Sample records for nadph-dependent oxidoreductase genes

  1. Characterization of the Kluyveromyces marxianus strain DMB1 YGL157w gene product as a broad specificity NADPH-dependent aldehyde reductase.

    PubMed

    Akita, Hironaga; Watanabe, Masahiro; Suzuki, Toshihiro; Nakashima, Nobutaka; Hoshino, Tamotsu

    2015-01-01

    The open reading frame YGL157w in the genome of the yeast Kluyveromyces marxianus strain DMB1 encodes a putative uncharacterized oxidoreductase. However, this protein shows 46% identity with the Saccharomyces cerevisiae S288c NADPH-dependent methylglyoxal reductase, which exhibits broad substrate specificity for aldehydes. In the present study, the YGL157w gene product (KmGRE2) was purified to homogeneity from overexpressing Escherichia coli cells and found to be a monomer. The enzyme was strictly specific for NADPH and was active with a wide variety of substrates, including aliphatic (branched-chain and linear) and aromatic aldehydes. The optimal pH for methylglyoxal reduction was 5.5. With methylglyoxal as a substrate, the optimal temperature for enzyme activity at pH 5.5 was 45°C. The enzyme retained more than 70% of its activity after incubation for 30 min at temperatures below 35°C or at pHs between 5.5 and 9.0. In addition, the KmGRE2-overexpressing E. coli showed improved growth when cultivated in cedar hydrolysate, as compared to cells not expressing the enzyme. Taken together, these results indicate that KmGRE2 is potentially useful as an inhibit decomposer in E. coli cells. PMID:25852994

  2. A novel NADPH-dependent aldehyde reductase gene from Saccharomyces cerevisiae NRRL Y-12632 involved in the detoxification of aldehyde inhibitors derived from lignocellulosic biomass conversion.

    PubMed

    Liu, Z Lewis; Moon, Jaewoong

    2009-10-01

    Aldehyde inhibitors such as furfural, 5-hydroxymethylfurfural, anisaldehyde, benzaldehyde, cinnamaldehyde, and phenylaldehyde are commonly generated during lignocellulosic biomass conversion process for low-cost cellulosic ethanol production that interferes with subsequent microbial growth and fermentation. In situ detoxification of the aldehyde inhibitors is possible by the tolerant ethanologenic yeast that involves multiple genes including numerous functional reductases. In this study, we report a novel aldehyde reductase gene clone Y63 from ethanologenic yeast Saccharomyces cerevisiae NRRL Y12632, representing the uncharacterized ORF YGL157W, which demonstrated NADPH-dependent reduction activities toward at least 14 aldehyde substrates. The identity of gene clone Y63 is the same with YGL157W of SGD since a variation of only 35 nucleotides in genomic sequence and three amino acid residues were observed between the two that share the same length of 347 residues in size. As one among the highly induced genes, YGL157W of Y-12632 showed significantly high levels of transcript abundance in response to furfural and HMF challenges. Based on the deduced amino acid sequence and the most conserved functional motif analyses including closely related reductases from five other yeast species to this date, YGL157W was identified as a member of the subclass 'intermediate' of the SDR (short-chain dehydrogenase/reductase) superfamily with the following typical characteristics: the most conserved catalytic site to lie at Tyr(169)-X-X-X-Lys(173); an indispensable reduction catalytic triad at Ser(131), Tyr(169), and Lys(173), and an approved cofactor-binding motif at Gly(11)-X-X-Gly(14)-X-X-Ala(17) near the N-terminus. YGL039W, YDR541C, and YOL151W (GRE2) appeared to be the similar type of enzymes falling into the same category of the intermediate subfamily. PMID:19577617

  3. Expression pattern of a chloroplast NADPH-dependent thioredoxin reductase in Chlorella vulgaris during hardening and its interaction with 2-Cys peroxiredoxin.

    PubMed

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

    2009-03-23

    A chloroplastic NADPH-dependent thioredoxin reductase gene was identified from Chlorella vulgaris and designated CvNTRC. Mature CvNTRC protein (mCvNTRC) was expressed in Escherichia coli, and it showed both NADPH-dependent thioredoxin reductase (NTR) and thioredoxin (Trx)-like dithiol-disulfide oxidoreductase activities. The transcript of CvNTRC increased throughout 24-h hardening, whereas the encoded protein amount and total NTR activity decreased once and then increased during hardening. By in vitro pull-down assay, a 21.2-kDa protein bound to mCvNTRC was isolated and identified as a 2-Cys peroxiredoxin (2-Cys Prx) based on the N-terminal sequence. These data suggest that CvNTRC is maintained at a constant level during hardening and functions as an antioxidant with 2-Cys Prx in the acquisition of freezing tolerance of Chlorella. PMID:19270395

  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 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. ...

  6. NADPH-dependent oxidation of benzidine by rat liver

    Microsoft Academic Search

    Vijaya M. Lakshmi; Nathan T. Zenser; F. F. Hsu; Michael B. Mattammal; Terry V. Zenser; Bernard B. Davis

    1996-01-01

    This study used liver microsomes from control and |}- naphthoflavone-treated rats to evaluate NADPH-dependent oxidation of benzidine. With microsomes from p^-naphtho- flavone-treated rats, the rates of formation of aqueous soluble metabolite (HPLC analysis) and protein and DNA binding were 835 ± 81, 14.5 ± 1.8 and 0.71 ± 0.14 pmol\\/ mg\\/min respectively. fJ-Naphthoflavone treatment elicited 12.3-, 1.8- and 14.2-fold increases

  7. An NADPH-dependent genetic switch regulates plant infection by the rice blast fungus.

    PubMed

    Wilson, Richard A; Gibson, Robert P; Quispe, Cristian F; Littlechild, Jennifer A; Talbot, Nicholas J

