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

    2015-05-01

    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. PMID:25809902

  3. 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 ?pfkA 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 ?pfkA 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 ?pfkA 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 ?pfkA 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 dehydrogenase. The differences in respiration rates can explain the suggested higher ArcAB activity in the reference strain. PMID:24771245

  4. Identification and characterization of NADPH-dependent cytochrome P450 reductase gene and cytochrome b? gene from Plutella xylostella: possible involvement in resistance to beta-cypermethrin.

    PubMed

    Chen, Xi'en; Zhang, Yalin

    2015-03-10

    NADPH-cytochrome P450 reductase (CPR) and cytochrome b5 (b5) are essential for cytochrome P450 mediated biological reactions. CPR and b5 in several insects have been found to be associated with insecticide resistance. However, CPR and b5 in the diamondback moth (DBM), Plutella xylostella, are not characterized and their roles remain undefined. A full-length cDNA of CPR encoding 678 amino acids and a full-length cDNA of b5 encoding 127 amino acids were cloned from DBM. Their deduced amino acid sequences shared high identities with those of other insects and showed characteristics of classical CPRs and b5s, respectively. The mRNAs of both genes were detectable in all developmental stages with the highest expression levels occurring in the 4th instar larvae. Tissue-specific expression analysis showed that their transcripts were most abundant in gut. Transcripts of CPR and b5 in the beta-cypermethrin resistant DBM strain were 13.2- and 2.84-fold higher than those in the beta-cypermethrin susceptible strain, respectively. The expression levels of CPR and b5 were enhanced by beta-cypermethrin at the concentration of 12 mg L(-1) (~LC10). The results indicate that CPR and b5 may play essential roles in the P450 mediated resistance of DBM to beta-cypermethrin or even other insecticides. PMID:25550052

  5. Isolation and characterization of a soluble NADPH-dependent Fe(III) reductase from Geobacter sulfurreducens.

    PubMed

    Kaufmann, F; Lovley, D R

    2001-08-01

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

  6. ISOLATION AND CHARACTERIZATION OF THE ALKANE-INDUCIBLE NADPH-CYTOCHROME P-450 OXIDOREDUCTASE GENE FROM CANDIDA TROPICALIS

    EPA Science Inventory

    The gene coding for the Candida tropicalis NADPH-cytochrome P-450 oxidoreductase (CPR, NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) was isolated by immunoscreening of a C. tropicalis gtll expression library and colony hybridization of a C. tropicalis genomic library. he C. ...

  7. Novel Nicotine Oxidoreductase-Encoding Gene Involved in Nicotine Degradation by Pseudomonas putida Strain S16

    Microsoft Academic Search

    Hongzhi Tang; Lijuan Wang; Xiangzhou Meng; Lanying Ma; Shuning Wang; Xiaofei He; Geng Wu; Ping Xu

    2009-01-01

    There are quite a few ongoing biochemical investigations of nicotine degradation in different organisms. In this work, we identified and sequenced a gene (designated nicA) involved in nicotine degradation by Pseudo- monas putida strain S16. The gene product, NicA, was heterologously expressed and characterized as a nicotine oxidoreductase catalyzing the initial steps of nicotine metabolism. Biochemical analyses using resting cells

  8. Novel Nicotine Oxidoreductase-Encoding Gene Involved in Nicotine Degradation by Pseudomonas putida Strain S16 ? †

    PubMed Central

    Tang, Hongzhi; Wang, Lijuan; Meng, Xiangzhou; Ma, Lanying; Wang, Shuning; He, Xiaofei; Wu, Geng; Xu, Ping

    2009-01-01

    There are quite a few ongoing biochemical investigations of nicotine degradation in different organisms. In this work, we identified and sequenced a gene (designated nicA) involved in nicotine degradation by Pseudomonas putida strain S16. The gene product, NicA, was heterologously expressed and characterized as a nicotine oxidoreductase catalyzing the initial steps of nicotine metabolism. Biochemical analyses using resting cells and the purified enzyme suggested that nicA encodes an oxidoreductase, which converts nicotine to 3-succinoylpyridine through pseudooxynicotine. Based on enzymatic reactions and direct evidence obtained using H218O labeling, the process may consist of enzyme-catalyzed dehydrogenation, followed by spontaneous hydrolysis and then repetition of the dehydrogenation and hydrolysis steps. Sequence comparisons revealed that the gene showed 40% similarity to genes encoding NADH dehydrogenase subunit I and cytochrome c oxidase subunit I in eukaryotes. Our findings demonstrate that the molecular mechanism for nicotine degradation in strain S16 involves the pyrrolidine pathway and is similar to the mechanism in mammals, in which pseudooxynicotine, the direct precursor of a potent tobacco-specific lung carcinogen, is produced. PMID:19060159

  9. Glutaric acidemia type II: gene structure and mutations of the electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO) gene

    Microsoft Academic Search

    Stephen I Goodman; Robert J Binard; Michael R Woontner; Frank E Frerman

    2002-01-01

    Glutaric acidemia type II is a human inborn error of metabolism which can be due to defects in either subunit of electron transfer flavoprotein (ETF) or in ETF:ubiquinone oxidoreductase (ETF:QO), but few disease-causing mutations have been described. The ETF:QO gene is located on 4q33, and contains 13 exons. Primers to amplify these exons are presented, together with mutations identified by

  10. Human carbonyl reductase 4 is a mitochondrial NADPH-dependent quinone reductase.

    PubMed

    Endo, Satoshi; Matsunaga, Toshiyuki; Kitade, Yukio; Ohno, Satoshi; Tajima, Kazuo; El-Kabbani, Ossama; Hara, Akira

    2008-12-26

    A protein encoded in the gene Cbr4 on human chromosome 4q32.3 belongs to the short-chain dehydrogenase/reductase family. Contrary to the functional annotation as carbonyl reductase 4 (CBR4), we show that the recombinant tetrameric protein, composed of 25-kDa subunits, exhibits NADPH-dependent reductase activity for o- and p-quinones, but not for other aldehydes and ketones. The enzyme was insensitive to dicumarol and quercetin, potent inhibitors of cytosolic quinone reductases. The 25-kDa CBR4 was detected in human liver, kidney and cell lines on Western blotting using anti-CBR4 antibodies. The overexpression of CBR4 in bovine endothelial cells reveals that the enzyme has a non-cleavable mitochondrial targeting signal. We further demonstrate that the in vitro quinone reduction by CBR4 generates superoxide through the redox cycling, and suggest that the enzyme may be involved in the induction of apoptosis by cytotoxic 9,10-phenanthrenequinone. PMID:19000905

  11. 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. PMID:26025518

  12. Regulation of expression of Na + -translocating NADH:quinone oxidoreductase genes in Vibrio harveyi and Klebsiella pneumoniae

    Microsoft Academic Search

    Maria S. Fadeeva; Evgenia A. Yakovtseva; Galina A. Belevich; Yulia V. Bertsova; Alexander V. Bogachev

    2007-01-01

    The expression of genes encoding sodium-translocating NADH:quinone oxidoreductase (Na+-NQR) was studied in the marine bacterium Vibrio harveyi and in the enterobacterium Klebsiella pneumoniae. It has been shown that such parameters as NaCl concentration, pH value, and presence of an uncoupler in the growth media\\u000a do not influence significantly the level of nqr expression. However, nqr expression depends on the growth

  13. Polymorphisms in the NAD( P) H: quinone oxidoreductase gene and small cell lung cancer risk in a UK population

    Microsoft Academic Search

    Sarah J Lewis; Nicola M Cherry; Robert McL Niven; Phillip V Barber; Andrew C Povey

    2001-01-01

    NAD(P)H: quinone oxidoreductase (NQO1) protects the cell against cytotoxicity by reducing the concentration of free quinone available for single electron reduction. The NQO1 gene is polymorphic and the variant protein exhibits just 2% of the enzymatic activity of the wildtype protein. In this study, we investigated NQO1 genotype in relation to lung cancer risk in patients attending a Manchester bronchoscopy

  14. Comparative Molecular Modeling Study of Arabidopsis NADPH-Dependent Thioredoxin Reductase and Its

    E-print Network

    Lee, Keun Woo

    of chloroplast proteins from oxidative damage. Arabidopsis NADPH-dependent thioredoxin reductase isotype C (AtNTRC) was identified as efficient electron donor for chloroplastic 2-Cys Prx-A. There are three isotypes (A, B, and C-terminal TrxR and C-terminal thioredoxin (Trx) domains. AtNTRC has various oligomer structures, and Trx domain

  15. Expression of the bile acid-inducible NADH:flavin oxidoreductase gene of Eubacterium sp. VPI 12708 in Escherichia coli

    Microsoft Academic Search

    Stephen F. Baron; Phillip B. Hylemon

    1995-01-01

    The intestinal microorganism Eubacterium sp. VPI 12708 synthesizes a bile acid-inducible NADH:flavin oxidoreductase (NADH:FOR) which presumably functions in the 7?-dehydroxylation of cholic acid to deoxycholic acid. The baiH gene encoding NADH:FOR was subcloned into an IPTG-inducible expression vector, pBaiH2.2. Escherichia coli DH5? cells transformed with pBaiH2.2 expressed 10-fold higher levels of NADH:.FOR upon induction with IPTG than did Eubacterium sp.

  16. Clostridium scindens baiCD and baiH genes encode stereo-specific 7?/7?-hydroxy-3-oxo-?4-cholenoic acid oxidoreductases *

    PubMed Central

    Kang, Dae-Joong; Ridlon, Jason M.; Moore, Doyle Ray; Barnes, Stephen; Hylemon, Phillip B.

    2008-01-01

    Secondary bile acids, formed by intestinal bacteria, are suggested to play a significant role in cancers of the gastrointestinal tract in humans. Bile acid 7?/?-dehydroxylation is carried out by a few species of intestinal clostridia which harbor a multi-gene bile acid inducible (bai) operon. Several genes encoding enzymes in this pathway have been cloned and characterized. However, no gene product(s) has yet been assigned to the production of 3-oxo-?4-cholenoic acid intermediates of cholic acid (CA), chenodeoxycholic acid (CDCA) or ursodeoxycholic acid (UDCA). We previously reported the baiH gene encodes an NADH:flavin oxidoreductase (NADH:FOR); however, the role of this protein in bile acid 7-dehydroxylation is unclear. Homology searches and secondary structural alignments suggest this protein to be similar to flavoproteins which reduce ?/?-unsaturated carbonyl compounds. The baiH gene product was expressed in E. coli, purified and discovered to be a stereospecific NAD(H)-dependent 7?-hydroxy-3-oxo-?4-cholenoic acid oxidoreductase. Additionally, high sequence similarity between the baiH and baiCD gene products suggests the baiCD gene may encode a 3-oxo-?4-cholenoic acid oxidoreductase specific for CDCA and CA. We tested this hypothesis using cell extracts prepared from E. coli overexpressing the baiCD gene and discovered that it encodes a stereo-specific NAD(H)-dependent 7?-hydroxy-3-oxo-?4-cholenoic acid oxidoreductase. PMID:18047844

  17. Pirfenidone inhibits NADPH-dependent microsomal lipid peroxidation and scavenges hydroxyl radicals

    Microsoft Academic Search

    Hara P. Misra; Christine Rabideau

    2000-01-01

    Pirfenidone (Pf), a new broad-spectrum anti-fibrotic agent, is known to offer protection against lung fibrosis in vivo in laboratory animals, and against mitogenesis and collagen formation by human lung fibroblasts in vitro. Because reactive oxygen species are thought to be involved in these events, we investigated the mechanism(s) by which Pf ameliorates oxidative stress and its effects on NADPH-dependent lipid

  18. Expression of the iorAB genes from Brevundimonas diminuta 7 encoding the molybdenum hydroxylase isoquinoline 1-oxidoreductase in Pseudomonas putida.

    PubMed

    Israel, Ilka; Sohni, Monika; Fetzner, Susanne

    2002-04-23

    Isoquinoline 1-oxidoreductase (Ior) from Brevundimonas diminuta 7, encoded by iorAB, is a molybdenum hydroxylase containing a molybdopterin cytosine dinucleotide molybdenum cofactor (Mo-MCD) and two distinct [2Fe2S] clusters. The iorAB genes were inserted into pJB653, generating pIL1. Pseudomonas putida KT2440, and P. putida 86 which produces a Mo-MCD-containing quinoline 2-oxidoreductase when grown on quinoline, were used as recipients for pIL1. Upon induction of gene expression, both clones produced Ior protein, but Ior activity was not detectable in P. putida KT2440 pIL1. In P. putida 86 pIL1, formation of catalytically active Ior required the presence of quinoline, suggesting that accessory gene(s) encoding product(s) essential for the assembly of catalytically competent Ior is (are) part of the quinoline regulon in P. putida 86. PMID:12023088

  19. Xanthine oxidoreductase

    Microsoft Academic Search

    Roger Harrison

    \\u000a Xanthine oxidoreductase (XOR) is a widely-distributed molybdenum-containing flavoenzyme, being particularly rich in mammary\\u000a epithelial cells and in capillary endothelium in a range of tissues [1,2]. While its conventionally accepted role is in purine catabolism, catalysing the oxidation of hypoxanthine to xanthine and\\u000a xanthine to uric acid, such a general housekeeping function is somewhat at odds with its specialised cellular distribution.

  20. The pc-1 phenotype of Chlamydomonas reinhardtii results from a deletion mutation in the nuclear gene for NADPH:protochlorophyllide oxidoreductase

    Microsoft Academic Search

    Jianming Li; Michael P. Tirnko

    1996-01-01

    The pc-1 mutant of Chlamydomonas reinhardtii has been shown to be incapable of protochlorophyllide photoconversion in vivo and is thought to be defective in light-dependent NADPH:protochlorophyllide oxidoreductase activity. We have isolated and characterized the nuclear genes encoding this enzyme from wild-type and pc-1 mutant Chlamydomonas cells. The wild-type CRlpcr-1 gene encodes a 397 amino acid polypeptide of which the N-terminal

  1. Stress defense mechanisms of NADPH-dependent thioredoxin reductases (NTRs) in plants.

    PubMed

    Cha, Joon-Yung; Barman, Dhirendra Nath; Kim, Min Gab; Kim, Woe-Yeon

    2015-05-01

    Plants establish highly and systemically organized stress defense mechanisms against unfavorable living conditions. To interpret these environmental stimuli, plants possess communication tools, referred as secondary messengers, such as Ca(2+) signature and reactive oxygen species (ROS) wave. Maintenance of ROS is an important event for whole lifespan of plants, however, in special cases, toxic ROS molecules are largely accumulated under excess stresses and diverse enzymes played as ROS scavengers. Arabidopsis and rice contain 3 NADPH-dependent thioredoxin reductases (NTRs) which transfer reducing power to Thioredoxin/Peroxiredoxin (Trx/Prx) system for scavenging ROS. However, due to functional redundancy between cytosolic and mitochondrial NTRs (NTRA and NTRB, respectively), their functional involvements under stress conditions have not been well characterized. Recently, we reported that cytosolic NTRA confers the stress tolerance against oxidative and drought stresses via regulation of ROS amounts using NTRA-overexpressing plants. With these findings, mitochondrial NTRB needs to be further elucidated. PMID:26039478

  2. Eicosanoids up-regulate production of reactive oxygen species by NADPH-dependent oxidase in Spodoptera exigua phagocytic hemocytes.

    PubMed

    Park, Youngjin; Stanley, David W; Kim, Yonggyun

    2015-08-01

    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 eicosanoids mediate ROS production by activating NADPH-dependent oxidase (NOX) and tested the idea in the model insect, Spodoptera exigua. A NOX gene (we named SeNOX4) was identified and cloned, yielding a full open reading frame encoding 547 amino acid residues with a predicted molecular weight of 63,410Da and an isoelectric point at 9.28. A transmembrane domain and a large intracellular domain containing NADPH and FAD-binding sites were predicted. Phylogenetic analysis indicated SeNOX4 clusters with other NOX4 genes. SeNOX4 was expressed in all life stages except eggs, and exclusively in hemocytes. Bacterial challenge and, separately, arachidonic acid (AA, a precursor of eicosanoid biosynthesis) injection increased its expression. The internalization step was assessed by counting hemocytes engulfing fluorescence-labeled bacteria. The phagocytic behavior was inhibited by dsRNA suppression of SeNOX4 expression and, separately by dexamethasone (DEX, a specific inhibitor of eicosanoid biosynthesis) treatments. However, injecting AA to dsSeNOX4-treated larvae did not rescue the phagocytic activity. Hemocytic ROS production increased following bacterial challenge, which was sharply reduced in dsSeNOX4-treated, and separately, in DEX-treated larvae. AA partially reversed the suppressed ROS production in dsSeNOX4-treated larvae. Treating larvae with either the ROS-suppressing dsSeNOX4 construct or DEX rendered experimental larvae unable to inhibit bacterial proliferation in their hemocoels. We infer that eicosanoids mediate ROS production during phagocytosis by inducing expression of SeNOX4. PMID:26071791

  3. 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

  4. Construction and Screening of Metagenomic Libraries Derived from Enrichment Cultures: Generation of a Gene Bank for Genes Conferring Alcohol Oxidoreductase Activity on Escherichia coli

    PubMed Central

    Knietsch, Anja; Waschkowitz, Tanja; Bowien, Susanne; Henne, Anke; Daniel, Rolf

    2003-01-01

    Enrichment of microorganisms with special traits and the construction of metagenomic libraries by direct cloning of environmental DNA have great potential for identifying genes and gene products for biotechnological purposes. We have combined these techniques to isolate novel genes conferring oxidation of short-chain (C2 to C4) polyols or reduction of the corresponding carbonyls. In order to favor the growth of microorganisms containing the targeted genes, samples collected from four different environments were incubated in the presence of glycerol and 1,2-propanediol. Subsequently, the DNA was extracted from the four samples and used to construct complex plasmid libraries. Approximately 100,000 Escherichia coli strains of each library per test substrate were screened for the production of carbonyls from polyols on indicator agar. Twenty-four positive E. coli clones were obtained during the initial screen. Sixteen of them contained a plasmid (pAK101 to pAK116) which conferred a stable carbonyl-forming phenotype. Eight of the positive clones exhibited NAD(H)-dependent alcohol oxidoreductase activity with polyols or carbonyls as the substrates in crude extracts. Sequencing revealed that the inserts of pAK101 to pAK116 encoded 36 complete and 17 incomplete presumptive protein-encoding genes. Fifty of these genes showed similarity to sequenced genes from a broad collection of different microorganisms. The genes responsible for the carbonyl formation of E. coli were identified for nine of the plasmids (pAK101, pAK102, pAK105, pAK107 to pAK110, pAK115, and pAK116). Analyses of the amino acid sequences deduced from these genes revealed that three (orf12, orf14, and orf22) encoded novel alcohol dehydrogenases of different types, four (orf5, sucB, fdhD, and yabF) encoded novel putative oxidoreductases belonging to groups distinct from alcohol dehydrogenases, one (glpK) encoded a putative glycerol kinase, and one (orf1) encoded a protein which showed no similarity to any other known gene product. PMID:12620823

  5. Randomly selected suppressor mutations in genes for NADH?:?quinone oxidoreductase-1, which rescue motility of a Salmonella ubiquinone-biosynthesis mutant strain.

    PubMed

    Barker, Clive S; Meshcheryakova, Irina V; Sasaki, Toshio; Roy, Michael C; Sinha, Prem Kumar; Yagi, Takao; Samatey, Fadel A

    2014-06-01

    The primary mobile electron-carrier in the aerobic respiratory chain of Salmonella is ubiquinone. Demethylmenaquinone and menaquinone are alternative electron-carriers involved in anaerobic respiration. Ubiquinone biosynthesis was disrupted in strains bearing deletions of the ubiA or ubiE genes. In soft tryptone agar both mutant strains swam poorly. However, the ubiA deletion mutant strain produced suppressor mutant strains with somewhat rescued motility and growth. Six independent suppressor mutants were purified and comparative genome sequence analysis revealed that they each bore a single new missense mutation, which localized to genes for subunits of NADH?:?quinone oxidoreductase-1. Four mutants bore an identical nuoG(Q297K) mutation, one mutant bore a nuoM(A254S) mutation and one mutant bore a nuoN(A444E) mutation. The NuoG subunit is part of the hydrophilic domain of NADH?:?quinone oxidoreductase-1 and the NuoM and NuoN subunits are part of the hydrophobic membrane-embedded domain. Respiration was rescued and the suppressed mutant strains grew better in Luria-Bertani broth medium and could use l-malate as a sole carbon source. The quinone pool of the cytoplasmic membrane was characterized by reversed-phase HPLC. Wild-type cells made ubiquinone and menaquinone. Strains with a ubiA deletion mutation made demethylmenaquinone and menaquinone and the ubiE deletion mutant strain made demethylmenaquinone and 2-octaprenyl-6-methoxy-1,4-benzoquinone; the total quinone pool was reduced. Immunoblotting found increased NADH?:?quinone oxidoreductase-1 levels for ubiquinone-biosynthesis mutant strains and enzyme assays measured electron transfer from NADH to demethylmenaquinone or menaquinone. Under certain growth conditions the suppressor mutations improved electron flow activity of NADH?:?quinone oxidoreductase-1 for cells bearing a ubiA deletion mutation. PMID:24692644

  6. Randomly selected suppressor mutations in genes for NADH?:?quinone oxidoreductase-1, which rescue motility of a Salmonella ubiquinone-biosynthesis mutant strain

    PubMed Central

    Meshcheryakova, Irina V.; Sasaki, Toshio; Roy, Michael C.; Sinha, Prem Kumar; Yagi, Takao

    2014-01-01

    The primary mobile electron-carrier in the aerobic respiratory chain of Salmonella is ubiquinone. Demethylmenaquinone and menaquinone are alternative electron-carriers involved in anaerobic respiration. Ubiquinone biosynthesis was disrupted in strains bearing deletions of the ubiA or ubiE genes. In soft tryptone agar both mutant strains swam poorly. However, the ubiA deletion mutant strain produced suppressor mutant strains with somewhat rescued motility and growth. Six independent suppressor mutants were purified and comparative genome sequence analysis revealed that they each bore a single new missense mutation, which localized to genes for subunits of NADH?:?quinone oxidoreductase-1. Four mutants bore an identical nuoG(Q297K) mutation, one mutant bore a nuoM(A254S) mutation and one mutant bore a nuoN(A444E) mutation. The NuoG subunit is part of the hydrophilic domain of NADH?:?quinone oxidoreductase-1 and the NuoM and NuoN subunits are part of the hydrophobic membrane-embedded domain. Respiration was rescued and the suppressed mutant strains grew better in Luria–Bertani broth medium and could use l-malate as a sole carbon source. The quinone pool of the cytoplasmic membrane was characterized by reversed-phase HPLC. Wild-type cells made ubiquinone and menaquinone. Strains with a ubiA deletion mutation made demethylmenaquinone and menaquinone and the ubiE deletion mutant strain made demethylmenaquinone and 2-octaprenyl-6-methoxy-1,4-benzoquinone; the total quinone pool was reduced. Immunoblotting found increased NADH?:?quinone oxidoreductase-1 levels for ubiquinone-biosynthesis mutant strains and enzyme assays measured electron transfer from NADH to demethylmenaquinone or menaquinone. Under certain growth conditions the suppressor mutations improved electron flow activity of NADH?:?quinone oxidoreductase-1 for cells bearing a ubiA deletion mutation. PMID:24692644

  7. Characterization of the baiH gene encoding a bile acid-inducible NADH:flavin oxidoreductase from Eubacterium sp. strain VPI 12708.

    PubMed Central

    Franklund, C V; Baron, S F; Hylemon, P B

    1993-01-01

    A cholate-inducible, NADH-dependent flavin oxidoreductase from the intestinal bacterium Eubacterium sp. strain VPI 12708 was purified 372-fold to apparent electrophoretic homogeneity. The subunit and native molecular weights were estimated to be 72,000 and 210,000, respectively, suggesting a homotrimeric organization. Three peaks of NADH:flavin oxidoreductase activity (forms I, II, and III) eluted from a DEAE-high-performance liquid chromatography column. Absorption spectra revealed that purified form III, but not form I, contained bound flavin, which dissociated during purification to generate form I. Enzyme activity was inhibited by sulfhydryl-reactive compounds, acriflavine, o-phenanthroline, and EDTA. Activity assays and Western blot (immunoblot) analysis confirmed that expression of the enzyme was cholate inducible. The first 25 N-terminal amino acid residues of purified NADH:flavin oxidoreductase were determined, and a corresponding oligonucleotide probe was synthesized for use in cloning of the associated gene, baiH. Restriction mapping, sequence data, and RNA blot analysis suggested that the baiH gene was located on a previously described, cholate-inducible operon > or = 10 kb long. The baiH gene encoded a 72,006-Da polypeptide containing 661 amino acids. The deduced amino acid sequence of the baiH gene was homologous to that of NADH oxidase from Thermoanaerobium brockii, trimethylamine dehydrogenase from methylotrophic bacterium W3A1, Old Yellow Enzyme from Saccharomyces carlsbergensis, and the product of the baiC gene of Eubacterium sp. strain VPI 12708, located upstream from the baiH gene in the cholate-inducible operon. Alignment of these five sequences revealed potential ligands for an iron-sulfur cluster, a putative flavin adenine dinucleotide-binding domain, and two other well-conserved domains of unknown function. Images PMID:8491719

  8. Reconstruction of an acetogenic 2,3-butanediol pathway involving a novel NADPH-dependent primary-secondary alcohol dehydrogenase.

    PubMed

    Köpke, Michael; Gerth, Monica L; Maddock, Danielle J; Mueller, Alexander P; Liew, FungMin; Simpson, Séan D; Patrick, Wayne M

    2014-06-01

    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 ?M 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

  9. 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 ?M 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

  10. Comparative molecular modeling study of Arabidopsis NADPH-dependent thioredoxin reductase and its hybrid protein.

    PubMed

    Lee, Yuno; Kim, Songmi; Lazar, Prettina; Moon, Jeong Chan; Hwang, Swan; Thangapandian, Sundarapandian; Shon, Youngsik; Lee, Kyun Oh; Lee, Sang Yeol; Lee, Keun Woo

    2012-01-01

    2-Cys peroxiredoxins (Prxs) play important roles in the protection of chloroplast proteins from oxidative damage. Arabidopsis NADPH-dependent thioredoxin reductase isotype C (AtNTRC) was identified as efficient electron donor for chloroplastic 2-Cys Prx-A. There are three isotypes (A, B, and C) of thioredoxin reductase (TrxR) in Arabidopsis. AtNTRA contains only TrxR domain, but AtNTRC consists of N-terminal TrxR and C-terminal thioredoxin (Trx) domains. AtNTRC has various oligomer structures, and Trx domain is important for chaperone activity. Our previous experimental study has reported that the hybrid protein (AtNTRA-(Trx-D)), which was a fusion of AtNTRA and Trx domain from AtNTRC, has formed variety of structures and shown strong chaperone activity. But, electron transfer mechanism was not detected at all. To find out the reason of this problem with structural basis, we performed two different molecular dynamics (MD) simulations on AtNTRC and AtNTRA-(Trx-D) proteins with same cofactors such as NADPH and flavin adenine dinucleotide (FAD) for 50 ns. Structural difference has found from superimposition of two structures that were taken relatively close to average structure. The main reason that AtNTRA-(Trx-D) cannot transfer the electron from TrxR domain to Trx domain is due to the difference of key catalytic residues in active site. The long distance between TrxR C153 and disulfide bond of Trx C387-C390 has been observed in AtNTRA-(Trx-D) because of following reasons: i) unstable and unfavorable interaction of the linker region, ii) shifted Trx domain, and iii) different or weak interface interaction of Trx domains. This study is one of the good examples for understanding the relationship between structure formation and reaction activity in hybrid protein. In addition, this study would be helpful for further study on the mechanism of electron transfer reaction in NADPH-dependent thioredoxin reductase proteins. PMID:23029461

  11. Synergistic effect of NADH on NADPH-dependent acetaminophen activation in liver microsomes and its inhibition by cyanide

    SciTech Connect

    Sato, Chifumi; Marumo, Fumiaki (Tokyo Medical and Dental Univ. (Japan))

    1991-01-01

    The effects of NADH and cyanide on NADPH-dependent acetaminophen activation in rat and mouse liver microsomes were studied. In both rat and mouse microsomes, NADPH-dependent acetaminophen-glutathione conjugate production was synergistically enhanced by the addition of NADH, whereas NADH alone did not initiate this reaction. The data suggest that the second electron in this reaction may be transferred from NADH. The present findings are different from a previous report in a reconstituted system that NADH decreases covalent binding of acetaminophen to proteins. This reaction was inhibited by low concentrations of sodium cyanide. The role of the cyanide sensitive factor in this reaction in liver microsomes remains to be further clarified.

  12. The housekeeping gene xanthine oxidoreductase is necessary for milk fat droplet enveloping and secretion: gene sharing in the lactating mammary gland

    PubMed Central

    Vorbach, Claudia; Scriven, Alistair; Capecchi, Mario R.

    2002-01-01

    Xanthine oxidoreductase (XOR) is the rate-limiting enzyme in purine catabolism occurring in most cell types. However, this housekeeping gene is expressed at very high levels in a number of mammalian tissues including the lactating mammary epithelium, suggesting additional roles for XOR in these tissues. Mice with targeted disruption of XOR were generated to assess these potential additional roles. XOR?/? mice are runted and do not live beyond 6 wk of age. Strikingly, however, XOR+/? females, although of healthy appearance and normal fertility, are unable to maintain lactation and their pups die of starvation 2 wk postpartum. Histological and whole-mount analyses showed that in XOR+/? females the mammary epithelium collapses, resulting in premature involution of the mammary gland. Electron microscopy showed that XOR is specifically required for enveloping milk fat droplets with the apical plasma membrane prior to secretion from the lactating mammary gland. We present evidence that XOR may have primarily a structural role, as a membrane-associated protein, in milk fat droplet secretion and thus XOR provides another example of “gene sharing”. About 5% of women experience primary lactation insufficiency. The above observations suggest that human females suffering from xanthinuria, a deficiency in XOR, are potential candidates for lactation problems. PMID:12502743

  13. 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

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

    SciTech Connect

    Kirkensgaard, Kristine G. [Carlsberg Laboratory (Denmark); Enzyme and Protein Chemistry, Department of Systems BioIogy, Technical University of Denmark (Denmark); Hägglund, Per; Finnie, Christine; Svensson, Birte [Enzyme and Protein Chemistry, Department of Systems BioIogy, Technical University of Denmark (Denmark); Henriksen, Anette, E-mail: anette@crc.dk [Carlsberg Laboratory (Denmark)

    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.

  15. A comparison of primer sets for detecting 16S rRNA and hydrazine oxidoreductase genes of anaerobic ammonium-oxidizing bacteria in marine sediments.

    PubMed

    Li, Meng; Hong, Yiguo; Klotz, Martin Gunter; Gu, Ji-Dong

    2010-03-01

    Published polymerase chain reaction primer sets for detecting the genes encoding 16S rRNA gene and hydrazine oxidoreductase (hzo) in anammox bacteria were compared by using the same coastal marine sediment samples. While four previously reported primer sets developed to detect the 16S rRNA gene showed varying specificities between 12% and 77%, an optimized primer combination resulted in up to 98% specificity, and the recovered anammox 16S rRNA gene sequences were >95% sequence identical to published sequences from anammox bacteria in the Candidatus "Scalindua" group. Furthermore, four primer sets used in detecting the hzo gene of anammox bacteria were highly specific (up to 92%) and efficient, and the newly designed primer set in this study amplified longer hzo gene segments suitable for phylogenetic analysis. The optimized primer set for the 16S rRNA gene and the newly designed primer set for the hzo gene were successfully applied to identify anammox bacteria from marine sediments of aquaculture zone, coastal wetland, and deep ocean where the three ecosystems form a gradient of anthropogenic impact. Results indicated a broad distribution of anammox bacteria with high niche-specific community structure within each marine ecosystem. PMID:20107988

  16. Vibrio harveyi NADPH-flavin oxidoreductase: cloning, sequencing and overexpression of the gene and purification and characterization of the cloned enzyme.

    PubMed Central

    Lei, B; Liu, M; Huang, S; Tu, S C

    1994-01-01

    NAD(P)H-flavin oxidoreductases (flavin reductases) from luminous bacteria catalyze the reduction of flavin by NAD(P)H and are believed to provide the reduced form of flavin mononucleotide (FMN) for luciferase in the bioluminescence reaction. By using an oligonucleotide probe based on the partial N-terminal amino acid sequence of the Vibrio harveyi NADPH-FMN oxidoreductase (flavin reductase P), a recombinant plasmid, pFRP1, was obtained which contained the frp gene encoding this enzyme. The DNA sequence of the frp gene was determined; the deduced amino acid sequence for flavin reductase P consists of 240 amino acid residues with a molecular weight of 26,312. The frp gene was overexpressed, apparently through induction, in Escherichia coli JM109 cells harboring pFRP1. The cloned flavin reductase P was purified to homogeneity by following a new and simple procedure involving FMN-agarose chromatography as a key step. The same chromatography material was also highly effective in concentrating diluted flavin reductase P. The purified enzyme is a monomer and is unusual in having a tightly bound FMN cofactor. Distinct from the free FMN, the bound FMN cofactor showed a diminished A375 peak and a slightly increased 8-nm red-shifted A453 peak and was completely or nearly nonfluorescent. The Kms for FMN and NADPH and the turnover number of this flavin reductase were determined. In comparison with other flavin reductases and homologous proteins, this flavin reductase P shows a number of distinct features with respect to primary sequence, redox center, and/or kinetic mechanism. Images PMID:8206832

  17. Characterization of the Rhodococcus sp. NI86/21 gene encoding alcohol: N,N'-dimethyl-4-nitrosoaniline oxidoreductase inducible by atrazine and thiocarbamate herbicides.

    PubMed

    Nagy, I; Verheijen, S; De Schrijver, A; Van Damme, J; Proost, P; Schoofs, G; Vanderleyden, J; De Mot, R

    1995-06-01

    A protein with a mol.mass of 51,000 (ThcE) that was induced in Rhodococcus sp. NI86/21 during assimilation of thiocarbamate herbicides, atrazine, ethanol, propanol, glycerol, propionaldehyde or ethanolamine was identified by two-dimensional electrophoresis. The thcE gene was cloned and sequenced. The deduced amino acid sequence revealed ThcE as a member of group III alcohol dehydrogenases. ThcE displayed strong homology with sequenced subunit fragments of the homodecameric N,N'-dimethyl-4-nitrosoaniline-dependent alcohol oxidoreductases (MNO) of Amycolatopsis methanolica and Mycobacterium gastri. N-Terminal sequence analysis of purified MNO from Rhodococcus sp. NI86/21 confirmed the identity with ThcE. When overproduced in Escherichia coli, ThcE was insoluble and no MNO activity was detected. PMID:7575099

  18. Five-gene cluster in Clostridium thermoaceticum consisting of two divergent operons encoding rubredoxin oxidoreductase- rubredoxin and rubrerythrin-type A flavoprotein- high-molecular-weight rubredoxin.

    PubMed

    Das, A; Coulter, E D; Kurtz, D M; Ljungdahl, L G

    2001-03-01

    A five-gene cluster encoding four nonheme iron proteins and a flavoprotein from the thermophilic anaerobic bacterium Clostridium thermoaceticum (Moorella thermoacetica) was cloned and sequenced. Based on analysis of deduced amino acid sequences, the genes were identified as rub (rubredoxin), rbo (rubredoxin oxidoreductase), rbr (rubrerythrin), fprA (type A flavoprotein), and a gene referred to as hrb (high-molecular-weight rubredoxin). Northern blot analysis demonstrated that the five-gene cluster is organized as two subclusters, consisting of two divergently transcribed operons, rbr-fprA-hrb and rbo-rub. The rbr, fprA, and rub genes were expressed in Escherichia coli, and their encoded recombinant proteins were purified. The molecular masses, UV-visible absorption spectra, and cofactor contents of the recombinant rubrerythrin, rubredoxin, and type A flavoprotein were similar to those of respective homologs from other microorganisms. Antibodies raised against Desulfovibrio vulgaris Rbr reacted with both native and recombinant Rbr from C. thermoaceticum, indicating that this protein was expressed in the native organism. Since Rbr and Rbo have been recently implicated in oxidative stress protection in several anaerobic bacteria and archaea, we suggest a similar function of these proteins in oxygen tolerance of C. thermoaceticum. PMID:11160086

  19. Molecular genetics evidence for the in vivo roles of the two major NADPH-dependent disulfide reductases in the malaria parasite.

    PubMed

    Buchholz, Kathrin; Putrianti, Elyzana D; Rahlfs, Stefan; Schirmer, R Heiner; Becker, Katja; Matuschewski, Kai

    2010-11-26

    Malaria-associated pathology is caused by the continuous expansion of Plasmodium parasites inside host erythrocytes. To maintain a reducing intracellular milieu in an oxygen-rich environment, malaria parasites have evolved a complex antioxidative network based on two central electron donors, glutathione and thioredoxin. Here, we dissected the in vivo roles of both redox pathways by gene targeting of the respective NADPH-dependent disulfide reductases. We show that Plasmodium berghei glutathione reductase and thioredoxin reductase are dispensable for proliferation of the pathogenic blood stages. Intriguingly, glutathione reductase is vital for extracellular parasite development inside the insect vector, whereas thioredoxin reductase is dispensable during the entire parasite life cycle. Our findings suggest that glutathione reductase is the central player of the parasite redox network, whereas thioredoxin reductase fulfils a specialized and dispensable role for P. berghei. These results also indicate redundant roles of the Plasmodium redox pathways during the pathogenic blood phase and query their suitability as promising drug targets for antimalarial intervention strategies. PMID:20852334

  20. Deletion of chloroplast NADPH-dependent thioredoxin reductase results in inability to regulate starch synthesis and causes stunted growth under short-day photoperiods

    PubMed Central

    Lepistö, Anna; Pakula, Eveliina; Toivola, Jouni; Krieger-Liszkay, Anja; Vignols, Florence; Rintamäki, Eevi

    2013-01-01

    Plastid-localized NADPH-dependent thioredoxin reductase C (NTRC) is a unique NTR enzyme containing both reductase and thioredoxin domains in a single polypeptide. Arabidopsis thaliana NTRC knockout lines (ntrc) show retarded growth, especially under short-day (SD) photoperiods. This study identified chloroplast processes that accounted for growth reduction in SD-acclimated ntrc. The strongest reduction in ntrc growth occurred under photoperiods with nights longer than 14h, whereas knockout of the NTRC gene did not alter the circadian-clock-controlled growth of Arabidopsis. Lack of NTRC modulated chloroplast reactive oxygen species (ROS) metabolism, but oxidative stress was not the primary cause of retarded growth of SD-acclimated ntrc. Scarcity of starch accumulation made ntrc leaves particularly vulnerable to photoperiods with long nights. Direct interaction of NTRC and ADP-glucose pyrophosphorylase, a key enzyme in starch synthesis, was confirmed by yeast two-hybrid analysis. The ntrc line was not able to maximize starch synthesis during the light period, which was particularly detrimental under SD conditions. Acclimation of Arabidopsis to SD conditions also involved an inductive rise of ROS production in illuminated chloroplasts that was not counterbalanced by the activation of plastidial anti-oxidative systems. It is proposed that knockout of NTRC challenges redox regulation of starch synthesis, resulting in stunted growth of the mutant lines acclimated to the SD photoperiod. PMID:23881397

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

    PubMed

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

    2014-11-01

    The apple tree (Malus sp.) is an agriculturally and economically important source of food and beverages. Many of the health beneficial properties of apples are due to (poly)phenolic metabolites that they contain, including various dihydrochalcones. Although many of the genes and enzymes involved in polyphenol biosynthesis are known in many plant species, the specific reactions that lead to the biosynthesis of the dihydrochalcone precursor, p-dihydrocoumaroyl-CoA (3), are unknown. To identify genes involved in the synthesis of these metabolites, existing genome databases of the Rosaceae were screened for apple genes with significant sequence similarity to Arabidopsis alkenal double bond reductases. Herein described are the isolation and characterization of a Malus hydroxycinnamoyl-CoA double bond reductase, which catalyzed the NADPH-dependent reduction of p-coumaroyl-CoA and feruloyl-CoA to p-dihydrocoumaroyl-CoA and dihydroferuloyl-CoA, respectively. Its apparent Km values for p-coumaroyl-CoA, feruloyl-CoA and NADPH were 96.6, 92.9 and 101.3?M, respectively. The Malus double bond reductase preferred feruloyl-CoA to p-coumaroyl-CoA as a substrate by a factor of 2.1 when comparing catalytic efficiencies in vitro. Expression analysis of the hydroxycinnamoyl-CoA double bond reductase gene revealed that its transcript levels showed significant variation in tissues of different developmental stages, but was expressed when expected for involvement in dihydrochalcone formation. Thus, the hydroxycinnamoyl-CoA double bond reductase appears to be responsible for the reduction of the ?,?-unsaturated double bond of p-coumaroyl-CoA, the first step of dihydrochalcone biosynthesis in apple tissues, and may be involved in the production of these compounds. PMID:25152451

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

    PubMed

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

    2009-06-01

    (R)-3-Quinuclidinol is a useful compound that is applicable to the synthesis of various pharmaceuticals. The NADPH-dependent carbonyl reductase 3-quinuclidinone reductase from Rhodotorula rubra catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol and is expected to be utilized in industrial production of this alcohol. 3-Quinuclidinone reductase from R. rubra was expressed in Escherichia coli and purified using Ni-affinity and ion-exchange column chromatography. Crystals of the protein were obtained by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant. The crystals belonged to space group P4(1)2(1)2, with unit-cell parameters a = b = 91.3, c = 265.4 A, and diffracted X-rays to 2.2 A resolution. The asymmetric unit contained four molecules of the protein and the solvent content was 48.4%. PMID:19478454

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

    PubMed Central

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

    2009-01-01

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

  4. Beta Hydroxylation of Glycolipids from Ustilago maydis and Pseudozyma flocculosa by an NADPH-Dependent ?-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 ? position but differ in the long fatty acid, which is hydroxylated at the ? position in UA and at the ? 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 ?-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 ?uhd1 mutants. Fhd1 was able to restore wild-type UA production, indicating that Fhd1 is responsible for ? 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 ?ahd1 mutant strains. P. flocculosa Ahd1 neither complemented the U. maydis ?ahd1 phenotype nor resulted in the production of ?-hydroxylated UA. This suggests that P. flocculosa Ahd1 is not involved in flocculosin hydroxylation. PMID:21926207

  5. Cell-specific expression of tryptophan decarboxylase and 10-hydroxygeraniol oxidoreductase, key genes involved in camptothecin biosynthesis in Camptotheca acuminata Decne (Nyssaceae)

    PubMed Central

    2010-01-01

    Background Camptotheca acuminata is a major natural source of the terpenoid indole alkaloid camptothecin (CPT). At present, little is known about the cellular distribution of the biosynthesis of CPT, which would be useful knowledge for developing new strategies and technologies for improving alkaloid production. Results The pattern of CPT accumulation was compared with the expression pattern of some genes involved in CPT biosynthesis in C. acuminata [i.e., Ca-TDC1 and Ca-TDC2 (encoding for tryptophan decarboxylase) and Ca-HGO (encoding for 10-hydroxygeraniol oxidoreductase)]. Both CPT accumulation and gene expression were investigated in plants at different degrees of development and in plantlets subjected to drought-stress. In all organs, CPT accumulation was detected in epidermal idioblasts, in some glandular trichomes, and in groups of idioblast cells localized in parenchyma tissues. Drought-stress caused an increase in CPT accumulation and in the number of glandular trichomes containing CPT, whereas no increase in epidermal or parenchymatous idioblasts was observed. In the leaf, Ca-TDC1 expression was detected in some epidermal cells and in groups of mesophyll cells but not in glandular trichomes; in the stem, it was observed in parenchyma cells of the vascular tissue; in the root, no expression was detected. Ca-TDC2 expression was observed exclusively in leaves of plantlets subjected to drought-stress, in the same sites described for Ca-TDC1. In the leaf, Ca-HGO was detected in all chlorenchyma cells; in the stem, it was observed in the same sites described for Ca-TDC1; in the root, no expression was detected. Conclusions The finding that the sites of CPT accumulation are not consistently the same as those in which the studied genes are expressed demonstrates an organ-to-organ and cell-to-cell translocation of CPT or its precursors. PMID:20403175

  6. Identification of the gene encoding the major NAD(P)H-flavin oxidoreductase of the bioluminescent bacterium Vibrio fischeri ATCC 7744.

    PubMed Central

    Zenno, S; Saigo, K; Kanoh, H; Inouye, S

    1994-01-01

    The gene encoding the major NAD(P)H-flavin oxidoreductase (flavin reductase) of the luminous bacterium Vibrio fischeri ATCC 7744 was isolated by using synthetic oligonucleotide probes corresponding to the N-terminal amino acid sequence of the enzyme. Nucleotide sequence analysis suggested that the major flavin reductase of V. fischeri consisted of 218 amino acids and had a calculated molecular weight of 24,562. Cloned flavin reductase expressed in Escherichia coli was purified virtually to homogeneity, and its basic biochemical properties were examined. As in the major flavin reductase in crude extracts of V. fischeri, cloned flavin reductase showed broad substrate specificity and served well as a catalyst to supply reduced flavin mononucleotide (FMNH2) to the bioluminescence reaction. The major flavin reductase of V. fischeri not only showed significant similarity in amino acid sequence to oxygen-insensitive NAD(P)H nitroreductases of Salmonella typhimurium, Enterobacter cloacae, and E. coli but also was associated with a low level of nitroreductase activity. The major flavin reductase of V. fischeri and the nitroreductases of members of the family Enterobacteriaceae would thus appear closely related in evolution and form a novel protein family. Images PMID:8206830

  7. THE AFLATOXIN BIOSYNTHESIS CLUSTER GENE, AFLX, ENCODES AN OXIDOREDUCTASE INVOLVED IN CONVERSION OF VERSICOLORIN A TO DEMETHYLSTERIGMATOCYSTIN

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biosynthesis of the toxic and carcinogenic aflatoxins is a complicated process involving more that 27 enzymes and regulatory factors encoded by genes in a gene cluster. Previous studies found that three enzymes are required for conversion of versicolorin A (VA), to demethylsterigmatocystin (DMST), ...

  8. Induction, isolation, and some properties of the NADPH-dependent glutamate dehydrogenase from the nonheterocystous cyanobacterium Phormidium laminosum.

    PubMed Central

    Martinez-Bilbao, M; Martinez, A; Urkijo, I; Llama, M J; Serra, J L

    1988-01-01

    The level of the NADPH-dependent glutamate dehydrogenase activity (EC 1.4.1.4) from nitrate-grown cells of the thermophilic non-N2-fixing cyanobacterium Phormidium laminosum OH-1-p.Cl1 could be significantly enhanced by the presence of ammonium or nitrite, as well as by L-methionine-DL-sulfoximine and other sources of organic nitrogen (L-Glu, L-Gln, and methylamine). The enzyme was purified more than 4,400-fold by ultracentrifugation, ion-exchange chromatography, and affinity chromatography, and at 30 degrees C it showed a specific activity of 32.9 mumol of NADPH oxidized per min per mg of protein. The purified enzyme showed no aminotransferase activity and catalyzed the amination of 2-oxoglutarate preferentially to the reverse catabolic reaction. The enzyme was very specific for its substrates 2-oxoglutarate (Km = 1.25 mM) and NADPH (Km = 64 microM), for which hyperbolic kinetics were obtained. However, negative cooperativity (Hill coefficient h = 0.89) and [S]0.5 of 18.2 mM were observed for ammonium. The mechanism of the aminating reaction was of a random type with independent sites. The purified enzyme showed its maximal activity at 60 degrees C (Ea = 5.1 kcal/mol [21.3 kJ/mol]) and optimal pH values of 8.0 and 7.5 when assayed in Tris hydrochloride and potassium phosphate buffers, respectively. The native molecular mass of the enzyme was about 280 kilodaltons. The possible physiological role of the enzyme in ammonia assimilation is discussed. PMID:3139639

  9. Identification of a five-oxidoreductase-gene cluster from Acetobacter pasteurianus conferring ethanol-dependent acidification in Escherichia coli

    PubMed Central

    Garcia-Armisen, Tamara; Vercammen, Ken; Rimaux, Tom; Vrancken, Gino; Vuyst, Luc De; Cornelis, Pierre

    2012-01-01

    Acetobacter pasteurianus, a Gram-negative bacterium belonging to the ?-divison of Proteobacteria, produces acetic acid through ethanol oxidation. A genomic bank of A. pasteurianus 386B DNA was cloned in the low-copy cosmid pRG930Cm vector and the resulting clones were screened for the production of protease using the skimmed-milk agar assay whereby a clearing zone around the inoculated spots indicates casein degradation. Several positive clones were selected and restriction analysis revealed that many contained the same inserts. One clone was further analyzed and the cosmid DNA subjected to in vitro transposon insertion. After electroporation, several clones having lost the capacity to cause casein degradation were isolated and the sequence of the transposon-flanking regions analyzed. The majority of insertions mapped to one gene encoding an NAD(P)+-dependent aldehyde dehydrogenase (ALDH) of the PNTB superfamily, whereas one insert was found upstream in a gene encoding an ethanol dehydrogenase. Addition of phenol red to the medium confirmed the ethanol-dependent acidification around the inoculated spots of the clones without transposon insertion, suggesting that casein degradation is due to the production of acetic acid as a result of the combined activities of the alcohol dehydrogenase and ALDH. Quantitative data and pH measurements confirmed a significant acidification, and the presence of acetic acid. PMID:22950009

  10. Identification of two Arabidopsis genes encoding a peroxisomal oxidoreductase-like protein and an acyl-CoA synthetase-like protein that are required for responses to pro-auxins

    Microsoft Academic Search

    Andrew A. G. Wiszniewski; Wenxu Zhou; Steven M. Smith; John D. Bussell

    2009-01-01

    Indole-3-butyric acid (IBA) and 2,4-dichlorophenoxybutyric acid (2,4-DB) are metabolised by peroxisomal ?-oxidation to active\\u000a auxins that inhibit root growth. We screened Arabidopsis mutants for resistance to IBA and 2,4-DB and identified two new 2,4-DB\\u000a resistant mutants. The mutant genes encode a putative oxidoreductase (SDRa) and a putative acyl-activating enzyme (AAE18).\\u000a Both proteins are localised to peroxisomes. SDRa is coexpressed with

  11. Construction and Screening of Metagenomic Libraries Derived from Enrichment Cultures: Generation of a Gene Bank for Genes Conferring Alcohol Oxidoreductase Activity on Escherichia coli

    Microsoft Academic Search

    Anja Knietsch; Tanja Waschkowitz; Susanne Bowien; Anke Henne; Rolf Daniel

    2003-01-01

    Enrichment of microorganisms with special traits and the construction of metagenomic libraries by direct cloning of environmental DNA have great potential for identifying genes and gene products for biotechnological purposes. We have combined these techniques to isolate novel genes conferring oxidation of short-chain (C2 to C4) polyols or reduction of the corresponding carbonyls. In order to favor the growth of

  12. Molecular cloning of the isoquinoline 1-oxidoreductase genes from Pseudomonas diminuta 7, structural analysis of iorA and iorB, and sequence comparisons with other molybdenum-containing hydroxylases.

    PubMed

    Lehmann, M; Tshisuaka, B; Fetzner, S; Lingens, F

    1995-06-16

    The iorA and iorB genes from the isoquinoline-degrading bacterium Pseudomonas diminuta 7, encoding the heterodimeric molybdo-iron-sulfur-protein isoquinoline 1-oxidoreductase, were cloned and sequenced. The deduced amino acid sequences IorA and IorB showed homologies (i) to the small (gamma) and large (alpha) subunits of complex molybdenum-containing hydroxylases (alpha beta gamma/alpha 2 beta 2 gamma 2) possessing a pterin molybdenum cofactor with a monooxo-monosulfido-type molybdenum center, (ii) to the N- and C-terminal regions of aldehyde oxidoreductase from Desulfovibrio gigas, and (iii) to the N- and C-terminal domains of eucaryotic xanthine dehydrogenases, respectively. The closest similarity to IorB was shown by aldehyde dehydrogenase (Adh) from the acetic acid bacterium Acetobacter polyoxogenes. Five conserved domains of IorB were identified by multiple sequence alignments. Whereas IorB and Adh showed an identical sequential arrangement of these conserved domains, in all other molybdenum-containing hydroxylases the relative position of "domain A" differed. IorA contained eight conserved cysteine residues. The amino acid pattern harboring the four cysteine residues proposed to ligate the Fe/S I cluster was homologous to the consensus binding site of bacterial and chloroplast-type [2Fe-2S] ferredoxins, whereas the pattern including the four cysteines assumed to ligate the Fe/S II center showed no similarities to any described [2Fe-2S] binding motif. The N-terminal region of IorB comprised a putative signal peptide similar to typical leader peptides, indicating that isoquinoline 1-oxidoreductase is associated with the cell membrane. PMID:7782304

  13. NADPH-dependent flavoenzymes catalyze one electron reduction of metal ions and molecular oxygen and generate hydroxyl radicals.

    PubMed

    Shi, X L; Dalal, N S

    1990-12-10

    This study reports a new property of the important NAD(P)H-dependent flavoenzymes, glutathione reductase, lipoyl dehydrogenase and ferredoxin-NADP+ oxidoreductase, that can catalyze a one electron reduction of metal ions such as chromium(VI) and vanadium(V). During the enzymatic reduction process, molecular oxygen is reduced to H2O2, which reacts with the reduced metal complexes to generate hydroxyl radicals. Since the hydroxyl radicals have been suggested to play an important role in Cr(VI) toxicity, this study provides a basis for a recent observation that Cr(VI) mutagenesis is strongly oxygen dependent. These results also point to an enzymatic pathway for the metabolism of some metal ions and concomitant generation of hydroxyl radicals. PMID:2176163

  14. Lipids and NADPH-dependent superoxide production in plasma membrane vesicles from roots of wheat grown under copper deficiency or excess.

    PubMed

    Quartacci, M F; Cosi, E; Navari-Izzo, F

    2001-01-01

    The effects of in vivo copper on the lipid composition of root plasma membrane and the activities of membrane-bound enzymes, such as NADPH-dependent oxidases and lipoxygenase, were studied. Plants were grown in hydroponic culture for 11 d without Cu supply or in the presence of 50 microM Cu. Control plants were supplied with 0.3 microM Cu. Growth of roots was severely affected in the 50 microM Cu-grown plants, whereas roots grown in Cu-deficient solution did not show any difference in comparison with the control. The 50 microM Cu concentration caused an increase in the leakage of K(+) ions as well. Excess metal supply resulted in a decrease in the total lipid content of plasma membrane, a higher phospholipid amount and a reduction of steryl lipids (free sterols, steryl glycosides and acylated steryl glycosides). Cu depletion in the growth solution had only a slight effect on the plasma membrane lipid composition. In comparison with the control, only the excess of Cu caused a decrease in the lipid to protein ratio as well as a change in the phospholipid composition, with a lower phosphatidylcholine to phosphatidylethanolamine ratio. The degree of unsaturation of root plasma membranes decreased following the 0 Cu treatment and even more after the 50 microM Cu supply. Plasma membranes of wheat grown under metal deficiency and excess showed increased NADPH-dependent superoxide-producing oxidase activities, whereas membrane-bound lipoxygenase was not increased or activated due to Cu treatments. The consequences of changes in plasma membrane lipid composition and activated oxygen production as a result of Cu treatments are discussed. PMID:11181715

  15. 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 Central

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

    2013-01-01

    NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with ?-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. PMID:23913421

  16. Evolution of FOXRED1, an FAD-dependent oxidoreductase necessary for NADH:ubiquinone oxidoreductase (Complex I) assembly.

    PubMed

    Lemire, Bernard D

    2015-01-01

    Complex I (NADH:ubiquinone oxidoreductase) is the major entry point for electrons into the respiratory chains of bacteria and mitochondria. Mammalian complex I is composed of 45 subunits and harbors FMN and iron-sulfur cluster cofactors. A heterogeneous disease profile is associated with complex I deficiency. In a large fraction of complex I deficiencies, the primary defect is not in any of the genes encoding a subunit. The proper assembly and function of complex I require the participation of at least 12 assembly factors or chaperones. FOXRED1 encodes a complex I-specific assembly factor and mutations in this gene result in complex I deficiency, infantile onset encephalomyopathy and Leigh syndrome. The human FOXRED1 protein is a mitochondria-targeted 486-amino acid FAD-dependent oxidoreductase. It is most closely related to N-methyl amino acid dehydrogenases. FOXRED1 orthologs are present in archaea, bacteria and eukaryotes. Fungal FOXRED1 orthologs were likely acquired from alphaproteobacteria by horizontal gene transfer. The phylogenetic profile of FOXRED1 orthologs does not parallel the phylogenetic profile of complex I, strongly suggesting that, at least in some organisms, FOXRED1 has a function unrelated to complex I. The only large clade where all members investigated contain both FOXRED1 and complex I is the metazoans. Some bacterial FOXRED1 genes are present in metabolic operons related to amino acid metabolism. FOXRED1 phylogenetic distribution and gene organization suggest a metabolic role for FOXRED1 in complex I biogenesis should be considered. PMID:25681241

  17. 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.

  18. Systems-Based Design of Bi-Ligand Inhibitors of Oxidoreductases

    Microsoft Academic Search

    Daniel S Sem; Bonnie Bertolaet; Brian Baker; Edcon Chang; Aurora D Costache; Stephen Coutts; Qing Dong; Mark Hansen; Victor Hong; Xuemei Huang; Richard M Jack; Richard Kho; Henk Lang; Chen-Ting Ma; David Meininger; Maurizio Pellecchia; Fabrice Pierre; Hugo Villar; Lin Yu

    2004-01-01

    Genomics-driven growth in the number of enzymes of unknown function has created a need for better strategies to characterize them. Since enzyme inhibitors have traditionally served this purpose, we present here an efficient systems-based inhibitor design strategy, enabled by bioinformatic and NMR structural developments. First, we parse the oxidoreductase gene family into structural subfamilies termed pharmacofamilies, which share pharmacophore features

  19. Close genetic relationship between Nitrobacter hamburgensis nitrite oxidoreductase and Escherichia coli nitrate reductases

    Microsoft Academic Search

    Karin Kirstein; Eberhard Bock

    1993-01-01

    The nitrite oxidoreductase (NOR) from the facultative nitrite-oxidizing bacterium Nitrobacter hamburgensis X14 was investigated genetically. In order to develop a probe for the gene norB, the N-terminal amino acid sequence of the NOR ß-subunit (NorB) was determined. Based on that amino acid sequence, an oligo-nucleotide was derived that was used for the identification and cloning of gene norB. Sequence analysis

  20. Oxidoreductases that Act as Conditional Virulence Suppressors in Salmonella enterica Serovar Typhimurium

    PubMed Central

    Anwar, Naeem; Sem, Xiao Hui; Rhen, Mikael

    2013-01-01

    In Salmonella enterica serovar Typhimurium, oxidoreductases of the thioredoxin superfamily contribute to bacterial invasiveness, intracellular replication and to the virulence in BALB/c mice as well as in the soil nematode Caenorhabditis elegans. The scsABCD gene cluster, present in many but not all enteric bacteria, codes for four putative oxidoreductases of the thioredoxin superfamily. Here we have analyzed the potential role of the scs genes in oxidative stress tolerance and virulence in S. Typhimurium. An scsABCD deletion mutant showed moderate sensitization to the redox-active transition metal ion copper and increased protein carbonylation upon exposure to hydrogen peroxide. Still, the scsABCD mutant was not significantly affected for invasiveness or intracellular replication in respectively cultured epithelial or macrophage-like cells. However, we noted a significant copper chloride sensitivity of SPI1 T3SS mediated invasiveness that strongly depended on the presence of the scs genes. The scsABCD deletion mutant was not attenuated in animal infection models. In contrast, the mutant showed a moderate increase in its competitive index upon intraperitoneal challenge and enhanced invasiveness in small intestinal ileal loops of BALB/c mice. Moreover, deletion of the scsABCD genes restored the invasiveness of a trxA mutant in epithelial cells and its virulence in C. elegans. Our findings thus demonstrate that the scs gene cluster conditionally affects virulence and underscore the complex interactions between oxidoreductases of the thioredoxin superfamily in maintaining host adaptation of S. Typhimurium. PMID:23750221

  1. [Characterization of some thiol oxidoreductase family members].

    PubMed

    Varlamova, E G; Gol'tiaev, M V; Novoselov, S V; Novoselov, V I; Fecenko, E E

    2013-01-01

    There are no doubt about the important role of free radicals and reactive oxygen species in the processes of cell activity. The disturbances of intracellular redox processes are often accompanied with the development of such common pathologies as diabetes, myocardial infarction, neurodegeneration, broncho-pulmonary diseases, cancer, etc. To date, there are a large number of antioxidant enzymes related to different redox biology systems, the key role among them is played by enzymes belong to the thiol oxidoreductases superfamily, which consists of thioredoxin, glutaredoxin, peroxiredoxin, protein disulfidizomeraz, glutathione peroxidase families, and a number of other proteins. In addition to the antioxidant function, thiol oxidoreductases display the ability to recycle of hydroperoxide to form specific disulfide bonds within and between proteins that significantly extends the range of their functionality. Therefore, biochemical characterization and elucidation of functional mechanisms of the superfamily proteins is a highly actual problem of redox biology. PMID:24466746

  2. Regulation of P450 oxidoreductase by gonadotropins in rat ovary and its effect on estrogen production

    Microsoft Academic Search

    Yoshihiko Inaoka; Takashi Yazawa; Tetsuya Mizutani; Koichi Kokame; Kenji Kangawa; Miki Uesaka; Akihiro Umezawa; Kaoru Miyamoto

    2008-01-01

    BACKGROUND: P450 oxidoreductase (POR) catalyzes electron transfer to microsomal P450 enzymes. Its deficiency causes Antley-Bixler syndrome (ABS), and about half the patients with ABS have ambiguous genitalia and\\/or impaired steroidogenesis. POR mRNA expression is up-regulated when mesenchymal stem cells (MSCs) differentiate into steroidogenic cells, suggesting that the regulation of POR gene expression is important for steroidogenesis. In this context we

  3. Expression of WW domain–containing oxidoreductase WWOX in pterygium

    PubMed Central

    Huang, Yi-Hsun; Chang, Nan-Shan

    2015-01-01

    Purpose Pterygium was traditionally regarded as a degenerative disease, but certain characteristics suggest that pterygium is probably premalignant tissue. The human WWOX gene, encoding the WW domain containing oxidoreductase (WWOX, FOR, or WOX1), is a candidate tumor suppressor gene. In this study, we investigated the WWOX gene and protein expression in pterygium. Methods Pterygium tissues were obtained from patients (n=16, primary=8, recurrent=8) who received surgical excisions. Each tissue sample was further divided into head and body regions. The WWOX gene and protein expression were examined with immunohistochemistry, western blot, and quantitative PCR. For comparison, normal superior temporal bulbar conjunctivas were used as controls. Results Compared to the controls, upregulation of WWOX and its Tyr33 phosphorylation was observed in the head region of all pterygium specimens. In the head and body of the pterygium specimens, WWOX expression was significantly higher than in the controls. In addition, WWOX expression was stronger in recurrent pterygia than in primary pterygia. Conclusions Increased WWOX expression, especially in the head region, is probably due to the invasiveness of the pterygium. Our results indicate that WWOX may play a role in pterygium progression and recurrence. PMID:26120275

  4. Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase.

    PubMed Central

    Leadbeater, C; McIver, L; Campopiano, D J; Webster, S P; Baxter, R L; Kelly, S M; Price, N C; Lysek, D A; Noble, M A; Chapman, S K; Munro, A W

    2000-01-01

    The structure of the Escherichia coli flavodoxin NADP(+) oxidoreductase (FLDR) places three arginines (R144, R174 and R184) in the proposed NADPH-binding site. Mutant enzymes produced by site-directed mutagenesis, in which each arginine was replaced by neutral alanine, were characterized. All mutants exhibited decreased NADPH-dependent cytochrome c reductase activity (R144A, 241.6 min(-1); R174A, 132.1 min(-1); R184A, 305.5 min(-1) versus wild type, 338.9 min(-1)) and increased K(m) for NADPH (R144A, 5.3 microM; R174A, 20.2 microM; R184A, 54.4 microM versus wild type, 3.9 microM). The k(cat) value for NADH-dependent cytochrome c reduction was increased for R174A (42.3 min(-1)) and R184A (50.4 min(-1)) compared with the wild type (33.0 min(-1)), consistent with roles for R174 and R184 in discriminating between NADPH/NADH by interaction with the adenosine ribose 2'-phosphate. Stopped-flow studies indicated that affinity (K(d)) for NADPH was markedly reduced in mutants R144A (635 microM) and R184A (2.3 mM) compared with the wild type (<5 microM). Mutant R184A displays the greatest change in pyridine nucleotide preference, with the NADH/NADPH K(d) ratio >175-fold lower than for wild-type FLDR. The rate constant for hydride transfer from NADPH to flavin was lowest for R174A (k(red)=8.82 s(-1) versus 22.63 s(-1) for the wild type), which also exhibited tertiary structure perturbation, as evidenced by alterations in CD and fluorescence spectra. Molecular modelling indicated that movement of the C-terminal tryptophan (W248) of FLDR is necessary to permit close approach of the nicotinamide ring of NADPH to the flavin. The positions of NADPH phosphates in the modelled structure are consistent with the kinetic data, with R174 and R184 located close to the adenosine ribose 2'-phosphate group, and R144 likely to interact with the nicotinamide ribose 5'-phosphate group. PMID:11085917

  5. Differential expression of plastome-encoded ndh genes in mesophyll and bundle-sheath chloroplasts of the C 4 plant Sorghum bicolor indicates that the complex I-homologous NAD(P)H-plastoquinone oxidoreductase is involved in cyclic electron transport

    Microsoft Academic Search

    Andreas Kubicki; Edgar Funk; Peter Westhoff; Klaus Steinmiiller

    1996-01-01

    Cyanobacteria and plastids harbor a putative NAD(P)H- or ferredoxin-plastoquinone oxidoreductase that is homologous to the NADH-ubiquinone oxidoreductase (complex I) of mitochondria and eubacteria. The enzyme is a multimeric protein complex that consists of at least 11 subunits (NDH-A-K) and is localized in the stroma lamellae of the thylakoid membrane system. We investigated the expression of the different subunits of the

  6. Endoplasmic reticulum chaperones and oxidoreductases: critical regulators of tumor cell survival and immunorecognition.

    PubMed

    Gutiérrez, Tomás; Simmen, Thomas

    2014-01-01

    Endoplasmic reticulum (ER) chaperones and oxidoreductases are abundant enzymes that mediate the production of fully folded secretory and transmembrane proteins. Resisting the Golgi and plasma membrane-directed "bulk flow," ER chaperones and oxidoreductases enter retrograde trafficking whenever they are pulled outside of the ER by their substrates. Solid tumors are characterized by the increased production of reactive oxygen species (ROS), combined with reduced blood flow that leads to low oxygen supply and ER stress. Under these conditions, hypoxia and the unfolded protein response upregulate their target genes. When this occurs, ER oxidoreductases and chaperones become important regulators of tumor growth. However, under these conditions, these proteins not only promote the folding of proteins, but also alter the properties of the plasma membrane and hence modulate tumor immune recognition. For instance, high levels of calreticulin serve as an "eat-me" signal on the surface of tumor cells. Conversely, both intracellular and surface BiP/GRP78 promotes tumor growth. Other ER folding assistants able to modulate the properties of tumor tissue include protein disulfide isomerase (PDI), Ero1? and GRP94. Understanding the roles and mechanisms of ER chaperones in regulating tumor cell functions and immunorecognition will lead to important insight for the development of novel cancer therapies. PMID:25386408

  7. Lipid-membrane modified electrodes to study quinone oxidoreductases

    PubMed Central

    Weiss, Sophie A.; Jeuken, Lars J. C.

    2013-01-01

    Quinone oxidoreductases are a class of membrane enzymes that catalyse the oxidation or reduction of membrane-bound quinols/quinones. The conversion of quinone/quinol by these enzymes is difficult to study due to the hydrophobic nature of the enzymes and their substrates. We describe some biochemical properties of quinones and quinone oxidoreductases and then look in more detail at two model membranes that can be used to study quinone oxidoreductases in a native-like membrane environment with their native lipophylic quinone substrates. The results obtained with these model membranes are compared to classical enzyme assays that use water-soluble quinone analogues. PMID:19614580

  8. Characterization of a Thermostable Short-Chain Alcohol Dehydrogenase from the Hyperthermophilic Archaeon Thermococcus sibiricus?

