-bi rapid-equilibrium mechanism in which NADPH binding precedes carbonyl binding. Significant primary kinetic isotope effects (2.0 – 3.1) were observed under single and multiple turnover conditions, indicating that the bond-breaking chemical step is rate-limiting. Structure-activity relationships with a series of para-substituted benzaldehydes indicated that the electronic interactions predominate during substrate binding and that no significant charge develops during the transition state. These data strengthen the view that Kvβ proteins are catalytically-active AKRs that impart redox-sensitivity to Kv channels. PMID:18672894

  2. Oscillatory shear stress stimulates endothelial production of O2- from p47phox-dependent NAD(P)H oxidases, leading to monocyte adhesion

    NASA Technical Reports Server (NTRS)

    Hwang, Jinah; Saha, Aniket; Boo, Yong Chool; Sorescu, George P.; McNally, J. Scott; Holland, Steven M.; Dikalov, Sergei; Giddens, Don P.; Griendling, Kathy K.; Harrison, David G.; hide

    2003-01-01

    Arterial regions exposed to oscillatory shear (OS) in branched arteries are lesion-prone sites of atherosclerosis, whereas those of laminar shear (LS) are relatively well protected. Here, we examined the hypothesis that OS and LS differentially regulate production of O2- from the endothelial NAD(P)H oxidase, which, in turn, is responsible for their opposite effects on a critical atherogenic event, monocyte adhesion. We used aortic endothelial cells obtained from C57BL/6 (MAE-C57) and p47phox-/- (MAE-p47-/-) mice, which lack a component of NAD(P)H oxidase. O2- production was determined by dihydroethidium staining and an electron spin resonance using an electron spin trap methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine. Chronic exposure (18 h) to an arterial level of OS (+/- 5 dynes/cm2) increased O2- (2-fold) and monocyte adhesion (3-fold) in MAE-C57 cells, whereas chronic LS (15 dynes/cm2, 18 h) significantly decreased both monocyte adhesion and O2- compared with static conditions. In contrast, neither LS nor OS were able to induce O2- production and monocyte adhesion to MAE-p47-/-. Treating MAE-C57 with a cell-permeable superoxide dismutase compound, polyethylene glycol-superoxide dismutase, also inhibited OS-induced monocyte adhesion. In addition, over-expressing p47phox in MAE-p47-/- restored OS-induced O2- production and monocyte adhesion. These results suggest that chronic exposure of endothelial cells to OS stimulates O2- and/or its derivatives produced from p47phox-dependent NAD(P)H oxidase, which, in turn, leads to monocyte adhesion, an early and critical atherogenic event.

  3. Myeloperoxidase amplified high glucose-induced endothelial dysfunction in vasculature: Role of NADPH oxidase and hypochlorous acid.

    PubMed

    Tian, Rong; Ding, Yun; Peng, Yi-Yuan; Lu, Naihao

    2017-03-11

    Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H 2 O 2 ), have emerged as important molecules in the pathogenesis of diabetic endothelial dysfunction. Additionally, neutrophils-derived myeloperoxidase (MPO) and MPO-catalyzed hypochlorous acid (HOCl) play important roles in the vascular injury. However, it is unknown whether MPO can use vascular-derived ROS to induce diabetic endothelial dysfunction. In the present study, we demonstrated that NADPH oxidase was the main source of ROS formation in high glucose-cultured human umbilical vein endothelial cells (HUVECs), and played a critical role in high glucose-induced endothelial dysfunction such as cell apoptosis, loss of cell viability and reduction of nitric oxide (NO). However, the addition of MPO could amplify the high glucose-induced endothelial dysfunction which was inhibited by the presence of apocynin (NADPH oxidase inhibitor), catalase (H 2 O 2 scavenger), or methionine (HOCl scavenger), demonstrating the contribution of NADPH oxidase-H 2 O 2 -MPO-HOCl pathway in the MPO/high glucose-induced vascular injury. In high glucose-incubated rat aortas, MPO also exacerbated the NADPH oxidase-induced impairment of endothelium-dependent relaxation. Consistent with these in vitro data, in diabetic rat aortas, both MPO expresion and NADPH oxidase activity were increased while the endothelial function was simultaneously impaired. The results suggested that vascular-bound MPO could amplify high glucose-induced vascular injury in diabetes. MPO-NADPH oxidase-HOCl may represent an important pathogenic pathway in diabetic vascular diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Microbial synthesis of propane by engineering valine pathway and aldehyde-deformylating oxygenase.

    PubMed

    Zhang, Lei; Liang, Yajing; Wu, Wei; Tan, Xiaoming; Lu, Xuefeng

    2016-01-01

    Propane, a major component of liquid petroleum gas (LPG) derived from fossil fuels, has widespread applications in vehicles, cooking, and ambient heating. Given the concerns about fossil fuel depletion and carbon emission, exploiting alternative and renewable source of propane have become attractive. In this study, we report the construction of a novel propane biosynthetic pathway in Escherichia coli. We constructed an aldehyde reductases (ALR)-deprived E. coli strain BW25113(DE3) Δ13 via genetic engineering, which produced sufficient isobutyraldehyde precursors and finally achieved de novo synthesis of propane (91 μg/L) by assembling the engineered valine pathway and cyanobacterial aldehyde-deformylating oxygenase (ADO). Additionally, after extensive screening of ADO mutants generated by engineering the active center to accommodate branched-chain isobutyraldehyde, we identified two ADO mutants (I127G, I127G/A48G) which exhibited higher catalytic activity for isobutyraldehyde and improved propane productivity by three times (267 μg/L). The propane biosynthetic pathway constructed here through the engineered valine pathway can produce abundant isobutyraldehyde for ADO and overcome the low availability of precursors in propane production. Furthermore, the rational design aiming at the ADO active center illustrates the plasticity and catalytic potential of ADO. These results together highlight the potential for developing a microbial biomanufacturing platform for propane.

  5. Structural comparison of chromosomal and exogenous dihydrofolate reductase from Staphylococcus aureus in complex with the potent inhibitor trimethoprim

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

    Heaslet, Holly; Harris, Melissa; Fahnoe, Kelly

    2010-09-02

    Dihydrofolate reductase (DHFR) is the enzyme responsible for the NADPH-dependent reduction of 5,6-dihydrofolate to 5,6,7,8-tetrahydrofolate, an essential cofactor in the synthesis of purines, thymidylate, methionine, and other key metabolites. Because of its importance in multiple cellular functions, DHFR has been the subject of much research targeting the enzyme with anticancer, antibacterial, and antimicrobial agents. Clinically used compounds targeting DHFR include methotrexate for the treatment of cancer and diaminopyrimidines (DAPs) such as trimethoprim (TMP) for the treatment of bacterial infections. DAP inhibitors of DHFR have been used clinically for >30 years and resistance to these agents has become widespread. Methicillin-resistant Staphylococcusmore » aureus (MRSA), the causative agent of many serious nosocomial and community acquired infections, and other gram-positive organisms can show resistance to DAPs through mutation of the chromosomal gene or acquisition of an alternative DHFR termed 'S1 DHFR.' To develop new therapies for health threats such as MRSA, it is important to understand the molecular basis of DAP resistance. Here, we report the crystal structure of the wild-type chromosomal DHFR from S. aureus in complex with NADPH and TMP. We have also solved the structure of the exogenous, TMP resistant S1 DHFR, apo and in complex with TMP. The structural and thermodynamic data point to important molecular differences between the two enzymes that lead to dramatically reduced affinity of DAPs to S1 DHFR. These differences in enzyme binding affinity translate into reduced antibacterial activity against strains of S. aureus that express S1 DHFR.« less

  6. Ethanol increases matrix metalloproteinase-12 expression via NADPH oxidase-dependent ROS production in macrophages

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

    Kim, Mi Jin; Nepal, Saroj; Lee, Eung-Seok

    2013-11-15

    Matrix metalloproteinase-12 (MMP-12), an enzyme responsible for degradation of extracellular matrix, plays an important role in the progression of various diseases, including inflammation and fibrosis. Although most of those are pathogenic conditions induced by ethanol ingestion, the effect of ethanol on MMP-12 has not been explored. In the present study, we investigated the effect of ethanol on MMP-12 expression and its potential mechanisms in macrophages. Here, we demonstrated that ethanol treatment increased MMP-12 expression in primary murine peritoneal macrophages and RAW 264.7 macrophages at both mRNA and protein levels. Ethanol treatment also significantly increased the activity of nicotinamide adenine dinucleotidemore » (NADPH) oxidase and the expression of NADPH oxidase-2 (Nox2). Pretreatment with an anti-oxidant (N-acetyl cysteine) or a selective inhibitor of NADPH oxidase (diphenyleneiodonium chloride (DPI)) prevented ethanol-induced MMP-12 expression. Furthermore, knockdown of Nox2 by small interfering RNA (siRNA) prevented ethanol-induced ROS production and MMP-12 expression in RAW 264.7 macrophages, indicating a critical role for Nox2 in ethanol-induced intracellular ROS production and MMP-12 expression in macrophages. We also showed that ethanol-induced Nox2 expression was suppressed by transient transfection with dominant negative IκB-α plasmid or pretreatment with Bay 11-7082, a selective inhibitor of NF-κB, in RAW 264.7 macrophages. In addition, ethanol-induced Nox2 expression was also attenuated by treatment with a selective inhibitor of p38 MAPK, suggesting involvement of p38 MAPK/NF-κB pathway in ethanol-induced Nox2 expression. Taken together, these results demonstrate that ethanol treatment elicited increase in MMP-12 expression via increase in ROS production derived from Nox2 in macrophages. - Highlights: • Ethanol increases ROS production through up-regulation of Nox2 in macrophages. • Enhanced oxidative stress contributes to

  7. Histamine H4-Receptors Inhibit Mast Cell Renin Release in Ischemia/Reperfusion via Protein Kinase Cε-Dependent Aldehyde Dehydrogenase Type-2 Activation

    PubMed Central

    Aldi, Silvia; Takano, Ken-ichi; Tomita, Kengo; Koda, Kenichiro; Chan, Noel Y.-K.; Marino, Alice; Salazar-Rodriguez, Mariselis; Thurmond, Robin L.

    2014-01-01

    Renin released by ischemia/reperfusion (I/R) from cardiac mast cells (MCs) activates a local renin-angiotensin system (RAS) causing arrhythmic dysfunction. Ischemic preconditioning (IPC) inhibits MC renin release and consequent activation of this local RAS. We postulated that MC histamine H4-receptors (H4Rs), being Gαi/o-coupled, might activate a protein kinase C isotype–ε (PKCε)–aldehyde dehydrogenase type-2 (ALDH2) cascade, ultimately eliminating MC-degranulating and renin-releasing effects of aldehydes formed in I/R and associated arrhythmias. We tested this hypothesis in ex vivo hearts, human mastocytoma cells, and bone marrow–derived MCs from wild-type and H4R knockout mice. We found that activation of MC H4Rs mimics the cardioprotective anti-RAS effects of IPC and that protection depends on the sequential activation of PKCε and ALDH2 in MCs, reducing aldehyde-induced MC degranulation and renin release and alleviating reperfusion arrhythmias. These cardioprotective effects are mimicked by selective H4R agonists and disappear when H4Rs are pharmacologically blocked or genetically deleted. Our results uncover a novel cardioprotective pathway in I/R, whereby activation of H4Rs on the MC membrane, possibly by MC-derived histamine, leads sequentially to PKCε and ALDH2 activation, reduction of toxic aldehyde-induced MC renin release, prevention of RAS activation, reduction of norepinephrine release, and ultimately to alleviation of reperfusion arrhythmias. This newly discovered protective pathway suggests that MC H4Rs may represent a new pharmacologic and therapeutic target for the direct alleviation of RAS-induced cardiac dysfunctions, including ischemic heart disease and congestive heart failure. PMID:24696042

  8. 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

  9. 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

  10. 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

  11. 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

  12. Hypertonic Saline Suppresses NADPH Oxidase-Dependent Neutrophil Extracellular Trap Formation and Promotes Apoptosis.

    PubMed

    Nadesalingam, Ajantha; Chen, Jacky H K; Farahvash, Armin; Khan, Meraj A

    2018-01-01

    Tonicity of saline (NaCl) is important in regulating cellular functions and homeostasis. Hypertonic saline is administered to treat many inflammatory diseases, including cystic fibrosis. Excess neutrophil extracellular trap (NET) formation, or NETosis, is associated with many pathological conditions including chronic inflammation. Despite the known therapeutic benefits of hypertonic saline, its underlying mechanisms are not clearly understood. Therefore, we aimed to elucidate the effects of hypertonic saline in modulating NETosis. For this purpose, we purified human neutrophils and induced NETosis using agonists such as diacylglycerol mimetic phorbol myristate acetate (PMA), Gram-negative bacterial cell wall component lipopolysaccharide (LPS), calcium ionophores (A23187 and ionomycin from Streptomyces conglobatus ), and bacteria ( Pseudomonas aeruginosa and Staphylococcus aureus ). We then analyzed neutrophils and NETs using Sytox green assay, immunostaining of NET components and apoptosis markers, confocal microscopy, and pH sensing reagents. This study found that hypertonic NaCl suppresses nicotinamide adenine dinucleotide phosphate oxidase (NADPH2 or NOX2)-dependent NETosis induced by agonists PMA, Escherichia coli LPS (0111:B4 and O128:B12), and P. aeruginosa . Hypertonic saline also suppresses LPS- and PMA- induced reactive oxygen species production. It was determined that supplementing H 2 O 2 reverses the suppressive effect of hypertonic saline on NOX2-dependent NETosis. Many of the aforementioned suppressive effects were observed in the presence of equimolar concentrations of choline chloride and osmolytes (d-mannitol and d-sorbitol). This suggests that the mechanism by which hypertonic saline suppresses NOX2-dependent NETosis is via neutrophil dehydration. Hypertonic NaCl does not significantly alter the intracellular pH of neutrophils. We found that hypertonic NaCl induces apoptosis while suppressing NOX2-dependent NETosis. In contrast, hypertonic

  13. 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

  14. 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.

  15. Mechanistic Insights from Reaction of α-Oxiranyl-Aldehydes with Cyanobacterial Aldehyde Deformylating Oxygenase

    PubMed Central

    Das, Debasis; Ellington, Benjamin; Paul, Bishwajit; Marsh, E. Neil G.

    2014-01-01

    The biosynthesis of long-chain aliphatic hydrocarbons, which are derived from fatty acids, is widespread in Nature. The last step in this pathway involves the decarbonylation of fatty aldehydes to the corresponding alkanes or alkenes. In cyanobacteria this is catalyzed by an aldehyde deformylating oxygenase. We have investigated the mechanism of this enzyme using substrates bearing an oxirane ring adjacent to the aldehyde carbon. The enzyme catalyzed the deformylation of these substrates to produce the corresponding oxiranes. Performing the reaction in D2O allowed the facial selectivity of proton addition to be examined by 1H-NMR spectroscopy. The proton is delivered with equal probability to either face of the oxirane ring, indicating the formation of an oxiranyl radical intermediate that is free to rotate during the reaction. Unexpectedly, the enzyme also catalyzes a side reaction in which oxiranyl-aldehydes undergo tandem deformylation to furnish alkanes two carbons shorter. We present evidence that this involves the rearrangement of the intermediate oxiranyl radical formed in the first step, resulting an aldehyde that is further deformylated in a second step. These observations provide support for a radical mechanism for deformylation and, furthermore, allow the lifetime of the radical intermediate to be estimated based on prior measurements of rate constants for the rearrangement of oxiranyl radicals. PMID:24313866

  16. Deciphering the key residues in Plasmodium falciparum beta-ketoacyl acyl carrier protein reductase responsible for interactions with Plasmodium falciparum acyl carrier protein.

    PubMed

    Karmodiya, Krishanpal; Modak, Rahul; Sahoo, Nirakar; Sajad, Syed; Surolia, Namita

    2008-10-01

    The type II fatty acid synthase (FAS) pathway of Plasmodium falciparum is a validated unique target for developing novel antimalarials, due to its intrinsic differences from the typeI pathway operating in humans. beta-Ketoacyl acyl carrier protein (ACP) reductase (FabG) performs the NADPH-dependent reduction of beta-ketoacyl-ACP to beta-hydroxyacyl-ACP, the first reductive step in the elongation cycle of fatty acid biosynthesis. In this article, we report intensive studies on the direct interactions of Plasmodium FabG and Plasmodium ACP in solution, in the presence and absence of its cofactor, NADPH, by monitoring the change in intrinsic fluorescence of P.falciparum FabG (PfFabG) and by surface plasmon resonance. To address the issue of the importance of the residues involved in strong, specific and stoichiometric binding of PfFabG to P.falciparum ACP (PfACP), we mutated Arg187, Arg190 and Arg230 of PfFabG. The activities of the mutants were assessed using both an ACP-dependent and an ACP-independent assay. The affinities of all the PfFabG mutants for acetoacetyl-ACP (the physiological substrate) were reduced to different extents as compared to wild-type PfFabG, but were equally active in biochemical assays with the substrate analog acetoacetyl-CoA. Kinetic analysis and studies of direct binding between PfFabG and PfACP confirmed the identification of Arg187 and Arg230 as critical residues for the PfFabG-PfACP interactions. Our studies thus reveal the significance of the positively charged/hydrophobic patch located adjacent to the active site cavities of PfFabG for interactions with PfACP.

