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Sample records for nadph-dependent oxidoreductase genes

  1. 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

  2. Structure, Activity and Stereoselectivity of NADPH-Dependent Oxidoreductases Catalysing the S-Selective Reduction of the Imine Substrate 2-Methylpyrroline.

    PubMed

    Man, Henry; Wells, Elizabeth; Hussain, Shahed; Leipold, Friedemann; Hart, Sam; Turkenburg, Johan P; Turner, Nicholas J; Grogan, Gideon

    2015-05-04

    Oxidoreductases from Streptomyces sp. GF3546 [3546-IRED], Bacillus cereus BAG3X2 (BcIRED) and Nocardiopsis halophila (NhIRED) each reduce prochiral 2-methylpyrroline (2MPN) to (S)-2-methylpyrrolidine with >95 % ee and also a number of other imine substrates with good selectivity. Structures of BcIRED and NhIRED have helped to identify conserved active site residues within this subgroup of imine reductases that have S selectivity towards 2MPN, including a tyrosine residue that has a possible role in catalysis and superimposes with an aspartate in related enzymes that display R selectivity towards the same substrate. Mutation of this tyrosine residue-Tyr169-in 3546-IRED to Phe resulted in a mutant of negligible activity. The data together provide structural evidence for the location and significance of the Tyr residue in this group of imine reductases, and permit a comparison of the active sites of enzymes that reduce 2MPN with either R or S selectivity.

  3. Characterization of the Saccharomyces cerevisiae YMR318C (ADH6) gene product as a broad specificity NADPH-dependent alcohol dehydrogenase: relevance in aldehyde reduction.

    PubMed Central

    Larroy, Carol; Fernández, M Rosario; González, Eva; Parés, Xavier; Biosca, Josep A

    2002-01-01

    YMR318C represents an open reading frame from Saccharomyces cerevisiae with unknown function. It possesses a conserved sequence motif, the zinc-containing alcohol dehydrogenase (ADH) signature, specific to the medium-chain zinc-containing ADHs. In the present study, the YMR318C gene product has been purified to homogeneity from overexpressing yeast cells, and found to be a homodimeric ADH, composed of 40 kDa subunits and with a pI of 5.0-5.4. The enzyme was strictly specific for NADPH and was active with a wide variety of substrates, including aliphatic (linear and branched-chain) and aromatic primary alcohols and aldehydes. Aldehydes were processed with a 50-fold higher catalytic efficiency than that for the corresponding alcohols. The highest k(cat)/K(m) values were found with pentanal>veratraldehyde > hexanal > 3-methylbutanal >cinnamaldehyde. Taking into consideration the substrate specificity and sequence characteristics of the YMR318C gene product, we have proposed this gene to be called ADH6. The disruption of ADH6 was not lethal for the yeast under laboratory conditions. Although S. cerevisiae is considered a non lignin-degrading organism, the catalytic activity of ADHVI can direct veratraldehyde and anisaldehyde, arising from the oxidation of lignocellulose by fungal lignin peroxidases, to the lignin biodegradation pathway. ADHVI is the only S. cerevisiae enzyme able to significantly reduce veratraldehyde in vivo, and its overexpression allowed yeast to grow under toxic concentrations of this aldehyde. The enzyme may also be involved in the synthesis of fusel alcohols. To our knowledge this is the first NADPH-dependent medium-chain ADH to be characterized in S. cerevisiae. PMID:11742541

  4. NADPH-dependent reductive biotransformation with Escherichia coli and its pfkA deletion mutant: influence on global gene expression and role of oxygen supply.

    PubMed

    Siedler, Solvej; Bringer, Stephanie; Polen, Tino; Bott, Michael

    2014-10-01

    An Escherichia coli ΔpfkA mutant lacking the major phosphofructokinase possesses a partially cyclized pentose phosphate pathway leading to an increased NADPH per glucose ratio. This effect decreases the amount of glucose required for NADPH regeneration in reductive biotransformations, such as the conversion of methyl acetoacetate (MAA) to (R)-methyl 3-hydroxybutyrate (MHB) by an alcohol dehydrogenase from Lactobacillus brevis. Here, global transcriptional analyses were performed to study regulatory responses during reductive biotransformation. DNA microarray analysis revealed amongst other things increased expression of soxS, supporting previous results indicating that a high NADPH demand contributes to the activation of SoxR, the transcriptional activator of soxS. Furthermore, several target genes of the ArcAB two-component system showed a lower mRNA level in the reference strain than in the ΔpfkA mutant, pointing to an increased QH2 /Q ratio in the reference strain. This prompted us to analyze yields and productivities of MAA reduction to MHB under different oxygen regimes in a bioreactor. Under anaerobic conditions, the specific MHB production rates of both strains were comparable (7.4 ± 0.2 mmolMHB  h(-1)  gcdw (-1) ) and lower than under conditions of 15% dissolved oxygen, where those of the reference strain (12.8 mmol h(-1)  gcdw (-1) ) and of the ΔpfkA mutant (11.0 mmol h(-1)  gcdw (-1) ) were 73% and 49% higher. While the oxygen transfer rate (OTR) of the reference strain increased after the addition of MAA, presumably due to the oxidation of the acetate accumulated before MAA addition, the OTR of the ΔpfkA strain strongly decreased, indicating a very low respiration rate despite sufficient oxygen supply. The latter effect can likely be attributed to a restricted conversion of NADPH into NADH via the soluble transhydrogenase SthA, as the enzyme is outcompeted in the presence of MAA by the recombinant NADPH-dependent alcohol

  5. A NADPH-dependent (S)-imine reductase (SIR) from Streptomyces sp. GF3546 for asymmetric synthesis of optically active amines: purification, characterization, gene cloning, and expression.

    PubMed

    Mitsukura, Koichi; Kuramoto, Tatsuya; Yoshida, Toyokazu; Kimoto, Norihiro; Yamamoto, Hiroaki; Nagasawa, Toru

    2013-09-01

    A NADPH-dependent (S)-imine reductase (SIR) was purified to be homogeneous from the cell-free extract of Streptomyces sp. GF3546. SIR appeared to be a homodimer protein with subunits of 30.5 kDa based on SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It also catalyzed the (S)-enantioselective reduction of not only 2-methyl-1-pyrroline (2-MPN) but also 1-methyl-3,4-dihydroisoquinoline and 6,7-dimethoxy-1-methyl-3,4-dihydroisoquinoline. Specific activities for their imines were 130, 44, and 2.6 nmol min(-1) mg(-1), and their optical purities were 92.7 % ee, 96.4 % ee, and >99 % ee, respectively. Using a NADPH-regenerating system, 10 mM 2-MPN was converted to amine with 100 % conversion and 92 % ee after 24 h. The amino acid sequence analysis revealed that SIR showed about 60 % identity to 6-phosphogluconate dehydrogenase. However, it showed only 37 % identity with Streptomyces sp. GF3587 (R)-imine reductase. Expression of SIR in Escherichia coli was achieved, and specific activity of the cell-free extract was about two times higher than that of the cell-free extract of Streptomyces sp. GF3546.

  6. Functional characterization of three genes encoding putative oxidoreductases required for cercosporin toxin biosynthesis in the fungus Cercospora nicotianae.

    PubMed

    Chen, Hui-Qin; Lee, Miin-Huey; Chung, Kuang-Ren

    2007-08-01

    Cercosporin is a non-host-selective, photoactivated polyketide toxin produced by many phytopathogenic Cercospora species, which plays a crucial role during pathogenesis on host plants. Upon illumination, cercosporin converts oxygen molecules to toxic superoxide and singlet oxygen that damage various cellular components and induce lipid peroxidation and electrolyte leakage. Three genes (CTB5, CTB6 and CTB7) encoding putative FAD/FMN- or NADPH-dependent oxidoreductases in the cercosporin toxin biosynthetic pathway of C. nicotianae were functionally analysed. Replacement of each gene via double recombination was utilized to create null mutant strains that were completely impaired in cercosporin production as a consequence of specific interruption at the CTB5, CTB6 or CTB7 locus. Expression of CTB1, CTB5, CTB6, CTB7 and CTB8 was drastically reduced or nearly abolished when CTB5, CTB6 or CTB7 was disrupted. Production of cercosporin was revived when a functional gene cassette was introduced into the respective mutants. All ctb5, ctb6 and ctb7 null mutants retained wild-type levels of resistance against toxicity of cercosporin or singlet-oxygen-generating compounds, indicating that none of the genes plays a role in self-protection.

  7. NADPH-dependent Reductases Involved in the Detoxification of Reactive Carbonyls in Plants*

    PubMed Central

    Yamauchi, Yasuo; Hasegawa, Ayaka; Taninaka, Ai; Mizutani, Masaharu; Sugimoto, Yukihiro

    2011-01-01

    Reactive carbonyls, especially α,β-unsaturated carbonyls produced through lipid peroxidation, damage biomolecules such as proteins and nucleotides; elimination of these carbonyls is therefore essential for maintaining cellular homeostasis. In this study, we focused on an NADPH-dependent detoxification of reactive carbonyls in plants and explored the enzyme system involved in this detoxification process. Using acrolein (CH2 = CHCHO) as a model α,β-unsaturated carbonyl, we purified a predominant NADPH-dependent acrolein-reducing enzyme from cucumber leaves, and we identified the enzyme as an alkenal/one oxidoreductase (AOR) catalyzing reduction of an α,β-unsaturated bond. Cloning of cDNA encoding AORs revealed that cucumber contains two distinct AORs, chloroplastic AOR and cytosolic AOR. Homologs of cucumber AORs were found among various plant species, including Arabidopsis, and we confirmed that a homolog of Arabidopsis (At1g23740) also had AOR activity. Phylogenetic analysis showed that these AORs belong to a novel class of AORs. They preferentially reduced α,β-unsaturated ketones rather than α,β-unsaturated aldehydes. Furthermore, we selected candidates of other classes of enzymes involved in NADPH-dependent reduction of carbonyls based on the bioinformatic information, and we found that an aldo-keto reductase (At2g37770) and aldehyde reductases (At1g54870 and At3g04000) were implicated in the reduction of an aldehyde group of saturated aldehydes and methylglyoxal as well as α,β-unsaturated aldehydes in chloroplasts. These results suggest that different classes of NADPH-dependent reductases cooperatively contribute to the detoxification of reactive carbonyls. PMID:21169366

  8. Identification of a gene sequence encoding a putative pyruvate oxidoreductase in Serpulina pilosicoli.

    PubMed

    Rayment, S J; Lee, B J; Hampson, D J; Livesley, M A

    1998-09-01

    Serpulina pilosicoli is a recently described species of intestinal spirochaete which can be identified using a species-specific monoclonal antibody BJL/AC1 reactive with a 29-kDa protein located in the cell envelope. A genomic library of the type strain of S. pilosicoli P43/6/78T was created in lambda zap express and screened using BJL/AC1. Single positive clones were isolated and excised into the phagemid vector pBK-CMV. Phagemid DNA was purified and a single clone was selected for sequencing. The size of spirochaetal DNA insert was determined by digestion with restriction endonucleases EcoRI and PstI as being approximately 2.6 kb. The nucleotide sequence of the gene encoding the protein with which the antibody reacted was determined by cycle sequencing. The insert contained an open reading frame of 285 nucleotides. Translation of the nucleotide sequence into amino acid (aa) residues showed a sequence of 275 aa. Comparison of this sequence with databases revealed homology to pyruvate oxidoreductases from various organisms found in the gastroinestinal tract. These included the pyruvate ferredoxin oxidoreductase (POR) alpha submit of Helicobacter pylori (38.8% identity in 250 aa), pyruvate-flavodoxin oxidoreductase of Escherichia coli (28.7% identify in 258 aa) and Giardia intestinalis (25.1% identity in 251 aa). A significant level of homology was also observed with hyperthermophilic bacteria such as the POR of Thermatoga maritima (38.6% in 254 aa) and the 2-ketovalerate-ferredoxin oxidoreductase of Pyrococcus furiosus (34% in 262 aa).

  9. Update of the NAD(P)H:quinone oxidoreductase (NQO) gene family

    PubMed Central

    2006-01-01

    The NAD(P)H:quinone acceptor oxidoreductase (NQO) gene family belongs to the flavoprotein clan and, in the human genome, consists of two genes (NQO1 and NQO2). These two genes encode cytosolic flavoenzymes that catalyse the beneficial two-electron reduction of quinones to hydroquinones. This reaction prevents the unwanted one-electron reduction of quinones by other quinone reductases; one-electron reduction results in the formation of reactive oxygen species, generated by redox cycling of semiquinones in the presence of molecular oxygen. Both the mammalian NQO1 and NQO2 genes are upregulated as a part of the oxidative stress response and are inexplicably overexpressed in particular types of tumours. A non-synonymous mutation in the NQO1 gene, leading to absence of enzyme activity, has been associated with an increased risk of myeloid leukaemia and other types of blood dyscrasia in workers exposed to benzene. NQO2 has a melatonin-binding site, which may explain the anti-oxidant role of melatonin. An ancient NQO3 subfamily exists in eubacteria and the authors suggest that there should be additional divisions of the NQO family to include the NQO4 subfamily in fungi and NQO5 subfamily in archaebacteria. Interestingly, no NQO genes could be identified in the worm, fly, sea squirt or plants; because these taxa carry quinone reductases capable of one- and two-electron reductions, there has been either convergent evolution or redundancy to account for the appearance of these enzyme functions whenever they have been needed during evolution. PMID:16595077

  10. Fur activates expression of the 2-oxoglutarate oxidoreductase genes (oorDABC) in Helicobacter pylori.

    PubMed

    Gilbreath, Jeremy J; West, Abby L; Pich, Oscar Q; Carpenter, Beth M; Michel, Sarah; Merrell, D Scott

    2012-12-01

    Helicobacter pylori is a highly successful pathogen that colonizes the gastric mucosa of ∼50% of the world's population. Within this colonization niche, the bacteria encounter large fluctuations in nutrient availability. As such, it is critical that this organism regulate expression of key metabolic enzymes so that they are present when environmental conditions are optimal for growth. One such enzyme is the 2-oxoglutarate (α-ketoglutarate) oxidoreductase (OOR), which catalyzes the conversion of α-ketoglutarate to succinyl coenzyme A (succinyl-CoA) and CO(2). Previous studies from our group suggested that the genes that encode the OOR are activated by iron-bound Fur (Fe-Fur); microarray analysis showed that expression of oorD, oorA, and oorC was altered in a fur mutant strain of H. pylori. The goal of the present work was to more thoroughly characterize expression of the oorDABC genes in H. pylori as well as to define the role of Fe-Fur in this process. Here we show that these four genes are cotranscribed as an operon and that expression of the operon is decreased in a fur mutant strain. Transcriptional start site mapping and promoter analysis revealed the presence of a canonical extended -10 element but a poorly conserved -35 element upstream of the +1. Additionally, we identified a conserved Fur binding sequence ∼130 bp upstream of the transcriptional start site. Transcriptional analysis using promoter fusions revealed that this binding sequence was required for Fe-Fur-mediated activation. Finally, fluorescence anisotropy assays indicate that Fe-Fur specifically bound this Fur box with a relatively high affinity (dissociation constant [K(d)] = 200 nM). These findings provide novel insight into the genetic regulation of a key metabolic enzyme and add to our understanding of the diverse roles Fur plays in gene regulation in H. pylori.

  11. NADPH-dependent glutamate dehydrogenase in Penicillium chrysogenum is involved in regulation of beta-lactam production.

    PubMed

    Thykaer, Jette; Rueksomtawin, Kanchana; Noorman, Henk; Nielsen, Jens

    2008-04-01

    The interactions between the ammonium assimilatory pathways and beta-lactam production were investigated by disruption of the NADPH-dependent glutamate dehydrogenase gene (gdhA) in two industrial beta-lactam-producing strains of Penicillium chrysogenum. The strains used were an adipoyl-7-ADCA- and a penicillin-producing strain. The gdhA gene disruption caused a decrease in maximum specific growth rate of 26 % and 35 % for the adipoyl-7-ADCA-producing strain and the penicillin-producing strain, respectively, compared to the corresponding reference strains. Interestingly, no beta-lactam production was detected in either of the DeltagdhA strains. Supplementation with glutamate restored growth but no beta-lactam production was detected for the constructed strains. Cultures with high ammonium concentrations (repressing conditions) and with proline as nitrogen source (de-repressed conditions) showed continued beta-lactam production for the reference strains whereas the DeltagdhA strains remained non-productive under all conditions. By overexpressing the NAD-dependent glutamate dehydrogenase, the specific growth rate could be restored, but still no beta-lactam production was detected. The results indicate that the NADPH-dependent glutamate dehydrogenase may be directly or indirectly involved in the regulation of beta-lactam production in industrial strains of P. chrysogenum.

  12. Biochemical and physiological analyses of NADPH-dependent thioredoxin reductase isozymes in Euglena gracilis.

    PubMed

    Tamaki, Shun; Maruta, Takanori; Sawa, Yoshihiro; Shigeoka, Shigeru; Ishikawa, Takahiro

    2015-07-01

    At least four peroxiredoxins that are coupled with the thioredoxin (Trx) system have been shown to play a key role in redox metabolism in the unicellular phytoflagellate Euglena gracilis. In order to clarify Trx-mediated redox regulation in this alga, we herein identified three NADPH-dependent thioredoxin reductases (NTRs) using a homologous search and characterized their enzymatic properties and physiological roles. Each Euglena NTR protein belonged to the small, large, and NTRC types, and were named EgNTR1, EgNTR2, and EgNTRC, respectively. EgNTR2 was phylogenetically different from the known NTRs in eukaryotic algae. EgNTR1 was predicted to be localized in mitochondria, EgNTR2 in the cytosol, and EgNTRC in plastids. The catalytic efficiency of EgNTR2 for NADPH was 30-46-fold higher than those of EgNTR1 and truncated form of EgNTRC, suggested that large type EgNTR2 reduced Trx more efficiently. The silencing of EgNTR2 gene expression resulted in significant growth inhibition and cell hypertrophy in Euglena cells. These results suggest that EgNTRs function in each cellular compartment and are physiologically important, particularly in the cytosol.

  13. The involvement of superoxide and iron ions in the NADPH-dependent lipid peroxidation in human placental mitochondria.

    PubMed

    Klimek, J

    1988-01-19

    Incubation of human term placental mitochondria with Fe2+ and a NADPH-generating system initiated high levels of lipid peroxidation, as measured by the production of malondialdehyde. Malondialdehyde formation was accompanied by a corresponding decrease of the unsaturated fatty acid content. This NADPH-dependent lipid peroxidation was strongly inhibited by superoxide dismutase and singlet oxygen scavengers, markedly stimulated by paraquat, but was not affected by hydroxyl radical scavengers. Catalase enhanced the production of malondialdehyde by placental mitochondria. The effects of catalase and hydroxyl radical scavengers suggest that the initiation of NADPH-dependent lipid peroxidation is not dependent upon the hydroxyl radical produced via an iron-catalyzed Fenton reaction. These studies provide evidence that hydrogen peroxide strongly inhibits NADPH-dependent mitochondrial lipid peroxidation. The inhibitory effect of superoxide dismutase and stimulatory effect of paraquat, which was abolished by the addition of superoxide dismutase, suggests that superoxide may promote NADPH-dependent lipid peroxidation in human placental mitochondria.

  14. ord1, an oxidoreductase gene responsible for conversion of O-methylsterigmatocystin to aflatoxin in Aspergillus flavus.

    PubMed Central

    Prieto, R; Woloshuk, C P

    1997-01-01

    Among the enzymatic steps in the aflatoxin biosynthetic pathway, the conversion of O-methylsterigmatocystin to aflatoxin has been proposed to be catalyzed by an oxidoreductase. Transformants of Aspergillus flavus 649WAF2 containing a 3.3-kb genomic DNA fragment and the aflatoxin biosynthesis regulatory gene aflR converted exogenously supplied O-methylsterigmatocystin to aflatoxin B1. A gene, ord1, corresponding to a transcript of about 2 kb was identified within the 3.3-kb DNA fragment. The promoter region presented a putative AFLR binding site and a TATA sequence. The nucleotide sequence of the gene revealed an open reading frame encoding a protein of 528 amino acids with a deduced molecular mass of 60.2 kDa. The gene contained six introns and seven exons. Heterologous expression of the ord1 open reading frame under the transcriptional control of the Saccharomyces cerevisiae galactose-inducible gal1 promoter results in the ability to convert O-methylsterigmatocystin to aflatoxin B1. The data indicate that ord1 is sufficient to accomplish the last step of the aflatoxin biosynthetic pathway. A search of various databases for similarity indicated that ord1 encodes a cytochrome P-450-type monooxygenase, and the gene has been assigned to a new P-450 gene family named CYP64. PMID:9143099

  15. Cloning, sequencing, and analysis of a gene cluster from Chelatobacter heintzii ATCC 29600 encoding nitrilotriacetate monooxygenase and NADH:flavin mononucleotide oxidoreductase.

    PubMed Central

    Xu, Y; Mortimer, M W; Fisher, T S; Kahn, M L; Brockman, F J; Xun, L

    1997-01-01

    Nitrilotriacetate (NTA) is an important chelating agent in detergents and has also been used extensively in processing radionuclides. In Chelatobacter heintzii ATCC 29600, biodegradation of NTA is initiated by NTA monooxygenase that oxidizes NTA to iminodiacetate and glyoxylate. The NTA monooxygenase activity requires two component proteins, component A and component B, but the function of each component is unclear. We have cloned and sequenced a gene cluster encoding components A and B (nmoA and nmoB) and two additional open reading frames, nmoR and nmoT, downstream of nmoA. Based on sequence similarities, nmoR and nmoT probably encode a regulatory protein and a transposase, respectively. The NmoA sequence was similar to a monooxygenase that uses reduced flavin mononucleotide (FMNH2) as reductant; NmoB was similar to an NADH:flavin mononucleotide (FMN) oxidoreductase. On the basis of this information, we tested the function of each component. Purified component B was shown to be an NADH:FMN oxidoreductase, and its activity could be separated from that of component A. When the Photobacterium fischeri NADH:FMN oxidoreductase was substituted for component B in the complete reaction, NTA was oxidized, showing that the substrate specificity of the reaction resides in component A. Component A is therefore an NTA monooxygenase that uses FMNH2 and O2 to oxidize NTA, and component B is an NADH:FMN oxidoreductase that provides FMNH2 for NTA oxidation. PMID:9023192

  16. Boost in bioethanol production using recombinant Saccharomyces cerevisiae with mutated strictly NADPH-dependent xylose reductase and NADP(+)-dependent xylitol dehydrogenase.

    PubMed

    Khattab, Sadat Mohammad Rezq; Saimura, Masayuki; Kodaki, Tsutomu

    2013-06-10

    The xylose-fermenting recombinant Saccharomyces cerevisiae and its improvement have been studied extensively. The redox balance between xylose reductase (XR) and xylitol dehydrogenase (XDH) is thought to be an important factor in effective xylose fermentation. Using protein engineering, we previously successfully reduced xylitol accumulation and improved ethanol production by reversing the dependency of XDH from NAD(+) to NADP(+). We also constructed a set of novel strictly NADPH-dependent XR from Pichia stipitis by site-directed mutagenesis. In the present study, we constructed a set of recombinant S. cerevisiae carrying a novel set of mutated strictly NADPH-dependent XR and NADP(+)-dependent XDH genes with overexpression of endogenous xylulokinase (XK) to study the effects of complete NADPH/NADP(+) recycling on ethanol fermentation and xylitol accumulation. All mutated strains demonstrated reduced xylitol accumulation, ranging 34.4-54.7% compared with the control strain. Moreover, compared with the control strain, the two strains showed 20% and 10% improvement in ethanol production.

  17. The NADPH-dependent O-.2-generating oxidase from human neutrophils.

    PubMed

    Gabig, T G

    1983-05-25

    A subcellular particulate fraction from normal neutrophils that was enriched in NADPH-dependent O-.2-generating activity (Gabig, T. G., Schervish, E. W., and Santinga, J. T. (1982) J. Biol. Chem. 257, 4114-4119) has been further characterized. This preparation contained 0.25 +/- 0.02 nmol of flavin adenine dinucleotide/mg of protein and 0.28 +/- 0.01 nmol of cytochrome b/mg of protein. Measurable amounts of riboflavin or flavin mononucleotide were not present. The flavoprotein was completely resolved from the cytochrome b by selective bile salt extraction of the particulate oxidase fraction. The identical subcellular particulate fraction was studied in the neutrophils from two male patients with chronic granulomatous disease. The neutrophil oxidase fraction from one of the chronic granulomatous disease patients had a cytochrome b component that was spectrally abnormal, but a normal content of flavin adenine dinucleotide. The fraction from this patient's neutrophils corresponding to the resolved flavoprotein from normal cells had fluorescence excitation and emission spectra that were identical to the normal flavoprotein. The neutrophil oxidase fraction from the second chronic granulomatous disease patient had a quantitatively and spectrally normal cytochrome b but less than 8% of the normal amount of flavin adenine dinucleotide. The fraction from the latter patient's neutrophils corresponding to the resolved flavoprotein from normal cells had no detectable flavoprotein by fluorescence excitation and emission spectroscopy. It is postulated that these two patients represent distinct mutants in two separate components of the neutrophil NADPH-dependent O-.2-generating oxidase system, flavoprotein and cytochrome b.

  18. Molecular and structural characterization of NADPH-dependent d-glycerate dehydrogenase from the enteric parasitic protist Entamoeba histolytica.

    PubMed Central

    Ali, Vahab; Shigeta, Yasuo; Nozaki, Tomoyoshi

    2003-01-01

    Putative NADPH-dependent GDH (L-glycerate dehydrogenase) of the protozoan parasite Entamoeba histolytica (EhGDH) has been characterized. The EhGDH gene encodes a protein of 318 amino acids with a calculated isoelectric point of 6.29 and a molecular mass of 35.8 kDa. EhGDH showed highest identities with GDH from epsilon-proteobacteria. This close kinship was also supported by phylogenetic analyses, suggesting possible lateral transfer of the gene from epsilon-proteobacteria to E. histolytica. In contrast with the implications from protein alignment and phylogenetic analysis, kinetic studies revealed that the amoebic GDH showed biochemical properties similar to those of mammalian GDH, i.e. a preference for NADPH as cofactor and higher affinities towards NADPH and beta-hydroxypyruvate than towards NADP+ and L-glycerate. Whereas the amino acids involved in nucleotide binding and catalysis are totally conserved in EhGDH, substitution of a negatively charged amino acid with a non-charged hydroxy-group-containing amino acid is probably responsible for the observed high affinity of EhGDH for NADP+/NADPH. In addition, the amoebic GDH, dissimilar to the bacterial and mammalian GDHs, lacks glyoxylate reductase activity. Native and recombinant EhGDH showed comparable subunit structure, kinetic parameters and elution profiles on anion-exchange chromatography. We propose that the GDH enzyme is likely to be involved in regulation of the intracellular concentration of serine, and, thus, also in controlling cysteine biosynthesis located downstream of serine metabolic pathways in this protist. PMID:12877657

  19. Reconstruction of an Acetogenic 2,3-Butanediol Pathway Involving a Novel NADPH-Dependent Primary-Secondary Alcohol Dehydrogenase

    PubMed Central

    Köpke, Michael; Gerth, Monica L.; Maddock, Danielle J.; Mueller, Alexander P.; Liew, FungMin

    2014-01-01

    Acetogenic bacteria use CO and/or CO2 plus H2 as their sole carbon and energy sources. Fermentation processes with these organisms hold promise for producing chemicals and biofuels from abundant waste gas feedstocks while simultaneously reducing industrial greenhouse gas emissions. The acetogen Clostridium autoethanogenum is known to synthesize the pyruvate-derived metabolites lactate and 2,3-butanediol during gas fermentation. Industrially, 2,3-butanediol is valuable for chemical production. Here we identify and characterize the C. autoethanogenum enzymes for lactate and 2,3-butanediol biosynthesis. The putative C. autoethanogenum lactate dehydrogenase was active when expressed in Escherichia coli. The 2,3-butanediol pathway was reconstituted in E. coli by cloning and expressing the candidate genes for acetolactate synthase, acetolactate decarboxylase, and 2,3-butanediol dehydrogenase. Under anaerobic conditions, the resulting E. coli strain produced 1.1 ± 0.2 mM 2R,3R-butanediol (23 μM h−1 optical density unit−1), which is comparable to the level produced by C. autoethanogenum during growth on CO-containing waste gases. In addition to the 2,3-butanediol dehydrogenase, we identified a strictly NADPH-dependent primary-secondary alcohol dehydrogenase (CaADH) that could reduce acetoin to 2,3-butanediol. Detailed kinetic analysis revealed that CaADH accepts a range of 2-, 3-, and 4-carbon substrates, including the nonphysiological ketones acetone and butanone. The high activity of CaADH toward acetone led us to predict, and confirm experimentally, that C. autoethanogenum can act as a whole-cell biocatalyst for converting exogenous acetone to isopropanol. Together, our results functionally validate the 2,3-butanediol pathway from C. autoethanogenum, identify CaADH as a target for further engineering, and demonstrate the potential of C. autoethanogenum as a platform for sustainable chemical production. PMID:24657865

  20. Recombinant expression and biochemical characterization of an NADPH:flavin oxidoreductase from Entamoeba histolytica.

    PubMed Central

    Bruchhaus, I; Richter, S; Tannich, E

    1998-01-01

    The gene encoding a putative NADPH:flavin oxidoreductase of the protozoan parasite Entamoeba histolytica (Eh34) was recombinantly expressed in Escherichia coli. The purified recombinant protein (recEh34) has a molecular mass of about 35 kDa upon SDS/PAGE analysis, exhibits a flavoprotein-like absorption spectrum and contains 1 mol of non-covalently bound FMN per mol of protein. RecEh34 reveals two different enzymic activities. It catalyses the NADPH-dependent reduction of oxygen to hydrogen peroxide (H2O2), as well as of disulphides such as 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and cystine. The disulphide reductase but not the H2O2-forming NADPH oxidase activity is inhibitable by sulphydryl-active compounds, indicating that a thiol component is part of the active site for the disulphide reductase activity, whereas for the H2O2-forming NADPH oxidase activity only the flavin is required. Compared with the recombinant protein, similar activities are present in amoebic extracts. Native Eh34 is active in a monomeric as well as in a dimeric state. In contrast to recEh34, no flavin was associated with the native protein. However, both NADPH oxidase as well as DTNB reductase activity were found to be dependent on the addition of FAD or FMN. PMID:9494088

  1. Effects of NADH kinase on NADPH-dependent biotransformation processes in Escherichia coli.

    PubMed

    Lee, Won-Heong; Kim, Jin-Woo; Park, Eun-Hee; Han, Nam Soo; Kim, Myoung-Dong; Seo, Jin-Ho

    2013-02-01

    Sufficient supply of NADPH is one of the most important factors affecting the productivity of biotransformation processes. In this study, construction of an efficient NADPH-regenerating system was attempted using direct phosphorylation of NADH by NADH kinase (Pos5p) from Saccharomyces cerevisiae for producing guanosine diphosphate (GDP)-L-fucose and ε-caprolactone in recombinant Escherichia coli. Expression of Pos5p in a fed-batch culture of recombinant E. coli producing GDP-L-fucose resulted in a maximum GDP-L-fucose concentration of 291.5 mg/l, which corresponded to a 51 % enhancement compared with the control strain. In a fed-batch Baeyer-Villiger (BV) oxidation of cyclohexanone using recombinant E. coli expressing Pos5p, a maximum ε-caprolactone concentration of 21.6 g/l was obtained, which corresponded to a 96 % enhancement compared with the control strain. Such an increase might be due to the enhanced availability of NADPH in recombinant E. coli expressing Pos5p. These results suggested that efficient regeneration of NADPH was possible by functional expression of Pos5p in recombinant E. coli, which can be applied to other NADPH-dependent biotransformation processes in E. coli.

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

    SciTech Connect

    Kirkensgaard, Kristine G.; Hägglund, Per; Finnie, Christine; Svensson, Birte; Henriksen, Anette

    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, 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.

  3. Comparative study of the tissue distribution of NADH and NADPH-dependent chloral hydrate reducing enzymes in the rat

    SciTech Connect

    Ogino, Keiki; Hobara, Tatsuya; Kobayashi, Haruo; Iwamoto, Susumu )

    1990-03-01

    Chloral hydrate (CH), an intermediate metabolite of trichloroethylene, is reduced to trichloroethanol (TCE) by alcohol dehydrogenase and aldehyde reductase. Alcohol dehydrogenase requires reduced nicotinamide adenine dinucleotide (NADH), and aldehyde reductase requires reduced nicotinamide adenine dinucleotide phosphate (NADPH). No reports have appeared concerning comparative studies of the tissue distribution of CH-reducing enzymes. In this report, NADH and NADPH-dependent CH-reducing activities were investigated in various organs of the rat.

  4. Irreversible inactivation of macrophage and brain nitric oxide synthase by L-NG-methylarginine requires NADPH-dependent hydroxylation.

    PubMed

    Feldman, P L; Griffith, O W; Hong, H; Stuehr, D J

    1993-02-19

    L-NG-Methylarginine (NMA) is an established mechanism-based inactivator of murine macrophage nitric oxide synthase (mNOS). In this report, NMA is shown to irreversibly inhibit both mNOS (k(inact) = 0.08 min-1) and the recombinant constitutive brain NOS (bNOS). For both NOS isoforms, metabolism of NMA parallels that of the natural substrate L-arginine (ARG), in that it undergoes a regiospecific, NADPH-dependent hydroxylation to form L-NG-hydroxy-NG-methylarginine (NOHNMA). This intermediate then undergoes further NADPH-dependent oxidation to form L-citrulline (CIT). Authentic NOHNMA, synthesized from L-ornithine, irreversibly inhibited both mNOS (k(inact) = 0.10 min-1) and bNOS in an NADPH-dependent reaction. The conversion of either NMA or NOHNMA to CIT correlated with irreversible enzyme inactivation. Thus, the data suggest that enzyme inhibition occurs as a consequence of oxidative metabolism of the intermediate, NOHNMA. A unified mechanism is proposed that accounts for NO biosynthesis from ARG, for the inactivation of NOS by NMA and for the intermediacy of hydroxylated ARG or NMA derivatives in these processes.

  5. 1,4-Naphthoquinones and Others NADPH-Dependent Glutathione Reductase-Catalyzed Redox Cyclers as Antimalarial Agents

    PubMed Central

    Belorgey, Didier; Lanfranchi, Don Antoine; Davioud-Charvet, Elisabeth

    2013-01-01

    The homodimeric flavoenzyme glutathione reductase catalyzes NADPH-dependent glutathione disulfide reduction. This reaction is important for keeping the redox homeostasis in human cells and in the human pathogen Plasmodium falciparum. Different types of NADPH-dependent disulfide reductase inhibitors were designed in various chemical series to evaluate the impact of each inhibition mode on the propagation of the parasites. Against malaria parasites in cultures the most potent and specific effects were observed for redox-active agents acting as subversive substrates for both glutathione reductases of the Plasmodium-infected red blood cells. In their oxidized form, these redox-active compounds are reduced by NADPH-dependent flavoenzyme-catalyzed reactions in the cytosol of infected erythrocytes. In their reduced forms, these compounds can reduce molecular oxygen to reactive oxygen species, or reduce oxidants like methemoglobin, the major nutrient of the parasite, to indigestible hemoglobin. Furthermore, studies on a fluorinated suicide-substrate of the human glutathione reductase indicate that the glutathione reductase-catalyzed bioactivation of 3-benzylnaphthoquinones to the corresponding reduced 3-benzoyl metabolites is essential for the observed antimalarial activity. In conclusion, the antimalarial lead naphthoquinones are suggested to perturb the major redox equilibria of the targeted cells. These effects result in development arrest of the parasite and contribute to the removal of the parasitized erythrocytes by macrophages. PMID:23116403

  6. New method for RNA isolation from actinomycetes: application to the transcriptional analysis of the alcohol oxidoreductase gene thcE in Rhodococcus and Mycobacterium.

    PubMed

    Nagy, I; Schoofs, G; De Schrijver, A; Vanderleyden, J; De Mot, R

    1997-07-01

    A new protocol for the isolation of RNA from Rhodococcus and other actinomycetes such as Mycobacterium and Amycolatopsis was developed. The method is based on rapid lysis of cells in a high-speed reciprocal shaker using small abrasive particles followed by spin column purification of the lysate. This quick procedure yields RNA preparations suitable for functional studies. This was shown for the thcE gene of R. erythropolis NI86/21, which encodes a N,N'-dimethyl-4-nitrosoaniline-dependent alcohol oxidoreductase. The thcE transcript was detected by Northern hybridization in R. erythropolis, R. fascians, Mycobacterium chlorophenolicum and Mycobacterium smegmatis. The transcriptional start point of the gene was determined by primer extension of the R. erythropolis mRNA.

  7. The nuclear genes encoding the internal (KlNDI1) and external (KlNDE1) alternative NAD(P)H:ubiquinone oxidoreductases of mitochondria from Kluyveromyces lactis.

    PubMed

    Tarrío, N; Díaz Prado, S; Cerdán, M E; González Siso, M I

    2005-01-01

    Cloning, sequence and functional analyses of the Kluyveromyces lactis genes KlNDI1 and KlNDE1 are reported. These genes encode for proteins with high homology to the mitochondrial internal (Ndi1p) and external (Nde1p) alternative NADH:ubiquinone oxidoreductases from Saccharomyces cerevisiae and complement the respective mutations. Analysis of KlNDI1 transcriptional regulation showed that expression of this gene is lower in 2% glucose than in 0.5% glucose or non-fermentable carbon sources. Beta-galactosidase activity values, shown by lacZ fusions of KlNDI1 promoter deletions, suggested that two Adr1p binding sites mediate this carbon source regulation of KlNDI1. The expression of the KlNDE1 gene in S. cerevisiae mutant strains and measurement of respiration with isolated mitochondria showed that the protein encoded by KlNDE1 oxidizes NADPH, this being an important difference with respect to the conventional yeast S. cerevisiae. Moreover, Northern blot experiments using a phosphoglucose isomerase mutant showed that KlNDE1 gene transcription increases with glucose metabolism through the pentose phosphate pathway.

  8. Toxic-selenium and low-selenium transcriptomes in Caenorhabditis elegans: toxic selenium up-regulates oxidoreductase and down-regulates cuticle-associated genes.

    PubMed

    Boehler, Christopher J; Raines, Anna M; Sunde, Roger A

    2014-01-01

    Selenium (Se) is an element that in trace quantities is both essential in mammals but also toxic to bacteria, yeast, plants and animals, including C. elegans. Our previous studies showed that selenite was four times as toxic as selenate to C. elegans, but that deletion of thioredoxin reductase did not modulate Se toxicity. To characterize Se regulation of the full transcriptome, we conducted a microarray study in C. elegans cultured in axenic media supplemented with 0, 0.05, 0.1, 0.2, and 0.4 mM Se as selenite. C. elegans cultured in 0.2 and 0.4 mM Se displayed a significant delay in growth as compared to 0, 0.05, or 0.1 mM Se, indicating Se-induced toxicity, so worms were staged to mid-L4 larval stage for these studies. Relative to 0.1 mM Se treatment, culturing C. elegans at these Se concentrations resulted in 1.9, 9.7, 5.5, and 2.3%, respectively, of the transcriptome being altered by at least 2-fold. This toxicity altered the expression of 295 overlapping transcripts, which when filtered against gene sets for sulfur and cadmium toxicity, identified a dataset of 182 toxic-Se specific genes that were significantly enriched in functions related to oxidoreductase activity, and significantly depleted in genes related to structural components of collagen and the cuticle. Worms cultured in low Se (0 mM Se) exhibited no signs of deficiency, but low Se was accompanied by a transcriptional response of 59 genes changed ≥2-fold when compared to all other Se concentrations, perhaps due to decreases in Se-dependent TRXR-1 activity. Overall, these results suggest that Se toxicity in C. elegans causes an increase in ROS and stress responses, marked by increased expression of oxidoreductases and reduced expression of cuticle-associated genes, which together underlie the impaired growth observed in these studies.

  9. Functional genetic variant in the Kozak sequence of WW domain-containing oxidoreductase (WWOX) gene is associated with oral cancer risk

    PubMed Central

    Su, Chun-Wen; Su, Shih-Chi; Chen, Mu-Kuan; Yang, Shun-Fa; Lin, Chiao-Wen

    2016-01-01

    In Taiwan, oral cancer is the fourth leading cancer in males and is associated with exposure to environmental carcinogens. WW domain-containing oxidoreductase (WWOX), a tumor suppressor gene, is associated with the development of various cancers. We hypothesized that genetic variants of WWOX influence the susceptibility to oral cancer. Five polymorphisms of WWOX gene from 761 male patients with oral cancer and 1199 male cancer-free individuals were genotyped. We observed that individuals carrying the polymorphic allele of WWOX rs11545028 are more susceptible to oral cancer. Furthermore, patients with advanced-stage oral cancer were associated with a higher frequency of WWOX rs11545028 polymorphisms with the variant genotype TT than did patients with the wild-type gene. An additional integrated in silico analysis confirmed that rs11545028 affects WWOX expression, which significantly correlates with tumor expression and subsequently with tumor development and aggressiveness. In conclusion, genetic variants of WWOX contribute to the occurrence of oral cancer, and the findings regarding these biomarkers provided a prediction model for risk assessment. PMID:27655721

  10. Catalytic properties of the resolved flavoprotein and cytochrome B components of the NADPH dependent O2- . generating oxidase from human neutrophils.

    PubMed

    Gabig, T G; Lefker, B A

    1984-01-30

    The resolved flavoprotein and cytochrome b559 components of the NADPH dependent O2- . generating oxidase from human neutrophils were the subject of further study. The resolved flavoprotein, depleted of cytochrome b559, was reduced by NADPH under anaerobic conditions and reoxidized by oxygen. NADPH dependent O2- . generation by the resolved flavoprotein fraction was not detectable, however it was competent in the transfer of electrons from NADPH to artificial electron acceptors. The resolved cytochrome b559, depleted of flavoprotein, demonstrated no measureable NADPH dependent O2- . generating activity and was not reduced by NADPH under anaerobic conditions. The dithionite reduced form of the resolved cytochrome b559 was rapidly oxidized by oxygen, as was the cytochrome b559 in the intact oxidase.

  11. Interindividual variability of CYP2C19-catalyzed drug metabolism due to differences in gene diplotypes and cytochrome P450 oxidoreductase content.

    PubMed

    Shirasaka, Y; Chaudhry, A S; McDonald, M; Prasad, B; Wong, T; Calamia, J C; Fohner, A; Thornton, T A; Isoherranen, N; Unadkat, J D; Rettie, A E; Schuetz, E G; Thummel, K E

    2016-08-01

    Large interindividual variability has been observed in the metabolism of CYP2C19 substrates in vivo. The study aimed to evaluate sources of this variability in CYP2C19 activity, focusing on CYP2C19 diplotypes and the cytochrome P450 oxidoreductase (POR). CYP2C19 gene analysis was carried out on 347 human liver samples. CYP2C19 activity assayed using human liver microsomes confirmed a significant a priori predicted rank order for (S)-mephenytoin hydroxylase activity of CYP2C19*17/*17 > *1B/*17 > *1B/*1B > *2A/*17 > *1B/*2A > *2A/*2A diplotypes. In a multivariate analysis, the CYP2C19*2A allele and POR protein content were associated with CYP2C19 activity. Further analysis indicated a strong effect of the CYP2C19*2A, but not the *17, allele on both metabolic steps in the conversion of clopidogrel to its active metabolite. The present study demonstrates that interindividual variability in CYP2C19 activity is due to differences in both CYP2C19 protein content associated with gene diplotypes and the POR concentration.The Pharmacogenomics Journal advance online publication, 1 September 2015; doi:10.1038/tpj.2015.58.

  12. Interindividual variability of CYP2C19-catalyzed drug metabolism due to differences in gene diplotypes and cytochrome P450 oxidoreductase content

    PubMed Central

    Shirasaka, Y; Chaudhry, A S; McDonald, M; Prasad, B; Wong, T; Calamia, J C; Fohner, A; Thornton, T A; Isoherranen, N; Unadkat, J D; Rettie, A E; Schuetz, E G; Thummel, K E

    2016-01-01

    Large interindividual variability has been observed in the metabolism of CYP2C19 substrates in vivo. The study aimed to evaluate sources of this variability in CYP2C19 activity, focusing on CYP2C19 diplotypes and the cytochrome P450 oxidoreductase (POR). CYP2C19 gene analysis was carried out on 347 human liver samples. CYP2C19 activity assayed using human liver microsomes confirmed a significant a priori predicted rank order for (S)-mephenytoin hydroxylase activity of CYP2C19*17/*17 > *1B/*17 > *1B/*1B > *2A/*17 > *1B/*2A > *2A/*2A diplotypes. In a multivariate analysis, the CYP2C19*2A allele and POR protein content were associated with CYP2C19 activity. Further analysis indicated a strong effect of the CYP2C19*2A, but not the *17, allele on both metabolic steps in the conversion of clopidogrel to its active metabolite. The present study demonstrates that interindividual variability in CYP2C19 activity is due to differences in both CYP2C19 protein content associated with gene diplotypes and the POR concentration. PMID:26323597

  13. 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.

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

    PubMed Central

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

    2015-01-01

    Background and Aims 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. Methods 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. Key Results 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. Conclusions 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. PMID:26229066

  15. RNAi based simultaneous silencing of all forms of light-dependent NADPH:protochlorophyllide oxidoreductase genes result in the accumulation of protochlorophyllide in tobacco (Nicotiana tabacum).

    PubMed

    Talaat, Neveen B

    2013-10-01

    Conversion of protochlorophyllide (Pchlide) into chlorophyllide (Chlide), a key step in chlorophyll biosynthesis, is mediated by a light-dependent NADPH:protochlorophyllide oxidoreductase (POR). POR exists in multiple isoforms that share high degree of homology. RNAi-mediated gene silencing approach was used to suppress the expression of POR genes in order to study its role in the Chls biosynthesis in tobacco (Nicotiana tabacum L.). The transgenic plants were devoid of chlorophyll pigments and resembled albino plants. Northern blot analysis confirmed the degradation of POR transcripts into 21-23 bp fragments. Pigment analysis showed the accumulation of various intermediate compounds of Chl biosynthesis pathway including Pchlide. However, no trace of chlorophyll was observed. As compared to wild type, POR-silenced plants accumulated larger (60%) amounts of Pchlide from its endogenous substrate. When leaf discs of WT and POR-silenced plants were treated with exogenous ALA both WT and POR-silenced plants accumulated large amounts of tetrapyrrolic intermediates demonstrating that Pchlide biosynthesis potential was not suppressed in POR-silenced plants. Upon illumination, WT plants photo-transformed large amounts of Pchlide to Chlide. However, POR-silenced plants almost completely failed to do so. Results demonstrate that the antisense approaches to drop expression of individual POR isoforms have provided valuable insights into the role of distinct PORs during greening. Moreover, data illustrate that the POR is the only enzyme that can convert the Pchlide to Chlide and there is no alternate enzyme that can substitute the POR in higher plants. Thus, this investigation describes ideal mechanism for the silencing of POR isozymes in tobacco.

  16. Disruption of the NADPH-dependent glutamate dehydrogenase affects the morphology of two industrial strains of Penicillium chrysogenum.

    PubMed

    Thykaer, Jette; Rueksomtawin, Kanchana; Noorman, Henk; Nielsen, Jens

    2009-02-23

    New morphological aspects of Penicillium chrysogenum were found during physiological characterisation of two NADPH-dependent glutamate dehydrogenase mutant strains. A morphological characterisation of the previously constructed strains, together with the two beta-lactam producing industrial recipient strains, was conducted. The reference strains showed a compact structure with highly branched hyphal elements whereas the morphology of the DeltagdhA strains consisting of long elongated hyphal elements with few branches. On solid medium, the hyphal growth unit (length) increased from an average of 47 microm tip(-1) in the reference strains to 117 microm tip(-1) in the DeltagdhA strains and in submerged cultures a decrease of 18% in branching frequency was measured due to the gdhA deletion. P. chrysogenum Wis 54-1255, the ancestor of most production strains was also characterised and this strain showed morphology similar to the industrial strains. Interestingly, the constructed strains showed morphology similar to wild type Aspergillus nidulans another species carrying the penicillin biosynthetic cluster. Thus, the results showed that elimination of glutamate dehydrogenase activity in high producing strains of P. chrysogenum has a radical impact on morphology.

  17. Thyroid Ca2+/NADPH-dependent H2O2 generation is partially inhibited by propylthiouracil and methimazole.

    PubMed

    Ferreira, Andrea C Freitas; de Carvalho Cardoso, Luciene; Rosenthal, Doris; de Carvalho, Denise Pires

    2003-06-01

    H2O2 generation is a limiting step in thyroid hormone biosynthesis. Biochemical studies have confirmed that H2O2 is generated by a thyroid Ca2+/NADPH-dependent oxidase. Decreased H2O2 availability may be another mechanism of inhibition of thyroperoxidase activity produced by thioureylene compounds, as propylthiouracil (PTU) and methimazole (MMI) are antioxidant agents. Therefore, we analyzed whether PTU or MMI could scavenge H2O2 or inhibit thyroid NADPH oxidase activity in vitro. Our results show that PTU and thiourea did not significantly scavenge H2O2. However, MMI significantly scavenged H2O2 at high concentrations. Only MMI was able to decrease the amount of H2O2 generated by the glucose-glucose oxidase system. On the other hand, both PTU and MMI were able to partially inhibit thyroid NADPH oxidase activity in vitro. As PTU did not scavenge H2O2 under the conditions used here, we presume that this drug may directly inhibit thyroid NADPH oxidase. Also, at the concentration necessary to inhibit NADPH oxidase activity, MMI did not scavenge H2O2, also suggesting a direct effect of MMI on thyroid NADPH oxidase. In conclusion, this study shows that MMI, but not PTU, is able to scavenge H2O2 in the micromolar range and that both PTU and MMI can impair thyroid H2O2 generation in addition to their potent thyroperoxidase inhibitory effects.

  18. A Novel NADPH-dependent flavoprotein reductase from Bacillus megaterium acts as an efficient cytochrome P450 reductase.

    PubMed

    Milhim, Mohammed; Gerber, Adrian; Neunzig, Jens; Hannemann, Frank; Bernhardt, Rita

    2016-08-10

    Cytochromes P450 (P450s) require electron transfer partners to catalyze substrate conversions. With regard to biotechnological approaches, the elucidation of novel electron transfer proteins is of special interest, as they can influence the enzymatic activity and specificity of the P450s. In the current work we present the identification and characterization of a novel soluble NADPH-dependent diflavin reductase from Bacillus megaterium with activity towards a bacterial (CYP106A1) and a microsomal (CYP21A2) P450 and, therefore, we referred to it as B. megaterium cytochrome P450 reductase (BmCPR). Sequence analysis of the protein revealed besides the conserved FMN-, FAD- and NADPH-binding motifs, the presence of negatively charged cluster, which is thought to represent the interaction domain with P450s and/or cytochrome c. BmCPR was expressed and purified to homogeneity in Escherichia coli. The purified BmCPR exhibited a characteristic diflavin reductase spectrum, and showed a cytochrome c reducing activity. Furthermore, in an in vitro reconstituted system, the BmCPR was able to support the hydroxylation of testosterone and progesterone with CYP106A1 and CYP21A2, respectively. Moreover, in view of the biotechnological application, the BmCPR is very promising, as it could be successfully utilized to establish CYP106A1- and CYP21A2-based whole-cell biotransformation systems, which yielded 0.3g/L hydroxy-testosterone products within 8h and 0.16g/L 21-hydroxyprogesterone within 6h, respectively. In conclusion, the BmCPR reported herein owns a great potential for further applications and studies and should be taken into consideration for bacterial and/or microsomal CYP-dependent bioconversions.

  19. A novel NADPH-dependent reductase of Sulfobacillus acidophilus TPY phenol hydroxylase: expression, characterization, and functional analysis.

    PubMed

    Li, Meng; Guo, Wenbin; Chen, Xinhua

    2016-12-01

    The reductase component (MhpP) of the Sulfobacillus acidophilus TPY multicomponent phenol hydroxylase exhibits only 40 % similarity to Pseudomonas sp. strain CF600 phenol hydroxylase reductase. Amino acid sequence alignment analysis revealed that four cysteine residues (Cys-X 4 -Cys-X 2 -Cys-X 29-35 -Cys) are conserved in the N terminus of MhpP for [2Fe-2S] cluster binding, and two other motifs (RXYS and GXXS/T) are conserved in the C terminus for binding the isoalloxazine and phosphate groups of flavin adenine dinucleotide (FAD). Two motifs (S/T-R and yXCGp) responsible for binding to reduce nicotinamide adenine dinucleotide phosphate (NADPH) are also conserved in MhpP, although some residues differ. To confirm the function of this reductase, MhpP was heterologously expressed in Escherichia coli BL21(DE3) and purified. UV-visible spectroscopy and electron paramagnetic resonance spectroscopy revealed that MhpP contains a [2Fe-2S] cluster. MhpP mutants in which the four cysteine residues were substituted via site-directed mutagenesis lost the ability to bind the [2Fe-2S] cluster, resulting in a decrease in enzyme-specific oxidation of NADPH. Thin-layer chromatography revealed that MhpP contains FAD. Substrate specificity analyses confirmed that MhpP uses NADPH rather than NADH as an electron donor. MhpP oxidizes NADPH using cytochrome c, potassium ferricyanide, or nitro blue tetrazolium as an electron acceptor, with a specific activity of 1.7 ± 0.36, 0.78 ± 0.13, and 0.16 ± 0.06 U/mg, respectively. Thus, S. acidophilus TPY MhpP is a novel NADPH-dependent reductase component of phenol hydroxylase that utilizes FAD and a [2Fe-2S] cluster as cofactors.

  20. The effect of allopurinol administration on mitochondrial respiration and gene expression of xanthine oxidoreductase, inducible nitric oxide synthase, and inflammatory cytokines in selected tissues of broiler chickens.

    PubMed

    Settle, T; Falkenstein, E; Klandorf, H

    2015-10-01

    Birds have a remarkable longevity for their body size despite an increased body temperature, higher metabolic rate, and increased blood glucose concentrations compared to most mammals. As the end-product of purine degradation, uric acid (UA) is generated in the xanthine/hypoxanthine reactions catalyzed by xanthine oxidoreductase (XOR). In the first study, Cobb × Cobb broilers (n = 12; 4 weeks old) were separated into 2 treatments (n = 6); control (CON) and allopurinol (AL) 35 mg/kg BW (ALLO). The purpose of this study was to assess mitochondrial function in broiler chickens in response to potential oxidative stress generated from the administration of AL for 1 wk. There was a significant reduction in state 3 respiration (P = 0.01) and state 4 respiration (P = 0.007) in AL-treated birds compared to the controls. The purpose of the second study was to assess the effect of AL on gene expression of inflammatory cytokines interferon-γ (IFN)-γ, IL-1β, IL-6, and IL-12p35, as well as inducible nitric oxide synthase and XOR in liver tissue. Cobb × Cobb broilers were separated into two groups at 4 wk age (n = 10); CON and ALLO. After 1 wk AL treatment, half of the birds in each group (CON 1 and ALLO 1) were euthanized while the remaining birds continued on AL treatment for an additional week (CON 2 and ALLO 2). A significant increase in gene expression of XOR, IFN-γ, IL-1β, and IL-12p35 in ALLO 2 birds as compared to birds in CON 2 was detected. Liver UA content was significantly decreased in both ALLO 1(P = 0.003) and ALLO 2 (P = 0.012) birds when compared to CON 1 and CON 2, respectively. The AL reduced liver UA concentrations and increased expression of inflammatory cytokines. Additional studies are needed to determine if AL causes a direct effect on mitochondria or if mitochondrial dysfunction observed in liver mitochondria was due indirectly through increased oxidative stress or increased inflammation.

  1. Coupling prokaryotic cell fate and division control with a bifunctional and oscillating oxidoreductase homolog.

    PubMed

    Radhakrishnan, Sunish Kumar; Pritchard, Sean; Viollier, Patrick H

    2010-01-19

    NAD(H)-binding proteins play important roles in cell-cycle and developmental signaling in eukaryotes. We identified a bifunctional NAD(H)-binding regulator (KidO) that integrates cell-fate signaling with cytokinesis in the bacterium Caulobacter crescentus. KidO stimulates the DivJ kinase and directly acts on the cytokinetic tubulin, FtsZ, to tune cytokinesis with the cell cycle. At the G1-->S transition, DivJ concomitantly signals the ClpXP-dependent degradation of KidO and CtrA, a cell-cycle transcriptional regulator/DNA replication inhibitor. This proteolytic event directs KidO and CtrA into oscillatory cell-cycle abundance patterns that coordinately license replication and cytokinesis. KidO resembles NAD(P)H-dependent oxidoreductases, and conserved residues in the KidO NAD(H)-binding pocket are critical for regulation of FtsZ, but not for DivJ. Since NADPH-dependent regulation by a KidO-like oxidoreductase also occurs in humans, organisms from two domains of life exploit the enzymatic fold of an ancestral oxidoreductase potentially to coordinate cellular or developmental activities with the availability of the metabolic currency, NAD(P)H.

  2. Expression of constitutive androstane receptor, hepatic nuclear factor 4 alpha, and P450 oxidoreductase genes determines interindividual variability in basal expression and activity of a broad scope of xenobiotic metabolism genes in the human liver.

    PubMed

    Wortham, Matthew; Czerwinski, Maciej; He, Lin; Parkinson, Andrew; Wan, Yu-Jui Yvonne

    2007-09-01

    Identification of genetic variation predictive of clearance rate of a wide variety of prescription drugs could lead to cost-effective personalized medicine. Here we identify regulatory genes whose variable expression level among individuals may have widespread effects upon clearance rate of a variety of drugs. Twenty liver samples with variable CYP3A activity were profiled for expression level and activity of xenobiotic metabolism genes as well as genes involved in the regulation thereof. Regulatory genes whose expression level accounted for the highest degree of collinearity among expression levels of xenobiotic metabolism genes were identified as possible master regulators of drug clearance rate. Significant linear correlations (p < 0.05) were identified among mRNA levels of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, MRP2, OATP2, P450 oxidoreductase (POR), and UDP-glucuronosyltranferase 1A1, suggesting that these xenobiotic metabolism genes are coregulated at the transcriptional level. Using partial regression analysis, constitutive androstane receptor (CAR) and hepatic nuclear factor 4 alpha (HNF4 alpha) were identified as the nuclear receptors whose expression levels are most strongly associated with expression of coregulated xenobiotic metabolism genes. POR expression level, which is also associated with CAR and HNF4 alpha expression level, was found to be strongly associated with the activity of many cytochromes P450. Thus, interindividual variation in the expression level of CAR, HNF4 alpha, and POR probably determines variation in expression and activity of a broad scope of xenobiotic metabolism genes and, accordingly, clearance rate of a variety of xenobiotics. Identification of polymorphisms in these candidate master regulator genes that account for their variable expression among individuals may yield readily detectable biomarkers that could serve as predictors of xenobiotic clearance rate.

  3. Gene-specific disruption in the filamentous fungus Cercospora nicotianae using a split-marker approach.

    PubMed

    You, Bang-Jau; Lee, Miin-Huey; Chung, Kuang-Ren

    2009-07-01

    To determine if DNA configuration, gene locus, and flanking sequences will affect homologous recombination in the phytopathogenic fungus Cercospora nicotianae, we evaluated and compared disruption efficiency targeting four cercosporin toxin biosynthetic genes encoding a polyketide synthase (CTB1), a monooxygenase/O-methyltransferase (CTB3), a NADPH-dependent oxidoreductase (CTB5), and a FAD/FMN-dependent oxidoreductase (CTB7). Transformation of C. nicotianae using a circular plasmid resulted in low disruption frequency. The use of endonucleases or a selectable marker DNA fragment flanked by homologous sequence either at one end or at both ends in the transformation procedures, increased disruption efficiency in some but not all CTB genes. A split-marker approach, using two DNA fragments overlapping within the selectable marker, increased the frequency of targeted gene disruption and homologous integration as high as 50%, depending on the target gene and on the length of homologous DNA sequence flanking the selectable marker. The results indicate that the split-marker approach favorably decreased ectopic integration and thus, greatly facilitated targeted gene disruption in this important fungal pathogen.

  4. Crystallization and preliminary X-ray diffraction analysis of NADPH-dependent thioredoxin reductase I from Saccharomyces cerevisiae

    SciTech Connect

    Oliveira, Marcos Antonio de; Discola, Karen Fulan; Alves, Simone Vidigal; Barbosa, João Alexandre Ribeiro Gonçalves; Medrano, Francisco Javier; Netto, Luis Eduardo Soares; Guimarães, Beatriz Gomes

    2005-04-01

    Thioredoxin reductase 1 (Trr1) from S. cerevisiae is a component of the thioredoxin system, which is involved in several biological processes, including the reduction of disulfide bonds and response to oxidative stress. The expression, purification, crystallization and preliminary X-ray crystallographic studies of yeast Trr1 are reported. Thioredoxin reductase 1 (Trr1) from Saccharomyces cerevisiae is a member of the family of pyridine nucleotide-disulfide oxidoreductases capable of reducing the redox-active disulfide bond of the cytosolic thioredoxin 1 (Trx1) and thioredoxin 2 (Trx2). NADPH, Trr1 and Trx1 (or Trx2) comprise the thioredoxin system, which is involved in several biological processes, including the reduction of disulfide bonds and response to oxidative stress. Recombinant Trr1 was expressed in Escherichia coli as a His{sub 6}-tagged fusion protein and purified by nickel-affinity chromatography. The protein was crystallized using the hanging-drop vapour-diffusion method in the presence of PEG 3000 as precipitant after treatment with hydrogen peroxide. X-ray diffraction data were collected to a maximum resolution of 2.4 Å using a synchrotron-radiation source. The crystal belongs to the centred monoclinic space group C2, with unit-cell parameters a = 127.97, b = 135.41, c = 75.81 Å, β = 89.95°. The crystal structure was solved by molecular-replacement methods and structure refinement is in progress.

  5. Cloning and expression of the ccdA-associated thiol-disulfide oxidoreductase (catA) gene from Brevibacillus choshinensis: stimulation of human epidermal growth factor production.

    PubMed

    Tanaka, Ryoichi; Mizukami, Makoto; Ishibashi, Matsujiro; Tokunaga, Hiroko; Tokunaga, Masao

    2003-06-12

    Brevibacillus choshinensis (Bacillus brevis) is a protein-hyperproducing bacterium with a useful host-vector system for the production of recombinant proteins. Here, we cloned the ccdA-catA (cmacr;cdA āssociated thioredoxin-like tmacr;hiol-disulfide oxidoreductase) locus of B. choshinensis HPD31-S5. CatA protein (molecular weight, 19664) contains a thioredoxin-like motif, Cys-Gly-Pro-Cys. It was successfully expressed in B. choshinensis extracellularly ( approximately 100 microg x ml(-1) culture) using the secretion vector pNCMO2, and in Escherichia coli intracellularly ( approximately 350 microg x ml(-1) culture) with an amino-terminal His-tag. Both recombinant proteins showed thiol-disulfide oxidoreductase activity. Incubation of non-native human epidermal growth factor (hEGF) containing incorrect disulfide bonds with B. choshinensis cells secreting CatA protein resulted in the stimulation of the conversion of non-native hEGF to the native form. Furthermore, co-expression of CatA protein with recombinant hEGF in the B. choshinensis production system increased the yield of native hEGF.

  6. Thioredoxin f1 and NADPH-Dependent Thioredoxin Reductase C Have Overlapping Functions in Regulating Photosynthetic Metabolism and Plant Growth in Response to Varying Light Conditions1[OPEN

    PubMed Central

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

    2015-01-01

    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. PMID:26338951

  7. 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.

  8. Beta Hydroxylation of Glycolipids from Ustilago maydis and Pseudozyma flocculosa by an NADPH-Dependent β-Hydroxylase▿

    PubMed Central

    Teichmann, Beate; Lefebvre, François; Labbé, Caroline; Bölker, Michael; Linne, Uwe; Bélanger, Richard R.

    2011-01-01

    Flocculosin and ustilagic acid (UA), two highly similar antifungal cellobiose lipids, are respectively produced by Pseudozyma flocculosa, a biocontrol agent, and Ustilago maydis, a plant pathogen. Both glycolipids contain a short-chain fatty acid hydroxylated at the β position but differ in the long fatty acid, which is hydroxylated at the α position in UA and at the β position in flocculosin. In both organisms, the biosynthesis genes are arranged in large clusters. The functions of most genes have already been characterized, but those of the P. flocculosa fhd1 gene and its homolog from U. maydis, uhd1, have remained undefined. The deduced amino acid sequences of these genes show homology to those of short-chain dehydrogenases and reductases (SDR). We disrupted the uhd1 gene in U. maydis and analyzed the secreted UA. uhd1 deletion strains produced UA lacking the β-hydroxyl group of the short-chain fatty acid. To analyze the function of P. flocculosa Fhd1, the corresponding gene was used to complement U. maydis Δuhd1 mutants. Fhd1 was able to restore wild-type UA production, indicating that Fhd1 is responsible for β hydroxylation of the flocculosin short-chain fatty acid. We also investigated a P. flocculosa homolog of the U. maydis long-chain fatty-acid alpha hydroxylase Ahd1. The P. flocculosa ahd1 gene, which does not reside in the flocculosin gene cluster, was introduced into U. maydis Δahd1 mutant strains. P. flocculosa Ahd1 neither complemented the U. maydis Δahd1 phenotype nor resulted in the production of β-hydroxylated UA. This suggests that P. flocculosa Ahd1 is not involved in flocculosin hydroxylation. PMID:21926207

  9. 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

  10. Cloning and analysis of structural genes from Streptomyces pristinaespiralis encoding enzymes involved in the conversion of pristinamycin IIB to pristinamycin IIA (PIIA): PIIA synthase and NADH:riboflavin 5'-phosphate oxidoreductase.

    PubMed Central

    Blanc, V; Lagneaux, D; Didier, P; Gil, P; Lacroix, P; Crouzet, J

    1995-01-01

    In Streptomyces pristinaespiralis, two enzymes are necessary for conversion of pristinamycin IIB (PIIB) to pristinamycin IIA (PIIA), the major component of pristinamycin (D. Thibaut, N. Ratet, D. Bisch, D. Faucher, L. Debussche, and F. Blanche, J. Bacteriol. 177:5199-5205, 1995); these enzymes are PIIA synthase, a heterodimer composed of the SnaA and SnaB proteins, which catalyzes the oxidation of PIIB to PIIA, and the NADH:riboflavin 5'-phosphate oxidoreductase (hereafter called FMN reductase), the SnaC protein, which provides the reduced form of flavin mononucleotide for the reaction. By using oligonucleotide probes designed from limited peptide sequence information of the purified proteins, the corresponding genes were cloned from a genomic library of S. pristinaespiralis. SnaA and SnaB showed no significant similarity with proteins from databases, but SnaA and SnaB had similar protein domains. Disruption of the snaA gene in S. pristinaespiralis led to accumulation of PIIB. Complementation of a S. pristinaespiralis PIIA-PIIB+ mutant with the snaA and snaB genes, cloned in a low-copy-number plasmid, partially restored production of PIIA. The deduced amino acid sequence of the snaC gene showed no similarity to the sequences of other FMN reductases but was 39% identical with the product of the actVB gene of the actinorhodin cluster of Streptomyces coelicolor A(3)2, likely to be involved in the dimerization step of actinorhodin biosynthesis. Furthermore, an S. coelicolor A(3)2 mutant blocked in this step was successfully complemented by the snaC gene, restoring the production of actinorhodin. PMID:7665509

  11. Directed evolution and structural analysis of NADPH-dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) reductase from Ralstonia eutropha reveals two mutations responsible for enhanced kinetics.

    PubMed

    Matsumoto, Ken'ichiro; Tanaka, Yoshikazu; Watanabe, Tsuyoshi; Motohashi, Ren; Ikeda, Koji; Tobitani, Kota; Yao, Min; Tanaka, Isao; Taguchi, Seiichi

    2013-10-01

    NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with β-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from Ralstonia eutropha was engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based in vivo screening system using Escherichia coli. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibited kcat values that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played an important role in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex with NADPH and acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity.

  12. Deficient flavoprotein component of the NADPH-dependent O2-.-generating oxidase in the neutrophils from three male patients with chronic granulomatous disease.

    PubMed Central

    Gabig, T G; Lefker, B A

    1984-01-01

    The NADPH-dependent O2-.-generating oxidase in subcellular fractions from the neutrophils of three male patients with chronic granulomatous disease was compared with the corresponding preparations from normal neutrophils. The oxidase from normal neutrophils contained flavin adenine dinucleotide in an approximately 0.9:1 molar ratio with cytochrome b559. Each of the three chronic granulomatous disease patients had decreased amounts of the flavoprotein component of the oxidase fraction. The oxidase from two chronic granulomatous disease patients had undetectable amounts of cytochrome b559 whereas the third patient had a normal content of cytochrome b559, which was spectrally indistinguishable from the normal. The intrinsic cytochrome b559 in the oxidase fraction from stimulated neutrophils of the latter chronic granulomatous disease patient was not reduced by NADPH under anaerobic conditions, in distinction with the previously reported reduction of the normal cytochrome b559 under identical conditions. We conclude that the flavoprotein component of the oxidase may mediate transfer of electrons from NADPH to the cytochrome b559 in normal neutrophils, and that deficiency of this flavoprotein is associated with the chronic granulomatous disease phenotype in the three patients studied. PMID:6707199

  13. Deficient flavoprotein component of the NADPH-dependent O2-.-generating oxidase in the neutrophils from three male patients with chronic granulomatous disease.

    PubMed

    Gabig, T G; Lefker, B A

    1984-03-01

    The NADPH-dependent O2-.-generating oxidase in subcellular fractions from the neutrophils of three male patients with chronic granulomatous disease was compared with the corresponding preparations from normal neutrophils. The oxidase from normal neutrophils contained flavin adenine dinucleotide in an approximately 0.9:1 molar ratio with cytochrome b559. Each of the three chronic granulomatous disease patients had decreased amounts of the flavoprotein component of the oxidase fraction. The oxidase from two chronic granulomatous disease patients had undetectable amounts of cytochrome b559 whereas the third patient had a normal content of cytochrome b559, which was spectrally indistinguishable from the normal. The intrinsic cytochrome b559 in the oxidase fraction from stimulated neutrophils of the latter chronic granulomatous disease patient was not reduced by NADPH under anaerobic conditions, in distinction with the previously reported reduction of the normal cytochrome b559 under identical conditions. We conclude that the flavoprotein component of the oxidase may mediate transfer of electrons from NADPH to the cytochrome b559 in normal neutrophils, and that deficiency of this flavoprotein is associated with the chronic granulomatous disease phenotype in the three patients studied.

  14. Exposure to low- vs iso-osmolar contrast agents reduces NADPH-dependent reactive oxygen species generation in a cellular model of renal injury.

    PubMed

    Netti, Giuseppe Stefano; Prattichizzo, Clelia; Montemurno, Eustacchio; Simone, Simona; Cafiero, Cesira; Rascio, Federica; Stallone, Giovanni; Ranieri, Elena; Grandaliano, Giuseppe; Gesualdo, Loreto

    2014-03-01

    Contrast-induced nephropathy represents the third cause of hospital-acquired acute renal failure. This study investigated the effects of low- vs iso-osmolar contrast medium (CM) exposure on NADPH-dependent reactive oxygen species (ROS) generation by tubular cells. X-ray attenuation of iohexol, iopamidol, and iodixanol was assessed at equimolar iodine concentrations and their effects on human renal proximal tubular cells (PTCs) were evaluated with equally attenuating solutions of each CM. Cytotoxicity, apoptosis, and necrosis were investigated by trypan blue exclusion, MTT assay, and annexin V/propidium iodide assay, respectively. ROS production was assessed by DCF assay, NADPH oxidase activity by the lucigenin-enhanced chemiluminescence method, and Nox4 expression by immunoblot. Yielding the same X-ray attenuation, CM cytotoxicity was assessed in PTCs at equimolar iodine concentrations. More necrosis was present after incubation with iohexol and iopamidol than after incubation with equal concentrations of iodixanol. Iohexol and iodixanol at low iodine concentrations induced less cytotoxicity than iopamidol. Moreover, both iohexol and iopamidol induced more apoptosis than iodixanol, with a dose-dependent effect. ROS generation was significantly higher with iopamidol and iohexol compared to iodixanol. NADPH oxidase activity and Nox4 protein expression significantly increased after exposure to iopamidol and iohexol, with a dose-dependent effect, compared with iodixanol. CM-induced Nox4 expression and activity depended upon Src activation. In conclusion, at angiographic concentrations, iodixanol induces fewer cytotoxic effects on cultured tubular cells than iohexol and iopamidol along with a lower induction of Nox4-dependent ROS generation. This enzyme may, thus, represent a potential therapeutic target to prevent iodinated CM-related oxidative stress.

  15. The Saccharomyces cerevisiae YMR315W Gene Encodes an NADP(H)-Specific Oxidoreductase Regulated by the Transcription Factor Stb5p in Response to NADPH Limitation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Engineered xylose-metabolizing cells grown on xylose show increased expression of YMR315W at both the mRNA and protein levels. Additionally, the YMR315W promoter contains a putative binding site for the transcription factor Stb5p, which has been shown to regulate genes involved in nicotinamide aden...

  16. The gene sml0013 of Synechocystis species strain PCC 6803 encodes for a novel subunit of the NAD(P)H oxidoreductase or complex I that is ubiquitously distributed among Cyanobacteria.

    PubMed

    Schwarz, Doreen; Schubert, Hendrik; Georg, Jens; Hess, Wolfgang R; Hagemann, Martin

    2013-11-01

    The NAD(P)H oxidoreductase or complex I (NDH1) complex participates in many processes such as respiration, cyclic electron flow, and inorganic carbon concentration in the cyanobacterial cell. Despite immense progress in our understanding of the structure-function relation of the cyanobacterial NDH1 complex, the subunits catalyzing NAD(P)H docking and oxidation are still missing. The gene sml0013 of Synechocystis 6803 encodes for a small protein of unknown function for which homologs exist in all completely known cyanobacterial genomes. The protein exhibits weak similarities to the NDH-dependent flow6 (NDF6) protein, which was reported from Arabidopsis (Arabidopsis thaliana) chloroplasts as a NDH subunit. An sml0013 inactivation mutant of Synechocystis 6803 was generated and characterized. It showed only weak differences regarding growth and pigmentation in various culture conditions; most remarkably, it exhibited a glucose-sensitive phenotype in the light. The genome-wide expression pattern of the Δsml0013::Km mutant was almost identical to the wild type when grown under high CO2 conditions as well as after shifts to low CO2 conditions. However, measurements of the photosystem I redox kinetic in cells of the Δsml0013::Km mutant revealed differences, such as a decreased capability of cyclic electron flow as well as electron flow into respiration in comparison with the wild type. These results suggest that the Sml0013 protein (named NdhP) represents a novel subunit of the cyanobacterial NDH1 complex, mediating its coupling either to the respiratory or the photosynthetic electron flow.

  17. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  18. Engineering class I cytochrome P450 by gene fusion with NADPH-dependent reductase and S. avermitilis host development for daidzein biotransformation.

    PubMed

    Choi, Kwon-Young; Jung, EunOk; Yun, Hyungdon; Yang, Yung-Hun; Kim, Byung-Gee

    2014-10-01

    Daidzein C6 hydroxylase (6-DH, nfa12130), which is a class I type of cytochrome P450 enzyme, catalyzes a hydroxylation reaction at the C6-position of the daidzein A-ring and requires auxiliary electron transfer proteins. Current utilization of cytochrome P450 (CYP) enzymes is limited by low coupling efficiency, which necessitates extramolecular electron transfers, and low driving forces, which derive electron flows from tightly regulated NADPH redox balances into the heterogeneous CYP catalytic cycle. To overcome such limitations, the heme domain of the 6-DH enzyme was genetically fused with the NADPH-reductase domain of self-sufficient CYP102D1 to enhance electron transfer efficiencies through intramolecular electron transfer and switching cofactor preference from NADH into NADPH. 6-DH-reductase fusion enzyme displayed distinct spectral properties of both flavoprotein and heme proteins and catalyzed daidzein hydroxylation more efficiently with a k cat/K m value of 120.3 ± 11.5 [10(3) M(-1) s(-1)], which was about three times higher than that of the 6-DH-FdxC-FdrA reconstituted system. Moreover, to obtain a higher redox driving force, a Streptomyces avermitilis host system was developed for heterologous expression of fusion 6-DH enzyme and whole cell biotransformation of daidzein. The whole cell reaction using the final recombinant strain, S. avermitilisΔcyp105D7::fusion 6-DH (nfa12130), resulted in 8.3 ± 1.4 % of 6-OHD yield from 25.4 mg/L of daidzein.

  19. Lactic acid-producing yeast cells having nonfunctional L- or D-lactate:ferricytochrome C oxidoreductase cells

    DOEpatents

    Miller, Matthew [Boston, MA; Suominen, Pirkko [Maple Grove, MN; Aristidou, Aristos [Highland Ranch, CO; Hause, Benjamin Matthew [Currie, MN; Van Hoek, Pim [Camarillo, CA; Dundon, Catherine Asleson [Minneapolis, MN

    2012-03-20

    Yeast cells having an exogenous lactate dehydrogenase gene ae modified by reducing L- or D-lactate:ferricytochrome c oxidoreductase activity in the cell. This leads to reduced consumption of lactate by the cell and can increase overall lactate yields in a fermentation process. Cells having the reduced L- or D-lactate:ferricytochrome c oxidoreductase activity can be screened for by resistance to organic acids such as lactic or glycolic acid.

  20. Xenobiotic induction of quinone oxidoreductase activity in lens epithelial cells.

    PubMed

    Tumminia, S J; Rao, P V; Zigler, J S; Russell, P

    1993-12-08

    Xenobiotic regulatory elements have been identified for enzymes which ameliorate oxidative damage in cells. Zeta (zeta)-crystallin, a taxon-specific enzyme/crystallin shown to be a novel NADPH-dependent quinone reductase, is found in a number of tissues and cell types. This study shows that zeta-crystallin is present in mouse lens epithelium, as well as in the alpha TN4 mouse lens epithelial cell line. To determine whether zeta-crystallin is an inducible quinone reductase, cell cultures were exposed to the xenobiotics, 1,2-naphthoquinone and beta-naphthoflavone. Assays of cellular homogenates showed that quinone reductase activity was stimulated greater than 70% and 90%, respectively, over the control cells. This observed activity was sensitive to dicumarol, a potent inhibitor of quinone reductase activity. 1,2-Naphthoquinone- and beta-naphthoflavone-exposed cells were found to exhibit 1.47- and 1.68-fold increases, respectively, in zeta-crystallin protein concentration. A comparable increase in zeta-crystallin mRNA was indicative of an induction in zeta-crystallin expression in response to naphthalene challenge. Lens epithelial cells were also checked for DT-diaphorase, a well-known cellular protective enzyme which can catalyze the two-electron reduction of quinones. Slot blot analyses indicated that alpha TN4 cells exposed to 1,2-naphthoquinone and beta-naphthoflavone exhibited 2.71- and 6.81-fold increases in DT-diaphorase concentration when compared to the control cells. The data suggest that while DT-diaphorase is most likely responsible for the majority of the observed increase in quinone reductase activity, the zeta-crystallin gene also undergoes activation which is apparently mediated by a xenobiotic-responsive element.

  1. Cytochrome P450 oxidoreductase participates in nitric oxide consumption by rat brain.

    PubMed

    Hall, Catherine N; Keynes, Robert G; Garthwaite, John

    2009-04-15

    In low nanomolar concentrations, NO (nitric oxide) functions as a transmitter in brain and other tissues, whereas near-micromolar NO concentrations are associated with toxicity and cell death. Control of the NO concentration, therefore, is critical for proper brain function, but, although its synthesis pathway is well-characterized, the major route of breakdown of NO in brain is unclear. Previous observations indicate that brain cells actively consume NO at a high rate. The mechanism of this consumption was pursued in the present study. NO consumption by a preparation of central glial cells was abolished by cell lysis and recovered by addition of NADPH. NADPH-dependent consumption of NO localized to cell membranes and was inhibited by proteinase K, indicating the involvement of a membrane-bound protein. Purification of this activity yielded CYPOR (cytochrome P450 oxidoreductase). Antibodies against CYPOR inhibited NO consumption by brain membranes and the amount of CYPOR in several cell types correlated with their rate of NO consumption. NO was also consumed by purified CYPOR but this activity was found to depend on the presence of the vitamin E analogue Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), included in the buffer as a precaution against inadvertent NO consumption by lipid peroxidation. In contrast, NO consumption by brain membranes was independent of Trolox. Hence, it appears that, during the purification process, CYPOR becomes separated from a partner needed for NO consumption. Cytochrome P450 inhibitors inhibited NO consumption by brain membranes, making these proteins likely candidates.

  2. Discovering the electronic circuit diagram of life: structural relationships among transition metal binding sites in oxidoreductases

    PubMed Central

    Kim, J. Dongun; Senn, Stefan; Harel, Arye; Jelen, Benjamin I.; Falkowski, Paul G.