    PubMed Central

    Stekhanova, Tatiana N.; Mardanov, Andrey V.; Bezsudnova, Ekaterina Y.; Gumerov, Vadim M.; Ravin, Nikolai V.; Skryabin, Konstantin G.; Popov, Vladimir O.

    2010-01-01

    Short-chain alcohol dehydrogenase, encoded by the gene Tsib_0319 from the hyperthermophilic archaeon Thermococcus sibiricus, was expressed in Escherichia coli, purified and characterized as an NADPH-dependent enantioselective oxidoreductase with broad substrate specificity. The enzyme exhibits extremely high thermophilicity, thermostability, and tolerance to organic solvents and salts. PMID:20418438

  9. Origin and Evolution of the Sodium -Pumping NADH: Ubiquinone Oxidoreductase

    PubMed Central

    Reyes-Prieto, Adrian; Barquera, Blanca; Juárez, Oscar

    2014-01-01

    The sodium -pumping NADH: ubiquinone oxidoreductase (Na+-NQR) is the main ion pump and the primary entry site for electrons into the respiratory chain of many different types of pathogenic bacteria. This enzymatic complex creates a transmembrane gradient of sodium that is used by the cell to sustain ionic homeostasis, nutrient transport, ATP synthesis, flagellum rotation and other essential processes. Comparative genomics data demonstrate that the nqr operon, which encodes all Na+-NQR subunits, is found in a large variety of bacterial lineages with different habitats and metabolic strategies. Here we studied the distribution, origin and evolution of this enzymatic complex. The molecular phylogenetic analyses and the organizations of the nqr operon indicate that Na+-NQR evolved within the Chlorobi/Bacteroidetes group, after the duplication and subsequent neofunctionalization of the operon that encodes the homolog RNF complex. Subsequently, the nqr operon dispersed through multiple horizontal transfer events to other bacterial lineages such as Chlamydiae, Planctomyces and ?, ?, ? and ? -proteobacteria. Considering the biochemical properties of the Na+-NQR complex and its physiological role in different bacteria, we propose a detailed scenario to explain the molecular mechanisms that gave rise to its novel redox- dependent sodium -pumping activity. Our model postulates that the evolution of the Na+-NQR complex involved a functional divergence from its RNF homolog, following the duplication of the rnf operon, the loss of the rnfB gene and the recruitment of the reductase subunit of an aromatic monooxygenase. PMID:24809444

  10. Overexpression of protochlorophyllide oxidoreductase C regulates oxidative stress in Arabidopsis.

    PubMed

    Pattanayak, Gopal K; Tripathy, Baishnab C

    2011-01-01

    Light absorbed by colored intermediates of chlorophyll biosynthesis is not utilized in photosynthesis; instead, it is transferred to molecular oxygen, generating singlet oxygen ((1)O(2)). As there is no enzymatic detoxification mechanism available in plants to destroy (1)O(2), its generation should be minimized. We manipulated the concentration of a major chlorophyll biosynthetic intermediate i.e., protochlorophyllide in Arabidopsis by overexpressing the light-inducible protochlorophyllide oxidoreductase C (PORC) that effectively phototransforms endogenous protochlorophyllide to chlorophyllide leading to minimal accumulation of the photosensitizer protochlorophyllide in light-grown plants. In PORC overexpressing (PORCx) plants exposed to high-light, the (1)O(2) generation and consequent malonedialdehyde production was minimal and the maximum quantum efficiency of photosystem II remained unaffected demonstrating that their photosynthetic apparatus and cellular organization were intact. Further, PORCx plants treated with 5-aminolevulinicacid when exposed to light, photo-converted over-accumulated protochlorophyllide to chlorophyllide, reduced the generation of (1)O(2) and malonedialdehyde production and reduced plasma membrane damage. So PORCx plants survived and bolted whereas, the 5-aminolevulinicacid-treated wild-type plants perished. Thus, overexpression of PORC could be biotechnologically exploited in crop plants for tolerance to (1)O(2)-induced oxidative stress, paving the use of 5-aminolevulinicacid as a selective commercial light-activated biodegradable herbicide. Reduced protochlorophyllide content in PORCx plants released the protochlorophyllide-mediated feed-back inhibition of 5-aminolevulinicacid biosynthesis that resulted in higher 5-aminolevulinicacid production. Increase of 5-aminolevulinicacid synthesis upregulated the gene and protein expression of several downstream chlorophyll biosynthetic enzymes elucidating a regulatory net work of expression of genes involved in 5-aminolevulinicacid and tetrapyrrole biosynthesis. PMID:22031838

  11. Glyceraldehyde-3-phosphate ferredoxin oxidoreductase from Methanococcus maripaludis.

    PubMed

    Park, Myong-Ok; Mizutani, Taeko; Jones, Patrik R

    2007-10-01

    The genome sequence of the non-sugar-assimilating mesophile Methanococcus maripaludis contains three genes encoding enzymes: a nonphosphorylating NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR); all these enzymes are potentially capable of catalyzing glyceraldehyde-3-phosphate (G3P) metabolism. GAPOR, whose homologs have been found mainly in archaea, catalyzes the reduction of ferredoxin coupled with oxidation of G3P. GAPOR has previously been isolated and characterized only from a sugar-assimilating hyperthermophile, Pyrococcus furiosus (GAPOR(Pf)), and contains the rare metal tungsten as an irreplaceable cofactor. Active recombinant M. maripaludis GAPOR (GAPOR(Mm)) was purified from Escherichia coli grown in minimal medium containing 100 muM sodium molybdate. In contrast, GAPOR(Mm) obtained from cells grown in medium containing tungsten (W) and W and molybdenum (Mo) or in medium without added W and Mo did not display any activity. Activity and transcript analysis of putative G3P-metabolizing enzymes and corresponding genes were performed with M. maripaludis cultured under autotrophic conditions in chemically defined medium. The activity of GAPOR(Mm) was constitutive throughout the culture period and exceeded that of GAPDH at all time points. As GAPDH activity was detected in only the gluconeogenic direction and GAPN activity was completely absent, only GAPOR(Mm) catalyzes oxidation of G3P in M. maripaludis. Recombinant GAPOR(Mm) is posttranscriptionally regulated as it exhibits pronounced and irreversible substrate inhibition and is completely inhibited by 1 muM ATP. With support from flux balance analysis, it is concluded that the major physiological role of GAPOR(Mm) in M. maripaludis most likely involves only nonoptimal growth conditions. PMID:17704226

  12. Overexpression of Protochlorophyllide Oxidoreductase C Regulates Oxidative Stress in Arabidopsis

    PubMed Central

    Pattanayak, Gopal K.; Tripathy, Baishnab C.

    2011-01-01

    Light absorbed by colored intermediates of chlorophyll biosynthesis is not utilized in photosynthesis; instead, it is transferred to molecular oxygen, generating singlet oxygen (1O2). As there is no enzymatic detoxification mechanism available in plants to destroy 1O2, its generation should be minimized. We manipulated the concentration of a major chlorophyll biosynthetic intermediate i.e., protochlorophyllide in Arabidopsis by overexpressing the light-inducible protochlorophyllide oxidoreductase C (PORC) that effectively phototransforms endogenous protochlorophyllide to chlorophyllide leading to minimal accumulation of the photosensitizer protochlorophyllide in light-grown plants. In PORC overexpressing (PORCx) plants exposed to high-light, the 1O2 generation and consequent malonedialdehyde production was minimal and the maximum quantum efficiency of photosystem II remained unaffected demonstrating that their photosynthetic apparatus and cellular organization were intact. Further, PORCx plants treated with 5-aminolevulinicacid when exposed to light, photo-converted over-accumulated protochlorophyllide to chlorophyllide, reduced the generation of 1O2 and malonedialdehyde production and reduced plasma membrane damage. So PORCx plants survived and bolted whereas, the 5-aminolevulinicacid-treated wild-type plants perished. Thus, overexpression of PORC could be biotechnologically exploited in crop plants for tolerance to 1O2-induced oxidative stress, paving the use of 5-aminolevulinicacid as a selective commercial light-activated biodegradable herbicide. Reduced protochlorophyllide content in PORCx plants released the protochlorophyllide-mediated feed-back inhibition of 5-aminolevulinicacid biosynthesis that resulted in higher 5-aminolevulinicacid production. Increase of 5-aminolevulinicacid synthesis upregulated the gene and protein expression of several downstream chlorophyll biosynthetic enzymes elucidating a regulatory net work of expression of genes involved in 5-aminolevulinicacid and tetrapyrrole biosynthesis. PMID:22031838

  13. 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. PMID:23361186

  14. Assignment of Electron Transfer Flavoprotein-Ubiquinone Oxidoreductase (ETF-QO) to Human Chromosome 4q33 by Fluorescence in Situ Hybridization and Somatic Cell Hybridization

    Microsoft Academic Search

    Elaine B. Spector; William K. Seltzer; Stephen I. Goodman

    1999-01-01

    Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a nuclear-encoded protein located in the inner mitochondrial membrane. Inherited defects of ETF-QO cause glutaric acidemia type II. We here describe the localization of the ETF-QO gene to human chromosome 4q33 by somatic cell hybridization and fluorescence in situ hybridization.

  15. A Molybdopterin Oxidoreductase Is Involved in H2 Oxidation in Desulfovibrio desulfuricans G20? †

    PubMed Central

    Li, Xiangzhen; Luo, Qingwei; Wofford, Neil Q.; Keller, Kimberly L.; McInerney, Michael J.; Wall, Judy D.; Krumholz, Lee R.

    2009-01-01

    Three mutants deficient in hydrogen/formate uptake were obtained through screening of a transposon mutant library containing 5,760 mutants of Desulfovibrio desulfuricans G20. Mutations were in the genes encoding the type I tetraheme cytochrome c3 (cycA), Fe hydrogenase (hydB), and molybdopterin oxidoreductase (mopB). Mutations did not decrease the ability of cells to produce H2 or formate during growth. Complementation of the cycA and mopB mutants with a plasmid carrying the intact cycA and/or mopB gene and the putative promoter from the parental strain allowed the recovery of H2 uptake ability, showing that these specific genes are involved in H2 oxidation. The mop operon encodes a periplasm-facing transmembrane protein complex which may shuttle electrons from periplasmic cytochrome c3 to the menaquinone pool. Electrons can then be used for sulfate reduction in the cytoplasm. PMID:19233927

  16. The Bifunctional Pyruvate Decarboxylase/Pyruvate Ferredoxin Oxidoreductase from Thermococcus guaymasensis

    PubMed Central

    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 ?-keto acids ferredoxin oxidoreductases are bifunctional, catalyzing the activities of nonoxidative and oxidative decarboxylation of the corresponding ?-keto acids. PMID:24982594

  17. Phycocyanobilin:Ferredoxin Oxidoreductase of Anabaena sp. PCC 7120

    Microsoft Academic Search

    Nicole Frankenberg; J. Clark Lagarias

    In cyanobacteria, the biosynthesis of the phycobili- protein and phytochrome chromophore precursor phy- cocyanobilin is catalyzed by the ferredoxin-dependent enzyme phycocyanobilin:ferredoxin oxidoreductase (PcyA), which mediates an atypical four-electron reduc- tion of biliverdin IX. Here we describe the expression, affinity purification, and biochemical characterization of recombinant PcyA from Anabaena sp. PCC 7120. A monomeric protein with a native Mr of 30,400

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

    PubMed

    Johnson, E A; Lin, E C

    1987-05-01

    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. PMID:3553154

  19. 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

  20. An Anaerobic-Type ?-Ketoglutarate Ferredoxin Oxidoreductase Completes the Oxidative Tricarboxylic Acid Cycle of Mycobacterium tuberculosis

    PubMed Central

    Baughn, Anthony D.; Garforth, Scott J.; Vilchèze, Catherine; Jacobs, William R.

    2009-01-01

    Aerobic organisms have a tricarboxylic acid (TCA) cycle that is functionally distinct from those found in anaerobic organisms. Previous reports indicate that the aerobic pathogen Mycobacterium tuberculosis lacks detectable ?-ketoglutarate (KG) dehydrogenase activity and drives a variant TCA cycle in which succinyl-CoA is replaced by succinic semialdehyde. Here, we show that M. tuberculosis expresses a CoA-dependent KG dehydrogenase activity, albeit one that is typically found in anaerobic bacteria. Unlike most enzymes of this family, the M. tuberculosis KG: ferredoxin oxidoreductase (KOR) is extremely stable under aerobic conditions. This activity is absent in a mutant strain deleted for genes encoding a previously uncharacterized oxidoreductase, and this strain is impaired for aerobic growth in the absence of sufficient amounts of CO2. Interestingly, inhibition of the glyoxylate shunt or exclusion of exogenous fatty acids alleviates this growth defect, indicating the presence of an alternate pathway that operates in the absence of ?-oxidation. Simultaneous disruption of KOR and the first enzyme of the succinic semialdehyde pathway (KG decarboxylase; KGD) results in strict dependence upon the glyoxylate shunt for growth, demonstrating that KG decarboxylase is also functional in M. tuberculosis intermediary metabolism. These observations demonstrate that unlike most organisms M. tuberculosis utilizes two distinct TCA pathways from KG, one that functions concurrently with ?-oxidation (KOR-dependent), and one that functions in the absence of ?-oxidation (KGD-dependent). As these pathways are regulated by metabolic cues, we predict that their differential utilization provides an advantage for growth in different environments within the host. PMID:19936047

  1. Biochemical and functional characterization of a periplasmic disulfide oxidoreductase from Neisseria meningitidis essential for meningococcal viability.

    PubMed

    Gand, Adeline; Selme-Roussel, Laure; Collin, Sabrina; Branlant, Guy; Jacob, Christophe; Boschi-Muller, Sandrine

    2015-06-01

    TlpAs (thioredoxin-like proteins) are bacterial thioredoxin-like periplasmic disulfide oxidoreductases generally involved in cytochrome c maturation (Ccm) process. They contain a characteristic CXXC active site motif involved in disulfide exchange reaction. In the human pathogenic Neisseria meningitidis species, no TlpA has been characterized so far. In the present study, using an in silico analysis, we identified a putative periplasmic TlpA, called TlpA2. Biochemical and kinetic characterizations of the soluble form of TlpA2, tTlpA2 (truncated TlpA2), were performed. A reduction potential of -0.230 V at pH 7 was calculated, suggesting that TlpA2 acts as a reductant in the oxidative environment of the periplasm. Using a second-order reactive probe, high pKapp (apparent pKa) values were determined for the two cysteines of the SCXXC motif. The tTlpA2 was shown to be efficiently reduced by the N-terminal domain of the DsbD, whereas tTlpA2 reduced a mimetic peptide of cytochrome c' with a catalytic efficiency similar to that observed with other disulfide oxidoreductase like ResA. Moreover, the corresponding gene tlpA2 was shown to be essential for the pathogen viability and able to partially complement a Bordetella pertussis CcsX mutant. Together, these data support an essential role of TlpA2 in the Ccm process in N. meningitidis. PMID:25826614

  2. The highly expressed and inducible endogenous NAD(P)H:quinone oxidoreductase 1 in cardiovascular cells acts as a potential superoxide scavenger.

    PubMed

    Zhu, Hong; Jia, Zhenquan; Mahaney, James E; Ross, David; Misra, Hara P; Trush, Michael A; Li, Yunbo

    2007-01-01

    It has recently been demonstrated that purified NAD(P)H:quinone oxidoreductase 1 (NQO1) is able to scavenge superoxide (O2(.-)) though the rate of reaction of O2(.-) with NQO1 is much lower than the rate of enzymatic dismutation catalyzed by superoxide dismutase (SOD). This study was undertaken to determine if the endogenously expressed NQO1 in cardiovascular cells could scavenge O2(.-). We observed that NQO1 was highly expressed in cardiovascular cells, including rat aortic smooth muscle A10 and cardiac H9c2 cells, as well as normal human aortic smooth muscle and endothelial cells. NQO1, but not SOD in the cardiovascular cells was highly inducible by 3H-1,2-dithiole-3-thione (D3T). Cytosols from H9c2 and human aortic smooth muscle cells (HASMCs) were isolated to determine the O2(.-) scavenging ability of the endogenously expressed NQO1 by using pyrogallol autooxidation assay. We showed that cytosols from the above cells inhibited pyrogallol autooxidation in an NADPH or NADH-dependent manner. The NADH/NADPH-dependent inhibition of pyrogallol autooxidation by the cytosols was completely abolished by the NQO1-specific inhibitor, ES936, suggesting that the endogenously expressed NQO1 could scavenge O2(.-). In the presence of NADH/NADPH, cytosols from D3T-treated cells showed increased ability to scavenge O2(.-) as compared to cytosols from untreated cells. This increased ability to scavenge O2(.-) was also completely reversed by ES936. 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide spin-trapping experiments using potassium superoxide as a O2(.-) generator further confirmed the ability of NQO1 from HASMCs to scavenge O2(.-). The spin-trapping experiments also showed that induction of NQO1 by D3T in HASMCs augmented the O2(.-) scavenging ability. Taken together, these results demonstrate that the highly expressed and inducible endogenous NQO1 in cardiovascular cells may act as a potential O2(.-) scavenger. PMID:17901563

  3. An NAD(P)H-nicotine blue oxidoreductase is part of the nicotine regulon and may protect Arthrobacter nicotinovorans from oxidative stress during nicotine catabolism.

    PubMed

    Mihasan, Marius; Chiribau, Calin-Bogdan; Friedrich, Thorsten; Artenie, Vlad; Brandsch, Roderich

    2007-04-01

    An NAD(P)H-nicotine blue (quinone) oxidoreductase was discovered as a member of the nicotine catabolic pathway of Arthrobacter nicotinovorans. Transcriptional analysis and electromobility shift assays showed that the enzyme gene was expressed in a nicotine-dependent manner under the control of the transcriptional activator PmfR and thus was part of the nicotine regulon of A. nicotinovorans. The flavin mononucleotide-containing enzyme uses NADH and, with lower efficiency, NADPH to reduce, by a two-electron transfer, nicotine blue to the nicotine blue leuco form (hydroquinone). Besides nicotine blue, several other quinones were reduced by the enzyme. The NAD(P)H-nicotine blue oxidoreductase may prevent intracellular one-electron reductions of nicotine blue which may lead to semiquinone radicals and potentially toxic reactive oxygen species. PMID:17293530

  4. Structure of the Biliverdin Radical Intermediate in Phycocyanobilin:Ferredoxin Oxidoreductase Identified by

    E-print Network

    Fisher, Andrew J.

    Structure of the Biliverdin Radical Intermediate in Phycocyanobilin:Ferredoxin Oxidoreductase@ucdavis.edu (S.S.); rdbritt@ucdavis.edu (R.D.B.) Abstract: The cyanobacterial enzyme phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the two- step four-electron reduction of biliverdin IXR to phycocyanobilin

  5. 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

    ...2012-07-01 2012-07-01 false Glyphosate Oxidoreductase GOX or GOXv247 in all...Tolerance Exemptions § 174.524 Glyphosate Oxidoreductase GOX or GOXv247 in all...requirement of a tolerance. Residues of the Glyphosate Oxidoreductase GOX or GOXv247...

  6. 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

    ...2013-07-01 2013-07-01 false Glyphosate Oxidoreductase GOX or GOXv247 in all...Tolerance Exemptions § 174.524 Glyphosate Oxidoreductase GOX or GOXv247 in all...requirement of a tolerance. Residues of the Glyphosate Oxidoreductase GOX or GOXv247...

  7. 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

    ...2014-07-01 2014-07-01 false Glyphosate Oxidoreductase GOX or GOXv247 in all...Tolerance Exemptions § 174.524 Glyphosate Oxidoreductase GOX or GOXv247 in all...requirement of a tolerance. Residues of the Glyphosate Oxidoreductase GOX or GOXv247...

  8. Congenital adrenal hyperplasia and P450 oxidoreductase deficiency.

    PubMed

    Krone, Nils; Dhir, Vivek; Ivison, Hannah E; Arlt, Wiebke

    2007-02-01

    Congenital adrenal hyperplasia (CAH) comprises a group of autosomal recessive disorders, which are usually due to inactivating mutations in single enzymes involved in adrenal steroid biosynthesis. The characteristics of the biochemical and clinical phenotype depend on the specific enzymatic defect. In 21-hydroxylase and 11beta-hydroxylase deficiency only adrenal steroidogenesis is affected, whereas a defect in 3beta-hydroxysteroid dehydrogenase or 17alpha-hydroxylase also involves gonadal steroid biosynthesis. Recently, mutations in the electron donor enzyme P450 oxidoreductase were identified as the cause of CAH with apparent combined 17alpha-hydroxylase and 21-hydroxylase deficiency, thereby illustrating the impact of redox regulation enzymes on steroidogenesis. P450 oxidoreductase deficiency (ORD) has a complex phenotype including two unique features not observed in any other CAH variant, skeletal malformations and severe genital ambiguity in both sexes. Despite invariably low circulating androgens, females with ORD may present with virilized genitalia and mothers may suffer from virilization during pregnancy. This apparently contradictory finding may be explained by the existence of an alternative pathway in human androgen biosynthesis, with important implications for physiology and pathophysiology. This review discusses the biochemical and clinical presentation and the genetic and functional basis of the currently known CAH variants, with a specific focus on ORD. PMID:17223983

  9. Carbon monoxide:methylene blue oxidoreductase from Pseudomonas carboxydovorans.

    PubMed Central

    Meyer, O; Schlegel, H G

    1980-01-01

    The enzyme carbon monoxide:methylene blue oxidoreductase from CO autotrophically grown cells of Pseudomonas carboxydovorans strain OM5, was purified to homogeneity. The enzyme was obtained in 26% yield and was purified 36-fold. The enzyme was stable for at least 6 days, had a molecular weight of 230,000, gave a single protein and activity band on polyacrylamide gel electrophoresis, and was homogeneous by the criterion of sedimentation equilibrium. Sodium dodecyl sulfate gel electrophoresis revealed a single band of molecular weight 107,000. Carbon monoxide:methylene blue oxidoreductase did not catalyze reduction of pyridine or flavin nucleotides but catalyzed the oxidation of CO to CO2 in the presence of methylene blue, thionine, toluylene blue, dichlorophenolindophenol, or pyocyanine under strictly anaerobic conditions. The visible spectrum revealed maxima at 405 and 470 nm. The millimolar extinction coefficients were 43.9 (405 nm) and 395.5 (275 nm), respectively. Absorption at 470 nm decreased in the presence of dithionite, and the spectrum was not affected by the substrate CO. Maximum reaction rates were found at pH 7.0 and 63 degrees C; temperature dependence followed the Arrhenius equation, with an activation energy (delta H degree) of 36.8 kJ/mol (8.8 kcal/mol). The apparent Km was 53 microM for CO. The purified enzyme was incapable of oxidizing methane, methanol, or formaldehyde in the presence of methylene blue as electron acceptor. Images PMID:7354006

  10. 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.

  11. Untargeted Plasma Metabolite Profiling Reveals the Broad Systemic Consequences of Xanthine Oxidoreductase Inactivation in Mice

    PubMed Central

    Chen, Qiuying; Park, Hyeong-Cheon; Goligorsky, Michael S.; Chander, Praveen; Fischer, Steven M.; Gross, Steven S.

    2012-01-01

    A major challenge in systems biology is integration of molecular findings for individual enzyme activities into a cohesive high-level understanding of cellular metabolism and physiology/pathophysiology. However, meaningful prediction for how a perturbed enzyme activity will globally impact metabolism in a cell, tissue or intact organisms is precluded by multiple unknowns, including in vivo enzymatic rates, subcellular distribution and pathway interactions. To address this challenge, metabolomics offers the potential to simultaneously survey changes in thousands of structurally diverse metabolites within complex biological matrices. The present study assessed the capability of untargeted plasma metabolite profiling to discover systemic changes arising from inactivation of xanthine oxidoreductase (XOR), an enzyme that catalyzes the final steps in purine degradation. Using LC-MS coupled with a multivariate statistical data analysis platform, we confidently surveyed >3,700 plasma metabolites (50–1,000 Da) for differential expression in XOR wildtype vs. mice with inactivated XOR, arising from gene deletion or pharmacological inhibition. Results confirmed the predicted derangements in purine metabolism, but also revealed unanticipated perturbations in metabolism of pyrimidines, nicotinamides, tryptophan, phospholipids, Krebs and urea cycles, and revealed kidney dysfunction biomarkers. Histochemical studies confirmed and characterized kidney failure in xor-nullizygous mice. These findings provide new insight into XOR functions and demonstrate the power of untargeted metabolite profiling for systemic discovery of direct and indirect consequences of gene mutations and drug treatments. PMID:22723833

  12. NAD(P)H: Quinone Oxidoreductase 1 Is Induced by Progesterone in Cardiomyocytes

    PubMed Central

    Morrissy, Stephen; Strom, Joshua; Purdom-Dickinson, Sally; Chen, Qin M.

    2014-01-01

    NAD(P)H: Quinone Oxidoreductase 1 (NQO1) is a ubiquitous flavo-enzyme that catalyzes two-electron reduction of various quinones by utilizing NAD(P)H as an electron donor. Our previous study found that progesterone (PG) can protect cardiomyocytes from apoptosis induced by Doxorubicin (Dox). Microarray analyses of genes induced by PG had let to the discovery of induction of NQO1 mRNA. We report here that PG induces NQO1 protein and activity in a dose dependent manner. Whereas NQO1 is well known as a target gene of Nrf2 transcription factor due to the presence of Antioxidant Response Element (ARE) in the promoter, PG did not activate the ARE, suggesting Nrf2 independent induction of NQO1. To address the role of NQO1 induction in PG induced cytoprotection, we tested the effect of NQO1 inducer ?-naphthoflavone and inhibitor dicoumarol. Induction of NQO1 by ?-naphthoflavone decreased Dox induced apoptosis and potentiated the protective effect of PG as measured by caspase-3 activity. PG induced NQO1 activity was inhibited with dicoumarol, which did not affect PG induced cytoprotection. Dicoumarol treatment alone potentiated Dox induced caspase-3 activity. These data suggest that while NQO1 plays a role in PG induced cytoprotection, there are additional components contributing to PG induced cytoprotection. PMID:21947872

  13. A Single-Electron Reducing Quinone Oxidoreductase Is Necessary to Induce Haustorium Development in the Root Parasitic Plant Triphysaria[C][W

    PubMed Central

    Bandaranayake, Pradeepa C.G.; Filappova, Tatiana; Tomilov, Alexey; Tomilova, Natalya B.; Jamison-McClung, Denneal; Ngo, Quy; Inoue, Kentaro; Yoder, John I.

    2010-01-01

    Parasitic plants in the Orobanchaceae develop haustoria in response to contact with host roots or chemical haustoria-inducing factors. Experiments in this manuscript test the hypothesis that quinolic-inducing factors activate haustorium development via a signal mechanism initiated by redox cycling between quinone and hydroquinone states. Two cDNAs were previously isolated from roots of the parasitic plant Triphysaria versicolor that encode distinct quinone oxidoreductases. QR1 encodes a single-electron reducing NADPH quinone oxidoreductase similar to ?-crystallin. The QR2 enzyme catalyzes two electron reductions typical of xenobiotic detoxification. QR1 and QR2 transcripts are upregulated in a primary response to chemical-inducing factors, but only QR1 was upregulated in response to host roots. RNA interference technology was used to reduce QR1 and QR2 transcripts in Triphysaria roots that were evaluated for their ability to form haustoria. There was a significant decrease in haustorium development in roots silenced for QR1 but not in roots silenced for QR2. The infrequent QR1 transgenic roots that did develop haustoria had levels of QR1 similar to those of nontransgenic roots. These experiments implicate QR1 as one of the earliest genes on the haustorium signal transduction pathway, encoding a quinone oxidoreductase necessary for the redox bioactivation of haustorial inducing factors. PMID:20424175

  14. Complementation of phytochrome chromophore-deficient Arabidopsis by expression of phycocyanobilin:ferredoxin oxidoreductase

    PubMed Central

    Kami, Chitose; Mukougawa, Keiko; Muramoto, Takuya; Yokota, Akiho; Shinomura, Tomoko; Lagarias, J. Clark; Kohchi, Takayuki

    2004-01-01

    The covalently bound phytochromobilin (P?B) prosthetic group is required for the diverse photoregulatory activities of all members of the phytochrome family in vascular plants, whereas by contrast, green algal and cyanobacterial phytochromes use the more reduced linear tetrapyrrole pigment phycocyanobilin (PCB). To assess the functional consequence of the substitution of P?B with PCB in plants, the phytochrome chromophore-deficient hy2 mutant of Arabidopsis was transformed with a constitutively expressed pcyA gene that encodes the cyanobacterial enzyme, PCB:ferredoxin oxidoreductase. Spectroscopic analyses of extracts from etiolated seedlings revealed that PcyA expression restored photoactive phytochrome to WT levels, albeit with blue-shifted absorption maxima, while also restoring light lability to phytochrome A. Photobiological measurements indicated that PcyA expression rescued phytochrome-mediated red high-irradiance responses, low-fluence red/far-red (FR) photoreversible responses, and very-low-fluence responses, thus confirming that PCB can functionally substitute for P?B for these photoregulatory activities. Although PcyA expression failed to rescue phytochrome A-mediated FR high-irradiance responsivity to that of WT, our studies indicate that the FR high-irradiance response is fully functional in pcyA-expressing plants but shifted to shorter wavelengths, indicating that PCB can functionally complement this phytochrome-mediated response in vascular plants. PMID:14722358

  15. Does molecular docking reveal alternative chemopreventive mechanism of activation of oxidoreductase by sulforaphane isothiocyanates?

    PubMed

    Mazur, Pawel; Magdziarz, Tomasz; Bak, Andrzej; Chilmonczyk, Zdzislaw; Kasprzycka-Guttman, Teresa; Misiewicz-Krzemi?ska, Irena; Skupi?ska, Katarzyna; Polanski, Jaroslaw

    2010-07-01

    Isothiocyanates (ITC) are well-known chemopreventive agents extracted from vegetables. This activity results from the activation of human oxidoreductase. In this letter, the uncompetitive activatory mechanism of ITC was investigated using docking and molecular dynamics simulations. This indicates that NAD(P)H:quinone oxidoreductase can efficiently improve enzyme-substrate recognition within the catalytic site if the ITC activator supports the interaction in the uncompetitive binding site. PMID:20024690

  16. Does molecular docking reveal alternative chemopreventive mechanism of activation of oxidoreductase by sulforaphane isothiocyanates?

    Microsoft Academic Search

    Pawel Mazur; Tomasz Magdziarz; Andrzej Bak; Zdzislaw Chilmonczyk; Teresa Kasprzycka-Guttman; Irena Misiewicz-Krzemi?ska; Katarzyna Skupi?ska; Jaroslaw Polanski

    2010-01-01

    Isothiocyanates (ITC) are well-known chemopreventive agents extracted from vegetables. This activity results from the activation\\u000a of human oxidoreductase. In this letter, the uncompetitive activatory mechanism of ITC was investigated using docking and\\u000a molecular dynamics simulations. This indicates that NAD(P)H:quinone oxidoreductase can efficiently improve enzyme-substrate\\u000a recognition within the catalytic site if the ITC activator supports the interaction in the uncompetitive binding

  17. Noncoupled NADH:Ubiquinone Oxidoreductase of Azotobacter vinelandii Is Required for Diazotrophic Growth at High Oxygen Concentrations

    PubMed Central

    Bertsova, Yulia V.; Bogachev, Alexander V.; Skulachev, Vladimir P.

    2001-01-01

    The gene encoding the noncoupled NADH:ubiquinone oxidoreductase (NDH II) from Azotobacter vinelandii was cloned, sequenced, and used to construct an NDH II-deficient mutant strain. Compared to the wild type, this strain showed a marked decrease in respiratory activity. It was unable to grow diazotrophically at high aeration, while it was fully capable of growth at low aeration or in the presence of NH4+. This result suggests that the role of NDH II is as a vital component of the respiratory protection mechanism of the nitrogenase complex in A. vinelandii. It was also found that the oxidation of NADPH in the A. vinelandii respiratory chain is catalyzed solely by NDH II. PMID:11698376

  18. Chlorophyllide a Oxidoreductase Works as One of the Divinyl Reductases Specifically Involved in Bacteriochlorophyll a Biosynthesis*

    PubMed Central

    Harada, Jiro; Mizoguchi, Tadashi; Tsukatani, Yusuke; Yokono, Makio; Tanaka, Ayumi; Tamiaki, Hitoshi

    2014-01-01

    Bacteriochlorophyll a is widely distributed among anoxygenic photosynthetic bacteria. In bacteriochlorophyll a biosynthesis, the reduction of the C8 vinyl group in 8-vinyl-chlorophyllide a is catalyzed to produce chlorophyllide a by an 8-vinyl reductase called divinyl reductase (DVR), which has been classified into two types, BciA and BciB. However, previous studies demonstrated that mutants lacking the DVR still synthesize normal bacteriochlorophyll a with the C8 ethyl group and suggested the existence of an unknown “third” DVR. Meanwhile, we recently observed that chlorophyllide a oxidoreductase (COR) of a purple bacterium happened to show the 8-vinyl reduction of 8-vinyl-chlorophyllide a in vitro. In this study, we made a double mutant lacking BciA and COR of the purple bacterium Rhodobacter sphaeroides in order to investigate whether the mutant still produces pigments with the C8 ethyl group or if COR actually works as the third DVR. The single mutant deleting BciA or COR showed production of the C8 ethyl group pigments, whereas the double mutant accumulated 8-vinyl-chlorophyllide, indicating that there was no enzyme other than BciA and COR functioning as the unknown third DVR in Rhodobacter sphaeroides (note that this bacterium has no bciB gene). Moreover, some COR genes derived from other groups of anoxygenic photosynthetic bacteria were introduced into the double mutant, and all of the complementary strains produced normal bacteriochlorophyll a. This observation indicated that COR of these bacteria performs two functions, reductions of the C8 vinyl group and the C7=C8 double bond, and that such an activity is probably conserved in the widely ranging groups. PMID:24637023

  19. Elementary tetrahelical protein design for diverse oxidoreductase functions

    PubMed Central

    Lichtenstein, Bruce R; Sheehan, Molly M; Fry, Bryan A; Bialas, Chris; Ennist, Nathan M; Siedlecki, Jessica A; Zhao, Zhenyu; Stetz, Matthew A; Valentine, Kathleen G; Anderson, J L Ross; Wand, A Joshua; Discher, Bohdana M; Moser, Christopher C; Dutton, P Leslie

    2014-01-01

    Emulating functions of natural enzymes in man-made constructs has proven challenging. Here we describe a man-made protein platform that reproduces many of the diverse functions of natural oxidoreductases without importing the complex and obscure interactions common to natural proteins. Our design is founded on an elementary, structurally stable 4-?-helix protein monomer with a minimalist interior malleable enough to accommodate various light- and redox-active cofactors and with an exterior tolerating extensive charge patterning for modulation of redox cofactor potentials and environmental interactions. Despite its modest size, the construct offers several independent domains for functional engineering that targets diverse natural activities, including dioxygen binding and superoxide and peroxide generation, interprotein electron transfer to natural cytochrome c and light-activated intraprotein energy transfer and charge separation approximating the core reactions of photosynthesis, cryptochrome and photolyase. The highly stable, readily expressible and biocompatible characteristics of these open-ended designs promise development of practical in vitro and in vivo applications. PMID:24121554

  20. Biosynthesis of poly(3-hydroxybutyrate- co -3-hydroxyalkanoates) by recombinant bacteria expressing the PHA synthase gene phaC1 from Pseudomonas sp. 61-3

    Microsoft Academic Search

    H. Matsusaki; H. Abe; K. Taguchi; T. Fukui; Y. Doi

    2000-01-01

    Pseudomonas sp. 61-3 accumulated a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer consisting of 3-hydroxyalkanoate\\u000a (3HA) units of 4–12 carbon atoms. The genes encoding ?-ketothiolase (PhbARe) and NADPH-dependent acetoacetyl-CoA reductase (PhbBRe) from Ralstoniaeutropha were expressed under the control of promoters for Pseudomonas sp. 61-3 pha locus or R. eutropha phb operon together with phaC1\\u000a Ps gene (PHA synthase

  1. Specificity of Human Aldo-Keto Reductases, NAD(P)H: Quinone Oxidoreductase and Carbonyl Reductases to Redox-Cycle Polycyclic Aromatic Hydrocarbon Diones and 4-Hydroxyequilenin-o-Quinone

    PubMed Central

    Shultz, Carol A.; Quinn, Amy M.; Park, Jong-Heum; Harvey, Ronald G.; Bolton, Judy L; Maser, Edmund; Penning, Trevor M.

    2011-01-01

    Polycyclic aromatic hydrocarbons (PAH) are suspect human lung carcinogens and can be metabolically activated to remote quinones, e.g. benzo[a]pyrene-1,6-dione (B[a]P-1,6-dione) and B[a]P-3,6-dione by the action of either P450 monooxygenase or peroxidases and to non-K region o-quinones by aldo-keto reductases (AKRs). B[a]P-7,8-dione also structurally resembles 4-hydroxyequilenin o-quinone. These three classes of quinones can redox cycle, generate reactive oxygen species (ROS) and produce the mutagenic lesion 8-oxo-dGuo, and may contribute to PAH- and estrogen-induced carcinogenesis. We compared the ability of a complete panel of human recombinant AKRs to catalyze reduction of PAH o-quinones in the phenanthrene, chrysene, pyrene and anthracene series. The specific activities for NADPH-dependent quinone reduction were often 100-1,000 times greater than the ability of the same AKR isoform to oxidize the cognate PAH-trans-dihydrodiol. However, the AKR with the highest quinone reductase activity for a particular PAH o-quinone was not always identical to the AKR isoform with the highest dihydrodiol dehydrogenase activity for the respective PAH-trans-dihydrodiol. Discrete AKRs also catalyzed the reduction of B[a]P-1,6-dione, B[a]P-3,6-dione and 4-hydroxyequilenin o-quinone. Concurrent measurements of oxygen consumption, superoxide anion and hydrogen peroxide formation established that ROS were produced as a result of the redox-cycling. When compared with human recombinant NAD(P)H: quinone oxidoreductase (NQO1) and carbonyl reductases (CBR1 and CBR3), NQO1 was a superior catalyst of these reactions followed by AKRs and lastly CBR1 and CBR3. In A549 cells two-electron reduction of PAH o-quinones causes intracellular ROS formation. ROS formation was unaffected by the addition of dicumarol suggesting that NQO1 is not responsible for the two-electron reduction observed and does not offer protection against ROS formation from PAH o-quinones. PMID:21910479

  2. Roles of the Sodium-Translocating NADH:Quinone Oxidoreductase (Na+-NQR) on Vibrio cholerae Metabolism, Motility and Osmotic Stress Resistance

    PubMed Central

    Minato, Yusuke; Halang, Petra; Quinn, Matthew J.; Faulkner, Wyatt J.; Aagesen, Alisha M.; Steuber, Julia; Stevens, Jan F.; Häse, Claudia C.

    2014-01-01

    The Na+ translocating NADH:quinone oxidoreductase (Na+-NQR) is a unique respiratory enzyme catalyzing the electron transfer from NADH to quinone coupled with the translocation of sodium ions across the membrane. Typically, Vibrio spp., including Vibrio cholerae, have this enzyme but lack the proton-pumping NADH:ubiquinone oxidoreductase (Complex I). Thus, Na+-NQR should significantly contribute to multiple aspects of V. cholerae physiology; however, no detailed characterization of this aspect has been reported so far. In this study, we broadly investigated the effects of loss of Na+-NQR on V. cholerae physiology by using Phenotype Microarray (Biolog), transcriptome and metabolomics analyses. We found that the V. cholerae ?nqrA-F mutant showed multiple defects in metabolism detected by Phenotype Microarray. Transcriptome analysis revealed that the V. cholerae ?nqrA-F mutant up-regulates 31 genes and down-regulates 55 genes in both early and mid-growth phases. The most up-regulated genes included the cadA and cadB genes, encoding a lysine decarboxylase and a lysine/cadaverine antiporter, respectively. Increased CadAB activity was further suggested by the metabolomics analysis. The down-regulated genes include sialic acid catabolism genes. Metabolomic analysis also suggested increased reductive pathway of TCA cycle and decreased purine metabolism in the V. cholerae ?nqrA-F mutant. Lack of Na+-NQR did not affect any of the Na+ pumping-related phenotypes of V. cholerae suggesting that other secondary Na+ pump(s) can compensate for Na+ pumping activity of Na+-NQR. Overall, our study provides important insights into the contribution of Na+-NQR to V. cholerae physiology. PMID:24811312

  3. Roles of the sodium-translocating NADH:quinone oxidoreductase (Na+-NQR) on vibrio cholerae metabolism, motility and osmotic stress resistance.

    PubMed

    Minato, Yusuke; Fassio, Sara R; Kirkwood, Jay S; Halang, Petra; Quinn, Matthew J; Faulkner, Wyatt J; Aagesen, Alisha M; Steuber, Julia; Stevens, Jan F; Häse, Claudia C

    2014-01-01

    The Na+ translocating NADH:quinone oxidoreductase (Na+-NQR) is a unique respiratory enzyme catalyzing the electron transfer from NADH to quinone coupled with the translocation of sodium ions across the membrane. Typically, Vibrio spp., including Vibrio cholerae, have this enzyme but lack the proton-pumping NADH:ubiquinone oxidoreductase (Complex I). Thus, Na+-NQR should significantly contribute to multiple aspects of V. cholerae physiology; however, no detailed characterization of this aspect has been reported so far. In this study, we broadly investigated the effects of loss of Na+-NQR on V. cholerae physiology by using Phenotype Microarray (Biolog), transcriptome and metabolomics analyses. We found that the V. cholerae ?nqrA-F mutant showed multiple defects in metabolism detected by Phenotype Microarray. Transcriptome analysis revealed that the V. cholerae ?nqrA-F mutant up-regulates 31 genes and down-regulates 55 genes in both early and mid-growth phases. The most up-regulated genes included the cadA and cadB genes, encoding a lysine decarboxylase and a lysine/cadaverine antiporter, respectively. Increased CadAB activity was further suggested by the metabolomics analysis. The down-regulated genes include sialic acid catabolism genes. Metabolomic analysis also suggested increased reductive pathway of TCA cycle and decreased purine metabolism in the V. cholerae ?nqrA-F mutant. Lack of Na+-NQR did not affect any of the Na+ pumping-related phenotypes of V. cholerae suggesting that other secondary Na+ pump(s) can compensate for Na+ pumping activity of Na+-NQR. Overall, our study provides important insights into the contribution of Na+-NQR to V. cholerae physiology. PMID:24811312

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

    SciTech Connect

    Conway, T. (Univ. of Nebraska, Lincoln (USA)); Ingram, L.O. (Univ. of Florida, Gainesville (USA))

    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.

  5. Suicide inactivation of hydroxylamine oxidoreductase of Nitrosomonas europaea by organohydrazines.

    PubMed

    Logan, M S; Hooper, A B

    1995-07-18

    In the presence of a suitable electron acceptor such as mammalian cytochrome c, hydroxylamine oxidoreductase (HAO) from the chemolithotrophic bacterium Nitrosomonas europaea catalyzes the oxidation of hydroxylamine or hydrazine to nitrite or dinitrogen, respectively. Each subunit of HAO contains 7 c-hemes and a chromophore of the active site called heme P460, a c-heme bridged from a methylene carbon to a ring carbon of a tyrosine of the peptide chain. Reaction with either substrate results in reduction of several c-hemes of HAO. The reaction of organohydrazines with HAO was investigated in this work. HAO was inactivated by (phenyl-, (methyl-, or (hydroxyethyl)hydrazine. The process followed first order kinetics and was inhibited by the substrates, hydroxylamine or hydrazine. Complete loss of enzyme activity and absorbancy characteristic of native heme P460 of HAO occurred at a 1:1 ratio of phenylhydrazine and HAO. HAO was covalently derivatized by two molecules of [14C]-phenylhydrazine per subunit. Heme P460 was derivatized with high affinity, and an amino acid residue was derivatized with lower affinity. c-Hemes were not derivatized except for the partial reaction of (hydroxyethyl)hydrazine with one heme. As with hydroxylamine and hydrazine, incubation with organohydrazines resulted in reduction of c-heme of HAO. Derivatized minus native optical difference spectra of ferric or ferrous HAO revealed changes in the optical properties of heme P460 which were generally similar to shifts seen in the reaction of the heme of other hemoproteins with organohydrazines.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7619827

  6. A second isoform of the ferredoxin:NADP oxidoreductase generated by an in-frame initiation of translation

    PubMed Central

    Thomas, Jean-Claude; Ughy, Bettina; Lagoutte, Bernard; Ajlani, Ghada

    2006-01-01

    Ferredoxin:NADP oxidoreductases (FNRs) constitute a family of flavoenzymes that catalyze the exchange of reducing equivalents between one-electron carriers and the two-electron-carrying NADP(H). The main role of FNRs in cyanobacteria and leaf plastids is to provide the NADPH for photoautotrophic metabolism. In root plastids, a distinct FNR isoform is found that has been postulated to function in the opposite direction, providing electrons for nitrogen assimilation at the expense of NADPH generated by heterotrophic metabolism. A multiple gene family encodes FNR isoenzymes in plants, whereas there is only one FNR gene (petH) in cyanobacteria. Nevertheless, we detected two FNR isoforms in the cyanobacterium Synechocystis sp. strain PCC6803. One of them (FNRS ?34 kDa) is similar in size to the plastid FNR and specifically accumulates under heterotrophic conditions, whereas the other one (FNRL ?46 kDa) contains an extra N-terminal domain that allows its association with the phycobilisome. Site-directed mutants allowed us to conclude that the smaller isoform, FNRS, is produced from an internal ribosome entry site within the petH ORF. Thus we have uncovered a mechanism by which two isoforms are produced from a single gene, which is, to our knowledge, novel in photosynthetic bacteria. Our results strongly suggest that FNRL is an NADP+ reductase, whereas FNRS is an NADPH oxidase. PMID:17116880

  7. P450 Oxidoreductase Deficiency: A Disorder of Steroidogenesis with Multiple Clinical Manifestations

    NSDL National Science Digital Library

    Walter L. Miller (San Francisco; University of California REV)

    2012-10-23

    Cytochrome P450 enzymes catalyze the biosynthesis of steroid hormones and metabolize drugs. There are seven human type I P450 enzymes in mitochondria and 50 type II enzymes in endoplasmic reticulum. Type II enzymes, including both drug-metabolizing and some steroidogenic enzymes, require electron donation from a two-flavin protein, P450 oxidoreductase (POR). Although knockout of the POR gene causes embryonic lethality in mice, we discovered human POR deficiency as a disorder of steroidogenesis associated with the Antley-Bixler skeletal malformation syndrome and found mild POR mutations in phenotypically normal adults with infertility. Assay results of mutant forms of POR using the traditional but nonphysiologic assay (reduction of cytochrome c) did not correlate with patient phenotypes; assays based on the 17,20 lyase activity of P450c17 (CYP17) correlated with clinical phenotypes. The POR sequence in 842 normal individuals revealed many polymorphisms; amino acid sequence variant A503V is encoded by ~28% of human alleles. POR A503V has about 60% of wild-type activity in assays with CYP17, CYP2D6, and CYP3A4, but nearly wild-type activity with P450c21, CYP1A2, and CYP2C19. Activity of a particular POR variant with one P450 enzyme will not predict its activity with another P450 enzyme: Each POR-P450 combination must be studied individually. Human POR transcription, initiated from an untranslated exon, is regulated by Smad3/4, thyroid receptors, and the transcription factor AP-2. A promoter polymorphism reduces transcription to 60% in liver cells and to 35% in adrenal cells. POR deficiency is a newly described disorder of steroidogenesis, and POR variants may account for some genetic variation in drug metabolism.

  8. NADPH:Quinone Oxidoreductase 1 Regulates Host Susceptibility to Ozone via Isoprostane Generation*

    PubMed Central

    Kummarapurugu, Apparao B.; Fischer, Bernard M.; Zheng, Shuo; Milne, Ginger L.; Ghio, Andrew J.; Potts-Kant, Erin N.; Foster, W. Michael; Soderblom, Erik J.; Dubois, Laura G.; Moseley, M. Arthur; Thompson, J. Will; Voynow, Judith A.

    2013-01-01

    NADPH:quinone oxidoreductase 1 (NQO1) is recognized as a major susceptibility gene for ozone-induced pulmonary toxicity. In the absence of NQO1 as can occur by genetic mutation, the human airway is protected from harmful effects of ozone. We recently reported that NQO1-null mice are protected from airway hyperresponsiveness and pulmonary inflammation following ozone exposure. However, NQO1 regenerates intracellular antioxidants and therefore should protect the individual from oxidative stress. To explain this paradox, we tested whether in the absence of NQO1 ozone exposure results in increased generation of A2-isoprostane, a cyclopentenone isoprostane that blunts inflammation. Using GC-MS, we found that NQO1-null mice had greater lung tissue levels of D2- and E2-isoprostanes, the precursors of J2- and A2-isoprostanes, both at base line and following ozone exposure compared with congenic wild-type mice. We confirmed in primary cultures of normal human bronchial epithelial cells that A2-isoprostane inhibited ozone-induced NF-?B activation and IL-8 regulation. Furthermore, we determined that A2-isoprostane covalently modified the active Cys179 domain in inhibitory ?B kinase in the presence of ozone in vitro, thus establishing the biochemical basis for A2-isoprostane inhibition of NF-?B. Our results demonstrate that host factors may regulate pulmonary susceptibility to ozone by regulating the generation of A2-isoprostanes in the lung. These observations provide the biochemical basis for the epidemiologic observation that NQO1 regulates pulmonary susceptibility to ozone. PMID:23275341

  9. WW-Domain-Containing Oxidoreductase Is Associated with Low Plasma HDL-C Levels

    PubMed Central

    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

    2008-01-01

    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 × 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 × 10?4 to 2 × 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. PMID:18674750

  10. NAD(P)H:Flavin Mononucleotide Oxidoreductase Inactivation during 2,4,6Trinitrotoluene Reduction

    Microsoft Academic Search

    R. Guy Riefler; Barth F. Smets

    2002-01-01

    Bacteria readily transform 2,4,6-trinitrotoluene (TNT), a contaminant frequently found at military bases and munitions production facilities, by reduction of the nitro group substituents. In this work, the kinetics of nitroreduction were investigated by using a model nitroreductase, NAD(P)H:flavin mononucleotide (FMN) oxidoreductase. Under mediation by NAD(P)H:FMN oxidoreductase, TNT rapidly reacted with NADH to form 2-hydroxylamino-4,6-dinitrotoluene and 4-hydroxylamino-2,6-dinitrotoluene, whereas 2-amino-4,6-dinitrotolu- ene and

  11. Gene replacement in Penicillium roqueforti.

    PubMed

    Goarin, Anne; Silar, Philippe; Malagnac, Fabienne

    2015-05-01

    Most cheese-making filamentous fungi lack suitable molecular tools to improve their biotechnology potential. Penicillium roqueforti, a species of high industrial importance, would benefit from functional data yielded by molecular genetic approaches. This work provides the first example of gene replacement by homologous recombination in P. roqueforti, demonstrating that knockout experiments can be performed in this fungus. To do so, we improved the existing transformation method to integrate transgenes into P. roqueforti genome. In the meantime, we cloned the PrNiaD gene, which encodes a NADPH-dependent nitrate reductase that reduces nitrate to nitrite. Then, we performed a deletion of the PrNiaD gene from P. roqueforti strain AGO. The ?PrNiaD mutant strain is more resistant to chlorate-containing medium than the wild-type strain, but did not grow on nitrate-containing medium. Because genomic data are now available, we believe that generating selective deletions of candidate genes will be a key step to open the way for a comprehensive exploration of gene function in P. roqueforti. PMID:25315520

  12. 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 ?-TrCP. However, in aripiprazole-treated cells, the relative amount of Nrf2 anchored to Keap1 or ?-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. PMID:25504008

  13. Regulation of P450 oxidoreductase by gonadotropins in rat ovary and its effect on estrogen production

    PubMed Central

    Inaoka, Yoshihiko; Yazawa, Takashi; Mizutani, Tetsuya; Kokame, Koichi; Kangawa, Kenji; Uesaka, Miki; Umezawa, Akihiro; Miyamoto, Kaoru

    2008-01-01

    Background P450 oxidoreductase (POR) catalyzes electron transfer to microsomal P450 enzymes. Its deficiency causes Antley-Bixler syndrome (ABS), and about half the patients with ABS have ambiguous genitalia and/or impaired steroidogenesis. POR mRNA expression is up-regulated when mesenchymal stem cells (MSCs) differentiate into steroidogenic cells, suggesting that the regulation of POR gene expression is important for steroidogenesis. In this context we examined the regulation of POR expression in ovarian granulosa cells by gonadotropins, and its possible role in steroidogenesis. Methods Changes in gene expression in MSCs during differentiation into steroidogenic cells were examined by DNA microarray analysis. Changes in mRNA and protein expression of POR in the rat ovary or in granulosa cells induced by gonadotropin treatment were examined by reverse transcription-polymerase chain reaction and western blotting. Effects of transient expression of wild-type or mutant (R457H or V492E) POR proteins on the production of estrone in COS-7 cells were examined in vitro. Effects of POR knockdown were also examined in estrogen producing cell-line, KGN cells. Results POR mRNA was induced in MSCs following transduction with the SF-1 retrovirus, and was further increased by cAMP treatment. Expression of POR mRNA, as well as Cyp19 mRNA, in the rat ovary were induced by equine chorionic gonadotropin and human chorionic gonadotropin. POR mRNA and protein were also induced by follicle stimulating hormone in primary cultured rat granulosa cells, and the induction pattern was similar to that for aromatase. Transient expression of POR in COS-7 cells, which expressed a constant amount of aromatase protein, greatly increased the rate of conversion of androstenedione to estrone, in a dose-dependent manner. The expression of mutant POR proteins (R457H or V492E), such as those found in ABS patients, had much less effect on aromatase activity than expression of wild-type POR proteins. Knockdown of endogenous POR protein in KGN human granulosa cells led to reduced estrone production, indicating that endogenous POR affected aromatase activity. Conclusion We demonstrated that the expression of POR, together with that of aromatase, was regulated by gonadotropins, and that its induction could up-regulate aromatase activity in the ovary, resulting in a coordinated increase in estrogen production. PMID:19077323

  14. Tungsten-dependent formaldehyde ferredoxin oxidoreductase: Reaction mechanism from quantum chemical calculations

    E-print Network

    Liao, Rongzhen

    theory Enzyme catalysis Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus is a tungsten-ray crystal structure of the native enzyme. Based on the calculations, we propose a new mechanism in which, and the nature of the axial ligands, tungsten enzymes have been divided into three different families: aldehyde

  15. Quinoline oxidoreductase from Pseudomonas putida 86: an improved purification procedure and electron paramagnetic resonance spectroscopy.

    PubMed

    Tshisuaka, B; Kappl, R; Hüttermann, J; Lingens, F

    1993-11-30

    Quinoline oxidoreductase, an iron-sulfur molybdenum flavoprotein containing flavin adenine dinucleotide and molybdopterin cytosine dinucleotide, was purified from Pseudomonas putida 86 to homogeneity. The various electron-transfer centers of the purified enzyme were examined by electron paramagnetic resonance spectroscopy. Quinoline deuterated at position 2 was prepared by deuterodecarboxylation of 2-quinolinecarboxylic acid. Quinoline added to the enzyme elicited the Mo(V) "rapid" type Q signal arising from the complex of enzyme and substrate, whereas in oxidized quinoline oxidoreductase a Mo(V) "resting" signal was observed. EPR spectroscopy at helium temperatures below 70 K revealed the existence of two types of iron-sulfur centers, Fe-S I and Fe-S II. An organic free radical appeared upon reduction with sodium dithionite. Inactivation of the enzyme by cyanide led to the inactive desulfo quinoline oxidoreductase, which yielded another Mo(V) signal designated "slow" type Q upon reduction with dithionite. Desulfo quinoline oxidoreductase was partially reactivated by incubation with sulfide. PMID:8251516

  16. Xanthine oxido-reductase, free radicals and cardiovascular disease. A critical review.

    PubMed

    Robert, A M; Robert, L

    2014-01-01

    Free radical mediated pathologies occupy a special place in medical semiology and in mechanistic interpretation of diseases. Free radicals, or better reactive oxygen species (ROS) or reactive nitrogen species (RNS) play also an important role in cell signaling. This is the basis of the ambivalent (Jekyll-Hyde) situation of ROS in biology and pathology. Aging itself is attributed by a popular theory to free radicals. A number of ROS-scavenging substances and procedures were described without however reaching credibility for their therapeutic value. An interesting exception is the xanthine oxido-reductase produced ROS and their role in cardiovascular disease. Allopurinol inhibition of xanthine oxido-reductase was shown to be efficient in some cases of cardiovascular diseases. Another important aspect of xanthine oxido-reductase produced ROS is their antibacterial capacity considered to be of importance with newborns fed on milk rich in this enzyme as well as at the gastrointestinal barrier. This ambivalent role of xanthine oxido-reductase justifies this review on the basic enzymatic mechanisms involved, derived ROS production, their role in the above mentioned biological processes and especially the interest of the inhibition of this enzyme as a preventive or curative measure in some cardiovascular pathologies. PMID:24127160

  17. The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria

    Microsoft Academic Search

    Lothar Kussmaul; Judy Hirst

    2006-01-01

    NADH:ubiquinone oxidoreductase (complex I) is a major source of reactive oxygen species in mitochondria and a significant contributor to cellular oxidative stress. Here, we describe the kinetic and molecular mechanism of superoxide production by complex I isolated from bovine heart mitochondria and confirm that it produces predominantly superoxide, not hydrogen peroxide. Redox titrations and electron paramagnetic resonance spectroscopy exclude the

  18. Ferredoxin:NADP Oxidoreductase Is a Subunit of the Chloroplast Cytochrome b6f Complex*

    E-print Network

    Cramer, William A.

    Ferredoxin:NADP Oxidoreductase Is a Subunit of the Chloroplast Cytochrome b6f Complex* Received of California, Los Angeles, California 90095-1569 Purified detergent-soluble cytochrome b6f complex from of 0.9 (cytochrome f ) 1 . The extra polypeptide did not stain for heme and was much more accessible

  19. 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...

  20. 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

  1. NAD(P)H cytochrome b5 oxidoreductase deficiency in Leishmania major results in impaired linoleate synthesis followed by increased oxidative stress and cell death.

    PubMed

    Mukherjee, Supratim; Sen Santara, Sumit; Das, Shantanabha; Bose, Moumita; Roy, Jayasree; Adak, Subrata

    2012-10-12

    NAD(P)H cytochrome b(5) oxidoreductase (Ncb5or), comprising cytochrome b(5) and cytochrome b(5) reductase domains, is widely distributed in eukaryotic organisms. Although Ncb5or plays a crucial role in lipid metabolism of mice, so far no Ncb5or gene has been reported in the unicellular parasitic protozoa Leishmania species. We have cloned, expressed, and characterized Ncb5or gene from Leishmania major. Steady state catalysis and spectral studies show that NADH can quickly reduce the ferric state of the enzyme to the ferrous state and is able to donate an electron(s) to external acceptors. To elucidate its exact physiological role in Leishmania, we attempted to create NAD(P)H cytochrome b(5) oxidoreductase from L. major (LmNcb5or) knock-out mutants by targeted gene replacement technique. A free fatty acid profile in knock-out (KO) cells reveals marked deficiency in linoleate and linolenate when compared with wild type (WT) or overexpressing cells. KO culture has a higher percentage of dead cells compared with both WT and overexpressing cells. Increased O(2) uptake, uncoupling and ATP synthesis, and loss of mitochondrial membrane potential are evident in KO cells. Flow cytometric analysis reveals the presence of a higher concentration of intracellular H(2)O(2), indicative of increased oxidative stress in parasites lacking LmNcb5or. Cell death is significantly reduced when the KO cells are pretreated with BSA bound linoleate. Real time PCR studies demonstrate a higher ?12 desaturase, superoxide dismutase, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA with a concomitant fall in ?9 desaturase mRNA expression in LmNcb5or null cell line. Together these findings suggest that decreased linoleate synthesis, and increased oxidative stress and apoptosis are the major consequences of LmNcb5or deficiency in Leishmania. PMID:22923617

  2. 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.

  3. Rhodobacter sphaeroides mutants overexpressing chlorophyllide a oxidoreductase of Blastochloris viridis elucidate functions of enzymes in late bacteriochlorophyll biosynthetic pathways

    PubMed Central

    Tsukatani, Yusuke; Harada, Jiro; Nomata, Jiro; Yamamoto, Haruki; Fujita, Yuichi; Mizoguchi, Tadashi; Tamiaki, Hitoshi

    2015-01-01

    In previous studies we have demonstrated that chlorophyllide a oxidoreductases (CORs) from bacteriochlorophyll (BChl) a-producing Rhodobacter species and BChl b-producing Blastochloris viridis show distinct substrate recognition and different catalytic hydrogenation reactions, and that these two types of CORs therefore cause committed steps for BChls a and b biosynthesis. In this study, COR genes from B.?viridis were incorporated and overexpressed in a series of Rhodobacter sphaeroides mutants. We found that the following two factors are essential in making R. sphaeroides produce BChl b: the loss of functions of both intrinsic COR and 8-vinyl reductase (BciA) in the host R. sphaeroides strain; and expression of the BchYZ catalytic components of COR from B. viridis, not the complete set of COR (BchXYZ), in the host strain. In addition, we incorporated bchYZ of B. viridis into the R. sphaeroides mutant lacking BchJ and BciA, resulting in the strain accumulating both BChl a and BChl b. This is the first example of an anoxygenic photosynthetic bacterium producing BChls a and b together. The results suggest that BchJ enhances activity of the intrinsic COR. The physiological significance of BchJ in pigment biosynthetic pathways will be discussed. PMID:25978726

  4. A Conserved Histidine-Aspartate Pair Is Required for Exovinyl Reduction of Biliverdin by a Cyanobacterial Phycocyanobilin:Ferredoxin Oxidoreductase

    Microsoft Academic Search

    Shih-Long Tu; Wesley Sughrue; R. David Britt; J. Clark Lagarias

    2005-01-01

    Phycocyanobilin:ferredoxin oxidoreductase is a member of the ferredoxin-dependent bilin reductase family and catalyzes two vinyl reductions of biliverdin IX to produce phycocyanobilin, the pig- ment precursor of both phytochrome and phycobiliprotein chro- mophores in cyanobacteria. Atypically for ferredoxin-dependent enzymes, phycocyanobilin:ferredoxin oxidoreductase mediates direct electron transfers from reduced ferredoxin to its tetrapyrrole substrate without metal ion or organic cofactors. We previously

  5. Identification of a Cytochrome b-Type NAD(P)H Oxidoreductase Ubiquitously Expressed in Human Cells

    Microsoft Academic Search

    Hao Zhu; Huawei Qiu; Hae-Won Patricia Yoon; Shuning Huang; H. Franklin Bunn

    1999-01-01

    Cytochrome b-type NAD(P)H oxidoreductases are involved in many physiological processes, including iron uptake in yeast, the respiratory burst, and perhaps oxygen sensing in mammals. We have identified a cytosolic cytochrome b-type NAD(P)H oxidoreductase in mammals, a flavohemoprotein (b5+b5R) containing cytochrome b5 (b5) and b5 reductase (b5R) domains. A genetic approach, using BLAST searches against DBEST for FAD-, NAD(P)H-binding sequences followed

  6. Towards a systematic analysis of human short-chain dehydrogenases/reductases (SDR): Ligand identification and structure-activity relationships.

    PubMed

    Bhatia, Chitra; Oerum, Stephanie; Bray, James; Kavanagh, Kathryn L; Shafqat, Naeem; Yue, Wyatt; Oppermann, Udo

    2015-06-01

    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. PMID:25526675

  7. Transcriptional regulation of nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase in murine hepatoma cells by 6-(methylsufinyl)hexyl isothiocyanate, an active principle of wasabi (Eutrema wasabi Maxim).

    PubMed

    Hou, D X; Fukuda, M; Fujii, M; Fuke, Y

    2000-12-20

    Wasabi is a very popular pungent spice in Japan. This study examined the ability of 6-(methylsufinyl)hexyl isothiocyanate (6-MITC), an active principle of wasabi, to induce the cellular expression of nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase (QR) in Hepa 1c1c7 cells. The cells were treated with various concentrations of 6-MITC, and were then assessed for cell growth, QR activity and QR mRNA expression. The induction of QR activity and QR mRNA expression was time- and dose-responsive over a narrow range of 0.1-5 microM, with declining induction at higher concentrations due to cell toxicity. Furthermore, transfection studies demonstrated that the induction of transcription of the QR gene by 6-MITC involved an antioxidant/electrophile-responsive element (ARE/EpRE) activation. Our results suggest a novel mechanism by which dietary wasabi 6-MITC may be implicated in cancer chemoprevention. PMID:11090969

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

    SciTech Connect

    Fernandes, Andreia S. [Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Av. da Republica, Apartado 127, 2784-505 Oeiras (Portugal); Konstantinov, Alexander A. [Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Av. da Republica, Apartado 127, 2784-505 Oeiras (Portugal); A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119992 Moscow (Russian Federation); Teixeira, Miguel [Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Av. da Republica, Apartado 127, 2784-505 Oeiras (Portugal); Pereira, Manuela M. [Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Av. da Republica, Apartado 127, 2784-505 Oeiras (Portugal)]. 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.