  17. Five Fatty Aldehyde Dehydrogenase Enzymes from Marinobacter and Acinetobacter spp. and Structural Insights into the Aldehyde Binding Pocket

    PubMed Central

    Bertram, Jonathan H.; Mulliner, Kalene M.; Shi, Ke; Plunkett, Mary H.; Nixon, Peter; Serratore, Nicholas A.; Douglas, Christopher J.; Aihara, Hideki

    2017-01-01

    ABSTRACT Enzymes involved in lipid biosynthesis and metabolism play an important role in energy conversion and storage and in the function of structural components such as cell membranes. The fatty aldehyde dehydrogenase (FAldDH) plays a central function in the metabolism of lipid intermediates, oxidizing fatty aldehydes to the corresponding fatty acid and competing with pathways that would further reduce the fatty aldehydes to fatty alcohols or require the fatty aldehydes to produce alkanes. In this report, the genes for four putative FAldDH enzymes from Marinobacter aquaeolei VT8 and an additional enzyme from Acinetobacter baylyi were heterologously expressed in Escherichia coli and shown to display FAldDH activity. Five enzymes (Maqu_0438, Maqu_3316, Maqu_3410, Maqu_3572, and the enzyme reported under RefSeq accession no. WP_004927398) were found to act on aldehydes ranging from acetaldehyde to hexadecanal and also acted on the unsaturated long-chain palmitoleyl and oleyl aldehydes. A comparison of the specificities of these enzymes with various aldehydes is presented. Crystallization trials yielded diffraction-quality crystals of one particular FAldDH (Maqu_3316) from M. aquaeolei VT8. Crystals were independently treated with both the NAD+ cofactor and the aldehyde substrate decanal, revealing specific details of the likely substrate binding pocket for this class of enzymes. A likely model for how catalysis by the enzyme is accomplished is also provided. IMPORTANCE This study provides a comparison of multiple enzymes with the ability to oxidize fatty aldehydes to fatty acids and provides a likely picture of how the fatty aldehyde and NAD+ are bound to the enzyme to facilitate catalysis. Based on the information obtained from this structural analysis and comparisons of specificities for the five enzymes that were characterized, correlations to the potential roles played by specific residues within the structure may be drawn. PMID:28389542

  18. Five Fatty Aldehyde Dehydrogenase Enzymes from Marinobacter and Acinetobacter spp. and Structural Insights into the Aldehyde Binding Pocket

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

    Bertram, Jonathan H.; Mulliner, Kalene M.; Shi, Ke

    ABSTRACT Enzymes involved in lipid biosynthesis and metabolism play an important role in energy conversion and storage and in the function of structural components such as cell membranes. The fatty aldehyde dehydrogenase (FAldDH) plays a central function in the metabolism of lipid intermediates, oxidizing fatty aldehydes to the corresponding fatty acid and competing with pathways that would further reduce the fatty aldehydes to fatty alcohols or require the fatty aldehydes to produce alkanes. In this report, the genes for four putative FAldDH enzymes fromMarinobacter aquaeoleiVT8 and an additional enzyme fromAcinetobacter baylyiwere heterologously expressed inEscherichia coliand shown to display FAldDH activity.more » Five enzymes (Maqu_0438, Maqu_3316, Maqu_3410, Maqu_3572, and the enzyme reported under RefSeq accession no.WP_004927398) were found to act on aldehydes ranging from acetaldehyde to hexadecanal and also acted on the unsaturated long-chain palmitoleyl and oleyl aldehydes. A comparison of the specificities of these enzymes with various aldehydes is presented. Crystallization trials yielded diffraction-quality crystals of one particular FAldDH (Maqu_3316) fromM. aquaeoleiVT8. Crystals were independently treated with both the NAD +cofactor and the aldehyde substrate decanal, revealing specific details of the likely substrate binding pocket for this class of enzymes. A likely model for how catalysis by the enzyme is accomplished is also provided. IMPORTANCEThis study provides a comparison of multiple enzymes with the ability to oxidize fatty aldehydes to fatty acids and provides a likely picture of how the fatty aldehyde and NAD +are bound to the enzyme to facilitate catalysis. Based on the information obtained from this structural analysis and comparisons of specificities for the five enzymes that were characterized, correlations to the potential roles played by specific residues within the structure may be drawn.« less

  19. Four distinct types of E.C. 1.2.1.30 enzymes can catalyze the reduction of carboxylic acids to aldehydes.

    PubMed

    Stolterfoht, Holly; Schwendenwein, Daniel; Sensen, Christoph W; Rudroff, Florian; Winkler, Margit

    2017-09-10

    Increasing demand for chemicals from renewable resources calls for the development of new biotechnological methods for the reduction of oxidized bio-based compounds. Enzymatic carboxylate reduction is highly selective, both in terms of chemo- and product selectivity, but not many carboxylate reductase enzymes (CARs) have been identified on the sequence level to date. Thus far, their phylogeny is unexplored and very little is known about their structure-function-relationship. CARs minimally contain an adenylation domain, a phosphopantetheinylation domain and a reductase domain. We have recently identified new enzymes of fungal origin, using similarity searches against genomic sequences from organisms in which aldehydes were detected upon incubation with carboxylic acids. Analysis of sequences with known CAR functionality and CAR enzymes recently identified in our laboratory suggests that the three-domain architecture mentioned above is modular. The construction of a distance tree with a subsequent 1000-replicate bootstrap analysis showed that the CAR sequences included in our study fall into four distinct subgroups (one of bacterial origin and three of fungal origin, respectively), each with a bootstrap value of 100%. The multiple sequence alignment of all experimentally confirmed CAR protein sequences revealed fingerprint sequences of residues which are likely to be involved in substrate and co-substrate binding and one of the three catalytic substeps, respectively. The fingerprint sequences broaden our understanding of the amino acids that might be essential for the reduction of organic acids to the corresponding aldehydes in CAR proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Callitriche cophocarpa (water starwort) proteome under chromate stress: evidence for induction of a quinone reductase.

    PubMed

    Kaszycki, Paweł; Dubicka-Lisowska, Aleksandra; Augustynowicz, Joanna; Piwowarczyk, Barbara; Wesołowski, Wojciech

    2018-03-01

    Chromate-induced physiological stress in a water-submerged macrophyte Callitriche cophocarpa Sendtn. (water starwort) was tested at the proteomic level. The oxidative stress status of the plant treated with 1 mM Cr(VI) for 3 days revealed stimulation of peroxidases whereas catalase and superoxide dismutase activities were similar to the control levels. Employing two-dimensional electrophoresis, comparative proteomics enabled to detect five differentiating proteins subjected to identification with mass spectrometry followed by an NCBI database search. Cr(VI) incubation led to induction of light harvesting chlorophyll a/b binding protein with a concomitant decrease of accumulation of ribulose bisphosphate carboxylase (RuBisCO). The main finding was, however, the identification of an NAD(P)H-dependent dehydrogenase FQR1, detectable only in Cr(VI)-treated plants. The FQR1 flavoenzyme is known to be responsive to oxidative stress and to act as a detoxification protein by protecting the cells against oxidative damage. It exhibits the in vitro quinone reductase activity and is capable of catalyzing two-electron transfer from NAD(P)H to several substrates, presumably including Cr(VI). The enhanced accumulation of FQR1 was chromate-specific since other stressful conditions, such as salt, temperature, and oxidative stresses, all failed to induce the protein. Zymographic analysis of chromate-treated Callitriche shoots showed a novel enzymatic protein band whose activity was attributed to the newly identified enzyme. We suggest that Cr(VI) phytoremediation with C. cophocarpa can be promoted by chromate reductase activity produced by the induced quinone oxidoreductase which might take part in Cr(VI) → Cr(III) bioreduction process and thus enable the plant to cope with the chromate-generated oxidative stress.

  1. Direct catalytic asymmetric alpha-amination of aldehydes.

    PubMed

    List, Benjamin

    2002-05-22

    The first direct catalytic asymmetric alpha-amination of aldehydes is described herein. alpha-Unbranched aldehydes react in this novel proline-catalyzed reaction with dialkyl azodicarboxylates to give alpha-amino aldehydes in excellent yields and enantioselectivities.

  2. Thermostable NADP+-Dependent Medium-Chain Alcohol Dehydrogenase from Acinetobacter sp. Strain M-1: Purification and Characterization and Gene Expression in Escherichia coli

    PubMed Central

    Tani, Akio; Sakai, Yasuyoshi; Ishige, Takeru; Kato, Nobuo

    2000-01-01

    NADPH-dependent alkylaldehyde reducing enzyme, which was greatly induced by n-hexadecane, from Acinetobacter sp. strain M-1 was purified and characterized. The purified enzyme had molecular masses of 40 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 160 kDa as determined by gel filtration chromatography. The enzyme, which was shown to be highly thermostable, was most active toward n-heptanal and could use n-alkylaldehydes ranging from C2 to C14 and several substituted benzaldehydes, including the industrially important compounds cinnamyl aldehyde and anisaldehyde, as substrates. The alrA gene coding for this enzyme was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the alrA gene exhibited homology to the amino acid sequences of zinc-containing alcohol dehydrogenases from various sources. The gene could be highly expressed in Escherichia coli, and the product was purified to homogeneity by simpler procedures from the recombinant than from the original host. Our results show that this enzyme can be used for industrial bioconversion of useful alcohols and aldehydes. PMID:11097895

  3. 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

  4. 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

  5. Peptide/laccase cocatalyzed asymmetric α-oxyamination of aldehydes.

    PubMed

    Akagawa, Kengo; Kudo, Kazuaki

    2011-07-01

    An asymmetric α-oxyamination could be successfully performed by a peptide catalyst and laccase. The combination of peptide catalysis and enzymatic air oxidation promoted the reaction smoothly in water without employing a metal reagent. The oxyaminated compounds could be obtained as both aldehyde and carboxylic acid products depending on the reaction conditions.

  6. 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.

  7. Calcium mobilization and Rac1 activation are required for VCAM-1 (vascular cell adhesion molecule-1) stimulation of NADPH oxidase activity.

    PubMed Central

    Cook-Mills, Joan M; Johnson, Jacob D; Deem, Tracy L; Ochi, Atsuo; Wang, Lei; Zheng, Yi

    2004-01-01

    VCAM-1 (vascular cell adhesion molecule-1) plays an important role in the regulation of inflammation in atherosclerosis, asthma, inflammatory bowel disease and transplantation. VCAM-1 activates endothelial cell NADPH oxidase, and this oxidase activity is required for VCAM-1-dependent lymphocyte migration. We reported previously that a mouse microvascular endothelial cell line promotes lymphocyte migration that is dependent on VCAM-1, but not on other known adhesion molecules. Here we have investigated the signalling mechanisms underlying VCAM-1 function. Lymphocyte binding to VCAM-1 on the endothelial cell surface activated an endothelial cell calcium flux that could be inhibited with anti-alpha4-integrin and mimicked by anti-VCAM-1-coated beads. VCAM-1 stimulation of calcium responses could be blocked by an inhibitor of intracellular calcium mobilization, a calcium channel inhibitor or a calcium chelator, resulting in the inhibition of NADPH oxidase activity. Addition of ionomycin overcame the calcium channel blocker suppression of VCAM-1-stimulated NADPH oxidase activity, but could not reverse the inhibitory effect imposed by intracellular calcium blockage, indicating that both intracellular and extracellular calcium mobilization are required for VCAM-1-mediated activation of NADPH oxidase. Furthermore, VCAM-1 specifically activated the Rho-family GTPase Rac1, and VCAM-1 activation of NADPH oxidase was blocked by a dominant negative Rac1. Thus VCAM-1 stimulates the mobilization of intracellular and extracellular calcium and Rac1 activity that are required for the activation of NADPH oxidase. PMID:14594451

  8. RDH13L, an enzyme responsible for the aldehyde-alcohol redox coupling reaction (AL-OL coupling reaction) to supply 11-cis retinal in the carp cone retinoid cycle.

    PubMed

    Sato, Shinya; Miyazono, Sadaharu; Tachibanaki, Shuji; Kawamura, Satoru

    2015-01-30

    Cone photoreceptors require effective pigment regeneration mechanisms to maintain their sensitivity in the light. Our previous studies in carp cones suggested the presence of an unconventional and very effective mechanism to produce 11-cis retinal, the necessary component in pigment regeneration. In this reaction (aldehyde-alcohol redox coupling reaction, AL-OL coupling reaction), formation of 11-cis retinal, i.e. oxidation of 11-cis retinol is coupled to reduction of an aldehyde at a 1:1 molar ratio without exogenous NADP(H) which is usually required in this kind of reaction. Here, we identified carp retinol dehydrogenase 13-like (RDH13L) as an enzyme catalyzing the AL-OL coupling reaction. RDH13L was partially purified from purified carp cones, identified as a candidate protein, and its AL-OL coupling activity was confirmed using recombinant RDH13L. We further examined the substrate specificity, subcellular localization, and expression level of RDH13L. Based on these results, we concluded that RDH13L contributes to a significant part, but not all, of the AL-OL coupling activity in carp cones. RDH13L contained tightly bound NADP(+) which presumably functions as a cofactor in the reaction. Mouse RDH14, a mouse homolog of carp RDH13L, also showed the AL-OL coupling activity. Interestingly, although carp cone membranes, carp RDH13L and mouse RDH14 all showed the coupling activity at 15-37 °C, they also showed a conventional NADP(+)-dependent 11-cis retinol oxidation activity above 25 °C without addition of aldehydes. This dual mechanism of 11-cis retinal synthesis attained by carp RDH13L and mouse RDH14 probably contribute to effective pigment regeneration in cones that function in the light. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Chiral aldehydes in hydrocarbons: diastereoselective nucleophilic addition, NMR, and CD spectroscopy reveal dynamic solvation effects.

    PubMed

    Cainelli, Gianfranco; Galletti, Paola; Pieraccini, Silvia; Quintavalla, Arianna; Giacomini, Daria; Piero Spada, Gian

    2004-01-01

    Temperature-dependent studies on the diastereoselective nucleophilic addition of n- BuLi to alpha-chiral aldehydes as (S)-O-(t-butyl-dimethylsilyl)lactal, (S)-O-(t-butyl-dimethylsilyl) mandelic aldehyde, and (R)-2-phenylpropanal in n-decane and n-dodecane reveal dynamic solvation phenomena with the presence of inversion temperatures (T(inv)) in the Eyring plots of ln (anti/syn) vs. 1/ T. These dynamic solvent effects were disclosed by temperature-dependent studies of the (13)C NMR, CD, and UV spectra of the starting aldehydes in solution of n-decane and n-dodecane. The concomitant presence of three peculiar temperatures T(CD), T(UV), and T(NMR), whose values are identical and match T(inv), clearly confirms our earlier interpretation of the solvent-dependent nature of T(inv). The inversion temperature, as well as T(CD), T(UV), and T(NMR) represents the interconversion temperature of two different solvation clusters which act as two different supramolecules with different stereoselectivities. Copyright 2003 Wiley-Liss, Inc.

  10. Myoglobin-Catalyzed Olefination of Aldehydes.

    PubMed

    Tyagi, Vikas; Fasan, Rudi

    2016-02-12

    The olefination of aldehydes constitutes a most valuable and widely adopted strategy for constructing carbon-carbon double bonds in organic chemistry. While various synthetic methods have been made available for this purpose, no biocatalysts are known to mediate this transformation. Reported herein is that engineered myoglobin variants can catalyze the olefination of aldehydes in the presence of α-diazoesters with high catalytic efficiency (up to 4,900 turnovers) and excellent E diastereoselectivity (92-99.9 % de). This transformation could be applied to the olefination of a variety of substituted benzaldehydes and heteroaromatic aldehydes, also in combination with different alkyl α-diazoacetate reagents. This work provides a first example of biocatalytic aldehyde olefination and extends the spectrum of synthetically valuable chemical transformations accessible using metalloprotein-based catalysts. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Deodorants: an experimental provocation study with cinnamic aldehyde.

    PubMed

    Bruze, Magnus; Johansen, J D; Andersen, K E; Frosch, P; Lepoittevin, J-P; Rastogi, S; Wakelin, S; White, I; Menné, T

    2003-02-01

    Axillary dermatitis is common and overrepresented in individuals with contact allergy to fragrances. Many individuals suspect their deodorants to be the incriminating products. Our aim was to investigate the significance of cinnamic aldehyde in deodorants for the development of axillary dermatitis when used by individuals with and without contact allergy to cinnamic aldehyde. Patch tests with deodorants and ethanol solutions with cinnamic aldehyde, and repeated open application tests with roll-on deodorants without and with cinnamic aldehyde at different concentrations, were performed in 37 patients with dermatitis, 20 without and 17 with contact allergy to cinnamic aldehyde. A repeated open application test with positive findings was noted only in patients hypersensitive to cinnamic aldehyde (P <.001) and only in the axilla to which the deodorants containing cinnamic aldehyde had been applied (P <.001). Deodorants containing cinnamic aldehyde in the concentration range 0.01% to 0.32%, used twice daily on healthy skin, can elicit axillary dermatitis within a few weeks.

  12. Relative importance of redox buffers GSH and NAD(P)H in age-related neurodegeneration and Alzheimer disease-like mouse neurons.

    PubMed

    Ghosh, Debolina; Levault, Kelsey R; Brewer, Gregory J

    2014-08-01

    Aging, a major risk factor in Alzheimer's disease (AD), is associated with an oxidative redox shift, decreased redox buffer protection, and increased free radical reactive oxygen species (ROS) generation, probably linked to mitochondrial dysfunction. While NADH is the ultimate electron donor for many redox reactions, including oxidative phosphorylation, glutathione (GSH) is the major ROS detoxifying redox buffer in the cell. Here, we explored the relative importance of NADH and GSH to neurodegeneration in aging and AD neurons from nontransgenic and 3xTg-AD mice by inhibiting their synthesis to determine whether NADH can compensate for the GSH loss to maintain redox balance. Neurons stressed by either depleting NAD(P)H or GSH indicated that NADH redox control is upstream of GSH levels. Further, although depletion of NAD(P)H or GSH correlated linearly with neuron death, compared with GSH depletion, higher neurodegeneration was observed when NAD(P)H was extrapolated to zero, especially in old age, and in the 3xTg-AD neurons. We also observed an age-dependent loss of gene expression of key redox-dependent biosynthetic enzymes, NAMPT (nicotinamide phosphoribosyltransferase), and NNT (nicotinamide nucleotide transhydrogenase). Moreover, age-related correlations between brain NNT or NAMPT gene expression and NADPH levels suggest that these genes contribute to the age-related declines in NAD(P)H. Our data indicate that in aging and more so in AD-like neurons, NAD(P)H redox control is upstream of GSH and an oxidative redox shift that promotes neurodegeneration. Thus, NAD(P)H generation may be a more efficacious therapeutic target upstream of GSH and ROS. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  13. 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.

  14. 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.

  15. Inhibitory activity and mechanism of inhibition of the N-[[(4-benzoylamino)phenyl]sulfonyl]amino acid aldose reductase inhibitors.

    PubMed

    DeRuiter, J; Mayfield, C A

    1990-11-15

    A series of substituted N-[[(4-benzoylamino)phenyl]sulfonyl]amino acids (BAPS-amino acids) were synthesized by established methods, and the stereochemistry of the products was confirmed by HPLC analysis after chiral derivatization. When tested against aldose reductase (alditol:NADP+ oxidoreductase; EC 1.1.1.21; ALR2) isolated from rat lens, all of the BAPS-amino acids were determined to be significantly more inhibitory than the corresponding N-(phenylsulfonyl)amino acids. Structure-inhibition and enzyme kinetic analyses suggest that the BAPS-amino acids inhibit ALR2 by a mechanism similar to the N-(phenylsulfonyl)amino acids. However, multiple inhibition analyses indicate that the increased inhibitory activity of the BAPS-amino acids is a result of interaction with multiple sites present on ALR2. Enzyme specificity studies with several of the BAPS-amino acids demonstrated that these compounds do not produce significant inhibition of other nucleotide-requiring enzymes including aldehyde reductase (alcohol: NADP+ oxidoreductase; EC 1.1.1.2; ALR1).