    2010-12-14

    To cause rice blast disease, the fungus Magnaporthe oryzae breaches the tough outer cuticle of the rice leaf by using specialized infection structures called appressoria. These cells allow the fungus to invade the host plant and proliferate rapidly within leaf tissue. Here, we show that a unique NADPH-dependent genetic switch regulates plant infection in response to the changing nutritional and redox conditions encountered by the pathogen. The biosynthetic enzyme trehalose-6-phosphate synthase (Tps1) integrates control of glucose-6-phosphate metabolism and nitrogen source utilization by regulating the oxidative pentose phosphate pathway, the generation of NADPH, and the activity of nitrate reductase. We report that Tps1 directly binds to NADPH and, thereby, regulates a set of related transcriptional corepressors, comprising three proteins, Nmr1, Nmr2, and Nmr3, which can each bind NADP. Targeted deletion of any of the Nmr-encoding genes partially suppresses the nonpathogenic phenotype of a ?tps1 mutant. Tps1-dependent Nmr corepressors control the expression of a set of virulence-associated genes that are derepressed during appressorium-mediated plant infection. When considered together, these results suggest that initiation of rice blast disease by M. oryzae requires a regulatory mechanism involving an NADPH sensor protein, Tps1, a set of NADP-dependent transcriptional corepressors, and the nonconsuming interconversion of NADPH and NADP acting as signal transducer. PMID:21115813

  8. Coregulated genes link sulfide:quinone oxidoreductase and arsenic metabolism in Synechocystis sp. strain PCC6803.

    PubMed

    Nagy, Csaba I; Vass, Imre; Rákhely, Gábor; Vass, István Zoltán; Tóth, András; Duzs, Agnes; Peca, Loredana; Kruk, Jerzy; Kós, Péter B

    2014-10-01

    Although the biogeochemistry of the two environmentally hazardous compounds arsenic and sulfide has been extensively investigated, the biological interference of these two toxic but potentially energy-rich compounds has only been hypothesized and indirectly proven. Here we provide direct evidence for the first time that in the photosynthetic model organism Synechocystis sp. strain PCC6803 the two metabolic pathways are linked by coregulated genes that are involved in arsenic transport, sulfide oxidation, and probably in sulfide-based alternative photosynthesis. Although Synechocystis sp. strain PCC6803 is an obligate photoautotrophic cyanobacterium that grows via oxygenic photosynthesis, we discovered that specific genes are activated in the presence of sulfide or arsenite to exploit the energy potentials of these chemicals. These genes form an operon that we termed suoRSCT, located on a transposable element of type IS4 on the plasmid pSYSM of the cyanobacterium. suoS (sll5036) encodes a light-dependent, type I sulfide:quinone oxidoreductase. The suoR (sll5035) gene downstream of suoS encodes a regulatory protein that belongs to the ArsR-type repressors that are normally involved in arsenic resistance. We found that this repressor has dual specificity, resulting in 200-fold induction of the operon upon either arsenite or sulfide exposure. The suoT gene encodes a transmembrane protein similar to chromate transporters but in fact functioning as an arsenite importer at permissive concentrations. We propose that the proteins encoded by the suoRSCT operon might have played an important role under anaerobic, reducing conditions on primordial Earth and that the operon was acquired by the cyanobacterium via horizontal gene transfer. PMID:25022856

  9. Coregulated Genes Link Sulfide:Quinone Oxidoreductase and Arsenic Metabolism in Synechocystis sp. Strain PCC6803

    PubMed Central

    Nagy, Csaba I.; Vass, Imre; Rákhely, Gábor; Vass, István Zoltán; Tóth, András; Duzs, Ágnes; Peca, Loredana; Kruk, Jerzy

    2014-01-01

    Although the biogeochemistry of the two environmentally hazardous compounds arsenic and sulfide has been extensively investigated, the biological interference of these two toxic but potentially energy-rich compounds has only been hypothesized and indirectly proven. Here we provide direct evidence for the first time that in the photosynthetic model organism Synechocystis sp. strain PCC6803 the two metabolic pathways are linked by coregulated genes that are involved in arsenic transport, sulfide oxidation, and probably in sulfide-based alternative photosynthesis. Although Synechocystis sp. strain PCC6803 is an obligate photoautotrophic cyanobacterium that grows via oxygenic photosynthesis, we discovered that specific genes are activated in the presence of sulfide or arsenite to exploit the energy potentials of these chemicals. These genes form an operon that we termed suoRSCT, located on a transposable element of type IS4 on the plasmid pSYSM of the cyanobacterium. suoS (sll5036) encodes a light-dependent, type I sulfide:quinone oxidoreductase. The suoR (sll5035) gene downstream of suoS encodes a regulatory protein that belongs to the ArsR-type repressors that are normally involved in arsenic resistance. We found that this repressor has dual specificity, resulting in 200-fold induction of the operon upon either arsenite or sulfide exposure. The suoT gene encodes a transmembrane protein similar to chromate transporters but in fact functioning as an arsenite importer at permissive concentrations. We propose that the proteins encoded by the suoRSCT operon might have played an important role under anaerobic, reducing conditions on primordial Earth and that the operon was acquired by the cyanobacterium via horizontal gene transfer. PMID:25022856

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

    PubMed Central

    2006-01-01

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

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

    Microsoft Academic Search

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

    2010-01-01

    Published polymerase chain reaction primer sets for detecting the genes encoding 16S rRNA gene and hydrazine oxidoreductase\\u000a (hzo) in anammox bacteria were compared by using the same coastal marine sediment samples. While four previously reported primer\\u000a sets developed to detect the 16S rRNA gene showed varying specificities between 12% and 77%, an optimized primer combination\\u000a resulted in up to 98%