    2013-01-01

    Oxidoreductases play a central role in catalysing enzymatic electron-transfer reactions across the tree of life. To first order, the equilibrium thermodynamic properties of these proteins are governed by protein folds associated with specific transition metals and ligands at the active site. A global analysis of holoenzyme structures and functions suggests that there are fewer than approximately 500 fundamental oxidoreductases, which can be further clustered into 35 unique groups. These catalysts evolved in prokaryotes early in the Earth's history and are largely responsible for the emergence of non-equilibrium biogeochemical cycles on the planet's surface. Although the evolutionary history of the amino acid sequences in the oxidoreductases is very difficult to reconstruct due to gene duplication and horizontal gene transfer, the evolution of the folds in the catalytic sites can potentially be used to infer the history of these enzymes. Using a novel, yet simple analysis of the secondary structures associated with the ligands in oxidoreductases, we developed a structural phylogeny of these enzymes. The results of this ‘composome’ analysis suggest an early split from a basal set of a small group of proteins dominated by loop structures into two families of oxidoreductases, one dominated by α-helices and the second by β-sheets. The structural evolutionary patterns in both clades trace redox gradients and increased hydrogen bond energy in the active sites. The overall pattern suggests that the evolution of the oxidoreductases led to decreased entropy in the transition metal folds over approximately 2.5 billion years, allowing the enzymes to use increasingly oxidized substrates with high specificity. PMID:23754810

  3. Occurrence of ferredoxin:NAD+ oxidoreductase activity and its ion specificity in several Gram-positive and Gram-negative bacteria

    PubMed Central

    Hess, Verena; Gallegos, Rene; Jones, J Andrew; Barquera, Blanca; Malamy, Michael H

    2016-01-01

    A ferredoxin:NAD+ oxidoreductase was recently discovered as a redox-driven ion pump in the anaerobic, acetogenic bacterium Acetobacterium woodii. The enzyme is assumed to be encoded by the rnf genes. Since these genes are present in the genomes of many bacteria, we tested for ferredoxin:NAD+ oxidoreductase activity in cytoplasmic membranes from several different Gram-positive and Gram-negative bacteria that have annotated rnf genes. We found this activity in Clostridium tetanomorphum, Clostridium ljungdahlii, Bacteroides fragilis, and Vibrio cholerae but not in Escherichia coli and Rhodobacter capsulatus. As in A. woodii, the activity was Na+-dependent in C. tetanomorphum and B. fragilis but Na+-independent in C. ljungdahlii and V. cholerae. We deleted the rnf genes from B. fragilis and demonstrated that the mutant has greatly reduced ferredoxin:NAD+ oxidoreductase activity. This is the first genetic proof that the rnf genes indeed encode the reduced ferredoxin:NAD+ oxidoreductase activity. PMID:26793417

  4. The Study of NADPH-Dependent Flavoenzyme-Catalyzed Reduction of Benzo[1,2-c]1,2,5-oxadiazole N-Oxides (Benzofuroxans)

    PubMed Central

    Šarlauskas, Jonas; Misevičienė, Lina; Marozienė, Audronė; Karvelis, Laimonas; Stankevičiūtė, Jonita; Krikštopaitis, Kastis; Čėnas, Narimantas; Yantsevich, Aleksey; Laurynėnas, Audrius; Anusevičius, Žilvinas

    2014-01-01

    The enzymatic reactivity of a series of benzo[1,2-c]1,2,5-oxadiazole N-oxides (benzofuroxans; BFXs) towards mammalian single-electron transferring NADPH:cytochrome P-450 reductase (P-450R) and two-electron (hydride) transferring NAD(P)H:quinone oxidoreductase (NQO1) was examined in this work. Since the =N+ (→O)O− moiety of furoxan fragments of BFXs bears some similarity to the aromatic nitro-group, the reactivity of BFXs was compared to that of nitro-aromatic compounds (NACs) whose reduction mechanisms by these and other related flavoenzymes have been extensively investigated. The reduction of BFXs by both P-450R and NQO1 was accompanied by O2 uptake, which was much lower than the NADPH oxidation rate; except for annelated BFXs, whose reduction was followed by the production of peroxide. In order to analyze the possible quantitative structure-activity relationships (QSARs) of the enzymatic reactivity of the compounds, their electron-accepting potency and other reactivity indices were assessed by quantum mechanical methods. In P-450R-catalyzed reactions, both BFXs and NACs showed the same reactivity dependence on their electron-accepting potency which might be consistent with an “outer sphere” electron transfer mechanism. In NQO1-catalyzed two-electron (hydride) transferring reactions, BFXs acted as more efficient substrates than NACs, and the reduction efficacy of BFXs by NQO1 was in general higher than by single-electron transferring P-450R. In NQO1-catalyzed reactions, QSARs obtained showed that the reduction efficacy of BFXs, as well as that of NACs, was determined by their electron-accepting potency and could be influenced by their binding mode in the active center of NQO1 and by their global softness as their electronic characteristic. The reductive conversion of benzofuroxan by both flavoenzymes yielded the same reduction product of benzofuroxan, 2,3-diaminophenazine, with the formation of o-benzoquinone dioxime as a putative primary reductive

  5. Purification of the enzyme NADPH: protochlorophyllide oxidoreductase.

    PubMed

    Beer, N S; Griffiths, W T

    1981-04-01

    A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.

  6. An oxidoreductase is involved in cercosporin degradation by the bacterium Xanthomonas campestris pv. zinniae.

    PubMed

    Taylor, Tanya V; Mitchell, Thomas K; Daub, Margaret E

    2006-09-01

    The polyketide toxin cercosporin plays a key role in pathogenesis by fungal species of the genus Cercospora. The bacterium Xanthomonas campestris pv. zinniae is able to rapidly degrade this toxin. Growth of X. campestris pv. zinniae strains in cercosporin-containing medium leads to the breakdown of cercosporin and to the formation of xanosporic acid, a nontoxic breakdown product. Five non-cercosporin-degrading mutants of a strain that rapidly degrades cercosporin (XCZ-3) were generated by ethyl methanesulfonate mutagenesis and were then transformed with a genomic library from the wild-type strain. All five mutants were complemented with the same genomic clone, which encoded a putative transcriptional regulator and an oxidoreductase. Simultaneous expression of these two genes was necessary to complement the mutant phenotype. Sequence analysis of the mutants showed that all five mutants had point mutations in the oxidoreductase gene and no mutations in the regulator. Quantitative reverse transcription-PCR (RT-PCR) showed that the expression of both of these genes in the wild-type strain is upregulated after exposure to cercosporin. Both the oxidoreductase and transcriptional regulator genes were transformed into three non-cercosporin-degrading bacteria to determine if they are sufficient for cercosporin degradation. Quantitative RT-PCR analysis confirmed that the oxidoreductase was expressed in all transconjugants. However, none of the transconjugants were able to degrade cercosporin, suggesting that additional factors are required for cercosporin degradation. Further study of cercosporin degradation in X. campestris pv. zinniae may allow for the engineering of Cercospora-resistant plants by using a suite of genes.

  7. Esculetin-induced protection of human hepatoma HepG2 cells against hydrogen peroxide is associated with the Nrf2-dependent induction of the NAD(P)H: Quinone oxidoreductase 1 gene

    SciTech Connect

    Subramaniam, Sudhakar R.; Ellis, Elizabeth M.

    2011-01-15

    Esculetin (6,7-dihydroxy coumarin), is a potent antioxidant that is present in several plant species. The aim of this study was to investigate the mechanism of protection of esculetin in human hepatoma HepG2 cells against reactive oxygen species (ROS) induced by hydrogen peroxide. Cell viability, cell integrity, intracellular glutathione levels, generation of reactive oxygen species and expression of antioxidant enzymes were used as markers to measure cellular oxidative stress and response to ROS. The protective effect of esculetin was compared to a well-characterized chemoprotective compound quercetin. Pre-treatment of HepG2 cells with sub-lethal (10-25 {mu}M) esculetin for 8 h prevented cell death and maintained cell integrity following exposure to 0.9 mM hydrogen peroxide. An increase in the generation of ROS following hydrogen peroxide treatment was significantly attenuated by 8 h pre-treatment with esculetin. In addition, esculetin ameliorated the decrease in intracellular glutathione caused by hydrogen peroxide exposure. Moreover, treatment with 25 {mu}M esculetin for 8 h increased the expression of NAD(P)H: quinone oxidoreductase (NQO1) at both protein and mRNA levels significantly, by 12-fold and 15-fold, respectively. Esculetin treatment also increased nuclear accumulation of Nrf2 by 8-fold indicating that increased NQO1 expression is Nrf2-mediated. These results indicate that esculetin protects human hepatoma HepG2 cells from hydrogen peroxide induced oxidative injury and that this protection is provided through the induction of protective enzymes as part of an adaptive response mediated by Nrf2 nuclear accumulation.

  8. Thioredoxin-thioredoxin reductase system of Streptomyces clavuligerus: sequences, expression, and organization of the genes.

    PubMed Central

    Cohen, G; Yanko, M; Mislovati, M; Argaman, A; Schreiber, R; Av-Gay, Y; Aharonowitz, Y

    1993-01-01

    The genes that encode thioredoxin and thioredoxin reductase of Streptomyces clavuligerus were cloned, and their DNA sequences were determined. Previously, we showed that S. clavuligerus possesses a disulfide reductase with broad substrate specificity that biochemically resembles the thioredoxin oxidoreductase system and may play a role in the biosynthesis of beta-lactam antibiotics. It consists consists of two components, a 70-kDa NADPH-dependent flavoprotein disulfide reductase with two identical subunits and a 12-kDa heat-stable protein general disulfide reductant. In this study, we found, by comparative analysis of their predicted amino acid sequences, that the 35-kDa protein is in fact thioredoxin reductase; it shares 48.7% amino acid sequence identity with Escherichia coli thioredoxin reductase, the 12-kDa protein is thioredoxin, and it shares 28 to 56% amino acid sequence identity with other thioredoxins. The streptomycete thioredoxin reductase has the identical cysteine redox-active region--Cys-Ala-Thr-Cys--and essentially the same flavin adenine dinucleotide- and NADPH dinucleotide-binding sites as E. coli thioredoxin reductase and is partially able to accept E. coli thioredoxin as a substrate. The streptomycete thioredoxin has the same cysteine redox-active segment--Trp-Cys-Gly-Pro-Cys--that is present in virtually all eucaryotic and procaryotic thioredoxins. However, in vivo it is unable to donate electrons to E. coli methionine sulfoxide reductase and does not serve as a substrate in vitro for E. coli thioredoxin reductase. The S. clavuligerus thioredoxin (trxA) and thioredoxin reductase (trxB) genes are organized in a cluster. They are transcribed in the same direction and separated by 33 nucleotides. In contrast, the trxA and trxB genes of E. coli, the only other organism in which both genes have been characterized, are physically widely separated. Images PMID:8349555

  9. Genetics Home Reference: cytochrome P450 oxidoreductase deficiency

    MedlinePlus

    ... hormones, which are needed for normal development and reproduction. The hormonal changes associated with cytochrome P450 oxidoreductase ... which are essential for normal sexual development and reproduction; corticosteroids, which are involved in the body's response ...

  10. Light-Dependent Protochlorophyllide Oxidoreductase: Phylogeny, Regulation, and Catalytic Properties.

    PubMed

    Gabruk, Michal; Mysliwa-Kurdziel, Beata

    2015-09-01

    This Current Topic focuses on light-dependent protochlorophyllide oxidoreductase (POR, EC 1.3.1.33). POR catalyzes the penultimate reaction of chlorophyll biosynthesis, i.e., the light-triggered reduction of protochlorophyllide to chlorophyllide. In this reaction, the chlorin ring of the chlorophyll molecule is formed, which is crucial for photosynthesis. POR is one of very few enzymes that are driven by light; however, it is unique in the need for its substrate to absorb photons to induce the conformational changes in the enzyme, which are required for its catalytic activation. Moreover, the enzyme is also involved in the negative feedback of the chlorophyll biosynthesis pathway and controls chlorophyll content via its light-dependent activity. Even though it has been almost 70 years since the first isolation of active POR complexes, our knowledge of them has markedly advanced in recent years. In this review, we summarize the current state of knowledge of POR, including the phylogenetic roots of POR, the mechanisms of the regulation of POR genes expression, the regulation of POR activity, the import of POR into plastids, the role of POR in PLB formation, and the molecular mechanism of protochlorophyllide reduction by POR. To the best of our knowledge, no previous review has compiled such a broad set of recent findings about POR.

  11. Targeted ablation of the WW domain-containing oxidoreductase tumor suppressor leads to impaired steroidogenesis.

    PubMed

    Aqeilan, Rami I; Hagan, John P; de Bruin, Alain; Rawahneh, Maysoon; Salah, Zaidoun; Gaudio, Eugenio; Siddiqui, Hasan; Volinia, Stefano; Alder, Hansjuerg; Lian, Jane B; Stein, Gary S; Croce, Carlo M

    2009-03-01

    The WW domain-containing oxidoreductase (WWOX) gene encodes a 46-kDa tumor suppressor. The Wwox protein contains two N-terminal WW domains that interact with several transcriptional activators containing proline-tyrosine motifs and a central short-chain dehydrogenase/reductase domain that has been suggested to play a role in steroid metabolism. Recently, we have shown that targeted deletion of the Wwox gene in mice leads to postnatal lethality and defects in bone growth. Here, we report that Wwox-deficient mice display impaired steroidogenesis. Mutant homozygous mice are born with gonadal abnormalities, including failure of Leydig cell development in testis and reduced theca cell proliferation in ovary. Furthermore, Wwox(-/-) mice displayed impaired gene expression of key steroidogenesis enzymes. Affymetrix microarray gene analysis revealed differentially expressed related genes in steroidogenesis in knockout mice testis and ovary as compared with control mice. These results demonstrate the essential requirement for the Wwox tumor suppressor in proper steroidogenesis.

  12. Efficient production of 1,3-propanediol from glycerol upon constitutive expression of the 1,3-propanediol oxidoreductase gene in engineered Klebsiella pneumoniae with elimination of by-product formation.

    PubMed

    Oh, Baek-Rock; Seo, Jeong-Woo; Heo, Sun-Yeon; Luo, Lian Hua; Hong, Won-Kyung; Park, Don-Hee; Kim, Chul-Ho

    2013-06-01

    In the present study, we developed an efficient method of 1,3-propanediol (1,3-PD) production from glycerol by genetic engineering of Klebsiella pneumoniae AK mutant strains. The proposed approach eliminated by-product formation and IPTG induction resulted in maximal production of 1,3-PD. A series of recombinant strains was designed to constitutively express the dhaB and/or dhaT genes, using the bacteriophage T5 P(DE20) promoter and the rho-independent transcription termination signal of the Rahnella aquatilis levansucrase gene. Among these strains, AK/pConT expressing dhaT alone gave the highest yield of 1,3-PD. Fed-batch fermentation resulted in efficient production of 1,3-PD from either pure or crude glycerol, without by-product formation.

  13. The roles of thiol oxidoreductases in yeast replicative aging.

    PubMed

    Hacioglu, Elise; Esmer, Isil; Fomenko, Dmitri E; Gladyshev, Vadim N; Koc, Ahmet

    2010-01-01

    Thiol-based redox reactions are involved in the regulation of a variety of biological functions, such as protection against oxidative stress, signal transduction and protein folding. Some proteins involved in redox regulation have been shown to modulate life span in organisms from yeast to mammals. To assess the role of thiol oxidoreductases in aging on a genome-wide scale, we analyzed the replicative life span of yeast cells lacking known and candidate thiol oxidoreductases. The data suggest the role of several pathways in controlling yeast replicative life span, including thioredoxin reduction, protein folding and degradation, peroxide reduction, PIP3 signaling, and ATP synthesis.

  14. Novel processing and localization of catA, ccdA associated thiol-disulfide oxidoreductase, in protein hyper-producing bacterium Brevibacillus choshinensis.

    PubMed

    Tanaka, Ryoichi; Mizukami, Makoto; Tokunaga, Masao

    2005-01-01

    Previously, we have cloned ccdA and its associated thiol-disulfide oxidoreductase gene, catA, in Brevibacillus choshinensis. CcdA is known to be an integral membrane protein and its associated oxidoreductase homologues are believed to be membrane anchoring proteins, both providing reducing equivalents across the membrane to control correct disulfide bond formation. Here, we found that CatA is first localized as a membrane bound form and then slowly released into the cellular periphery and culture medium with cleavage at a novel processing site.

  15. Study of the thiol/disulfide redox systems of the anaerobe Desulfovibrio vulgaris points out pyruvate:ferredoxin oxidoreductase as a new target for thioredoxin 1.

    PubMed

    Pieulle, Laetitia; Stocker, Pierre; Vinay, Manon; Nouailler, Matthieu; Vita, Nicolas; Brasseur, Gaël; Garcin, Edwige; Sebban-Kreuzer, Corinne; Dolla, Alain

    2011-03-11

    Sulfate reducers have developed a multifaceted adaptative strategy to survive against oxidative stresses. Along with this oxidative stress response, we recently characterized an elegant reversible disulfide bond-dependent protective mechanism in the pyruvate:ferredoxin oxidoreductase (PFOR) of various Desulfovibrio species. Here, we searched for thiol redox systems involved in this mechanism. Using thiol fluorescent labeling, we show that glutathione is not the major thiol/disulfide balance-controlling compound in four different Desulfovibrio species and that no other plentiful low molecular weight thiol can be detected. Enzymatic analyses of two thioredoxins (Trxs) and three thioredoxin reductases allow us to propose the existence of two independent Trx systems in Desulfovibrio vulgaris Hildenborough (DvH). The TR1/Trx1 system corresponds to the typical bacterial Trx system. We measured a TR1 apparent K(m) value for Trx1 of 8.9 μM. Moreover, our results showed that activity of TR1 was NADPH-dependent. The second system named TR3/Trx3 corresponds to an unconventional Trx system as TR3 used preferentially NADH (K(m) for NADPH, 743 μM; K(m) for NADH, 5.6 μM), and Trx3 was unable to reduce insulin. The K(m) value of TR3 for Trx3 was 1.12 μM. In vitro experiments demonstrated that the TR1/Trx1 system was the only one able to reactivate the oxygen-protected form of Desulfovibrio africanus PFOR. Moreover, ex vivo pulldown assays using the mutant Trx1(C33S) as bait allowed us to capture PFOR from the DvH extract. Altogether, these data demonstrate that PFOR is a new target for Trx1, which is probably involved in the protective switch mechanism of the enzyme.

  16. The bifunctional pyruvate decarboxylase/pyruvate ferredoxin oxidoreductase from Thermococcus guaymasensis.

    PubMed

    Eram, Mohammad S; Oduaran, Erica; Ma, Kesen

    2014-01-01

    The hyperthermophilic archaeon Thermococcus guaymasensis produces ethanol as a metabolic end product, and an alcohol dehydrogenase (ADH) catalyzing the reduction of acetaldehyde to ethanol has been purified and characterized. However, the enzyme catalyzing the formation of acetaldehyde has not been identified. In this study an enzyme catalyzing the production of acetaldehyde from pyruvate was purified and characterized from T. guaymasensis under strictly anaerobic conditions. The enzyme had both pyruvate decarboxylase (PDC) and pyruvate ferredoxin oxidoreductase (POR) activities. It was oxygen sensitive, and the optimal temperatures were 85°C and >95°C for the PDC and POR activities, respectively. The purified enzyme had activities of 3.8 ± 0.22 U mg(-1) and 20.2 ± 1.8 U mg(-1), with optimal pH-values of 9.5 and 8.4 for each activity, respectively. Coenzyme A was essential for both activities, although it did not serve as a substrate for the former. Enzyme kinetic parameters were determined separately for each activity. The purified enzyme was a heterotetramer. The sequences of the genes encoding the subunits of the bifunctional PDC/POR were determined. It is predicted that all hyperthermophilic β -keto acids ferredoxin oxidoreductases are bifunctional, catalyzing the activities of nonoxidative and oxidative decarboxylation of the corresponding β -keto acids.

  17. Archaeal Mo-Containing Glyceraldehyde Oxidoreductase Isozymes Exhibit Diverse Substrate Specificities through Unique Subunit Assemblies

    PubMed Central

    Miyake, Masayuki; Fushinobu, Shinya

    2016-01-01

    Archaea use glycolytic pathways distinct from those found in bacteria and eukaryotes, where unique enzymes catalyze each reaction step. In this study, we isolated three isozymes of glyceraldehyde oxidoreductase (GAOR1, GAOR2 and GAOR3) from the thermoacidophilic archaeon Sulfolobus tokodaii. GAOR1–3 belong to the xanthine oxidoreductase superfamily, and are composed of a molybdo-pyranopterin subunit (L), a flavin subunit (M), and an iron-sulfur subunit (S), forming an LMS hetero-trimer unit. We found that GAOR1 is a tetramer of the STK17810/STK17830/STK17820 hetero-trimer, GAOR2 is a dimer of the STK23390/STK05620/STK05610 hetero-trimer, and GAOR3 is the STK24840/STK05620/STK05610 hetero-trimer. GAOR1–3 exhibited diverse substrate specificities for their electron donors and acceptors, due to their different L-subunits, and probably participate in the non-phosphorylative Entner-Doudoroff glycolytic pathway. We determined the crystal structure of GAOR2, as the first three-dimensional structure of an archaeal molybdenum-containing hydroxylase, to obtain structural insights into their substrate specificities and subunit assemblies. The gene arrangement and the crystal structure suggested that the M/S-complex serves as a structural scaffold for the binding of the L-subunit, to construct the three enzymes with different specificities. Collectively, our findings illustrate a novel principle of a prokaryotic multicomponent isozyme system. PMID:26808202

  18. Klebsiella pneumoniae 1,3-propanediol:NAD+ oxidoreductase.

    PubMed Central

    Johnson, E A; Lin, E C

    1987-01-01

    Fermentative utilization of glycerol, a more reduced carbohydrate than aldoses and ketoses, requires the disposal of the two extra hydrogen atoms. This is accomplished by sacrificing an equal quantity of glycerol via an auxiliary pathway initiated by glycerol dehydratase. The product, 3-hydroxypropionaldehyde, is then reduced by 1,3-propanediol NAD+:oxidoreductase (1,3-propanediol dehydrogenase; EC 1.1.1.202), resulting in the regeneration of NAD+ from NADH. The pathway for the assimilation of glycerol is initiated by an NAD-linked dehydrogenase. In Klebsiella pneumoniae the two pathways are encoded by the dha regulon which is inducible only anaerobically. In this study 1,3-propanediol:NAD+ oxidoreductase was purified from cells grown anaerobically on glycerol. The enzyme was immunochemically distinct from the NAD-linked glycerol dehydrogenase and was an octamer or hexamer of a polypeptide of 45,000 +/- 3,000 daltons. When tested as a dehydrogenase, only 1,3-propanediol served as a substrate; no activity was detected with ethanol, 1-propanol, 1,2-propanediol, glycerol, or 1,4-butanediol. The enzyme was inhibited by chelators of divalent cations. An enzyme preparation inhibited by alpha,alpha'-dipyridyl was reactivated by the addition of Fe2+ or Mn2+ after removal of the chelator by gel filtration. As for glycerol dehydrogenase, 1,3-propanediol oxidoreductase is apparently inactivated by oxidation during aerobic metabolism, under which condition the enzyme becomes superfluous. Images PMID:3553154

  19. WW domain-containing oxidoreductase's role in myriad cancers: clinical significance and future implications.

    PubMed

    Gardenswartz, Aliza; Aqeilan, Rami I

    2014-03-01

    The WW domain-containing oxidoreductase (WWOX) gene, encodes a tumor suppressor located on 16q23.1, spanning FRA16D, one of the most active common fragile sites in the human genome, that is altered in numerous types of cancer. WWOX's alteration in these myriad cancers is due to disparate mechanisms including loss of heterozygosity, homozygous deletion and epigenetic changes. In vitro, WWOX has been found to be reduced or absent in numerous cancer cell lines and WWOX restoration has been found to inhibit tumor cell growth and invasion. Wwox knockout mice developed femoral focal lesions resembling osteosarcomas within one month of their life and aging Wwox heterozygous mice have an increased incidence of spontaneous lung and mammary tumors as well as B-cell lymphomas. We herein review WWOX's role that has been unearthed thus far in different types of malignancies, its clinical significance and future implications.

  20. NADPH: Protochlorophyllide Oxidoreductase-Structure, Catalytic Function, and Role in Prolamellar Body Formation and Morphogenesis

    SciTech Connect

    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 of both the POR and LIPOR catalyzed processes.

  1. Pharmacogenetics of P450 oxidoreductase: implications in drug metabolism and therapy.

    PubMed

    Hu, Lei; Zhuo, Wei; He, Yi-Jing; Zhou, Hong-Hao; Fan, Lan

    2012-11-01

    The redox reaction of cytochrome P450 enzymes (CYP) is an important physiological and biochemical reaction in the human body, as it is involved in the oxidative metabolism of both endogenous and exogenous substrates. Cytochrome P450 oxidoreductase (POR) is the only obligate electron donor for all of the hepatic microsomal CYP enzymes. It plays a crucial role in drug metabolism and treatment by not only acting as an electron donor involved in drug metabolism mediated by CYP enzymes but also by directly inducing the transformation of some antitumor precursors. Studies have found that the gene encoding human POR is highly polymorphic, which is of considerable clinical significance as it affects the metabolism and curative effects of clinically used drugs. This review aims to discuss the effect of POR and its genetic polymorphisms on drug metabolism and therapy, as well as the potential mechanisms of POR pharmacogenetics.

  2. LATERAL BRANCHING OXIDOREDUCTASE acts in the final stages of strigolactone biosynthesis in Arabidopsis

    PubMed Central

    Yoneyama, Kaori; Filardo, Fiona; Meyers, Emma; Scaffidi, Adrian; Frickey, Tancred; Akiyama, Kohki; Seto, Yoshiya; Dun, Elizabeth A.; Cremer, Julia E.; Kerr, Stephanie C.; Waters, Mark T.; Flematti, Gavin R.; Mason, Michael G.; Weiller, Georg; Yamaguchi, Shinjiro; Nomura, Takahito; Smith, Steven M.; Yoneyama, Koichi; Beveridge, Christine A.

    2016-01-01

    Strigolactones are a group of plant compounds of diverse but related chemical structures. They have similar bioactivity across a broad range of plant species, act to optimize plant growth and development, and promote soil microbe interactions. Carlactone, a common precursor to strigolactones, is produced by conserved enzymes found in a number of diverse species. Versions of the MORE AXILLARY GROWTH1 (MAX1) cytochrome P450 from rice and Arabidopsis thaliana make specific subsets of strigolactones from carlactone. However, the diversity of natural strigolactones suggests that additional enzymes are involved and remain to be discovered. Here, we use an innovative method that has revealed a missing enzyme involved in strigolactone metabolism. By using a transcriptomics approach involving a range of treatments that modify strigolactone biosynthesis gene expression coupled with reverse genetics, we identified LATERAL BRANCHING OXIDOREDUCTASE (LBO), a gene encoding an oxidoreductase-like enzyme of the 2-oxoglutarate and Fe(II)-dependent dioxygenase superfamily. Arabidopsis lbo mutants exhibited increased shoot branching, but the lbo mutation did not enhance the max mutant phenotype. Grafting indicated that LBO is required for a graft-transmissible signal that, in turn, requires a product of MAX1. Mutant lbo backgrounds showed reduced responses to carlactone, the substrate of MAX1, and methyl carlactonoate (MeCLA), a product downstream of MAX1. Furthermore, lbo mutants contained increased amounts of these compounds, and the LBO protein specifically converts MeCLA to an unidentified strigolactone-like compound. Thus, LBO function may be important in the later steps of strigolactone biosynthesis to inhibit shoot branching in Arabidopsis and other seed plants. PMID:27194725

  3. [Forms of xanthine oxidoreductase in the tissues of Japanese quail].

    PubMed

    Jankela, J; Baranovská, M; Antalíková, J

    1993-01-01

    The Japanese quail tissues--liver, kidney and pancreas were analysed for the presence of forms of xanthine oxidoreductase utilised cofactors NAD+, molecular oxygen or artificial acceptor--methylene blue, as well as for the validity of correlation between enzymatic activity and diet protein content. Four groups of animals with the experimental diets, the formulae of which are given in Tab. I, and control group with a commercial mash were fed for ten days. For enzyme preparation, the rough purification of cytoplasmic fraction with subsequent dialysis was used. The xanthine oxidoreductase utilised NAD+ (XOR-NAD) was detected in all examined tissues (Fig. 1), whereby the correlation of enzymatic activity with diet protein content was shown only in liver, according our previous findings (Jankela; 1978; Baranovská and Gazo, 1990). The values in liver and pancreas of animals fed a commercial mash were somewhat out of the range of linearity, probably because of the presence of nonprotein substances in mash, which affected the XOR activity in these organs (Jankela, 1992). The XOR utilised O2 (Fig. 2) was only detected in liver and kidney with certain activity in animals fed free protein diet. The percentage of this enzyme form was below 18% of the total activity (Fig. 5). The xanthine oxidoreductase utilised methylene blue (XOR-MM) was detected in liver, kidney and pancreas (Fig. 3). The correlation of enzymatic activity with diet protein content was linear in liver and kidney. The percentage of XOR-MM activity was very high, it amounted to 55% of the total activity (Fig. 4).(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Structural Basis of Biological NO Generation by Octaheme Oxidoreductases*

    PubMed Central

    Maalcke, Wouter J.; Dietl, Andreas; Marritt, Sophie J.; Butt, Julea N.; Jetten, Mike S. M.; Keltjens, Jan T.; Barends, Thomas R. M.; Kartal, Boran

    2014-01-01

    Nitric oxide is an important molecule in all domains of life with significant biological functions in both pro- and eukaryotes. Anaerobic ammonium-oxidizing (anammox) bacteria that contribute substantially to the release of fixed nitrogen into the atmosphere use the oxidizing power of NO to activate inert ammonium into hydrazine (N2H4). Here, we describe an enzyme from the anammox bacterium Kuenenia stuttgartiensis that uses a novel pathway to make NO from hydroxylamine. This new enzyme is related to octaheme hydroxylamine oxidoreductase, a key protein in aerobic ammonium-oxidizing bacteria. By a multiphasic approach including the determination of the crystal structure of the K. stuttgartiensis enzyme at 1.8 Å resolution and refinement and reassessment of the hydroxylamine oxidoreductase structure from Nitrosomonas europaea, both in the presence and absence of their substrates, we propose a model for NO formation by the K. stuttgartiensis enzyme. Our results expand the understanding of the functions that the widespread family of octaheme proteins have. PMID:24302732

  5. CRYSTAL STRUCTURE ANALYSIS OF A PUTATIVE OXIDOREDUCTASE FROM KLEBSIELLA PNEUMONIAE

    SciTech Connect

    Baig, M.; Brown, A.; Eswaramoorthy, S.; Swaminathan, S.

    2009-01-01

    Klebsiella pneumoniae, a gram-negative enteric bacterium, is found in nosocomial infections which are acquired during hospital stays for about 10% of hospital patients in the United States. The crystal structure of a putative oxidoreductase from K. pneumoniae has been determined. The structural information of this K. pneumoniae protein was used to understand its function. Crystals of the putative oxidoreductase enzyme were obtained by the sitting drop vapor diffusion method using Polyethylene glycol (PEG) 3350, Bis-Tris buffer, pH 5.5 as precipitant. These crystals were used to collect X-ray data at beam line X12C of the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory (BNL). The crystal structure was determined using the SHELX program and refi ned with CNS 1.1. This protein, which is involved in the catalysis of an oxidation-reduction (redox) reaction, has an alpha/beta structure. It utilizes nicotinamide adenine dinucleotide phosphate (NADP) or nicotine adenine dinucleotide (NAD) to perform its function. This structure could be used to determine the active and co-factor binding sites of the protein, information that could help pharmaceutical companies in drug design and in determining the protein’s relationship to disease treatment such as that for pneumonia and other related pathologies.

  6. Genetic variations in NADPH-CYP450 oxidoreductase in a Czech Slavic cohort

    PubMed Central

    Tomková, Mária; Panda, Satya Prakash; Šeda, Ondřej; Baxová, Alice; Hůlková, Martina; Masters, Bettie Sue Siler; Martásek, Pavel

    2015-01-01

    Background Gene polymorphisms encoding the enzyme NADPH–cytochrome P450 oxidoreductase (POR) contribute to inter-individual differences in drug response. Aim To estimate polymorphic allele frequencies of the POR gene in a Czech Slavic population. Materials & Methods The gene POR was analyzed in 322 Czech Slavic individuals from a control cohort by sequencing and HRM analysis. Results Twenty-five SNP genetic variations were identified. Of these variants, 7 were new, unreported SNPs, including two SNPs in the 5´flanking region (g.4965 C>T and g.4994 G>T), one intronic variant (c.1899 −20C>T), one synonymous SNP (p.20Ala=) and three nonsynonymous SNPs (p.Thr29Ser, p.Pro384Leu and p.Thr529Met). The p.Pro384Leu variant exhibited reduced enzymatic activities compared to wild type. Conclusion New POR variant identification indicates that the number of uncommon variants might be specific for each subpopulation being investigated, particularly germane to the singular role that POR plays in providing reducing equivalents to all CYPs in the endoplasmic reticulum. PMID:25712184

  7. Enzymatic product formation impairs both the chloroplast receptor- binding function as well as translocation competence of the NADPH: protochlorophyllide oxidoreductase, a nuclear-encoded plastid precursor protein

    PubMed Central

    1995-01-01

    The key enzyme of chlorophyll biosynthesis in higher plants, the light- dependent NADPH:protochlorophyllide oxidoreductase (POR, EC 1.6.99.1), is a nuclear-encoded plastid protein. Its posttranslational transport into plastids of barley depends on the intraplastidic availability of one of its substrates, protochlorophyllide (PChlide). The precursor of POR (pPOR), synthesized from a corresponding full-length barley cDNA clone by coupling in vitro transcription and translation, is enzymatically active and converts PChlide to chlorophyllide (Chlide) in a light- and NADPH-dependent manner. Chlorophyllide formed catalytically remains tightly but noncovalently bound to the precursor protein and stabilizes a transport-incompetent conformation of pPOR. As shown by in vitro processing experiments, the chloroplast transit peptide in the Chlide-pPOR complex appears to be masked and thus is unable to physically interact with the outer plastid envelope membrane. In contrast, the chloroplast transit peptide in the naked pPOR (without its substrates and its product attached to it) and in the pPOR- substrate complexes, such as pPOR-PChlide or pPOR-PChlide-NADPH, seems to react independently of the mature region of the polypeptide, and thus is able to bind to the plastid envelope. When envelope-bound pPOR- PChlide-NADPH complexes were exposed to light during a short preincubation, the enzymatically produced Chlide slowed down the actual translocation step, giving rise to the sequential appearance of two partially processed translocation intermediates. However, ongoing translocation induced by feeding the chloroplasts delta-aminolevulinic acid, a precursor of PChlide, was able to override these two early blocks in translocation, suggesting that the plastid import machinery has a substantial capacity to denature a tightly folded, envelope-bound precursor protein. Together, our results show that pPOR with Chlide attached to it is impaired both in the ATP-dependent step of binding to a

  8. Xanthine oxidoreductase and its inhibitors: relevance for gout.

    PubMed

    Day, Richard O; Kamel, Bishoy; Kannangara, Diluk R W; Williams, Kenneth M; Graham, Garry G

    2016-12-01

    Xanthine oxidoreductase (XOR) is the rate-limiting enzyme in purine catabolism and converts hypoxanthine to xanthine, and xanthine into uric acid. When concentrations of uric acid exceed its biochemical saturation point, crystals of uric acid, in the form of monosodium urate, emerge and can predispose an individual to gout, the commonest form of inflammatory arthritis in men aged over 40 years. XOR inhibitors are primarily used in the treatment of gout, reducing the formation of uric acid and thereby, preventing the formation of monosodium urate crystals. Allopurinol is established as first-line therapy for gout; a newer alternative, febuxostat, is used in patients unable to tolerate allopurinol. This review provides an overview of gout, a detailed analysis of the structure and function of XOR, discussion on the pharmacokinetics and pharmacodynamics of XOR inhibitors-allopurinol and febuxostat, and the relevance of XOR in common comorbidities of gout.

  9. Recent progress on the characterization of aldonolactone oxidoreductases.

    PubMed

    Aboobucker, Siddique I; Lorence, Argelia

    2016-01-01

    L-Ascorbic acid (ascorbate, AsA, vitamin C) is essential for animal and plant health. Despite our dependence on fruits and vegetables to fulfill our requirement for this vitamin, the metabolic network leading to its formation in plants is just being fully elucidated. There is evidence supporting the operation of at least four biosynthetic pathways leading to AsA formation in plants. These routes use D-mannose/L-galactose, L-gulose, D-galacturonate, and myo-inositol as the main precursors. This review focuses on aldonolactone oxidoreductases, a subgroup of the vanillyl alcohol oxidase (VAO; EC 1.1.3.38) superfamily, enzymes that catalyze the terminal step in AsA biosynthesis in bacteria, protozoa, animals, and plants. In this report, we review the properties of well characterized aldonolactone oxidoreductases to date. A shared feature in these proteins is the presence of a flavin cofactor as well as a thiol group. The flavin cofactor in many cases is bound to the N terminus of the enzymes or to a recently discovered HWXK motif in the C terminus. The binding between the flavin moiety and the protein can be either covalent or non-covalent. Substrate specificity and subcellular localization differ among the isozymes of each kingdom. All oxidases among these enzymes possess dehydrogenase activity, however, exclusive dehydrogenases are also found. We also discuss recent evidence indicating that plants have both L-gulono-1,4-lactone oxidases and L-galactono-1,4-lactone dehydrogenases involved in AsA biosynthesis.

  10. Recent Progress on the Characterization of Aldonolactone Oxidoreductases

    PubMed Central

    Aboobucker, Siddique I; Lorence, Argelia

    2015-01-01

    l-Ascorbic acid (ascorbate, AsA, vitamin C) is essential for animal and plant health. Despite our dependence on fruits and vegetables to fulfill our requirement for this vitamin, the metabolic network leading to its formation in plants is just being fully elucidated. There is evidence supporting the operation of at least four biosynthetic pathways leading to AsA formation in plants. These routes use d-mannose/l-Galactose, l-gulose, d-galacturonate, and myo-inositol as the main precursors. This review focuses on aldonolactone oxidoreductases, a subgroup of the vanillyl alcohol oxidase (VAO; EC 1.1.3.38) superfamily, enzymes that catalyze the terminal step in AsA biosynthesis in bacteria, protozoa, animals, and plants. In this report, we review the properties of well characterized aldonolactone oxidoreductases to date. A shared feature in these proteins is the presence of a flavin cofactor as well as a thiol group. The flavin cofactor in many cases is bound to the N terminus of the enzymes or to a recently discovered HWXK motif in the C terminus. The binding between the flavin moiety and the protein can be either covalent or non-covalent. Substrate specificity and subcellular localization differ among the isozymes of each kingdom. All oxidases among these enzymes possess dehydrogenase activity, however, exclusive dehydrogenases are also found. We also discuss recent evidence indicating that plants have both l-gulono-1,4-lactone oxidases and l-Galactono-1,4-lactone dehydrogenases involved in AsA biosynthesis. PMID:26696130

  11. Catalytic properties of NAD(P)H:quinone oxidoreductase-2 (NQO2), a dihydronicotinamide riboside dependent oxidoreductase.

    PubMed

    Wu, K; Knox, R; Sun, X Z; Joseph, P; Jaiswal, A K; Zhang, D; Deng, P S; Chen, S

    1997-11-15

    Human NAD(P)H:quinone acceptor oxidoreductase-2 (NQO2) has been prepared using an Escherichia coli expression method. NQO2 is thought to be an isoform of DT-diaphorase (EC 1.6.99.2) [also referred to as NAD(P)H:quinone acceptor oxidoreductase] because there is a 49% identity between their amino acid sequences. The present investigation has revealed that like DT-diaphorase, NQO2 is a dimer enzyme with one FAD prosthetic group per subunit. Interestingly, NQO2 uses dihydronicotinamide riboside (NRH) rather than NAD(P)H as an electron donor. It catalyzes a two-electron reduction of quinones and oxidation-reduction dyes. One-electron acceptors, such as potassium ferricyanide, cannot be reduced by NQO2. This enzyme also catalyzes a four-electron reduction, using methyl red as the electron acceptor. The NRH-methyl red reductase activity of NQO2 is 11 times the NADH-methyl red reductase activity of DT-diaphorase. In addition, through a four-electron reduction reaction, NQO2 can catalyze nitroreduction of cytotoxic compound CB 1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. NQO2 is 3000 times more effective than DT-diaphorase in the reduction of CB 1954. Therefore, NQO2 is a NRH-dependent oxidoreductase which catalyzes two- and four-electron reduction reactions. NQO2 is resistant to typical inhibitors of DT-diaphorase, such as dicumarol, Cibacron blue, and phenindone. Flavones are inhibitors of NQO2. However, structural requirements of flavones for the inhibition of NQO2 are different from those for DT-diaphorase. The most potent flavone inhibitor tested so far is quercetin (3,5,7,3',4'-. 6pentahydroxyflavone). It has been found that quercetin is a competitive inhibitor with respect to NRH (Ki = 21 nM). NQO2 is 43 amino acids shorter than DT-diaphorase, and it has been suggested that the carboxyl terminus of DT-diaphorase plays a role in substrate binding (S. Chen et al., Protein Sci. 3, 51-57, 1994). In order to understand better the basis of catalytic differences between

  12. Arabidopsis light-dependent protochlorophyllide oxidoreductase A (PORA) is essential for normal plant growth and development.

    PubMed

    Paddock, Troy; Lima, Daniel; Mason, Mary E; Apel, Klaus; Armstrong, Gregory A

    2012-03-01

    During skotomorphogenesis in angiosperms, NADPH:protochlorophyllide oxidoreductase (POR) forms an aggregate of photolabile NADPH-POR-protochlorophyllide (Pchlide) ternary complexes localized to the prolamellar bodies within etioplasts. During photomorphogenesis, POR catalyzes the light-dependent reduction of Pchlide a to chlorophyllide (Chlide) a, which is subsequently converted to chlorophyll (Chl). In Arabidopsis there are three structurally related POR genes, denoted PORA, PORB and PORC. The PORA and PORB proteins accumulate during skotomorphogenesis. During illumination, PORA is only transiently expressed, whereas PORB and PORC persist and are responsible for bulk Chl synthesis throughout plant development. Here we have tested whether PORA is important for skotomorphogenesis by assisting in etioplast development, and normal photomorphogenic development. Using reverse genetic approaches, we have identified the porA-1 null mutant, which contains an insertion of the maize Dissociation transposable element in the PORA gene. Additionally, we have characterized PORA RNAi lines. The porA-1 and PORA RNAi lines display severe photoautotrophic growth defects, which can be partially rescued on sucrose-supplemented growth media. Elimination of PORA during skotomorphogenesis results in reductions in the volume and frequency of prolamellar bodies, and in photoactive Pchlide conversion. The porA-1 mutant characterization thus establishes a quantitative requirement for PORA in etioplast development by demonstrating significant membrane ultrastructural and biochemical defects, in addition to suggesting PORA-specific functions in photomorphogenesis and plant development.

  13. Ferredoxin:NAD + Oxidoreductase of Thermoanaerobacterium saccharolyticum and Its Role in Ethanol Formation

    DOE PAGES

    Tian, Liang; Lo, Jonathan; Shao, Xiongjun; ...

    2016-09-30

    Ferredoxin:NAD+oxidoreductase (NADH-FNOR) catalyzes the transfer of electrons from reduced ferredoxin to NAD+. This enzyme has been hypothesized to be the main enzyme responsible for ferredoxin oxidization in the NADH-based ethanol pathway inThermoanaerobacterium saccharolyticum; however, the corresponding gene has not yet been identified. Here, we identified the Tsac_1705 protein as a candidate FNOR based on the homology of its functional domains. We then confirmed its activityin vitrowith a ferredoxin-based FNOR assay. To determine its role in metabolism, thetsac_1705gene was deleted in different strains ofT. saccharolyticum. In wild-typeT. saccharolyticum, deletion oftsac_1705resulted in a 75% loss of NADH-FNOR activity, which indicated thatmore » Tsac_1705 is the main NADH-FNOR inT.saccharolyticum. When both NADH- and NADPH-linked FNOR genes were deleted, the ethanol titer decreased and the ratio of ethanol to acetate approached unity, indicative of the absence of FNOR activity. Finally, we tested the effect of heterologous expression of Tsac_1705 inClostridium thermocellumand found improvements in both the titer and the yield of ethanol. IMPORTANCERedox balance plays a crucial role in many metabolic engineering strategies. Ferredoxins are widely used as electron carriers for anaerobic microorganism and plants. This study identified the gene responsible for electron transfer from ferredoxin to NAD+, a key reaction in the ethanol production pathway of this organism and many other metabolic pathways. Identification of this gene is an important step in transferring the ethanol production ability of this organism to other organisms.« less

  14. Thiol-disulfide Oxidoreductases TRX1 and TMX3 Decrease Neuronal Atrophy in a Lentiviral Mouse Model of Huntington's Disease.

    PubMed

    Fox, Jonathan; Lu, Zhen; Barrows, Lorraine

    2015-11-06

    Huntington's disease (HD) is caused by a trinucleotide CAG repeat in the huntingtin gene (HTT) that results in expression of a polyglutamine-expanded mutant huntingtin protein (mHTT). N-terminal fragments of mHTT accumulate in brain neurons and glia as soluble monomeric and oligomeric species as well as insoluble protein aggregates and drive the disease process. Decreasing mHTT levels in brain provides protection and reversal of disease signs in HD mice making mHTT a prime target for disease modification. There is evidence for aberrant thiol oxidation within mHTT and other proteins in HD models. Based on this, we hypothesized that a specific thiol-disulfide oxidoreductase exists that decreases mHTT levels in cells and provides protection in HD mice. We undertook an in-vitro genetic screen of key thiol-disulfide oxidoreductases then completed secondary screens to identify those with mHTT decreasing properties. Our in-vitro experiments identified thioredoxin 1 and thioredoxin-related transmembrane protein 3 as proteins that decrease soluble mHTT levels in cultured cells. Using a lentiviral mouse model of HD we tested the effect of these proteins in striatum. Both proteins decreased mHTT-induced striatal neuronal atrophy. Findings provide evidence for a role of dysregulated protein-thiol homeostasis in the pathogenesis of HD.

  15. Transcript Analysis of Multiple Copies of amo (Encoding Ammonia Monooxygenase) and hao (Encoding Hydroxylamine Oxidoreductase) in Nitrosomonas europaea

    PubMed Central

    Hommes, Norman G.; Sayavedra-Soto, Luis A.; Arp, Daniel J.

    2001-01-01

    The genes encoding ammonia monooxygenase (amoCAB), hydroxylamine oxidoreductase (hao), and the c-type cytochrome c-554 (hcy) are present in multiple copies in the genome of Nitrosomonas europaea. The upstream regions of the two copies of amoC, the three copies of hao, and one copy of hcy were cloned and sequenced. Primer extension reactions were done to identify transcription start sites for these genes, as well as for amoA. Putative ς70 promoter sequences were found associated with all but one of the mapped transcription start sites. Primer extensions were done with amoC primers using RNA harvested from cells incubated with and without ammonium. The experiments suggested that N. europaea cells may be able to use different promoters in the presence and absence of ammonium. PMID:11208810

  16. Photoprotective role of NADPH:protochlorophyllide oxidoreductase A

    PubMed Central

    Buhr, Frank; El Bakkouri, Majida; Valdez, Oscar; Pollmann, Stephan; Lebedev, Nikolai; Reinbothe, Steffen; Reinbothe, Christiane

    2008-01-01

    A homology model of NADPH:protochlorophyllide (Pchlide) oxidoreductase A (POR; E.C. 1.3.33.1) of barley is developed and verified by site-directed mutagenesis. PORA is considered a globular protein consisting of nine α-helices and seven β-strands. The model predicts the presence of two functionally distinctive Pchlide binding sites where the pigment is coordinated by cystein residues. The pigment bound to the first, high-affinity Pchlide binding site is used for the formation of the photoactive state of the enzyme. The pigment bound to the second, low-affinity Pchlide binding site is involved in the PORA:PORB interaction, allowing for resonance energy transfer between the neighboring PORs in the complex. In the in vitro reconstituted light-harvesting POR:Pchlide complex (LHPP), light absorbed by PORA-bound Pchlide b is transferred to PORB-bound Pchlide a. That induces the conversion of Pchlide a to chlorophyllide (Chlide) a. This energy transfer eliminates the possibility of Pchlide b photoreduction and prevents that excited triplet states of either Pchlides a or b accumulate and provoke singlet oxygen production. Together, our results provide a photoprotective role of PORA during greening. PMID:18723681

  17. Purification and partial characterisation of camel milk xanthine oxidoreductase.

    PubMed

    Baghiani, A; Harrison, R; Benboubetra, M

    2003-12-01

    Xanthine oxidoreductase (XOR) was purified in the presence of dithiothrietol from camel milk with yields of up to 22.2mg/l that were comparable to those obtained from bovine and human milk sources. On SDS-PAGE, the freshly purified camel milk XOR had a protein flavin (A280/A450) ratio of 5.3 +/- 0.4 and appeared homogenous with a single major band of approximately Mr 145.3 KDa. Surprisingly, in all the batches (n = 8) purified camel milk XOR showed no detectable activity towards xanthine or NADH. The molybdenum content of camel XOR was comparable to human and goat milk enzymes. After resulphuration, camel milk XOR gave a specific activity of 1.1 nmol/min/mg and 13.0 nmol/min/mg enzyme towards pterin (fluorimetric assay) and xanthine (spectrophotometric assay) respectively. This activity was markedly lower than that of human, bovine and goat enzymes obtained under the same conditions. These findings suggest that the molybdo-form of camel enzyme is totally under desulpho inactive form. It is possible that camel neonates are equipped with an enzymic system that reactivates XOR in their gut and consequently generates antibacterial reactive oxygen species.

  18. Xanthine Oxidoreductase-Derived Reactive Species: Physiological and Pathological Effects

    PubMed Central

    Bortolotti, Massimo

    2016-01-01

    Xanthine oxidoreductase (XOR) is the enzyme that catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid and is widely distributed among species. In addition to this housekeeping function, mammalian XOR is a physiological source of superoxide ion, hydrogen peroxide, and nitric oxide, which can function as second messengers in the activation of various pathways. This review intends to address the physiological and pathological roles of XOR-derived oxidant molecules. The cytocidal action of XOR products has been claimed in relation to tissue damage, in particular damage induced by hypoxia and ischemia. Attempts to exploit this activity to eliminate unwanted cells via the construction of conjugates have also been reported. Moreover, different aspects of XOR activity related to phlogosis, endothelial activation, leukocyte activation, and vascular tone regulation, have been taken into consideration. Finally, the positive and negative outcomes concerning cancer pathology have been analyzed because XOR products may induce mutagenesis, cell proliferation, and tumor progression, but they are also associated with apoptosis and cell differentiation. In conclusion, XOR activity generates free radicals and other oxidant reactive species that may result in either harmful or beneficial outcomes. PMID:26823950

  19. Xanthine oxidoreductase in atherosclerosis pathogenesis: not only oxidative stress.

    PubMed

    Battelli, Maria Giulia; Polito, Letizia; Bolognesi, Andrea

    2014-12-01

    Endothelial xanthine oxidoreductase (XOR) together with NAD(P)H oxidase and nitric oxide (NO) synthase plays a physiologic role in inflammatory signalling, the regulation of NO production and vascular function. The oxidative stress generated by these enzymes may induce endothelial dysfunction, leading to atherosclerosis, cardiovascular diseases and metabolic syndrome. XOR activity creates both oxidant and anti-oxidant products that are implicated in the development of hypertension, smoking vascular injury, dyslipidemia and diabetes, which are the main risk factors of atherosclerosis. In particular, uric acid may have a protective as well as a detrimental role in vascular alterations, thus justifying the multi-directional effects of XOR inhibition. Moreover, XOR products are associated with cell differentiation, leading to adipogenesis and foam cell formation, as well as to the production of monocyte chemoattractant protein-1 from arterial smooth muscle cells, after proliferation and migration. The role of XOR in adipogenesis is also connected with insulin resistance and obesity, two main features of type 2 diabetes.

  20. Dual targeted poplar ferredoxin NADP(+) oxidoreductase interacts with hemoglobin 1.

    PubMed

    Jokipii-Lukkari, Soile; Kastaniotis, Alexander J; Parkash, Vimal; Sundström, Robin; Leiva-Eriksson, Nélida; Nymalm, Yvonne; Blokhina, Olga; Kukkola, Eija; Fagerstedt, Kurt V; Salminen, Tiina A; Läärä, Esa; Bülow, Leif; Ohlmeier, Steffen; Hiltunen, J Kalervo; Kallio, Pauli T; Häggman, Hely

    2016-06-01

    Previous reports have connected non-symbiotic and truncated hemoglobins (Hbs) to metabolism of nitric oxide (NO), an important signalling molecule involved in wood formation. We have studied the capability of poplar (Populus tremula × tremuloides) Hbs PttHb1 and PttTrHb proteins alone or with a flavin-protein reductase to relieve NO cytotoxicity in living cells. Complementation tests in a Hb-deficient, NO-sensitive yeast (Saccharomyces cerevisiae) Δyhb1 mutant showed that neither PttHb1 nor PttTrHb alone protected cells against NO. To study the ability of Hbs to interact with a reductase, ferredoxin NADP(+) oxidoreductase PtthFNR was characterized by sequencing and proteomics. To date, by far the greatest number of the known dual-targeted plant proteins are directed to chloroplasts and mitochondria. We discovered a novel variant of hFNR that lacks the plastid presequence and resides in cytosol. The coexpression of PttHb1 and PtthFNR partially restored NO resistance of the yeast Δyhb1 mutant, whereas PttTrHb coexpressed with PtthFNR failed to rescue growth. YFP fusion proteins confirmed the interaction between PttHb1 and PtthFNR in plant cells. The structural modelling results indicate that PttHb1 and PtthFNR are able to interact as NO dioxygenase. This is the first report on dual targeting of central plant enzyme FNR to plastids and cytosol.

  1. Functional characterization of the chloroplast ferric chelate oxidoreductase enzyme.

    PubMed

    Solti, Adám; Müller, Brigitta; Czech, Viktória; Sárvári, Éva; Fodor, Ferenc

    2014-05-01

    Iron (Fe) has an essential role in the biosynthesis of chlorophylls and redox cofactors, and thus chloroplast iron uptake is a process of special importance. The chloroplast ferric chelate oxidoreductase (cFRO) has a crucial role in this process but it is poorly characterized. To study the localization and mechanism of action of cFRO, sugar beet (Beta vulgaris cv Orbis) chloroplast envelope fractions were isolated by gradient ultracentrifugation, and their purity was tested by western blotting against different marker proteins. The ferric chelate reductase (FCR) activity of envelope fractions was studied in the presence of NAD(P)H (reductants) and FAD coenzymes. Reduction of Fe(III)-ethylenediaminetetraacetic acid was monitored spectrophotometrically by the Fe(II)-bathophenanthroline disulfonate complex formation. FCR activity, that is production of free Fe(II) for Fe uptake, showed biphasic saturation kinetics, and was clearly associated only to chloroplast inner envelope (cIE) vesicles. The reaction rate was > 2.5 times higher with NADPH than with NADH, which indicates the natural coenzyme preference of cFRO activity and its dependence on photosynthesis. FCR activity of cIE vesicles isolated from Fe-deficient plants also showed clear biphasic kinetics, where the KM of the low affinity component was elevated, and thus this component was down-regulated.

  2. Thermodynamics of Enzyme-Catalyzed Reactions: Part 1. Oxidoreductases

    NASA Astrophysics Data System (ADS)

    Goldberg, Robert N.; Tewari, Yadu B.; Bell, Donna; Fazio, Kari; Anderson, Ellen

    1993-03-01

    Equilibrium constants and enthalpy changes for reactions catalyzed by oxidoreductases have been compiled. For each reaction the following information is given: the reference for the data; the reaction studied; the name of the enzyme used and its Enzyme Commission number; the method of measurement; the conditions of measurement (temperature, pH, ionic strength, and the buffer(s) and cofactor(s) used); the data and an evaluation of it; and, sometimes, commentary on the data and on any corrections which have been applied to it. The thermodynamic conventions pertinent to the tabulation of equilibrium data are discussed. A distinction is made between those thermodynamic quantities which pertain to the overall biochemical reaction and those which pertain to a reference reaction that involves specific species. The data from 205 references have been examined and evaluated. Chemical Abstract Service Registry Numbers have been assigned to the substances involved in these various reactions. There is a cross reference between the substances and the Enzyme Commission numbers of the enzymes used to catalyze the reactions in which the substances participated.

  3. NAD(P)H:quinone oxidoreductase 1 inducer activity of some novel anilinoquinazoline derivatives

    PubMed Central

    Ghorab, Mostafa M; Alsaid, Mansour S; Higgins, Maureen; Dinkova-Kostova, Albena T; Shahat, Abdelaaty A; Elghazawy, Nehal H; Arafa, Reem K

    2016-01-01

    The Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response elements pathway enables cells to survive oxidative stress conditions through regulating the expression of cytoprotective enzymes such as NAD(P)H:quinone oxidoreductase 1 (NQO1). This work presents the design and synthesis of novel anilinoquinazoline derivatives (2–16a) and evaluation of their NQO1 inducer activity in murine cells. Molecular docking of the new compounds was performed to assess their ability to inhibit Keap1–Nrf2 protein–protein interaction through occupying the Keap1–Nrf2-binding domain, which leads to Nrf2 accumulation and enhanced gene expression of NQO1. Docking results showed that all compounds can potentially interact with Keap1; however, 1,5-dimethyl-2-phenyl-4-(2-phenylquinazolin-4-ylamino)-1,2-dihydropyrazol-3-one (9), the most potent inducer, showed the largest number of interactions with key amino acids in the binding pocket (Arg483, Tyr525, and Phe478) compared to the native ligand or any other compound in this series. PMID:27540279

  4. Assembly of the Escherichia coli NADH:ubiquinone oxidoreductase (respiratory complex I).

    PubMed

    Friedrich, Thorsten; Dekovic, Doris Kreuzer; Burschel, Sabrina

    2016-03-01

    Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, couples the electron transfer from NADH to ubiquinone with the translocation of four protons across the membrane. The Escherichia coli complex I is made up of 13 different subunits encoded by the so-called nuo-genes. The electron transfer is catalyzed by nine cofactors, a flavin mononucleotide and eight iron-sulfur (Fe/S)-clusters. The individual subunits and the cofactors have to be assembled together in a coordinated way to guarantee the biogenesis of the active holoenzyme. Only little is known about the assembly of the bacterial complex compared to the mitochondrial one. Due to the presence of so many Fe/S-clusters the assembly of complex I is intimately connected with the systems responsible for the biogenesis of these clusters. In addition, a few other proteins have been reported to be required for an effective assembly of the complex in other bacteria. The proposed role of known bacterial assembly factors is discussed and the information from other bacterial species is used in this review to draw an as complete as possible model of bacterial complex I assembly. In addition, the supramolecular organization of the complex in E. coli is briefly described. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Prof. Conrad Mullineaux.

  5. Functional analysis of three sulfide:quinone oxidoreductase homologs in Chlorobaculum tepidum.

    PubMed

    Chan, Leong-Keat; Morgan-Kiss, Rachael M; Hanson, Thomas E

    2009-02-01

    Sulfide:quinone oxidoreductase (SQR) catalyzes sulfide oxidation during sulfide-dependent chemo- and phototrophic growth in bacteria. The green sulfur bacterium Chlorobaculum tepidum (formerly Chlorobium tepidum) can grow on sulfide as the sole electron donor and sulfur source. C. tepidum contains genes encoding three SQR homologs: CT0117, CT0876, and CT1087. This study examined which, if any, of the SQR homologs possess sulfide-dependent ubiquinone reduction activity and are required for growth on sulfide. In contrast to CT0117 and CT0876, transcripts of CT1087 were detected only when cells actively oxidized sulfide. Mutation of CT0117 or CT1087 in C. tepidum decreased SQR activity in membrane fractions, and the CT1087 mutant could not grow with >or=6 mM sulfide. Mutation of both CT0117 and CT1087 in C. tepidum completely abolished SQR activity, and the double mutant failed to grow with >or=4 mM sulfide. A C-terminal His(6)-tagged CT1087 protein was membrane localized, as was SQR activity. Epitope-tagged CT1087 was detected only when sulfide was actively consumed by cells. Recombinantly produced CT1087 and CT0117 proteins had SQR activity, while CT0876 did not. In summary, we conclude that, under the conditions tested, both CT0117 and CT1087 function as SQR proteins in C. tepidum. CT0876 may support the growth of C. tepidum at low sulfide concentrations, but no evidence was found for SQR activity associated with this protein.

  6. Physiological roles of pyruvate ferredoxin oxidoreductase and pyruvate formate-lyase in Thermoanaerobacterium saccharolyticum JW/SL-YS485

    SciTech Connect

    Zhou, Jilai; Olson, Daniel G.; Lanahan, Anthony A.; Tian, Liang; Murphy, Sean Jean-Loup; Lo, Jonathan; Lynd, Lee R.

    2015-09-15

    We report that Thermoanaerobacter saccharolyticum is a thermophilic microorganism that has been engineered to produce ethanol at high titer (30–70 g/L) and greater than 90 % theoretical yield. However, few genes involved in pyruvate to ethanol production pathway have been unambiguously identified. In T. saccharolyticum, the products of six putative pfor gene clusters and one pfl gene may be responsible for the conversion of pyruvate to acetyl-CoA. To gain insights into the physiological roles of PFOR and PFL, we studied the effect of deletions of several genes thought to encode these activities. We found that that pyruvate ferredoxin oxidoreductase enzyme (PFOR) is encoded by the pforA gene and plays a key role in pyruvate dissimilation. We further demonstrated that pyruvate formate-lyase activity (PFL) is encoded by the pfl gene. Although the pfl gene is normally expressed at low levels, it is crucial for biosynthesis in T. saccharolyticum. In pforA deletion strains, pfl expression increased and was able to partially compensate for the loss of PFOR activity. Deletion of both pforA and pfl resulted in a strain that required acetate and formate for growth and produced lactate as the primary fermentation product, achieving 88 % theoretical lactate yield. PFOR encoded by Tsac_0046 and PFL encoded by Tsac_0628 are only two routes for converting pyruvate to acetyl-CoA in T. saccharolyticum. The physiological role of PFOR is pyruvate dissimilation, whereas that of PFL is supplying C1 units for biosynthesis.

  7. Protein Conformational Gating of Enzymatic Activity in Xanthine Oxidoreductase

    SciTech Connect

    Ishikita, Hiroshi; Eger, Bryan T.; Okamoto, Ken; Nishino, Takeshi; Pai, Emil F.

    2012-05-24

    In mammals, xanthine oxidoreductase can exist as xanthine dehydrogenase (XDH) and xanthine oxidase (XO). The two enzymes possess common redox active cofactors, which form an electron transfer (ET) pathway terminated by a flavin cofactor. In spite of identical protein primary structures, the redox potential difference between XDH and XO for the flavin semiquinone/hydroquinone pair (E{sub sq/hq}) is {approx}170 mV, a striking difference. The former greatly prefers NAD{sup +} as ultimate substrate for ET from the iron-sulfur cluster FeS-II via flavin while the latter only accepts dioxygen. In XDH (without NAD{sup +}), however, the redox potential of the electron donor FeS-II is 180 mV higher than that for the acceptor flavin, yielding an energetically uphill ET. On the basis of new 1.65, 2.3, 1.9, and 2.2 {angstrom} resolution crystal structures for XDH, XO, the NAD{sup +}- and NADH-complexed XDH, E{sub sq/hq} were calculated to better understand how the enzyme activates an ET from FeS-II to flavin. The majority of the E{sub sq/hq} difference between XDH and XO originates from a conformational change in the loop at positions 423-433 near the flavin binding site, causing the differences in stability of the semiquinone state. There was no large conformational change observed in response to NAD{sup +} binding at XDH. Instead, the positive charge of the NAD{sup +} ring, deprotonation of Asp429, and capping of the bulk surface of the flavin by the NAD{sup +} molecule all contribute to altering E{sub sq/hq} upon NAD{sup +} binding to XDH.

  8. Disarming Burkholderia pseudomallei: Structural and Functional Characterization of a Disulfide Oxidoreductase (DsbA) Required for Virulence In Vivo

    PubMed Central

    McMahon, Róisín M.; Marshall, Laura E.; Halili, Maria; Furlong, Emily; Tay, Stephanie; Sarkar-Tyson, Mitali

    2014-01-01

    Abstract Aims: The intracellular pathogen Burkholderia pseudomallei causes the disease melioidosis, a major source of morbidity and mortality in southeast Asia and northern Australia. The need to develop novel antimicrobials is compounded by the absence of a licensed vaccine and the bacterium's resistance to multiple antibiotics. In a number of clinically relevant Gram-negative pathogens, DsbA is the primary disulfide oxidoreductase responsible for catalyzing the formation of disulfide bonds in secreted and membrane-associated proteins. In this study, a putative B. pseudomallei dsbA gene was evaluated functionally and structurally and its contribution to infection assessed. Results: Biochemical studies confirmed the dsbA gene encodes a protein disulfide oxidoreductase. A dsbA deletion strain of B. pseudomallei was attenuated in both macrophages and a BALB/c mouse model of infection and displayed pleiotropic phenotypes that included defects in both secretion and motility. The 1.9 Å resolution crystal structure of BpsDsbA revealed differences from the classic member of this family Escherichia coli DsbA, in particular within the region surrounding the active site disulfide where EcDsbA engages with its partner protein E. coli DsbB, indicating that the interaction of BpsDsbA with its proposed partner BpsDsbB may be distinct from that of EcDsbA-EcDsbB. Innovation: This study has characterized BpsDsbA biochemically and structurally and determined that it is required for virulence of B. pseudomallei. Conclusion: These data establish a critical role for BpsDsbA in B. pseudomallei infection, which in combination with our structural characterization of BpsDsbA will facilitate the future development of rationally designed inhibitors against this drug-resistant organism. Antioxid. Redox Signal. 20, 606–617. PMID:23901809

  9. Cellobiose quinone oxidoreductase from the white rot fungus Phanerochaete chrysosporium is produced by intracellular proteolysis of cellobiose dehydrogenase.

    PubMed

    Raíces, Manuel; Montesino, Raquel; Cremata, José; García, Bianca; Perdomo, Walmer; Szabó, István; Henriksson, Gunnar; Hallberg, B Martin; Pettersson, Göran; Johansson, Gunnar

    2002-06-07

    The fungus Phanerochaete chrysosporium was grown in a 10-l automatic fermenter using cellobiose as carbon source to monitor the induction of cellobiose dehydrogenase (CDH) and cellobiose quinone oxidoreductase (CBQ) enzymes, and to search for tentative cbq and cdh genes and their transcriptional products. After 24 h of induction, CDH was detected in the culture supernatant and a protein was recognized by a specific anti-CDH polyclonal antibody in the sonicated biomass. Northern blot experiments performed with several fungal RNA samples showed, after 24 h of induction, only one single species of an mRNA transcript corresponding in size to the cdh gene (2.5 kb) The relative amount of this transcript decreased as a function of time. Southern blot experiments done with genomic DNA and database search in the recently available genome information also ruled out the presence in this strain of a separate cbq gene distinct from the cdh gene. Taken together, these results demonstrated that CBQ originates from the cdh gene. Furthermore, it is not produced by differential splicing but by a posttranslational, predominantly intracellular, proteolytic cleavage.

  10. Cooperative Protein Folding by Two Protein Thiol Disulfide Oxidoreductases and ERO1 in Soybean1[OPEN

    PubMed Central

    Okuda, Aya; Masuda, Taro; Koishihara, Katsunori; Mita, Ryuta; Iwasaki, Kensuke; Hara, Kumiko; Naruo, Yurika; Hirose, Akiho; Tsuchi, Yuichiro

    2016-01-01

    Most proteins produced in the endoplasmic reticulum (ER) of eukaryotic cells fold via disulfide formation (oxidative folding). Oxidative folding is catalyzed by protein disulfide isomerase (PDI) and PDI-related ER protein thiol disulfide oxidoreductases (ER oxidoreductases). In yeast and mammals, ER oxidoreductin-1s (Ero1s) supply oxidizing equivalent to the active centers of PDI. In this study, we expressed recombinant soybean Ero1 (GmERO1a) and found that GmERO1a oxidized multiple soybean ER oxidoreductases, in contrast to mammalian Ero1s having a high specificity for PDI. One of these ER oxidoreductases, GmPDIM, associated in vivo and in vitro with GmPDIL-2, was unable to be oxidized by GmERO1a. We therefore pursued the possible cooperative oxidative folding by GmPDIM, GmERO1a, and GmPDIL-2 in vitro and found that GmPDIL-2 synergistically accelerated oxidative refolding. In this process, GmERO1a preferentially oxidized the active center in the a′ domain among the a, a′, and b domains of GmPDIM. A disulfide bond introduced into the active center of the a′ domain of GmPDIM was shown to be transferred to the active center of the a domain of GmPDIM and the a domain of GmPDIM directly oxidized the active centers of both the a or a′ domain of GmPDIL-2. Therefore, we propose that the relay of an oxidizing equivalent from one ER oxidoreductase to another may play an essential role in cooperative oxidative folding by multiple ER oxidoreductases in plants. PMID:26645455

  11. WW-domain-containing oxidoreductase is associated with low plasma HDL-C levels.

    PubMed

    Lee, Jenny C; Weissglas-Volkov, Daphna; Kyttälä, Mira; Dastani, Zari; Cantor, Rita M; Sobel, Eric M; Plaisier, Christopher L; Engert, James C; van Greevenbroek, Marleen M J; Kane, John P; Malloy, Mary J; Pullinger, Clive R; Huertas-Vazquez, Adriana; Aguilar-Salinas, Carlos A; Tusie-Luna, Teresa; de Bruin, Tjerk W A; Aouizerat, Bradley E; van der Kallen, Carla C J; Croce, Carlo M; Aqeilan, Rami I; Marcil, Michel; Viikari, Jorma S A; Lehtimäki, Terho; Raitakari, Olli T; Kuusisto, Johanna; Laakso, Markku; Taskinen, Marja-Riitta; Genest, Jacques; Pajukanta, Päivi

    2008-08-01

    Low serum HDL-cholesterol (HDL-C) is a major risk factor for coronary artery disease. We performed targeted genotyping of a 12.4 Mb linked region on 16q to test for association with low HDL-C by using a regional-tag SNP strategy. We identified one SNP, rs2548861, in the WW-domain-containing oxidoreductase (WWOX) gene with region-wide significance for low HDL-C in dyslipidemic families of Mexican and European descent and in low-HDL-C cases and controls of European descent (p = 6.9 x 10(-7)). We extended our investigation to the population level by using two independent unascertained population-based Finnish cohorts, the cross-sectional METSIM cohort of 4,463 males and the prospective Young Finns cohort of 2,265 subjects. The combined analysis provided p = 4 x 10(-4) to 2 x 10(-5). Importantly, in the prospective cohort, we observed a significant longitudinal association of rs2548861 with HDL-C levels obtained at four different time points over 21 years (p = 0.003), and the T risk allele explained 1.5% of the variance in HDL-C levels. The rs2548861 resides in a highly conserved region in intron 8 of WWOX. Results from our in vitro reporter assay and electrophoretic mobility-shift assay demonstrate that this region functions as a cis-regulatory element whose associated rs2548861 SNP has a specific allelic effect and that the region forms an allele-specific DNA-nuclear-factor complex. In conclusion, analyses of 9,798 subjects show significant association between HDL-C and a WWOX variant with an allele-specific cis-regulatory function.

  12. Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency

    SciTech Connect

    Xia, Chuanwu; Panda, Satya P.; Marohnic, Christopher C.; Martásek, Pavel; Masters, Bettie Sue; Kim, Jung-Ja P.

    2012-03-15

    NADPH-cytochrome P450 oxidoreductase (CYPOR) is essential for electron donation to microsomal cytochrome P450-mediated monooxygenation in such diverse physiological processes as drug metabolism (approximately 85-90% of therapeutic drugs), steroid biosynthesis, and bioactive metabolite production (vitamin D and retinoic acid metabolites). Expressed by a single gene, CYPOR's role with these multiple redox partners renders it a model for understanding protein-protein interactions at the structural level. Polymorphisms in human CYPOR have been shown to lead to defects in bone development and steroidogenesis, resulting in sexual dimorphisms, the severity of which differs significantly depending on the degree of CYPOR impairment. The atomic structure of human CYPOR is presented, with structures of two naturally occurring missense mutations, V492E and R457H. The overall structures of these CYPOR variants are similar to wild type. However, in both variants, local disruption of H bonding and salt bridging, involving the FAD pyrophosphate moiety, leads to weaker FAD binding, unstable protein, and loss of catalytic activity, which can be rescued by cofactor addition. The modes of polypeptide unfolding in these two variants differ significantly, as revealed by limited trypsin digestion: V492E is less stable but unfolds locally and gradually, whereas R457H is more stable but unfolds globally. FAD addition to either variant prevents trypsin digestion, supporting the role of the cofactor in conferring stability to CYPOR structure. Thus, CYPOR dysfunction in patients harboring these particular mutations may possibly be prevented by riboflavin therapy in utero, if predicted prenatally, or rescued postnatally in less severe cases.

  13. Clinical, Genetic, and Enzymatic Characterization of P450 Oxidoreductase Deficiency in Four Patients

    PubMed Central

    Sahakitrungruang, Taninee; Huang, Ningwu; Tee, Meng Kian; Agrawal, Vishal; Russell, William E.; Crock, Patricia; Murphy, Nuala; Migeon, Claude J.; Miller, Walter L.

    2009-01-01

    Context: P450 oxidoreductase (POR) deficiency causes disordered steroidogenesis; severe mutations cause genital ambiguity in both sexes plus the Antley-Bixler skeletal malformation syndrome, whereas mild mutations can cause adult infertility. Objective: We describe four patients with POR deficiency and identify and characterize the activities of their mutations. A 46,XY male with micropenis and two 46,XX female infants with genital ambiguity presented with skeletal malformations, and a 46,XX adolescent presented with primary amenorrhea, elevated 17α-hydroxyprogesterone, and low sex steroids. Methods: The coding regions of the POR gene were sequenced, and the identified mutations were recreated in human POR cDNA expression vectors lacking 27 N-terminal residues. POR and human P450c17 were expressed in bacteria. POR activity was measured by four assays: reduction of cytochrome c, oxidation of reduced nicotinamide adenine dinucleotide phosphate, and support of the 17α-hydroxylase and 17,20 lyase activities of P450c17. Results: All four patients were compound heterozygotes for POR mutations, including five novel mutations: L577R, N185K, delE217, and frameshift mutations 1363delC and 697–698insGAAC. N185K and delE217 lacked measurable activity in the assays based on P450c17 but retained partial activity in the assays based on cytochrome c. As assessed by Vmax/Km, L577R supported 46% of 17α-hydroxylase activity but only 27% of 17,20 lyase activity. Computational modeling of these novel mutants revealed the structural basis for their reduced or absent activities. Conclusion: These patients illustrate the broad clinical spectrum of POR deficiency, including amenorrhea and infertility as the sole manifestation. POR assays based on P450c17 correlate well with hormonal and clinical phenotypes. PMID:19837910

  14. 40 CFR 174.524 - Glyphosate Oxidoreductase GOX or GOXv247 in all plants; exemption from the requirement of a...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Glyphosate Oxidoreductase GOX or... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.524 Glyphosate... Glyphosate Oxidoreductase GOX or GOXv247 enzyme in all plants are exempt from the requirement of a...

  15. 40 CFR 174.524 - Glyphosate Oxidoreductase GOX or GOXv247 in all plants; exemption from the requirement of a...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Glyphosate Oxidoreductase GOX or... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.524 Glyphosate... Glyphosate Oxidoreductase GOX or GOXv247 enzyme in all plants are exempt from the requirement of a...

  16. 40 CFR 174.524 - Glyphosate Oxidoreductase GOX or GOXv247 in all plants; exemption from the requirement of a...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Glyphosate Oxidoreductase GOX or... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.524 Glyphosate... Glyphosate Oxidoreductase GOX or GOXv247 enzyme in all plants are exempt from the requirement of a...

  17. 40 CFR 174.524 - Glyphosate Oxidoreductase GOX or GOXv247 in all plants; exemption from the requirement of a...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Glyphosate Oxidoreductase GOX or... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.524 Glyphosate... Glyphosate Oxidoreductase GOX or GOXv247 enzyme in all plants are exempt from the requirement of a...

  18. 40 CFR 174.524 - Glyphosate Oxidoreductase GOX or GOXv247 in all plants; exemption from the requirement of a...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Glyphosate Oxidoreductase GOX or... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.524 Glyphosate... Glyphosate Oxidoreductase GOX or GOXv247 enzyme in all plants are exempt from the requirement of a...

  19. Ferredoxin:NAD+ oxidoreductase of Thermoanaerobacterium saccharolyticum and its role in ethanol formation [Identification of a ferredoxin:NAD+ oxidoreductase of Thermoanaerobacterium saccharolyticum and its role in ethanol formation

    DOE PAGES

    Tian, Liang; Lo, Jonathan; Shao, Xiongjun; ...

    2016-09-30

    Ferredoxin:NAD+ oxidoreductase (NADH-FNOR) catalyzes the transfer of electrons from reduced ferredoxin to NAD+. This enzyme has been hypothesized to be the main enzyme responsible for ferredoxin oxidization in the NADH-based ethanol pathway in Thermoanaerobacterium saccharolyticum; however, the corresponding gene has not yet been identified. Here, we identified the Tsac_1705 protein as a candidate FNOR based on the homology of its functional domains. We then confirmed its activity in vitro with a ferredoxin-based FNOR assay. To determine its role in metabolism, the tsac_1705 gene was deleted in different strains of T. saccharolyticum. In wild-type T. saccharolyticum, deletion of tsac_1705 resulted inmore » a 75% loss of NADH-FNOR activity, which indicated that Tsac_1705 is the main NADH-FNOR in T. saccharolyticum. When both NADH- and NADPH-linked FNOR genes were deleted, the ethanol titer decreased and the ratio of ethanol to acetate approached unity, indicative of the absence of FNOR activity. As a result, we tested the effect of heterologous expression of Tsac_1705 in Clostridium thermocellum and found improvements in both the titer and the yield of ethanol.« less

  20. Physiological roles of pyruvate ferredoxin oxidoreductase and pyruvate formate-lyase in Thermoanaerobacterium saccharolyticum JW/SL-YS485

    DOE PAGES

    Zhou, Jilai; Olson, Daniel G.; Lanahan, Anthony A.; ...

    2015-09-15

    We report that Thermoanaerobacter saccharolyticum is a thermophilic microorganism that has been engineered to produce ethanol at high titer (30–70 g/L) and greater than 90 % theoretical yield. However, few genes involved in pyruvate to ethanol production pathway have been unambiguously identified. In T. saccharolyticum, the products of six putative pfor gene clusters and one pfl gene may be responsible for the conversion of pyruvate to acetyl-CoA. To gain insights into the physiological roles of PFOR and PFL, we studied the effect of deletions of several genes thought to encode these activities. We found that that pyruvate ferredoxin oxidoreductase enzymemore » (PFOR) is encoded by the pforA gene and plays a key role in pyruvate dissimilation. We further demonstrated that pyruvate formate-lyase activity (PFL) is encoded by the pfl gene. Although the pfl gene is normally expressed at low levels, it is crucial for biosynthesis in T. saccharolyticum. In pforA deletion strains, pfl expression increased and was able to partially compensate for the loss of PFOR activity. Deletion of both pforA and pfl resulted in a strain that required acetate and formate for growth and produced lactate as the primary fermentation product, achieving 88 % theoretical lactate yield. PFOR encoded by Tsac_0046 and PFL encoded by Tsac_0628 are only two routes for converting pyruvate to acetyl-CoA in T. saccharolyticum. The physiological role of PFOR is pyruvate dissimilation, whereas that of PFL is supplying C1 units for biosynthesis.« less

  1. Benzofuran-, benzothiophene-, indazole- and benzisoxazole- quinones: excellent substrates for NAD(P)H:quinone oxidoreductase 1

    PubMed Central

    Newsome, Jeffery J.; Hassani, Mary; Swann, Elizabeth; Bibby, Jane M.; Beall, Howard D.; Moody, Christopher J.

    2013-01-01

    A series of heterocyclic quinones based on benzofuran, benzothiophene, indazole and benzisoxazole has been synthesized, and evaluated for their ability to function as substrates for recombinant human NAD(P)H:quinone oxidoreductase (NQO1), a two-electron reductase upregulated in tumor cells. Overall, the quinones are excellent substrates for NQO1, approaching the reduction rates observed for menadione PMID:23635904

  2. Similarity of Escherichia coli propanediol oxidoreductase (fucO product) and an unusual alcohol dehydrogenase from Zymomonas mobilis and Saccharomyces cerevisiae

    SciTech Connect

    Conway, T. ); Ingram, L.O. )

    1989-07-01

    The gene that encodes 1,2-propanediol oxidoreductase (fucO) from Escherichia coli was sequenced. The reading frame specified a protein of 383 amino acids (including the N-terminal methionine), with an aggregate molecular weight of 40,642. The induction of fucO transcription, which occurred in the presence of fucose, was confirmed by Northern blot analysis. In E. coli, the primary fucO transcript was approximately 2.1 kilobases in length. The 5{prime} end of the transcript began more than 0.7 kilobase upstream of the fucO start codon within or beyond the fucA gene. Propanediol oxidoreductase exhibited 41.7% identity with the iron-containing alcohol dehydrogenase II from Zymomonas mobilis and 39.5% identity with ADH4 from Saccharomyces cerevisiae. These three proteins did not share homology with either short-chain or long-chain zinc-containing alcohol dehydrogenase enzymes. We propose that these three unusual alcohol dehydrogenases define a new family of enzymes.

  3. Differential regulation of duplicate light-dependent protochlorophyllide oxidoreductases in the diatom Phaeodactylum tricornutum

    DOE PAGES

    Hunsperger, Heather M.; Ford, Christopher J.; Miller, James S.; ...

    2016-07-01

    Diatoms (Bacilliariophyceae) encode two light-dependent protochlorophyllide oxidoreductases (POR1 and POR2) that catalyze the penultimate step of chlorophyll biosynthesis in the light. Algae live in dynamic environments whose changing light levels induce photoacclimative metabolic shifts, including altered cellular chlorophyll levels. We hypothesized that the two POR proteins may be differentially adaptive under varying light conditions. Using the diatom Phaeodactylum tricornutum as a test system, differences in POR protein abundance and por gene expression were examined when this organism was grown on an alternating light:dark cycles at different irradiances; exposed to continuous light; and challenged by a significant decrease in light availability.more » As a result, for cultures maintained on a 12h light: 12h dark photoperiod at 200μEm–2 s–1 (200L/D), both por genes were up-regulated during the light and down-regulated in the dark, though por1 transcript abundance rose and fell earlier than that of por2. Little concordance occurred between por1 mRNA and POR1 protein abundance. In contrast, por2 mRNA and POR2 protein abundances followed similar diurnal patterns. When 200L/D P. tricornutum cultures were transferred to continuous light (200L/L), the diurnal regulatory pattern of por1 mRNA abundance but not of por2 was disrupted, and POR1 but not POR2 protein abundance dropped steeply. Under 1200μEm–2 s–1 (1200L/D), both por1 mRNA and POR1 protein abundance displayed diurnal oscillations. A compromised diel por2 mRNA response under 1200L/D did not impact the oscillation in POR2 abundance. When cells grown at 1200L/D were then shifted to 50μEm–2 s–1 (50L/D), por1 and por2 mRNA levels decreased swiftly but briefly upon light reduction. Thereafter, POR1 but not POR2 protein levels rose significantly in response to this light stepdown.« less

  4. Differential Regulation of Duplicate Light-Dependent Protochlorophyllide Oxidoreductases in the Diatom Phaeodactylum tricornutum

    PubMed Central

    Hunsperger, Heather M.; Cattolico, Rose Ann

    2016-01-01

    Background Diatoms (Bacilliariophyceae) encode two light-dependent protochlorophyllide oxidoreductases (POR1 and POR2) that catalyze the penultimate step of chlorophyll biosynthesis in the light. Algae live in dynamic environments whose changing light levels induce photoacclimative metabolic shifts, including altered cellular chlorophyll levels. We hypothesized that the two POR proteins may be differentially adaptive under varying light conditions. Using the diatom Phaeodactylum tricornutum as a test system, differences in POR protein abundance and por gene expression were examined when this organism was grown on an alternating light:dark cycles at different irradiances; exposed to continuous light; and challenged by a significant decrease in light availability. Results For cultures maintained on a 12h light: 12h dark photoperiod at 200μE m−2 s−1 (200L/D), both por genes were up-regulated during the light and down-regulated in the dark, though por1 transcript abundance rose and fell earlier than that of por2. Little concordance occurred between por1 mRNA and POR1 protein abundance. In contrast, por2 mRNA and POR2 protein abundances followed similar diurnal patterns. When 200L/D P. tricornutum cultures were transferred to continuous light (200L/L), the diurnal regulatory pattern of por1 mRNA abundance but not of por2 was disrupted, and POR1 but not POR2 protein abundance dropped steeply. Under 1200μE m−2 s−1 (1200L/D), both por1 mRNA and POR1 protein abundance displayed diurnal oscillations. A compromised diel por2 mRNA response under 1200L/D did not impact the oscillation in POR2 abundance. When cells grown at 1200L/D were then shifted to 50μE m−2 s−1 (50L/D), por1 and por2 mRNA levels decreased swiftly but briefly upon light reduction. Thereafter, POR1 but not POR2 protein levels rose significantly in response to this light stepdown. Conclusion Given the sensitivity of diatom por1/POR1 to real-time light cues and adherence of por2/POR2 regulation to

  5. Toward high-throughput screening of NAD(P)-dependent oxidoreductases using boron-doped diamond microelectrodes and microfluidic devices.