  16. NADPH Oxidase-Driven Phagocyte Recruitment Controls Candida albicans Filamentous Growth and Prevents Mortality

    PubMed Central

    Brothers, Kimberly M.; Gratacap, Remi L.; Barker, Sarah E.; Newman, Zachary R.; Norum, Ashley; Wheeler, Robert T.

    2013-01-01

    Candida albicans is a human commensal and clinically important fungal pathogen that grows as both yeast and hyphal forms during human, mouse and zebrafish infection. Reactive oxygen species (ROS) produced by NADPH oxidases play diverse roles in immunity, including their long-appreciated function as microbicidal oxidants. Here we demonstrate a non-traditional mechanistic role of NADPH oxidase in promoting phagocyte chemotaxis and intracellular containment of fungi to limit filamentous growth. We exploit the transparent zebrafish model to show that failed NADPH oxidase-dependent phagocyte recruitment to C. albicans in the first four hours post-infection permits fungi to germinate extracellularly and kill the host. We combine chemical and genetic tools with high-resolution time-lapse microscopy to implicate both phagocyte oxidase and dual-specific oxidase in recruitment, suggesting that both myeloid and non-myeloid cells promote chemotaxis. We show that early non-invasive imaging provides a robust tool for prognosis, strongly connecting effective early immune response with survival. Finally, we demonstrate a new role of a key regulator of the yeast-to-hyphal switching program in phagocyte-mediated containment, suggesting that there are species-specific methods for modulation of NADPH oxidase-independent immune responses. These novel links between ROS-driven chemotaxis and fungal dimorphism expand our view of a key host defense mechanism and have important implications for pathogenesis. PMID:24098114

  17. 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.

  18. 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.

  19. 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

  20. New insights into ergot alkaloid biosynthesis in Claviceps purpurea: an agroclavine synthase EasG catalyses, via a non-enzymatic adduct with reduced glutathione, the conversion of chanoclavine-I aldehyde to agroclavine.

    PubMed

    Matuschek, Marco; Wallwey, Christiane; Xie, Xiulan; Li, Shu-Ming

    2011-06-07

    Ergot alkaloids are indole derivatives with diverse structures and biological activities. They are produced by a wide range of fungi with Claviceps purpurea as the most important producer for medical use. Chanoclavine-I aldehyde is proposed as a branch point via festuclavine or pyroclavine to clavine-type alkaloids in Trichocomaceae and via agroclavine to ergoamides and ergopeptines in Clavicipitaceae. Here we report the conversion of chanoclavine-I aldehyde to agroclavine by EasG from Claviceps purpurea, a homologue of the festuclavine synthase FgaFS in Aspergillus fumigatus, in the presence of reduced glutathione and NADPH. EasG comprises 290 amino acids with a molecular mass of about 31.9 kDa. The soluble monomeric His(6)-EasG was purified after overproduction in E. coli by affinity chromatography and used for enzyme assays. The structure of agroclavine was unequivocally elucidated by NMR and MS analyses.

  1. Role of the NAD(P)H quinone oxidoreductase NQR and the cytochrome b AIR12 in controlling superoxide generation at the plasma membrane.

    PubMed

    Biniek, Catherine; Heyno, Eiri; Kruk, Jerzy; Sparla, Francesca; Trost, Paolo; Krieger-Liszkay, Anja

    2017-04-01

    The quinone reductase NQR and the b-type cytochrome AIR12 of the plasma membrane are important for the control of reactive oxygen species in the apoplast. AIR12 and NQR are two proteins attached to the plant plasma membrane which may be important for generating and controlling levels of reactive oxygen species in the apoplast. AIR12 (Auxin Induced in Root culture) is a single gene of Arabidopsis that codes for a mono-heme cytochrome b. The NADPH quinone oxidoreductase NQR is a two-electron-transferring flavoenzyme that contributes to the generation of O 2 •- in isolated plasma membranes. A. thaliana double knockout plants of both NQR and AIR12 generated more O 2 •- and germinated faster than the single mutant affected in AIR12. To test whether NQR and AIR12 are able to interact functionally, recombinant purified proteins were added to plasma membranes isolated from soybean hypocotyls. In vitro NADH-dependent O 2 •- production at the plasma membrane in the presence of NQR was reduced upon addition of AIR12. Electron donation from semi-reduced menadione to AIR12 was shown to take place. Biochemical analysis showed that purified plasma membrane from soybean hypocotyls or roots contained phylloquinone and menaquinone-4 as redox carriers. This is the first report on the occurrence of menaquinone-4 in eukaryotic photosynthetic organisms. We propose that NQR and AIR12 interact via the quinone, allowing an electron transfer from cytosolic NAD(P)H to apoplastic monodehydroascorbate and control thereby the level of reactive oxygen production and the redox state of the apoplast.

  2. 40 CFR 721.639 - Amine aldehyde condensate.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Amine aldehyde condensate. 721.639... Substances § 721.639 Amine aldehyde condensate. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as an amine aldehyde condensate (PMN P-94-1810...

  3. 40 CFR 721.639 - Amine aldehyde condensate.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Amine aldehyde condensate. 721.639... Substances § 721.639 Amine aldehyde condensate. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as an amine aldehyde condensate (PMN P-94-1810...

  4. 40 CFR 721.639 - Amine aldehyde condensate.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Amine aldehyde condensate. 721.639... Substances § 721.639 Amine aldehyde condensate. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as an amine aldehyde condensate (PMN P-94-1810...

  5. 40 CFR 721.639 - Amine aldehyde condensate.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Amine aldehyde condensate. 721.639... Substances § 721.639 Amine aldehyde condensate. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as an amine aldehyde condensate (PMN P-94-1810...

  6. 40 CFR 721.639 - Amine aldehyde condensate.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Amine aldehyde condensate. 721.639... Substances § 721.639 Amine aldehyde condensate. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as an amine aldehyde condensate (PMN P-94-1810...

  7. Sepiapterin Reductase Mediates Chemical Redox Cycling in Lung Epithelial Cells*

    PubMed Central

    Yang, Shaojun; Jan, Yi-Hua; Gray, Joshua P.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2013-01-01

    In the lung, chemical redox cycling generates highly toxic reactive oxygen species that can cause alveolar inflammation and damage to the epithelium, as well as fibrosis. In this study, we identified a cytosolic NADPH-dependent redox cycling activity in mouse lung epithelial cells as sepiapterin reductase (SPR), an enzyme important for the biosynthesis of tetrahydrobiopterin. Human SPR was cloned and characterized. In addition to reducing sepiapterin, SPR mediated chemical redox cycling of bipyridinium herbicides and various quinones; this activity was greatest for 1,2-naphthoquinone followed by 9,10-phenanthrenequinone, 1,4-naphthoquinone, menadione, and 2,3-dimethyl-1,4-naphthoquinone. Whereas redox cycling chemicals inhibited sepiapterin reduction, sepiapterin had no effect on redox cycling. Additionally, inhibitors such as dicoumarol, N-acetylserotonin, and indomethacin blocked sepiapterin reduction, with no effect on redox cycling. Non-redox cycling quinones, including benzoquinone and phenylquinone, were competitive inhibitors of sepiapterin reduction but noncompetitive redox cycling inhibitors. Site-directed mutagenesis of the SPR C-terminal substrate-binding site (D257H) completely inhibited sepiapterin reduction but had minimal effects on redox cycling. These data indicate that SPR-mediated reduction of sepiapterin and redox cycling occur by distinct mechanisms. The identification of SPR as a key enzyme mediating chemical redox cycling suggests that it may be important in generating cytotoxic reactive oxygen species in the lung. This activity, together with inhibition of sepiapterin reduction by redox-active chemicals and consequent deficiencies in tetrahydrobiopterin, may contribute to tissue injury. PMID:23640889

  8. Purification and characterization of two enone reductases from Saccharomyces cerevisiae.

    PubMed

    Wanner, P; Tressl, R

    1998-07-01

    Two enone reductases catalyzing irreversibly the enantioselective reduction of alpha,beta-unsaturated carbonyls have been purified 165-fold and 257-fold, respectively, from the cytosolic fraction of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, blue Sepharose CL-6B chromatography and Superdex 200 preparation-grade filtration. One enzyme (E I) was NADPH-dependent, showed a molecular mass of 75 kDa and decomposed under denaturing electrophoretic conditions into two subunits of 34 kDa and 37 kDa. The other enzyme (E II) was NADH linked and the molecular mass estimated by means of Superdex 200 preparation-grade filtration, was 130 kDa. The enzyme decomposed into subunits of 56 kDa and 64 kDa on SDS/PAGE. Both enzymes were most active at pH 4.8 and accepted 1-octen-3-one, 1-hexen-3-one, 3-alken-2-ones, 4-alken-3-ones, 2-cyclohexen-1-ones, 2-alkenals, 2,4-alkadienals, 2-phenyl-2-alkenals, and 2-alkyl-2-alkenals as substrates. Both enzymes showed their highest activities with 1-hexen-3-one and 1-octen-3-one, respectively. The Km values for 1-octen-3-one were estimated as 0.54 mM (E I) and 0.20 mM (E II), respectively. Both enzymes catalyzed the enantioselective reduction of cis- and trans-2-phenyl-2-butenal into (R)-2-phenylbutanal.

  9. 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.

  10. Coupling Drosophila melanogaster Cryptochrome Light Activation and Oxidation of the Kvβ Subunit Hyperkinetic NADPH Cofactor.

    PubMed

    Hong, Gongyi; Pachter, Ruth; Ritz, Thorsten

    2018-06-28

    Motivated by the observations on the involvement of light-induced processes in the Drosophila melanogaster cryptochrome (DmCry) in regulation of the neuronal firing rate, which is achieved by a redox-state change of its voltage-dependent K + channel Kvβ subunit hyperkinetic (Hk) reduced nicotinamide adenine dinucleotide phosphate (NADPH) cofactor, we propose in this work two hypothetical pathways that may potentially enable such coupling. In the first pathway, triggered by blue-light-induced formation of a radical pair [FAD •- TRP •+ ] in DmCry, the hole (TRP •+ ) may hop to Hk, for example, through a tryptophan chain and oxidize NADPH, possibly leading to inhibition of the N-terminus inactivation in the K + channel. In a second possible pathway, DmCry's FAD •- is reoxidized by molecular oxygen, producing H 2 O 2 , which then diffuses to Hk and oxidizes NADPH. In this work, by applying a combination of quantum and empirical-based methods for free-energy calculations, we find that the oxidation of NADPH by TRP •+ or H 2 O 2 and the reoxidation of FAD •- by O 2 are thermodynamically feasible. Our results may have an implication in identifying a magnetic sensing signal transduction pathway, specifically upon Drosophila's Hk NADPH cofactor oxidation, with a subsequent inhibition of the K + channel N-terminus inactivation gate, permitting K + flux.

  11. 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.

  12. 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.

  13. 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

  14. Ebselen and congeners inhibit NADPH oxidase 2-dependent superoxide generation by interrupting the binding of regulatory subunits.

    PubMed

    Smith, Susan M E; Min, Jaeki; Ganesh, Thota; Diebold, Becky; Kawahara, Tsukasa; Zhu, Yerun; McCoy, James; Sun, Aiming; Snyder, James P; Fu, Haian; Du, Yuhong; Lewis, Iestyn; Lambeth, J David

    2012-06-22

    NADPH oxidases (Nox) are a primary source of reactive oxygen species (ROS), which function in normal physiology and, when overproduced, in pathophysiology. Recent studies using mice deficient in Nox2 identify this isoform as a novel target against Nox2-implicated inflammatory diseases. Nox2 activation depends on the binding of the proline-rich domain of its heterodimeric partner p22phox to p47phox. A high-throughput screen that monitored this interaction via fluorescence polarization identified ebselen and several of its analogs as inhibitors. Medicinal chemistry was performed to explore structure-activity relationships and to optimize potency. Ebselen and analogs potently inhibited Nox1 and Nox2 activity but were less effective against other isoforms. Ebselen also blocked translocation of p47phox to neutrophil membranes. Thus, ebselen and its analogs represent a class of compounds that inhibit ROS generation by interrupting the assembly of Nox2-activating regulatory subunits. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. 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.

  16. Molecular Dynamics Simulations Reveal Proton Transfer Pathways in Cytochrome C-Dependent Nitric Oxide Reductase

    PubMed Central

    Pisliakov, Andrei V.; Hino, Tomoya; Shiro, Yoshitsugu; Sugita, Yuji

    2012-01-01

    Nitric oxide reductases (NORs) are membrane proteins that catalyze the reduction of nitric oxide (NO) to nitrous oxide (N2O), which is a critical step of the nitrate respiration process in denitrifying bacteria. Using the recently determined first crystal structure of the cytochrome c-dependent NOR (cNOR) [Hino T, Matsumoto Y, Nagano S, Sugimoto H, Fukumori Y, et al. (2010) Structural basis of biological N2O generation by bacterial nitric oxide reductase. Science 330: 1666–70.], we performed extensive all-atom molecular dynamics (MD) simulations of cNOR within an explicit membrane/solvent environment to fully characterize water distribution and dynamics as well as hydrogen-bonded networks inside the protein, yielding the atomic details of functionally important proton channels. Simulations reveal two possible proton transfer pathways leading from the periplasm to the active site, while no pathways from the cytoplasmic side were found, consistently with the experimental observations that cNOR is not a proton pump. One of the pathways, which was newly identified in the MD simulation, is blocked in the crystal structure and requires small structural rearrangements to allow for water channel formation. That pathway is equivalent to the functional periplasmic cavity postulated in cbb 3 oxidase, which illustrates that the two enzymes share some elements of the proton transfer mechanisms and confirms a close evolutionary relation between NORs and C-type oxidases. Several mechanisms of the critical proton transfer steps near the catalytic center are proposed. PMID:22956904

  17. Inactivation of CYP2A6 by the Dietary Phenylpropanoid trans-Cinnamic Aldehyde (Cinnamaldehyde) and Estimation of Interactions with Nicotine and Letrozole

    PubMed Central

    Chan, Jeannine; Oshiro, Tyler; Thomas, Sarah; Higa, Allyson; Black, Stephen; Todorovic, Aleksandar; Elbarbry, Fawzy

    2016-01-01

    Human exposure to trans-cinnamic aldehyde [t-CA; cinnamaldehyde; cinnamal; (E)-3-phenylprop-2-enal] is common through diet and through the use of cinnamon powder for diabetes and to provide flavor and scent in commercial products. We evaluated the likelihood of t-CA to influence metabolism by inhibition of P450 enzymes. IC50 values from recombinant enzymes indicated that an interaction is most probable for CYP2A6 (IC50 = 6.1 µM). t-CA was 10.5-fold more selective for human CYP2A6 than for CYP2E1; IC50 values for P450s 1A2, 2B6, 2C9, 2C19, 2D6, and 3A4 were 15.8-fold higher or more. t-CA is a type I ligand for CYP2A6 (KS = 14.9 µM). Inhibition of CYP2A6 by t-CA was metabolism-dependent; inhibition required NADPH and increased with time. Glutathione lessened the extent of inhibition modestly and statistically significantly. The carbon monoxide binding spectrum was dramatically diminished after exposure to NADPH and t-CA, suggesting degradation of the heme or CYP2A6 apoprotein. Using a static model and mechanism-based inhibition parameters (KI = 18.0 µM; kinact = 0.056 minute−1), changes in the area under the concentration-time curve (AUC) for nicotine and letrozole were predicted in the presence of t-CA (0.1 and 1 µM). The AUC fold-change ranged from 1.1 to 3.6. In summary, t-CA is a potential source of pharmacokinetic variability for CYP2A6 substrates due to metabolism-dependent inhibition, especially in scenarios when exposure to t-CA is elevated due to high dietary exposure, or when cinnamon is used as a treatment of specific disease states (e.g., diabetes). PMID:26851241

  18. Molecular Insights of p47phox Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production*

    PubMed Central

    Meijles, Daniel N.; Fan, Lampson M.; Howlin, Brendan J.; Li, Jian-Mei

    2014-01-01

    Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47phox is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47phox−/− coronary microvascular cells. Compared with wild-type p47phox cDNA transfected cells, the single mutation of S379A completely blocked p47phox membrane translocation, binding to p22phox and endothelial O2⨪ production in response to acute stimulation of PKC. p47phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47phox conformational changes and NADPH oxidase-dependent superoxide production by cells. PMID:24970888

  19. ALDEHYDE DEHYDROGENASES EXPRESSION DURING POSTNATAL DEVELOPMENT: LIVER VS. LUNG

    EPA Science Inventory

    Aldehydes are highly reactive molecules present in the environment, and can be produced during biotransformation of xenobiotics. Although the lung can be a major target for aldehyde toxicity, development of aldehyde dehydrogenases (ALDHs), which detoxify aldehydes, in lung has be...

  20. 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.