  12. Diversity and Spatial Distribution of Hydrazine Oxidoreductase (hzo) Gene in the Oxygen Minimum Zone Off Costa Rica

    PubMed Central

    Kong, Liangliang; Jing, Hongmei; Kataoka, Takafumi; Buchwald, Carolyn; Liu, Hongbin

    2013-01-01

    Anaerobic ammonia oxidation (anammox) as an important nitrogen loss pathway has been reported in marine oxygen minimum zones (OMZs), but the community composition and spatial distribution of anammox bacteria in the eastern tropical North Pacific (ETNP) OMZ are poorly determined. In this study, anammox bacterial communities in the OMZ off Costa Rica (CRD-OMZ) were analyzed based on both hydrazine oxidoreductase (hzo) genes and their transcripts assigned to cluster 1 and 2. The anammox communities revealed by hzo genes and proteins in CRD-OMZ showed a low diversity. Gene quantification results showed that hzo gene abundances peaked in the upper OMZs, associated with the peaks of nitrite concentration. Nitrite and oxygen concentrations may therefore colimit the distribution of anammox bacteria in this area. Furthermore, transcriptional activity of anammox bacteria was confirmed by obtaining abundant hzo mRNA transcripts through qRT-PCR. A novel hzo cluster 2x clade was identified by the phylogenetic analysis and these novel sequences were abundant and widely distributed in this environment. Our study demonstrated that both cluster 1 and 2 anammox bacteria play an active role in the CRD-OMZ, and the cluster 1 abundance and transcriptional activity were higher than cluster 2 in both free-living and particle-attached fractions at both gene and transcriptional levels. PMID:24205176

  13. Development of an NADPH-dependent homophenylalanine dehydrogenase by protein engineering.

    PubMed

    Li, Han; Liao, James C

    2014-01-17

    l-Homophenylalanine is a nonproteinogenic amino acid and can be used as a versatile pharmaceutical intermediate. Production of l-homophenylalanine involves amination of the keto acid precursor 2-oxo-4-phenylbutyric acid (2-OPBA), which can be accomplished by bioenzymatic processes. Current biocatalysts for this reaction include transaminases and NADH-dependent phenylalanine dehydrogenases, which are not optimal for metabolic engineering of whole-cell biocatalysis. Here, we report the development of an NADPH-dependent homophenylalanine dehydrogenase by engineering the NADPH-dependent glutamate dehydrogenase (GDH) from Escherichia coli, which provides a new tool for in vitro catalysis and in vivo metabolic engineering. We took a stepwise substrate walking strategy: the first round directed evolution switched GDH's substrate specificity from its natural substrate 2-ketoglutarate to the intermediate target phenylpyruvate, which has similar structure as 2-OPBA; and the second round further improved the enzyme's catalytic efficiency toward the final target 2-OPBA. Compared to wild type GDH, the catalytic efficiency (kcat/Km) of the final mutant was ?100 fold higher for 2-OPBA and ?3000 fold lower for the original substrate 2-ketoglutarate. When overexpressed in E. coli, the engineered GDH aminated 2-OPBA to l-homophenylalanine more effectively than the transaminases and NADH-dependent phenylalanine dehydrogenase, possibly because it utilizes the strong anabolic driving force NADPH under aerobic condition. PMID:24053171

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

    PubMed

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

    2013-06-10

    The xylose-fermenting recombinant Saccharomyces cerevisiae and its improvement have been studied extensively. The redox balance between xylose reductase (XR) and xylitol dehydrogenase (XDH) is thought to be an important factor in effective xylose fermentation. Using protein engineering, we previously successfully reduced xylitol accumulation and improved ethanol production by reversing the dependency of XDH from NAD(+) to NADP(+). We also constructed a set of novel strictly NADPH-dependent XR from Pichia stipitis by site-directed mutagenesis. In the present study, we constructed a set of recombinant S. cerevisiae carrying a novel set of mutated strictly NADPH-dependent XR and NADP(+)-dependent XDH genes with overexpression of endogenous xylulokinase (XK) to study the effects of complete NADPH/NADP(+) recycling on ethanol fermentation and xylitol accumulation. All mutated strains demonstrated reduced xylitol accumulation, ranging 34.4-54.7% compared with the control strain. Moreover, compared with the control strain, the two strains showed 20% and 10% improvement in ethanol production. PMID:23578809

  15. 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

  16. 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

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

    PubMed

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

    2013-02-01

    Sufficient supply of NADPH is one of the most important factors affecting the productivity of biotransformation processes. In this study, construction of an efficient NADPH-regenerating system was attempted using direct phosphorylation of NADH by NADH kinase (Pos5p) from Saccharomyces cerevisiae for producing guanosine diphosphate (GDP)-L-fucose and ?-caprolactone in recombinant Escherichia coli. Expression of Pos5p in a fed-batch culture of recombinant E. coli producing GDP-L-fucose resulted in a maximum GDP-L-fucose concentration of 291.5 mg/l, which corresponded to a 51 % enhancement compared with the control strain. In a fed-batch Baeyer-Villiger (BV) oxidation of cyclohexanone using recombinant E. coli expressing Pos5p, a maximum ?-caprolactone concentration of 21.6 g/l was obtained, which corresponded to a 96 % enhancement compared with the control strain. Such an increase might be due to the enhanced availability of NADPH in recombinant E. coli expressing Pos5p. These results suggested that efficient regeneration of NADPH was possible by functional expression of Pos5p in recombinant E. coli, which can be applied to other NADPH-dependent biotransformation processes in E. coli. PMID:23053084

  18. 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

  19. Three-dimensional Structure and Enzymatic Function of Proapoptotic Human p53-inducible Quinone Oxidoreductase PIG3*

    PubMed Central

    Porté, Sergio; Valencia, Eva; Yakovtseva, Evgenia A.; Borràs, Emma; Shafqat, Naeem; Debreczeny, Judit É.; Pike, Ashley C. W.; Oppermann, Udo; Farrés, Jaume; Fita, Ignacio; Parés, Xavier