    PubMed

    Oyobiki, Ryo; Kato, Taisuke; Katayama, Michinobu; Sugitani, Ai; Watanabe, Takeshi; Einaga, Yasuaki; Matsumoto, Yoshinori; Horisawa, Kenichi; Doi, Nobuhide

    2014-10-07

    Although oxidoreductases are widely used in many applications, such as biosensors and biofuel cells, improvements in the function of existing oxidoreductases or the discovery of novel oxidoreductases with greater activities is desired. To increase the activity of oxidoreductases by directed evolution, a powerful screening technique for oxidoreductases is required. In this study, we demonstrate the utility of boron-doped diamond (BDD) microelectrodes for quantitative and potentially high-throughput measurement of the activity of NAD(P)-dependent oxidoreductases. We first confirmed that BDD microelectrodes can quantify the activity of low concentrations (10-100 pM) of glucose-6-phosphate dehydrogenase and alcohol dehydrogenase with a measuring time of 1 ms per sample. In addition, we found that poisoning of BDD microelectrodes can be repressed by optimizing the pH and by adding l-arginine to the enzyme solution as an antiaggregation agent. Finally, we fabricated a microfluidic device containing a BDD electrode for the first time and observed the elevation of the oxidation current of NADH with increasing flow rate. These results imply that the combination of a BDD microelectrode and microfluidics can be used for high-throughput screening of an oxidoreductase library containing a large number (>10(6)) of samples, each with a small (nanoliter) sample volume.

  6. Long-term follow-up of a female with congenital adrenal hyperplasia due to P450-oxidoreductase deficiency.

    PubMed

    Bonamichi, Beatriz D S F; Santiago, Stella L M; Bertola, Débora R; Kim, Chong A; Alonso, Nivaldo; Mendonca, Berenice B; Bachega, Tania A S S; Gomes, Larissa G

    2016-10-01

    P450 oxidoreductase deficiency (PORD) is a variant of congenital adrenal hyperplasia that is caused by POR gene mutations. The POR gene encodes a flavor protein that transfers electrons from nicotinamide adenine dinucleotide phosphate (NADPH) to all microsomal cytochrome P450 type II (including 21-hydroxylase, 17α-hydroxylase 17,20 lyase and aromatase), which is fundamental for their enzymatic activity. POR mutations cause variable impairments in steroidogenic enzyme activities that result in wide phenotypic variability ranging from 46,XX or 46,XY disorders of sexual differentiation, glucocorticoid deficiency, with or without skeletal malformations similar to Antley-Bixler syndrome to asymptomatic newborns diagnosed during neonatal screening test. Little is known about the PORD long-term evolution. We described a 46,XX patient with mild atypical genitalia associated with severe bone malformation, who was diagnosed after 13 years due to sexual infantilism. She developed large ovarian cysts and late onset adrenal insufficiency during follow-up, both of each regressed after hormone replacement therapies. We also described a late surgical approach for the correction of facial hypoplasia in a POR patient.

  7. Aripiprazole increases NAD(P)H-quinone oxidoreductase-1 and heme oxygenase-1 in PC12 cells.

    PubMed

    Kaneko, Yoko S; Takayanagi, Takeshi; Nagasaki, Hiroshi; Kodani, Yu; Nakashima, Akira; Mori, Keiji; Suzuki, Atsushi; Itoh, Mitsuyasu; Kondo, Kazunao; Nagatsu, Toshiharu; Ota, Miyuki; Ota, Akira

    2015-06-01

    We previously showed that aripiprazole increases intracellular NADPH and glucose-6-phosphate dehydrogenase mRNA in PC12 cells. Aripiprazole presumably activates a system that concurrently detoxifies reactive oxygen species and replenishes NADPH. Nrf2, a master transcriptional regulator of redox homeostasis genes, also activates the pentose phosphate pathway, including NADPH production. Therefore, our aim was to determine whether aripiprazole activates Nrf2 in PC12 cells. Aripiprazole increased mRNA expression of Nrf2-dependent genes (NAD(P)H-quinone oxidoreductase-1, Nqo1; heme oxygenase-1, HO1; and glutamate-cysteine ligase catalytic subunit) and protein expression of Nqo1 and HO1 in these cells (p < 0.05). To maintain increased Nrf2 activity, it is necessary to inhibit Nrf2 degradation; this is done by causing Nrf2 to dissociate from Keap1 or β-TrCP. However, in aripiprazole-treated cells, the relative amount of Nrf2 anchored to Keap1 or β-TrCP was unaffected and Nrf2 in the nuclear fraction decreased (p < 0.05). Aripiprazole did not affect phosphorylation of Nrf2 at Ser40 and decreased the relative amount of acetylated Nrf2 (p < 0.05). The increase in Nqo1 and HO1 in aripiprazole-treated cells cannot be explained by the canonical Nrf2-degrading pathways. Further experiments are needed to determine the biochemical mechanisms underlying the aripiprazole-induced increase in these enzymes.

  8. Thiol-disulfide Oxidoreductases TRX1 and TMX3 Decrease Neuronal Atrophy in a Lentiviral Mouse Model of Huntington’s Disease

    PubMed Central

    Fox, Jonathan; Lu, Zhen; Barrows, Lorraine

    2015-01-01

    Huntington’s disease (HD) is caused by a trinucleotide CAG repeat in the huntingtin gene (HTT) that results in expression of a polyglutamine-expanded mutant huntingtin protein (mHTT). N-terminal fragments of mHTT accumulate in brain neurons and glia as soluble monomeric and oligomeric species as well as insoluble protein aggregates and drive the disease process. Decreasing mHTT levels in brain provides protection and reversal of disease signs in HD mice making mHTT a prime target for disease modification. There is evidence for aberrant thiol oxidation within mHTT and other proteins in HD models. Based on this, we hypothesized that a specific thiol-disulfide oxidoreductase exists that decreases mHTT levels in cells and provides protection in HD mice. We undertook an in-vitro genetic screen of key thiol-disulfide oxidoreductases then completed secondary screens to identify those with mHTT decreasing properties. Our in-vitro experiments identified thioredoxin 1 and thioredoxin-related transmembrane protein 3 as proteins that decrease soluble mHTT levels in cultured cells. Using a lentiviral mouse model of HD we tested the effect of these proteins in striatum. Both proteins decreased mHTT-induced striatal neuronal atrophy. Findings provide evidence for a role of dysregulated protein-thiol homeostasis in the pathogenesis of HD. PMID:26664998

  9. Heterologous production, isolation, characterization and crystallization of a soluble fragment of the NADH:ubiquinone oxidoreductase (complex I) from Aquifex aeolicus.

    PubMed

    Kohlstädt, Markus; Dörner, Katerina; Labatzke, Ramona; Koç, Cengiz; Heilscher, Ruth; Schiltz, Emile; Einsle, Oliver; Hellwig, Petra; Friedrich, Thorsten

    2008-12-09

    The proton-pumping NADH:ubiquinone oxidoreductase (complex I) is the first enzyme complex of the respiratory chains in many bacteria and most eukaryotes. It is the least understood of all, due to its enormous size and unique energy conversion mechanism. The bacterial complex is in general made up of 14 different subunits named NuoA-N. Subunits NuoE, -F, and -G comprise the electron input part of the complex. We have cloned these genes from the hyperthermophilic bacterium Aquifex aeolicus and expressed them heterologously in Escherichia coli. A soluble subcomplex made up of NuoE and NuoF and containing the NADH binding site, the primary electron acceptor flavin mononucleotide (FMN), the binuclear iron-sulfur cluster N1a, and the tetranuclear iron-sulfur cluster N3 was isolated by chromatographic methods. The proteins were identified by N-terminal sequencing and mass spectrometry; the cofactors were characterized by UV/vis and EPR spectroscopy. Subunit NuoG was not produced in this strain. The preparation was thermostable and exhibited maximum NADH/ferricyanide oxidoreductase activity at 85 degrees C. Analytical size-exclusion chromatography and dynamic light scattering revealed the homogeneity of the preparation. First attempts to crystallize the preparation led to crystals diffracting more than 2 A.

  10. The human B22 subunit of the NADH-ubiquinone oxidoreductase maps to the region of chromosome 8 involved in Branchio-oto-renal syndrome

    SciTech Connect

    Gu, J.Z.; Lin, Xin; Wells, D.E.

    1996-07-01

    To identify candidate genes for Branchio-oto-renal (BOR) syndrome, we have made use of a set of cosmids that map to 8q13.3, which has previously been shown to be involved in this syndrome. These cosmids were used as genomic clones in the attempts to isolate corresponding cDNAs using a modified hybrid selection technique. cDNAs using a modified hybrid selection technique. cDNAs from the region were identified and used to search for sequence similarity in human or other species. One cDNA clone was found to have 89% sequence similarity to the bovine B22 subunit of NADH-ubiquinone oxidoreductase, a mitochondrial protein in the respiratory electron transport chain. Given the history of other mitochondrial mutations being involved in hearing loss syndromes, this gene should be considered a strong candidate for involvement in BOR.

  11. Role for Ferredoxin:NAD(P)H Oxidoreductase (FprA) in Sulfate Assimilation and Siderophore Biosynthesis in Pseudomonads

    PubMed Central

    Glassing, Angela; Harper, Justin; Franklin, Michael J.

    2013-01-01

    Pyridine-2,6-bis(thiocarboxylate) (PDTC), produced by certain pseudomonads, is a sulfur-containing siderophore that binds iron, as well as a wide range of transition metals, and it affects the net hydrolysis of the environmental contaminant carbon tetrachloride. The pathway of PDTC biosynthesis has not been defined. Here, we performed a transposon screen of Pseudomonas putida DSM 3601 to identify genes necessary for PDTC production (Pdt phenotype). Transposon insertions within genes for sulfate assimilation (cysD, cysNC, and cysG [cobA2]) dominated the collection of Pdt mutations. In addition, two insertions were within the gene for the LysR-type transcriptional activator FinR (PP1637). Phenotypic characterization indicated that finR mutants were cysteine bradytrophs. The Pdt phenotype of finR mutants could be complemented by the known target of FinR regulation, fprA (encoding ferredoxin:NADP+ oxidoreductase), or by Escherichia coli cysJI (encoding sulfite reductase). These data indicate that fprA is necessary for effective sulfate assimilation by P. putida and that the effect of finR mutation on PDTC production was due to deficient expression of fprA and sulfite reduction. fprA expression in both P. putida and P. aeruginosa was found to be regulated by FinR, but in a manner dependent upon reduced sulfur sources, implicating FinR in sulfur regulatory physiology. The genes and phenotypes identified in this study indicated a strong dependence upon intracellular reduced sulfur/cysteine for PDTC biosynthesis and that pseudomonads utilize sulfite reduction enzymology distinct from that of E. coli and possibly similar to that of chloroplasts and other proteobacteria. PMID:23794620

  12. Plant science. Morphinan biosynthesis in opium poppy requires a P450-oxidoreductase fusion protein.

    PubMed

    Winzer, Thilo; Kern, Marcelo; King, Andrew J; Larson, Tony R; Teodor, Roxana I; Donninger, Samantha L; Li, Yi; Dowle, Adam A; Cartwright, Jared; Bates, Rachel; Ashford, David; Thomas, Jerry; Walker, Carol; Bowser, Tim A; Graham, Ian A

    2015-07-17

    Morphinan alkaloids from the opium poppy are used for pain relief. The direction of metabolites to morphinan biosynthesis requires isomerization of (S)- to (R)-reticuline. Characterization of high-reticuline poppy mutants revealed a genetic locus, designated STORR [(S)- to (R)-reticuline] that encodes both cytochrome P450 and oxidoreductase modules, the latter belonging to the aldo-keto reductase family. Metabolite analysis of mutant alleles and heterologous expression demonstrate that the P450 module is responsible for the conversion of (S)-reticuline to 1,2-dehydroreticuline, whereas the oxidoreductase module converts 1,2-dehydroreticuline to (R)-reticuline rather than functioning as a P450 redox partner. Proteomic analysis confirmed that these two modules are contained on a single polypeptide in vivo. This modular assembly implies a selection pressure favoring substrate channeling. The fusion protein STORR may enable microbial-based morphinan production.

  13. Quinone reduction by Rhodothermus marinus succinate:menaquinone oxidoreductase is not stimulated by the membrane potential

    SciTech Connect

    Fernandes, Andreia S.; Konstantinov, Alexander A.; Teixeira, Miguel; Pereira, Manuela M. . E-mail: mpereira@itqb.unl.pt

    2005-05-06

    Succinate:quinone oxidoreductase (SQR), a di-haem enzyme purified from Rhodothermus marinus, reveals an HQNO-sensitive succinate:quinone oxidoreductase activity with several menaquinone analogues as electron acceptors that decreases with lowering the redox midpoint potential of the quinones. A turnover with the low-potential 2,3-dimethyl-1,4-naphthoquinone that is the closest analogue of menaquinone, although low, can be detected in liposome-reconstituted SQR. Reduction of the quinone is not stimulated by an imposed K{sup +}-diffusion membrane potential of a physiological sign (positive inside the vesicles). Nor does the imposed membrane potential increase the reduction level of the haems in R. marinus SQR poised with the succinate/fumarate redox couple. The data do not support a widely discussed hypothesis on the electrogenic transmembrane electron transfer from succinate to menaquinone catalysed by di-haem SQRs. The role of the membrane potential in regulation of the SQR activity is discussed.

  14. Identification and characterization of oxalate oxidoreductase, a novel thiamine pyrophosphate-dependent 2-oxoacid oxidoreductase that enables anaerobic growth on oxalate.

    PubMed

    Pierce, Elizabeth; Becker, Donald F; Ragsdale, Stephen W

    2010-12-24

    Moorella thermoacetica is an anaerobic acetogen, a class of bacteria that is found in the soil, the animal gastrointestinal tract, and the rumen. This organism engages the Wood-Ljungdahl pathway of anaerobic CO(2) fixation for heterotrophic or autotrophic growth. This paper describes a novel enzyme, oxalate oxidoreductase (OOR), that enables M. thermoacetica to grow on oxalate, which is produced in soil and is a common component of kidney stones. Exposure to oxalate leads to the induction of three proteins that are subunits of OOR, which oxidizes oxalate coupled to the production of two electrons and CO(2) or bicarbonate. Like other members of the 2-oxoacid:ferredoxin oxidoreductase family, OOR contains thiamine pyrophosphate and three [Fe(4)S(4)] clusters. However, unlike previously characterized members of this family, OOR does not use coenzyme A as a substrate. Oxalate is oxidized with a k(cat) of 0.09 s(-1) and a K(m) of 58 μM at pH 8. OOR also oxidizes a few other 2-oxoacids (which do not induce OOR) also without any requirement for CoA. The enzyme transfers its reducing equivalents to a broad range of electron acceptors, including ferredoxin and the nickel-dependent carbon monoxide dehydrogenase. In conjunction with the well characterized Wood-Ljungdahl pathway, OOR should be sufficient for oxalate metabolism by M. thermoacetica, and it constitutes a novel pathway for oxalate metabolism.

  15. 6-Phospho-D-gluconate:NAD+ 2-oxidoreductase (decarboxylating) from slow-growing Rhizobia.

    PubMed Central

    Martínez-Drets, G; Gardiol, A; Arias, A

    1977-01-01

    6-Phospho-D-gluconate:NAD+ 2-oxidoreductase (decarboxylating) (NAD+-6PGD) was detected in several slow-growing strains of rhizobia, and no activity involving NADP+ was found in the same extracts. By contrast, fast-growing strains of rhizobia had NADP+-6PGD activity; most of them also had NAD+-6PGD activity. NAD+-6PGD was partially purified from the slow-growing strain Rhizobium japonicum 5006. The reaction was shown to be an oxidative decarboxylation. PMID:16867

  16. NqrM (DUF539) Protein Is Required for Maturation of Bacterial Na+-Translocating NADH:Quinone Oxidoreductase

    PubMed Central

    Kostyrko, Vitaly A.; Bertsova, Yulia V.; Serebryakova, Marina V.; Baykov, Alexander A.

    2015-01-01

    ABSTRACT Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) catalyzes electron transfer from NADH to ubiquinone in the bacterial respiratory chain, coupled with Na+ translocation across the membrane. Na+-NQR maturation involves covalent attachment of flavin mononucleotide (FMN) residues, catalyzed by flavin transferase encoded by the nqr-associated apbE gene. Analysis of complete bacterial genomes has revealed another putative gene (duf539, here renamed nqrM) that usually follows the apbE gene and is present only in Na+-NQR-containing bacteria. Expression of the Vibrio harveyi nqr operon alone or with the associated apbE gene in Escherichia coli, which lacks its own Na+-NQR, resulted in an enzyme incapable of Na+-dependent NADH or reduced nicotinamide hypoxanthine dinucleotide (dNADH) oxidation. However, fully functional Na+-NQR was restored when these genes were coexpressed with the V. harveyi nqrM gene. Furthermore, nqrM lesions in Klebsiella pneumoniae and V. harveyi prevented production of functional Na+-NQR, which could be recovered by an nqrM-containing plasmid. The Na+-NQR complex isolated from the nqrM-deficient strain of V. harveyi lacks several subunits, indicating that nqrM is necessary for Na+-NQR assembly. The protein product of the nqrM gene, NqrM, contains a single putative transmembrane α-helix and four conserved Cys residues. Mutating one of these residues (Cys33 in V. harveyi NqrM) to Ser completely prevented Na+-NQR maturation, whereas mutating any other Cys residue only decreased the yield of the mature protein. These findings identify NqrM as the second specific maturation factor of Na+-NQR in proteobacteria, which is presumably involved in the delivery of Fe to form the (Cys)4[Fe] center between subunits NqrD and NqrE. IMPORTANCE Na+-translocating NADH:quinone oxidoreductase complex (Na+-NQR) is a unique primary Na+ pump believed to enhance the vitality of many bacteria, including important pathogens such as Vibrio cholerae, Vibrio

  17. Characterization of an 8-hydroxy-5-deazaflavin:NADPH oxidoreductase from Streptomyces griseus.

    PubMed

    Eker, A P; Hessels, J K; Meerwaldt, R

    1989-01-27

    An 8-hydroxy-5-deazaflavin-dependent oxidoreductase was isolated from the actinomycete Streptomyces griseus and purified 590-fold with 72% overall yield. The enzyme catalyzes electron transfer between 8-hydroxy-5-deazaflavins and NADPH. It seems to be more specific than methanogenic oxidoreductase as it has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate. A molecular weight of 42,000 was found with gel permeation chromatography, while SDS gel electrophoresis indicated the presence of two identical subunits. Maximal enzymatic activity was found at 0.32 M NaCl and pH 5.9 for reduction of 8-hydroxy-5-deazaflavin and pH 7.9 for the reverse reaction. From the kinetic constants it was estimated that the main function of this oxidoreductase is probably to provide cells with reduced 8-hydroxy-5-deazaflavin to be used in specific reduction reactions. These results indicate the occurrence of 8-hydroxy-5-deazaflavin-dependent electron transfer in microorganisms not belonging to the archaebacteria.

  18. Bioelectrocatalysts: engineered oxidoreductase system for utilization of fumarate reductase in chemical synthesis, detection, and fuel cells.

    PubMed

    Park, Doo Hyun; Vieille, C; Zeikus, J G

    2003-10-01

    Fumarate reductase was used as a model oxidoreductase to demonstrate continuous electrical cofactor reduction-oxidation during the bioelectrochemical synthesis and detection of chemicals. The enzyme preparation was immobilized onto a graphite felt electrode that was modified with carboxymethylcellulose (CMC). Nicotinamide adenine dinucleotide (NAD), neutral red, and fumarate reductase (which contained menaquinone) were covalently linked by peptide bonds to the CMC. The electron mediator neutral red allowed NAD and menaquinone to be recycled electrically during enzymatic chemical synthesis. Succinate detection by the bioelectrocatalyst was linear from 5 microM to 10 mM succinate. Fumarate synthesis using this bioelectrode was dependent on succinate utilization and resulted in proportional production of electricity and fumarate. Succinate synthesis using this bioelectrocatalyst was dependent on current and fumarate concentration. This bioelectrocatalyst system may enhance the utility of menaquinone- and/or pyridine nucleotide-linked oxidoreductases in diverse enzymatic fuel cells and sensors. It may also enhance the utility of oxidoreductase-based chemical synthesis systems because it eliminates the problem of cofactor recycling.

  19. Role of the Na(+)-translocating NADH:quinone oxidoreductase in voltage generation and Na(+) extrusion in Vibrio cholerae.

    PubMed

    Vorburger, Thomas; Nedielkov, Ruslan; Brosig, Alexander; Bok, Eva; Schunke, Emina; Steffen, Wojtek; Mayer, Sonja; Götz, Friedrich; Möller, Heiko M; Steuber, Julia

    2016-04-01

    For Vibrio cholerae, the coordinated import and export of Na(+) is crucial for adaptation to habitats with different osmolarities. We investigated the Na(+)-extruding branch of the sodium cycle in this human pathogen by in vivo (23)Na-NMR spectroscopy. The Na(+) extrusion activity of cells was monitored after adding glucose which stimulated respiration via the Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR). In a V. cholerae deletion mutant devoid of the Na(+)-NQR encoding genes (nqrA-F), rates of respiratory Na(+) extrusion were decreased by a factor of four, but the cytoplasmic Na(+) concentration was essentially unchanged. Furthermore, the mutant was impaired in formation of transmembrane voltage (ΔΨ, inside negative) and did not grow under hypoosmotic conditions at pH8.2 or above. This growth defect could be complemented by transformation with the plasmid encoded nqr operon. In an alkaline environment, Na(+)/H(+) antiporters acidify the cytoplasm at the expense of the transmembrane voltage. It is proposed that, at alkaline pH and limiting Na(+) concentrations, the Na(+)-NQR is crucial for generation of a transmembrane voltage to drive the import of H(+) by electrogenic Na(+)/H(+) antiporters. Our study provides the basis to understand the role of the Na(+)-NQR in pathogenicity of V. cholerae and other pathogens relying on this primary Na(+) pump for respiration.

  20. ArxA, a new clade of arsenite oxidase within the DMSO reductase family of molybdenum oxidoreductases

    USGS Publications Warehouse

    Zargar, Kamrun; Conrad, Alison; Bernick, David L.; Lowe, Todd M.; Stolc, Viktor; Hoeft, Shelley; Oremland, Ronald S.; Stolz, John; Saltikov, Chad W.

    2012-01-01

    Arsenotrophy, growth coupled to autotrophic arsenite oxidation or arsenate respiratory reduction, occurs only in the prokaryotic domain of life. The enzymes responsible for arsenotrophy belong to distinct clades within the DMSO reductase family of molybdenum-containing oxidoreductases: specifically arsenate respiratory reductase, ArrA, and arsenite oxidase, AioA (formerly referred to as AroA and AoxB). A new arsenite oxidase clade, ArxA, represented by the haloalkaliphilic bacterium Alkalilimnicola ehrlichii strain MLHE-1 was also identified in the photosynthetic purple sulfur bacterium Ectothiorhodospira sp. strain PHS-1. A draft genome sequence of PHS-1 was completed and an arx operon similar to MLHE-1 was identified. Gene expression studies showed that arxA was strongly induced with arsenite. Microbial ecology investigation led to the identification of additional arxA-like sequences in Mono Lake and Hot Creek sediments, both arsenic-rich environments in California. Phylogenetic analyses placed these sequences as distinct members of the ArxA clade of arsenite oxidases. ArxA-like sequences were also identified in metagenome sequences of several alkaline microbial mat environments of Yellowstone National Park hot springs. These results suggest that ArxA-type arsenite oxidases appear to be widely distributed in the environment presenting an opportunity for further investigations of the contribution of Arx-dependent arsenotrophy to the arsenic biogeochemical cycle.

  1. Xanthine oxidoreductase is required for genotoxic stress-induced NKG2D ligand expression and gemcitabine-mediated antitumor activity

    PubMed Central

    Xu, Xiulong; Rao, Geetha; Li, Yi

    2016-01-01

    MICA/B (the major histocompatibility antigen-related chain A and B) and Rae I are stress-inducible ligands for the immune-receptor NKG2D. Mechanisms by which genotoxic stress and DNA damage induce the expression of NKG2D ligands remain incompletely understood. Here, we report that inhibition of xanthine oxidoreductase (XOR) activity by allopurinol or inhibition of XOR expression by gene knockdown abrogated genotoxic stress-induced expression of MICA/B and Rae I in three tumor cell lines. XOR knockdown also blocked gemcitabine-mediated antitumor activity in an orthotopic syngeneic mouse model of breast cancer. As a rate-limiting enzyme in the purine catabolic pathway, XOR generates two end-products, uric acid and reactive oxygen species (ROS). ROS scavenging had an insignificant effect on genotoxic drug-induced MICA/B expression but modestly inhibited radiation-induced MICA/B expression. Exogenous uric acid (in the form of monosodium urate) induced MICA/B expression by activating the MAP kinase pathway. Allopurinol blocked genotoxic stress-induced MAP kinase activation. Our study provides mechanistic insights into genotoxic stress-induced activation of the MAP kinase pathway and suggests that XOR is required for genotoxic stress-induced NKG2D ligand expression and gemcitabine-mediated antitumor activity. PMID:27494876

  2. Cholesterol metabolism: the main pathway acting downstream of cytochrome P450 oxidoreductase in skeletal development of the limb.

    PubMed

    Schmidt, Katy; Hughes, Catherine; Chudek, J A; Goodyear, Simon R; Aspden, Richard M; Talbot, Richard; Gundersen, Thomas E; Blomhoff, Rune; Henderson, Colin; Wolf, C Roland; Tickle, Cheryll

    2009-05-01

    Cytochrome P450 oxidoreductase (POR) is the obligate electron donor for all microsomal cytochrome P450 enzymes, which catalyze the metabolism of a wide spectrum of xenobiotic and endobiotic compounds. Point mutations in POR have been found recently in patients with Antley-Bixler-like syndrome, which includes limb skeletal defects. In order to study P450 function during limb and skeletal development, we deleted POR specifically in mouse limb bud mesenchyme. Forelimbs and hind limbs in conditional knockout (CKO) mice were short with thin skeletal elements and fused joints. POR deletion occurred earlier in forelimbs than in hind limbs, leading additionally to soft tissue syndactyly and loss of wrist elements and phalanges due to changes in growth, cell death, and skeletal segmentation. Transcriptional analysis of E12.5 mouse forelimb buds demonstrated the expression of P450s involved in retinoic acid, cholesterol, and arachidonic acid metabolism. Biochemical analysis of CKO limbs confirmed retinoic acid excess. In CKO limbs, expression of genes throughout the whole cholesterol biosynthetic pathway was upregulated, and cholesterol deficiency can explain most aspects of the phenotype. Thus, cellular POR-dependent cholesterol synthesis is essential during limb and skeletal development. Modulation of P450 activity could contribute to susceptibility of the embryo and developing organs to teratogenesis.

  3. Pyruvate : NADP+ oxidoreductase from the mitochondrion of Euglena gracilis and from the apicomplexan Cryptosporidium parvum: a biochemical relic linking pyruvate metabolism in mitochondriate and amitochondriate protists.

    PubMed

    Rotte, C; Stejskal, F; Zhu, G; Keithly, J S; Martin, W

    2001-05-01

    Most eukaryotes perform the oxidative decarboxylation of pyruvate in mitochondria using pyruvate dehydrogenase (PDH). Eukaryotes that lack mitochondria also lack PDH, using instead the O(2)-sensitive enzyme pyruvate : ferredoxin oxidoreductase (PFO), which is localized either in the cytosol or in hydrogenosomes. The facultatively anaerobic mitochondria of the photosynthetic protist Euglena gracilis constitute a hitherto unique exception in that these mitochondria oxidize pyruvate with the O(2)-sensitive enzyme pyruvate : NADP oxidoreductase (PNO). Cloning and analysis of Euglena PNO revealed that the cDNA encodes a mitochondrial transit peptide followed by an N-terminal PFO domain that is fused to a C-terminal NADPH-cytochrome P450 reductase (CPR) domain. Two independent 5.8-kb full-size cDNAs for Euglena mitochondrial PNO were isolated; the gene was expressed in cultures supplied with 2% CO(2) in air and with 2% CO(2) in N(2). The apicomplexan Cryptosporidium parvum was also shown to encode and express the same PFO-CPR fusion, except that, unlike E. gracilis, no mitochondrial transit peptide for C. parvum PNO was found. Recombination-derived remnants of PNO are conserved in the genomes of Saccharomyces cerevisiae and Schizosaccharomyces pombe as proteins involved in sulfite reduction. Notably, Trypanosoma brucei was found to encode homologs of both PFO and all four PDH subunits. Gene organization and phylogeny revealed that eukaryotic nuclear genes for mitochondrial, hydrogenosomal, and cytosolic PFO trace to a single eubacterial acquisition. These findings suggest a common ancestry of PFO in amitochondriate protists with Euglena mitochondrial PNO and Cryptosporidium PNO. They are also consistent with the view that eukaryotic PFO domains are biochemical relics inherited from a facultatively anaerobic, eubacterial ancestor of mitochondria and hydrogenosomes.

  4. Arabidopsis Root-Type Ferredoxin:NADP(H) Oxidoreductase 2 is Involved in Detoxification of Nitrite in Roots.

    PubMed

    Hachiya, Takushi; Ueda, Nanae; Kitagawa, Munenori; Hanke, Guy; Suzuki, Akira; Hase, Toshiharu; Sakakibara, Hitoshi

    2016-11-01

    Ferredoxin:NADP(H) oxidoreductase (FNR) plays a key role in redox metabolism in plastids. Whereas leaf FNR (LFNR) is required for photosynthesis, root FNR (RFNR) is believed to provide electrons to ferredoxin (Fd)-dependent enzymes, including nitrite reductase (NiR) and Fd-glutamine-oxoglutarate aminotransferase (Fd-GOGAT) in non-photosynthetic conditions. In some herbal species, however, most nitrate reductase activity is located in photosynthetic organs, and ammonium in roots is assimilated mainly by Fd-independent NADH-GOGAT. Therefore, RFNR might have a limited impact on N assimilation in roots grown with nitrate or ammonium nitrogen sources. AtRFNR genes are rapidly induced by application of toxic nitrite. Thus, we tested the hypothesis that RFNR could contribute to nitrite reduction in roots by comparing Arabidopsis thaliana seedlings of the wild type with loss-of-function mutants of RFNR2 When these seedlings were grown under nitrate, nitrite or ammonium, only nitrite nutrition caused impaired growth and nitrite accumulation in roots of rfnr2 Supplementation of nitrite with nitrate or ammonium as N sources did not restore the root growth in rfnr2 Also, a scavenger for nitric oxide (NO) could not effectively rescue the growth impairment. Thus, nitrite toxicity, rather than N depletion or nitrite-dependent NO production, probably causes the rfnr2 root growth defect. Our results strongly suggest that RFNR2 has a major role in reduction of toxic nitrite in roots. A specific set of genes related to nitrite reduction and the supply of reducing power responded to nitrite concomitantly, suggesting that the products of these genes act co-operatively with RFNR2 to reduce nitrite in roots.

  5. An ancient oxidoreductase making differential use of its cofactors.

    PubMed

    Blüher, Doreen; Reinhardt-Tews, Annekathrin; Hey, Martin; Lilie, Hauke; Golbik, Ralph; Breunig, Karin D; Anders, Alexander

    2014-07-01

    Abstract Many transcription factors contribute to cellular homeostasis by integrating multiple signals. Signaling via the yeast Gal80 protein, a negative regulator of the prototypic transcription activator Gal4, is primarily regulated by galactose. ScGal80 from Saccharomyces cerevisiae has been reported to bind NAD(P). Here, we show that the ability to bind these ligands is conserved in KlGal80, a Gal80 homolog from the distantly related yeast Kluyveromyces lactis. However, the homologs apparently have diverged with respect to response to the dinucleotide. Strikingly, ScGal80 binds NAD(P) and NAD(P)H with more than 50-fold higher affinity than KlGal80. In contrast to ScGal80, where NAD is neutral, NAD and NADP have a negative effect in KlGal80 on its interaction with a KlGal4-peptide in vitro. Swapping a loop in the NAD(P) binding Rossmann-fold of ScGal80 into KlGal80 increases the affinity for NAD(P) and has a significant impact on KlGal4 regulation in vivo. Apparently, dinucleotide binding allows coupling of the metabolic state of the cell to regulation of the GAL/LAC genes. The particular sequences involved in binding determine how exactly the metabolic state is sensed and integrated by Gal80 to regulate Gal4.

  6. The tumor suppressor WW domain-containing oxidoreductase modulates cell metabolism.

    PubMed

    Abu-Remaileh, Muhannad; Aqeilan, Rami I

    2015-03-01

    The WW domain-containing oxidoreductase (WWOX) encodes a tumor suppressor that is frequently altered in cancer. WWOX binds several proteins and thus is postulated to be involved in a variety of cellular processes. Interestingly, Wwox-knockout mice develop normally in utero but succumb to hypoglycemia and other metabolic defects early in life resulting in their death by 3-4 weeks of age. Cumulative evidence has linked WWOX with cellular metabolism including steroid metabolism, high-density lipoprotein cholesterol (HDL-C) metabolism, bone metabolism and, more recently, glucose metabolism. In this review, we discuss these evolving functions for WWOX and how its deletion affects cellular metabolism and neoplastic progression.

  7. Characteristics of endogenous flavin fluorescence of Photobacterium leiognathi luciferase and Vibrio fischeri NAD(P)H:FMN-oxidoreductase.

    PubMed

    Vetrova, E V; Kudryasheva, N S; Visser, A J W G; van Hoek, A

    2005-01-01

    The bioluminescent bacterial enzyme system NAD(P)H:FMN-oxidoreductase-luciferase has been used as a test system for ecological monitoring. One of the modes to quench bioluminescence is the interaction of xenobiotics with the enzymes, which inhibit their activity. The use of endogenous flavin fluorescence for investigation of the interactions of non-fluorescent compounds with the bacterial luciferase from Photobacterium leiognathi and NAD(P)H:FMN-oxidoreductase from Vibrio fischeri has been proposed. Fluorescence spectroscopy methods have been used to study characteristics of endogenous flavin fluorescence (fluorophore lifetime, the rotational correlation time). The fluorescence anisotropy behaviour of FMN has been analysed and compared to that of the enzyme-bound flavin. The fluorescence characteristics of endogenous flavin of luciferase and NAD(P)H:FMN-oxidoreductase have been shown to be applicable in studying enzymes' interactions with non-fluorescent compounds.

  8. Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity

    PubMed Central

    Hernandez-Fernaud, Juan R.; Ruengeler, Elena; Casazza, Andrea; Neilson, Lisa J.; Pulleine, Ellie; Santi, Alice; Ismail, Shehab; Lilla, Sergio; Dhayade, Sandeep; MacPherson, Iain R.; McNeish, Iain; Ennis, Darren; Ali, Hala; Kugeratski, Fernanda G.; Al Khamici, Heba; van den Biggelaar, Maartje; van den Berghe, Peter V.E.; Cloix, Catherine; McDonald, Laura; Millan, David; Hoyle, Aoisha; Kuchnio, Anna; Carmeliet, Peter; Valenzuela, Stella M.; Blyth, Karen; Yin, Huabing; Mazzone, Massimiliano; Norman, Jim C.; Zanivan, Sara

    2017-01-01

    The secretome of cancer and stromal cells generates a microenvironment that contributes to tumour cell invasion and angiogenesis. Here we compare the secretome of human mammary normal and cancer-associated fibroblasts (CAFs). We discover that the chloride intracellular channel protein 3 (CLIC3) is an abundant component of the CAF secretome. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive angiogenesis and increases invasiveness of cancer cells both in vivo and in 3D cell culture models, and this requires active transglutaminase-2 (TGM2). CLIC3 acts as a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels correlate with poor clinical outcome. This work reveals a previously undescribed invasive mechanism whereby the secretion of a glutathione-dependent oxidoreductase drives angiogenesis and cancer progression by promoting TGM2-dependent invasion. PMID:28198360

  9. Another unusual type of citric acid cycle enzyme in Helicobacter pylori: the malate:quinone oxidoreductase.

    PubMed

    Kather, B; Stingl, K; van der Rest, M E; Altendorf, K; Molenaar, D

    2000-06-01

    The only enzyme of the citric acid cycle for which no open reading frame (ORF) was found in the Helicobacter pylori genome is the NAD-dependent malate dehydrogenase. Here, it is shown that in this organism the oxidation of malate to oxaloacetate is catalyzed by a malate:quinone oxidoreductase (MQO). This flavin adenine dinucleotide-dependent membrane-associated enzyme donates electrons to quinones of the electron transfer chain. Similar to succinate dehydrogenase, it is part of both the electron transfer chain and the citric acid cycle. MQO activity was demonstrated in isolated membranes of H. pylori. The enzyme is encoded by the ORF HP0086, which is shown by the fact that expression of the HP0086 sequence from a plasmid induces high MQO activity in mqo deletion mutants of Escherichia coli or Corynebacterium glutamicum. Furthermore, this plasmid was able to complement the phenotype of the C. glutamicum mqo deletion mutant. Interestingly, the protein predicted to be encoded by this ORF is only distantly related to known or postulated MQO sequences from other bacteria. The presence of an MQO shown here and the previously demonstrated presence of a 2-ketoglutarate:ferredoxin oxidoreductase and a succinyl-coenzyme A (CoA):acetoacetyl-CoA transferase indicate that H. pylori possesses a complete citric acid cycle, but one which deviates from the standard textbook example in three steps.

  10. NADPH-Cytochrome P450 Oxidoreductase: Prototypic Member of the Diflavin Reductase Family

    PubMed Central

    Iyanagi, Takashi; Xia, Chuanwu; Kim, Jung-Ja P.

    2012-01-01

    NADPH-cytochrome P450 oxidoreductase (CYPOR) and nitric oxide synthase (NOS), two members of the diflavin oxidoreductase family, are multi-domain enzymes containing distinct FAD and FMN domains connected by a flexible hinge. FAD accepts a hydride ion from NADPH, and reduced FAD donates electrons to FMN, which in turn transfers electrons to the heme center of cytochrome P450 or NOS oxygenase domain. Structural analysis of CYPOR, the prototype of this enzyme family, has revealed the exact nature of the domain arrangement and the role of residues involved in cofactor binding. Recent structural and biophysical studies of CYPOR have shown that the two flavin domains undergo large domain movements during catalysis. NOS isoforms contain additional regulatory elements within the reductase domain that control electron transfer through Ca2+-dependent calmodulin (CaM) binding. The recent crystal structure of an iNOS Ca2+/CaM-FMN construct, containing the FMN domain in complex with Ca2+/CaM, provided structural information on the linkage between the reductase and oxgenase domains of NOS, making it possible to model the holo iNOS structure. This review summarizes recent advances in our understanding of the dynamics of domain movements during CYPOR catalysis and the role of the NOS diflavin reductase domain in the regulation of NOS isozyme activities. PMID:22982532

  11. Transient Kinetic Analysis of Hydrogen Sulfide Oxidation Catalyzed by Human Sulfide Quinone Oxidoreductase.

    PubMed

    Mishanina, Tatiana V; Yadav, Pramod K; Ballou, David P; Banerjee, Ruma

    2015-10-09

    The first step in the mitochondrial sulfide oxidation pathway is catalyzed by sulfide quinone oxidoreductase (SQR), which belongs to the family of flavoprotein disulfide oxidoreductases. During the catalytic cycle, the flavin cofactor is intermittently reduced by sulfide and oxidized by ubiquinone, linking H2S oxidation to the electron transfer chain and to energy metabolism. Human SQR can use multiple thiophilic acceptors, including sulfide, sulfite, and glutathione, to form as products, hydrodisulfide, thiosulfate, and glutathione persulfide, respectively. In this study, we have used transient kinetics to examine the mechanism of the flavin reductive half-reaction and have determined the redox potential of the bound flavin to be -123 ± 7 mV. We observe formation of an unusually intense charge-transfer (CT) complex when the enzyme is exposed to sulfide and unexpectedly, when it is exposed to sulfite. In the canonical reaction, sulfide serves as the sulfur donor and sulfite serves as the acceptor, forming thiosulfate. We show that thiosulfate is also formed when sulfide is added to the sulfite-induced CT intermediate, representing a new mechanism for thiosulfate formation. The CT complex is formed at a kinetically competent rate by reaction with sulfide but not with sulfite. Our study indicates that sulfide addition to the active site disulfide is preferred under normal turnover conditions. However, under pathological conditions when sulfite concentrations are high, sulfite could compete with sulfide for addition to the active site disulfide, leading to attenuation of SQR activity and to an alternate route for thiosulfate formation.

  12. Structural insights into the functional versatility of WW domain-containing oxidoreductase tumor suppressor

    PubMed Central

    2015-01-01

    Recent work on WW domain-containing oxidoreductase (WWOX) tumor suppressor is beginning to shed new light on both the molecular mechanism of action of its WW domains as well as the contiguous catalytic domain. Herein, the structural basis underlying the ability of WW1 domain to bind to various physiological ligands and how the orphan WW2 tandem partner synergizes its ligand binding in the context of WW1–WW2 tandem module of WWOX is discussed. Notably, the WW domains within the WW1–WW2 tandem module physically associate so as to adopt a fixed spatial orientation relative to each other. In this manner, the association of WW2 domain with WW1 hinders ligand binding to the latter. Consequently, ligand binding to WW1 domain not only results in the displacement of WW2 lid but also disrupts the fixed orientation of WW domains in the liganded conformation. Equally importantly, structure-guided functional approach suggests that the catalytic domain of WWOX likely serves as a retinal oxidoreductase that catalyzes the reversible oxidation and reduction of all-trans-retinal. Collectively, this review provides structural insights into the functional versatility of a key signaling protein with important implications on its biology. PMID:25662954

  13. Structural insights into the functional versatility of WW domain-containing oxidoreductase tumor suppressor.

    PubMed

    Farooq, Amjad

    2015-03-01

    Recent work on WW domain-containing oxidoreductase (WWOX) tumor suppressor is beginning to shed new light on both the molecular mechanism of action of its WW domains as well as the contiguous catalytic domain. Herein, the structural basis underlying the ability of WW1 domain to bind to various physiological ligands and how the orphan WW2 tandem partner synergizes its ligand binding in the context of WW1-WW2 tandem module of WWOX is discussed. Notably, the WW domains within the WW1-WW2 tandem module physically associate so as to adopt a fixed spatial orientation relative to each other. In this manner, the association of WW2 domain with WW1 hinders ligand binding to the latter. Consequently, ligand binding to WW1 domain not only results in the displacement of WW2 lid but also disrupts the fixed orientation of WW domains in the liganded conformation. Equally importantly, structure-guided functional approach suggests that the catalytic domain of WWOX likely serves as a retinal oxidoreductase that catalyzes the reversible oxidation and reduction of all-trans-retinal. Collectively, this review provides structural insights into the functional versatility of a key signaling protein with important implications on its biology.

  14. Molecular genetics of 3beta-hydroxy-Delta5-C27-steroid oxidoreductase deficiency in 16 patients with loss of bile acid synthesis and liver disease.

    PubMed

    Cheng, Jeffrey B; Jacquemin, Emmanuel; Gerhardt, Marie; Nazer, Hisham; Cresteil, Danièle; Heubi, James E; Setchell, Kenneth D R; Russell, David W

    2003-04-01

    The 3beta-hydroxy-Delta(5)-C(27)-steroid oxidoreductase (C(27) 3beta-HSD) is a membrane-bound enzyme of the endoplasmic reticulum that catalyzes an early step in the synthesis of bile acids from cholesterol. Subjects with autosomal recessive mutations in the encoding gene, HSD3B7, on chromosome 16p11.2-12 fail to synthesize bile acids and develop a form of progressive liver disease characterized by cholestatic jaundice and malabsorption of lipids and lipid-soluble vitamins from the gastrointestinal tract. The gene encoding the human C(27) 3beta-HSD enzyme was isolated previously, and a 2-bp deletion in exon 6 of HSD3B7 was identified in a well characterized subject with this disorder. Here, we report a molecular analysis of 15 additional patients from 13 kindreds with C(27) 3beta-HSD deficiency. Twelve different mutations were identified in the HSD3B7 gene on chromosome 16p11.2-12. Ten mutations were studied in detail and shown to cause complete loss of enzyme activity and, in two cases, alterations in the size or amount of the transcribed mRNA. Mutations were inherited in homozygous form in 13 subjects from 10 families and compound heterozygous form in four subjects from three families. We conclude that a diverse spectrum of mutations in the HSD3B7 gene underlies this rare form of neonatal cholestasis.

  15. The role of oxidoreductases in determining the function of the neisserial lipid A phosphoethanolamine transferase required for resistance to polymyxin.

    PubMed

    Piek, Susannah; Wang, Zhirui; Ganguly, Jhuma; Lakey, Adam M; Bartley, Stephanie N; Mowlaboccus, Shakeel; Anandan, Anandhi; Stubbs, Keith A; Scanlon, Martin J; Vrielink, Alice; Azadi, Parastoo; Carlson, Russell W; Kahler, Charlene M

    2014-01-01

    The decoration of the lipid A headgroups of the lipooligosaccharide (LOS) by the LOS phosphoethanolamine (PEA) transferase (LptA) in Neisseria spp. is central for resistance to polymyxin. The structure of the globular domain of LptA shows that the protein has five disulphide bonds, indicating that it is a potential substrate of the protein oxidation pathway in the bacterial periplasm. When neisserial LptA was expressed in Escherichia coli in the presence of the oxidoreductase, EcDsbA, polymyxin resistance increased 30-fold. LptA decorated one position of the E. coli lipid A headgroups with PEA. In the absence of the EcDsbA, LptA was degraded in E. coli. Neisseria spp. express three oxidoreductases, DsbA1, DsbA2 and DsbA3, each of which appear to donate disulphide bonds to different targets. Inactivation of each oxidoreductase in N. meningitidis enhanced sensitivity to polymyxin with combinatorial mutants displaying an additive increase in sensitivity to polymyxin, indicating that the oxidoreductases were required for multiple pathways leading to polymyxin resistance. Correlates were sought between polymyxin sensitivity, LptA stability or activity and the presence of each of the neisserial oxidoreductases. Only meningococcal mutants lacking DsbA3 had a measurable decrease in the amount of PEA decoration on lipid A headgroups implying that LptA stability was supported by the presence of DsbA3 but did not require DsbA1/2 even though these oxidoreductases could oxidise the protein. This is the first indication that DsbA3 acts as an oxidoreductase in vivo and that multiple oxidoreductases may be involved in oxidising the one target in N. meningitidis. In conclusion, LptA is stabilised by disulphide bonds within the protein. This effect was more pronounced when neisserial LptA was expressed in E. coli than in N. meningitidis and may reflect that other factors in the neisserial periplasm have a role in LptA stability.

  16. A Transcriptome-proteome Integrated Network Identifies Endoplasmic Reticulum thiol oxidoreductase (ERp57) as a Hub that Mediates Bone Metastasis*

    PubMed Central

    Santana-Codina, Naiara; Carretero, Rafael; Sanz-Pamplona, Rebeca; Cabrera, Teresa; Guney, Emre; Oliva, Baldo; Clezardin, Philippe; Olarte, Omar E.; Loza-Alvarez, Pablo; Méndez-Lucas, Andrés; Perales, Jose Carlos; Sierra, Angels

    2013-01-01

    Bone metastasis is the most common distant relapse in breast cancer. The identification of key proteins involved in the osteotropic phenotype would represent a major step toward the development of new prognostic markers and therapeutic improvements. The aim of this study was to characterize functional phenotypes that favor bone metastasis in human breast cancer. We used the human breast cancer cell line MDA-MB-231 and its osteotropic BO2 subclone to identify crucial proteins in bone metastatic growth. We identified 31 proteins, 15 underexpressed and 16 overexpressed, in BO2 cells compared with parental cells. We employed a network-modeling approach in which these 31 candidate proteins were prioritized with respect to their potential in metastasis formation, based on the topology of the protein-protein interaction network and differential expression. The protein-protein interaction network provided a framework to study the functional relationships between biological molecules by attributing functions to genes whose functions had not been characterized. The combination of expression profiles and protein interactions revealed an endoplasmic reticulum-thiol oxidoreductase, ERp57, functioning as a hub that retained four down-regulated nodes involved in antigen presentation associated with the human major histocompatibility complex class I molecules, including HLA-A, HLA-B, HLA-E, and HLA-F. Further analysis of the interaction network revealed an inverse correlation between ERp57 and vimentin, which influences cytoskeleton reorganization. Moreover, knockdown of ERp57 in BO2 cells confirmed its bone organ-specific prometastatic role. Altogether, ERp57 appears as a multifunctional chaperone that can regulate diverse biological processes to maintain the homeostasis of breast cancer cells and promote the development of bone metastasis. PMID:23625662

  17. Tenebrionid secretions and a fungal benzoquinone oxidoreductase form competing components of an arms race between a host and pathogen

    PubMed Central

    Pedrini, Nicolás; Ortiz-Urquiza, Almudena; Huarte-Bonnet, Carla; Fan, Yanhua; Juárez, M. Patricia; Keyhani, Nemat O.

    2015-01-01

    Entomopathogenic fungi and their insect hosts represent a model system for examining invertebrate-pathogen coevolutionary selection processes. Here we report the characterization of competing components of an arms race consisting of insect protective antimicrobial compounds and evolving fungal mechanisms of detoxification. The insect pathogenic fungus Beauveria bassiana has a remarkably wide host range; however, some insects are resistant to fungal infection. Among resistant insects is the tenebrionid beetle Tribolium castaneum that produces benzoquinone-containing defensive secretions. Reduced fungal germination and growth was seen in media containing T. castaneum dichloromethane extracts or synthetic benzoquinone. In response to benzoquinone exposure, the fungus expresses a 1,4-benzoquinone oxidoreductase, BbbqrA, induced >40-fold. Gene knockout mutants (ΔBbbqrA) showed increased growth inhibition, whereas B. bassiana overexpressing BbbqrA (Bb::BbbqrAO) displayed increased resistance to benzoquinone compared with wild type. Increased benzoquinone reductase activity was detected in wild-type cells exposed to benzoquinone and in the overexpression strain. Heterologous expression and purification of BbBqrA in Escherichia coli confirmed NAD(P)H-dependent benzoquinone reductase activity. The ΔBbbqrA strain showed decreased virulence toward T. castaneum, whereas overexpression of BbbqrA increased mortality versus T. castaneum. No change in virulence was seen for the ΔBbbqrA or Bb::BbbqrAO strains when tested against the greater wax moth Galleria mellonella or the beetle Sitophilus oryzae, neither of which produce significant amounts of cuticular quinones. The observation that artificial overexpression of BbbqrA results in increased virulence only toward quinone-secreting insects implies the lack of strong selection or current failure of B. bassiana to counteradapt to this particular host defense throughout evolution. PMID:26056261

  18. Interplay Between the Oxidoreductase PDIA6 and microRNA-322 Controls the Response to Disrupted Endoplasmic Reticulum Calcium Homeostasis

    PubMed Central

    Groenendyk, Jody; Peng, Zhenling; Dudek, Elzbieta; Fan, Xiao; Mizianty, Marcin J.; Dufey, Estefanie; Urra, Hery; Sepulveda, Denisse; Rojas-Rivera, Diego; Lim, Yunki; Kim, Do Han; Baretta, Kayla; Srikanth, Sonal; Gwack, Yousang; Ahnn, Joohong; Kaufman, Randal J.; Lee, Sun-Kyung; Hetz, Claudio; Kurgan, Lukasz; Michalak, Marek

    2016-01-01

    The disruption of the energy or nutrient balance triggers endoplasmic reticulum (ER) stress, a process that mobilizes various strategies, collectively called the unfolded protein response (UPR), which reestablish homeostasis of the ER and cell. Activation of the UPR stress sensor IRE1α (inositol-requiring enzyme 1α) stimulates its endoribonuclease activity, leading to the generation of the mRNA encoding the transcription factor XBP1 (X-box binding protein 1), which regulates the transcription of genes encoding factors involved in controlling the quality and folding of proteins. We found that the activity of IRE1α was regulated by the ER oxidoreductase PDIA6 (protein disulfide isomerase A6) and the microRNA miR-322 in response to disruption of ER Ca2+ homeostasis. PDIA6 interacted with IRE1α and enhanced IRE1α activity as monitored by phosphorylation of IRE1α and XBP1 mRNA splicing, but PDIA6 did not substantially affect the activity of other pathways that mediate responses to ER stress. ER Ca2+ depletion and activation of store operated Ca2+ entry reduced the abundance of the microRNA miR-322, which increased PDIA6 mRNA stability and consequently IRE1α activity during the ER stress response. In vivo experiments with mice and worms showed that the induction of ER stress correlated with decreased miR-322 abundance, increased PDIA6 mRNA abundance, or both. Together these findings demonstrated that ER Ca2+, PDIA6, IRE1α, and miR-322 function in a dynamic feedback loop modulating the UPR under conditions of disrupted ER Ca2+ homeostasis. PMID:24917591

  19. Legionella pneumophila utilizes a single-player disulfide-bond oxidoreductase system to manage disulfide bond formation and isomerization.

    PubMed

    Kpadeh, Zegbeh Z; Day, Shandra R; Mills, Brandy W; Hoffman, Paul S

    2015-03-01

    Legionella pneumophila uses a single homodimeric disulfide bond (DSB) oxidoreductase DsbA2 to catalyze extracytoplasmic protein folding and to correct DSB errors through protein-disulfide isomerase (PDI) activity. In Escherichia coli, these functions are separated to avoid futile cycling. In L. pneumophila, DsbA2 is maintained as a mixture of disulfides (S-S) and free thiols (SH), but when expressed in E. coli, only the SH form is observed. We provide evidence to suggest that structural differences in DsbB oxidases (LpDsbB1 and LpDsbB2) and DsbD reductases (LpDsbD1 and LpDsbD2) (compared with E. coli) permit bifunctional activities without creating a futile cycle. LpdsbB1 and LpdsbB2 partially complemented an EcdsbB mutant while neither LpdsbD1 nor LpdsbD2 complemented an EcdsbD mutant unless DsbA2 was also expressed. When the dsb genes of E. coli were replaced with those of L. pneumophila, motility was restored and DsbA2 was present as a mixture of redox forms. A dominant-negative approach to interfere with DsbA2 function in L. pneumophila determined that DSB oxidase activity was necessary for intracellular multiplication and assembly/function of the Dot/Icm Type IVb secretion system. Our studies show that a single-player system may escape the futile cycle trap by limiting transfer of reducing equivalents from LpDsbDs to DsbA2.

  20. Selenoprotein T Exerts an Essential Oxidoreductase Activity That Protects Dopaminergic Neurons in Mouse Models of Parkinson's Disease

    PubMed Central

    Boukhzar, Loubna; Hamieh, Abdallah; Cartier, Dorthe; Tanguy, Yannick; Alsharif, Ifat; Castex, Matthieu; Arabo, Arnaud; Hajji, Sana El; Bonnet, Jean-Jacques; Errami, Mohammed; Falluel-Morel, Anthony; Chagraoui, Abdeslam; Lihrmann, Isabelle

    2016-01-01

    Abstract Aims: Oxidative stress is central to the pathogenesis of Parkinson's disease (PD), but the mechanisms involved in the control of this stress in dopaminergic cells are not fully understood. There is increasing evidence that selenoproteins play a central role in the control of redox homeostasis and cell defense, but the precise contribution of members of this family of proteins during the course of neurodegenerative diseases is still elusive. Results: We demonstrated first that selenoprotein T (SelT) whose gene disruption is lethal during embryogenesis, exerts a potent oxidoreductase activity. In the SH-SY5Y cell model of dopaminergic neurons, both silencing and overexpression of SelT affected oxidative stress and cell survival. Treatment with PD-inducing neurotoxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or rotenone triggered SelT expression in the nigrostriatal pathway of wild-type mice, but provoked rapid and severe parkinsonian-like motor defects in conditional brain SelT-deficient mice. This motor impairment was associated with marked oxidative stress and neurodegeneration and decreased tyrosine hydroxylase activity and dopamine levels in the nigrostriatal system. Finally, in PD patients, we report that SelT is tremendously increased in the caudate putamen tissue. Innovation: These results reveal the activity of a novel selenoprotein enzyme that protects dopaminergic neurons against oxidative stress and prevents early and severe movement impairment in animal models of PD. Conclusions: Our findings indicate that selenoproteins such as SelT play a crucial role in the protection of dopaminergic neurons against oxidative stress and cell death, providing insight into the molecular underpinnings of this stress in PD. Antioxid. Redox Signal. 24, 557–574. PMID:26866473

  1. The end of the line: can ferredoxin and ferredoxin NADP(H) oxidoreductase determine the fate of photosynthetic electrons?

    PubMed

    Goss, Tatjana; Hanke, Guy

    2014-01-01

    At the end of the linear photosynthetic electron transfer (PET) chain, the small soluble protein ferredoxin (Fd) transfers electrons to Fd:NADP(H) oxidoreductase (FNR), which can then reduce NADP+ to support C assimilation. In addition to this linear electron flow (LEF), Fd is also thought to mediate electron flow back to the membrane complexes by different cyclic electron flow (CEF) pathways: either antimycin A sensitive, NAD(P)H complex dependent, or through FNR located at the cytochrome b6f complex. Both Fd and FNR are present in higher plant genomes as multiple gene copies, and it is now known that specific Fd iso-proteins can promote CEF. In addition, FNR iso-proteins vary in their ability to dynamically interact with thylakoid membrane complexes, and it has been suggested that this may also play a role in CEF. We will highlight work on the different Fd-isoproteins and FNR-membrane association found in the bundle sheath (BSC) and mesophyll (MC) cell chloroplasts of the C4 plant maize. These two cell types perform predominantly CEF and LEF, and the properties and activities of Fd and FNR in the BSC and MC are therefore specialized for CEF and LEF respectively. A diversity of Fd isoproteins and dynamic FNR location has also been recorded in C3 plants, algae and cyanobacteria. This indicates that the principles learned from the extreme electron transport situations in the BSC and MC of maize might be usefully applied to understanding the dynamic transition between these states in other systems.

  2. The End of the Line: Can Ferredoxin and Ferredoxin NADP(H) Oxidoreductase Determine the Fate of Photosynthetic Electrons?

    PubMed Central

    Goss, Tatjana; Hanke, Guy

    2014-01-01

    At the end of the linear photosynthetic electron transfer (PET) chain, the small soluble protein ferredoxin (Fd) transfers electrons to Fd:NADP(H) oxidoreductase (FNR), which can then reduce NADP+ to support C assimilation. In addition to this linear electron flow (LEF), Fd is also thought to mediate electron flow back to the membrane complexes by different cyclic electron flow (CEF) pathways: either antimycin A sensitive, NAD(P)H complex dependent, or through FNR located at the cytochrome b6f complex. Both Fd and FNR are present in higher plant genomes as multiple gene copies, and it is now known that specific Fd iso-proteins can promote CEF. In addition, FNR iso-proteins vary in their ability to dynamically interact with thylakoid membrane complexes, and it has been suggested that this may also play a role in CEF. We will highlight work on the different Fd-isoproteins and FNR-membrane association found in the bundle sheath (BSC) and mesophyll (MC) cell chloroplasts of the C4 plant maize. These two cell types perform predominantly CEF and LEF, and the properties and activities of Fd and FNR in the BSC and MC are therefore specialized for CEF and LEF respectively. A diversity of Fd isoproteins and dynamic FNR location has also been recorded in C3 plants, algae and cyanobacteria. This indicates that the principles learned from the extreme electron transport situations in the BSC and MC of maize might be usefully applied to understanding the dynamic transition between these states in other systems. PMID:24678667

  3. Chlamydomonas reinhardtii Chloroplasts Contain a Homodimeric Pyruvate:Ferredoxin Oxidoreductase That Functions with FDX11[W][OA

    PubMed Central

    van Lis, Robert; Baffert, Carole; Couté, Yohann; Nitschke, Wolfgang; Atteia, Ariane

    2013-01-01

    Eukaryotic algae have long been known to live in anoxic environments, but interest in their anaerobic energy metabolism has only recently gained momentum, largely due to their utility in biofuel production. Chlamydomonas reinhardtii figures remarkably in this respect, because it efficiently produces hydrogen and its genome harbors many genes for anaerobic metabolic routes. Central to anaerobic energy metabolism in many unicellular eukaryotes (protists) is pyruvate:ferredoxin oxidoreductase (PFO), which decarboxylates pyruvate and forms acetyl-coenzyme A with concomitant reduction of low-potential ferredoxins or flavodoxins. Here, we report the biochemical properties of the homodimeric PFO of C. reinhardtii expressed in Escherichia coli. Electron paramagnetic resonance spectroscopy of the recombinant enzyme (Cr-rPFO) showed three distinct [4Fe-4S] iron-sulfur clusters and a thiamine pyrophosphate radical upon reduction by pyruvate. Purified Cr-rPFO exhibits a specific decarboxylase activity of 12 µmol pyruvate min−1 mg−1 protein using benzyl viologen as electron acceptor. Despite the fact that the enzyme is very oxygen sensitive, it localizes to the chloroplast. Among the six known chloroplast ferredoxins (FDX1–FDX6) in C. reinhardtii, FDX1 and FDX2 were the most efficient electron acceptors from Cr-rPFO, with comparable apparent Km values of approximately 4 µm. As revealed by immunoblotting, anaerobic conditions that lead to the induction of CrPFO did not increase levels of either FDX1 or FDX2. FDX1, being by far the most abundant ferredoxin, is thus likely the partner of PFO in C. reinhardtii. This finding postulates a direct link between CrPFO and hydrogenase and provides new opportunities to better study and engineer hydrogen production in this protist. PMID:23154536

  4. Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu and pyruvate:ferredoxin oxidoreductase of C. perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium-related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by react...

  5. Benzofuran-, benzothiophene-, indazole- and benzisoxazole-quinones: excellent substrates for NAD(P)H:quinone oxidoreductase 1.

    PubMed

    Newsome, Jeffery J; Hassani, Mary; Swann, Elizabeth; Bibby, Jane M; Beall, Howard D; Moody, Christopher J

    2013-06-01

    A series of heterocyclic quinones based on benzofuran, benzothiophene, indazole and benzisoxazole has been synthesized, and evaluated for their ability to function as substrates for recombinant human NAD(P)H:quinone oxidoreductase (NQO1), a two-electron reductase upregulated in tumor cells. Overall, the quinones are excellent substrates for NQO1, approaching the reduction rates observed for menadione.

  6. The tumor suppressor WW domain-containing oxidoreductase modulates cell metabolism

    PubMed Central

    Abu-Remaileh, Muhannad

    2015-01-01

    The WW domain-containing oxidoreductase (WWOX) encodes a tumor suppressor that is frequently altered in cancer. WWOX binds several proteins and thus is postulated to be involved in a variety of cellular processes. Interestingly, Wwox-knockout mice develop normally in utero but succumb to hypoglycemia and other metabolic defects early in life resulting in their death by 3–4 weeks of age. Cumulative evidence has linked WWOX with cellular metabolism including steroid metabolism, high-density lipoprotein cholesterol (HDL-C) metabolism, bone metabolism and, more recently, glucose metabolism. In this review, we discuss these evolving functions for WWOX and how its deletion affects cellular metabolism and neoplastic progression. PMID:25491415

  7. 1,3-Propanediol:NAD+ oxidoreductases of Lactobacillus brevis and Lactobacillus buchneri.

    PubMed

    Veiga-da-Cunha, M; Foster, M A

    1992-06-01

    In the cofermentation of glycerol with a sugar by Lactobacillus brevis and Lactobacillus buchneri, a 1,3-propanediol:NAD+ oxidoreductase provides an additional method of NADH disposal. The enzyme has been purified from both L. brevis B22 and L. buchneri B190 and found to have properties very similar to those reported for the enzyme from Klebsiella pneumoniae. The enzymes required Mn2+ and are probably octamers with a molecular mass of 350 kDa. Although not absolutely specific for 1,3-propanediol when tested as dehydrogenases, the enzymes have less than 10% activity with glycerol, ethanol, and 1,2-propanediol. These properties contrast sharply with those of a protein isolated from another Lactobacillus species (L. reuteri) that ferments glycerol with glucose and previously designated a 1,3-propanediol dehydrogenase.

  8. 1,3-Propanediol:NAD+ oxidoreductases of Lactobacillus brevis and Lactobacillus buchneri.

    PubMed Central

    Veiga-da-Cunha, M; Foster, M A

    1992-01-01

    In the cofermentation of glycerol with a sugar by Lactobacillus brevis and Lactobacillus buchneri, a 1,3-propanediol:NAD+ oxidoreductase provides an additional method of NADH disposal. The enzyme has been purified from both L. brevis B22 and L. buchneri B190 and found to have properties very similar to those reported for the enzyme from Klebsiella pneumoniae. The enzymes required Mn2+ and are probably octamers with a molecular mass of 350 kDa. Although not absolutely specific for 1,3-propanediol when tested as dehydrogenases, the enzymes have less than 10% activity with glycerol, ethanol, and 1,2-propanediol. These properties contrast sharply with those of a protein isolated from another Lactobacillus species (L. reuteri) that ferments glycerol with glucose and previously designated a 1,3-propanediol dehydrogenase. Images PMID:1622279

  9. Induction of D-aldohexoside:cytochrome c oxidoreductase in Agrobacterium tumefaciens.

    PubMed Central

    Nakamura, L K; Tyler, D D

    1977-01-01

    D-Aldohexopyranoside:cytochrome c oxidoreductase (ACO) was strongly induced by cellobiose, alpha-methylglucoside, beta-methylglucoside, kojibiose, and sophorose. Induction was rapid, and ACO was readily detectable within 10 min after addition of cellobiose as inducer. Although not measurable for 30 to 40 min after addition of inducer, once started, the rate of induction with alpha-methylglucoside equaled or even exceeded that obtained with cellobiose. Induction by sucrose, maltose, alpha-alpha-trehalose, melibiose, and lactose was weak. In general, the active ACO inducers were poor glycosidase inducers; the converse also appeared to be true. Although ACO induction was not repressed by D-glucose, it was repressed by succinate, malate, and fumarate. PMID:838689

  10. Crystallization and preliminary crystallographic analysis of the pyruvate-ferredoxin oxidoreductase from Desulfovibrio africanus.

    PubMed

    Pieulle, L; Chabrière, E; Hatchikian, C; Fontecilla-Camps, J C; Charon, M H

    1999-01-01

    For the first time, crystals of a pyruvate-ferredoxin oxidoreductase (PFOR) suitable for X-ray analysis have been obtained. This enzyme catalyzes, in anaerobic organisms, the crucial energy-yielding reaction of pyruvate decarboxylation to acetylCoA. Polyethylene glycol and divalent metal cations have been used to crystallize the PFOR from the sulfate-reducing bacterium Desulfovibrio africanus. Two different orthorhombic (P212121 ) crystal forms have been grown with unit-cell dimensions a = 86.1, b = 146.7, c = 212.5 A and a = 84.8, b = 144.9, c = 203.0 A. Both crystals diffract to 2.3 A resolution using synchrotron radiation.

  11. Structural domains in NADPH: Protochlorophyllide oxidoreductases involved in catalysis and substrate binding. Final report

    SciTech Connect

    Timko, Michael P.

    1999-09-24

    Until recently little direct information was available about specific structural determinants within the light-dependent NADPH: protochlorophyllide oxidoreductases (PORs) required for substrate and cofactor binding, catalytic activity, and thylakoid membrane localization. Based on our previous DOE-funded studies, during the past year we brought to fruition a number of ongoing experiments, initiated several new avenues of investigations, and overall have made considerable progress towards establishing the basic structural parameters governing POR function. Our studies to date have defined residues and domains involved in substrate and cofactor binding and catalysis, and elaborated on the mechanism for membrane localization of POR in developing plastids. Our results and their significance, as well as our work in progress, are detailed.

  12. Pyruvate Oxidoreductases Involved in Glycolytic Anaerobic Metabolism of Polychaetes from the Continental Shelf off Central-South Chile

    NASA Astrophysics Data System (ADS)

    González, R. R.; Quiñones, R. A.

    2000-10-01

    The presence of low oxygen conditions in extensive areas of the continental shelf off central-south Chile has important effects on the biochemical adaptations of the organisms living in this ecosystem. Polychaetes assemblages cohabit on the shelf with an extensively distributed prokaryotic community made up of giant filamentous sulfur bacteria (mainly Thioploca sp.). The aim of this research was to characterize the pyruvate oxidoreductases enzymes involved in the biochemical adaptation of these benthic polychaetes. Nine polychaete species ( Paraprionospio pinnata, Nephtys ferruginea, Glycera americana, Haploscoloplos sp., Lumbrineris composita, Sigambra bassi, Aricidea pigmentata , Cossura chilensis, and Pectinaria chilensis) were assayed for lactic dehydrogenase (LDH), octopine dehydrogenase (OPDH), strombine dehydrogenase (STRDH) and alanopine dehydrogenase (ALPDH). Each species had a characteristic number of the pyruvate oxidoreductases assayed ranging from 4 in Paraprionospio pinnata to 1 in Pectinaria chilensis . The pyruvate saturation curves obtained for the enzymes from all species analysed, except L. composita, suggest that NADH can be oxidized at different rates depending on the amino acid used in the reaction with pyruvate. Our results indicate that organisms having more that one pyruvate oxidoreductase present a greater metabolic capacity to cope with functional and environmental hypoxia because these enzymes would better regulate the pyruvate consumption rate during the transition period. Thus, the dominance of Paraprionospio pinnata in the study area and its worldwide distribution is consistent with its higher number of pyruvate oxidoreductases with different pyruvate consumption rates involved in anaerobic metabolism. Finally, a positive allometric relationship was found between body size and the specific activity of ALPDH, STRDH, and maximum pyruvate oxidoreductase specific activity. This latter result suggests a positive scaling of the specific

  13. Molybdenum(VI) salts convert the xanthine oxidoreductase apoprotein into the active enzyme in mouse L929 fibroblastic cells.

    PubMed Central

    Falciani, F; Terao, M; Goldwurm, S; Ronchi, A; Gatti, A; Minoia, C; Li Calzi, M; Salmona, M; Cazzaniga, G; Garattini, E

    1994-01-01

    The mouse L929 fibroblastic cell line presents low, but detectable, levels of the mRNA encoding xanthine oxidoreductase under basal conditions, and it responds to type I and type II interferons by inducing the expression of the transcript [Falciani, Ghezzi, Terao, Cazzaniga, and Garattini (1992) Biochem. J. 285, 1001-1008]. This cell line, however, does not show any detectable amount of xanthine oxidoreductase enzymic activity, either before or after treatment with the cytokines. Molybdenum(VI) salts, in the millimolar range, are capable of activating xanthine oxidoreductase in L929 cells both under basal conditions and after treatment with interferon-alpha. The increase is observed in mouse L929 as well as in clones derived from it, but not in many other human and mouse cell lines. The induction observed in L929 cells is post-translational in nature and it is insensitive to cycloheximide, indicating that the molybdenum ion converts a pool of inactive xanthine oxidoreductase apoenzyme into its holoenzymic form. When grown in the absence of sodium molybdate, the L929 cell line has undetectable intracellular levels of the molybdenum cofactor, since the cell extracts are unable to complement the nitrate reductase defect of the nit-1 mutant of Neurospora crassa. L929 cells grown in the presence of millimolar concentrations of sodium molybdate, however, become competent to complement the nit-1 defect. L929 cells accumulate molybdenum ion inside the intracellular compartment as efficiently as TEnd cells, a mouse endothelial cell line that expresses xanthine oxidoreductase activity both under basal conditions and after treatment with interferon-gamma, suggesting that L929 cells have a defect in one or more of the metabolic steps leading to the synthesis of the molybdenum cofactor. Images Figure 1 Figure 2 Figure 4 PMID:8129733

  14. A novel aldose-aldose oxidoreductase for co-production of D-xylonate and xylitol from D-xylose with Saccharomyces cerevisiae.

    PubMed

    Wiebe, Marilyn G; Nygård, Yvonne; Oja, Merja; Andberg, Martina; Ruohonen, Laura; Koivula, Anu; Penttilä, Merja; Toivari, Mervi

    2015-11-01

    An open reading frame CC1225 from the Caulobacter crescentus CB15 genome sequence belongs to the Gfo/Idh/MocA protein family and has 47 % amino acid sequence identity with the glucose-fructose oxidoreductase from Zymomonas mobilis (Zm GFOR). We expressed the ORF CC1225 in the yeast Saccharomyces cerevisiae and used a yeast strain expressing the gene coding for Zm GFOR as a reference. Cell extracts of strains overexpressing CC1225 (renamed as Cc aaor) showed some Zm GFOR type of activity, producing D-gluconate and D-sorbitol when a mixture of D-glucose and D-fructose was used as substrate. However, the activity in Cc aaor expressing strain was >100-fold lower compared to strains expressing Zm gfor. Interestingly, C. crescentus AAOR was clearly more efficient than the Zm GFOR in converting in vitro a single sugar substrate D-xylose (10 mM) to xylitol without an added cofactor, whereas this type of activity was very low with Zm GFOR. Furthermore, when cultured in the presence of D-xylose, the S. cerevisiae strain expressing Cc aaor produced nearly equal concentrations of D-xylonate and xylitol (12.5 g D-xylonate l(-1) and 11.5 g D-xylitol l(-1) from 26 g D-xylose l(-1)), whereas the control strain and strain expressing Zm gfor produced only D-xylitol (5 g l(-1)). Deletion of the gene encoding the major aldose reductase, Gre3p, did not affect xylitol production in the strain expressing Cc aaor, but decreased xylitol production in the strain expressing Zm gfor. In addition, expression of Cc aaor together with the D-xylonolactone lactonase encoding the gene xylC from C. crescentus slightly increased the final concentration and initial volumetric production rate of both D-xylonate and D-xylitol. These results suggest that C. crescentus AAOR is a novel type of oxidoreductase able to convert the single aldose substrate D-xylose to both its oxidized and reduced product.

  15. Expression of 1,3-propanediol oxidoreductase and its isoenzyme in Klebsiella pneumoniae for bioconversion of glycerol into 1,3-propanediol.

    PubMed

    Zhuge, Bin; Zhang, Cheng; Fang, Huiying; Zhuge, Jian; Permaul, Kugen

    2010-08-01

    In the Klebsiella pneumoniae reduction pathway for 1,3-propanediol (1,3-PD) synthesis, glycerol is first dehydrated to 3-hydroxypropionaldehyde (3-HPA) and then reduced to 1,3-PD with NADH consumption. Rapid conversion of 3-HPA to 1,3-PD is one of the ways to improve the yield of 1,3-PD from glycerol and to avoid 3-HPA accumulation, which depends on enzyme activity of the reaction and the amount of reducing equivalents available from the oxidative pathway of glycerol. In the present study, the yqhD gene, encoding 3-propanediol oxidoreductase isoenzyme from Escherichia coli and the dhaT gene, encoding 3-propanediol oxidoreductase from K. pneumoniae were expressed individually and co-expressed in K. pneumoniae using the double tac promoter expression plasmid pEtac-dhaT-tac-yqhD. The three resultant recombinant strains (K. pneumoniae/pEtac-yqhD, K. pneumoniae/pEtac-dhaT, and K. pneumoniae/pEtac-dhaT-tac-yqhD) were used for fermentation studies. Experimental results showed that the peak values for 3-HPA production in broth of the three recombinant strains were less than 25% of that of the parent strain. Expression of dhaT reduced formation of by-products (ethanol and lactic acid) and increased molar yield of 1,3-PD slightly, while expression of yqhD did not enhance molar yield of 1,3-PD, but increased ethanol concentration in broth as NADPH participation in transforming 3-HPA to 1,3-PD allowed more cellular NADH to be used to produce ethanol. Co-expression of both genes therefore decreased by-products and increased the molar yield of 1,3-PD by 11.8%, by catalyzing 3-HPA conversion to 1,3-propanediol using two cofactors (NADH and NADPH). These results have important implications for further studies involving use of YqhD and DhaT for bioconversion of glycerol into 1,3-PD.

  16. Crystal structures of archaeal 2-oxoacid:ferredoxin oxidoreductases from Sulfolobus tokodaii

    PubMed Central

    Yan, Zhen; Maruyama, Akane; Arakawa, Takatoshi; Fushinobu, Shinya; Wakagi, Takayoshi

    2016-01-01

    As the first three-dimensional structure of the two-subunit type 2-oxoacid:ferredoxin oxidoreductases (OFOR) from archaea, we solved the crystal structures of STK_23000/STK_22980 (StOFOR1) and STK_24350/STK_24330 (StOFOR2) from Sulfolobus tokodaii. They showed similar overall structures, consisting of two a- and b-subunit heterodimers containing thiamin pyrophosphate (TPP) cofactor and [4Fe-4S] cluster, but lack an intramolecular ferredoxin domain. Unlike other OFORs, StOFORs can utilize both pyruvate and 2-oxoglutarate, playing a key role in the central metabolism. In the structure of StOFOR2 in unreacted pyruvate complex form, carboxylate group of pyruvate is recognized by Arg344 and Thr257 from the a-subunit, which are conserved in pyruvate:ferredoxin oxidoreductase from Desulfovbrio africanus (DaPFOR). In the structure of StOFOR1 co-crystallized with 2-oxobutyrate, electron density corresponding to a 1-hydroxypropyl group (post-decarboxylation state) was observed at the thiazole ring of TPP. The binding pockets of the StOFORs surrounding the methyl or propyl group of the ligands are wider than that of DaPFOR. Mutational analyses indicated that several residues were responsible for the broad 2-oxoacid specificity of StOFORs. We also constructed a possible complex structural model by placing a Zn2+-containing dicluster ferredoxin of S. tokodaii into the large pocket of StOFOR2, providing insight into the electron transfer between the two redox proteins. PMID:27619895

  17. (The interaction of ferredoxin:NADP sup + oxidoreductase and ferredoxin:thioredoxin reductase with substrates)

    SciTech Connect

    Not Available

    1992-01-01

    We seek to map the ferredoxin-binding sites on three soluble enzymes located in spinach chloroplasts which utilize ferredoxin as an electron donor:Ferredoxin:NADP{sup +}oxidoreductase (FNR); ferredoxin:thioredoxin reductase (FTR) and glutamate synthase. As the availability of amino acid sequences for the enzymes are important in such studies, that the amino acid sequence of glutamate synthase needs be determined, the amino acid sequences of FNR, FTR and ferredoxin are already known. Related to an aim elucidate the binding sites for ferredoxin to determine whether there is a common binding site on all of these ferredoxin-dependent chloroplast enzymes and, if so, to map it. Additionally thioredoxin binding by FTR needs be determine to resolve whether the same site on FTR is involved in binding both ferredoxin and thioredoxin. Considerable progress is reported on the prosthetic groups of glutamate synthase, in establishing the role of arginine and lysine residues in ferredoxin binding by, ferredoxin:nitrite oxidoreductase nitrite reductase, labelling carboxyl groups on ferredoxin with taurine and labelling lysine residues biotinylation, and low potential heme proteins have been isolated and characterized from a non-photosynthetic plant tissue. Although the monoclonal antibodies raised against FNR turned out not to be useful for mapping the FNR/ferredoxin or FNR/NADPinteraction domains, good progress has been made on mapping the FNR/ferredoxin interaction domains by an alternative technique. The techniques developed for differential chemical modification of these two proteins - taurine modification of aspartate and glutamate residues and biotin modification of lysine residues - should be useful for mapping the interaction domains of many proteins that associate through electrostatic interactions.

  18. Regulation of gap junction function and Connexin 43 expression by cytochrome P450 oxidoreductase (CYPOR)

    SciTech Connect

    Polusani, Srikanth R.; Kar, Rekha; Riquelme, Manuel A.; Masters, Bettie Sue; Panda, Satya P.

    2011-08-05

    Highlights: {yields} Humans with severe forms of cytochrome P450 oxidoreductase (CYPOR) mutations show bone defects as observed in Antley-Bixler Syndrome. {yields} First report showing knockdown of CYPOR in osteoblasts decreased Connexin 43 (Cx43) protein levels. Cx43 is known to play an important role in bone modeling. {yields} Knockdown of CYPOR decreased Gap Junctional Intercellular Communication and hemichannel activity. {yields} Knockdown of CYPOR decreased Cx43 in mouse primary calvarial osteoblasts. {yields} Decreased Cx43 expression was observed at the transcriptional level. -- Abstract: Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron-transferring enzyme containing both FAD and FMN as co-factors, which provides the reducing equivalents to various redox partners, such as cytochromes P450 (CYPs), heme oxygenase (HO), cytochrome b{sub 5} and squalene monooxygenase. Human patients with severe forms of CYPOR mutation show bone defects such as cranio- and humeroradial synostoses and long bone fractures, known as Antley-Bixler-like Syndrome (ABS). To elucidate the role of CYPOR in bone, we knocked-down CYPOR in multiple osteoblast cell lines using RNAi technology. In this study, knock-down of CYPOR decreased the expression of Connexin 43 (Cx43), known to play a critical role in bone formation, modeling, and remodeling. Knock-down of CYPOR also decreased Gap Junction Intercellular Communication (GJIC) and hemichannel activity. Promoter luciferase assays revealed that the decrease in expression of Cx43 in CYPOR knock-down cells was due to transcriptional repression. Primary osteoblasts isolated from bone specific Por knock-down mice calvariae confirmed the findings in the cell lines. Taken together, our study provides novel insights into the regulation of gap junction function by CYPOR and suggests that Cx43 may play an important role(s) in CYPOR-mediated bone defects seen in patients.