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

    Microsoft Academic Search

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

    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 5weeks. Chicks were weighed twice weekly and leukocyte oxidative activity

  10. Iron (III) reduction: A novel activity of the human NAD(P)H:oxidoreductase

    Microsoft Academic Search

    Rob U. Onyenwoke; Juergen. Wiegel

    2007-01-01

    NAD(P)H:quinone oxidoreductase (NQO1; EC 1.6.99.2) catalyzes a two-electron transfer involved in the protection of cells from reactive oxygen species. These reactive oxygen species are often generated by the one-electron reduction of quinones or quinone analogs. We report here on the previously unreported Fe(III) reduction activity of human NQO1. Under steady state conditions with Fe(III) citrate, the apparent Michaelis–Menten constant (Kmapp)

  11. Mutant P450 oxidoreductase causes disordered steroidogenesis with and without Antley-Bixler syndrome

    Microsoft Academic Search

    C. E. Fluck; Toshihro Tajima; A. V. Pandey; Wiebke Arlt; Kouji Okuhara; Charles F Verge; Ethylin Wang Jabs; B. B. Mendonca; Kenji Fujieda; Walter L Miller

    2004-01-01

    Deficient activities of multiple steroidogenic enzymes have been reported without and with Antley-Bixler syndrome (ABS), but mutations of corresponding cytochrome P450 enzymes have not been found. We identified mutations in POR, encoding P450 oxidoreductase, the obligate electron donor for these enzymes, in a woman with amenorrhea and three children with ABS, even though knock-out of POR is embryonically lethal in

  12. Crystal Structure of the Xanthine Oxidase-Related Aldehyde Oxido-Reductase from D. gigas

    Microsoft Academic Search

    Maria J. Romao; Margarida Archer; Isabel Moura; Jose J. G. Moura; Jean Legall; Richard Engh; Monika Schneider; Peter Hof; Robert Huber

    1995-01-01

    The crystal structure of the aldehyde oxido-reductase (Mop) from the sulfate reducing anaerobic Gram-negative bacterium Desulfovibrio gigas has been determined at 2.25 overset{circ}{mathrm A} resolution by multiple isomorphous replacement and refined. The protein, a homodimer of 907 amino acid residues subunits, is a member of the xanthine oxidase family. The protein contains a molybdopterin cofactor (Mo-co) and two different [2Fe-2S

  13. Amiloride inhibition of the proton-translocating NADH-quinone oxidoreductase of mammals and bacteria

    Microsoft Academic Search

    Eiko Nakamaru-Ogiso; Byoung Boo Seo; Takao Yagi; Akemi Matsuno-Yagi

    2003-01-01

    The proton-translocating NADH-quinone oxidoreductase in mitochondria (complex I) and bacteria (NDH-1) was shown to be inhibited by amiloride derivatives that are known as specific inhibitors for Na+\\/H+ exchangers. In bovine submitochondrial particles, the effective concentrations were about the same as those for the Na+\\/H+ exchangers, whereas in bacterial membranes the inhibitory potencies were lower. These results together with our earlier

  14. The proton-pumping NADH:ubiquinone oxidoreductase (complex I) of Aquifex aeolicus

    Microsoft Academic Search

    Dierk Scheide; Robert Huber; Thorsten Friedrich

    2002-01-01

    The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first energy-transducing complex of many respiratory chains. Homologues of complex I are present in the three domains of life. Here, we report the properties of complex I in membranes of the hyperthermophilic bacterium Aquifex aeolicus. The complex reacted with NADH but not with NADPH and F420H2 as electron donors. Short-chain

  15. Reaction of electron-transfer flavoprotein with electron-transfer flavoprotein-ubiquinone oxidoreductase

    Microsoft Academic Search

    Joe D. Beckmann; Frank E. Frerman

    1985-01-01

    The oxidative half-reaction of electron-transfer flavoprotein (ETF), electron transfer from ETF to electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), is dependent on complementary surface charges on the two proteins. ETF is the positively charged member of the redox pair. The evidence is based on the pH and ionic strength dependencies of the comproportionation of oxidized ETF and ETF hydroquinone catalyzed by ETF-QO and

  16. NAD(P)H-utilizing oxidoreductases of the plasma membrane An overview of presently purified proteins

    Microsoft Academic Search

    A. Bérczi; H. Asard

    1995-01-01

    Summary A considerable number of studies have demonstrated the presence of NAD(P)-oxidoreductases in the plant and animal cell plasma membranes. Recently several attempts on the isolation and purification of these proteins have been presented. The results indicate the presence of distinct NAD(P)H-utilizing enzymes in the plasma membrane of several species. Proteins with molecular masses of 27 kDa, 31 kDa, 36–39

  17. EPR Studies of Xanthine Oxidoreductase and Other Molybdenum-Containing Hydroxylases

    Microsoft Academic Search

    Russ Hille

    \\u000a A summary is provided both of early work establishing the different types of EPR signals manifested by the molybdenum centers\\u000a of enzymes such as xanthine oxidoreductase and of more recent studies, frequently employing more advanced EPR-related methods,\\u000a which have contributed significantly to our understanding of the physical and electronic structures of the enzyme active site.\\u000a Structures for the signal-giving species,

  18. Purification and Characterization of an NAD(P)H:Quinone Oxidoreductase from Glycine Max Seedlings

    Microsoft Academic Search

    A. Rescigno; F. Sollai; S. Masala; M. C. Porcu; E. Sanjust; A. C. Rinaldi; N. Curreli; D. Grifi; Augusto Rinaldi

    1995-01-01

    An NAD(P)H:(quinone acceptor) oxidoreductase (EC 1.6.99.2) was purified from Glycine max seedlings by means of chromatographic procedures. After 1371-fold purification, the enzyme showed a single band in IEF corresponding to an isoelectric point of 6.1. A single band was also found in native-PAGE both by activity staining and Coomassie brilliant blue staining. The molecular mass determined in SDS-PAGE was 21900

  19. Strategies to develop malic acid biosensors based on malate quinone oxidoreductase (MQO)

    Microsoft Academic Search

    Bogdan Bucur; Elena Mallat; Ana-Maria Gurban; Yana Gocheva; Carine Velasco; Jean-Louis Marty; Thierry Noguer

    2006-01-01

    An amperometric biosensor based on malate quinone oxidoreductase (MQO) was developed for monitoring of the malolactic fermentation of wines. Screen-printed electrodes coupled with appropriate mediators were used as transducers for this novel biosensor. MQO was immobilized by physical entrapment in a photo-cross-linkable poly(vinyl alcohol) polymer (PVA-SbQ) on the surface of the working electrode. Several electrochemical mediators were studied in order

  20. Two binding sites of inhibitors in NADH: ubiquinone oxidoreductase (complex I). Relationship of one site with the ubiquinone-binding site of bacterial glucose:ubiquinone oxidoreductase.

    PubMed

    Friedrich, T; van Heek, P; Leif, H; Ohnishi, T; Forche, E; Kunze, B; Jansen, R; Trowitzsch-Kienast, W; Höfle, G; Reichenbach, H

    1994-01-15

    The effect of ten naturally occurring and two synthetic inhibitors of NADH:ubiquinone oxidoreductase (complex I) of bovine heart, Neurospora crassa and Escherichia coli and glucose:ubiquinone oxidoreductase (glucose dehydrogenase) of Gluconobacter oxidans was investigated. These inhibitors could be divided into two classes with regard to their specificity and mode of action. Class I inhibitors, including the naturally occurring piericidin A, annonin VI, phenalamid A2, aurachins A and B, thiangazole and the synthetic fenpyroximate, inhibit complex I from all three species in a partially competitive manner and glucose dehydrogenase in a competitive manner, both with regard to ubiquinone. Class II inhibitors including the naturally occurring rotenone, phenoxan, aureothin and the synthetic benzimidazole inhibit complex I from all species in an non-competitive manner, but have no effect on the glucose dehydrogenase. Myxalamid PI could not be classified as above because it inhibits only the mitochondrial complex I and in a competitive manner. All inhibitors affect the electron-transfer step from the high-potential iron-sulphur cluster to ubiquinone. Class I inhibitors appear to act directly at the ubiquinone-catalytic site which is related in complex I and glucose dehydrogenase. PMID:8307034

  1. 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 ?M 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. PMID:20956531

  2. Identification and Characterization of Oxalate Oxidoreductase, a Novel Thiamine Pyrophosphate-dependent 2-Oxoacid Oxidoreductase That Enables Anaerobic Growth on Oxalate*

    PubMed Central

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

    2010-01-01

    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 CO2 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 CO2 or bicarbonate. Like other members of the 2-oxoacid:ferredoxin oxidoreductase family, OOR contains thiamine pyrophosphate and three [Fe4S4] clusters. However, unlike previously characterized members of this family, OOR does not use coenzyme A as a substrate. Oxalate is oxidized with a kcat of 0.09 s?1 and a Km of 58 ?m 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. PMID:20956531

  3. The Structure of an Oxalate Oxidoreductase Provides Insight into Microbial 2-Oxoacid Metabolism.

    PubMed

    Gibson, Marcus I; Brignole, Edward J; Pierce, Elizabeth; Can, Mehmet; Ragsdale, Stephen W; Drennan, Catherine L

    2015-07-01

    Thiamine pyrophosphate (TPP), a derivative of vitamin B1, is a versatile and ubiquitous cofactor. When coupled with [4Fe-4S] clusters in microbial 2-oxoacid:ferredoxin oxidoreductases (OFORs), TPP is involved in catalyzing low-potential redox reactions that are important for the synthesis of key metabolites and the reduction of N2, H(+), and CO2. We have determined the high-resolution (2.27 Å) crystal structure of the TPP-dependent oxalate oxidoreductase (OOR), an enzyme that allows microbes to grow on oxalate, a widely occurring dicarboxylic acid that is found in soil and freshwater and is responsible for kidney stone disease in humans. OOR catalyzes the anaerobic oxidation of oxalate, harvesting the low-potential electrons for use in anaerobic reduction and fixation of CO2. We compare the OOR structure to that of the only other structurally characterized OFOR family member, pyruvate:ferredoxin oxidoreductase. This side-by-side structural analysis highlights the key similarities and differences that are relevant for the chemistry of this entire class of TPP-utilizing enzymes. PMID:26061898

  4. Diversity and Function of Mutations in P450 Oxidoreductase in Patients with Antley-Bixler Syndrome and Disordered Steroidogenesis

    PubMed Central

    Huang, Ningwu; Pandey, Amit V.; Agrawal, Vishal; Reardon, William; Lapunzina, Pablo D.; Mowat, David; Jabs, Ethylin Wang; Vliet, Guy Van; Sack, Joseph; Flück, Christa E.; Miller, Walter L.

    2005-01-01

    P450 oxidoreductase (POR) is the obligatory flavoprotein intermediate that transfers electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to all microsomal cytochrome P450 enzymes. Although mouse Por gene ablation causes embryonic lethality, POR missense mutations cause disordered steroidogenesis, ambiguous genitalia, and Antley-Bixler syndrome (ABS), which has also been attributed to fibroblast growth factor receptor 2 (FGFR2) mutations. We sequenced the POR gene and FGFR2 exons 8 and 10 in 32 individuals with ABS and/or hormonal findings that suggested POR deficiency. POR and FGFR2 mutations segregated completely. Fifteen patients carried POR mutations on both alleles, 4 carried mutations on only one allele, 10 carried FGFR2 or FGFR3 mutations, and 3 patients carried no mutations. The 34 affected POR alleles included 10 with A287P (all from whites) and 7 with R457H (four Japanese, one African, two whites); 17 of the 34 alleles carried 16 “private” mutations, including 9 missense and 7 frameshift mutations. These 11 missense mutations, plus 10 others found in databases or reported elsewhere, were recreated by site-directed mutagenesis and were assessed by four assays: reduction of cytochrome c, oxidation of NADPH, support of 17?-hydroxylase activity, and support of 17,20 lyase using human P450c17. Assays that were based on cytochrome c, which is not a physiologic substrate for POR, correlated poorly with clinical phenotype, but assays that were based on POR’s support of catalysis by P450c17—the enzyme most closely associated with the hormonal phenotype—provided an excellent genotype/phenotype correlation. Our large survey of patients with ABS shows that individuals with an ABS-like phenotype and normal steroidogenesis have FGFR mutations, whereas those with ambiguous genitalia and disordered steroidogenesis should be recognized as having a distinct new disease: POR deficiency. PMID:15793702

  5. Characterizing a monotopic membrane enzyme. Biochemical, enzymatic and crystallization studies on Aquifex aeolicus sulfide:quinone oxidoreductase.

    PubMed

    Marcia, Marco; Langer, Julian D; Parcej, David; Vogel, Vitali; Peng, Guohong; Michel, Hartmut

    2010-11-01

    Monotopic membrane proteins are membrane proteins that interact with only one leaflet of the lipid bilayer and do not possess transmembrane spanning segments. They are endowed with important physiological functions but until now only few of them have been studied. Here we present a detailed biochemical, enzymatic and crystallographic characterization of the monotopic membrane protein sulfide:quinone oxidoreductase. Sulfide:quinone oxidoreductase is a ubiquitous enzyme involved in sulfide detoxification, in sulfide-dependent respiration and photosynthesis, and in heavy metal tolerance. It may also play a crucial role in mammals, including humans, because sulfide acts as a neurotransmitter in these organisms. We isolated and purified sulfide:quinone oxidoreductase from the native membranes of the hyperthermophilic bacterium Aquifex aeolicus. We studied the pure and solubilized enzyme by denaturing and non-denaturing polyacrylamide electrophoresis, size-exclusion chromatography, cross-linking, analytical ultracentrifugation, visible and ultraviolet spectroscopy, mass spectrometry and electron microscopy. Additionally, we report the characterization of its enzymatic activity before and after crystallization. Finally, we discuss the crystallization of sulfide:quinone oxidoreductase in respect to its membrane topology and we propose a classification of monotopic membrane protein crystal lattices. Our data support and complement an earlier description of the three-dimensional structure of A. aeolicus sulfide:quinone oxidoreductase (M. Marcia, U. Ermler, G. Peng, H. Michel, Proc Natl Acad Sci USA, 106 (2009) 9625-9630) and may serve as a reference for further studies on monotopic membrane proteins. PMID:20691146

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

    PubMed

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

    2015-07-14

    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 (?BbbqrA) showed increased growth inhibition, whereas B. bassiana overexpressing BbbqrA (Bb::BbbqrA(O)) 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 ?BbbqrA strain showed decreased virulence toward T. castaneum, whereas overexpression of BbbqrA increased mortality versus T. castaneum. No change in virulence was seen for the ?BbbqrA or Bb::BbbqrA(O) 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

  7. Simultaneous Involvement of a Tungsten-Containing Aldehyde:Ferredoxin Oxidoreductase and a Phenylacetaldehyde Dehydrogenase in Anaerobic Phenylalanine Metabolism

    PubMed Central

    Debnar-Daumler, Carlotta; Seubert, Andreas; Schmitt, Georg

    2014-01-01

    Anaerobic phenylalanine metabolism in the denitrifying betaproteobacterium Aromatoleum aromaticum is initiated by conversion of phenylalanine to phenylacetate, which is further metabolized via benzoyl-coenzyme A (CoA). The formation of phenylacetate is catalyzed by phenylalanine transaminase, phenylpyruvate decarboxylase, and a phenylacetaldehyde-oxidizing enzyme. The presence of these enzymes was detected in extracts of cells grown with phenylalanine and nitrate. We found that two distinct enzymes are involved in the oxidation of phenylacetaldehyde to phenylacetate, an aldehyde:ferredoxin oxidoreductase (AOR) and a phenylacetaldehyde dehydrogenase (PDH). Based on sequence comparison, growth studies with various tungstate concentrations, and metal analysis of the enriched enzyme, AOR was shown to be a tungsten-containing enzyme, necessitating specific cofactor biosynthetic pathways for molybdenum- and tungsten-dependent enzymes simultaneously. We predict from the genome sequence that most enzymes of molybdopterin biosynthesis are shared, while the molybdate/tungstate uptake systems are duplicated and specialized paralogs of the sulfur-inserting MoaD and the metal-inserting MoeA proteins seem to be involved in dedicating biosynthesis toward molybdenum or tungsten cofactors. We also characterized PDH biochemically and identified both NAD+ and NADP+ as electron acceptors. We identified the gene coding for the enzyme and purified a recombinant Strep-tagged PDH variant. The homotetrameric enzyme is highly specific for phenylacetaldehyde, has cooperative kinetics toward the substrate, and shows considerable substrate inhibition. Our data suggest that A. aromaticum utilizes PDH as the primary enzyme during anaerobic phenylalanine degradation, whereas AOR is not essential for the metabolic pathway. We hypothesize a function as a detoxifying enzyme if high aldehyde concentrations accumulate in the cytoplasm, which would lead to substrate inhibition of PDH. PMID:24214948

  8. Human sulfide:quinone oxidoreductase catalyzes the first step in hydrogen sulfide metabolism and produces a sulfane sulfur metabolite.

    PubMed

    Jackson, Michael R; Melideo, Scott L; Jorns, Marilyn Schuman

    2012-08-28

    Sulfide:quinone oxidoreductase (SQOR) is a membrane-bound enzyme that catalyzes the first step in the mitochondrial metabolism of H(2)S. Human SQOR is successfully expressed at low temperature in Escherichia coli by using an optimized synthetic gene and cold-adapted chaperonins. Recombinant SQOR contains noncovalently bound FAD and catalyzes the two-electron oxidation of H(2)S to S(0) (sulfane sulfur) using CoQ(1) as an electron acceptor. The prosthetic group is reduced upon anaerobic addition of H(2)S in a reaction that proceeds via a long-wavelength-absorbing intermediate (?(max) = 673 nm). Cyanide, sulfite, or sulfide can act as the sulfane sulfur acceptor in reactions that (i) exhibit pH optima at 8.5, 7.5, or 7.0, respectively, and (ii) produce thiocyanate, thiosulfate, or a putative sulfur analogue of hydrogen peroxide (H(2)S(2)), respectively. Importantly, thiosulfate is a known intermediate in the oxidation of H(2)S by intact animals and the major product formed in glutathione-depleted cells or mitochondria. Oxidation of H(2)S by SQOR with sulfite as the sulfane sulfur acceptor is rapid and highly efficient at physiological pH (k(cat)/K(m,H(2)S) = 2.9 × 10(7) M(-1) s(-1)). A similar efficiency is observed with cyanide, a clearly artificial acceptor, at pH 8.5, whereas a 100-fold lower value is seen with sulfide as the acceptor at pH 7.0. The latter reaction is unlikely to occur in healthy individuals but may become significant under certain pathological conditions. We propose that sulfite is the physiological acceptor of the sulfane sulfur and that the SQOR reaction is the predominant source of the thiosulfate produced during H(2)S oxidation by mammalian tissues. PMID:22852582

  9. 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

  10. Purification and characterization of pyruvate:NADP+ oxidoreductase in Euglena gracilis.

    PubMed

    Inui, H; Ono, K; Miyatake, K; Nakano, Y; Kitaoka, S

    1987-07-01

    Pyruvate:NADP+ oxidoreductase was homogeneously purified from crude extract of Euglena gracilis. The Mr of the enzyme was estimated to be 309,000 by gel filtration. The enzyme migrated as a single protein band with Mr of 166,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the enzyme consists of two identical polypeptides. The absorption spectrum of the native enzyme exhibited maxima at 278, 380, and 430 nm, and a broad shoulder was observed around 480 nm; the maximum at 430 nm was eliminated by reduction of the enzyme with dithionite. Reduction of the enzyme with pyruvate and CoA and reoxidation with NADP+ were proved from changes of absorption spectra. The enzyme contained 2 molecules of FAD and 8 molecules of iron. It was also indicated that the enzyme was thiamine pyrophosphate-dependent. The enzyme was oxygen-sensitive, and the reaction was affected by the presence of oxygen. Pyruvate was the most active substrate, but the enzyme was slightly active for 2-oxobutyrate, 3-hydroxypyruvate, and oxalacetate, but not for glyoxylate and 2-oxoglutarate. The native electron acceptor was NADP+, whereas NAD+ was completely inactive. Methyl viologen, benzyl viologen, FAD, and FMN were utilized as artificial electron acceptors, whereas spinach and Clostridium ferredoxins were inactive. Pyruvate synthesis by reductive carboxylation of acetyl-CoA with NADPH as the electron donor occurred by the reverse reaction of the enzyme. The enzyme also catalyzed a pyruvate-CO2 exchange reaction and electron-transfer reaction from NADPH to other electron acceptors like methyl viologen. These results indicate that pyruvate:NADP+ oxidoreductase in E. gracilis is clearly distinct from either the pyruvate dehydrogenase multienzyme complex or pyruvate:ferredoxin oxidoreductase. PMID:3110154

  11. Insight into the Radical Mechanism of Phycocyanobilin-Ferredoxin Oxidoreductase (PcyA) Revealed by X-ray Crystallography and Biochemical Measurements

    E-print Network

    Fisher, Andrew J.

    Insight into the Radical Mechanism of Phycocyanobilin-Ferredoxin Oxidoreductase (PcyA) Revealed-ray crystal structure of the substrate-free form of phycocyanobilin (PCB)-ferredoxin oxidoreductase (PcyA; EC-electron reduction of biliverdin IXR to 3E/3Z-phycocyanobilin via enzyme-bound bilin radical intermediates

  12. WrbA bridges bacterial flavodoxins and eukaryotic NAD(P)H:quinone oxidoreductases.

    PubMed

    Carey, Jannette; Brynda, Jiri; Wolfová, Julie; Grandori, Rita; Gustavsson, Tobias; Ettrich, Rüdiger; Smatanová, Ivana Kutá

    2007-10-01

    The crystal structure of the flavodoxin-like protein WrbA with oxidized FMN bound reveals a close relationship to mammalian NAD(P)H:quinone oxidoreductase, Nqo1. Structural comparison of WrbA, flavodoxin, and Nqo1 indicates how the twisted open-sheet fold of flavodoxins is elaborated to form multimers that extend catalytic function from one-electron transfer between protein partners using FMN to two-electron reduction of xenobiotics using FAD. The structure suggests a novel physiological role for WrbA and Nqo1. PMID:17893367

  13. Characterization of a soluble oxidoreductase from the thermophilic bacterium Carboxydothermus ferrireducens

    Microsoft Academic Search

    Rob Uche Onyenwoke; R. Geyer; Juergen Wiegel

    2009-01-01

    An NAD(P)H-dependent oxidoreductase has been purified approximately 40-fold from the soluble protein fraction of the dissimilatory\\u000a iron-reducing, anaerobic, thermophilic bacterium Carboxydothermus ferrireducens. The enzyme, a flavoprotein, has broad-substrate specificity—reducing Fe3+, Cr6+, and AQDS with rates of 0.31, 0.33, and 3.3 U mg?1 protein and calculated NADH oxidation turnover numbers of 0.25, 0.25, and 2.5 s?1, respectively. Numerous quinones are reduced via a

  14. Amiloride inhibition of the proton-translocating NADH-quinone oxidoreductase of mammals and bacteria.

    PubMed

    Nakamaru-Ogiso, Eiko; Seo, Byoung Boo; Yagi, Takao; Matsuno-Yagi, Akemi

    2003-08-14

    The proton-translocating NADH-quinone oxidoreductase in mitochondria (complex I) and bacteria (NDH-1) was shown to be inhibited by amiloride derivatives that are known as specific inhibitors for Na(+)/H(+) exchangers. In bovine submitochondrial particles, the effective concentrations were about the same as those for the Na(+)/H(+) exchangers, whereas in bacterial membranes the inhibitory potencies were lower. These results together with our earlier observation that the amiloride analogues prevent labeling of the ND5 subunit of complex I with a fenpyroximate analogue suggest the involvement of ND5 in H(+) (Na(+)) translocation and no direct involvement of electron carriers in H(+) (Na(+)) translocation. PMID:12914922

  15. Crystal structures of Pseudomonas syringae pv. tomato DC3000 quinone oxidoreductase and its complex with NADPH.

    PubMed

    Pan, Xiaowei; Zhang, Hongmei; Gao, Yu; Li, Mei; Chang, Wenrui

    2009-12-18

    Zeta-crystallin-like quinone oxidoreductase is NAD(P)H-dependent and catalyzes one-electron reduction of certain quinones to generate semiquinone. Here we present the crystal structures of zeta-crystallin-like quinone oxidoreductase from Pseudomonas syringae pv. tomato DC3000 (PtoQOR) and its complexes with NADPH determined at 2.4 and 2.01A resolutions, respectively. PtoQOR forms as a homologous dimer, each monomer containing two domains. In the structure of the PtoQOR-NADPH complex, NADPH locates in the groove between the two domains. NADPH binding causes obvious conformational changes in the structure of PtoQOR. The putative substrate-binding site of PtoQOR is wider than that of Escherichia coli and Thermus thermophilus HB8. Activity assays show that PtoQOR has weak 1,4-benzoquinone catalytic activity, and very strong reduction activity towards large substrates such as 9,10-phenanthrenequinone. We propose a model to explain the conformational changes which take place during reduction reactions catalyzed by PtoQOR. PMID:19818736

  16. Glucagon activation of the thiol:protein disulfide oxidoreductase in isolated, rat, hepatic microsomes

    SciTech Connect

    McConkey, D.J.; Crankshaw, D.L.; Holtzman, J.L.

    1986-05-01

    Thiol:protein disulfide oxidoreductase catalyzes the GSH reduction of protein disulfides to sulfhydryl groups. The authors determined this activity in washed rat hepatic microsomes (1) by a coupled reaction in which GSSG is reduced by GSH reductase and NADPH is oxidized and (2) by the cleavage of (/sup 125/I)-insulin (insulinase). Physiological concentrations of glucagon (GLU)(1 nM) with GSH (1 mM) increased both activities (NADPH oxidae - 1.1 nmol/min-mg prot (control)(C) to 4.3 (GLU); insulinase - 36 (C) to 83 (GLU)). For both assays stimulation was only seen with low protein concentrations (< 100 ..mu..g/ml), probably due to nonspecific GLU binding rather than proteolysis of the GLU since both reactions were linear for at least 30 min. The stimulation of NADPH oxidase had a P50 for GLU of 0.78 nM. GLU stimulation of insulinase was only observed in the presence of a GSH reducing system. Basal insulinase activity was unaffected by GSH reductase. These two observation suggest that the stimulation may be inhibited by the presence of GSSG. This effect was not due to depletion of GSH since the same effect was observed with higher GSH (5 mM). Although the effect on NADPH oxidase could represent activation of a GSH peroxidase, the insulinase data support the hypothesis that GLU may act by stimulating the thiol:protein disulfide oxidoreductase catalyzed reduction of protein disulfides.

  17. Role of human sulfide: quinone oxidoreductase in H2S metabolism.

    PubMed

    Jackson, Michael R; Melideo, Scott L; Jorns, Marilyn Schuman

    2015-01-01

    The first step in the mammalian metabolism of H2S is catalyzed by sulfide:quinone oxidoreductase (SQOR). Human SQOR is an integral membrane protein, which presumably interacts with the inner mitochondrial membrane in a monotopic fashion. The enzyme is a member of a family of flavoprotein disulfide oxidoreductases (e.g., glutathione reductase) that utilize a Cys-S-S-Cys disulfide bridge as an additional redox center. SQOR catalyzes a two-electron oxidation of H2S to sulfane sulfur using coenzyme Q as electron acceptor. The enzyme also requires a third substrate to act as the acceptor of the sulfane sulfur from a cysteine persulfide intermediate. Here, we describe a method for the bacterial expression of human SQOR as a catalytically active membrane-bound protein, procedures for solubilization and purification of the recombinant protein to >95% homogeneity, and spectrophotometric assays to monitor SQOR-mediated H2S oxidation in reactions with different sulfane sulfur acceptors. PMID:25725526

  18. Alcohol Dehydrogenase Genes & Proteins In Grapevine

    Microsoft Academic Search

    C. Tesniere; P. Abbal

    Alcohol dehydrogenase (ADH; alcohol: NAD oxidoreductase; EC 1.1.1.1) is a dimeric-zinc enzyme which catalyses the inter-conversion\\u000a of acetaldehyde to ethanol, using NAD\\/NADH as a cofactor. This is the terminal step of glycolysis, leading to fermentative\\u000a metabolism in anaerobic conditions. In this context, the evolution of this enzyme’s activity, and gene expression have been\\u000a widely investigated in response to anaerobiosis in

  19. Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Relaxation of functional constraint on light-independent protochlorophyllide oxidoreductase in Thuja.

    PubMed

    Kusumi, Junko; Sato, Aya; Tachida, Hidenori

    2006-05-01

    The light-independent protochlorophyllide oxidoreductase (DPOR) plays a key role in the ability of nonflowering plants and algae to synthesize chlorophyll in darkness. This enzyme consists of three subunits encoded by the chlB, chlL, and chlN genes in the plastid genome. Previously, we found a high nonsynonymous substitution rate (dN) of the chlL gene in the lineage of Thuja standishii, a conifer belonging to the Cupressaceae. Here we revealed that the acceleration of dN in the chlL occurred as well in other species of Thuja, Thuja occidentalis and Thuja plicata. In addition, dark-grown seedlings of T. occidentalis were found to exhibit a pale yellowish color, and their chlorophyll concentration was much lower than that of other species of Cupressaceae. The results suggested that the species of Thuja have lost the ability to synthesize chlorophyll in darkness, and the functional constraint on the DPOR would thus be expected to be relaxed in this genus. Therefore, we expected to find that the evolutionary rates of all subunits of DPOR would in this case be accelerated. Sequence analyses of the chlN and chlB (encoding the other subunits of DPOR) in 18 species of Cupressaceae revealed that the dN of the chlN gene was accelerated in Thuja as was the dN of the chlL gene, but the dN of the chlB gene did not appear to differ significantly among the species of Cupressaceae. Sequencing of reverse transcription-polymerase chain reaction (RT-PCR) products of these genes showed that RNA editing was rare and unlikely to have contributed to the acceleration. Moreover, the RT-PCR analysis indicated that all chl genes were still transcriptionally active in T. occidentalis. Based on these results, it appears that species of Thuja still bear the DPOR protein, although the enzyme has lost its activity because of nonsynonymous mutations of some of the chl genes. The lack of acceleration of the dN of the chlB gene might be accounted for by various unknown functions of its gene product. PMID:16428257

  20. Coordinated Production and Utilization of FADH2 by NAD(P)H-Flavin Oxidoreductase and 4-Hydroxyphenylacetate 3-Monooxygenase

    E-print Network

    Xie, Xiaoliang Sunney

    to be an NAD(P)H-flavin oxidoreductase (also known as flavin reductase) (3) that supplies FADH2 to HpaB. The 2,4,6-trichlorophenol 4-monooxygenase of Ralstonia eutropha JMP134 is the second characterized FADH2- utilizing

  1. 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...

  2. Lumen Thiol Oxidoreductase1, a Disulfide Bond-Forming Catalyst, Is Required for the Assembly of Photosystem II

    E-print Network

    Hamel, Patrice

    needed to reduce the active site of a disulfide reductase involved in the assembly of cytochromes c is that the CXXCH motif is first oxidized by the Dsb machinery and then reduced by the disulfide reductaseLumen Thiol Oxidoreductase1, a Disulfide Bond-Forming Catalyst, Is Required for the Assembly

  3. The Rate-limiting Step in the Cytochrome bc1 Complex (Ubiquinol-Cytochrome c Oxidoreductase) Is Not Changed by

    E-print Network

    Trumpower, Bernard L.

    The Rate-limiting Step in the Cytochrome bc1 Complex (Ubiquinol-Cytochrome c Oxidoreductase) Is Not Changed by Inhibition of Cytochrome b-dependent Deprotonation IMPLICATIONS FOR THE MECHANISM OF UBIQUINOL Hampshire 03755 Quinol oxidation at center P of the cytochrome bc1 complex involves bifurcated electron

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

    DOE PAGESBeta

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

    2015-01-01

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

    SciTech Connect

    Hunsperger, Heather M [Univ. of Washington, Seattle, WA (United States); Randhawa, Tejinder [Univ. of Washington, Seattle, WA (United States); Cattolico, Rose [Univ. of Washington, Seattle, WA (United States)

    2015-01-01

    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.

  6. 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

  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. PMID:1622279

  8. Proton pumping by NADH:ubiquinone oxidoreductase. A redox driven conformational change mechanism?

    PubMed

    Brandt, Ulrich; Kerscher, Stefan; Dröse, Stefan; Zwicker, Klaus; Zickermann, Volker

    2003-06-12

    The modular evolutionary origin of NADH:ubiquinone oxidoreductase (complex I) provides useful insights into its functional organization. Iron-sulfur cluster N2 and the PSST and 49 kDa subunits were identified as key players in ubiquinone reduction and proton pumping. Structural studies indicate that this 'catalytic core' region of complex I is clearly separated from the membrane. Complex I from Escherichia coli and Klebsiella pneumoniae was shown to pump sodium ions rather than protons. These new insights into structure and function of complex I strongly suggest that proton or sodium pumping in complex I is achieved by conformational energy transfer rather than by a directly linked redox pump. PMID:12788486

  9. Biochemical and genetic analysis of NAD(P)H:quinone oxidoreductase 1 (NQO1).

    PubMed

    Siegel, David; Kepa, Jadwiga K; Ross, David

    2007-01-01

    NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaphorase, E.C. 1.6.99.2) is an FAD containing obligate two-electron reductase that catalyzes the reduction of a broad range of substrates. This unit will describe methods for the detection of NQO1 protein in formalin-fixed, paraffin-embedded tissues by immunohistochemistry; detection of NQO1 protein in fresh tissues or cell lines by immunoblot analysis; measurement of NQO1 catalytic activity in fresh and frozen tissues and cell lines using spectrophotometric assays based upon the reduction of 2,6-dichlorophenol-indophenol (DCPIP) or coupled menadione-cytochrome reduction; and determination of the NQO1*2 polymorphism by PCR-RFLP. PMID:23045144

  10. A novel method for preparation of MNP@CS-tethered coenzyme for coupled oxidoreductase system.

    PubMed

    Chen, Guo; Wu, Zhichao; Ma, Yunhui

    2015-02-20

    The immobilized cofactor NAD(H) is easily recovered from the reaction bulk, which is essential for repeated use of NAD(H) in the bioprocess catalyzed by NAD(H)-dependent oxidoreductase. Here, a magnetic nanoparticle platform was designed to immobilize both of the NADH and the NAD(+). The design was based on chitosan-coated magnetic nanoparticles (MNP@CS) which was activated by the EDC/NHS with the aid of azelaic acid as spacer. Interestingly, the succinimide group at the end of spacer arm catalyzed direct coupling of a carboxyl-terminal to the 6-amino group of the adenine residue of NAD(H). Our results indicated that 150 ?mol NADH and 50 ?mol NAD(+) was effectively attached to 1g MNP@CS at 25°C in 120 min and the prepared MNP@CS-NAD(H) showed good activity according to the coupling reaction of benzyl alcohol and acetaldehyde catalyzed by alcohol dehydrogenase. PMID:25617681

  11. MICALs, a family of conserved flavoprotein oxidoreductases, function in plexin-mediated axonal repulsion.

    PubMed

    Terman, Jonathan R; Mao, Tianyi; Pasterkamp, R Jeroen; Yu, Hung-Hsiang; Kolodkin, Alex L

    2002-06-28

    Members of the semaphorin family of secreted and transmembrane proteins utilize plexins as neuronal receptors to signal repulsive axon guidance. It remains unknown how plexin proteins are directly linked to the regulation of cytoskeletal dynamics. Here, we show that Drosophila MICAL, a large, multidomain, cytosolic protein expressed in axons, interacts with the neuronal plexin A (PlexA) receptor and is required for Semaphorin 1a (Sema-1a)-PlexA-mediated repulsive axon guidance. In addition to containing several domains known to interact with cytoskeletal components, MICAL has a flavoprotein monooxygenase domain, the integrity of which is required for Sema-1a-PlexA repulsive axon guidance. Vertebrate orthologs of Drosophila MICAL are neuronally expressed and also interact with vertebrate plexins, and monooxygenase inhibitors abrogate semaphorin-mediated axonal repulsion. These results suggest a novel role for oxidoreductases in repulsive neuronal guidance. PMID:12110185

  12. Role of NAD(P)H:quinone oxidoreductase 1 (DT diaphorase) in protection against quinone toxicity.

    PubMed

    Joseph, P; Long, D J; Klein-Szanto, A J; Jaiswal, A K

    2000-07-15

    NQO1-/- mice, along with Chinese hamster ovary (CHO) cells, were used to determine the in vivo role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in cellular protection against quinone cytotoxicity, membrane damage, DNA damage, and carcinogenicity. CHO cells permanently expressing various levels of cDNA-derived P450 reductase and NQO1 were produced. Treatment of CHO cells overexpressing P450 reductase with menadione, benzo[a]pyrene-3,6-quinone (BPQ), and benzoquinone led to increased cytotoxicity as compared with CHO cells expressing endogenous P450 reductase. In a similar experiment, overexpression of NQO1 significantly protected CHO cells against the cytotoxicity of these quinones. Knockout (NQO1-/-) mice deficient in NQO1 protein and activity had been generated previously in our laboratory and were used in the present studies. Wild-type (NQO1+/+) and knockout (NQO1-/-) mice were given i.p. injections of menadione and BPQ, followed by analysis of membrane damage and DNA damage. Both menadione and BPQ induced lipid peroxidation in hepatic and non-hepatic tissues, indicating increased membrane damage. Exposure to BPQ also resulted in increased hepatic DNA adducts in NQO1-/- mice as compared with NQO1+/+ mice. The skin application of BPQ alone and BPQ + 12-O-tetradecanoylphorbol-13-acetate (TPA) failed to induce papillomas, or other lesions, for up to 50 weeks in either NQO1+/+ or NQO1-/- mice. The various results from CHO cells and NQO1-/- mice indicated that NQO1 protects against quinone-induced cytotoxicity, as well as DNA and membrane damage. The absence of BPQ-induced skin carcinogenicity in NQO1-/- mice may be related to the strain (C57BL/6) of mice used in the present study and/or due to poor BPQ absorption into the skin and/or due to detoxification of BPQ by cytosolic NRH:quinone oxidoreductase 2 (NQO2). PMID:10825465

  13. Iron-sulfur cluster-dependent catalysis of chlorophyllide a oxidoreductase from Roseobacter denitrificans.

    PubMed

    Kiesel, Svenja; Wätzlich, Denise; Lange, Christiane; Reijerse, Edward; Bröcker, Markus J; Rüdiger, Wolfhart; Lubitz, Wolfgang; Scheer, Hugo; Moser, Jürgen; Jahn, Dieter

    2015-01-01

    Bacteriochlorophyll a biosynthesis requires the stereo- and regiospecific two electron reduction of the C7-C8 double bond of chlorophyllide a by the nitrogenase-like multisubunit metalloenzyme, chlorophyllide a oxidoreductase (COR). ATP-dependent COR catalysis requires interaction of the protein subcomplex (BchX)2 with the catalytic (BchY/BchZ)2 protein to facilitate substrate reduction via two redox active iron-sulfur centers. The ternary COR enzyme holocomplex comprising subunits BchX, BchY, and BchZ from the purple bacterium Roseobacter denitrificans was trapped in the presence of the ATP transition state analog ADP·AlF4(-). Electron paramagnetic resonance experiments revealed a [4Fe-4S] cluster of subcomplex (BchX)2. A second [4Fe-4S] cluster was identified on (BchY/BchZ)2. Mutagenesis experiments indicated that the latter is ligated by four cysteines, which is in contrast to the three cysteine/one aspartate ligation pattern of the closely related dark-operative protochlorophyllide a oxidoreductase (DPOR). In subsequent mutagenesis experiments a DPOR-like aspartate ligation pattern was implemented for the catalytic [4Fe-4S] cluster of COR. Artificial cluster formation for this inactive COR variant was demonstrated spectroscopically. A series of chemically modified substrate molecules with altered substituents on the individual pyrrole rings and the isocyclic ring were tested as COR substrates. The COR enzyme was still able to reduce the B ring of substrates carrying modified substituents on ring systems A, C, and E. However, substrates with a modification of the distantly located propionate side chain were not accepted. A tentative substrate binding mode was concluded in analogy to the related DPOR system. PMID:25422320

  14. Synthetic Seleno-Glutaredoxin 3 Analogs are Highly Reducing Oxidoreductases with Enhanced Catalytic Efficiency

    PubMed Central

    Metanis, Norman; Keinan, Ehud; Davison, Philip E.

    2008-01-01

    Selenoenzymes have a central role in maintaining cellular redox potential. These enzymes have selenenylsulfide bonds in their active sites that catalyze the reduction of peroxides, sulfoxides and disulfides. The selenol/disufide exchange reaction is common to all of these enzymes and the active site redox potential reflects the ratio between the forward and reverse rates of this reaction. The preparation of enzymes containing selenocysteine (Sec) is experimentally challenging. As a result, little is known about the kinetic role of selenols in enzyme active sites, and the redox potential of a selenenylsulfide or diselenide bond in a protein has not been experimentally determined. In order to fully evaluate the effects of Sec on oxidoreductase redox potential and kinetics, glutaredoxin 3 (Grx3) and all three Sec variants of its conserved 11CXX14C active site were chemically synthesized. Grx3, Grx3(C11U) and Grx3(C14U) exhibited redox potentials of ?194, ?260 and ?275 mV, respectively. The position of redox equilibrium between Grx3(C11U-C14U) (?309 mV) and thioredoxin (Trx) (?270 mV) suggests a possible role for diselenide bonds in biological systems. Kinetic analysis is consistent with the hypothesis that the lower redox potentials of the Sec variants result primarily from the greater nucleophilicity of the active site selenium rather than its role as either a leaving group or a ‘central atom’ in the exchange reaction. The 102 to 104-fold increase in the rate of Trx reduction by the seleno-Grx3 analogs demonstrates that Oxidoreductases containing either selenenylsulfide or diselenide bonds can have physiologically compatible redox potentials and enhanced reduction kinetics in comparison with their sulfide counterparts. PMID:17177418

  15. Reaction Mechanism of Single Subunit NADH-Ubiquinone Oxidoreductase (Ndi1) from Saccharomyces cerevisiae

    PubMed Central

    Yang, Yu; Yamashita, Tetsuo; Nakamaru-Ogiso, Eiko; Hashimoto, Takeshi; Murai, Masatoshi; Igarashi, Junsuke; Miyoshi, Hideto; Mori, Nozomu; Matsuno-Yagi, Akemi; Yagi, Takao; Kosaka, Hiroaki

    2011-01-01

    The flavoprotein rotenone-insensitive internal NADH-ubiquinone (UQ) oxidoreductase (Ndi1) is a member of the respiratory chain in Saccharomyces cerevisiae. We reported previously that bound UQ in Ndi1 plays a key role in preventing the generation of reactive oxygen species. Here, to elucidate this mechanism, we investigated biochemical properties of Ndi1 and its mutants in which highly conserved amino acid residues (presumably involved in NADH and/or UQ binding sites) were replaced. We found that wild-type Ndi1 formed a stable charge transfer (CT) complex (around 740 nm) with NADH, but not with NADPH, under anaerobic conditions. The intensity of the CT absorption band was significantly increased by the presence of bound UQ or externally added n-decylbenzoquinone. Interestingly, however, when Ndi1 was exposed to air, the CT band transiently reached the same maximum level regardless of the presence of UQ. This suggests that Ndi1 forms a ternary complex with NADH and UQ, but the role of UQ in withdrawing an electron can be substitutable with oxygen. Proteinase K digestion analysis showed that NADH (but not NADPH) binding induces conformational changes in Ndi1. The kinetic study of wild-type and mutant Ndi1 indicated that there is no overlap between NADH and UQ binding sites. Moreover, we found that the bound UQ can reversibly dissociate from Ndi1 and is thus replaceable with other quinones in the membrane. Taken together, unlike other NAD(P)H-UQ oxidoreductases, the Ndi1 reaction proceeds through a ternary complex (not a ping-pong) mechanism. The bound UQ keeps oxygen away from the reduced flavin. PMID:21220430

  16. 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 17?-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 pregnancy, steroid ratios were only slightly elevated and estriol excretion was normal. Conclusion: Prenatal diagnosis in PORD is readily established via urinary steroid metabolite analysis of maternal urine. Visible malformations at prenatal ultrasound predict a severe malformation phenotype. PMID:23365120

  17. 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 [The University of Texas Health Science Center at San Antonio, Department of Biochemistry, San Antonio, TX 78229 (United States)] [The University of Texas Health Science Center at San Antonio, Department of Biochemistry, San Antonio, TX 78229 (United States); Panda, Satya P., E-mail: panda@uthscsa.edu [The University of Texas Health Science Center at San Antonio, Department of Biochemistry, San Antonio, TX 78229 (United States)

    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.

  18. (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.

  19. Genes encoding mercuric reductases from selected gram-negative aquatic bacteria have a low degree of homology with merA of transposon Tn501.

    PubMed Central

    Barkay, T; Gillman, M; Liebert, C

    1990-01-01

    An investigation of the Hg2+ resistance mechanism of four freshwater and four coastal marine bacteria that did not hybridize with a mer operonic probe was conducted (T. Barkay, C. Liebert, and M. Gillman, Appl. Environ. Microbiol. 55:1196-1202, 1989). Hybridization with a merA probe, the gene encoding the mercuric reductase polypeptide, at a stringency of hybridization permitting hybrid formation between evolutionarily distant merA genes (as exists between gram-positive and -negative bacteria), detected merA sequences in the genomes of all tested strains. Inducible Hg2+ volatilization was demonstrated for all eight organisms, and NADPH-dependent mercuric reductase activities were detected in crude cell extracts of six of the strains. Because these strains represented random selections of bacteria from three aquatic environments, it is concluded that merA encodes a common molecular mechanism for Hg2+ resistance and volatilization in aerobic heterotrophic aquatic communities. Images PMID:2166470

  20. Discovery of a Eukaryotic Pyrroloquinoline Quinone-Dependent Oxidoreductase Belonging to a New Auxiliary Activity Family in the Database of Carbohydrate-Active Enzymes

    PubMed Central

    Sugimoto, Naohisa; Ishida, Takuya; Samejima, Masahiro; Ohno, Hiroyuki; Yoshida, Makoto; Igarashi, Kiyohiko; Nakamura, Nobuhumi

    2014-01-01

    Pyrroloquinoline quinone (PQQ) is a redox cofactor utilized by a number of prokaryotic dehydrogenases. Not all prokaryotic organisms are capable of synthesizing PQQ, even though it plays important roles in the growth and development of many organisms, including humans. The existence of PQQ-dependent enzymes in eukaryotes has been suggested based on homology studies or the presence of PQQ-binding motifs, but there has been no evidence that such enzymes utilize PQQ as a redox cofactor. However, during our studies of hemoproteins, we fortuitously discovered a novel PQQ-dependent sugar oxidoreductase in a mushroom, the basidiomycete Coprinopsis cinerea. The enzyme protein has a signal peptide for extracellular secretion and a domain for adsorption on cellulose, in addition to the PQQ-dependent sugar dehydrogenase and cytochrome domains. Although this enzyme shows low amino acid sequence homology with known PQQ-dependent enzymes, it strongly binds PQQ and shows PQQ-dependent activity. BLAST search uncovered the existence of many genes encoding homologous proteins in bacteria, archaea, amoebozoa, and fungi, and phylogenetic analysis suggested that these quinoproteins may be members of a new family that is widely distributed not only in prokaryotes, but also in eukaryotes. PMID:25121592

  1. Discovery of a eukaryotic pyrroloquinoline quinone-dependent oxidoreductase belonging to a new auxiliary activity family in the database of carbohydrate-active enzymes.

    PubMed

    Matsumura, Hirotoshi; Umezawa, Kiwamu; Takeda, Kouta; Sugimoto, Naohisa; Ishida, Takuya; Samejima, Masahiro; Ohno, Hiroyuki; Yoshida, Makoto; Igarashi, Kiyohiko; Nakamura, Nobuhumi

    2014-01-01

    Pyrroloquinoline quinone (PQQ) is a redox cofactor utilized by a number of prokaryotic dehydrogenases. Not all prokaryotic organisms are capable of synthesizing PQQ, even though it plays important roles in the growth and development of many organisms, including humans. The existence of PQQ-dependent enzymes in eukaryotes has been suggested based on homology studies or the presence of PQQ-binding motifs, but there has been no evidence that such enzymes utilize PQQ as a redox cofactor. However, during our studies of hemoproteins, we fortuitously discovered a novel PQQ-dependent sugar oxidoreductase in a mushroom, the basidiomycete Coprinopsis cinerea. The enzyme protein has a signal peptide for extracellular secretion and a domain for adsorption on cellulose, in addition to the PQQ-dependent sugar dehydrogenase and cytochrome domains. Although this enzyme shows low amino acid sequence homology with known PQQ-dependent enzymes, it strongly binds PQQ and shows PQQ-dependent activity. BLAST search uncovered the existence of many genes encoding homologous proteins in bacteria, archaea, amoebozoa, and fungi, and phylogenetic analysis suggested that these quinoproteins may be members of a new family that is widely distributed not only in prokaryotes, but also in eukaryotes. PMID:25121592

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

    Microsoft Academic Search

    Josa-Marie Wehrfritz; Jon P. Carter; Stephen Spiro; David J. Richardson

    1997-01-01

    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

  3. Light-induced changes in the distribution of the 36000-M r polypeptide of NADPH-protochlorophyllide oxidoreductase within different cellular compartments of barley ( Hordeum vulgare L.)

    Microsoft Academic Search

    K. Dehesh; M. Klaas; I. Häuser; K. Apel

    1986-01-01

    Changes in the relative content of NADPH-protochlorophyllide oxidoreductase during the light-induced greening of barley plants were measured both in the total leaf extract as well as in intact and broken plastids. The enzyme protein was identified by its apparent molecular weight and its immunological crossreactivity with an antiserum directed against the NADPH-protochlorophyllide oxidoreductase. The monospecificity of the antiserum was tested

  4. Analytical ultracentrifugation and preliminary X-ray studies of the chloroplast envelope quinone oxidoreductase homologue from Arabidopsis thaliana.

    PubMed

    Mas y mas, Sarah; Giustini, Cécile; Ferrer, Jean Luc; Rolland, Norbert; Curien, Gilles; Cobessi, David

    2015-04-01

    Quinone oxidoreductases reduce a broad range of quinones and are widely distributed among living organisms. The chloroplast envelope quinone oxidoreductase homologue (ceQORH) from Arabidopsis thaliana binds NADPH, lacks a classical N-terminal and cleavable chloroplast transit peptide, and is transported through the chloroplast envelope membrane by an unknown alternative pathway without cleavage of its internal chloroplast targeting sequence. To unravel the fold of this targeting sequence and its substrate specificity, ceQORH from A. thaliana was overexpressed in Escherichia coli, purified and crystallized. Crystals of apo ceQORH were obtained and a complete data set was collected at 2.34?Å resolution. The crystals belonged to space group C222?, with two molecules in the asymmetric unit. PMID:25849509

  5. Purification and characterization of isoquinoline 1-oxidoreductase from Pseudomonas diminuta 7, a novel molybdenum-containing hydroxylase.

    PubMed

    Lehmann, M; Tshisuaka, B; Fetzner, S; Röger, P; Lingens, F

    1994-04-15

    Isoquinoline 1-oxidoreductase, which catalyzes the hydroxylation of isoquinoline to 1-oxo-1,2-dihydroisoquinoline with concomitant reduction of a suitable electron acceptor, was purified from the isoquinoline degrading bacterium Pseudomonas diminuta 7 to apparent homogeneity. The native enzyme was a heterodimer with a molecular mass of 95 kDa consisting of a 16- and a 80-kDa subunit. It contained 0.85 g atom molybdenum, 3.95 g atom iron, 3.9 g atom acid-labile sulfur, 2.1 mol of phosphate, and 1 mol of CMP/mol of enzyme. CMP and phosphate are suggested to originate from molybdopterin cytosine dinucleotide of the pterin molybdenum cofactor. It is assumed that the iron and the acid-labile sulfur are arranged in two (2Fe-2S) clusters. The isoelectric point of the isoquinoline 1-oxidoreductase was within the range of pH 6.2 to 6.8. Cytochrome c, ferricyanide, and several non-physiological electron acceptors served as oxidizing substrates, whereas O2 and NAD were not used. Isoquinoline 1-oxidoreductase revealed a high specificity toward the reducing substrates isoquinoline, 5-hydroxyisoquinoline, quinazoline, and phthalazine. Isoquinoline 1-oxidoreductase was inactivated by methanol, arsenite, p-hydroxymercuribenzoate, 1,10-phenanthroline, and cyanide. Additionally, the enzyme was inactivated upon incubation with its substrates isoquinoline, which slowly inhibited the enzyme in the absence of an electron acceptor, and 5-hydroxy-isoquinoline, which rapidly and very effectively inactivated the enzyme in the presence as well as in the absence of the electron acceptors iodonitrotetrazolium chloride, phenazine methosulfate, or ferricyanide. PMID:8157655

  6. Immunodetection of the Ferredoxin-NADP+ Oxidoreductase-Binding Protein Complex in Thylakoids of Different Higher Plant Species 1

    PubMed Central

    Soncini, Fernando C.; Vallejos, Rubén H.

    1989-01-01

    Monospecific polyclonal antibodies against thylakoid ferredoxin-NADP+ oxidoreductase and its binding protein from Spinacia oleracea were used to detect the presence of these proteins in different higher plants, including C3, C4, and Crassulacean acid metabolism species. A remarkable conservation of antigenic determinants in all the species analyzed was demonstrated for both the reductase and its binding protein. The association of these polypeptides in a complex was detected by immunoprecipitation. Images Figure 1 Figure 2 PMID:16666777

  7. Effectors of the mammalian plasma membrane NADH-oxidoreductase system. Short-chain ubiquinone analogues as potent stimulators

    Microsoft Academic Search

    FranÇois Vaillant; Jari A. Larm; Gabrielle L. McMullen; Ernst J. Wolvetang; Alfons Lawen I

    1996-01-01

    In the presence of effectors variations in the two recognized activities of the plasma membrane NADH-oxidoreductase system were studied in separate, specificin vitro assays. We report here that ubiquinone analogues that contain a short, less hydrophobic side chain than coenzyme Q-10 dramatically stimulate the NADH-oxidase activity of isolated rat liver plasma membranes whereas they show no effect on the reductase

  8. Increase of plasma membrane oxidoreductase activity is not correlated with the production of extracellular Superoxide radicals in human Namalwa cells

    Microsoft Academic Search

    J. A. Larm; E. J. Wolvetang; F. Vaillant; R. D. Martinus; A. Lawen; A. W. Linnane

    1995-01-01

    Summary We have considered the regulatory interrelationship of the plasma membrane oxidoreductase (PMOR) system and the mitochondrial respiratory capacity of human Namalwa (lymphoblastoid) cells. To this end, we made use of mitchondrially respiratory competent (?+) cells and ?0 cells, which lack mitochondrial DNA (mtDNA) and consequently mitochondrial respiratory activity. NADH-fer-ricyanide reductase activity of the PMOR system is increased 3-fold in

  9. The Critical Role of Arabidopsis Electron-Transfer Flavoprotein:Ubiquinone Oxidoreductase during Dark-Induced Starvation

    Microsoft Academic Search

    Kimitsune Ishizaki; Tony R. Larson; Nicolas Schauer; Alisdair R. Fernie; Ian A. Graham; Christopher J. Leavera

    2005-01-01

    In mammals, electron-transfer flavoprotein:ubiquinone oxidoreductase (ETFQO) and electron-transfer flavoprotein (ETF) are functionally associated, and ETF accepts electrons from at least nine mitochondrial matrix flavoprotein dehydrogenases and transfers them to ubiquinone in the inner mitochondrial membrane. In addition, the mammalian ETF\\/ETFQO system plays a key role in b-oxidation of fatty acids and catabolism of amino acids and choline. By contrast, nothing

  10. Specific Modification of a Na+ Binding Site in NADH:Quinone Oxidoreductase from Klebsiella pneumoniae with Dicyclohexylcarbodiimide

    Microsoft Academic Search

    Irini Vgenopoulou; Anja C. Gemperli; Julia Steuber

    2006-01-01

    Received 16 September 2005\\/Accepted 13 February 2006 The respiratory NADH:quinone oxidoreductase (complex I) (NDH-1) is a multisubunit enzyme that trans- locates protons (or in some cases Na) across energy-conserving membranes from bacteria or mitochondria. We studied the reaction of the Na-translocating complex I from the enterobacterium Klebsiella pneumoniae with N,N-dicyclohexylcarbodiimide (DCCD), with the aim of identifying a subunit critical for

  11. Two-electron reduction of quinones by rat liver NAD(P)H:quinone oxidoreductase: quantitative structure–activity relationships

    Microsoft Academic Search

    Žilvinas Anusevi?ius; Jonas Šarlauskas

    2002-01-01

    Mammalian NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2) catalyzes the two-electron reduction of quinones and plays one of the main roles in the bioactivation of quinoidal drugs. In order to understand the enzyme substrate specificity, we have examined the reactions of rat NQO1 with a number of quinones with available potentials of single-electron (E17) reduction and pKa of their semiquinones. The

  12. The Crystal Structure of NAD(P)H Quinone Oxidoreductase 1 in Complex with Its Potent Inhibitor Dicoumarol †

    Microsoft Academic Search

    Gad Asher; Orly Dym; Peter Tsvetkov; Julia Adler; Yosef Shaul

    2006-01-01

    NAD(P)H quinone oxidoreductase 1 (NQO1) is a ubiquitous flavoenzyme that catalyzes two- electron reduction of quinones to hydroquinones utilizing NAD(P)H as an electron donor. NQO1 binds and stabilizes several short-lived proteins including the tumor suppressors p53 and p73 and the enzyme ornithine decarboxylase (ODC). Dicoumarol is a widely used potent competitive inhibitor of NQO1 enzymatic activity, which competes with NAD(P)H

  13. NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), a multifunctional antioxidant enzyme and exceptionally versatile cytoprotector

    Microsoft Academic Search

    Albena T. Dinkova-Kostova; Paul Talalay

    2010-01-01

    NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1) is a widely-distributed FAD-dependent flavoprotein that promotes obligatory 2-electron reductions of quinones, quinoneimines, nitroaromatics, and azo dyes, at rates that are comparable with NADH or NADPH. These reductions depress quinone levels and thereby minimize opportunities for generation of reactive oxygen intermediates by redox cycling, and for depletion of intracellular thiol pools. NQO1 is a highly-inducible

  14. NADH-Quinone Oxidoreductase: PSST Subunit Couples Electron Transfer from Iron-Sulfur Cluster N2 to Quinone

    Microsoft Academic Search

    Franz Schuler; Takahiro Yano; Salvatore di Bernardo; Takao Yagi; Victoria Yankovskaya; Thomas P. Singer; John E. Casida

    1999-01-01

    The proton-translocating NADH-quinone oxidoreductase (EC 1.6.99.3) is the largest and least understood enzyme complex of the respiratory chain. The mammalian mitochondrial enzyme (also called complex I) contains more than 40 subunits, whereas its structurally simpler bacterial counterpart (NDH-1) in Paracoccus denitrificans and Thermus thermophilus HB-8 consists of 14 subunits. A major unsolved question is the location and mechanism of the

  15. Non-specific peroxidase (donor: H2O2-oxidoreductase, EC 1.11.1.7) activity in multiple sclerosis.

    PubMed

    Najbauer, J; Vécsei, L; Pálffy, G

    1990-01-01

    The non-specific peroxidase (donor: H2O2-oxidoreductase, EC 1.11.1.7) activity of red blood cells in patients with multiple sclerosis, patients with other neurological diseases, and healthy control individuals was investigated. To this end, a simple method was developed. No significant difference was found in the non-specific peroxidase activity of red blood cells from patients with multiple sclerosis and controls. PMID:1966628

  16. Antiparasitic Drug Nitazoxanide Inhibits the Pyruvate Oxidoreductases of Helicobacter pylori, Selected Anaerobic Bacteria and Parasites, and Campylobacter jejuni

    Microsoft Academic Search

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

    2007-01-01

    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 (Ki ,2t o 10 M) 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

  17. Localization-controlled specificity of FAD:threonine flavin transferases in Klebsiella pneumoniae and its implications for the mechanism of Na(+)-translocating NADH:quinone oxidoreductase.

    PubMed

    Bertsova, Yulia V; Kostyrko, Vitaly A; Baykov, Alexander A; Bogachev, Alexander V

    2014-07-01

    The Klebsiella pneumoniae genome contains genes for two putative flavin transferase enzymes (ApbE1 and ApbE2) that add FMN to protein Thr residues. ApbE1, but not ApbE2, has a periplasm-addressing signal sequence. The genome also contains genes for three target proteins with the Dxx(s/t)gAT flavinylation motif: two subunits of Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR), and a 99.5kDa protein, KPK_2907, with a previously unknown function. We show here that KPK_2907 is an active cytoplasmically-localized fumarate reductase. K. pneumoniae cells with an inactivated kpk_2907 gene lack cytoplasmic fumarate reductase activity, while retaining this activity in the membrane fraction. Complementation of the mutant strain with a kpk_2907-containing plasmid resulted in a complete recovery of cytoplasmic fumarate reductase activity. KPK_2907 produced in Escherichia coli cells contains 1mol/mol each of covalently bound FMN, noncovalently bound FMN and noncovalently bound FAD. Lesion in the ApbE1 gene in K. pneumoniae resulted in inactive Na(+)-NQR, but cytoplasmic fumarate reductase activity remained unchanged. On the contrary, lesion in the ApbE2 gene abolished the fumarate reductase but not the Na(+)-NQR activity. Both activities could be restored by transformation of the ApbE1- or ApbE2-deficient K. pneumoniae strains with plasmids containing the Vibrio cholerae apbE gene with or without the periplasm-directing signal sequence, respectively. Our data thus indicate that ApbE1 and ApbE2 bind FMN to Na(+)-NQR and fumarate reductase, respectively, and that, contrary to the presently accepted view, the FMN residues are on the periplasmic side of Na(+)-NQR. A new, "electron loop" mechanism is proposed for Na(+)-NQR, involving an electroneutral Na(+)/electron symport. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. PMID:24361839

  18. WOR5, a Novel Tungsten-Containing Aldehyde Oxidoreductase from Pyrococcus furiosus with a Broad Substrate Specificity

    PubMed Central

    Bevers, Loes E.; Bol, Emile; Hagedoorn, Peter-Leon; Hagen, Wilfred R.

    2005-01-01

    WOR5 is the fifth and last member of the family of tungsten-containing oxidoreductases purified from the hyperthermophilic archaeon Pyrococcus furiosus. It is a homodimeric protein (subunit, 65 kDa) that contains one [4Fe-4S] cluster and one tungstobispterin cofactor per subunit. It has a broad substrate specificity with a high affinity for several substituted and nonsubstituted aliphatic and aromatic aldehydes with various chain lengths. The highest catalytic efficiency of WOR5 is found for the oxidation of hexanal (Vmax = 15.6 U/mg, Km = 0.18 mM at 60°C). Hexanal-incubated enzyme exhibits S = 1/2 electron paramagnetic resonance signals from [4Fe-4S]1+ (g values of 2.08, 1.93, and 1.87) and W5+ (g values of 1.977, 1.906, and 1.855). Cyclic voltammetry of ferredoxin and WOR5 on an activated glassy carbon electrode shows a catalytic wave upon addition of hexanal, suggesting that ferredoxin can be a physiological redox partner. The combination of WOR5, formaldehyde oxidoreductase, and aldehyde oxidoreductase forms an efficient catalyst for the oxidation of a broad range of aldehydes in P. furiosus. PMID:16199576

  19. NAD(P)H:Quinone Oxidoreductase 1 (NQO1) in the Sensitivity and Resistance to Antitumor Quinones

    PubMed Central

    Siegel, David; Yan, Chao; Ross, David

    2012-01-01

    The quinone pharmacophore is present in many drug classes but is particularly common among antitumor drugs. Many quinones serve essentially as pro-drugs and exert their activities after reduction. Reduction of quinones may generate semiquinones or hydroquinones with subsequent generation of reactive oxygen radicals and oxidative stress, quinones can be designed so they lose a leaving group when reduced to the hydroquinone generating a reactive electrophile or the hydroquinone form of the molecule may have greater pharmacological activity than the parent quinone against a particular target. Enzyme systems that reduce quinones therefore become critically important in the pharmacological activity of this class of drugs. There are a number of enzyme systems that can catalyze reduction of quinones including cytochrome P450 reductase, cytochrome b5 reductase, NAD(P)H:quinone oxidoreductase 1 (NQO1), NAD(P)H:quinone oxidoreductase 2 (NQO2), carbonyl reductases, and thioredoxin reductase. In this context, one of the most extensively studied reductases has been NAD(P)H:quinone oxidoreductase 1 (NQO1). In this review we will focus on the role of NQO1 in the bioactivation of clinically important quinones mitomycin C, ?-lapachone and 17AAG as well as the influence of the NQO1*2 polymorphism on the sensitivity and resistance to these agents. PMID:22209713

  20. Na + translocation by bacterial NADH:quinone oxidoreductases: an extension to the complex-I family of primary redox pumps 1 1 Dedicated to Peter Dimroth on the occasion of his 60th birthday

    Microsoft Academic Search

    Julia Steuber

    2001-01-01

    The current knowledge on the Na+-translocating NADH:ubiquinone oxidoreductase of the Na+-NQR type from Vibrio alginolyticus, and on Na+ transport by the electrogenic NADH:Q oxidoreductases from Escherichia coli and Klebsiella pneumoniae (complex I, or NDH-I) is summarized. A general mode of redox-linked Na+ transport by NADH:Q oxidoreductases is proposed that is based on the electrostatic attraction of a positively charged Na+

  1. Characterization of two-step deglycosylation via oxidation by glycoside oxidoreductase and defining their subfamily.

    PubMed

    Kim, Eun-Mi; Seo, Joo-Hyun; Baek, Kiheon; Kim, Byung-Gee

    2015-01-01

    Herein, we report a two-step deglycosylation mediated by the oxidation of glycoside which is different from traditional glycoside hydrolase (GH) mechanism. Previously, we reported a novel flavin adenine dinucleotide (FAD)-dependent glycoside oxidoreductase (FAD-GO) having deglycosylation activity. Various features of the reaction of FAD-GO such as including mechanism and catalytic residue and substrate specificity were studied. In addition, classification of novel FAD-GO subfamily was attempted. Deglycosylation of glycoside was performed spontaneously via oxidation of 3-OH of glycone moiety by FAD-GO mediated oxidation reaction. His493 residue was identified as a catalytic residue for the oxidation step. Interestingly, this enzyme has broad glycone and aglycon specificities. For the classification of FAD-GO enzyme subfamily, putative FAD-GOs were screened based on the FAD-GO from Rhizobium sp. GIN611 (gi 365822256) using BLAST search. The homologs of R. sp. GIN611 included the putative FAD-GOs from Stenotrophomonas strains, Sphingobacterium strains, Agrobacterium tumefaciens str. C58, and etc. All the cloned FAD-GOs from the three strains catalyzed the deglycosylation via enzymatic oxidation. Based on their substrate specificities, deglycosylation and oxidation activities to various ginsenosides, the FAD-GO subfamily members can be utilized as novel biocatalysts for the production of various aglycones. PMID:26057169

  2. A disulfide-bond A oxidoreductase-like protein (DsbA-L) regulates adiponectin multimerization

    PubMed Central

    Liu, Meilian; Zhou, Lijun; Xu, Aimin; Lam, Karen S. L.; Wetzel, Michael D.; Xiang, Ruihua; Zhang, Jingjing; Xin, Xiaoban; Dong, Lily Q.; Liu, Feng

    2008-01-01

    Impairments in adiponectin multimerization lead to defects in adiponectin secretion and function and are associated with diabetes, yet the underlying mechanisms remain largely unknown. We have identified an adiponectin-interacting protein, previously named GST-kappa, by yeast 2-hybrid screening. The adiponectin-interacting protein contains 2 thioredoxin domains and has very little sequence similarity to other GST isoforms. However, this protein shares high sequence and secondary structure homology to bacterial disulfide-bond A oxidoreductase (DsbA) and is thus renamed DsbA-like protein (DsbA-L). DsbA-L is highly expressed in adipose tissue, and its expression level is negatively correlated with obesity in mice and humans. DsbA-L expression in 3T3-L1 adipocytes is stimulated by the insulin sensitizer rosiglitazone and inhibited by the inflammatory cytokine TNF?. Overexpression of DsbA-L promoted adiponectin multimerization while suppressing DsbA-L expression by RNAi markedly and selectively reduced adiponectin levels and secretion in 3T3-L1 adipocytes. Our results identify DsbA-L as a key regulator for adiponectin biosynthesis and uncover a potential new target for developing therapeutic drugs for the treatment of insulin resistance and its associated metabolic disorders. PMID:19011089

  3. Monitoring Oxidative Folding of a Single Protein Catalyzed by the Disulfide Oxidoreductase DsbA.

    PubMed

    Kahn, Thomas B; Fernández, Julio M; Perez-Jimenez, Raul

    2015-06-01

    Oxidative folding, the process by which proteins fold and acquire disulfide bonds concurrently, is of critical importance for a wide range of biological processes. Generally, this process is catalyzed by oxidoreductase enzymes that facilitate oxidation and also bear chaperone functionality. Although this process has been well described qualitatively, fine yet important details remain obscured by a limited quantitative perspective, arising from the limitations in the application of bulk biochemical methods to the study of oxidative folding. In this work, we have applied single molecule force spectroscopy techniques to monitor in real time the process of oxidative folding as catalyzed by DsbA, the enzyme solely responsible for the catalysis of oxidative folding in the bacterial periplasm. We provide a quantitative and detailed description of the catalytic mechanism utilized by DsbA that offers insight into the entire sequence of events that occurs in the periplasm from the unfolded-reduced state to the folded-oxidized protein. We have compared our results with those of protein disulfide-isomerase, the eukaryotic counterpart of DsbA, allowing us to devise a general mechanism for oxidative folding that also reflects upon the physiological functions and demands of these enzymes in vivo. PMID:25897077

  4. Cytochrome P450 oxidoreductase deficiency: rare congenital disorder leading to skeletal malformations and steroidogenic defects.