  1. Ultrafine carbon particles promote rotenone-induced dopamine neuronal loss through activating microglial NADPH oxidase

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

    Wang, Yinxi; Liu, Dan; Zhang, Huifeng

    Background: Atmospheric ultrafine particles (UFPs) and pesticide rotenone were considered as potential environmental risk factors for Parkinson's disease (PD). However, whether and how UFPs alone and in combination with rotenone affect the pathogenesis of PD remains largely unknown. Methods: Ultrafine carbon black (ufCB, a surrogate of UFPs) and rotenone were used individually or in combination to determine their roles in chronic dopaminergic (DA) loss in neuron-glia, and neuron-enriched, mix-glia cultures. Immunochemistry using antibody against tyrosine hydroxylase was performed to detect DA neuronal loss. Measurement of extracellular superoxide and intracellular reactive oxygen species (ROS) were performed to examine activation of NADPHmore » oxidase. Genetic deletion and pharmacological inhibition of NADPH oxidase and MAC-1 receptor in microglia were employed to examine their role in DA neuronal loss triggered by ufCB and rotenone. Results: In rodent midbrain neuron-glia cultures, ufCB and rotenone alone caused neuronal death in a dose-dependent manner. In particularly, ufCB at doses of 50 and 100 μg/cm{sup 2} induced significant loss of DA neurons. More importantly, nontoxic doses of ufCB (10 μg/cm{sup 2}) and rotenone (2 nM) induced synergistic toxicity to DA neurons. Microglial activation was essential in this process. Furthermore, superoxide production from microglial NADPH oxidase was critical in ufCB/rotenone-induced neurotoxicity. Studies in mix-glia cultures showed that ufCB treatment activated microglial NADPH oxidase to induce superoxide production. Firstly, ufCB enhanced the expression of NADPH oxidase subunits (gp91{sup phox}, p47{sup phox} and p40{sup phox}); secondly, ufCB was recognized by microglial surface MAC-1 receptor and consequently promoted rotenone-induced p47{sup phox} and p67{sup phox} translocation assembling active NADPH oxidase. Conclusion: ufCB and rotenone worked in synergy to activate NADPH oxidase in microglia, leading to

  2. A possible role of NADPH-dependent cytochrome P450nor isozyme in glycolysis under denitrifying conditions.

    PubMed

    Watsuji, Tomo-o; Takaya, Naoki; Nakamura, Akira; Shoun, Hirofumi

    2003-05-01

    The denitrifying fungus Cylindrocarpon tonkinense contains two isozymes of cytochrome P450nor. One isozyme, P450nor1, uses NADH specifically as its electron donor whereas the other isozyme P450nor2 prefers NADPH to NADH. Here we show that P450nor1 is localized in both cytosol and mitochondria, like P450nor of Fusarium oxysporum, while P450nor2 is exclusively in cytosol. We also found that the addition of glucose as a carbon source to the culture media leads to the production of much more P450nor2 in the fungal cells than a non-fermentable substrate (glycerol or acetate) does. These results suggest that the NADP-dependent pentose phosphate cycle acts predominantly in C. tonkinense as the glycolysis pathway under the denitrifying conditions, which was confirmed by the observation that glucose induced enzyme activities involved in the cycle. These results showed that P450nor2 should act as the electron sink under anaerobic, denitrifying conditions to regenerate NADP+ for the pentose phosphate cycle.

  3. Overproduction of a rice aldo-keto reductase increases oxidative and heat stress tolerance by malondialdehyde and methylglyoxal detoxification.

    PubMed

    Turóczy, Zoltán; Kis, Petra; Török, Katalin; Cserháti, Mátyás; Lendvai, Agnes; Dudits, Dénes; Horváth, Gábor V

    2011-03-01

    The accumulation of toxic compounds generated by the interaction between reactive oxygen species and polyunsaturated fatty acids of membrane lipids can significantly damage plant cells. A plethora of enzymes act on these reactive carbonyls, reducing their toxicity. Based on the chromosomal localization and on their homology with other stress-induced aldo-keto reductases (AKRs) we have selected three rice AKR genes. The transcription level of OsAKR1 was greatly induced by abscisic acid and various stress treatments; the other two AKR genes tested were moderately stress-inducible. The OsAKR1 recombinant protein exhibited a high nicotinamide adenine dinucleotide phosphate-dependent catalytic activity to reduce toxic aldehydes including glycolysis-derived methylglyoxal (MG) and lipid peroxidation-originated malondialdehyde (MDA). The function of this enzyme in MG detoxification was demonstrated in vivo in E. coli and in transgenic plants overproducing the OsAKR1 protein. Heterologous synthesis of the OsAKR1 enzyme in transgenic tobacco plants resulted in increased tolerance against oxidative stress generated by methylviologen (MV) and improved resistance to high temperature. In these plants lower levels of MDA were detected both following MV and heat treatment due to the activity of the OsAKR1 enzyme. The transgenic tobaccos also exhibited higher AKR activity and accumulated less MG in their leaves than the wild type plants; both in the presence and absence of heat stress. These results support the positive role of OsAKR1 in abiotic stress-related reactive aldehyde detoxification pathways and its use for improvement of stress tolerance in plants.

  4. Respiratory arsenate reductase as a bidirectional enzyme

    USGS Publications Warehouse

    Richey, C.; Chovanec, P.; Hoeft, S.E.; Oremland, R.S.; Basu, P.; Stolz, J.F.

    2009-01-01

    The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe–S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.

  5. Triggering the approach of an arene or heteroarene towards an aldehyde via Lewis acid-aldehyde communication.

    PubMed

    Pratihar, Sanjay

    2016-03-14

    The present work reports a combined experimental/computational study of the Lewis acid promoted hydroxyalkylation reaction involving aldehyde and arene/heteroarene and reveals a mechanism in which the rate determining aldehyde to alcohol formation via a four-member cyclic transition state (TS) involves a transfer of hydrogen from arene/heteroarene C-H to aldehyde oxygen with the breaking of the C-H bond and formation of C-C and O-H bonds. The effect of different Sn(iv) derivatives on the hydroxyalkylation reaction from different in situ NMR and computational studies reveals that although the exergonic formation of the intermediate and its gained electrophilicity at the carbonyl carbon drive the reaction in SnCl4 compared to other Sn(iv) derivatives, the overall reaction is low yielding because of its stable intermediate. With respect to different aldehydes, LA promoted hydroxylation was found to be more feasible for an electron withdrawing aldehyde compared to electron rich aldehyde because of lower stability, enhanced electrophilicity gained at the aldehyde center, and a lower activation barrier between its intermediate and TS in the former as compared to the latter. The relative stability of the LA-aldehyde adduct decreases in the order SnCl4 > AlCl3 > InCl3 > BF3 > ZnCl2 > TiCl4 > SiCl4, while the activation barrier (ΔG(#)) between intermediate and transition states increases in the order AlCl3 < SnCl4 < InCl3 < BF3 < TiCl4 < ZnCl2 < SiCl4. On the other hand, the activation barriers in the case of different arenes/heteroarenes are in the order of indole < furan < anisole < thiophene < toluene < benzene < chlorobenzene < cyanobenzene, which suggests a facile reaction in the case of indole and the most difficult reaction in the case of cyanobenzene. The ease of formation of the corresponding diaryl methyl carbocation from the alcohol-LA intermediate is responsible for the determination of the undesired product and is found to be more viable in the case of strong

  6. 27 CFR 24.183 - Use of distillates containing aldehydes.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... containing aldehydes. 24.183 Section 24.183 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX... distillates containing aldehydes. Distillates containing aldehydes may be received on wine premises for use in... fermentation of wine made from a different kind of fruit. Distillates containing aldehydes which are received...

  7. 27 CFR 24.183 - Use of distillates containing aldehydes.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... containing aldehydes. 24.183 Section 24.183 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX... distillates containing aldehydes. Distillates containing aldehydes may be received on wine premises for use in... fermentation of wine made from a different kind of fruit. Distillates containing aldehydes which are received...

  8. 27 CFR 24.183 - Use of distillates containing aldehydes.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... containing aldehydes. 24.183 Section 24.183 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX... distillates containing aldehydes. Distillates containing aldehydes may be received on wine premises for use in... fermentation of wine made from a different kind of fruit. Distillates containing aldehydes which are received...

  9. 27 CFR 24.183 - Use of distillates containing aldehydes.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... containing aldehydes. 24.183 Section 24.183 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX... distillates containing aldehydes. Distillates containing aldehydes may be received on wine premises for use in... fermentation of wine made from a different kind of fruit. Distillates containing aldehydes which are received...

  10. Response surface methodology as an approach to determine the optimal activities of xylose reductase and xylitol dehydrogenase enzymes from Candida Mogii.

    PubMed

    Mayerhoff, Zea D V L; Roberto, Inês C; Franco, Telma T

    2006-05-01

    A central composite experimental design leading to a set of 16 experiments with different combinations of pH and temperature was performed to attain the optimal activities of xylose reductase (XR) and xylitol dehydrogenase (XDH) enzymes from Candida mogii cell extract. Under optimized conditions (pH 6.5 and 38 degrees C), the XR and XDH activities were found to be 0.48 U/ml and 0.22 U/ml, respectively, resulting in an XR to XDH ratio of 2.2. Stability, cofactor specificity and kinetic parameters of the enzyme XR were also evaluated. XR activity remained stable for 3 h under 4 and 38 degrees C and for 4 months of storage at -18 degrees C. Studies on cofactor specificity showed that only NADPH-dependent XR was obtained under the cultivation conditions employed. The XR present in C. mogii extracts showed a superior Km value for xylose when compared with other yeast strains. Besides, this parameter was not modified after enzyme extraction by aqueous two-phase system.

  11. Pulsed corona discharge oxidation of aqueous lignin: decomposition and aldehydes formation.

    PubMed

    Panorel, Iris; Kaijanen, Laura; Kornev, Iakov; Preis, Sergei; Louhi-Kultanen, Marjatta; Sirén, Heli

    2014-01-01

    Lignin is the mass waste product of pulp and paper industry mostly incinerated for energy recovery. Lignin is, however, a substantial source of raw material for derivatives currently produced in costly wet oxidation processes. The pulsed corona discharge (PCD) for the first time was applied to lignin oxidation aiming a cost-effective environmentally friendly lignin removal and transformation to aldehydes. The experimental research into treatment of coniferous kraft lignin aqueous solutions was undertaken to establish the dependence of lignin oxidation and aldehyde formation on the discharge parameters, initial concentration of lignin and gas phase composition. The rate and the energy efficiency of lignin oxidation increased with increasing oxygen concentration reaching up to 82 g kW-1 h-1 in 89% vol. oxygen. Oxidation energy efficiency in PCD treatment exceeds the one for conventional ozonation by the factor of two under the experimental conditions. Oxidation at low oxygen concentrations showed a tendency of the increasing aldehydes and glyoxylic acid formation yield.

  12. Aldehyde-containing urea-absorbing polysaccharides

    NASA Technical Reports Server (NTRS)

    Mueller, W. A.; Hsu, G. C.; Marsh, H. E., Jr. (Inventor)

    1977-01-01

    A novel aldehyde containing polymer (ACP) is prepared by reaction of a polysaccharide with periodate to introduce aldehyde groups onto the C2 - C3 carbon atoms. By introduction of ether and ester groups onto the pendant primary hydroxyl solubility characteristics are modified. The ACP is utilized to absorb nitrogen bases such as urea in vitro or in vivo.

  13. Null Mutation of 5α-Reductase Type I Gene Alters Ethanol Consumption Patterns in a Sex-Dependent Manner

    PubMed Central

    Nickel, Jeffrey D.; Kaufman, Moriah N.; Finn, Deborah A.

    2014-01-01

    The neuroactive steroid allopregnanolone (ALLO) is a positive modulator of GABAA receptors, and manipulation of neuroactive steroid levels via injection of ALLO or the 5α-reductase inhibitor finasteride alters ethanol self-administration patterns in male, but not female, mice. The Srd5a1 gene encodes the enzyme 5α-reductase-1, which is required for the synthesis of ALLO. The current studies investigated the influence of Srd5a1 deletion on voluntary ethanol consumption in male and female wildtype (WT) and knockout (KO) mice. Under a continuous access condition, 6 and 10 % ethanol intake was significantly greater in KO versus WT females, but significantly lower in KO versus WT males. In 2-h limited access sessions, Srd5a1 deletion retarded acquisition of 10 % ethanol intake in female mice, but facilitated it in males, versus respective WT mice. The present findings demonstrate that the Srd5a1 gene modulates ethanol consumption in a sex-dependent manner that is also contingent upon ethanol access condition and concentration. PMID:25416204

  14. Null mutation of 5α-reductase type I gene alters ethanol consumption patterns in a sex-dependent manner.

    PubMed

    Ford, Matthew M; Nickel, Jeffrey D; Kaufman, Moriah N; Finn, Deborah A

    2015-05-01

    The neuroactive steroid allopregnanolone (ALLO) is a positive modulator of GABAA receptors, and manipulation of neuroactive steroid levels via injection of ALLO or the 5α-reductase inhibitor finasteride alters ethanol self-administration patterns in male, but not female, mice. The Srd5a1 gene encodes the enzyme 5α-reductase-1, which is required for the synthesis of ALLO. The current studies investigated the influence of Srd5a1 deletion on voluntary ethanol consumption in male and female wildtype (WT) and knockout (KO) mice. Under a continuous access condition, 6 and 10 % ethanol intake was significantly greater in KO versus WT females, but significantly lower in KO versus WT males. In 2-h limited access sessions, Srd5a1 deletion retarded acquisition of 10 % ethanol intake in female mice, but facilitated it in males, versus respective WT mice. The present findings demonstrate that the Srd5a1 gene modulates ethanol consumption in a sex-dependent manner that is also contingent upon ethanol access condition and concentration.

  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. Covalent modification and time-dependent inhibition of human CYP2E1 by the meta-isomer of acetaminophen.

    PubMed

    Harrelson, John P; Stamper, Brendan D; Chapman, John D; Goodlett, David R; Nelson, Sidney D

    2012-08-01

    The hypothesis that N-acetyl-m-aminophenol (AMAP), the meta isomer of acetaminophen, will covalently bind to and inhibit human CYP2E1 in a time- and NADPH-dependent manner was investigated. Liquid chromatography/electrospray ionization-mass spectrometry analysis indicated that AMAP metabolites (i.e., AMAP*) selectively and covalently modified CYP2E1 apoprotein in a ratio of 1.4:1 (AMAP*/CYP2E1) in a reconstituted system. The deconvoluted spectra of CYP2E1 apoprotein from incubations containing NADPH and AMAP displayed mass shifts of 167.2 ± 7.1 and 334.4 ± 6.5 Da, suggesting the addition of one and two hydroxylated AMAP metabolites to CYP2E1, respectively. Mass shifts in cytochrome P450 reductase, cytochrome b(5), and heme from these samples were not observed. CYP2E1 inhibition by AMAP increased with time in the presence of NADPH; a reversible inhibition component was also observed. The results support a bioactivation process that involves formation of a hydroquinone metabolite that undergoes further oxidation to a quinone, which reacts with CYP2E1 nucleophilic residues. The data are consistent with evidence from previous studies that identified hydroxylated AMAP glutathione conjugates collected from mice and indicate that cysteine residues are the most likely sites for adduct formation. This study reports the first direct evidence of AMAP-derived hydroquinone metabolites bound to human CYP2E1.

  17. 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

  18. Molecular Mechanisms of Aldehyde Toxicity: A Chemical Perspective

    PubMed Central

    2015-01-01

    Aldehydes are electrophilic compounds to which humans are pervasively exposed. Despite a significant health risk due to exposure, the mechanisms of aldehyde toxicity are poorly understood. This ambiguity is likely due to the structural diversity of aldehyde derivatives and corresponding differences in chemical reactions and biological targets. To gain mechanistic insight, we have used parameters based on the hard and soft, acids and bases (HSAB) theory to profile the different aldehyde subclasses with respect to electronic character (softness, hardness), electrophilic reactivity (electrophilic index), and biological nucleophilic targets. Our analyses indicate that short chain aldehydes and longer chain saturated alkanals are hard electrophiles that cause toxicity by forming adducts with hard biological nucleophiles, e.g., primary nitrogen groups on lysine residues. In contrast, α,β-unsaturated carbonyl derivatives, alkenals, and the α-oxoaldehydes are soft electrophiles that preferentially react with soft nucleophilic thiolate groups on cysteine residues. The aldehydes can therefore be grouped into subclasses according to common electronic characteristics (softness/hardness) and molecular mechanisms of toxicity. As we will discuss, the toxic potencies of these subgroups are generally related to corresponding electrophilicities. For some aldehydes, however, predictions of toxicity based on electrophilicity are less accurate due to inherent physicochemical variables that limit target accessibility, e.g., steric hindrance and solubility. The unsaturated aldehydes are also members of the conjugated type-2 alkene chemical class that includes α,β-unsaturated amide, ketone, and ester derivatives. Type-2 alkenes are electrophiles of varying softness and electrophilicity that share a common mechanism of toxicity. Therefore, exposure to an environmental mixture of unsaturated carbonyl derivatives could cause “type-2 alkene toxicity” through additive interactions

  19. Oleic, Linoleic and Linolenic Acids Increase ROS Production by Fibroblasts via NADPH Oxidase Activation

    PubMed Central

    Hatanaka, Elaine; Dermargos, Alexandre; Hirata, Aparecida Emiko; Vinolo, Marco Aurélio Ramirez; Carpinelli, Angelo Rafael; Newsholme, Philip; Armelin, Hugo Aguirre; Curi, Rui

    2013-01-01

    The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of β-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47phox phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47phox mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts. PMID:23579616

  20. One-Pot Amide Bond Formation from Aldehydes and Amines via a Photoorganocatalytic Activation of Aldehydes.

    PubMed

    Papadopoulos, Giorgos N; Kokotos, Christoforos G

    2016-08-19

    A mild, one-pot, and environmentally friendly synthesis of amides from aldehydes and amines is described. Initially, a photoorganocatalytic reaction of aldehydes with di-isopropyl azodicarboxylate leads to an intermediate carbonyl imide, which can react with a variety of amines to afford the desired amides. The initial visible light-mediated activation of a variety of monosubstituted or disubstituted aldehydes is usually fast, occurring in a few hours. Following the photocatalytic reaction, addition of the primary amine at room temperature or the secondary amine at elevated temperatures leads to the corresponding amide from moderate to excellent yields without epimerization. This methodology was applied in the synthesis of Moclobemide, a drug against depression and social anxiety.

  1. The use of tomato aminoaldehyde dehydrogenase 1 for the detection of aldehydes in fruit distillates.

    PubMed

    Frömmel, Jan; Tarkowski, Petr; Kopečný, David; Šebela, Marek

    2016-09-25

    Plant NAD(+)-dependent aminoaldehyde dehydrogenases (AMADHs, EC 1.2.1.19) belong to the family 10 of aldehyde dehydrogenases. They participate in the metabolism of polyamines or osmoprotectants. The enzymes are characterized by their broad substrate specificity covering ω-aminoaldehydes, aliphatic and aromatic aldehydes as well as nitrogen-containing heterocyclic aldehydes. The isoenzyme 1 from tomato (Solanum lycopersicum; SlAMADH1) oxidizes aliphatic aldehydes very efficiently and converts also furfural, its derivatives or benzaldehyde, which are present at low concentrations in alcoholic distillates such as fruit brandy. In this work, SlAMADH1 was examined as a bioanalytical tool for their detection. These aldehydes arise from fermentation processes or thermal degradation of sugars and their presence is related to health complications after consumption including nausea, emesis, sweating, decrease in blood pressure, hangover headache, among others. Sixteen samples of slivovitz (plum brandy) from local producers in Moravia, Czech Republic, were analyzed for their aldehyde content using a spectrophotometric activity assay with SlAMADH1. In all cases, there were oxidative responses observed when monitoring NADH production in the enzymatic reaction. Aldehydes in the distillate samples were also subjected to a standard determination using reversed-phase HPLC with spectrophotometric and tandem mass spectrometric detection after a derivatization with 2,4-dinitrophenylhydrazine. Results obtained by both methods were found to correlate well for a majority of the analyzed samples. The possible applicability of SlAMADH1 for the evaluation of aldehyde content in food and beverages has now been demonstrated. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. 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.