  19. Prenatal Diagnosis of Congenital Adrenal Hyperplasia Caused by P450 Oxidoreductase Deficiency

    PubMed Central

    Reisch, Nicole; Idkowiak, Jan; Hughes, Beverly A.; Ivison, Hannah E.; Abdul-Rahman, Omar A.; Hendon, Laura G.; Olney, Ann Haskins; Nielsen, Shelly; Harrison, Rachel; Blair, Edward M.; Dhir, Vivek; Krone, Nils; Shackleton, Cedric H. L.

    2013-01-01

    Context: Mutations in the electron donor enzyme P450 oxidoreductase (POR) result in congenital adrenal hyperplasia with apparent combined 17α-hydroxylase/17,20 lyase and 21-hydroxylase deficiencies, also termed P450 oxidoreductase deficiency (PORD). Major clinical features present in PORD are disordered sex development in affected individuals of both sexes, glucocorticoid deficiency, and multiple skeletal malformations. Objective: The objective of the study was to establish a noninvasive approach to prenatal diagnosis of PORD including assessment of malformation severity to facilitate optimized prenatal diagnosis and timely treatment. Design: We analyzed 20 pregnancies with children homozygous or compound heterozygous for disease-causing POR mutations and 1 pregnancy with a child carrying a heterozygous POR mutation by recording clinical and biochemical presentations and fetal ultrasound findings. In 4 of the pregnancies (3 homozygous and 1 heterozygous for disease-causing POR mutations), prenatal analysis of steroid metabolite excretion in maternal urine was carried out by gas chromatography/mass spectrometry during gestational weeks 11–23. Results: Pregnancy complications in our cohort included maternal virilization (6 of 20) with onset in the second trimester. Seven pregnant women presented with low unconjugated estriol at prenatal screening (triple or quadruple antenatal screening test). Overt dysmorphic features were noted in 19 of the 20 babies at birth but observed in only 5 by prenatal ultrasound. These 5 had the most severe malformation phenotypes and poor outcome, whereas the other babies showed normal development. Steroid profiling of maternal urine revealed significantly increased steroids of fetal origin, namely the pregnenolone metabolite epiallopregnanediol and the androgen metabolite androsterone, with concomitant low values for estriol. Diagnostic steroid ratios conclusively indicated PORD as early as gestational week 12. In the heterozygous

  20. Genotype-Phenotype Analysis in Congenital Adrenal Hyperplasia due to P450 Oxidoreductase Deficiency

    PubMed Central

    Krone, Nils; Reisch, Nicole; Idkowiak, Jan; Dhir, Vivek; Ivison, Hannah E.; Hughes, Beverly A.; Rose, Ian T.; O'Neil, Donna M.; Vijzelaar, Raymon; Smith, Matthew J.; MacDonald, Fiona; Cole, Trevor R.; Adolphs, Nicolai; Barton, John S.; Blair, Edward M.; Braddock, Stephen R.; Collins, Felicity; Cragun, Deborah L.; Dattani, Mehul T.; Day, Ruth; Dougan, Shelley; Feist, Miriam; Gottschalk, Michael E.; Gregory, John W.; Haim, Michaela; Harrison, Rachel; Haskins Olney, Ann; Hauffa, Berthold P.; Hindmarsh, Peter C.; Hopkin, Robert J.; Jira, Petr E.; Kempers, Marlies; Kerstens, Michiel N.; Khalifa, Mohamed M.; Köhler, Birgit; Maiter, Dominique; Nielsen, Shelly; O'Riordan, Stephen M.; Roth, Christian L.; Shane, Kate P.; Silink, Martin; Stikkelbroeck, Nike M. M. L.; Sweeney, Elizabeth; Szarras-Czapnik, Maria; Waterson, John R.; Williamson, Lori; Hartmann, Michaela F.; Taylor, Norman F.; Wudy, Stefan A.; Malunowicz, Ewa M.; Shackleton, Cedric H. L.

    2012-01-01

    Context: P450 oxidoreductase deficiency (PORD) is a unique congenital adrenal hyperplasia variant that manifests with glucocorticoid deficiency, disordered sex development (DSD), and skeletal malformations. No comprehensive data on genotype-phenotype correlations in Caucasian patients are available. Objective: The objective of the study was to establish genotype-phenotype correlations in a large PORD cohort. Design: The design of the study was the clinical, biochemical, and genetic assessment including multiplex ligation-dependent probe amplification (MLPA) in 30 PORD patients from 11 countries. Results: We identified 23 P450 oxidoreductase (POR) mutations (14 novel) including an exonic deletion and a partial duplication detected by MLPA. Only 22% of unrelated patients carried homozygous POR mutations. p.A287P was the most common mutation (43% of unrelated alleles); no other hot spot was identified. Urinary steroid profiling showed characteristic PORD metabolomes with variable impairment of 17α-hydroxylase and 21-hydroxylase. Short cosyntropin testing revealed adrenal insufficiency in 89%. DSD was present in 15 of 18 46,XX and seven of 12 46,XY individuals. Homozygosity for p.A287P was invariably associated with 46,XX DSD but normal genitalia in 46,XY individuals. The majority of patients with mild to moderate skeletal malformations, assessed by a novel scoring system, were compound heterozygous for missense mutations, whereas nearly all patients with severe malformations carried a major loss-of-function defect on one of the affected alleles. Conclusions: We report clinical, biochemical, and genetic findings in a large PORD cohort and show that MLPA is a useful addition to POR mutation analysis. Homozygosity for the most frequent mutation in Caucasians, p.A287P, allows for prediction of genital phenotype and moderate malformations. Adrenal insufficiency is frequent, easily overlooked, but readily detected by cosyntropin testing. PMID:22162478

  1. Differential regulation of duplicate light-dependent protochlorophyllide oxidoreductases in the diatom Phaeodactylum tricornutum

    SciTech Connect

    Hunsperger, Heather M.; Ford, Christopher J.; Miller, James S.; Cattolico, Rose Ann; Ianora, Adrianna

    2016-07-01

    Diatoms (Bacilliariophyceae) encode two light-dependent protochlorophyllide oxidoreductases (POR1 and POR2) that catalyze the penultimate step of chlorophyll biosynthesis in the light. Algae live in dynamic environments whose changing light levels induce photoacclimative metabolic shifts, including altered cellular chlorophyll levels. We hypothesized that the two POR proteins may be differentially adaptive under varying light conditions. Using the diatom Phaeodactylum tricornutum as a test system, differences in POR protein abundance and por gene expression were examined when this organism was grown on an alternating light:dark cycles at different irradiances; exposed to continuous light; and challenged by a significant decrease in light availability. As a result, for cultures maintained on a 12h light: 12h dark photoperiod at 200μEm–2 s–1 (200L/D), both por genes were up-regulated during the light and down-regulated in the dark, though por1 transcript abundance rose and fell earlier than that of por2. Little concordance occurred between por1 mRNA and POR1 protein abundance. In contrast, por2 mRNA and POR2 protein abundances followed similar diurnal patterns. When 200L/D P. tricornutum cultures were transferred to continuous light (200L/L), the diurnal regulatory pattern of por1 mRNA abundance but not of por2 was disrupted, and POR1 but not POR2 protein abundance dropped steeply. Under 1200μEm–2 s–1 (1200L/D), both por1 mRNA and POR1 protein abundance displayed diurnal oscillations. A compromised diel por2 mRNA response under 1200L/D did not impact the oscillation in POR2 abundance. When cells grown at 1200L/D were then shifted to 50μEm–2 s–1 (50L/D), por1 and por2 mRNA levels decreased swiftly but briefly upon light reduction. Thereafter, POR1 but not POR2 protein levels rose significantly in

  2. 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.

  3. Functional and phylogenetic analysis of the Aspergillus ochraceoroseus aflQ (ordA) gene ortholog

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Within the Aspergillus parasiticus and A. flavus aflatoxin (AF) biosynthetic gene cluster the aflQ (ordA) and aflP (omtA) genes encode an oxidoreductase and methyltransferase, respectively. These genes are required for the final steps in the conversion of sterigmatocystin (ST) to aflatoxin B1 (AFB1...

  4. Hydroxylamine oxidation in heterotrophic nitrate-reducing soil bacteria and purification of a hydroxylamine-cytochrome c oxidoreductase from a Pseudomonas species.

    PubMed

    Wehrfritz, J; Carter, J P; Spiro, S; Richardson, D J

    1996-12-01

    Hydroxylamine oxidation was measured in four recently isolated heterotrophic nitrate-reducing bacteria belonging to the genera Pseudomonas, Moraxella, Arthrobacter and Aeromonas. A hydroxylamine-cytochrome c oxidoreductase activity was detected in periplasmic fractions of the Pseudomonas and Aeromonas spp. and in total soluble fractions of the Arthrobacter sp. A monomeric 19-kDa non-haem iron hydroxylamine-cytochrome c oxidoreductase was purified from the Pseudomonas species and shown to be similar to hydroxylamine-cytochrome c oxidoreductase of Paracoccus denitrificans.

  5. Biochemical characterization of NfsA, the Escherichia coli major nitroreductase exhibiting a high amino acid sequence homology to Frp, a Vibrio harveyi flavin oxidoreductase.

    PubMed Central

    Zenno, S; Koike, H; Kumar, A N; Jayaraman, R; Tanokura, M; Saigo, K

    1996-01-01

    We identified the nfsA gene, encoding the major oxygen-insensitive nitroreductase in Escherichia coli, and determined its position on the E. coli map to be 19 min. We also purified its gene product, NfsA, to homogeneity. It was suggested that NfsA is a nonglobular protein with a molecular weight of 26,799 and is associated tightly with a flavin mononucleotide. Its amino acid sequence is highly similar to that of Frp, a flavin oxidoreductase from Vibrio harveyi (B. Lei, M. Liu, S. Huang, and S.-C. Tu, J. Bacteriol. 176:3552-3558, 1994), an observation supporting the notion that E. coli nitroreductase and luminescent-bacterium flavin reductase families are intimately related in evolution. Although no appreciable sequence similarity was detected between two E. coli nitroreductases, NfsA and NfsB, NfsA exhibited a low level of the flavin reductase activity and a broad electron acceptor specificity similar to those of NfsB. NfsA reduced nitrofurazone by a ping-pong Bi-Bi mechanism possibly to generate a two-electron transfer product. PMID:8755878

  6. Potential plant growth-promoting strain Bacillus sp. SR-2-1/1 decolorized azo dyes through NADH-ubiquinone:oxidoreductase activity.

    PubMed

    Mahmood, Faisal; Shahid, Muhammad; Hussain, Sabir; Shahzad, Tanvir; Tahir, Muhammad; Ijaz, Muhammad; Hussain, Athar; Mahmood, Khalid; Imran, Muhammad; Babar, Shahid Ali Khan

    2017-03-22

    In this study, a bacterial strain SR-2-1/1 was isolated from textile wastewater-irrigated soil for its concurrent potential of plant growth promotion and azo-dye decolorization. Analysis of 16S rRNA gene sequence confirmed its identity as Bacillus sp. The strain tolerated high concentrations (i.e. up to 1000mgL(-1)) of metals (Ni(2+), Cd(2+), Co(2+), Zn(2+), and Cr(6+)) and efficiently decolorized the azo dyes (i.e. reactive black-5, reactive red-120, direct blue-1 and congo red). It also demonstrated considerable in vitro phosphate solubilizing and 1-aminocyclopropane-1-carboxylic acid deaminase abilities at high metal and salt levels. Bioinformatics analysis of its 537bp azoreductase gene and deduced protein revealed that it decolorized azo dyes through NADH-ubiquinone:oxidoreductase enzyme activity. The deduced protein was predicted structurally and functionally different to those of its closely related database proteins. Thus, the strain SR-2-1/1 is a powerful bioinoculant for bioremediation of textile wastewater contaminated soils in addition to stimulation of plant growth.

  7. Correlation between NAD(P)H: quinone oxidoreductase 1 C609T polymorphism and increased risk of esophageal cancer: evidence from a meta-analysis

    PubMed Central

    Diao, Jingfang; Bao, Jie; Peng, Jianxin; Mo, Jiaqiang; Ye, Qing; He, Junming

    2016-01-01

    NAD(P)H: quinone oxidoreductase 1 (NQO1) C609T gene polymorphisms have been reported to influence the risk for esophageal cancer (EC) in many studies. However, the results remain controversial and ambiguous. We performed a meta-analysis, which included 13 independent studies with a total of 2357 subjects, to examine the association between NQO1 C609T polymorphism and EC. The association was assessed by five different gene models. The overall analysis suggested that the variant allele and genotypes were significantly related to increased risk of EC (odds ratio [OR] T versus C = 1.15, 95% confidence interval [CI] 0.95–1.40, probability of rejection [POR] = 0.014; OR TT versus CC = 1.32, 95% CI 1.01–1.73, POR = 0.045; OR TC versus CC = 1.32, 95% CI 0.98–1.21, POR = 0.128; OR TT + TC versus CC = 1.10, 95% CI 1.00–1.20, POR = 0.05; OR TT versus CC + TC = 1.26, 95% CI 0.95–1.57, POR = 0.103). Sensitivity analysis confirmed the reliability of these findings. Our study shows that individuals carrying the NQO1 C609T variant allele and genotypes are more susceptible to EC. PMID:28203294

  8. Ferredoxin:NAD+ oxidoreductase of Thermoanaerobacterium saccharolyticum and its role in ethanol formation [Identification of a ferredoxin:NAD+ oxidoreductase of Thermoanaerobacterium saccharolyticum and its role in ethanol formation

    SciTech Connect

    Tian, Liang; Lo, Jonathan; Shao, Xiongjun; Zheng, Tianyong; Olson, Daniel G.; Lynd, Lee R.

    2016-09-30

    Ferredoxin:NAD+ oxidoreductase (NADH-FNOR) catalyzes the transfer of electrons from reduced ferredoxin to NAD+. This enzyme has been hypothesized to be the main enzyme responsible for ferredoxin oxidization in the NADH-based ethanol pathway in Thermoanaerobacterium saccharolyticum; however, the corresponding gene has not yet been identified. Here, we identified the Tsac_1705 protein as a candidate FNOR based on the homology of its functional domains. We then confirmed its activity in vitro with a ferredoxin-based FNOR assay. To determine its role in metabolism, the tsac_1705 gene was deleted in different strains of T. saccharolyticum. In wild-type T. saccharolyticum, deletion of tsac_1705 resulted in a 75% loss of NADH-FNOR activity, which indicated that Tsac_1705 is the main NADH-FNOR in T. saccharolyticum. When both NADH- and NADPH-linked FNOR genes were deleted, the ethanol titer decreased and the ratio of ethanol to acetate approached unity, indicative of the absence of FNOR activity. As a result, we tested the effect of heterologous expression of Tsac_1705 in Clostridium thermocellum and found improvements in both the titer and the yield of ethanol.

  9. Over-expression of a putative oxidoreductase (UcpA) for increasing furfural or 5-hydroxymethylfurfural tolerance

    DOEpatents

    Wang, Xuan; Miller, Elliot N.; Yomano, Lorraine P.; Shanmugam, Keelnatham T.; Ingram, Lonnie O'Neal

    2016-05-24

    The subject invention pertains to overexpression of a putative oxidoreductase (ucpA) for increasing furfural tolerance in genetically modified microorganisms. Genetically modified microorganisms capable of overexpressing UcpA are also provided. Increased expression of ucpA was shown to increase furfural tolerance by 50%, and to permit the fermentation of sugars to products in the presence of 15 mM furfural.

  10. Pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon, Pyrococcus furiosus, functions as a CoA-dependent pyruvate decarboxylase.

    PubMed

    Ma, K; Hutchins, A; Sung, S J; Adams, M W

    1997-09-02

    Pyruvate ferredoxin oxidoreductase (POR) has been previously purified from the hyperthermophilic archaeon, Pyrococcus furiosus, an organism that grows optimally at 100 degrees C by fermenting carbohydrates and peptides. The enzyme contains thiamine pyrophosphate and catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2 and reduces P. furiosus ferredoxin. Here we show that this enzyme also catalyzes the formation of acetaldehyde from pyruvate in a CoA-dependent reaction. Desulfocoenzyme A substituted for CoA showing that the cofactor plays a structural rather than a catalytic role. Ferredoxin was not necessary for the pyruvate decarboxylase activity of POR, nor did it inhibit acetaldehyde production. The apparent Km values for CoA and pyruvate were 0.11 mM and 1.1 mM, respectively, and the optimal temperature for acetaldehyde formation was above 90 degrees C. These data are comparable to those previously determined for the pyruvate oxidation reaction of POR. At 80 degrees C (pH 8.0), the apparent Vm value for pyruvate decarboxylation was about 40% of the apparent Vm value for pyruvate oxidation rate (using P. furiosus ferredoxin as the electron acceptor). Tentative catalytic mechanisms for these two reactions are presented. In addition to POR, three other 2-keto acid ferredoxin oxidoreductases are involved in peptide fermentation by hyperthermophilic archaea. It is proposed that the various aldehydes produced by these oxidoreductases in vivo are used by two aldehyde-utilizing enzymes, alcohol dehydrogenase and aldehyde ferredoxin oxidoreductase, the physiological roles of which were previously unknown.

  11. Over-expression of NADH-dependent oxidoreductase (fucO) for increasing furfural or 5-hydroxymethylfurfural tolerance

    SciTech Connect

    Miller, Elliot N.; Zhang, Xueli; Yomano, Lorraine P.; Wang, Xuan; Shanmugam, Keelnatham T.; Ingram, Lonnie O'Neal

    2015-10-13

    The subject invention pertains to the discovery that the NADH-dependent propanediol oxidoreductase (FucO) can reduce furfural. This allows for a new approach to improve furfural tolerance in bacterial and/or yeast cells used to produce desired products. Thus, novel biocatalysts (bacterial, fungal or yeast cells) exhibiting increased tolerance to furfural and 5-hydroxymethylfurfural (5-HMF) are provided as are methods of making and using such biocatalysts for the production of a desired product.

  12. Structure and mechanism of a eukaryotic transmembrane ascorbate-dependent oxidoreductase.

    PubMed

    Lu, Peilong; Ma, Dan; Yan, Chuangye; Gong, Xinqi; Du, Mingjian; Shi, Yigong

    2014-02-04

    Vitamin C, also known as ascorbate, is required in numerous essential metabolic reactions in eukaryotes. The eukaryotic ascorbate-dependent oxidoreductase cytochrome b561 (Cyt b561), a family of highly conserved transmembrane enzymes, plays an important role in ascorbate recycling and iron absorption. Although Cyt b561 was identified four decades ago, its atomic structure and functional mechanism remain largely unknown. Here, we report the high-resolution crystal structures of cytochrome b561 from Arabidopsis thaliana in both substrate-free and substrate-bound states. Cyt b561 forms a homodimer, with each protomer consisting of six transmembrane helices and two heme groups. The negatively charged substrate ascorbate, or monodehydroascorbate, is enclosed in a positively charged pocket on either side of the membrane. Two highly conserved amino acids, Lys(81) and His(106), play an essential role in substrate recognition and catalysis. Our structural and biochemical analyses allow the proposition of a general electron transfer mechanism for members of the Cyt b561 family.

  13. Mitochondrial Sulfide Quinone Oxidoreductase Prevents Activation of the Unfolded Protein Response in Hydrogen Sulfide*

    PubMed Central

    Horsman, Joseph W.

    2016-01-01

    Hydrogen sulfide (H2S) is an endogenously produced gaseous molecule with important roles in cellular signaling. In mammals, exogenous H2S improves survival of ischemia/reperfusion. We have previously shown that exposure to H2S increases the lifespan and thermotolerance in Caenorhabditis elegans, and improves protein homeostasis in low oxygen. The mitochondrial SQRD-1 (sulfide quinone oxidoreductase) protein is a highly conserved enzyme involved in H2S metabolism. SQRD-1 is generally considered important to detoxify H2S. Here, we show that SQRD-1 is also required to maintain protein translation in H2S. In sqrd-1 mutant animals, exposure to H2S leads to phosphorylation of eIF2α and inhibition of protein synthesis. In contrast, global protein translation is not altered in wild-type animals exposed to lethally high H2S or in hif-1(ia04) mutants that die when exposed to low H2S. We demonstrate that both gcn-2 and pek-1 kinases are involved in the H2S-induced phosphorylation of eIF2α. Both ER and mitochondrial stress responses are activated in sqrd-1 mutant animals exposed to H2S, but not in wild-type animals. We speculate that SQRD-1 activity in H2S may coordinate proteostasis responses in multiple cellular compartments. PMID:26677221

  14. Posttranslational Modifications of FERREDOXIN-NADP+ OXIDOREDUCTASE in Arabidopsis Chloroplasts1[W][OPEN

    PubMed Central

    Lehtimäki, Nina; Koskela, Minna M.; Dahlström, Käthe M.; Pakula, Eveliina; Lintala, Minna; Scholz, Martin; Hippler, Michael; Hanke, Guy T.; Rokka, Anne; Battchikova, Natalia; Salminen, Tiina A.; Mulo, Paula

    2014-01-01

    Rapid responses of chloroplast metabolism and adjustments to photosynthetic machinery are of utmost importance for plants’ survival in a fluctuating environment. These changes may be achieved through posttranslational modifications of proteins, which are known to affect the activity, interactions, and localization of proteins. Recent studies have accumulated evidence about the crucial role of a multitude of modifications, including acetylation, methylation, and glycosylation, in the regulation of chloroplast proteins. Both of the Arabidopsis (Arabidopsis thaliana) leaf-type FERREDOXIN-NADP+ OXIDOREDUCTASE (FNR) isoforms, the key enzymes linking the light reactions of photosynthesis to carbon assimilation, exist as two distinct forms with different isoelectric points. We show that both AtFNR isoforms contain multiple alternative amino termini and undergo light-responsive addition of an acetyl group to the α-amino group of the amino-terminal amino acid of proteins, which causes the change in isoelectric point. Both isoforms were also found to contain acetylation of a conserved lysine residue near the active site, while no evidence for in vivo phosphorylation or glycosylation was detected. The dynamic, multilayer regulation of AtFNR exemplifies the complex regulatory network systems controlling chloroplast proteins by a range of posttranslational modifications, which continues to emerge as a novel area within photosynthesis research. PMID:25301888

  15. P450 oxidoreductase deficiency - a new form of congenital adrenal hyperplasia.

    PubMed

    Flück, Christa E; Pandey, Amit V; Huang, Ningwu; Agrawal, Vishal; Miller, Walter L

    2008-01-01

    Patients with adrenal insufficiency, genital anomalies and bony malformations resembling the Antley- Bixler syndrome (a craniosynostosis syndrome), are likely to have P450 oxidoreductase (POR) deficiency. Since our first report in 2004, about 26 recessive POR mutations have been identified in 50 patients. POR is the obligate electron donor to all microsomal (type II) P450 enzymes, including the steroidogenic enzymes CYP17A1, CYP21A2 and CYP19A1. POR deficiency may cause disordered sexual development manifested as genital undervirilization in 46,XY newborns as well as overvirilization in those who are 46,XX. This may be explained by impaired aromatization of fetal androgens which may also lead to maternal virilization and low urinary estriol levels during pregnancy. A role for the alternate 'backdoor' pathway of androgen biosynthesis, leading to dihydrotestosterone production bypassing androstenedione and testosterone, has been suggested in POR deficiency but remains unclear. POR variants may play an important role in drug metabolism, as most drugs are metabolized by hepatic microsomal P450 enzymes. However, functional assays studying the effects of specific POR mutations on steroidogenesis showed that several POR variants impaired CYP17A1, CYP21A2 and CYP19A1 activities to different degrees, indicating that each POR variant must be studied separately for each potential target P450 enzyme. Thus, the impact of POR mutations on drug metabolism by hepatic P450s requires further investigation.

  16. Subcellular localization of ferredoxin-NADP(+) oxidoreductase in phycobilisome retaining oxygenic photosysnthetic organisms.

    PubMed

    Morsy, Fatthy Mohamed; Nakajima, Masato; Yoshida, Takayuki; Fujiwara, Tatsuki; Sakamoto, Toshio; Wada, Keishiro

    2008-01-01

    Ferredoxin-NADP(+) oxidoreductase (FNR) catalyzing the terminal step of the linear photosynthetic electron transport was purified from the cyanobacterium Spirulina platensis and the red alga Cyanidium caldarium. FNR of Spirulina consisted of three domains (CpcD-like domain, FAD-binding domain, and NADP(+)-binding domain) with a molecular mass of 46 kDa and was localized in either phycobilisomes or thylakoid membranes. The membrane-bound FNR with 46 kDa was solublized by NaCl and the solublized FNR had an apparent molecular mass of 90 kDa. FNR of Cyanidium consisted of two domains (FAD-binding domain and NADP(+)-binding domain) with a molecular mass of 33 kDa. In Cyanidium, FNR was found on thylakoid membranes, but there was no FNR on phycobilisomes. The membrane-bound FNR of Cyanidium was not solublized by NaCl, suggesting the enzyme is tightly bound in the membrane. Although both cyanobacteria and red algae are photoautotrophic organisms bearing phycobilisomes as light harvesting complexes, FNR localization and membrane-binding characteristics were different. These results suggest that FNR binding to phycobilisomes is not characteristic for all phycobilisome retaining oxygenic photosynthetic organisms, and that the rhodoplast of red algae had possibly originated from a cyanobacterium ancestor, whose FNR lacked the CpcD-like domain.

  17. On the importance of anion-π interactions in the mechanism of sulfide:quinone oxidoreductase.

    PubMed

    Bauzá, Antonio; Quiñonero, David; Deyà, Pere M; Frontera, Antonio

    2013-11-01

    Sulfide:quinone oxidoreductase (SQR) is a flavin-dependent enzyme that plays a physiological role in two important processes. First, it is responsible for sulfide detoxification by oxidizing sulfide ions (S(2-) and HS(-)) to elementary sulfur and the electrons are first transferred to flavin adenine dinucleotide (FAD), which in turn passes them to the quinone pool in the membrane. Second, in sulfidotrophic bacteria, SQRs play a key role in the sulfide-dependent respiration and anaerobic photosynthesis, deriving energy for their growth from reduced sulfur. Two mechanisms of action for SQR have been proposed: first, nucleophilic attack of a Cys residue on the C4 of FAD, and second, an alternate anionic radical mechanism by direct electron transfer from Cys to the isoalloxazine ring of FAD. Both mechanisms involve a common anionic intermediate that it is stabilized by a relevant anion-π interaction and its previous formation (from HS(-) and Cys-S-S-Cys) is also facilitated by reducing the transition-state barrier, owing to an interaction that involves the π system of FAD. By analyzing the X-ray structures of SQRs available in the Protein Data Bank (PDB) and using DFT calculations, we demonstrate the relevance of the anion-π interaction in the enzymatic mechanism.

  18. Structural and Biochemical Characterization of the Oxidoreductase NmDsbA3 from Neisseria meningitidis

    SciTech Connect

    Vivian, Julian P.; Scoullar, Jessica; Robertson, Amy L.; Bottomley, Stephen P.; Horne, James; Chin, Yanni; Wielens, Jerome; Thompson, Philip E.; Velkov, Tony; Piek, Susannah; Byres, Emma; Beddoe, Travis; Wilce, Matthew C.J.; Kahler, Charlene M.; Rossjohn, Jamie; Scanlon, Martin J.

    2009-09-02

    DsbA is an enzyme found in the periplasm of Gram-negative bacteria that catalyzes the formation of disulfide bonds in a diverse array of protein substrates, many of which are involved in bacterial pathogenesis. Although most bacteria possess only a single essential DsbA, Neisseria meningitidis is unusual in that it possesses three DsbAs, although the reason for this additional redundancy is unclear. Two of these N. meningitidis enzymes (NmDsbA1 and NmDsbA2) play an important role in meningococcal attachment to human epithelial cells, whereas NmDsbA3 is considered to have a narrow substrate repertoire. To begin to address the role of DsbAs in the pathogenesis of N. meningitidis, we have determined the structure of NmDsbA3 to 2.3-{angstrom} resolution. Although the sequence identity between NmDsbA3 and other DsbAs is low, the NmDsbA3 structure adopted a DsbA-like fold. Consistent with this finding, we demonstrated that NmDsbA3 acts as a thiol-disulfide oxidoreductase in vitro and is reoxidized by Escherichia coli DsbB (EcDsbB). However, pronounced differences in the structures between DsbA3 and EcDsbA, which are clustered around the active site of the enzyme, suggested a structural basis for the unusual substrate specificity that is observed for NmDsbA3.

  19. Structure and mechanism of a eukaryotic transmembrane ascorbate-dependent oxidoreductase

    PubMed Central

    Lu, Peilong; Ma, Dan; Yan, Chuangye; Gong, Xinqi; Du, Mingjian; Shi, Yigong

    2014-01-01

    Vitamin C, also known as ascorbate, is required in numerous essential metabolic reactions in eukaryotes. The eukaryotic ascorbate-dependent oxidoreductase cytochrome b561 (Cyt b561), a family of highly conserved transmembrane enzymes, plays an important role in ascorbate recycling and iron absorption. Although Cyt b561 was identified four decades ago, its atomic structure and functional mechanism remain largely unknown. Here, we report the high-resolution crystal structures of cytochrome b561 from Arabidopsis thaliana in both substrate-free and substrate-bound states. Cyt b561 forms a homodimer, with each protomer consisting of six transmembrane helices and two heme groups. The negatively charged substrate ascorbate, or monodehydroascorbate, is enclosed in a positively charged pocket on either side of the membrane. Two highly conserved amino acids, Lys81 and His106, play an essential role in substrate recognition and catalysis. Our structural and biochemical analyses allow the proposition of a general electron transfer mechanism for members of the Cyt b561 family. PMID:24449903

  20. Functional Characterization of the FoxE Iron Oxidoreductase from the Photoferrotroph Rhodobacter ferrooxidans SW2*

    PubMed Central

    Saraiva, Ivo H.; Newman, Dianne K.; Louro, Ricardo O.

    2012-01-01

    Photoferrotrophy is presumed to be an ancient type of photosynthetic metabolism in which bacteria use the reducing power of ferrous iron to drive carbon fixation. In this work the putative iron oxidoreductase of the photoferrotroph Rhodobacter ferrooxidans SW2 was cloned, purified, and characterized for the first time. This protein, FoxE, was characterized using spectroscopic, thermodynamic, and kinetic techniques. It is a c-type cytochrome that forms a trimer or tetramer in solution; the two hemes of each monomer are hexacoordinated by histidine and methionine. The hemes have positive reduction potentials that allow downhill electron transfer from many geochemically relevant ferrous iron forms to the photosynthetic reaction center. The reduction potentials of the hemes are different and are cross-assigned to fast and slow kinetic phases of ferrous iron oxidation in vitro. Lower reactivity was observed at high pH and may contribute to prevent ferric iron precipitation inside or at the surface of the cell. These results help fill in the molecular details of a metabolic process that likely contributed to the deposition of precambrian banded iron formations, globally important sedimentary rocks that are found on every continent today. PMID:22661703

  1. Hyperuricemia-Related Diseases and Xanthine Oxidoreductase (XOR) Inhibitors: An Overview

    PubMed Central

    Chen, Changyi; Lü, Jian-Ming; Yao, Qizhi

    2016-01-01

    Uric acid is the final oxidation product of purine metabolism in humans. Xanthine oxidoreductase (XOR) catalyzes oxidative hydroxylation of hypoxanthine to xanthine to uric acid, accompanying the production of reactive oxygen species (ROS). Uric acid usually forms ions and salts known as urates and acid urates in serum. Clinically, overproduction or under-excretion of uric acid results in the elevated level of serum uric acid (SUA), termed hyperuricemia, which has long been established as the major etiologic factor in gout. Accordingly, urate-lowering drugs such as allopurinol, an XOR-inhibitor, are extensively used for the treatment of gout. In recent years, the prevalence of hyperuricemia has significantly increased and more clinical investigations have confirmed that hyperuricemia is an independent risk factor for cardiovascular disease, hypertension, diabetes, and many other diseases. Urate-lowering therapy may also play a critical role in the management of these diseases. However, current XOR-inhibitor drugs such as allopurinol and febuxostat may have significant adverse effects. Therefore, there has been great effort to develop new XOR-inhibitor drugs with less or no toxicity for the long-term treatment or prevention of these hyperuricemia-related diseases. In this review, we discuss the mechanism of uric acid homeostasis and alterations, updated prevalence, therapeutic outcomes, and molecular pathophysiology of hyperuricemia-related diseases. We also summarize current discoveries in the development of new XOR inhibitors. PMID:27423335

  2. Influence of 120 kDa Pyruvate:Ferredoxin Oxidoreductase on Pathogenicity of Trichomonas vaginalis.

    PubMed

    Song, Hyun-Ouk

    2016-02-01

    Trichomonas vaginalis is a flagellate protozoan parasite and commonly infected the lower genital tract in women and men. Iron is a known nutrient for growth of various pathogens, and also reported to be involved in establishment of trichomoniasis. However, the exact mechanism was not clarified. In this study, the author investigated whether the 120 kDa protein of T. vaginalis may be involved in pathogenicity of trichomonads. Antibodies against 120 kDa protein of T. vaginalis, which was identified as pyruvate:ferredoxin oxidoreductase (PFOR) by peptide analysis of MALDI-TOF-MS, were prepared in rabbits. Pretreatment of T. vaginalis with anti-120 kDa Ab decreased the proliferation and adherence to vaginal epithelial cells (MS74) of T. vaginalis. Subcutaneous tissue abscess in anti-120 kDa Ab-treated T. vaginalis-injected mice was smaller in size than that of untreated T. vaginalis-infected mice. Collectively, the 120 kDa protein expressed by iron may be involved in proliferation, adhesion to host cells, and abscess formation, thereby may influence on the pathogenicity of T. vaginalis.

  3. Human NADH:ubiquinone oxidoreductase deficiency: radical changes in mitochondrial morphology?

    PubMed

    Koopman, Werner J H; Verkaart, Sjoerd; Visch, Henk Jan; van Emst-de Vries, Sjenet; Nijtmans, Leo G J; Smeitink, Jan A M; Willems, Peter H G M

    2007-07-01

    Malfunction of NADH:ubiquinone oxidoreductase or complex I (CI), the first and largest complex of the mitochondrial oxidative phosphorylation system, has been implicated in a wide variety of human disorders. To demonstrate a quantitative relationship between CI amount and activity and mitochondrial shape and cellular reactive oxygen species (ROS) levels, we recently combined native electrophoresis and confocal and video microscopy of dermal fibroblasts of healthy control subjects and children with isolated CI deficiency. Individual mitochondria appeared fragmented and/or less branched in patient fibroblasts with a severely reduced CI amount and activity (class I), whereas patient cells in which these latter parameters were only moderately reduced displayed a normal mitochondrial morphology (class II). Moreover, cellular ROS levels were significantly more increased in class I compared with class II cells. We propose a mechanism in which a mutation-induced decrease in the cellular amount and activity of CI leads to enhanced ROS levels, which, in turn, induce mitochondrial fragmentation when not appropriately counterbalanced by the cell's antioxidant defense systems.

  4. Pyruvate:NADP+ oxidoreductase is stabilized by its cofactor, thiamin pyrophosphate, in mitochondria of Euglena gracilis.

    PubMed

    Nakazawa, Masami; Takenaka, Shigeo; Ueda, Mitsuhiro; Inui, Hiroshi; Nakano, Yoshihisa; Miyatake, Kazutaka

    2003-03-15

    Pyruvate:NADP(+) oxidoreductase (PNO) is a thiamin pyrophosphate (TPP)-dependent enzyme that plays a central role in the respiratory metabolism of Euglena gracilis, which requires thiamin for growth. When thiamin was depleted in Euglena cells, PNO protein level was greatly reduced, but its mRNA level was barely changed. In addition, a large part of PNO occurred as an apoenzyme lacking TPP in the deficient cells. The PNO protein level increased rapidly, without changes in the mRNA level, after supplementation of thiamin into its deficient cells. In the deficient cells, in contrast to the sufficient ones, a steep decrease in the PNO protein level was induced when the cells were incubated with cycloheximide. Immunofluorescence microscopy indicated that most of the PNO localized in the mitochondria in either the sufficient or the deficient cells. These findings suggest that PNO is readily degraded when TPP is not provided in mitochondria, and consequently the PNO protein level is greatly reduced by thiamin deficiency in E. gracilis.

  5. Xanthine Oxidoreductase in Drug Metabolism: Beyond a Role as a 
Detoxifying Enzyme

    PubMed Central

    Battelli, Maria Giulia; Polito, Letizia; Bortolotti, Massimo; Bolognesi, Andrea

    2016-01-01

    The enzyme xanthine oxidoreductase (XOR) catalyzes the last two steps of purine catabolism in the highest uricotelic primates. XOR is an enzyme with dehydrogenase activity that, in mammals, may be converted into oxidase activity under a variety of pathophysiologic conditions. XOR activity is highly regulated at the transcriptional and post-translational levels and may generate reactive oxygen and nitrogen species, which trigger different consequences, ranging from cytotoxicity to inflammation. The low specificity for substrates allows XOR to metabolize a number of endogenous metabolites and a variety of exogenous compounds, including drugs. The present review focuses on the role of XOR as a drug-metabolizing enzyme, specifically for drugs with anticancer, antimicrobial, antiviral, immunosuppressive or vasodilator activities, as well as drugs acting on metabolism or inducing XOR expression. XOR has an activating role that is essential to the pharmacological action of quinone drugs, cyadox, antiviral nucleoside analogues, allopurinol, nitrate and nitrite. XOR activity has a degradation function toward thiopurine nucleotides, pyrazinoic acid, methylxanthines and tolbutamide, whose half-life may be prolonged by the use of XOR inhibitors. In conclusion, to avoid potential drug interaction risks, such as a toxic excess of drug bioavailability or a loss of drug efficacy, caution is suggested in the use of XOR inhibitors, as in the case of hyperuricemic patients affected by gout or tumor lysis syndrome, when it is necessary to simultaneously administer therapeutic substances that are activated or degraded by the drug-metabolizing activity of XOR. PMID:27458036

  6. Mitochondrial Ca2+ homeostasis in human NADH:ubiquinone oxidoreductase deficiency.

    PubMed

    Willems, Peter H G M; Valsecchi, Federica; Distelmaier, Felix; Verkaart, Sjoerd; Visch, Henk-Jan; Smeitink, Jan A M; Koopman, Werner J H

    2008-07-01

    NADH:ubiquinone oxidoreductase or complex I is a large multisubunit assembly of the mitochondrial inner membrane that channels high-energy electrons from metabolic NADH into the electron transport chain (ETC). Its dysfunction is associated with a range of progressive neurological disorders, often characterized by a very early onset and short devastating course. To better understand the cytopathological mechanisms of these disorders, we use live cell luminometry and imaging microscopy of patient skin fibroblasts with mutations in nuclear-encoded subunits of the complex. Here, we present an overview of our recent work, showing that mitochondrial membrane potential, Ca(2+) handling and ATP production are to a variable extent impaired among a large cohort of patient fibroblast lines. From the results obtained, the picture emerges that a reduction in cellular complex I activity leads to a depolarization of the mitochondrial membrane potential, resulting in a decreased supply of mitochondrial ATP to the Ca(2+)-ATPases of the intracellular stores and thus to a reduced Ca(2+) content of these stores. As a consequence, the increase in cytosolic Ca(2+) concentration evoked by a Ca(2+) mobilizing stimulus is decreased, leading to a reduction in mitochondrial Ca(2+) accumulation and ensuing ATP production and thus to a hampered energization of stimulus-induced cytosolic processes.

  7. Effects of allopurinol on uric acid concentrations, xanthine oxidoreductase activity and oxidative stress in broiler chickens.

    PubMed

    Carro, M D; Falkenstein, E; Radke, W J; Klandorf, H

    2010-01-01

    The purpose of this study was to determine the effects of allopurinol (AL) on xanthine oxidoreductase (XOR) activity and uric acid (UA) levels in chickens. Thirty 5-week-old broilers were divided into three groups and fed 0 (control), 25 (AL25) or 50 (AL50) mg AL per kg of body mass for 5 weeks. Chicks were weighed twice weekly and leukocyte oxidative activity (LOA) and plasma purine levels were determined weekly in five birds per group. Chicks were sacrificed after 2 or 5 weeks, and samples from tissues were taken for analysis of XOR activity. Plasma UA concentrations were lower (P<0.001) and xanthine and hypoxanthine concentrations were greater (P<0.001) in AL25 and AL50 birds compared to controls, whereas no differences (P=0.904) were detected in allantoin concentrations. By week 5, body mass was reduced (P<0.001) to 84.0 and 65.1% of that in controls for AL25 and AL50 broilers, respectively, and LOA was 4.1 times greater (P<0.05) in AL25 compared to control birds. Liver XOR activity was increased by 1.1 and 1.2 times in AL25 and AL50 birds, but there was no change (P>0.05) in XOR activity in the pancreas and intestine. These results suggest that AL effect on XOR activity is tissue dependent.

  8. Cation transport by the respiratory NADH:quinone oxidoreductase (complex I): facts and hypotheses.

    PubMed

    Steffen, Wojtek; Steuber, Julia

    2013-10-01

    The respiratory complex I (electrogenic NADH:quinone oxidoreductase) has been considered to act exclusively as a H+ pump. This was questioned when the search for the NADH-driven respiratory Na+ pump in Klebsiella pneumoniae initiated by Peter Dimroth led to the discovery of a Na+-translocating complex in this enterobacterium. The 3D structures of complex I from different organisms support the idea that the mechanism of cation transport by complex I involves conformational changes of the membrane-bound NuoL, NuoM and NuoN subunits. In vitro methods to follow Na+ transport were compared with in vivo approaches to test whether complex I, or its individual NuoL, NuoM or NuoN subunits, extrude Na+ from the cytoplasm to the periplasm of bacterial host cells. The truncated NuoL subunit of the Escherichia coli complex I which comprises amino acids 1-369 exhibits Na+ transport activity in vitro. This observation, together with an analysis of putative cation channels in NuoL, suggests that there exists in NuoL at least one continuous pathway for cations lined by amino acid residues from transmembrane segments 3, 4, 5, 7 and 8. Finally, we discuss recent studies on Na+ transport by mitochondrial complex I with respect to its putative role in the cycling of Na+ ions across the inner mitochondrial membrane.

  9. Novel Insights into the PKCβ-dependent Regulation of the Oxidoreductase p66Shc*

    PubMed Central

    Haller, Martina; Khalid, Sana; Kremser, Leopold; Fresser, Friedrich; Furlan, Tobias; Hermann, Martin; Guenther, Julia; Drasche, Astrid; Leitges, Michael; Giorgio, Marco; Baier, Gottfried; Lindner, Herbert; Troppmair, Jakob

    2016-01-01

    Dysfunctional mitochondria contribute to the development of many diseases and pathological conditions through the excessive production of reactive oxygen species (ROS), and, where studied, ablation of p66Shc (p66) was beneficial. p66 translocates to the mitochondria and oxidizes cytochrome c to yield H2O2, which in turn initiates cell death. PKCβ-mediated phosphorylation of serine 36 in p66 has been implicated as a key regulatory step preceding mitochondrial translocation, ROS production, and cell death, and PKCβ thus may provide a target for therapeutic intervention. We performed a reassessment of PKCβ regulation of the oxidoreductase activity of p66. Although our experiments did not substantiate Ser36 phosphorylation by PKCβ, they instead provided evidence for Ser139 and Ser213 as PKCβ phosphorylation sites regulating the pro-oxidant and pro-apoptotic function of p66. Mutation of another predicted PKCβ phosphorylation site also located in the phosphotyrosine binding domain, threonine 206, had no phenotype. Intriguingly, p66 with Thr206 and Ser213 mutated to glutamic acid showed a gain-of-function phenotype with significantly increased ROS production and cell death induction. Taken together, these data argue for a complex mechanism of PKCβ-dependent regulation of p66 activation involving Ser139 and a motif surrounding Ser213. PMID:27624939

  10. Relaxing the coenzyme specificity of 1,3-propanediol oxidoreductase from Klebsiella pneumoniae by rational design.

    PubMed

    Ma, Chengwei; Zhang, Le; Dai, Jianying; Xiu, Zhilong

    2010-04-15

    1,3-Propanediol has wide applications for large volume markets, particularly in the polymer business. Microbial production of 1,3-propanediol has been considered as a competitor to the traditional petrochemical routes. However, the formation of 1,3-propanediol is limited by the amount of NADH supplied by the oxidative pathway of glycerol dismutation. Previous metabolic flux analysis revealed that relaxation of the coenzyme specificity of 1,3-propanediol oxidoreductase for both NADH and NADPH would increase the production of 1,3-propanediol as well as maintaining the NADH-NAD(+) circle. This work tried to accomplish such a relaxation by rational protein design. Overall binding free energy indicated that the electrostatic energy was the major force discriminating NADH from NADPH. Computational alanine-scanning mutagenesis of the active site residues illustrated that Asp41 was the key residue responsible for the coenzyme specificity. Compared with Asp41Ala, Asp41Gly could further weaken the repulsion between Asp41 and the phosphate group esterified to the 2'-hydroxyl group of the ribose at the adenine end of NADPH. Site-directed mutagenesis was conducted and the relaxation was successfully realized.

  11. Relevance of renal-specific oxidoreductase in tubulogenesis during mammalian nephron development.

    PubMed

    Kanwar, Yashpal S; Yang, Qiwei; Tian, Yufeng; Lin, Sun; Wada, Jun; Chugh, Sumant; Srivastava, Satish K

    2002-04-01

    Renal-specific oxidoreductase (RSOR), an enzyme relevant to diabetic nephropathy, is exclusively expressed in renal tubules. Studies were initiated to determine whether, like other tubule-specific proteins, it selectively modulates tubulogenesis. Northern blot analyses revealed a approximately 1.5-kb transcript, and RSOR expression was detectable in mice embryonic kidneys at day 13, gradually increased by day 17, and extended into neo- and postnatal periods. RSOR mRNA and protein expression was confined to proximal tubules, commencing at gestational day 17 and increasing subsequently, but remained absent in glomeruli and medulla. Treatment with RSOR antisense oligodeoxynucleotide resulted in a dose-dependent dysmorphogenesis of metanephric explants harvested at gestational day 13. The explants were smaller and had expanded mesenchyme, and the population of tubules was markedly decreased. The glomeruli were unaffected, as assessed by mRNA expression of glomerular epithelial protein 1 and reactivity with wheat germ agglutinin. Antisense treatment led to a selective reduction of RSOR mRNA. Immunoprecipitation also indicated a selective translational blockade of RSOR. These findings suggest that RSOR is developmentally regulated, exhibits a distinct spatiotemporal distribution, and probably plays a role in tubulogenesis.

  12. Strategies of oncogenic microbes to deal with WW domain-containing oxidoreductase

    PubMed Central

    Lan, Yu-Yan; Hsiao, Jenn-Ren; Chang, Nan-Shan

    2015-01-01

    WW domain-containing oxidoreductase (WWOX) is a well-documented tumor suppressor protein that controls growth, survival, and metastasis of malignant cells. To counteract WWOX’s suppressive effects, cancer cells have developed many strategies either to downregulate WWOX expression or to functionally inactivate WWOX. Relatively unknown is, in the context of those cancers associated with certain viruses or bacteria, how the oncogenic pathogens deal with WWOX. Here we review recent studies showing different strategies utilized by three cancer-associated pathogens. Helicobactor pylori reduces WWOX expression through promoter hypermethylation, an epigenetic mechanism also occurring in many other cancer cells. WWOX has a potential to block canonical NF-κB activation and tumorigenesis induced by Tax, an oncoprotein of human T-cell leukemia virus. Tax successfully overcomes the blockage by inhibiting WWOX expression through activation of the non-canonical NF-κB pathway. On the other hand, latent membrane protein 2A of Epstein–Barr virus physically interacts with WWOX and redirects its function to trigger a signaling pathway that upregulates matrix metalloproteinase 9 and cancer cell invasion. These reports may be just “the tip of the iceberg” regarding multiple interactions between WWOX and oncogenic microbes. Further studies in this direction should expand our understanding of infection-driven oncogenesis. PMID:25488911

  13. A Complex of Htm1 and the Oxidoreductase Pdi1 Accelerates Degradation of Misfolded Glycoproteins.

    PubMed

    Pfeiffer, Anett; Stephanowitz, Heike; Krause, Eberhard; Volkwein, Corinna; Hirsch, Christian; Jarosch, Ernst; Sommer, Thomas

    2016-06-03

    A quality control system in the endoplasmic reticulum (ER) efficiently discriminates polypeptides that are in the process of productive folding from conformers that are trapped in an aberrant state. Only the latter are transported into the cytoplasm and degraded in a process termed ER-associated protein degradation (ERAD). In the ER, an enzymatic cascade generates a specific N-glycan structure of seven mannosyl and two N-acetylglucosamine residues (Man7GlcNAc2) on misfolded glycoproteins to facilitate their disposal. We show that a complex encompassing the yeast lectin-like protein Htm1 and the oxidoreductase Pdi1 converts Man8GlcNAc2 on glycoproteins into the Man7GlcNAc2 signal. In vitro the Htm1-Pdi1 complex processes both unfolded and native proteins albeit with a preference for the former. In vivo, elevated expression of HTM1 causes glycan trimming on misfolded and folded proteins, but only degradation of the non-native species is accelerated. Thus, modification with a Man7GlcNAc2 structure does not inevitably commit a protein for ER-associated protein degradation. The function of Htm1 in ERAD relies on its association with Pdi1, which appears to regulate the access to substrates. Our data support a model in which the balanced activities of Pdi1 and Htm1 are crucial determinants for the efficient removal of misfolded secretory glycoproteins.

  14. The mechanism of catalysis by type-II NADH:quinone oxidoreductases

    PubMed Central

    Blaza, James N.; Bridges, Hannah R.; Aragão, David; Dunn, Elyse A.; Heikal, Adam; Cook, Gregory M.; Nakatani, Yoshio; Hirst, Judy

    2017-01-01

    Type II NADH:quinone oxidoreductase (NDH-2) is central to the respiratory chains of many organisms. It is not present in mammals so may be exploited as an antimicrobial drug target or used as a substitute for dysfunctional respiratory complex I in neuromuscular disorders. NDH-2 is a single-subunit monotopic membrane protein with just a flavin cofactor, yet no consensus exists on its mechanism. Here, we use steady-state and pre-steady-state kinetics combined with mutagenesis and structural studies to determine the mechanism of NDH-2 from Caldalkalibacillus thermarum. We show that the two substrate reactions occur independently, at different sites, and regardless of the occupancy of the partner site. We conclude that the reaction pathway is determined stochastically, by the substrate/product concentrations and dissociation constants, and can follow either a ping-pong or ternary mechanism. This mechanistic versatility provides a unified explanation for all extant data and a new foundation for the development of therapeutic strategies. PMID:28067272

  15. Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of C. perfringens.

    PubMed

    Lee, Kyungwoo; Lillehoj, Hyun S; Li, Guangxing; Park, Myeong-Seon; Jang, Seung I; Jeong, Wooseog; Jeoung, Hye-Young; An, Dong-Jun; Lillehoj, Erik P

    2011-12-01

    Clostridium-related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by reaction with immune sera from commercial meat-type chickens with clinical outbreak of Clostridium infections. In addition to the genes encoding EF-Tu and PFO, C. perfringens alpha-toxin and necrotic enteritis B-like (NetB) toxin were also expressed in Escherichia coli and their corresponding recombinant proteins were purified. Using the four recombinant proteins as target antigens in ELISA immunoassays, high serum antibody titers were observed not only in chickens with clinical signs of Clostridium infections, but also in apparently healthy animals from the same disease-endemic farm. By contrast, no antibodies against any of the proteins were present in the serum of a specific pathogen-free bird. In ELISA using recombinant proteins of C. perfringens, the levels of anti-bacterial protein antibodies were also higher in chickens which were experimentally induced to show NE clinical signs after co-infection with C. perfringens and Eimeria maxima compared with uninfected controls. These results show that two antigenic C. perfringens proteins, EF-Tu and PFO can be useful detection antigens for C. perfringens-afflicted infections in commercial poultry.

  16. Kinetic and product distribution analysis of NO* reductase activity in Nitrosomonas europaea hydroxylamine oxidoreductase.

    PubMed

    Kostera, Joshua; Youngblut, Matthew D; Slosarczyk, Jeffrey M; Pacheco, A Andrew

    2008-09-01

    Hydroxylamine oxidoreductase (HAO) from the ammonia-oxidizing bacterium Nitrosomonas europaea normally catalyzes the four-electron oxidation of hydroxylamine to nitrite, which is the second step in ammonia-dependent respiration. Here we show that, in the presence of methyl viologen monocation radical (MV(red)), HAO can catalyze the reduction of nitric oxide to ammonia. The process is analogous to that catalyzed by cytochrome c nitrite reductase, an enzyme found in some bacteria that use nitrite as a terminal electron acceptor during anaerobic respiration. The availability of a reduction pathway to ammonia is an important factor to consider when designing in vitro studies of HAO, and may also have some physiological relevance. The reduction of nitric oxide to ammonia proceeds in two kinetically distinct steps: nitric oxide is first reduced to hydroxylamine, and then hydroxylamine is reduced to ammonia at a tenfold slower rate. The second step was investigated independently in solutions initially containing hydroxylamine, MV(red), and HAO. Both steps show first-order dependence on nitric oxide and HAO concentrations, and zero-order dependence on MV(red) concentration. The rate constants governing each reduction step were found to have values of (4.7 +/- 0.3) x 10(5) and (2.06 +/- 0.04) x 10(4) M(-1) s(-1), respectively. A second reduction pathway, with second-order dependence on nitric oxide, may become available as the concentration of nitric oxide is increased. Such a pathway might lead to production of nitrous oxide. We estimate a maximum value of (1.5 +/- 0.05) x 10(10) M(-2) s(-1) for the rate constant of the alternative pathway, which is small and suggests that the pathway is not physiologically important.

  17. Mechanistic reappraisal of early stage photochemistry in the light-driven enzyme protochlorophyllide oxidoreductase.

    PubMed

    Heyes, Derren J; Hardman, Samantha J O; Mansell, David; Gardiner, John M; Scrutton, Nigel S

    2012-01-01

    The light-driven enzyme protochlorophyllide oxidoreductase (POR) catalyzes the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide). This reaction is a key step in the biosynthesis of chlorophyll. Ultrafast photochemical processes within the Pchlide molecule are required for catalysis and previous studies have suggested that a short-lived excited-state species, known as I675*, is the first catalytic intermediate in the reaction and is essential for capturing excitation energy to drive subsequent hydride and proton transfers. The chemical nature of the I675* excited state species and its role in catalysis are not known. Here, we report time-resolved pump-probe spectroscopy measurements to study the involvement of the I675* intermediate in POR photochemistry. We show that I675* is not unique to the POR-catalyzed photoreduction of Pchlide as it is also formed in the absence of the POR enzyme. The I675* species is only produced in samples that contain both Pchlide substrate and Chlide product and its formation is dependent on the pump excitation wavelength. The rate of formation and the quantum yield is maximized in 50∶50 mixtures of the two pigments (Pchlide and Chlide) and is caused by direct energy transfer between Pchlide and neighboring Chlide molecules, which is inhibited in the polar solvent methanol. Consequently, we have re-evaluated the mechanism for early stage photochemistry in the light-driven reduction of Pchlide and propose that I675* represents an excited state species formed in Pchlide-Chlide dimers, possibly an excimer. Contrary to previous reports, we conclude that this excited state species has no direct mechanistic relevance to the POR-catalyzed reduction of Pchlide.

  18. Engineering of Helicobacter pylori Dimeric Oxidoreductase DsbK (HP0231)

    PubMed Central

    Bocian-Ostrzycka, Katarzyna M.; Grzeszczuk, Magdalena J.; Banaś, Anna M.; Jastrząb, Katarzyna; Pisarczyk, Karolina; Kolarzyk, Anna; Łasica, Anna M.; Collet, Jean-François; Jagusztyn-Krynicka, Elżbieta K.

    2016-01-01

    The formation of disulfide bonds that are catalyzed by proteins of the Dsb (disulfide bond) family is crucial for the correct folding of many extracytoplasmic proteins. Thus, this formation plays an essential, pivotal role in the assembly of many virulence factors. The Helicobacter pylori disulfide bond-forming system is uncomplicated compared to the best-characterized Escherichia coli Dsb pathways. It possesses only two extracytoplasmic Dsb proteins named HP0377 and HP0231. As previously shown, HP0377 is a reductase involved in the process of cytochrome c maturation. Additionally, it also possesses disulfide isomerase activity. HP0231 was the first periplasmic dimeric oxidoreductase involved in disulfide generation to be described. Although HP0231 function is critical for oxidative protein folding, its structure resembles that of dimeric EcDsbG, which does not confer this activity. However, the HP0231 catalytic motifs (CXXC and the so-called cis-Pro loop) are identical to that of monomeric EcDsbA. To understand the functioning of HP0231, we decided to study the relations between its sequence, structure and activity through an extensive analysis of various HP0231 point mutants, using in vivo and in vitro strategies. Our work shows the crucial role of the cis-Pro loop, as changing valine to threonine in this motif completely abolishes the protein function in vivo. Functioning of HP0231 is conditioned by the combination of CXXC and the cis-Pro loop, as replacing the HP0231 CXXC motif by the motif from EcDsbG or EcDsbC results in bifunctional protein, at least in E. coli. We also showed that the dimerization domain of HP0231 ensures contact with its substrates. Moreover, the activity of this oxidase is independent on the structure of the catalytic domain. Finally, we showed that HP0231 chaperone activity is independent of its redox function. PMID:27507968

  19. Decreased xanthine oxidoreductase is a predictor of poor prognosis in early‐stage gastric cancer

    PubMed Central

    Linder, N; Haglund, C; Lundin, M; Nordling, S; Ristimäki, A; Kokkola, A; Mrena, J; Wiksten, J‐P; Lundin, J

    2006-01-01

    Background Xanthine oxidoreductase (XOR) is a key enzyme in the degradation of DNA, RNA and high‐energy phosphates. About half of the patients with breast cancer have a decrease in XOR expression. Patients with breast cancer with unfavourable prognosis are independently identified by the loss of XOR. Aim To assess the clinical relevance of XOR expression in gastric cancer. Methods XOR levels were studied by immunohistochemistry in tissue microarray specimens of 337 patients with gastric cancer and the relation between XOR expression and a series of clinicopathological variables, as well as disease‐specific survival, was assessed. Results XOR was moderately decreased in 41% and was undetectable in another 14% of the tumours compared with the corresponding normal tissue. Decreased XOR was associated with advanced stage, deep tumour penetration, diffusely spread tumour location, positive lymph node status, large tumour size, non‐curative disease, cellular aneuploidy, high S‐phase fraction and high cyclooxygenase‐2 expression, but not with p53 expression or Borrmann classification. Down regulation of XOR was associated with unfavourable outcome, and the cumulative 5‐year gastric cancer‐specific survival in patients with strong XOR expression was 47%, compared with 22% in those with moderate to negative expression (p<0.001). This was also true in patients with stage I–II (p = 0.01) and lymph node‐negative (p = 0.02) disease, as well as in patients with smaller (⩽5 cm) tumours (p = 0.02). Conclusion XOR expression in gastric cancer may be a new marker for a more aggressive gastric cancer biology, similar to that previously reported for breast cancer. PMID:16935971

  20. Mechanistic Reappraisal of Early Stage Photochemistry in the Light-Driven Enzyme Protochlorophyllide Oxidoreductase

    PubMed Central

    Heyes, Derren J.; Hardman, Samantha J. O.; Mansell, David; Gardiner, John M.; Scrutton, Nigel S.

    2012-01-01

    The light-driven enzyme protochlorophyllide oxidoreductase (POR) catalyzes the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide). This reaction is a key step in the biosynthesis of chlorophyll. Ultrafast photochemical processes within the Pchlide molecule are required for catalysis and previous studies have suggested that a short-lived excited-state species, known as I675*, is the first catalytic intermediate in the reaction and is essential for capturing excitation energy to drive subsequent hydride and proton transfers. The chemical nature of the I675* excited state species and its role in catalysis are not known. Here, we report time-resolved pump-probe spectroscopy measurements to study the involvement of the I675* intermediate in POR photochemistry. We show that I675* is not unique to the POR-catalyzed photoreduction of Pchlide as it is also formed in the absence of the POR enzyme. The I675* species is only produced in samples that contain both Pchlide substrate and Chlide product and its formation is dependent on the pump excitation wavelength. The rate of formation and the quantum yield is maximized in 50∶50 mixtures of the two pigments (Pchlide and Chlide) and is caused by direct energy transfer between Pchlide and neighboring Chlide molecules, which is inhibited in the polar solvent methanol. Consequently, we have re-evaluated the mechanism for early stage photochemistry in the light-driven reduction of Pchlide and propose that I675* represents an excited state species formed in Pchlide-Chlide dimers, possibly an excimer. Contrary to previous reports, we conclude that this excited state species has no direct mechanistic relevance to the POR-catalyzed reduction of Pchlide. PMID:23049830

  1. Enhanced vasodilator activity of nitrite in hypertension: critical role for erythrocytic xanthine oxidoreductase and translational potential.

    PubMed

    Ghosh, Suborno M; Kapil, Vikas; Fuentes-Calvo, Isabel; Bubb, Kristen J; Pearl, Vanessa; Milsom, Alexandra B; Khambata, Rayomand; Maleki-Toyserkani, Sheiva; Yousuf, Mubeen; Benjamin, Nigel; Webb, Andrew J; Caulfield, Mark J; Hobbs, Adrian J; Ahluwalia, Amrita

    2013-05-01

    Elevation of circulating nitrite (NO2(-)) levels causes vasodilatation and lowers blood pressure in healthy volunteers. Whether these effects and the underpinning mechanisms persist in hypertension is unknown. Therefore, we investigated the consequences of systemic nitrite elevation in spontaneously hypertensive rats and conducted proof-of-principle studies in patients. Nitrite caused dose-dependent blood pressure-lowering that was profoundly enhanced in spontaneously hypertensive rats versus normotensive Wistar Kyoto controls. This effect was virtually abolished by the xanthine oxidoreductase (XOR) inhibitor, allopurinol, and associated with hypertension-specific XOR-dependent nitrite reductase activity localized to the erythrocyte but not the blood vessel wall. To determine whether these pathways translate to human hypertension, we investigated the effects of elevation of circulating nitrite levels in 15 drug naïve grade 1 hypertensives. To elevate nitrite, we used a dose of dietary nitrate (≈ 3.5 mmol) that elevated nitrite levels ≈ 1.5-fold (P<0.01); a rise shown previously to exert no significant blood pressure-lowering effects in normotensives. This dose caused substantial reductions in systolic (≈ 12 mm Hg) and diastolic blood pressures (P<0.001) and pulse wave velocity (P<0.05); effects associated with elevations in erythrocytic XOR expression and XOR-dependent nitrite reductase activity. Our observations demonstrate the improved efficacy of inorganic nitrate and nitrite in hypertension as a consequence of increased erythrocytic XOR nitrite reductase activity and support the concept of dietary nitrate supplementation as an effective, but simple and inexpensive, antihypertensive strategy.

  2. Enzyme activation and catalysis: characterisation of the vibrational modes of substrate and product in protochlorophyllide oxidoreductase.

    PubMed

    Sytina, Olga A; Alexandre, Maxime T; Heyes, Derren J; Hunter, C Neil; Robert, Bruno; van Grondelle, Rienk; Groot, Marie Louise

    2011-02-14

    The light-dependent reduction of protochlorophyllide, a key step in the synthesis of chlorophyll, is catalyzed by the enzyme protochlorophyllide oxidoreductase (POR) and requires two photons (O. A. Sytina et al., Nature, 2008, 456, 1001-1008). The first photon activates the enzyme-substrate complex, a subsequent second photon initiates the photochemistry by triggering the formation of a catalytic intermediate. These two events are characterized by different spectral changes in the infra-red spectral region. Here, we investigate the vibrational frequencies of the POR-bound and unbound substrate, and product, and thus provide a detailed assignment of the spectral changes in the 1800-1250 cm(-1) region associated with the catalytic conversion of PChlide:NADPH:TyrOH into Chlide:NADP(+):TyrO(-). Fluorescence line narrowed spectra of the POR-bound Pchlide reveal a C=O keto group downshifted by more than 20 cm(-1) to a relatively low vibrational frequency of 1653 cm(-1), as compared to the unbound Pchlide, indicating that binding of the chromophore to the protein occurs via strong hydrogen bond(s). The frequencies of the C=C vibrational modes are consistent with a six-coordinated state of the POR-bound Pchlide, suggesting that there are two coordination interactions between the central Mg atom of the chromophore and protein residues, and/or a water molecule. The frequencies of the C=C vibrational modes of Chlide are consistent with a five-coordinated state, indicating a single interaction between the central Mg atom of the chromophore and a water molecule. Rapid-scan FTIR measurements on the Pchlide:POR:NADPH complex at 4 cm(-1) spectral resolution reveal a new band in the 1670 cm(-1) region. The FTIR spectra of the enzyme activation phase indicate involvement of a nucleotide-binding structural motif, and an increased exposure of the protein to solvent after activation.

  3. Biotransformation of arsenic by bacterial strains mediated by oxido-reductase enzyme system.

    PubMed

    Vishnoi, N; Singh, D P

    2014-12-24

    The present study deals with the enzyme mediated biotransformation of arsenic in five arsenic tolerant strains (Bacillus subtilis, Bacillus megaterium, Bacillus pumilus, Paenibacillus macerans and Escherichia coli). Biotransformation ability of these isolates was evaluated by monitoring arsenite oxidase and arsenate reductase activity. Results showed that arsenic oxidase activity was exclusively present in P. macerans and B. pumilus while B. subtilis, B. megaterium and E. coli strains showed presence of Arsenic oxido-reductase enzyme. The reversible nature of arsenic oxido- reductase suggested that same enzyme can carry out oxidation and reduction of arsenic depending upon the relative concentration of arsenic species. Lineweaver-Burk plot of the arsenite oxidase activity in P. macerans showed highest Km value (Km- 200 μM) and lower Vmax (0.012 μmol mg-1 protein min-1) indicating lowest affinity of the enzyme for arsenite. On the contrary, E. coli showed the lower Km value ( Km- 38.46 μM) and higher Vmax (0.044 μmol mg-1 protein min-1) suggesting for higher affinity for the arsenite. Lineweaver-Burk plot of arsenate reductase activity showed the presence of this enzyme in B. subtilis, B. megaterium and E. coli which were in the range of 200-360 μM Km and Vmax value between 0.256- 0.129 mmol mg-1 protein min-1. These results suggested that affinity of the as reductase enzyme is lowest for arsenate than that for the arsenite. Thus, arsenite oxidase system appears to be a predominant mechanism of cellular defense in these bacterial strains.

  4. Characterization of new DsbB-like thiol-oxidoreductases of Campylobacter jejuni and Helicobacter pylori and classification of the DsbB family based on phylogenomic, structural and functional criteria.

    PubMed

    Raczko, Anna M; Bujnicki, Janusz M; Pawlowski, Marcin; Godlewska, Renata; Lewandowska, Magdalena; Jagusztyn-Krynicka, Elzbieta K

    2005-01-01

    In Gram-negative bacterial cells, disulfide bond formation occurs in the oxidative environment of the periplasm and is catalysed by Dsb (disulfide bond) proteins found in the periplasm and in the inner membrane. In this report the identification of a new subfamily of disulfide oxidoreductases encoded by a gene denoted dsbI, and functional characterization of DsbI proteins from Campylobacter jejuni and Helicobacter pylori, as well as DsbB from C. jejuni, are described. The N-terminal domain of DsbI is related to DsbB proteins and comprises five predicted transmembrane segments, while the C-terminal domain is predicted to locate to the periplasm and to fold into a beta-propeller structure. The dsbI gene is co-transcribed with a small ORF designated dba (dsbI-accessory). Based on a series of deletion and complementation experiments it is proposed that DsbB can complement the lack of DsbI but not the converse. In the presence of DsbB, the activity of DsbI was undetectable, hence it probably acts only on a subset of possible substrates of DsbB. To reconstruct the principal events in the evolution of DsbB and DsbI proteins, sequences of all their homologues identifiable in databases were analysed. In the course of this study, previously undetected variations on the common thiol-oxidoreductase theme were identified, such as development of an additional transmembrane helix and loss or migration of the second pair of Cys residues between two distinct periplasmic loops. In conjunction with the experimental characterization of two members of the DsbI lineage, this analysis has resulted in the first comprehensive classification of the DsbB/DsbI family based on structural, functional and evolutionary criteria.

  5. NAD(P)H-dependent quinone oxidoreductase 1 (NQO1) and cytochrome P450 oxidoreductase (CYP450OR) differentially regulate menadione-mediated alterations in redox status, survival and metabolism in pancreatic β-cells.

    PubMed

    Gray, Joshua P; Karandrea, Shpetim; Burgos, Delaine Zayasbazan; Jaiswal, Anil A; Heart, Emma A

    2016-11-16

    NQO1 (NAD(P)H-quinone oxidoreductase 1) reduces quinones and xenobiotics to less-reactive compounds via 2-electron reduction, one feature responsible for the role of NQO1 in antioxidant defense in several tissues. In contrast, NADPH cytochrome P450 oxidoreductase (CYP450OR), catalyzes the 1-electron reduction of quinones and xenobiotics, resulting in enhanced superoxide formation. However, to date, the roles of NQO1 and CYP450OR in pancreatic β-cell metabolism under basal conditions and oxidant challenge have not been characterized. Using NQO1 inhibition, over-expression and knock out, we have demonstrated that, in addition to protection of β-cells from toxic concentrations of the redox cycling quinone menadione, NQO1 also regulates the basal level of reduced-to-oxidized nucleotides, suggesting other role(s) beside that of an antioxidant enzyme. In contrast, over-expression of NADPH cytochrome P450 oxidoreductase (CYP450OR) resulted in enhanced redox cycling activity and decreased cellular viability, consistent with the enhanced generation of superoxide and H2O2. Basal expression of NQO1 and CYP450OR was comparable in isolated islets and liver. However, NQO1, but not CYP450OR, was strongly induced in β-cells exposed to menadione. NQO1 and CYP450OR exhibited a reciprocal preference for reducing equivalents in β-cells: while CYP450OR preferentially utilized NADPH, NQO1 primarily utilized NADH. Together, these results demonstrate that NQO1 and CYP450OR reciprocally regulate oxidant metabolism in pancreatic β-cells.

  6. Glucose oxidase from Penicillium amagasakiense. Primary structure and comparison with other glucose-methanol-choline (GMC) oxidoreductases.

    PubMed

    Kiess, M; Hecht, H J; Kalisz, H M

    1998-02-15

    The complete amino acid sequence of glucose oxidase from Penicillium amagasakiense was determined by Edman degradation and mass spectrometry of peptide fragments derived from three different specific proteolytic digests and a cyanogen bromide cleavage. The complete sequence of each monomer comprises 587 amino acid residues, contains three cysteine residues, and seven potential N-glycosylation sites, of which at least five were confirmed to be glycosylated. Glucose oxidase from P. amagasakiense shows a high degree of identity (66%) and 79% similarity to glucose oxidase from Aspergillus niger, and is a member of the glucose-methanol-choline (GMC) oxidoreductase family. The tertiary structures of glucose oxidase from A. niger and cholesterol oxidase from Brevibacterium sterolicum were superimposed to provide a template for the sequence comparison of members of the GMC family. The general topology of the GMC oxidoreductases is conserved, with the exception of the presence of an active site lid in cholesterol oxidase and the insertion of additional structural elements in the substrate-binding domain of alcohol oxidase. The overall structure can be divided into five distinct sequence regions: FAD-binding domain, extended FAD-binding domain, flavin attachment loop and intermediate region, FAD covering lid, and substrate-binding domain. The FAD-binding and the extended FAD-binding domains are composed of several separate sequence regions. The other three regions each comprise a single contiguous sequence. Four major consensus patterns have been identified, including the nucleotide-binding consensus sequence close to their N-termini. The functions of the two motifs recently selected by the Genetics Computer Group, Madison, Wisconsin, as additional signature patterns of the GMC oxidoreductases are discussed. The other consensus patterns belong to either the FAD-binding or the extended FAD-binding domain. In addition, the roles of conserved residues are discussed wherever

  7. Identification of NAD(P)H quinone oxidoreductase activity in azoreductases from P. aeruginosa: azoreductases and NAD(P)H quinone oxidoreductases belong to the same FMN-dependent superfamily of enzymes.

    PubMed

    Ryan, Ali; Kaplan, Elise; Nebel, Jean-Christophe; Polycarpou, Elena; Crescente, Vincenzo; Lowe, Edward; Preston, Gail M; Sim, Edith

    2014-01-01

    Water soluble quinones are a group of cytotoxic anti-bacterial compounds that are secreted by many species of plants, invertebrates, fungi and bacteria. Studies in a number of species have shown the importance of quinones in response to pathogenic bacteria of the genus Pseudomonas. Two electron reduction is an important mechanism of quinone detoxification as it generates the less toxic quinol. In most organisms this reaction is carried out by a group of flavoenzymes known as NAD(P)H quinone oxidoreductases. Azoreductases have previously been separate from this group, however using azoreductases from Pseudomonas aeruginosa we show that they can rapidly reduce quinones. Azoreductases from the same organism are also shown to have distinct substrate specificity profiles allowing them to reduce a wide range of quinones. The azoreductase family is also shown to be more extensive than originally thought, due to the large sequence divergence amongst its members. As both NAD(P)H quinone oxidoreductases and azoreductases have related reaction mechanisms it is proposed that they form an enzyme superfamily. The ubiquitous and diverse nature of azoreductases alongside their broad substrate specificity, indicates they play a wide role in cellular survival under adverse conditions.

  8. Identification of NAD(P)H Quinone Oxidoreductase Activity in Azoreductases from P. aeruginosa: Azoreductases and NAD(P)H Quinone Oxidoreductases Belong to the Same FMN-Dependent Superfamily of Enzymes

    PubMed Central

    Ryan, Ali; Kaplan, Elise; Nebel, Jean-Christophe; Polycarpou, Elena; Crescente, Vincenzo; Lowe, Edward; Preston, Gail M.; Sim, Edith

    2014-01-01

    Water soluble quinones are a group of cytotoxic anti-bacterial compounds that are secreted by many species of plants, invertebrates, fungi and bacteria. Studies in a number of species have shown the importance of quinones in response to pathogenic bacteria of the genus Pseudomonas. Two electron reduction is an important mechanism of quinone detoxification as it generates the less toxic quinol. In most organisms this reaction is carried out by a group of flavoenzymes known as NAD(P)H quinone oxidoreductases. Azoreductases have previously been separate from this group, however using azoreductases from Pseudomonas aeruginosa we show that they can rapidly reduce quinones. Azoreductases from the same organism are also shown to have distinct substrate specificity profiles allowing them to reduce a wide range of quinones. The azoreductase family is also shown to be more extensive than originally thought, due to the large sequence divergence amongst its members. As both NAD(P)H quinone oxidoreductases and azoreductases have related reaction mechanisms it is proposed that they form an enzyme superfamily. The ubiquitous and diverse nature of azoreductases alongside their broad substrate specificity, indicates they play a wide role in cellular survival under adverse conditions. PMID:24915188

  9. Role of xanthine oxidoreductase in the anti-thrombotic effects of nitrite in rats in vivo.

    PubMed

    Kramkowski, K; Leszczynska, A; Przyborowski, K; Kaminski, T; Rykaczewska, U; Sitek, B; Zakrzewska, A; Proniewski, B; Smolenski, R T; Chabielska, E; Buczko, W; Chlopicki, S

    2016-01-01

    The mechanisms underlying nitrite-induced effects on thrombosis and hemostasis in vivo are not clear. The goal of the work described here was to investigate the role of xanthine oxidoreductase (XOR) in the anti-platelet and anti-thrombotic activities of nitrite in rats in vivo. Arterial thrombosis was induced electrically in rats with renovascular hypertension by partial ligation of the left renal artery. Sodium nitrite (NaNO2, 0.17 mmol/kg twice daily for 3 days, p.o) was administered with or without one of the XOR-inhibitors: allopurinol (ALLO) and febuxostat (FEB) (100 and 5 mg/kg, p.o., for 3 days). Nitrite treatment (0.17 mmol/kg), which was associated with a significant increase in NOHb, nitrite/nitrate plasma concentration, resulted in a substantial decrease in thrombus weight (TW) (0.48 ± 0.03 mg vs. vehicle [VEH] 0.88 ± 0.08 mg, p < 0.001) without a significant hypotensive effect. The anti-thrombotic effect of nitrite was partially reversed by FEB (TW = 0.63 ± 0.06 mg, p < 0.05 vs. nitrites), but not by ALLO (TW = 0.43 ± 0.02 mg). In turn, profound anti-platelet effect of nitrite measured ex vivo using collagen-induced whole-blood platelet aggregation (70.5 ± 7.1% vs. VEH 100 ± 4.5%, p < 0.05) and dynamic thromboxaneB2 generation was fully reversed by both XOR-inhibitors. In addition, nitrite decreased plasminogen activator inhibitor-1 concentration (0.47 ± 0.13 ng/ml vs. VEH 0.62 ± 0.04 ng/ml, p < 0.05) and FEB/ALLO reversed this effect. In vitro the anti-platelet effect of nitrite (1 mM) was reversed by FEB (0.1 mM) under hypoxia (0.5%O2) and normoxia (20%O2). Nitrite treatment had no effect on coagulation parameters. In conclusion, the nitrite-induced anti-platelet effect in rats in vivo is mediated by XOR, but XOR does not fully account for the anti-thrombotic effects of nitrite.

  10. Purification and properties of an unusual UDP-glucose dehydrogenase, NADPH-dependent, from Xanthomonas albilineans.

    PubMed

    Blanch, María; Legaz, María-Estrella; Vicente, C

    2008-01-01

    Xanthomonas albilineans produces a UDP-glucose dehydrogenase growing on sucrose. The enzyme oxidizes UDP-glucose to UDP-glucuronic acid by using molecular oxygen and NADPH. Kinetics of enzymatic oxydation of NADPH is linearly dependent on the amount of oxygen supplied. The enzyme has been purified at homogeneity. The value of pI of the purified enzyme is 8.98 and its molecular mass has been estimated as about 14 kDa. The enzyme shows a michaelian kinetics for UDP-glucose concentrations. The value of K(m) for UDP-glucose is 0.87 mM and 0.26 mM for NADPH, although the enzyme has three different sites to interact with NADPH. The enzyme is inhibited by UDP-glucose concentrations higher than 1.3 mM. N-Terminal sequence has been determined as IQPYNH.

  11. Rhamnose-induced propanediol oxidoreductase in Escherichia coli: purification, properties, and comparison with the fucose-induced enzyme.

    PubMed Central

    Boronat, A; Aguilar, J

    1979-01-01

    Escherichia coli are capable of growing anaerobically on L-rhamnose as a sole source of carbon and energy and without any exogenous hydrogen acceptor. When grown under such condition, synthesis of a nicotinamide adenine dinucleotide-linked L-lactaldehydepropanediol oxidoreductase is induced. The functioning of this enzyme results in the regeneration of nicotinamide adenine dinucleotide. The enzyme was purified to electrophoretic homogeneity. It has a molecular weight of 76,000, with two subunits that are indistinguishable by electrophoretic mobility. The enzyme reduces L-lactaldehyde to L-1,2-propanediol with reduced nicotinamide adenine dinucleotide as a cofactor. The Km were 0.035 mM L-lactaldehyde and 1.25 mM L-1,2-propanediol, at pH 7.0 and 9.5, respectively. The enzyme acts only on the L-isomers. Strong substrate inhibition was observed with L-1,2-propanediol (above 25 mM) in the dehydrogenase reaction. The enzyme has a pH optimum of 6.5 for the reduction of L-lactaldehyde and of 9.5 for the dehydrogenation of L-1,2-propanediol. The enzyme is, according to the parameters presented in this report, indistinguishable from the propanediol oxidoreductase induced by anaerobic growth on fucose. Images PMID:40956

  12. Convenient microtiter plate-based, oxygen-independent activity assays for flavin-dependent oxidoreductases based on different redox dyes

    PubMed Central

    Brugger, Dagmar; Krondorfer, Iris; Zahma, Kawah; Stoisser, Thomas; Bolivar, Juan M; Nidetzky, Bernd; Peterbauer, Clemens K; Haltrich, Dietmar

    2014-01-01

    Flavin-dependent oxidoreductases are increasingly recognized as important biocatalysts for various industrial applications. In order to identify novel activities and to improve these enzymes in engineering approaches, suitable screening methods are necessary. We developed novel microtiter-plate-based assays for flavin-dependent oxidases and dehydrogenases using redox dyes as electron acceptors for these enzymes. 2,6-dichlorophenol-indophenol, methylene green, and thionine show absorption changes between their oxidized and reduced forms in the visible range, making it easy to judge visually changes in activity. A sample set of enzymes containing both flavoprotein oxidases and dehydrogenases – pyranose 2-oxidase, pyranose dehydrogenase, cellobiose dehydrogenase, d-amino acid oxidase, and l-lactate oxidase – was selected. Assays for these enzymes are based on a direct enzymatic reduction of the redox dyes and not on the coupled detection of a reaction product as in the frequently used assays based on hydrogen peroxide formation. The different flavoproteins show low Michaelis constants with these electron acceptor substrates, and therefore these dyes need to be added in only low concentrations to assure substrate saturation. In conclusion, these electron acceptors are useful in selective, reliable and cheap MTP-based screening assays for a range of flavin-dependent oxidoreductases, and offer a robust method for library screening, which could find applications in enzyme engineering programs. PMID:24376171

  13. Structural and Functional insights into the catalytic mechanism of the Type II NADH:quinone oxidoreductase family

    PubMed Central

    Marreiros, Bruno C.; Sena, Filipa V.; Sousa, Filipe M.; Oliveira, A. Sofia F.; Soares, Cláudio M.; Batista, Ana P.; Pereira, Manuela M.

    2017-01-01

    Type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains. These proteins contribute indirectly to the establishment of the transmembrane difference of electrochemical potential by catalyzing the reduction of quinone by oxidation of NAD(P)H. NDH-2s are widespread enzymes being present in the three domains of life. In this work, we explored the catalytic mechanism of NDH-2 by investigating the common elements of all NDH-2s, based on the rationale that conservation of such elements reflects their structural/functional importance. We observed conserved sequence motifs and structural elements among 1762 NDH-2s. We identified two proton pathways possibly involved in the protonation of the quinone. Our results led us to propose the first catalytic mechanism for NDH-2 family, in which a conserved glutamate residue, E172 (in NDH-2 from Staphylococcus aureus) plays a key role in proton transfer to the quinone pocket. This catalytic mechanism may also be extended to the other members of the two-Dinucleotide Binding Domains Flavoprotein (tDBDF) superfamily, such as sulfide:quinone oxidoreductases. PMID:28181562

  14. Screening of microorganisms producing cold-active oxidoreductases to be applied in enantioselective alcohol oxidation. An Antarctic survey.