    2009-01-01

    Tumor suppressor p53 regulates the expression of p53-induced genes (PIG) that trigger apoptosis. PIG3 or TP53I3 is the only known member of the medium chain dehydrogenase/reductase superfamily induced by p53 and is used as a proapoptotic marker. Although the participation of PIG3 in the apoptotic pathway is proven, the protein and its mechanism of action were never characterized. We analyzed human PIG3 enzymatic function and found NADPH-dependent reductase activity with ortho-quinones, which is consistent with the classification of PIG3 in the quinone oxidoreductase family. However, the activity is much lower than that of ?-crystallin, a better known quinone oxidoreductase. In addition, we report the crystallographic structure of PIG3, which allowed the identification of substrate- and cofactor-binding sites, with residues fully conserved from bacteria to human. Tyr-59 in ?-crystallin (Tyr-51 in PIG3) was suggested to participate in the catalysis of quinone reduction. However, kinetics of Tyr/Phe and Tyr/Ala mutants of both enzymes demonstrated that the active site Tyr is not catalytic but may participate in substrate binding, consistent with a mechanism based on propinquity effects. It has been proposed that PIG3 contribution to apoptosis would be through oxidative stress generation. We found that in vitro activity and in vivo overexpression of PIG3 accumulate reactive oxygen species. Accordingly, an inactive PIG3 mutant (S151V) did not produce reactive oxygen species in cells, indicating that enzymatically active protein is necessary for this function. This supports that PIG3 action is through oxidative stress produced by its enzymatic activity and provides essential knowledge for eventual control of apoptosis. PMID:19349281

  20. The Mycobacterium avium subsp. paratuberculosis MAP3464 gene encodes an oxidoreductase involved in invasion of bovine epithelial cells through the activation of host cell Cdc42.

    PubMed

    Alonso-Hearn, Marta; Patel, Dilip; Danelishvili, Lia; Meunier-Goddik, Lisbeth; Bermudez, Luiz E

    2008-01-01

    Mycobacterium avium subsp. paratuberculosis infection of cattle takes place through the intestinal mucosa. To identify M. avium subsp. paratuberculosis genes associated with the invasion of bovine epithelial cells in vitro, we screened a library of transposon mutants. Several mutants of M. avium subsp. paratuberculosis were identified which invaded Madin-Darby bovine kidney (MDBK) epithelial cells less efficiently than wild-type (wt) M. avium subsp. paratuberculosis. The deltaOx mutant had the transposon located in the MAP3464 gene, a putative oxidoreductase gene whose expression is upregulated upon bacterial contact with MDBK cells. Complete restoration of invasion comparable to that for the wt bacterium was achieved by introducing a copy of the complete oxidoreductase operon into the deltaOx mutant. Immunoprecipitation and Western blot analysis indicated that wt M. avium subsp. paratuberculosis activates Cdc42 and RhoA pathways of internalization 15 and 60 min after infection of the host cell, respectively. The deltaOx mutant, however, failed to activate the Cdc42 pathway. To determine whether an M. avium subsp. paratuberculosis protein delivered to the host cell mediates the entry of the wt bacterium by activation of the Cdc42 pathway, affinity precipitation of active Cdc42 from MDBK-infected cells followed by mass spectrometry was carried out. We identified a 17-amino-acid bacterial peptide associated with the Cdc42 of cells infected with wt M. avium subsp. paratuberculosis but not with the deltaOx mutant. The sequence of the peptide matches MAP3985c, a hypothetical protein, possibly functioning as a putative Cdc42 effector. These findings reveal a novel signaling pathway activated during M. avium subsp. paratuberculosis entry that links the product of MAP3464 gene to activation of Cdc42 in the host cell. PMID:17938223

  1. Marked increases in hepatic NAD(P)H:oxidoreductase gene transcription and mRNA levels correlated with a mouse chromosome 7 deletion.

    PubMed Central

    Petersen, D D; Gonzalez, F J; Rapic, V; Kozak, C A; Lee, J Y; Jones, J E; Nebert, D W

    1989-01-01

    The NAD(P)H:menadione oxidoreductase gene (Nmo-1) codes for a quinone reductase (also called DT diaphorase; EC 1.6.99.2) believed to play a central role in protection against oxidative stress. We have studied mice with a radiation-induced chromosomal deletion involving the albino locus (c) on chromosome 7 and found that Nmo-1 mRNA levels and the rate of Nmo-1 gene transcription are markedly increased (greater than 100-fold and greater than 12-fold, respectively) in the untreated c14CoS/c14CoS deletion homozygote, compared with the untreated Cch/Cch wild-type and the Cch/C14CoS heterozygote. These data suggest that a gene located on chromosome 7 encodes a trans-acting regulatory factor that might be a negative effector of the Nmo-1 gene, which we show here is located on chromosome 8 approximately 1.4 centimorgans (about 1000 kilobase pairs) from the Es-2 gene. Conversely, there are no detectable basal levels of cytochrome P1450 (Cyp1a1 gene) or cytochrome P3450 (Cyp1a2 gene) mRNA, indicating that the regulation of basal expression of the Cyp1a1 and Cyp1a2 genes is distinct from that of the Nmo-1 gene. Moreover, the Cyp1a1 and Cyp1a2 genes and the Nmo-1 gene are induced by tetrachlorodibenzo-p-dioxin in the cch/cch, cch/c14CoS, and c14CoS/c14CoS mice. The mechanism of tetrachlorodibenzo-p-dioxin inducibility of the Cyp1a1, Cyp1a2, and Nmo-1 genes is, therefore, independent of the mechanism of Nmo-1 gene activation in untreated c14CoS/c14CoS mice. Images PMID:2505256