    PubMed

    Fukami, Maki; Ogata, Tsutomu

    2014-12-01

    Cytochrome P450 oxidoreductase (POR) deficiency (PORD) is a newly characterized disorder. PORD is caused by homozygous or compound heterozygous mutations in POR encoding an electron donor for several microsomal enzymes such as CYP21A2, CYP17A1, CYP19A1, CYP51A1, and CYP26A1-C1. Molecular defects of PORD include a Japanese founder mutation p.R457H, as well as various missense, nonsense, frameshift, and splice-site mutations and exonic deletions. PORD leads to unique skeletal malformations referred to as Antley-Bixler syndrome, in addition to 46,XX and 46,XY disorders of sex development, pubertal failure, adrenal dysfunction, and maternal virilization during pregnancy. Such clinical features are ascribable to impaired activities of the POR-dependent microsomal enzymes. PORD represents one form of congenital adrenal hyperplasia, although it can occur as a congenital malformation syndrome and a disorder of sex development. Phenotypic severity of PORD is highly variable and only partly depends on the residual activity of the mutant proteins. It is possible that PORD remains undiagnosed in several patients. Detailed hormonal assessment and molecular analysis are useful for diagnosis of PORD. PMID:25294558

  5. Pigment-free NADPH:protochlorophyllide oxidoreductase from Avena sativa L. Purification and substrate specificity.

    PubMed

    Klement, H; Helfrich, M; Oster, U; Schoch, S; Rüdiger, W

    1999-11-01

    The enzyme NADPH:protochlorophyllide oxidoreductase (POR) is the key enzyme for light-dependent chlorophyll biosynthesis. It accumulates in dark-grown plants as the ternary enzyme-substrate complex POR-protochlorophyllide a-NADPH. Here, we describe a simple procedure for purification of pigment-free POR from etioplasts of Avena sativa seedlings. The procedure implies differential solubilization with n-octyl-beta-D-glucoside and one chromatographic step with DEAE-cellulose. We show, using pigment and protein analysis, that etioplasts contain a one-to-one complex of POR and protochlorophyllide a. The preparation of 13 analogues of protochlorophyllide a is described. The analogues differ in the side chains of the macrocycle and in part contain zinc instead of the central magnesium. Six analogues with different side chains at rings A or B are active substrates, seven analogues with different side chains at rings D or E are not accepted as substrates by POR. The kinetics of the light-dependent reaction reveals three groups of substrate analogues with a fast, medium and slow reaction. To evaluate the kinetic data, the molar extinction coefficients in the reaction buffer had to be determined. At concentrations above 2 mole substrate/mole enzyme, inhibition was found for protochlorophyllide a and for the analogues. PMID:10518779

  6. Reduction of nitric oxide catalyzed by hydroxylamine oxidoreductase from an anammox bacterium.

    PubMed

    Irisa, Tatsuya; Hira, Daisuke; Furukawa, Kenji; Fujii, Takao

    2014-12-01

    The hydroxylamine oxidoreductase (HAO) from the anammox bacterium, Candidatus Kuenenia stuttgartiensis has been reported to catalyze the oxidation of hydroxylamine (NH2OH) to nitric oxide (NO) by using bovine cytochrome c as an oxidant. In contrast, we investigated whether the HAO from anammox bacterium strain KSU-1 could catalyze the reduction of NO with reduced benzyl viologen (BVred) and the NO-releasing reagent, NOC 7. The reduction proceeded, resulting in the formation of NH2OH as a product. The oxidation rate of BVred was proportional to the concentration of BVred itself for a short period in each experiment, a situation that was termed quasi-steady state. The analyses of the states at various concentrations of HAO allowed us to determine the rate constant for the catalytic reaction, (2.85 ± 0.19) × 10(5) M(-1) s(-1), governing NO reduction by BVred and HAO, which was comparable to that reported for the HAO from the ammonium oxidizer, Nitrosomonas with reduced methyl viologen. These results suggest that the anammox HAO functions to adjust anammox by inter-conversion of NO and NH2OH depending on the redox potential of the physiological electron transfer protein in anammox bacteria. PMID:24996970

  7. Iron (III) reduction: A novel activity of the human NAD(P)H:oxidoreductase

    SciTech Connect

    Onyenwoke, Rob U. [Department of Microbiology, University of Georgia, 1000 Cedar Street, Athens, GA 30602-2605 (United States); Wiegel, Juergen [Department of Microbiology, University of Georgia, 1000 Cedar Street, Athens, GA 30602-2605 (United States)]. E-mail: jwiegel@uga.edu

    2007-02-09

    NAD(P)H:quinone oxidoreductase (NQO1; EC 1.6.99.2) catalyzes a two-electron transfer involved in the protection of cells from reactive oxygen species. These reactive oxygen species are often generated by the one-electron reduction of quinones or quinone analogs. We report here on the previously unreported Fe(III) reduction activity of human NQO1. Under steady state conditions with Fe(III) citrate, the apparent Michaelis-Menten constant (K{sub m}{sup app}) was {approx}0.3nM and the apparent maximum velocity (V{sub max}{sup app}) was 16Umg{sup -1}. Substrate inhibition was observed above 5nM. NADH was the electron donor, K{sub m}{sup app}=340{mu}M and V{sub max}{sup app}=46Umg{sup -1}. FAD was also a cofactor with a K{sub m}{sup app} of 3.1{mu}M and V{sub max}{sup app} of 89Umg{sup -1}. The turnover number for NADH oxidation was 25s{sup -1}. Possible physiological roles of the Fe(III) reduction by this enzyme are discussed.

  8. Iron (III) reduction: A novel activity of the human NAD(P)H:oxidoreductase.

    PubMed

    Onyenwoke, Rob U; Wiegel, Juergen

    2007-02-01

    NAD(P)H:quinone oxidoreductase (NQO1; EC 1.6.99.2) catalyzes a two-electron transfer involved in the protection of cells from reactive oxygen species. These reactive oxygen species are often generated by the one-electron reduction of quinones or quinone analogs. We report here on the previously unreported Fe(III) reduction activity of human NQO1. Under steady state conditions with Fe(III) citrate, the apparent Michaelis-Menten constant (Km(app)) was approximately 0.3 nM and the apparent maximum velocity (Vmax(app)) was 16 U mg(-1). Substrate inhibition was observed above 5 nM. NADH was the electron donor, Km(app)= 340 microM and Vmax(app) = 46 Umg(-1). FAD was also a cofactor with a Km(app) of 3.1 microM and Vmax(app) of 89 U mg(-1). The turnover number for NADH oxidation was 25 s(-1). Possible physiological roles of the Fe(III) reduction by this enzyme are discussed. PMID:17178108

  9. Structure of the V. cholerae Na+-pumping NADH:quinone oxidoreductase.

    PubMed

    Steuber, Julia; Vohl, Georg; Casutt, Marco S; Vorburger, Thomas; Diederichs, Kay; Fritz, Günter

    2014-12-01

    NADH oxidation in the respiratory chain is coupled to ion translocation across the membrane to build up an electrochemical gradient. The sodium-translocating NADH:quinone oxidoreductase (Na(+)-NQR), a membrane protein complex widespread among pathogenic bacteria, consists of six subunits, NqrA, B, C, D, E and F. To our knowledge, no structural information on the Na(+)-NQR complex has been available until now. Here we present the crystal structure of the Na(+)-NQR complex at 3.5 Å resolution. The arrangement of cofactors both at the cytoplasmic and the periplasmic side of the complex, together with a hitherto unknown iron centre in the midst of the membrane-embedded part, reveals an electron transfer pathway from the NADH-oxidizing cytoplasmic NqrF subunit across the membrane to the periplasmic NqrC, and back to the quinone reduction site on NqrA located in the cytoplasm. A sodium channel was localized in subunit NqrB, which represents the largest membrane subunit of the Na(+)-NQR and is structurally related to urea and ammonia transporters. On the basis of the structure we propose a mechanism of redox-driven Na(+) translocation where the change in redox state of the flavin mononucleotide cofactor in NqrB triggers the transport of Na(+) through the observed channel. PMID:25471880

  10. The use of amphipathic polymers for cryo electron microscopy of NADH:ubiquinone oxidoreductase (complex I).

    PubMed

    Flötenmeyer, Matthias; Weiss, Hanns; Tribet, Christophe; Popot, Jean-Luc; Leonard, Kevin

    2007-09-01

    In the three-dimensional (3D) structure determination of macromolecules, cryo electron microscopy (cryo-EM) is an important method for obtaining micrographs of unstained specimens for the single-particle reconstruction approach. For cryo-EM, proteins are fixed in a frozen hydrated state by quick-freezing in a thin water layer on a holey carbon film. Cryo-EM of detergent-solubilized membrane proteins is hindered by the fact that detergents reduce the surface tension of water, so that it is difficult to control the ice thickness and the distribution of protein. Amphipols are a new class of amphipathic polymers designed to handle membrane proteins in aqueous solutions under particularly mild conditions. Amphipol A8-35 stabilizes NADH:ubiquinone oxidoreductase (complex I) from Neurospora crassa and keeps it water-soluble in the absence of free detergent. Electron microscope images of quick-frozen complex I/A8-35 samples were used for computer-based single-particle averaging and 3D reconstruction, and the reconstruction of unstained frozen-hydrated particles compared with previous detergent-based reconstructions. The potential of amphipols for cryo-EM is discussed. PMID:17760617

  11. A new structure-based classification of sulfide:quinone oxidoreductases.

    PubMed

    Marcia, Marco; Ermler, Ulrich; Peng, Guohong; Michel, Hartmut

    2010-04-01

    Sulfide:quinone oxidoreductases (SQR) are ubiquitous membrane-bound flavoproteins involved in sulfide detoxification, in sulfide-dependent energy conservation processes and potenatially in the homeostasis of the neurotransmitter sulfide. The first 2 structures of SQRs from the bacterium Aquifex aeolicus (Marcia et al., Proc Natl Acad Sci USA 2009; 106:9625-9630) and the archaeon Acidianus ambivalens (Brito et al., Biochemistry 2009; 48:5613-5622) were determined recently by X-ray crystallography revealing unexpected differences in the active sites and in flavin adenine dinucleotide binding. Besides the reciprocal differences, they show a different conformation of the active site compared with another sulfide oxidizing enzyme, the flavocytochrome c:sulfide dehydrogenase (FCSD) from Allochromatium vinosum (protein data bank id: 1FCD). In addition to the new structural data, the number of available SQR-like protein sequences is continuously increasing (Pham et al., Microbiology 2008; 154:3112-3121) and the SQR activity of new members of this protein family was recently proven too (Chan et al., J Bacteriol 2009; 191:1026-1034). In the light of the new data, here we revisit the previously proposed contradictory SQR classification and we define new structure-based sequence fingerprints that support a subdivision of the SQR family into six groups. Our report summarizes the state-of-art knowledge about SQRs and highlights the questions that still remain unanswered. Despite two decades of work already done on these enzymes, new and most exciting discoveries can be expected in the future. PMID:20077566

  12. Localization of Ubiquinone-8 in the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae*

    PubMed Central

    Casutt, Marco S.; Nedielkov, Ruslan; Wendelspiess, Severin; Vossler, Sara; Gerken, Uwe; Murai, Masatoshi; Miyoshi, Hideto; Möller, Heiko M.; Steuber, Julia

    2011-01-01

    Na+ is the second major coupling ion at membranes after protons, and many pathogenic bacteria use the sodium-motive force to their advantage. A prominent example is Vibrio cholerae, which relies on the Na+-pumping NADH:quinone oxidoreductase (Na+-NQR) as the first complex in its respiratory chain. The Na+-NQR is a multisubunit, membrane-embedded NADH dehydrogenase that oxidizes NADH and reduces quinone to quinol. Existing models describing redox-driven Na+ translocation by the Na+-NQR are based on the assumption that the pump contains four flavins and one FeS cluster. Here we show that the large, peripheral NqrA subunit of the Na+-NQR binds one molecule of ubiquinone-8. Investigations of the dynamic interaction of NqrA with quinones by surface plasmon resonance and saturation transfer difference NMR reveal a high affinity, which is determined by the methoxy groups at the C-2 and C-3 positions of the quinone headgroup. Using photoactivatable quinone derivatives, it is demonstrated that ubiquinone-8 bound to NqrA occupies a functional site. A novel scheme of electron transfer in Na+-NQR is proposed that is initiated by NADH oxidation on subunit NqrF and leads to quinol formation on subunit NqrA. PMID:21885438

  13. 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. PMID:24059520

  14. Secondary structure of NADPH: protochlorophyllide oxidoreductase examined by circular dichroism and prediction methods.

    PubMed Central

    Birve, S J; Selstam, E; Johansson, L B

    1996-01-01

    To study the secondary structure of the enzyme NADPH: protochlorophyllide oxidoreductase (PCOR), a novel method of enzyme isolation was developed. The detergent isotridecyl poly-(ethylene glycol) ether (Genapol X-080) selectively solubilizes the enzyme from a prolamellar-body fraction isolated from wheat (Triticum aestivum L.). The solubilized fraction was further purified by ion-exchange chromatography. The isolated enzyme was studied by fluorescence spectroscopy at 77 K, and by CD spectroscopy. The fluorescence-emission spectra revealed that the binding properties of the substrate and co-substrate were preserved and that photo-reduction occurred. The CD spectra of PCOR were analysed for the relative amounts of the secondary structures, alpha-helix, beta-sheet, turn and random coil. The secondary structure composition was estimated to be 33% alpha-helix, 19% beta-sheet, 20% turn and 28% random coil. These values are in agreement with those predicted by the Predict Heidelberg Deutschland and self-optimized prediction method from alignments methods. The enzyme has some amino acid identity with other NADPH-binding enzymes containing the Rossmann fold. The Rossmann-fold fingerprint motif is localized in the N-terminal region and at the expected positions in the predicted secondary structure. It is suggested that PCOR is anchored to the interfacial region of the membrane by either a beta-sheet or an alpha-helical region containing tryptophan residues. A hydrophobic loop-region could also be involved in membrane anchoring. PMID:8713084

  15. Chemical Nature and Reaction Mechanisms of the Molybdenum Cofactor of Xanthine Oxidoreductase

    PubMed Central

    Okamoto, Ken; Kusano, Teruo; Nishino, Takeshi

    2013-01-01

    Xanthine oxidoreductase (XOR), a complex flavoprotein, catalyzes the metabolic reactions leading from hypoxanthine to xanthine and from xanthine to urate, and both reactions take place at the molybdenum cofactor. The enzyme is a target of drugs for therapy of gout or hyperuricemia. We review the chemical nature and reaction mechanisms of the molybdenum cofactor of XOR, focusing on molybdenum-dependent reactions of actual or potential medical importance, including nitric oxide (NO) synthesis. It is now generally accepted that XOR transfers the water-exchangeable -OH ligand of the molybdenum atom to the substrate. The hydroxyl group at OH-Mo(IV) can be replaced by urate, oxipurinol and FYX-051 derivatives and the structures of these complexes have been determined by x-ray crystallography under anaerobic conditions. Although formation of NO from nitrite or formation of xanthine from urate by XOR is chemically feasible, it is not yet clear whether these reactions have any physiological significance since the reactions are catalyzed at a slow rate even under anaerobic conditions. PMID:23116398

  16. High ratio of bacteriochlorophyll biosynthesis genes to chlorophyll biosynthesis genes in bacteria of humic lakes.

    PubMed

    Eiler, Alexander; Beier, Sara; Säwström, Christin; Karlsson, Jan; Bertilsson, Stefan

    2009-11-01

    Recent studies highlight the diversity and significance of marine phototrophic microorganisms such as picocyanobacteria, phototrophic picoeukaryotes, and bacteriochlorophyll- and rhodopsin-holding phototrophic bacteria. To assess if freshwater ecosystems also harbor similar phototroph diversity, genes involved in the biosynthesis of bacteriochlorophyll and chlorophyll were targeted to explore oxygenic and aerobic anoxygenic phototroph composition in a wide range of lakes. Partial dark-operative protochlorophyllide oxidoreductase (DPOR) and chlorophyllide oxidoreductase (COR) genes in bacteria of seven lakes with contrasting trophic statuses were PCR amplified, cloned, and sequenced. Out of 61 sequences encoding the L subunit of DPOR (L-DPOR), 22 clustered with aerobic anoxygenic photosynthetic bacteria, whereas 39 L-DPOR sequences related to oxygenic phototrophs, like cyanobacteria, were observed. Phylogenetic analysis revealed clear separation of these freshwater L-DPOR genes as well as 11 COR gene sequences from their marine counterparts. Terminal restriction fragment length analysis of L-DPOR genes was used to characterize oxygenic aerobic and anoxygenic photosynthesizing populations in 20 lakes differing in physical and chemical characteristics. Significant differences in L-DPOR community composition were observed between dystrophic lakes and all other systems, where a higher proportion of genes affiliated with aerobic anoxygenic photosynthetic bacteria was observed than in other systems. Our results reveal a significant diversity of phototrophic microorganisms in lakes and suggest niche partitioning of oxygenic and aerobic anoxygenic phototrophs in these systems in response to trophic status and coupled differences in light regime. PMID:19801478

  17. Plasma membrane NADH-oxidoreductase system: a critical review of the structural and functional data.

    PubMed

    Baker, M A; Lawen, A

    2000-01-01

    The observation in the early 1970s that ferricyanide can replace transferrin as a growth factor highlighted the major role plasma membrane proteins can play within a mammalian cell. Ferricyanide, being impermeant to the cell, was assumed to act at the level of the plasma membrane. Since that time, several enzymes isolated from the plasma membrane have been described, which, using NADH as the intracellular electron donor, are capable of reducing ferricyanide. However, their exact modes of action, and their physiological substrates and functions have not been solved to date. Numerous hypotheses have been proposed for the role of such redox enzymes within the plasma membrane. Examples include the regulation of cell signaling, cell growth, apoptosis, proton pumping, and ion channels. All of these roles may be a result of the function of these enzymes as cellular redox sensors. The emergence of many diverse roles for ferricyanide utilizing redox enzymes present in the plasma membrane might also, in part, be due to the numerous redox enzymes present within the membrane; the poor molecular characterization of the enzymes may be the reason for some of the diverging results reported in the literature as various researchers may be working on different enzymes. Here we review the diverse proposals given for structure and function to the plasma membrane NADH-oxidoreductase system(s) with a specific focus on those enzyme activities which can couple ferricyanide and NADH. Although they are still ill-defined enzymes, evidence is rising that they are of utmost significance for cellular regulation. PMID:11229526

  18. Structural insights into vinyl reduction regiospecificity of phycocyanobilin:ferredoxin oxidoreductase (PcyA).

    PubMed

    Hagiwara, Yoshinori; Sugishima, Masakazu; Khawn, Htoi; Kinoshita, Hideki; Inomata, Katsuhiko; Shang, Lixia; Lagarias, J Clark; Takahashi, Yasuhiro; Fukuyama, Keiichi

    2010-01-01

    Phycocyanobilin:ferredoxin oxidoreductase (PcyA) is the best characterized member of the ferredoxin-dependent bilin reductase family. Unlike other ferredoxin-dependent bilin reductases that catalyze a two-electron reduction, PcyA sequentially reduces D-ring (exo) and A-ring (endo) vinyl groups of biliverdin IXalpha (BV) to yield phycocyanobilin, a key pigment precursor of the light-harvesting antennae complexes of red algae, cyanobacteria, and cryptophytes. To address the structural basis for the reduction regiospecificity of PcyA, we report new high resolution crystal structures of bilin substrate complexes of PcyA from Synechocystis sp. PCC6803, all of which lack exo-vinyl reduction activity. These include the BV complex of the E76Q mutant as well as substrate-bound complexes of wild-type PcyA with the reaction intermediate 18(1),18(2)-dihydrobiliverdin IXalpha (18EtBV) and with biliverdin XIIIalpha (BV13), a synthetic substrate that lacks an exo-vinyl group. Although the overall folds and the binding sites of the U-shaped substrates of all three complexes were similar with wild-type PcyA-BV, the orientation of the Glu-76 side chain, which was in close contact with the exo-vinyl group in PcyA-BV, was rotated away from the bilin D-ring. The local structures around the A-rings in the three complexes, which all retain the ability to reduce the A-ring of their bound pigments, were nearly identical with that of wild-type PcyA-BV. Consistent with the proposed proton-donating role of the carboxylic acid side chain of Glu-76 for exo-vinyl reduction, these structures reveal new insight into the reduction regiospecificity of PcyA. PMID:19887371

  19. Xanthine crystals induced by topiroxostat, a xanthine oxidoreductase inhibitor, in rats, cause transitional cell tumors.

    PubMed

    Shimo, Takeo; Moto, Mitsuyoshi; Ashizawa, Naoki; Matsumoto, Koji; Iwanaga, Takashi; Saito, Kazuhiro

    2014-04-01

    The present study was performed to elucidate the underlying mechanism of transitional cell tumors found in the carcinogenicity testing of topiroxostat, a xanthine oxidoreductase inhibitor, in which topiroxostat was orally given to F344 rats at 0.3, 1, and 3 mg/kg for 2 years. In the urinary bladder, transitional cell papillomas and/or carcinomas were seen in males receiving 0.3, 1, and 3 mg/kg (1/49, 3/49, and 10/50, respectively). In the kidney, transitional cell papillomas and/or carcinomas in the pelvis were seen in 2/50 males and 1/50 females receiving 3 mg/kg. In the mechanistic study by 52-week oral treatment with topiroxostat at 3 mg/kg to F344 male rats, with and without citrate, simple and papillary transitional cell hyperplasias of the urinary bladder epithelium were observed in 5/17 in the topiroxostat-alone treatment group, along with xanthine-induced nephropathy, in contrast to neither xanthine crystals nor lesions in urinary organs by co-treatment group with citrate. As for sex differences of urinary bladder tumors, the BrdU labeling index for epithelial cells of the urinary bladder by 5-week oral treatment with topiroxostat at 10 mg/kg to F344 rats was increased in males only, showing consistency with histopathological findings. Therefore, the present study indicates that transitional cell tumors induced by topiroxostat in rats were due to physical stimulation to transitional cells of xanthine crystals/calculi and provides that other factors were not implicated in this tumorigenesis. Furthermore, the present study suggests that such tumors do not predict for humans since topiroxostat-induced xanthine deposition is a rodent-specific event. PMID:24448833

  20. Spatially programmed assembling of oxidoreductases with single-stranded DNA for cofactor-required reactions.

    PubMed

    Wang, Tianwen David; Ma, Fei; Ma, Xingyuan; Wang, Ping

    2015-04-01

    Cofactor is especially important for biotransformation catalyzed by oxidoreductases. Many attempts in enhancing performance of the reactions by improving cofactor utilization have been reported. In this study, efficiency of cofactor-requiring biocatalysis was enhanced by improving cofactor recycling via spatially programmed assembling glycerol dehydrogenase (GlyDH, Escherichia coli MG1655) and glutamate dehydrogenase (GluDH, Bacillus subtilis str168), with the aid of single-stranded DNA (ssDNA). The two enzymes were first independently expressed as molecules fused with a phage protein A* that could covalently link ssDNA with certain features. After an enzymatic cross-linking reaction taking place under mild conditions, the conjugate of fused enzyme and ssDNA was assembled into desired structures through base pairing enabled by the ssDNA. Results showed that, to some extent, the fusion with protein A* could improve the specific activity of the enzymes (GlyDH-A*/GlyDH?=?116.0 %; GluDH-A*/GluDH?=?105.2 %). Additionally, in the coupled reaction system with glycerol and ?-ketoglutaric acid as substrates, regarding the production of glutamic acid based on HPLC analysis, the efficiency of cofactor utilization was significantly enhanced (by 23.8- to 41.9-folds), indicating the existence of a substrate-channeling mechanism for cofactor utilization in the assembled reaction system due to the proximity effects. The degree of substrate channeling was calculated as from 1.65 to 1.73. Furthermore, the efficiency of cofactor utilization was influenced in an architecture-dependent manner when complexes with different stoichiometry of GlyDH and GluDH were utilized in biotransformation. This study demonstrated a novel strategy of cofactor recycling for enhanced performance of coupled oxidoreductive reactions. PMID:25363557

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

    PubMed

    Vishnoi, N; Singh, D P

    2014-01-01

    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 ?M) and lower Vmax (0.012 ?mol 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 ?M) and higher Vmax (0.044 ?mol 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 ?M 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. PMID:25535706

  2. Molecular study of the rat liver NADH: cytochrome c oxidoreductase complex during development and ageing.

    PubMed

    Torres-Mendoza, C E; Albert, A; de la Cruz Arriaga, M J

    1999-05-01

    The mechanisms involved in ageing are yet to be fully understood but it is thought that changes produced in energy transfer pathways occurring in the mitochondria may be responsible for the lack of energy typical of the later stages of life. The aim of the present investigation was to determine the enzymatic activity of the liver NADH cytochrome c oxidoreductase complex (Complex I-III) in mitochondria isolated from the liver of rats of 3 different age groups: lactating, animals (15-17 days), adult females (3-5 months) and old animals (26-30 months). The activities of the unbound Complexes I and III were also determined. An increase in Complex I-III activity was detected during development (142 +/- 10 vs. 447 +/- 23 micromol cyt. c/mg/min, p < 0.001) ang ageing (447 +/- 23 vs. 713 +/- 45 micromol cyt. c/mg/min, p < 0.001). However, unbound Complex I showed a reduction in activity during the ageing period whilst Complex III activity moderately increased. Immunological studies indicated only a moderate increase in the amount of Complex I-III and studies on the purified complex suggested that the increase in activity was due to effects other than an increase in enzyme quantity. The analysis of protein bands and the quantification of prosthetic groups showed particular reductions in the relative concentrations of Complex I subunits including the 51 kDa unit, which binds FMN, confirmed by a similar reduction in levels of the nucleotide. In contrast, 4 of the 5 subunits which increased during the lifetime of the animals corresponded to those of Complex III. These subunits are responsible for the binding of catalytic groups. The results suggest that, in addition to the increase in the amount of enzyme, binding factors between Complexes I and III may also play an important role in the observed increase in Complex I-III activity. PMID:10395077

  3. 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

  4. Arsenite and cadmium, but not chromium, induce NAD(P)H:quinone oxidoreductase 1 through transcriptional mechanisms, in spite of post-transcriptional modifications.

    PubMed

    Elbekai, Reem H; El-Kadi, Ayman O S

    2008-08-01

    NAD(P)H:quinone oxidoreductase (Nqo1)-mediated detoxification of quinones plays a critical role in cancer prevention. Metals alter the carcinogenicity of AhR ligands, such as TCDD, by modulating the induction of Nqo1, but the mechanism(s) remain unresolved. To decipher the molecular mechanisms involved in the alteration of Nqo1, we analyzed the effect of the metals As3+ (5 microM), Cd2+ (5 microM), and Cr6+ (25 microM) on the transcriptional activation of the Nqo1 gene and post-transcriptional modifications, in Hepa 1c1c7 cells. Both As3+ and Cd2+ induced Nqo1 mRNA in a time-dependent manner and potentiated TCDD-induced Nqo1 mRNA. Cr6+ on the other hand, completely inhibited the induction of Nqo1 mRNA by TCDD. The induction of Nqo1 mRNA by the metals was completely inhibited with the DNA transcription inhibitor actinomycin-D, indicating a requirement for de novo mRNA synthesis for the induction. Furthermore, the protein synthesis inhibitor cycloheximide decreased Nqo1 mRNA induction, suggesting a role for a labile protein in the transcriptional induction of Nqo1 mRNA by metals. Surprisingly, all three metals decreased Nqo1 mRNA stability while having no effect on Nqo1 protein half-life. Meanwhile, As3+ and Cd2+ induced constitutive Nqo1 activity and potentiated the induction by increasing concentrations of TCDD. On the other hand, Cr6+ inhibited inducible Nqo1 activity. It is apparent that metals alter Nqo1 expression at the transcriptional level, through a labile protein-mediated pathway. PMID:18474416

  5. Endothelial NADH\\/NADPH-dependent enzymatic sources of superoxide production: relationship to endothelial dysfunction

    Microsoft Academic Search

    Leszek Kalinowski; Tadeusz Malinski

    2004-01-01

    There is growing evidence that endothelial dysfunction, which is often defined as the decreased endothelial-derived nitric oxide (NO) bioavailability, is a crucial factor leading to vascular disease states such as hypertension, diabetes, atherosclerosis, heart failure and cigarette smoking. This is due to the fact that the lack of NO in en- dothelium-dependent vascular disorders contributes to impaired vascular relax- ation,

  6. Cytochrome P450/NADPH-dependent formation of trans epoxides from trans-arachidonic acids.

    PubMed

    Roy, Uzzal; Loreau, Olivier; Balazy, Michael

    2004-02-23

    Trans-arachidonic acids (trans-AA) are products of cis-trans isomerization of arachidonic acid by nitrogen dioxide radical (NO(2)), and occur in vivo, but their metabolism is unknown. We found that hepatic microsomes oxidized trans-AA via cytochrome P450/NADPH system to epoxides, which were hydrolyzed by epoxide hydrolase to diols (DiHETEs). 14,15-trans-AA produced one erythro diol and three threo diols each having one trans double bond. PMID:15013014

  7. Utilization of Glycerol as a Hydrogen Acceptor by Lactobacillus reuteri: Purification of 1,3-Propanediol:NAD+ Oxidoreductase

    PubMed Central

    Talarico, Todd L.; Axelsson, Lars T.; Novotny, James; Fiuzat, Mitra; Dobrogosz, Walter J.

    1990-01-01

    Lactobacillus reuteri utilizes exogenously added glycerol as a hydrogen acceptor during carbohydrate fermentations, resulting in higher growth rates and cell yields than those obtained during growth on carbohydrates alone. Glycerol is first converted to 3-hydroxypropionaldehyde by a coenzyme B12-dependent glycerol dehydratase and then reduced to 1,3-propanediol by an NAD+ -dependent oxidoreductase. The latter enzyme was purified and determined to have a molecular weight of 180,000; it is predicted to exist as a tetramer of identical 42,000-molecular-weight subunits. Images PMID:16348177

  8. NAD(P)H:Quinone Oxidoreductase 1 and its Potential Protective Role in Cardiovascular Diseases and Related Conditions

    Microsoft Academic Search

    Hong Zhu; Yunbo Li

    NAD(P)H:quinone oxidoreductase (NQO) represents a family of flavoproteins that catalyze the two-electron reduction of quinones\\u000a and their derivatives. In mammalian systems, there are two members of NQO, namely, NQO1 and NQO2. NQO1 utilizes NAD(P)H, whereas\\u000a NQO2 employs dihydronicotinamide riboside (NRH) as the electron donors. In addition to the well-documented action in reducing\\u000a quinone compounds and preventing the formation of reactive

  9. NAD(P)H:Quinone Oxidoreductase 1 Deficiency Increases Susceptibility to Benzo(a)pyrene-induced Mouse Skin Carcinogenesis1

    Microsoft Academic Search

    Delwin J. Long II; Rebekah L. Waikel; Xiao-Jing Wang; Laszlo Perlaky; Dennis R. Roop; Anil K. Jaiswal

    NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoprotein that catalyzes the metabolic detoxification of quinones and their derivatives. This protects cells against quinone-induced oxidative stress, cytotoxicity, and mutagenicity. C57BL6 NQO12\\/2 mice, deficient in NQO1 RNA and protein, were generated in our laboratory. To investigate the role of NQO1 in chemical carcinogenesis, the dorsal skin of NQO1-deficient (NQO12\\/2) and wild-type (NQO11\\/1) mice

  10. 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

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

    NASA Astrophysics Data System (ADS)

    Szczepaniak, Krzysztof; Worch, Remigiusz; Grzyb, Joanna

    2013-05-01

    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.

  12. 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. PMID:21742892

  13. 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

  14. 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

  15. Probing the Transmembrane Structure and Dynamics of Microsomal NADPH-cytochrome P450 oxidoreductase by Solid-State NMR

    PubMed Central

    Huang, Rui; Yamamoto, Kazutoshi; Zhang, Meng; Popovych, Nataliya; Hung, Ivan; Im, Sang-Choul; Gan, Zhehong; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2014-01-01

    NADPH-cytochrome P450 oxidoreductase (CYPOR) is an essential redox partner of the cytochrome P450 (cyt P450) superfamily of metabolic enzymes. In the endoplasmic reticulum of liver cells, such enzymes metabolize ?75% of the pharmaceuticals in use today. It is known that the transmembrane domain of CYPOR plays a crucial role in aiding the formation of a complex between CYPOR and cyt P450. Here we present the transmembrane structure, topology, and dynamics of the FMN binding domain of CYPOR in a native membrane-like environment. Our solid-state NMR results reveal that the N-terminal transmembrane domain of CYPOR adopts an ?-helical conformation in the lipid membrane environment. Most notably, we also show that the transmembrane helix is tilted ?13° from the lipid bilayer normal, and exhibits motions on a submillisecond timescale including rotational diffusion of the whole helix and fluctuation of the helical director axis. The approaches and the information reported in this study would enable further investigations on the structure and dynamics of the full-length NADPH-cytochrome P450 oxidoreductase and its interaction with other membrane proteins in a membrane environment. PMID:24853741

  16. Chemical constituents from the rice fermented with the edible mushroom Pleurotus eryngii and their quinone oxidoreductase 1 inducing effect.

    PubMed

    Liu, Shun; Dong, Yanan; Li, Yongxia; Bao, Li; Liu, Hongwei; Li, Heran

    2013-12-01

    The fruiting bodies or mycelia of mushrooms have been used as food and food-flavoring material for centuries due to their nutritional and medicinal values and the diversity of their bioactive components. The present research was the first to study the chemical components in rice fermented with the edible mushroom Pleurotus eryngii and the quinone oxidoreductase 1 inducing effect of these compounds. Through chemical investigation, one new compound, ((6S,7S)-6,7-dihydroxy-6-methyl-2-(3-methylbutanoyl)-4,5,6,7-tetrahydrobenzofuran-3-yl)methyl acetate (1) and eight known compounds (2-9) were isolated from the P. eryngii-fermented rice. All of these compounds were isolated from rice fermented with the edible mushroom P. eryngii for the first time. Their structures were elucidated by MS and NMR data analyses. Alternariol-5-O-methyl ether (2) showed strong quinone oxidoreductase 1 inducing effect with an IR value of 2.58 at the concentration of 20 ?g/ml. The content of adenosine (8) in the fermented rice (175.64 ?g/g) is much higher than that of non-fermented rice (14.38 ?g/g). PMID:23933238

  17. High-Yield Expression of a Catalytically Active Membrane-Bound Protein: Human P450 Oxidoreductase

    PubMed Central

    Sandee, Duanpen

    2011-01-01

    P450 oxidoreductase (POR) is a two-flavin protein that reduces microsomal P450 enzymes and some other proteins. Preparation of active bacterially expressed human POR for biochemical studies has been difficult because membrane-bound proteins tend to interact with column matrices. To reduce column-protein interactions and permit more vigorous washing, human POR lacking 27 N-terminal residues (N-27 POR) was modified to carry a C-terminal Gly3His6-tag (N-27 POR-G3H6). When expressed in Escherichia coli, N-27 POR-G3H6 could be purified to apparent homogeneity by a modified, single-step nickel-nitrilotriacetic acid affinity chromatography, yielding 31 mg POR per liter of culture, whereas standard purification of native N-27 POR required multiple steps, yielding 5 mg POR per liter. Both POR proteins had absorption maxima at 375 and 453 nm and both reduced cytochrome c with indistinguishable specific activities. Using progesterone as substrate for bacterially expressed purified human P450c17, the Michaelis constant for 17?-hydroxylase activity supported by N-27 POR or N-27 POR-G3H6 were 1.73 or 1.49 ?m, and the maximal velocity was 0.029 or 0.026 pmol steroids per picomole P450 per minute, respectively. Using 17-hydroxypregnenolone as the P450c17 substrate, the Michaelis constant for 17,20 lyase activity using N-27 POR or N-27 POR-G3H6 was 1.92 or 1.89 ?m and the maximal velocity was 0.041 or 0.042 pmol steroid per picomole P450 per minute, respectively. Thus, N-27 POR-G3H6 is equally active as native N-27 POR. This expression and purification system permits the rapid preparation of large amounts of highly pure, biologically active POR and may be generally applicable for the preparation of membrane-bound proteins. PMID:21586563

  18. Interaction of the mitochondria-targeted antioxidant MitoQ with phospholipid bilayers and ubiquinone oxidoreductases.

    PubMed

    James, Andrew M; Sharpley, Mark S; Manas, Abdul-Rahman B; Frerman, Frank E; Hirst, Judy; Smith, Robin A J; Murphy, Michael P

    2007-05-18

    MitoQ(10) is a ubiquinone that accumulates within mitochondria driven by a conjugated lipophilic triphenylphosphonium cation (TPP(+)). Once there, MitoQ(10) is reduced to its active ubiquinol form, which has been used to prevent mitochondrial oxidative damage and to infer the involvement of reactive oxygen species in signaling pathways. Here we show MitoQ(10) is effectively reduced by complex II, but is a poor substrate for complex I, complex III, and electron-transferring flavoprotein (ETF):quinone oxidoreductase (ETF-QOR). This differential reactivity could be explained if the bulky TPP(+) moiety sterically hindered access of the ubiquinone group to enzyme active sites with a long, narrow access channel. Using a combination of molecular modeling and an uncharged analog of MitoQ(10) with similar sterics (tritylQ(10)), we infer that the interaction of MitoQ(10) with complex I and ETF-QOR, but not complex III, is inhibited by its bulky TPP(+) moiety. To explain its lack of reactivity with complex III we show that the TPP(+) moiety of MitoQ(10) is ineffective at quenching pyrene fluorophors deeply buried within phospholipid bilayers and thus is positioned near the membrane surface. This superficial position of the TPP(+) moiety, as well as the low solubility of MitoQ(10) in non-polar organic solvents, suggests that the concentration of the entire MitoQ(10) molecule in the membrane core is very limited. As overlaying MitoQ(10) onto the structure of complex III indicates that MitoQ(10) cannot react with complex III without its TPP(+) moiety entering the low dielectric of the membrane core, we conclude that the TPP(+) moiety does anchor the tethered ubiquinol group out of reach of the active site(s) of complex III, thus explaining its slow oxidation. In contrast the ubiquinone moiety of MitoQ(10) is able to quench fluorophors deep within the membrane core, indicating a high concentration of the ubiquinone moiety within the membrane and explaining its good anti-oxidant efficacy. These findings will facilitate the rational design of future mitochondria-targeted molecules. PMID:17369262

  19. 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...

  20. Anti-cancer analogues ME-143 and ME-344 exert toxicity by directly inhibiting mitochondrial NADH: ubiquinone oxidoreductase (Complex I)

    PubMed Central

    Lim, Sze Chern; Carey, Kirstyn T; McKenzie, Matthew

    2015-01-01

    Isoflavonoids have been shown to inhibit tumor proliferation and metastasis by activating cell death pathways. As such, they have been widely studied as potential therapies for cancer prevention. The second generation synthetic isoflavan analogues ME-143 and ME-344 also exhibit anti-cancer effects, however their specific molecular targets have not been completely defined. To identify these targets, we examined the effects of ME-143 and ME-344 on cellular metabolism and found that they are potent inhibitors of mitochondrial oxidative phosphorylation (OXPHOS) complex I (NADH: ubiquinone oxidoreductase) activity. In isolated HEK293T mitochondria, ME-143 and ME-344 reduced complex I activity to 14.3% and 28.6% of control values respectively. In addition to the inhibition of complex I, ME-344 also significantly inhibited mitochondrial complex III (ubiquinol: ferricytochrome-c oxidoreductase) activity by 10.8%. This inhibition of complex I activity (and to a lesser extent complex III activity) was associated with a reduction in mitochondrial oxygen consumption. In permeabilized HEK293T cells, ME-143 and ME-344 significantly reduced the maximum ADP-stimulated respiration rate to 62.3% and 70.0% of control levels respectively in the presence of complex I-linked substrates. Conversely, complex II-linked respiration was unaffected by either drug. We also observed that the inhibition of complex I-linked respiration caused the dissipation of the mitochondrial membrane potential (??m). Blue native (BN-PAGE) analysis revealed that prolonged loss of ??m results in the destabilization of the native OXPHOS complexes. In particular, treatment of 143B osteosarcoma, HeLa and HEK293T human embryonic kidney cells with ME-344 for 4 h resulted in reduced steady-state levels of mature complex I. Degradation of the complex I subunit NDUFA9, as well as the complex IV (ferrocytochrome c: oxygen oxidoreductase) subunit COXIV, was also evident. The identification of OXPHOS complex I as a target of ME-143 and ME-344 advances our understanding of how these drugs induce cell death by disrupting mitochondrial metabolism, and will direct future work to maximize the anti-cancer capacity of these and other isoflavone-based compounds. PMID:25973307

  1. Anti-cancer analogues ME-143 and ME-344 exert toxicity by directly inhibiting mitochondrial NADH: ubiquinone oxidoreductase (Complex I).

    PubMed

    Lim, Sze Chern; Carey, Kirstyn T; McKenzie, Matthew

    2015-01-01

    Isoflavonoids have been shown to inhibit tumor proliferation and metastasis by activating cell death pathways. As such, they have been widely studied as potential therapies for cancer prevention. The second generation synthetic isoflavan analogues ME-143 and ME-344 also exhibit anti-cancer effects, however their specific molecular targets have not been completely defined. To identify these targets, we examined the effects of ME-143 and ME-344 on cellular metabolism and found that they are potent inhibitors of mitochondrial oxidative phosphorylation (OXPHOS) complex I (NADH: ubiquinone oxidoreductase) activity. In isolated HEK293T mitochondria, ME-143 and ME-344 reduced complex I activity to 14.3% and 28.6% of control values respectively. In addition to the inhibition of complex I, ME-344 also significantly inhibited mitochondrial complex III (ubiquinol: ferricytochrome-c oxidoreductase) activity by 10.8%. This inhibition of complex I activity (and to a lesser extent complex III activity) was associated with a reduction in mitochondrial oxygen consumption. In permeabilized HEK293T cells, ME-143 and ME-344 significantly reduced the maximum ADP-stimulated respiration rate to 62.3% and 70.0% of control levels respectively in the presence of complex I-linked substrates. Conversely, complex II-linked respiration was unaffected by either drug. We also observed that the inhibition of complex I-linked respiration caused the dissipation of the mitochondrial membrane potential (??m). Blue native (BN-PAGE) analysis revealed that prolonged loss of ??m results in the destabilization of the native OXPHOS complexes. In particular, treatment of 143B osteosarcoma, HeLa and HEK293T human embryonic kidney cells with ME-344 for 4 h resulted in reduced steady-state levels of mature complex I. Degradation of the complex I subunit NDUFA9, as well as the complex IV (ferrocytochrome c: oxygen oxidoreductase) subunit COXIV, was also evident. The identification of OXPHOS complex I as a target of ME-143 and ME-344 advances our understanding of how these drugs induce cell death by disrupting mitochondrial metabolism, and will direct future work to maximize the anti-cancer capacity of these and other isoflavone-based compounds. PMID:25973307

  2. Role of the O-antigen of lipopolysaccharide, and possible roles of growth rate and of NADH:ubiquinone oxidoreductase (nuo) in competitive tomato root-tip colonization by Pseudomonas fluorescens WCS365.

    PubMed

    Dekkers, L C; van der Bij, A J; Mulders, I H; Phoelich, C C; Wentwoord, R A; Glandorf, D C; Wijffelman, C A; Lugtenberg, B J

    1998-08-01

    Colonization-defective, transposon-induced mutants of the efficient root colonizer Pseudomonas fluorescens WCS365 were identified with a gnotobiotic system. Most mutants were impaired in known colonization traits, i.e., prototrophy for amino acids, motility, and synthesis of the O-antigen of LPS (lipopolysaccharide). Mutants lacking the O-antigen of LPS were impaired in both colonization and competitive growth whereas one mutant (PCL1205) with a shorter O-antigen chain was defective only in colonization ability, suggesting a role for the intact O-antigen of LPS in colonization. Eight competitive colonization mutants that were not defective in the above-mentioned traits colonized the tomato root tip well when inoculated alone, but were defective in competitive root colonization of tomato, radish, and wheat, indicating they contained mutations affecting host range. One of these eight mutants (PCL1201) was further characterized and contains a mutation in a gene that shows homology to the Escherichia coli nuo4 gene, which encodes a subunit of one of two known NADH:ubiquinone oxidoreductases. Competition experiments in an oxygen-poor medium between mutant PCL1201 and its parental strain showed a decreased growth rate of mutant PCL1201. The requirement of the nuo4 gene homolog for optimal growth under conditions of oxygen limitation suggests that the root-tip environment is micro-aerobic. A mutant characterized by a slow growth rate (PCL1216) was analyzed further and contained a mutation in a gene with similarity to the E. coli HtrB protein, a lauroyl transferase that functions in lipid A biosynthesis. PMID:9675892

  3. Regulation of Gene Expression of Class I Alcohol Dehydrogenase by Glucocorticoids

    Microsoft Academic Search

    Yu Dong; Lorenz Poellinger; Sam Okret; Jan-Olov Hoog; Hedvig von Bahr-Lindstrom; Hans Jornvall; Jan-Ake Gustafsson

    1988-01-01

    The effect of glucocorticoids on gene expression of rat class I alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) was investigated. A cDNA clone for the beta -subunit of human ADH (ADH2) was used to analyze class I ADH mRNA levels in rat hepatoma cells, which are known to contain a functional glucocorticoid receptor. RNA gel blot analysis of total cellular

  4. Murine and Human b Locus Pigmentation Genes Encode a Glycoprotein (gp75) with Catalase Activity

    Microsoft Academic Search

    Ruth Halaban; Gisela Moellmann

    1990-01-01

    Melanogenesis is regulated in large part by tyrosinase (monophenol monooxygenase; monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1), and defective tyrosinase leads to albinism. The mechanisms for other pigmentation determinants (e.g., those operative in tyrosinase-positive albinism and in murine coat-color mutants) are not yet known. One murine pigmentation gene, the brown (b) locus, when mutated leads to a brown (b\\/b) or hypopigmented (Blt\\/Blt)

  5. Simultaneous enzymatic synthesis of gluconic acid and sorbitol. Continuous process development using glucose-fructose oxidoreductase from Zymomonas mobilis.

    PubMed

    Silva-Martinez, M; Haltrich, D; Novalic, S; Kulbe, K D; Nidetzky, B

    1998-01-01

    The production of sorbitol and gluconic acid by isolated glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis has been studied in a convective, 100-mL loop reactor with tangential ultrafiltration. Using a dilution rate of 0.04/h and 5 kU/L GFOR, substrate conversion (3 M sugar) in a single stage was >85%, and productivities of 126 g sorbitol/(L x d) were obtained. At a constant recycle rate (3/min) and a membrane area of 50 cm2, the dilution rates (and thus productivities) were however limited by a more than 30-fold reduction of the permeate flow in the presence of high sugar and protein concentrations (5 g/L). Protein was added, together with 10 mM dithiothreitol, to improve the stability of GFOR during substrate turnover and crossflow filtration, thus leading to a stable operation of the enzyme reactor for at least 5 d. PMID:18576049

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

    PubMed Central

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

    2014-01-01

    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. PMID:24631540

  7. Purification and characterization of electron-transfer flavoprotein: rhodoquinone oxidoreductase from anaerobic mitochondria of the adult parasitic nematode, Ascaris suum.

    PubMed

    Ma, Y C; Funk, M; Dunham, W R; Komuniecki, R

    1993-09-25

    Electron-transfer flavoprotein:rhodoquinone oxidoreductase (ETF-RO) was purified to homogeneity from anaerobic mitochondria of the parasitic nematode, Ascaris suum. The enzyme has a subunit molecular mass of 64.5 kDa and is similar in many respects to the electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF-UO) characterized in mammalian tissues. EPR spectroscopy of the purified enzyme revealed signals at g = 2.076, 1,936, and 1.883, arising from an iron-sulfur center, as well as signals attributable to a flavin semiquinone. Potentiometric titration on the enzyme with dithionite yielded an oxidation-reduction midpoint potential (Em) for the iron-sulfur center of +25 mV at pH 7.4. The reduction of flavin occurred in two distinct steps, with a flavin semiquinone radical detected as an intermediate. The Em values for the two steps in the complete reduction of flavin were +15 mV and -9 mV, respectively. Physiologically, the ascarid ETF-RO accepts electrons from a low potential quinone, rhodoquinone, and functions in a direction opposite to that of the ETF-UO. Incubations of A. suum submitochondrial particles with NADH, 2-methylcrotonyl-CoA, purified A. suum electron-transfer flavoprotein and 2-methyl branched-chain enoyl-CoA reductase resulted in significant 2-methylbutyryl-CoA formation, which was inhibited by both rotenone and antisera to the purified ETF-RO. Quinone extraction of the submitchondrial particles with dry pentane resulted in almost the complete loss of 2-MBCoA formation by the system. However, the reincorporation of rhodoquinone, but not ubiquinone restored over 50% of the NADH-dependent 2-MBCoA formation. PMID:8376393

  8. Crystal Structures of NADH:FMN Oxidoreductase (EmoB) at Different Stages of Catalysis*S??

    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 ?/?-single-domain monomers of five parallel ?-strands flanked by five ?-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

  9. Human NAD(P)H:quinone oxidoreductase type I (hNQO1) activation of quinone propionic acid trigger groups†

    PubMed Central

    Mendoza, Maria F.; Hollabaugh, Nicole M.; Hettiarachchi, Suraj U.; McCarley, Robin L.

    2012-01-01

    NAD(P)H:quinone oxidoreductase type I (NQO1) is a target enzyme for triggered delivery of drugs at inflamed tissue and tumor sites, particularly those that challenge traditional therapies. Prodrugs, macromolecules, and molecular assemblies possessing trigger groups that can be cleaved by environmental stimuli are vehicles with the potential to yield active drug only at prescribed sites. Furthermore, quinone propionic acids (QPAs) covalently attached to prodrugs or liposome surfaces can be removed by application of a reductive trigger stimulus, such as that from NQO1; their rates of reductive activation should be tunable via QPA structure. We explored in detail the recombinant human NAD(P)H:quinone oxidoreductase type I (rhNQO1)-catalyzed NADH reduction of a family of substituted QPAs and obtained high precision kinetic parameters. It is found that small changes in QPA structure—in particular, single atom and function group substitutions on the quinone ring at R1—lead to significant impacts on the Michaelis constant (Km), maximum velocity (Vmax), catalytic constant (kcat), and catalytic efficiency (kcat/Km). Molecular docking simulations demonstrate that alterations in QPA structure result in large changes in QPA alignment and placement with respect to the flavin isoalloxazine ring in the active site of rhNQO1; a qualitative relationship exists between the kinetic parameters and the depth of QPA penetration into the rhNQO1 active site. From a quantitative perspective, a very good correlation is observed between log(kcat/Km) and the molecular-docking-derived distance between flavin hydride donor site and quinone hydride acceptor site in the QPAs, an observation that is in agreement with developing theories. The comprehensive kinetic and molecular modeling knowledge obtained for the interaction of recombinant human NQO1 with the quinone propionic acid analogues provides insight into the design and implementation of the QPA trigger groups for drug delivery applications. PMID:22989153

  10. A macrophage migration inhibitory factor like oxidoreductase from pearl oyster Pinctada fucata involved in innate immune responses.

    PubMed

    Cui, Shuge; Zhang, Dianchang; Jiang, Shigui; Pu, Hanlin; Hu, Yuting; Guo, Huayang; Chen, Mingqiang; Su, Tanfeng; Zhu, Caiyan

    2011-08-01

    Macrophage migration inhibitory factor (MIF) is an important cytokine and plays a crucial role as a pivotal regulator of innate immunity. In this study, a MIF cDNA was identified and characterized from the pearl oyster Pinctada fucata (designated as PoMIF). The full-length of PoMIF was 1544 bp and consisted of a 5'-untranslated region (UTR) of 45 bp, a 3'-UTR of 1139 bp with a polyadenylation signal (AATAAA) at 12 nucleotides upstream of the poly (A) tail. The open reading frame (ORF) of PoMIF was 360 bp which encoded a polypeptide of 120 amino acids with an estimated molecular mass of 13.3 kDa and a predicted pI of 6.1. SMART analysis showed that PoMIF contained the catalytic-sites P² and K³³ for tautomerase activity, a motif C??GSV?? for oxidoreductase activity and a MIF family signature D??PCGSVEVYSIGALG??. Homology analysis revealed that the PoMIF shared 40.3-65.5% similarity and 26.9-45.0% identity to other known MIF sequences. PoMIF mRNA was constitutively expressed in seven selected tissues of healthy pearl oysters, with the highest expression level in digestive gland. Eight hours after P. fucata was injected with Vibrio alginolyticus, the expression of PoMIF mRNA was significantly up-regulated in digestive gland, gills, hemocytes and intestine. The cDNA fragment encoding mature protein of PoMIF was subcloned to expression vector pRSET and transformed into Escherichia coli BL21 (DE3). The recombinant PoMIF (rPoMIF) was expressed and purified under optimized conditions. Function analysis showed that rPoMIF had oxidoreductase activity and could utilize dithiothreitol (DTT) as reductant to reduce insulin. PMID:21496487

  11. The structure of Aquifex aeolicus sulfide:quinone oxidoreductase, a basis to understand sulfide detoxification and respiration

    PubMed Central

    Marcia, Marco; Ermler, Ulrich; Peng, Guohong; Michel, Hartmut

    2009-01-01

    Sulfide:quinone oxidoreductase (SQR) is a flavoprotein with homologues in all domains of life except plants. It plays a physiological role both in sulfide detoxification and in energy transduction. We isolated the protein from native membranes of the hyperthermophilic bacterium Aquifex aeolicus, and we determined its X-ray structure in the “as-purified,” substrate-bound, and inhibitor-bound forms at resolutions of 2.3, 2.0, and 2.9 ?, respectively. The structure is composed of 2 Rossmann domains and 1 attachment domain, with an overall monomeric architecture typical of disulfide oxidoreductase flavoproteins. A. aeolicus SQR is a surprisingly trimeric, periplasmic integral monotopic membrane protein that inserts about 12 ? into the lipidic bilayer through an amphipathic helix–turn–helix tripodal motif. The quinone is located in a channel that extends from the si side of the FAD to the membrane. The quinone ring is sandwiched between the conserved amino acids Phe-385 and Ile-346, and it is possibly protonated upon reduction via Glu-318 and/or neighboring water molecules. Sulfide polymerization occurs on the re side of FAD, where the invariant Cys-156 and Cys-347 appear to be covalently bound to polysulfur fragments. The structure suggests that FAD is covalently linked to the polypeptide in an unusual way, via a disulfide bridge between the 8-methyl group and Cys-124. The applicability of this disulfide bridge for transferring electrons from sulfide to FAD, 2 mechanisms for sulfide polymerization and channeling of the substrate, S2?, and of the product, Sn, in and out of the active site are discussed. PMID:19487671

  12. The structure of Aquifex aeolicus sulfide:quinone oxidoreductase, a basis to understand sulfide detoxification and respiration.

    PubMed

    Marcia, Marco; Ermler, Ulrich; Peng, Guohong; Michel, Hartmut

    2009-06-16

    Sulfide:quinone oxidoreductase (SQR) is a flavoprotein with homologues in all domains of life except plants. It plays a physiological role both in sulfide detoxification and in energy transduction. We isolated the protein from native membranes of the hyperthermophilic bacterium Aquifex aeolicus, and we determined its X-ray structure in the "as-purified," substrate-bound, and inhibitor-bound forms at resolutions of 2.3, 2.0, and 2.9 A, respectively. The structure is composed of 2 Rossmann domains and 1 attachment domain, with an overall monomeric architecture typical of disulfide oxidoreductase flavoproteins. A. aeolicus SQR is a surprisingly trimeric, periplasmic integral monotopic membrane protein that inserts about 12 A into the lipidic bilayer through an amphipathic helix-turn-helix tripodal motif. The quinone is located in a channel that extends from the si side of the FAD to the membrane. The quinone ring is sandwiched between the conserved amino acids Phe-385 and Ile-346, and it is possibly protonated upon reduction via Glu-318 and/or neighboring water molecules. Sulfide polymerization occurs on the re side of FAD, where the invariant Cys-156 and Cys-347 appear to be covalently bound to polysulfur fragments. The structure suggests that FAD is covalently linked to the polypeptide in an unusual way, via a disulfide bridge between the 8-methyl group and Cys-124. The applicability of this disulfide bridge for transferring electrons from sulfide to FAD, 2 mechanisms for sulfide polymerization and channeling of the substrate, S(2-), and of the product, S(n), in and out of the active site are discussed. PMID:19487671

  13. Mechanism of Porcine Liver Xanthine Oxidoreductase Mediated N-Oxide Reduction of Cyadox as Revealed by Docking and Mutagenesis Studies

    PubMed Central

    Hao, Haihong; Dai, Menghong; Wang, Xu; Huang, Lingli; Liu, Zhenli; Yuan, Zonghui

    2013-01-01

    Xanthine oxidoreductase (XOR) is a cytoplasmic molybdenum-containing oxidoreductase, catalyzing both endogenous purines and exogenous compounds. It is suggested that XOR in porcine hepatocytes catalyzes the N-oxide reduction of quinoxaline 1,4-di-N-oxides (QdNOs). To elucidate the molecular mechanism underlying this metabolism, the cDNA of porcine XOR was cloned and heterologously expressed in Spodoptera frugiperda insect cells. The bovine XOR, showing sequence identity of 91% to porcine XOR, was employed as template for homology modeling. By docking cyadox, a representative compound of QdNOs, into porcine XOR model, eight amino acid residues, Gly47, Asn352, Ser360, Arg427, Asp430, Asp431, Ser1227 and Lys1230, were located at distances of less than 4Å to cyadox. Site-directed mutagenesis was performed to analyze their catalytic functions. Compared with wild type porcine XOR, G47A, S360P, D431A, S1227A, and K1230A displayed altered kinetic parameters in cyadox reduction, similarly to that in xanthine oxidation, indicating these mutations influenced electron-donating process of xanthine before subsequent electron transfer to cyadox to fulfill the N-oxide reduction. Differently, R427E and D430H, both located in the 424–434 loop, exhibited a much lower Km and a decreased Vmax respectively in cyadox reduction. Arg427 may be related to the substrate binding of porcine XOR to cyadox, and Asp430 is suggested to be involved in the transfer of electron to cyadox. This study initially reveals the possible catalytic mechanism of porcine XOR in cyadox metabolism, providing with novel insights into the structure-function relationship of XOR in the reduction of exogenous di-N-oxides. PMID:24040113

  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. Biophysical and structural studies of novel F420-dependent oxidoreductases in Mycobacterium tuberculosis

    E-print Network

    Mashalidis, Ellene H.

    2013-10-08

    -phosphate dehydrogenase FMN flavin mononucleotide GST glutathione S-transferase His polyhistidine tag HTS high-throughput screening HPLC high-pressure liquid chromatography IC50 half maximal inhibitory concentration iNOS nitric oxide synthase IPTG isopropyl... , there is only one gene for PNPOx106,107. It is an FMN-dependent enzyme encoded by the gene pdxH that oxidises either pyridoxine 5’-phosphate (1), a primary alcohol, or pyridoxamine 5’-phosphate (3), a primary amine, into pyridoxal 5’-phosphate (2)108 (Figure 2...

  16. 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 histories. These adaptations were likely shaped by ancient, as well as contemporary, intimate associations with monocot hosts. PMID:18373860

  17. NRH:Quinone Oxidoreductase 2 (NQO2) Protein Competes with the 20 S Proteasome to Stabilize Transcription Factor CCAAT Enhancer-binding Protein ? (C/EBP?), Leading to Protection against ? Radiation-induced Myeloproliferative Disease*

    PubMed Central

    Xu, Junkang; Patrick, Brad Allen; Jaiswal, Anil K.

    2013-01-01

    NRH:quinone oxidoreductase 2 (NQO2) is a flavoprotein that protects cells against radiation and chemical-induced oxidative stress. Disruption of the NQO2 gene in mice leads to ? radiation-induced myeloproliferative diseases. In this report, we showed that the 20 S proteasome and NQO2 both interact with myeloid differentiation factor CCAAT-enhancer-binding protein ? (C/EBP?). The interaction of the 20 S proteasome with C/EBP? led to the degradation of C/EBP?. NQO2, in the presence of its cofactor NRH, protected C/EBP? against 20 S degradation. Deletion and site-directed mutagenesis demonstrated that NQO2 and 20 S competed for the same binding region of S(268)GAGAGKAKKSV(279) in C/EBP?. Exposure of mice and HL-60 cells to ? radiation enhanced the levels of NQO2, which led to an increased NQO2 interaction with C/EBP? and decreased 20 S interaction with C/EBP?. NQO2 stabilization of C/EBP? was independent of NQO1, even though both interacted with the same C/EBP? domain. NQO2?/? mice, deficient in NQO2, failed to stabilize C/EBP?. This contributed to the development of ? radiation-induced myeloproliferative disease in NQO2?/? mice. PMID:24142791

  18. Glycogen Metabolic Genes Are Involved in Trehalose-6-Phosphate Synthase-Mediated Regulation of Pathogenicity by the Rice Blast Fungus Magnaporthe oryzae

    PubMed Central

    Wilson, Richard A.; Wang, Zheng-Yi; Kershaw, Michael J.; Talbot, Nicholas J.

    2013-01-01

    The filamentous fungus Magnaporthe oryzae is the causal agent of rice blast disease. Here we show that glycogen metabolic genes play an important role in plant infection by M. oryzae. Targeted deletion of AGL1 and GPH1, which encode amyloglucosidase and glycogen phosphorylase, respectively, prevented mobilisation of glycogen stores during appressorium development and caused a significant reduction in the ability of M. oryzae to cause rice blast disease. By contrast, targeted mutation of GSN1, which encodes glycogen synthase, significantly reduced the synthesis of intracellular glycogen, but had no effect on fungal pathogenicity. We found that loss of AGL1 and GPH1 led to a reduction in expression of TPS1 and TPS3, which encode components of the trehalose-6-phosphate synthase complex, that acts as a genetic switch in M. oryzae. Tps1 responds to glucose-6-phosphate levels and the balance of NADP/NADPH to regulate virulence-associated gene expression, in association with Nmr transcriptional inhibitors. We show that deletion of the NMR3 transcriptional inhibitor gene partially restores virulence to a ?agl1?gph1 mutant, suggesting that glycogen metabolic genes are necessary for operation of the NADPH-dependent genetic switch in M. oryzae. PMID:24098112

  19. Regulation of the photosynthetic electron transport during dark-light transitions by activation of the ferredoxin-NADP + -oxidoreductase in higher plants

    Microsoft Academic Search

    W. Rühle; R. Pschorn; A. Wild

    1987-01-01

    Absorbance changes associated with the oxidation and reduction of cytochrome f belong to the classical observations about the interaction of the two photosystems. A complex induction pattern of cytochrome f oxidation results, if both photosystems are excited simultaneously. This indicates a light-modulated regulation of the photosynthetic electron transport, which we examined for intact biological systems of decreasing complexity. The ferredoxin-NADP+-oxidoreductase

  20. From the Cover: Crystal structure of phycocyanobilin:ferredoxin oxidoreductase in complex with biliverdin IX, a key enzyme in the biosynthesis of phycocyanobilin

    Microsoft Academic Search

    Yoshinori Hagiwara; Masakazu Sugishima; Yasuhiro Takahashi; Keiichi Fukuyama

    2006-01-01

    Phytobilins (light harvesting and photoreceptor pigments in higher plants, algae, and cyanobacteria) are synthesized from biliverdin IX (BV) by ferredoxin-dependent bilin reductases (FDBRs). Phycocyanobilin:ferredoxin oxidoreductase (PcyA), one such FDBR, is a new class of radical enzymes that require neither cofactors nor metals and serially reduces the vinyl group of the D-ring and A-ring of BV using four electrons from ferredoxin

  1. The Highly Expressed and Inducible Endogenous NAD(P)H:quinone Oxidoreductase 1 in Cardiovascular Cells Acts as a Potential Superoxide Scavenger

    Microsoft Academic Search

    Hong Zhu; Zhenquan Jia; James E. Mahaney; David Ross; Hara P. Misra; Michael A. Trush; Yunbo Li

    2007-01-01

    It has recently been demonstrated that purified NAD(P)H:quinone oxidoreductase 1 (NQO1) is able to scavenge superoxide (O2•?) though the rate of reaction of O2•? with NQO1 is much lower than the rate of enzymatic dismutation catalyzed by superoxide dismutase (SOD). This study was undertaken\\u000a to determine if the endogenously expressed NQO1 in cardiovascular cells could scavenge O2•?. We observed that

  2. Electron spin relaxation enhancement measurements of interspin distances in human, porcine, and Rhodobacter electron transfer flavoprotein–ubiquinone oxidoreductase (ETF–QO)

    Microsoft Academic Search

    Alistair J. Fielding; Robert J. Usselman; Nicholas Watmough; Martin Simkovic; Frank E. Frerman; Gareth R. Eaton; Sandra S. Eaton

    2008-01-01

    Electron transfer flavoprotein–ubiquinone oxidoreductase (ETF–QO) is a membrane-bound electron transfer protein that links primary flavoprotein dehydrogenases with the main respiratory chain. Human, porcine, and Rhodobacter sphaeroides ETF–QO each contain a single [4Fe–4S]2+,1+ cluster and one equivalent of FAD, which are diamagnetic in the isolated enzyme and become paramagnetic on reduction with the enzymatic electron donor or with dithionite. The anionic

  3. Selective localisation of P450 enzymes and NADPH-P450 oxidoreductase in rat basal ganglia using anti-peptide antisera

    Microsoft Academic Search

    Andrew G Riedl; Paul M Watts; Robert J Edwards; Alan R Boobis; Peter Jenner; C. David Marsden

    1996-01-01

    Environmental or endogenous toxins may cause nigral cell death in Parkinson's disease (PD) due to altered expression of P450 enzymes. In rat brain, immunohistochemistry using anti-peptide antisera showed NADPH-P450 oxidoreductase and CYP2B1\\/2 in various hypothalamic nuclei and CYP1A1 in the globus pallidus, but neither enzyme was expressed in substantia nigra. No specific immunoreactivity to CYP2D1 or CYP3A1 was found in

  4. Expression of the Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae and its potential applications in wine production

    Microsoft Academic Search

    D. F. Malherbe; M. du Toit; R. R. Cordero Otero; P. van Rensburg; I. S. Pretorius

    2003-01-01

    There is a growing consumer demand for wines containing lower levels of alcohol and chemical preservatives. The objectives of this study were to express the Aspergillus niger gene encoding a glucose oxidase (GOX; ?-d-glucose:oxygen oxidoreductase, EC 1.1.3.4) in Saccharomyces cerevisiae and to evaluate the transformants for lower alcohol production and inhibition of wine spoilage organisms, such as acetic acid bacteria

  5. Enzymes Are Enriched in Bacterial Essential Genes

    PubMed Central

    Gao, Feng; Zhang, Randy Ren

    2011-01-01

    Essential genes, those indispensable for the survival of an organism, play a key role in the emerging field, synthetic biology. Characterization of functions encoded by essential genes not only has important practical implications, such as in identifying antibiotic drug targets, but can also enhance our understanding of basic biology, such as functions needed to support cellular life. Enzymes are critical for almost all cellular activities. However, essential genes have not been systematically examined from the aspect of enzymes and the chemical reactions that they catalyze. Here, by comprehensively analyzing essential genes in 14 bacterial genomes in which large-scale gene essentiality screens have been performed, we found that enzymes are enriched in essential genes. Essential enzymes have overrepresented ligases (especially those forming carbon-oxygen bonds and carbon-nitrogen bonds), nucleotidyltransferases and phosphotransferases, while have underrepresented oxidoreductases. Furthermore, essential enzymes tend to associate with more gene ontology domains. These results, from the aspect of chemical reactions, provide further insights into the understanding of functions needed to support natural cellular life, as well as synthetic cells, and provide additional parameters that can be integrated into gene essentiality prediction algorithms. PMID:21738765

  6. Biphasic Kinetic Behavior of E. coli WrbA, an FMN-Dependent NAD(P)H:Quinone Oxidoreductase

    PubMed Central

    Kishko, Iryna; Harish, Balasubramanian; Zayats, Vasilina; Reha, David; Tenner, Brian; Beri, Dhananjay; Gustavsson, Tobias; Ettrich, Rüdiger; Carey, Jannette

    2012-01-01

    The E. coli protein WrbA is an FMN-dependent NAD(P)H:quinone oxidoreductase that has been implicated in oxidative defense. Three subunits of the tetrameric enzyme contribute to each of four identical, cavernous active sites that appear to accommodate NAD(P)H or various quinones, but not simultaneously, suggesting an obligate tetramer with a ping-pong mechanism in which NAD departs before oxidized quinone binds. The present work was undertaken to evaluate these suggestions and to characterize the kinetic behavior of WrbA. Steady-state kinetics results reveal that WrbA conforms to a ping-pong mechanism with respect to the constancy of the apparent Vmax to Km ratio with substrate concentration. However, the competitive/non-competitive patterns of product inhibition, though consistent with the general class of bi-substrate reactions, do not exclude a minor contribution from additional forms of the enzyme. NMR results support the presence of additional enzyme forms. Docking and energy calculations find that electron-transfer-competent binding sites for NADH and benzoquinone present severe steric overlap, consistent with the ping-pong mechanism. Unexpectedly, plots of initial velocity as a function of either NADH or benzoquinone concentration present one or two Michaelis-Menten phases depending on the temperature at which the enzyme is held prior to assay. The effect of temperature is reversible, suggesting an intramolecular conformational process. WrbA shares these and other details of its kinetic behavior with mammalian DT-diaphorase, an FAD-dependent NAD(P)H:quinone oxidoreductase. An extensive literature review reveals several other enzymes with two-plateau kinetic plots, but in no case has a molecular explanation been elucidated. Preliminary sedimentation velocity analysis of WrbA indicates a large shift in size of the multimer with temperature, suggesting that subunit assembly coupled to substrate binding may underlie the two-plateau behavior. An additional aim of this report is to bring under wider attention the apparently widespread phenomenon of two-plateau Michaelis-Menten plots. PMID:22952804

  7. Mechanism of the Clostridium thermoaceticum pyruvate:ferredoxin oxidoreductase: evidence for the common catalytic intermediacy of the hydroxyethylthiamine pyropyrosphate radical.