    PubMed

    Araújo, Lidiane S; Kagohara, Edna; Garcia, Thaís P; Pellizari, Vivian H; Andrade, Leandro H

    2011-01-01

    Several microorganisms were isolated from soil/sediment samples of Antarctic Peninsula. The enrichment technique using (RS)-1-(phenyl)ethanol as a carbon source allowed us to isolate 232 psychrophile/psychrotroph microorganisms. We also evaluated the enzyme activity (oxidoreductases) for enantioselective oxidation reactions, by using derivatives of (RS)-1-(phenyl)ethanol as substrates. Among the studied microorganisms, 15 psychrophile/psychrotroph strains contain oxidoreductases that catalyze the (S)-enantiomer oxidation from racemic alcohols to their corresponding ketones. Among the identified microorganisms, Flavobacterium sp. and Arthrobacter sp. showed excellent enzymatic activity. These new bacteria strains were selected for optimization study, in which the (RS)-1-(4-methyl-phenyl)ethanol oxidation was evaluated in several reaction conditions. From these studies, it was observed that Flavobacterium sp. has an excellent enzymatic activity at 10 °C and Arthrobacter sp. at 15 and 25 °C. We have also determined the growth curves of these bacteria, and both strains showed optimum growth at 25 °C, indicating that these bacteria are psychrotroph.

  15. Comprehensively Characterizing the Thioredoxin Interactome In Vivo Highlights the Central Role Played by This Ubiquitous Oxidoreductase in Redox Control.

    PubMed

    Arts, Isabelle S; Vertommen, Didier; Baldin, Francesca; Laloux, Géraldine; Collet, Jean-François

    2016-06-01

    Thioredoxin (Trx) is a ubiquitous oxidoreductase maintaining protein-bound cysteine residues in the reduced thiol state. Here, we combined a well-established method to trap Trx substrates with the power of bacterial genetics to comprehensively characterize the in vivo Trx redox interactome in the model bacterium Escherichia coli Using strains engineered to optimize trapping, we report the identification of a total 268 Trx substrates, including 201 that had never been reported to depend on Trx for reduction. The newly identified Trx substrates are involved in a variety of cellular processes, ranging from energy metabolism to amino acid synthesis and transcription. The interaction between Trx and two of its newly identified substrates, a protein required for the import of most carbohydrates, PtsI, and the bacterial actin homolog MreB was studied in detail. We provide direct evidence that PtsI and MreB contain cysteine residues that are susceptible to oxidation and that participate in the formation of an intermolecular disulfide with Trx. By considerably expanding the number of Trx targets, our work highlights the role played by this major oxidoreductase in a variety of cellular processes. Moreover, as the dependence on Trx for reduction is often conserved across species, it also provides insightful information on the interactome of Trx in organisms other than E. coli.

  16. KefF, the regulatory subunit of the potassium efflux system KefC, shows quinone oxidoreductase activity.

    PubMed

    Lyngberg, Lisbeth; Healy, Jessica; Bartlett, Wendy; Miller, Samantha; Conway, Stuart J; Booth, Ian R; Rasmussen, Tim

    2011-09-01

    Escherichia coli and many other Gram-negative pathogenic bacteria protect themselves from the toxic effects of electrophilic compounds by using a potassium efflux system (Kef). Potassium efflux is coupled to the influx of protons, which lowers the internal pH and results in immediate protection. The activity of the Kef system is subject to complex regulation by glutathione and its S conjugates. Full activation of KefC requires a soluble ancillary protein, KefF. This protein has structural similarities to oxidoreductases, including human quinone reductases 1 and 2. Here, we show that KefF has enzymatic activity as an oxidoreductase, in addition to its role as the KefC activator. It accepts NADH and NADPH as electron donors and quinones and ferricyanide (in addition to other compounds) as acceptors. However, typical electrophilic activators of the Kef system, e.g., N-ethyl maleimide, are not substrates. If the enzymatic activity is disrupted by site-directed mutagenesis while retaining structural integrity, KefF is still able to activate the Kef system, showing that the role as an activator is independent of the enzyme activity. Potassium efflux assays show that electrophilic quinones are able to activate the Kef system by forming S conjugates with glutathione. Therefore, it appears that the enzymatic activity of KefF diminishes the redox toxicity of quinones, in parallel with the protection afforded by activation of the Kef system.

  17. Purification and characterization of 2-oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6.

    PubMed Central

    Yoon, K S; Ishii, M; Igarashi, Y; Kodama, T

    1996-01-01

    2-Oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately autotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, was purified to homogeneity by precipitation with ammonium sulfate and by fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The purified enzyme had a molecular mass of about 105 kDa and comprised two subunits (70 kDa and 35 kDa). The activity of the 2-oxoglutarate:ferredoxin oxidoreductase was detected by the use of 2-oxoglutarate, coenzyme A, and one of several electron acceptors in substrate amounts (ferredoxin isolated from H. thermophilus, flavin adenine dinucleotide, flavin mononucleotide, or methyl viologen). NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective. The enzyme was extremely thermostable; the temperature optimum for 2-oxoglutarate oxidation was above 80 degrees C, and the time for a 50% loss of activity at 70 degrees C under anaerobic conditions was 22 h. The optimum pH for a 2-oxoglutarate oxidation reaction was 7.6 to 7.8. The apparent Km values for 2-oxoglutarate and coenzyme A at 70 degrees C were 1.42 mM and 80 microM, respectively. PMID:8655524

  18. The effects of allopurinol, uric acid, and inosine administration on xanthine oxidoreductase activity and uric acid concentrations in broilers.

    PubMed

    Settle, T; Carro, M D; Falkenstein, E; Radke, W; Klandorf, H

    2012-11-01

    The purpose of these studies was to determine the effects of uric acid (UA) and inosine administration on xanthine oxidoreductase activity in broilers. In experiment one, 25 broilers were assigned to 5 treatment groups: control, AL (25 mg of allopurinol/kg of body mass), AR (AL for 2 wk followed by allopurinol withdrawal over wk 3), UAF (AL plus 6.25 g of UA sodium salt/kg of feed), and UAI (AL plus 120 mg of UA sodium salt injected daily). The UA administration had no effect on plasma concentration of UA (P > 0.05), and all allopurinol-treated birds had lower (P < 0.05) UA levels than controls. The UA concentrations were restored in both plasma and kidney of AR birds at wk 3, but liver UA concentrations remained lower. Whereas xanthine oxidoreductase (XOR) activity in the liver (LXOR) was reduced (P < 0.05) by allopurinol treatment, XOR activity in the kidney (KXOR) was not affected (P = 0.05). In experiment two, 3 groups of 5 birds each were fed 0 (control), 0.6 M inosine/kg of feed (INO), or INO plus 50 mg of allopurinol/kg of body mass (INOAL). The INOAL birds showed lower total LXOR activity, but KXOR activity was not affected. Both INO and INOAL birds had higher plasma and kidney UA concentrations than controls. The results suggest that regulation of UA production is tissue dependent.

  19. Inhibitory Effects of Tart Cherry (Prunus cerasus) Juice on Xanthine Oxidoreductase Activity and its Hypouricemic and Antioxidant Effects on Rats.

    PubMed

    Haidari, F; Mohammad Shahi, M; Keshavarz, S A; Rashidi, M R

    2009-03-01

    The aim of this study was to investigate the effect of tart cherry juice on serum uric acid levels, hepatic xanthine oxidoreductase activity and two non-invasive biomarkers of oxidative stress (total antioxidant capacity and malondialdehyde concentration), in normal and hyperuricemic rats. Tart cherry juice (5 ml/kg) was given by oral gavage to rats for 2 weeks. Allopurinol (5 mg/kg) was used as a positive control and was also given by oral gavage. Data showed that tart cherry juice treatment did not cause any significant reduction in the serum uric acid levels in normal rats, but significantly reduced (P<0.05) the serum uric acid levels of hyperuricemic rats in a time-dependent manner. Tart cherry juice treatment also inhibited hepatic xanthine oxidase/dehydrogenase activity. Moreover, a significant increase (P<0.05) in serum total antioxidant capacity was observed in tart cherry juice treated-rats in both normal and hyperuricemic groups. The oral administration of tart cherry juice also led to a significant reduction (P<0.05) in MDA concentration in the hyperuricemic rats. Although the hypouricemic effect of allopurinol, as a putative inhibitor of xanthine oxidoreductase, was much higher than that of tart cherry, it could not significantly change anti-oxidative parameters. These features of tart cherry make it an attractive candidate for the prophylactic treatment of hyperuricaemia, particularly if it is to be taken on a long-term basis. Further investigations to define its clinical efficacy would be highly desirable.

  20. Ferredoxin:NADP+ oxidoreductase in junction with CdSe/ZnS quantum dots: characteristics of an enzymatically active nanohybrid.

    PubMed

    Szczepaniak, Krzysztof; Worch, Remigiusz; Grzyb, Joanna

    2013-05-15

    Ferredoxin:NADP(+) oxidoreductase (FNR) is a plant and cyanobacterial photosynthetic enzyme, also found in non-photosynthetic tissues, where it is involved in redox reactions of biosynthetic pathways. In vivo it transfers electrons to nicotinamide adenine dinucleotide phosphate (NADP(+)), forming its reduced version, NADPH, while in vitro it can also use NADPH to reduce several substrates, such as ferricyanide, various quinones and nitriles. As an oxidoreductase catalyzing reaction of a broad range of substrates, FNR may be used in biotechnological processes. Quantum dots are semiconductor nanocrystals of a few to several nanometers diameter, having very useful luminescent properties. We present the spectroscopic and functional characteristics of a covalent conjugation of FNR and CdSe/ZnS quantum dots. Two types of quantum dots, of different diameter and emission maximum (550 and 650 nm), were used for comparison. Steady-state fluorescence and gel electrophoresis confirmed efficient conjugation, while fluorescence correlation spectroscopy (FCS) allowed for determination of the conjugates' radii. The nanohybrids sustained enzymatic activity; however, changes in maximal reaction rates and Michaelis constant were found. Detailed analysis of the kinetic parameters showed that the changes in the enzyme activity depend on the substrate used for activity measurement but also on the size of the quantum dots. The presented nanohybrids, as the first example using plant and photosynthetic enzyme as a protein partner, may became a tool to study photosynthesis as well as other biosynthetic and biotechnological processes, involving enzymatically catalyzed electron transfer.

  1. Structural and Functional insights into the catalytic mechanism of the Type II NADH:quinone oxidoreductase family.

    PubMed

    Marreiros, Bruno C; Sena, Filipa V; Sousa, Filipe M; Oliveira, A Sofia F; Soares, Cláudio M; Batista, Ana P; Pereira, Manuela M

    2017-02-09

    Type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains. These proteins contribute indirectly to the establishment of the transmembrane difference of electrochemical potential by catalyzing the reduction of quinone by oxidation of NAD(P)H. NDH-2s are widespread enzymes being present in the three domains of life. In this work, we explored the catalytic mechanism of NDH-2 by investigating the common elements of all NDH-2s, based on the rationale that conservation of such elements reflects their structural/functional importance. We observed conserved sequence motifs and structural elements among 1762 NDH-2s. We identified two proton pathways possibly involved in the protonation of the quinone. Our results led us to propose the first catalytic mechanism for NDH-2 family, in which a conserved glutamate residue, E172 (in NDH-2 from Staphylococcus aureus) plays a key role in proton transfer to the quinone pocket. This catalytic mechanism may also be extended to the other members of the two-Dinucleotide Binding Domains Flavoprotein (tDBDF) superfamily, such as sulfide:quinone oxidoreductases.

  2. Arabidopsis Tic62 and Ferredoxin-NADP(H) Oxidoreductase Form Light-Regulated Complexes That Are Integrated into the Chloroplast Redox Poise[C][W

    PubMed Central

    Benz, J.P.; Stengel, A.; Lintala, M.; Lee, Y.-H.; Weber, A.; Philippar, K.; Gügel, I.L.; Kaieda, S.; Ikegami, T.; Mulo, P.; Soll, J.; Bölter, B.

    2009-01-01

    Translocation of nuclear-encoded preproteins across the inner envelope of chloroplasts is catalyzed by the Tic translocon, consisting of Tic110, Tic40, Tic62, Tic55, Tic32, Tic20, and Tic22. Tic62 was proposed to act as a redox sensor of the complex because of its redox-dependent shuttling between envelope and stroma and its specific interaction with the photosynthetic protein ferredoxin-NADP(H) oxidoreductase (FNR). However, the nature of this close relationship so far remained enigmatic. A putative additional localization of Tic62 at the thylakoids mandated further studies examining how this feature might be involved in the respective redox sensing pathway and the interaction with its partner protein. Therefore, both the association with FNR and the physiological role of the third, thylakoid-bound pool of Tic62 were investigated in detail. Coexpression analysis indicates that Tic62 has similar expression patterns as genes involved in photosynthetic functions and protein turnover. At the thylakoids, Tic62 and FNR form high molecular weight complexes that are not involved in photosynthetic electron transfer but are dynamically regulated by light signals and the stromal pH. Structural analyses reveal that Tic62 binds to FNR in a novel binding mode for flavoproteins, with a major contribution from hydrophobic interactions. Moreover, in absence of Tic62, membrane binding and stability of FNR are drastically reduced. We conclude that Tic62 represents a major FNR interaction partner not only at the envelope and in the stroma, but also at the thylakoids of Arabidopsis thaliana and perhaps all flowering plants. Association with Tic62 stabilizes FNR and is involved in its dynamic and light-dependent membrane tethering. PMID:20040542

  3. Membrane-Associated Glucose-Methanol-Choline Oxidoreductase Family Enzymes PhcC and PhcD Are Essential for Enantioselective Catabolism of Dehydrodiconiferyl Alcohol

    PubMed Central

    Takahashi, Kenji; Hirose, Yusaku; Kamimura, Naofumi; Hishiyama, Shojiro; Hara, Hirofumi; Araki, Takuma; Kasai, Daisuke; Kajita, Shinya; Katayama, Yoshihiro; Fukuda, Masao

    2015-01-01

    Sphingobium sp. strain SYK-6 is able to degrade various lignin-derived biaryls, including a phenylcoumaran-type compound, dehydrodiconiferyl alcohol (DCA). In SYK-6 cells, the alcohol group of the B-ring side chain of DCA is initially oxidized to the carboxyl group to generate 3-(2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-7-methoxy-2,3-dihydrobenzofuran-5-yl) acrylic acid (DCA-C). Next, the alcohol group of the A-ring side chain of DCA-C is oxidized to the carboxyl group, and then the resulting metabolite is catabolized through vanillin and 5-formylferulate. In this study, the genes involved in the conversion of DCA-C were identified and characterized. The DCA-C oxidation activities in SYK-6 were enhanced in the presence of flavin adenine dinucleotide and an artificial electron acceptor and were induced ca. 1.6-fold when the cells were grown with DCA. Based on these observations, SLG_09480 (phcC) and SLG_09500 (phcD), encoding glucose-methanol-choline oxidoreductase family proteins, were presumed to encode DCA-C oxidases. Analyses of phcC and phcD mutants indicated that PhcC and PhcD are essential for the conversion of (+)-DCA-C and (−)-DCA-C, respectively. When phcC and phcD were expressed in SYK-6 and Escherichia coli, the gene products were mainly observed in their membrane fractions. The membrane fractions of E. coli that expressed phcC and phcD catalyzed the specific conversion of DCA-C into the corresponding carboxyl derivatives. In the oxidation of DCA-C, PhcC and PhcD effectively utilized ubiquinone derivatives as electron acceptors. Furthermore, the transcription of a putative cytochrome c gene was significantly induced in SYK-6 grown with DCA. The DCA-C oxidation catalyzed by membrane-associated PhcC and PhcD appears to be coupled to the respiratory chain. PMID:26362985

  4. Extensive horizontal gene transfer, duplication, and loss of chlorophyll synthesis genes in the algae

    DOE PAGES

    Hunsperger, Heather M.; Randhawa, Tejinder; Cattolico, Rose Ann

    2015-02-10

    Two non-homologous, isofunctional enzymes catalyze the penultimate step of chlorophyll a synthesis in oxygenic photosynthetic organisms such as cyanobacteria, eukaryotic algae and land plants: the light independent (LIPOR) and light-dependent (POR) protochlorophyllide oxidoreductases. Whereas the distribution of these enzymes in cyanobacteria and land plants is well understood, the presence, loss, duplication, and replacement of these genes have not been surveyed in the polyphyletic and remarkably diverse eukaryotic algal lineages.

  5. Laboratory Prototype of Bioreactor for Oxidation of Toxic D-Lactate Using Yeast Cells Overproducing D-Lactate Cytochrome c Oxidoreductase

    PubMed Central

    Karkovska, Maria

    2016-01-01

    D-lactate is a natural component of many fermented foods like yogurts, sour milk, cheeses, and pickles vegetable products. D-lactate in high concentrations is toxic for children and people with short bowel syndrome and provokes encephalopathy. These facts convincingly demonstrate a need for effective tools for the D-lactate removal from some food products. The main idea of investigation is focused on application of recombinant thermotolerant methylotrophic yeast Hansenula polymorpha “tr6,” overproducing D-lactate: cytochrome c oxidoreductase (EC 1.1.2.4, D-lactate cytochrome c oxidoreductase, D-lactate dehydrogenase (cytochrome), DLDH). In addition to 6-fold overexpression of DLDH under a strong constitutive promoter (prAOX), the strain of H. polymorpha “tr6” (gcr1 catX/Δcyb2, prAOX_DLDH) is characterized by impairment in glucose repression of AOX promoter, devoid of catalase and L-lactate-cytochrome c oxidoreductase activities. Overexpression of DLDH coupling with the deletion of L-lactate-cytochrome c oxidoreductase activity opens possibility for usage of the strain as a base for construction of bioreactor for removing D-lactate from fermented products due to oxidation to nontoxic pyruvate. A laboratory prototype of column-type bioreactor for removing a toxic D-lactate from model solution based on permeabilized cells of the H. polymorpha “tr6” and alginate gel was constructed and efficiency of this process was tested. PMID:27446952

  6. Identification and cloning of two immunogenic C. perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of Clostridium perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic C. perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by reaction with...

  7. Crystallization and preliminary X-ray crystallographic analysis of a new crystal form of hydroxylamine oxidoreductase from Nitrosomonas europaea.

    PubMed

    Cedervall, Peder E; Hooper, Alan B; Wilmot, Carrie M

    2009-12-01

    Hydroxylamine oxidoreductase (HAO) from Nitrosomonas europaea is a homotrimeric protein that catalyzes the oxidation of hydroxylamine to nitrite. Each monomer, with a molecular weight of 67.1 kDa, contains seven c-type hemes and one heme P460, the porphyrin ring of which is covalently linked to a tyrosine residue from an adjacent subunit. HAO was first crystallized and structurally characterized at a resolution of 2.8 A in 1997. The structure was solved in space group P6(3) and suffered from merohedral twinning. Here, a crystallization procedure is presented that yielded untwinned crystals belonging to space group P2(1)2(1)2, which diffracted to 2.25 A resolution and contained one trimer in the asymmetric unit. The unit-cell parameters were a = 140.7, b = 142.6, c = 107.4 A.

  8. The two common polymorphic forms of human NRH-quinone oxidoreductase 2 (NQO2) have different biochemical properties.

    PubMed

    Megarity, Clare F; Gill, James R E; Caraher, M Clare; Stratford, Ian J; Nolan, Karen A; Timson, David J

    2014-05-02

    There are two common forms of NRH-quinone oxidoreductase 2 (NQO2) in the human population resulting from SNP rs1143684. One has phenylalanine at position 47 (NQO2-F47) and the other leucine (NQO2-L47). Using recombinant proteins, we show that these variants have similar steady state kinetic parameters, although NQO2-L47 has a slightly lower specificity constant. NQO2-L47 is less stable towards proteolytic digestion and thermal denaturation than NQO2-F47. Both forms are inhibited by resveratrol, but NQO2-F47 shows negative cooperativity with this inhibitor. Thus these data demonstrate, for the first time, clear biochemical differences between the variants which help explain previous biomedical and epidemiological findings.

  9. P5-type sulfhydryl oxidoreductase promotes the sorting of proteins to protein body I in rice endosperm cells

    PubMed Central

    Onda, Yayoi; Kawagoe, Yasushi

    2013-01-01

    In rice (Oryza sativa) endosperm cells, oxidative protein folding is necessary for the sorting of storage proteins to protein bodies, PB-I and PB-II. Here we examined the role of sulfhydryl oxidoreductase PDIL2;3 (a human P5 ortholog) in the endoplasmic reticulum (ER), using GFP-AB, a PB-I marker in which the N-terminal region (AB) of α-globulin is fused to green fluorescent protein (GFP). RNAi knockdown of PDIL2;3 inhibited the accumulation of GFP-AB in PB-I and promoted its exit from the ER. We discuss the role of PDIL2;3 in retaining proteins within the ER and specifying their localization to PB-I through disulfide bond formation. PMID:23299424

  10. Submitochondrial fragments of brain mitochondria: general characteristics and catalytic properties of NADH:ubiquinone oxidoreductase (complex I).

    PubMed

    Kalashnikov, D S; Grivennikova, V G; Vinogradov, A D

    2011-02-01

    A number of genetic or drug-induced pathophysiological disorders, particularly neurodegenerative diseases, have been reported to correlate with catalytic impairments of NADH:ubiquinone oxidoreductase (mitochondrial complex I). The vast majority of the data on catalytic properties of this energy-transducing enzyme have been accumulated from studies on bovine heart complex I preparations of different degrees of resolution, whereas almost nothing is known about the functional activities of the enzyme in neuronal tissues. Here a procedure for preparation of coupled inside-out submitochondrial particles from brain is described and their NADH oxidase activity is characterized. The basic characteristics of brain complex I, particularly the parameters of A/D-transition are found to be essentially the same as those previously reported for heart enzyme. The results show that coupled submitochondrial particles prepared from either heart or brain can equally be used as a model system for in vitro studies aimed to delineate neurodegenerative-associated defects of complex I.

  11. Thermodynamic contribution to the regulation of electron transfer in the Na(+)-pumping NADH:quinone oxidoreductase from Vibrio cholerae.

    PubMed

    Neehaul, Yashvin; Juárez, Oscar; Barquera, Blanca; Hellwig, Petra

    2012-05-15

    The Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR) is a fundamental enzyme of the oxidative phosphorylation metabolism and ionic homeostasis in several pathogenic and marine bacteria. To understand the mechanism that couples electron transfer with sodium translocation in Na(+)-NQR, the ion dependence of the redox potential of the individual cofactors was studied using a spectroelectrochemical approach. The redox potential of one of the FMN cofactors increased 90 mV in the presence of Na(+) or Li(+), compared to the redox potentials measured in the presence of other cations that are not transported by the enzyme, such as K(+), Rb(+), and NH(4)(+). This shift in redox potential of one FMN confirms the crucial role of the FMN anionic radicals in the Na(+) pumping mechanism and demonstrates that the control of the electron transfer rate has both kinetic (via conformational changes) and thermodynamic components.

  12. Flexibility of thiamine diphosphate revealed by kinetic crystallographic studies of the reaction of pyruvate-ferredoxin oxidoreductase with pyruvate.

    PubMed

    Cavazza, Christine; Contreras-Martel, Carlos; Pieulle, Laetitia; Chabrière, Eric; Hatchikian, E Claude; Fontecilla-Camps, Juan C

    2006-02-01

    Pyruvate-ferredoxin oxidoreductases (PFOR) are unique among thiamine pyrophosphate (ThDP)-containing enzymes in giving rise to a rather stable cofactor-based free-radical species upon the decarboxylation of their first substrate, pyruvate. We have obtained snapshots of unreacted and partially reacted (probably as a tetrahedral intermediate) pyruvate-PFOR complexes at different time intervals. We conclude that pyruvate decarboxylation involves very limited substrate-to-product movements but a significant displacement of the thiazolium moiety of ThDP. In this respect, PFOR seems to differ substantially from other ThDP-containing enzymes, such as transketolase and pyruvate decarboxylase. In addition, exposure of PFOR to oxygen in the presence of pyruvate results in significant inhibition of catalytic activity, both in solution and in the crystals. Examination of the crystal structure of inhibited PFOR suggests that the loss of activity results from oxime formation at the 4' amino substituent of the pyrimidine moiety of ThDP.

  13. Genes

    MedlinePlus

    ... Search Search MedlinePlus GO GO About MedlinePlus Site Map FAQs Customer Support Health Topics Drugs & Supplements Videos & Tools Español You Are Here: Home → Medical Encyclopedia → Genes URL of this page: //medlineplus.gov/ency/article/ ...

  14. [The interaction of ferredoxin:NADP{sup +} oxidoreductase and ferredoxin:thioredoxin reductase with substrates]. Progress report

    SciTech Connect

    Not Available

    1992-09-01

    We seek to map the ferredoxin-binding sites on three soluble enzymes located in spinach chloroplasts which utilize ferredoxin as an electron donor:Ferredoxin:NADP{sup +}oxidoreductase (FNR); ferredoxin:thioredoxin reductase (FTR) and glutamate synthase. As the availability of amino acid sequences for the enzymes are important in such studies, that the amino acid sequence of glutamate synthase needs be determined, the amino acid sequences of FNR, FTR and ferredoxin are already known. Related to an aim elucidate the binding sites for ferredoxin to determine whether there is a common binding site on all of these ferredoxin-dependent chloroplast enzymes and, if so, to map it. Additionally thioredoxin binding by FTR needs be determine to resolve whether the same site on FTR is involved in binding both ferredoxin and thioredoxin. Considerable progress is reported on the prosthetic groups of glutamate synthase, in establishing the role of arginine and lysine residues in ferredoxin binding by, ferredoxin:nitrite oxidoreductase nitrite reductase, labelling carboxyl groups on ferredoxin with taurine and labelling lysine residues biotinylation, and low potential heme proteins have been isolated and characterized from a non-photosynthetic plant tissue. Although the monoclonal antibodies raised against FNR turned out not to be useful for mapping the FNR/ferredoxin or FNR/NADPinteraction domains, good progress has been made on mapping the FNR/ferredoxin interaction domains by an alternative technique. The techniques developed for differential chemical modification of these two proteins - taurine modification of aspartate and glutamate residues and biotin modification of lysine residues - should be useful for mapping the interaction domains of many proteins that associate through electrostatic interactions.

  15. Crystal Structures of NADH:FMN Oxidoreductase (EmoB) at Different Stages of Catalysis*S⃞♦

    PubMed Central

    Nissen, Mark S.; Youn, Buhyun; Knowles, Benjamin D.; Ballinger, Jordan W.; Jun, Se-Young; Belchik, Sara M.; Xun, Luying; Kang, ChulHee

    2008-01-01

    EDTA has become a major organic pollutant in the environment because of its extreme usage and resistance to biodegradation. Recently, two critical enzymes, EDTA monooxygenase (EmoA) and NADH:FMN oxidoreductase (EmoB), belonging to the newly established two-component flavin-diffusible monooxygenase family, were identified in the EDTA degradation pathway in Mesorhizobium sp. BNC1. EmoA is an FMNH2-dependent enzyme that requires EmoB to provide FMNH2 for the conversion of EDTA to ethylenediaminediacetate. To understand the molecular basis of this FMN-mediated reaction, the crystal structures of the apo-form, FMN·FMN complex, and FMN·NADH complex of EmoB were determined at 2.5Å resolution. The structure of EmoB is a homotetramer consisting of four α/β-single-domain monomers of five parallel β-strands flanked by five α-helices, which is quite different from those of other known two-component flavin-diffusible monooxygenase family members, such as PheA2 and HpaC, in terms of both tertiary and quaternary structures. For the first time, the crystal structures of both the FMN·FMN and FMN·NADH complexes of an NADH:FMN oxidoreductase were determined. Two stacked isoalloxazine rings and nicotinamide/isoalloxazine rings were at a proper distance for hydride transfer. The structures indicated a ping-pong reaction mechanism, which was confirmed by activity assays. Thus, the structural data offer detailed mechanistic information for hydride transfer between NADH to an enzyme-bound FMN and between the bound FMNH2 and a diffusible FMN. PMID:18701448

  16. Contribution of rubredoxin:oxygen oxidoreductases and hybrid cluster proteins of Desulfovibrio vulgaris Hildenborough to survival under oxygen and nitrite stress.

    PubMed

    Yurkiw, Marcy A; Voordouw, Johanna; Voordouw, Gerrit

    2012-10-01

    A genomic island (GEI) of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, found to be able to migrate between two tRNA-Met loci of the genome, contains genes for rubredoxin:oxygen oxidoreductase-1 (roo1) and hybrid cluster protein-1 (hcp1) with additional copies for these genes (roo2 and hcp2) being found elsewhere on the chromosome. A suite of mutants was created in which roo2 and/or hcp2 and/or the GEI were either present or missing. The GEI and roo2 increased survival under microaerobic conditions and allowed growth in closer proximity to the air-water interface of soft agar tubes, two properties which appeared to be closely linked. When Hcp2(+) GEI(+) or Hcp2(-) GEI(+) cells, harbouring cytochrome c nitrite reductase (NrfHA) and growing on lactate and sulfate, were amended with 10 mM nitrite at mid-log phase (8-10 mM sulfide), all nitrite was reduced within 30 h with a rate of 3.0 mmol (g biomass)(-1)  h(-1) after which sulfate reduction resumed. However, Hcp2(+) GEI(-) or Hcp2(-) GEI(-) cells were unable to use lactate, causing sulfide to be used as electron donor for nitrite reduction at a sixfold lower rate. Complementation studies indicated that hcp1, not roo1, enhanced the rate of nitrite reduction under these conditions. Hcp2 enhanced the rate of nitrite reduction when, in addition to lactate, hydrogen was also present as an electron donor. These results indicate a critical role of Hcps in alleviating nitrite stress in D. vulgaris Hildenborough by maintaining the integrity of electron transport chains from lactate or H(2) to NrfHA through removal of reactive nitrogen species. It thus appears that the GEI contributes considerably to the fitness of the organism, allowing improved growth in microaerobic environments found in sulfide-oxygen gradients and in environments, containing both sulfide and nitrite, through the action of Roo1 and Hcp1 respectively.

  17. The Rnf Complex of Clostridium ljungdahlii Is a Proton-Translocating Ferredoxin:NAD(+) Oxidoreductase Essential for Autotrophic Growth

    SciTech Connect

    Tremblay, PL; Zhang, T; Dar, SA; Leang, C; Lovley, DR

    2012-12-26

    It has been predicted that the Rnf complex of Clostridium ljungdahlii is a proton-translocating ferredoxin: NAD(+) oxidoreductase which contributes to ATP synthesis by an H+-translocating ATPase under both autotrophic and heterotrophic growth conditions. The recent development of methods for genetic manipulation of C. ljungdahlii made it possible to evaluate the possible role of the Rnf complex in energy conservation. Disruption of the C. ljungdahlii rnf operon inhibited autotrophic growth. ATP synthesis, proton gradient, membrane potential, and proton motive force collapsed in the Rnf-deficient mutant with H-2 as the electron source and CO2 as the electron acceptor. Heterotrophic growth was hindered in the absence of a functional Rnf complex, as ATP synthesis, proton gradient, and proton motive force were significantly reduced with fructose as the electron donor. Growth of the Rnf-deficient mutant was also inhibited when no source of fixed nitrogen was provided. These results demonstrate that the Rnf complex of C. ljungdahlii is responsible for translocation of protons across the membrane to elicit energy conservation during acetogenesis and is a multifunctional device also implicated in nitrogen fixation. IMPORTANCE Mechanisms for energy conservation in the acetogen Clostridium ljungdahlii are of interest because of its potential value as a chassis for the production of biocommodities with novel electron donors such as carbon monoxide, syngas, and electrons derived from electrodes. Characterizing the components implicated in the chemiosmotic ATP synthesis during acetogenesis by C. ljungdahlii is a prerequisite for the development of highly productive strains. The Rnf complex has been considered the prime candidate to be the pump responsible for the formation of an ion gradient coupled with ATP synthesis in multiple acetogens. However, experimental evidence for a proton-pumping Rnf complex has been lacking. This study establishes the C. ljungdahlii Rnf complex as

  18. The disulfide oxidoreductase SdbA is active in Streptococcus gordonii using a single C-terminal cysteine of the CXXC motif.

    PubMed

    Davey, Lauren; Cohen, Alejandro; LeBlanc, Jason; Halperin, Scott A; Lee, Song F

    2016-01-01

    Recently, we identified a novel disulfide oxidoreductase, SdbA, in the oral bacterium Streptococcus gordonii. Disulfide oxidoreductases form disulfide bonds in nascent proteins using a CXXC catalytic motif. Typically, the N-terminal cysteine interacts with substrates, whereas the C-terminal cysteine is buried and only reacts with the first cysteine of the motif. In this study, we investigated the SdbA C(86) P(87) D(88) C(89) catalytic motif. In vitro, SdbA single cysteine variants at the N or C-terminal position (SdbAC86P and SdbAC89A ) were active but displayed different susceptibility to oxidation, and N-terminal cysteine was prone to sulfenylation. In S. gordonii, mutants with a single N-terminal cysteine were inactive and formed unstable disulfide adducts with other proteins. Activity was partially restored by inactivation of pyruvate oxidase, a hydrogen peroxide generator. Presence of the C-terminal cysteine alone (in the SdbAC86P variant) could complement the ΔsdbA mutant and restore disulfide bond formation in recombinant and natural protein substrates. These results provide evidence that certain disulfide oxidoreductases can catalyze disulfide bond formation using a single cysteine of the CXXC motif, including the buried C-terminal cysteine.

  19. Response surface methodology to optimize partition and purification of two recombinant oxidoreductase enzymes, glucose dehydrogenase and d-galactose dehydrogenase in aqueous two-phase systems.

    PubMed

    Shahbaz Mohammadi, Hamid; Mostafavi, Seyede Samaneh; Soleimani, Saeideh; Bozorgian, Sajad; Pooraskari, Maryam; Kianmehr, Anvarsadat

    2015-04-01

    Oxidoreductases are an important family of enzymes that are used in many biotechnological processes. An experimental design was applied to optimize partition and purification of two recombinant oxidoreductases, glucose dehydrogenase (GDH) from Bacillus subtilis and d-galactose dehydrogenase (GalDH) from Pseudomonas fluorescens AK92 in aqueous two-phase systems (ATPS). Response surface methodology (RSM) with a central composite rotatable design (CCRD) was performed to optimize critical factors like polyethylene glycol (PEG) concentration, concentration of salt and pH value. The best partitioning conditions was achieved in an ATPS composed of 12% PEG-6000, 15% K2HPO4 with pH 7.5 at 25°C, which ensured partition coefficient (KE) of 66.6 and 45.7 for GDH and GalDH, respectively. Under these experimental conditions, the activity of GDH and GalDH was 569.5U/ml and 673.7U/ml, respectively. It was found that these enzymes preferentially partitioned into the top PEG-rich phase and appeared as single bands on SDS-PAGE gel. Meanwhile the validity of the response model was confirmed by a good agreement between predicted and experimental results. Collectively, according to the obtained data it can be inferred that the ATPS optimization using RSM approach can be applied for recovery and purification of any enzyme from oxidoreductase family.

  20. Structures of almond hydroxynitrile lyase isoenzyme 5 provide a rationale for the lack of oxidoreductase activity in flavin dependent HNLs.

    PubMed

    Pavkov-Keller, Tea; Bakhuis, Janny; Steinkellner, Georg; Jolink, Fenneke; Keijmel, Esther; Birner-Gruenberger, Ruth; Gruber, Karl

    2016-10-10

    Hydroxynitrile lyases (HNLs) catalyze the asymmetric addition of HCN to aldehydes producing enantiomerically pure cyanohydrins. These enzymes can be heterologously expressed in large quantities making them interesting candidates for industrial applications. The HNLs from Rosaceae evolved from flavin dependent dehydrogenase/oxidase structures. Here we report the high resolution X-ray structure of the highly glycosylated Prunus amygdalus HNL isoenzyme5 (PaHNL5 V317A) expressed in Aspergillus niger and its complex with benzyl alcohol. A comparison with the structure of isoenzyme PaHNL1 indicates a higher accessibility to the active site and a larger cavity for PaHNL5. Additionally, the PaHNL5 complex structure with benzyl alcohol was compared with the structurally related aryl-alcohol oxidase (AAO). Even though both enzymes contain an FAD-cofactor and histidine residues at crucial positions in the active site, PaHNL5 lacks the oxidoreductase activity. The structures indicate that in PaHNLs benzyl alcohol is bound too far away from the FAD cofactor in order to be oxidized.

  1. Insights into Flavin-based Electron Bifurcation via the NADH-dependent Reduced Ferredoxin:NADP Oxidoreductase Structure*

    PubMed Central

    Demmer, Julius K.; Huang, Haiyan; Wang, Shuning; Demmer, Ulrike; Thauer, Rudolf K.; Ermler, Ulrich

    2015-01-01

    NADH-dependent reduced ferredoxin:NADP oxidoreductase (NfnAB) is found in the cytoplasm of various anaerobic bacteria and archaea. The enzyme reversibly catalyzes the endergonic reduction of ferredoxin with NADPH driven by the exergonic transhydrogenation from NADPH onto NAD+. Coupling is most probably accomplished via the mechanism of flavin-based electron bifurcation. To understand this process on a structural basis, we heterologously produced the NfnAB complex of Thermotoga maritima in Escherichia coli, provided kinetic evidence for its bifurcating behavior, and determined its x-ray structure in the absence and presence of NADH. The structure of NfnAB reveals an electron transfer route including the FAD (a-FAD), the [2Fe-2S] cluster of NfnA and the FAD (b-FAD), and the two [4Fe-4S] clusters of NfnB. Ferredoxin is presumably docked onto NfnB close to the [4Fe-4S] cluster distal to b-FAD. NAD(H) binds to a-FAD and NADP(H) consequently to b-FAD, which is positioned in the center of the NfnAB complex and the site of electron bifurcation. Arg187 is hydrogen-bonded to N5 and O4 of the bifurcating b-FAD and might play a key role in adjusting a low redox potential of the FADH•/FAD pair required for ferredoxin reduction. A mechanism of FAD-coupled electron bifurcation by NfnAB is proposed. PMID:26139605

  2. EPR spectroscopic and computational characterization of the hydroxyethylidene-thiamine pyrophosphate radical intermediate of pyruvate:ferredoxin oxidoreductase.

    PubMed

    Mansoorabadi, Steven O; Seravalli, Javier; Furdui, Cristina; Krymov, Vladimir; Gerfen, Gary J; Begley, Tadhg P; Melnick, Jonathan; Ragsdale, Stephen W; Reed, George H

    2006-06-13

    The radical intermediate of pyruvate:ferredoxin oxidoreductase (PFOR) from Moorella thermoacetica was characterized using electron paramagnetic resonance (EPR) spectroscopy at X-band and D-band microwave frequencies. EPR spectra, obtained with various combinations of isotopically labeled substrate (pyruvate) and coenzyme (thiamine pyrophosphate (TPP)), were analyzed by spectral simulations. Parameters obtained from the simulations were compared with those predicted from electronic structure calculations on various radical structures. The g-values and 14N/15N-hyperfine splittings obtained from the spectra are consistent with a planar, hydroxyethylidene-thiamine pyrophosphate (HE-TPP) pi-radical, in which spin is delocalized onto the thiazolium sulfur and nitrogen atoms. The 1H-hyperfine splittings from the methyl group of pyruvate and the 13C-hyperfine splittings from C2 of both pyruvate and TPP are consistent with a model in which the pyruvate-derived oxygen atom of the HE-TPP radical forms a hydrogen bond. The hyperfine splitting constants and g-values are not compatible with those predicted for a nonplanar, sigma/n-type cation radical.

  3. Automated resonance assignment of the 21 kDa stereo-array isotope labeled thioldisulfide oxidoreductase DsbA

    NASA Astrophysics Data System (ADS)

    Schmidt, Elena; Ikeya, Teppei; Takeda, Mitsuhiro; Löhr, Frank; Buchner, Lena; Ito, Yutaka; Kainosho, Masatsune; Güntert, Peter

    2014-12-01

    The automated chemical shift assignment algorithm FLYA has been extended for use with stereo-array isotope labeled (SAIL) proteins to determine the sequence-specific resonance assignments of large proteins. Here we present the assignment of the backbone and sidechain chemical shifts of the 21 kDa thioldisulfide oxidoreductase DsbA from Escherichia coli that were determined with the SAIL-FLYA algorithm in conjunction with automated peak picking. No manual corrections of peak lists or assignments were applied. The assignments agreed with manually determined reference assignments in 95.4% of the cases if 16 input spectra were used, 94.1% if only 3D 13C/15N-resolved NOESY, CBCA(CO)NH, and 2D [13C/15N,1H]-HSQC were used, and 86.8% if exclusively 3D 13C/15N-resolved NOESY spectra were used. Considering only the assignments that are classified as reliable by the SAIL-FLYA algorithm, the degrees of agreement increased to 97.5%, 96.5%, and 94.2%, respectively. With our approach it is thus possible to automatically obtain almost complete and correct assignments of proteins larger than 20 kDa.

  4. Insecticidal quinazoline derivatives with (trifluoromethyl)diazirinyl and azido substituents as NADH:ubiquinone oxidoreductase inhibitors and candidate photoaffinity probes.

    PubMed

    Latli, B; Wood, E; Casida, J E

    1996-03-01

    Two candidate photoaffinity probes are designed from 4-substituted quinazolines known to be potent insecticides/acaricides and NADH:ubiquinone oxidoreductase inhibitors acting at or near the rotenone site. 4-(11-Azidoundecyl-2-amino)quinazoline, based on the undecylamino analog SAN 548A as a prototype, was synthesized in 18% overall yield from ethyl 10-undecenoate by oxidation of the terminal double bond, reductive amination, coupling to 4-chloroquinazoline, and functional group manipulation of the terminal ethyl ester to an alcohol, a mesylate and finally nucleophilic displacement with azide ions. 4-(4-(3-(Trifluoromethyl)-3H-diazirin-3-yl)phenethoxy)quinaz oline [the (trifluoromethyl)diazirinyl analog of fenazaquin insecticide/acaricide] was prepared from 4-bromophenethyl alcohol in 31% overall yield by first introducing the trifluoromethylketone moiety followed by its conversion to the (trifluoromethyl)-diazirine and finally coupling to 4-chloroquinazoline as above. Both candidate photoaffinity probes have the inhibitory potency of rotenone (IC50 of 3-4 nM in each case). The azidoundecylamino compound has inadequate photoreactivity whereas that of the (trifluoromethyl)diazirinyl analog is ideal at 350 nm. Radiosynthesis of the latter photoaffinity ligand included introduction of the diazirinyl moiety as the carbene precursor, oxidation of (trifluoromethyl)diazirinylphenethyl alcohol to the corresponding acid with Jones' reagent, and reduction of the phenacetyl chloride intermediate with sodium borotritide to incorporate tritium.

  5. The Crystal Structure and Mechanism of an Unusual Oxidoreductase, GilR, Involved in Gilvocarcin V Biosynthesis

    SciTech Connect

    Noinaj, Nicholas; Bosserman, Mary A.; Schickli, M. Alexandra; Piszczek, Grzegorz; Kharel, Madan K.; Pahari, Pallab; Buchanan, Susan K.; Rohr, Jürgen

    2012-11-26

    GilR is a recently identified oxidoreductase that catalyzes the terminal step of gilvocarcin V biosynthesis and is a unique enzyme that establishes the lactone core of the polyketide-derived gilvocarcin chromophore. Gilvocarcin-type compounds form a small distinct family of anticancer agents that are involved in both photo-activated DNA-alkylation and histone H3 cross-linking. High resolution crystal structures of apoGilR and GilR in complex with its substrate pregilvocarcin V reveals that GilR belongs to the small group of a relatively new type of the vanillyl-alcohol oxidase flavoprotein family characterized by bicovalently tethered cofactors. GilR was found as a dimer, with the bicovalently attached FAD cofactor mediated through His-65 and Cys-125. Subsequent mutagenesis and functional assays indicate that Tyr-445 may be involved in reaction catalysis and in mediating the covalent attachment of FAD, whereas Tyr-448 serves as an essential residue initiating the catalysis by swinging away from the active site to accommodate binding of the 6R-configured substrate and consequently abstracting the proton of the hydroxyl residue of the substrate hemiacetal 6-OH group. These studies lay the groundwork for future enzyme engineering to broaden the substrate specificity of this bottleneck enzyme of the gilvocarcin biosynthetic pathway for the development of novel anti-cancer therapeutics.

  6. Structural and functional features of the NAD(P) dependent Gfo/Idh/MocA protein family oxidoreductases.

    PubMed

    Taberman, Helena; Parkkinen, Tarja; Rouvinen, Juha

    2016-04-01

    The Gfo/Idh/MocA protein family contains a number of different proteins, which almost exclusively consist of NAD(P)-dependent oxidoreductases that have a diverse set of substrates, typically pyranoses. In this study, to clarify common structural features that would contribute to their function, the available crystal structures of the members of this family have been analyzed. Despite a very low sequence identity, the central features of the three-dimensional structures of the proteins are surprisingly similar. The members of the protein family have a two-domain structure consisting of a N-terminal nucleotide-binding domain and a C-terminal α/β-domain. The C-terminal domain contributes to the substrate binding and catalysis, and contains a βα-motif with a central α-helix carrying common essential amino acid residues. The β-sheet of the α/β-domain contributes to the oligomerization in most of the proteins in the family.

  7. Molecular and biochemical characterization of bifunctional pyruvate decarboxylases and pyruvate ferredoxin oxidoreductases from Thermotoga maritima and Thermotoga hypogea.

    PubMed

    Eram, Mohammad S; Wong, Alton; Oduaran, Erica; Ma, Kesen

    2015-12-01

    Hyperthermophilic bacteria Thermotoga maritima and Thermotoga hypogea produce ethanol as a metabolic end product, which is resulted from acetaldehyde reduction catalysed by an alcohol dehydrogenase (ADH). However, the enzyme that is involved in the production of acetaldehyde from pyruvate is not well characterized. An oxygen sensitive and coenzyme A-dependent pyruvate decarboxylase (PDC) activity was found to be present in cell free extracts of T. maritima and T. hypogea. Both enzymes were purified and found to have pyruvate ferredoxin oxidoreductase (POR) activity, indicating their bifunctionality. Both PDC and POR activities from each of the purified enzymes were characterized in regards to their optimal assay conditions including pH dependency, oxygen sensitivity, thermal stability, temperature dependency and kinetic parameters. The close relatedness of the PORs that was shown by sequence analysis could be an indication of the presence of such bifunctionality in other hyperthermophilic bacteria. This is the first report of a bifunctional PDC/POR enzyme in hyperthermophilic bacteria. The PDC and the previously reported ADHs are most likely the key enzymes catalysing the production of ethanol from pyruvate in bacterial hyperthermophiles.

  8. Inactivation of corticosteroids in intestinal mucosa by 11 beta-hydroxysteroid: NADP oxidoreductase (EC 1. 1. 1. 146)

    SciTech Connect

    Burton, A.F.; Anderson, F.H.

    1983-10-01

    Activity of the enzyme 11 beta-hydroxysteroid:NADP oxidoreductase (EC 1.1.1.146) in human intestinal mucosa was determined by incubating scraped mucosa with /sup 3/H-cortisone and /sup 14/C-cortisol; these steroids were then extracted, separated chromatographically, and the radioactivity assayed to determine simultaneously both reductase and dehydrogenase activities. This was the only significant metabolic alteration which the substrate underwent. Only two cases had slight (5 and 13%) reductase activity. In 35 patients, 16 male and 19 female, including seven cases of Crohn's disease, three ulcerative colitis, five diverticulitis, two undergoing surgery for repair of injuries and 18 for carcinoma of colon or rectum, cortisol was converted to cortisone in 15 min with a wide range of values distributed uniformly up to 85% dehydrogenation, with a mean of 42%. When tissue homogenates were fortified with coenzymes, excess NADPH lowered dehydrogenase activity 81%; excess NADP increased dehydrogenase activity 2-fold in three cases. It is possible that a value is characteristic of an individual but perhaps more likely enzyme activity varies with metabolic events involving changes in the coenzyme levels in mucosa, and a random sampling might be expected to yield such a distribution of values. In any event, where activity is high most of the cortisol is inactivated within minutes. It is suggested that synthetic corticoids which escape such metabolic alteration might, except during pregnancy, prove superior in the treatment of conditions such as inflammatory bowel disease.

  9. The oxidoreductase ERp57 efficiently reduces partially folded in preference to fully folded MHC class I molecules

    PubMed Central

    Antoniou, Antony N.; Ford, Stuart; Alphey, Magnus; Osborne, Andrew; Elliott, Tim; Powis, Simon J.

    2002-01-01

    The oxidoreductase ERp57 is an integral component of the peptide loading complex of major histocompatibility complex (MHC) class I molecules, formed during their chaperone-assisted assembly in the endoplasmic reticulum. Misfolded MHC class I molecules or those denied suitable peptides are retrotranslocated and degraded in the cytosol. The presence of ERp57 during class I assembly suggests it may be involved in the reduction of intrachain disulfides prior to retrotranslocation. We have studied the ability of ERp57 to reduce MHC class I molecules in vitro. Recombinant ERp57 specifically reduced partially folded MHC class I molecules, whereas it had little or no effect on folded and peptide-loaded MHC class I molecules. Reductase activity was associated with cysteines at positions 56 and 405 of ERp57, the N-terminal residues of the active CXXC motifs. Our data suggest that the reductase activity of ERp57 may be involved during the unfolding of MHC class I molecules, leading to targeting for degradation. PMID:12032078

  10. Multiple active site residues are important for photochemical efficiency in the light-activated enzyme protochlorophyllide oxidoreductase (POR).

    PubMed

    Menon, Binuraj R K; Hardman, Samantha J O; Scrutton, Nigel S; Heyes, Derren J

    2016-08-01

    Protochlorophyllide oxidoreductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide), an essential, regulatory step in chlorophyll biosynthesis. The unique requirement of the enzyme for light has provided the opportunity to investigate how light energy can be harnessed to power biological catalysis and enzyme dynamics. Excited state interactions between the Pchlide molecule and the protein are known to drive the subsequent reaction chemistry. However, the structural features of POR and active site residues that are important for photochemistry and catalysis are currently unknown, because there is no crystal structure for POR. Here, we have used static and time-resolved spectroscopic measurements of a number of active site variants to study the role of a number of residues, which are located in the proposed NADPH/Pchlide binding site based on previous homology models, in the reaction mechanism of POR. Our findings, which are interpreted in the context of a new improved structural model, have identified several residues that are predicted to interact with the coenzyme or substrate. Several of the POR variants have a profound effect on the photochemistry, suggesting that multiple residues are important in stabilizing the excited state required for catalysis. Our work offers insight into how the POR active site geometry is finely tuned by multiple active site residues to support enzyme-mediated photochemistry and reduction of Pchlide, both of which are crucial to the existence of life on Earth.

  11. A New Class of Tungsten-Containing Oxidoreductase in Caldicellulosiruptor, a Genus of Plant Biomass-Degrading Thermophilic Bacteria.

    PubMed

    Scott, Israel M; Rubinstein, Gabe M; Lipscomb, Gina L; Basen, Mirko; Schut, Gerrit J; Rhaesa, Amanda M; Lancaster, W Andrew; Poole, Farris L; Kelly, Robert M; Adams, Michael W W

    2015-10-01

    Caldicellulosiruptor bescii grows optimally at 78°C and is able to decompose high concentrations of lignocellulosic plant biomass without the need for thermochemical pretreatment. C. bescii ferments both C5 and C6 sugars primarily to hydrogen gas, lactate, acetate, and CO2 and is of particular interest for metabolic engineering applications given the recent availability of a genetic system. Developing optimal strains for technological use requires a detailed understanding of primary metabolism, particularly when the goal is to divert all available reductant (electrons) toward highly reduced products such as biofuels. During an analysis of the C. bescii genome sequence for oxidoreductase-type enzymes, evidence was uncovered to suggest that the primary redox metabolism of C. bescii has a completely uncharacterized aspect involving tungsten, a rarely used element in biology. An active tungsten utilization pathway in C. bescii was demonstrated by the heterologous production of a tungsten-requiring, aldehyde-oxidizing enzyme (AOR) from the hyperthermophilic archaeon Pyrococcus furiosus. Furthermore, C. bescii also contains a tungsten-based AOR-type enzyme, here termed XOR, which is phylogenetically unique, representing a completely new member of the AOR tungstoenzyme family. Moreover, in C. bescii, XOR represents ca. 2% of the cytoplasmic protein. XOR is proposed to play a key, but as yet undetermined, role in the primary redox metabolism of this cellulolytic microorganism.

  12. Synthesis and Antimicrobial Evaluation of Amixicile-Based Inhibitors of the Pyruvate-Ferredoxin Oxidoreductases of Anaerobic Bacteria and Epsilonproteobacteria

    PubMed Central

    Kennedy, Andrew J.; Bruce, Alexandra M.; Gineste, Catherine; Ballard, T. Eric; Olekhnovich, Igor N.; Macdonald, Timothy L.

    2016-01-01

    Amixicile is a promising derivative of nitazoxanide (an antiparasitic therapeutic) developed to treat systemic infections caused by anaerobic bacteria, anaerobic parasites, and members of the Epsilonproteobacteria (Campylobacter and Helicobacter). Amixicile selectively inhibits pyruvate-ferredoxin oxidoreductase (PFOR) and related enzymes by inhibiting the function of the vitamin B1 cofactor (thiamine pyrophosphate) by a novel mechanism. Here, we interrogate the amixicile scaffold, guided by docking simulations, direct PFOR inhibition assays, and MIC tests against Clostridium difficile, Campylobacter jejuni, and Helicobacter pylori. Docking simulations revealed that the nitro group present in nitazoxanide interacts with the protonated N4′-aminopyrimidine of thiamine pyrophosphate (TPP). The ortho-propylamine on the benzene ring formed an electrostatic interaction with an aspartic acid moiety (B456) of PFOR that correlated with improved PFOR-inhibitory activity and potency by MIC tests. Aryl substitution with electron-withdrawing groups and substitutions of the propylamine with other alkyl amines or nitrogen-containing heterocycles both improved PFOR inhibition and, in many cases, biological activity against C. difficile. Docking simulation results correlate well with mechanistic enzymology and nuclear magnetic resonance (NMR) studies that show members of this class of antimicrobials to be specific inhibitors of vitamin B1 function by proton abstraction, which is both novel and likely to limit mutation-based drug resistance. PMID:27090174

  13. Dark-operative protochlorophyllide oxidoreductase generates substrate radicals by an iron-sulphur cluster in bacteriochlorophyll biosynthesis

    PubMed Central

    Nomata, Jiro; Kondo, Toru; Mizoguchi, Tadashi; Tamiaki, Hitoshi; Itoh, Shigeru; Fujita, Yuichi

    2014-01-01

    Photosynthesis converts solar energy to chemical energy using chlorophylls (Chls). In a late stage of biosynthesis of Chls, dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR), a nitrogenase-like enzyme, reduces the C17 = C18 double bond of Pchlide and drastically changes the spectral properties suitable for photosynthesis forming the parental chlorin ring for Chl a. We previously proposed that the spatial arrangement of the proton donors determines the stereospecificity of the Pchlide reduction based on the recently resolved structure of the DPOR catalytic component, NB-protein. However, it was not clear how the two-electron and two-proton transfer events are coordinated in the reaction. In this study, we demonstrate that DPOR initiates a single electron transfer reaction from a [4Fe-4S]-cluster (NB-cluster) to Pchlide, generating Pchlide anion radicals followed by a single proton transfer, and then, further electron/proton transfer steps transform the anion radicals into chlorophyllide (Chlide). Thus, DPOR is a unique iron-sulphur enzyme to form substrate radicals followed by sequential proton- and electron-transfer steps with the protein folding very similar to that of nitrogenase. This novel radical-mediated reaction supports the biosynthesis of Chl in a wide variety of photosynthetic organisms. PMID:24965831

  14. Atomic-resolution structure of the phycocyanobilin:ferredoxin oxidoreductase I86D mutant in complex with fully protonated biliverdin.

    PubMed

    Hagiwara, Yoshinori; Wada, Kei; Irikawa, Teppei; Sato, Hideaki; Unno, Masaki; Yamamoto, Ken; Fukuyama, Keiichi; Sugishima, Masakazu

    2016-10-01

    Phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the reduction of biliverdin (BV) to produce phycocyanobilin, a linear tetrapyrrole pigment used for light harvesting and light sensing. Spectroscopic and HPLC analyses inidicate that BV bound to the I86D mutant of PcyA is fully protonated (BVH(+) ) and can accept an electron, but I86D is unable to donate protons for the reduction; therefore, compared to the wild-type PcyA, the I86D mutant stabilizes BVH(+) . To elucidate the structural basis of the I86D mutation, we determined the atomic-resolution structure of the I86D-BVH(+) complex and the protonation states of the essential residues Asp105 and Glu76 in PcyA. Our study revealed that Asp105 adopted a fixed conformation in the I86D mutant, although it had dual conformations in wild-type PcyA which reflected the protonation states of BV. Taken together with biochemical/spectroscopic results, our analysis of the I86D-BVH(+) structure supports the hypothesis that flexibility of Asp105 is essential for the catalytic activity of PcyA.

  15. Insights into MHC class I peptide loading from the structure of the Tapasin-ERp57 thiol oxidoreductase heterodimer

    SciTech Connect

    Dong, G.; Wearsch, P.A.; Peaper, D.R.; Cresswell, P.; Reinisch, K.M.

    2009-03-02

    Tapasin is a glycoprotein critical for loading major histocompatibility complex (MHC) class I molecules with high-affinity peptides. It functions within the multimeric peptide-loading complex (PLC) as a disulfide-linked, stable heterodimer with the thiol oxidoreductase ERp57, and this covalent interaction is required to support optimal PLC activity. Here, we present the 2.6 {angstrom} resolution structure of the tapasin-ERp57 core of the PLC. The structure revealed that tapasin interacts with both ERp57 catalytic domains, accounting for the stability of the heterodimer, and provided an example of a protein disulfide isomerase family member interacting with substrate. Mutational analysis identified a conserved surface on tapasin that interacted with MHC class I molecules and was critical for peptide loading and editing functions of the tapasin-ERp57 heterodimer. By combining the tapasin-ERp57 structure with those of other defined PLC components, we present a molecular model that illuminates the processes involved in MHC class I peptide loading.

  16. Staphylococcus aureus DsbA is a membrane-bound lipoprotein with thiol-disulfide oxidoreductase activity.

    PubMed

    Dumoulin, Alexis; Grauschopf, Ulla; Bischoff, Markus; Thöny-Meyer, Linda; Berger-Bächi, Brigitte

    2005-11-01

    DsbA proteins, the primary catalysts of protein disulfide bond formation, are known to affect virulence and penicillin resistance in Gram-negative bacteria. We identified a putative DsbA homologue in the Gram-positive pathogen Staphylococcus aureus that was able to restore the motility phenotype of an Escherichia coli dsbA mutant and thus demonstrated a functional thiol oxidoreductase activity. The staphylococcal DsbA (SaDsbA) had a strong oxidative redox potential of -131 mV. The persistence of the protein throughout the growth cycle despite its predominant transcription during exponential growth phase suggested a rather long half-life for the SaDsbA. SaDsbA was found to be a membrane localised lipoprotein, supporting a role in disulfide bond formation. But so far, neither in vitro nor in vivo phenotype could be identified in a staphylococcal dsbA mutant, leaving its physiological role unknown. The inability of SaDsbA to interact with the E. coli DsbB and the lack of an apparent staphylococcal DsbB homologue suggest an alternative re-oxidation pathway for the SaDsbA.

  17. Dark-operative protochlorophyllide oxidoreductase generates substrate radicals by an iron-sulphur cluster in bacteriochlorophyll biosynthesis.

    PubMed

    Nomata, Jiro; Kondo, Toru; Mizoguchi, Tadashi; Tamiaki, Hitoshi; Itoh, Shigeru; Fujita, Yuichi

    2014-06-26

    Photosynthesis converts solar energy to chemical energy using chlorophylls (Chls). In a late stage of biosynthesis of Chls, dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR), a nitrogenase-like enzyme, reduces the C17 = C18 double bond of Pchlide and drastically changes the spectral properties suitable for photosynthesis forming the parental chlorin ring for Chl a. We previously proposed that the spatial arrangement of the proton donors determines the stereospecificity of the Pchlide reduction based on the recently resolved structure of the DPOR catalytic component, NB-protein. However, it was not clear how the two-electron and two-proton transfer events are coordinated in the reaction. In this study, we demonstrate that DPOR initiates a single electron transfer reaction from a [4Fe-4S]-cluster (NB-cluster) to Pchlide, generating Pchlide anion radicals followed by a single proton transfer, and then, further electron/proton transfer steps transform the anion radicals into chlorophyllide (Chlide). Thus, DPOR is a unique iron-sulphur enzyme to form substrate radicals followed by sequential proton- and electron-transfer steps with the protein folding very similar to that of nitrogenase. This novel radical-mediated reaction supports the biosynthesis of Chl in a wide variety of photosynthetic organisms.

  18. An Oxidoreductase AioE is Responsible for Bacterial Arsenite Oxidation and Resistance

    PubMed Central

    Wang, Qian; Han, Yushan; Shi, Kaixiang; Fan, Xia; Wang, Lu; Li, Mingshun; Wang, Gejiao

    2017-01-01

    Previously, we found that arsenite (AsIII) oxidation could improve the generation of ATP/NADH to support the growth of Agrobacterium tumefaciens GW4. In this study, we found that aioE is induced by AsIII and located in the arsenic island near the AsIII oxidase genes aioBA and co-transcripted with the arsenic resistant genes arsR1-arsC1-arsC2-acr3-1. AioE belongs to TrkA family corresponding the electron transport function with the generation of NADH and H+. An aioE in-frame deletion strain showed a null AsIII oxidation and a reduced AsIII resistance, while a cytC mutant only reduced AsIII oxidation efficiency. With AsIII, aioE was directly related to the increase of NADH, while cytC was essential for ATP generation. In addition, cyclic voltammetry analysis showed that the redox potential (ORP) of AioBA and AioE were +0.297 mV vs. NHE and +0.255 mV vs. NHE, respectively. The ORP gradient is AioBA > AioE > CytC (+0.217 ~ +0.251 mV vs. NHE), which infers that electron may transfer from AioBA to CytC via AioE. The results indicate that AioE may act as a novel AsIII oxidation electron transporter associated with NADH generation. Since AsIII oxidation contributes AsIII detoxification, the essential of AioE for AsIII resistance is also reasonable. PMID:28128323

  19. Dynamic function of the alkyl spacer of acetogenins in their inhibitory action with mitochondrial complex I (NADH-ubiquinone oxidoreductase).

    PubMed

    Abe, Masato; Murai, Masatoshi; Ichimaru, Naoya; Kenmochi, Atsushi; Yoshida, Takehiko; Kubo, Akina; Kimura, Yuka; Moroda, Aki; Makabe, Hidefumi; Nishioka, Takaaki; Miyoshi, Hideto

    2005-11-15

    Studies on the inhibitory mechanism of acetogenins, the most potent inhibitors of mitochondrial complex I (NADH-ubiquinone oxidoreductase), are useful for elucidating the structural and functional features of the terminal electron transfer step of this enzyme. Previous studies of the structure-activity relationship revealed that except for the alkyl spacer linking the two toxophores (i.e., the hydroxylated THF and the gamma-lactone rings), none of the multiple functional groups of these inhibitors is essential for potent inhibition. To elucidate the function of the alkyl spacer, two sets of systematically selected analogues were synthesized. First, the length of the spacer was varied widely. Second, the local flexibility of the spacer was specifically reduced by introducing multiple bond(s) into different regions of the spacer. The optimal length of the spacer for inhibition was approximately 13 carbon atoms. The decrease in the strength of the inhibitory effect caused by elongating the spacer from 13 carbons was much more drastic than that caused by shortening. Local flexibility in a specific region of the spacer was not important for the inhibition. These observations indicate that the active conformation of the spacer is not an extended form, and is not necessarily restricted to a certain rigid shape. Moreover, an analogue in which a spacer covering 10 carbon atoms was hardened into a rodlike shape still maintained a potent inhibitory effect. Our results strongly suggest that the spacer portion is free from steric congestion arising from the putative binding site probably because there is no cavity-like binding site for the spacer portion. The manner of acetogenin binding to the enzyme may not be explained by a simple "key and keyhole" analogy.

  20. Phylogenomic Analysis and Predicted Physiological Role of the Proton-Translocating NADH:Quinone Oxidoreductase (Complex I) Across Bacteria

    PubMed Central

    Spero, Melanie A.; Aylward, Frank O.; Currie, Cameron R.

    2015-01-01

    ABSTRACT The proton-translocating NADH:quinone oxidoreductase (complex I) is a multisubunit integral membrane enzyme found in the respiratory chains of both bacteria and eukaryotic organelles. Although much research has focused on the enzyme’s central role in the mitochondrial respiratory chain, comparatively little is known about its role in the diverse energetic lifestyles of different bacteria. Here, we used a phylogenomic approach to better understand the distribution of complex I across bacteria, the evolution of this enzyme, and its potential roles in shaping the physiology of different bacterial groups. By surveying 970 representative bacterial genomes, we predict complex I to be present in ~50% of bacteria. While this includes bacteria with a wide range of energetic schemes, the presence of complex I is associated with specific lifestyles, including aerobic respiration and specific types of phototrophy (bacteria with only a type II reaction center). A phylogeny of bacterial complex I revealed five main clades of enzymes whose evolution is largely congruent with the evolution of the bacterial groups that encode complex I. A notable exception includes the gammaproteobacteria, whose members encode one of two distantly related complex I enzymes predicted to participate in different types of respiratory chains (aerobic versus anaerobic). Comparative genomic analyses suggest a broad role for complex I in reoxidizing NADH produced from various catabolic reactions, including the tricarboxylic acid (TCA) cycle and fatty acid beta-oxidation. Together, these findings suggest diverse roles for complex I across bacteria and highlight the importance of this enzyme in shaping diverse physiologies across the bacterial domain. PMID:25873378

  1. Higher activity of polymorphic NAD(P)H:quinone oxidoreductase in liver cytosols from blacks compared to whites.

    PubMed

    Covarrubias, Vanessa Gonzalez; Lakhman, Sukhwinder S; Forrest, Alan; Relling, Mary V; Blanco, Javier G

    2006-07-14

    In human liver, the two-electron reduction of quinone compounds, such as menadione is catalyzed by cytosolic carbonyl reductase (CBR) and NAD(P)H:quinone oxidoreductase (NQO1) activities. We assessed the relative contributions of CBR and NQO1 activities to the total menadione reducing capacity in liver cytosols from black (n=31) and white donors (n=63). Maximal menadione reductase activities did not differ between black (13.0+/-5.0 nmol/min mg), and white donors (11.4+/-6.6 nmol/min mg; p=0.208). In addition, both groups presented similar levels of CBR activities (CBR(blacks)=10.9+/-4.1 nmol/min mg) versus CBR(whites)=10.5+/-5.8 nmol/min mg; p=0.708). In contrast, blacks showed higher NQO1 activities (two-fold) than whites (NQO1(blacks)=2.1+/-3.0 nmol/min mg versus NQO1(whites)=0.9+/-1.6 nmol/min mg, p<0.01). To further explore this disparity, we tested whether NQO1 activity was associated with the common NQO1(*)2 genetic polymorphism by using paired DNA samples for genotyping. Cytosolic NQO1 activities differed significantly by NQO1 genotype status in whites (NQO1(whites[NQO1*1/*1])=1.3+/-1.7 nmol/min mg versus NQO1(whites[NQO1*1/*2+NQO1*2/*2])=0.5+/-0.7 nmol/min mg, p<0.01), but not in blacks (NQO1(blacks[NQO1*1/*1])=2.6+/-3.4 nmol/min mg versus NQO1(blacks[NQO1*1/*2])=1.1+/-1.2 nmol/min mg, p=0.134). Our findings pinpoint the presence of significant interethnic differences in polymorphic hepatic NQO1 activity.

  2. Manganese ions enhance mitochondrial H2O2 emission from Krebs cycle oxidoreductases by inducing permeability transition.

    PubMed

    Bonke, Erik; Siebels, Ilka; Zwicker, Klaus; Dröse, Stefan

    2016-10-01

    Manganese-induced toxicity has been linked to mitochondrial dysfunction and an increased generation of reactive oxygen species (ROS). We could recently show in mechanistic studies that Mn(2+) ions induce hydrogen peroxide (H2O2) production from the ubiquinone binding site of mitochondrial complex II (IIQ) and generally enhance H2O2 formation by accelerating the rate of superoxide dismutation. The present study with intact mitochondria reveals that manganese additionally enhances H2O2 emission by inducing mitochondrial permeability transition (mPT). In mitochondria fed by NADH-generating substrates, the combination of Mn(2+) and different respiratory chain inhibitors led to a dynamically increasing H2O2emission which was sensitive to the mPT inhibitor cyclosporine A (CsA) as well as Ru-360, an inhibitor of the mitochondrial calcium uniporter (MCU). Under these conditions, flavin-containing enzymes of the mitochondrial matrix, e.g. the mitochondrial 2-oxoglutaratedehydrogenase (OGDH), were major sources of ROS. With succinate as substrate, Mn(2+) stimulated ROS production mainly at complex II, whereby the applied succinate concentration had a marked effect on the tendency for mPT. Also Ca(2+) increased the rate of H2O2 emission by mPT, while no direct effect on ROS-production of complex II was observed. The present study reveals a complex scenario through which manganese affects mitochondrial H2O2 emission: stimulating its production from distinct sites (e.g. site IIQ), accelerating superoxide dismutation and enhancing the emission via mPT which also leads to the loss of soluble components of the mitochondrial antioxidant systems and favors the ROS production from flavin-containing oxidoreductases of the Krebs cycle.

  3. NAD(P)H: Quinone Oxidoreductase 1 Deficiency Conjoint with Marginal Vitamin C Deficiency Causes Cigarette Smoke Induced Myelodysplastic Syndromes

    PubMed Central

    Das, Archita; Dey, Neekkan; Ghosh, Arunava; Das, Tanusree; Chatterjee, Indu B.

    2011-01-01

    Background The etiology of myelodysplastic syndromes (MDS) is largely unknown. Exposure to cigarette smoke (CS) is reported to be associated with MDS risk. There is inconsistent evidence that deficiency of NAD(P)H-quinone: oxidoreductase 1 (NQO1) increases the risk of MDS. Earlier we had shown that CS induces toxicity only in marginal vitamin C-deficient guinea pigs but not in vitamin C-sufficient ones. We therefore considered that NQO1 deficiency along with marginal vitamin C deficiency might produce MDS in CS-exposed guinea pigs. Methodology and Principal Findings Here we show that CS exposure for 21 days produces MDS in guinea pigs having deficiency of NQO1 (fed 3 mg dicoumarol/day) conjoint with marginal vitamin C deficiency (fed 0.5 mg vitamin C/day). As evidenced by morphology, histology and cytogenetics, MDS produced in the guinea pigs falls in the category of refractory cytopenia with unilineage dysplasia (RCUD): refractory anemia; refractory thrombocytopenia that is associated with ring sideroblasts, micromegakaryocytes, myeloid hyperplasia and aneuploidy. MDS is accompanied by increased CD34(+) cells and oxidative stress as shown by the formation of protein carbonyls and 8-oxodeoxyguanosine. Apoptosis precedes MDS but disappears later with marked decrease in the p53 protein. MDS produced in the guinea pigs are irreversible. MDS and all the aforesaid pathophysiological events do not occur in vitamin C-sufficient guinea pigs. However, after the onset of MDS vitamin C becomes ineffective. Conclusions and Significance CS exposure causes MDS in guinea pigs having deficiency of NQO1 conjoint with marginal vitamin C deficiency. The syndromes are not produced in singular deficiency of NQO1 or marginal vitamin C deficiency. Our results suggest that human smokers having NQO1 deficiency combined with marginal vitamin C deficiency are likely to be at high risk for developing MDS and that intake of a moderately large dose of vitamin C would prevent MDS. PMID:21655231

  4. Activities of Secreted Aryl Alcohol Quinone Oxidoreductases from Pycnoporus cinnabarinus Provide Insights into Fungal Degradation of Plant Biomass.

    PubMed

    Mathieu, Yann; Piumi, Francois; Valli, Richard; Aramburu, Juan Carro; Ferreira, Patricia; Faulds, Craig B; Record, Eric

    2016-04-01

    Auxiliary activities family 3 subfamily 2 (AA3_2) from the CAZy database comprises various functions related to ligninolytic enzymes, such as fungal aryl alcohol oxidases (AAO) and glucose oxidases, both of which are flavoenzymes. The recent study of the Pycnoporus cinnabarinus CIRM BRFM 137 genome combined with its secretome revealed that four AA3_2 enzymes are secreted during biomass degradation. One of these AA3_2 enzymes, scf184803.g17, has recently been produced heterologously in Aspergillus niger Based on the enzyme's activity and specificity, it was assigned to the glucose dehydrogenases (PcinnabarinusGDH [PcGDH]). Here, we analyze the distribution of the other three AA3_2 enzymes (scf185002.g8, scf184611.g7, and scf184746.g13) to assess their putative functions. These proteins showed the highest homology with aryl alcohol oxidase from Pleurotus eryngii Biochemical characterization demonstrated that they were also flavoenzymes harboring flavin adenine dinucleotide (FAD) as a cofactor and able to oxidize a wide variety of phenolic and nonphenolic aryl alcohols and one aliphatic polyunsaturated primary alcohol. Though presenting homology with fungal AAOs, these enzymes exhibited greater efficiency in reducing electron acceptors (quinones and one artificial acceptor) than molecular oxygen and so were defined as aryl-alcohol:quinone oxidoreductases (AAQOs) with two enzymes possessing residual oxidase activity (PcAAQO2 and PcAAQO3). Structural comparison of PcAAQO homology models with P. eryngii AAO demonstrated a wider substrate access channel connecting the active-site cavity to the solvent, explaining the absence of activity with molecular oxygen. Finally, the ability of PcAAQOs to reduce radical intermediates generated by laccase from P. cinnabarinus was demonstrated, shedding light on the ligninolytic system of this fungus.

  5. Activities of Secreted Aryl Alcohol Quinone Oxidoreductases from Pycnoporus cinnabarinus Provide Insights into Fungal Degradation of Plant Biomass

    PubMed Central

    Piumi, Francois; Valli, Richard; Aramburu, Juan Carro; Ferreira, Patricia; Faulds, Craig B.; Record, Eric

    2016-01-01

    Auxiliary activities family 3 subfamily 2 (AA3_2) from the CAZy database comprises various functions related to ligninolytic enzymes, such as fungal aryl alcohol oxidases (AAO) and glucose oxidases, both of which are flavoenzymes. The recent study of the Pycnoporus cinnabarinus CIRM BRFM 137 genome combined with its secretome revealed that four AA3_2 enzymes are secreted during biomass degradation. One of these AA3_2 enzymes, scf184803.g17, has recently been produced heterologously in Aspergillus niger. Based on the enzyme's activity and specificity, it was assigned to the glucose dehydrogenases (P. cinnabarinus GDH [PcGDH]). Here, we analyze the distribution of the other three AA3_2 enzymes (scf185002.g8, scf184611.g7, and scf184746.g13) to assess their putative functions. These proteins showed the highest homology with aryl alcohol oxidase from Pleurotus eryngii. Biochemical characterization demonstrated that they were also flavoenzymes harboring flavin adenine dinucleotide (FAD) as a cofactor and able to oxidize a wide variety of phenolic and nonphenolic aryl alcohols and one aliphatic polyunsaturated primary alcohol. Though presenting homology with fungal AAOs, these enzymes exhibited greater efficiency in reducing electron acceptors (quinones and one artificial acceptor) than molecular oxygen and so were defined as aryl-alcohol:quinone oxidoreductases (AAQOs) with two enzymes possessing residual oxidase activity (PcAAQO2 and PcAAQO3). Structural comparison of PcAAQO homology models with P. eryngii AAO demonstrated a wider substrate access channel connecting the active-site cavity to the solvent, explaining the absence of activity with molecular oxygen. Finally, the ability of PcAAQOs to reduce radical intermediates generated by laccase from P. cinnabarinus was demonstrated, shedding light on the ligninolytic system of this fungus. PMID:26873317

  6. Suppression of Chloroplastic Alkenal/One Oxidoreductase Represses the Carbon Catabolic Pathway in Arabidopsis Leaves during Night1[OPEN

    PubMed Central

    Ifuku, Kentaro; Ikeda, Ken-ichi; Inoue, Kanako Ikeda; Park, Pyoyun; Tamoi, Masahiro; Inoue, Hironori; Sakamoto, Katsuhiko; Saito, Ryota

    2016-01-01

    Lipid-derived reactive carbonyl species (RCS) possess electrophilic moieties and cause oxidative stress by reacting with cellular components. Arabidopsis (Arabidopsis thaliana) has a chloroplast-localized alkenal/one oxidoreductase (AtAOR) for the detoxification of lipid-derived RCS, especially α,β-unsaturated carbonyls. In this study, we aimed to evaluate the physiological importance of AtAOR and analyzed AtAOR (aor) mutants, including a transfer DNA knockout, aor (T-DNA), and RNA interference knockdown, aor (RNAi), lines. We found that both aor mutants showed smaller plant sizes than wild-type plants when they were grown under day/night cycle conditions. To elucidate the cause of the aor mutant phenotype, we analyzed the photosynthetic rate and the respiration rate by gas-exchange analysis. Subsequently, we found that both wild-type and aor (RNAi) plants showed similar CO2 assimilation rates; however, the respiration rate was lower in aor (RNAi) than in wild-type plants. Furthermore, we revealed that phosphoenolpyruvate carboxylase activity decreased and starch degradation during the night was suppressed in aor (RNAi). In contrast, the phenotype of aor (RNAi) was rescued when aor (RNAi) plants were grown under constant light conditions. These results indicate that the smaller plant sizes observed in aor mutants grown under day/night cycle conditions were attributable to the decrease in carbon utilization during the night. Here, we propose that the detoxification of lipid-derived RCS by AtAOR in chloroplasts contributes to the protection of dark respiration and supports plant growth during the night. PMID:26884484

  7. Antiparasitic drug nitazoxanide inhibits the pyruvate oxidoreductases of Helicobacter pylori, selected anaerobic bacteria and parasites, and Campylobacter jejuni.

    PubMed

    Hoffman, Paul S; Sisson, Gary; Croxen, Matthew A; Welch, Kevin; Harman, W Dean; Cremades, Nunilo; Morash, Michael G

    2007-03-01

    Nitazoxanide (NTZ) exhibits broad-spectrum activity against anaerobic bacteria and parasites and the ulcer-causing pathogen Helicobacter pylori. Here we show that NTZ is a noncompetitive inhibitor (K(i), 2 to 10 microM) of the pyruvate:ferredoxin/flavodoxin oxidoreductases (PFORs) of Trichomonas vaginalis, Entamoeba histolytica, Giardia intestinalis, Clostridium difficile, Clostridium perfringens, H. pylori, and Campylobacter jejuni and is weakly active against the pyruvate dehydrogenase of Escherichia coli. To further mechanistic studies, the PFOR operon of H. pylori was cloned and overexpressed in E. coli, and the multisubunit complex was purified by ion-exchange chromatography. Pyruvate-dependent PFOR activity with NTZ, as measured by a decrease in absorbance at 418 nm (spectral shift from 418 to 351 nm), unlike the reduction of viologen dyes, did not result in the accumulation of products (acetyl coenzyme A and CO(2)) and pyruvate was not consumed in the reaction. NTZ did not displace the thiamine pyrophosphate (TPP) cofactor of PFOR, and the 351-nm absorbing form of NTZ was inactive. Optical scans and (1)H nuclear magnetic resonance analyses determined that the spectral shift (A(418) to A(351)) of NTZ was due to protonation of the anion (NTZ(-)) of the 2-amino group of the thiazole ring which could be generated with the pure compound under acidic solutions (pK(a) = 6.18). We propose that NTZ(-) intercepts PFOR at an early step in the formation of the lactyl-TPP transition intermediate, resulting in the reversal of pyruvate binding prior to decarboxylation and in coordination with proton transfer to NTZ. Thus, NTZ might be the first example of an antimicrobial that targets the "activated cofactor" of an enzymatic reaction rather than its substrate or catalytic sites, a novel mechanism that may escape mutation-based drug resistance.

  8. Structural and Biochemical Characterization of Chlamydia trachomatis DsbA Reveals a Cysteine-Rich and Weakly Oxidising Oxidoreductase

    PubMed Central

    Byriel, Karl; Huston, Wilhelmina M.; Furlong, Emily; Heras, Begoña; Martin, Jennifer L.