  2. 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

  3. Beta hydroxylation of glycolipids from Ustilago maydis and Pseudozyma flocculosa by an NADPH-dependent ?-hydroxylase.

    PubMed

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

    2011-11-01

    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

  4. Nrf1 and Nrf2 Positively and c-Fos and Fral Negatively Regulate the Human Antioxidant Response Element-Mediated Expression of NAD(P)H:Quinone oxidoreductase1 Gene

    Microsoft Academic Search

    Radjendirane Venugopal; Anil K. Jaiswal

    1996-01-01

    Twenty-four base pairs of the human antioxidant response element (hARE) are required for high basal transcription of the NAD(P)H:quinone oxidoreductase1 (NQO1) gene and its induction in response to xenobiotics and antioxidants. hARE is a unique cis-element that contains one perfect and one imperfect AP1 element arranged as inverse repeats separated by 3 bp, followed by a ``GC'' box. We report

  5. Oxidative stress in tardive dyskinesia: Genetic association study and meta-analysis of NADPH quinine oxidoreductase 1 (NQO1) and Superoxide dismutase 2 (SOD2, MnSOD) genes

    Microsoft Academic Search

    Clement C. Zai; Arun K. Tiwari; Vincenzo Basile; Vincenzo de Luca; Daniel J. Müller; Aristotle N. Voineskos; Gary Remington; Herbert Y. Meltzer; Jeffrey A. Lieberman; Steven G. Potkin; James L. Kennedy

    2010-01-01

    IntroductionTardive dyskinesia (TD) is a potentially irreversible side effect of antipsychotic medication treatment that occurs in approximately 25% of chronically treated schizophrenia patients. Oxidative stress has been one of the proposed mechanisms influencing TD risk. Pae et al. (2004) originally reported a significant association between TD and the NADPH quinine oxidoreductase 1 (NQO1) gene Pro187Ser (C609T, rs1800566) polymorphism in Korean

  6. 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

  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. The oxen gene of Drosophila encodes a homolog of subunit 9 of yeast ubiquinol-cytochrome c oxidoreductase complex: evidence for modulation of gene expression in response to mitochondrial activity.

    PubMed Central

    Frolov, M V; Benevolenskaya, E V; Birchler, J A

    2000-01-01

    A P-element insertion in the oxen gene, ox(1), has been isolated in a search for modifiers of white gene expression. The mutation preferentially exerts a negative dosage effect upon the expression of three genes encoding ABC transporters involved in pigment precursor transport, white, brown, and scarlet. A precise excision of the P element reverts the mutant phenotype. Five different transcription units were identified around the insertion site. To distinguish a transcript responsible for the mutant phenotype, a set of deletions within the oxen region was generated. Analysis of gene expression within the oxen region in the case of deletions as well as generation of transgenic flies allowed us to identify the transcript responsible for oxen function. It encodes a 6.6-kD homolog of mitochondrial ubiquinol cytochrome c oxidoreductase (QCR9), subunit 9 of the bc(1) complex in yeast. In addition to white, brown, and scarlet, oxen regulates the expression of three of seven tested genes. Thus, our data provide additional evidence for a cellular response to changes in mitochondrial function. The oxen mutation provides a model for the genetic analysis in multicellular organisms of the effect of mitochondrial activity on nuclear gene expression. PMID:11102369

  9. The oxen gene of Drosophila encodes a homolog of subunit 9 of yeast ubiquinol-cytochrome c oxidoreductase complex: evidence for modulation of gene expression in response to mitochondrial activity.

    PubMed

    Frolov, M V; Benevolenskaya, E V; Birchler, J A

    2000-12-01

    A P-element insertion in the oxen gene, ox(1), has been isolated in a search for modifiers of white gene expression. The mutation preferentially exerts a negative dosage effect upon the expression of three genes encoding ABC transporters involved in pigment precursor transport, white, brown, and scarlet. A precise excision of the P element reverts the mutant phenotype. Five different transcription units were identified around the insertion site. To distinguish a transcript responsible for the mutant phenotype, a set of deletions within the oxen region was generated. Analysis of gene expression within the oxen region in the case of deletions as well as generation of transgenic flies allowed us to identify the transcript responsible for oxen function. It encodes a 6.6-kD homolog of mitochondrial ubiquinol cytochrome c oxidoreductase (QCR9), subunit 9 of the bc(1) complex in yeast. In addition to white, brown, and scarlet, oxen regulates the expression of three of seven tested genes. Thus, our data provide additional evidence for a cellular response to changes in mitochondrial function. The oxen mutation provides a model for the genetic analysis in multicellular organisms of the effect of mitochondrial activity on nuclear gene expression. PMID:11102369

  10. The superoxide-generating oxidase of leucocytes. NADPH-dependent reduction of flavin and cytochrome b in solubilized preparations.