    PubMed

    Menon, S; Ragsdale, S W

    1997-07-15

    The cofactor content and mechanism of pyruvate:ferredoxin oxidoreductase (PFOR) are controversial. By using rapid freeze-quench EPR and stopped-flow spectroscopy, the elementary steps that constitute the first half-reaction of the Clostridiumthermoaceticum PFOR mechanism were elucidated. A hydroxyethyl-TPP (HE-TPP) radical was identified and characterized as a transient intermediate, and for the first time, the kinetic competence of this substrate-derived radical was demonstrated. When the C. thermoaceticum PFOR was reacted with pyruvate and CoA, it had a lifetime of only approximately 100 ms. The results described here suggest that this radical intermediate is often not detected in studies of alpha-ketoacid oxidoreductases because it rapidly decays. It is postulated here that the HE-TPP radical is an intermediate in the mechanism of all PFORs irrespective of the number of 4Fe-4S clusters and will be detected in all PFORs when rapid mixing methods are used. The C. thermoaceticum PFOR was shown to contain two 4Fe-4S clusters, as concluded earlier [Wahl, R. C., & Orme-Johnson, W. H. (1987) J. Biol. Chem. 262, 10489-10496]. The first reductive half-reaction was shown to involve the following steps: (i) reaction with pyruvate with PFOR to form the hydroxyethylidene-TPP intermediate; (ii) one-electron transfer to reduce one of the two Fe4S4 clusters and yield the HE-TPP radical; and, (iii) reaction with CoA resulting in formation of acetyl-CoA, rapid decay of the HE-TPP radical intermediate, and reduction of the second Fe4S4 cluster. Thus, at the end of the first half-reaction, the two Fe4S4 clusters are fully reduced. The rate of the third step was found to depend on the CoA concentration (k = 35 per s at saturating concentrations of CoA); however, in its absence, this step was slower by approximately 4400-fold. PMID:9214293

  8. Identification of Novel Ssl0352 Protein (NdhS), Essential for Efficient Operation of Cyclic Electron Transport around Photosystem I, in NADPH:plastoquinone Oxidoreductase (NDH-1) Complexes of Synechocystis sp. PCC 6803*

    PubMed Central

    Battchikova, Natalia; Wei, Lanzhen; Du, Lingyu; Bersanini, Luca; Aro, Eva-Mari; Ma, Weimin

    2011-01-01

    Cyanobacterial NADPH:plastoquinone oxidoreductase, or type I NAD(P)H dehydrogenase, or the NDH-1 complex is involved in plastoquinone reduction and cyclic electron transfer (CET) around photosystem I. CET, in turn, produces extra ATP for cell metabolism particularly under stressful conditions. Despite significant achievements in the study of cyanobacterial NDH-1 complexes during the past few years, the entire subunit composition still remains elusive. To identify missing subunits, we screened a transposon-tagged library of Synechocystis 6803 cells grown under high light. Two NDH-1-mediated CET (NDH-CET)-defective mutants were tagged in the same ssl0352 gene encoding a short unknown protein. To clarify the function of Ssl0352, the ssl0352 deletion mutant and another mutant with Ssl0352 fused to yellow fluorescent protein (YFP) and the His6 tag were constructed. Immunoblotting, mass spectrometry, and confocal microscopy analyses revealed that the Ssl0352 protein resides in the thylakoid membrane and associates with the NDH-1L and NDH-1M complexes. We conclude that Ssl0352 is a novel subunit of cyanobacterial NDH-1 complexes and designate it NdhS. Deletion of the ssl0352 gene considerably impaired the NDH-CET activity and also retarded cell growth under high light conditions, indicating that NdhS is essential for efficient operation of NDH-CET. However, the assembly of the NDH-1L and NDH-1M complexes and their content in the cells were not affected in the mutant. NdhS contains a Src homology 3-like domain and might be involved in interaction of the NDH-1 complex with an electron donor. PMID:21880717

  9. Ipsdienol dehydrogenase (IDOLDH): a novel oxidoreductase important for Ips pini pheromone production

    Microsoft Academic Search

    Rubi Figueroa-Teran; William H. Welch; Gary J. Blomquist; Claus Tittiger

    Ipsdienone (2-methyl-6-methylene-2,7-octadien-4-one) is an important intermediate in the biosynthesis of pheromonal ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) and ipsenol (2-methyl-6-methylene-7-octen-4-ol) in male pine engraver beetles, Ips pini (Say). A novel ipsdienol dehydrogenase (IDOLDH) with a pheromone biosynthetic gene expression pattern was cloned, expressed, functionally characterized, and its cellular localization analyzed. The cDNA has a 762 nt ORF encoding a 253 amino acid predicted translation

  10. EPR Spectroscopic and Computational Characterization of the Hydroxyethylidene-Thiamine Pyrophosphate Radical Intermediate of Pyruvate:Ferredoxin Oxidoreductase

    PubMed Central

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

    2008-01-01

    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) ?-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 non-planar, ?/n-type cation radical. PMID:16752902

  11. Catalytic Mechanism of Short Ethoxy Chain Nonylphenol Dehydrogenase Belonging to a Polyethylene Glycol Dehydrogenase Group in the GMC Oxidoreductase Family

    PubMed Central

    Liu, Xin; Ohta, Takeshi; Kawabata, Takeshi; Kawai, Fusako

    2013-01-01

    Ethoxy (EO) chain nonylphenol dehydrogenase (NPEO-DH) from Ensifer sp. AS08 and EO chain octylphenol dehydrogenase from Pseudomonas putida share common molecular characteristics with polyethylene glycol (PEG) dehydrogenases (PEG-DH) and comprise a PEG-DH subgroup in the family of glucose-methanol-choline (GMC) oxidoreductases that includes glucose/alcohol oxidase and glucose/choline dehydrogenase. Three-dimensional (3D) molecular modeling suggested that differences in the size, secondary structure and hydropathy in the active site caused differences in their substrate specificities toward EO chain alkylphenols and free PEGs. Based on 3D molecular modeling, site-directed mutagenesis was utilized to introduce mutations into potential catalytic residues of NPEO-DH. From steady state and rapid kinetic characterization of wild type and mutant NPEO-DHs, we can conclude that His465 and Asn507 are directly involved in the catalysis. Asn507 mediates the transfer of proton from a substrate to FAD and His465 transfers the same proton from the reduced flavin to an electron acceptor. PMID:23306149

  12. Effectors of the mammalian plasma membrane NADH-oxidoreductase system. Short-chain ubiquinone analogues as potent stimulators.

    PubMed

    Vaillant, F; Larm, J A; McMullen, G L; Wolvetang, E J; Lawen, A

    1996-12-01

    In the presence of effectors variations in the two recognized activities of the plasma membrane NADH-oxidoreductase system were studied in separate, specific in vitro assays. We report here that ubiquinone analogues that contain a short, less hydrophobic side chain than coenzyme Q-10 dramatically stimulate the NADH-oxidase activity of isolated rat liver plasma membranes whereas they show no effect on the reductase activity of isolated membranes. If measured in assays of the NADH:ferricyanide reductase of living cultured cells these compounds have only a limited effect; the oxidase activity of whole cells is not measurable in our hands. We have furthermore identified selective inhibitors of both enzyme activities. In particular, the NADH-oxidase activity can be significantly inhibited by structural analogues of ubiquinone, such as capsaicin and resiniferatoxin. The NADH:ferricyanide reductase, on the other hand, is particularly sensitive to pCMBS, indicating the presence of a sulfhydryl group of groups at its active site. The identification of these specific effectors of the different enzyme activities of the PMOR yields further insights into the function of this system. PMID:8953385

  13. The secretome of Trametes versicolor grown on tomato juice medium and purification of the secreted oxidoreductases including a versatile peroxidase.

    PubMed

    Carabajal, Maira; Kellner, Harald; Levin, Laura; Jehmlich, Nico; Hofrichter, Martin; Ullrich, René

    2013-10-10

    The present work was carried out with the aim to analyze the secretome of Trametes versicolor BAFC 2234 grown on tomato juice medium supplemented with copper and manganese. T. versicolor BAFC 2234 was selected among diverse wood dwelling agaricomycetes from Argentina by its ability to cause a strong white rot on hardwood and in addition to show high tolerance toward phenolic compounds. A considerable number of the identified proteins were related to the degradation/modification of lignocelluloses. Hydrolases, peroxidases and phenoloxidases were the most abundant enzymes produced under the above-mentioned culture conditions. The lignin-modifying oxidoreductases laccase, manganese peroxidase (MnP) and versatile peroxidase (VP) were successfully purified - the latter for the first time from T. versicolor. The native VP protein has a molecular mass of 45kDa and an isoelectric point of pH 3.7. The study clearly shows that complex plant-based media being rich in phenolics, such as tomato juice, can stimulate the secretion of a broad set of extracellular lignocellulolytic enzymes. Using such natural products as fungal culture media may give the opportunity to investigate plant biomass decomposition as well as the biodegradation of organic pollutants in an environment close to nature. PMID:23948257

  14. Insight Into the Radical Mechanism of Phycocyanobilin-Ferredoxin Oxidoreductase (Pcya) Revealed By X-Ray Crystallography And Biochemical Measurements

    SciTech Connect

    Tu, S.-L.; Rockwell, N.; Lagarias, J.C.; Fisher, A.J.; /Inst. Plant Microb. Biol., Taipei /UC, Davis

    2007-07-13

    The X-ray crystal structure of the substrate-free form of phycocyanobilin (PCB)-ferredoxin oxidoreductase (PcyA; EC 1.3.7.5) from the cyanobacterium Nostoc sp. PCC7120 has been solved at 2.5 angstrom resolution. A comparative analysis of this structure with those recently reported for substrate-bound and substrate-free forms of PcyA from the cyanobacterium Synechocystis sp. PCC6803 (Hagiwara et al. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 27-32; Hagiwara et al. (2006) FEBS Lett. 580, 3823-3828) provides a compelling picture of substrate-induced changes in the PcyA enzyme and the chemical basis of PcyA's catalytic activity. On the basis of these structures and the biochemical analysis of site-directed mutants of Nostoc PcyA, including mutants reported in recent studies (Tu et al. (2006) J. Biol. Chem. 281, 3127-3136) as well as mutants described in this study, a revised mechanism for the PcyA-mediated four-electron reduction of biliverdin IX{alpha} to 3E/3Z-phycocyanobilin via enzyme-bound bilin radical intermediates is proposed. The mechanistic insight of these studies, along with homology modeling, have provided new insight into the catalytic mechanisms of other members of the ferredoxin-dependent bilin reductase family that are widespread in oxygenic photosynthetic organisms.

  15. Human complex II (succinate-ubiquinone oxidoreductase): cDNA cloning of iron sulfur (Ip) subunit of liver mitochondria.

    PubMed

    Kita, K; Oya, H; Gennis, R B; Ackrell, B A; Kasahara, M

    1990-01-15

    Complex II (succinate-ubiquinone oxidoreductase) is an important enzyme complex of both the tricarboxylic acid cycle and of the aerobic respiratory chains of mitochondria in eukaryotic cell and prokaryotic organisms. In this study, the amino acid sequence of iron sulfur-subunit in human liver mitochondria was deduced from cDNA which was isolated by immunoscreening a human liver lambda gtll cDNA library. An isolated clone contains an open reading frame of 786 nucleotides and encodes a mature protein of 252 amino acids with a molecular weight of 28,804. The amino acid sequence was highly homologous with that of bovine heart (94.1%) which has been determined from the purified peptide and that of Escherichia coli sdh B product (50.8%). Striking sequence conservation was found around the three cysteine-rich clusters which have been thought to comprise the iron-sulfur centers of the enzyme. This is the first report on the cDNA sequence of mitochondrial complex II. PMID:2302193

  16. NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), a multifunctional antioxidant enzyme and exceptionally versatile cytoprotector.

    PubMed

    Dinkova-Kostova, Albena T; Talalay, Paul

    2010-09-01

    NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1) is a widely-distributed FAD-dependent flavoprotein that promotes obligatory 2-electron reductions of quinones, quinoneimines, nitroaromatics, and azo dyes, at rates that are comparable with NADH or NADPH. These reductions depress quinone levels and thereby minimize opportunities for generation of reactive oxygen intermediates by redox cycling, and for depletion of intracellular thiol pools. NQO1 is a highly-inducible enzyme that is regulated by the Keap1/Nrf2/ARE pathway. Evidence for the importance of the antioxidant functions of NQO1 in combating oxidative stress is provided by demonstrations that induction of NQO1 levels or their depletion (knockout, or knockdown) are associated with decreased and increased susceptibilities to oxidative stress, respectively. Furthermore, benzene genotoxicity is markedly enhanced when NQO1 activity is compromised. Not surprisingly, human polymorphisms that suppress NQO1 activities are associated with increased predisposition to disease. Recent studies have uncovered protective roles for NQO1 that apparently are unrelated to its enzymatic activities. NQO1 binds to and thereby stabilizes the important tumor suppressor p53 against proteasomal degradation. Indeed, NQO1 appears to regulate the degradative fate of other proteins. These findings suggest that NQO1 may exercise a selective "gatekeeping" role in regulating the proteasomal degradation of specific proteins, thereby broadening the cytoprotective role of NQO1 far beyond its highly effective antioxidant functions. PMID:20361926

  17. Arabidopsis tic62 trol mutant lacking thylakoid-bound ferredoxin-NADP+ oxidoreductase shows distinct metabolic phenotype.

    PubMed

    Lintala, Minna; Schuck, Natalie; Thormählen, Ina; Jungfer, Andreas; Weber, Katrin L; Weber, Andreas P M; Geigenberger, Peter; Soll, Jürgen; Bölter, Bettina; Mulo, Paula

    2014-01-01

    Ferredoxin-NADP+ oxidoreductase (FNR), functioning in the last step of the photosynthetic electron transfer chain, exists both as a soluble protein in the chloroplast stroma and tightly attached to chloroplast membranes. Surface plasmon resonance assays showed that the two FNR isoforms, LFNR1 and LFNR2, are bound to the thylakoid membrane via the C-terminal domains of Tic62 and TROL proteins in a pH-dependent manner. The tic62 trol double mutants contained a reduced level of FNR, exclusively found in the soluble stroma. Although the mutant plants showed no visual phenotype or defects in the function of photosystems under any conditions studied, a low ratio of NADPH/NADP+ was detected. Since the CO? fixation capacity did not differ between the tic62 trol plants and wild-type, it seems that the plants are able to funnel reducing power to most crucial reactions to ensure survival and fitness of the plants. However, the activity of malate dehydrogenase was down-regulated in the mutant plants. Apparently, the plastid metabolism is able to cope with substantial changes in directing the electrons from the light reactions to stromal metabolism and thus only few differences are visible in steady-state metabolite pool sizes of the tic62 trol plants. PMID:24043709

  18. 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. PMID:16752902

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

    PubMed Central

    Tremblay, Pier-Luc; Zhang, Tian; Dar, Shabir A.; Leang, Ching; Lovley, Derek R.

    2012-01-01

    ABSTRACT 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 H2 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. PMID:23269825

  20. Identification and Characterization of the Rhizobium sp. Strain GIN611 Glycoside Oxidoreductase Resulting in the Deglycosylation of Ginsenosides

    PubMed Central

    Kim, Eun-Mi; Kim, Juhan; Seo, Joo-Hyun; Park, Jun-Seong; Kim, Duck-Hee

    2012-01-01

    Using enrichment culture, Rhizobium sp. strain GIN611 was isolated as having activity for deglycosylation of a ginsenoside, compound K (CK). The purified heterodimeric protein complex from Rhizobium sp. GIN611 consisted of two subunits with molecular masses of 63.5 kDa and 17.5 kDa. In the genome, the coding sequence for the small subunit was located right after the sequence for the large subunit, with one nucleotide overlapping. The large subunit showed CK oxidation activity, and the deglycosylation of compound K was performed via oxidation of ginsenoside glucose by glycoside oxidoreductase. Coexpression of the small subunit helped soluble expression of the large subunit in recombinant Escherichia coli. The purified large subunit also showed oxidation activity against other ginsenoside compounds, such as Rb1, Rb2, Rb3, Rc, F2, CK, Rh2, Re, F1, and the isoflavone daidzin, but at a much lower rate. When oxidized CK was extracted and incubated in phosphate buffer with or without enzyme, (S)-protopanaxadiol [PPD(S)] was detected in both cases, which suggests that deglycosylation of oxidized glucose is spontaneous. PMID:22020506

  1. 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.

  2. 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.

  3. Identification and characterization of the Rhizobium sp. strain GIN611 glycoside oxidoreductase resulting in the deglycosylation of ginsenosides.

    PubMed

    Kim, Eun-Mi; Kim, Juhan; Seo, Joo-Hyun; Park, Jun-Seong; Kim, Duck-Hee; Kim, Byung-Gee

    2012-01-01

    Using enrichment culture, Rhizobium sp. strain GIN611 was isolated as having activity for deglycosylation of a ginsenoside, compound K (CK). The purified heterodimeric protein complex from Rhizobium sp. GIN611 consisted of two subunits with molecular masses of 63.5 kDa and 17.5 kDa. In the genome, the coding sequence for the small subunit was located right after the sequence for the large subunit, with one nucleotide overlapping. The large subunit showed CK oxidation activity, and the deglycosylation of compound K was performed via oxidation of ginsenoside glucose by glycoside oxidoreductase. Coexpression of the small subunit helped soluble expression of the large subunit in recombinant Escherichia coli. The purified large subunit also showed oxidation activity against other ginsenoside compounds, such as Rb1, Rb2, Rb3, Rc, F2, CK, Rh2, Re, F1, and the isoflavone daidzin, but at a much lower rate. When oxidized CK was extracted and incubated in phosphate buffer with or without enzyme, (S)-protopanaxadiol [PPD(S)] was detected in both cases, which suggests that deglycosylation of oxidized glucose is spontaneous. PMID:22020506

  4. Myocardial Overexpression of Mecr, a Gene of Mitochondrial FAS II Leads to Cardiac Dysfunction in Mouse

    PubMed Central

    Chen, Zhijun; Leskinen, Hanna; Liimatta, Erkki; Sormunen, Raija T.; Miinalainen, Ilkka J.; Hassinen, Ilmo E.; Hiltunen, J. Kalervo

    2009-01-01

    It has been recently recognized that mammalian mitochondria contain most, if not all, of the components of fatty acid synthesis type II (FAS II). Among the components identified is 2-enoyl thioester reductase/mitochondrial enoyl-CoA reductase (Etr1/Mecr), which catalyzes the NADPH-dependent reduction of trans-2-enoyl thioesters, generating saturated acyl-groups. Although the FAS type II pathway is highly conserved, its physiological role in fatty acid synthesis, which apparently occurs simultaneously with breakdown of fatty acids in the same subcellular compartment in mammals, has remained an enigma. To study the in vivo function of the mitochondrial FAS in mammals, with special reference to Mecr, we generated mice overexpressing Mecr under control of the mouse metallothionein-1 promoter. These Mecr transgenic mice developed cardiac abnormalities as demonstrated by echocardiography in vivo, heart perfusion ex vivo, and electron microscopy in situ. Moreover, the Mecr transgenic mice showed decreased performance in endurance exercise testing. Our results showed a ventricular dilatation behind impaired heart function upon Mecr overexpression, concurrent with appearance of dysmorphic mitochondria. Furthermore, the data suggested that inappropriate expression of genes of FAS II can result in the development of hereditary cardiomyopathy. PMID:19440339

  5. Gene expression of sternohyoid and diaphragm muscles in type 2 diabetic rats

    PubMed Central

    2013-01-01

    Background Type 2 diabetes differs from type 1 diabetes in its pathogenesis. Type 1 diabetic diaphragm has altered gene expression which includes lipid and carbohydrate metabolism, ubiquitination and oxidoreductase activity. The objectives of the present study were to assess respiratory muscle gene expression changes in type 2 diabetes and to determine whether they are greater for the diaphragm than an upper airway muscle. Methods Diaphragm and sternohyoid muscle from Zucker diabetic fatty (ZDF) rats were analyzed with Affymetrix gene expression arrays. Results The two muscles had 97 and 102 genes, respectively, with at least ± 1.5-fold significantly changed expression with diabetes, and these were assigned to gene ontology groups based on over-representation analysis. Several significantly changed groups were common to both muscles, including lipid metabolism, carbohydrate metabolism, muscle contraction, ion transport and collagen, although the number of genes and the specific genes involved differed considerably for the two muscles. In both muscles there was a shift in metabolism gene expression from carbohydrate metabolism toward lipid metabolism, but the shift was greater and involved more genes in diabetic diaphragm than diabetic sternohyoid muscle. Groups present in only diaphragm were blood circulation and oxidoreductase activity. Groups present in only sternohyoid were immune & inflammation and response to stress & wounding, with complement genes being a prominent component. Conclusion Type 2 diabetes-induced gene expression changes in respiratory muscles has both similarities and differences relative to previous data on type 1 diabetes gene expression. Furthermore, the diabetic alterations in gene expression differ between diaphragm and sternohyoid. PMID:24199937

  6. The structural and functional basis of catalysis mediated by NAD(P)H:acceptor Oxidoreductase (FerB) of Paracoccus denitrificans.

    PubMed

    Sedlá?ek, Vojt?ch; Klumpler, Tomáš; Marek, Jaromír; Ku?era, Igor

    2014-01-01

    FerB from Paracoccus denitrificans is a soluble cytoplasmic flavoprotein that accepts redox equivalents from NADH or NADPH and transfers them to various acceptors such as quinones, ferric complexes and chromate. The crystal structure and small-angle X-ray scattering measurements in solution reported here reveal a head-to-tail dimer with two flavin mononucleotide groups bound at the opposite sides of the subunit interface. The dimers tend to self-associate to a tetrameric form at higher protein concentrations. Amino acid residues important for the binding of FMN and NADH and for the catalytic activity are identified and verified by site-directed mutagenesis. In particular, we show that Glu77 anchors a conserved water molecule in close proximity to the O2 of FMN, with the probable role of facilitating flavin reduction. Hydride transfer is shown to occur from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring. When using deuterated NADH, this process exhibits a kinetic isotope effect of about 6 just as does the NADH-dependent quinone reductase activity of FerB; the first, reductive half-reaction of flavin cofactor is thus rate-limiting. Replacing the bulky Arg95 in the vicinity of the active site with alanine substantially enhances the activity towards external flavins that obeys the standard bi-bi ping-pong reaction mechanism. The new evidence for a cryptic flavin reductase activity of FerB justifies the previous inclusion of this enzyme in the protein family of NADPH-dependent FMN reductases. PMID:24817153

  7. Discovery of the first light-dependent protochlorophyllide oxidoreductase in anoxygenic phototrophic bacteria.

    PubMed

    Kaschner, Marco; Loeschcke, Anita; Krause, Judith; Minh, Bui Quang; Heck, Achim; Endres, Stephan; Svensson, Vera; Wirtz, Astrid; von Haeseler, Arndt; Jaeger, Karl-Erich; Drepper, Thomas; Krauss, Ulrich

    2014-09-01

    In all photosynthetic organisms, chlorophylls function as light-absorbing photopigments allowing the efficient harvesting of light energy. Chlorophyll biosynthesis recurs in similar ways in anoxygenic phototrophic proteobacteria as well as oxygenic phototrophic cyanobacteria and plants. Here, the biocatalytic conversion of protochlorophyllide to chlorophyllide is catalysed by evolutionary and structurally distinct protochlorophyllide reductases (PORs) in anoxygenic and oxygenic phototrophs. It is commonly assumed that anoxygenic phototrophs only contain oxygen-sensitive dark-operative PORs (DPORs), which catalyse protochlorophyllide reduction independent of the presence of light. In contrast, oxygenic phototrophs additionally (or exclusively) possess oxygen-insensitive but light-dependent PORs (LPORs). Based on this observation it was suggested that light-dependent protochlorophyllide reduction first emerged as a consequence of increased atmospheric oxygen levels caused by oxygenic photosynthesis in cyanobacteria. Here, we provide experimental evidence for the presence of an LPOR in the anoxygenic phototrophic ?-proteobacterium Dinoroseobacter shibae?DFL12(T). In vitro and in vivo functional assays unequivocally prove light-dependent protochlorophyllide reduction by this enzyme and reveal that LPORs are not restricted to cyanobacteria and plants. Sequence-based phylogenetic analyses reconcile our findings with current hypotheses about the evolution of LPORs by suggesting that the light-dependent enzyme of D. shibae?DFL12(T) might have been obtained from cyanobacteria by horizontal gene transfer. PMID:25039543

  8. Ipsdienol dehydrogenase (IDOLDH): a novel oxidoreductase important for Ips pini pheromone production.

    PubMed

    Figueroa-Teran, Rubi; Welch, William H; Blomquist, Gary J; Tittiger, Claus

    2012-02-01

    Ipsdienone (2-methyl-6-methylene-2,7-octadien-4-one) is an important intermediate in the biosynthesis of pheromonal ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) and ipsenol (2-methyl-6-methylene-7-octen-4-ol) in male pine engraver beetles, Ips pini (Say). A novel ipsdienol dehydrogenase (IDOLDH) with a pheromone-biosynthetic gene expression pattern was cloned, expressed, functionally characterized, and its cellular localization analyzed. The cDNA has a 762nt ORF encoding a 253 amino acid predicted translation product of 28kDa and pI 5.8. The protein has conserved motifs of the Cp2 subfamily of "classical" short-chain dehydrogenases. Transcript levels were highest in pheromone producing tissue: the anterior midgut of fed males. The protein was detected only in male midguts and localized in the cytosolic fraction of midgut cells. Recombinant IDOLDH was produced in Sf9 cells using a baculovirus expression system. Enzyme assays of protein preparations showed IDOLDH used both NAD? and NADP? as coenzymes with specific activities in the nanomole range. Enzyme assays and GC/MS analysis showed that IDOLDH catalyzed the oxidation of racemic ipsdienol and (4R)-(-)-ipsdienol to form ipsdienone, while (4S)-(+)-ipsdienol was not a substrate. These data strongly implicate IDOLDH as an enzyme involved in terminal pheromone-biosynthetic steps, likely functioning to "tune" ipsdienol enantiomeric ratios. PMID:22101251

  9. Residence of Habitat-Specific Anammox Bacteria in the Deep-Sea Subsurface Sediments of the South China Sea: Analyses of Marker Gene Abundance with Physical Chemical Parameters

    Microsoft Academic Search

    Yi-Guo Hong; Meng Li; Huiluo Cao; Ji-Dong Gu

    Anaerobic ammonium oxidation (anammox) has been recognized as an important process for the global nitrogen cycle. In this\\u000a study, the occurrence and diversity of anammox bacteria in the deep-sea subsurface sediments of the South China Sea (SCS)\\u000a were investigated. Results indicated that the anammox bacterial sequences recovered from this habitat by amplifying both 16S\\u000a rRNA gene and hydrazine oxidoreductase encoding

  10. The yeast Saccharomyces cerevisiae contains two glutaredoxin genes that are required for protection against reactive oxygen species.

    PubMed

    Luikenhuis, S; Perrone, G; Dawes, I W; Grant, C M

    1998-05-01

    Glutaredoxins are small heat-stable proteins that act as glutathione-dependent disulfide oxidoreductases. Two genes, designated GRX1 and GRX2, which share 40-52% identity and 61-76% similarity with glutaredoxins from bacterial and mammalian species, were identified in the yeast Saccharomyces cerevisiae. Strains deleted for both GRX1 and GRX2 were viable but lacked heat-stable oxidoreductase activity using beta-hydroxyethylene disulfide as a substrate. Surprisingly, despite the high degree of homology between Grx1 and Grx2 (64% identity), the grx1 mutant was unaffected in oxidoreductase activity, whereas the grx2 mutant displayed only 20% of the wild-type activity, indicating that Grx2 accounted for the majority of this activity in vivo. Expression analysis indicated that this difference in activity did not arise as a result of differential expression of GRX1 and GRX2. In addition, a grx1 mutant was sensitive to oxidative stress induced by the superoxide anion, whereas a strain that lacked GRX2 was sensitive to hydrogen peroxide. Sensitivity to oxidative stress was not attributable to altered glutathione metabolism or cellular redox state, which did not vary between these strains. The expression of both genes was similarly elevated under various stress conditions, including oxidative, osmotic, heat, and stationary phase growth. Thus, Grx1 and Grx2 function differently in the cell, and we suggest that glutaredoxins may act as one of the primary defenses against mixed disulfides formed following oxidative damage to proteins. PMID:9571241

  11. The role of short-chain dehydrogenase/oxidoreductase, induced by salt stress, on host interaction of B. pseudomallei

    PubMed Central

    2014-01-01

    Background Burkholderia pseudomallei is the causative agent of melioidosis, a frequently occurring disease in northeastern Thailand, where soil and water high in salt content are common. Using microarray analysis, we previously showed that B. pseudomallei up-regulated a short-chain dehydrogenase/oxidoreductase (SDO) under salt stress. However, the importance of SDO in B. pseudomallei infection is unknown. This study aimed to explore the function of B. pseudomallei SDO, and to investigate its role in interactions between B. pseudomallei and host cells. Results Bioinformatics analysis of B. pseudomallei SDO structure, based on homology modeling, revealed a NAD+ cofactor domain and a catalytic triad containing Ser149, Tyr162, and Lys166. This is similar to Bacillus megaterium glucose 1-dehydrogenase. To investigate the role of this protein, we constructed a B. pseudomallei SDO defective mutant, measured glucose dehydrogenase (GDH) activity, and tested the interactions with host cells. The B. pseudomallei K96243 wild type exhibited potent GDH activity under condition containing 300 mM NaCl, while the mutant showed activity levels 15 times lower. Both invasion into the A549 cell line and early intracellular survival within the J774A.1 macrophage cell were impaired in the mutant. Complementation of SDO was able to restore the mutant ability to produce GDH activity, invade epithelial cells, and survive in macrophages. Conclusions Our data suggest that induced SDO activity during salt stress may facilitate B. pseudomallei invasion and affect initiation of successful intracellular infection. Identifying the role of B. pseudomallei SDO provides a better understanding of the association between bacterial adaptation and pathogenesis in melioidosis. PMID:24382268

  12. 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

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

    SciTech Connect

    Flueck, Christa E., E-mail: christa.flueck@dkf.unibe.ch [Pediatric Endocrinology, Diabetology and Metabolism, University Children's Hospital, Bern (Switzerland); Mallet, Delphine [Service d'Endocrinologie Moleculaire et Maladies Rares, Hospices Civils de Lyon, Bron (France)] [Service d'Endocrinologie Moleculaire et Maladies Rares, Hospices Civils de Lyon, Bron (France); Hofer, Gaby [Pediatric Endocrinology, Diabetology and Metabolism, University Children's Hospital, Bern (Switzerland)] [Pediatric Endocrinology, Diabetology and Metabolism, University Children's Hospital, Bern (Switzerland); Samara-Boustani, Dinane [Hopital Necker-Enfants malades, Paris (France)] [Hopital Necker-Enfants malades, Paris (France); Leger, Juliane [Hopital Robert Debre, Paris (France)] [Hopital Robert Debre, Paris (France); Polak, Michel [Hopital Necker-Enfants malades, Paris (France)] [Hopital Necker-Enfants malades, Paris (France); Morel, Yves [Service d'Endocrinologie Moleculaire et Maladies Rares, Hospices Civils de Lyon, Bron (France)] [Service d'Endocrinologie Moleculaire et Maladies Rares, Hospices Civils de Lyon, Bron (France); Pandey, Amit V., E-mail: amit@pandeylab.org [Pediatric Endocrinology, Diabetology and Metabolism, University Children's Hospital, Bern (Switzerland)

    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.

  14. NRH:quinone oxidoreductase 2 (NQO2) catalyzes metabolic activation of quinones and anti-tumor drugs.

    PubMed

    Celli, Claudia M; Tran, Namphuong; Knox, Richard; Jaiswal, Anil K

    2006-07-28

    NRH:quinone oxidoreductase 2 (NQO2) is a cytosolic flavoprotein that utilizes NRH as electron donor. The present studies investigate the role of NQO2 in metabolic detoxification/activation of quinones and quinone based anti-tumor drugs. Chinese hamster ovary (CHO) cells stably overexpressing cDNA derived mouse NQO2 and mouse keratinocytes from DMBA-induced skin tumors in wild-type and NQO2-null mice were generated. The CHO cells overexpressing NQO2 and mouse keratinocytes expressing or deficient in NQO2 were treated with varying concentrations of mitomycin C (MMC), CB1954, MMC analog BMY25067, EO9, menadione and BP-3,6-quinone, in the absence and presence of NRH. The cytotoxicity of the drugs was evaluated by colony formation. The CHO cells overexpressing higher levels of mouse NQO2 showed significantly increased cytotoxicity to menadione, BP-3,6-quinone and to the anti-tumor drugs MMC and CB1954 when compared to CHO cells expressing endogenous NQO2. The cytotoxicity increased in presence of NRH. Similar results were also observed with BMY25067 and EO9 treatments, but to a lesser extent. The results on keratinocytes deficient in NQO2 supported the data from CHO cells. The inclusion of NRH had no effect on cytotoxicity of quinones and drugs in keratinocytes deficient in NQO2. Mouse NQO2 protein was expressed in bacteria, purified and used to study the role of NQO2 in MMC-induced DNA cross-linking. Bacterially expressed and purified NQO2 efficiently catalyzed MMC activation that led to DNA cross-linking. These results concluded that NQO2 plays a significant role in the metabolic activation of both quinones and anti-tumor drugs leading to cytotoxicity and cell death. PMID:16765324

  15. Suppression of NAD(P)H-quinone oxidoreductase 1 enhanced the susceptibility of cholangiocarcinoma cells to chemotherapeutic agents

    PubMed Central

    2014-01-01

    Background Cholangiocarcinoma (CCA) is highly resistant to most of the known chemotherapeutic treatments. NAD(P)H-quinone oxidoreductase 1 (NQO1) is an antioxidant/detoxifying enzyme recently recognized as an important contributor to chemoresistance in some human cancers. However, the contribution of NQO1 to chemotherapy resistance in CCA is unknown. Methods Two CCA cell lines, KKU-100 and KKU-M214, with high and low NQO1 expression levels, respectively, were used to evaluate the sensitivity to chemotherapeutic agents; 5-fluorouracil (5-FU), doxorubicin (Doxo), and gemcitabine (Gem). NQO1 and/or p53 expression in KKU-100 cells were knocked down by siRNA. NQO1 was over-expressed in KKU-M214 cells by transfection with pCMV6-XL5-NQO1 expression vector. CCA cells with modulated NQO1 and/or p53 expression were treated with chemotherapeutic agents, and the cytotoxicity was assessed by SRB assay. The mechanism of enhanced chemosensitivity was evaluated by Western blot analysis. Results When NQO1 was knocked down, KKU-100 cells became more susceptible to all chemotherapeutic agents. Conversely, with over-expression of NQO1 made KKU-M214 cells more resistant to chemotherapeutic agents. Western blot analysis suggested that enhanced chemosensitivity was probably due to the activation of p53-mediated cell death. Enhanced susceptibility to chemotherapeutic agents by NQO1 silencing was abolished by knockdown of p53. Conclusions These results suggest that inhibition of NQO1 could enhance the susceptibility of CCA to an array of chemotherapeutic agents. PMID:24460787

  16. Electron Transfer in Subunit NuoI (TYKY) of Escherichia coli NADH:Quinone Oxidoreductase (NDH-1)*

    PubMed Central

    Sinha, Prem Kumar; Nakamaru-Ogiso, Eiko; Torres-Bacete, Jesus; Sato, Motoaki; Castro-Guerrero, Norma; Ohnishi, Tomoko; Matsuno-Yagi, Akemi; Yagi, Takao

    2012-01-01

    Bacterial proton-translocating NADH:quinone oxidoreductase (NDH-1) consists of a peripheral and a membrane domain. The peripheral domain catalyzes the electron transfer from NADH to quinone through a chain of seven iron-sulfur (Fe/S) clusters. Subunit NuoI in the peripheral domain contains two [4Fe-4S] clusters (N6a and N6b) and plays a role in bridging the electron transfer from cluster N5 to the terminal cluster N2. We constructed mutants for eight individual Cys-coordinating Fe/S clusters. With the exception of C63S, all mutants had damaged architecture of NDH-1, suggesting that Cys-coordinating Fe/S clusters help maintain the NDH-1 structure. Studies of three mutants (C63S-coordinating N6a, P110A located near N6a, and P71A in the vicinity of N6b) were carried out using EPR measurement. These three mutations did not affect the EPR signals from [2Fe-2S] clusters and retained electron transfer activities. Signals at gz = 2.09 disappeared in C63S and P110A but not in P71A. Considering our data together with the available information, gz,x = 2.09, 1.88 signals are assigned to cluster N6a. It is of interest that, in terms of gz,x values, cluster N6a is similar to cluster N4. In addition, we investigated the residues (Ile-94 and Ile-100) that are predicted to serve as electron wires between N6a and N6b and between N6b and N2, respectively. Replacement of Ile-100 and Ile-94 with Ala/Gly did not affect the electron transfer activity significantly. It is concluded that conserved Ile-100 and Ile-94 are not essential for the electron transfer. PMID:22474289

  17. Characterization of the Ubiquinone Binding Site in Alternative NADH-Quinone Oxidoreductase of Saccharomyces cerevisiae by Photoaffinity Labeling†

    PubMed Central

    Murai, Masatoshi; Yamashita, Tetsuo; Senoh, Mai; Mashimo, Yuko; Kataoka, Michihiko; Kosaka, Hiroaki; Matsuno-Yagi, Akemi; Yagi, Takao; Miyoshi, Hideto

    2010-01-01

    The Ndi1 enzyme found in the mitochondrial membrane of Saccharomyces cerevisiae is an NDH-2-type alternative NADH-quinone (Q) oxidoreductase. As Ndi1 is expected to be a possible remedy for complex I defects of mammalian mitochondria, a detailed biochemical characterization of the enzyme is needed. To identify the ubiquinone (UQ) binding site in Ndi1, we carried out photoaffinity labeling using a photoreactive biotinylated UQ mimic (compound 2) synthesized following a concept of least modification of the substituents on the quinone ring possible. Cleavage with CNBr of Ndi1 cross-linked by 2 revealed the UQ-ring of 2 to be specifically cross-linked to the region Phe281-Met410 (130 amino acids). Digestion of the CNBr fragment with V8 protease and lysylendopeptidase (Lys C) gave ~8 kDa and ~4 kDa peptides, respectively. The ~8 kDa V8 digest was identified as Thr329-Glu399 (71 amino acids) by an N-terminal sequence analysis. Although the ~4 kDa Lys C digest could not be identified by N-terminal sequence analysis, the band was thought to cover the region Gly374-Lys405 (32 amino acids). Taken together, the binding site of the Q-ring of 2 must be located in a common region of the V8 and the Lys C digests Gly374-Glu399 (26 amino acids). Superimposition of the Ndi1 sequence onto a 3D-structural model of NDH-2 from Escherichia coli suggested that the C-terminal portion of this region is close to the isoalloxazine ring of FAD. PMID:20192260

  18. NAD(P)H Quinone Oxidoreductase Protects TAp63? from Proteasomal Degradation and Regulates TAp63?-Dependent Growth Arrest

    PubMed Central

    Hershkovitz Rokah, Oshrat; Shpilberg, Ofer; Granot, Galit

    2010-01-01

    Background p63 is a member of the p53 transcription factor family. p63 is expressed from two promoters resulting in proteins with opposite functions: the transcriptionally active TAp63 and the dominant-negative ?Np63. Similar to p53, the TAp63 isoforms induce cell cycle arrest and apoptosis. The ?Np63 isoforms are dominant-negative variants opposing the activities of p53, TAp63 and TAp73. To avoid unnecessary cell death accompanied by proper response to stress, the expression of the p53 family members must be tightly regulated. NAD(P)H quinone oxidoreductase (NQO1) has recently been shown to interact with and inhibit the degradation of p53. Due to the structural similarities between p53 and p63, we were interested in studying the ability of wild-type and polymorphic, inactive NQO1 to interact with and stabilize p63. We focused on TAp63?, as it is the most potent transcription activator and it is expected to have a role in tumor suppression. Principal Findings We show that TAp63? can be degraded by the 20S proteasomes. Wild-type but not polymorphic, inactive NQO1 physically interacts with TAp63?, stabilizes it and protects it from this degradation. NQO1-mediated TAp63? stabilization was especially prominent under stress. Accordingly, we found that downregulation of NQO1 inhibits TAp63?-dependant p21 upregulation and TAp63?-induced growth arrest stimulated by doxorubicin. Conclusions/Significance Our report is the first to identify this new mechanism demonstrating a physical and functional relationship between NQO1 and the most potent p63 isoform, TAp63?. These findings appoint a direct role for NQO1 in the regulation of TAp63? expression, especially following stress and may therefore have clinical implications for tumor development and therapy. PMID:20613985

  19. NAD(P)H:Quinone Oxidoreductase 1 (NQO1) P187S Polymorphism and Prostate Cancer Risk in Caucasians

    PubMed Central

    Stoehr, Christine G.; Nolte, Elke; Wach, Sven; Wieland, Wolf F.; Hofstaedter, Ferdinand; Hartmann, Arndt; Stoehr, Robert

    2012-01-01

    NAD(P)H:quinone oxidoreductase 1 (NQO1) catalyses the reduction of quinoid compounds to hydroquinones, preventing the generation of free radicals and reactive oxygen. A “C” to “T” transversion at position 609 of NQO1, leading to a nonsynonymous amino acid change (Pro187Ser, P187S), results in an altered enzyme activity. No NQO1 protein activity was detected in NQO1 609TT genotype, and low to intermediate activity was detected in NQO1 609CT genotype compared with 609CC genotype. Thus, this polymorphism may result in altered cancer predisposition. For prostate cancer, only sparse data are available. We therefore analyzed the distribution of the NQO1 P187S SNP (single nucleotide polymorphism) in prostate cancer patients and a healthy control group. Allelic variants were determined using RFLP analysis. Overall, 232 patients without any malignancy and 119 consecutive prostate cancer patients were investigated. The genotype distribution in our cohorts followed the Hardy–Weinberg equilibrium in cases and controls. The distribution of the NQO1 codon 187 SNP did not differ significantly between prostate cancer patients and the control group (p = 0.242). There was also no association between the allelic variants and stage or Gleason score of the tumors. The NQO1 P187S SNP was not significantly associated with an increased prostate cancer risk in our cohorts. The SNP has also no influence on histopathological characteristics of the tumors. A combined analysis of all available data from published European studies also showed no significant differences in the genotype distribution between controls and prostate cancer patients. Our data suggest a minor role of the NQO1 nucleotide 609 polymorphism in prostate carcinogenesis. PMID:23109831

  20. NAD(P)H:quinone oxidoreductase 1 (NQO1) localizes to the mitotic spindle in human cells.

    PubMed

    Siegel, David; Kepa, Jadwiga K; Ross, David

    2012-01-01

    NAD(P)H:quinone oxidoreductase 1 (NQO1) is an FAD containing quinone reductase that catalyzes the 2-electron reduction of a broad range of quinones. The 2-electron reduction of quinones to hydroquinones by NQO1 is believed to be a detoxification process since this reaction bypasses the formation of the highly reactive semiquinone. NQO1 is expressed at high levels in normal epithelium, endothelium and adipocytes as well as in many human solid tumors. In addition to its function as a quinone reductase NQO1 has been shown to reduce superoxide and regulate the 20 S proteasomal degradation of proteins including p53. Biochemical studies have indicated that NQO1 is primarily located in the cytosol, however, lower levels of NQO1 have also been found in the nucleus. In these studies we demonstrate using immunocytochemistry and confocal imaging that NQO1 was found associated with mitotic spindles in cells undergoing division. The association of NQO1 with the mitotic spindles was observed in many different human cell lines including nontransformed cells (astrocytes, HUVEC) immortalized cell lines (HBMEC, 16HBE) and cancer (pancreatic adenocarcinoma, BXPC3). Confocal analysis of double-labeling experiments demonstrated co-localization of NQO1with alpha-tubulin in mitotic spindles. In studies with BxPc-3 human pancreatic cancer cells the association of NQO1 with mitotic spindles appeared to be unchanged in the presence of NQO1 inhibitors ES936 or dicoumarol suggesting that NQO1 can associate with the mitotic spindle and still retain catalytic activity. Analysis of archival human squamous lung carcinoma tissue immunostained for NQO1 demonstrated positive staining for NQO1 in the spindles of mitotic cells. The purpose of this study is to demonstrate for the first time the association of the quinone reductase NQO1 with the mitotic spindle in human cells. PMID:22984577

  1. NAD(P)H:Quinone Oxidoreductase 1 (NQO1) Localizes to the Mitotic Spindle in Human Cells

    PubMed Central

    Siegel, David; Kepa, Jadwiga K.; Ross, David

    2012-01-01

    NAD(P)H:quinone oxidoreductase 1 (NQO1) is an FAD containing quinone reductase that catalyzes the 2-electron reduction of a broad range of quinones. The 2-electron reduction of quinones to hydroquinones by NQO1 is believed to be a detoxification process since this reaction bypasses the formation of the highly reactive semiquinone. NQO1 is expressed at high levels in normal epithelium, endothelium and adipocytes as well as in many human solid tumors. In addition to its function as a quinone reductase NQO1 has been shown to reduce superoxide and regulate the 20 S proteasomal degradation of proteins including p53. Biochemical studies have indicated that NQO1 is primarily located in the cytosol, however, lower levels of NQO1 have also been found in the nucleus. In these studies we demonstrate using immunocytochemistry and confocal imaging that NQO1 was found associated with mitotic spindles in cells undergoing division. The association of NQO1 with the mitotic spindles was observed in many different human cell lines including nontransformed cells (astrocytes, HUVEC) immortalized cell lines (HBMEC, 16HBE) and cancer (pancreatic adenocarcinoma, BXPC3). Confocal analysis of double-labeling experiments demonstrated co-localization of NQO1with alpha-tubulin in mitotic spindles. In studies with BxPc-3 human pancreatic cancer cells the association of NQO1 with mitotic spindles appeared to be unchanged in the presence of NQO1 inhibitors ES936 or dicoumarol suggesting that NQO1 can associate with the mitotic spindle and still retain catalytic activity. Analysis of archival human squamous lung carcinoma tissue immunostained for NQO1 demonstrated positive staining for NQO1 in the spindles of mitotic cells. The purpose of this study is to demonstrate for the first time the association of the quinone reductase NQO1 with the mitotic spindle in human cells. PMID:22984577

  2. Amixicile, a novel inhibitor of pyruvate: ferredoxin oxidoreductase, shows efficacy against Clostridium difficile in a mouse infection model.

    PubMed

    Warren, Cirle A; van Opstal, Edward; Ballard, T Eric; Kennedy, Andrew; Wang, Xia; Riggins, Mary; Olekhnovich, Igor; Warthan, Michelle; Kolling, Glynis L; Guerrant, Richard L; Macdonald, Timothy L; Hoffman, Paul S

    2012-08-01

    Clostridium difficile infection (CDI) is a serious diarrheal disease that often develops following prior antibiotic usage. One of the major problems with current therapies (oral vancomycin and metronidazole) is the high rate of recurrence. Nitazoxanide (NTZ), an inhibitor of pyruvate:ferredoxin oxidoreductase (PFOR) in anaerobic bacteria, parasites, Helicobacter pylori, and Campylobacter jejuni, also shows clinical efficacy against CDI. From a library of ?250 analogues of NTZ, we identified leads with increased potency for PFOR. MIC screens indicated in vitro activity in the 0.05- to 2-?g/ml range against C. difficile. To improve solubility, we replaced the 2-acetoxy group with propylamine, producing amixicile, a soluble (10 mg/ml), nontoxic (cell-based assay) lead that produced no adverse effects in mice by oral or intraperitoneal (i.p.) routes at 200 mg/kg of body weight/day. In initial efficacy testing in mice treated (20 mg/kg/day, 5 days each) 1 day after receiving a lethal inoculum of C. difficile, amixicile showed slightly less protection than did vancomycin by day 5. However, in an optimized CDI model, amixicile showed equivalence to vancomycin and fidaxomicin at day 5 and there was significantly greater survival produced by amixicile than by the other drugs on day 12. All three drugs were comparable by measures of weight loss/gain and severity of disease. Recurrence of CDI was common for mice treated with vancomycin or fidaxomicin but not for mice receiving amixicile or NTZ. These results suggest that gut repopulation with beneficial (non-PFOR) bacteria, considered essential for protection against CDI, rebounds much sooner with amixicile therapy than with vancomycin or fidaxomicin. If the mouse model is indeed predictive of human CDI disease, then amixicile, a novel PFOR inhibitor, appears to be a very promising new candidate for treatment of CDI. PMID:22585229

  3. Nucleotide sequence of a cDNA coding for the NADPH-protochlorophyllide oxidoreductase (PCR) of barley ( Hordeum vulgare L.) and its expression in Escherichia coli

    Microsoft Academic Search

    Riidiger Schulz; Klaus Steinmiiller; Manfred Klaas; Christoph Forreiter; Siiren Rasmussen; Claudia Hiller; Klaus Apel

    1989-01-01

    Summary  The primary structure of the NADPH-protochlorophyllide oxidoreductase of barley has been deduced from the nucleotide sequence\\u000a of a cloned full-length cDNA. This cDNA hybridizes to a 1.7 kb RNA whose steady-state level in dark-grown seedlings is drastically\\u000a reduced upon illumination. The predicted amino acid sequence (388 residues in length) includes a transit peptide of 74 amino\\u000a acids whose end point

  4. 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. (Universite de Montreal (Canada)); Malfoy, B. (Institut Curie Section de Biologie, Paris (France)); Forrest, G.L. (Beckman Research Institute at the City of Hope, Duarte, CA (United States))

    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.

  5. 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 ? 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. PMID:24813672

  6. 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

    2011-01-01

    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. PMID:22112775

  7. New properties of Bacillus subtilis succinate dehydrogenase altered at the active site. The apparent active site thiol of succinate oxidoreductases is dispensable for succinate oxidation.

    PubMed Central

    Hederstedt, L; Hedén, L O

    1989-01-01

    Mammalian and Escherichia coli succinate dehydrogenase (SDH) and E. coli fumarate reductase apparently contain an essential cysteine residue at the active site, as shown by substrate-protectable inactivation with thiol-specific reagents. Bacillus subtilis SDH was found to be resistant to this type of reagent and contains an alanine residue at the amino acid position equivalent to the only invariant cysteine in the flavoprotein subunit of E. coli succinate oxidoreductases. Substitution of this alanine, at position 252 in the flavoprotein subunit of B. subtilis SDH, by cysteine resulted in an enzyme sensitive to thiol-specific reagents and protectable by substrate. Other biochemical properties of the redesigned SDH were similar to those of the wild-type enzyme. It is concluded that the invariant cysteine in the flavoprotein of E. coli succinate oxidoreductases corresponds to the active site thiol. However, this cysteine is most likely not essential for succinate oxidation and seemingly lacks an assignable specific function. An invariant arginine in juxtaposition to Ala-252 in the flavoprotein of B. subtilis SDH, and to the invariant cysteine in the E. coli homologous enzymes, is probably essential for substrate binding. PMID:2504145

  8. 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

  9. Ubiquinol-cytochrome c oxidoreductase of higher plants. Isolation and characterization of the bc1 complex from potato tuber mitochondria.

    PubMed

    Berry, E A; Huang, L S; DeRose, V J

    1991-05-15

    A procedure is described for isolation of active ubiquinol-cytochrome c oxidoreductase (bc1 complex) from potato tuber mitochondria using dodecyl maltoside extraction and ion exchange chromatography. The same procedure works well with mitochondria from red beet and sweet potato. The potato complex has at least 10 subunits resolvable by gel electrophoresis in the presence of dodecyl sulfate. The fifth subunit carries covalently bound heme. The two largest ("core") subunits either show heterogeneity or include a third subunit. The purified complex contains about 4 mumol of cytochrome c1, 8 mumol of cytochrome b, and 20 mumol of iron/g of protein. The complex is highly delipidated, with 1-6 mol of phospholipid and about 0.2 mol of ubiquinone/mol of cytochrome c1. Nonetheless it catalyzes electron transfer from a short chain ubiquinol analog to equine cytochrome c with a turnover number of 50-170 mol of cytochrome c reduced per mol of cytochrome c1 per s, as compared with approximately 220 in whole mitochondria. The enzymatic activity is stable for weeks at 4 degrees C in phosphate buffer and for months at -20 degrees C in 50% glycerol. The activity is inhibited by antimycin, myxothiazol, and funiculosin. The complex is more resistant to funiculosin and diuron than the beef heart enzyme. The optical difference spectra of the cytochromes were resolved by analysis of full-spectrum redox titrations. The alpha-band absorption maxima are 552 nm (cytochrome c1), 560 nm (cytochrome b-560), and 557.5 + 565.5 nm (cytochrome b-566, which has a split alpha-band). Extinction coefficients appropriate for the potato cytochromes are estimated. Despite the low lipid and ubiquinone content of the purified complex, the midpoint potentials of the cytochromes (257, 51, and -77 mV for cytochromes c1, b-560, and b-566, respectively) are not very different from values reported for whole mitochondria. EPR spectroscopy shows the presence of a Rieske-type iron sulfur center, and the absence of centers associated with succinate and NADH dehydrogenases. The complex shows characteristics associated with a Q-cycle mechanism of redox-driven proton translocation, including two pathways for reduction of b cytochromes by quinols and oxidant-induced reduction of b cytochromes in the presence of antimycin. PMID:1851164

  10. Two-electron reduction of quinones by rat liver NAD(P)H:quinone oxidoreductase: quantitative structure-activity relationships.

    PubMed

    Anusevicius, Zilvinas; Sarlauskas, Jonas; Cenas, Narimantas

    2002-08-15

    Mammalian NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2) catalyzes the two-electron reduction of quinones and plays one of the main roles in the bioactivation of quinoidal drugs. In order to understand the enzyme substrate specificity, we have examined the reactions of rat NQO1 with a number of quinones with available potentials of single-electron (E(1)(7)) reduction and pK(a) of their semiquinones. The hydride transfer potentials (E(7)(H(-))) were calculated from the midpoint potentials of quinones and pK(a) of hydroquinones. Our findings imply that benzo- and naphthoquinones with a van der Waals volume (VdWvol) < or = 200 A(3) are much more reactive than glutathionyl-substituted naphthoquinones, polycyclic quinones, and FMN (VdWvol>200 A(3)) with the same reduction potentials. The entropies of activation (DeltaS(not equal)) in the reduction of "fast" oxidants are equal to -84 to -76 J mol(-1) K(-1), whereas in the reduction of "slow" oxidants Delta S(not equal)=-36 to -11 J mol(-1) K(-1). The large negative Delta S(not equal) in the reduction of fast oxidants may be explained by their better electronic coupling with reduced FAD or the formation of charge-transfer complexes, since fast oxidants bind at the dicumarol binding site, whereas the binding of some slow oxidants outside it has been demonstrated. The reactivity of quinones may be equally well described in terms of the three-step (e(-),H(+),e(-)) hydride transfer, using E(1)(7), pK(a)(QH*), and VdWvol as correlation parameters, or in terms of single-step (H(-)) hydride transfer, using E(7)(H(-)) and VdWvol in the correlation. The analysis of NQO1 reactions with single-electron acceptors and quinones using an "outer-sphere" electron transfer model points to the possibility of a three-step hydride transfer. PMID:12147263

  11. Fenpyroximate binds to the interface between PSST and 49 kDa subunits in mitochondrial NADH-ubiquinone oxidoreductase.

    PubMed

    Shiraishi, Yusuke; Murai, Masatoshi; Sakiyama, Naoto; Ifuku, Kentaro; Miyoshi, Hideto

    2012-03-01

    Using a photoaffinity labeling technique, Nakamaru-Ogiso et al. demonstrated that fenpyroximate, a strong inhibitor of bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I), binds to the ND5 subunit [Nakamaru-Ogiso, E., et al. (2003) Biochemistry 42, 746-754]. Considering that the main body of the ND5 subunit composed of transmembrane helixes 1-15 is located at the distal end of the membrane domain [Efremov, R. G., et al. (2010) Nature 465, 441-445], however, their result may be questionable. Because establishing the number and location of inhibitors and/or quinone binding sites in the membrane domain is necessary to elucidate the function of the enzyme, it is critical to clarify whether there is an additional inhibitor and/or quinone binding site besides the interface between the hydrophilic and membrane domains. We therefore performed photoaffinity labeling experiments using two newly synthesized fenpyroximate derivatives [[(125)I]-4-azidophenyl fenpyroximate ([(125)I]APF) and [(125)I]-3-azido-5-iodobenzyl fenpyroximate ([(125)I]AIF)] possessing a photoreactive azido group at and far from the pharmacophoric core moiety, respectively. Doubled sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that [(125)I]APF and [(125)I]AIF bind to the PSST and 49 kDa subunits, respectively. Careful examination of the fragmentation patterns of the labeled PSST and 49 kDa subunits generated by limited proteolysis indicated that the residues labeled by [(125)I]APF and [(125)I]AIF are located in the Ser43-Arg66 (PSST) and Asp160-Arg174 (49 kDa) regions, respectively, which face the supposed quinone-binding pocket formed at the interface of the PSST, 49 kDa, and ND1 subunits. We conclude that fenpyroximate does not bind to the distal end of the membrane domain but rather resides at the interface between the two domains in a manner such that the pharmacophoric pyrazole ring and side chain of the inhibitor orient toward the PSST and 49 kDa subunits, respectively. This study answers a critical question relating to complex I. PMID:22353032

  12. Differences in the secretion pattern of oxidoreductases from Bjerkandera adusta induced by a phenolic olive mill extract.

    PubMed

    Reina, Rocío; Kellner, Harald; Jehmlich, Nico; Ullrich, René; García-Romera, Inmaculada; Aranda, Elisabet; Liers, Christiane

    2014-11-01

    The secretome of the white-rot fungus Bjerkandera adusta produced in synthetic Kirk medium was compared to that supplemented with an aqueous phenol-rich extract of dry olive mill residues (ADOR). Distinct changes in the protein composition of oxidoreductases, namely diverse class-II peroxidases and aryl alcohol oxidases were found. In the ADOR-supplemented medium (ASC), 157 distinct proteins were identified by the secretome analysis, whereas only 59 of them were identified without ADOR supplementation (Kirk medium culture; KM). Proteome analysis indicated that the number of peroxidases produced in ASC was more than doubled (from 4 to 11) compared to KM. Two short manganese peroxidases (MnP1 and MnP6) and one versatile peroxidase (VP1) represented 29% of the relative abundance (NSAF) in ASC. Two of them (MnP1 and VP1) were also detected in KM at a relative abundance (NSAF) of only 3%. Further peroxidases present in ASC were one lignin peroxidase (LiP2), one generic peroxidase (GP) and three dye-decolorizing peroxidases (DyPs). The relative abundance of DyPs and aryl alcohol oxidases (AAO) were lower in ASC in comparison to KM. In addition to peptide sequence analysis, the secretion of Mn(2+)-oxidizing peroxidases as well as AAOs were followed by enzyme measurement. The Mn(2+)-oxidizing activity increased nearly 30-fold (from 10 to 281Ul(-1)) after ADOR addition. Two enzymes responsible for that activity were successfully purified (BadVPI and BadVPII). To prove a potential involvement of these enzymes in the degradation of aromatic compounds, BadVPI was tested for its ability to degrade the recalcitrant dehydrogenated polymer (DHP, synthetic lignin). These results show that natural phenol-rich materials act as secretome-stimulating additives. Applying these substances enables us to investigate fungal degradation and detoxification processes and gives more insight into the complexity of fungal secretomes, e.g. of white-rot fungi. PMID:25069088

  13. Molecular Characterization of an NADPH-Dependent Acetoin Reductase/2,3-Butanediol Dehydrogenase from Clostridium beijerinckii NCIMB 8052

    PubMed Central

    Raedts, John; Siemerink, Marco A. J.; Levisson, Mark; van der Oost, John

    2014-01-01

    Acetoin reductase is an important enzyme for the fermentative production of 2,3-butanediol, a chemical compound with a very broad industrial use. Here, we report on the discovery and characterization of an acetoin reductase from Clostridium beijerinckii NCIMB 8052. An in silico screen of the C. beijerinckii genome revealed eight potential acetoin reductases. One of them (CBEI_1464) showed substantial acetoin reductase activity after expression in Escherichia coli. The purified enzyme (C. beijerinckii acetoin reductase [Cb-ACR]) was found to exist predominantly as a homodimer. In addition to acetoin (or 2,3-butanediol), other secondary alcohols and corresponding ketones were converted as well, provided that another electronegative group was attached to the adjacent C-3 carbon. Optimal activity was at pH 6.5 (reduction) and 9.5 (oxidation) and around 68°C. Cb-ACR accepts both NADH and NADPH as electron donors; however, unlike closely related enzymes, NADPH is preferred (Km, 32 ?M). Cb-ACR was compared to characterized close homologs, all belonging to the “threonine dehydrogenase and related Zn-dependent dehydrogenases” (COG1063). Metal analysis confirmed the presence of 2 Zn2+ atoms. To gain insight into the substrate and cofactor specificity, a structural model was constructed. The catalytic zinc atom is likely coordinated by Cys37, His70, and Glu71, while the structural zinc site is probably composed of Cys100, Cys103, Cys106, and Cys114. Residues determining NADP specificity were predicted as well. The physiological role of Cb-ACR in C. beijerinckii is discussed. PMID:24441158

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

    PubMed

    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

    2015-08-01

    Overexpression of AtNTRC (AtNTRC(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. PMID:26086110

  15. Respiratory Epithelial Gene Expression in Patients with Mild and Severe Cystic Fibrosis Lung Disease

    PubMed Central

    Wright, Jerry M.; Merlo, Christian A.; Reynolds, Jeffrey B.; Zeitlin, Pamela L.; Garcia, Joe G. N.; Guggino, William B.; Boyle, Michael P.

    2006-01-01

    Despite having identical cystic fibrosis transmembrane conductance regulator genotypes, individuals with ?F508 homozygous cystic fibrosis (CF) demonstrate significant variability in severity of pulmonary disease. This investigation used high-density oligonucleotide microarray analysis of nasal respiratory epithelium to investigate the molecular basis of phenotypic differences in CF by (1) identifying differences in gene expression between ?F508 homozygotes in the most severe 20th percentile of lung disease by forced expiratory volume in 1 s and those in the most mild 20th percentile of lung disease and (2) identifying differences in gene expression between ?F508 homozygotes and age-matched non-CF control subjects. Microarray results from 23 participants (12 CF, 11 non-CF) met the strict quality control guidelines and were used for final data analysis. A total of 652 of the 11,867 genes identified as present in 75% of the samples were significantly differentially expressed in one of the three disease phenotypes: 30 in non-CF, 53 in mild CF, and 569 in severe CF. An analysis of genes differentially expressed by severity of CF lung disease demonstrated significant upregulation in severe CF of genes involved in protein ubiquination (P < 0.04), mitochondrial oxidoreductase activity (P < 0.01), and lipid metabolism (P < 0.03). Analysis of genes with decreased expression in patients with CF compared with control subjects demonstrated significant downregulation of genes involved in airway defense (P < 0.047) and protein metabolism (P < 0.048). This study suggests that differences in CF lung phenotype are associated with differences in expression of genes involving airway defense, protein ubiquination, and mitochondrial oxidoreductase activity and identifies specific new candidate modifiers of the CF phenotype. PMID:16614352

  16. A secondary mode of action of polymyxins against Gram-negative bacteria involves the inhibition of NADH-quinone oxidoreductase activity.

    PubMed

    Deris, Zakuan Z; Akter, Jesmin; Sivanesan, Sivashangarie; Roberts, Kade D; Thompson, Philip E; Nation, Roger L; Li, Jian; Velkov, Tony

    2014-02-01

    Polymyxin B and colistin were examined for their ability to inhibit the type II NADH-quinone oxidoreductases (NDH-2) of three species of Gram-negative bacteria. Polymyxin B and colistin inhibited the NDH-2 activity in preparations from all of the isolates in a concentration-dependent manner. The mechanism of NDH-2 inhibition by polymyxin B was investigated in detail with Escherichia coli inner membrane preparations and conformed to a mixed inhibition model with respect to ubiquinone-1 and a non-competitive inhibition model with respect to NADH. These suggest that the inhibition of vital respiratory enzymes in the bacterial inner membrane represents one of the secondary modes of action for polymyxins. PMID:24169795

  17. Crystallization and preliminary analysis of the NqrA and NqrC subunits of the Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio cholerae.

    PubMed

    Vohl, Georg; Nedielkov, Ruslan; Claussen, Björn; Casutt, Marco S; Vorburger, Thomas; Diederichs, Kay; Möller, Heiko M; Steuber, Julia; Fritz, Günter

    2014-07-01

    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 C222? and P2? 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

  18. 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...

  19. 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. PMID:24117886

  20. RNA-Seq Analysis Reveals a Six-Gene SoxR Regulon in Streptomyces coelicolor

    PubMed Central

    Naseer, Nawar; Shapiro, Joshua A.; Chander, Monica

    2014-01-01

    The redox-regulated transcription factor SoxR is conserved in diverse bacteria, but emerging studies suggest that this protein plays distinct physiological roles in different bacteria. SoxR regulates a global oxidative stress response (involving >100 genes) against exogenous redox-cycling drugs in Escherichia coli and related enterics. In the antibiotic producers Streptomyces coelicolor and Pseudomonas aeruginosa, however, SoxR regulates a smaller number of genes that encode membrane transporters and proteins with homology to antibiotic-tailoring enzymes. In both S. coelicolor and P. aeruginosa, SoxR-regulated genes are expressed in stationary phase during the production of endogenously-produced redox-active antibiotics. These observations suggest that SoxR evolved to sense endogenous secondary metabolites and activate machinery to process and transport them in antibiotic-producing bacteria. Previous bioinformatics analysis that searched the genome for SoxR-binding sites in putative promoters defined a five-gene SoxR regulon in S. coelicolor including an ABC transporter, two oxidoreductases, a monooxygenase and an epimerase/dehydratase. Since this in silico screen may have missed potential SoxR-targets, we conducted a whole genome transcriptome comparison of wild type S. coelicolor and a soxR-deficient mutant in stationary phase using RNA-Seq. Our analysis revealed a sixth SoxR-regulated gene in S. coelicolor that encodes a putative quinone oxidoreductase. Knowledge of the full complement of genes regulated by SoxR will facilitate studies to elucidate the function of this regulatory molecule in antibiotic producers. PMID:25162599

  1. 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 ?g/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

  2. A super-family of medium-chain dehydrogenases/reductases (MDR). Sub-lines including zeta-crystallin, alcohol and polyol dehydrogenases, quinone oxidoreductase enoyl reductases, VAT-1 and other proteins.