    2016-01-01

    The Gram negative bacteria Chlamydia trachomatis is an obligate intracellular human pathogen that can cause pelvic inflammatory disease, infertility and blinding trachoma. C. trachomatis encodes a homolog of the dithiol oxidoreductase DsbA. Bacterial DsbA proteins introduce disulfide bonds to folding proteins providing structural bracing for secreted virulence factors, consequently these proteins are potential targets for antimicrobial drugs. Despite sharing functional and structural characteristics, the DsbA enzymes studied to date vary widely in their redox character. In this study we show that the truncated soluble form of the predicted membrane anchored protein C. trachomatis DsbA (CtDsbA) has oxidase activity and redox properties broadly similar to other characterized DsbA proteins. However CtDsbA is distinguished from other DsbAs by having six cysteines, including a second disulfide bond, and an unusual dipeptide sequence in its catalytic motif (Cys-Ser-Ala-Cys). We report the 2.7 Å crystal structure of CtDsbA revealing a typical DsbA fold, which is most similar to that of DsbA-II type proteins. Consistent with this, the catalytic surface of CtDsbA is negatively charged and lacks the hydrophobic groove found in EcDsbA and DsbAs from other enterobacteriaceae. Biochemical characterization of CtDsbA reveals it to be weakly oxidizing compared to other DsbAs and with only a mildly destabilizing active site disulfide bond. Analysis of the crystal structure suggests that this redox character is consistent with a lack of contributing factors to stabilize the active site nucleophilic thiolate relative to more oxidizing DsbA proteins. PMID:28030602

  9. Application of nanodisc technology for direct electrochemical investigation of plant cytochrome P450s and their NADPH P450 oxidoreductase

    PubMed Central

    Bavishi, Krutika; Laursen, Tomas; Martinez, Karen L.; Møller, Birger Lindberg; Della Pia, Eduardo Antonio

    2016-01-01

    Direct electrochemistry of cytochrome P450 containing systems has primarily focused on investigating enzymes from microbes and animals for bio-sensing applications. Plant P450s receive electrons from NADPH P450 oxidoreductase (POR) to orchestrate the bio-synthesis of a plethora of commercially valuable compounds. In this report, full length CYP79A1, CYP71E1 and POR of the dhurrin pathway in Sorghum bicolor were reconstituted individually in nanoscale lipid patches, “nanodiscs”, and directly immobilized on unmodified gold electrodes. Cyclic voltammograms of CYP79A1 and CYP71E1 revealed reversible redox peaks with average midpoint potentials of 80 ± 5 mV and 72 ± 5 mV vs. Ag/AgCl, respectively. POR yielded two pairs of redox peaks with midpoint potentials of 90 ± 5 mV and −300 ± 10 mV, respectively. The average heterogeneous electron transfer rate constant was calculated to be ~1.5 s−1. POR was electro-catalytically active while the P450s generated hydrogen peroxide (H2O2). These nanodisc-based investigations lay the prospects and guidelines for construction of a simplified platform to perform mediator-free, direct electrochemistry of non-engineered cytochromes P450 under native-like conditions. It is also a prelude for driving plant P450 systems electronically for simplified and cost-effective screening of potential substrates/inhibitors and fabrication of nano-bioreactors for synthesis of high value natural products. PMID:27386958

  10. Deletion of P399{sub E}401 in NADPH cytochrome P450 oxidoreductase results in partial mixed oxidase deficiency

    SciTech Connect

    Flueck, Christa E.; Mallet, Delphine; Hofer, Gaby; Samara-Boustani, Dinane; Leger, Juliane; Polak, Michel; Morel, Yves; Pandey, Amit V.

    2011-09-09

    Highlights: {yields} Mutations in human POR cause congenital adrenal hyperplasia. {yields} We are reporting a novel 3 amino acid deletion mutation in POR P399{sub E}401del. {yields} POR mutation P399{sub E}401del decreased P450 activities by 60-85%. {yields} Impairment of steroid metabolism may be caused by multiple hits. {yields} Severity of aromatase inhibition is related to degree of in utero virilization. -- Abstract: P450 oxidoreductase (POR) is the electron donor for all microsomal P450s including steroidogenic enzymes CYP17A1, CYP19A1 and CYP21A2. We found a novel POR mutation P399{sub E}401del in two unrelated Turkish patients with 46,XX disorder of sexual development. Recombinant POR proteins were produced in yeast and tested for their ability to support steroid metabolizing P450 activities. In comparison to wild-type POR, the P399{sub E}401del protein was found to decrease catalytic efficiency of 21-hydroxylation of progesterone by 68%, 17{alpha}-hydroxylation of progesterone by 76%, 17,20-lyase action on 17OH-pregnenolone by 69%, aromatization of androstenedione by 85% and cytochrome c reduction activity by 80%. Protein structure analysis of the three amino acid deletion P399{sub E}401 revealed reduced stability and flexibility of the mutant. In conclusion, P399{sub E}401del is a novel mutation in POR that provides valuable genotype-phenotype and structure-function correlation for mutations in a different region of POR compared to previous studies. Characterization of P399{sub E}401del provides further insight into specificity of different P450s for interaction with POR as well as nature of metabolic disruptions caused by more pronounced effect on specific P450s like CYP17A1 and aromatase.

  11. Roles of bound quinone in the single subunit NADH-quinone oxidoreductase (Ndi1) from Saccharomyces cerevisiae.

    PubMed

    Yamashita, Tetsuo; Nakamaru-Ogiso, Eiko; Miyoshi, Hideto; Matsuno-Yagi, Akemi; Yagi, Takao

    2007-03-02

    To understand the biochemical basis for the function of the rotenone-insensitive internal NADH-quinone (Q) oxidoreductase (Ndi1), we have overexpressed mature Ndi1 in Escherichia coli membranes. The Ndi1 purified from the membranes contained one FAD and showed enzymatic activities comparable with the original Ndi1 isolated from Saccharomyces cerevisiae. When extracted with Triton X-100, the isolated Ndi1 did not contain Q. The Q-bound form was easily reconstituted by incubation of the Q-free Ndi1 enzyme with ubiquinone-6. We compared the properties of Q-bound Ndi1 enzyme with those of Q-free Ndi1 enzyme, with higher activity found in the Q-bound enzyme. Although both are inhibited by low concentrations of AC0-11 (IC(50) = 0.2 microm), the inhibitory mode of AC0-11 on Q-bound Ndi1 was distinct from that of Q-free Ndi1. The bound Q was slowly released from Ndi1 by treatment with NADH or dithionite under anaerobic conditions. This release of Q was prevented when Ndi1 was kept in the reduced state by NADH. When Ndi1 was incorporated into bovine heart submitochondrial particles, the Q-bound form, but not the Q-free form, established the NADH-linked respiratory activity, which was insensitive to piericidin A but inhibited by KCN. Furthermore, Ndi1 produces H(2)O(2) as isolated regardless of the presence of bound Q, and this H(2)O(2) was eliminated when the Q-bound Ndi1, but not the Q-free Ndi1, was incorporated into submitochondrial particles. The data suggest that Ndi1 bears at least two distinct Q sites: one for bound Q and the other for catalytic Q.

  12. Contribution of xanthine oxidoreductase to mammary epithelial and breast cancer cell differentiation in part modulates inhibitor of differentiation-1.

    PubMed

    Fini, Mehdi A; Monks, Jenifer; Farabaugh, Susan M; Wright, Richard M

    2011-09-01

    Loss of xanthine oxidoreductase (XOR) has been linked to aggressive breast cancer in vivo and to breast cancer cell aggressiveness in vitro. In the present study, we hypothesized that the contribution of XOR to the development of the normal mammary gland may underlie its capacity to modulate breast cancer. We contrasted in vitro and in vivo developmental systems by differentiation marker and microarray analyses. Human breast cancer microarray was used for clinical outcome studies. The role of XOR in differentiation and proliferation was examined in human breast cancer cells and in a mouse xenograft model. Our data show that XOR was required for functional differentiation of mammary epithelial cells both in vitro and in vivo. Poor XOR expression was observed in a mouse ErbB2 breast cancer model, and pharmacologic inhibition of XOR increased breast cancer tumor burden in mouse xenograft. mRNA microarray analysis of human breast cancer revealed that low XOR expression was significantly associated with time to tumor relapse. The opposing expression of XOR and inhibitor of differentiation-1 (Id1) during HC11 differentiation and mammary gland development suggested a potential functional relationship. While overexpression of Id1 inhibited HC11 differentiation and XOR expression, XOR itself modulated expression of Id1 in differentiating HC11 cells. Overexpression of XOR both inhibited Id1-induced proliferation and -stimulated differentiation of Heregulin-β1-treated human breast cancer cells. These results show that XOR is an important functional component of differentiation whose diminished expression contributes to breast cancer aggressiveness, and they support XOR as both a breast cancer biomarker and a target for pharmacologic activation in therapeutic management of aggressive breast cancer.

  13. Q-band ENDOR spectra of the Rieske protein from Rhodobacter capsulatus ubiquinol-cyctochrome c oxidoreductase show two histidines coordinated to the (2Fe-2S) cluster

    SciTech Connect

    Gurbiel, R.J. Jagiellonian Univ., Krakow ); Ohnishi, Tomoko; Robertson, D.E.; Daldal, F. ); Hoffman, B.M. )

    1991-12-10

    Electron nuclear double resonance (ENDOR) experiments were performed on {sup 14}N (natural abundance) and {sup 15}N-enriched iron-sulfur Rieske protein in the ubiquinol-cytochrome c{sub 2} oxidoreductase from Rhodobactor capsulatus. The experiments proved that two distinct nitrogenous ligands, histidines, are undoubtedly ligated to the Rieske (2Fe-2S) center. The calculations of hyperfine tensors give values similar but not identical to those of the Rieske-type cluster in phthalate dioxygenase of Pseudomonas cepacia and suggest a slightly different geometry of the iron-sulfur cluster in the two proteins.

  14. Augmenter of Liver Regeneration: Substrate Specificity of a Flavin-dependent Oxidoreductase from the Mitochondrial Intermembrane Space†

    PubMed Central

    Daithankar, Vidyadhar N.; Farrell, Scott R.; Thorpe, Colin

    2009-01-01

    Augmenter of liver regeneration (ALR) is both a growth factor and a sulfhydryl oxidase that binds FAD in an unusual helix-rich domain containing a redox-active CxxC disulfide proximal to the flavin ring. In addition to the cytokine form of ALR (sfALR) that circulates in serum, a longer form, lfALR, is believed to participate in oxidative trapping of reduced proteins entering the mitochondrial intermembrane space (IMS). This longer form has an 80-residue N-terminal extension containing an additional, distal, CxxC motif. This work presents the first enzymological characterization of human lfALR. The N-terminal region conveys no catalytic advantage towards the oxidation of the model substrate dithiothreitol (DTT). In addition, C71A or C74A mutations of the distal disulfide do not increase the turnover number towards DTT. Unlike Erv1p, the yeast homolog of lfALR, static spectrophotometric experiments of the human oxidase provide no evidence for communication between distal and proximal disulfides. An N-terminal his-tagged version of human Mia40, a resident oxidoreductase of the IMS and a putative physiological reductant of lfALR, was subcloned and expressed in Escherichia coli BL21 DE3 cells. Mia40, as isolated, shows a visible spectrum characteristic of an Fe/S center and contains 0.56 ± 0.02 atoms of iron per subunit. Treatment of Mia40 with guanidine hydrochloride and triscarboxyethylphosphine hydrochloride during purification removed this chromophore. The resulting protein, with a reduced CxC motif, was a good substrate of lfALR. However, neither sfALR, nor lfALR mutants lacking the distal disulfide, could oxidize reduced Mia40 efficiently. Thus, catalysis involves a flow of reducing equivalents from the reduced CxC motif of Mia40, to distal- and then proximal CxxC motifs of lfALR, to the flavin ring, and, finally, to cytochrome c or molecular oxygen. PMID:19397338

  15. The Na+-Translocating NADH:Quinone Oxidoreductase Enhances Oxidative Stress in the Cytoplasm of Vibrio cholerae

    PubMed Central

    Muras, Valentin; Dogaru-Kinn, Paul; Minato, Yusuke; Häse, Claudia C.

    2016-01-01

    ABSTRACT We searched for a source of reactive oxygen species (ROS) in the cytoplasm of the human pathogen Vibrio cholerae and addressed the mechanism of ROS formation using the dye 2′,7′-dichlorofluorescein diacetate (DCFH-DA) in respiring cells. By comparing V. cholerae strains with or without active Na+-translocating NADH:quinone oxidoreductase (Na+-NQR), this respiratory sodium ion redox pump was identified as a producer of ROS in vivo. The amount of cytoplasmic ROS detected in V. cholerae cells producing variants of Na+-NQR correlated well with rates of superoxide formation by the corresponding membrane fractions. Membranes from wild-type V. cholerae showed increased superoxide production activity (9.8 ± 0.6 μmol superoxide min−1 mg−1 membrane protein) compared to membranes from the mutant lacking Na+-NQR (0.18 ± 0.01 μmol min−1 mg−1). Overexpression of plasmid-encoded Na+-NQR in the nqr deletion strain resulted in a drastic increase in the formation of superoxide (42.6 ± 2.8 μmol min−1 mg−1). By analyzing a variant of Na+-NQR devoid of quinone reduction activity, we identified the reduced flavin adenine dinucleotide (FAD) cofactor of cytoplasmic NqrF subunit as the site for intracellular superoxide formation in V. cholerae. The impact of superoxide formation by the Na+-NQR on the virulence of V. cholerae is discussed. IMPORTANCE In several studies, it was demonstrated that the Na+-NQR in V. cholerae affects virulence in a yet unknown manner. We identified the reduced FAD cofactor in the NADH-oxidizing NqrF subunit of the Na+-NQR as the site of superoxide formation in the cytoplasm of V. cholerae. Our study provides the framework to understand how reactive oxygen species formed during respiration could participate in the regulated expression of virulence factors during the transition from aerobic to microaerophilic (intestinal) habitats. This hypothesis may turn out to be right for many other pathogens which, like V. cholerae, depend on

  16. Functional and Bioinformatics Analysis of Two Campylobacter jejuni Homologs of the Thiol-Disulfide Oxidoreductase, DsbA

    PubMed Central

    Grabowska, Anna D.; Wywiał, Ewa; Dunin-Horkawicz, Stanislaw; Łasica, Anna M.; Wösten, Marc M. S. M.; Nagy-Staroń, Anna; Godlewska, Renata; Bocian-Ostrzycka, Katarzyna; Pieńkowska, Katarzyna; Łaniewski, Paweł; Bujnicki, Janusz M.; van Putten, Jos P. M.; Jagusztyn-Krynicka, E. Katarzyna

    2014-01-01

    Background Bacterial Dsb enzymes are involved in the oxidative folding of many proteins, through the formation of disulfide bonds between their cysteine residues. The Dsb protein network has been well characterized in cells of the model microorganism Escherichia coli. To gain insight into the functioning of the Dsb system in epsilon-Proteobacteria, where it plays an important role in the colonization process, we studied two homologs of the main Escherichia coli Dsb oxidase (EcDsbA) that are present in the cells of the enteric pathogen Campylobacter jejuni, the most frequently reported bacterial cause of human enteritis in the world. Methods and Results Phylogenetic analysis suggests the horizontal transfer of the epsilon-Proteobacterial DsbAs from a common ancestor to gamma-Proteobacteria, which then gave rise to the DsbL lineage. Phenotype and enzymatic assays suggest that the two C. jejuni DsbAs play different roles in bacterial cells and have divergent substrate spectra. CjDsbA1 is essential for the motility and autoagglutination phenotypes, while CjDsbA2 has no impact on those processes. CjDsbA1 plays a critical role in the oxidative folding that ensures the activity of alkaline phosphatase CjPhoX, whereas CjDsbA2 is crucial for the activity of arylsulfotransferase CjAstA, encoded within the dsbA2-dsbB-astA operon. Conclusions Our results show that CjDsbA1 is the primary thiol-oxidoreductase affecting life processes associated with bacterial spread and host colonization, as well as ensuring the oxidative folding of particular protein substrates. In contrast, CjDsbA2 activity does not affect the same processes and so far its oxidative folding activity has been demonstrated for one substrate, arylsulfotransferase CjAstA. The results suggest the cooperation between CjDsbA2 and CjDsbB. In the case of the CjDsbA1, this cooperation is not exclusive and there is probably another protein to be identified in C. jejuni cells that acts to re-oxidize CjDsbA1. Altogether

  17. The transfer of reduced flavin mononucleotide from LuxG oxidoreductase to luciferase occurs via free diffusion.

    PubMed

    Tinikul, Ruchanok; Pitsawong, Warintra; Sucharitakul, Jeerus; Nijvipakul, Sarayut; Ballou, David P; Chaiyen, Pimchai

    2013-10-01

    Bacterial luciferase (LuxAB) is a two-component flavin mononucleotide (FMN)-dependent monooxygenase that catalyzes the oxidation of reduced FMN (FMNH(-)) and a long-chain aliphatic aldehyde by molecular oxygen to generate oxidized FMN, the corresponding aliphatic carboxylic acid, and concomitant emission of light. The LuxAB reaction requires a flavin reductase to generate FMNH(-) to serve as a luciferin in its reaction. However, FMNH(-) is unstable and can react with oxygen to generate H2O2, so that it is important to transfer it efficiently to LuxAB. Recently, LuxG has been identified as a NADH:FMN oxidoreductase that supplies FMNH(-) to luciferase in vivo. In this report, the mode of transfer of FMNH(-) between LuxG from Photobacterium leiognathi TH1 and LuxABs from both P. leiognathi TH1 and Vibrio campbellii (PlLuxAB and VcLuxAB, respectively) was investigated using single-mixing and double-mixing stopped-flow spectrophotometry. The oxygenase component of p-hydroxyphenylacetate hydroxylase (C2) from Acinetobacter baumannii, which has no structural similarity to LuxAB, was used to measure the kinetics of release of FMNH(-) from LuxG. With all FMNH(-) acceptors used (C2, PlLuxAB, and VcLuxAB), the kinetics of FMN reduction on LuxG were the same, showing that LuxG releases FMNH(-) with a rate constant of 4.5-6 s(-1). Our data showed that the kinetics of binding of FMNH(-)to PlLuxAB and VcLuxAB and the subsequent reactions with oxygen were the same with either free FMNH(-) or FMNH(-) generated in situ by LuxG. These results strongly suggest that no complexes between LuxG and the various species are necessary to transfer FMNH(-) to the acceptors. The kinetics of the overall reactions and the individual rate constants correlate well with a free diffusion model for the transfer of FMNH(-) from LuxG to either LuxAB.

  18. Modulation of Sonic hedgehog signaling and WW domain containing oxidoreductase WOX1 expression enhances radiosensitivity of human glioblastoma cells.

    PubMed

    Chiang, Ming-Fu; Chen, Hsin-Hong; Chi, Chih-Wen; Sze, Chun-I; Hsu, Ming-Ling; Shieh, Hui-Ru; Lin, Chin-Ping; Tsai, Jo-Ting; Chen, Yu-Jen

    2015-03-01

    WW domain containing oxidoreductase, designated WWOX, FOR or WOX1, is a known pro-apoptotic factor when ectopically expressed in various types of cancer cells, including glioblastoma multiforme (GBM). The activation of sonic hedgehog (Shh) signaling, especially paracrine Shh secretion in response to radiation, is associated with impairing the effective irradiation of cancer cells. Here, we examined the role of Shh signaling and WOX1 overexpression in the radiosensitivity of human GBM cells. Our results showed that ionizing irradiation (IR) increased the cytoplasmic Shh and nuclear Gli-1 content in GBM U373MG and U87MG cells. GBM cells with exogenous Shh treatment exhibited similar results. Pretreatment with Shh peptides protected U373MG and U87MG cells against IR in a dose-dependent manner. Cyclopamine, a Hedgehog/Smoothened (SMO) inhibitor, reversed the protective effect of Shh in U87MG cells. Cyclopamine increased Shh plus IR-induced H2AX, a marker of DNA double-strand breaks, in these cells. To verify the role of Shh signaling in the radiosensitivity of GBM cells, we tested the effect of the Gli family zinc finger 1 (Gli-1) inhibitor zerumbone and found that it could sensitize GBM cells to IR. We next examined the role of WOX1 in radiosensitivity. Overexpression of WOX1 enhanced the radiosensitivity of U87MG (possessing wild type p53 or WTp53) but not U373MG (harboring mutant p53 or MTp53) cells. Pretreatment with Shh peptides protected both WOX1-overexpressed U373MG and U87MG cells against IR and increased the cytoplasmic Shh and nuclear Gli-1 content. Zerumbone enhanced the radiosensitivity of WOX1-overexpressed U373MG and U87MG cells. In conclusion, overexpression of WOX1 preferentially sensitized human GBM cells possessing wild type p53 to radiation therapy. Blocking of Shh signaling may enhance radiosensitivity independently of the expression of p53 and WOX1. The crosstalk between Shh signaling and WOX1 expression in human glioblastoma warrants further

  19. The antidote effect of quinone oxidoreductase 2 inhibitor against paraquat-induced toxicity in vitro and in vivo

    PubMed Central

    Janda, Elzbieta; Parafati, Maddalena; Aprigliano, Serafina; Carresi, Cristina; Visalli, Valeria; Sacco, Iolanda; Ventrice, Domenica; Mega, Tiziana; Vadalá, Nuria; Rinaldi, Stefano; Musolino, Vincenzo; Palma, Ernesto; Gratteri, Santo; Rotiroti, Domenicantonio; Mollace, Vincenzo

    2013-01-01

    BACKGROUND AND PURPOSE The mechanisms of paraquat (PQ)-induced toxicity are poorly understood and PQ poisoning is often fatal due to a lack of effective antidotes. In this study we report the effects of N-[2-(2-methoxy-6H-dipyrido{2,3-a:3,2-e}pyrrolizin-11-yl)ethyl]-2-furamide (NMDPEF), a melatonin-related inhibitor of quinone oxidoreductase2 (QR2) on the toxicity of PQ in vitro & in vivo. EXPERIMENTAL APPROACH Prevention of PQ-induced toxicity was tested in different cells, including primary pneumocytes and astroglial U373 cells. Cell death and reactive oxygen species (ROS) were analysed by flow cytometry and fluorescent probes. QR2 silencing was achieved by lentiviral shRNAs. PQ (30 mg·kg−1) and NMDPEF were administered i.p. to Wistar rats and animals were monitored for 28 days. PQ toxicity in the substantia nigra (SN) was tested by a localized microinfusion and electrocorticography. QR2 activity was measured by fluorimetry of N-benzyldihydronicotinamide oxidation. KEY RESULTS NMDPEF potently antagonized non-apoptotic PQ-induced cell death, ROS generation and inhibited cellular QR2 activity. In contrast, the cytoprotective effect of melatonin and apocynin was limited and transient compared with NMDPEF. Silencing of QR2 attenuated PQ-induced cell death and reduced the efficacy of NMDPEF. Significantly, NMDPEF (4.5 mg·kg−1) potently antagonized PQ-induced systemic toxicity and animal mortality. Microinfusion of NMDPEF into SN prevented severe behavioural and electrocortical effects of PQ which correlated with inhibition of malondialdehyde accumulation in cells and tissues. CONCLUSIONS AND IMPLICATIONS NMDPEF protected against PQ-induced toxicity in vitro and in vivo, suggesting a key role for QR2 in the regulation of oxidative stress. LINKED ARTICLE This article is commented on by Baltazar et al., pp. 44–45 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.02017.x PMID:22289031

  20. The role of human xanthine oxidoreductase (HXOR), anti-HXOR antibodies, and microorganisms in synovial fluid of patients with joint inflammation.

    PubMed

    Al-Muhtaseb, Najah; Al-Kaissi, Elham; Thawaini, Abdul Jalil; Eldeen, Zuhair Muhi; Al-Muhtaseb, Sabah; Al-Saleh, Badiee

    2012-08-01

    This work is to investigate the levels of human xanthine oxidoreductase (HXOR), its antibodies, and microorganisms in synovial fluid of patients with untreated rheumatoid joint diseases. Synovial fluids were collected from sixty-four patients with rheumatoid joint diseases. Sixty-four age-matched individuals were included as control. Xanthine oxidoreductase (XOR) proteins level and anti-XOR antibodies were determined in the blood and synovial fluid, using human XOR as antigen, by enzyme-linked immunosorbent (ELISA) assay. Synovial fluids were cultured for bacteria and fungi. The titers of XOR protein in the synovial fluid of patients with rheumatoid arthritis were 90.43 ± 23.37 μg/ml (mean ± SD, n = 29) and up to 62.42 ± 8.74 μg/ml (mean ± SD, n = 35) in other joint inflammation. Anti-HXOR antibodies titers in patients were 167.72 ± 23.64 μg/ml, n = 64, which was significantly higher in rheumatoid arthritis patients. The results indicated that anti-HXOR antibodies in synovial fluids have a protective role as high concentrations against XOR were detected in inflammatory arthritis. These antibodies play a role in eliminating XOR from synovial fluids. However, immune complex formation could activate complement and participate in propagating the inflammatory cycle. Synovial aspirate ordinary microbial cultures were negative for any bacteria or fungi, but that does not exclude organisms of special culture requirements.

  1. A thiol-disulfide oxidoreductase of the Gram-positive pathogen Corynebacterium diphtheriae is essential for viability, pilus assembly, toxin production and virulence.

    PubMed

    Reardon-Robinson, Melissa E; Osipiuk, Jerzy; Jooya, Neda; Chang, Chungyu; Joachimiak, Andrzej; Das, Asis; Ton-That, Hung

    2015-12-01

    The Gram-positive pathogen Corynebacterium diphtheriae exports through the Sec apparatus many extracellular proteins that include the key virulence factors diphtheria toxin and the adhesive pili. How these proteins attain their native conformations after translocation as unfolded precursors remains elusive. The fact that the majority of these exported proteins contain multiple cysteine residues and that several membrane-bound oxidoreductases are encoded in the corynebacterial genome suggests the existence of an oxidative protein-folding pathway in this organism. Here we show that the shaft pilin SpaA harbors a disulfide bond in vivo and alanine substitution of these cysteines abrogates SpaA polymerization and leads to the secretion of degraded SpaA peptides. We then identified a thiol-disulfide oxidoreductase (MdbA), whose structure exhibits a conserved thioredoxin-like domain with a CPHC active site. Remarkably, deletion of mdbA results in a severe temperature-sensitive cell division phenotype. This mutant also fails to assemble pilus structures and is greatly defective in toxin production. Consistent with these defects, the ΔmdbA mutant is attenuated in a guinea pig model of diphtheritic toxemia. Given its diverse cellular functions in cell division, pilus assembly and toxin production, we propose that MdbA is a component of the general oxidative folding machine in C. diphtheriae.

  2. Type-II NADH:quinone oxidoreductase from Staphylococcus aureus has two distinct binding sites and is rate limited by quinone reduction.

    PubMed

    Sena, Filipa V; Batista, Ana P; Catarino, Teresa; Brito, José A; Archer, Margarida; Viertler, Martin; Madl, Tobias; Cabrita, Eurico J; Pereira, Manuela M

    2015-10-01

    A prerequisite for any rational drug design strategy is understanding the mode of protein-ligand interaction. This motivated us to explore protein-substrate interaction in Type-II NADH:quinone oxidoreductase (NDH-2) from Staphylococcus aureus, a worldwide problem in clinical medicine due to its multiple drug resistant forms. NDHs-2 are involved in respiratory chains and recognized as suitable targets for novel antimicrobial therapies, as these are the only enzymes with NADH:quinone oxidoreductase activity expressed in many pathogenic organisms. We obtained crystal and solution structures of NDH-2 from S. aureus, showing that it is a dimer in solution. We report fast kinetic analyses of the protein and detected a charge-transfer complex formed between NAD(+) and the reduced flavin, which is dissociated by the quinone. We observed that the quinone reduction is the rate limiting step and also the only half-reaction affected by the presence of HQNO, an inhibitor. We analyzed protein-substrate interactions by fluorescence and STD-NMR spectroscopies, which indicate that NADH and the quinone bind to different sites. In summary, our combined results show the presence of distinct binding sites for the two substrates, identified quinone reduction as the rate limiting step and indicate the establishment of a NAD(+)-protein complex, which is released by the quinone.

  3. Biotransformation of pineapple juice sugars into dietetic derivatives by using a cell free oxidoreductase from Zymomonas mobilis together with commercial invertase.

    PubMed

    Aziz, M G; Michlmayr, H; Kulbe, K D; Del Hierro, A M

    2011-01-05

    An easy procedure for cell free biotransformation of pineapple juice sugars into dietetic derivatives was accomplished using a commercial invertase and an oxidoreductase from Zymomonas mobilis. First, pineapple juice sucrose was quantitatively converted into glucose and fructose by invertase, thus increasing the concentration of each monosaccharide in the original juice to almost twice. In a second step, glucose-fructose oxidoreductase (GFOR) transformed glucose into gluconolactone, and fructose into the low calorie sweetener sorbitol. The advantage of using GFOR is simultaneous reduction of fructose and oxidation of glucose, allowing the continuous regeneration of the essential coenzyme NADP(H), that is tightly bound to the enzyme. The yield of GFOR catalyzed sugar conversion depends on initial pH and control of pH during the reaction. At optimal conditions (pH control at 6.2) a maximum of 80% (w/v) sugar conversion was obtained. Without pH control, GFOR is inactivated rapidly due to gluconic acid formation. Therefore, conversion yields are relatively low at the natural pH of pineapple juice. The application of this process might be more advantageous on juices of other tropical fruits (papaya, jackfruit, mango) due to their naturally given higher pH.

  4. A Mutation in the Flavin Adenine Dinucleotide-Dependent Oxidoreductase FOXRED1 Results in Cell-Type-Specific Assembly Defects in Oxidative Phosphorylation Complexes I and II

    PubMed Central

    Zurita Rendón, Olga; Antonicka, Hana; Horvath, Rita

    2016-01-01

    Complex I (NADH ubiquinone oxidoreductase) is a large multisubunit enzyme that catalyzes the first step in oxidative phosphorylation (OXPHOS). In mammals, complex I biogenesis occurs in a stepwise manner, a process that requires the participation of several nucleus-encoded accessory proteins. The FAD-dependent oxidoreductase-containing domain 1 (FOXRED1) protein is a complex I assembly factor; however, its specific role in the assembly pathway remains poorly understood. We identified a homozygous missense mutation, c.1308 G→A (p.V421M) in FOXRED1 in a patient who presented with epilepsy and severe psychomotor retardation. A patient myoblast line showed a severe reduction in complex I, associated with the accumulation of subassemblies centered around ∼340 kDa, and a milder decrease in complex II, all of which were rescued by retroviral expression of wild-type FOXRED1. Two additional assembly factors, AIFM1 and ACAD9, coimmunoprecipitated with FOXRED1, and all were associated with a 370-kDa complex I subassembly that, together with a 315-kDa subassembly, forms the 550-kDa subcomplex. Loss of FOXRED1 function prevents efficient formation of this midassembly subcomplex. Although we could not identify subassemblies of complex II, our results establish that FOXRED1 function is both broader than expected, involving the assembly of two flavoprotein-containing OXPHOS complexes, and cell type specific. PMID:27215383

  5. Directed Evolution and Resolution Mechanism of 1, 3-Propanediol Oxidoreductase from Klebsiella pneumoniae toward Higher Activity by Error-Prone PCR and Bioinformatics.

    PubMed

    Jiang, Wei; Zhuang, Yuan; Wang, Shizhen; Fang, Baishan

    2015-01-01

    1, 3-propanediol oxidoreductase (PDOR) is a key enzyme in glycerol bioconversion to 1,3-propanediol (1, 3-PD) which is a valuable chemical and one of the six new petrochemical products. We used error-prone PCR and activity screening to identify mutants of Klebsiella pneumoniae (K. pneumoniae) PDOR with improved activity. The activity of one of the identified mutants, PDOR'-24, which includes a single mutation, A199S, was 48 U/mg, 4.9 times that of the wild-type enzyme. Molecular docking was performed to analyze the identified mutants; and amino acids S103, H271, N366, D106, N262 and D364 were predicted to bond with NADH. The origins of the improved activity of PDOR'-24, as well as three other mutants were analyzed by simulating the interaction mechanism of the mutants with the substrate and coenzyme, respectively. This research provides useful information about the use of safranine O plate screening for the directed evolution of oxidoreductases, identifies interesting sites for improving PDOR activity, and demonstrates the utility of using molecular docking to analyze the interaction mechanism of the mutants with the substrate and coenzyme, respectively.

  6. A region of the C-terminal part of the 11-kDa subunit of ubiquinol-cytochrome-c oxidoreductase of the yeast Saccharomyces cerevisiae contributes to the structure of the Qout reaction domain.

    PubMed

    Hemrika, W; Berden, J A; Grivell, L A

    1993-08-01

    QCR8, the gene encoding the 11-kDa subunit of ubiquinol-cytochrome-c oxidoreductase of the yeast Saccharomyces cerevisiae has been resequenced in the course of a search for mutants disturbed in subunit function. Resequencing shows that the previously published sequence [Maarse A.C. & Grivell L.A. (1987) Eur. J. Biochem 155, 419-425] lacks a C at position 185 of the coding sequence. As a result of this extra nucleotide, the reading frame now contains 285 base pairs and it codes for a protein of 94 amino acids with a calculated molecular mass of 11.0 kDa. Despite the altered C-terminus, similarity to the corresponding beef heart subunit is not significantly altered. One mutant (LTN1), arising from hydroxylamine mutagenesis, has been studied in detail: Assembly of the enzyme appears to be normal, as judged from the levels of the subunits observed in Western blots, while spectral analysis showed that only holo-cytochrome b was lowered to 70% of that of the wildtype. Measurement of the specific activity and calculation of the turnover number of the enzyme showed that these were 45% and 56% of that of the wild type, respectively. Further analysis of the mutant showed that the affinity for the inhibitor myxothiazol was decreased, that the 11-kDa subunit stabilises the enzyme once assembly has occurred, and that the reduction of cytochrome b via the Qout site is impaired. Sequence analysis showed that this mutant carries a deletion of 12 nucleotides at position 206-217 of the coding sequence, resulting in the replacement of residues 69-73 (WWKNG) by a cysteine. These results are discussed in terms of the 11-kDa subunit contributing to the conformation of the Qout binding domain.

  7. Modulation of Biofilm-Formation in Salmonella enterica Serovar Typhimurium by the Periplasmic DsbA/DsbB Oxidoreductase System Requires the GGDEF-EAL Domain Protein STM3615

    PubMed Central

    Römling, Ute; Rhen, Mikael

    2014-01-01

    In Salmonella enterica serovar Typhimurium (S. Typhimurium), biofilm-formation is controlled by the cytoplasmic intracellular small-molecular second messenger cyclic 3′, 5′-di- guanosine monophosphate (c-di-GMP) through the activities of GGDEF and EAL domain proteins. Here we describe that deleting either dsbA or dsbB, respectively encoding a periplasmic protein disulfide oxidase and a cytoplasmic membrane disulfide oxidoreductase, resulted in increased biofilm-formation on solid medium. This increased biofilm-formation, defined as a red, dry and rough (rdar) colony morphotype, paralleled with enhanced expression of the biofilm master regulator CsgD and the biofilm-associated fimbrial subunit CsgA. Deleting csgD in either dsb mutant abrogated the enhanced biofilm-formation. Likewise, overexpression of the c-di-GMP phosphodiesterase YhjH, or mutationally inactivating the CsgD activator EAL-domain protein YdiV, reduced biofilm-formation in either of the dsb mutants. Intriguingly, deleting the GGDEF-EAL domain protein gene STM3615 (yhjK), previously not connected to rdar morphotype development, also abrogated the escalated rdar morphotype formation in dsb mutant backgrounds. Enhanced biofilm-formation in dsb mutants was furthermore annulled by exposure to the protein disulfide catalyst copper chloride. When analyzed for the effect of exogenous reducing stress on biofilm-formation, both dsb mutants initially showed an escalated rdar morphotype development that later dissolved to reveal a smooth mucoid colony morphotype. From these results we conclude that biofilm-development in S. Typhimurium is affected by periplasmic protein disulphide bond status through CsgD, and discuss the involvement of selected GGDEF/EAL domain protein(s) as signaling mediators. PMID:25153529

  8. 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.

  9. Physiological properties of a mutant of Pachysolen tannophilus deficient in NADPH-dependent D-xylose reductase

    SciTech Connect

    Schneider, H.; James, A.P. ); Lee, Hung; Barbosa, M. De F.S. Univ. of Guelph, Ontario ); Kubicek, C.P. )

    1989-11-01

    A D-xylose reductase mutant of Pachysolen tannophilus was isolated on the basis of its poor growth on D-xylose but normal growth on xylitol and D-glucose. Fractionation of cell extracts indicated that the mutant was deficient in D-cylose reductase activity that used NADPH exclusively as a cofactor, but not in activity that used both NADH and NADPH. Mutant cultures grown on D-xylose as the sole carbon source exhibited some properties that would be desired in improved strains. Growth rate, growth yield, and D-xylose consumption rate of the mutant were less sensitive than those of the wild type to changes in aeration rate. D-Xylose was utilized more efficiently in that less of a by-product, xylitol, was produced. In addition, under low aeration conditions, more ethanol was produced. A disadvantage was a relatively slow rate of D-xylose utilization.

  10. 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

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

    SciTech Connect

    Chung, Daehwan; Verbeke, Tobin J.; Cross, Karissa L.; Westpheling, Janet; Elkins, James G.

    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 component in making biofuels production from lignocellulosic biomass feasible.

  12. Aqueous soluble tetrazolium/formazan MTS as an indicator of NADH- and NADPH-dependent dehydrogenase activity.

    PubMed

    Dunigan, D D; Waters, S B; Owen, T C

    1995-10-01

    Recently a new tetrazolium was described for the use of monitoring cell viability in culture. This tetrazolium, commonly referred to as MTS [3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt], has the unusual property that it can be reduced to a water-soluble formazan. beta-Nicotinamide adenine dinucleotide/reduced (NADH) and beta-nicotinamide adenine dinucleotide phosphate/reduced (NADPH) are examples of physiologically important reducing agents. In cell-free studies, MTS was reduce to the soluble formazan in the presence of NADH and NADPH, and reaction were compared to those with dithiothreitol (DTT) or 2-mercaptoethanol (2-ME). The efficiency of these reactions was enhanced 1000-fold by the presence of phenazine methosulfate. Selectivity in the electron transfer from NADPH was slightly greater than NADH, and NADPH or NADH was much greater than the thiols DTT or 2-ME. Generation of either NADH or NADPH in solution by malate dehydrogenase or isocitrate dehydrogenase, respectively, was monitored by the MTS reduction reaction. The rate of formazan formation was comparable to the formation of NADH or NADPH. This system represents a useful tool for evaluating reaction kinetics in solutions of NAD- or NADP-dependent dehydrogenase enzymes, and these reactions can be performed in typical biological buffers containing reducing agents without significant interference to the MTS/formazan system.

  13. Purification and biochemical characterization of a moderately halotolerant NADPH dependent xylose reductase from Debaryomyces nepalensis NCYC 3413.

    PubMed

    Kumar, Sawan; Gummadi, Sathyanarayana N

    2011-10-01

    A Xylose reductase (XR) from the halotolerant yeast, Debaryomyces nepalensis NCYC 3413 was purified to apparent homogeneity. The enzyme has a molecular mass of 74 kDa with monomeric subunit of 36.4 kDa (MALDI-TOF/MS) and pI of 6.0. The enzyme exhibited its maximum activity at pH 7.0 and 45 °C (21.2U/mg). In situ gel digestion and peptide mass fingerprinting analysis showed 12-22% sequence homology with XR from other yeasts. Inhibition of the enzyme by DEPC (diethylpyrocarbonate) confirmed the presence of histidine residue in its active site. The enzyme exhibited high preference for pentoses over hexoses with greater catalytic efficiency for arabinose than xylose. The enzyme also showed absolute specificity with NADPH over NADH. The enzyme retained 90% activity with 100 mM of NaCl or KCl and 40% activity with 1 M KCl which suggest that the enzyme is moderately halotolerant and can be utilized for commercial production of xylitol under conditions where salts are present.

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

    SciTech Connect

    Moon, Jeong Chan; Lee, Sangmin; Shin, Su Young; Chae, Ho Byoung; Jung, Young Jun; Jung, Hyun Suk; Lee, Kyun Oh; Lee, Jung Ro; Lee, Sang Yeol

    2015-08-07

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

  15. Eicosanoids up-regulate production of reactive oxygen species by NADPH-dependent oxidase in Spodoptera exigua phagocytic hemocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Eicosanoids mediate cellular immune responses in insects, including phagocytosis of invading microbes. Phagocytosis entails two major steps, the internalization of microbes and the subsequent killing of them via formation of reactive oxygen species (ROS). Here, we posed the hypothesis that eicosanoi...

  16. The subcellular particulate NADPH-dependent O2.(-)-generating oxidase from human blood monocytes: comparison to the neutrophil system.

    PubMed

    Chaudhry, A N; Santinga, J T; Gabig, T G

    1982-10-01

    Highly purified preparations of normal human monocytes obtained from peripheral blood were shown to contain a subcellular particulate O2.(-)-generating oxidase system. This O2.(-)-generating activity was present in particulate preparations from monocytes that had been previously stimulated with phorbol myristate acetate but was low or absent in control preparations from unstimulated monocytes or stimulated monocytes from a patient with chronic granulomatous disease. In the stimulated preparations from normal monocytes, O2.(-)-generation was linearly proportional to cell protein concentration, insensitive to inhibition by azide, and dependent on NADPH as substrate. These characteristics are similar to the O2.(-)-generating oxidase system from human neutrophils. A significant difference in the apparent Km for NADPH was shown between preparations from stimulated monocytes and neutrophils (monocyte 83 +/- 16 microM, neutrophil 31 +/- 5 microM, mean +/- SE). Additionally, affinity of the stimulated monocyte particulate preparation for NADH was unmeasurably low.

  17. Nanosecond ligand migration and functional protein relaxation in ba3 oxidoreductase: Structures of the B0, B1 and B2 intermediate states.

    PubMed

    Nicolaides, Antonis; Soulimane, Tewfik; Varotsis, Constantinos

    2016-09-01

    Nanosecond time-resolved step-scan FTIR spectroscopy (nTRS (2) -FTIR) has been applied to literally probe the active site of the carbon monoxide (CO)-bound thermophilic ba3 heme-copper oxidoreductase as it executes its function. The nTRS (2) - snapshots of the photolysed heme a3 Fe-CO/CuB species captured a "transition state" whose side chains prevent the photolysed CO to enter the docking cavity. There are three sets of ba3 photoproduct bands of docked CO with different orientation exhibiting different kinetics. The trajectories of the "docked" CO at 2122, 2129 and 2137cm(-1) is referred to in the literature as B2, B1 and B0 intermediate states, respectively. The present data provided direct evidence for the role of water in controlling ligand orientation in an intracavity protein environment.

  18. Crystallization and preliminary analysis of the NqrA and NqrC subunits of the Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio cholerae

    PubMed Central

    Vohl, Georg; Nedielkov, Ruslan; Claussen, Björn; Casutt, Marco S.; Vorburger, Thomas; Diederichs, Kay; Möller, Heiko M.; Steuber, Julia; Fritz, Günter

    2014-01-01

    The Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio cholerae is a membrane protein complex consisting of six different subunits NqrA–NqrF. The major domains of the NqrA and NqrC subunits were heterologously expressed in Escherichia coli and crystallized. The structure of NqrA1–377 was solved in space groups C2221 and P21 by SAD phasing and molecular replacement at 1.9 and 2.1 Å resolution, respectively. NqrC devoid of the transmembrane helix was co-expressed with ApbE to insert the flavin mononucleotide group covalently attached to Thr225. The structure was determined by molecular replacement using apo-NqrC of Parabacteroides distasonis as search model at 1.8 Å resolution. PMID:25005105

  19. Light-independent and light-dependent protochlorophyllide-reducing activities and two distinct NADPH-protochlorophyllide oxidoreductase polypeptides in mountain pine (Pinus mugo).

    PubMed

    Forreiter, C; Apel, K

    1993-01-01

    Lower plants and gymnosperms synthesize chlorophyll and develop photosynthetically competent chloroplasts even when grown in the dark. In cell-free extracts of pine (Pinus mugo, Turra, ssp. mugo) seedlings, light-independent and light-dependent protochlorophyllide-reducing activities are present. Two distinct NADPH-protochlorophyllide-oxidoreductase (POR) polypeptides can be detected immunologically with an antiserum raised against the POR of barley. The subcellular localization and amounts of the two POR polypeptides are differentially affected by light: one of them is predominantly present in prolamellar bodies of etiochloroplasts and its abundance rapidly declines once the pine seedlings are exposed to light; the other is found in thylakoid membranes and its amount does not change during illumination of dark-grown seedlings. Two types of cDNA sequences are identified that encode two distinct POR polypeptides in pine. The relevance of these POR polypeptides for the two chlorophyll biosynthetic pathways active in gymnosperms is discussed.

  20. Wiring of the aldehyde oxidoreductase PaoABC to electrode surfaces via entrapment in low potential phenothiazine-modified redox polymers.

    PubMed

    Pinyou, Piyanut; Ruff, Adrian; Pöller, Sascha; Alsaoub, Sabine; Leimkühler, Silke; Wollenberger, Ulla; Schuhmann, Wolfgang

    2016-06-01

    Phenothiazine-modified redox hydrogels were synthesized and used for the wiring of the aldehyde oxidoreductase PaoABC to electrode surfaces. The effects of the pH value and electrode surface modification on the biocatalytic activity of the layers were studied in the presence of vanillin as the substrate. The enzyme electrodes were successfully employed as bioanodes in vanillin/O2 biofuel cells in combination with a high potential bilirubin oxidase biocathode. Open circuit voltages of around 700 mV could be obtained in a two compartment biofuel cell setup. Moreover, the use of a rather hydrophobic polymer with a high degree of crosslinking sites ensures the formation of stable polymer/enzyme films which were successfully used as bioanode in membrane-less biofuel cells.

  1. Time-lapse anomalous X-ray diffraction shows how Fe2+ substrate ions move through ferritin protein nanocages to oxidoreductase sites

    PubMed Central

    Pozzi, Cecilia; Di Pisa, Flavio; Lalli, Daniela; Rosa, Camilla; Theil, Elizabeth; Turano, Paola; Mangani, Stefano

    2015-01-01

    Ferritin superfamily protein cages reversibly synthesize internal biominerals, Fe2O3·H2O. Fe2+ and O2 (or H2O2) substrates bind at oxidoreductase sites in the cage, initiating biomineral synthesis to concentrate iron and prevent potentially toxic reactions products from Fe2+and O2 or H2O2 chemistry. By freezing ferritin crystals of Rana catesbeiana ferritin M (RcMf) at different time intervals after exposure to a ferrous salt, a series of high-resolution anomalous X-ray diffraction data sets were obtained that led to crystal structures that allowed the direct observation of ferrous ions entering, moving along and binding at enzyme sites in the protein cages. The ensemble of crystal structures from both aerobic and anaerobic conditions provides snapshots of the iron substrate bound at different cage locations that vary with time. The observed differential occupation of the two iron sites in the enzyme oxidoreductase centre (with Glu23 and Glu58, and with Glu58, His61 and Glu103 as ligands, respectively) and other iron-binding sites (with Glu53, His54, Glu57, Glu136 and Asp140 as ligands) reflects the approach of the Fe2+ substrate and its progression before the enzymatic cycle 2Fe2+ + O2 → Fe3+—O—O—Fe3+ → Fe3+—O(H)—Fe3+ and turnover. The crystal structures also revealed different Fe2+ coordination compounds bound to the ion channels located at the threefold and fourfold symmetry axes of the cage. PMID:25849404

  2. Time-lapse anomalous X-ray diffraction shows how Fe(2+) substrate ions move through ferritin protein nanocages to oxidoreductase sites.

    PubMed

    Pozzi, Cecilia; Di Pisa, Flavio; Lalli, Daniela; Rosa, Camilla; Theil, Elizabeth; Turano, Paola; Mangani, Stefano

    2015-04-01

    Ferritin superfamily protein cages reversibly synthesize internal biominerals, Fe2O3·H2O. Fe(2+) and O2 (or H2O2) substrates bind at oxidoreductase sites in the cage, initiating biomineral synthesis to concentrate iron and prevent potentially toxic reactions products from Fe(2+)and O2 or H2O2 chemistry. By freezing ferritin crystals of Rana catesbeiana ferritin M (RcMf) at different time intervals after exposure to a ferrous salt, a series of high-resolution anomalous X-ray diffraction data sets were obtained that led to crystal structures that allowed the direct observation of ferrous ions entering, moving along and binding at enzyme sites in the protein cages. The ensemble of crystal structures from both aerobic and anaerobic conditions provides snapshots of the iron substrate bound at different cage locations that vary with time. The observed differential occupation of the two iron sites in the enzyme oxidoreductase centre (with Glu23 and Glu58, and with Glu58, His61 and Glu103 as ligands, respectively) and other iron-binding sites (with Glu53, His54, Glu57, Glu136 and Asp140 as ligands) reflects the approach of the Fe(2+) substrate and its progression before the enzymatic cycle 2Fe(2+) + O2 → Fe(3+)-O-O-Fe(3+) → Fe(3+)-O(H)-Fe(3+) and turnover. The crystal structures also revealed different Fe(2+) coordination compounds bound to the ion channels located at the threefold and fourfold symmetry axes of the cage.

  3. FeS/S/FeS(2) redox system and its oxidoreductase-like chemistry in the iron-sulfur world.

    PubMed

    Wang, Wei; Yang, Bin; Qu, Youpeng; Liu, Xiaoyang; Su, Wenhui

    2011-06-01

    The iron-sulfur world (ISW) theory is an intriguing prediction regarding the origin of life on early Earth. It hypothesizes that life arose as a geochemical process from inorganic starting materials on the surface of sulfide minerals in the vicinity of deep-sea hot springs. During the last two decades, many experimental studies have been carried out on this topic, and some interesting results have been achieved. Among them, however, the processes of carbon/nitrogen fixation and biomolecular assembly on the mineral surface have received an inordinate amount of attention. To the present, an abiotic model for the oxidation-reduction of intermediates participating in metabolic pathways has been ignored. We examined the oxidation-reduction effect of a prebiotic FeS/S/FeS(2) redox system on the interconversion between several pairs of α-hydroxy acids and α-keto acids (i.e., lactate/pyruvate, malate/oxaloacetate, and glycolate/glyoxylate). We found that, in the absence of FeS, elemental sulfur (S) oxidized α-hydroxy acids to form corresponding keto acids only at a temperature higher than its melting point (113°C); in the presence of FeS, such reactions occurred more efficiently through a coupled reaction mechanism, even at a temperature below the phase transition point of S. On the other hand, FeS was shown to have the capacity to reversibly reduce the keto acids. Such an oxidoreductase-like chemistry of the FeS/S/FeS(2) redox system suggests that it can determine the redox homeostasis of metabolic intermediates in the early evolutionary phase of life. The results provide a possible pathway for the development of primordial redox biochemistry in the iron-sulfur world. Key Words: Iron-sulfur world-FeS/S/FeS(2) redox system-Oxidoreductase-like chemistry. Astrobiology 11, 471-476.

  4. Homologous npdGI genes in 2,4-dinitrophenol- and 4-nitrophenol-degrading Rhodococcus spp.

    PubMed

    Heiss, Gesche; Trachtmann, Natalie; Abe, Yoshikatsu; Takeo, Masahiro; Knackmuss, Hans-Joachim

    2003-05-01

    Rhodococcus (opacus) erythropolis HL PM-1 grows on 2,4,6-trinitrophenol or 2,4-dinitrophenol (2,4-DNP) as a sole nitrogen source. The NADPH-dependent F(420) reductase (NDFR; encoded by npdG) and the hydride transferase II (HTII; encoded by npdI) of the strain were previously shown to convert both nitrophenols to their respective hydride Meisenheimer complexes. In the present study, npdG and npdI were amplified from six 2,4-DNP degrading Rhodococcus spp. The genes showed sequence similarities of 86 to 99% to the respective npd genes of strain HL PM-1. Heterologous expression of the npdG and npdI genes showed that they were involved in 2,4-DNP degradation. Sequence analyses of both the NDFRs and the HTIIs revealed conserved domains which may be involved in binding of NADPH or F(420). Phylogenetic analyses of the NDFRs showed that they represent a new group in the family of F(420)-dependent NADPH reductases. Phylogenetic analyses of the HTIIs revealed that they form an additional group in the family of F(420)-dependent glucose-6-phosphate dehydrogenases and F(420)-dependent N(5),N(10)-methylenetetrahydromethanopterin reductases. Thus, the NDFRs and the HTIIs may each represent a novel group of F(420)-dependent enzymes involved in catabolism.

  5. Cytosolic T3-binding protein modulates dynamic alteration of T3-mediated gene expression in cells.

    PubMed

    Takeshige, Keiko; Sekido, Takashi; Kitahara, Jun-ichirou; Ohkubo, Yousuke; Hiwatashi, Dai; Ishii, Hiroaki; Nishio, Shin-ichi; Takeda, Teiji; Komatsu, Mitsuhisa; Suzuki, Satoru

    2014-01-01

    μ-Crystallin (CRYM) is also known as NADPH-dependent cytosolic T3-binding protein. A study using CRYM-null mice suggested that CRYM stores triiodothyronine (T3) in tissues. We previously established CRYM-expressing cells derived from parental GH3 cells. To examine the precise regulation of T3-responsive genes in the presence of CRYM, we evaluated serial alterations of T3-responsive gene expression by changing pericellular T3 concentrations in the media. We estimated the constitutive expression of three T3-responsive genes, growth hormone (GH), deiodinase 1 (Dio1), and deiodinase 2 (Dio2), in two cell lines. Subsequently, we measured the responsiveness of these three genes at 4, 8, 16, and 24 h after adding various concentrations of T3. We also estimated the levels of these mRNAs 24 and 48 h after removing T3. The levels of constitutive expression of GH and Dio1 were low and high in C8 cells, respectively, while Dio2 expression was not significantly different between GH3 and C8 cells. When treated with T3, Dio2 expression was significantly enhanced in C8 cells, while there were no differences in GH or Dio1 expression between GH3 and C8 cell lines. In contrast, removal of T3 retained the mRNA expression of GH and Dio2 in C8 cells. These results suggest that CRYM expression increases and sustains the T3 responsiveness of genes in cells, especially with alteration of the pericellular T3 concentration. The heterogeneity of T3-related gene expression is dependent on cellular CRYM expression in cases of dynamic changes in pericellular T3 concentration.

  6. NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The nuclear factor E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) pathway responds to oxidative stress via control of the expression of several antioxidant genes. Recent efforts demonstrate that Nrf2 modulates development of adiposity and adipogenesis. However little is kno...

  7. Altered patterns of gene duplication and differential gene gain and loss in fungal pathogens

    PubMed Central

    Powell, Amy J; Conant, Gavin C; Brown, Douglas E; Carbone, Ignazio; Dean, Ralph A

    2008-01-01

    Background Duplication, followed by fixation or random loss of novel genes, contributes to genome evolution. Particular outcomes of duplication events are possibly associated with pathogenic life histories in fungi. To date, differential gene gain and loss have not been studied at genomic scales in fungal pathogens, despite this phenomenon's known importance in virulence in bacteria and viruses. Results To determine if patterns of gene duplication differed between pathogens and non-pathogens, we identified gene families across nine euascomycete and two basidiomycete species. Gene family size distributions were fit to power laws to compare gene duplication trends in pathogens versus non-pathogens. Fungal phytopathogens showed globally altered patterns of gene duplication, as indicated by differences in gene family size distribution. We also identified sixteen examples of gene family expansion and five instances of gene family contraction in pathogenic lineages. Expanded gene families included those predicted to be important in melanin biosynthesis, host cell wall degradation and transport functions. Contracted families included those encoding genes involved in toxin production, genes with oxidoreductase activity, as well as subunits of the vacuolar ATPase complex. Surveys of the functional distribution of gene duplicates indicated that pathogens show enrichment for gene duplicates associated with receptor and hydrolase activities, while euascomycete pathogens appeared to have not only these differences, but also significantly more duplicates associated with regulatory and carbohydrate binding functions. Conclusion Differences in the overall levels of gene duplication in phytopathogenic species versus non-pathogenic relatives implicate gene inventory flux as an important virulence-associated process in fungi. We hypothesize that the observed patterns of gene duplicate enrichment, gene family expansion and contraction reflect adaptation within pathogenic life

  8. An NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme responsive nanocarrier based on mesoporous silica nanoparticles for tumor targeted drug delivery in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Gayam, Srivardhan Reddy; Venkatesan, Parthiban; Sung, Yi-Ming; Sung, Shuo-Yuan; Hu, Shang-Hsiu; Hsu, Hsin-Yun; Wu, Shu-Pao

    2016-06-01

    The synthesis and characterization of an NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme responsive nanocarrier based on mesoporous silica nanoparticles (MSNPs) for on-command delivery applications has been described in this paper. Gatekeeping of MSNPs is achieved by the integration of mechanically interlocked rotaxane nanovalves on the surface of MSNPs. The rotaxane nanovalve system is composed of a linear stalk anchoring on the surface of MSNPs, an α-cyclodextrin ring that encircles it and locks the payload ``cargo'' molecules in the mesopores, and a benzoquinone stopper incorporated at the end of the stalk. The gate opening and controlled release of the cargo are triggered by cleavage of the benzoquinone stopper using an endogenous NQO1 enzyme. In addition to having efficient drug loading and controlled release mechanisms, this smart biocompatible carrier system showed obvious uptake and consequent release of the drug in tumor cells, could selectively induce the tumor cell death and enhance the capability of inhibition of tumor growth in vivo. The controlled drug delivery system demonstrated its use as a potential theranostic material.The synthesis and characterization of an NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme responsive nanocarrier based on mesoporous silica nanoparticles (MSNPs) for on-command delivery applications has been described in this paper. Gatekeeping of MSNPs is achieved by the integration of mechanically interlocked rotaxane nanovalves on the surface of MSNPs. The rotaxane nanovalve system is composed of a linear stalk anchoring on the surface of MSNPs, an α-cyclodextrin ring that encircles it and locks the payload ``cargo'' molecules in the mesopores, and a benzoquinone stopper incorporated at the end of the stalk. The gate opening and controlled release of the cargo are triggered by cleavage of the benzoquinone stopper using an endogenous NQO1 enzyme. In addition to having efficient drug loading and controlled release mechanisms, this

  9. PAH Particles Perturb Prenatal Processes and Phenotypes: Protection from Deficits in Object Discrimination Afforded by Dampening of Brain Oxidoreductase Following In Utero Exposure to Inhaled Benzo(a)pyrene

    PubMed Central

    Chadalapaka, Gayathri; Ramesh, Aramandla; Khoshbouei, Habibeh; Maguire, Mark; Safe, Stephen; Rhoades, Raina E.; Clark, Ryan; Jules, George; McCallister, Monique; Aschner, Michael; Hood, Darryl B.

    2012-01-01

    The wild-type (WT) Cprlox/lox (cytochrome P450 oxidoreductase, Cpr) mouse is an ideal model to assess the contribution of P450 enzymes to the metabolic activation and disposition of environmental xenobiotics. In the present study, we examined the effect of in utero exposure to benzo(a)pyrene [B(a)P] aerosol on Sp4 and N-methyl-D-aspartate (NMDA)–dependent systems as well as a resulting behavioral phenotype (object discrimination) in Cpr offspring. Results from in utero exposure of WT Cprlox/lox mice were compared with in utero exposed brain-Cpr-null offspring mice. Null mice were used as they do not express brain cytochrome P4501B1–associated NADPH oxidoreductase (CYP1B1-associated NADPH oxidoreductase), thus reducing their capacity to produce neural B(a)P metabolites. Subsequent to in utero (E14–E17) exposure to B(a)P (100 μg/m3), Cprlox/lox offspring exhibited: (1) elevated B(a)P metabolite and F2-isoprostane neocortical tissue burdens, (2) elevated concentrations of cortical glutamate, (3) premature developmental expression of Sp4, (4) decreased subunit ratios of NR2B:NR2A, and (5) deficits in a novelty discrimination phenotype monitored to in utero exposed brain-Cpr-null offspring. Collectively, these findings suggest that in situ generation of metabolites by CYP1B1-associated NADPH oxidoreductase promotes negative effects on NMDA-mediated signaling processes during the period when synapses are first forming as well as effects on a subsequent behavioral phenotype. PMID:21987461

  10. Identification of rice genes associated with cosmic-ray response via co-expression gene network analysis.

    PubMed

    Hwang, Sun-Goo; Kim, Dong Sub; Hwang, Jung Eun; Han, A-Reum; Jang, Cheol Seong

    2014-05-15

    In order to better understand the biological systems that are affected in response to cosmic ray (CR), we conducted weighted gene co-expression network analysis using the module detection method. By using the Pearson's correlation coefficient (PCC) value, we evaluated complex gene-gene functional interactions between 680 CR-responsive probes from integrated microarray data sets, which included large-scale transcriptional profiling of 1000 microarray samples. These probes were divided into 6 distinct modules that contained 20 enriched gene ontology (GO) functions, such as oxidoreductase activity, hydrolase activity, and response to stimulus and stress. In particular, modules 1 and 2 commonly showed enriched annotation categories such as oxidoreductase activity, including enriched cis-regulatory elements known as ROS-specific regulators. These results suggest that the ROS-mediated irradiation response pathway is affected by CR in modules 1 and 2. We found 243 ionizing radiation (IR)-responsive probes that exhibited similarities in expression patterns in various irradiation microarray data sets. The expression patterns of 6 randomly selected IR-responsive genes were evaluated by quantitative reverse transcription polymerase chain reaction following treatment with CR, gamma rays (GR), and ion beam (IB); similar patterns were observed among these genes under these 3 treatments. Moreover, we constructed subnetworks of IR-responsive genes and evaluated the expression levels of their neighboring genes following GR treatment; similar patterns were observed among them. These results of network-based analyses might provide a clue to understanding the complex biological system related to the CR response in plants.

  11. The Bioinformatics Report of Mutation Outcome on NADPH Flavin Oxidoreductase Protein Sequence in Clinical Isolates of H. pylori.

    PubMed

    Mirzaei, Nasrin; Poursina, Farkhondeh; Moghim, Sharareh; Ghaempanah, Abdol Majid; Safaei, Hajieh Ghasemian

    2016-05-01

    frxA gene has been implicated in the metronidazole nitro reduction by H. pylori. Alternatively, frxA is expected to contribute to the protection of urease and to the in vivo survival of H. pylori. The aim of present study is to report the mutation effects on the frxA protein sequence in clinical isolates of H. pylori in our community. Metronidazole resistance was proven in 27 of 48 isolates. glmM and frxA genes were used for molecular confirmation of H. pylori isolates. The primer set for detection of whole sequence of frxA gene for the effect of mutation on protein sequence was used. DNA and protein sequence evaluation and analysis were done by blast, Clustal Omega, and T COFFEE programs. Then, FrxA protein sequences from six metronidazole-resistant clinical isolates were analyzed by web-based bioinformatics tools. The result of six metronidazole-resistant clinical isolates in comparison with strain 26695 showed ten missense mutations. The result with the STRING program revealed that no change was seen after alterations in these sequences. According to consensus data involving four methods, residue substitutions at 40, 13, and 141 increase the stability of protein sequence after mutation, while other alterations decrease. Residue substitutions at 40, 43, 141, 138, 169, and 179 are deleterious, while, V7I, Q10R, V34I, and V96I alterations are neutral. As FrxA contribute to survival of bacterium and in regard to the effect of mutations on protein function, it might affect the survival and bacterium phenotype and it need to be studied more. Also, none of the stability prediction tool is perfect; iStable is the best predictor method among all methods.

  12. Electron microscopic analysis and structural characterization of novel NADP(H)-containing methanol: N,N'-dimethyl-4-nitrosoaniline oxidoreductases from the gram-positive methylotrophic bacteria Amycolatopsis methanolica and Mycobacterium gastri MB19.

    PubMed Central

    Bystrykh, L V; Vonck, J; van Bruggen, E F; van Beeumen, J; Samyn, B; Govorukhina, N I; Arfman, N; Duine, J A; Dijkhuizen, L

    1993-01-01

    The quaternary protein structure of two methanol:N,N'-dimethyl-4-nitrosoaniline (NDMA) oxidoreductases purified from Amycolatopsis methanolica and Mycobacterium gastri MB19 was analyzed by electron microscopy and image processing. The enzymes are decameric proteins (displaying fivefold symmetry) with estimated molecular masses of 490 to 500 kDa based on their subunit molecular masses of 49 to 50 kDa. Both methanol:NDMA oxidoreductases possess a tightly but noncovalently bound NADP(H) cofactor at an NADPH-to-subunit molar ratio of 0.7. These cofactors are redox active toward alcohol and aldehyde substrates. Both enzymes contain significant amounts of Zn2+ and Mg2+ ions. The primary amino acid sequences of the A. methanolica and M. gastri MB19 methanol:NDMA oxidoreductases share a high degree of identity, as indicated by N-terminal sequence analysis (63% identity among the first 27 N-terminal amino acids), internal peptide sequence analysis, and overall amino acid composition. The amino acid sequence analysis also revealed significant similarity to a decameric methanol dehydrogenase of Bacillus methanolicus C1. Images PMID:8449887

  13. NAD(P)H:Quinone Oxidoreductase-1 Expression Sensitizes Malignant Melanoma Cells to the HSP90 Inhibitor 17-AAG

    PubMed Central

    Kasai, Shuya; Arakawa, Nobuyuki; Okubo, Ayaka; Shigeeda, Wataru; Yasuhira, Shinji; Masuda, Tomoyuki; Akasaka, Toshihide; Shibazaki, Masahiko; Maesawa, Chihaya

    2016-01-01

    The KEAP1-NRF2 pathway regulates cellular redox homeostasis by transcriptional induction of genes associated with antioxidant synthesis and detoxification in response to oxidative stress. Previously, we reported that KEAP1 mutation elicits constitutive NRF2 activation and resistance to cisplatin (CDDP) and dacarbazine (DTIC) in human melanomas. The present study was conducted to clarify whether an HSP90 inhibitor, 17-AAG, efficiently eliminates melanoma with KEAP1 mutation, as the NRF2 target gene, NQO1, is a key enzyme in 17-AAG bioactivation. In melanoma and non-small cell lung carcinoma cell lines with or without KEAP1 mutations, NQO1 expression and 17-AAG sensitivity are inversely correlated. NQO1 is highly expressed in normal melanocytes and in several melanoma cell lines despite the presence of wild-type KEAP1, and the NQO1 expression is dependent on NRF2 activation. Because either CDDP or DTIC produces reactive oxygen species that activate NRF2, we determined whether these agents would sensitize NQO1-low melanoma cells to 17-AAG. Synergistic cytotoxicity of the 17-AAG and CDDP combination was detected in four out of five NQO1-low cell lines, but not in the cell line with KEAP1 mutation. These data indicate that 17-AAG could be a potential chemotherapeutic agent for melanoma with KEAP1 mutation or NQO1 expression. PMID:27045471

  14. Arabidopsis light-dependent NADPH: protochlorophyllide oxidoreductase A (PORA) is essential for normal plant growth and development: an addendum.

    PubMed

    Kim, Chanhong; Apel, Klaus

    2012-09-01

    Recently the porA-1 null mutant of Arabidopsis thaliana has been identified, which contains an insertion of the Dissociation (Ds) element in the PORA gene (Paddock et al. in Plant Mol Biol 78:447-460, 2012). Light-grown porA-1 seedlings suffer from a drastically reduced chlorophyll content and a developmental arrest beyond the cotyledon stage, suggesting that PORA is not only transiently involved in initiating chlorophyll synthesis during illumination of etiolated seedlings but is also essential for normal growth and plant development. Here we report the presence of a second Ds element in this porA-1 mutant line that inactivates the Speechless gene required for stomata formation. Similar to porA-1, speechless seedlings are severely impaired in their development. Our results suggest that the lack of stomata in porA-1 may contribute to the dwarfed phenotype of the mutant and thus emphasizes the need to re-address the proposed role of PORA during plant development by studying a porA mutant that retains its stomata formation.

  15. Involvement of Xanthine Oxidoreductase-related Oxidative Stress in a Dermatophagoides farinae-induced Asthma Model of NC/Nga Mice.

    PubMed

    Setiawan, Heri; Nagaoka, Kenjiro; Kubo, Masayuki; Fujikura, Yoshihisa; Ogino, Keiki

    2016-06-01

    Oxidative stress is widely known to play a role in asthma. However, the contribution of xanthine oxidoreductase (XOR) as a source of the superoxide anion radical (O2-) in oxidative stress associated with asthma has not yet been examined in detail. Here we investigated pathophysiological changes in XOR in an experimental model of asthma induced by the house dust mite Dermatophagoides farinae (Df). In the lungs of Df-treated mice, the production of O2 - from XOR increased and the nitrite concentrations decreased, whereas the protein expression of XOR remained unchanged. Moreover, the protein expression levels of XOR and the hydrogen peroxide (H2O2) concentrations in bronchoalveolar lavage fluid (BALF) were higher in the Df-treated mice than in saline-treated mice. Immunohistochemically, although XOR was highly localized in the bronchial epithelial cells of the saline-treated mice, immunostaining for XOR was absent in the bronchial epithelium of Df-treated mice. These results suggest that oxidative stress is up-regulated by increases in the conversion of the dehydrogenase form (xanthine dehydrogenase; XDH) of XOR to the oxidase form (xanthine oxidase; XOD).

  16. Mutation of the Streptococcus gordonii Thiol-Disulfide Oxidoreductase SdbA Leads to Enhanced Biofilm Formation Mediated by the CiaRH Two-Component Signaling System

    PubMed Central

    Davey, Lauren; Halperin, Scott A.; Lee, Song F.

    2016-01-01

    Streptococcus gordonii is a commensal inhabitant of human oral biofilms. Previously, we identified an enzyme called SdbA that played an important role in biofilm formation by S. gordonii. SdbA is thiol-disulfide oxidoreductase that catalyzes disulfide bonds in secreted proteins. Surprisingly, inactivation of SdbA results in enhanced biofilm formation. In this study we investigated the basis for biofilm formation by the ΔsdbA mutant. The results revealed that biofilm formation was mediated by the interaction between the CiaRH and ComDE two-component signalling systems. Although it did not affect biofilm formation by the S. gordonii parent strain, CiaRH was upregulated in the ΔsdbA mutant and it was essential for the enhanced biofilm phenotype. The biofilm phenotype was reversed by inactivation of CiaRH or by the addition of competence stimulating peptide, the production of which is blocked by CiaRH activity. Competition assays showed that the enhanced biofilm phenotype also corresponded to increased oral colonization in mice. Thus, the interaction between SdbA, CiaRH and ComDE affects biofilm formation both in vitro and in vivo. PMID:27846284

  17. Novel lavendamycin analogues as antitumor agents: synthesis, in vitro cytotoxicity, structure-metabolism, and computational molecular modeling studies with NAD(P)H:quinone oxidoreductase 1.

    PubMed

    Hassani, Mary; Cai, Wen; Holley, David C; Lineswala, Jayana P; Maharjan, Babu R; Ebrahimian, G Reza; Seradj, Hassan; Stocksdale, Mark G; Mohammadi, Farahnaz; Marvin, Christopher C; Gerdes, John M; Beall, Howard D; Behforouz, Mohammad

    2005-12-01

    Novel lavendamycin analogues with various substituents were synthesized and evaluated as potential NAD(P)H:quinone oxidoreductase (NQO1)-directed antitumor agents. Pictet-Spengler condensation of quinoline- or quninoline-5,8-dione aldehydes with tryptamine or tryptophans yielded the lavendamycins. Metabolism studies with recombinant human NQO1 revealed that addition of NH2 and CH2OH groups at the quinolinedione-7-position and indolopyridine-2'-position had the greatest positive impact on substrate specificity. The best and poorest substrates were 37 (2'-CH2OH-7-NH2 derivative) and 31 (2'-CONH2-7-NHCOC3H7-n derivative) with reduction rates of 263 +/- 30 and 0.1 +/- 0.1 micromol/min/mg NQO1, respectively. Cytotoxicity toward human colon adenocarcinoma cells was determined for the lavendamycins. The best substrates for NQO1 were also the most selectively toxic to the NQO1-rich BE-NQ cells compared to NQO1-deficient BE-WT cells with 37 as the most selective. Molecular docking supported a model in which the best substrates were capable of efficient hydrogen-bonding interactions with key residues of the active site along with hydride ion reception.

  18. Upregulation of NAD(P)H:Quinone Oxidoreductase By Radiation Potentiates the Effect of Bioreductive β-Lapachone on Cancer Cells1

    PubMed Central

    Choi, Eun K; Terai, Kaoru; Ji, In-Mi; Kook, Yeon H; Park, Kyung H; Oh, Eun T; Griffin, Robert J; Lim, Byung U; Kim, Jin-Seok; Lee, Doo S; Boothman, David A; Loren, Melissa; Song, Chang W; Park, Heon Joo

    2007-01-01

    We found that β-lapachone (β-lap), a novel bioreductive drug, caused rapid apoptosis and clonogenic cell death in A549 human lung epithelial cancer cells in vitro in a dose-dependent manner. The clonogenic cell death caused by β-lap could be significantly inhibited by dicoumarol, an inhibitor of NAD(P)H:quinone oxido-reductase (NQO1), and also by siRNA for NQO1, demonstrating that NQO1-induced bioreduction of β-lap is an essential step in β-lap-induced cell death. Irradiation of A549 cells with 4 Gy caused a long-lasting upregulation of NQO1, thereby increasing NQO1-mediated β-lap-induced cell deaths. Although the direct cause of β-lap-induced apoptosis is not yet clear, β-lap treatment reduced the expression of p53 and NF-κB, whereas it increased cytochrome C release, caspase-3 activity, and γH2AX foci formation. Importantly, β-lap treatment immediately after irradiation enhanced radiation-induced cell death, indicating that β-lap sensitizes cancer cells to radiation, in addition to directly killing some of the cells. The growth of A549 tumors induced in immunocompromised mice could be markedly suppressed by local radiation therapy when followed by β-lap treatment. This is the first study to demonstrate that combined radiotherapy and β-lap treatment can have a significant effect on human tumor xenografts. PMID:17786182

  19. Identification, design and biological evaluation of heterocyclic quinolones targeting Plasmodium falciparum type II NADH:quinone oxidoreductase (PfNDH2).

    PubMed

    Leung, Suet C; Gibbons, Peter; Amewu, Richard; Nixon, Gemma L; Pidathala, Chandrakala; Hong, W David; Pacorel, Bénédicte; Berry, Neil G; Sharma, Raman; Stocks, Paul A; Srivastava, Abhishek; Shone, Alison E; Charoensutthivarakul, Sitthivut; Taylor, Lee; Berger, Olivier; Mbekeani, Alison; Hill, Alasdair; Fisher, Nicholas E; Warman, Ashley J; Biagini, Giancarlo A; Ward, Stephen A; O'Neill, Paul M

    2012-03-08

    Following a program undertaken to identify hit compounds against NADH:ubiquinone oxidoreductase (PfNDH2), a novel enzyme target within the malaria parasite Plasmodium falciparum, hit to lead optimization led to identification of CK-2-68, a molecule suitable for further development. In order to reduce ClogP and improve solubility of CK-2-68 incorporation of a variety of heterocycles, within the side chain of the quinolone core, was carried out, and this approach led to a lead compound SL-2-25 (8b). 8b has IC(50)s in the nanomolar range versus both the enzyme and whole cell P. falciparum (IC(50) = 15 nM PfNDH2; IC(50) = 54 nM (3D7 strain of P. falciparum) with notable oral activity of ED(50)/ED(90) of 1.87/4.72 mg/kg versus Plasmodium berghei (NS Strain) in a murine model of malaria when formulated as a phosphate salt. Analogues in this series also demonstrate nanomolar activity against the bc(1) complex of P. falciparum providing the potential added benefit of a dual mechanism of action. The potent oral activity of 2-pyridyl quinolones underlines the potential of this template for further lead optimization studies.

  20. Strong pH dependence of coupling efficiency of the Na+ - translocating NADH:quinone oxidoreductase (Na+-NQR) of Vibrio cholerae.

    PubMed

    Toulouse, Charlotte; Claussen, Björn; Muras, Valentin; Fritz, Günter; Steuber, Julia

    2017-02-01

    The Na+-translocating NADH:quinone oxidoreductase (NQR) is the entry site for electrons into the respiratory chain of Vibrio cholerae, the causative agent of cholera disease. NQR couples the electron transfer from NADH to ubiquinone to the translocation of sodium ions across the membrane. We investigated the pH dependence of electron transfer and generation of a transmembrane voltage (ΔΨ) by NQR reconstituted in liposomes with Na+ or Li+ as coupling cation. ΔΨ formation was followed with the voltage-sensitive dye oxonol. With Na+, ΔΨ was barely influenced by pH (6.5-8.5), while Q reduction activity exhibited a maximum at pH 7.5-8.0. With Li+, ΔΨ was generally lower, and the pH profile of electron transfer activity did not reveal a pronounced maximum. We conclude that the coupling efficiency of NQR is influenced by the nature of the transported cation, and by the concentration of protons. The 3D structure of NQR reveals a transmembrane channel in subunit NqrB. It is proposed that partial uncoupling of the NQR observed with the smaller Li+, or with Na+ at pH 7.5-8.0, is caused by the backflow of the coupling cation through the channel in NqrB.

  1. Sorbitol promotes growth of Zymomonas mobilis in environments with high concentrations of sugar: evidence for a physiological function of glucose-fructose oxidoreductase in osmoprotection.

    PubMed Central

    Loos, H; Krämer, R; Sahm, H; Sprenger, G A

    1994-01-01

    The gram-negative ethanologenic bacterium Zymomonas mobilis is able to grow in media containing high concentrations of glucose or other sugars. A novel compatible solute for bacteria, sorbitol, which enhances growth of Z. mobilis at glucose concentrations exceeding 0.83 M (15%), is described. Added sorbitol was accumulated intracellularly up to 1 M to counteract high external glucose concentrations (up to 1.66 M or 30%). Accumulation of sorbitol was triggered by a glucose upshift (e.g., from 0.33 to 1.27 M or 6 to 23%) and was prevented by the uncoupler CCCP (carbonyl cyanide m-chlorophenylhydrazone; 100 microM). The sorbitol transport system followed Michaelis-Menten kinetics, with an apparent Km of 34 mM and a Vmax of 11.2 nmol.min-1.mg-1 (dry mass). Sorbitol was produced by the cells themselves and was accumulated when growing on sucrose (1 M or 36%) by the action of the periplasmic enzyme glucose-fructose oxidoreductase, which converts glucose and fructose to gluconolactone and sorbitol. Thus, Z. mobilis can form and accumulate the compatible solute sorbitol from a natural carbon source, sucrose, in order to overcome osmotic stress in high-sugar media. No other major compatible solute (betaine, proline, glutamate, or trehalose) was detected. PMID:8002594

  2. Identification, Design and Biological Evaluation of Heterocyclic Quinolones Targeting Plasmodium falciparum Type II NADH:Quinone Oxidoreductase (PfNDH2)

    PubMed Central

    2012-01-01

    Following a program undertaken to identify hit compounds against NADH:ubiquinone oxidoreductase (PfNDH2), a novel enzyme target within the malaria parasite Plasmodium falciparum, hit to lead optimization led to identification of CK-2-68, a molecule suitable for further development. In order to reduce ClogP and improve solubility of CK-2-68 incorporation of a variety of heterocycles, within the side chain of the quinolone core, was carried out, and this approach led to a lead compound SL-2-25 (8b). 8b has IC50s in the nanomolar range versus both the enzyme and whole cell P. falciparum (IC50 = 15 nM PfNDH2; IC50 = 54 nM (3D7 strain of P. falciparum) with notable oral activity of ED50/ED90 of 1.87/4.72 mg/kg versus Plasmodium berghei (NS Strain) in a murine model of malaria when formulated as a phosphate salt. Analogues in this series also demonstrate nanomolar activity against the bc1 complex of P. falciparum providing the potential added benefit of a dual mechanism of action. The potent oral activity of 2-pyridyl quinolones underlines the potential of this template for further lead optimization studies. PMID:22364417

  3. Inhibition of the sodium-translocating NADH-ubiquinone oxidoreductase [Na+-NQR] decreases cholera toxin production in Vibrio cholerae O1 at the late exponential growth phase.

    PubMed

    Minato, Yusuke; Fassio, Sara R; Reddekopp, Rylan L; Häse, Claudia C

    2014-01-01

    Two virulence factors produced by Vibrio cholerae, cholera toxin (CT) and toxin-corregulated pilus (TCP), are indispensable for cholera infection. ToxT is the central regulatory protein involved in activation of CT and TCP expression. We previously reported that lack of a respiration-linked sodium-translocating NADH-ubiquinone oxidoreductase (Na(+)-NQR) significantly increases toxT transcription. In this study, we further characterized this link and found that Na(+)-NQR affects toxT expression only at the early-log growth phase, whereas lack of Na(+)-NQR decreases CT production after the mid-log growth phase. Such decreased CT production was independent of toxT and ctxB transcription. Supplementing a respiratory substrate, l-lactate, into the growth media restored CT production in the nqrA-F mutant, suggesting that decreased CT production in the Na(+)-NQR mutant is dependent on electron transport chain (ETC) activity. This notion was supported by the observations that two chemical inhibitors, a Na(+)-NQR specific inhibitor 2-n-Heptyl-4-hydroxyquinoline N-oxide (HQNO) and a succinate dehydrogenase (SDH) inhibitor, thenoyltrifluoroacetone (TTFA), strongly inhibited CT production in both classical and El Tor biotype strains of V. cholerae. Accordingly, we propose the main respiratory enzyme of V. cholerae, as a potential drug target to treat cholera because human mitochondria do not contain Na(+)-NQR orthologs.

  4. LIGHT-INDUCED RICE1 Regulates Light-Dependent Attachment of LEAF-TYPE FERREDOXIN-NADP+ OXIDOREDUCTASE to the Thylakoid Membrane in Rice and Arabidopsis

    PubMed Central

    Yang, Chao; Lin, Hongwei; Wang, Lingling; He, Yi; Ding, Xiaomeng; Grabsztunowicz, Magda; Chen, Tao; Liu, Yu; Wu, Zhongchang; Wu, Yunrong; Wu, Ping; Mo, Xiaorong

    2016-01-01

    LIR1 (LIGHT-INDUCED RICE1) encodes a 13-kD, chloroplast-targeted protein containing two nearly identical motifs of unknown function. LIR1 is present in the genomes of vascular plants, mosses, liverworts, and algae, but not in cyanobacteria. Using coimmunoprecipitation assays, pull-down assays, and yeast two-hybrid analyses, we showed that LIR1 interacts with LEAF-TYPE FERREDOXIN-NADP+ OXIDOREDUCTASE (LFNR), an essential chloroplast enzyme functioning in the last step of photosynthetic linear electron transfer. LIR1 and LFNR formed high molecular weight thylakoid protein complexes with the TIC62 and TROL proteins, previously shown to anchor LFNR to the membrane. We further showed that LIR1 increases the affinity of LFNRs for TIC62 and that the rapid light-triggered degradation of the LIR1 coincides with the release of the LFNR from the thylakoid membrane. Loss of LIR1 resulted in a marked decrease in the accumulation of LFNR-containing thylakoid protein complexes without a concomitant decrease in total LFNR content. In rice (Oryza sativa), photosynthetic capacity of lir1 plants was slightly impaired, whereas no such effect was observed in Arabidopsis thaliana knockout mutants. The consequences of LIR1 deficiency in different species are discussed. PMID:26941088

  5. Characterization of two quinone radicals in the NADH:ubiquinone oxidoreductase from Escherichia coli by a combined fluorescence spectroscopic and electrochemical approach.

    PubMed

    Hielscher, Ruth; Yegres, Michelle; Voicescu, Mariana; Gnandt, Emmanuel; Friedrich, Thorsten; Hellwig, Petra

    2013-12-17

    The NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. It was proposed that the electron transfer involves quinoid groups localized at the end of the electron transfer chain. To identify these groups, fluorescence excitation and emission spectra of Escherichia coli complex I and its fragments, namely, the NADH dehydrogenase fragment containing the flavin mononucleotide and six iron-sulfur (Fe-S) clusters, and the quinone reductase fragment containing three Fe-S clusters were measured. Signals sensitive to reduction by either NADH or dithionite were detected within the complex and the quinone reductase fragment and attributed to the redox transition of protonated ubiquinone radicals. A fluorescence spectroscopic electrochemical redox titration revealed midpoint potentials of -37 and- 235 mV (vs the standard hydrogen electrode) for the redox transitions of the quinone radicals in complex I at pH 6 with an absorption around 325 nm and a fluorescence emission at 460/475 nm. The role of these cofactor(s) for electron transfer is discussed.