    PubMed Central

    Cross, A R; Parkinson, J F; Jones, O T

    1984-01-01

    An NADPH-dependent O2.- -generating oxidase was solubilized from phorbol 12-myristate 13-acetate-activated pig neutrophils by using a mixture of detergents. Recovery of oxidase was approx. 40%. The extract contained cytochrome b-245 (331 pmol/mg of protein) and FAD (421 pmol/mg of protein); approx. 30% of each was reduced within 60s when NADPH was added to anaerobic incubations. Three different additives, quinacrine, p-chloromercuribenzoate and cetyltrimethylammonium bromide, strongly inhibited O2.- generation; they also inhibited the reduction by NADPH of cytochrome b at the same low concentrations. In the presence of p-chloromercuribenzoate cytochrome b reduction was strongly inhibited and flavin reduction was less inhibited. A detergent extract prepared from non-stimulated neutrophils also contained flavin and cytochrome b, but its rate of O2.- production was less than 1% of that from activated cells; its initial rate of cytochrome b and flavin reduction was low, although the state of reduction at equilibrium was similar to that of extracts of activated cells. Even in the non-activated cell extract the reduction of flavin and cytochrome was made fast and complete when Methyl Viologen was added to the anaerobic incubations. The oxidase was temperature-sensitive, with a sharp maximum at 25 degrees C; temperatures above this caused loss of O2.- generation, and this coincided with loss of the characteristic cytochrome b spectrum, indicate of denaturation of the cytochrome. The cytochrome b formed a complex with butyl isocyanide (close to 100% binding at 10mM); butyl isocyanide also inhibited the oxidase activity of stimulated whole neutrophils (22.5% inhibition at 10mM). Photoreduced FMN stimulated O2 uptake by the oxidase. The results support a scheme of electron transport within the oxidase complex involving NADPH, FAD, cytochrome b-245 and O2 in that sequence. PMID:6497852

  11. 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

  12. 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

  13. The P450 oxidoreductase, RedA, controls development beyond the mound stage in Dictyostelium discoideum

    Microsoft Academic Search

    Daniela C Gonzalez-Kristeller; Layla Farage; Leonardo C Fiorini; William F Loomis; Aline M da Silva

    2008-01-01

    BACKGROUND: NADPH-cytochrome-P450 oxidoreductase (CPR) is a ubiquitous enzyme that belongs to a family of diflavin oxidoreductases and is required for activity of the microsomal cytochrome-P450 monooxygenase system. CPR gene-disruption experiments have demonstrated that absence of this enzyme causes developmental defects both in mouse and insect. RESULTS: Annotation of the sequenced genome of D. discoideum revealed the presence of three genes

  14. Soil oxidoreductases and FDA hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The oxidoreductases (E.C. 1.) comprise the largest enzyme group and consist of enzymes that catalyze reactions between two compounds, one of which is oxidized (the donor) while reducing the other (the acceptor) (Dixon and Webb, 1979). In common with all redox reactions, the reaction mechanism involv...

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

    PubMed Central

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

    1995-01-01

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

  16. NADPH-dependent covalent binding of [3H]paroxetine to human liver microsomes and S-9 fractions: identification of an electrophilic quinone metabolite of paroxetine.

    PubMed

    Zhao, Sabrina X; Dalvie, Deepak K; Kelly, Joan M; Soglia, John R; Frederick, Kosea S; Smith, Evan B; Obach, R Scott; Kalgutkar, Amit S

    2007-11-01

    The primary pathway of clearance of the methylenedioxyphenyl-containing compound and selective serotonin reuptake inhibitor paroxetine in humans involves P450 2D6-mediated demethylenation to a catechol intermediate. The process of demethylenation also results in the mechanism-based inactivation of the P450 isozyme. While the link between P450 2D6 inactivation and pharmacokinetic interactions of paroxetine with P450 2D6 substrates has been firmly established, there is a disconnect in terms of paroxetine's excellent safety record despite the potential for bioactivation. In the present study, we have systematically assessed the NADPH-dependent covalent binding of [(3)H]paroxetine to human liver microsomes and S-9 preparations in the absence and presence of cofactors of the various phase II drug-metabolizing enzymes involved in the downstream metabolism/detoxification of the putative paroxetine-catechol intermediate. Incubation of [(3)H]paroxetine with human liver microsomes and S-9 preparations resulted in irreversible binding of radioactive material to macromolecules by a process that was NADPH-dependent. The addition of reduced glutathione (GSH) to the microsomal and S-9 incubations resulted in a dramatic reduction of covalent binding. Following incubations with NADPH- and GSH-supplemented human liver microsomes and S-9, three sulfydryl conjugates with MH(+) ions at 623 Da (GS1), 779 Da (GS2), and 928 Da (GS3), respectively, were detected by LC-MS/MS. The collision-induced dissociation spectra allowed an insight into the structure of the GSH conjugates, based on which, bioactivation pathways were proposed. The formation of GS 1 was consistent with Michael addition of GSH to the quinone derived from two-electron oxidation of paroxetine-catechol. GS 3 was formed by the addition of a second molecule of GSH to the quinone species obtained via the two-electron oxidation of GS 1. The mechanism of formation of GS 2 can be rationalized via (i) further two-electron oxidation of the catechol motif in GS 3 to the ortho-quinone, (ii) loss of a glutamic acid residue from one of the adducted GSH molecules, and (iii) condensation of a cysteine-NH 2 with an adjacent carbonyl of the ortho-quinone to yield an ortho-benzoquinoneimine structure. Inclusion of the catechol-O-methyltransferase cofactor S-adenosylmethionine (SAM) in S-9 incubations also dramatically reduced the covalent binding of [(3)H]paroxetine, a finding that was consistent with O-methylation of the paroxetine-catechol metabolite to the corresponding guaiacol regioisomers in S-9 incubations. While the NADPH-dependent covalent binding was attenuated by GSH and SAM, these reagents did not alter paroxetine's ability to inactivate P450 2D6, suggesting that the reactive intermediate responsible for P450 inactivation did not leave the active site to react with other proteins. The results of our studies indicate that in addition to the low once-a-day dosing regimen (20 mg) of paroxetine, efficient scavenging of the catechol and quinone metabolites by SAM and GSH, respectively, serves as an explanation for the excellent safety record of paroxetine despite the fact that it undergoes bioactivation. PMID:17907785

  17. Meta-Analyses of Dehalococcoides mccartyi Strain 195 Transcriptomic Profiles Identify a Respiration Rate-Related Gene Expression Transition Point and Interoperon Recruitment of a Key Oxidoreductase Subunit

    PubMed Central

    Mansfeldt, Cresten B.; Rowe, Annette R.; Heavner, Gretchen L. W.; Zinder, Stephen H.