  3. Respiratory arsenate reductase as a bidirectional enzyme

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

    Richey, Christine; Chovanec, Peter; Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA 15282

    2009-05-01

    The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function asmore » a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe-S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.« less

  4. Salivary aldehyde dehydrogenase - temporal and population variability, correlations with drinking and smoking habits and activity towards aldehydes contained in food.

    PubMed

    Giebułtowicz, Joanna; Dziadek, Marta; Wroczyński, Piotr; Woźnicka, Katarzyna; Wojno, Barbara; Pietrzak, Monika; Wierzchowski, Jacek

    2010-01-01

    Fluorimetric method based on oxidation of the fluorogenic 6-methoxy-2-naphthaldehyde was applied to evaluate temporal and population variability of the specific activity of salivary aldehyde dehydrogenase (ALDH) and the degree of its inactivation in healthy human population. Analyzed was also its dependence on drinking and smoking habits, coffee consumption, and its sensitivity to N-acetylcysteine. Both the specific activity of salivary ALDH and the degree of its inactivation were highly variable during the day, with the highest activities recorded in the morning hours. The activities were also highly variable both intra- and interpersonally, and negatively correlated with age, and this correlation was stronger for the subgroup of volunteers declaring abstinence from alcohol and tobacco. Moderately positive correlations of salivary ALDH specific activity with alcohol consumption and tobacco smoking were also recorded (r(s) ~0.27; p=0.004 and r(s) =0.30; p=0.001, respectively). Moderate coffee consumption correlated positively with the inactivation of salivary ALDH, particularly in the subgroup of non-drinking and non-smoking volunteers. It was found that mechanical stimulation of the saliva flow increases the specific activity of salivary ALDH. The specific activity of the salivary ALDH was strongly and positively correlated with that of superoxide dismutase, and somewhat less with salivary peroxidase. The antioxidant-containing drug N-acetylcysteine increased activity of salivary ALDH presumably by preventing its inactivation in the oral cavity. Some food-related aldehydes, mainly cinnamic aldehyde and anisaldehyde, were excellent substrates of the salivary ALDH3A1 enzyme, while alkenals, particularly those with short chain, were characterized by lower affinity towards this enzyme but high catalytic constants. The protective role of salivary ALDH against aldehydes in food and those found in the cigarette smoke is discussed, as well as its participation in

  5. Biosynthesis of the phytoalexin pisatin. [Pisum sativum

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

    Preisig, C.L.; Bell, J.N.; Matthews, D.E.

    1990-11-01

    NADPH-dependent reduction of 2{prime},7-dihydroxy-4{prime},5{prime}-methylenedioxyisoflavone to the isoflavanone sophorol, a proposed intermediate step in pisatin biosynthesis, was detected in extracts of Pisum sativum. This isoflavone reductase activity was inducible by treatment of pea seedlings with CuCl{sub 2}. The timing of induction coincided with that of the 6a-hydroxymaackiain 3-O-methyltransferase, which catalyzes the terminal biosynthetic step. Neither enzyme was light inducible. Further NADPH-dependent metabolism of sophorol by extracts of CuCl{sub 2}-treated seedlings was also observed; three products were radiolabeled when ({sup 3}H)sophorol was the substrate, one of which is tentatively identified as maackiain.

  6. [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.

  7. Aldehyde dehydrogenase (ALDH) superfamily in plants: gene nomenclature and comparative genomics.

    PubMed

    Brocker, Chad; Vasiliou, Melpomene; Carpenter, Sarah; Carpenter, Christopher; Zhang, Yucheng; Wang, Xiping; Kotchoni, Simeon O; Wood, Andrew J; Kirch, Hans-Hubert; Kopečný, David; Nebert, Daniel W; Vasiliou, Vasilis

    2013-01-01

    In recent years, there has been a significant increase in the number of completely sequenced plant genomes. The comparison of fully sequenced genomes allows for identification of new gene family members, as well as comprehensive analysis of gene family evolution. The aldehyde dehydrogenase (ALDH) gene superfamily comprises a group of enzymes involved in the NAD(+)- or NADP(+)-dependent conversion of various aldehydes to their corresponding carboxylic acids. ALDH enzymes are involved in processing many aldehydes that serve as biogenic intermediates in a wide range of metabolic pathways. In addition, many of these enzymes function as 'aldehyde scavengers' by removing reactive aldehydes generated during the oxidative degradation of lipid membranes, also known as lipid peroxidation. Plants and animals share many ALDH families, and many genes are highly conserved between these two evolutionarily distinct groups. Conversely, both plants and animals also contain unique ALDH genes and families. Herein we carried out genome-wide identification of ALDH genes in a number of plant species-including Arabidopsis thaliana (thale crest), Chlamydomonas reinhardtii (unicellular algae), Oryza sativa (rice), Physcomitrella patens (moss), Vitis vinifera (grapevine) and Zea mays (maize). These data were then combined with previous analysis of Populus trichocarpa (poplar tree), Selaginella moellindorffii (gemmiferous spikemoss), Sorghum bicolor (sorghum) and Volvox carteri (colonial algae) for a comprehensive evolutionary comparison of the plant ALDH superfamily. As a result, newly identified genes can be more easily analyzed and gene names can be assigned according to current nomenclature guidelines; our goal is to clarify previously confusing and conflicting names and classifications that might confound results and prevent accurate comparisons between studies.

  8. Antimicrobial activity and physical characterization of silver nanoparticles green synthesized using nitrate reductase from Fusarium oxysporum.

    PubMed

    Gholami-Shabani, Mohammadhassan; Akbarzadeh, Azim; Norouzian, Dariush; Amini, Abdolhossein; Gholami-Shabani, Zeynab; Imani, Afshin; Chiani, Mohsen; Riazi, Gholamhossein; Shams-Ghahfarokhi, Masoomeh; Razzaghi-Abyaneh, Mehdi

    2014-04-01

    Nanostructures from natural sources have received major attention due to wide array of biological activities and less toxicity for humans, animals, and the environment. In the present study, silver nanoparticles were successfully synthesized using a fungal nitrate reductase, and their biological activity was assessed against human pathogenic fungi and bacteria. The enzyme was isolated from Fusarium oxysporum IRAN 31C after culturing on malt extract-glucose-yeast extract-peptone (MGYP) medium. The enzyme was purified by a combination of ultrafiltration and ion exchange chromatography on DEAE Sephadex and its molecular weight was estimated by gel filtration on Sephacryl S-300. The purified enzyme had a maximum yield of 50.84 % with a final purification of 70 folds. With a molecular weight of 214 KDa, it is composed of three subunits of 125, 60, and 25 KDa. The purified enzyme was successfully used for synthesis of silver nanoparticles in a way dependent upon NADPH using gelatin as a capping agent. The synthesized silver nanoparticles were characterized by X-ray diffraction, dynamic light scattering spectroscopy, and transmission and scanning electron microscopy. These stable nonaggregating nanoparticles were spherical in shape with an average size of 50 nm and a zeta potential of -34.3. Evaluation of the antimicrobial effects of synthesized nanoparticles by disk diffusion method showed strong growth inhibitory activity against all tested human pathogenic fungi and bacteria as evident from inhibition zones that ranged from 14 to 25 mm. Successful green synthesis of biologically active silver nanoparticles by a nitrate reductase from F. oxysporum in the present work not only reduces laborious downstream steps such as purification of nanoparticle from interfering cellular components, but also provides a constant source of safe biologically-active nanomaterials with potential application in agriculture and medicine.

  9. 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

  10. N-triflylthiophosphoramide catalyzed enantioselective Mukaiyama aldol reaction of aldehydes with silyl enol ethers of ketones.

    PubMed

    Cheon, Cheol Hong; Yamamoto, Hisashi

    2010-06-04

    The first Brønsted acid catalyzed asymmetric Mukaiyama aldol reaction of aldehydes using silyl enol ethers of ketones as nucleophiles has been reported. A variety of aldehydes and silyl enol ethers of ketones afforded the aldol products in excellent yields and good to excellent enantioselectivities. Mechanistic studies revealed that the actual catalyst may be changed from the silylated Brønsted acid to the Brønsted acid itself depending on the reaction temperature.

  11. 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

  12. NADPH oxidase activity and reactive oxygen species production in brain and kidney of adult male hypertensive Ren-2 transgenic rats.

    PubMed

    Vokurková, M; Rauchová, H; Řezáčová, L; Vaněčková, I; Zicha, J

    2015-01-01

    Hypothalamic paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM) play an important role in brain control of blood pressure (BP). One of the important mechanisms involved in the pathogenesis of hypertension is the elevation of reactive oxygen species (ROS) production by nicotine adenine dinucleotide phosphate (NADPH) oxidase. The aim of our present study was to investigate NADPH oxidase-mediated superoxide (O(2)(-)) production and to search for the signs of lipid peroxidation in hypothalamus and medulla oblongata as well as in renal medulla and cortex of hypertensive male rats transgenic for the murine Ren-2 renin gene (Ren-2 TGR) and their age-matched normotensive controls - Hannover Sprague Dawley rats (HanSD). We found no difference in the activity of NADPH oxidase measured as a lucigenin-mediated O(2)(-) production in the hypothalamus and medulla oblongata. However, we observed significantly elevated NADPH oxidase in both renal cortex and medulla of Ren-2 TGR compared with HanSD. Losartan (LOS) treatment (10 mg/kg body weight/day) for 2 months (Ren-2 TGR+LOS) did not change NADPH oxidase-dependent O(2)(-) production in the kidney. We detected significantly elevated indirect markers of lipid peroxidation measured as thiobarbituric acid-reactive substances (TBARS) in Ren-2 TGR, while they were significantly decreased in Ren-2 TGR+LOS. In conclusion, the present study shows increased NADPH oxidase activities in renal cortex and medulla with significantly increased TBARS in renal cortex. No significant changes of NADPH oxidase and markers of lipid peroxidation were detected in the studied brain regions.

  13. 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.

  14. Process for producing furan from furfural aldehyde

    DOEpatents

    Diebold, James P.; Evans, Robert J.

    1988-01-01

    A process of producing furan and derivatives thereof is disclosed. The process includes generating furfural aldehyde vapors and then passing those vapors over a zeolite catalyst at a temperature and for a residence time effective to decarbonylate the furfural aldehydes to form furans and derivatives thereof. The resultant furan vapors and derivatives are then separated. In a preferred form, the furfural aldehyde vapors are generated during the process of converting biomass materials to liquid and gaseous fuels.

  15. Process for producing furan from furfural aldehyde

    DOEpatents

    Diebold, J.P.; Evans, R.J.

    1987-04-06

    A process of producing furan and derivatives thereof as disclosed. The process includes generating furfural aldehyde vapors and then passing those vapors over a zeolite catalyst at a temperature and for a residence time effective to decarbonylate the furfural aldehydes to form furans and derivatives thereof. The resultant furan vapors and derivatives are then separated. In a preferred form, the furfural aldehyde vapors are generated during the process of converting biomass materials to liquid and gaseous fuels.

  16. Microsphere coated substrate containing reactive aldehyde groups

    NASA Technical Reports Server (NTRS)

    Yen, Richard C. K. (Inventor); Rembaum, Alan (Inventor)

    1984-01-01

    A synthetic organic resin is coated with a continuous layer of contiguous, tangential, individual microspheres having a uniform diameter preferably between 100 Angstroms and 2000 Angstroms. The microspheres are an addition polymerized polymer of an unsaturated aldehyde containing 4 to 20 carbon atoms and are covalently bonded to the substrate by means of high energy radiation grafting. The microspheres contain reactive aldehyde groups and can form conjugates with proteins such as enzymes or other aldehyde reactive materials.

  17. Nickel-Catalyzed Coupling of Alkenes, Aldehydes, and Silyl Triflates

    PubMed Central

    Ng, Sze-sze; Ho, Chun-Yu; Jamison, Timothy F.

    2011-01-01

    A full account of two recently developed nickel-catalyzed coupling reactions of alkenes, aldehydes and silyl triflates is presented. These reactions provide either allylic alcohol or homoallylic alcohol derivatives selectively, depending on the ligand employed. These processes are believed to be mechanistically distinct from Lewis acid-catalyzed carbonyl-ene reactions, and several lines of evidence supporting this hypothesis are discussed. PMID:16939275

  18. [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.

  19. 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.

  20. Very-long-chain aldehydes induce appressorium formation in ascospores of the wheat powdery mildew fungus Blumeria graminis.

    PubMed

    Zhu, Mo; Riederer, Markus; Hildebrandt, Ulrich

    2017-08-01

    Asexually produced conidia of the wheat powdery mildew fungus Blumeria graminis f. sp. tritici (Bgt) are known to perceive cuticular very-long-chain aldehydes as signal substances strongly stimulating germination and differentiation of infection structures in a concentration- and chain-length-dependent manner. Conidial germination and appressorium formation are widely prevented by the presence of free water on the host surface. However, sexually produced ascospores can differentiate immersed in water. Applying a Formvar ® -based in vitro-system showed that ascospore appressorium formation was strongly induced by the presence of wheat leaf cuticular wax. Similar to conidia, ascospore appressorium formation is triggered by the presence of very-long-chain aldehydes in a chain-length-dependent manner with n-octacosanal as the most inducing aldehyde. Surface hydrophobicity positively affected ascospore germination but not appressorium formation. Ascospores required significantly more time to complete the differentiation of appressoria and exhibited a more distinct dependence on the availability of free water than their conidial counterparts. Unlike conidia, ascospores showed a more variable germination and differentiation pattern even with a single germ tube differentiating an appressorium. Despite these differences our results demonstrate that a host surface recognition principle based on cuticular very-long-chain aldehydes is a common feature of B. graminis f. sp. tritici ascospores and conidia. Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  1. Dynamics of the active site architecture in plant-type ferredoxin-NADP(+) reductases catalytic complexes.

    PubMed

    Sánchez-Azqueta, Ana; Catalano-Dupuy, Daniela L; López-Rivero, Arleth; Tondo, María Laura; Orellano, Elena G; Ceccarelli, Eduardo A; Medina, Milagros

    2014-10-01

    Kinetic isotope effects in reactions involving hydride transfer and their temperature dependence are powerful tools to explore dynamics of enzyme catalytic sites. In plant-type ferredoxin-NADP(+) reductases the FAD cofactor exchanges a hydride with the NADP(H) coenzyme. Rates for these processes are considerably faster for the plastidic members (FNR) of the family than for those belonging to the bacterial class (FPR). Hydride transfer (HT) and deuteride transfer (DT) rates for the NADP(+) coenzyme reduction of four plant-type FNRs (two representatives of the plastidic type FNRs and the other two from the bacterial class), and their temperature dependences are here examined applying a full tunnelling model with coupled environmental fluctuations. Parameters for the two plastidic FNRs confirm a tunnelling reaction with active dynamics contributions, but isotope effects on Arrhenius factors indicate a larger contribution for donor-acceptor distance (DAD) dynamics in the Pisum sativum FNR reaction than in the Anabaena FNR reaction. On the other hand, parameters for bacterial FPRs are consistent with passive environmental reorganisation movements dominating the HT coordinate and no contribution of DAD sampling or gating fluctuations. This indicates that active sites of FPRs are more organised and rigid than those of FNRs. These differences must be due to adaptation of the active sites and catalytic mechanisms to fulfil their particular metabolic roles, establishing a compromise between protein flexibility and functional optimisation. Analysis of site-directed mutants in plastidic enzymes additionally indicates the requirement of a minimal optimal architecture in the catalytic complex to provide a favourable gating contribution. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. N-Triflylthiophosphoramide Catalyzed Enantioselective Mukaiyama Aldol Reaction of Aldehydes with Silyl Enol Ethers of Ketones

    PubMed Central

    Cheon, Cheol Hong; Yamamoto, Hisashi

    2010-01-01

    The first Brønsted acid catalyzed asymmetric Mukaiyama aldol reaction of aldehydes using silyl enol ethers of ketones as nucleophiles has been reported. A variety of aldehydes and silyl enol ethers of ketones afforded the aldol products in excellent yields and good to excellent enantioselectivities. Mechanistic studies revealed that the actual catalyst may be changed from the silylated Brønsted acid to Brønsted acid itself depending on the reaction temperature. PMID:20465277

  3. Glutathione-dependent extracellular ferric reductase activities in dimorphic zoopathogenic fungi

    PubMed Central

    Zarnowski, Robert; Woods, Jon P.

    2009-01-01

    In this study, extracellular glutathione-dependent ferric reductase (GSH-FeR) activities in different dimorphic zoopathogenic fungal species were characterized. Supernatants from Blastomyces dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis and Sporothrix schenckii strains grown in their yeast form were able to reduce iron enzymically with glutathione as a cofactor. Some variations in the level of reduction were noted amongst the strains. This activity was stable in acidic, neutral and slightly alkaline environments and was inhibited when trivalent aluminium and gallium ions were present. Using zymography, single bands of GSH-FeRs with apparent molecular masses varying from 430 to 460 kDa were identified in all strains. The same molecular mass range was determined by size exclusion chromatography. These data demonstrate that dimorphic zoopathogenic fungi produce and secrete a family of similar GSH-FeRs that may be involved in the acquisition and utilization of iron. Siderophore production by these and other fungi has sometimes been considered to provide a full explanation of iron acquisition in these organisms. Our work reveals an additional common mechanism that may be biologically and pathogenically important. Furthermore, while some characteristics of these enzymes such as extracellular location, cofactor utilization and large size are not individually unique, when considered together and shared across a range of fungi, they represent an important novel physiological feature. PMID:16000713

  4. Cytosolic NADPH Homeostasis in Glucose-starved Procyclic Trypanosoma brucei Relies on Malic Enzyme and the Pentose Phosphate Pathway Fed by Gluconeogenic Flux*

    PubMed Central

    Allmann, Stefan; Morand, Pauline; Ebikeme, Charles; Gales, Lara; Biran, Marc; Hubert, Jane; Brennand, Ana; Mazet, Muriel; Franconi, Jean-Michel; Michels, Paul A. M.; Portais, Jean-Charles; Boshart, Michael; Bringaud, Frédéric

    2013-01-01

    All living organisms depend on NADPH production to feed essential biosyntheses and for oxidative stress defense. Protozoan parasites such as the sleeping sickness pathogen Trypanosoma brucei adapt to different host environments, carbon sources, and oxidative stresses during their infectious life cycle. The procyclic stage develops in the midgut of the tsetse insect vector, where they rely on proline as carbon source, although they prefer glucose when grown in rich media. Here, we investigate the flexible and carbon source-dependent use of NADPH synthesis pathways in the cytosol of the procyclic stage. The T. brucei genome encodes two cytosolic NADPH-producing pathways, the pentose phosphate pathway (PPP) and the NADP-dependent malic enzyme (MEc). Reverse genetic blocking of those pathways and a specific inhibitor (dehydroepiandrosterone) of glucose-6-phosphate dehydrogenase together established redundancy with respect to H2O2 stress management and parasite growth. Blocking both pathways resulted in ∼10-fold increase of susceptibility to H2O2 stress and cell death. Unexpectedly, the same pathway redundancy was observed in glucose-rich and glucose-depleted conditions, suggesting that gluconeogenesis can feed the PPP to provide NADPH. This was confirmed by (i) a lethal phenotype of RNAi-mediated depletion of glucose-6-phosphate isomerase (PGI) in the glucose-depleted Δmec/Δmec null background, (ii) an ∼10-fold increase of susceptibility to H2O2 stress observed for the Δmec/Δmec/RNAiPGI double mutant when compared with the single mutants, and (iii) the 13C enrichment of glycolytic and PPP intermediates from cells incubated with [U-13C]proline, in the absence of glucose. Gluconeogenesis-supported NADPH supply may also be important for nucleotide and glycoconjugate syntheses in the insect host. PMID:23665470

  5. 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

  6. Aldehyde dehydrogenase (ALDH) superfamily in plants: gene nomenclature and comparative genomics

    PubMed Central

    Brocker, Chad; Vasiliou, Melpomene; Carpenter, Sarah; Carpenter, Christopher; Zhang, Yucheng; Wang, Xiping; Kotchoni, Simeon O.; Wood, Andrew J.; Kirch, Hans-Hubert; Kopečný, David; Nebert, Daniel W.