    PubMed

    Persson, B; Zigler, J S; Jörnvall, H

    1994-11-15

    The protein super-family of medium-chain alcohol dehydrogenases (and glutathione-dependent formaldehyde dehydrogenase), polyol dehydrogenases, threonine dehydrogenase, archaeon glucose dehydrogenase, and eye lens reductase-active zeta-crystallins also includes Escherichia coli quinone oxidoreductase, Torpedo VAT-1 protein, and enoyl reductases of mammalian fatty acid and yeast erythronolide synthases. In addition, two proteins with hitherto unknown function are shown to belong to this super-family of medium-chain dehydrogenases and reductases (MDR). Alignment of zeta-crystallins/quinone oxidoreductases/VAT-1 reveals 38 strictly conserved residues, of which approximately half are glycine residues, including those at several space-restricted turn positions and critical coenzyme-binding positions in the alcohol dehydrogenases. This indicates a conserved three-dimensional structure at the corresponding parts of these distantly related proteins and a conserved binding of a coenzyme in the two proteins with hitherto unknown function, thus ascribing a likely oxidoreductase function to these proteins. When all forms are aligned, including enoyl reductases, a zeta-crystallin homologue from Leishmania and the two proteins with hitherto unknown function, only three residues are strictly conserved among the 106 proteins characterised within the superfamily, and significantly these residues are all glycines, corresponding to Gly66, Gly86 and Gly201 of mammalian class I alcohol dehydrogenase. Notably, these residues are located in different domains. Hence, a distant origin and divergent functions, but related forms and interactions, appear to apply to the entire chains of the many prokaryotic and eukaryotic members. Additionally, in the zeta-crystallins/quinone oxidoreductases, a highly conserved tyrosine residue is found. This residue, in the three-dimensional structure of the homologous alcohol dehydrogenase, is positioned at the subunit cleft that contains the active site and could therefore be involved in catalysis. If so, this residue and its role may resemble the pattern of a conserved tyrosine residue in the different family of short-chain dehydrogenases/reductases (SDR). PMID:7957243

  3. 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

  4. 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

  5. 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 Central

    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. PMID:24847713

  6. Were there any "misassignments" among iron-sulfur clusters N4, N5 and N6b in NADH-quinone oxidoreductase (complex I)?

    PubMed

    Ohnishi, Tomoko; Nakamaru-Ogiso, Eiko

    2008-01-01

    NADH-quinone oxidoreductase (complex I) in bovine heart mitochondria has a molecular weight of approximately 1 million Da composed of 45 distinct subunits. It is the largest energy transducing complex so far known. Bacterial complex I is simpler and smaller, but the essential redox components and the basic mechanisms of electron and proton translocation are the same. Over the past three decades, Ohnishi et al. have pursued extensive EPR studies near liquid helium temperatures and characterized most of the iron-sulfur clusters in complex I. Recently, Yakovlev et al. [G. Yakovlev, T. Reda, J. Hirst, Reevaluating the relationship between EPR spectra and enzyme structure for the iron-sulfur clusters in NADH:quinone oxidoreductase, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 12720-12725] challenged Ohnishi's group by claiming that there were EPR "misassignments" among clusters N4, N5 and N6b (in order to prevent confusion, we used current consensus nomenclature, as the nickname). They claimed that we misassigned EPR signals arising from cluster N5 to cluster N4, and signals from cluster N6b to cluster N4. They also proposed that cluster N5 has (4Cys)-ligands. Based on the accumulated historical data and recent results of our site-specific mutagenesis experiments, we confirmed that cluster N5 has (1His+3Cys)-ligands as we had predicted. We revealed that E. coli cluster N5 signals could be clearly detected at the sample temperature around 3 K with microwave power higher than 5 mW. Thus Hirst's group could not detect N5 signals under any of their EPR conditions, reported in their PNAS paper. It seems that they misassigned the signals from cluster N4 to N5. As to the claim of "misassignment" between clusters N4 and N6b, that was not a possibility because our mutagenesis systems did not contain cluster N6b. Therefore, we believe that we have not made any "misassignment" in our work. PMID:18486592

  7. 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

    1998-02-01

    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. PMID:9448329

  8. Localization and Function of the Membrane-bound Riboflavin in the Na+-translocating NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio cholerae*

    PubMed Central

    Casutt, Marco S.; Huber, Tamara; Brunisholz, René; Tao, Minli; Fritz, Günter; Steuber, Julia

    2010-01-01

    The sodium ion-translocating NADH:quinone oxidoreductase (Na+-NQR) from the human pathogen Vibrio cholerae is a respiratory membrane protein complex that couples the oxidation of NADH to the transport of Na+ across the bacterial membrane. The Na+-NQR comprises the six subunits NqrABCDEF, but the stoichiometry and arrangement of these subunits are unknown. Redox-active cofactors are FAD and a 2Fe-2S cluster on NqrF, covalently attached FMNs on NqrB and NqrC, and riboflavin and ubiquinone-8 with unknown localization in the complex. By analyzing the cofactor content and NADH oxidation activity of subcomplexes of the Na+-NQR lacking individual subunits, the riboflavin cofactor was unequivocally assigned to the membrane-bound NqrB subunit. Quantitative analysis of the N-terminal amino acids of the holo-complex revealed that NqrB is present in a single copy in the holo-complex. It is concluded that the hydrophobic NqrB harbors one riboflavin in addition to its covalently attached FMN. The catalytic role of two flavins in subunit NqrB during the reduction of ubiquinone to ubiquinol by the Na+-NQR is discussed. PMID:20558724

  9. Interplay of Flavin's Redox States and Protein Dynamics: An Insight from QM/MM Simulations of Dihydronicotinamide Riboside Quinone Oxidoreductase 2

    PubMed Central

    Mueller, Robyn M.; North, Michael A.; Yang, Chee; Hati, Sanchita; Bhattacharyya, Sudeep

    2011-01-01

    Dihydronicotinamide riboside quinone oxidoreductase 2 is known to catalyze a two-electron reduction of quinone to hydroquinone using its cofactor, flavin adenine dinucleotide. Using quantum mechanical/molecular mechanical simulations, we have computed the reorganization free energies of the electron and proton transfer processes of flavin in the free state as well as when it is bound in the active site of the enzyme. The calculated energetics for electron transfer processes demonstrate that the enzyme active site lowers the reorganization energy for the redox process as compared to the enzyme-free aqueous state. This is most apparent in the two electron reduction step, which eliminates the possibility of flavosemiquinone generation. In addition, essential dynamics study of the simulated motions revealed spectacular changes in the principal components of atomic fluctuations upon reduction of flavin. This alteration of active site dynamics provides an insight into the `ping-pong' kinetics exhibited by the enzyme upon a change in the redox state of the enzyme-bound flavin. A charge perturbation analysis provides further support that the observed change in dynamics is correlated with the change in energetics due to the altered electrostatic interactions between the flavin ring and the active site residues. This study shows that the effect of electrostatic preorganization goes beyond the chemical catalysis as it strongly impacts the post-catalytic intrinsic protein dynamics. PMID:21410212

  10. E3 Ligase STUB1/CHIP Regulates NAD(P)H:Quinone Oxidoreductase 1 (NQO1) Accumulation in Aged Brain, a Process Impaired in Certain Alzheimer Disease Patients*

    PubMed Central

    Tsvetkov, Peter; Adamovich, Yaarit; Elliott, Evan; Shaul, Yosef

    2011-01-01

    NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoenzyme that is important in maintaining the cellular redox state and regulating protein degradation. The NQO1 polymorphism C609T has been associated with increased susceptibility to various age-related pathologies. We show here that NQO1 protein level is regulated by the E3 ligase STUB1/CHIP (C terminus of Hsc70-interacting protein). NQO1 binds STUB1 via the Hsc70-interacting domain (tetratricopeptide repeat domain) and undergoes ubiquitination and degradation. We demonstrate here that the product of the C609T polymorphism (P187S) is a stronger STUB1 interactor with increased susceptibility to ubiquitination by the E3 ligase STUB1. Furthermore, age-dependent decrease of STUB1 correlates with increased NQO1 accumulation. Remarkably, examination of hippocampi from Alzheimer disease patients revealed that in half of the cases examined the NQO1 protein level was undetectable due to C609T polymorphism, suggesting that the age-dependent accumulation of NQO1 is impaired in certain Alzheimer disease patients. PMID:21220432

  11. Sulindac Compounds Facilitate the Cytotoxicity of ?-Lapachone by Up-Regulation of NAD(P)H Quinone Oxidoreductase in Human Lung Cancer Cells

    PubMed Central

    Kung, Hsiu-Ni; Lu, Kuo-Shyan; Chau, Yat-Pang

    2014-01-01

    ?-lapachone, a major component in an ethanol extract of Tabebuia avellanedae bark, is a promising potential therapeutic drug for various tumors, including lung cancer, the leading cause of cancer-related deaths worldwide. In the first part of this study, we found that apoptotic cell death induced in lung cancer cells by high concentrations of ?-lapachone was mediated by increased activation of the pro-apoptotic factor JNK and decreased activation of the cell survival/proliferation factors PI3K, AKT, and ERK. In addition, ?-lapachone toxicity was positively correlated with the expression and activity of NAD(P)H quinone oxidoreductase 1 (NQO1) in the tumor cells. In the second part, we found that the FDA-approved non-steroidal anti-inflammatory drug sulindac and its metabolites, sulindac sulfide and sulindac sulfone, increased NQO1 expression and activity in the lung adenocarcinoma cell lines CL1-1 and CL1-5, which have lower NQO1 levels and lower sensitivity to ?-lapachone treatment than the A549 cell lines, and that inhibition of NQO1 by either dicoumarol treatment or NQO1 siRNA knockdown inhibited this sulindac-induced increase in ?-lapachone cytotoxicity. In conclusion, sulindac and its metabolites synergistically increase the anticancer effects of ?-lapachone primarily by increasing NQO1 activity and expression, and these two drugs may provide a novel combination therapy for lung cancers. PMID:24505400

  12. Reduction of nitroaromatic compounds by NAD(P)H:quinone oxidoreductase (NQO1): the role of electron-accepting potency and structural parameters in the substrate specificity.

    PubMed

    Miseviciene, Lina; Anusevicius, Zilvinas; Sarlauskas, Jonas; Cenas, Narimantas

    2006-01-01

    We aimed to elucidate the role of electronic and structural parameters of nitroaromatic compounds in their two-electron reduction by NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2). The multiparameter regression analysis shows that the reactivity of nitroaromatic compounds (n=38) increases with an increase in their single-electron reduction potential and the torsion angle between nitrogroup(s) and the aromatic ring. The binding efficiency of nitroaromatics in the active center of NQO1 exerted a less evident role in their reactivity. The reduction of nitroaromatics is characterized by more positive entropies of activation than the reduction of quinones. This points to a less efficient electronic coupling of nitroaromatics with the reduced isoalloxazine ring of FAD, and may explain their lower reactivity as compared to quinones. Another important but poorly understood factor enhancing the reactivity of nitroaromatics is their ability to bind at the dicumarol/quinone binding site in the active center of NQO1. PMID:16929382

  13. Possible Roles of Two Quinone Molecules in Direct and Indirect Proton Pumps of Bovine Heart NADH-quinone Oxidoreductase (Complex I)

    PubMed Central

    Ohnishi, S. Tsuyoshi; Salerno, John C.; Ohnishi, Tomoko

    2010-01-01

    In many energy transducing systems which couple electron and proton transport, for example, bacterial photosynthetic reaction center, cytochrome bc1-complex (complex III) and E. coli quinol-oxidase (cytochrome bo3 complex), two protein-associated quinone molecules are known to work together. T. Ohnishi and her collaborators reported that two distinct semiquinone species also play important roles in NADH-ubiquinone oxidoreductase (complex I). They were called SQNf (fast relaxing semiquinone) and SQNs (slow relaxing semiquinone). It was proposed that QNf serves as a “direct” proton carrier in the semiquinone-gated proton pump (Ohnishi and Salerno, FEBS Letters 579 (2005) 4555), while QNs works as a converter between one-electron and two electron transport processes. This communication presents a revised hypothesis in which QNf plays a role in a “direct” redox-driven proton pump, while QNs triggers an “indirect” conformation-driven proton pump. QNf and Q together serve as (1e?/2e?) converter, for the transfer of reducing equivalent to the Q-pool. PMID:20599678

  14. The MoxR ATPase RavA and Its Cofactor ViaA Interact with the NADH:Ubiquinone Oxidoreductase I in Escherichia coli

    PubMed Central

    Wong, Keith S.; Snider, Jamie D.; Graham, Chris; Greenblatt, Jack F.; Emili, Andrew; Babu, Mohan; Houry, Walid A.

    2014-01-01

    MoxR ATPases are widespread throughout bacteria and archaea. The experimental evidence to date suggests that these proteins have chaperone-like roles in facilitating the maturation of dedicated protein complexes that are functionally diverse. In Escherichia coli, the MoxR ATPase RavA and its putative cofactor ViaA are found to exist in early stationary-phase cells at 37°C at low levels of about 350 and 90 molecules per cell, respectively. Both proteins are predominantly localized to the cytoplasm, but ViaA was also unexpectedly found to localize to the cell membrane. Whole genome microarrays and synthetic lethality studies both indicated that RavA-ViaA are genetically linked to Fe-S cluster assembly and specific respiratory pathways. Systematic analysis of mutant strains of ravA and viaA indicated that RavA-ViaA sensitizes cells to sublethal concentrations of aminoglycosides. Furthermore, this effect was dependent on RavA's ATPase activity, and on the presence of specific subunits of NADH:ubiquinone oxidoreductase I (Nuo Complex, or Complex I). Importantly, both RavA and ViaA were found to physically interact with specific Nuo subunits. We propose that RavA-ViaA facilitate the maturation of the Nuo complex. PMID:24454883

  15. Structural Data on the Periplasmic Aldehyde Oxidoreductase PaoABC from Escherichia coli: SAXS and Preliminary X-ray Crystallography Analysis

    PubMed Central

    Otrelo-Cardoso, Ana Rita; da Silva Correia, Márcia Alexandra; Schwuchow, Viola; Svergun, Dmitri I.; Romão, Maria João; Leimkühler, Silke; Santos-Silva, Teresa

    2014-01-01

    The periplasmic aldehyde oxidoreductase PaoABC from Escherichia coli is a molybdenum enzyme involved in detoxification of aldehydes in the cell. It is an example of an ??? heterotrimeric enzyme of the xanthine oxidase family of enzymes which does not dimerize via its molybdenum cofactor binding domain. In order to structurally characterize PaoABC, X-ray crystallography and small angle X-ray scattering (SAXS) have been carried out. The protein crystallizes in the presence of 20% (w/v) polyethylene glycol 3350 using the hanging-drop vapour diffusion method. Although crystals were initially twinned, several experiments were done to overcome twinning and lowering the crystallization temperature (293 K to 277 K) was the solution to the problem. The non-twinned crystals used to solve the structure diffract X-rays to beyond 1.80 Å and belong to the C2 space group, with cell parameters a = 109.42 Å, b = 78.08 Å, c = 151.77 Å, ? = 99.77°, and one molecule in the asymmetric unit. A molecular replacement solution was found for each subunit separately, using several proteins as search models. SAXS data of PaoABC were also collected showing that, in solution, the protein is also an ??? heterotrimer. PMID:24492481

  16. 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

  17. Electron transport chain of Saccharomyces cerevisiae mitochondria is inhibited by H2O2 at succinate-cytochrome c oxidoreductase level without lipid peroxidation involvement

    PubMed Central

    CORTÉS-ROJO, CHRISTIAN; CALDERÓN-CORTÉS, ELIZABETH; CLEMENTE-GUERRERO, MÓNICA; MANZO-ÁVALOS, SALVADOR; URIBE, SALVADOR; BOLDOGH, ISTVAN; SAAVEDRA-MOLINA, ALFREDO

    2011-01-01

    The deleterious effects of H2O2 on the electron transport chain of yeast mitochondria and on mitochondrial lipid peroxidation were evaluated. Exposure to H2O2 resulted in inhibition of the oxygen consumption in the uncoupled and phosphorylating states to 69% and 65%, respectively. The effect of H2O2 on the respiratory rate was associated with an inhibition of succinate-ubiquinone and succinate-DCIP oxidoreductase activities. Inhibitory effect of H2O2 on respiratory complexes was almost completely recovered by ?-mercaptoethanol treatment. H2O2 treatment resulted in full resistance to QO site inhibitor myxothiazol and thus it is suggested that the quinol oxidase site (QO) of complex III is the target for H2O2. H2O2 did not modify basal levels of lipid peroxidation in yeast mitochondria. However, H2O2 addition to rat brain and liver mitochondria induced an increase in lipid peroxidation. These results are discussed in terms of the known physiological differences between mammalian and yeast mitochondria. PMID:17907001

  18. Structure and Function of an NADPH-Cytochrome P450 Oxidoreductase in an Open Conformation Capable of Reducing Cytochrome P450

    SciTech Connect

    Hamdane, Djemel; Xia, Chuanwu; Im, Sang-Choul; Zhang, Haoming; Kim, Jung-Ja P.; Waskell, Lucy; (MCW); (Michigan-Med)

    2010-01-20

    NADPH-cytochrome P450 oxidoreductase (CYPOR) catalyzes the transfer of electrons to all known microsomal cytochromes P450. A CYPOR variant, with a 4-amino acid deletion in the hinge connecting the FMN domain to the rest of the protein, has been crystallized in three remarkably extended conformations. The variant donates an electron to cytochrome P450 at the same rate as the wild-type, when provided with sufficient electrons. Nevertheless, it is defective in its ability to transfer electrons intramolecularly from FAD to FMN. The three extended CYPOR structures demonstrate that, by pivoting on the C terminus of the hinge, the FMN domain of the enzyme undergoes a structural rearrangement that separates it from FAD and exposes the FMN, allowing it to interact with its redox partners. A similar movement most likely occurs in the wild-type enzyme in the course of transferring electrons from FAD to its physiological partner, cytochrome P450. A model of the complex between an open conformation of CYPOR and cytochrome P450 is presented that satisfies mutagenesis constraints. Neither lengthening the linker nor mutating its sequence influenced the activity of CYPOR. It is likely that the analogous linker in other members of the diflavin family functions in a similar manner.

  19. Mycobacterium tuberculosis Type II NADH-Menaquinone Oxidoreductase Catalyzes Electron Transfer through a Two-Site Ping-Pong Mechanism and Has Two Quinone-Binding Sites

    PubMed Central

    2015-01-01

    Type II NADH-quinone oxidoreductase (NDH-2) catalyzes the transfer electrons from NADH to the quinone pool and plays an essential role in the oxidative phosphorylation system of Mycobacterium tuberculosis (Mtb). The absence of NDH-2 in the mammalian mitochondrial electron transport chain makes this enzyme an attractive target for antibiotic development. To fully establish the kinetic properties of this enzyme, we studied the interaction of Mtb NDH-2 with substrates, NADH, and various quinone analogues and their products in both membrane and soluble environments. These studies, and comparative analyses of the kinetics with thio-NAD+ and quinone electron acceptors, provided evidence that Mtb NDH-2 catalyzes the transfer electrons from NADH to quinone substrates by a nonclassical, two-site ping-pong kinetic mechanism whereby substrate quinones bind to a site that is distinct from the NADH-binding site. Furthermore, the effects of quinols on Mtb NDH-2 catalytic activity demonstrate the presence of two binding sites for quinone ligands, one favoring the reduced form and the other favoring the oxidized form. PMID:24447297

  20. Identification and Characterization of Catabolic para-Nitrophenol 4-Monooxygenase and para-Benzoquinone Reductase from Pseudomonas sp. Strain WBC-3?

    PubMed Central

    Zhang, Jun-Jie; Liu, Hong; Xiao, Yi; Zhang, Xian-En; Zhou, Ning-Yi

    2009-01-01

    Pseudomonas sp. strain WBC-3 utilizes para-nitrophenol (PNP) as a sole source of carbon, nitrogen, and energy. In order to identify the genes involved in this utilization, we cloned and sequenced a 12.7-kb fragment containing a conserved region of NAD(P)H:quinone oxidoreductase genes. Of the products of the 13 open reading frames deduced from this fragment, PnpA shares 24% identity to the large component of a 3-hydroxyphenylacetate hydroxylase from Pseudomonas putida U and PnpB is 58% identical to an NAD(P)H:quinone oxidoreductase from Escherichia coli. Both PnpA and PnpB were purified to homogeneity as His-tagged proteins, and they were considered to be a monomer and a dimer, respectively, as determined by gel filtration. PnpA is a flavin adenine dinucleotide-dependent single-component PNP 4-monooxygenase that converts PNP to para-benzoquinone in the presence of NADPH. PnpB is a flavin mononucleotide-and NADPH-dependent p-benzoquinone reductase that catalyzes the reduction of p-benzoquinone to hydroquinone. PnpB could enhance PnpA activity, and genetic analyses indicated that both pnpA and pnpB play essential roles in PNP mineralization in strain WBC-3. Furthermore, the pnpCDEF gene cluster next to pnpAB shares significant similarities with and has the same organization as a gene cluster responsible for hydroquinone degradation (hapCDEF) in Pseudomonas fluorescens ACB (M. J. Moonen, N. M. Kamerbeek, A. H. Westphal, S. A. Boeren, D. B. Janssen, M. W. Fraaije, and W. J. van Berkel, J. Bacteriol. 190:5190-5198, 2008), suggesting that the genes involved in PNP degradation are physically linked. PMID:19218392

  1. Cloning and characterization of the nucleoredoxin gene that encodes a novel nuclear protein related to thioredoxin

    SciTech Connect

    Kurooka, Hisanori; Kato, Keizo; Minoguchi, Shigeru [Kyoto Univ. (Japan)] [and others] [Kyoto Univ. (Japan); and others

    1997-02-01

    In a yeast artificial chromosome contig close to the nude locus on mouse chromosome 11, we identified a novel gene, nucleoredoxin, that encodes a protein with similarity to the active site of thioredoxins. Nucleoredoxin is conserved between mammalian species, and two homologous genes were found in Caenorhabditis elegans. The nucleoredoxin transcripts are expressed in all adult tissues examined, but restricted to the nervous system and the limb buds in Day 10.5-11.5 embryos. The nucleoredoxin protein is predominantly localized in the nucleus of cells transfected with the nucleoredoxin expression construct. Since the bacterially expressed protein of nucleoredoxin showed oxidoreductase activity of the insulin disulfide bonds with kinetics similar to that of thioredoxin, it may be a redox regulator of the nuclear proteins, such as transcription factors. 40 refs., 6 figs.

  2. Isolation and characterization of the human tyrosine hydroxylase gene: identification of 5' alternative splice sites responsible for multiple mRNAs

    SciTech Connect

    O'Malley, K.L.; Anhalt, M.J.; Martin, B.M.; Kelsoe, J.R.; Winfield, S.L.; Ginns, E.I.

    1987-11-03

    A full-length genomic clone for human tyrosine hydroxylase (L-tyrosine, tetrahydropteridine:oxygen oxidoreductase, EC 1.14.16.2) has been isolated. A human brain genomic library constructed in EMBL3 was screened by using a rat cDNA for tyrosine hydroxylase as a probe. Out of one million recombinant phage, one clone was identified that hybridized to both 5' and 3' rat cDNA probes. Restriction endonuclease mapping, Southern blotting, and sequence analysis revealed that, like its rodent counterpart, the human gene is single copy, contains 13 primary exons, and spans approximately 8 kilobases (kb). In contrast to the rat gene, human tyrosine hydroxylase undergoes alternative RNA processing within intron 1, generating at least three distinct mRNAs. A comparison of the human tyrosine hydroxylase and phenylalanine hydroxylase genes indicates that although both probably evolved from a common ancestral gene, major changes in the size of introns have occurred since their divergence.

  3. Bromodomain and extraterminal proteins suppress NF-E2-related factor 2-mediated antioxidant gene expression.

    PubMed

    Michaeloudes, Charalambos; Mercado, Nicolas; Clarke, Colin; Bhavsar, Pankaj K; Adcock, Ian M; Barnes, Peter J; Chung, Kian Fan

    2014-05-15

    Oxidative stress, a pathogenetic factor in many conditions, including chronic obstructive pulmonary disease, arises due to accumulation of reactive oxygen species and defective antioxidant defenses in the lungs. The latter is due, at least in part, to impaired activation of NF-E2-related factor 2 (Nrf2), a transcription factor involved in the activation of antioxidant and cytoprotective genes. The bromodomain and extraterminal (BET) proteins, Brd2, Brd3, Brd4, and BrdT, bind to acetylated lysine residues on histone or nonhistone proteins recruiting transcriptional regulators and thus activating or repressing gene transcription. We investigated whether BET proteins modulate the regulation of Nrf2-dependent gene expression in primary human airway smooth muscle cells and the human monocytic cell line, THP-1. Inhibition of BET protein bromodomains using the inhibitor JQ1+ or attenuation of Brd2 and Brd4 expression using small interfering RNA led to activation of Nrf2-dependent transcription and expression of the antioxidant proteins heme oxygenase-1, NADPH quinone oxidoreductase 1, and glutamate-cysteine ligase catalytic subunit. Also, JQ1+ prevented H2O2-induced intracellular reactive oxygen species production. By coimmunoprecipitation, BET proteins were found to be complexed with Nrf2, whereas chromatin-immunoprecipitation studies indicated recruitment of Brd2 and Brd4 to Nrf2-binding sites on the promoters of heme oxygenase-1 and NADPH quinone oxidoreductase 1. BET proteins, particularly Brd2 and Brd4, may play a key role in the regulation of Nrf2-dependent antioxidant gene transcription and are hence an important target for augmenting antioxidant responses in oxidative stress-mediated diseases. PMID:24733848

  4. Two genes encoding steroid 21-hydroxylase are located near the genes encoding the fourth component of complement in man.

    PubMed Central

    White, P C; Grossberger, D; Onufer, B J; Chaplin, D D; New, M I; Dupont, B; Strominger, J L

    1985-01-01

    Two genes encoding steroid 21-hydroxylase [21-OHase; steroid 21-monooxygenase; steroid, hydrogen-donor: oxygen oxidoreductase (21-hydroxylating); EC 1.14.99.10], a cytochrome P-450 enzyme, have been located within the HLA major histocompatibility complex. Congenital adrenal hyperplasia due to 21-OHase deficiency is a common inherited disorder of cortisol biosynthesis which is in genetic linkage disequilibrium with certain extended HLA haplotypes. These haplotypes include characteristic serum complement allotypes. A series of cosmid clones was isolated from a human genomic library by using a probe encoding part of the fourth component of complement, C4. These clones also hybridized with a probe encoding most of human 21-OHase. Restriction mapping and hybridization analysis showed that there are two 21-OHase genes, each located near the 3' end of one of the two C4 genes. Hybridization with probes specific for the 5' and 3' ends of the 21-OHase gene showed that the 21-OHase and C4 genes all have the same orientation. The 21-OHase genes 3' to C4A and C4B carry T aq I fragments of 3.2 and 3.7 kilobases (kb), respectively. Both of these fragments are found in genomic DNA of most individuals. In DNA from an individual with the severe, "salt-wasting" form of 21-OHase deficiency who was homozygous for HLA-A3;Bw47;C4A*1;C4B*Q0(null); DR7, the 3.7-kb Taq I fragment is absent, whereas hormonally normal individuals homozygous for HLA-A1;B8;C4A*Q0;C4B*1;DR3 do not carry the 3.2-kb Taq I fragment. These data suggest that the 21-OHase "B" gene (3.7-kb Taq I fragment) is functional, but the 21-OHase "A" gene (3.2-kb Taq I fragment) is not. Images PMID:2983330

  5. Alternative splicing isoform in succinate dehydrogenase complex, subunit C causes downregulation of succinate-coenzyme Q oxidoreductase activity in mitochondria

    PubMed Central

    SATOH, NANA; YOKOYAMA, CHIKAKO; ITAMURA, NORIAKI; MIYAJIMA-NAKANO, YOSHIHARU; HISATOMI, HISASHI

    2015-01-01

    Mitochondrial succinate dehydrogenase (SDH) is localized to the inner mitochondrial membrane and is responsible for the redox of succinic acid. SDH is a tetrameric iron-sulfur flavoprotein of the tricarboxylic acid cycle and respiratory chain. The SDH complex, subunit C (SDHC) transcript has deletion-type alternative splicing sites. Generally, alternative splicing produces variant proteins and expression patterns, as products of different genes. In certain cases, specific alternative splicing variants (ASVs) have been associated with human disease. Due to a frameshift mutation causing loss of the heme binding region, the SDHC ?5 isoform (lacking exon 5) exhibits no SDHC activity. To investigate whether the SDHC splicing variants can function as dominant-negative inhibitors, SDHC ASVs were overexpressed in HCT-15 human colorectal cancer cells. Using real-time reverse transcription-polymerase chain reaction, a dominant-negative effect of the ?5 isoform on SDHC mRNA was shown. In addition, ?5 overexpression increased the levels of reactive oxygen species. Furthermore, in the ?5 isoform-overexpressing cells, SDH activity was reduced. SDHC activation is a significant event during the electron transport chain, and the function of the SDHC ?5 variant may be significant for the differentiation of tumor cells. PMID:25435987

  6. Murine and human b locus pigmentation genes encode a glycoprotein (gp75) with catalase activity.

    PubMed Central

    Halaban, R; Moellmann, G

    1990-01-01

    Melanogenesis is regulated in large part by tyrosinase (monophenol monooxygenase; monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1), and defective tyrosinase leads to albinism. The mechanisms for other pigmentation determinants (e.g., those operative in tyrosinase-positive albinism and in murine coat-color mutants) are not yet known. One murine pigmentation gene, the brown (b) locus, when mutated leads to a brown (b/b) or hypopigmented (Blt/Blt) coat versus the wild-type black (B/B). We show that the b locus codes for a glycoprotein with the activity of a catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase, EC 1.11.1.6) (catalase B). Only the c locus protein is a tyrosinase. Because peroxides may be by-products of melanogenic activity and hydrogen peroxide in particular is known to destroy melanin precursors and melanin, we conclude that pigmentation is controlled not only by tyrosinase but also by a hydroperoxidase. Our studies indicate that catalase B is identical with gp75, a known human melanosomal glycoprotein; that the b mutation is in a heme-associated domain; and that the Blt mutation renders the protein susceptible to rapid proteolytic degradation. Images PMID:1693779

  7. Plasma membrane NADH-oxidoreductase in cells carrying mitochondrial DNA G11778A mutation and in cells devoid of mitochondrial DNA (rho0).

    PubMed

    Malik, Safarina G; Vaillant, François; Lawen, Alfons

    2004-01-01

    The mammalian plasma membrane (PM) NADH-oxidoreductase (PMOR) system is a multi-enzyme complex located in the plasma membrane of all eukaryotic cells, harboring at least two distinct activities, the plasma membrane NADH-ferricyanide reductase and the NADH-oxidase. To assess the behaviour of the two activities of the PMOR system, we measured the NADH-ferricyanide reductase and NADH-oxidase activities in fibroblast cell lines derived from patients carrying a mitochondrial DNA (mtDNA) G11778A mutation. We also measured the two activities in other cell lines, the HL-60 and HeLa (S3) lines, as well as in rho0 cells (cells devoid of mtDNA) generated from those lines and the fibroblast cells. These rho0 cells consequently lack oxidative phosphorylation and rely on anaerobic glycolysis for their ATP need. We have proposed that in rho0 cells, at least in part, up-regulation of the PMOR is a necessity to maintain the NAD+/NADH ratio, and a pre-requisite for cell growth and viability. We show here that the PM NADH-ferricyanide reductase activity was up-regulated in HL-AV2 (HL-60 rho0) cell lines, but not in the other rho0 and mtDNA mutant lines. The plasma membrane NADH oxidase activity was found to be up-regulated in both HL-AV2 and HeLa rho0 cell lines, but not significantly in the fibroblast rho0 and G11778A lines. PMID:15706055

  8. 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 ?, ?-unsaturated ?-lactam moiety. Structurally, they were elucidated to be 9?-hydroxy-13(14)-labden-16,15-amide (2) and 6?-acetoxy-9?-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-?-D-glucoside (9), luteolin-7-O-glucoside (10), 5-hydroxy-3,6,7,4?-tetramethoxyflavone (11), casticin (12), artemetin (13), aucubin (14), agnuside (15), ?-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

  9. 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

  10. FeS/S/FeS2 Redox System and Its Oxidoreductase-like Chemistry in the Iron-Sulfur World

    NASA Astrophysics Data System (ADS)

    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/FeS2 redox system on the interconversion between several pairs of ±-hydroxy acids and ±-keto acids (i.e., lactate/pyruvate, malate/oxaloacetate, and glycolate/glyoxylate). We found that, in the absence of FeS, elemental sulfur (S) oxidized ±-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/FeS2 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.

  11. Indolequinone inhibitors of NRH:quinone oxidoreductase 2. Characterization of the mechanism of inhibition in both cell-free and cellular systems.

    PubMed

    Yan, Chao; Dufour, Marine; Siegel, David; Reigan, Philip; Gomez, Joe; Shieh, Biehuoy; Moody, Christopher J; Ross, David

    2011-08-01

    We describe a series of indolequinones as efficient mechanism-based inhibitors of NRH:quinone oxidoreductase 2 (NQO2) for use either in cellular or cell-free systems. Compounds were designed to be reduced in the active site of the enzyme leading to loss of a substituted phenol leaving group and generation of a reactive iminium electrophile. Inhibition of NQO2 activity was assessed in both cell-free systems and the human leukemia K562 cell line. Inhibition of recombinant human NQO2 by the indolequinones was NRH-dependent, with kinetic parameters characteristic of mechanism-based inhibition and partition ratios as low as 2.0. Indolequinones inhibited NQO2 activity in K562 cells at nanomolar concentrations that did not inhibit NQO1 and were nontoxic to cells. Computation-based molecular modeling simulations demonstrated favorable conformations of indolequinones positioned directly above and in parallel with the isoalloxazine ring of FAD, and mass spectrometry extended our previous finding of adduction of the FAD in the active site of NQO2 by an indolequinone-derived iminium electrophile to the wider series of indolequinone inhibitors. Modeling combined with biochemical testing identified key structural parameters for effective inhibition, including a 5-aminoalkylamino side chain. Hydrogen bonding of the terminal amine nitrogen in the aminoalkylamino side chain was found to be critical for the correct orientation of the inhibitors in the active site. These indolequinones were irreversible inhibitors and were found to be at least 1 order of magnitude more potent than any previously documented competitive inhibitors of NQO2 and represent the first mechanism-based inhibitors of NQO2 to be characterized in cellular systems. PMID:21718050

  12. Indolequinone Inhibitors of NRH:Quinone Oxidoreductase 2 (NQO2). Characterization of Mechanism of Inhibition in both Cell-free and Cellular Systems. †

    PubMed Central

    Yan, Chao; Dufour, Marine; Siegel, David; Reigan, Philip; Gomez, Joe; Shieh, Biehuoy; Moody, Christopher J.; Ross, David

    2011-01-01

    We describe a series of indolequinones as efficient mechanism-based inhibitors of NRH:quinone oxidoreductase 2 (NQO2) for use either in cellular or cell-free systems. Compounds were designed to be reduced in the active site of the enzyme leading to loss of a substituted phenol leaving group and generation of a reactive iminium electrophile. Inhibition of NQO2 activity was assessed in both cell-free systems and in the human leukemia K562 cell line. Inhibition of recombinant human NQO2 by the indolequinones was NRH-dependent with kinetic parameters characteristic of mechanism-based inhibition and partition ratios as low as 2.0. Indolequinones inhibited NQO2 activity in K562 cells at nanomolar concentrations which did not inhibit NQO1 and were non-toxic to cells. Computational-based molecular modeling simulations demonstrated favorable conformations of indolequinones positioned directly above and in parallel to the isoalloxazine ring of FAD and mass spectrometry extended our previous finding of adduction of the FAD in the active site of NQO2 by an indolequinone-derived iminium electrophile to the wider series of indolequinone inhibitors. Modeling combined with biochemical testing identified key structural parameters for effective inhibition including a 5-aminoalkyamino side chain. Hydrogen bonding of the terminal amine nitrogen in the aminoalkylamino side chain was found to be critical for correct orientation of the inhibitors in the active site. These indolequinones were irreversible inhibitors and were found to be at least an order of magnitude more potent than any previously documented competitive inhibitors of NQO2 and represent the first mechanism-based inhibitors of NQO2 to be characterized in cellular systems. PMID:21718050

  13. NMR reveals double occupancy of quinone-type ligands in the catalytic quinone binding site of the Na+-translocating NADH:Quinone oxidoreductase from Vibrio cholerae.

    PubMed

    Nedielkov, Ruslan; Steffen, Wojtek; Steuber, Julia; Möller, Heiko M

    2013-10-18

    The sodium ion-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from the pathogen Vibrio cholerae exploits the free energy liberated during oxidation of NADH with ubiquinone to pump sodium ions across the cytoplasmic membrane. The Na(+)-NQR consists of four membrane-bound subunits NqrBCDE and the peripheral NqrF and NqrA subunits. NqrA binds ubiquinone-8 as well as quinones with shorter prenyl chains (ubiquinone-1 and ubiquinone-2). Here we show that the quinone derivative 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), a known inhibitor of the bc1 and b6f complexes found in mitochondria and chloroplasts, also inhibits quinone reduction by the Na(+)-NQR in a mixed inhibition mode. Tryptophan fluorescence quenching and saturation transfer difference NMR experiments in the presence of Na(+)-NQR inhibitor (DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide) indicate that two quinone analog ligands are bound simultaneously by the NqrA subunit with very similar interaction constants as observed with the holoenzyme complex. We conclude that the catalytic site of quinone reduction is located on NqrA. The two ligands bind to an extended binding pocket in direct vicinity to each other as demonstrated by interligand Overhauser effects between ubiquinone-1 and DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide, respectively. We propose that a similar spatially close arrangement of the native quinone substrates is also operational in vivo, enhancing the catalytic efficiency during the final electron transfer steps in the Na(+)-NQR. PMID:24003222

  14. Characterization of the inhibitor binding site in mitochondrial NADH-ubiquinone oxidoreductase by photoaffinity labeling using a quinazoline-type inhibitor.

    PubMed

    Murai, Masatoshi; Sekiguchi, Koji; Nishioka, Takaaki; Miyoshi, Hideto

    2009-02-01

    The diverse inhibitors of bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase) are believed to share a common large binding domain with partially overlapping sites, though it remains unclear how these binding sites relate to each other. To obtain new insight into the inhibitor binding domain in complex I, we synthesized a photoreactive azidoquinazoline {[(125)I]-6-azido-4-(4-iodophenethylamino)quinazoline, [(125)I]AzQ}, in which a photolabile azido group was introduced into the toxophoric quinazoline ring to allow specific cross-linking, and carried out a photoaffinity labeling study using bovine heart submitochondrial particles. Analysis of the photo-cross-linked proteins by peptide mass fingerprinting and immunoblotting revealed that [(125)I]AzQ specifically binds to the 49 kDa and ND1 subunits with a frequency of approximately 4:1. The cross-linking was completely blocked by excess amounts of other inhibitors such as acetogenin and fenpyroximate. Considerable cross-linking was also detected in the ADP/ATP carrier and 3-hydroxybutyrate dehydrogenase, though it was not associated with dysfunction of the two proteins. The partial proteolysis of the [(125)I]AzQ-labeled 49 kDa subunit by V8-protease and N-terminal sequencing of the resulting peptides revealed that the amino acid residue cross-linked by [(125)I]AzQ is within the sequence region Thr25-Glu143 (118 amino acids). Furthermore, examination of fragment patterns generated by exhaustive digestion of the [(125)I]AzQ-labeled 49 kDa subunit by V8-protease, lysylendopeptidase, or trypsin strongly suggested that the cross-linked residue is located within the region Asp41-Arg63 (23 amino acids). The present study has revealed, for the first time, the inhibitor binding site in complex I at the sub-subunit level. PMID:19128036

  15. Reduction of Hydrophilic Ubiquinones by the Flavin in Mitochondrial NADH:Ubiquinone Oxidoreductase (Complex I) and Production of Reactive Oxygen Species†

    PubMed Central

    2009-01-01

    NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a complicated, energy-transducing, membrane-bound enzyme that contains 45 different subunits, a non-covalently bound flavin mononucleotide, and eight iron?sulfur clusters. The mechanisms of NADH oxidation and intramolecular electron transfer by complex I are gradually being defined, but the mechanism linking ubiquinone reduction to proton translocation remains unknown. Studies of ubiquinone reduction by isolated complex I are problematic because the extremely hydrophobic natural substrate, ubiquinone-10, must be substituted with a relatively hydrophilic analogue (such as ubiquinone-1). Hydrophilic ubiquinones are reduced by an additional, non-energy-transducing pathway (which is insensitive to inhibitors such as rotenone and piericidin A). Here, we show that inhibitor-insensitive ubiquinone reduction occurs by a ping-pong type mechanism, catalyzed by the flavin mononucleotide cofactor in the active site for NADH oxidation. Moreover, semiquinones produced at the flavin site initiate redox cycling reactions with molecular oxygen, producing superoxide radicals and hydrogen peroxide. The ubiquinone reactant is regenerated, so the NADH:Q reaction becomes superstoichiometric. Idebenone, an artificial ubiquinone showing promise in the treatment of Friedreich’s Ataxia, reacts at the flavin site. The factors which determine the balance of reactivity between the two sites of ubiquinone reduction (the energy-transducing site and the flavin site) and the implications for mechanistic studies of ubiquinone reduction by complex I are discussed. Finally, the possibility that the flavin site in complex I catalyzes redox cycling reactions with a wide range of compounds, some of which are important in pharmacology and toxicology, is discussed. PMID:19220002

  16. 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 ?mol/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

  17. Roles of Subunit NuoK (ND4L) in the Energy-transducing Mechanism of Escherichia coli NDH-1 (NADH:Quinone Oxidoreductase)*

    PubMed Central

    Torres-Bacete, Jesus; Sinha, Prem Kumar; Sato, Motoaki; Patki, Gaurav; Kao, Mou-Chieh; Matsuno-Yagi, Akemi; Yagi, Takao

    2012-01-01

    The bacterial H+-translocating NADH:quinone oxidoreductase (NDH-1) catalyzes electron transfer from NADH to quinone coupled with proton pumping across the cytoplasmic membrane. The NuoK subunit (counterpart of the mitochondrial ND4L subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (TM1–3). Two glutamic residues located in the adjacent transmembrane helices of NuoK are important for the energy coupled activity of NDH-1. In particular, mutation of the highly conserved carboxyl residue (KGlu-36 in TM2) to Ala led to a complete loss of the NDH-1 activities. Mutation of the second conserved carboxyl residue (KGlu-72 in TM3) moderately reduced the activities. To clarify the contribution of NuoK to the mechanism of proton translocation, we relocated these two conserved residues. When we shifted KGlu-36 along TM2 to positions 32, 38, 39, and 40, the mutants largely retained energy transducing NDH-1 activities. According to the recent structural information, these positions are located in the vicinity of KGlu-36, present in the same helix phase, in an immediately before and after helix turn. In an earlier study, a double mutation of two arginine residues located in a short cytoplasmic loop between TM1 and TM2 (loop-1) showed a drastic effect on energy transducing activities. Therefore, the importance of this cytosolic loop of NuoK (KArg-25, KArg-26, and KAsn-27) for the energy transducing activities was extensively studied. The probable roles of subunit NuoK in the energy transducing mechanism of NDH-1 are discussed. PMID:23105119

  18. Down-regulation of the detoxifying enzyme NAD(P)H:quinone oxidoreductase 1 by vanadium in Hepa 1c1c7 cells

    SciTech Connect

    Anwar-Mohamed, Anwar [Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, T6G 2N8 (Canada); El-Kadi, Ayman O.S. [Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, T6G 2N8 (Canada)], E-mail: aelkadi@pharmacy.ualberta.ca

    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.

  19. Compounds from the Fruits of the Popular European Medicinal Plant Vitex agnus-castus in Chemoprevention via NADP(H):Quinone Oxidoreductase Type 1 Induction.

    PubMed

    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 ? , ? -unsaturated ? -lactam moiety. Structurally, they were elucidated to be 9 ? -hydroxy-13(14)-labden-16,15-amide (2) and 6 ? -acetoxy-9 ? -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- ? -D-glucoside (9), luteolin-7-O-glucoside (10), 5-hydroxy-3,6,7,4'-tetramethoxyflavone (11), casticin (12), artemetin (13), aucubin (14), agnuside (15), ? -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

  20. A new family of enzymes catalyzing the first committed step of the methylerythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in bacteria

    PubMed Central

    Sangari, Félix J.; Pérez-Gil, Jordi; Carretero-Paulet, Lorenzo; García-Lobo, Juan M.; Rodríguez-Concepción, Manuel

    2010-01-01

    Isoprenoids are a large family of compounds with essential functions in all domains of life. Most eubacteria synthesize their isoprenoids using the methylerythritol 4-phosphate (MEP) pathway, whereas a minority uses the unrelated mevalonate pathway and only a few have both. Interestingly, Brucella abortus and some other bacteria that only use the MEP pathway lack deoxyxylulose 5-phosphate (DXP) reductoisomerase (DXR), the enzyme catalyzing the NADPH-dependent production of MEP from DXP in the first committed step of the pathway. Fosmidomycin, a specific competitive inhibitor of DXR, inhibited growth of B. abortus cells expressing the Escherichia coli GlpT transporter (required for fosmidomycin uptake), confirming that a DXR-like (DRL) activity exists in these bacteria. The B. abortus DRL protein was found to belong to a family of uncharacterized proteins similar to homoserine dehydrogenase. Subsequent experiments confirmed that DRL and DXR catalyze the same biochemical reaction. DRL homologues shown to complement a DXR-deficient E. coli strain grouped within the same phylogenetic clade. The scattered taxonomic distribution of sequences from the DRL clade and the occurrence of several paralogues in some bacterial strains might be the result of lateral gene transfer and lineage-specific gene duplications and/or losses, similar to that described for typical mevalonate and MEP pathway genes. These results reveal the existence of a novel class of oxidoreductases catalyzing the conversion of DXP into MEP in prokaryotic cells, underscoring the biochemical and genetic plasticity achieved by bacteria to synthesize essential compounds such as isoprenoids. PMID:20660776

  1. A new family of enzymes catalyzing the first committed step of the methylerythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in bacteria.

    PubMed

    Sangari, Félix J; Pérez-Gil, Jordi; Carretero-Paulet, Lorenzo; García-Lobo, Juan M; Rodríguez-Concepción, Manuel

    2010-08-10

    Isoprenoids are a large family of compounds with essential functions in all domains of life. Most eubacteria synthesize their isoprenoids using the methylerythritol 4-phosphate (MEP) pathway, whereas a minority uses the unrelated mevalonate pathway and only a few have both. Interestingly, Brucella abortus and some other bacteria that only use the MEP pathway lack deoxyxylulose 5-phosphate (DXP) reductoisomerase (DXR), the enzyme catalyzing the NADPH-dependent production of MEP from DXP in the first committed step of the pathway. Fosmidomycin, a specific competitive inhibitor of DXR, inhibited growth of B. abortus cells expressing the Escherichia coli GlpT transporter (required for fosmidomycin uptake), confirming that a DXR-like (DRL) activity exists in these bacteria. The B. abortus DRL protein was found to belong to a family of uncharacterized proteins similar to homoserine dehydrogenase. Subsequent experiments confirmed that DRL and DXR catalyze the same biochemical reaction. DRL homologues shown to complement a DXR-deficient E. coli strain grouped within the same phylogenetic clade. The scattered taxonomic distribution of sequences from the DRL clade and the occurrence of several paralogues in some bacterial strains might be the result of lateral gene transfer and lineage-specific gene duplications and/or losses, similar to that described for typical mevalonate and MEP pathway genes. These results reveal the existence of a novel class of oxidoreductases catalyzing the conversion of DXP into MEP in prokaryotic cells, underscoring the biochemical and genetic plasticity achieved by bacteria to synthesize essential compounds such as isoprenoids. PMID:20660776

  2. Occurrence, phylogeny, structure, and function of catalases and peroxidases in cyanobacteria.

    PubMed

    Bernroitner, Margit; Zamocky, Marcel; Furtmüller, Paul G; Peschek, Günter A; Obinger, Christian

    2009-01-01

    Cyanobacteria have evolved approximately 3x10(9) years ago from ancient phototrophic microorganisms that already lived on our planet Earth. By opening the era of an aerobic, oxygen-containing biosphere, they are the true pacemakers of geological and biological evolution. Cyanobacteria must have been among the first organisms to elaborate mechanisms for the detoxification of partially reduced oxygen species including (hydrogen) peroxide. Since there is still an suprising lack of knowledge on the type, role, and mechanism(s) of peroxide-degrading enzymes in these bacteria, all 44 fully or partially sequenced genomes for haem and non-haem catalases and peroxidases have been critically analysed based on well known structure-function relationships of the corresponding oxidoreductases. It is demonstrated that H(2)O(2)-dismutating enzymes are mainly represented by bifunctional (haem) catalase-peroxidases and (binuclear) manganese catalases, with the latter being almost exclusively found in diazotrophic species. Several strains even lack a gene that encodes an enzyme with catalase activity. Two groups of peroxidases are found. Genes encoding putative (primordial) haem peroxidases (with homology to corresponding mammalian enzymes) and vanadium-containing iodoperoxidases are found only in a few species, whereas genes encoding peroxiredoxins (1-Cys, 2-Cys, type II, and Q-type) are ubiquitous in cyanobacteria. In addition, approximately 70% contain NADPH-dependent glutathione peroxidase-like proteins. The occurrence and phylogeny of these enzymes is discussed, as well as the present knowledge of their physiological role(s). PMID:19129167

  3. 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 [Department of Biochemistry, Faculty of Science, Charles University, Prague (Czech Republic)] [Department of Biochemistry, Faculty of Science, Charles University, Prague (Czech Republic); Nebert, Daniel W. [Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati (United States)] [Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati (United States); Phillips, David H. [Analytical and Environmental Sciences Division, MRC-HPA Centre for Environment and Health, King's College London, London (United Kingdom)] [Analytical and Environmental Sciences Division, MRC-HPA Centre for Environment and Health, King's College London, London (United Kingdom); Frei, Eva [Division of Preventive Oncology, National Center for Tumor Diseases, German Cancer Research Center (DKFZ), Heidelberg (Germany)] [Division of Preventive Oncology, National Center for Tumor Diseases, German Cancer Research Center (DKFZ), Heidelberg (Germany); Schmeiser, Heinz H. [Research Group Genetic Alterations in Carcinogenesis, German Cancer Research Center (DKFZ), Heidelberg (Germany)] [Research Group Genetic Alterations in Carcinogenesis, German Cancer Research Center (DKFZ), Heidelberg (Germany); Arlt, Volker M. [Analytical and Environmental Sciences Division, MRC-HPA Centre for Environment and Health, King's College London, London (United Kingdom)] [Analytical and Environmental Sciences Division, MRC-HPA Centre for Environment and Health, King's College London, London (United Kingdom); Stiborova, Marie, E-mail: stiborov@natur.cuni.cz [Department of Biochemistry, Faculty of Science, Charles University, Prague (Czech Republic)] [Department of Biochemistry, Faculty of Science, Charles University, Prague (Czech Republic)

    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.

  4. Cold-Induced Changes in Gene Expression in Brown Adipose Tissue, White Adipose Tissue and Liver

    PubMed Central

    Shore, Andrew M.; Karamitri, Angeliki; Kemp, Paul; Speakman, John R.; Graham, Neil S.; Lomax, Michael A.

    2013-01-01

    Cold exposure imposes a metabolic challenge to mammals that is met by a coordinated response in different tissues to prevent hypothermia. This study reports a transcriptomic analysis in brown adipose tissue (BAT), white adipose (WAT) and liver of mice in response to 24 h cold exposure at 8°C. Expression of 1895 genes were significantly (P<0.05) up- or down-regulated more than two fold by cold exposure in all tissues but only 5 of these genes were shared by all three tissues, and only 19, 14 and 134 genes were common between WAT and BAT, WAT and liver, and BAT and liver, respectively. We confirmed using qRT-PCR, the increased expression of a number of characteristic BAT genes during cold exposure. In both BAT and the liver, the most common direction of change in gene expression was suppression (496 genes in BAT and 590 genes in liver). Gene ontology analysis revealed for the first time significant (P<0.05) down regulation in response to cold, of genes involved in oxidoreductase activity, lipid metabolic processes and protease inhibitor activity, in both BAT and liver, but not WAT. The results reveal an unexpected importance of down regulation of cytochrome P450 gene expression and apolipoprotein, in both BAT and liver, but not WAT, in response to cold exposure. Pathway analysis suggests a model in which down regulation of the nuclear transcription factors HNF4? and PPAR? in both BAT and liver may orchestrate the down regulation of genes involved in lipoprotein and steroid metabolism as well as Phase I enzymes belonging to the cytochrome P450 group in response to cold stress in mice. We propose that the response to cold stress involves decreased gene expression in a range of cellular processes in order to maximise pathways involved in heat production. PMID:23894377

  5. Sodium arsenite-induced stress-related gene expression in normal human epidermal, HaCaT, and HEL30 keratinocytes.

    PubMed

    Trouba, Kevin J; Geisenhoffer, Kristen M; Germolec, Dori R

    2002-10-01

    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

  6. A human alcohol dehydrogenase gene (ADH6) encoding an additional class of isozyme.

    PubMed Central

    Yasunami, M; Chen, C S; Yoshida, A

    1991-01-01

    The human alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) gene family consists of five known loci (ADH1-ADH5), which have been mapped close together on chromosome 4 (4q21-25). ADH isozymes encoded by these genes are grouped in three distinct classes in terms of their enzymological properties. A moderate structural similarity is observed between the members of different classes. We isolated an additional member of the ADH gene family by means of cross-hybridization with the ADH2 (class I) cDNA probe. cDNA clones corresponding to this gene were derived from PCR-amplified libraries as well. The coding sequence of a 368-amino-acid-long open reading frame was interrupted by introns into eight exons and spanned approximately 17 kilobases on the genome. The gene contains a glucocorticoid response element at the 5' region. The transcript was detected in the stomach and liver. The deduced amino acid sequence of the open reading frame showed about 60% positional identity with known human ADHs. This extent of homology is comparable to interclass similarity in the human ADH family. Thus, the newly identified gene, which is designated ADH6, governs the synthesis of an enzyme that belongs to another class of ADHs presumably with a distinct physiological role. Images PMID:1881901

  7. Fusarium wilt (Fusarium oxysporum f. sp. vasinfectum) genes expressed during infection of cotton (Gossypium hirsutum)dagger.

    PubMed

    McFadden, Helen G; Wilson, Iain W; Chapple, Robin M; Dowd, Caitriona

    2006-03-01

    SUMMARY We sought to identify Fusarium oxysporum f. sp. vasinfectum (Fov) genes that may be associated with pathogenicity. Initially we utilized microarray and Q-PCR technology to identify Fov genes expressed in root and hypocotyl tissues during a compatible infection of cotton. We identified 218 fungal clones representing 174 Fov non-redundant genes as expressed in planta. The majority of the expressed sequences were expressed in infected roots, with only six genes detected in hypocotyl tissue. The Fov genes identified were predominately of unknown function or associated with fungal growth and energy production. We then analysed the expression of the identified fungal genes in infected roots and in saprophytically grown mycelia and identified 11 genes preferentially expressed in plant tissue. A putative oxidoreductase gene (with homology to AtsC) was found to be highly preferentially expressed in planta. In Agrobacterium tumefaciens, AtsC is associated with virulence. Inoculation of a susceptible and a partially resistant cotton cultivar with either a pathogenic or a non-pathogenic isolate of Fov revealed that the expression of the Fov AtsC homologue was associated with pathogenicity and disease symptom formation. PMID:20507430

  8. Identification of the Genes Involved in the Fruiting Body Production and Cordycepin Formation of Cordyceps militaris Fungus.

    PubMed

    Zheng, Zhuang-Li; Qiu, Xue-Hong; Han, Ri-Chou

    2015-03-01

    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

  9. Resolution of the aerobic respiratory system of the thermoacidophilic archaeon, Sulfolobus sp. strain 7. III. The archaeal novel respiratory complex II (succinate:caldariellaquinone oxidoreductase complex) inherently lacks heme group.

    PubMed

    Iwasaki, T; Wakagi, T; Oshima, T

    1995-12-29

    An active respiratory complex II (succinate:quinone oxidoreductase) has been purified from tetraether lipid membranes of the thermoacidophilic archaeon, Sulfolobus sp. strain 7. It consists of four different subunits with apparent molecular masses of 66, 37, 33, and 12 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 66-kDa subunit contains a covalently bound flavin, the 37-kDa subunit is a possible iron-sulfur protein carrying three distinct types of EPR-visible FeS cluster, and the 33- and 12-kDa subunits are putative membrane-anchor subunits, respectively. While no heme group is detected in the purified complex II, it catalyzes succinate-dependent reduction of ubiquinone-1 and 2,6-dichlorophenolindophenol in the absence of phenazine methosulfate. The respiratory complex II of Sulfolobus sp. strain 7 appears to be novel in that it functions as a true succinate:caldariellaquinone oxidoreductase, although inherently lacking any heme group. This further indicates that the heme group of several respiratory complexes II may not be involved in the redox intermediates of the electron transfer from succinate to quinone. PMID:8537344

  10. 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 [Research Laboratories 2, Fuji Yakuhin Co., Ltd., 636-1 Iidashinden, Nishi-ku, Saitama 331-0068 (Japan)]. E-mail: L2-26899@fujiyakuhin.co.jp; Shimo, Takeo [Research Laboratories 2, Fuji Yakuhin Co., Ltd., 636-1 Iidashinden, Nishi-ku, Saitama 331-0068 (Japan); Matsumoto, Koji [Research Laboratories 2, Fuji Yakuhin Co., Ltd., 636-1 Iidashinden, Nishi-ku, Saitama 331-0068 (Japan); Oba, Kazuhiko [Research Laboratories 2, Fuji Yakuhin Co., Ltd., 636-1 Iidashinden, Nishi-ku, Saitama 331-0068 (Japan); Nakazawa, Takashi [Research Laboratories 2, Fuji Yakuhin Co., Ltd., 636-1 Iidashinden, Nishi-ku, Saitama 331-0068 (Japan); Nagata, Osamu [Research Laboratories 2, Fuji Yakuhin Co., Ltd., 636-1 Iidashinden, Nishi-ku, Saitama 331-0068 (Japan)

    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.

  11. NAD(P)H:quinone Oxidoreductase 1-Compromised Human Bone Marrow Endothelial Cells Exhibit Decreased Adhesion Molecule Expression and CD34+ Hematopoietic Cell Adhesion

    PubMed Central

    Zhou, Hongfei; Dehn, Donna; Kepa, Jadwiga K.; Siegel, David; Scott, Devon E.; Tan, Wei

    2010-01-01

    NAD(P)H:quinone oxidoreductase 1 (NQO1) deficiency resulting from a homozygous NQO1*2 polymorphism has been associated with an increased risk of benzene-induced myeloid toxicity and a variety of de novo and therapy-induced leukemias. Endothelial cells in human bone marrow form one of the two known hematopoietic stem cell microenvironments and are one of the major cell types that express NQO1 in bone marrow. We have used a transformed human bone marrow endothelial cell (TrHBMEC) line to study the potential impact of a lack of NQO1 activity on adhesion molecule [endothelial leukocyte adhesion molecule 1 (E-selectin), vascular cell adhesion molecule (VCAM)-1, and intercellular adhesion molecule (ICAM)-1] expression and functional adhesion to bone marrow progenitor cells. We used both 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936), a mechanism-based inhibitor of NQO1, and anti-NQO1 small interfering RNA to abrogate NQO1 activity. Real-time reverse transcription-polymerase chain reaction data demonstrated a significant inhibition of tumor necrosis factor (TNF)?-induced E-selectin mRNA levels after ES936 pretreatment. Immunoblot assays demonstrated a significant reduction in TNF?-stimulated E-selectin, VCAM-1, and ICAM-1 proteins after inhibition or knockdown of NQO1. The mechanisms underlying this effect remain undefined, but modulation of nuclear factor-?B (p65), c-Jun, and activating transcription factor 2, transcriptional regulators of adhesion molecules, were observed after inhibition or knockdown of NQO1. Decreased level of E-selectin, VCAM-1, and ICAM-1 also resulted in a functional deficit in adhesion. A parallel plate flow chamber study demonstrated a marked reduction in CD34+ cell (KG1a) adhesion to NQO1-deficient TrHBMECs relative to controls. The reduced adhesive ability of TrHBMECs may affect the function of the vascular stem cell niche and also may contribute to the increased susceptibility of polymorphic individuals lacking NQO1 to leukemias and hematotoxicants such as benzene. PMID:20378716

  12. Four structural subclasses of the antivirulence drug target disulfide oxidoreductase DsbA provide a platform for design of subclass-specific inhibitors.

    PubMed

    McMahon, Róisín M; Premkumar, Lakshmanane; Martin, Jennifer L

    2014-08-01

    By catalyzing oxidative protein folding, the bacterial disulfide bond protein A (DsbA) plays an essential role in the assembly of many virulence factors. Predictably, DsbA disruption affects multiple downstream effector molecules, resulting in pleiotropic effects on the virulence of important human pathogens. These findings mark DsbA as a master regulator of virulence, and identify the enzyme as a target for a new class of antivirulence agents that disarm pathogenic bacteria rather than killing them. The purpose of this article is to discuss and expand upon recent findings on DsbA and to provide additional novel insights into the druggability of this important disulfide oxidoreductase by comparing the structures and properties of 13 well-characterized DsbA enzymes. Our structural analysis involved comparison of the overall fold, the surface properties, the conformations of three loops contributing to the binding surface and the sequence identity of residues contributing to these loops. Two distinct structural classes were identified, classes I and II, which are differentiated by their central ?-sheet arrangements and which roughly separate the DsbAs produced by Gram-negative from Gram-positive organisms. The classes can be further subdivided into a total of four subclasses on the basis of surface features. Class Ia is equivalent to the Enterobacteriaceae class that has been defined previously. Bioinformatic analyses support the classification of DsbAs into 3 of the 4 subclasses, but did not pick up the 4th subclass which is only apparent from analysis of DsbA electrostatic surface properties. In the context of inhibitor development, the discrete structural subclasses provide a platform for developing DsbA inhibitory scaffolds with a subclass-wide spectrum of activity. We expect that more DsbA classes are likely to be identified, as enzymes from other pathogens are explored, and we highlight the issues associated with structure-based inhibitor development targeting this pivotal mediator of bacterial virulence. This article is part of a Special Issue entitled: Thiol-Based Redox Processes. PMID:24487020

  13. High-level chromate resistance in Arthrobacter sp. strain FB24 requires previously uncharacterized accessory genes

    PubMed Central

    2009-01-01

    Background The genome of Arthrobacter sp. strain FB24 contains a chromate resistance determinant (CRD), consisting of 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. To delineate the contribution of the CRD genes to the FB24 chromate [Cr(VI)] response, we evaluated the growth of mutant strains bearing regions of the CRD and transcript expression levels in response to Cr(VI) challenge. Results 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 possible regulatory roles. Conclusion Our findings indicate that chromate resistance in Arthrobacter sp. strain FB24 is due to chromate efflux through the ChrA transport protein. More importantly, new genes have been identified as having significant roles in chromate resistance. Collectively, the functional predictions of these additional genes suggest the involvement of a signal transduction system in the regulation of chromate efflux and warrants further study. PMID:19758450

  14. Piper betle induces phase I & II genes through Nrf2/ARE signaling pathway in mouse embryonic fibroblasts derived from wild type and Nrf2 knockout cells

    PubMed Central

    2014-01-01

    Background Nuclear factor-erythroid 2 p45 related factor 2 (Nrf2) is a primary transcription factor, protecting cells from oxidative stress by regulating a number of antioxidants and phase II detoxifying enzymes. Dietary components such as sulforaphane in broccoli and quercetin in onions have been shown to be inducers of Nrf2. Piper betle (PB) grows well in tropical climate and the leaves are used in a number of traditional remedies for the treatment of stomach ailments and infections among Asians. The aim of this study was to elucidate the effect of Piper betle (PB) leaves extract in Nrf2 signaling pathway by using 2 types of cells; mouse embryonic fibroblasts (MEFs) derived from wild-type (WT) and Nrf2 knockout (N0) mice. Methods WT and N0 cells were treated with 5 and 10 ?g/ml of PB for 10 and 12-h for the determination of nuclear translocation of Nrf2 protein. Luciferase reporter gene activity was performed to evaluate the antioxidant response element (ARE)-induction by PB. Real-time PCR and Western blot were conducted on both WT and N0 cells after PB treatment for the determination of antioxidant enzymes [superoxide dismutase (SOD1) and heme-oxygenase (HO-1)], phase I oxidoreductase enzymes [NAD(P)H: quinone oxidoreductase (NQO1)] and phase II detoxifying enzyme [glutathione S-transferase (GST)]. Results Nuclear translocation of Nrf2 by PB in WT cells was better after 10 h incubation compared to 12 h. Real time PCR and Western blot analysis showed increased expressions of Nrf2, NQO1 and GSTA1 genes with corresponding increases in glutathione, NQO1 and HO-1 proteins in WT cells. Reporter gene ARE was stimulated by PB as shown by ARE/luciferase assay. Interestingly, PB induced SOD1 gene and protein expressions in N0 cells but not in WT cells. Conclusion The results of this study confirmed that PB activated Nrf2-ARE signaling pathway which subsequently induced some phase I oxidoreductase, phase II detoxifying and antioxidant genes expression via ARE reporter gene involved in the Nrf2 pathway with the exception of SOD1 which may not be dependent on this pathway. PMID:24559113

  15. Gene Therapy

    PubMed Central

    Scheller, E.L.; Krebsbach, P.H.

    2009-01-01

    Gene therapy is defined as the treatment of disease by transfer of genetic material into cells. This review will explore methods available for gene transfer as well as current and potential applications for craniofacial regeneration, with emphasis on future development and design. Though non-viral gene delivery methods are limited by low gene transfer efficiency, they benefit from relative safety, low immunogenicity, ease of manufacture, and lack of DNA insert size limitation. In contrast, viral vectors are nature’s gene delivery machines that can be optimized to allow for tissue-specific targeting, site-specific chromosomal integration, and efficient long-term infection of dividing and non-dividing cells. In contrast to traditional replacement gene therapy, craniofacial regeneration seeks to use genetic vectors as supplemental building blocks for tissue growth and repair. Synergistic combination of viral gene therapy with craniofacial tissue engineering will significantly enhance our ability to repair and replace tissues in vivo. PMID:19641145

  16. 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

  17. 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

  18. Gene Concepts, Gene Talk, and Gene Patents

    E-print Network

    Torrance, Andrew W.

    2010-01-01

    Since the existence of a discrete unit of heredity was first proposed by Gregor Mendel, scientific concepts of the “gene” have undergone rapid evolution. Beyond obvious epistemic and operational importance to the scientific ...

  19. Involvement of NADPH-Dependent and cAMP-PKA Sensitive H+ Channels in the Chorda Tympani Nerve Responses to Strong Acids

    PubMed Central

    DeSimone, John A.; T. Phan, Tam-Hao; Heck, Gerard L.; Ren, ZuoJun; Coleman, Jamison; Mummalaneni, Shobha; Melone, Pamela

    2011-01-01

    To investigate if chorda tympani (CT) taste nerve responses to strong (HCl) and weak (CO2 and acetic acid) acidic stimuli are dependent upon NADPH oxidase–linked and cAMP-sensitive proton conductances in taste cell membranes, CT responses were monitored in rats, wild-type (WT) mice, and gp91phox knockout (KO) mice in the absence and presence of blockers (Zn2+ and diethyl pyrocarbonate [DEPC]) or activators (8-(4-chlorophenylthio)-cAMP; 8-CPT-cAMP) of proton channels and activators of the NADPH oxidase enzyme (phorbol 12-myristate 13-acetate [PMA], H2O2, and nitrazepam). Zn2+ and DEPC inhibited and 8-CPT-cAMP, PMA, H2O2, and nitrazepam enhanced the tonic CT responses to HCl without altering responses to CO2 and acetic acid. In KO mice, the tonic HCl CT response was reduced by 64% relative to WT mice. The residual CT response was insensitive to H2O2 but was blocked by Zn2+. Its magnitude was further enhanced by 8-CPT-cAMP treatment, and the enhancement was blocked by 8-CPT-adenosine-3?-5?-cyclic monophospho-rothioate, a protein kinase A (PKA) inhibitor. Under voltage-clamp conditions, before cAMP treatment, rat tonic HCl CT responses demonstrated voltage-dependence only at ±90 mV, suggesting the presence of H+ channels with voltage-dependent conductances. After cAMP treatment, the tonic HCl CT response had a quasi-linear dependence on voltage, suggesting that the cAMP-dependent part of the HCl CT response has a quasi-linear voltage dependence between +60 and ?60 mV, only becoming sigmoidal when approaching +90 and ?90 mV. The results suggest that CT responses to HCl involve 2 proton entry pathways, an NADPH oxidase–dependent proton channel, and a cAMP-PKA sensitive proton channel. PMID:21339339

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

    Microsoft Academic Search

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

    2009-01-01

    Wax esters, ester-linked fatty acids and long-chain alcohols, are important energy storage compounds in select bacteria. The synthesis of wax esters from fatty acids is proposed to require the action of a four-enzyme pathway. An essential step in the pathway is the reduction of a fatty aldehyde to the corresponding fatty alcohol, although the enzyme responsible for catalyzing this reaction

  1. Enzymatic test for the detection of a riboflavin deficiency. NADPH-dependent glutathione reductase of red blood cells and its activation by FAD in vitro

    Microsoft Academic Search

    D. Glatzle; F. Weber; O. Wiss

    1968-01-01

    Zusammenfassung Die NADPH-spezifische Glutathion-reduktaseaktivität hämolysierter Erythrozyten erscheint brauchbar als Parameter zur Erfassung von Riboflavin-mangelzuständen. Die Enzymaktivität wird in vitro durch einen Zusatz von FAD bei Riboflavinmangelratten wesentlich stärker erhöht als bei Kontrollratten. Untersuchungen mit menschlichen Erythrozyten deuten darauf hin, dass auch hier die Aktivierbarkeit des Enzyms durch FAD vom Riboflavinstatus abhängt.