    2014-01-01

    A cDNA-microarray was designed and used to monitor the transcriptomic profile of Dehalococcoides mccartyi strain 195 (in a mixed community) respiring various chlorinated organics, including chloroethenes and 2,3-dichlorophenol. The cultures were continuously fed in order to establish steady-state respiration rates and substrate levels. The organization of array data into a clustered heat map revealed two major experimental partitions. This partitioning in the data set was further explored through principal component analysis. The first two principal components separated the experiments into those with slow (1.6 ± 0.6 ?M Cl?/h)- and fast (22.9 ± 9.6 ?M Cl?/h)-respiring cultures. Additionally, the transcripts with the highest loadings in these principal components were identified, suggesting that those transcripts were responsible for the partitioning of the experiments. By analyzing the transcriptomes (n = 53) across experiments, relationships among transcripts were identified, and hypotheses about the relationships between electron transport chain members were proposed. One hypothesis, that the hydrogenases Hup and Hym and the formate dehydrogenase-like oxidoreductase (DET0186-DET0187) form a complex (as displayed by their tight clustering in the heat map analysis), was explored using a nondenaturing protein separation technique combined with proteomic sequencing. Although these proteins did not migrate as a single complex, DET0112 (an FdhB-like protein encoded in the Hup operon) was found to comigrate with DET0187 rather than with the catalytic Hup subunit DET0110. On closer inspection of the genome annotations of all Dehalococcoides strains, the DET0185-to-DET0187 operon was found to lack a key subunit, an FdhB-like protein. Therefore, on the basis of the transcriptomic, genomic, and proteomic evidence, the place of the missing subunit in the DET0185-to-DET0187 operon is likely filled by recruiting a subunit expressed from the Hup operon (DET0112). PMID:25063656

  18. A novel cytosolic NADH:quinone oxidoreductase from Methanothermobacter marburgensis

    PubMed Central

    Ullmann, Eva; Tan, Tien Chye; Gundinger, Thomas; Herwig, Christoph; Divne, Christina; Spadiut, Oliver

    2014-01-01

    Methanothermobacter marburgensis is a strictly anaerobic, thermophilic methanogenic archaeon that uses methanogenesis to convert H2 and CO2 to energy. M. marburgensis is one of the best-studied methanogens, and all genes required for methanogenic metabolism have been identified. Nonetheless, the present study describes a gene (Gene ID 9704440) coding for a putative NAD(P)H:quinone oxidoreductase that has not yet been identified as part of the metabolic machinery. The gene product, MmNQO, was successfully expressed, purified and characterized biochemically, as well as structurally. MmNQO was identified as a flavin-dependent NADH:quinone oxidoreductase with the capacity to oxidize NADH in the presence of a wide range of electron acceptors, whereas NADPH was oxidized with only three acceptors. The 1.50 Å crystal structure of MmNQO features a homodimeric enzyme where each monomer comprises 196 residues folding into flavodoxin-like ?/? domains with non-covalently bound FMN (flavin mononucleotide). The closest structural homologue is the modulator of drug activity B from Streptococcus mutans with 1.6 Å root-mean-square deviation on 161 C? atoms and 28% amino-acid sequence identity. The low similarity at sequence and structural level suggests that MmNQO is unique among NADH:quinone oxidoreductases characterized to date. Based on preliminary bioreactor experiments, MmNQO could provide a useful tool to prevent overflow metabolism in applications that require cells with high energy demand. PMID:25372605

  19. Contribution of NAD(P)H:quinone oxidoreductase 1 to protection against carcinogenesis, and regulation of its gene by the Nrf2 basic-region leucine zipper and the arylhydrocarbon receptor basic helix-loop-helix transcription factors

    Microsoft Academic Search

    Paul Nioi; John D. Hayes

    2004-01-01

    NAD(P)H:quinone oxidoreductase 1 (NQO1) is a key enzyme involved in defence against reactive forms of oxygen and inhibition of neoplasia. Under conditions of oxidative stress, expression of NQO1 is induced, and the resulting increase in oxidoreductase protein provides the cell with multiple layers of protection against environmental insults. Firstly, the catalytic activity of NQO1 is directed towards the complete reduction

  20. The NADH-binding subunit of the energy-transducing NADH-ubiquinone oxidoreductase of Paracoccus denitrificans: Gene cloning and deduced primary structure

    SciTech Connect

    Xu, Xuemin; Matsuno-Yagi, Akemi; Yagi, Takao (Research Inst. of Scripps Clinic, La Jolla, CA (United States))

    1991-07-02

    The NADH dehydrogenase complex isolated from Paracoccus denitrificans is composed of approximately 10 unlike polypeptides and contains noncovalently bound FMN, non-heme iron, and acid-labile sulfide. The NADH-binding subunit of this enzyme complex was identified by direct photoaffinity labeling with ({sup 32}P)NADH. primers were synthesized on the basis of the N-terminal amino acid sequency of this polypeptide, and these primers were used to synthesize an oligonucleotide probe by the polymerase chain reaction. This probe was utilized to isolate the gene encoding the NADH-binding subunit from a genomic library of P. denitrificans. The nucleotide sequence of the gene and the deduced amino acid sequence of the entire NADH-binding subunit were determined. The NADH-binding subunit has 431 amino acid residues and a calculated molecular weight of 47 191. The encoded protein contains a putative NAD(H)-binding and an iron-sulfur cluster-binding consensus sequence. The deduced amino acid sequence of the Paracoccus NADH-binding subunit shows remarkable similarity to the {alpha} subunit of the NAD-linked hydrogenase of Alcaligenes eutrophus H16. When partial DNA sequencing of the regions surrounding the gene encoding the NADH-binding subunit was carried out, sequences homologous to the 24-, 49-, and 75-kDa polypeptides of bovine complex 1 were detected, suggesting that the structural genes of the Paracoccus NADH dehydrogenase complex constitute a gene cluster.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  2. Functional characterization and role of INrf2 in antioxidant response element-mediated expression and antioxidant induction of NAD(P)H:quinone oxidoreductase1 gene