    2012-01-01

    In recent years, there has been a significant increase in the number of completely sequenced plant genomes. The comparison of fully sequenced genomes allows for identification of new gene family members, as well as comprehensive analysis of gene family evolution. The aldehyde dehydrogenase (ALDH) gene superfamily comprises a group of enzymes involved in the NAD+- or NADP+-dependent conversion of various aldehydes to their corresponding carboxylic acids. ALDH enzymes are involved in processing many aldehydes that serve as biogenic intermediates in a wide range of metabolic pathways. In addition, many of these enzymes function as ‘aldehyde scavengers’ by removing reactive aldehydes generated during the oxidative degradation of lipid membranes, also known as lipid peroxidation. Plants and animals share many ALDH families, and many genes are highly conserved between these two evolutionarily distinct groups. Conversely, both plants and animals also contain unique ALDH genes and families. Herein we carried outgenome-wide identification of ALDH genes in a number of plant species—including Arabidopsis thaliana (thale crest), Chlamydomonas reinhardtii (unicellular algae), Oryza sativa (rice), Physcomitrella patens (moss), Vitis vinifera (grapevine) and Zea mays (maize). These data were then combined with previous analysis of Populus trichocarpa (poplar tree), Selaginella moellindorffii (gemmiferous spikemoss), Sorghum bicolor (sorghum) and Volvox carteri (colonial algae) for a comprehensive evolutionary comparison of the plant ALDH superfamily. As a result, newly identified genes can be more easily analyzed and gene names can be assigned according to current nomenclature guidelines; our goal is to clarify previously confusing and conflicting names and classifications that might confound results and prevent accurate comparisons between studies. PMID:23007552

  7. 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.

  8. Synthesis of robalzotan, ebalzotan, and rotigotine precursors via the stereoselective multienzymatic cascade reduction of α,β-unsaturated aldehydes.

    PubMed

    Brenna, Elisabetta; Gatti, Francesco G; Malpezzi, Luciana; Monti, Daniela; Parmeggiani, Fabio; Sacchetti, Alessandro

    2013-05-17

    A stereoselective synthesis of bicyclic primary or secondary amines, based on tetralin or chroman structural moieties, is reported. These amines are precursors of important active pharmaceutical ingredients such as rotigotine (Neupro), robalzotan, and ebalzotan. The key step is based on a multienzymatic reduction of an α,β-unsaturated aldehyde or ketone to give the saturated primary or secondary alcohol, in a high yield and with a high ee. The catalytic system consists of the combination of an ene-reductase (ER; i.e., OYE2 or OYE3 belonging to the Old Yellow Enzyme family) with an alcohol dehydrogenase (ADH), applying the in situ substrate feeding product removal technology. By this system the formation of the allylic alcohol side product and the racemization of the chirally unstable α-substituted aldehyde intermediate are minimized. The primary alcohols were elaborated via a Curtius rearrangement. The combination of OYE2 with a Prelog or an anti-Prelog ADH allowed the preparation of the secondary alcohols with ee > 99% and de > 87%. The absolute configuration of the primary amines was unambiguously assigned by comparison with authentic samples. The stereochemistry of secondary alcohols was assigned by X-ray crystal structure and NMR analysis of Mosher esters.

  9. Beyond benzoin condensation: trimerization of aldehydes via metal-free aerobic oxidative esterification of aldehydes with benzoin products in the presence of cyanide.

    PubMed

    Kim, Yoo-Jin; Kim, Na Yeun; Cheon, Cheol-Hong

    2014-05-02

    An unusual trimerization of aldehydes in the presence of cyanide via metal-free aerobic oxidative esterification under ambient conditions is described. Various aromatic aldehydes provided the corresponding oxidative esterification products in good to excellent yields. Mechanistic studies suggested that this reaction would proceed via a two-step sequence: cyanide-catalyzed benzoin condensation of aldehydes and subsequent aerobic oxidative esterification of aldehydes with the resultant benzoin products. The usefulness of this protocol was further demonstrated by converting the resulting trimeric products into other biologically important compounds.

  10. Rosuvastatin prevents angiotensin II-induced vascular changes by inhibition of NAD(P)H oxidase and COX-1

    PubMed Central

    Colucci, Rocchina; Fornai, Matteo; Duranti, Emiliano; Antonioli, Luca; Rugani, Ilaria; Aydinoglu, Fatma; Ippolito, Chiara; Segnani, Cristina; Bernardini, Nunzia; Taddei, Stefano; Blandizzi, Corrado; Virdis, Agostino

    2013-01-01

    Background and Purpose NAD(P)H oxidase and COX-1 participate in vascular damage induced by angiotensin II. We investigated the effect of rosuvastatin on endothelial dysfunction, vascular remodelling, changes in extracellular matrix components and mechanical properties of small mesenteric arteries from angiotensin II-infused rats. Experimental Approach Male rats received angiotensin II (120 ng·kg−1·min−1, subcutaneously) for 14 days with or without rosuvastatin (10 mg·kg−1·day−1, oral gavage) or vehicle. Vascular functions and morphological parameters were assessed by pressurized myography. Key Results In angiotensin II-infused rats, ACh-induced relaxation was attenuated compared with controls, less sensitive to L-NAME, enhanced by SC-560 (COX-1 inhibitor) or SQ-29548 (prostanoid TP receptor antagonist), and normalized by the antioxidant ascorbic acid or NAD(P)H oxidase inhibitors. After rosuvastatin, relaxations to ACh were normalized, fully sensitive to L-NAME, and no longer affected by SC-560, SQ-29548 or NAD(P)H oxidase inhibitors. Angiotensin II enhanced intravascular superoxide generation, eutrophic remodelling, collagen and fibronectin depositions, and decreased elastin content, resulting in increased vessel stiffness. All these changes were prevented by rosuvastatin. Angiotensin II increased phosphorylation of NAD(P)H oxidase subunit p47phox and its binding to subunit p67phox, effects inhibited by rosuvastatin. Rosuvastatin down-regulated vascular Nox4/NAD(P)H isoform and COX-1 expression, attenuated the vascular release of 6-keto-PGF1α, and enhanced copper/zinc-superoxide dismutase expression. Conclusion and Implications Rosuvastatin prevents angiotensin II-induced alterations in resistance arteries in terms of function, structure, mechanics and composition. These effects depend on restoration of NO availability, prevention of NAD(P)H oxidase-derived oxidant excess, reversal of COX-1 induction and its prostanoid production, and stimulation of

  11. RP-HPLC-fluorescence analysis of aliphatic aldehydes: application to aldehyde-generating enzymes HACL1 and SGPL1

    PubMed Central

    Mezzar, Serena; de Schryver, Evelyn; Van Veldhoven, Paul P.

    2014-01-01

    Long-chain aldehydes are commonly produced in various processes, such as peroxisomal α-oxidation of long-chain 3-methyl-branched and 2-hydroxy fatty acids and microsomal breakdown of phosphorylated sphingoid bases. The enzymes involved in the aldehyde-generating steps of these processes are 2-hydroxyacyl-CoA lyase (HACL1) and sphingosine-1-phosphate lyase (SGPL1), respectively. In the present work, nonradioactive assays for these enzymes were developed employing the Hantzsch reaction. Tridecanal (C13-al) and heptadecanal (C17-al) were selected as model compounds and cyclohexane-1,3-dione as 1,3-diketone, and the fluorescent derivatives were analyzed by reversed phase (RP)-HPLC. Assay mixture composition, as well as pH and heating, were optimized for C13-al and C17-al. Under optimized conditions, these aldehydes could be quantified in picomolar range and different long-chain aldehyde derivatives were well resolved with a linear gradient elution by RP-HPLC. Aldehydes generated by recombinant enzymes could easily be detected via this method. Moreover, the assay allowed to document activity or deficiency in tissue homogenates and fibroblast lysates without an extraction step. In conclusion, a simple, quick, and cheap assay for the study of HACL1 and SGPL1 activities was developed, without relying on expensive mass spectrometric detectors or radioactive substrates. PMID:24323699

  12. A LED-based method for monitoring NAD(P)H and FAD fluorescence in cell cultures and brain slices.

    PubMed

    Rösner, Jörg; Liotta, Agustin; Schmitz, Dietmar; Heinemann, Uwe; Kovács, Richard

    2013-01-30

    Nicotinamide- and flavine-adenine-dinucleotides (NAD(P)H and FADH₂) are electron carriers involved in cellular energy metabolism and in a multitude of enzymatic processes. As reduced NAD(P)H and oxidised FAD molecules are fluorescent, changes in tissue auto-fluorescence provide valuable information on the cellular redox state and energy metabolism. Since fluorescence excitation, by mercury arc lamps (HBO) is inherently coupled to photo-bleaching and photo-toxicity, microfluorimetric monitoring of energy metabolism might benefit from the replacement of HBO lamps by light emitting diodes (LEDs). Here we describe a LED-based custom-built setup for monitoring NAD(P)H and FAD fluorescence at the level of single cells (HEK293) and of brain slices. We compared NAD(P)H bleaching characteristics with two light sources (HBO lamp and LED) as well as sensitivity and signal to noise ratio of three different detector types (multi-pixel photon counter (MPPC), photomultiplier tube (PMT) and photodiode). LED excitation resulted in reduced photo-bleaching at the same fluorescence output in comparison to excitation with the HBO lamp. Transiently increasing LED power resulted in reversible bleaching of NAD(P)H fluorescence. Recovery kinetics were dependent on metabolic substrates indicating coupling of NAD(P)H fluorescence to metabolism. Electrical stimulation of brain slices induced biphasic redox changes, as indicated by NAD(P)H/FAD fluorescence transients. Increasing the gain of PMT and decreasing the LED power resulted in similar sensitivity as obtained with the MPPC and the photodiode, without worsening the signal to noise ratio. In conclusion, replacement of HBO lamp with LED might improve conventional PMT based microfluorimetry of tissue auto-fluorescence. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Cellular fatty acids and aldehydes of oral Eubacterium.

    PubMed

    Itoh, U; Sato, M; Tsuchiya, H; Namikawa, I

    1995-02-01

    The cellular fatty acids and aldehydes of oral Eubacterium species were determined by gas chromatography-mass spectrometry. E. brachy and E. lentum contained mainly branched-chain fatty acids, whereas the others contained straight-chain acids. E. brachy, E. lentum, E. yurii ssp. yurii, E. yurii spp. margaretiae, E. limosum, E. plauti and E. aerofaciens also contained aldehydes with even carbon numbers. In addition to species-specific components, the compositional ratios of fatty acids and aldehydes characterized each individual species. The 10 species tested were divided into 5 groups by the principal component analysis. Cellular fatty acids and aldehydes would be chemical markers for interspecies differentiation of oral Eubacterium.

  14. 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.

  15. Ribonucleotide reductase activity is regulated by proliferating cell nuclear antigen (PCNA)

    PubMed Central

    Salguero, Israel; Guarino, Estrella; Shepherd, Marianne; Deegan, Tom; Havens, Courtney G.; MacNeill, Stuart A.; Walter, Johannes C.; Kearsey, Stephen E.

    2014-01-01

    Summary Synthesis of dNTPs is required for both DNA replication and DNA repair and is catalyzed by ribonucleotide reductases (RNR), which convert ribonucleotides to their deoxy forms [1, 2]. Maintaining the correct levels of dNTPs for DNA synthesis is important for minimising the mutation rate [3-7], and this is achieved by tight regulation of ribonucleotide reductase [2, 8, 9]. In fission yeast, ribonucleotide reductase is regulated in part by a small protein inhibitor, Spd1, which is degraded in S phase and after DNA damage to allow up-regulation of dNTP supply [10-12]. Spd1 degradation is mediated by the activity of the CRL4Cdt2 ubiquitin ligase complex [5, 13, 14]. This has been reported to be dependent on modulation of Cdt2 levels which are cell cycle regulated, peaking in S phase, and which also increase after DNA damage in a checkpoint-dependent manner [7, 13]. We show here that Cdt2 levels fluctuations are not sufficient to regulate Spd1 proteolysis and that the key step in this event is the interaction of Spd1 with the polymerase processivity factor PCNA, complexed onto DNA. This mechanism thus provides a direct link between DNA synthesis and ribonucleotide reductase regulation. PMID:22464192

  16. 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.

  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. Regulation of the NADPH Oxidase RBOHD During Plant Immunity.

    PubMed

    Kadota, Yasuhiro; Shirasu, Ken; Zipfel, Cyril

    2015-08-01

    Pathogen recognition induces the production of reactive oxygen species (ROS) by NADPH oxidases in both plants and animals. ROS have direct antimicrobial properties, but also serve as signaling molecules to activate further immune outputs. However, ROS production has to be tightly controlled to avoid detrimental effects on host cells, but yet must be produced in the right amount, at the right place and at the right time upon pathogen perception. Plant NADPH oxidases belong to the respiratory burst oxidase homolog (RBOH) family, which contains 10 members in the model plant Arabidopsis thaliana. The perception of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) leads to a rapid, specific and strong production of ROS, which is dependent on RBOHD. RBOHD is mainly controlled by Ca(2+) via direct binding to EF-hand motifs and phosphorylation by Ca(2+)-dependent protein kinases. Recent studies have, however, revealed a critical role for a Ca(2+)-independent regulation of RBOHD. The plasma membrane-associated cytoplasmic kinase BIK1 (BOTRYTIS-INDUCED KINASE1), which is a direct substrate of the PRR complex, directly interacts with and phosphorylates RBOHD upon PAMP perception. Impairment of these phosphorylation events completely abolishes the function of RBOHD in immunity. These results suggest that RBOHD activity is tightly controlled by multilayered regulations. In this review, we summarize recent advances in our understanding of the regulatory mechanisms controlling RBOHD activation. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  19. 40 CFR 721.5762 - Aromatic aldehyde phenolic resin (generic).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Aromatic aldehyde phenolic resin... Specific Chemical Substances § 721.5762 Aromatic aldehyde phenolic resin (generic). (a) Chemical substance... aromatic aldehyde phenolic resin (PMN P-01-573) is subject to reporting under this section for the...

  20. 40 CFR 721.5762 - Aromatic aldehyde phenolic resin (generic).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Aromatic aldehyde phenolic resin... Specific Chemical Substances § 721.5762 Aromatic aldehyde phenolic resin (generic). (a) Chemical substance... aromatic aldehyde phenolic resin (PMN P-01-573) is subject to reporting under this section for the...

  1. 40 CFR 721.5762 - Aromatic aldehyde phenolic resin (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Aromatic aldehyde phenolic resin... Specific Chemical Substances § 721.5762 Aromatic aldehyde phenolic resin (generic). (a) Chemical substance... aromatic aldehyde phenolic resin (PMN P-01-573) is subject to reporting under this section for the...

  2. 40 CFR 721.5762 - Aromatic aldehyde phenolic resin (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Aromatic aldehyde phenolic resin... Specific Chemical Substances § 721.5762 Aromatic aldehyde phenolic resin (generic). (a) Chemical substance... aromatic aldehyde phenolic resin (PMN P-01-573) is subject to reporting under this section for the...

  3. 40 CFR 721.5762 - Aromatic aldehyde phenolic resin (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Aromatic aldehyde phenolic resin... Specific Chemical Substances § 721.5762 Aromatic aldehyde phenolic resin (generic). (a) Chemical substance... aromatic aldehyde phenolic resin (PMN P-01-573) is subject to reporting under this section for the...

  4. 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.

  5. Isolation of 2-deoxy-scyllo-inosose (DOI)-assimilating yeasts and cloning and characterization of the DOI reductase gene of Cryptococcus podzolicus ND1.