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

    PubMed

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

    2014-11-15

    Nitric Oxide (NO) and Reactive oxygen species (ROS) are endogenous regulators of angiogenesis-related events as endothelial cell proliferation and survival, but NO/ROS defect or unbalance contribute to cancers. We recently designed a novel photoactive inhibitor of NO-Synthases (NOS) called NS1, which binds their NADPH site in vitro. Here, we show that NS1 inhibited NO formed in aortic rings. NS1-induced NO decrease led to an inhibition of angiogenesis in a model of VEGF-induced endothelial tubes formation. Beside this effect, NS1 reduced ROS levels in endothelial and melanoma A375 cells and in aorta. In metastatic melanoma cells, NS1 first induced a strong decrease of VEGF and blocked melanoma cell cycle at G2/M. NS1 decreased NOX(4) and ROS levels that could lead to a specific proliferation arrest and cell death. In contrast, NS1 did not perturb melanocytes growth. Altogether, NS1 revealed a possible cross-talk between eNOS- and NOX(4) -associated pathways in melanoma cells via VEGF, Erk and Akt modulation by NS1 that could be targeted to stop proliferation. NS1 thus constitutes a promising tool that modulates NO and redox stresses by targeting and directly inhibiting eNOS and, at least indirectly, NADPH oxidase(s), with great potential to control angiogenesis. PMID:25296975

  3. Changes in gene expression linked to methamphetamine-induced dopaminergic neurotoxicity.

    PubMed

    Xie, Tao; Tong, Liqiong; Barrett, Tanya; Yuan, Jie; Hatzidimitriou, George; McCann, Una D; Becker, Kevin G; Donovan, David M; Ricaurte, George A

    2002-01-01

    The purpose of these studies was to examine the role of gene expression in methamphetamine (METH)-induced dopamine (DA) neurotoxicity. First, the effects of the mRNA synthesis inhibitor, actinomycin-D, and the protein synthesis inhibitor, cycloheximide, were examined. Both agents afforded complete protection against METH-induced DA neurotoxicity and did so independently of effects on core temperature, DA transporter function, or METH brain levels, suggesting that gene transcription and mRNA translation play a role in METH neurotoxicity. Next, microarray technology, in combination with an experimental approach designed to facilitate recognition of relevant gene expression patterns, was used to identify gene products linked to METH-induced DA neurotoxicity. This led to the identification of several genes in the ventral midbrain associated with the neurotoxic process, including genes for energy metabolism [cytochrome c oxidase subunit 1 (COX1), reduced nicotinamide adenine dinucleotide ubiquinone oxidoreductase chain 2, and phosphoglycerate mutase B], ion regulation (members of sodium/hydrogen exchanger and sodium/bile acid cotransporter family), signal transduction (adenylyl cyclase III), and cell differentiation and degeneration (N-myc downstream-regulated gene 3 and tau protein). Of these differentially expressed genes, we elected to further examine the increase in COX1 expression, because of data implicating energy utilization in METH neurotoxicity and the known role of COX1 in energy metabolism. On the basis of time course studies, Northern blot analyses, in situ hybridization results, and temperature studies, we now report that increased COX1 expression in the ventral midbrain is linked to METH-induced DA neuronal injury. The precise role of COX1 and other genes in METH neurotoxicity remains to be elucidated. PMID:11756511

  4. YNL134C from Saccharomyces cerevisiae encodes a novel protein with aldehyde reductase activity for detoxification of furfural derived from lignocellulosic biomass.

    PubMed

    Zhao, Xianxian; Tang, Juan; Wang, Xu; Yang, Ruoheng; Zhang, Xiaoping; Gu, Yunfu; Li, Xi; Ma, Menggen

    2015-05-01

    Furfural and 5-hydroxymethylfurfural (HMF) are the two main aldehyde compounds derived from pentoses and hexoses, respectively, during lignocellulosic biomass pretreatment. These two compounds inhibit microbial growth and interfere with subsequent alcohol fermentation. Saccharomyces cerevisiae has the in situ ability to detoxify furfural and HMF to the less toxic 2-furanmethanol (FM) and furan-2,5-dimethanol (FDM), respectively. Herein, we report that an uncharacterized gene, YNL134C, was highly up-regulated under furfural or HMF stress and Yap1p and Msn2/4p transcription factors likely controlled its up-regulated expression. Enzyme activity assays showed that YNL134C is an NADH-dependent aldehyde reductase, which plays a role in detoxification of furfural to FM. However, no NADH- or NADPH-dependent enzyme activity was observed for detoxification of HMF to FDM. This enzyme did not catalyse the reverse reaction of FM to furfural or FDM to HMF. Further studies showed that YNL134C is a broad-substrate aldehyde reductase, which can reduce multiple aldehydes to their corresponding alcohols. Although YNL134C is grouped into the quinone oxidoreductase family, no quinone reductase activity was observed using 1,2-naphthoquinone or 9,10-phenanthrenequinone as a substrate, and phylogenetic analysis indicates that it is genetically distant to quinone reductases. Proteins similar to YNL134C in sequence from S. cerevisiae and other microorganisms were phylogenetically analysed. Copyright © 2015 John Wiley & Sons, Ltd. PMID:25656244

  5. Identification of a new member (ZNF183) of the Ring finger gene family in Xq24-25.

    PubMed

    Frattini, A; Faranda, S; Bagnasco, L; Patrosso, C; Nulli, P; Zucchi, I; Vezzoni, P

    1997-06-19

    Four genes were mapped to the Xq24-25 region by searching the EST and the non-redundant database with short tracts of genomic sequences. These were random STSs present in the STS database or sequences derived from CpG islands (EagI-based STSs). One of the four matches corresponded to the full length transcript from the intronless glutamate dehydrogenase gene. The second was the human homolog of the bovine NADH ubiquinone oxidoreductase MWFE subunit gene (GDB symbol: NDUFA1). The other two, ZNF183 and ITBA4, were novel genes whose function cannot directly be inferred from their sequence analysis. However, a known motif, the C3HC4 Ring finger domain, shared by various tumor suppressors, DNA repair genes and cytokine receptor-associated molecules, is present at the C terminus of the ubiquitously expressed ZNF183 gene. ITBA4 is expressed at various levels in different tissues and is alternatively processed in brain. Similarity search did not detect any significant match in databases. These results, together with others previously reported by our laboratory, suggest that comparison of genomic and transcribed sequences which are continuously accumulating in databases, can provide 'virtual' mapping of a substantial number of ESTs to the specific genomic region which the STSs have been derived from. PMID:9224902

  6. Screening differentially expressed genes in an amphipod (Hyalella azteca) exposed to fungicide vinclozolin by suppression subtractive hybridization.

    PubMed

    Wu, Yun H; Wu, Tsung M; Hong, Chwan Y; Wang, Yei S; Yen, Jui H

    2014-01-01

    Vinclozolin, a dicarboximide fungicide, is an endocrine disrupting chemical that competes with an androgenic endocrine disruptor compound. Most research has focused on the epigenetic effect of vinclozolin in humans. In terms of ecotoxicology, understanding the effect of vinclozolin on non-target organisms is important. The expression profile of a comprehensive set of genes in the amphipod Hyalella azteca exposed to vinclozolin was examined. The expressed sequence tags in low-dose vinclozolin-treated and -untreated amphipods were isolated and identified by suppression subtractive hybridization. DNA dot blotting was used to confirm the results and establish a subtracted cDNA library for comparing all differentially expressed sequences with and without vinclozolin treatment. In total, 494 differentially expressed genes, including hemocyanin, heatshock protein, cytochrome, cytochrome oxidase and NADH dehydrogenase were detected. Hemocyanin was the most abundant gene. DNA dot blotting revealed 55 genes with significant differential expression. These genes included larval serum protein 1 alpha, E3 ubiquitin-protein ligase, mitochondrial cytochrome c oxidase, mitochondrial protein, proteasome inhibitor, hemocyanin, zinc-finger-containing protein, mitochondrial NADH-ubiquinone oxidoreductase and epididymal sperm-binding protein. Vinclozolin appears to upregulate stress-related genes and hemocyanin, related to immunity. Moreover, vinclozolin downregulated NADH dehydrogenase, related to respiration. Thus, even a non-lethal concentration of vinclozolin still has an effect at the genetic level in H. azteca and presents a potential risk, especially as it would affect non-target organism hormone metabolism. PMID:25190560

  7. Pseudomonas aeruginosa LysR PA4203 regulator NmoR acts as a repressor of the PA4202 nmoA gene, encoding a nitronate monooxygenase.

    PubMed

    Vercammen, Ken; Wei, Qing; Charlier, Daniel; Dötsch, Andreas; Haüssler, Susanne; Schulz, Sebastian; Salvi, Francesca; Gadda, Giovanni; Spain, Jim; Rybtke, Morten Levin; Tolker-Nielsen, Tim; Dingemans, Jozef; Ye, Lumeng; Cornelis, Pierre

    2015-03-01

    The PA4203 gene encodes a LysR regulator and lies between the ppgL gene (PA4204), which encodes a periplasmic gluconolactonase, and, in the opposite orientation, the PA4202 (nmoA) gene, coding for a nitronate monooxygenase, and ddlA (PA4201), encoding a d-alanine alanine ligase. The intergenic regions between PA4203 and ppgL and between PA4203 and nmoA are very short (79 and 107 nucleotides, respectively). Here we show that PA4203 (nmoR) represses its own transcription and the expression of nmoA. A chromatin immunoprecipitation analysis showed the presence of a single NmoR binding site between nmoA and nmoR, which was confirmed by electrophoretic mobility shift assays (EMSAs) with the purified NmoR protein. Despite this observation, a transcriptome analysis revealed more genes to be affected in an nmoR mutant, including genes known to be part of the MexT LysR activator regulon. The PA1225 gene, encoding a quinone oxidoreductase, was the most highly upregulated gene in the nmoR deletion mutant, independently of MexT. Finally, deletion of the nmoA gene resulted in an increased sensitivity of the cells to 3-nitropropionic acid (3-NPA), confirming the role of the nitronate monooxygenase protein in the detoxification of nitronate. PMID:25384477

  8. 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 Central

    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. PMID:24250417

  9. 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. PMID:24714576

  10. Gene Therapy

    PubMed Central

    Baum, Bruce J

    2014-01-01

    Applications of gene therapy have been evaluated in virtually every oral tissue, and many of these have proved successful at least in animal models. While gene therapy will not be used routinely in the next decade, practitioners of oral medicine should be aware of the potential of this novel type of treatment that doubtless will benefit many patients with oral diseases. PMID:24372817

  11. Trichoderma genes

    DOEpatents

    Foreman, Pamela (Los Altos, CA); Goedegebuur, Frits (Vlaardingen, NL); Van Solingen, Pieter (Naaldwijk, NL); Ward, Michael (San Francisco, CA)

    2012-06-19

    Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

  12. Gene Identification

    NSDL National Science Digital Library

    Chuck Daniels

    This module will examine the "Language" of genes and illustrate how basic statistical methods can be applied to the problem of gene prediction. The merger of computational sciences with biology, and the challenges facing BioinFormatics, will also be explored through the use of analysis tools available at the National Center for Biotechnology InFormation (NCBI).

  13. Gene Control

    NSDL National Science Digital Library

    WGBH Educational Foundation

    2003-09-26

    The development of creatures that appear to have nothing in common is directed by a surprisingly small number of genes. In this video segment, learn about the power of master control genes. Footage from The Secret of Life: Birth, Sex & Death.

  14. Molecular characterization of berberine bridge enzyme genes from opium poppy.

    PubMed Central

    Facchini, P J; Penzes, C; Johnson, A G; Bull, D

    1996-01-01

    In Papaver somniferum (opium poppy) and related species, (S)-reticuline serves as a branch-point intermediate in the biosynthesis of numerous isoquinoline alkaloids. The berberine bridge enzyme (BBE) ([S]-reticuline:oxygen oxidoreductase [methylene bridge forming], EC 1.5.3.9) catalyzes the stereospecific conversion of the N-methyl moiety of (S)-reticuline into the berberine bridge carbon of (S)-scoulerine and represents the first committed step in the pathway leading to the antimicrobial alkaloid sanguinarine. Three unique genomic clones (bbe1, bbe2, and bbe3) similar to a BBE cDNA from Eschscholtzia californica (California poppy) were isolated from opium poppy. Two clones (bbe2 and bbe3) contained frame-shift mutations of which bbe2 was identified as a putative, nonexpressed pseudogene by RNA blot hybridization using a gene-specific probe and by the lack of transient expression of a chimeric gene fusion between the bbe2 5' flanking region and a beta-glucuronidase reporter gene. Similarly, bbe1 was shown to be expressed in opium poppy plants and cultured cells. Genomic DNA blot-hybridization data were consistent with a limited number of bbe homologs. RNA blot hybridization showed that bbe genes are expressed in roots and stems of mature plants and in seedlings within 3 d after germination. Rapid and transient BBE mRNA accumulation also occurred after treatment with a fungal elicitor or with methyl jasmonate. However, sanguinarine was found only in roots, seedlings, and fungal elicitor-treated cell cultures. PMID:8972604

  15. Metabolic gene variants associated with chromosomal aberrations in healthy humans.

    PubMed

    Hemminki, Kari; Frank, Christoph; Försti, Asta; Musak, Ludovit; Kazimirova, Alena; Barancokova, Magdalena; Horska, Alexandra; Vymetalkova, Veronika; Smerhovsky, Zdenek; Naccarati, Alessio; Soucek, Pavel; Vodickova, Ludmila; Buchancova, Janka; Smolkova, Bozena; Dusinska, Maria; Vodicka, Pavel

    2015-04-01

    Nonspecific chromosomal aberrations (CAs) are found in about 1% of lymphocytes drawn from healthy individuals. They include chromosome-type aberrations (CSAs), which are increased in exposure to ionizing radiation, and chromatid-type aberrations (CTAs) which in experimental systems are formed by DNA binding carcinogens and mutagens. The frequency of CAs is associated with the risk of cancer, but the causes of CAs in general population are unknown. Here, we want to test whether variants in metabolic genes associate with CAs in healthy volunteers. Cases were considered those whose total CA (CAtot) frequency was >2% and for CSA and CTA the limit was >1%. Controls had lower frequencies of CAs. Functional polymorphisms in seven genes were selected for analysis: cytochrome P450 1B1 (CYP1B1), epoxide hydrolase 1 (EPHX1), NAD(P)H:quinone oxidoreductase 1 (NQO1), each coding for phase 1 enzymes, and glutathione S-transferase P1 (GSTP1), glutathione S-transferases M1 (GSTM1) and T1 (GSTT1), coding for enzymes which conjugate reactive metabolites, that is, phase 2 enzymes. The number of volunteers genotyped for each gene varied from 550 to 1,500. Only EPHX1 was individually associated with CAtot; high activity genotypes decreased CAtot. A total of six significant (P < 0.01) pair-wise interactions were observed, most including a GST variant as one of the pair. In all genotype combinations with significant odds ratios for CAs a GST variant was involved. The present data provide evidence that variants in genes coding for metabolic enzymes, which individually have small effects, interact and are associated with CA frequencies in peripheral lymphocytes of healthy volunteers. PMID:25622915

  16. A novel whole exon deletion in WWOX gene causes early epilepsy, intellectual disability and optic atrophy.

    PubMed

    Ben-Salem, Salma; Al-Shamsi, Aisha M; John, Anne; Ali, Bassam R; Al-Gazali, Lihadh

    2015-05-01

    Recent studies have implicated the WW domain-containing oxidoreductase encoding gene (WWOX) in a severe form of autosomal recessive neurological disorder. This condition showed an overlapping spectrum of clinical features including spinocerebellar ataxia associated with generalized seizures and delayed psychomotor development to growth retardation, spasticity, and microcephaly. We evaluated a child from a consanguineous Emirati family that presented at birth with growth retardation, microcephaly, epileptic seizures, and later developed spasticity and delayed psychomotor development. Screening for deletions and duplications using whole-chromosomal microarray analysis identified a novel homozygous microdeletion encompassing exon 5 of the WWOX gene. Analysis of parental DNA indicated that this deletion was inherited from both parents and lies within a large region of homozygosity. Sanger sequencing of the cDNA showed that the deletion resulted in exon 5 skipping leading to a frame-shift and creating a premature stop codon at amino acid position 212. Quantification of mRNA revealed striking low level of WWOX expression in the child and moderate level of expression in the mother compared to a healthy control. To the best of our knowledge, this is the first homozygous germline structural variation in WWOX gene resulting in truncated transcripts that were presumably subject to NMD pathway. Our findings extend the clinical and genetic spectrum of WWOX mutations and support a crucial role of this gene in neurological development. PMID:25403906

  17. Transcriptome analysis of integument differentially expressed genes in the pigment mutant (quail) during molting of silkworm, Bombyx mori.

    PubMed

    Nie, Hongyi; Liu, Chun; Cheng, Tingcai; Li, Qiongyan; Wu, Yuqian; Zhou, Mengting; Zhang, Yinxia; Xia, Qingyou

    2014-01-01

    In the silkworm Bombyx mori, pigment mutants with diverse body colors have been maintained throughout domestication for about 5000 years. The silkworm larval body color is formed through the mutual interaction of melanin, ommochromes, pteridines and uric acid. These pigments/compounds are synthesized by the cooperative action of various genes and enzymes. Previous reports showed that melanin, ommochrome and pteridine are increased in silkworm quail (q) mutants. To understand the pigment increase and alterations in pigment synthesis in q mutant, transcriptome profiles of the silkworm integument were investigated at 16 h after head capsule slippage in the fourth molt in q mutants and wild-type (Dazao). Compared to the wild-type, 1161 genes were differentially expressed in the q mutant. Of these modulated genes, 62.4% (725 genes) were upregulated and 37.6% (436 genes) were downregulated in the q mutant. The molecular function of differently expressed genes was analyzed by Blast2GO. The results showed that upregulated genes were mainly involved in protein binding, small molecule binding, transferase activity, nucleic acid binding, specific DNA-binding transcription factor activity and chromatin binding, while exclusively down-expressed genes functioned in oxidoreductase activity, cofactor binding, tetrapyrrole binding, peroxidase activity and pigment binding. We focused on genes related to melanin, pteridine and ommochrome biosynthesis; transport of uric acid; and juvenile hormone metabolism because of their importance in integument coloration during molting. This study identified differently expressed genes implicated in silkworm integument formation and pigmentation using silkworm q mutant. The results estimated the number and types of genes that drive new integument formation. PMID:24718369

  18. Gene Cloning

    NSDL National Science Digital Library

    WGBH Educational Foundation

    2009-12-07

    This interactive activity adapted from the University of Nebraska's Library of Crop Technologies details the steps involved in producing clones of genes that can then be used to transform the characteristics of an organism.

  19. Transcriptomic analysis of Arabidopsis developing stems: a close-up on cell wall genes

    PubMed Central

    Minic, Zoran; Jamet, Elisabeth; San-Clemente, Hélène; Pelletier, Sandra; Renou, Jean-Pierre; Rihouey, Christophe; Okinyo, Denis PO; Proux, Caroline; Lerouge, Patrice; Jouanin, Lise

    2009-01-01

    Background Different strategies (genetics, biochemistry, and proteomics) can be used to study proteins involved in cell biogenesis. The availability of the complete sequences of several plant genomes allowed the development of transcriptomic studies. Although the expression patterns of some Arabidopsis thaliana genes involved in cell wall biogenesis were identified at different physiological stages, detailed microarray analysis of plant cell wall genes has not been performed on any plant tissues. Using transcriptomic and bioinformatic tools, we studied the regulation of cell wall genes in Arabidopsis stems, i.e. genes encoding proteins involved in cell wall biogenesis and genes encoding secreted proteins. Results Transcriptomic analyses of stems were performed at three different developmental stages, i.e., young stems, intermediate stage, and mature stems. Many genes involved in the synthesis of cell wall components such as polysaccharides and monolignols were identified. A total of 345 genes encoding predicted secreted proteins with moderate or high level of transcripts were analyzed in details. The encoded proteins were distributed into 8 classes, based on the presence of predicted functional domains. Proteins acting on carbohydrates and proteins of unknown function constituted the two most abundant classes. Other proteins were proteases, oxido-reductases, proteins with interacting domains, proteins involved in signalling, and structural proteins. Particularly high levels of expression were established for genes encoding pectin methylesterases, germin-like proteins, arabinogalactan proteins, fasciclin-like arabinogalactan proteins, and structural proteins. Finally, the results of this transcriptomic analyses were compared with those obtained through a cell wall proteomic analysis from the same material. Only a small proportion of genes identified by previous proteomic analyses were identified by transcriptomics. Conversely, only a few proteins encoded by genes having moderate or high level of transcripts were identified by proteomics. Conclusion Analysis of the genes predicted to encode cell wall proteins revealed that about 345 genes had moderate or high levels of transcripts. Among them, we identified many new genes possibly involved in cell wall biogenesis. The discrepancies observed between results of this transcriptomic study and a previous proteomic study on the same material revealed post-transcriptional mechanisms of regulation of expression of genes encoding cell wall proteins. PMID:19149885

  20. Temperature and Development Impacts on Housekeeping Gene Expression in Cowpea Aphid, Aphis craccivora (Hemiptera: Aphidiae)

    PubMed Central

    Liu, Yong; Zhou, Xuguo

    2015-01-01

    Quantitative real-time PCR (qRT-PCR) is a powerful technique to quantify gene expression. To standardize gene expression studies and obtain more accurate qRT-PCR analysis, normalization relative to consistently expressed housekeeping genes (HKGs) is required. In this study, ten candidate HKGs including elongation factor 1 ? (EF1A), ribosomal protein L11 (RPL11), ribosomal protein L14 (RPL14), ribosomal protein S8 (RPS8), ribosomal protein S23 (RPS23), NADH-ubiquinone oxidoreductase (NADH), vacuolar-type H+-ATPase (ATPase), heat shock protein 70 (HSP70), 18S ribosomal RNA (18S), and 12S ribosomal RNA (12S) from the cowpea aphid, Aphis craccivora Koch were selected. Four algorithms, geNorm, Normfinder, BestKeeper, and the ?Ct method were employed to evaluate the expression profiles of these HKGs as endogenous controls across different developmental stages and temperature regimes. Based on RefFinder, which integrates all four analytical algorithms to compare and rank the candidate HKGs, RPS8, RPL14, and RPL11 were the three most stable HKGs across different developmental stages and temperature conditions. This study is the first step to establish a standardized qRT-PCR analysis in A. craccivora following the MIQE guideline. Results from this study lay a foundation for the genomics and functional genomics research in this sap-sucking insect pest with substantial economic impact. PMID:26090683

  1. Cloning and sequencing of Coxiella burnetii outer membrane protein gene com1.

    PubMed Central

    Hendrix, L R; Mallavia, L P; Samuel, J E

    1993-01-01

    The gene for an approximately 27-kDa outer membrane-associated, immunoreactive protein was cloned from the rickettsial pathogen Coxiella burnetii. The gene, designated com1 for Coxiella outer membrane protein 1, was expressed in Escherichia coli, presumably by its own promoter. The complete nucleotide sequence of the gene was determined. The deduced amino acid sequence of 252 residues includes a putative leader sequence. The leader sequence is recognized in and removed by E. coli on the basis of the difference in the molecular mass of the protein produced in an in vitro transcription-translation system (27.6 kDa) and that of the protein immunoprecipitated from an iodinated E. coli clone (25.7 kDa). The Com1 protein expressed in E. coli was proteinase K sensitive in nondisrupted cells and soluble in 1% Sarkosyl, suggesting a loose association with the outer membrane. While the complete predicted sequence of the Com1 protein does not show any overall similarity to those of previously described proteins, a region which includes the only two cysteines in Com1 is homologous to the catalytic site of protein disulfide oxidoreductases. Images PMID:8423075

  2. Genome-Wide Functional Profiling Reveals Genes Required for Tolerance to Benzene Metabolites in Yeast

    PubMed Central

    North, Matthew; Tandon, Vickram J.; Thomas, Reuben; Loguinov, Alex; Gerlovina, Inna; Hubbard, Alan E.; Zhang, Luoping; Smith, Martyn T.; Vulpe, Chris D.

    2011-01-01

    Benzene is a ubiquitous environmental contaminant and is widely used in industry. Exposure to benzene causes a number of serious health problems, including blood disorders and leukemia. Benzene undergoes complex metabolism in humans, making mechanistic determination of benzene toxicity difficult. We used a functional genomics approach to identify the genes that modulate the cellular toxicity of three of the phenolic metabolites of benzene, hydroquinone (HQ), catechol (CAT) and 1,2,4-benzenetriol (BT), in the model eukaryote Saccharomyces cerevisiae. Benzene metabolites generate oxidative and cytoskeletal stress, and tolerance requires correct regulation of iron homeostasis and the vacuolar ATPase. We have identified a conserved bZIP transcription factor, Yap3p, as important for a HQ-specific response pathway, as well as two genes that encode putative NAD(P)H:quinone oxidoreductases, PST2 and YCP4. Many of the yeast genes identified have human orthologs that may modulate human benzene toxicity in a similar manner and could play a role in benzene exposure-related disease. PMID:21912624

  3. Genome-wide functional profiling reveals genes required for tolerance to benzene metabolites in yeast.

    PubMed

    North, Matthew; Tandon, Vickram J; Thomas, Reuben; Loguinov, Alex; Gerlovina, Inna; Hubbard, Alan E; Zhang, Luoping; Smith, Martyn T; Vulpe, Chris D

    2011-01-01

    Benzene is a ubiquitous environmental contaminant and is widely used in industry. Exposure to benzene causes a number of serious health problems, including blood disorders and leukemia. Benzene undergoes complex metabolism in humans, making mechanistic determination of benzene toxicity difficult. We used a functional genomics approach to identify the genes that modulate the cellular toxicity of three of the phenolic metabolites of benzene, hydroquinone (HQ), catechol (CAT) and 1,2,4-benzenetriol (BT), in the model eukaryote Saccharomyces cerevisiae. Benzene metabolites generate oxidative and cytoskeletal stress, and tolerance requires correct regulation of iron homeostasis and the vacuolar ATPase. We have identified a conserved bZIP transcription factor, Yap3p, as important for a HQ-specific response pathway, as well as two genes that encode putative NAD(P)H:quinone oxidoreductases, PST2 and YCP4. Many of the yeast genes identified have human orthologs that may modulate human benzene toxicity in a similar manner and could play a role in benzene exposure-related disease. PMID:21912624

  4. In vitro study of genes and molecular pathways differentially regulated by synchrotron microbeam radiotherapy.

    PubMed

    Yang, Yuqing; Crosbie, Jeffrey C; Paiva, Premila; Ibahim, Mohammad; Stevenson, Andrew; Rogers, Peter A W

    2014-12-01

    The aim of this study was to identify genes and molecular pathways differentially regulated by synchrotron-generated microbeam radiotherapy (MRT) versus conventional broadbeam radiotherapy (CRT) in vitro using cultured EMT6.5 cells. We hypothesized (based on previous findings) that gene expression and molecular pathway changes after MRT are different from those seen after CRT. We found that at 24 h postirradiation, MRT exerts a broader regulatory effect on multiple pathways than CRT. MRT regulated those pathways involved in gene transcription, translation initiation, macromolecule metabolism, oxidoreductase activity and signaling transduction in a different manner compared to CRT. We also found that MRT/CRT alone, or when combined with inflammatory factor lipopolysaccharide, upregulated expression of Ccl2, Ccl5 or Csf2, which are involved in host immune cell recruitment. Our findings demonstrated differences in the molecular pathway for MRT versus CRT in the cultured tumor cells, and were consistent with the idea that radiation plays a role in recruiting tumor-associated immune cells to the tumor. Our results also suggest that a combination of MRT/CRT with a treatment targeting CCL2 or CSF2 could repress the tumor-associated immune cell recruitment, delay tumor growth and/or metastasis and yield better tumor control than radiation alone. PMID:25409126

  5. Analyses of genome architecture and gene expression reveal novel candidate virulence factors in the secretome of Phytophthora infestans

    PubMed Central

    2010-01-01

    Background Phytophthora infestans is the most devastating pathogen of potato and a model organism for the oomycetes. It exhibits high evolutionary potential and rapidly adapts to host plants. The P. infestans genome experienced a repeat-driven expansion relative to the genomes of Phytophthora sojae and Phytophthora ramorum and shows a discontinuous distribution of gene density. Effector genes, such as members of the RXLR and Crinkler (CRN) families, localize to expanded, repeat-rich and gene-sparse regions of the genome. This distinct genomic environment is thought to contribute to genome plasticity and host adaptation. Results We used in silico approaches to predict and describe the repertoire of P. infestans secreted proteins (the secretome). We defined the "plastic secretome" as a subset of the genome that (i) encodes predicted secreted proteins, (ii) is excluded from genome segments orthologous to the P. sojae and P. ramorum genomes and (iii) is encoded by genes residing in gene sparse regions of P. infestans genome. Although including only ~3% of P. infestans genes, the plastic secretome contains ~62% of known effector genes and shows >2 fold enrichment in genes induced in planta. We highlight 19 plastic secretome genes induced in planta but distinct from previously described effectors. This list includes a trypsin-like serine protease, secreted oxidoreductases, small cysteine-rich proteins and repeat containing proteins that we propose to be novel candidate virulence factors. Conclusions This work revealed a remarkably diverse plastic secretome. It illustrates the value of combining genome architecture with comparative genomics to identify novel candidate virulence factors from pathogen genomes. PMID:21080964

  6. Inhibition of protein translation by the DISC1-Boymaw fusion gene from a Scottish family with major psychiatric disorders.

    PubMed

    Ji, Baohu; Higa, Kerin K; Kim, Minjung; Zhou, Lynn; Young, Jared W; Geyer, Mark A; Zhou, Xianjin

    2014-11-01

    The t(1; 11) translocation appears to be the causal genetic lesion with 70% penetrance for schizophrenia, major depression and other psychiatric disorders in a Scottish family. Molecular studies identified the disruption of the disrupted-in-schizophrenia 1 (DISC1) gene by chromosome translocation at chromosome 1q42. Our previous studies, however, revealed that the translocation also disrupted another gene, Boymaw (also termed DISC1FP1), on chromosome 11. After translocation, two fusion genes [the DISC1-Boymaw (DB7) and the Boymaw-DISC1 (BD13)] are generated between the DISC1 and Boymaw genes. In the present study, we report that expression of the DB7 fusion gene inhibits both intracellular NADH oxidoreductase activities and protein translation. We generated humanized DISC1-Boymaw mice with gene targeting to examine the in vivo functions of the fusion genes. Consistent with the in vitro studies on the DB7 fusion gene, protein translation activity is decreased in the hippocampus and in cultured primary neurons from the brains of the humanized mice. Expression of Gad67, Nmdar1 and Psd95 proteins are also reduced. The humanized mice display prolonged and increased responses to the NMDA receptor antagonist, ketamine, on various mouse genetic backgrounds. Abnormal information processing of acoustic startle and depressive-like behaviors are also observed. In addition, the humanized mice display abnormal erythropoiesis, which was reported to associate with depression in humans. Expression of the DB7 fusion gene may reduce protein translation to impair brain functions and thereby contribute to the pathogenesis of major psychiatric disorders. PMID:24908665

  7. The organization of the fuc regulon specifying L-fucose dissimilation in Escherichia coli K12 as determined by gene cloning.

    PubMed

    Chen, Y M; Zhu, Y; Lin, E C

    1987-12-01

    In Escherichia coli the six known genes specifying the utilization of L-fucose as carbon and energy source cluster at 60.2 min and constitute a regulon. These genes include fucP (encoding L-fucose permease), fucI (encoding L-fucose isomerase), fucK (encoding L-fuculose kinase), fucA (encoding L-fuculose 1-phosphate aldolase), fucO (encoding L-1,2-propanediol oxidoreductase), and fucR (encoding the regulatory protein). In this study the fuc genes were cloned and their positions on the chromosome were established by restriction endonuclease and complementation analyses. Clockwise, the gene order is: fucO-fucA-fucP-fucI-fucK-fucR. The operons comprising the structural genes and the direction of transcription were determined by complementation analysis and Southern blot hybridization. The fucPIK and fucA operons are transcribed clockwise. The fucO operon is transcribed counterclockwise. The fucR gene product activates the three structural operons in trans. PMID:3325779

  8. Gene and protein expression profiles of Shewanella oneidensis during anaerobic growth with different electron acceptors.

    SciTech Connect

    Beliaev, A. S.; Thompson, D. K.; Khare, T.; Lim, H.; Brandt, C. C.; Li, G.; Murray, A. E.; Heidelberg, J. F.; Giometti, C. S.; Yates, J., III; Nealson, K. H.; Tiedje, J. M.; Zhou, J.; Biosciences Division; ORNL; Scripps Research Inst.; Michigan State Univ.; The Inst. for Genomic Research; Jet Propulsion Laboratory; California Inst. of Tech.

    2002-01-01

    Changes in mRNA and protein expression profiles of Shewanella oneidenesis MR-1 during switch from aerobic to fumarate-, Fe(III)-, or nitrate-reducing conditions were examined using DNA microarrays and two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). In response to changes in growth conditions, 121 of the 691 arrayed genes displayed at least a two-fold difference in transcript abundance as determined by microarray analysis. Genes involved in aerobic respiration encoding cytochrome c and d oxidases and TCA cycle enzymes were repressed under anaerobic conditions. Genes induced during anaerobic respiration included those involved in cofactor biosynthesis and assembly (moaACE, ccmHF, nosD, cysG), substrate transport (cysUP, cysTWA, dcuB), and anaerobic energy metabolism (dmsAB, psrC, pshA, hyaABC, hydA). Transcription of genes encoding a periplasmic nitrate reductase (napBHGA), cytochrome c{sub 552}, and prismane was elevated 8- to 56-fold in response to the presence of nitrate, while cymA, ifcA, and frdA were specifically induced three- to eightfold under fumarate-reducing conditions. The mRNA levels for two oxidoreductase-like genes of unknown function and several cell envelope genes involved in multidrug resistance increased two- to fivefold specifically under Fe(III)-reducing conditions. Analysis of protein expression profiles under aerobic and anaerobic conditions revealed 14 protein spots that showed significant differences in abundance on 2-D gels. Protein identification by mass spectrometry indicated that the expression of prismane, dihydrolipoamide succinyltransferase, and alcaligin siderophore biosynthesis protein correlated with the microarray data.

  9. Vulnerability genes or plasticity genes?

    PubMed Central

    Belsky, J; Jonassaint, C; Pluess, M; Stanton, M; Brummett, B; Williams, R

    2009-01-01

    The classic diathesis–stress framework, which views some individuals as particularly vulnerable to adversity, informs virtually all psychiatric research on behavior–gene–environment (G × E) interaction. An alternative framework of ‘differential susceptibility' is proposed, one which regards those most susceptible to adversity because of their genetic make up as simultaneously most likely to benefit from supportive or enriching experiences—or even just the absence of adversity. Recent G × E findings consistent with this perspective and involving monoamine oxidase-A, 5-HTTLPR (5-hydroxytryptamine-linked polymorphic region polymorphism) and dopamine receptor D4 (DRD4) are reviewed for illustrative purposes. Results considered suggest that putative ‘vulnerability genes' or ‘risk alleles' might, at times, be more appropriately conceptualized as ‘plasticity genes', because they seem to make individuals more susceptible to environmental influences—for better and for worse. PMID:19455150

  10. Expression of FRA16D/WWOX and FRA3B/FHIT genes in hematopoietic malignancies.

    PubMed

    Ishii, Hideshi; Vecchione, Andrea; Furukawa, Yutaka; Sutheesophon, Krittaya; Han, Shuang-Yin; Druck, Teresa; Kuroki, Tamotsu; Trapasso, Francesco; Nishimura, Miki; Saito, Yasushi; Ozawa, Keiya; Croce, Carlo M; Huebner, Kay; Furukawa, Yusuke

    2003-11-01

    The WW domain containing oxidoreductase (WWOX) gene was recently identified as a candidate tumor suppressor gene at a common fragile site, FRA16D. Because the fragile histidine triad (FHIT) gene, a tumor suppressor gene encompassing the most active, common fragile site FRA3B, is frequently deleted in various cancers, we evaluated the expression of WWOX and FHIT in 74 cases of primary hematopoietic neoplasias and 20 leukemia cell lines. Aberration or absence of WWOX transcripts was detected in 51% of the primary cases and 55% of cell lines, and three WWOX nucleotide variants were detected among the leukemia cell lines. FHIT expression was absent or altered in 36% of the primary cases and 15% of cell lines. The occurrence of aberrant FHIT reverse transcription-PCR products correlated significantly with the occurrence of WWOX alterations. Wild-type transcripts of both genes were expressed in normal hematopoiesis along with a small fraction of short transcripts. A DNA blot study showed that WWOX and FHIT genes were deleted in 2 of 18 cases with primary acute leukemias; both genes were not expressed in the 2 cases. Furthermore, treatment of cells with a demethylating or histone acetylating agent in culture resulted in increased expression of WWOX and FHIT mRNA in leukemia cells. Conclusions are that WWOX expression is frequently altered or absent in hematopoietic disorders, often in association with FHIT alterations, and that alterations of these fragile genes may result not only from genomic deletions but also from epigenetic modifications associated with expression of fragility. PMID:14638866

  11. A mechanistic study on SMOB-ADP1: an NADH:flavin oxidoreductase of the two-component styrene monooxygenase of Acinetobacter baylyi ADP1.

    PubMed

    Gröning, Janosch A D; Kaschabek, Stefan R; Schlömann, Michael; Tischler, Dirk

    2014-12-01

    Two styrene monooxygenase types, StyA/StyB and StyA1/StyA2B, have been described each consisting of an epoxidase and a reductase. A gene fusion which led to the chimeric reductase StyA2B and the occurrence in different phyla are major differences. Identification of SMOA/SMOB-ADP1 of Acinetobacter baylyi ADP1 may enlighten the gene fusion event since phylogenetic analysis indicated both proteins to be more related to StyA2B than to StyA/StyB. SMOB-ADP1 is classified like StyB and StyA2B as HpaC-like reductase. Substrate affinity and turnover number of the homo-dimer SMOB-ADP1 were determined for NADH (24 µM, 64 s(-1)) and FAD (4.4 µM, 56 s(-1)). SMOB-ADP1 catalysis follows a random sequential mechanism, and FAD fluorescence is quenched upon binding to SMOB-ADP1 (K d = 1.8 µM), which clearly distinguishes that reductase from StyB of Pseudomonas. In summary, this study confirmes made assumptions and provides phylogenetic and biochemical data for the differentiation of styrene monooxygenase-related flavin reductases. PMID:25116410

  12. c-Fos proteasomal degradation is activated by a default mechanism, and its regulation by NAD(P)H:quinone oxidoreductase 1 determines c-Fos serum response kinetics.

    PubMed

    Adler, Julia; Reuven, Nina; Kahana, Chaim; Shaul, Yosef

    2010-08-01

    The short-lived proto-oncoprotein c-Fos is a component of the activator protein 1 (AP-1) transcription factor. A large region of c-Fos is intrinsically unstructured and susceptible to a recently characterized proteasomal ubiquitin-independent degradation (UID) pathway. UID is active by a default mechanism that is inhibited by NAD(P)H:quinone oxidoreductase 1 (NQO1), a 20S proteasome gatekeeper. Here, we show that NQO1 binds and induces robust c-Fos accumulation by blocking the UID pathway. c-Jun, a partner of c-Fos, also protects c-Fos from proteasomal degradation by default. Our findings suggest that NQO1 protects monomeric c-Fos from proteasomal UID, a function that is fulfilled later by c-Jun. We show that this process regulates c-Fos homeostasis (proteostasis) in response to serum stimulation, phosphorylation, nuclear translocation, and transcription activity. In addition, we show that NQO1 is important to ensure immediate c-Fos accumulation in response to serum, since a delayed response was observed under low NQO1 expression. These data suggest that in vivo, protein unstructured regions determine the kinetics and the homeostasis of regulatory proteins. Our data provide evidence for another layer of regulation of key regulatory proteins that functions at the level of protein degradation and is designed to ensure optimal formation of functional complexes such as AP-1. PMID:20498278

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

    Microsoft Academic Search

    AKIO TANI; YASUYOSHI SAKAI; TAKERU ISHIGE; NOBUO KATO

    2000-01-01

    NADPH-dependent alkylaldehyde reducing enzyme, which was greatly induced by n-hexadecane, from Acin- etobacter sp. strain M-1 was purified and characterized. The purified enzyme had molecular masses of 40 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 160 kDa as determined by gel filtration chromatography. The enzyme, which was shown to be highly thermostable, was most active toward n-heptanal

  14. Characterization of the Community Structure of a Dechlorinating Mixed Culture and Comparisons of Gene Expression in Planktonic and Biofloc-Associated “Dehalococcoides” and Methanospirillum Species? †

    PubMed Central

    Rowe, Annette R.; Lazar, Brendan J.; Morris, Robert M.; Richardson, Ruth E.

    2008-01-01

    This study sought to characterize bacterial and archaeal populations in a perchloroethene- and butyrate-fed enrichment culture containing hydrogen-consuming “Dehalococcoides ethenogenes” strain 195 and a Methanospirillum hungatei strain. Phylogenetic characterization of this microbial community was done via 16S rRNA gene clone library and gradient gel electrophoresis analyses. Fluorescence in situ hybridization was used to quantify populations of “Dehalococcoides” and Archaea and to examine the colocalization of these two groups within culture bioflocs. A technique for enrichment of planktonic and biofloc-associated biomass was developed and used to assess differences in population distribution and gene expression patterns following provision of substrate. On a per-milliliter-of-culture basis, most D. ethenogenes genes (the hydrogenase gene hupL; the highly expressed gene for an oxidoreductase of unknown function, fdhA; the RNA polymerase subunit gene rpoB; and the 16S rRNA gene) showed no statistical difference in expression between planktonic and biofloc enrichments at either time point studied (1 to 2 and 6 h postfeeding). Normalization of transcripts to ribosome (16S rRNA) levels supported that planktonic and biofloc-associated D. ethenogenes had similar gene expression profiles, with one notable exception; planktonic D. ethenogenes showed higher expression of tceA relative to biofloc-associated cells at 6 h postfeeding. These trends were compared to those for the hydrogen-consuming methanogen in the culture, M. hungatei. The vast majority of M. hungatei cells, ribosomes (16S rRNA), and transcripts of the hydrogenase gene mvrD and the housekeeping gene rpoE were observed in the biofloc enrichments. This suggests that, unlike the comparable activity of D. ethenogenes from both enrichments, planktonic M. hungatei is responsible for only a small fraction of the hydrogenotrophic methanogenesis in this culture. PMID:18776027

  15. Characterization of the community structure of a dechlorinating mixed culture and comparisons of gene expression in planktonic and biofloc-associated "Dehalococcoides" and Methanospirillum species.

    PubMed

    Rowe, Annette R; Lazar, Brendan J; Morris, Robert M; Richardson, Ruth E

    2008-11-01

    This study sought to characterize bacterial and archaeal populations in a perchloroethene- and butyrate-fed enrichment culture containing hydrogen-consuming "Dehalococcoides ethenogenes" strain 195 and a Methanospirillum hungatei strain. Phylogenetic characterization of this microbial community was done via 16S rRNA gene clone library and gradient gel electrophoresis analyses. Fluorescence in situ hybridization was used to quantify populations of "Dehalococcoides" and Archaea and to examine the colocalization of these two groups within culture bioflocs. A technique for enrichment of planktonic and biofloc-associated biomass was developed and used to assess differences in population distribution and gene expression patterns following provision of substrate. On a per-milliliter-of-culture basis, most D. ethenogenes genes (the hydrogenase gene hupL; the highly expressed gene for an oxidoreductase of unknown function, fdhA; the RNA polymerase subunit gene rpoB; and the 16S rRNA gene) showed no statistical difference in expression between planktonic and biofloc enrichments at either time point studied (1 to 2 and 6 h postfeeding). Normalization of transcripts to ribosome (16S rRNA) levels supported that planktonic and biofloc-associated D. ethenogenes had similar gene expression profiles, with one notable exception; planktonic D. ethenogenes showed higher expression of tceA relative to biofloc-associated cells at 6 h postfeeding. These trends were compared to those for the hydrogen-consuming methanogen in the culture, M. hungatei. The vast majority of M. hungatei cells, ribosomes (16S rRNA), and transcripts of the hydrogenase gene mvrD and the housekeeping gene rpoE were observed in the biofloc enrichments. This suggests that, unlike the comparable activity of D. ethenogenes from both enrichments, planktonic M. hungatei is responsible for only a small fraction of the hydrogenotrophic methanogenesis in this culture. PMID:18776027

  16. Residence of habitat-specific anammox bacteria in the deep-sea subsurface sediments of the South China Sea: analyses of marker gene abundance with physical chemical parameters.

    PubMed

    Hong, Yi-Guo; Li, Meng; Cao, Huiluo; Gu, Ji-Dong

    2011-07-01

    Anaerobic ammonium oxidation (anammox) has been recognized as an important process for the global nitrogen cycle. In this study, the occurrence and diversity of anammox bacteria in the deep-sea subsurface sediments of the South China Sea (SCS) were investigated. Results indicated that the anammox bacterial sequences recovered from this habitat by amplifying both 16S rRNA gene and hydrazine oxidoreductase encoding hzo gene were all closely related to the Candidatus Scalindua genus. A total of 96 16S rRNA gene sequences from 346 clones were grouped into five subclusters: two subclusters affiliated with the brodae and arabica species, while three new subclusters named zhenghei-I, -II, and -III showed ? 97.4% nucleic acid sequence identity with other known Candidatus Scalindua species. Meanwhile, 88 hzo gene sequences from the sediments also formed five distant subclusters within hzo cluster 1c. Through fluorescent real-time PCR analysis, the abundance of anammox bacteria in deep-sea subsurface sediment was quantified by hzo genes, which ranged from 1.19 × 10(4) to 7.17 × 10(4) copies per gram of dry sediments. Combining all the information from this study, diverse Candidatus Scalindua anammox bacteria were found in the deep-sea subsurface sediments of the SCS, and they could be involved in the nitrogen loss from the fixed inventory in the habitat. PMID:21491114

  17. Genome-wide investigation of the genes involved in nicotine metabolism in Pseudomonas putida J5 by Tn5 transposon mutagenesis.

    PubMed

    Xia, Zhenyuan; Zhang, Wei; Lei, Liping; Liu, Xingzhong; Wei, Hai-Lei

    2015-08-01

    Pseudomonas putida J5 is an efficient nicotine-degrading bacterial strain isolated from the tobacco rhizosphere. We successfully performed a comprehensive whole-genome analysis of nicotine metabolism-associated genes by Tn5 transposon mutagenesis in P. putida J5. A total of 18 mutants with unique insertions screened from 16,324 Tn5-transformants failed to use nicotine as the sole carbon source. Flanking sequences of the Tn5 transposon were cloned with a shotgun method from all of the nicotine-growth-deficient mutants. The potentially essential products of mutated gene were classified as follows: oxidoreductases, protein and metal transport systems, proteases and peptidases, transcriptional and translational regulators, and unknown proteins. Bioinformatic analysis of the Tn5 insertion sites indicated that the nicotine metabolic genes were separated and widely distributed in the genome. One of the mutants, M2022, was a Tn5 insert into a gene encoding a homolog of 6-hydroxy-L-nicotine oxidase, the second enzyme of nicotine metabolism in Arthrobacter nicotinovorans. Genetic and biochemical analysis confirmed that three open reading frames (ORFs) from an approximately 13-kb fragment recovered from the mutant M2022 were responsible for the transformation of nicotine to 3-succinoyl-pyridine via pseudooxynicotine and 3-succinoyl semialdehyde-pyridine, the first three steps of nicotine degradation. Further research on these mutants and the Tn5-inserted genes will help us characterize nicotine metabolic processes in P. putida J5. PMID:25808517

  18. Lower citrinin production by gene disruption of ctnB involved in citrinin biosynthesis in Monascus aurantiacus Li AS3.4384.

    PubMed

    Li, Yan-Ping; Pan, Yi-Feng; Zou, Le-Hua; Xu, Yang; Huang, Zhi-Bing; He, Qing-Hua

    2013-07-31

    The filamentous fungi Monascus spp. have been used in the production of food colorants and health remedies for more than 1000 years in Asia. However, greater attention has been given to the safety of Monascus products because they contain citrinin, which is harmful to the hepatic and renal systems. The citrinin biosynthetic gene cluster has been characterized in Monasucs aurantiacus . The ctnB gene encoding an oxidoreductase is located between pksCT and ctnA. In this study, a ctnB replacement vector (pCTNB-HPH) was constructed to disrupt the ctnB gene with a hygromycin resistance gene as the selection marker. The linear vector was transformed into M. aurantiacus using the protoplast CaCl2/polyethylene glycol (PEG) method. Three ctnB-disrupted strains were obtained by homologous recombination. In comparison to the parental strain, the ?ctnB mutants barely produced citrinin. These data confirmed that the ctnB gene is directly involved in citrinin biosynthesis. Moreover, the yields of the pigments of two disruptants were similar to that of the wild-type strain, but the yield of another mutant was slightly higher than that of the latter strain. These results indicate that the production of the mycotoxin citrinin was successfully eliminated through genetic engineering. PMID:23841779

  19. Quantification of Yeast and Bacterial Gene Transcripts in Retail Cheeses by Reverse Transcription-Quantitative PCR

    PubMed Central

    Straub, Cécile; Castellote, Jessie; Onesime, Djamila; Bonnarme, Pascal; Irlinger, Françoise

    2013-01-01

    The cheese microbiota contributes to a large extent to the development of the typical color, flavor, and texture of the final product. Its composition is not well defined in most cases and varies from one cheese to another. The aim of the present study was to establish procedures for gene transcript quantification in cheeses by reverse transcription-quantitative PCR. Total RNA was extracted from five smear-ripened cheeses purchased on the retail market, using a method that does not involve prior separation of microbial cells. 16S rRNA and malate:quinone oxidoreductase gene transcripts of Corynebacterium casei, Brevibacterium aurantiacum, and Arthrobacter arilaitensis and 26S rRNA and beta tubulin gene transcripts of Geotrichum candidum and Debaryomyces hansenii could be detected and quantified in most of the samples. Three types of normalization were applied: against total RNA, against the amount of cheese, and against a reference gene. For the first two types of normalization, differences of reverse transcription efficiencies from one sample to another were taken into account by analysis of exogenous control mRNA. No good correlation was found between the abundances of target mRNA or rRNA transcripts and the viable cell concentration of the corresponding species. However, in most cases, no mRNA transcripts were detected for species that did not belong to the dominant species. The applications of gene expression measurement in cheeses containing an undefined microbiota, as well as issues concerning the strategy of normalization and the assessment of amplification specificity, are discussed. PMID:23124230

  20. Burkholderia cenocepacia ShvR-Regulated Genes That Influence Colony Morphology, Biofilm Formation, and Virulence ?

    PubMed Central

    Subramoni, Sujatha; Nguyen, David T.; Sokol, Pamela A.

    2011-01-01

    Burkholderia cenocepacia is an opportunistic pathogen that primarily infects cystic fibrosis (CF) patients. Previously, we reported that ShvR, a LysR regulator, influences colony morphology, virulence, and biofilm formation and regulates the expression of an adjacent 24-kb genomic region encoding 24 genes. In this study, we report the functional characterization of selected genes in this region. A Tn5 mutant with shiny colony morphology was identified with a polar mutation in BCAS0208, predicted to encode an acyl-coenzyme A dehydrogenase. Mutagenesis of BCAS0208 and complementation analyses revealed that BCAS0208 is required for rough colony morphology, biofilm formation, and virulence on alfalfa seedlings. It was not possible to complement with BCAS0208 containing a mutation in the catalytic site. BCAS0201, encoding a putative flavin adenine dinucleotide (FAD)-dependent oxidoreductase, and BCAS0207, encoding a putative citrate synthase, do not influence colony morphology but are required for optimum levels of biofilm formation and virulence. Both BCAS0208 and BCAS0201 contribute to pellicle formation, although individual mutations in each of these genes had no appreciable effect on pellicle formation. A mutant with a polar insertion in BCAS0208 was significantly less virulent in a rat model of chronic lung infection as well as in the alfalfa model. Genes in this region were shown to influence utilization of branched-chain fatty acids, tricarboxylic acid cycle substrates, l-arabinose, and branched-chain amino acids. Together, our data show that the ShvR-regulated genes BCAS0208 to BCAS0201 are required for the rough colony morphotype, biofilm and pellicle formation, and virulence in B. cenocepacia. PMID:21690240

  1. CDDO-Im protects from acetaminophen hepatotoxicity through induction of Nrf2-dependent genes

    SciTech Connect

    Reisman, Scott A.; Buckley, David B.; Tanaka, Yuji [Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160-7417 (United States); Klaassen, Curtis D. [Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160-7417 (United States)], E-mail: cklaasse@kumc.edu

    2009-04-01

    CDDO-Im is a synthetic triterpenoid recently shown to induce cytoprotective genes through the Nrf2-Keap1 pathway, an important mechanism for the induction of cytoprotective genes in response to oxidative stress. Upon oxidative or electrophilic insult, the transcription factor Nrf2 translocates to the nucleus, heterodimerizes with small Maf proteins, and binds to antioxidant response elements (AREs) in the upstream promoter regions of various cytoprotective genes. To further elucidate the hepatoprotective effects of CDDO-Im, wild-type and Nrf2-null mice were pretreated with CDDO-Im (1 mg/kg, i.p.) or vehicle (DMSO), and then administered acetaminophen (500 mg/kg, i.p.). Pretreatment of wild-type mice with CDDO-Im reduced liver injury caused by acetaminophen. In contrast, hepatoprotection by CDDO-Im was not observed in Nrf2-null mice. CDDO-Im increased Nrf2 protein expression and Nrf2-ARE binding in wild-type, but not Nrf2-null mice. Furthermore, CDDO-Im increased the mRNA expression of the Nrf2 target genes NAD(P)H: quinone oxidoreductase-1 (Nqo1); glutamate-cysteine ligase, catalytic subunit (Gclc); and heme-oxygenase-1 (Ho-1), in both a dose- and time-dependent manner. Conversely, CDDO-Im did not induce Nqo1, Gclc, and Ho-1 mRNA expression in Nrf2-null mice. Collectively, the present study shows that CDDO-Im pretreatment induces Nrf2-dependent cytoprotective genes and protects the liver from acetaminophen-induced hepatic injury.

  2. Gene Ontology Driven Classification of Gene

    E-print Network

    Spang, Rainer

    expression patterns Gene Ontology · Structure knowledge about genes · Directed acyclic graph · Represents · No biological knowledge #12;Introduction 23-Jul-02 5 / 17Claudio Lottaz: GO driven classification of gene knowledge on · Molecular function · Bilogical process · Cellular component · Genes are annotated to nodes

  3. Gene Expression Mural: Visualizing Gene Expression Databases

    E-print Network

    Gene Expression Mural: Visualizing Gene Expression Databases Mathew Clement, Margaret Ellis, Josh@cs.vt.edu Abstract The Gene Expression Mural is a tool designed for managing the vast amount of information produced quantifying the expression level of gene sequences in a number of conditions [1]. Software tools integrating

  4. Mutations in a new cytochrome P450 gene in lamellar ichthyosis type 3.

    PubMed

    Lefèvre, Caroline; Bouadjar, Bakar; Ferrand, Véronique; Tadini, Gianluca; Mégarbané, André; Lathrop, Mark; Prud'homme, Jean-François; Fischer, Judith

    2006-03-01

    We report the identification of mutations in a non-syndromic autosomal recessive congenital ichthyosis (ARCI) in a new gene mapping within a previously identified locus on chromosome 19p12-q12, which has been defined as LI3 in the OMIM database (MIM 604777). The phenotype usually presents as lamellar ichthyosis and hyperlinearity of palms and soles. Seven homozygous mutations including five missense mutations and two deletions were identified in a new gene, FLJ39501, on chromosome 19p12 in 21 patients from 12 consanguineous families from Algeria, France, Italy and Lebanon. FLJ39501 encodes a protein which was found to be a cytochrome P450, family 4, subfamily F, polypeptide 2 homolog of the leukotriene B4-omega-hydroxylase (CYP4F2) and could catalyze the 20-hydroxylation of trioxilin A3 from the 12(R)-lipoxygenase pathway. Further oxidation of this substrate by the fatty alcohol:nicotinamide-adenine dinucleotide oxidoreductase (FAO) enzyme complex, in which one component, ALDH3A2, is known to be mutated in Sjögren-Larsson syndrome (characterized by ichthyosis and spastic paraplegia), would lead to 20-carboxy-(R)-trioxilin A3. This compound could be involved in skin hydration and would be the essential missing product in most forms of ARCI. Its chiral homolog, 20-carboxy-(S)-trioxilin A3, could be implicated in spastic paraplegia and in the maintenance of neuronal integrity. PMID:16436457

  5. The electron transfer flavoprotein fixABCX gene products from Azospirillum brasilense show a NifA-dependent promoter regulation.

    PubMed

    Sperotto, Raul Antonio; Gross, Jeferson; Vedoy, Cleber; Passaglia, Luciane Maria Pereira; Schrank, Irene Silveira

    2004-10-01

    The complete nucleotide sequence of the A. brasilense fixA, fixB, fixC, and fixX genes is reported here. Sequence similarities between the protein sequences deduced from fixABCX genes and many electron transfer flavoproteins (ETFs) have been noted. Comparison of the amino acid sequences of both subunits of ETF with the A. brasilense fixA and fixB gene products exhibits an identity of 30%. The amino acid sequence of the other two genes, fixC and fixX, revealed similarity with the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase (ETF-QO). Using site-directed mutagenesis, mutations were introduced in the fixA promoter element of the A. brasilense fixABCX operon and chimeric p fixA-lacZ reporter gene fusions were constructed. The activation of the fixA promoter of A. brasilense is dependent upon the presence of the NifA protein being approximately 7 times less active than the A. brasilense nifH promoter. These results indicate that NifA from Klebsiella pneumoniae activates the fix promoter of A. brasilense and provide further evidence in support of the regulatory model of NifA activation in A. brasilense. Although no specific function has been assigned to the fixABCX gene products they are apparently required for symbiotic nitrogen fixation. An electron-transferring capacity in the nitrogen fixation pathway has been suggested for the fix gene products based on sequence homologies to the ETFs and ETF-QO proteins and by the absence of orthologous electron transfer proteins NifJ and NifF in A. brasilense. PMID:15386115

  6. The aryl hydrocarbon receptor and estrogen receptor alpha differentially modulate nuclear factor erythroid-2-related factor 2 transactivation in MCF-7 breast cancer cells

    SciTech Connect

    Lo, Raymond; Matthews, Jason, E-mail: jason.matthews@utoronto.ca

    2013-07-15

    Nuclear factor erythroid-2-related factor 2 (NRF2; NFE2L2) plays an important role in mediating cellular protection against reactive oxygen species. NRF2 signaling is positively modulated by the aryl hydrocarbon receptor (AHR) but inhibited by estrogen receptor alpha (ER?). In this study we investigated the crosstalk among NRF2, AHR and ER? in MCF-7 breast cancer cells treated with the NRF2 activator sulforaphane (SFN), a dual AHR and ER? activator, 3,3?-diindolylmethane (DIM), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 17?-estradiol (E2). SFN-dependent increases in NADPH-dependent oxidoreductase 1 (NQO1) and heme oxygenase I (HMOX1) mRNA levels were significantly reduced after co-treatment with E2. E2-dependent repression of NQO1 and HMOX1 was associated with increased ER? but reduced p300 recruitment and reduced histone H3 acetylation at both genes. In contrast, DIM + SFN or TCDD + SFN induced NQO1 and HMOX1 mRNA expression to levels higher than SFN alone, which was prevented by RNAi-mediated knockdown of AHR. DIM + SFN but not TCDD + SFN also induced recruitment of ER? to NQO1 and HMOX1. However, the presence of AHR at NQO1 and HMOX1 restored p300 recruitment and histone H3 acetylation, thereby reversing the ER?-dependent repression of NRF2. Taken together, our study provides further evidence of functional interplay among NRF2, AHR and ER? signaling pathways through altered p300 recruitment to NRF2-regulated target genes. - Highlights: • We examined crosstalk among ER?, AHR, and NRF2 in MCF-7 breast cancer cells. • AHR enhanced the mRNA expression levels of two NRF2 target genes – HMOX1 and NQO1. • ER? repressed HMOX1 and NQO1 expression via decreased histone acetylation. • AHR prevented ER?-dependent repression of HMOX1 and NQO1.

  7. Gene Switches

    NSDL National Science Digital Library

    2013-07-30

    In this activity, learners explore how genetic switches function and the role of genetic switches in the process of evolution. To make these concepts less abstract and more understandable, learners first view a series of video clips and animations from the HHMI DVD (or online) "Evolution: Constant Change and Common Threads." Then, learners construct a model of a gene switch using craft materials or FridgiGears (magnetic gears). This activity can be done as a demonstration, a student inquiry activity, or a combination of the two.

  8. Characterisation of the flavin-free oxygen-tolerant azoreductase from Xenophilus azovorans KF46F in comparison to flavin-containing azoreductases

    Microsoft Academic Search

    Sibylle Bürger; Andreas Stolz

    2010-01-01

    The flavin-free azoreductase from Xenophilus azovorans KF46F (AzoB), which has been the very first characterized oxygen-tolerant azoreductase, was analyzed in comparison to various\\u000a recently described flavin-containing azoreductases from different bacterial sources. Sequence comparisons demonstrated that\\u000a the azoreductase from X. azovorans KF46F is a member of the NmrA family of proteins and demonstrates 30% sequence identity with a NADPH-dependent quinone oxidoreductase

  9. Replacement of Tyr50 stacked on the si-face of the isoalloxazine ring of the flavin adenine dinucleotide prosthetic group modulates Bacillus subtilis ferredoxin-NADP(+) oxidoreductase activity toward NADPH.

    PubMed

    Seo, Daisuke; Naito, Hiroshi; Nishimura, Erika; Sakurai, Takeshi

    2015-08-01

    Ferredoxin-NAD(P)(+) oxidoreductases ([EC 1.18.1.2], [EC 1.18.1.3], FNRs) from green sulfur bacteria, purple non-sulfur bacteria and most of Firmicutes, such as Bacillus subtilis (BsFNR) are homo-dimeric flavoproteins homologous to bacterial NADPH-thioredoxin reductase. These FNRs contain two unique aromatic residues stacked on the si- and re-face of the isoalloxazine ring moiety of the FAD prosthetic group whose configurations are often found among other types of flavoproteins including plant-type FNR and flavodoxin, but not in bacterial NADPH-thioredoxin reductase. To investigate the role of the si-face Tyr50 residue in BsFNR, we replaced Tyr50 with Gly, Ser, and Trp and examined its spectroscopic properties and enzymatic activities in the presence of NADPH and ferredoxin (Fd) from B. subtilis (BsFd). The replacement of Tyr50 to Gly (Y50G), Ser (Y50S), and Trp (Y50W) in BsFNR resulted in a blue shift of the FAD transition bands. The Y50G and Y50S mutations enhanced the FAD fluorescence emission, whereas those of the wild type and Y50W mutant were quenched. All three mutants decreased thermal stabilities compared to wild type. Using a diaphorase assay, the k cat values for the Y50G and Y50S mutants in the presence of NADPH and ferricyanide were decreased to less than 5 % of the wild type activity. The Y50W mutant retained approximately 20 % reactivity in the diaphorase assay and BsFd-dependent cytochrome c reduction assay relative to wild type. The present results suggest that Tyr50 modulates the electronic properties and positioning of the prosthetic group. PMID:25698107

  10. Increased levels of NAD(P)H: quinone oxidoreductase 1 (NQO1) in pancreatic tissues from smokers and pancreatic adenocarcinomas: A potential biomarker of early damage in the pancreas.

    PubMed

    Lyn-Cook, B D; Yan-Sanders, Y; Moore, S; Taylor, S; Word, B; Hammons, G J

    2006-03-01

    NAD(P)H:quinone oxidoreductase 1 (NQO1) is elevated in several human tumors. This study was conducted to determine whether increased levels of NQO1 expression also occur in human pancreatic tumor tissue, and to compare expression levels in nontumorous tissue from smokers with those in nonsmokers. The expression of NQO1 was examined in pancreatic tissue samples from 82 human donors. These samples included normal (n = 20), smokers (n = 25), pancreatitis (n = 7), and adenocarcinomas of the pancreas (n = 30). Genotyping for the C609T polymorphism in NQO1 by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was also performed. Polymorphic variants were confirmed by automatic sequencing. Higher levels of NQO1 expression were demonstrated in pancreatic adenocarcinomas (0.831 +/- 0.021) compared to those in nontumorous tissues from nonsmokers (0.139 +/- 0.024). These high levels were also found in smokers (0.729 +/- 0.167) and in pancreatitis tissues (0.923 +/- 0.184). NQO1 activity was also higher in smokers (2.43 +/- 0.61 nmol/min per mg protein) compared to nonsmokers (0.44 +/- 0.05 nmol/min per mg protein; p < 0.05). No differences were found in genotype distribution and frequencies of the variant alleles between normal and cancer tissues in this relatively small sample pool. Seventy-five percent of the normal pancreatic tissues showed 609(C/C) and 25% 609(C/T). In pancreatic adenocarcinomas the frequency distribution was 65% C/C, 30% C/T and 5% T/T. The increased expression in noncancer pancreatic tissue from smokers and the fact that smoking is a moderate risk factor for pancreatic cancer suggest that NQO1 expression may be a good candidate as a biomarker for pancreatic cancer, especially in risk groups such as smokers. PMID:16532285

  11. 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.; (UWA); (Monash)

    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.

  12. Zinc Finger Nuclease Knock-out of NADPH:Cytochrome P450 Oxidoreductase (POR) in Human Tumor Cell Lines Demonstrates That Hypoxia-activated Prodrugs Differ in POR Dependence*

    PubMed Central

    Su, Jiechuang; Gu, Yongchuan; Pruijn, Frederik B.; Smaill, Jeff B.; Patterson, Adam V.; Guise, Christopher P.; Wilson, William R.

    2013-01-01

    Hypoxia, a ubiquitous feature of tumors, can be exploited by hypoxia-activated prodrugs (HAP) that are substrates for one-electron reduction in the absence of oxygen. NADPH:cytochrome P450 oxidoreductase (POR) is considered one of the major enzymes responsible, based on studies using purified enzyme or forced overexpression in cell lines. To examine the role of POR in HAP activation at endogenous levels of expression, POR knock-outs were generated in HCT116 and SiHa cells by targeted mutation of exon 8 using zinc finger nucleases. Absolute quantitation by proteotypic peptide mass spectrometry of DNA sequence-confirmed multiallelic mutants demonstrated expression of proteins with residual one-electron reductase activity in some clones and identified two (Hko2 from HCT116 and S2ko1 from SiHa) that were functionally null by multiple criteria. Sensitivities of the clones to 11 HAP (six nitroaromatics, three benzotriazine N-oxides, and two quinones) were compared with wild-type and POR-overexpressing cells. All except the quinones were potentiated by POR overexpression. Knocking out POR had a marked effect on antiproliferative activity of the 5-nitroquinoline SN24349 in both genetic backgrounds after anoxic exposure but little or no effect on activity of most other HAP, including the clinical stage 2-nitroimidazole mustard TH-302, dinitrobenzamide mustard PR-104A, and benzotriazine N-oxide SN30000. Clonogenic cell killing and reductive metabolism of PR-104A and SN30000 under anoxia also showed little change in the POR knock-outs. Thus, although POR expression is a potential biomarker of sensitivity to some HAP, identification of other one-electron reductases responsible for HAP activation is needed for their rational clinical development. PMID:24196959

  13. Essential Bacillus subtilis genes

    Microsoft Academic Search

    K. Kobayashi; S. D. Ehrlichb; A. Albertini; G. Amati; K. Asaig Arnaudf; M. Arnaud; K. Asai; S. Ashikaga; S. Aymerich; P. Bessieres; F. Boland; S. C. Brignell; S. Bron; K. Bunai; J. Chapuis; L. C. Christiansen; A. Danchin; M. Débarbouillé; E. Dervyn; E. Deuerling; K. Devine; S. K. Devine; O. Dreesen; J. Errington; S. Fillinger; S. J. Foster; Y. Fujita; A. Galizzi; R. Gardan; C. Eschevins; T. Fukushima; K. Haga; C. R. Harwood; M. Hecker; D. Hosoya; M. F. Hullo; H. Kakeshita; D. Karamata; Y. Kasahara; F. Kawamura; K. Koga; P. Koski; R. Kuwana; D. Imamura; M. Ishimaru; S. Ishikawa; I. Ishio; D. Le Coq; A. Masson; C. Mauël; R. Meima; R. P. Mellado; A. Moir; S. Moriya; E. Nagakawa; H. Nanamiya; S. Nakai; P. Nygaard; M. Ogura; T. Ohanan; M. O'Reilly; M. O'Rourke; Z. Pragai; H. M. Pooley; G. Rapoport; J. P. Rawlins; L. A. Rivas; C. Rivolta; A. Sadaie; Y. Sadaie; M. Sarvas; T. Sato; H. H. Saxild; E. Scanlan; W. Schumann; J. F. Seegers; J. Sekiguchi; A. Sekowska; S. J. Seror; M. Simon; P. Stragier; R. Studer; H. Takamatsu; T. Tanaka; M. Takeuchi; H. B. Thomaides; V. Vagner; J. M. van Dijl; K. Watabe; A. Wipat; H. Yamamoto; M. Yamamoto; Y. Yamamoto; K. Yamane; K. Yata; K. Yoshida; H. Yoshikawa; U. Zuber; N. Ogasawara

    2003-01-01

    To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among 4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively

  14. Identification and molecular characterization of the aco genes encoding the Pelobacter carbinolicus acetoin dehydrogenase enzyme system.