  6. Deficiency of the iron-sulfur clusters of mitochondrial reduced nicotinamide-adenine dinucleotide-ubiquinone oxidoreductase (complex I) in an infant with congenital lactic acidosis.

    PubMed

    Moreadith, R W; Batshaw, M L; Ohnishi, T; Kerr, D; Knox, B; Jackson, D; Hruban, R; Olson, J; Reynafarje, B; Lehninger, A L

    1984-09-01

    We report the case of an infant with hypoglycemia, progressive lactic acidosis, an increased serum lactate/pyruvate ratio, and elevated plasma alanine, who had a moderate to profound decrease in the ability of mitochondria from four organs to oxidize pyruvate, malate plus glutamate, citrate, and other NAD+-linked respiratory substrates. The capacity to oxidize the flavin adenine dinucleotide-linked substrate, succinate, was normal. The most pronounced deficiency was in skeletal muscle, the least in kidney mitochondria. Enzymatic assays on isolated mitochondria ruled out defects in complexes II, III, and IV of the respiratory chain. Further studies showed that the defect was localized in the inner membrane mitochondrial NADH-ubiquinone oxidoreductase (complex I). When ferricyanide was used as an artificial electron acceptor, complex I activity was normal, indicating that electrons from NADH could reduce the flavin mononucleotide cofactor. However, electron paramagnetic resonance spectroscopy performed on liver submitochondrial particles showed an almost total loss of the iron-sulfur clusters characteristic of complex I, whereas normal signals were noted for other mitochondrial iron-sulfur clusters. This infant is presented as the first reported case of congenital lactic acidosis caused by a deficiency of the iron-sulfur clusters of complex I of the mitochondrial electron transport chain.

  7. Phytonutrients Differentially Stimulate NAD(P)H:Quinone Oxidoreductase, Inhibit Proliferation, and Trigger Mitotic Catastrophe in Hepa1c1c7 Cells

    PubMed Central

    Singletary, Keith W.; Murphy, Laura L.; Venema, Richard C.; Young, Andrew J.

    2016-01-01

    Abstract Phytonutrients have rapidly emerged as natural food chemicals possessing multifaceted biological actions that may support beneficial health outcomes. Among the vast array of phytonutrients currently being studied, sulforaphane, curcumin, quercetin, and resveratrol have been frequently reported to stimulate the expression of endogenous detoxification enzymes and may thereby facilitate the neutralization of otherwise harmful environmental agents. Some of these same phytonutrients, however, have also been implicated in disrupting normal cell proliferation and hence may possess toxic properties in and of themselves. In this study, we characterize the respective minimum threshold concentrations of the aforementioned phytonutrients in Hepa1c1c7 cells that stimulate NAD(P)H:quinone oxidoreductase (NQO1), a key enzyme in the hepatic neutralization of menadione, other biological oxidants, and some environmental carcinogens. Moreover, our findings demonstrate that relatively low concentrations of either sulforaphane or curcumin significantly (P < .05) increase NQO1 protein expression and activity without triggering G2/M cell cycle arrest or mitotic catastrophe. The minimal quercetin concentration inducing NQO1, however, was 100-fold higher than that which disrupted mitosis. Also, while resveratrol modestly stimulated NQO1, the minimally effective resveratrol concentration concomitantly induced evidence of cellular apoptosis. Taken together, these findings indicate that only particular phytonutrients are likely efficacious in upregulating NQO1 activity without also leading to hepatic cytotoxicity. PMID:26623679

  8. Reference genes for gene expression studies in wheat flag leaves grown under different farming conditions

    PubMed Central

    2011-01-01

    Background Internal control genes with highly uniform expression throughout the experimental conditions are required for accurate gene expression analysis as no universal reference genes exists. In this study, the expression stability of 24 candidate genes from Triticum aestivum cv. Cubus flag leaves grown under organic and conventional farming systems was evaluated in two locations in order to select suitable genes that can be used for normalization of real-time quantitative reverse-transcription PCR (RT-qPCR) reactions. The genes were selected among the most common used reference genes as well as genes encoding proteins involved in several metabolic pathways. Findings Individual genes displayed different expression rates across all samples assayed. Applying geNorm, a set of three potential reference genes were suitable for normalization of RT-qPCR reactions in winter wheat flag leaves cv. Cubus: TaFNRII (ferredoxin-NADP(H) oxidoreductase; AJ457980.1), ACT2 (actin 2; TC234027), and rrn26 (a putative homologue to RNA 26S gene; AL827977.1). In addition of these three genes that were also top-ranked by NormFinder, two extra genes: CYP18-2 (Cyclophilin A, AY456122.1) and TaWIN1 (14-3-3 like protein, AB042193) were most consistently stably expressed. Furthermore, we showed that TaFNRII, ACT2, and CYP18-2 are suitable for gene expression normalization in other two winter wheat varieties (Tommi and Centenaire) grown under three treatments (organic, conventional and no nitrogen) and a different environment than the one tested with cv. Cubus. Conclusions This study provides a new set of reference genes which should improve the accuracy of gene expression analyses when using wheat flag leaves as those related to the improvement of nitrogen use efficiency for cereal production. PMID:21951810

  9. Digital Gene Expression Analysis Provides Insight into the Transcript Profile of the Genes Involved in Aporphine Alkaloid Biosynthesis in Lotus (Nelumbo nucifera)

    PubMed Central

    Yang, Mei; Zhu, Lingping; Li, Ling; Li, Juanjuan; Xu, Liming; Feng, Ji; Liu, Yanling

    2017-01-01

    The predominant alkaloids in lotus leaves are aporphine alkaloids. These are the most important active components and have many pharmacological properties, but little is known about their biosynthesis. We used digital gene expression (DGE) technology to identify differentially-expressed genes (DEGs) between two lotus cultivars with different alkaloid contents at four leaf development stages. We also predicted potential genes involved in aporphine alkaloid biosynthesis by weighted gene co-expression network analysis (WGCNA). Approximately 335 billion nucleotides were generated; and 94% of which were aligned against the reference genome. Of 22 thousand expressed genes, 19,000 were differentially expressed between the two cultivars at the four stages. Gene Ontology (GO) enrichment analysis revealed that catalytic activity and oxidoreductase activity were enriched significantly in most pairwise comparisons. In Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, dozens of DEGs were assigned to the categories of biosynthesis of secondary metabolites, isoquinoline alkaloid biosynthesis, and flavonoid biosynthesis. The genes encoding norcoclaurine synthase (NCS), norcoclaurine 6-O-methyltransferase (6OMT), coclaurine N-methyltransferase (CNMT), N-methylcoclaurine 3′-hydroxylase (NMCH), and 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′OMT) in the common pathways of benzylisoquinoline alkaloid biosynthesis and the ones encoding corytuberine synthase (CTS) in aporphine alkaloid biosynthetic pathway, which have been characterized in other plants, were identified in lotus. These genes had positive effects on alkaloid content, albeit with phenotypic lag. The WGCNA of DEGs revealed that one network module was associated with the dynamic change of alkaloid content. Eleven genes encoding proteins with methyltransferase, oxidoreductase and CYP450 activities were identified. These were surmised to be genes involved in aporphine alkaloid biosynthesis. This

  10. Role of farnesoid X receptor in establishment of ontogeny of phase-I drug metabolizing enzyme genes in mouse liver.

    PubMed

    Peng, Lai; Piekos, Stephanie; Guo, Grace L; Zhong, Xiao-Bo

    2016-09-01

    The expression of phase-I drug metabolizing enzymes in liver changes dramatically during postnatal liver maturation. Farnesoid X receptor (FXR) is critical for bile acid and lipid homeostasis in liver. However, the role of FXR in regulating ontogeny of phase-I drug metabolizing genes is not clear. Hence, we applied RNA-sequencing to quantify the developmental expression of phase-I genes in both Fxr-null and control (C57BL/6) mouse livers during development. Liver samples of male C57BL/6 and Fxr-null mice at 6 different ages from prenatal to adult were used. The Fxr-null showed an overall effect to diminish the "day-1 surge" of phase-I gene expression, including cytochrome P450s at neonatal ages. Among the 185 phase-I genes from 12 different families, 136 were expressed, and differential expression during development occurred in genes from all 12 phase-I families, including hydrolysis: carboxylesterase (Ces), paraoxonase (Pon), and epoxide hydrolase (Ephx); reduction: aldoketo reductase (Akr), quinone oxidoreductase (Nqo), and dihydropyrimidine dehydrogenase (Dpyd); and oxidation: alcohol dehydrogenase (Adh), aldehyde dehydrogenase (Aldh), flavin monooxygenases (Fmo), molybdenum hydroxylase (Aox and Xdh), cytochrome P450 (P450), and cytochrome P450 oxidoreductase (Por). The data also suggested new phase-I genes potentially targeted by FXR. These results revealed an important role of FXR in regulation of ontogeny of phase-I genes.

  11. Human carbonyl reductase (CBR) localized to band 21q22. 1 by high-resolution fluorescence in situ hybridization displays gene dosage effects in trisomy 21 cells

    SciTech Connect

    Lemieux, N. ); Malfoy, B. ); Forrest, G.L. )

    1993-01-01

    Human carbonyl reductase (CBR) belongs to a group of NADPH-dependent enzymes called aldo-keto reductases. The enzyme can function as an aldo-keto reductase or as a quinone reductase with potential for modulating quinone-mediated oxygen free radicals. The CBR gene was mapped by high-resolution fluorescence in situ hybridization to band 21q22.12, very close to the SOD1 locus at position 2lq22.11. CBR displayed gene dosage effects in trisomy 21 human lymphoblasts at the DNA and mRNA levels. Lymphoblasts with increasing chromosome 21 ploidy also showed increased aldo-keto reductase activity and increased quinone reductase activity. Both aldo-keto reductase activity and quinone reductase activity have been shown to be associated with carbonyl reductase. The location of CBR near SOD1 and the increased enzyme activity and potential for free radical modulation in trisomy 21 cells implicate CBR as a candidate for contributing to the pathology of certain diseases such as Down syndrome and Alzheimer disease. 28 refs., 1 fig., 1 tab.

  12. Defining redox centers in human electron transfer flavoprotein: ubiquinone oxidoreductase (ETF:QO) by expression in Saccharomyces cerevisiae

    SciTech Connect

    Frerman, F.E.; Beard, S.; Goodman, S.I.

    1994-09-01

    Mutations in ETF or ETC:QO cause glutaric acidemia type II (GA2). ETF:QO is an iron-sulfur flavoprotein in the inner mitochondrial membrane which transfers electrons from ETF in the mitochondrial matrix to ubiquinone (Q). The human ETF:QO gene is on chromosome 4q32{r_arrow}qter, and encodes a 617 amino acid precursor which is processed to the 64 kDa mature form in the mitochondrion. One ETF:QO mutation in GA2 is a G{r_arrow}T transversion in a donor splice site, deleting the 222 bp upstream exon from the transcript. The deleted 74 amino acids are near the carboxyl terminus just beyond a predicted membrane helix, and include C561, one of four cysteine residues predicted to ligate the 4Fe4S cluster. The mutant protein is not stable in patient fibroblasts. We have expressed cDNAs encoding wild type (wt) ETF:QO, ETF:QO with the 74 amino acid deletion, and ETFF:QO with only a C561A mutation, in S cerevisiae. In all instances, precursor and mature ETF:QOs were stably inserted into the mitochondrial membrane. ETF:QO (C561A) is extracted from the membrane under the same conditions as wt ETF:QO, but ETF:QO with the deletion is much more difficult to extract. Wt ETF:QO accepts electrons from ETF and reduces Q but, while both mutant proteins accept electrons from ETF, neither of them reduces Q. This work demonstrates that C561 in human ETF:QO is essential for Q reduction (probably because it ligands the 4Fe4S cluster), that mutant proteins that are unstable in man may be stable in other systems, that cleavage of signal peptide from precursor proteins can occur within the inner mitochondrial membrane, and the general usefulness of expressing human mitochondrial proteins in yeast.

  13. Genes and Gene Therapy

    MedlinePlus

    ... correctly, a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... or prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

  14. Chlorophyll Synthesis in a Deetiolated (det340) Mutant of Arabidopsis without NADPH-Protochlorophyllide (PChlide) Oxidoreductase (POR) A and Photoactive PChlide-F655.

    PubMed Central

    Lebedev, N.; Van Cleve, B.; Armstrong, G.; Apel, K.

    1995-01-01

    Chlorophyll (Chl) synthesis in Arabidopsis is controlled by two light-dependent NADPH-protochlorophyllide (PChlide) oxidoreductases (PORs), one (POR A) that is active transiently in etiolated seedlings at the beginning of illumination and another (POR B) that also operates in green plants. The function of these two enzymes during the light-induced greening of dark-grown seedlings has been studied in the wild type and a deetiolated (det340) mutant of Arabidopsis. One of the consequences of the det mutation is that POR A is constitutively down-regulated, and therefore, synthesis of the POR A enzyme is shut off. When grown in the dark, the det340 mutant lacks POR A and the photoactive PChlide-F655 species but maintains the second PChlide reductase, POR B. Previously, photoactive PChlide-F655 has often been considered to be the only PChlide form that leads to Chl formation. Despite its deficiency in POR A and photoactive PChlide-F655, the det340 mutant is able to green when placed in the light. Chl accumulation, however, proceeds abnormally. At the beginning of illumination, seedlings of det340 mutants are extremely susceptible to photooxidative damage and accumulate Chl only at extremely low light intensities. They form core complexes of photosystems I and II but are almost completely devoid of light-harvesting structures. The results of this study demonstrate that in addition to the route of Chl synthesis that has been studied extensively in illuminated dark-grown wild-type plants, a second branch of Chl synthesis exists that is driven by POR B and does not require POR A. PMID:12242369

  15. Identification of the Binding Sites for Ubiquinone and Inhibitors in the Na(+)-Pumping NADH-Ubiquinone Oxidoreductase of Vibrio cholerae by Photoaffinity Labeling.

    PubMed

    Ito, Takeshi; Murai, Masatoshi; Ninokura, Satoshi; Kitazumi, Yuki; Mezic, Katherine G; Cress, Brady F; Koffas, Mattheos A G; Morgan, Joel E; Barquera, Blanca; Miyoshi, Hideto

    2017-03-15

    The Na(+)-pumping NADH-quinone oxidoreductase (Na(+)-NQR) is the first enzyme of the respiratory chain and the main ion transporter in many marine and pathogenic bacteria, including Vibrio cholerae The V. cholerae Na(+)-NQR has been extensively studied, but its binding sites for ubiquinone and inhibitors remain controversial. Here, using a photoreactive ubiquinone PUQ-3 as well as two aurachin-type inhibitors [(125)I]PAD-1 and [(125)I]PAD-2 and photoaffinity labeling experiments on the isolated enzyme, we demonstrate that the ubiquinone ring binds to the NqrA subunit in the regions Leu32-Met39 and Phe131-Lys138, encompassing the rear wall of a predicted ubiquinone-binding cavity. The quinolone ring and alkyl side chain of aurachin bound to the NqrB subunit in the regions Arg43-Lys54 and Trp23-Gly89, respectively. These results indicate that the binding sites for ubiquinone and aurachin-type inhibitors are in close proximity but do not overlap with one another. Unexpectedly, while the inhibitory effects of PAD-1 and PAD-2 were almost completely abolished by certain mutations in NqrB (i.e. Gly140Ala and Glu144Cys), the binding reactivities of [(125)I]PAD-1 and [(125)I]PAD-2 to the mutated enzymes were unchanged compared to those of the wild-type enzyme. We also found that photoaffinity labeling by [(125)I]PAD-1 and [(125)I]PAD-2 rather than being competitively suppressed in the presence of other inhibitors, is enhanced under some experimental conditions. To explain these apparently paradoxical results, we propose models for the catalytic reaction of Na(+)-NQR and its interactions with inhibitors on the basis of the biochemical and biophysical results reported here and in previous work.

  16. Structural and Functional Investigation of Flavin Binding Center of the NqrC Subunit of Sodium-Translocating NADH:Quinone Oxidoreductase from Vibrio harveyi

    PubMed Central

    Bertsova, Yulia; Polovinkin, Vitaly; Gushchin, Ivan; Ishchenko, Andrii; Kovalev, Kirill; Mishin, Alexey; Kachalova, Galina; Popov, Alexander; Bogachev, Alexander; Gordeliy, Valentin

    2015-01-01

    Na+-translocating NADH:quinone oxidoreductase (NQR) is a redox-driven sodium pump operating in the respiratory chain of various bacteria, including pathogenic species. The enzyme has a unique set of redox active prosthetic groups, which includes two covalently bound flavin mononucleotide (FMN) residues attached to threonine residues in subunits NqrB and NqrC. The reason of FMN covalent bonding in the subunits has not been established yet. In the current work, binding of free FMN to the apo-form of NqrC from Vibrio harveyi was studied showing very low affinity of NqrC to FMN in the absence of its covalent bonding. To study structural aspects of flavin binding in NqrC, its holo-form was crystallized and its 3D structure was solved at 1.56 Å resolution. It was found that the isoalloxazine moiety of the FMN residue is buried in a hydrophobic cavity and that its pyrimidine ring is squeezed between hydrophobic amino acid residues while its benzene ring is extended from the protein surroundings. This structure of the flavin-binding pocket appears to provide flexibility of the benzene ring, which can help the FMN residue to take the bended conformation and thus to stabilize the one-electron reduced form of the prosthetic group. These properties may also lead to relatively weak noncovalent binding of the flavin. This fact along with periplasmic location of the FMN-binding domains in the vast majority of NqrC-like proteins may explain the necessity of the covalent bonding of this prosthetic group to prevent its loss to the external medium. PMID:25734798

  17. GLYCOLATE OXIDASE3, a Glycolate Oxidase Homolog of Yeast l-Lactate Cytochrome c Oxidoreductase, Supports l-Lactate Oxidation in Roots of Arabidopsis1

    PubMed Central

    Engqvist, Martin K.M.; Schmitz, Jessica; Gertzmann, Anke; Florian, Alexandra; Jaspert, Nils; Arif, Muhammad; Balazadeh, Salma; Mueller-Roeber, Bernd; Fernie, Alisdair R.; Maurino, Veronica G.

    2015-01-01

    In roots of Arabidopsis (Arabidopsis thaliana), l-lactate is generated by the reduction of pyruvate via l-lactate dehydrogenase, but this enzyme does not efficiently catalyze the reverse reaction. Here, we identify the Arabidopsis glycolate oxidase (GOX) paralogs GOX1, GOX2, and GOX3 as putative l-lactate-metabolizing enzymes based on their homology to CYB2, the l-lactate cytochrome c oxidoreductase from the yeast Saccharomyces cerevisiae. We found that GOX3 uses l-lactate with a similar efficiency to glycolate; in contrast, the photorespiratory isoforms GOX1 and GOX2, which share similar enzymatic properties, use glycolate with much higher efficiencies than l-lactate. The key factor making GOX3 more efficient with l-lactate than GOX1 and GOX2 is a 5- to 10-fold lower Km for the substrate. Consequently, only GOX3 can efficiently metabolize l-lactate at low intracellular concentrations. Isotope tracer experiments as well as substrate toxicity tests using GOX3 loss-of-function and overexpressor plants indicate that l-lactate is metabolized in vivo by GOX3. Moreover, GOX3 rescues the lethal growth phenotype of a yeast strain lacking CYB2, which cannot grow on l-lactate as a sole carbon source. GOX3 is predominantly present in roots and mature to aging leaves but is largely absent from young photosynthetic leaves, indicating that it plays a role predominantly in heterotrophic rather than autotrophic tissues, at least under standard growth conditions. In roots of plants grown under normoxic conditions, loss of function of GOX3 induces metabolic rearrangements that mirror wild-type responses under hypoxia. Thus, we identified GOX3 as the enzyme that metabolizes l-lactate to pyruvate in vivo and hypothesize that it may ensure the sustainment of low levels of l-lactate after its formation under normoxia. PMID:26246447

  18. 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.

  19. The relationship between different spectral forms of the protochlorophyllide oxidoreductase complex and the structural organisation of prolamellar bodies isolated from Zea mays.

    PubMed

    Selstam, Eva; Brain, Anthony P R; Williams, W Patrick

    2011-05-01

    Incubation of prolamellar bodies (PLB) in high-salt media leads to changes in PLB structure and properties of their protochlorophyllide oxidoreductase-protochlorophyllide (POR-PChlide) complex. The paracrystalline organisation typical of PLB is disrupted and NADPH dissociates from photoconvertible POR-PChlide, with absorption maxima at 640 and 650 nm (POR-PChlide (640/650)), and a non-photoconvertible form, with absorption maxima at 635 nm (POR-PChlide (635)), is formed. These effects are strongly dependent on the valence of the cation of the perturbing salt, indicating that they involve surface double layers effects. They are also influenced by the nature of the anion and by high concentrations of non-electrolytes, suggesting the involvement of surface hydration effects. The structural changes are largely, if not entirely, independent of the presence of excess NADPH. Changes to the POR-PChlide complex, however, are strongly inhibited by excess NADPH suggesting that the two sets of changes may not be causally linked. As long as the disruption is not too great, the structural changes seen on incubation of PLB in high salt media lacking excess NADPH are reversed on removal of the high salt perturbation. This reversal is independent of the presence or absence of added NADPH. Reformation of photoconvertible POR-PChlide, however, requires the presence of NADPH. The reformation of paracrystalline PLB in the absence of NADPH strongly indicates that preservation of PLB structure, in isolated PLB preparations at least, is independent of the presence or absence of POR-PChlide (650).

  20. Identification, design and biological evaluation of bisaryl quinolones targeting Plasmodium falciparum type II NADH:quinone oxidoreductase (PfNDH2).

    PubMed

    Pidathala, Chandrakala; Amewu, Richard; Pacorel, Bénédicte; Nixon, Gemma L; Gibbons, Peter; Hong, W David; Leung, Suet C; Berry, Neil G; Sharma, Raman; Stocks, Paul A; Srivastava, Abhishek; Shone, Alison E; Charoensutthivarakul, Sitthivut; Taylor, Lee; Berger, Olivier; Mbekeani, Alison; Hill, Alasdair; Fisher, Nicholas E; Warman, Ashley J; Biagini, Giancarlo A; Ward, Stephen A; O'Neill, Paul M

    2012-03-08

    A program was undertaken to identify hit compounds against NADH:ubiquinone oxidoreductase (PfNDH2), a dehydrogenase of the mitochondrial electron transport chain of the malaria parasite Plasmodium falciparum. PfNDH2 has only one known inhibitor, hydroxy-2-dodecyl-4-(1H)-quinolone (HDQ), and this was used along with a range of chemoinformatics methods in the rational selection of 17 000 compounds for high-throughput screening. Twelve distinct chemotypes were identified and briefly examined leading to the selection of the quinolone core as the key target for structure-activity relationship (SAR) development. Extensive structural exploration led to the selection of 2-bisaryl 3-methyl quinolones as a series for further biological evaluation. The lead compound within this series 7-chloro-3-methyl-2-(4-(4-(trifluoromethoxy)benzyl)phenyl)quinolin-4(1H)-one (CK-2-68) has antimalarial activity against the 3D7 strain of P. falciparum of 36 nM, is selective for PfNDH2 over other respiratory enzymes (inhibitory IC(50) against PfNDH2 of 16 nM), and demonstrates low cytotoxicity and high metabolic stability in the presence of human liver microsomes. This lead compound and its phosphate pro-drug have potent in vivo antimalarial activity after oral administration, consistent with the target product profile of a drug for the treatment of uncomplicated malaria. Other quinolones presented (e.g., 6d, 6f, 14e) have the capacity to inhibit both PfNDH2 and P. falciparum cytochrome bc(1), and studies to determine the potential advantage of this dual-targeting effect are in progress.

  1. 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

  2. Kinetic and Structural Studies of Aldehyde Oxidoreductase from Desulfovibrio gigas Reveal a Dithiolene-Based Chemistry for Enzyme Activation and Inhibition by H2O2

    PubMed Central

    Brondino, Carlos D.; Moura, José J. G.; Romão, Maria J.; González, Pablo J.; Santos-Silva, Teresa

    2013-01-01

    Mononuclear Mo-containing enzymes of the xanthine oxidase (XO) family catalyze the oxidative hydroxylation of aldehydes and heterocyclic compounds. The molybdenum active site shows a distorted square-pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. The XO family member aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is an exception as presents in its catalytically competent form an equatorial oxo ligand instead of the sulfido ligand. Despite this structural difference, inactive samples of DgAOR can be activated upon incubation with dithionite plus sulfide, a procedure similar to that used for activation of desulfo-XO. The fact that DgAOR does not need a sulfido ligand for catalysis indicates that the process leading to the activation of inactive DgAOR samples is different to that of desulfo-XO. We now report a combined kinetic and X-ray crystallographic study to unveil the enzyme modification responsible for the inactivation and the chemistry that occurs at the Mo site when DgAOR is activated. In contrast to XO, which is activated by resulfuration of the Mo site, DgAOR activation/inactivation is governed by the oxidation state of the dithiolene moiety of the pyranopterin cofactor, which demonstrates the non-innocent behavior of the pyranopterin in enzyme activity. We also showed that DgAOR incubation with dithionite plus sulfide in the presence of dioxygen produces hydrogen peroxide not associated with the enzyme activation. The peroxide molecule coordinates to molybdenum in a η2 fashion inhibiting the enzyme activity. PMID:24391748

  3. Identification, Design and Biological Evaluation of Bisaryl Quinolones Targeting Plasmodium falciparum Type II NADH:Quinone Oxidoreductase (PfNDH2)

    PubMed Central

    2012-01-01

    A program was undertaken to identify hit compounds against NADH:ubiquinone oxidoreductase (PfNDH2), a dehydrogenase of the mitochondrial electron transport chain of the malaria parasite Plasmodium falciparum. PfNDH2 has only one known inhibitor, hydroxy-2-dodecyl-4-(1H)-quinolone (HDQ), and this was used along with a range of chemoinformatics methods in the rational selection of 17 000 compounds for high-throughput screening. Twelve distinct chemotypes were identified and briefly examined leading to the selection of the quinolone core as the key target for structure–activity relationship (SAR) development. Extensive structural exploration led to the selection of 2-bisaryl 3-methyl quinolones as a series for further biological evaluation. The lead compound within this series 7-chloro-3-methyl-2-(4-(4-(trifluoromethoxy)benzyl)phenyl)quinolin-4(1H)-one (CK-2-68) has antimalarial activity against the 3D7 strain of P. falciparum of 36 nM, is selective for PfNDH2 over other respiratory enzymes (inhibitory IC50 against PfNDH2 of 16 nM), and demonstrates low cytotoxicity and high metabolic stability in the presence of human liver microsomes. This lead compound and its phosphate pro-drug have potent in vivo antimalarial activity after oral administration, consistent with the target product profile of a drug for the treatment of uncomplicated malaria. Other quinolones presented (e.g., 6d, 6f, 14e) have the capacity to inhibit both PfNDH2 and P. falciparum cytochrome bc1, and studies to determine the potential advantage of this dual-targeting effect are in progress. PMID:22364416

  4. Semiquinone and Cluster N6 Signals in His-tagged Proton-translocating NADH:Ubiquinone Oxidoreductase (Complex I) from Escherichia coli*

    PubMed Central

    Narayanan, Madhavan; Gabrieli, David J.; Leung, Steven A.; Elguindy, Mahmoud M.; Glaser, Carl A.; Saju, Nitha; Sinha, Subhash C.; Nakamaru-Ogiso, Eiko

    2013-01-01

    NADH:ubiquinone oxidoreductase (complex I) pumps protons across the membrane using downhill redox energy. The Escherichia coli complex I consists of 13 different subunits named NuoA-N coded by the nuo operon. Due to the low abundance of the protein and some difficulty with the genetic manipulation of its large ∼15-kb operon, purification of E. coli complex I has been technically challenging. Here, we generated a new strain in which a polyhistidine sequence was inserted upstream of nuoE in the operon. This allowed us to prepare large amounts of highly pure and active complex I by efficient affinity purification. The purified complex I contained 0.94 ± 0.1 mol of FMN, 29.0 ± 0.37 mol of iron, and 1.99 ± 0.07 mol of ubiquinone/1 mol of complex I. The extinction coefficient of isolated complex I was 495 mm−1 cm−1 at 274 nm and 50.3 mm−1 cm−1 at 410 nm. NADH:ferricyanide activity was 219 ± 9.7 μmol/min/mg by using HEPES-Bis-Tris propane, pH 7.5. Detailed EPR analyses revealed two additional iron-sulfur cluster signals, N6a and N6b, in addition to previously assigned signals. Furthermore, we found small but significant semiquinone signal(s), which have been reported only for bovine complex I. The line width was ∼12 G, indicating its neutral semiquinone form. More than 90% of the semiquinone signal originated from the single entity with P½ (half-saturation power level) = 1.85 milliwatts. The semiquinone signal(s) decreased by 60% when with asimicin, a potent complex I inhibitor. The functional role of semiquinone and the EPR assignment of clusters N6a/N6b are discussed. PMID:23543743

  5. Down-regulation of the detoxifying enzyme NAD(P)H:quinone oxidoreductase 1 by vanadium in Hepa 1c1c7 cells

    SciTech Connect

    Anwar-Mohamed, Anwar; El-Kadi, Ayman O.S.

    2009-05-01

    Recent data suggest that vanadium (V{sup 5+}) compounds exert protective effects against chemical-induced carcinogenesis, mainly through modifying various xenobiotic metabolizing enzymes. In fact, we have shown that V{sup 5+} down-regulates the expression of Cyp1a1 at the transcriptional level through an ATP-dependent mechanism. However, incongruously, there is increasing evidence that V{sup 5+} is found in higher amounts in cancer cells and tissues than in normal cells or tissues. Therefore, the current study aims to address the possible effect of this metal on the regulation of expression of an enzyme that helps maintain endogenous antioxidants used to protect tissues/cells from mutagens, carcinogens, and oxidative stress damage, NAD(P)H:quinone oxidoreductase 1 (Nqo1). In an attempt to examine these effects, Hepa 1c1c7 cells and its AhR-deficient version, c12, were treated with increasing concentrations of V{sup 5+} in the presence of two distinct Nqo1 inducers, the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and isothiocyanate sulforaphane (SUL). Our results showed that V{sup 5+} inhibits the TCDD- and SUL-mediated induction of Nqo1 at mRNA, protein, and catalytic activity levels. At transcriptional level, V{sup 5+} was able to decrease the TCDD- and SUL-induced nuclear accumulation of Nrf2 and the subsequent binding to antioxidant responsive element (ARE) without affecting Nrf2 protein levels. Looking at post-transcriptional level; we found that V{sup 5+} did not affect Nqo1 mRNA transcripts turn-over rates. However, at the post-translational level V{sup 5+} increased Nqo1 protein half-life. In conclusion, the present study demonstrates that V{sup 5+} down-regulates Nqo1 at the transcriptional level, possibly through inhibiting the ATP-dependent activation of Nrf2.

  6. The transition between active and de-activated forms of NADH:ubiquinone oxidoreductase (Complex I) in the mitochondrial membrane of Neurospora crassa.

    PubMed Central

    Grivennikova, Vera G; Serebryanaya, Darya V; Isakova, Elena P; Belozerskaya, Tatyana A; Vinogradov, Andrei D

    2003-01-01

    The mammalian mitochondrial NADH:ubiquinone oxidoreductase (Complex I) has been shown to exist in two kinetically and structurally distinct slowly interconvertible forms, active (A) and de-activated (D) [Vinogradov and Grivennikova (2001) IUBMB Life 52, 129-134]. This work was undertaken to investigate the putative Complex I A-D transition in the mitochondrial membrane of the lower eukaryote Neurospora crassa and in plasma membrane of the prokaryote Paracoccus denitrificans, organisms that are eligible for molecular genetic manipulations. The potential interconversion between A and D forms was assessed by examination of the initial and steady-state rates of NADH oxidation catalysed by inside-out submitochondrial ( N. crassa ) and sub-bacterial ( P. denitrificans ) particles and their sensitivities to N -ethylmaleimide and Mg(2+). All diagnostic tests provide evidence that slow temperature- and turnover-dependent A-D transition is an explicit feature of eukaryotic N. crassa Complex I, whereas the phenomenon is not seen in the membranes of the prokaryote P. denitrificans. Significantly lower activation energy for A-to-D transition characterizes the N. crassa enzyme compared with that determined previously for the mammalian Complex I. Either a lag or a burst in the onset of the NADH oxidase assayed in the presence of Mg(2+) is seen when the reaction is initiated by the thermally de-activated or NADH-activated particles, whereas the delayed final activities of both preparations are the same. We conclude that continuous slow cycling between A and D forms occurs during the steady-state operation of Complex I in N. crassa mitochondria. PMID:12379145

  7. Electrical Wiring of the Aldehyde Oxidoreductase PaoABC with a Polymer Containing Osmium Redox Centers: Biosensors for Benzaldehyde and GABA

    PubMed Central

    Badalyan, Artavazd; Dierich, Marlen; Stiba, Konstanze; Schwuchow, Viola; Leimkühler, Silke; Wollenberger, Ulla

    2014-01-01

    Biosensors for the detection of benzaldehyde and γ−aminobutyric acid (GABA) are reported using aldehyde oxidoreductase PaoABC from Escherichia coli immobilized in a polymer containing bound low potential osmium redox complexes. The electrically connected enzyme already electrooxidizes benzaldehyde at potentials below −0.15 V (vs. Ag|AgCl, 1 M KCl). The pH-dependence of benzaldehyde oxidation can be strongly influenced by the ionic strength. The effect is similar with the soluble osmium redox complex and therefore indicates a clear electrostatic effect on the bioelectrocatalytic efficiency of PaoABC in the osmium containing redox polymer. At lower ionic strength, the pH-optimum is high and can be switched to low pH-values at high ionic strength. This offers biosensing at high and low pH-values. A “reagentless” biosensor has been formed with enzyme wired onto a screen-printed electrode in a flow cell device. The response time to addition of benzaldehyde is 30 s, and the measuring range is between 10–150 µM and the detection limit of 5 µM (signal to noise ratio 3:1) of benzaldehyde. The relative standard deviation in a series (n = 13) for 200 µM benzaldehyde is 1.9%. For the biosensor, a response to succinic semialdehyde was also identified. Based on this response and the ability to work at high pH a biosensor for GABA is proposed by coimmobilizing GABA-aminotransferase (GABA-T) and PaoABC in the osmium containing redox polymer. PMID:25587431

  8. Optimization of heme precursors for the expression of human cytochrome P450 2A13 and its co-expression with oxidoreductase in baculovirus/sf9 system.

    PubMed

    Lu, Hui-Yuan; Qiu, Liang-Lin; Yang, Xue-Jiao; Zhang, Xiao-Ming; Zhang, Zhan; Wang, Shou-Lin

    2013-06-01

    Human cytochrome P450 2A13 (CYP2A13), mainly expressed in respiratory tract, is active towards numerous toxicants. To establish the metabolism in vitro, we expressed CYP2A13 and NADPH-CYP450 oxidoreductase (POR) in a baculovirus/sf9 system. Due to the deficiency of sf9 cells in heme incorporation, we investigated the effects of different heme precursors on the expression of CYP2A13, POR and their co-expression. The present results showed that both CYP2A13 and POR were presented the highest expression levels or activity with 0.2 mM δ-aminolaevulinic acid (5-ALA), 0.02 mM Fe(3+) and 0.5-1.0 μg/ml hemin. The combination of 0.2 mM 5-ALA and 0.02 mM Fe(3+) significantly improved CYP2A13 expression and content compared with heme precursors alone, so was POR activity. A multiplicity of infection (MOI) value of 5 pfu/cell for CYP2A13 baculovirus particles induced very high CYP2A13 expression. When co-infected with different POR MOI values, a viral ratio of 5 : 2 was associated with the highest CYP2A13 activity, whereas POR activity dose dependently increased with POR MOI. Furthermore, the expressed CYP2A13 in the optimized conduction could eliminate its substrate aflatoxin B1 at a significantly higher than those in other condition (P < 0.01). Our results provide an efficient approach for expressing functionally characterized, highly active and homogeneous CYP2A13 proteins.

  9. Compounds from the Fruits of the Popular European Medicinal Plant Vitex agnus-castus in Chemoprevention via NADP(H):Quinone Oxidoreductase Type 1 Induction

    PubMed Central

    Li, Shenghong; Qiu, Shengxiang; Yao, Ping; Sun, Handong; Fong, Harry H. S.; Zhang, Hongjie

    2013-01-01

    As part of our continuing efforts in the search for potential biologically active compounds from medicinal plants, we have isolated 18 compounds including two novel nitrogen containing diterpenes from extracts of the fruits of Vitex agnus-castus. These isolates, along with our previously obtained novel compound vitexlactam A (1), were evaluated for potential biological effects, including cancer chemoprevention. Chemically, the nitrogenous isolates were found to be two labdane diterpene alkaloids, each containing an α, β-unsaturated γ-lactam moiety. Structurally, they were elucidated to be 9α-hydroxy-13(14)-labden-16,15-amide (2) and 6β-acetoxy-9α-hydroxy-13(14)-labden-15,16-amide (3), which were named vitexlactams B and C, respectively. The 15 known isolates were identified as vitexilactone (4), rotundifuran (5), 8-epi-manoyl oxide (6), vitetrifolin D (7), spathulenol (8), cis-dihydro-dehydro-diconiferylalcohol-9-O-β-D-glucoside (9), luteolin-7-O-glucoside (10), 5-hydroxy-3,6,7,4′-tetramethoxyflavone (11), casticin (12), artemetin (13), aucubin (14), agnuside (15), β-sitosterol (16), p-hydroxybenzoic acid (17), and p-hydroxybenzoic acid glucose ester (18). All compound structures were determined/identified on the basis of 1D and/or 2D NMR and mass spectrometry techniques. Compounds 6, 8, 9, and 18 were reported from a Vitex spieces for the first time. The cancer chemopreventive potentials of these isolates were evaluated for NADP(H):quinone oxidoreductase type 1 (QR1) induction activity. Compound 7 demonstrated promising QR1 induction effect, while the new compound vitexlactam (3) was only slightly active. PMID:23662135

  10. Classic and non-classic 21-hydroxylase deficiency can be discriminated from P450 oxidoreductase deficiency in Japanese infants by urinary steroid metabolites.

    PubMed

    Koyama, Yuhei; Homma, Keiko; Fukami, Maki; Miwa, Masayuki; Ikeda, Kazushige; Ogata, Tsutomu; Murata, Mitsuru; Hasegawa, Tomonobu

    2016-04-01

    We previously reported a two-step biochemical diagnosis to discriminate classic 21-hydroxylase deficiency (C21OHD) from P450 oxidoreductase deficiency (PORD) by using urinary steroid metabolites: the pregnanetriolone/tetrahydrocortisone ratio (Ptl / the cortisol metabolites 5α- and 5β-tetrahydrocortisone (sum of these metabolites termed THEs), and 11β-hydroxyandrosterone (11OHAn). The objective of this study was to investigate whether both C21OHD and non-classic 21OHD (C+NC21OHD) could be biochemically differentiated from PORD. We recruited 55 infants with C21OHD, 8 with NC21OHD, 16 with PORD, 57 with transient hyper-17α-hydroxyprogesteronemia (TH17OHP), and 2,473 controls. All infants were Japanese with ages between 0-180 d. In addition to Ptl, THEs, and 11OHAn, we measured urinary tetrahydroaldosterone (THAldo) and pregnenediol (PD5). The first step: by Ptl with the age-specific cutoffs 0.06 mg/g creatinine (0-10 d of age) and 0.3 mg/g creatinine (11-180 d of age), we were able to differentiate C+NC21OHD and PORD from TH17OHP and controls (0-10 d of age: 0.065-31 vs. < 0.001-0.052, 11-180 d of age: 0.40-42 vs. < 0.001-0.086) with 100% sensitivity and specificity. The second step: by the 11OHAn/THAldo or 11OHAn/PD5 ratio with a cutoff of 0.80 or 1.0, we were able to discriminate between C+NC21OHD and PORD (1.0-720 vs. 0.021-0.61 or 1.8-160 vs. 0.005-0.32, respectively) with 100% sensitivity and specificity. Ptl, 11OHAn/THAldo, and 11OHAn/PD5 could differentiate between C+NC21OHD and PORD in Japanese infants.

  11. Arsenite pretreatment enhances the cytotoxicity of mitomycin C in human cancer cell lines via increased NAD(P)H quinone oxidoreductase 1 expression

    SciTech Connect

    Lin Yiling; Ho, I-C.; Su, P.-F.; Lee, T.-C. . E-mail: bmtcl@ibms.sinica.edu.tw

    2006-08-01

    Arsenic is an effective therapeutic agent for the treatment of patients with refractory or relapsed acute promyelocytic leukemia. The use of arsenic for treating solid tumors, particularly in combination with other chemotherapeutic agents, has been extensively studied. Here, we report that arsenite-resistant human lung cancer CL3R15 cells constitutively overexpress NAD(P)H quinone oxidoreductase 1 (NQO1), an enzyme responsible for activation of mitomycin C (MMC), and are more susceptible to MMC cytotoxicity than parental CL3 cells. The effects of arsenite pretreatment on NQO1 induction were examined in CL3, H1299, H460, and MC-T2 cells. Arsenite pretreatment significantly enhanced the expression of NQO1 and susceptibility to MMC in CL3, H1299, and MC-T2 cells, but not in H460 cells that express high endogenous levels of NQO1. Alternatively, arsenic pretreatment reduced adriamycin sensitivity of CL3 cells. Arsenite-mediated MMC susceptibility was abrogated by dicumarol (DIC), an NQO1 inhibitor, indicating that NQO1 is one of the key regulators of arsenite-mediated MMC susceptibility. Various cancer cell lines showed different basal levels of NQO1 activity and a different capacity for NQO1 induction in response to arsenite treatment. However, overall, there was a positive correlation between induced NQO1 activity and MMC susceptibility in cells pretreated with various doses of arsenite. These results suggest that arsenite may increase NQO1 activity and thus enhance the antineoplastic activity of MMC. In addition, our results also showed that inhibition of NQO1 activity by DIC reversed the arsenite resistance of CL3R15 cells.

  12. Gypenosides Inhibits Xanthine Oxidoreductase and Ameliorates Urate Excretion in Hyperuricemic Rats Induced by High Cholesterol and High Fat Food (Lipid Emulsion)

    PubMed Central

    Pang, Minxia; Fang, Yingying; Chen, Suhong; Zhu, Xuexin; Shan, Chaowen; Su, Jie; Yu, Jingjing; Li, Bo; Yang, Yao; Chen, Bo; Liang, Kailun; Hu, Huiming; Lv, Guiyuan

    2017-01-01

    Background The aim of this study was to study the effects of gypenosides (GPS) on lowering uric acid (UA) levels in hyperuricemic rats induced by lipid emulsion (LE) and the related mechanisms. GPS are natural saponins extracted from Gynostemma pentaphyllum. Material/Methods Forty-eight male SD rats were randomly divided into six groups: normal, model, two positive controls, and two GPS treated groups (two different doses of GPS). The normal group rats were fed a basic diet, and the other rats were orally pretreated with LE. Urine and blood were collected at regular intervals. Full automatic biochemical analyzer was used to detect the concentration levels of serum UA (SUA), serum creatinine (SCr), BUN, and urine UA (UUA), and urine creatinine (UCr) and fractional excretion of UA (FEUA). ELISA kits were used to detect enzymes activities: xanthine oxidase (XOD), adenosime deaminase (ADA), guanine deaminase (GDA), and xanthine dehydrogenase (XDH). Immunohistochemistry was used to observe kidney changes and protein (URAT1, GLUT9, and OAT1) expression levels. RT-PCR was used to detect the relevant mRNA expression levels. Results Treatment with GPS significantly reduced the SUA, prevented abnormal weight loss caused by LE, and improved kidney pathomorphology. Treatment with GPS also decreased the levels of XOD, ADA, and XDH expression, increased the kidney index and FEUA, downregulated URAT1 and GLUT9 expression and upregulated OAT1 expression in the kidney. Conclusions GPS may be an effective treatment for hyperuricemia via a decrease in xanthine oxidoreductase through the XOD/XDH system; and via an increase in urate excretion through regulating URAT1, GLUT9, and OAT1 transporters. PMID:28258276

  13. Sequence analysis of the oxidase/reductase genes upstream of the Rhodococcus erythropolis aldehyde dehydrogenase gene thcA reveals a gene organisation different from Mycobacterium tuberculosis.

    PubMed

    Nagy, I; De Mot, R

    1999-01-01

    The sequence of the DNA region upstream of the thiocarbamate-inducible aldehyde dehydrogenase gene thcA of Rhodococcus erythropolis NI86/21 was determined. Most of the predicted ORFs are related to various oxidases/reductases, including short-chain oxidases/reductases, GMC oxidoreductases, alpha-hydroxy acid oxidases (subfamily 1 flavin oxidases/dehydrogenases), and subfamily 2 flavin oxidases/dehydrogenases. One ORF is related to enzymes involved in biosynthesis of PQQ or molybdopterin cofactors. In addition, a putative member of the TetR family of regulatory proteins was identified. The substantial sequence divergence from functionally characterized enzymes precludes a reliable prediction about the probable function of these proteins at this stage. In Mycobacterium tuberculosis H37Rv, most of these ORFs have homologs that are also clustered in the genome, but some striking differences in gene organization were observed between Rhodococcus and Mycobacterium.

  14. H2S exposure elicits differential expression of candidate genes in fish adapted to sulfidic and non-sulfidic environments.

    PubMed

    Tobler, Michael; Henpita, Chathurika; Bassett, Brandon; Kelley, Joanna L; Shaw, Jennifer H

    2014-09-01

    Disentangling the effects of plasticity, genetic variation, and their interactions on organismal responses to environmental stressors is a key objective in ecological physiology. We quantified the expression of five candidate genes in response to hydrogen sulfide (H2S) exposure in fish (Poecilia mexicana, Poeciliidae) from a naturally sulfide-rich environment as well as an ancestral, non-sulfidic population to test for constitutive and environmentally dependent population differences in gene expression patterns. Common garden raised individuals that had never encountered environmental H2S during their lifetime were subjected to short or long term H2S exposure treatments or respective non-sulfidic controls. The expression of genes involved in responses to H2S toxicity (cytochrome c oxidase, vascular endothelial growth factor, and cytochrome P450-2J6), H2S detoxification (sulfide:quinone oxidoreductase), and endogenous H2S production (cystathionine γ lyase) was determined in both gill and liver tissues by real time PCR. The results indicated complex changes in expression patterns that--depending on the gene--not only differed between organs and populations, but also on the type of H2S exposure. Populations differences, both constitutive and H2S exposure dependent (i.e., plastic), in gene expression were particularly evident for sulfide:quinone oxidoreductase, vascular endothelial growth factor, and to a lesser degree for cytochrome P450-2J6. Our study uncovered putatively adaptive modifications in gene regulation that parallel previously documented adaptive changes in phenotypic traits.

  15. Isolation of a gene encoding a glycosylated cytokinin oxidase from maize.

    PubMed

    Morris, R O; Bilyeu, K D; Laskey, J G; Cheikh, N N

    1999-02-16

    The major cytokinin oxidase in immature maize kernels was purified to homogeneity. Selected tryptic peptides were used to design degenerate oligonucleotide primers for PCR isolation of a fragment of the oxidase gene. Hybridization of the PCR fragment to a maize genomic library allowed isolation of a full-length cytokinin oxidase gene (ckx1). The gene encodes a protein of approximately 57 kDa that possesses a signal peptide, eight consensus N-glycosylation sequences and a consensus FAD binding sequence. Expression of ckx1 in Pichia caused secretion of active glycosylated cytokinin oxidase that contains a substrate-reducible FAD. The gene displays sequence homology with a putative oxidoreductase from Arabidopsis thaliana and with the fas5 gene from Rhodococcus fascians.

  16. The two CcdA proteins of Bacillus anthracis differentially affect virulence gene expression and sporulation.

    PubMed

    Han, Hesong; Wilson, Adam C

    2013-12-01

    The cytochrome c maturation system influences the expression of virulence factors in Bacillus anthracis. B. anthracis carries two copies of the ccdA gene, encoding predicted thiol-disulfide oxidoreductases that contribute to cytochrome c maturation, while the closely related organism Bacillus subtilis carries only one copy of ccdA. To investigate the roles of the two ccdA gene copies in B. anthracis, strains were constructed without each ccdA gene, and one strain was constructed without both copies simultaneously. Loss of both ccdA genes results in a reduction of cytochrome c production, an increase in virulence factor expression, and a reduction in sporulation efficiency. Complementation and expression analyses indicate that ccdA2 encodes the primary CcdA in B. anthracis, active in all three pathways. While CcdA1 retains activity in cytochrome c maturation and virulence control, it has completely lost its activity in the sporulation pathway. In support of this finding, expression of ccdA1 is strongly reduced when cells are grown under sporulation-inducing conditions. When the activities of CcdA1 and CcdA2 were analyzed in B. subtilis, neither protein retained activity in cytochrome c maturation, but CcdA2 could still function in sporulation. These observations reveal the complexities of thiol-disulfide oxidoreductase function in pathways relevant to virulence and physiology.

  17. Penicillium roqueforti PR toxin gene cluster characterization.

    PubMed

    Hidalgo, Pedro I; Poirier, Elisabeth; Ullán, Ricardo V; Piqueras, Justine; Meslet-Cladière, Laurence; Coton, Emmanuel; Coton, Monika

    2017-03-01

    PR toxin is a well-known isoprenoid mycotoxin almost solely produced by Penicillium roqueforti after growth on food or animal feed. This mycotoxin has been described as the most toxic produced by this species. In this study, an in silico analysis allowed identifying for the first time a 22.4-kb biosynthetic gene cluster involved in PR toxin biosynthesis in P. roqueforti. The pathway contains 11 open reading frames encoding for ten putative proteins including the major fungal terpene cyclase, aristolochene synthase, involved in the first farnesyl-diphosphate cyclization step as well as an oxidoreductase, an oxidase, two P450 monooxygenases, a transferase, and two dehydrogenase enzymes. Gene silencing was used to study three genes (ORF5, ORF6, and ORF8 encoding for an acetyltransferase and two P450 monooxygenases, respectively) and resulted in 20 to 40% PR toxin production reductions in all transformants proving the involvement of these genes and the corresponding enzyme activities in PR toxin biosynthesis. According to the considered silenced gene target, eremofortin A and B productions were also affected suggesting their involvement as biosynthetic intermediates in this pathway. A PR toxin biosynthesis pathway is proposed based on the most recent and available data.

  18. IMP2, a gene involved in the expression of glucose-repressible genes in Saccharomyces cerevisiae.

    PubMed

    Lodi, T; Goffrini, P; Ferrero, I; Donnini, C

    1995-09-01

    Two mutants carrying different deletions of the IMP2 coding sequence of Saccharomyces cerevisiae, delta T1, which encodes a protein lacking the last 26 C-terminal amino acids, and delta T2, which completely lacks the coding region, were analysed for derepression of glucose-repressible maltose, galactose, raffinose and ethanol utilization pathways in response to glucose limitation. The role of the IMP2 gene product in the regulation of carbon catabolite repressible enzymes maltase, invertase, alcohol dehydrogenase, NAD-dependent glutamate dehydrogenase (NAD-GDH) and L-lactate:ferricytochrome-c oxidoreductase (L-LCR) was also analysed. The IMP2 gene product is required for the rapid glucose derepression of all above-mentioned carbon source utilization pathways and of all the enzymes except for L-LCR. NAD-GDH is regulated by IMP2 in the opposite way and, in fact, this enzyme was released at higher levels in both imp2 mutants than in the wild-type strain. Therefore, the product of IMP2 appears to be involved in positive and negative regulation. Both deletions result in growth and catalytic defects; in some cases partial modification of the gene product yielded more dramatic effects than its complete absence. Moreover, evidence is provided that the IMP2 gene product regulates galactose- and maltose-inducible genes at the transcriptional level and is a positive regulator of maltase, maltose permease and galactose permease gene expression.

  19. NAD(P)H:quinone oxidoreductase expression in Cyp1a-knockout and CYP1A-humanized mouse lines and its effect on bioactivation of the carcinogen aristolochic acid I

    SciTech Connect

    Levova, Katerina; Moserova, Michaela; Nebert, Daniel W.; Phillips, David H.; Frei, Eva; Schmeiser, Heinz H.; Arlt, Volker M.; Stiborova, Marie

    2012-12-15

    Aristolochic acid causes a specific nephropathy (AAN), Balkan endemic nephropathy, and urothelial malignancies. Using Western blotting suitable to determine protein expression, we investigated in several transgenic mouse lines expression of NAD(P)H:quinone oxidoreductase (NQO1)—the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI). The mouse tissues used were from previous studies [Arlt et al., Chem. Res. Toxicol. 24 (2011) 1710; Stiborova et al., Toxicol. Sci. 125 (2012) 345], in which the role of microsomal cytochrome P450 (CYP) enzymes in AAI metabolism in vivo had been determined. We found that NQO1 levels in liver, kidney and lung of Cyp1a1(−/−), Cyp1a2(−/−) and Cyp1a1/1a2(−/−) knockout mouse lines, as well as in two CYP1A-humanized mouse lines harboring functional human CYP1A1 and CYP1A2 and lacking the mouse Cyp1a1/1a2 orthologs, differed from NQO1 levels in wild-type mice. NQO1 protein and enzymic activity were induced in hepatic and renal cytosolic fractions isolated from AAI-pretreated mice, compared with those in untreated mice. Furthermore, this increase in hepatic NQO1 enzyme activity was associated with bioactivation of AAI and elevated AAI-DNA adduct levels in ex vivo incubations of cytosolic fractions with DNA and AAI. In conclusion, AAI appears to increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential. Highlights: ► NAD(P)H:quinone oxidoreductase expression in Cyp1a knockout and humanized CYP1A mice ► Reductive activation of the nephrotoxic and carcinogenic aristolochic acid I (AAI) ► NAD(P)H:quinone oxidoreductase is induced in mice treated with AAI. ► Induced hepatic enzyme activity resulted in elevated AAI-DNA adduct levels.

  20. High or low correlation between co-occuring gene clusters and 16S rRNA gene phylogeny.

    PubMed

    Rudi, Knut; Sekelja, Monika

    2013-02-01

    Ribosomal RNA (rRNA) genes are universal for all living organisms. Yet, the correspondence between genome composition and rRNA phylogeny remains poorly known. The aim of this study was to use the information from genome sequence databases to address the correlation between rRNA gene phylogeny and total gene composition in bacteria. This was done by analysing 327 genomes with TIGRFAM functional gene annotations. Our approach consisted of two steps. First, we searched for discriminatory clusters of co-occurring genes. Using a multivariate statistical approach, we identified 11 such clusters which contain genes that were co-occurring only in a subset of genomes and contributed to explain the gene content differences between genome subsets. Second, we mapped the discovered clusters to 16S rRNA-based phylogeny and calculated the correlation between co-occuring genes and phylogeny. Six of the 11 clusters exhibited significant correlation with 16S rRNA gene phylogeny. The most distinct phylogenetic finding was a high correlation between iron-sulfur oxidoreductases in combination with carbon nitrogen ligases and Chlorobium. The other correlations identified covered relatively large phylogroups: Actinobacteria were positively associated with kinases, while Gammaproteobacteria were positively associated with methylases and acyltransferases. The suggested functional differences between higher phylogroups, however, need experimental verification.

  1. Influence of Various Polymorphic Variants of Cytochrome P450 Oxidoreductase (POR) on Drug Metabolic Activity of CYP3A4 and CYP2B6

    PubMed Central

    Naranmandura, Hua; Zeng, Su; Chen, Shu Qing

    2012-01-01

    Cytochrome P450 oxidoreductase (POR) is known as the sole electron donor in the metabolism of drugs by cytochrome P450 (CYP) enzymes in human. However, little is known about the effect of polymorphic variants of POR on drug metabolic activities of CYP3A4 and CYP2B6. In order to better understand the mechanism of the activity of CYPs affected by polymorphic variants of POR, six full-length mutants of POR (e.g., Y181D, A287P, K49N, A115V, S244C and G413S) were designed and then co-expressed with CYP3A4 and CYP2B6 in the baculovirus-Sf9 insect cells to determine their kinetic parameters. Surprisingly, both mutants, Y181D and A287P in POR completely inhibited the CYP3A4 activity with testosterone, while the catalytic activity of CYP2B6 with bupropion was reduced to approximately ∼70% of wild-type activity by Y181D and A287P mutations. In addition, the mutant K49N of POR increased the CLint (Vmax/Km) of CYP3A4 up to more than 31% of wild-type, while it reduced the catalytic efficiency of CYP2B6 to 74% of wild-type. Moreover, CLint values of CYP3A4-POR (A115V, G413S) were increased up to 36% and 65% of wild-type respectively. However, there were no appreciable effects observed by the remaining two mutants of POR (i.e., A115V and G413S) on activities of CYP2B6. In conclusion, the extent to which the catalytic activities of CYP were altered did not only depend on the specific POR mutations but also on the isoforms of different CYP redox partners. Thereby, we proposed that the POR-mutant patients should be carefully monitored for the activity of CYP3A4 and CYP2B6 on the prescribed medication. PMID:22719896

  2. Strain differences in the responsiveness between Sprague-Dawley and Fischer rats to nephropathy induced by FYX-051, a xanthine oxidoreductase inhibitor

    SciTech Connect

    Ashizawa, Naoki . E-mail: L2-26899@fujiyakuhin.co.jp; Shimo, Takeo; Matsumoto, Koji; Oba, Kazuhiko; Nakazawa, Takashi; Nagata, Osamu

    2006-12-15

    To determine a rat strain appropriate for carcinogenicity testing of FYX-051, a xanthine oxidoreductase inhibitor, we performed a 4-week oral toxicity study by administering 0.3, 1 and 3 mg/kg, and 1, 3 and 10 mg/kg of FYX-051 to male Sprague-Dawley (SD) and Fischer (F344) rats, respectively. Histopathology revealed that the degree of FYX-051-induced nephropathy was 3-fold stronger in SD rats than in F344 rats. Our previous study demonstrated that the key factor of species differences in FYX-051-induced nephropathy is purine metabolism. This observation led us to examine the involvement of purine metabolism in differences among two strains of rats. However, purine metabolism was proven not to be implicated as an important factor. Subsequently, other factors responsible for the strain differences were examined. FYX-051-induced increases in plasma xanthine concentrations were higher in SD rats than in F344 rats, suggesting more remarkable effects on pharmacodynamics in the former than the latter. Urinary volume was greater in F344 rats administered 10 mg/kg of FYX-051 (6.8 ml/h/kg) than in SD rats administered 3 mg/kg of FYX-051 (5.0 ml/h/kg), implying easier xanthine excretion in the former. Urinary xanthine solubility was 55 mg/dl in F344 rats aged 6 weeks, in contrast to 38 mg/dl in SD rats of the same age. Also, there were no significant differences in exposure levels at the same dose between SD and F344 rats. The outcomes of exposure levels and renal histopathology in both rats suggest the possibility that F344 rats could be exposed to a 3-fold higher amount of drug than SD rats in a carcinogenicity bioassay. The present study, therefore, suggested that strain differences of nephrotoxicity were caused by the combined effects of pharmacodynamics, xanthine excretion capacity, and urinary xanthine solubility. Furthermore, these results indicate that F344 rats would be a suitable strain for the carcinogenicity study of FYX-051.

  3. Determination of xanthine oxidoreductase activity in broilers: effect of pH and temperature of the assay and distribution in tissues.

    PubMed

    Carro, M D; Falkenstein, E; Blemings, K P; Klandorf, H

    2009-11-01

    Xanthine oxidoreductase (XOR) is the enzyme responsible for the synthesis of uric acid, which exists primarily in the dehydrogenase form in birds. Uric acid is the major end product of the metabolism of nitrogen-containing compounds in birds and it functions as an antioxidant to reduce oxidative stress. Despite the importance of this enzyme, the tissue distribution of XOR in physiologically normal chickens is not well known. In this study, we analyzed XOR activity in extracts of 8 tissues from broilers at 7 and 10 wk of age. No differences in XOR activity due to the age were found in any tissue. Liver and kidney showed the greatest activity, that in the kidney being about 89% of the activity in the liver. Enzyme activity in intestine and pancreas was about 60 and 37% of that in the liver. All breast muscle, heart, and lung samples showed enzyme activity, but values were only 3.0, 1.2, and 0.6% of those found in the liver. Traces of enzyme activity were also detected in 3 out of 10 brain samples, and no activity was found in the plasma. Our results show that XOR distribution in chickens differs from that in mammals, in which the highest levels have been found in liver and intestine. An additional objective was the evaluation of the effect of pH (7.2, 7.7, 8.2, and 8.7) and temperature (25 and 41 degrees C) on the enzyme activity in liver and kidney samples. Temperature had a similar effect on both tissues, with the activity at 25 degrees C being about 30% of that measured at 41 degrees C. At 41 degrees C, the enzyme activity in liver and kidney decreased quadratically as pH decreased from 8.7 to 7.2. The highest activity in kidney was measured at pH 8.2, although there were no differences between enzyme activities at pH 8.7 or 8.2 in the liver. Our results indicate that the optimum pH of the enzyme in chicken liver and kidney is around 8.2.

  4. Classic and non-classic 21-hydroxylase deficiency can be discriminated from P450 oxidoreductase deficiency in Japanese infants by urinary steroid metabolites

    PubMed Central

    Koyama, Yuhei; Homma, Keiko; Fukami, Maki; Miwa, Masayuki; Ikeda, Kazushige; Ogata, Tsutomu; Murata, Mitsuru; Hasegawa, Tomonobu

    2016-01-01

    Abstract. We previously reported a two-step biochemical diagnosis to discriminate classic 21-hydroxylase deficiency (C21OHD) from P450 oxidoreductase deficiency (PORD) by using urinary steroid metabolites: the pregnanetriolone/tetrahydrocortisone ratio (Ptl / the cortisol metabolites 5α- and 5β-tetrahydrocortisone (sum of these metabolites termed THEs), and 11β-hydroxyandrosterone (11OHAn). The objective of this study was to investigate whether both C21OHD and non-classic 21OHD (C+NC21OHD) could be biochemically differentiated from PORD. We recruited 55 infants with C21OHD, 8 with NC21OHD, 16 with PORD, 57 with transient hyper-17α-hydroxyprogesteronemia (TH17OHP), and 2,473 controls. All infants were Japanese with ages between 0–180 d. In addition to Ptl, THEs, and 11OHAn, we measured urinary tetrahydroaldosterone (THAldo) and pregnenediol (PD5). The first step: by Ptl with the age-specific cutoffs 0.06 mg/g creatinine (0–10 d of age) and 0.3 mg/g creatinine (11–180 d of age), we were able to differentiate C+NC21OHD and PORD from TH17OHP and controls (0–10 d of age: 0.065–31 vs. < 0.001–0.052, 11–180 d of age: 0.40–42 vs. < 0.001–0.086) with 100% sensitivity and specificity. The second step: by the 11OHAn/THAldo or 11OHAn/PD5 ratio with a cutoff of 0.80 or 1.0, we were able to discriminate between C+NC21OHD and PORD (1.0–720 vs. 0.021–0.61 or 1.8–160 vs. 0.005–0.32, respectively) with 100% sensitivity and specificity. Ptl, 11OHAn/THAldo, and 11OHAn/PD5 could differentiate between C+NC21OHD and PORD in Japanese infants. PMID:27212795

  5. Induction of NAD(P)H:quinone oxidoreductase 1 (NQO1) by Glycyrrhiza species used for women's health: differential effects of the Michael acceptors isoliquiritigenin and licochalcone A

    PubMed Central

    Hajirahimkhan, Atieh; Simmler, Charlotte; Dong, Huali; Lantvit, Daniel D.; Li, Guannan; Chen, Shao-Nong; Nikolić, Dejan; Pauli, Guido F.; van Breemen, Richard B.; Dietz, Birgit M.; Bolton, Judy L.

    2016-01-01

    For the alleviation of menopausal symptoms, women frequently turn to botanical dietary supplements, such as licorice and hops. In addition to estrogenic properties, these botanicals could also have chemopreventive effects. We have previously shown that hops and its Michael acceptor xanthohumol (XH) induced the chemoprevention enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1), in vitro and in vivo. Licorice species could also induce NQO1, as they contain the Michael acceptors isoliquiritigenin (LigC) found in Glycyrrhiza glabra (GG), G. uralensis (GU), and G. inflata (GI) and licochalcone A (LicA) which is only found in GI. These licorice species and hops induced NQO1 activity in murine hepatoma (Hepa1c1c7) cells; hops >> GI > GG ≅ GU. Similar to the known chemopreventive compounds curcumin (turmeric), sulforaphane (broccoli), and XH, LigC and LicA were active dose-dependently; sulforaphane >> XH > LigC > LicA ≅ curcumin >> LigF. Induction of the antioxidant response element-luciferase in human hepatoma (Hep-G2-ARE-C8) cells suggested involvement of the Keap1-Nrf2 pathway. GG, GU, and LigC also induced NQO1 in non-tumorigenic breast epithelial MCF-10A cells. In female Sprague-Dawley rats treated with GG and GU, LigC and LigF were detected in the liver and mammary gland. GG weakly enhanced NQO1 activity in the mammary tissue but not in the liver. Treatment with LigC alone did not induce NQO1 in vivo most likely due to its conversion to LigF, extensive metabolism, and its low bioavailability in vivo. These data show the chemopreventive potential of licorice species in vitro could be due to LigC and LicA and emphasize the importance of chemical and biological standardization of botanicals used as dietary supplements. Although the in vivo effects in the rat model after four day treatment are minimal, it must be emphasized that menopausal women take these supplements for extended periods of time and long-term beneficial effects are quite possible. PMID:26473469

  6. Genome-wide analysis of glutathione reductase (GR) genes from rice and Arabidopsis.

    PubMed

    Trivedi, Dipesh Kumar; Gill, Sarvajeet Singh; Yadav, Sandep; Tuteja, Narendra

    2013-02-01

    Plant cells and tissues remain always on risk under abiotic and biotic stresses due to increased production of reactive oxygen species (ROS). Plants protect themselves against ROS induced oxidative damage by the upregulation of antioxidant machinery. Out of many components of antioxidant machinery, glutathione reductase (GR, EC 1.6.4.2) and glutathione (GSH, γ-Glu-Cys-Gly) play important role in the protection of cell against oxidative damage. In stress condition, the GR helps in maintaining the reduced glutathione pool for strengthening the antioxidative processes in plants. Present study investigates genome wide analysis of GR from rice and Arabidopsis. We were able to identify 3 rice GR genes (LOC_Os02 g56850, LOC_Os03 g06740, LOC_Os10 g28000) and 2 Arabidopsis GR genes (AT3G54660, AT3G24170) from their respective genomes on the basis of their annotation as well as the presence of pyridine nucleotide-disulphide oxidoreductases class-I active site. The evolutionary relationship of the GR genes from rice and Arabidopsis genomes was analyzed using the multiple sequence alignment and phylogenetic tree. This revealed evolutionary conserved pyridine nucleotide-disulphide oxidoreductases class-I active site among the GR protein in rice and Arabidopsis. This study should make an important contribution to our better understanding of the GR under normal and stress condition in plants.

  7. Aluminum Induces Oxidative Stress Genes in Arabidopsis thaliana1

    PubMed Central

    Richards, Keith D.; Schott, Eric J.; Sharma, Yogesh K.; Davis, Keith R.; Gardner, Richard C.

    1998-01-01

    Changes in gene expression induced by toxic levels of Al were characterized to investigate the nature of Al stress. A cDNA library was constructed from Arabidopsis thaliana seedlings treated with Al for 2 h. We identified five cDNA clones that showed a transient induction of their mRNA levels, four cDNA clones that showed a longer induction period, and two down-regulated genes. Expression of the four long-term-induced genes remained at elevated levels for at least 48 h. The genes encoded peroxidase, glutathione-S-transferase, blue copper-binding protein, and a protein homologous to the reticuline:oxygen oxidoreductase enzyme. Three of these genes are known to be induced by oxidative stresses and the fourth is induced by pathogen treatment. Another oxidative stress gene, superoxide dismutase, and a gene for Bowman-Birk protease inhibitor were also induced by Al in A. thaliana. These results suggested that Al treatment of Arabidopsis induces oxidative stress. In confirmation of this hypothesis, three of four genes induced by Al stress in A. thaliana were also shown to be induced by ozone. Our results demonstrate that oxidative stress is an important component of the plant's reaction to toxic levels of Al. PMID:9449849

  8. Gene duplication, exon gain and neofunctionalization of OEP16-related genes in land plants.

    PubMed

    Drea, Sinéad C; Lao, Nga T; Wolfe, Kenneth H; Kavanagh, Tony A

    2006-06-01

    OEP16, a channel protein of the outer membrane of chloroplasts, has been implicated in amino acid transport and in the substrate-dependent import of protochlorophyllide oxidoreductase A. Two major clades of OEP16-related sequences were identified in land plants (OEP16-L and OEP16-S), which arose by a gene duplication event predating the divergence of seed plants and bryophytes. Remarkably, in angiosperms, OEP16-S genes evolved by gaining an additional exon that extends an interhelical loop domain in the pore-forming region of the protein. We analysed the sequence, structure and expression of the corresponding Arabidopsis genes (atOEP16-S and atOEP16-L) and demonstrated that following duplication, both genes diverged in terms of expression patterns and coding sequence. AtOEP16-S, which contains multiple G-box ABA-responsive elements (ABREs) in the promoter region, is regulated by ABI3 and ABI5 and is strongly expressed during the maturation phase in seeds and pollen grains, both desiccation-tolerant tissues. In contrast, atOEP-L, which lacks promoter ABREs, is expressed predominantly in leaves, is induced strongly by low-temperature stress and shows weak induction in response to osmotic stress, salicylic acid and exogenous ABA. Our results indicate that gene duplication, exon gain and regulatory sequence evolution each played a role in the divergence of OEP16 homologues in plants.

  9. Isolation, identification and sequence analysis of a thioredoxin h gene, a member of subgroup III of h-type Trxs from grape (Vitis vinifera L. cv. Askari).

    PubMed

    Japelaghi, Reza Heidari; Haddad, Raheem; Garoosi, Ghasem-Ali

    2012-04-01

    Thioredoxins (Trxs) are small ubiquitous proteins which play a regulatory role in a variety of cellular processes. In contrast to other organisms, plants have a great number of Trx types, consisting of six well-defined groups: f, m, x, and y in chloroplasts, o in mitochondria, and h mainly in cytosol. A full-length cDNA, designated VvCxxS2, encoding Trx h polypeptide was isolated and cloned from grape (Vitis vinifera L. cv. Askari) berries organ by reverse transcription polymerase chain reaction (RT-PCR). The cDNA was 381 bp nucleotides in length with a deduced amino acid of 126 residues, possessing a WCIPS active site, which belongs to the subgroup III of h-type Trxs based on phylogenetic analysis. The calculated molecular mass and the predicted isoelectric point of the deduced polypeptide are 14.25 kDa and 4.68, respectively. Nucleotide sequence analysis of genomic DNA fragment of VvCxxS2 gene revealed that this gene possesses two introns at positions identical to the previously sequenced Trx h genes. A modeling analysis indicated that VvCxxS2 shares a common structure with other Trxs, and is preferably reduced by Grx rather than NADPH-dependent thioredoxin reductase (NTR). The deduced protein sequence showed a high similarity to Trx h from other plants, in particular from castor bean (Ricinus communis), Betula pendula and sweet orange (Citrus sinensis). Semiquantitative RT-PCR experiments indicated that the transcripts of VvCxxS2 gene are present in all plant organs and different developmental stages. In addition, the higher expression of the VvCxxS2 gene was observed in berry organ as compared to the other organs.

  10. The Thiol Reductase Activity of YUCCA6 Mediates Delayed Leaf Senescence by Regulating Genes Involved in Auxin Redistribution

    PubMed Central

    Cha, Joon-Yung; Kim, Mi R.; Jung, In J.; Kang, Sun B.; Park, Hee J.; Kim, Min G.; Yun, Dae-Jin; Kim, Woe-Yeon

    2016-01-01

    Auxin, a phytohormone that affects almost every aspect of plant growth and development, is biosynthesized from tryptophan via the tryptamine, indole-3-acetamide, indole-3-pyruvic acid, and indole-3-acetaldoxime pathways. YUCCAs (YUCs), flavin monooxygenase enzymes, catalyze the conversion of indole-3-pyruvic acid (IPA) to the auxin (indole acetic acid). Arabidopsis thaliana YUC6 also exhibits thiol-reductase and chaperone activity in vitro; these activities require the highly conserved Cys-85 and are essential for scavenging of toxic reactive oxygen species (ROS) in the drought tolerance response. Here, we examined whether the YUC6 thiol reductase activity also participates in the delay in senescence observed in YUC6-overexpressing (YUC6-OX) plants. YUC6 overexpression delays leaf senescence in natural and dark-induced senescence conditions by reducing the expression of SENESCENCE-ASSOCIATED GENE 12 (SAG12). ROS accumulation normally occurs during senescence, but was not observed in the leaves of YUC6-OX plants; however, ROS accumulation was observed in YUC6-OXC85S plants, which overexpress a mutant YUC6 that lacks thiol reductase activity. We also found that YUC6-OX plants, but not YUC6-OXC85S plants, show upregulation of three genes encoding NADPH-dependent thioredoxin reductases (NTRA, NTRB, and NTRC), and GAMMA-GLUTAMYLCYSTEINE SYNTHETASE 1 (GSH1), encoding an enzyme involved in redox signaling. We further determined that excess ROS accumulation caused by methyl viologen treatment or decreased glutathione levels caused by buthionine sulfoximine treatment can decrease the levels of auxin efflux proteins such as PIN2-4. The expression of PINs is also reduced in YUC6-OX plants. These findings suggest that the thiol reductase activity of YUC6 may play an essential role in delaying senescence via the activation of genes involved in redox signaling and auxin availability. PMID:27242830

  11. The thioredoxin system of Penicillium chrysogenum and its possible role in penicillin biosynthesis.

    PubMed Central

    Cohen, G; Argaman, A; Schreiber, R; Mislovati, M; Aharonowitz, Y

    1994-01-01

    Penicillium chrysogenum is an important producer of penicillin antibiotics. A key step in their biosynthesis is the oxidative cyclization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N by the enzyme isopenicillin N synthase (IPNS). bis-ACV, the oxidized disulfide form of ACV is, however, not a substrate for IPNS. We report here the characterization of a broad-range disulfide reductase from P. chrysogenum that efficiently reduces bis-ACV to the thiol monomer. When coupled in vitro with IPNS, it converts bis-ACV to isopenicillin N and may therefore play a role in penicillin biosynthesis. The disulfide reductase consists of two protein components, a 72-kDa NADPH-dependent reductase, containing two identical subunits, and a 12-kDa general disulfide reductant. The latter reduces disulfide bonds in low-molecular-weight compounds and in proteins. The genes coding for the reductase system were cloned and sequenced. Both possess introns. A comparative analysis of their predicted amino acid sequences showed that the 12-kDa protein shares 26 to 60% sequence identity with thioredoxins and that the 36-kDa protein subunit shares 44 to 49% sequence identity with the two known bacterial thioredoxin reductases. In addition, the P. chrysogenum NADPH-dependent reductase is able to accept thioredoxin as a substrate. These results establish that the P. chrysogenum broad-range disulfide reductase is a member of the thioredoxin family of oxidoreductases. This is the first example of the cloning of a eucaryotic thioredoxin reductase gene. Images PMID:8106340

  12. The thioredoxin system of Penicillium chrysogenum and its possible role in penicillin biosynthesis.

    PubMed

    Cohen, G; Argaman, A; Schreiber, R; Mislovati, M; Aharonowitz, Y

    1994-02-01

    Penicillium chrysogenum is an important producer of penicillin antibiotics. A key step in their biosynthesis is the oxidative cyclization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N by the enzyme isopenicillin N synthase (IPNS). bis-ACV, the oxidized disulfide form of ACV is, however, not a substrate for IPNS. We report here the characterization of a broad-range disulfide reductase from P. chrysogenum that efficiently reduces bis-ACV to the thiol monomer. When coupled in vitro with IPNS, it converts bis-ACV to isopenicillin N and may therefore play a role in penicillin biosynthesis. The disulfide reductase consists of two protein components, a 72-kDa NADPH-dependent reductase, containing two identical subunits, and a 12-kDa general disulfide reductant. The latter reduces disulfide bonds in low-molecular-weight compounds and in proteins. The genes coding for the reductase system were cloned and sequenced. Both possess introns. A comparative analysis of their predicted amino acid sequences showed that the 12-kDa protein shares 26 to 60% sequence identity with thioredoxins and that the 36-kDa protein subunit shares 44 to 49% sequence identity with the two known bacterial thioredoxin reductases. In addition, the P. chrysogenum NADPH-dependent reductase is able to accept thioredoxin as a substrate. These results establish that the P. chrysogenum broad-range disulfide reductase is a member of the thioredoxin family of oxidoreductases. This is the first example of the cloning of a eucaryotic thioredoxin reductase gene.

  13. The Diversity of Sulfide Oxidation and Sulfate Reduction Genes Expressed by the Bacterial Communities of the Cariaco Basin, Venezuela

    PubMed Central

    Rodriguez-Mora, Maria J.; Edgcomb, Virginia P.; Taylor, Craig; Scranton, Mary I.; Taylor, Gordon T.; Chistoserdov, Andrei Y.

    2016-01-01

    Qualitative expression of dissimilative sulfite reductase (dsrA), a key gene in sulfate reduction, and sulfide:quinone oxidoreductase (sqr), a key gene in sulfide oxidation was investigated. Neither of the two could be amplified from mRNA retrieved with Niskin bottles but were amplified from mRNA retrieved by the Deep SID. The sqr and sqr-like genes retrieved from the Cariaco Basin were related to the sqr genes from a Bradyrhizobium sp., Methylomicrobium alcaliphilum, Sulfurovum sp. NBC37-1, Sulfurimonas autotrophica, Thiorhodospira sibirica and Chlorobium tepidum. The dsrA gene sequences obtained from the redoxcline of the Cariaco Basin belonged to chemoorganotrophic and chemoautotrophic sulfate and sulfur reducers belonging to the class Deltaproteobacteria (phylum Proteobacteria) and the order Clostridiales (phylum Firmicutes). PMID:27651847

  14. The Diversity of Sulfide Oxidation and Sulfate Reduction Genes Expressed by the Bacterial Communities of the Cariaco Basin, Venezuela.

    PubMed

    Rodriguez-Mora, Maria J; Edgcomb, Virginia P; Taylor, Craig; Scranton, Mary I; Taylor, Gordon T; Chistoserdov, Andrei Y

    2016-01-01

    Qualitative expression of dissimilative sulfite reductase (dsrA), a key gene in sulfate reduction, and sulfide:quinone oxidoreductase (sqr), a key gene in sulfide oxidation was investigated. Neither of the two could be amplified from mRNA retrieved with Niskin bottles but were amplified from mRNA retrieved by the Deep SID. The sqr and sqr-like genes retrieved from the Cariaco Basin were related to the sqr genes from a Bradyrhizobium sp., Methylomicrobium alcaliphilum, Sulfurovum sp. NBC37-1, Sulfurimonas autotrophica, Thiorhodospira sibirica and Chlorobium tepidum. The dsrA gene sequences obtained from the redoxcline of the Cariaco Basin belonged to chemoorganotrophic and chemoautotrophic sulfate and sulfur reducers belonging to the class Deltaproteobacteria (phylum Proteobacteria) and the order Clostridiales (phylum Firmicutes).

  15. Identification of pathways, gene networks and paralogous gene families in Daphnia pulex responding to exposure to the toxic cyanobacterium Microcystis aeruginosa

    PubMed Central

    Asselman, Jana; De Coninck, Dieter IM; Glaholt, Stephen; Colbourne, John K; Janssen, Colin R; Shaw, Joseph R; De Schamphelaere, Karel AC

    2013-01-01

    Although cyanobacteria produce a wide range of natural toxins that impact aquatic organisms, food webs and water quality, the mechanisms of toxicity are still insufficiently understood. Here, we implemented a whole-genome expression microarray to identify pathways, gene networks and paralogous gene families responsive to Microcystis stress in Daphnia pulex. Therefore, neonates of a sensitive isolate were given a diet contaminated with Microcystis to contrast with those given a control diet for sixteen days. The microarray revealed 2247 differentially expressed (DE) genes (7.6% of the array) in response to Microcystis, of which 17% are lineage specific( i.e., these genes have no detectable homology to any other gene in currently available databases) and 49% are gene duplicates (paralogs). We identified four pathways/gene networks and eight paralogous gene families affected by Microcystis. Differential regulation of the ribosome, including 3 paralogous gene families encoding 40S, 60S and mitochondrial ribosomal proteins, suggests an impact of Microcystis on protein synthesis of D. pulex. In addition, differential regulation of the oxidative phosphorylation pathway (including the NADH ubquinone oxidoreductase gene family) and the trypsin paralogous gene family (a major component of the digestive system in D. pulex) could explain why fitness is reduced based on energy budget considerations. PMID:22799445

  16. Novel integrons and gene cassettes from a Cascadian submarine gas-hydrate-bearing core.

    PubMed

    Elsaied, Hosam; Stokes, Hatch W; Yoshioka, Hideyoshi; Mitani, Yasuo; Maruyama, Akihiko

    2014-02-01

    To determine whether integrons are present in a submarine gas hydrate community, metagenomic DNA was extracted from a gas-hydrate-bearing core, 150 m below the seafloor, from the Cascadian Margin. Integrons and gene cassettes were recovered by PCR from metagenomic DNA and sequenced. Thirty-seven integron integrase phylotypes were identified. The phylotypes were diverse and included members with homology to integrases from Methylomonas methanica, Desulfuromonas acetoxidans, Thermodesulfatator indicus, and marine uncultured bacteria. The gene cassette composition, 153 gene cassettes, was dominated by two types of encoded putative proteins. The first of these was predicted oxidoreductases, such as iron/sulfur cluster-binding proteins. A second type was alkyl transferases. Some cassette proteins showed homologies with those from methane-related archaea. These observations suggest that integrons may assist in the adaptation of microbial communities in this environment.