    PubMed

    Ara, Satoshi; Yamazaki, Harutake; Takaku, Hiroaki

    2018-04-01

    2-Deoxy-scyllo-inosose (DOI) is the first intermediate in the 2-deoxystreptamine-containing aminoglycoside antibiotic biosynthesis pathway and has a six-membered carbocycle structure. DOI is a valuable material because it is easily converted to aromatic compounds and carbasugar derivatives. In this study, we isolated yeast strains capable of assimilating DOI as a carbon source. One of the strains, Cryptococcus podzolicus ND1, mainly converted DOI to scyllo-quercitol and (-)-vibo-quercitol, which is a valuable compound used as an antihypoglycemia agent and as a heat storage material. An NADH-dependent DOI reductase coding gene, DOIR, from C. podzolicus ND1 was cloned and successfully overexpressed in Escherichia coli. The purified protein catalyzed the irreversible reduction of DOI with NADH and converted DOI into (-)-vibo-quercitol. The enzyme had an optimal pH of 8.5 and optimal temperature of 35°C, respectively. The k cat of this enzyme was 9.98 s -1 , and the K m values for DOI and NADH were 4.38 and 0.24 mM, respectively. The enzyme showed a strong preference for NADH and showed no activity with NADPH. Multiple-alignment analysis of DOI reductase revealed that it belongs to the GFO_IDH_MocA protein family and is an inositol dehydrogenase homolog in other fungi, such as Cryptococcus gattii, and bacteria, such as Bacillus subtilis. This is the first identification of a DOI-assimilating yeast and a gene involved in DOI metabolism in fungi. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  6. Inhibition of arsenic induced-rat liver injury by grape seed exact through suppression of NADPH oxidase and TGF-{beta}/Smad activation

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

    Pan Xinjuan; Dai Yujie; Li Xing

    2011-08-01

    Chronic arsenic exposure induces oxidative damage to liver leading to liver fibrosis. We aimed to define the effect of grape seed extract (GSE), an antioxidant dietary supplement, on arsenic-induced liver injury. First, Male Sprague-Dawley rats were exposed to a low level of arsenic in drinking water (30 ppm) with or without GSE (100 mg/kg, every other day by oral gavage) for 12 months and the effect of GSE on arsenic-induced hepatotoxicity was examined. The results from this study revealed that GSE co-treatment significantly attenuated arsenic-induced low antioxidant defense, oxidative damage, proinflammatory cytokines and fibrogenic genes. Moreover, GSE reduced arsenic-stimulated Smad2/3more » phosphorylation and protein levels of NADPH oxidase subunits (Nox2, Nox4 and p47phox). Next, we explored the molecular mechanisms underlying GSE inhibition of arsenic toxicity using cultured rat hepatic stellate cells (HSCs). From the in vitro study, we found that GSE dose-dependently reduced arsenic-stimulated ROS production and NADPH oxidase activities. Both NADPH oxidases flavoprotein inhibitor DPI and Nox4 siRNA blocked arsenic-induced ROS production, whereas Nox4 overexpression suppressed the inhibitory effects of GSE on arsenic-induced ROS production and NADPH oxidase activities, as well as expression of TGF-{beta}1, type I procollagen (Coll-I) and {alpha}-smooth muscle actin ({alpha}-SMA) mRNA. We also observed that GSE dose-dependently inhibited TGF-{beta}1-induced transactivation of the TGF-{beta}-induced smad response element p3TP-Lux, and that forced expression of Smad3 attenuated the inhibitory effects of GSE on TGF-{beta}1-induced mRNA expression of Coll-I and {alpha}-SMA. Collectively, GSE could be a potential dietary therapeutic agent for arsenic-induced liver injury through suppression of NADPH oxidase and TGF-{beta}/Smad activation. - Research Highlights: > GSE attenuated arsenic-induced low antioxidant defense, oxidative damage, proinflammatory cytokines

  7. Partial vinylphenol reductase purification and characterization from Brettanomyces bruxellensis.

    PubMed

    Tchobanov, Iavor; Gal, Laurent; Guilloux-Benatier, Michèle; Remize, Fabienne; Nardi, Tiziana; Guzzo, Jean; Serpaggi, Virginie; Alexandre, Hervé

    2008-07-01

    Brettanomyces is the major microbial cause for wine spoilage worldwide and causes significant economic losses. The reasons are the production of ethylphenols that lead to an unpleasant taint described as 'phenolic odour'. Despite its economic importance, Brettanomyces has remained poorly studied at the metabolic level. The origin of the ethylphenol results from the conversion of vinylphenols in ethylphenol by Brettanomyces hydroxycinnamate decarboxylase. However, no information is available on the vinylphenol reductase responsible for the conversion of vinylphenols in ethylphenols. In this study, a vinylphenol reductase was partially purified from Brettanomyces bruxellensis that was active towards 4-vinylguaiacol and 4-vinylphenol only among the substrates tested. First, a vinylphenol reductase activity assay was designed that allowed us to show that the enzyme was NADH dependent. The vinylphenol reductase was purified 152-fold with a recovery yield of 1.77%. The apparent K(m) and V(max) values for the hydrolysis of 4-vinylguaiacol were, respectively, 0.14 mM and 1900 U mg(-1). The optimal pH and temperature for vinylphenol reductase were pH 5-6 and 30 degrees C, respectively. The molecular weight of the enzyme was 26 kDa. Trypsic digest of the protein was performed and the peptides were sequenced, which allowed us to identify in Brettanomyces genome an ORF coding for a 210 amino acid protein.

  8. 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.

  9. P2x7 Receptor-NADPH Oxidase-Axis Mediates Protein radical Formation And Kupffer Cell Activation in Carbon Tetrachloride-Mediated Steatohepatitis in Obese Mice

    PubMed Central

    Chatterjee, Saurabh; Rana, Ritu; Corbett, Jean; Kadiiska, Maria B.; Goldstein, Joyce; Mason, Ronald P.

    2012-01-01

    While some studies show that carbon tetrachloride-mediated metabolic oxidative stress exacerbates steatohepatitic-like lesions in obese mice, the redox mechanisms that trigger the innate immune system and accentuate the inflammatory cascade remain unclear. Here we have explored the role of the purinergic receptor P2X7-NADPH oxidase axis as a primary event in recognizing the heightened release of extracellular ATP from CCl4-treated hepatocytes and generating redoxmediated Kupffer cell activation in obese mice. We found that an underlying condition of obesity led to the formation of protein radicals and post-translational nitration, primarily in Kupffer cells, at 24 h post-CCl4 administration. The free radical-mediated oxidation of cellular macromolecules, which was NADPH oxidase- and P2X7 receptor-dependent, correlated well with the release of TNF- α and MCP-2 from Kupffer cells. The Kupffer cells in CCl4-treated mice exhibited increased expression of MHC Class II proteins and showed an activated phenotype. Increased expression of MHC Class II was inhibited by the NADPH oxidase inhibitor apocynin , P2X7 receptor antagonist A438709 hydrochloride, and genetic deletions of the NADPH oxidase p47 phox subunit or the P2X7 receptor. The P2X7 receptor acted upstream of NADPH oxidase activation by up-regulating the expression of the p47 phox subunit and p47 phox binding to the membrane subunit, gp91 phox. We conclude that the P2X7 receptor is a primary mediator of oxidative stress-induced exacerbation of inflammatory liver injury in obese mice via NADPH oxidase-dependent mechanisms. PMID:22343416

  10. Role of Conserved Glycine in Zinc-dependent Medium Chain Dehydrogenase/Reductase Superfamily*

    PubMed Central

    Tiwari, Manish Kumar; Singh, Raushan Kumar; Singh, Ranjitha; Jeya, Marimuthu; Zhao, Huimin; Lee, Jung-Kul

    2012-01-01

    The medium-chain dehydrogenase/reductase (MDR) superfamily consists of a large group of enzymes with a broad range of activities. Members of this superfamily are currently the subject of intensive investigation, but many aspects, including the zinc dependence of MDR superfamily proteins, have not yet have been adequately investigated. Using a density functional theory-based screening strategy, we have identified a strictly conserved glycine residue (Gly) in the zinc-dependent MDR superfamily. To elucidate the role of this conserved Gly in MDR, we carried out a comprehensive structural, functional, and computational analysis of four MDR enzymes through a series of studies including site-directed mutagenesis, isothermal titration calorimetry, electron paramagnetic resonance (EPR), quantum mechanics, and molecular mechanics analysis. Gly substitution by other amino acids posed a significant threat to the metal binding affinity and activity of MDR superfamily enzymes. Mutagenesis at the conserved Gly resulted in alterations in the coordination of the catalytic zinc ion, with concomitant changes in metal-ligand bond length, bond angle, and the affinity (Kd) toward the zinc ion. The Gly mutants also showed different spectroscopic properties in EPR compared with those of the wild type, indicating that the binding geometries of the zinc to the zinc binding ligands were changed by the mutation. The present results demonstrate that the conserved Gly in the GHE motif plays a role in maintaining the metal binding affinity and the electronic state of the catalytic zinc ion during catalysis of the MDR superfamily enzymes. PMID:22500022

  11. 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.

  12. Electron spin resonance characterization of vascular xanthine and NAD(P)H oxidase activity in patients with coronary artery disease: relation to endothelium-dependent vasodilation.

    PubMed

    Spiekermann, Stephan; Landmesser, Ulf; Dikalov, Sergey; Bredt, Martin; Gamez, Graciela; Tatge, Helma; Reepschläger, Nina; Hornig, Burkhard; Drexler, Helmut; Harrison, David G

    2003-03-18

    Increased inactivation of nitric oxide by superoxide (O2*-) contributes to endothelial dysfunction in patients with coronary disease (CAD). We therefore characterized the vascular activities of xanthine oxidase and NAD(P)H oxidase, 2 major O2*--producing enzyme systems, and their relationship with flow-dependent, endothelium-mediated vasodilation (FDD) in patients with CAD. Xanthine- and NAD(P)H-mediated O*.- formation was determined in coronary arteries from 10 patients with CAD and 10 controls by using electron spin resonance spectroscopy. Furthermore, activity of endothelium-bound xanthine oxidase in vivo and FDD of the radial artery were determined in 21 patients with CAD and 10 controls. FDD was measured before and after infusion of the antioxidant vitamin C (25 mg/min i.a.) to determine the portion of FDD inhibited by radicals. In coronary arteries from patients with CAD, xanthine- and NAD(P)H-mediated O2*- formation was increased compared with controls (xanthine: 12+/-2 versus 7+/-1 nmol O2*-/ microg protein; NADH: 11+/-1 versus 7+/-1 nmol O2*-/ microg protein; and NADPH: 12+/-2 versus 9+/-1 nmol O2*-/ microg protein; each P<0.05). Endothelium-bound xanthine oxidase activity was increased by >200% in patients with CAD (25+/-4 versus 9+/-1 nmol O2*-/ microL plasma per min; P<0.05) and correlated inversely with FDD (r=-0.55; P<0.05) and positively with the effect of vitamin C on FDD (r=0.54; P<0.05). The present study represents the first electron spin resonance measurements of xanthine and NAD(P)H oxidase activity in human coronary arteries and supports the concept that increased activities of both enzymes contribute to increased vascular oxidant stress in patients with CAD. Furthermore, the present study suggests that increased xanthine oxidase activity contributes to endothelial dysfunction in patients with CAD and may thereby promote the atherosclerotic process.

  13. Betaine aldehyde dehydrogenase isozymes of spinach

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

    Hanson, A.D.; Weretilnyk, E.A.; Weigel, P.

    1986-04-01

    Betaine is synthesized in spinach chloroplasts via the pathway Choline ..-->.. Betaine Aldehyde ..-->.. Betaine; the second step is catalyzed by betaine aldehyde dehydrogenase (BADH). The subcellular distribution of BADH was determined in leaf protoplast lysates; BADH isozymes were separated by 6-9% native PAGE. The chloroplast stromal fraction contains a single BADH isozyme (number1) that accounts for > 80% of the total protoplast activity; the extrachloroplastic fraction has a minor isozyme (number2) which migrates more slowly than number1. Both isozymes appear specific for betaine aldehyde, are more active with NAD than NADP, and show a ca. 3-fold activity increase inmore » salinized leaves. The phenotype of a natural variant of isozyme number1 suggests that the enzyme is a dimer.« less

  14. 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.

  15. Cinnamic aldehyde: a survey of consumer patch-test sensitization.

    PubMed

    Danneman, P J; Booman, K A; Dorsky, J; Kohrman, K A; Rothenstein, A S; Sedlak, R I; Steltenkamp, R J; Thompson, G R

    1983-12-01

    The potential for cinnamic aldehyde, an important fragrance and flavour ingredient, to induce or to elicit delayed contact hypersensitivity reactions in man was evaluated by analysing patch-test data. Results of studies involving a total of 4117 patch tests on various consumer products and fragrance blends containing cinnamic aldehyde and on the material itself were collected from fragrance and formulator companies. The data indicate that cinnamic aldehyde contained in consumer products and fragrance blends at concentrations up to 6 X 10(-1)%, and patch-tested at concentrations up to 8 X 10(-3)%, has no detectable potential to induce hypersensitivity. Cinnamic aldehyde when tested alone induced a dose-related hypersensitivity response. According to published reports, cinnamic aldehyde elicited positive delayed hypersensitivity responses in dermatitic patients. However, results of the current survey show that when cinnamic aldehyde was tested alone or as part of a mixture in subjects in the general population, no pre-existing hypersensitivity reactions to the fragrance material were observed in any of the 4117 patch tests which constituted the survey. Cinnamic aldehyde at the concentrations contained in consumer products and fragrances, has a very low potential to induce hypersensitivity ('induced' reactions) or to elicit sensitization reactions ('elicited' reactions) in the general population.

  16. EMISSIONS OF ODOROUS ALDEHYDES FROM ALKYD PAINT

    EPA Science Inventory

    Aldehyde emissions are widely held responsible for the acrid after-odor of drying alkyd-based paint films. The aldehyde emissions from three different alkyd paints were measured in small environmental chambers. It was found that, for each alkyd paint applied, more than 2 mg of ...

  17. NADPH oxidase-mediated generation of reactive oxygen species is critically required for survival of undifferentiated human promyelocytic leukemia cell line HL-60.

    PubMed

    Dong, Jing-Mei; Zhao, Sheng-Guo; Huang, Guo-Yin; Liu, Qing

    2004-06-01

    Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) mediated generation of reactive oxygen species (ROS) was originally identified as the powerful host defense machinery against microorganism in phagocytes. But recent reports indicated that some non-phagocytic cells also have the NADPH oxidase activity, and the ROS produced by it may act as cell signal molecule. But as far as today, whether the NADPH oxidase also plays similar role in phagocyte has not been paid much attention. Utilizing the undifferentiated HL-60 promyelocytic leukemia cells as a model, the aim of the present study was to determine whether NADPH oxidase plays a role on ROS generation in undifferentiated HL-60, and the ROS mediated by it was essential for cell's survival. For the first time, we verified that the release of ROS in undifferentiated HL-60 was significantly increased by the stimulation with Calcium ionophore or opsonized zymosan, which are known to trigger respiration burst in phagocytes by NADPH oxidase pathway. Diphenylene iodonium (DPI) or apocynin (APO), two inhibitors of NADPH oxidase, significantly suppressed the increasing of ROS caused by opsonized zymosan. Cell survival assay and fluorescence double dyeing with acridine orange and ethidium bromide showed that DPI and APO, as well as superoxide dismutase (SOD) and catalase (CAT) concentration-dependently decreased the viability of undifferentiated HL-60 cells, whereas exogenous H2O2 can rescue the cells from death obviously. Our results suggested that the ROS, generated by NADPH oxidase play an essential role in the survival of undifferentiated HL-60 cells.

  18. Coniferyl aldehyde 5-hydroxylation and methylation direct syringyl lignin biosynthesis in angiosperms

    PubMed Central

    Osakabe, Keishi; Tsao, Cheng Chung; Li, Laigeng; Popko, Jacqueline L.; Umezawa, Toshiaki; Carraway, Daniel T.; Smeltzer, Richard H.; Joshi, Chandrashekhar P.; Chiang, Vincent L.

    1999-01-01

    A central question in lignin biosynthesis is how guaiacyl intermediates are hydroxylated and methylated to the syringyl monolignol in angiosperms. To address this question, we cloned cDNAs encoding a cytochrome P450 monooxygenase (LsM88) and a caffeate O-methyltransferase (COMT) from sweetgum (Liquidambar styraciflua) xylem. Mass spectrometry-based functional analysis of LsM88 in yeast identified it as coniferyl aldehyde 5-hydroxylase (CAld5H). COMT expressed in Escherichia coli methylated 5-hydroxyconiferyl aldehyde to sinapyl aldehyde. Together, CAld5H and COMT converted coniferyl aldehyde to sinapyl aldehyde, suggesting a CAld5H/COMT-mediated pathway from guaiacyl to syringyl monolignol biosynthesis via coniferyl aldehyde that contrasts with the generally accepted route to sinapate via ferulate. Although the CAld5H/COMT enzyme system can mediate the biosynthesis of syringyl monolignol intermediates through either route, kcat/Km of CAld5H for coniferyl aldehyde was ≈140 times greater than that for ferulate. More significantly, when coniferyl aldehyde and ferulate were present together, coniferyl aldehyde was a noncompetitive inhibitor (Ki = 0.59 μM) of ferulate 5-hydroxylation, thereby eliminating the entire reaction sequence from ferulate to sinapate. In contrast, ferulate had no effect on coniferyl aldehyde 5-hydroxylation. 5-Hydroxylation also could not be detected for feruloyl-CoA or coniferyl alcohol. Therefore, in the presence of coniferyl aldehyde, ferulate 5-hydroxylation does not occur, and the syringyl monolignol can be synthesized only from coniferyl aldehyde. Endogenous coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes were detected, consistent with in vivo operation of the CAld5H/COMT pathway from coniferyl to sinapyl aldehydes via 5-hydroxyconiferyl aldehyde for syringyl monolignol biosynthesis. PMID:10430877

  19. Cop9/signalosome subunits and Pcu4 regulate ribonucleotide reductase by both checkpoint-dependent and -independent mechanisms

    PubMed Central

    Liu, Cong; Powell, Kelly A.; Mundt, Kirsten; Wu, LeJung; Carr, Antony M.; Caspari, Thomas

    2003-01-01

    The signalosome is implicated in regulating cullin-dependent ubiquitin ligases. We find that two signalosome subunits, Csn1 and Csn2, are required to regulate ribonucleotide reductase (RNR) through the degradation of a small protein, Spd1, that acts to anchor the small RNR subunit in the nucleus. Spd1 destruction correlates with the nuclear export of the small RNR subunit, which, in turn, correlates with a requirement for RNR in replication and repair. Spd1 degradation is promoted by two separate CSN-dependent mechanisms. During unperturbed S phase, Spd1 degradation is independent of checkpoint proteins. In irradiated G2 cells, Spd1 degradation requires the DNA damage checkpoint. The signalosome copurifies with Pcu4 (cullin 4). Pcu4, Csn1, and Csn2 promote the degradation of Spd1, identifying a new function for the signalosome as a regulator of Pcu4-containing E3 ubiquitin ligase. PMID:12695334

  20. 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.

  1. 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.

  2. 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

  3. Improved strategies for electrochemical 1,4-NAD(P)H2 regeneration: A new era of bioreactors for industrial biocatalysis.