    PubMed Central

    Oppermann, F B; Steinbüchel, A

    1994-01-01

    Use of oligonucleotide probes, which were deduced from the N-terminal sequences of the purified enzyme components, identified the structural genes for the alpha and beta subunits of E1 (acetoin:2,6-dichlorophenolindophenol oxidoreductase), E2 (dihydrolipoamide acetyltransferase), and E3 (dihydrolipoamide dehydrogenase) of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system, which were designated acoA, acoB, acoC, and acoL, respectively. The nucleotide sequences of acoA (979 bp), acoB (1,014 bp), acoC (1,353 bp), and acoL (1,413 bp) as well as of acoS (933 bp), which encodes a protein with an M(r) of 34,421 exhibiting 64.7% amino acid identity to the Escherichia coli lipA gene product, were determined. These genes are clustered on a 6.1-kbp region. Heterologous expression of acoA, acoB, acoC, acoL, and acoS in E. coli was demonstrated. The amino acid sequences deduced from acoA, acoB, acoC, and acoL for E1 alpha (M(r), 34,854), E1 beta (M(r), 36,184), E2 (M(r), 47,281), and E3 (M(r), 49,394) exhibited striking similarities to the amino acid sequences of the components of the Alcaligenes eutrophus acetoin-cleaving system. Homologies of up to 48.7% amino acid identity to the primary structures of the enzyme components of various 2-oxo acid dehydrogenase complexes also were found. In addition, the respective genes of the 2-oxo acid dehydrogenase complexes and of the acetoin dehydrogenase enzyme system were organized very similarly, indicating a close relationship of the P. carbinolicus acetoin dehydrogenase enzyme system to 2-oxo acid dehydrogenase complexes. Images PMID:8110297

  15. The Evaluation of WBP2NL-Related Genes Expression in Breast Cancer.

    PubMed

    Nourashrafeddin, Seyedmehdi; Aarabi, Mahmoud; Modarressi, Mohammad Hosein; Rahmati, Marveh; Nouri, Mohammad

    2015-04-01

    Breast cancer is the most frequent cause of mortality in women all around the world; therefore, study on molecular aspects of breast cancer is necessary for finding new biomarkers. Recent studies have shown that WW Binding Protein 2 (WBP2) is an important protein for the oncogenic property of cancer. We have previously evaluated the WW Binding Protein 2 N-Terminal Like (WBP2NL) gene expression in cancerous cell line and breast tumor tissues, and reported changes in expression, which could increase tumorigenic cell growth. However, the molecular mechanisms of WBP2NL and its clinical relevance have not been investigated. In this study, the expression of WBP2NL-related genes in the invasive breast carcinoma and normal breast tissues was evaluated for the first time. Analysis of WBP2NL-related genes expression was performed with reverse transcription-PCR and real time-PCR detection method. The target genes studied were as follow: WW domain containing E3 ubiquitin protein ligase 1(WWP1), membrane associated guanylatekinase containing WW and PDZ domain-1 (MAGI1), neural precursor cell expressed developmentally down-regulated 4 (NEDD4), formin binding protein-4 (FNBP4), BCL2-associated athanogene-3 (BAG3), WW domain-containing oxidoreductase (WWOX), yes-associated protein-1 (YAP1), WW domain containing transcription regulator (WWTR1), member RAS oncogene family (RAB2A), and small G protein signaling modulator 3 (SGSM3). The expression of WWP1, BAG3, and WWTR1 was significantly increased in breast cancer. In contrast, the expression of WWOX, YAP1, RAB2A, and SGSM3 was significantly decreased. The MAGI1 and NEDD4 expression was increased, while the expression of FNBP4 was unchanged. These findings lead us to suggest that WBP2NL might play roles as an anti-apoptotic factor or co-activator to promote breast cancer cell survival and proliferation. PMID:25417742

  16. GENES 2000

    NSDL National Science Digital Library

    Benjamin Lewin provides this detailed educational site as the online (and continuously updated) version of the printed resource, GENES. Designed as a pilot project "for building an online site that will develop into a general resource for the life sciences, including molecular biology, cell biology, development, immunology, and neurobiology, at levels varying from introductory to advanced," this impressive site is intended to be useful to students at the undergraduate level (or above). First-time users must register (access is free, "at least temporarily") to gain access to chapters; registered users may conduct searches, build references, write notes, highlight specified information, or browse the full resource. Future plans include "better (and faster) searches on the text, searches on figures, identification of new material and recently added references, printing by chapter or by section, more animations, on line tests and problem sets." Content is detailed, well illustrated, and thorough; this looks to be an exceptional resource.

  17. Genes and Psoriasis

    MedlinePLUS

    ... Your Patients Corrona Psoriasis Registry Donate Genes and Psoriasis Genes hold the key to understanding how the ... Are some genes linked to specific kinds of psoriasis? At the University of Utah, Drs. Gerald Krueger ...

  18. Identification and In Vivo Functional Analysis by Gene Disruption of ctnA, an Activator Gene Involved in Citrinin Biosynthesis in Monascus purpureus? †

    PubMed Central

    Shimizu, Takeo; Kinoshita, Hiroshi; Nihira, Takuya

    2007-01-01

    Citrinin, a secondary fungal metabolite of polyketide origin, is moderately nephrotoxic to vertebrates, including humans. From the red-pigment producer Monascus purpureus, a 21-kbp region flanking pksCT, which encodes citrinin polyketide synthase, was cloned. Four open reading frames (ORFs) (orf1, orf2, orf3, and orf4) in the 5?-flanking region and one ORF (orf5) in the 3?-flanking region were identified in the vicinity of pksCT. orf1 to orf5 encode a homolog of a dehydrogenase (similarity, 46%), a regulator (similarity, 38%), an oxygenase (similarity, 41%), an oxidoreductase (similarity, 26%), and a transporter (similarity, 58%), respectively. orf2 (2,006 bp with four introns) encodes a 576-amino-acid protein containing a typical Zn(II)2Cys6 DNA binding motif at the N terminus and was designated ctnA. Although reverse transcriptase PCR analysis revealed that all of these ORFs, except for orf1, were transcribed with pksCT under citrinin production conditions, the disruption of ctnA caused large decreases in the transcription of pksCT and orf5, together with reduction of citrinin production to barely detectable levels, suggesting that these two genes are under control of the ctnA product. Complementation of the ctnA disruptant with intact ctnA on an autonomously replicating plasmid restored both transcription and citrinin production, indicating that CtnA is a major activator of citrinin biosynthesis. PMID:17586673

  19. Oxygen control of nif gene expression in Klebsiella pneumoniae depends on NifL reduction at the cytoplasmic membrane by electrons derived from the reduced quinone pool.

    PubMed

    Grabbe, Roman; Schmitz, Ruth A

    2003-04-01

    In Klebsiella pneumoniae, the flavoprotein, NifL regulates NifA mediated transcriptional activation of the N2-fixation (nif) genes in response to molecular O2 and ammonium. We investigated the influence of membrane-bound oxidoreductases on nif-regulation by biochemical analysis of purified NifL and by monitoring NifA-mediated expression of nifH'-'lacZ reporter fusions in different mutant backgrounds. NifL-bound FAD-cofactor was reduced by NADH only in the presence of a redox-mediator or inside-out vesicles derived from anaerobically grown K. pneumoniae cells, indicating that in vivo NifL is reduced by electrons derived from membrane-bound oxidoreductases of the anaerobic respiratory chain. This mechanism is further supported by three lines of evidence: First, K. pneumoniae strains carrying null mutations of fdnG or nuoCD showed significantly reduced nif-induction under derepressing conditions, indicating that NifL inhibition of NifA was not relieved in the absence of formate dehydrogenase-N or NADH:ubiquinone oxidoreductase. The same effect was observed in a heterologous Escherichia coli system carrying a ndh null allele (coding for NADH dehydrogenaseII). Second, studying nif-induction in K. pneumoniae revealed that during anaerobic growth in glycerol, under nitrogen-limitation, the presence of the terminal electron acceptor nitrate resulted in a significant decrease of nif-induction. The final line of evidence is that reduced quinone derivatives, dimethylnaphthoquinol and menadiol, are able to transfer electrons to the FAD-moiety of purified NifL. On the basis of these data, we postulate that under anaerobic and nitrogen-limited conditions, NifL inhibition of NifA activity is relieved by reduction of the FAD-cofactor by electrons derived from the reduced quinone pool, generated by anaerobic respiration, that favours membrane association of NifL. We further hypothesize that the quinol/quinone ratio is important for providing the signal to NifL. PMID:12654011

  20. Chronic stress and impaired glutamate function elicit a depressive-like phenotype and common changes in gene expression in the mouse frontal cortex.

    PubMed

    Tordera, R M; Garcia-García, A L; Elizalde, N; Segura, V; Aso, E; Venzala, E; Ramírez, M J; Del Rio, J

    2011-01-01

    Major depression might originate from both environmental and genetic risk factors. The environmental chronic mild stress (CMS) model mimics some environmental factors contributing to human depression and induces anhedonia and helplessness. Mice heterozygous for the synaptic vesicle protein (SVP) vesicular glutamate transporter 1 (VGLUT1) have been proposed as a genetic model of deficient glutamate function linked to depressive-like behaviour. Here, we aimed to identify, in these two experimental models, gene expression changes in the frontal cortex, common to stress and impaired glutamate function. Both VGLUT1(+/-) and CMS mice showed helpless and anhedonic-like behavior. Microarray studies in VGLUT1(+/-) mice revealed regulation of genes involved in apoptosis, neurogenesis, synaptic transmission, protein metabolic process or learning and memory. In addition, RT-PCR studies confirmed gene expression changes in several glutamate, GABA, dopamine and serotonin neurotransmitter receptors. On the other hand, CMS affected the regulation of 147 transcripts, some of them involved in response to stress and oxidoreductase activity. Interestingly, 52 genes were similarly regulated in both models. Specifically, a dowregulation in genes that promote cell proliferation (Anapc7), cell growth (CsnK1g1), cell survival (Hdac3), and inhibition of apoptosis (Dido1) was observed. Genes linked to cytoskeleton (Hspg2, Invs), psychiatric disorders (Grin1, MapK12) or an antioxidant enzyme (Gpx2) were also downregulated. Moreover, genes that inhibit the MAPK pathways (Dusp14), stimulate oxidative metabolism (Eif4a2) and enhance glutamate transmission (Rab8b) were upregulated. We suggest that these genes could form part of the altered "molecular context" underlying depressive-like behaviour in animal models. The clinical relevance of these findings is discussed. PMID:20937555

  1. Gene function, gene networks and the fate of duplicated genes.

    PubMed

    Shimeld, S M

    1999-10-01

    For both copies of a duplicated gene to become fixed in a population and subsequently maintained, selection must favour individuals with both genes over individuals with one. Here I review and assess some of the proposed ways that gene structure and function might affect the likelihood of both copies acquiring distinct functions and therefore positive selection. In particular I focus on the interacting pathways of genes that make up gene networks, and how these may affect genes duplicated both singly and en masse. Using the Wnt and hedgehog pathways as examples and data from developmental and genome analyses, I show that, while some of these theories may genuinely reflect what has occurred in animal evolution, there are still insufficient data to rigorously assess their relative importance. This, however, is likely to change in the near future. PMID:10597639

  2. Regulation of the rat glutathione S-transferase A2 gene by glucocorticoids: crosstalk through C/EBPs.

    PubMed

    Falkner, K Cameron; Prough, Russell A

    2007-01-01

    Regulation of the rat glutathione S-transferase A2 (GSTA2) gene by glucocorticoids is biphasic in its concentration dependence to glucocorticoids, with concentrations of 10-100 nM repressing gene activity (GR-dependent), and concentrations above 1 microM increasing transactivation (PXR-dependent) in adult rat hepatocytes or transient transfection assays. Over-expression of either C/EBP alpha or beta negatively regulates basal and inducible expression of a 1.65 Kb GSTA2 luciferase reporter, and synergizes the response to glucocorticoids (GC). C/EBP responsive elements have been identified in the GSTA2 5'-flanking sequence, associated with the palindrominic Glucocorticoid Responsive Element (GRE), the Ah receptor response elements, and the antioxidant response element. In reporters lacking the palindromic GRE, negative regulation by GC is observed only when C/EBP alpha is co-expressed. Co-transfection of C/EBP alpha/beta induced gene expression of the GSTA2 XRE reporter, but negatively regulated the GSTA2 ARE-reporter. In contrast, the ARE from the rat NAD(P)H quinone oxidoreductase gene was induced by co-transfection of C/EBPs, but was still negatively regulated by GC. PXR-induction of the GSTA2 reporter was partially ablated by co-transfection of C/EBP alpha and enhanced by co-transfection of C/EBPbeta. We conclude that C/EBP alpha and beta are involved in GC-dependent repression of GSTA2 gene expression and ARE sequences that bind C/EBPs appears to be critical for these responses. PMID:17786629

  3. Evaluation of Gene, Protein and Neurotrophin Expression in the Brain of Mice Exposed to Space Environment for 91 Days

    PubMed Central

    Santucci, Daniela; Kawano, Fuminori; Ohira, Takashi; Terada, Masahiro; Nakai, Naoya; Francia, Nadia; Alleva, Enrico; Aloe, Luigi; Ochiai, Toshimasa; Cancedda, Ranieri; Goto, Katsumasa; Ohira, Yoshinobu

    2012-01-01

    Effects of 3-month exposure to microgravity environment on the expression of genes and proteins in mouse brain were studied. Moreover, responses of neurobiological parameters, nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF), were also evaluated in the cerebellum, hippocampus, cortex, and adrenal glands. Spaceflight-related changes in gene and protein expression were observed. Biological processes of the up-regulated genes were related to the immune response, metabolic process, and/or inflammatory response. Changes of cellular components involving in microsome and vesicular fraction were also noted. Molecular function categories were related to various enzyme activities. The biological processes in the down-regulated genes were related to various metabolic and catabolic processes. Cellular components were related to cytoplasm and mitochondrion. The down-regulated molecular functions were related to catalytic and oxidoreductase activities. Up-regulation of 28 proteins was seen following spaceflight vs. those in ground control. These proteins were related to mitochondrial metabolism, synthesis and hydrolysis of ATP, calcium/calmodulin metabolism, nervous system, and transport of proteins and/or amino acids. Down-regulated proteins were related to mitochondrial metabolism. Expression of NGF in hippocampus, cortex, and adrenal gland of wild type animal tended to decrease following spaceflight. As for pleiotrophin transgenic mice, spaceflight-related reduction of NGF occured only in adrenal gland. Consistent trends between various portions of brain and adrenal gland were not observed in the responses of BDNF to spaceflight. Although exposure to real microgravity influenced the expression of a number of genes and proteins in the brain that have been shown to be involved in a wide spectrum of biological function, it is still unclear how the functional properties of brain were influenced by 3-month exposure to microgravity. PMID:22808101

  4. Clustering gene expression patterns

    Microsoft Academic Search

    Amir Ben-Dor; Zohar Yakhinit; Zohar Yakhini

    1999-01-01

    Recent advances in biotechnology allow researchers to measure expression levels for thousands of genes simultaneously, across different conditions and over time. Analysis of data produced by such experiments offers potential insight into gene function and regulatory mechanisms. A key step in the analysis of gene expression data is the detection of groups of genes that manifest similar expression patterns. The

  5. Autism and Genes

    ERIC Educational Resources Information Center

    National Institutes of Health, 2005

    2005-01-01

    This document defines and discusses autism and how genes play a role in the condition. Answers to the following questions are covered: (1) What are genes? (2) What is autism? (3) What causes autism? (4) Why study genes to learn about autism? (5) How do researchers look for the genes involved in autism? (screen the whole genome; conduct cytogenetic…

  6. Gene–environment interdependence

    Microsoft Academic Search

    Michael Rutter

    2012-01-01

    The modern understanding of genetic influences, of environmental effects, of mental disorder, and of heritabilities is noted. The practical utility of finding susceptibility genes with a very small effect is questioned. The empirical findings and implications of developmental perturbations, epigenetics, gene–environment correlations and interactions are then discussed. It is noted that the genes involved in gene–environment interactions may be concerned

  7. Clustering gene expression data

    E-print Network

    Shamir, Ron

    CG 1 Clustering gene expression data #12;CG 2 How Gene Expression Data Looks Expression levels-expression co-regulation). · Identifying disease profiles · Drug development ·... Analysis requires clustering of genes/conditions. #12;CG 5 Clustering: Objective Group elements (genes) to clusters satisfying

  8. Role for NAD(P)H:quinone Oxidoreductase 1 and Manganese-Dependent Superoxide Dismutase in 17-(Allylamino)-17-demethoxygeldanamycin-Induced Heat Shock Protein 90 Inhibition in Pancreatic Cancer CellsS?

    PubMed Central

    Shieh, Biehuoy; Yan, Chao; Kepa, Jadwiga K.; Ross, David

    2011-01-01

    Previous work demonstrated that NAD(P)H:quinone oxidoreductase 1 (NQO1) metabolized the heat shock protein 90 (Hsp90) inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17AAG) to the corresponding hydroquinone (17AAGH2). The formation of 17AAGH2 by NQO1 results in a molecule that binds with greater affinity to Hsp90 compared with the parent quinone. 17AAG induced substantial growth inhibition in human pancreatic cancer cell lines expressing NQO1. Growth inhibition induced by 17AAG could be reduced by pretreatment with 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]-indole-4,7-dione (ES936), a mechanism-based inhibitor of NQO1. After treatment with 17AAG, biomarkers of Hsp90 inhibition, including markers of cell-cycle arrest, were more pronounced in NQO1-expressing cells compared with NQO1-null cells. The intracellular concentrations of 17AAG and 17AAGH2 were measured in human pancreatic cancer cells, and it was observed that larger amounts of 17AAG and 17AAGH2 could be detected in cells with catalytically active NQO1 compared with cells lacking NQO1 activity or cells pretreated with ES936. These data demonstrate that, in addition to generating an inhibitor with greater affinity for Hsp90 (17AAGH2), reduction of 17AAG to 17AAGH2 by NQO1 leads to substantially greater intracellular concentrations of 17AAG and 17AAGH2. In addition, oxidation of 17AAGH2 could be prevented by superoxide dismutase (SOD), demonstrating that 17AAGH2 was sensitive to oxidation by superoxide. Stable transfection of manganese-dependent SOD into MiaPaCa-2 cells resulted in a significantly greater intracellular concentration of 17AAGH2 with a corresponding increase in growth inhibitory activity. These data confirm the role of NQO1 in sensitivity to 17AAG and demonstrate that SOD functions in conjunction with NQO1 to maintain intracellular levels of 17AAGH2, the active Hsp90 inhibitor derived from 17AAG. PMID:21156818

  9. Therapeutic gene targeting.

    PubMed

    Yáñez, R J; Porter, A C

    1998-02-01

    Gene targeting is the use of homologous recombination to make defined alterations to the genome. One of the possible outcomes of gene targeting is the accurate correction of genetic defects, and this would make it the ideal method of gene therapy for single gene disorder. While gene targeting has been achieved both in human cell lines and in nontransformed, primary human cells, its low efficiency has been a major limitation to its therapeutic potential. Gene therapy in vivo gene targeting is there for impractical without dramatic improvements in targeting efficiency. Ex vivo approaches might more realistically be considered, but would benefit from progress in the isolation and growth of somatic stem cells and improvements in targeting efficiency. We provide here a brief review of the challenges of gene therapy by gene targeting. This is followed by a critical overview of recent developments in gene targeting techniques, and in our understanding of the underlying processes of homologous and nonhomologous recombination. PMID:9578833

  10. GeneCards

    NSDL National Science Digital Library

    The Weizmann Institute of Science provides GeneCards, a database of "human genes, their products and their involvement in diseases." GeneCards contains concise information on the functions of "all human genes with an approved symbol" (e.g., genes named according to the Human Gene Nomenclature Committee), in addition to "selected others" -- collectively representing over 9,600 genes. The database is searchable, and its most current version (Version 2.14) features a new navigation support system (the "Guidance System") that facilitates information retrieval. Typical information on each Gene Card includes Synonyms, Chromosomal location, Proteins, Sequences, Similar genes in other organisms, Disorders & Mutations, Medical News, Research Articles, and Additional Sources of Information on the WWW. The GeneCards database is of particular use in the context of functional genomics and proteomics.

  11. Induction of a Unique Isoform of the NCOA7 Oxidation Resistance Gene by Interferon ?-1b

    PubMed Central

    Yu, Lijian; Croze, Ed; Yamaguchi, Ken D.; Tran, Tiffany; Reder, Anthony T.; Litvak, Vladimir

    2015-01-01

    We demonstrate that interferon (IFN)-?-1b induces an alternative-start transcript containing the C-terminal TLDc domain of nuclear receptor coactivator protein 7 (NCOA7), a member of the OXR family of oxidation resistance proteins. IFN-?-1b induces NCOA7-AS (alternative start) expression in peripheral blood mononuclear cells (PBMCs) obtained from healthy individuals and multiple sclerosis patients and human fetal brain cells, astrocytoma, neuroblastoma, and fibrosarcoma cells. NCOA7-AS is a previously undocumented IFN-?-inducible gene that contains only the last 5 exons of full-length NCOA7 plus a unique first exon (exon 10a) that is not found in longer forms of NCOA7. This exon encodes a domain closely related to an important class of bacterial aldo-keto oxido-reductase proteins that play a critical role in regulating redox activity. We demonstrate that NCOA7-AS is induced by IFN and LPS, but not by oxidative stress and exhibits, independently, oxidation resistance activity. We further demonstrate that induction of NCOA7-AS by IFN is dependent on IFN-receptor activation, the Janus kinase-signal transducers and activators of transcription (JAK-STAT) signaling pathway, and a canonical IFN-stimulated response element regulatory sequence upstream of exon 10a. We describe a new role for IFN-?s involving a mechanism of action that leads to an increase in resistance to inflammation-mediated oxidative stress. PMID:25330068

  12. Induction of a unique isoform of the NCOA7 oxidation resistance gene by interferon ?-1b.

    PubMed

    Yu, Lijian; Croze, Ed; Yamaguchi, Ken D; Tran, Tiffany; Reder, Anthony T; Litvak, Vladimir; Volkert, Michael R

    2015-03-01

    We demonstrate that interferon (IFN)-?-1b induces an alternative-start transcript containing the C-terminal TLDc domain of nuclear receptor coactivator protein 7 (NCOA7), a member of the OXR family of oxidation resistance proteins. IFN-?-1b induces NCOA7-AS (alternative start) expression in peripheral blood mononuclear cells (PBMCs) obtained from healthy individuals and multiple sclerosis patients and human fetal brain cells, astrocytoma, neuroblastoma, and fibrosarcoma cells. NCOA7-AS is a previously undocumented IFN-?-inducible gene that contains only the last 5 exons of full-length NCOA7 plus a unique first exon (exon 10a) that is not found in longer forms of NCOA7. This exon encodes a domain closely related to an important class of bacterial aldo-keto oxido-reductase proteins that play a critical role in regulating redox activity. We demonstrate that NCOA7-AS is induced by IFN and LPS, but not by oxidative stress and exhibits, independently, oxidation resistance activity. We further demonstrate that induction of NCOA7-AS by IFN is dependent on IFN-receptor activation, the Janus kinase-signal transducers and activators of transcription (JAK-STAT) signaling pathway, and a canonical IFN-stimulated response element regulatory sequence upstream of exon 10a. We describe a new role for IFN-?s involving a mechanism of action that leads to an increase in resistance to inflammation-mediated oxidative stress. PMID:25330068

  13. Aging: gene silencing or gene activation?

    PubMed

    Burzynski, Stanislaw R

    2005-01-01

    According to the author's theory of gene silencing, the key process in aging involves reduced expression of a number of genes. Silencing of genes has a complex mechanism, which involves methylation of DNA, histone modification and chromatin remodeling. In addition to deacetylation of the histones and methylation of DNA, recently described RNAi mechanism could initiate formation of silenced chromatin. Hypermethylation of the promoter will silence the gene. Genome-wide hypomethylation will induce genomic instability, amplification of oncogenes and also silencing of the genes through RNAi mechanism. Studies by different groups, conducted in yeast, worms, flies and mice, confirmed substantial changes in gene expression in aging. Among them, the most important was silencing of tumor suppressors and other genes involved in the control of cell cycle, apoptosis, detoxification, and cholesterol metabolism. There was also increased expression of the smaller group of oncogenes and other genes which are associated with typical diseases of old age. Caloric restriction normalizes expression of a substantial percentage of these genes. Animal studies confirmed importance of caloric restriction, which decreases signaling through the IGF-1/AKT pathway and expression of gene p53. These studies, however, cannot be directly applied to human aging. It is proposed that age management therapy should attempt to normalize gene expression in the older population to the level typical for young adults. This would require activation of silenced genes and normalization of overexpressed genes. Caloric restriction and exercise are helpful in decreasing the activity of important oncogenes and activation of silenced tumor suppressors, and may have a positive impact, not only on aging, but also on prevention of cancer. Dietary supplements containing phytochemicals should normalize increased expression of oncogenes. Examples are: genistein and EGCG, which effect signaling through the IGF-1/AKT pathway and resveratrol and limonen, which do so through the RAS pathway. A group of amino acid derivatives and organic acids of animal and human origin should activate silenced tumor suppressor genes (Aminocare A10, Aminocare Extra). Among them 3-phenylacetylamino-2, 6-piperidinedione intercalates specifically with DNA and protects sequences of tumor suppressor genes, which are vulnerable to the effects of carcinogens. Phenylacetate activates p53 and p21 through inhibition of methyltransferase and farnesylation of the RAS protein. Phenylbutyrate activates tumor suppressor genes through inhibition of histone deacetylation. Phenylacetylglutamine decreases genomic instability and expression of oncogenes and promotes apoptosis. The application of DNA microarray techniques to human studies should provide more information about differences in gene expression in different age groups and help design more effective age management regimens. PMID:15533642

  14. Insertion of mini-IS605 and deletion of adjacent sequences in the nitroreductase (rdxA) gene cause metronidazole resistance in Helicobacter pylori NCTC11637.

    PubMed

    Debets-Ossenkopp, Y J; Pot, R G; van Westerloo, D J; Goodwin, A; Vandenbroucke-Grauls, C M; Berg, D E; Hoffman, P S; Kusters, J G

    1999-11-01

    We found that NCTC11637, the type strain of Helicobacter pylori, the causative agent of peptic ulcer disease and an early risk factor for gastric cancer, is metronidazole resistant. DNA transformation, PCR-based restriction analysis, and DNA sequencing collectively showed that the metronidazole resistance of this strain was due to mutation in rdxA (gene HP0954 in the full genome sequence of H. pylori 26695) and that resistance did not depend on mutation in any of the other genes that had previously been suggested: catalase (katA), ferredoxin (fdx), flavodoxin (fldA), pyruvate:flavodoxin oxidoreductase (porgammadeltaalphabeta), RecA (recA), or superoxide dismutase (sodB). This is in accord with another recent study that attributed metronidazole resistance to point mutations in rdxA. However, the mechanism of rdxA inactivation that we found in NCTC11637 is itself also novel: insertion of mini-IS605, one of the endogenous transposable elements of H. pylori, and deletion of adjacent DNA sequences including 462 bp of the 851-bp-long rdxA gene. PMID:10543743

  15. LuSens: a keratinocyte based ARE reporter gene assay for use in integrated testing strategies for skin sensitization hazard identification.

    PubMed

    Ramirez, Tzutzuy; Mehling, Annette; Kolle, Susanne N; Wruck, Christoph J; Teubner, Wera; Eltze, Tobias; Aumann, Alexandra; Urbisch, Daniel; van Ravenzwaay, Ben; Landsiedel, Robert

    2014-12-01

    Allergic contact dermatitis can develop following repeated exposure to allergenic substances. To date, hazard identification is still based on animal studies as non-animal alternatives have not yet gained global regulatory acceptance. Several non-animal methods addressing key-steps of the adverse outcome pathway (OECD, 2012) will most likely be needed to fully address this effect. Among the initial cellular events is the activation of keratinocytes and currently only one method, the KeratinoSens™, has been formally validated to address this event. In this study, a further method, the LuSens assay, that uses a human keratinocyte cell line harbouring a reporter gene construct composed of the antioxidant response element (ARE) of the rat NADPH:quinone oxidoreductase 1 gene and the luciferase gene. The assay was validated in house using a selection of 74 substances which included the LLNA performance standards. The predictivity of the LuSens assay for skin sensitization hazard identification was comparable to other non-animal methods, in particular to the KeratinoSens™. When used as part of a testing battery based on the OECD adverse outcome pathway for skin sensitization, a combination of the LuSens assay, the DPRA and a dendritic cell line activation test attained predictivities similar to that of the LLNA. PMID:25172300

  16. Airway Mucin Genes and Gene Products

    Microsoft Academic Search

    Mary Callaghan Rose; Sandra J. Gendler

    \\u000a \\u000a MUC genes and their MUC gene products have been the subject of several reviews in the last few years [1-4] as expected in a field where the molec­ular tools to address fundamental and disease-related questions are becom­ing available.\\u000a A description and analysis of human mucin genes MUC\\/through MUC7 and of porcine canine murine rat and bovine mucin geneswere published in

  17. Protection against Photooxidative Injury of Tobacco Leaves by 2-Alkenal Reductase. Detoxication of Lipid Peroxide-Derived Reactive Carbonyls1

    PubMed Central

    Mano, Jun'ichi; Belles-Boix, Enric; Babiychuk, Elena; Inzé, Dirk; Torii, Yoshimitsu; Hiraoka, Eiji; Takimoto, Koichi; Slooten, Luit; Asada, Kozi; Kushnir, Sergei

    2005-01-01

    Degradation of lipid peroxides leads to the formation of cytotoxic 2-alkenals and oxenes (collectively designated reactive carbonyls). The novel NADPH-dependent oxidoreductase 2-alkenal reductase (AER; EC 1.3.1.74) from Arabidopsis (Arabidopsis thaliana), which is encoded by the gene At5g16970, catalyzes the reduction of the ?,?-unsaturated bond of reactive carbonyls, and hence is presumed to function in antioxidative defense in plants. Here we show that Arabidopsis AER (At-AER) has a broad substrate spectrum to biologically relevant reactive carbonyls. Besides 2-alkenals, the enzyme recognized as substrates the lipid peroxide-derived oxenes 9-oxo-octadeca-(10E),(12Z)-dienoic acid and 13-oxo-octadeca-(9E),(11Z)-dienoic acid, as well as the potent genotoxin 4-oxo-(2E)-nonenal, altogether suggesting AER has a key role in the detoxification of reactive carbonyls. To validate this conclusion by in vivo studies, transgenic tobacco (Nicotiana tabacum) plants that had 100- to 250-fold higher AER activity levels than control plants were generated. The engineered plants exhibited significantly less damage from either (1) the exogenously administered 4-hydroxy-(2E)-nonenal, (2) treatment with methyl viologen plus light, or (3) intense light. We further show that the At-AER protein fused with the Aequorea victoria green fluorescent protein localizes in cytosol and the nucleus in Bright-Yellow 2 cells. These results indicate that reactive carbonyls mediate photooxidative injury in leaf cells, and At-AER in the cytosol protects the cells by reducing the ?,?-unsaturated bond of the photoproduced reactive carbonyls. PMID:16299173

  18. Molecular characterization of thioredoxin-1 and thioredoxin reductase activity in mud crab Scylla paramamosain.

    PubMed

    Hu, J H; Zhang, F Y; Jiang, K J; Fang, Y B; Wang, J; Zhao, M; Qiao, Z G; Ma, L B

    2014-01-01

    The thioredoxin (Trx) system consists of thioredoxin reductase (TrxR), Trx, and nicotinamide adenine dinucleotide phosphate (NADPH). TrxR is an NADPH-dependent oxidoreductase. Trx is a ubiquitous small protein with a redox-active disulfide bridge that plays important regulatory roles in some vital metabolic reactions. In this study, a cDNA sequence (SpTrx1) showing high identity to the first Trx gene was isolated from a hepatopancreas cDNA library of the mud crab Scylla paramamosain. The full-length cDNA of SpTrx1 consisted of 672 bp and contained a complete open reading frame of 318 bp encoding a polypeptide of 105 amino acids. Quantitative real-time polymerase chain reaction analysis revealed that SpTrx1 expression was ubiquitous in various organs of S. paramamosain, including the gill, muscle, heart, hemolymph, testis, and hepatopancreas. SpTrx1 expression was upregulated significantly after Vibrio parahaemolyticus challenge: it obviously rose at 48 h and reached the highest level at 72 h. Furthermore, TrxR activity was detected in the gill, heart, muscle, hemolymph, and hepatopancreas. The relative TrxR activity in different tissues after V. parahaemolyticus injection had the same tendency in each tissue (P < 0.01) as SpTrx1 expression. The TrxR activity increased 2 h after injection, peaked at 8 h, slowly decreased from 12 to 24 h, and returned to normal levels at 48 h. The consistency of the expression between the Trx transcript and TrxR activity demonstrated that Trx was closely related to TrxR in the Trx system in S. paramamosain, suggesting that it may participate in the immune system of mud crabs. PMID:25501236

  19. Gene symbol precision.

    PubMed

    Bennani-Baiti, Barbara; Bennani-Baiti, Idriss M

    2012-01-10

    Several gene databases, including heavily used ones such as the National Center for Biotechnology Information (NCBI) database, erroneously assign, on occasion, literature references to genes or proteins. These mistakes are mostly due to an overlap in gene aliases, whereby two distinct genes share a pseudonym. This is particularly confusing when the gene products have also biological properties in common, are part of signaling pathways that cross-talk to one another, or are regulated by the same effectors. We present examples spanning several research fields including apoptosis, ubiquitin-dependent degradation, signaling by Notch, Wnt, and small G proteins, transporters of glutathione conjugates of electrophiles, and mitochondrial and ribosomal RNA genes. To solve the problem, we argue in favor of including Entrez gene numbers in papers submitted for publication as unique gene identifiers to allow precise identification of genes and species studied. PMID:22019431

  20. Studies on the enzymatic reduction of N-Boc-4S-amino-3-oxo-5-phenylpentanoic acid methylester.

    PubMed

    Nassenstein, A; Hemberger, J; Schwartz, H; Kula, M R

    1992-11-01

    The enzymatic reduction of N-Boc-4S-amino-3-oxo-5-phenylpentanoic acid methylester, the key intermediate in the stereoselective synthesis of a statinanalogue, was studied with Hansenula anomala and Hansenula silvicola. Using whole cells of H. anomala gives complete conversion and a diastereomeric excess of 88% of the desired 3S, 4S statinanalogue. The strain contains two NADPH-dependent oxidoreductases, that can be separated by ion exchange chromatography or gelfiltration, yielding the 3S, 4S or 3R, 4S stereoisomers, respectively, with > 99% diastereomeric excess (DE). In the crude extract the 3S, 4S oxidoreductase is very unstable and could be purified with < 1% yield only. In contrast, H. silvicola, which gave poor conversions using whole cells, exhibited about 80-fold higher specific activity in the crude extract than H. anomala. The NADPH-dependent oxidoreductase was purified 317-fold in 12% yield. A single enzyme of 54 kDa reduces the substrate with 97.4% DE. Besides the statinanalogue a wide range of other compounds could be reduced, most notably diones and chinones such as isatin or campherchinone. It was demonstrated that the enzymes often discussed for the reduction of beta-ketoesters with yeast e.g. L-3-hydroxyacyl CoA dehydrogenase (EC 1.1.1.35), the beta-ketoreductase of the fatty acid synthase complex and also the 3-hydroxy-3-methyl glutaryl-CoA dehydrogenase (EC 1.1.1.34) are separated during the purification steps from the oxidoreductase acting on N-Boc-4S-amino-3-oxo-5-phenylpentanoic acid methylester. The physiological role of the new enzyme is still unknown. PMID:1369149

  1. Murine mdr-1, mdr-2, and mdr-3 gene expression: no coinduction with the Cyp1a-1 and Nmo-1 genes in liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

    PubMed

    Teeter, L D; Petersen, D D; Nebert, D W; Kuo, M T

    1991-01-01

    Multidrug-resistance (MDR) genes are induced in the liver of rodents treated with a variety of foreign chemicals and hepatocarcinogens. It has been reported that 2,3,6,7-tetrachlorodibenzo-p-dioxin (TCDD) might increase hepatic MDR transcripts in the Fischer rat and the C57BL/6 (B6) inbred mouse strain having the high-affinity aromatic hydrocarbon (Ah) receptor, but not in the DBA/2 (D2) strain having the low-affinity Ah receptor. These intriguing results suggest that TCDD might activate MDR gene expression by way of an Ah receptor-mediated signal transduction pathway. We have attempted to confirm these data in four inbred mouse strains: two (B6 and BALB/c) having the high-affinity Ah receptor, and two (D2 and AKR) having the low-affinity Ah receptor. The RNase protection assay was used to distinguish between the MDR1, MDR2, and MDR3 mRNAs. TCDD treatment at high (100 micrograms/kg) and low (1 mu/kg) doses, a time course from 6 to 96 hr of TCDD treatment, progeny from the B6D2F1 x D2 backcross, and transcriptional run-on experiments were performed. The Cyp1a-1 (cytochrome P1450) and Nmo-1 [NAD(P)H:menadione oxidoreductase] genes, two members of the TCDD-inducible [Ah] battery, were used as positive controls. We were unable to detect significant coinduction of MDR1, MDR2, or MDR3 mRNA with CYP1A1 mRNA or with Cyp1a-1 or Nmo-1 transcription under any conditions. Therefore, we conclude that any effects that TCDD might have on MDR expression must be substantially different from TCDD effects on genes known to be induced via the Ah receptor. PMID:2069718

  2. Virtual Gene: Using Correlations Between Genes to Select Informative Genes on Microarray Datasets

    Microsoft Academic Search

    Xian Xu; Aidong Zhang

    2005-01-01

    Gene Selection is one class of most used data analysis algorithms on microarray datasets. The goal of gene selection algorithms is to filter out a small set of informative genes that best explains experimental variations. Traditional gene selection algorithms are mostly single-gene based. Some discriminative scores are calculated and sorted for each gene. Top ranked genes are then se- lected

  3. Structural Aspects of Plant Ferredoxin : NADP + Oxidoreductases

    Microsoft Academic Search

    P. Andrew Karplus; H. Richard Faber

    2004-01-01

    Ferredoxin reductase (FNR) is ubiquitous among photosynthetic organisms as the enzyme directly responsible for the generation\\u000a of NADPH. Structural studies over the last 15 years have generated over 30 crystal structures of wild-type and mutant FNRs\\u000a that have yielded a great deal of insight into its structure–function relations. These insights are summarized and combined\\u000a to propose a structurally informed cycle

  4. Fungal Oxidoreductases and Humification in Forest Soils

    Microsoft Academic Search

    A. G. Zavarzina; A. A. Lisov; A. A. Zavarzin; A. A. Leontievsky

    \\u000a Humification is aerobic, largely oxidative process of non-living organic matter biotransformation into recalcitrant humic\\u000a substances (HS). HS comprise up to 90% of soil organic matter and represent a long-time sink for atmospheric CO2 with mean residence time of 102–103 years. Wood- and soil-colonizing fungi are the major driving force in humification, being involved in transformation of plant\\u000a residues, synthesis, and

  5. Biosynthetic Gene Cluster of Cetoniacytone A, an Unusual Aminocyclitol from the Endosymbiotic Bacterium Actinomyces sp. Lu 9419

    PubMed Central

    Wu, Xiumei; Flatt, Patricia M.; Xu, Hui

    2011-01-01

    A gene cluster responsible for the biosynthesis of the antitumor agent cetoniacytone A was identified in Actinomyces sp. strain Lu 9419, an endosymbiotic bacteria isolated from the intestines of the rose chafer beetle (Cetonia aurata). The nucleotide sequence analysis of the 46 kb DNA region revealed the presence of 31 complete ORFs, including genes predicted to encode a 2-epi-5-epi-valiolone synthase (CetA), a glyoxalase/bleomycin resistance protein (CetB), an acyltransferase (CetD), an FAD-dependent dehydrogenase (CetF2), two oxidoreductases (CetF1 and CetG), two aminotransferases (CetH and CetM), and a pyranose oxidase (CetL). CetA has previously been demonstrated to catalyze the cyclization of sedoheptulose 7-phosphate to the cyclic intermediate, 2-epi-5-epi-valiolone. In this report, the glyoxalase/bleomycin resistance protein homolog CetB was identified as a 2-epi-5-epi-valiolone epimerase (EVE), a new member of the Vicinal Oxygen Chelate (VOC) superfamily. The 24 kDa recombinant histidine-tagged CetB was found to form a homodimer; each monomer contains two ????? scaffolds that form a metal binding site with two histidine and two glutamic acid residues. BLAST search using the newly isolated cet biosynthetic genes revealed an analogous suite of genes in the genome of Frankia alni ACN14a, suggesting that this plant symbiotic nitrogen-fixing bacterium is capable of producing a secondary metabolite related to the cetoniacytones. PMID:19101977

  6. Characterization of a Thermolabile Sulfite Reductase from Salmonella pullorum1

    PubMed Central

    Hoeksema, Walter D.; Schoenhard, Delbert E.

    1971-01-01

    The biochemical basis for sulfite accumulation by sulfate-using revertants of Salmonella pullorum was determined. All of the sulfate-using mutants isolated from wild-type S. pullorum accumulated sulfite when grown at 37 but not at 25 C. The specific activity of reduced nicotinamide adenine dinucleotide (NADPH)-dependent sulfite reductase (H 2S-NADP oxidoreductase, EC 1.8.1.2) and of reduced methyl viologen (MVH)-dependent sulfite reductase (H 2S-MV oxidoreductase), in extracts prepared from cells incubated at 37 C, declined as the incubation period lengthened. However, the specific activity of both reductases from cells incubated at 25 C did not decline. Thermolability of cell-free NADPH-dependent sulfite reductase from cells of S. pullorum incubated at 37 C was greater than the lability of this enzyme either from cells of S. typhimurium incubated at 37 C or from cells of S. pullorum incubated at 25 C. Cells cultured at 37 C continued to accumulate sulfite when the incubation temperature was shifted to 25 C; cells cultured at 25 C and shifted to 37 C accumulated no sulfite, whereas these cells shifted to 41 C accumulated sulfite. It was concluded that the configuration of the sulfite reductase of S. pullorum strain 6–18 is a function of the incubation temperature at which synthesis occurs. PMID:5122801

  7. Oxidative stress induced by HIV-1 reverse transcriptase modulates the enzyme’s performance in gene immunization

    PubMed Central

    Isaguliants, Maria; Smirnova, Olga; Ivanov, Alexander V.; Kilpelainen, Athina; Kuzmenko, Yulia; Petkov, Stefan; Latanova, Anastasia; Krotova, Olga; Engström, Gunnel; Karpov, Vadim; Kochetkov, Sergey; Wahren, Britta; Starodubova, Elizaveta

    2013-01-01

    HIV-1 infection induces chronic oxidative stress. The resultant neurotoxicity has been associated with Tat protein. Here, we for the first time describe the induction of oxidative stress by another HIV-1 protein, reverse transcriptase (RT). Expression of HIV-1 RT in human embryonic kidney cells generated potent production of the reactive oxygen species (ROS), detected by the fluorescence-based probes. Quantitative RT-PCR demonstrated that expression of RT in HEK293 cells induced a 10- to 15-fold increased transcription of the phase II detoxifying enzymes human NAD(P)H:quinone oxidoreductase (Nqo1) and heme oxygenase 1 (HO-1), indicating the induction of oxidative stress response. The capacity to induce oxidative stress and stress response appeared to be an intrinsic property of a vast variety of RTs: enzymatically active and inactivated, bearing mutations of drug resistance, following different routes of processing and presentation, expressed from viral or synthetic expression-optimized genes. The total ROS production induced by RT genes of the viral origin was found to be lower than that induced by the synthetic/expression-optimized or chimeric RT genes. However, the viral RT genes induced higher levels of ROS production and higher levels of HO-1 mRNA than the synthetic genes per unit of protein in the expressing cell. The capacity of RT genes to induce the oxidative stress and stress response was then correlated with their immunogenic performance. For this, RT genes were administered into BALB/c mice by intradermal injections followed by electroporation. Splenocytes of immunized mice were stimulated with the RT-derived and control antigens and antigen-specific proliferation was assessed by IFN-?/IL-2 Fluorospot. RT variants generating high total ROS levels induced significantly stronger IFN-? responses than the variants inducing lower total ROS, while high levels of ROS normalized per unit of protein in expressing cell were associated with a weak IFN-? response. Poor gene immunogenicity was also associated with a high (per unit of protein) transcription of antioxidant response element (ARE) dependent phase II detoxifying enzyme genes, specifically HO-1. Thus, we have revealed a direct link between the propensity of the microbial proteins to induce oxidative stress and their immunogenicity. PMID:23881028

  8. Bacterial avirulence genes.

    PubMed

    Leach, J E; White, F F

    1996-01-01

    Although more than 30 bacterial avirulence genes have been cloned and characterized, the function of the gene products in the elictitation of resistance is unknown in all cases but one. The product of avrD from Pseudomonas syringae pv. glycinea likely functions indirectly to elicit resistance in soybean, that is, evidence suggests the gene product is an enzyme involved in elicitor production. In most if not all cases, bacterial avirulence gene function is dependent on interactions with the hypersensitive response and pathogenicity (hrp) genes. Many hrp genes are similar to genes involved in delivery of pathogenicity factors in mammalian bacterial pathogens. Thus, analogies between mammalian and plant pathogens may provide needed clues to elucidate how virulence gene products control induction of resistance. PMID:15012539

  9. Molecular Cell Gene Regulation

    E-print Network

    Collins, James J.

    Molecular Cell Previews Gene Regulation: Hacking the Network on a Sugar High Tom Ellis,1,2 Xiao. The resulting input-function map reveals that gene network regulation follows non-Boolean, and often how its individual parts behave in response to different conditions. In the case of gene networks

  10. Evidence for Homosexuality Gene

    Microsoft Academic Search

    Robert Pool

    1993-01-01

    A genetic analysis of 40 pairs of homosexual brothers has uncovered a region on the X chromosome that appears to contain a gene or genes for homosexuality. When analyzing the pedigrees of homosexual males, the researcheres found evidence that the trait has a higher likelihood of being passed through maternal genes. This led them to search the X chromosome for

  11. Gene Testing Can Be Flawed

    MedlinePLUS

    ... HealthDay . All rights reserved. More Health News on: Genes and Gene Therapy Genetic Testing Recent Health News Related MedlinePlus Health Topics Genes and Gene Therapy Genetic Testing About MedlinePlus Site Map FAQs Contact ...

  12. Evolution of Gene Expression after Gene Amplification

    PubMed Central

    Garcia, Nelson; Zhang, Wei; Wu, Yongrui; Messing, Joachim

    2015-01-01

    We took a rather unique approach to investigate the conservation of gene expression of prolamin storage protein genes across two different subfamilies of the Poaceae. We took advantage of oat plants carrying single maize chromosomes in different cultivars, called oat–maize addition (OMA) lines, which permitted us to determine whether regulation of gene expression was conserved between the two species. We found that ?-zeins are expressed in OMA7.06, which carries maize chromosome 7 even in the absence of the trans-acting maize prolamin-box-binding factor (PBF), which regulates their expression. This is likely because oat PBF can substitute for the function of maize PBF as shown in our transient expression data, using a ?-zein promoter fused to green fluorescent protein (GFP). Despite this conservation, the younger, recently amplified prolamin genes in maize, absent in oat, are not expressed in the corresponding OMAs. However, maize can express the oldest prolamin gene, the wheat high-molecular weight glutenin Dx5 gene, even when maize Pbf is knocked down (through PbfRNAi), and/or another maize transcription factor, Opaque-2 (O2) is knocked out (in maize o2 mutant). Therefore, older genes are conserved in their regulation, whereas younger ones diverged during evolution and eventually acquired a new repertoire of suitable transcriptional activators. PMID:25912045

  13. The 380 kb pCMU01 Plasmid Encodes Chloromethane Utilization Genes and Redundant Genes for Vitamin B12- and Tetrahydrofolate-Dependent Chloromethane Metabolism in Methylobacterium extorquens CM4: A Proteomic and Bioinformatics Study

    PubMed Central

    Roselli, Sandro; Nadalig, Thierry; Vuilleumier, Stéphane; Bringel, Françoise

    2013-01-01

    Chloromethane (CH3Cl) is the most abundant volatile halocarbon in the atmosphere and contributes to the destruction of stratospheric ozone. The only known pathway for bacterial chloromethane utilization (cmu) was characterized in Methylobacterium extorquens CM4, a methylotrophic bacterium able to utilize compounds without carbon-carbon bonds such as methanol and chloromethane as the sole carbon source for growth. Previous work demonstrated that tetrahydrofolate and vitamin B12 are essential cofactors of cmuA- and cmuB-encoded methyltransferases of chloromethane dehalogenase, and that the pathway for chloromethane utilization is distinct from that for methanol. This work reports genomic and proteomic data demonstrating that cognate cmu genes are located on the 380 kb pCMU01 plasmid, which drives the previously defined pathway for tetrahydrofolate-mediated chloromethane dehalogenation. Comparison of complete genome sequences of strain CM4 and that of four other M. extorquens strains unable to grow with chloromethane showed that plasmid pCMU01 harbors unique genes without homologs in the compared genomes (bluB2, btuB, cobA, cbiD), as well as 13 duplicated genes with homologs of chromosome-borne genes involved in vitamin B12-associated biosynthesis and transport, or in tetrahydrofolate-dependent metabolism (folC2). In addition, the presence of both chromosomal and plasmid-borne genes for corrinoid salvaging pathways may ensure corrinoid coenzyme supply in challenging environments. Proteomes of M. extorquens CM4 grown with one-carbon substrates chloromethane and methanol were compared. Of the 49 proteins with differential abundance identified, only five (CmuA, CmuB, PurU, CobH2 and a PaaE-like uncharacterized putative oxidoreductase) are encoded by the pCMU01 plasmid. The mainly chromosome-encoded response to chloromethane involves gene clusters associated with oxidative stress, production of reducing equivalents (PntAA, Nuo complex), conversion of tetrahydrofolate-bound one-carbon units, and central metabolism. The mosaic organization of plasmid pCMU01 and the clustering of genes coding for dehalogenase enzymes and for biosynthesis of associated cofactors suggests a history of gene acquisition related to chloromethane utilization. PMID:23593113

  14. Cell surface modulation of gene expression in brain cells by down regulation of glucocorticoid receptors

    SciTech Connect

    McGinnis, J.F.; de Vellis, J.

    1981-02-01

    The concentration of glycerol-3-phosphate dehydrogenase (GPDH; sn-glycerol-3-phosphate:NAD/sup +/ 2-oxidoreductase, EC 1.1.1.8) had previously been determined to be regulated by glucocorticoids in rat brain cells in vivo and in cell culture. We now demonstrate that concanavalin A (Con A) can inhibit the induction of GPDH in a dose-dependent manner in C6 rat glioma cells and in primary cultures of rat brain oligodendrocytes. The inhibition specifically prevents the appearance of new molecules of GPDH, although Con A does not significantly inhibit protein synthesis in these cells, nor does it affect the activity of another solube enzyme, lactate dehydrogenase. The ability to block enzyme induction is not limited to Con A, because other lectins also inhibit induction. The molecular mechanism by which Con A inhibits GPDH induction appears to be by the down regulation of the cytoplasmic glucocorticoid receptors, because exposure to Con A results in the loss of more than 90% of the receptor activity. Con A does not inhibit the receptor assay and no direct interaction between the receptor and Con A could be demonstrated. This down regulation is not tumor cell specific and appears to be a general phenomenon, because it occurs in normal oligodendrocytes and even in normal astrocytes (a cell type in which the gene for GPDH is not expressed). The down regulation of glucocorticoid receptors in normal brain cells suggests two important corollaries. First, it demonstrates the existence of a rate-limiting step controlling the glucocorticoid-dependent gene expression in brain cells and possibly represents a regulatory site common to all glucocorticoid target cells. Second, it suggests that the response to glucocorticoids of oligodendrocytes and astrocytes can be regulated in vivo by cell surface contact with endogenous lectins, neighboring cells, or both.

  15. Gene Gateway: Exploring Genes and Genetic Disorders

    NSDL National Science Digital Library

    This collection of guides and tutorials is intended to help users take advantage of of online data sources from the Human Genome Project for learning about genetic disorders, genes, and proteins. Resources include the Gene Gateway Workbook, a downloadable tutorial consisting of activities with screenshots and instructions, that helps new users locate and use genetic-disorder and bioinformatics resources on the web. There are also resources for learning about genes and the proteins they encode; tips, tutorials, and terminology for using selected resources in the Genome Database Guide; a guide to nontechnical resources on genetic disorder descriptions and treatments; a human genome landmarks poster; and others.

  16. Gene hunting in autoinflammation

    PubMed Central

    2013-01-01

    Steady progress in our understanding of the genetic basis of autoinflammatory diseases has been made over the past 16 years. Since the discovery of the familial Mediterranean fever gene MEFV (also known as marenostrin) in 1997, 18 other genes responsible for monogenic autoinflammatory diseases have been identified to date. The discovery of these genes was made through the utilisation of many genetic mapping techniques, including next generation sequencing platforms. This review article clearly describes the gene hunting approaches, methods of data analysis and the technological platforms used, which has relevance to all those working within the field of gene discovery for Mendelian disorders. PMID:24070009

  17. Evidence for the bacterial origin of genes encoding fermentation enzymes of the amitochondriate protozoan parasite Entamoeba histolytica.

    PubMed Central

    Rosenthal, B; Mai, Z; Caplivski, D; Ghosh, S; de la Vega, H; Graf, T; Samuelson, J

    1997-01-01

    Entamoeba histolytica is an amitochondriate protozoan parasite with numerous bacterium-like fermentation enzymes including the pyruvate:ferredoxin oxidoreductase (POR), ferredoxin (FD), and alcohol dehydrogenase E (ADHE). The goal of this study was to determine whether the genes encoding these cytosolic E. histolytica fermentation enzymes might derive from a bacterium by horizontal transfer, as has previously been suggested for E. histolytica genes encoding heat shock protein 60, nicotinamide nucleotide transhydrogenase, and superoxide dismutase. In this study, the E. histolytica por gene and the adhE gene of a second amitochondriate protozoan parasite, Giardia lamblia, were sequenced, and their phylogenetic positions were estimated in relation to POR, ADHE, and FD cloned from eukaryotic and eubacterial organisms. The E. histolytica por gene encodes a 1,620-amino-acid peptide that contained conserved iron-sulfur- and thiamine pyrophosphate-binding sites. The predicted E. histolytica POR showed fewer positional identities to the POR of G. lamblia (34%) than to the POR of the enterobacterium Klebsiella pneumoniae (49%), the cyanobacterium Anabaena sp. (44%), and the protozoan Trichomonas vaginalis (46%), which targets its POR to anaerobic organelles called hydrogenosomes. Maximum-likelihood, neighbor-joining, and parsimony analyses also suggested as less likely E. histolytica POR sharing more recent common ancestry with G. lamblia POR than with POR of bacteria and the T. vaginalis hydrogenosome. The G. lamblia adhE encodes an 888-amino-acid fusion peptide with an aldehyde dehydrogenase at its amino half and an iron-dependent (class 3) ADH at its carboxy half. The predicted G. lamblia ADHE showed extensive positional identities to ADHE of Escherichia coli (49%), Clostridium acetobutylicum (44%), and E. histolytica (43%) and lesser identities to the class 3 ADH of eubacteria and yeast (19 to 36%). Phylogenetic analyses inferred a closer relationship of the E. histolytica ADHE to bacterial ADHE than to the G. lamblia ADHE. The 6-kDa FD of E. histolytica and G. lamblia were most similar to those of the archaebacterium Methanosarcina barkeri and the delta-purple bacterium Desulfovibrio desulfuricans, respectively, while the 12-kDa FD of the T. vaginalis hydrogenosome was most similar to the 12-kDa FD of gamma-purple bacterium Pseudomonas putida. E. histolytica genes (and probably G. lamblia genes) encoding fermentation enzymes therefore likely derive from bacteria by horizontal transfer, although it is not clear from which bacteria these amebic genes derive. These are the first nonorganellar fermentation enzymes of eukaryotes implicated to have derived from bacteria. PMID:9171424

  18. Genome-Wide Gene Expression Patterns and Growth Requirements Suggest that Pelobacter carbinolicus Reduces Fe(III) Indirectly via Sulfide Production? †

    PubMed Central

    Haveman, Shelley A.; DiDonato, Raymond J.; Villanueva, Laura; Shelobolina, Evgenya S.; Postier, Bradley L.; Xu, Bo; Liu, Anna; Lovley, Derek R.

    2008-01-01

    Although Pelobacter species are closely related to Geobacter species, recent studies suggested that Pelobacter carbinolicus may reduce Fe(III) via a different mechanism because it lacks the outer-surface c-type cytochromes that are required for Fe(III) reduction by Geobacter sulfurreducens. Investigation into the mechanisms for Fe(III) reduction demonstrated that P. carbinolicus had growth yields on both soluble and insoluble Fe(III) consistent with those of other Fe(III)-reducing bacteria. Comparison of whole-genome transcript levels during growth on Fe(III) versus fermentative growth demonstrated that the greatest apparent change in gene expression was an increase in transcript levels for four contiguous genes. These genes encode two putative periplasmic thioredoxins; a putative outer-membrane transport protein; and a putative NAD(FAD)-dependent dehydrogenase with homology to disulfide oxidoreductases in the N terminus, rhodanese (sulfurtransferase) in the center, and uncharacterized conserved proteins in the C terminus. Unlike G. sulfurreducens, transcript levels for cytochrome genes did not increase in P. carbinolicus during growth on Fe(III). P. carbinolicus could use sulfate as the sole source of sulfur during fermentative growth, but required elemental sulfur or sulfide for growth on Fe(III). The increased expression of genes potentially involved in sulfur reduction, coupled with the requirement for sulfur or sulfide during growth on Fe(III), suggests that P. carbinolicus reduces Fe(III) via an indirect mechanism in which (i) elemental sulfur is reduced to sulfide and (ii) the sulfide reduces Fe(III) with the regeneration of elemental sulfur. This contrasts with the direct reduction of Fe(III) that has been proposed for Geobacter species. PMID:18515480

  19. The ability to perceive changes in the internal and external environment and modify the activity of genes

    E-print Network

    Higgins, Darren

    , such as proteases, peptidyl-prolyl- isomerases (PPIases), chaperones and thiol:disulfide oxido-reductases (for example, protein disulfide isomer- ase II, which is involved in disulfide-bond formation), as well

  20. The p53-inducible gene 3 involved in flavonoid-induced cytotoxicity through the reactive oxygen species-mediated mitochondrial apoptotic pathway in human hepatoma cells.

    PubMed

    Zhang, Qiang; Cheng, Guangdong; Qiu, Hongbin; Zhu, Liling; Ren, Zhongjuan; Zhao, Wei; Zhang, Tao; Liu, Lei

    2015-05-13

    Flavonoids have been reported to exhibit prooxidant cytotoxicity against cancer cells, but the underlying mechanism is still poorly understood. Here we investigated the potential mechanism that p53-inducible gene 3 (PIG3), a NADPH:quinone oxidoreductase, mediated the prooxidant cytotoxicity of flavonoids on human hepatoma HepG2 cells. The results showed that flavonoids (apigenin, luteolin, kaempferol, and quercetin) inhibited the growth of HepG2 cells in a dosage- and time-dependent manner, and induced the morphological changes characteristic of apoptosis in HepG2 cells. We also found that expression of PIG3 was increased markedly in HepG2 cells treated with flavonoids at both mRNA and protein levels, which was accompanied by increased intracellular ROS production and a decreased mitochondrial membrane potential (??m). All these effects were largely reversed through knockdown of the PIG3 gene in HepG2 cells. Western blotting indicated that flavonoids increased cytochrome c release, upregulated the ratio of Bax/Bcl-2, and activated the caspases-9 and -3. Moreover, knockdown of PIG3 could reverse the changes of these apoptotic-related proteins. These results suggest that PIG3 plays an important role in regulating the prooxidant activity and apoptosis-inducing action of flavonoids on HepG2 cells though the ROS-triggered mitochondrial apoptotic pathway. PMID:25820747

  1. Genes encoding the same three subunits of respiratory complex II are present in the mitochondrial DNA of two phylogenetically distant eukaryotes.

    PubMed Central

    Burger, G; Lang, B F; Reith, M; Gray, M W

    1996-01-01

    Although mitochondrial DNA is known to encode a limited number (<20) of the polypeptide components of respiratory complexes I, III, IV, and V, genes for components of complex II [succinate dehydrogenase (ubiquinone); succinate:ubiquinone oxidoreductase, EC 1.3.5.1] are conspicuously lacking in mitochondrial genomes so far characterized. Here we show that the same three subunits of complex II are encoded in the mitochondrial DNA of two phylogenetically distant eukaryotes, Porphyra purpurea (a photosynthetic red alga) and Reclinomonas americana (a heterotrophic zooflagellate). These complex II genes, sdh2, sdh3, and sdh4, are homologs, respectively, of Escherichia coli sdhB, sdhC, and sdhD. In E. coli, sdhB encodes the iron-sulfur subunit of succinate dehydrogenase (SDH), whereas sdhC and sdhD specify, respectively, apocytochrome b558 and a hydrophobic 13-kDa polypeptide, which together anchor SDH to the inner mitochondrial membrane. Amino acid sequence similarities indicate that sdh2, sdh3, and sdh4 were originally encoded in the protomitochondrial genome and have subsequently been transferred to the nuclear genome in most eukaryotes. The data presented here are consistent with the view that mitochondria constitute a monophyletic lineage. Images Fig. 1 Fig. 3 Fig. 4 PMID:8637872

  2. Do Housekeeping Genes Exist?

    PubMed Central

    Sun, Bingyun

    2015-01-01

    The searching of human housekeeping (HK) genes has been a long quest since the emergence of transcriptomics, and is instrumental for us to understand the structure of genome and the fundamentals of biological processes. The resolved genes are frequently used in evolution studies and as normalization standards in quantitative gene-expression analysis. Within the past 20 years, more than a dozen HK-gene studies have been conducted, yet none of them sampled human tissues completely. We believe an integration of these results will help remove false positive genes owing to the inadequate sampling. Surprisingly, we only find one common gene across 15 examined HK-gene datasets comprising 187 different tissue and cell types. Our subsequent analyses suggest that it might not be appropriate to rigidly define HK genes as expressed in all tissue types that have diverse developmental, physiological, and pathological states. It might be beneficial to use more robustly identified HK functions for filtering criteria, in which the representing genes can be a subset of genome. These genes are not necessarily the same, and perhaps need not to be the same, everywhere in our body. PMID:25970694

  3. Essential Bacillus subtilis genes.

    PubMed

    Kobayashi, K; Ehrlich, S D; Albertini, A; Amati, G; Andersen, K K; Arnaud, M; Asai, K; Ashikaga, S; Aymerich, S; Bessieres, P; Boland, F; Brignell, S C; Bron, S; Bunai, K; Chapuis, J; Christiansen, L C; Danchin, A; Débarbouille, M; Dervyn, E; Deuerling, E; Devine, K; Devine, S K; Dreesen, O; Errington, J; Fillinger, S; Foster, S J; Fujita, Y; Galizzi, A; Gardan, R; Eschevins, C; Fukushima, T; Haga, K; Harwood, C R; Hecker, M; Hosoya, D; Hullo, M F; Kakeshita, H; Karamata, D; Kasahara, Y; Kawamura, F; Koga, K; Koski, P; Kuwana, R; Imamura, D; Ishimaru, M; Ishikawa, S; Ishio, I; Le Coq, D; Masson, A; Mauël, C; Meima, R; Mellado, R P; Moir, A; Moriya, S; Nagakawa, E; Nanamiya, H; Nakai, S; Nygaard, P; Ogura, M; Ohanan, T; O'Reilly, M; O'Rourke, M; Pragai, Z; Pooley, H M; Rapoport, G; Rawlins, J P; Rivas, L A; Rivolta, C; Sadaie, A; Sadaie, Y; Sarvas, M; Sato, T; Saxild, H H; Scanlan, E; Schumann, W; Seegers, J F M L; Sekiguchi, J; Sekowska, A; Séror, S J; Simon, M; Stragier, P; Studer, R; Takamatsu, H; Tanaka, T; Takeuchi, M; Thomaides, H B; Vagner, V; van Dijl, J M; Watabe, K; Wipat, A; Yamamoto, H; Yamamoto, M; Yamamoto, Y; Yamane, K; Yata, K; Yoshida, K; Yoshikawa, H; Zuber, U; Ogasawara, N

    2003-04-15

    To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximately 4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life. PMID:12682299

  4. Essential Bacillus subtilis genes

    PubMed Central

    Kobayashi, K.; Ehrlich, S. D.; Albertini, A.; Amati, G.; Andersen, K. K.; Arnaud, M.; Asai, K.; Ashikaga, S.; Aymerich, S.; Bessieres, P.; Boland, F.; Brignell, S. C.; Bron, S.; Bunai, K.; Chapuis, J.; Christiansen, L. C.; Danchin, A.; Débarbouillé, M.; Dervyn, E.; Deuerling, E.; Devine, K.; Devine, S. K.; Dreesen, O.; Errington, J.; Fillinger, S.; Foster, S. J.; Fujita, Y.; Galizzi, A.; Gardan, R.; Eschevins, C.; Fukushima, T.; Haga, K.; Harwood, C. R.; Hecker, M.; Hosoya, D.; Hullo, M. F.; Kakeshita, H.; Karamata, D.; Kasahara, Y.; Kawamura, F.; Koga, K.; Koski, P.; Kuwana, R.; Imamura, D.; Ishimaru, M.; Ishikawa, S.; Ishio, I.; Le Coq, D.; Masson, A.; Mauël, C.; Meima, R.; Mellado, R. P.; Moir, A.; Moriya, S.; Nagakawa, E.; Nanamiya, H.; Nakai, S.; Nygaard, P.; Ogura, M.; Ohanan, T.; O'Reilly, M.; O'Rourke, M.; Pragai, Z.; Pooley, H. M.; Rapoport, G.; Rawlins, J. P.; Rivas, L. A.; Rivolta, C.; Sadaie, A.; Sadaie, Y.; Sarvas, M.; Sato, T.; Saxild, H. H.; Scanlan, E.; Schumann, W.; Seegers, J. F. M. L.; Sekiguchi, J.; Sekowska, A.; Séror, S. J.; Simon, M.; Stragier, P.; Studer, R.; Takamatsu, H.; Tanaka, T.; Takeuchi, M.; Thomaides, H. B.; Vagner, V.; van Dijl, J. M.; Watabe, K.; Wipat, A.; Yamamoto, H.; Yamamoto, M.; Yamamoto, Y.; Yamane, K.; Yata, K.; Yoshida, K.; Yoshikawa, H.; Zuber, U.; Ogasawara, N.

    2003-01-01

    To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among ?4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden–Meyerhof–Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life. PMID:12682299

  5. Development of Signature-Tagged Mutagenesis in Burkholderia pseudomallei To Identify Genes Important in Survival and Pathogenesis?

    PubMed Central

    Cuccui, J.; Easton, A.; Chu, K. K.; Bancroft, G. J.; Oyston, P. C. F.; Titball, R. W.; Wren, B. W.

    2007-01-01

    Burkholderia pseudomallei, the causative agent of melioidosis, is an important human pathogen in Southeast Asia and northern Australia for which a vaccine is unavailable. A panel of 892 double signature-tagged mutants was screened for virulence using an intranasal BALB/c mouse model of infection. A novel DNA tag microarray identified 33 mutants as being attenuated in spleens, while 6 were attenuated in both lungs and spleens. The transposon insertion sites in spleen-attenuated mutants revealed genes involved in several stages of capsular polysaccharide biosynthesis and DNA replication and repair, a putative oxidoreductase, ABC transporters, and a lipoprotein that may be important in intercellular spreading. The six mutants identified as missing in both lungs and spleens were found to have insertions in recA involved in the SOS response and DNA repair; putative auxotrophs of leucine, thr