  17. Characterization of Pseudomonas putida Genes Responsive to Nutrient Limitation

    SciTech Connect

    Syn, Chris K.; Magnuson, Jon K.; Kingsley, Mark T.; Swarup, Sanjay

    2004-06-01

    The low bioavailability of nutrients and oxygen in the soil environment has hampered successful expression of biodegradation/biocontrol genes that are driven by promoters highly active during routine laboratory conditions of high nutrient- and oxygen-availability. Hence, in the present study, expression of the gus-tagged genes in 12 Tn5-gus mutants of the soil microbe Pseudomonas putida PNL-MK25 was examined under various conditions chosen to mimic the soil environment: low carbon, phosphate, nitrate, or oxygen, and in the rhizosphere. Based on their expression profiles, three nutrient-responsive mutant (NRM) strains, NRM5, NRM7, and NRM17, were selected for identification of the tagged genes. In the mutant strain NRM5, expression of the glutamate dehydrogenase (gdhA) gene was increased between 4.9- to 26.4-fold under various low nutrient conditions. In NRM7, expression of the novel NADPH:quinone oxidoreductase-like (nql) gene was consistently amongst the highest and was synergistically upregulated by low nutrient and anoxic conditions. The cyoD gene in NRM17, which encodes the fourth subunit of the cytochrome o ubiquinol oxidase complex, had decreased expression in low nutrient conditions but its absolute expression levels was still amongst the highest. Additionally, it was independent of oxygen availability, in contrast to that in E. coli.

  18. High-Level Chromate Resistance in Arthrobacter sp. strain FB24 Requires Previously Uncharacterized Accessory Genes

    SciTech Connect

    Henne, Kristene L.; Nakatsu, Cindy N.; Thompson, Dorothea K.; Konopka, Allan

    2009-09-24

    The annotated genome sequence of Arthrobacter sp. strain FB24 revealed a chromate resistance determinant (CRD): a cluster of 8 genes located on a 10.6 kb fragment of a 96 kb plasmid. The CRD includes chrA, which encodes a putative chromate efflux protein, and three genes with amino acid similarities to the amino and carboxy termini of ChrB, a putative regulatory protein. There are also three novel genes that have not been previously associated with chromate resistance in other bacteria; they encode an oxidoreductase (most similar to malate:quinone oxidoreductase), a functionally unknown protein with a WD40 repeat domain and a lipoprotein. A chromate-sensitive mutant (strain D11) was generated by curing FB24 of its 96-kb plasmid. Elemental analysis indicated that chromate-exposed cells of strain D11 accumulated three times more chromium than strain FB24. Introduction of the CRD into strain D11 conferred chromate resistance comparable to wild-type levels, whereas deletion of specific regions of the CRD led to decreased resistance. Using real-time reverse transcriptase PCR, we show that expression of each gene within the CRD is specifically induced in response to chromate but not by lead, hydrogen peroxide or arsenate. Higher levels of chrA expression were achieved when the chrB orthologs and the WD40 repeat domain genes were present, suggesting their regulatory roles. Collectively, our findings indicate that chromate resistance in strain FB24 is primarily achieved by plasmid-mediated chromate efflux with the contribution of previously unrecognized accessory genes.

  19. The Role of Glycine Residues 140 and 141 of Subunit B in the Functional Ubiquinone Binding Site of the Na+-pumping NADH:quinone Oxidoreductase from Vibrio cholerae*

    PubMed Central

    Juárez, Oscar; Neehaul, Yashvin; Turk, Erin; Chahboun, Najat; DeMicco, Jessica M.; Hellwig, Petra; Barquera, Blanca

    2012-01-01

    The Na+-pumping NADH:quinone oxidoreductase (Na+-NQR) is the main entrance for electrons into the respiratory chain of many marine and pathogenic bacteria. The enzyme accepts electrons from NADH and donates them to ubiquinone, and the free energy released by this redox reaction is used to create an electrochemical gradient of sodium across the cell membrane. Here we report the role of glycine 140 and glycine 141 of the NqrB subunit in the functional binding of ubiquinone. Mutations at these residues altered the affinity of the enzyme for ubiquinol. Moreover, mutations in residue NqrB-G140 almost completely abolished the electron transfer to ubiquinone. Thus, NqrB-G140 and -G141 are critical for the binding and reaction of Na+-NQR with its electron acceptor, ubiquinone. PMID:22645140

  20. Structure-based computational study of two disease resistance gene homologues (Hm1 and Hm2) in maize (Zea mays L.) with implications in plant-pathogen interactions.

    PubMed

    Dehury, Budheswar; Patra, Mahesh Chandra; Maharana, Jitendra; Sahu, Jagajjit; Sen, Priyabrata; Modi, Mahendra Kumar; Choudhury, Manabendra Dutta; Barooah, Madhumita

    2014-01-01

    The NADPH-dependent HC-toxin reductases (HCTR1 and 2) encoded by enzymatic class of disease resistance homologous genes (Hm1 and Hm2) protect maize by detoxifying a cyclic tetrapeptide, HC-toxin, secreted by the fungus Cochliobolus carbonum race 1(CCR1). Unlike the other classes' resistance (R) genes, HCTR-mediated disease resistance is an inimitable mechanism where the avirulence (Avr) component from CCR1 is not involved in toxin degradation. In this study, we attempted to decipher cofactor (NADPH) recognition and mode of HC-toxin binding to HCTRs through molecular docking, molecular dynamics (MD) simulations and binding free energy calculation methods. The rationality and the stability of docked complexes were validated by 30-ns MD simulation. The binding free energy decomposition of enzyme-cofactor complex was calculated to find the driving force behind cofactor recognition. The overall binding free energies of HCTR1-NADPH and HCTR2-NADPH were found to be -616.989 and -16.9749 kJ mol-1 respectively. The binding free energy decomposition revealed that the binding of NADPH to the HCTR1 is mainly governed by van der Waals and nonpolar interactions, whereas electrostatic terms play dominant role in stabilizing the binding mode between HCTR2 and NADPH. Further, docking analysis of HC-toxin with HCTR-NADPH complexes showed a distinct mode of binding and the complexes were stabilized by a strong network of hydrogen bond and hydrophobic interactions. This study is the first in silico attempt to unravel the biophysical and biochemical basis of cofactor recognition in enzymatic class of R genes in cereal crop maize.

  1. Novel insights into structure–function mechanism and tissue-specific expression profiling of full-length dxr gene from Cymbopogon winterianus

    PubMed Central

    Devi, Kamalakshi; Dehury, Budheswar; Phukon, Munmi; Modi, Mahendra Kumar; Sen, Priyabrata

    2015-01-01

    The 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR; EC1.1.1.267), an NADPH-dependent reductase, plays a pivotal role in the methylerythritol 4-phosphate pathway (MEP), in the conversion of 1-deoxy-d-xylulose-5-phosphate (DXP) into MEP. The sheath and leaf of citronella (Cymbopogon winterianus) accumulates large amount of terpenes and sesquiterpenes with proven medicinal value and economic uses. Thus, sequencing of full length dxr gene and its characterization seems to be a valuable resource in metabolic engineering to alter the flux of isoprenoid active ingredients in plants. In this study, full length DXR from citronella was characterized through in silico and tissue-specific expression studies to explain its structure–function mechanism, mode of cofactor recognition and differential expression. The modelled DXR has a three-domain architecture and its active site comprised of a cofactor (NADPH) binding pocket and the substrate-binding pocket. Molecular dynamics simulation studies indicated that DXR model retained most of its secondary structure during 10 ns simulation in aqueous solution. The modelled DXR superimposes well with its closest structural homolog but subtle variations in the charge distribution over the cofactor recognition site were noticed. Molecular docking study revealed critical residues aiding tight anchoring NADPH within the active pocket of DXR. Tissue-specific differential expression analysis using semi-quantitative RT-PCR and qRT-PCR in various tissues of citronella plant revealed distinct differential expression of DXR. To our knowledge, this is the first ever report on DXR from the important medicinal plant citronella and further characterization of this gene will open up better avenues for metabolic engineering of secondary metabolite pathway genes from medicinal plants in the near future. PMID:25941629

  2. Environmental pollutant and potent mutagen 3-nitrobenzanthrone forms DNA adducts after reduction by NAD(P)H:quinone oxidoreductase and conjugation by acetyltransferases and sulfotransferases in human hepatic cytosols.

    PubMed

    Arlt, Volker M; Stiborova, Marie; Henderson, Colin J; Osborne, Martin R; Bieler, Christian A; Frei, Eva; Martinek, Vaclav; Sopko, Bruno; Wolf, C Roland; Schmeiser, Heinz H; Phillips, David H

    2005-04-01

    3-Nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one, 3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and air pollution. We compared the ability of human hepatic cytosolic samples to catalyze DNA adduct formation by 3-NBA. Using the (32)P-postlabeling method, we found that 12/12 hepatic cytosols activated 3-NBA to form multiple DNA adducts similar to those formed in vivo in rodents. By comparing 3-NBA-DNA adduct formation in the presence of cofactors of NAD(P)H:quinone oxidoreductase (NQO1) and xanthine oxidase, most of the reductive activation of 3-NBA in human hepatic cytosols was attributed to NQO1. Inhibition of adduct formation by dicoumarol, an NQO1 inhibitor, supported this finding and was confirmed with human recombinant NQO1. When cofactors of N,O-acetyltransferases (NAT) and sulfotransferases (SULT) were added to cytosolic samples, 3-NBA-DNA adduct formation increased 10- to 35-fold. Using human recombinant NQO1 and NATs or SULTs, we found that mainly NAT2, followed by SULT1A2, NAT1, and, to a lesser extent, SULT1A1 activate 3-NBA. We also evaluated the role of hepatic NADPH:cytochrome P450 oxidoreductase (POR) in the activation of 3-NBA in vivo by treating hepatic POR-null mice and wild-type littermates i.p. with 0.2 or 2 mg/kg body weight of 3-NBA. No difference in DNA binding was found in any tissue examined (liver, lung, kidney, bladder, and colon) between null and wild-type mice, indicating that 3-NBA is predominantly activated by cytosolic nitroreductases rather than microsomal POR. Collectively, these results show the role of human hepatic NQO1 to reduce 3-NBA to species being further activated by NATs and SULTs.

  3. Effects of Parsley (Petroselinum crispum) and its Flavonol Constituents, Kaempferol and Quercetin, on Serum Uric Acid Levels, Biomarkers of Oxidative Stress and Liver Xanthine Oxidoreductase Aactivity inOxonate-Induced Hyperuricemic Rats.

    PubMed

    Haidari, Fatemeh; Keshavarz, Seid Ali; Mohammad Shahi, Majid; Mahboob, Soltan-Ali; Rashidi, Mohammad-Reza

    2011-01-01

    Increased serum uric acid is known to be a major risk related to the development of several oxidative stress diseases. The aim of this study was to investigate the effect of parsley, quercetin and kaempferol on serum uric acid levels, liver xanthine oxidoreductase activity and two non-invasive biomarkers of oxidative stress (total antioxidant capacity and malondialdehyde concentration) in normal and oxonate-induced hyperuricemic rats. A total of 60 male Wistar rats were randomly divided into ten equal groups; including 5 normal groups (vehicle, parsley, quercetin, kaempferol and allopurinol) and 5 hyperuricemic groups (vehicle, parsley, quercetin, kaempferol and allopurinol). Parsley (5 g/Kg), quercetin (5 mg/Kg), kaempferol (5 mg/Kg) and allopurinol (5 mg/Kg) were administrated to the corresponding groups by oral gavage once a day for 2 weeks. The results showed that parsley and its flavonol did not cause any significant reduction in the serum uric acid levels in normal rats, but significantly reduced the serum uric acid levels of hyperuricemic rats in a time-dependent manner. All treatments significantly inhibited liver xanthine oxidoreductase activity. Parsley, kaempferol and quercetin treatment led also to a significant increase in total antioxidant capacity and decrease in malondialdehyde concentration in hyperuricemic rats. Although the hypouricemic effect of allopurinol was much higher than that of parsley and its flavonol constituents, it could not significantly change oxidative stress biomarkers. These features of parsley and its flavonols make them as a possible alternative for allopurinol, or at least in combination therapy to minimize the side effects of allopurinol to treat hyperuricemia and oxidative stress diseases.

  4. Omeprazole Induces NAD(P)H Quinone Oxidoreductase 1 via Aryl Hydrocarbon Receptor-Independent Mechanisms: Role of the Transcription Factor Nuclear Factor Erythroid 2–Related Factor 2

    PubMed Central

    Zhang, Shaojie; Patel, Ananddeep; Moorthy, Bhagavatula; Shivanna, Binoy

    2015-01-01

    Activation of the aryl hydrocarbon receptor (AhR) transcriptionally induces phase I (cytochrome P450 (CYP) 1A1) and phase II (NAD(P)H quinone oxidoreductase 1 (NQO1) detoxifying enzymes. The effects of the classical and nonclassical AhR ligands on phase I and II enzymes are well studied in human hepatocytes. Additionally, we observed that the proton pump inhibitor, omeprazole (OM), transcriptionally induces CYP1A1 in the human adenocarcinoma cell line, H441 cells via AhR. Whether OM activates AhR and induces the phase II enzyme, NAD(P)H quinone oxidoreductase 1 (NQO1), in fetal primary human pulmonary microvascular endothelial cells (HPMEC) is unknown. Therefore, we tested the hypothesis that OM will induce NQO1 in HPMEC via the AhR. The concentrations of OM used in our experiments did not result in cytotoxicity. OM activated AhR as evident by increased CYP1A1 mRNA expression. However, contrary to our hypothesis, OM increased NQO1 mRNA and protein via an AhR-independent mechanism as AhR knockdown failed to abrogate OM-mediated increase in NQO1 expression. Interestingly, OM activated Nrf2 as evident by increased phosphoNrf2 (S40) expression in OM-treated compared to vehicle-treated cells. Furthermore, Nrf2 knockdown abrogated OM-mediated increase in NQO1 expression. In conclusion, we provide evidence that OM induces NQO1 via AhR-independent, but Nrf2-dependent mechanisms. PMID:26441083

  5. The nairovirus nairobi sheep disease virus/ganjam virus induces the translocation of protein disulphide isomerase-like oxidoreductases from the endoplasmic reticulum to the cell surface and the extracellular space.

    PubMed

    Lasecka, Lidia; Baron, Michael D

    2014-01-01

    Nairobi sheep disease virus (NSDV) of the genus Nairovirus causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%; the virus is found in East and Central Africa, and in India, where the virus is called Ganjam virus. NSDV is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus, which also causes a haemorrhagic disease. As with other nairoviruses, replication of NSDV takes place in the cytoplasm and the new virus particles bud into the Golgi apparatus; however, the effect of viral replication on cellular compartments has not been studied extensively. We have found that the overall structure of the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment and the Golgi were unaffected by infection with NSDV. However, we observed that NSDV infection led to the loss of protein disulphide isomerase (PDI), an oxidoreductase present in the lumen of the endoplasmic reticulum (ER) and which assists during protein folding, from the ER. Further investigation showed that NSDV-infected cells have high levels of PDI at their surface, and PDI is also secreted into the culture medium of infected cells. Another chaperone from the PDI family, ERp57, was found to be similarly affected. Analysis of infected cells and expression of individual viral glycoproteins indicated that the NSDV PreGn glycoprotein is involved in redistribution of these soluble ER oxidoreductases. It has been suggested that extracellular PDI can activate integrins and tissue factor, which are involved respectively in pro-inflammatory responses and disseminated intravascular coagulation, both of which manifest in many viral haemorrhagic fevers. The discovery of enhanced PDI secretion from NSDV-infected cells may be an important finding for understanding the mechanisms underlying the pathogenicity of haemorrhagic nairoviruses.

  6. Structure and sequence conservation of hao cluster genes of autotrophic ammonia-oxidizing bacteria: evidence for their evolutionary history.

    PubMed

    Bergmann, David J; Hooper, Alan B; Klotz, Martin G

    2005-09-01

    Comparison of the organization and sequence of the hao (hydroxylamine oxidoreductase) gene clusters from the gammaproteobacterial autotrophic ammonia-oxidizing bacterium (aAOB) Nitrosococcus oceani and the betaproteobacterial aAOB Nitrosospira multiformis and Nitrosomonas europaea revealed a highly conserved gene cluster encoding the following proteins: hao, hydroxylamine oxidoreductase; orf2, a putative protein; cycA, cytochrome c(554); and cycB, cytochrome c(m)(552). The deduced protein sequences of HAO, c(554), and c(m)(552) were highly similar in all aAOB despite their differences in species evolution and codon usage. Phylogenetic inference revealed a broad family of multi-c-heme proteins, including HAO, the pentaheme nitrite reductase, and tetrathionate reductase. The c-hemes of this group also have a nearly identical geometry of heme orientation, which has remained conserved during divergent evolution of function. High sequence similarity is also seen within a protein family, including cytochromes c(m)(552), NrfH/B, and NapC/NirT. It is proposed that the hydroxylamine oxidation pathway evolved from a nitrite reduction pathway involved in anaerobic respiration (denitrification) during the radiation of the Proteobacteria. Conservation of the hydroxylamine oxidation module was maintained by functional pressure, and the module expanded into two separate narrow taxa after a lateral gene transfer event between gamma- and betaproteobacterial ancestors of extant aAOB. HAO-encoding genes were also found in six non-aAOB, either singly or tandemly arranged with an orf2 gene, whereas a c(554) gene was lacking. The conservation of the hao gene cluster in general and the uniqueness of the c(554) gene in particular make it a suitable target for the design of primers and probes useful for molecular ecology approaches to detect aAOB.

  7. Structure and Sequence Conservation of hao Cluster Genes of Autotrophic Ammonia-Oxidizing Bacteria: Evidence for Their Evolutionary History

    PubMed Central

    Bergmann, David J.; Hooper, Alan B.; Klotz, Martin G.

    2005-01-01

    Comparison of the organization and sequence of the hao (hydroxylamine oxidoreductase) gene clusters from the gammaproteobacterial autotrophic ammonia-oxidizing bacterium (aAOB) Nitrosococcus oceani and the betaproteobacterial aAOB Nitrosospira multiformis and Nitrosomonas europaea revealed a highly conserved gene cluster encoding the following proteins: hao, hydroxylamine oxidoreductase; orf2, a putative protein; cycA, cytochrome c554; and cycB, cytochrome cm552. The deduced protein sequences of HAO, c554, and cm552 were highly similar in all aAOB despite their differences in species evolution and codon usage. Phylogenetic inference revealed a broad family of multi-c-heme proteins, including HAO, the pentaheme nitrite reductase, and tetrathionate reductase. The c-hemes of this group also have a nearly identical geometry of heme orientation, which has remained conserved during divergent evolution of function. High sequence similarity is also seen within a protein family, including cytochromes cm552, NrfH/B, and NapC/NirT. It is proposed that the hydroxylamine oxidation pathway evolved from a nitrite reduction pathway involved in anaerobic respiration (denitrification) during the radiation of the Proteobacteria. Conservation of the hydroxylamine oxidation module was maintained by functional pressure, and the module expanded into two separate narrow taxa after a lateral gene transfer event between gamma- and betaproteobacterial ancestors of extant aAOB. HAO-encoding genes were also found in six non-aAOB, either singly or tandemly arranged with an orf2 gene, whereas a c554 gene was lacking. The conservation of the hao gene cluster in general and the uniqueness of the c554 gene in particular make it a suitable target for the design of primers and probes useful for molecular ecology approaches to detect aAOB. PMID:16151127

  8. Mechanistic insights into human pre-mRNA splicing of human ultra-short introns: potential unusual mechanism identifies G-rich introns.

    PubMed

    Sasaki-Haraguchi, Noriko; Shimada, Makoto K; Taniguchi, Ichiro; Ohno, Mutsuhito; Mayeda, Akila

    2012-06-29

    It is unknown how very short introns (<65 nt; termed 'ultra-short' introns) could be spliced in a massive spliceosome (>2.7 MDa) without steric hindrance. By screening an annotated human transcriptome database (H-InvDB), we identified three model ultra-short introns: the 56-nt intron in the HNRNPH1 (hnRNP H1) gene, the 49-nt intron in the NDOR1 (NADPH dependent diflavin oxidoreductase 1) gene, and the 43-nt intron in the ESRP2 (epithelial splicing regulatory protein 2) gene. We verified that these endogenous ultra-short introns are spliced, and also recapitulated this in cultured cells transfected with the corresponding mini-genes. The splicing of these ultra-short introns was repressed by a splicing inhibitor, spliceostatin A, suggesting that SF3b (a U2 snRNP component) is involved in their splicing processes. The 56-nt intron containing a pyrimidine-rich tract was spliced out in a lariat form, and this splicing was inhibited by the disruption of U1, U2, or U4 snRNA. In contrast, the 49- and 43-nt introns were purine-rich overall without any pyrimidine-rich tract, and these lariat RNAs were not detectable. Remarkably, shared G-rich intronic sequences in the 49- and 43-nt introns were required for their splicing, suggesting that these ultra-short introns may recruit a novel auxiliary splicing mechanism linked to G-rich intronic splicing enhancers.

  9. The plastid ndh genes code for an NADH-specific dehydrogenase: isolation of a complex I analogue from pea thylakoid membranes.

    PubMed

    Sazanov, L A; Burrows, P A; Nixon, P J

    1998-02-03

    The plastid genomes of several plants contain ndh genes-homologues of genes encoding subunits of the proton-pumping NADH:ubiquinone oxidoreductase, or complex I, involved in respiration in mitochondria and eubacteria. From sequence similarities with these genes, the ndh gene products have been suggested to form a large protein complex (Ndh complex); however, the structure and function of this complex remains to be established. Herein we report the isolation of the Ndh complex from the chloroplasts of the higher plant Pisum sativum. The purification procedure involved selective solubilization of the thylakoid membrane with dodecyl maltoside, followed by two anion-exchange chromatography steps and one size-exclusion chromatography step. The isolated Ndh complex has an apparent total molecular mass of approximately 550 kDa and according to SDS/PAGE consists of at least 16 subunits including NdhA, NdhI, NdhJ, NdhK, and NdhH, which were identified by N-terminal sequencing and immunoblotting. The Ndh complex showed an NADH- and deamino-NADH-specific dehydrogenase activity, characteristic of complex I, when either ferricyanide or the quinones menadione and duroquinone were used as electron acceptors. This study describes the isolation of the chloroplast analogue of the respiratory complex I and provides direct evidence for the function of the plastid Ndh complex as an NADH:plastoquinone oxidoreductase. Our results are compatible with a dual role for the Ndh complex in the chlororespiratory and cyclic photophosphorylation pathways.

  10. Expression profiling in spondyloarthropathy synovial biopsies highlights changes in expression of inflammatory genes in conjunction with tissue remodelling genes

    PubMed Central

    2013-01-01

    Background In the spondyloarthropathies, the underlying molecular and cellular pathways driving disease are poorly understood. By undertaking a study in knee synovial biopsies from spondyloarthropathy (SpA) and ankylosing spondylitis (AS) patients we aimed to elucidate dysregulated genes and pathways. Methods RNA was extracted from six SpA, two AS, three osteoarthritis (OA) and four normal control knee synovial biopsies. Whole genome expression profiling was undertaken using the Illumina DASL system, which assays 24000 cDNA probes. Differentially expressed candidate genes were then validated using quantitative PCR and immunohistochemistry. Results Four hundred and sixteen differentially expressed genes were identified that clearly delineated between AS/SpA and control groups. Pathway analysis showed altered gene-expression in oxidoreductase activity, B-cell associated, matrix catabolic, and metabolic pathways. Altered "myogene" profiling was also identified. The inflammatory mediator, MMP3, was strongly upregulated (5-fold) in AS/SpA samples and the Wnt pathway inhibitors DKK3 (2.7-fold) and Kremen1 (1.5-fold) were downregulated. Conclusions Altered expression profiling in SpA and AS samples demonstrates that disease pathogenesis is associated with both systemic inflammation as well as local tissue alterations that may underlie tissue damaging modelling and remodelling outcomes. This supports the hypothesis that initial systemic inflammation in spondyloarthropathies transfers to and persists in the local joint environment, and might subsequently mediate changes in genes directly involved in the destructive tissue remodelling. PMID:24330574

  11. Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity

    PubMed Central

    Yang, Jianguo; Xie, Xiaqing; Yang, Mingxuan; Dixon, Ray; Wang, Yi-Ping

    2017-01-01

    A large number of genes are necessary for the biosynthesis and activity of the enzyme nitrogenase to carry out the process of biological nitrogen fixation (BNF), which requires large amounts of ATP and reducing power. The multiplicity of the genes involved, the oxygen sensitivity of nitrogenase, plus the demand for energy and reducing power, are thought to be major obstacles to engineering BNF into cereal crops. Genes required for nitrogen fixation can be considered as three functional modules encoding electron-transport components (ETCs), proteins required for metal cluster biosynthesis, and the “core” nitrogenase apoenzyme, respectively. Among these modules, the ETC is important for the supply of reducing power. In this work, we have used Escherichia coli as a chassis to study the compatibility between molybdenum and the iron-only nitrogenases with ETC modules from target plant organelles, including chloroplasts, root plastids, and mitochondria. We have replaced an ETC module present in diazotrophic bacteria with genes encoding ferredoxin–NADPH oxidoreductases (FNRs) and their cognate ferredoxin counterparts from plant organelles. We observe that the FNR–ferredoxin module from chloroplasts and root plastids can support the activities of both types of nitrogenase. In contrast, an analogous ETC module from mitochondria could not function in electron transfer to nitrogenase. However, this incompatibility could be overcome with hybrid modules comprising mitochondrial NADPH-dependent adrenodoxin oxidoreductase and the Anabaena ferredoxins FdxH or FdxB. We pinpoint endogenous ETCs from plant organelles as power supplies to support nitrogenase for future engineering of diazotrophy in cereal crops. PMID:28193863

  12. Adaptive response due to changes in gene regulation: a study with Drosophila.

    PubMed Central

    McDonald, J F; Chambers, G K; David, J; Ayala, F J

    1977-01-01

    In spite of the critical role of the process of adaptation in evolution, there are few detailed studies of the genotypic and molecular basis of the process. Drosophila melanogaster flies selected for increased tolerance to ethanol exhibited higher levels of alcohol dehydrogenase (alcohol:NAD+ oxidoreductase; EC 1.1.1.1) activity than unselected controls. A series of tests (electrophoresis, product inhibition, temperature stability, pH optima, substrate specificity, and Michaelis constants) gave no evidence of structural differences in the enzyme of the selected and the control flies. However, quantitative immunological assays showed that the selected flies contained significantly higher amounts of alcohol dehydrogenase. Adaptation of the selected flies to higher alcohol tolerance has most likely taken place by changes not in the structural gene locus coding for the enzyme, but by regulatory changes affecting the amount of gene product. Images PMID:412190

  13. Localizing gene regulation reveals a staggered wood decay mechanism for the brown rot fungus Postia placenta

    SciTech Connect

    Zhang, Jiwei; Presley, Gerald N.; Hammel, Kenneth E.; Ryu, Jae-San; Menke, Jon R.; Figueroa, Melania; Hu, Dehong; Orr, Galya; Schilling, Jonathan S.

    2016-09-12

    The fungi that cause brown rot of wood are essential contributors to biomass recycling in forest ecosystems. Their highly efficient cellulolytic systems, which may have practical applications, apparently depend on a combination of two mechanisms: nonselective oxidation of the lignocellulose by reactive oxygen species (ROS) coupled with hydrolysis of the polysaccharide components by a limited set of glycoside hydrolases (GHs). Since the production of strongly oxidizing ROS appears incompatible with the operation of GHs, it has been proposed that the fungi regulate ROS production by maintaining concentration gradients of the chelated metal ions they use to generate extracellular oxidants. However, calculations have indicated that this protective mechanism is physically infeasible. We examined a different hypothesis, that expression of ROS and GH components is temporally staggered by brown rot fungi in wood. We sectioned thin wafers of spruce and aspen that had been colonized directionally by Postia placenta and measured expression of relevant genes and some of the encoded enzymes, thus using the spatial distribution of fungal hyphae to resolve a fine-scale temporal sequence. Hierarchical clustering of gene expression for eight oxidoreductases thought to have a role in ROS production and of eight GHs revealed a zone of oxidoreductase upregulation at the hyphal front that persisted about 48 h before upregulation of the GHs. Additional evidence for differential expression was provided by localization of endoglucanase, xylanase, mannanase, and laccase activities in the colonized wood. Our results support a two-step mechanism for brown rot, in which substrate oxidation precedes enzymatic hydrolysis.

  14. Studying Genes

    MedlinePlus

    ... Area What are genes? Genes are sections of DNA that contain instructions for making the molecules—many ... material in an organism. This includes genes and DNA elements that control the activity of genes. Does ...

  15. Expression of an isoflavone reductase-like gene enhanced by pollen tube growth in pistils of Solanum tuberosum.

    PubMed

    van Eldik, G J; Ruiter, R K; Colla, P H; van Herpen, M M; Schrauwen, J A; Wullems, G J

    1997-03-01

    Successful sexual reproduction relies on gene products delivered by the pistil to create an environment suitable for pollen tube growth. These compounds are either produced before pollination or formed during the interactions between pistil and pollen tubes. Here we describe the pollination-enhanced expression of the cp100 gene in pistils of Solanum tuberosum. Temporal analysis of gene expression revealed an enhanced expression already one hour after pollination and lasts more than 72 h. Increase in expression also occurred after touching the stigma and was not restricted to the site of touch but spread into the style. The predicted CP100 protein shows similarity to leguminous isoflavone reductases (IFRs), but belongs to a family of IFR-like NAD(P)H-dependent oxidoreductases present in various plant species.

  16. Gene delivery to mitochondria by targeting modified adenoassociated virus suppresses Leber's hereditary optic neuropathy in a mouse model.

    PubMed

    Yu, Hong; Koilkonda, Rajeshwari D; Chou, Tsung-Han; Porciatti, Vittorio; Ozdemir, Sacide S; Chiodo, Vince; Boye, Sanford L; Boye, Shannon E; Hauswirth, William W; Lewin, Alfred S; Guy, John

    2012-05-15

    To introduce DNA into mitochondria efficiently, we fused adenoassociated virus capsid VP2 with a mitochondrial targeting sequence to carry the mitochondrial gene encoding the human NADH ubiquinone oxidoreductase subunit 4 (ND4). Expression of WT ND4 in cells with the G11778A mutation in ND4 led to restoration of defective ATP synthesis. Furthermore, with injection into the rodent eye, human ND4 DNA levels in mitochondria reached 80% of its mouse homolog. The construct expressed in most inner retinal neurons, and it also suppressed visual loss and optic atrophy induced by a mutant ND4 homolog. The adenoassociated virus cassette accommodates genes of up to ∼5 kb in length, thus providing a platform for introduction of almost any mitochondrial gene and perhaps even allowing insertion of DNA encompassing large deletions of mtDNA, some associated with aging, into the organelle of adults.

  17. Bioinformatics Prediction of Polyketide Synthase Gene Clusters from Mycosphaerella fijiensis

    PubMed Central

    Noar, Roslyn D.; Daub, Margaret E.

    2016-01-01

    Mycosphaerella fijiensis, causal agent of black Sigatoka disease of banana, is a Dothideomycete fungus closely related to fungi that produce polyketides important for plant pathogenicity. We utilized the M. fijiensis genome sequence to predict PKS genes and their gene clusters and make bioinformatics predictions about the types of compounds produced by these clusters. Eight PKS gene clusters were identified in the M. fijiensis genome, placing M. fijiensis into the 23rd percentile for the number of PKS genes compared to other Dothideomycetes. Analysis of the PKS domains identified three of the PKS enzymes as non-reducing and two as highly reducing. Gene clusters contained types of genes frequently found in PKS clusters including genes encoding transporters, oxidoreductases, methyltransferases, and non-ribosomal peptide synthases. Phylogenetic analysis identified a putative PKS cluster encoding melanin biosynthesis. None of the other clusters were closely aligned with genes encoding known polyketides, however three of the PKS genes fell into clades with clusters encoding alternapyrone, fumonisin, and solanapyrone produced by Alternaria and Fusarium species. A search for homologs among available genomic sequences from 103 Dothideomycetes identified close homologs (>80% similarity) for six of the PKS sequences. One of the PKS sequences was not similar (< 60% similarity) to sequences in any of the 103 genomes, suggesting that it encodes a unique compound. Comparison of the M. fijiensis PKS sequences with those of two other banana pathogens, M. musicola and M. eumusae, showed that these two species have close homologs to five of the M. fijiensis PKS sequences, but three others were not found in either species. RT-PCR and RNA-Seq analysis showed that the melanin PKS cluster was down-regulated in infected banana as compared to growth in culture. Three other clusters, however were strongly upregulated during disease development in banana, suggesting that they may encode

  18. Gene Expression Profiling in the Type 1 Diabetes Rat Diaphragm

    PubMed Central

    van Lunteren, Erik; Moyer, Michelle

    2009-01-01

    Background Respiratory muscle contractile performance is impaired by diabetes, mechanisms of which included altered carbohydrate and lipid metabolism, oxidative stress and changes in membrane electrophysiology. The present study examined to what extent these cellular perturbations involve changes in gene expression. Methodology/Principal Findings Diaphragm muscle from streptozotocin-diabetic rats was analyzed with Affymetrix gene expression arrays. Diaphragm from diabetic rats had 105 genes with at least ±2-fold significantly changed expression (55 increased, 50 decreased), and these were assigned to gene ontology groups based on over-representation analysis using DAVID software. There was increased expression of genes involved in palmitoyl-CoA hydrolase activity (a component of lipid metabolism) (P = 0.037, n = 2 genes, fold change 4.2 to 27.5) and reduced expression of genes related to carbohydrate metabolism (P = 0.000061, n = 8 genes, fold change −2.0 to −8.5). Other gene ontology groups among upregulated genes were protein ubiquitination (P = 0.0053, n = 4, fold change 2.2 to 3.4), oxidoreductase activity (P = 0.024, n = 8, fold change 2.1 to 6.0), and morphogenesis (P = 0.012, n = 10, fold change 2.1 to 4.3). Other downregulated gene groups were extracellular region (including extracellular matrix and collagen) (P = 0.00032, n = 13, fold change −2.2 to −3.7) and organogenesis (P = 0.032, n = 7, fold change −2.1 to −3.7). Real-time PCR confirmed the directionality of changes in gene expression for 30 of 31 genes tested. Conclusions/Significance These data indicate that in diaphragm muscle type 1 diabetes increases expression of genes involved in lipid energetics, oxidative stress and protein ubiquitination, decreases expression of genes involved in carbohydrate metabolism, and has little effect on expression of ion channel genes. Reciprocal changes in expression of genes involved in

  19. The Conformational Changes Induced by Ubiquinone Binding in the Na+-pumping NADH:Ubiquinone Oxidoreductase (Na+-NQR) Are Kinetically Controlled by Conserved Glycines 140 and 141 of the NqrB Subunit*

    PubMed Central

    Strickland, Madeleine; Juárez, Oscar; Neehaul, Yashvin; Cook, Darcie A.; Barquera, Blanca; Hellwig, Petra

    2014-01-01

    Na+-pumping NADH:ubiquinone oxidoreductase (Na+-NQR) is responsible for maintaining a sodium gradient across the inner bacterial membrane. This respiratory enzyme, which couples sodium pumping to the electron transfer between NADH and ubiquinone, is not present in eukaryotes and as such could be a target for antibiotics. In this paper it is shown that the site of ubiquinone reduction is conformationally coupled to the NqrB subunit, which also hosts the final cofactor in the electron transport chain, riboflavin. Previous work showed that mutations in conserved NqrB glycine residues 140 and 141 affect ubiquinone reduction and the proper functioning of the sodium pump. Surprisingly, these mutants did not affect the dissociation constant of ubiquinone or its analog HQNO (2-n-heptyl-4-hydroxyquinoline N-oxide) from Na+-NQR, which indicates that these residues do not participate directly in the ubiquinone binding site but probably control its accessibility. Indeed, redox-induced difference spectroscopy showed that these mutations prevented the conformational change involved in ubiquinone binding but did not modify the signals corresponding to bound ubiquinone. Moreover, data are presented that demonstrate the NqrA subunit is able to bind ubiquinone but with a low non-catalytically relevant affinity. It is also suggested that Na+-NQR contains a single catalytic ubiquinone binding site and a second site that can bind ubiquinone but is not active. PMID:25006248

  20. Indigofera suffruticosa Mill extracts up-regulate the expression of the π class of glutathione S-transferase and NAD(P)H: quinone oxidoreductase 1 in rat Clone 9 liver cells.

    PubMed

    Chen, Chun-Chieh; Liu, Chin-San; Li, Chien-Chun; Tsai, Chia-Wen; Yao, Hsien-Tsung; Liu, Te-Chung; Chen, Haw-Wen; Chen, Pei-Yin; Wu, Yu-Ling; Lii, Chong-Kuei; Liu, Kai-Li

    2013-09-01

    Because induction of phase II detoxification enzyme is important for chemoprevention, we study the effects of Indigofera suffruticosa Mill, a medicinal herb, on the expression of π class of glutathione S-transferase (GSTP) and NAD(P)H: quinone oxidoreductase 1 (NQO1) in rat Clone 9 liver cells. Both water and ethanolic extracts of I. suffruticosa significantly increased the expression and enzyme activities of GSTP and NQO1. I. suffruticosa extracts up-regulated GSTP promoter activity and the binding affinity of nuclear factor erythroid 2-related factor 2 (Nrf2) with the GSTP enhancer I oligonucleotide. Moreover, I. suffruticosa extracts increased nuclear Nrf2 accumulation as well as ARE transcriptional activity. The level of phospho-ERK was augmented by I. suffruticosa extracts, and the ERK inhibitor PD98059 abolished the I. suffruticosa extract-induced ERK activation and GSTP and NQO-1 expression. Moreover, I. suffruticosa extracts, especially the ethanolic extract increased the glutathione level in mouse liver and red blood cells as well as Clone 9 liver cells. The efficacy of I. suffruticosa extracts in induction of phase II detoxification enzymes and glutathione content implies that I. suffruticosa could be considered as a potential chemopreventive agent.

  1. Aspartic Acid 397 in Subunit B of the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae Forms Part of a Sodium-binding Site, Is Involved in Cation Selectivity, and Affects Cation-binding Site Cooperativity

    PubMed Central

    Shea, Michael E.; Juárez, Oscar; Cho, Jonathan; Barquera, Blanca

    2013-01-01

    The Na+-pumping NADH:quinone complex is found in Vibrio cholerae and other marine and pathogenic bacteria. NADH:ubiquinone oxidoreductase oxidizes NADH and reduces ubiquinone, using the free energy released by this reaction to pump sodium ions across the cell membrane. In a previous report, a conserved aspartic acid residue in the NqrB subunit at position 397, located in the cytosolic face of this protein, was proposed to be involved in the capture of sodium. Here, we studied the role of this residue through the characterization of mutant enzymes in which this aspartic acid was substituted by other residues that change charge and size, such as arginine, serine, lysine, glutamic acid, and cysteine. Our results indicate that NqrB-Asp-397 forms part of one of the at least two sodium-binding sites and that both size and charge at this position are critical for the function of the enzyme. Moreover, we demonstrate that this residue is involved in cation selectivity, has a critical role in the communication between sodium-binding sites, by promoting cooperativity, and controls the electron transfer step involved in sodium uptake (2Fe-2S → FMNC). PMID:24030824

  2. RNA-Seq Reveals OTA-Related Gene Transcriptional Changes in Aspergillus carbonarius.

    PubMed

    Gerin, Donato; De Miccolis Angelini, Rita M; Pollastro, Stefania; Faretra, Francesco

    2016-01-01

    Ochratoxin A (OTA) is a mycotoxin harmful for animals and humans. Aspergillus carbonarius is the main responsible for OTA contamination of grapes and derived products. Gene transcriptional profiling of 4 A. carbonarius strains was carried out by RNA-Seq analysis to study transcriptome changes associated with OTA production. By comparing OTA inducing (OTAI) vs. non-inducing (OTAN) cultural conditions, a total of 3,705 differentially expressed genes (DEGs) (fold change > |2| and FDR ≤ 0.05) were identified. Several genes involved in primary metabolic processes, with particular regard to carbohydrate and amino acid metabolisms, secondary metabolic processes, transport, response to stress and sporulation were up-regulated by OTAI conditions at all the analysed sampling times (4, 6 and 8 DAI) or starting from 6 DAI. Highly up-regulated DEGs encoding enzymes involved in biosynthesis of secondary metabolites, oxidoreductases, transporters and transcription factors were examined for their potential involvement in OTA biosynthesis and related metabolic pathways. Differential expression of genes encoding polyketide synthases (pks), non-ribosomal peptide synthetases (nrps) and chloroperoxidase (cpo) was validated by RT-qPCR. Among clusters of co-regulated genes involved in SM biosynthesis, one putative OTA-gene cluster, including both pks and nrps genes, was detected in the A. carbonarius genome.

  3. RNA-Seq Reveals OTA-Related Gene Transcriptional Changes in Aspergillus carbonarius

    PubMed Central

    Gerin, Donato; De Miccolis Angelini, Rita M.; Pollastro, Stefania; Faretra, Francesco

    2016-01-01

    Ochratoxin A (OTA) is a mycotoxin harmful for animals and humans. Aspergillus carbonarius is the main responsible for OTA contamination of grapes and derived products. Gene transcriptional profiling of 4 A. carbonarius strains was carried out by RNA-Seq analysis to study transcriptome changes associated with OTA production. By comparing OTA inducing (OTAI) vs. non-inducing (OTAN) cultural conditions, a total of 3,705 differentially expressed genes (DEGs) (fold change > |2| and FDR ≤ 0.05) were identified. Several genes involved in primary metabolic processes, with particular regard to carbohydrate and amino acid metabolisms, secondary metabolic processes, transport, response to stress and sporulation were up-regulated by OTAI conditions at all the analysed sampling times (4, 6 and 8 DAI) or starting from 6 DAI. Highly up-regulated DEGs encoding enzymes involved in biosynthesis of secondary metabolites, oxidoreductases, transporters and transcription factors were examined for their potential involvement in OTA biosynthesis and related metabolic pathways. Differential expression of genes encoding polyketide synthases (pks), non-ribosomal peptide synthetases (nrps) and chloroperoxidase (cpo) was validated by RT-qPCR. Among clusters of co-regulated genes involved in SM biosynthesis, one putative OTA-gene cluster, including both pks and nrps genes, was detected in the A. carbonarius genome. PMID:26765536

  4. Comparative mapping and genomic annotation of the bovine oncosuppressor gene WWOX.

    PubMed

    Manera, S; Bonfiglio, S; Malusà, A; Denis, C; Boussaha, M; Russo, V; Roperto, F; Perucatti, A; Di Meo, G P; Eggen, A; Ferretti, L

    2009-01-01

    WWOX (WW domain-containing oxidoreductase) is the gene mapping at FRA16D HSA16q23.1, the second most active common fragile site in the human genome. In this study we characterized at a detailed molecular level WWOX in the bovine genome. First, we sequenced cDNA from various tissues and obtained evidence in support of a 9-exon structure for the gene, similar to the human gene. Then, we recovered BACs using exon tags and annotated the gene to a >1-Mb genomic region of BTA18 using the Btau 4.0 genome assembly as a reference, thus resolving an issue related to exon 9, which is not included in the genomic annotation of the gene in the Entrez database. Finally, BACs spanning WWOX were used as FISH probes to obtain comparative mapping of the gene in Bos taurus, Bubalus bubalis, Ovis aries and Capra hircus to BTA18q12.1, BBU18q13, OAR14q12.1 and CHI18q12.1, respectively. Our data show that the chromosomal location of WWOX is conserved between man and 4 major domesticated species. Moreover, the annotation of the bovine gene also suggests a highly conserved genomic arrangement, including number and size of introns.

  5. Identification of genes involved in rice seed priming in the early imbibition stage.

    PubMed

    Cheng, J; Wang, L; Zeng, P; He, Y; Zhou, R; Zhang, H; Wang, Z

    2017-01-01

    Phase II of seed imbibition is a critical process during seed priming. To identify genes involved in rice seed priming, the altered proteins between the dry and imbibed (24 h) seeds were compared using a two-dimensional gel electrophoresis system in this study. Ten significantly changed proteins (fold change ≥ twofold; P < 0.01) were successfully identified, which could be categorised as carbohydrate and protein biosynthesis and metabolism-related, signalling-related, storage and stress-related proteins. A meta-analysis indicated that the highest expression of the identified genes was at the milk and dough stages and in the endosperm tissue. Quantitative real-time PCR analysis showed that there was significant variation in gene expression (except FAD-dependent oxidoreductase) in embryos during seed priming (0-48 h). The expression of genes associated with stress appeared at the early imbibition stage, while those associated with carbohydrate metabolism, protein synthesis and signalling increased at the late imbibition stage. Three identified proteins (glucose-1-phosphate adenylyltransferase large subunit, aminotransferase and prolamin precursor) had similar transcript and protein expression patterns in embryos. Based on phenotype and gene expression, the optimal stop time for seed priming is 24 h, when these three genes have relatively low expression, followed by significant induction during imbibition in embryos. These three genes are ideal candidate biomarkers for rice seed priming.

  6. Metronidazole activation and isolation of Clostridium acetobutylicum electron transport genes.

    PubMed Central

    Santangelo, J D; Jones, D T; Woods, D R

    1991-01-01

    An Escherichia coli F19 recA, nitrate reductase-deficient mutant was constructed by transposon mutagenesis and shown to be resistant to metronidazole. This mutant was a most suitable host for the isolation of Clostridium acetobutylicum genes on recombinant plasmids, which activated metronidazole and rendered the E. coli F19 strain sensitive to metronidazole. Twenty-five E. coli F19 clones containing different recombinant plasmids were isolated and classified into five groups on the basis of their sensitivity to metronidazole. The clones were tested for nitrate reductase, pyruvate-ferredoxin oxidoreductase, and hydrogenase activities. DNA hybridization and restriction endonuclease mapping revealed that four of the C. acetobutylicum insert DNA fragments on recombinant plasmids were linked in an 11.1-kb chromosomal fragment. DNA sequencing and amino acid homology studies indicated that this DNA fragment contained a flavodoxin gene which encoded a protein of 160 amino acids that activated metronidazole and made the E. coli F19 mutant very sensitive to metronidazole. The flavodoxin and hydrogenase genes which are involved in electron transfer systems were linked on the 11.1-kb DNA fragment from C. acetobutylicum. Images PMID:1991710

  7. H2O2 production rate in Lactobacillus johnsonii is modulated via the interplay of a heterodimeric flavin oxidoreductase with a soluble 28 Kd PAS domain containing protein

    PubMed Central

    Valladares, Ricardo B.; Graves, Christina; Wright, Kaitlyn; Gardner, Christopher L.; Lorca, Graciela L.; Gonzalez, Claudio F.

    2015-01-01

    Host and commensals crosstalk, mediated by reactive oxygen species (ROS), has triggered a growing scientific interest to understand the mechanisms governing such interaction. However, the majority of the scientific studies published do not evaluate the ROS production by commensals bacteria. In this context we recently showed that Lactobacillus johnsonii N6.2, a strain of probiotic value, modulates the activity of the critical enzymes 2,3-indoleamine dioxygenase via H2O2 production. L. johnsonii N6.2 by decreasing IDO activity, is able to modify the tryptophan/kynurenine ratio in the host blood with further systemic consequences. Understanding the mechanisms of H2O2 production is critical to predict the probiotic value of these strains and to optimize bacterial biomass production in industrial processes. We performed a transcriptome analysis to identify genes differentially expressed in L. johnsonii N6.2 cells collected from cultures grown under different aeration conditions. Herein we described the biochemical characteristics of a heterodimeric FMN reductase (FRedA/B) whose in vitro activity is controlled by LjPAS protein with a typical Per-Arnst-Sim (PAS) sensor domain. Interestingly, LjPAS is fused to the FMN reductase domains in other lactobacillaceae. In L. johnsonii, LjPAS is encoded by an independent gene which expression is repressed under anaerobic conditions (>3 fold). Purified LjPAS was able to slow down the FRedA/B initial activity rate when the holoenzyme precursors (FredA, FredB, and FMN) were mixed in vitro. Altogether the results obtained suggest that LjPAS module regulates the H2O2 production helping the cells to minimize oxidative stress in response to environmental conditions. PMID:26236298

  8. Sodium arsenite-induced stress-related gene expression in normal human epidermal, HaCaT, and HEL30 keratinocytes.

    PubMed Central

    Trouba, Kevin J; Geisenhoffer, Kristen M; Germolec, Dori R

    2002-01-01

    Arsenic is a carcinogen that poses a significant health risk in humans. Based on evidence that arsenic has differential effects on human, rodent, normal, and transformed cells, these studies addressed the relative merits of using normal human epidermal keratinocytes (NHEK) and immortalized human (HaCaT) and mouse (HEL30) keratinocytes when examining stress-induced gene expression that may contribute to carcinogenesis. We hypothesize that redox-related gene expression is differentially modulated by arsenic in normal versus immortalized keratinocytes. To test the hypothesis, we exposed keratinocytes to sodium arsenite for 4 or 24 hr, at which time serine threonine kinase-25 (stk25) and nicotine adenine dinucleotide phosphate [nad(p)h] quinone oxidoreductase gene expression were measured. The effect of glutathione reduction on arsenite-induced cytotoxicity and gene expression in NHEK also was evaluated by addition of l-buthionine-[S,R]-sulfoximine (BSO) to culture media. Results indicate the term LC(50) for arsenite is approximately 10-15 microM in NHEK and HEL30 keratinocytes and 30 microM in HaCaT keratinocytes. Compared with HaCaT and HEL30 keratinocytes, a nontoxic concentration of arsenite (2.5 microM) increases stk25 and nad(p)h quinone oxidoreductase gene expression in NHEK, an effect partially attenuated by BSO. These data indicate that NHEK and HaCaT/HEL30 keratinocytes have similar sensitivities toward arsenite-induced cytotoxicity but unique gene expression responses. They also suggest that arsenite modulates gene expression in NHEK involved in cellular signaling and other aspects of intermediary metabolism that may contribute to the carcinogenic process. PMID:12426128

  9. Identification of the Genes Involved in the Fruiting Body Production and Cordycepin Formation of Cordyceps militaris Fungus

    PubMed Central

    Zheng, Zhuang-li; Qiu, Xue-hong

    2015-01-01

    A mutant library of Cordyceps militaris was constructed by improved Agrobacterium tumefaciens-mediated transformation and screened for degradation features. Six mutants with altered characters in in vitro and in vivo fruiting body production, and cordycepin formation were found to contain a single copy T-DNA. T-DNA flanking sequences of these mutants were identified by thermal asymmetric interlaced-PCR approach. ATP-dependent helicase, cytochrome oxidase subunit I and ubiquitin-like activating enzyme were involved in in vitro fruiting body production, serine/threonine phosphatase involved in in vivo fruiting body production, while glucose-methanol-choline oxidoreductase and telomerase reverse transcriptase involved in cordycepin formation. These genes were analyzed by bioinformatics methods, and their molecular function and biology process were speculated by Gene Ontology (GO) analysis. The results provided useful information for the control of culture degeneration in commercial production of C. militaris. PMID:25892913

  10. Rapid expansion of the protein disulfide isomerase gene family facilitates the folding of venom peptides

    PubMed Central

    Safavi-Hemami, Helena; Li, Qing; Jackson, Ronneshia L.; Song, Albert S.; Boomsma, Wouter; Bandyopadhyay, Pradip K.; Gruber, Christian W.; Purcell, Anthony W.; Yandell, Mark; Olivera, Baldomero M.

    2016-01-01

    Formation of correct disulfide bonds in the endoplasmic reticulum is a crucial step for folding proteins destined for secretion. Protein disulfide isomerases (PDIs) play a central role in this process. We report a previously unidentified, hypervariable family of PDIs that represents the most diverse gene family of oxidoreductases described in a single genus to date. These enzymes are highly expressed specifically in the venom glands of predatory cone snails, animals that synthesize a remarkably diverse set of cysteine-rich peptide toxins (conotoxins). Enzymes in this PDI family, termed conotoxin-specific PDIs, significantly and differentially accelerate the kinetics of disulfide-bond formation of several conotoxins. Our results are consistent with a unique biological scenario associated with protein folding: The diversification of a family of foldases can be correlated with the rapid evolution of an unprecedented diversity of disulfide-rich structural domains expressed by venomous marine snails in the superfamily Conoidea. PMID:26957604

  11. Gene Therapy

    MedlinePlus

    ... cells in an effort to treat or stop disease. Genes contain your DNA — the code that controls much of your body's form and function, from making you grow taller to regulating your body systems. Genes that don't work properly can cause disease. Gene therapy replaces a faulty gene or adds ...

  12. Expression profile of early estradiol-responsive genes in cynomolgus macaque liver: implications for drug-metabolizing enzymes.

    PubMed

    Ise, Ryota; Kito, Go; Uno, Yasuhiro

    2012-01-01

    Estrogen plays important roles in estrogen-responsive tissues, such as mammary glands, ovaries, and the uterus. In the liver, the major drug metabolizing organ, estrogen is known to regulate expression of some drug-metabolizing enzymes. Due to the lack of information on the role of estrogen in hepatic gene expression in primate species, we previously investigated the late response of hepatic gene expression to estradiol in cynomolgus macaques. To understand the early response of hepatic gene expression to estradiol, in this study, microarray analysis was conducted using cynomolgus macaque liver samples collected at 1 h and 5 h after estradiol injection. Comparison of expression profiles in estradiol and solvent (control)-treated ovariectomized cynomolgus macaques revealed 27 differentially expressed genes (>2.0-fold), including 18 at 1 h and 9 at 5 h after estradiol injection. As indicated by Gene Ontology analysis, these genes were related to oxidoreductase activity and transferase activity, partly representing important aspects of drug-metabolizing enzymes. Further analysis by quantitative polymerase chain reaction revealed that estradiol down-regulated CYP2A24, CYP2C76, and CYP2E1 (>2.0-fold) at 1 h and up-regulated GSTM5 (>2.0-fold) at 5 h after estradiol injection. These results suggest that the short-term estradiol treatment influenced expression of hepatic genes, including drug-metabolizing enzyme genes, in cynomolgus macaque liver.

  13. Impact of high-fat diet on liver genes expression profiles in mice model of nonalcoholic fatty liver disease.

    PubMed

    Wang, Chunhua; Tao, Qimeng; Wang, Xinghe; Wang, Xiurong; Zhang, Xiuying

    2016-07-01

    Evidences have shown that NAFLD influences expression of some drug metabolic enzyme genes. This study aims to investigate the role of HFD-induced NAFLD in regulating the transcription of genes, particularly the drug metabolizing genes variation. Transcriptome analysis demonstrated that HFD feeding caused the 150 genes expression to change, most genes associated with lipid metabolism, inflammatory, oxidative stress and oxidoreductase activity up-regulated, whereas most genes involved in nucleic acid metabolism repressed. The genes involved in drug metabolism had 16 down-regulated and 21 up-regulated in NAFLD. The over-4-fold change genes included the down-regulation of Cyp8b1, Cyp7a1, Sult3a1, Sult1e1, Cyp17a1, Cyp3a41a, Gstt3, Cyp51, Cyp2c54 and Cyp4f14, and the up-regulation of Asns, Past1, Cyp2c55, Gstm2, Cyp2e1 and Gstaα1. In conclusion, significant alterations in the expression of drug metabolizing enzymes may affect the clearance of therapeutic drugs, with the potential to result in adverse drug reactions or drug toxicity in nonalcoholic fatty liver disease.

  14. 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.

  15. The environmental pollutant and carcinogen 3-nitrobenzanthrone and its human metabolite 3-aminobenzanthrone are potent inducers of rat hepatic cytochromes P450 1A1 and -1A2 and NAD(P)H:quinone oxidoreductase.

    PubMed

    Stiborová, Marie; Dracínská, Helena; Hájková, Jana; Kaderábková, Pavla; Frei, Eva; Schmeiser, Heinz H; Soucek, Pavel; Phillips, David H; Arlt, Volker M

    2006-08-01

    3-Nitrobenzanthrone (3-NBA), a suspected human carcinogen occurring in diesel exhaust and air pollution, and its human metabolite 3-aminobenzanthrone (3-ABA) were investigated for their ability to induce biotransformation enzymes in rat liver and the influence of such induction on DNA adduct formation by the compounds. Rats were treated (i.p.) with 0.4, 4, or 40 mg/kg body weight 3-NBA or 3-ABA. When hepatic cytosolic fractions from rats treated with 40 mg/kg body weight 3-NBA or 3-ABA were incubated with 3-NBA, DNA adduct formation, measured by 32P-postlabeling analysis, was 10-fold higher in incubations with cytosols from pretreated rats than with controls. The increase in 3-NBA-derived DNA adduct formation corresponded to a dose-dependent increase in protein levels and enzymatic activity of NAD(P)H:quinone oxidoreductase (NQO1). NQO1 is the major enzyme reducing 3-NBA in human and rat livers. Incubations of 3-ABA with hepatic microsomes of rats treated with 3-NBA or 3-ABA (40 mg/kg body weight) led to as much as a 12-fold increase in 3-ABA-derived DNA adduct formation compared with controls. The observed stimulation of DNA adduct formation by both compounds was attributed to their potential to induce protein expression and enzymatic activity of cytochromes P450 1A1 and/or -1A2 (CYP1A1/2), the major enzymes responsible for 3-ABA activation in human and rat livers. Collectively, these results demonstrate for the first time, to our knowledge, that by inducing hepatic NQO1 and CYP1A1/2, both 3-NBA and 3-ABA increase the enzymatic activation of these two compounds to reactive DNA adduct-forming species, thereby enhancing their own genotoxic potential.

  16. The environmental pollutant and carcinogen 3-nitrobenzanthrone induces cytochrome P450 1A1 and NAD(P)H:quinone oxidoreductase in rat lung and kidney, thereby enhancing its own genotoxicity.

    PubMed

    Stiborová, Marie; Dracínská, Helena; Mizerovská, Jana; Frei, Eva; Schmeiser, Heinz H; Hudecek, Jirí; Hodek, Petr; Phillips, David H; Arlt, Volker M

    2008-05-02

    3-Nitrobenzanthrone (3-NBA) is a carcinogen occurring in diesel exhaust and air pollution. Using the (32)P-postlabelling method, we found that 3-NBA and its human metabolite, 3-aminobenzanthrone (3-ABA), are activated to species forming DNA adducts by cytosols and/or microsomes isolated from rat lung, the target organ for 3-NBA carcinogenicity, and kidney. Each compound generated identical five DNA adducts. We have demonstrated the importance of pulmonary and renal NAD(P)H:quinone oxidoreductase (NQO1) to reduce 3-NBA to species that are further activated by N,O-acetyltransferases and sulfotransferases. Cytochrome P450 (CYP) 1A1 is the essential enzyme for oxidative activation of 3-ABA in microsomes of both organs, while cyclooxygenase plays a minor role. 3-NBA was also investigated for its ability to induce NQO1 and CYP1A1 in lungs and kidneys, and for the influence of such induction on DNA adduct formation by 3-NBA and 3-ABA. When cytosols from rats treated i.p. with 40mg/kg bw of 3-NBA were incubated with 3-NBA, DNA adduct formation was up to 2.1-fold higher than in incubations with cytosols from control animals. This increase corresponded to an increase in protein level and enzymatic activity of NQO1. Incubations of 3-ABA with microsomes of 3-NBA-treated rats led to up to a fivefold increase in DNA adduct formation relative to controls. The stimulation of DNA adduct formation correlated with the potential of 3-NBA to induce protein expression and activity of CYP1A1. These results demonstrate that 3-NBA is capable to induce NQO1 and CYP1A1 in lungs and kidney of rats thereby enhancing its own genotoxic and carcinogenic potential.

  17. The Structure of the Bacterial Oxidoreductase Enzyme DsbA in Complex with a Peptide Reveals a Basis for Substrate Specificity in the Catalytic Cycle of DsbA Enzymes

    SciTech Connect

    Paxman, Jason J.; Borg, Natalie A.; Horne, James; Thompson, Philip E.; Chin, Yanni; Sharma, Pooja; Simpson, Jamie S.; Wielens, Jerome; Piek, Susannah; Kahler, Charlene M.; Sakellaris, Harry; Pearce, Mary; Bottomley, Stephen P.; Rossjohn, Jamie; Scanlon, Martin J.

    2010-09-07

    Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the complex.

  18. The structure of the bacterial oxidoreductase enzyme DsbA in complex with a peptide reveals a basis for substrate specificity in the catalytic cycle of DsbA enzymes.

    PubMed

    Paxman, Jason J; Borg, Natalie A; Horne, James; Thompson, Philip E; Chin, Yanni; Sharma, Pooja; Simpson, Jamie S; Wielens, Jerome; Piek, Susannah; Kahler, Charlene M; Sakellaris, Harry; Pearce, Mary; Bottomley, Stephen P; Rossjohn, Jamie; Scanlon, Martin J

    2009-06-26

    Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the complex.

  19. The single NqrB and NqrC subunits in the Na(+)-translocating NADH: quinone oxidoreductase (Na(+)-NQR) from Vibrio cholerae each carry one covalently attached FMN.

    PubMed

    Casutt, Marco S; Schlosser, Andreas; Buckel, Wolfgang; Steuber, Julia

    2012-10-01

    The Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) is the prototype of a novel class of flavoproteins carrying a riboflavin phosphate bound to serine or threonine by a phosphodiester bond to the ribityl side chain. This membrane-bound, respiratory complex also contains one non-covalently bound FAD, one non-covalently bound riboflavin, ubiquinone-8 and a [2Fe-2S] cluster. Here, we report the quantitative analysis of the full set of flavin cofactors in the Na(+)-NQR and characterize the mode of linkage of the riboflavin phosphate to the membrane-bound NqrB and NqrC subunits. Release of the flavin by β-elimination and analysis of the cofactor demonstrates that the phosphate group is attached at the 5'-position of the ribityl as in authentic FMN and that the Na(+)-NQR contains approximately 1.7mol covalently bound FMN per mol non-covalently bound FAD. Therefore, each of the single NqrB and NqrC subunits in the Na(+)-NQR carries a single FMN. Elimination of the phosphodiester bond yields a dehydro-2-aminobutyrate residue, which is modified with β-mercaptoethanol by Michael addition. Proteolytic digestion followed by mass determination of peptide fragments reveals exclusive modification of threonine residues, which carry FMN in the native enzyme. The described reactions allow quantification and localization of the covalently attached FMNs in the Na(+)-NQR and in related proteins belonging to the Rhodobacter nitrogen fixation (RNF) family of enzymes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

  20. Induction of NAD(P)H-quinone oxidoreductase 1 by antioxidants in female ACI rats is associated with decrease in oxidative DNA damage and inhibition of estrogen-induced breast cancer.

    PubMed

    Singh, Bhupendra; Bhat, Nimee K; Bhat, Hari K

    2012-01-01

    Exact mechanisms underlying the initiation and progression of estrogen-related cancers are not clear. Literature, evidence and our studies strongly support the role of estrogen metabolism-mediated oxidative stress in estrogen-induced breast carcinogenesis. We have recently demonstrated that antioxidants vitamin C and butylated hydroxyanisole (BHA) or estrogen metabolism inhibitor α-naphthoflavone (ANF) inhibit 17β-estradiol (E2)-induced mammary tumorigenesis in female ACI rats. The objective of the current study was to identify the mechanism of antioxidant-mediated protection against E2-induced DNA damage and mammary tumorigenesis. Female ACI rats were treated with E2 in the presence or absence of vitamin C or BHA or ANF for up to 240 days. Nuclear factor erythroid 2-related factor 2 (NRF2) and NAD(P)H-quinone oxidoreductase 1 (NQO1) were suppressed in E2-exposed mammary tissue and in mammary tumors after treatment of rats with E2 for 240 days. This suppression was overcome by co-treatment of rats with E2 and vitamin C or BHA. Time course studies indicate that NQO1 levels tend to increase after 4 months of E2 treatment but decrease on chronic exposure to E2 for 8 months. Vitamin C and BHA significantly increased NQO1 levels after 120 days. 8-Hydroxydeoxyguanosine (8-OHdG) levels were higher in E2-exposed mammary tissue and in mammary tumors compared with age-matched controls. Vitamin C or BHA treatment significantly decreased E2-mediated increase in 8-OHdG levels in the mammary tissue. In vitro studies using silencer RNA confirmed the role of NQO1 in prevention of oxidative DNA damage. Our studies further demonstrate that NQO1 upregulation by antioxidants is mediated through NRF2.

  1. Isolation and partial characterization of the Drosophila alcohol dehydrogenase gene.

    PubMed Central

    Goldberg, D A

    1980-01-01

    The alcohol dehydrogenase (ADH; alcohol: NAD+ oxidoreductase, EC 1.1.1.1) gene (Adh) of Drosophila melanogaster was isolated by utilizing a mutant strain in which the Adh locus is deleted. Adult RNA from wild-type flies was enriched in ADH sequences by gel electrophoresis and then used to prepare labeled cDNA for screening a bacteriophage lambda library of genomic Drosophila DNA. Of the clones that hybridized in the initial screen, one clone was identified that hybridized with labeled cDNA prepared from a wild-type Drosophila strain but did not hybridize with cDNA prepared from an Adh deletion strain. This clone was shown to contain ADH structural gene sequences by three criteria: in situ hybridization, in vitro translation of mRNA selected by hybridization to the cloned DNA, and comparison of the ADH protein sequence with a nucleotide sequence derived from the cloned DNA. Comparison of the restriction site maps from clones of three different wild-type Drosophila strains revealed the presence of a 200-nucleotide sequence in one strain that was absent from the other two strains. The ADH mRNA sequences were located within the cloned DNA by hybridization mapping experiments. Two intervening sequences were identified within Adh by S1 nuclease mapping experiments. Images PMID:6777776

  2. Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae

    PubMed Central

    Gonzalez-Perez, David; Alcalde, Miguel

    2014-01-01

    The ligninolytic enzymatic consortium produced by white-rot fungi is one of the most efficient oxidative systems found in nature, with many potential applications that range from the production of 2nd generation biofuels to chemicals synthesis. In the current study, two high redox potential oxidoreductase fusion genes (laccase -Lac- and versatile peroxidase -Vp-) that had been evolved in the laboratory were re-assembled in Saccharomyces cerevisiae. First, cell viability and secretion were assessed after co-transforming the Lac and Vp genes into yeast. Several expression cassettes were inserted in vivo into episomal bi-directional vectors in order to evaluate inducible promoter and/or terminator pairs of different strengths in an individual and combined manner. The synthetic white-rot yeast model harboring Vp(GAL1/CYC1)-Lac(GAL10/ADH1) displayed up to 1000 and 100 Units per L of peroxidase and laccase activity, respectively, representing a suitable point of departure for future synthetic biology studies. PMID:24830983

  3. Single gene insertion drives bioalcohol production by a thermophilic archaeon

    PubMed Central

    Basen, Mirko; Schut, Gerrit J.; Nguyen, Diep M.; Lipscomb, Gina L.; Benn, Robert A.; Prybol, Cameron J.; Vaccaro, Brian J.; Poole, Farris L.; Kelly, Robert M.; Adams, Michael W. W.

    2014-01-01

    Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 °C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. By heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways. PMID:25368184

  4. Single gene insertion drives bioalcohol production by a thermophilic archaeon

    SciTech Connect

    Basen, M; Schut, GJ; Nguyen, DM; Lipscomb, GL; Benn, RA; Prybol, CJ; Vaccaro, BJ; Poole, FL; Kelly, RM; Adams, MWW

    2014-12-09

    Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 degrees C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. By heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways.

  5. Single gene insertion drives bioalcohol production by a thermophilic archaeon.

    PubMed

    Basen, Mirko; Schut, Gerrit J; Nguyen, Diep M; Lipscomb, Gina L; Benn, Robert A; Prybol, Cameron J; Vaccaro, Brian J; Poole, Farris L; Kelly, Robert M; Adams, Michael W W

    2014-12-09

    Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 °C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. By heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways.

  6. Gene Positioning

    PubMed Central

    Ferrai, Carmelo; de Castro, Inês Jesus; Lavitas, Liron; Chotalia, Mita; Pombo, Ana

    2010-01-01

    Eukaryotic gene expression is an intricate multistep process, regulated within the cell nucleus through the activation or repression of RNA synthesis, processing, cytoplasmic export, and translation into protein. The major regulators of gene expression are chromatin remodeling and transcription machineries that are locally recruited to genes. However, enzymatic activities that act on genes are not ubiquitously distributed throughout the nucleoplasm, but limited to specific and spatially defined foci that promote preferred higher-order chromatin arrangements. The positioning of genes within the nuclear landscape relative to specific functional landmarks plays an important role in gene regulation and disease. PMID:20484389

  7. Light and dark modulation of chlorophyll biosynthetic genes in response to temperature.

    PubMed

    Mohanty, Sasmita; Grimm, Bernhard; Tripathy, Baishnab C

    2006-08-01

    Temperature and light significantly influence chloroplast development and chlorophyll biosynthesis. To understand the mechanism of the modulation of chlorophyll biosynthesis, the levels of transcripts and proteins of many enzymatic steps of tetrapyrrole biosynthesis in wheat and cucumber were simultaneously examined. The effect of low (chill-stress) as well as high (heat-stress) temperatures on dark- and light-grown seedlings was monitored. The protochlorophyllide oxidoreductase (POR) content was greatly reduced in response to light in control and heat-stressed seedlings. However, the POR level was not reduced in light-exposed chill-stressed seedlings. The genes for glutamate semialdehyde aminotransferase (gsa; cucumber), glutamyl-tRNA reductase (GluTR; cucumber), 5-aminolevulinic acid dehydratase (Ala D; cucumber and wheat) and for a subunit of Mg-chelatase (Chl I; wheat) showed a reduced expression in cold stress compared to controls and heat-stress conditions. Although expression of the ferrochelatase gene (Fch) and geranylgeranyl reductase gene (Chl P) was upregulated in light, they were downregulated by both chill- and heat-stress. Interestingly, gsa and uroporphyrinogen decarboxylase gene (UroD) and gene product abundance was stimulated by light and heat-stress implying the presence of both light and heat-inducible elements in their promoters. This observation corroborates with the previous report of increased enzymatic activity of UroD in heat-stressed cucumber seedlings. The gsa and Uro D may play an important role in tolerance of the greening process of plants to heat-stress.

  8. Transcriptional profiling of biomass degradation-related genes during Trichoderma reesei growth on different carbon sources.

    PubMed

    Chen, Xiuzhen; Luo, Yingfeng; Yu, Hongtao; Sun, Yuhui; Wu, Hong; Song, Shuhui; Hu, Songnian; Dong, Zhiyang

    2014-03-10

    To identify all the gene products involved in cellulosic biomass degradation, we employed RNA sequencing technology to perform a genome-wide comparison of gene expression during growth of Trichoderma reesei QM9414 on cellulose or glucose. Due to their important role in lignocellulose decomposition, we focused on CAZymes and other secreted proteins. In total, 122 CAZymes showed at least a two-fold change in mRNA abundance, and 97 of those were highly induced by cellulose. Compared to the well-characterized cellulases and hemicellulases, a majority of the other upregulated CAZymes showed lower transcriptional levels. In addition, 64 secreted proteins, including oxidoreductases, exhibited at least two-fold upregulation on cellulose medium. To better understand the potential roles of low-abundance CAZymes in cellulose breakdown, we compared the expression patterns of 25 glycoside hydrolase genes under different conditions via real-time PCR. Substantial differences for the 25 genes were observed for individual strains grown on different carbon sources, and between QM9414 and RUTC30 when grown on the same carbon source. Moreover, we identified 3 genes that are coregulated with known cellulases. Collectively, this study highlights a comprehensive transcriptional profile for biomass degradation-related proteins and provides a first step toward the identification of candidates to construct optimized enzyme cocktails.

  9. Identification of methylmercury tolerance gene candidates in Drosophila.

    PubMed

    Mahapatra, Cecon T; Bond, Jeffrey; Rand, David M; Rand, Matthew D

    2010-07-01

    Methylmercury (MeHg) is a ubiquitous environmental contaminant that preferentially targets the developing nervous system. Variable outcomes of prenatal MeHg exposure within a population point to a genetic component that regulates MeHg toxicity. We therefore sought to identify fundamental MeHg tolerance genes using the Drosophila model for genetic and molecular dissection of a MeHg tolerance trait. We observe autosomal dominance in a MeHg tolerance trait (development on MeHg food) in both wild-derived and laboratory-selected MeHg-tolerant strains of flies. We performed whole-genome transcript profiling of larval brains of tolerant (laboratory selected) and nontolerant (control) strains in the presence and absence of MeHg stress. Pairwise transcriptome comparisons of four conditions (+/-selection and +/-MeHg) identified a "down-down-up" expression signature, whereby MeHg alone and selection alone resulted in a greater number of downregulated transcripts, and the combination of selection + MeHg resulted in a greater number of upregulated transcripts. Functional annotation cluster analyses showed enrichment for monooxygenases/oxidoreductases, which include cytochrome P450 (CYP) family members. Among the 10 CYPs upregulated with selection + MeHg in tolerant strains, CYP6g1, previously identified as the dichlorodiphenyl trichloroethane resistance allele in flies, was the most highly expressed and responsive to MeHg. Among all the genes, Turandot A (TotA), an immune pathway-regulated humoral response gene, showed the greatest upregulation with selection + MeHg. Neural-specific transgenic overexpression of TotA enhanced MeHg tolerance during pupal development. Identification of TotA and CYP genes as MeHg tolerance genes is an inroad to investigating the conserved function of immune signaling and phase I metabolism pathways in MeHg toxicity and tolerance in higher organisms.

  10. Digital gene expression profiling analysis of duodenum transcriptomes in SD rats administered ferrous sulfate or ferrous glycine chelate by gavage.

    PubMed

    Zhuo, Zhao; Fang, Shenglin; Hu, Qiaoling; Huang, Danping; Feng, Jie

    2016-11-30

    The absorption of different iron sources is a trending research topic. Many studies have revealed that organic iron exhibits better bioavailability than inorganic iron, but the concrete underlying mechanism is still unclear. In the present study, we examined the differences in bioavailability of ferrous sulfate and ferrous glycinate in the intestines of SD rats using Illumina sequencing technology. Digital gene expression analysis resulted in the generation of almost 128 million clean reads, with expression data for 17,089 unigenes. A total of 123 differentially expressed genes with a |log2(fold change)| >1 and q-value < 0.05 were identified between the FeSO4 and Fe-Gly groups. Gene Ontology functional analysis revealed that these genes were involved in oxidoreductase activity, iron ion binding, and heme binding. Kyoto Encyclopedia of Genes and Genomes pathway analysis also showed relevant important pathways. In addition, the expression patterns of 9 randomly selected genes were further validated by qRT-PCR, which confirmed the digital gene expression results. Our study showed that the two iron sources might share the same absorption mechanism, and that differences in bioavailability between FeSO4 and Fe-Gly were not only in the absorption process but also during the transport and utilization process.

  11. Digital gene expression profiling analysis of duodenum transcriptomes in SD rats administered ferrous sulfate or ferrous glycine chelate by gavage

    PubMed Central

    Zhuo, Zhao; Fang, Shenglin; Hu, Qiaoling; Huang, Danping; Feng, Jie

    2016-01-01

    The absorption of different iron sources is a trending research topic. Many studies have revealed that organic iron exhibits better bioavailability than inorganic iron, but the concrete underlying mechanism is still unclear. In the present study, we examined the differences in bioavailability of ferrous sulfate and ferrous glycinate in the intestines of SD rats using Illumina sequencing technology. Digital gene expression analysis resulted in the generation of almost 128 million clean reads, with expression data for 17,089 unigenes. A total of 123 differentially expressed genes with a |log2(fold change)| >1 and q-value < 0.05 were identified between the FeSO4 and Fe-Gly groups. Gene Ontology functional analysis revealed that these genes were involved in oxidoreductase activity, iron ion binding, and heme binding. Kyoto Encyclopedia of Genes and Genomes pathway analysis also showed relevant important pathways. In addition, the expression patterns of 9 randomly selected genes were further validated by qRT-PCR, which confirmed the digital gene expression results. Our study showed that the two iron sources might share the same absorption mechanism, and that differences in bioavailability between FeSO4 and Fe-Gly were not only in the absorption process but also during the transport and utilization process. PMID:27901057

  12. The ctnG gene encodes carbonic anhydrase involved in mycotoxin citrinin biosynthesis from Monascus aurantiacus.

    PubMed

    Li, Yan-Ping; Tang, Xiao; Wu, Wei; Xu, Yang; Huang, Zhi-Bing; He, Qing-Hua

    2015-01-01

    Citrinin, a fungal secondary metabolite of polyketide origin, is moderately nephrotoxic to vertebrates, including humans. Citrinin is synthesised by condensation of acetyl-CoA and malonyl-CoA. Six genes involved in the citrinin biosynthesis, including pksCT, ctnA and ctnB, have been cloned in Monascus purpureus. The pksCT gene encodes a polyketide synthase; ctnA is a regulatory factor; and ctnB encodes an oxidoreductase. When the three genes were respectively disrupted, the disruption strains drastically decreased citrinin production or barely produced citrinin. Ten new genes have been discovered in Monascus aurantiacus besides the above six genes. One of these gene displayed the highest similarity to the β-carbonic anhydrase gene from Aspergillus oryzae (74% similarity) and was designated ctnG. To learn more about the citrinin biosynthetic pathway, a ctnG-replacement vector was constructed to disrupt ctnG with the hygromycin resistance gene as the selection marker, then transformed into M. aurantiacus Li AS3.4384 by a protoplast-PEG method. The citrinin content of three disruptants was reduced to about 50%, meanwhile pigment production decreased by 23%, respectively, over those of the wild-type strains. ctnG was deduced to be involved in the formation of malonyl-CoA as a common precursor of red pigments and citrinin. Therefore, the disruption of the ctnG gene decreased citrinin and pigment production. M. aurantiacus Li AS3.4384 can produce higher concentrations of citrinin than other strains such as M. purpureus and M. ruber. Establishing the function of citrinin biosynthetic genes in M. aurantiacus is helpful in understanding the citrinin synthetic pathway and adopting some strategies to control contamination.

  13. Influence of sex on gene expression in human corneal epithelial cells

    PubMed Central

    Suzuki, Tomo; Richards, Stephen M.; Liu, Shaohui; Jensen, Roderick V.

    2009-01-01

    Purpose Sex-associated differences have been identified in the anatomy, physiology and pathophysiology of the human cornea. We hypothesize that many of these differences are due to fundamental variations in gene expression. Our objective in this study was to determine whether such differences exist in human corneal epithelial cells both in vivo and in vitro. Methods Human corneal epithelial cells were isolated from the corneoscleral rims of male and female donors. Cells were processed either directly for RNA extraction, or first cultured in phenol red-free keratinocyte serum-free media. The RNA samples were examined for differentially expressed mRNAs by using of CodeLink Bioarrays and Affymetrix GeneChips. Data were analyzed with GeneSifter.Net software. Results Our results demonstrate that sex significantly influences the expression of over 600 genes in human corneal epithelial cells in vivo. These genes are involved in a broad spectrum of biologic processes, molecular functions and cellular components, such as metabolic processes, DNA replication, cell migration, RNA binding, oxidoreductase activity and nucleoli. We also identified significant, sex-related effects on gene expression in human corneal epithelial cells in vitro. However, with few exceptions (e.g., X- and Y-linked genes), these sex-related differences in gene expression in vitro were typically different than those in vivo. Conclusions Our findings support our hypothesis that sex-related differences exist in the gene expression of human corneal epithelial cells. Variations in gene expression may contribute to sex-related differences in the prevalence of certain corneal diseases. PMID:20011627

  14. Gene doping.

    PubMed

    Azzazy, Hassan M E

    2010-01-01

    Gene doping abuses the legitimate approach of gene therapy. While gene therapy aims to correct genetic disorders by introducing a foreign gene to replace an existing faulty one or by manipulating existing gene(s) to achieve a therapeutic benefit, gene doping employs the same concepts to bestow performance advantages on athletes over their competitors. Recent developments in genetic engineering have contributed significantly to the progress of gene therapy research and currently numerous clinical trials are underway. Some athletes and their staff are probably watching this progress closely. Any gene that plays a role in muscle development, oxygen delivery to tissues, neuromuscular coordination, or even pain control is considered a candidate for gene dopers. Unfortunately, detecting gene doping is technically very difficult because the transgenic proteins expressed by the introduced genes are similar to their endogenous counterparts. Researchers today are racing the clock because assuring the continued integrity of sports competition depends on their ability to develop effective detection strategies in preparation for the 2012 Olympics, which may mark the appearance of genetically modified athletes.

  15. Gene therapy.

    PubMed

    Williamson, B

    1982-07-29

    Gene therapy is not yet possible, but may become feasible soon, particularly for well understood gene defects. Although treatment of a patient raises no ethical problems once it can be done well, changing the genes of an early embryo is more difficult, controversial and unlikely to be required clinically.

  16. Bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) by human NAD(P)H quinone oxidoreductase 2: a novel co-substrate-mediated antitumor prodrug therapy.

    PubMed

    Knox, R J; Jenkins, T C; Hobbs, S M; Chen, S; Melton, R G; Burke, P J

    2000-08-01

    A novel prodrug activation system, endogenous in human tumor cells, is described. A latent enzyme-prodrug system is switched on by a simple synthetic, small molecule co-substrate. This ternary system is inactive if any one of the components is absent. CB 1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] is an antitumor prodrug that is activated in certain rat tumors via its 4-hydroxylamine derivative to a potent bifunctional alkylating agent. However, human tumor cells are resistant to CB 1954 because they are unable to catalyze this bioactivation efficiently. A human enzyme has been discovered that can activate CB 1954, and it has been shown to be commonly present in human tumor cells. The enzyme is NQO2 [NAD(P)H quinone oxidoreductase 2], but its activity is normally latent, and a nonbiogenic co-substrate such as NRH [nicotinamide riboside (reduced)] is required for enzymatic activity. There is a very large (100-3000-fold) increase in CB 1954 cytotoxicity toward either NQO2-transfected rodent or nontransfected human tumor cell lines in the presence of NRH. Other reduced pyridinium compounds can also act as co-substrates for NQO2. Thus, the simplest quaternary salt of nicotinamide, 1-methyl-3-carboxamidopyridinium iodide, was a co-substrate for NQO2 when reduced to the corresponding 1,4-dihydropyridine derivative. Increased chain length and/or alkyl load at the 1-position of the dihydropyridine ring improved specific activity, and compounds more active than NRH were found. However, little activity was seen with either the 1-benzyl or 1-(2-phenylethyl) derivatives. A negatively charged substituent at the 3-position of the reduced pyridine ring also negated the ability of these compounds to act as cosubstrates for NQO2. In particular, 1-carbamoylmethyl-3-carbamoyl-1,4dihydropyridine was shown to be a co-substrate for NQO2 with greater stability than NRH, with the ability to enter cells and potentiate the cytotoxicity of CB 1954. Furthermore, this agent is synthetically

  17. Response of Chloroplast NAD(P)H Dehydrogen