    PubMed

    Morrison, Clifford S; Armiger, William B; Dodds, David R; Dordick, Jonathan S; Koffas, Mattheos A G

    Industrial enzymatic reactions requiring 1,4-NAD(P)H 2 to perform redox transformations often require convoluted coupled enzyme regeneration systems to regenerate 1,4-NAD(P)H 2 from NAD(P) and recycle the cofactor for as many turnovers as possible. Renewed interest in recycling the cofactor via electrochemical means is motivated by the low cost of performing electrochemical reactions, easy monitoring of the reaction progress, and straightforward product recovery. However, electrochemical cofactor regeneration methods invariably produce adventitious reduced cofactor side products which result in unproductive loss of input NAD(P). We review various literature strategies for mitigating adventitious product formation by electrochemical cofactor regeneration systems, and offer insight as to how a successful electrochemical bioreactor system could be constructed to engineer efficient 1,4-NAD(P)H 2 -dependent enzyme reactions of interest to the industrial biocatalysis community. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. NAD(P)H oxidase mediates the endothelial barrier dysfunction induced by TNF-alpha.

    PubMed

    Gertzberg, Nancy; Neumann, Paul; Rizzo, Victor; Johnson, Arnold

    2004-01-01

    We tested the hypothesis that the NAD(P)H oxidase-dependent generation of superoxide anion (O2-*) mediates tumor necrosis factor-alpha (TNF)-induced alterations in the permeability of pulmonary microvessel endothelial monolayers (PMEM). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. The NAD(P)H oxidase subcomponents p47phox and p22phox were assessed by immunofluorescent microscopy and Western blot. The reactive oxygen species O2-* was measured by the fluorescence of 6-carboxy-2',7'-dichlorodihydrofluorescein diacetatedi(acetoxymethyl ester), 5 (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate-acetyl ester, and dihydroethidium. TNF treatment (50 ng/ml for 4.0 h) induced 1) p47phox translocation, 2) an increase in p22phox protein, 3) increased localization of p47phox with p22phox, 4) O2-* generation, and 5) increased permeability to albumin. p22phox antisense oligonucleotide prevented the TNF-induced effect on p22phox, p47phox, O2-*, and permeability. The scrambled nonsense oligonucleotide had no effect. The TNF-induced increase in O2-* and permeability to albumin was also prevented by the O2-* scavenger Cu-Zn superoxide dismutase (100 U/ml). The results indicate that the activation of NAD(P)H oxidase, via the generation of O2-*, mediates TNF-induced barrier dysfunction in PMEM.

  5. 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.

  6. Sinorhizobium meliloti requires a cobalamin-dependent ribonucleotide reductase for symbiosis with its plant host

    PubMed Central

    Taga, Michiko E.; Walker, Graham C.

    2010-01-01

    Vitamin B12 (cobalamin) is a critical cofactor for animals and protists, yet its biosynthesis is limited to prokaryotes. We previously showed that the symbiotic nitrogen-fixing alphaproteobacterium Sinorhizobium meliloti requires cobalamin to establish a symbiotic relationship with its plant host, Medicago sativa (alfalfa). Here, the specific requirement for cobalamin in the S. meliloti-alfalfa symbiosis was investigated. Of the three known cobalamin-dependent enzymes in S. meliloti, the methylmalonyl CoA mutase (BhbA) does not affect symbiosis whereas disruption of the metH gene encoding the cobalamin-dependent methionine synthase causes a significant defect in symbiosis. Expression of the cobalamin-independent methionine synthase MetE alleviates this symbiotic defect, indicating that the requirement for methionine synthesis does not reflect a need for the cobalamin-dependent enzyme. To investigate the function of the cobalamin-dependent ribonucleotide reductase (RNR) encoded by nrdJ, S. meliloti was engineered to express an Escherichia coli cobalamin-independent (Class Ia) RNR instead of nrdJ. This strain is severely defective in symbiosis. Electron micrographs show that these cells can penetrate alfalfa nodules but are unable to differentiate into nitrogen-fixing bacteroids and instead are lysed in the plant cytoplasm. Flow cytometry analysis indicates that these bacteria are largely unable to undergo endoreduplication. These phenotypes may be due to the inactivation of the Class Ia RNR by reactive oxygen species and/or inadequate oxygen availability in the nodule. These results show that the critical role of the cobalamin-dependent RNR for survival of S. meliloti in its plant host can account for the considerable resources that S. meliloti dedicates to cobalamin biosynthesis. PMID:20698752

  7. Purification and characterization of a novel recombinant highly enantioselective short-chain NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus.

    PubMed

    Pennacchio, Angela; Pucci, Biagio; Secundo, Francesco; La Cara, Francesco; Rossi, Mosè; Raia, Carlo A

    2008-07-01

    The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the short-chain dehydrogenase/reductase (SDR) superfamily was identified in the extremely thermophilic, halotolerant gram-negative eubacterium Thermus thermophilus HB27. The T. thermophilus ADH gene (adh(Tt)) was heterologously overexpressed in Escherichia coli, and the protein (ADH(Tt)) was purified to homogeneity and characterized. ADH(Tt) is a tetrameric enzyme consisting of identical 26,961-Da subunits composed of 256 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to approximately 73 degrees C and a 30-min half-inactivation temperature of approximately 90 degrees C, as well as good tolerance to common organic solvents. ADH(Tt) has a strict requirement for NAD(H) as the coenzyme, a preference for reduction of aromatic ketones and alpha-keto esters, and poor activity on aromatic alcohols and aldehydes. This thermophilic enzyme catalyzes the following reactions with Prelog specificity: the reduction of acetophenone, 2,2,2-trifluoroacetophenone, alpha-tetralone, and alpha-methyl and alpha-ethyl benzoylformates to (S)-(-)-1-phenylethanol (>99% enantiomeric excess [ee]), (R)-alpha-(trifluoromethyl)benzyl alcohol (93% ee), (S)-alpha-tetralol (>99% ee), methyl (R)-(-)-mandelate (92% ee), and ethyl (R)-(-)-mandelate (95% ee), respectively, by way of an efficient in situ NADH-recycling system involving 2-propanol and a second thermophilic ADH. This study further supports the critical role of the D37 residue in discriminating NAD(H) from NADP(H) in members of the SDR superfamily.

  8. 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

  9. Chiral Brønsted Acid-Catalyzed Allylboration of Aldehydes

    PubMed Central

    Jain, Pankaj; Antilla, Jon C.

    2010-01-01

    The catalytic enantioselective allylation of aldehydes is a long-standing problem with considerable interest to the chemical community. We wish to disclose a new high yielding and highly enantioselective chiral Brønsted acid-catalyzed allylboration of aldehydes. The reaction is shown to be highly general, with broad substrate scope that covers aryl, heteroaryl, α,β-unsaturated, and aliphatic aldehydes. The reaction conditions were also shown to be effective for the catalytic enantioselective crotylation of aldehydes. We believe that the high reactivity of the allyl boronate is due to protonation of the boronate oxygen by the chiral phosphoric acid catalyst. PMID:20690662

  10. Aldehydes with high and low toxicities inactivate cells by damaging distinct cellular targets.

    PubMed

    Xie, Ming-Zhang; Shoulkamy, Mahmoud I; Salem, Amir M H; Oba, Shunya; Goda, Mizuki; Nakano, Toshiaki; Ide, Hiroshi

    2016-04-01

    Aldehydes are genotoxic and cytotoxic molecules and have received considerable attention for their associations with the pathogenesis of various human diseases. In addition, exposure to anthropogenic aldehydes increases human health risks. The general mechanism of aldehyde toxicity involves adduct formation with biomolecules such as DNA and proteins. Although the genotoxic effects of aldehydes such as mutations and chromosomal aberrations are directly related to DNA damage, the role of DNA damage in the cytotoxic effects of aldehydes is poorly understood because concurrent protein damage by aldehydes has similar effects. In this study, we have analysed how saturated and α,β-unsaturated aldehydes exert cytotoxic effects through DNA and protein damage. Interestingly, DNA repair is essential for alleviating the cytotoxic effect of weakly toxic aldehydes such as saturated aldehydes but not highly toxic aldehydes such as long α,β-unsaturated aldehydes. Thus, highly toxic aldehydes inactivate cells exclusively by protein damage. Our data suggest that DNA interstrand crosslinks, but not DNA-protein crosslinks and DNA double-strand breaks, are the critical cytotoxic DNA damage induced by aldehydes. Further, we show that the depletion of intracellular glutathione and the oxidation of thioredoxin 1 partially account for the DNA damage-independent cytotoxicity of aldehydes. On the basis of these findings, we have proposed a mechanistic model of aldehyde cytotoxicity mediated by DNA and protein damage. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Colorimetric Recognition of Aldehydes and Ketones.

    PubMed

    Li, Zheng; Fang, Ming; LaGasse, Maria K; Askim, Jon R; Suslick, Kenneth S

    2017-08-07

    A colorimetric sensor array has been designed for the identification of and discrimination among aldehydes and ketones in vapor phase. Due to rapid chemical reactions between the solid-state sensor elements and gaseous analytes, distinct color difference patterns were produced and digitally imaged for chemometric analysis. The sensor array was developed from classical spot tests using aniline and phenylhydrazine dyes that enable molecular recognition of a wide variety of aliphatic or aromatic aldehydes and ketones, as demonstrated by hierarchical cluster, principal component, and support vector machine analyses. The aldehyde/ketone-specific sensors were further employed for differentiation among and identification of ten liquor samples (whiskies, brandy, vodka) and ethanol controls, showing its potential applications in the beverage industry. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. 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.

  13. 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

  14. An aldo-keto reductase, Bbakr1, is involved in stress response and detoxification of heavy metal chromium but not required for virulence in the insect fungal pathogen, Beauveria bassiana.

    PubMed

    Wang, Huifang; He, Zhangjiang; Luo, Linli; Zhao, Xin; Lu, Zhuoyue; Luo, Tingying; Li, Min; Zhang, Yongjun

    2018-02-01

    The aldo-keto reductases (AKRs) belong to the NADP-dependent oxidoreductase superfamily, which play important roles in various physiological functions in prokaryotic and eukaryotic organisms. However, many AKR superfamily members remain uncharacterized. Here, a downstream target gene of the HOG1 MAPK pathways coding for an aldo-keto reductase, named Bbakr1, was characterized in the insect fungal pathogen, Beauveria bassiana. Bbakr1 expression increased in response to osmotic and salt stressors, and oxidative and heavy metal (chromium) stress. Deletion of Bbakr1 caused a reduction in conidiation, as well as delayed conidial germination. ΔBbakr1 displayed increased sensitivity to osmotic/high-salt stress with decreased compatible solute accumulation. In addition, the mutant was more sensitive to high concentrations of the heavy metal, chromium, and to oxidative stress than the wild type cells, with impaired ability to detoxify active aldehyde that might accumulate due to lipid peroxidation. However, over-expressing Bbakr1 in either the wild type strain or a ΔBbhog1 background did not cause any obvious changes in phenotypes as compared to their controls. Little effect on virulence was seen for either the ΔBbakr1 or overexpression strains in insect bioassays via cuticle infection or intrahemocoel injection assays, suggesting that Bbakr1 is not required for virulence. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Kinetics of Forming Aldehydes in Frying Oils and Their Distribution in French Fries Revealed by LC-MS-Based Chemometrics.

    PubMed

    Wang, Lei; Csallany, A Saari; Kerr, Brian J; Shurson, Gerald C; Chen, Chi

    2016-05-18

    In this study, the kinetics of aldehyde formation in heated frying oils was characterized by 2-hydrazinoquinoline derivatization, liquid chromatography-mass spectrometry (LC-MS) analysis, principal component analysis (PCA), and hierarchical cluster analysis (HCA). The aldehydes contributing to time-dependent separation of heated soybean oil (HSO) in a PCA model were grouped by the HCA into three clusters (A1, A2, and B) on the basis of their kinetics and fatty acid precursors. The increases of 4-hydroxynonenal (4-HNE) and the A2-to-B ratio in HSO were well-correlated with the duration of thermal stress. Chemometric and quantitative analysis of three frying oils (soybean, corn, and canola oils) and French fry extracts further supported the associations between aldehyde profiles and fatty acid precursors and also revealed that the concentrations of pentanal, hexanal, acrolein, and the A2-to-B ratio in French fry extracts were more comparable to their values in the frying oils than other unsaturated aldehydes. All of these results suggest the roles of specific aldehydes or aldehyde clusters as novel markers of the lipid oxidation status for frying oils or fried foods.

  16. Cigarette Filter Ventilation and Smoking Protocol Influence Aldehyde Smoke Yields

    PubMed Central

    2018-01-01

    The WHO study group on tobacco product regulation (TobReg) advised regulating and lowering toxicant levels in cigarette smoke. Aldehydes are one of the chemical classes on the TobReg smoke toxicants priority list. To provide insight in factors determining aldehyde yields, the levels of 12 aldehydes in mainstream cigarette smoke of 11 Dutch brands were quantified. Variations in smoking behavior and cigarette design affecting human exposure to aldehydes were studied by using four different machine testing protocols. Machine smoking was based on the International Standardization Organization (ISO) and Health Canada Intense (HCI) regime, both with and without taping the filter vents. The 11 cigarette brands differed in (i) design and blend characteristics; (ii) tar, nicotine, and carbon monoxide (TNCO) levels; (iii) popularity; and (iv) manufacturer. Cigarette smoke was trapped on a Cambridge filter pad and carboxen cartridge. After being dissolved in methanol/CS2 and derivatization with DNPH, the aldehyde yields were determined using HPLC-DAD. Using an intense smoking regime (increased puff volume, shorter puff interval) significantly increased aldehyde yields, following the pattern: ISO < ISO-taped < HCI-untaped < HCI. For all of the regimes, acetaldehyde and acrolein yields were strongly correlated (r = 0.804). The difference in TNCO and aldehyde levels between regular and highly ventilated low-TNCO cigarettes (as measured using ISO) diminished when smoking intensely; this effect is stronger when combined with taping filter vents. The highly ventilated low-TNCO brands showed six times more aldehyde production per mg nicotine for the intense smoking regimes. In conclusion, acetaldehyde and acrolein can be used as representatives for the class of volatile aldehydes for the different brands and smoking regimes. The aldehyde-to-nicotine ratio increased when highly ventilated cigarettes were smoked intensely, similar to real smokers. Thus, a smoker of highly ventilated

  17. Arginase Inhibition Suppresses Native Low-Density Lipoprotein-Stimulated Vascular Smooth Muscle Cell Proliferation by NADPH Oxidase Inactivation.

    PubMed

    Koo, Bon Hyeock; Yi, Bong Gu; Wang, Wi Kwang; Ko, In Young; Hoe, Kwang Lae; Kwon, Young Guen; Won, Moo Ho; Kim, Young Myeong; Lim, Hyun Kyo; Ryoo, Sungwoo

    2018-05-01

    Vascular smooth muscle cell (VSMC) proliferation induced by native low-density lipoprotein (nLDL) stimulation is dependent on superoxide production from activated NADPH oxidase. The present study aimed to investigate whether the novel arginase inhibitor limonin could suppress nLDL-induced VSMC proliferation and to examine related mechanisms. Isolated VSMCs from rat aortas were treated with nLDL, and cell proliferation was measured by WST-1 and BrdU assays. NADPH oxidase activation was evaluated by lucigenin-induced chemiluminescence, and phosphorylation of protein kinase C (PKC) βII and extracellular signal-regulated kinase (ERK) 1/2 was determined by western blot analysis. Mitochondrial reactive oxygen species (ROS) generation was assessed using MitoSOX-red, and intracellular L-arginine concentrations were determined by high-performance liquid chromatography (HPLC) in the presence or absence of limonin. Limonin inhibited arginase I and II activity in the uncompetitive mode, and prevented nLDL-induced VSMC proliferation in a p21Waf1/Cip1-dependent manner without affecting arginase protein levels. Limonin blocked PKCβII phosphorylation, but not ERK1/2 phosphorylation, and translocation of p47phox to the membrane was decreased, as was superoxide production in nLDL-stimulated VSMCs. Moreover, mitochondrial ROS generation was increased by nLDL stimulation and blocked by preincubation with limonin. Mitochondrial ROS production was responsible for the phosphorylation of PKCβII. HPLC analysis showed that arginase inhibition with limonin increases intracellular L-arginine concentrations, but decreases polyamine concentrations. L-Arginine treatment prevented PKCβII phosphorylation without affecting ERK1/2 phosphorylation. Increased L-arginine levels following limonin-dependent arginase inhibition prohibited NADPH oxidase activation in a PKCβII-dependent manner, and blocked nLDL-stimulated VSMC proliferation. © Copyright: Yonsei University College of Medicine 2018.

  18. Arginase Inhibition Suppresses Native Low-Density Lipoprotein-Stimulated Vascular Smooth Muscle Cell Proliferation by NADPH Oxidase Inactivation

    PubMed Central

    Wang, Wi-Kwang; Ko, In-Young; Hoe, Kwang-Lae; Kwon, Young-Guen; Won, Moo-Ho; Kim, Young-Myeong

    2018-01-01

    Purpose Vascular smooth muscle cell (VSMC) proliferation induced by native low-density lipoprotein (nLDL) stimulation is dependent on superoxide production from activated NADPH oxidase. The present study aimed to investigate whether the novel arginase inhibitor limonin could suppress nLDL-induced VSMC proliferation and to examine related mechanisms. Materials and Methods Isolated VSMCs from rat aortas were treated with nLDL, and cell proliferation was measured by WST-1 and BrdU assays. NADPH oxidase activation was evaluated by lucigenin-induced chemiluminescence, and phosphorylation of protein kinase C (PKC) βII and extracellular signal-regulated kinase (ERK) 1/2 was determined by western blot analysis. Mitochondrial reactive oxygen species (ROS) generation was assessed using MitoSOX-red, and intracellular L-arginine concentrations were determined by high-performance liquid chromatography (HPLC) in the presence or absence of limonin. Results Limonin inhibited arginase I and II activity in the uncompetitive mode, and prevented nLDL-induced VSMC proliferation in a p21Waf1/Cip1-dependent manner without affecting arginase protein levels. Limonin blocked PKCβII phosphorylation, but not ERK1/2 phosphorylation, and translocation of p47phox to the membrane was decreased, as was superoxide production in nLDL-stimulated VSMCs. Moreover, mitochondrial ROS generation was increased by nLDL stimulation and blocked by preincubation with limonin. Mitochondrial ROS production was responsible for the phosphorylation of PKCβII. HPLC analysis showed that arginase inhibition with limonin increases intracellular L-arginine concentrations, but decreases polyamine concentrations. L-Arginine treatment prevented PKCβII phosphorylation without affecting ERK1/2 phosphorylation. Conclusion Increased L-arginine levels following limonin-dependent arginase inhibition prohibited NADPH oxidase activation in a PKCβII-dependent manner, and blocked nLDL-stimulated VSMC proliferation. PMID

  19. 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

  20. 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.