    Microsoft Academic Search

    Saravanakumar Dhakshinamoorthy; Anil K Jaiswal

    2001-01-01

    Antioxidant response element (ARE) and nuclear transcription factor Nrf2 are known to regulate expression and coordinated induction of NQO1 and other detoxifying enzyme genes in response to antioxidants and xenobiotics. A cytosolic inhibitor of Nrf2, INrf2, that retains Nrf2 in the cytoplasm, was cloned and sequenced. Treatment of cells with antioxidants and xenobiotics results in the release of Nrf2 from

  3. 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

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

    SciTech Connect

    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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. NADH- and NADPH-dependent lipid peroxidation in bovine heart submitochondrial particles. Dependence on the rate of electron flow in the respiratory chain and an antioxidant role of ubiquinol.

    PubMed Central

    Takayanagi, R; Takeshige, K; Minakami, S

    1980-01-01

    Malondialdehyde formations by bovine heart submitochondrial particles supported by NADH or NADPH in the presence of ADP and FeCl3 was studied. The NADH-dependent reaction was maximal at very low rate of electron input from NADH to the respiratory chain and it decreased when the rate became high. The reaction was stimulated by rotenone and inhibited by antimycin A when the input was fast, whereas it was not affected by the inhibitors when the input was slow. The input rate of the electrons from NADPH was also so low that the reaction supported by NADPH was not affected by the inhibitors. Most of the endogenous ubiquinone in the particles treated with antimycin A was reduced by NADH even in the presence of ADP-Fe3+ chelate, but uniquinone was not reduced by NADPH when ADP-Fe3+ was present. Succinate strongly inhibited both NADH- and NADPH-dependent lipid peroxidation. The inhibition was abolished when uniquinone was removed from the particles, and it appeared again when uniquinone was reincorporated into the particles. Reduced uniquinone-2 also inhibited the peroxidation, but duroquinol, which reduces cytochrome b without reducing endogenous uniquinone, did not. Thus the malondialdehyde formation appeared to be inversely related to the extent of the reduction of endogenous uniquinone. These observations suggest that both NADH- and NADPH-dependent liquid-peroxidation reactions are closely related to the respiratory chain and that the peroxidation is controlled by the concentration of reduced ubiquinone. PMID:7236242

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

    PubMed Central

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

    1993-01-01

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

  8. NADPH-dependent reductive ortho dehalogenation of 2,4-dichlorobenzoic acid in Corynebacterium sepedonicum KZ-4 and Coryneform bacterium strainNTB-1 via 2,4-dichlorobenzoyl coenzyme A.

    PubMed Central

    Romanov, V; Hausinger, R P

    1996-01-01

    Corynebacterium sepedonicum KZ-4, described earlier as a strain capable of growth on 2,4-dichlorobenzoate (G.M. Zaitsev and Y.N. Karasevich, Mikrobiologiya 54:356-369, 1985), is known to metabolize this substrate via 4-hydroxybenzoate and protocatechuate, and evidence consistent with an initial reductive dechlorination step to form 4-chlorobenzoate was found in another coryneform bacterium, strain NTB-1 (W.J.J. van den Tweel, J.B. Kok, and J.A.M. de Bont, Appl. Environ. Microbiol. 53:810-815, 1987). 2-Chloro-4-fluorobenzoate was found to be converted stoichiometrically to 4-fluorobenzoate by resting cells of strain KZ-4, compatible with a reductive process. Experiments with cell extracts demonstrated that Mg - ATP and coenzyme A (CoA) were required to stimulate reductive dehalogenation, consistent with the intermediacy of 2-chloro-4-fluoro-benzoyl-CoA and 2,4-dichlorobenzoyl-CoA thioesters. 2,4-Dichlorobenzoyl-CoA was shown to be converted to 4-chlorobenzoyl-CoA in a novel NADPH-dependent reaction in extracts of both KZ-4 and NTB-1. In addition to the ligase and reductive dehalogenase activities, hydrolytic 4-chlorobenzoyl-CoA dehalogenase and thioesterase activities, 4-hydroxybenzoate 3-monooxygenase, and protocatechuate 3,4-dioxygenase activities were demonstrated to be present in the soluble fraction of KZ-4 extracts following ultracentrifugation. We propose that the pathway for 2,4-dichlorobenzoate catabolism in strains KZ-4 and NTB-1 involves formation of 2,4-dichlorobenzoyl-CoA, NADPH-dependent ortho dehalogenation yielding 4-chlorobenzoyl-CoA, hydrolytic removal of chlorine from the para position to generate 4-hydroxybenzoyl-CoA, hydrolysis to form 4-hydroxybenzoate, oxidation to yield protocatechuate, and oxidative ring cleavage. PMID:8626335

  9. 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

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

    PubMed

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

    2008-11-01

    Reactions requiring reducing equivalents, NAD(P)H, are of enormous importance for the synthesis of industrially valuable compounds such as carotenoids, polymers, antibiotics and chiral alcohols among others. The use of whole-cell biocatalysis can reduce process cost by acting as catalyst and cofactor regenerator at the same time; however, product yields might be limited by cofactor availability within the cell. Thus, our study focussed on the genetic manipulation of a whole-cell system by modifying metabolic pathways and enzymes to improve the overall production process. In the present work, we genetically engineered an Escherichia coli strain to increase NADPH availability to improve the productivity of products that require NADPH in its biosynthesis. The approach involved an alteration of the glycolysis step where glyceraldehyde-3-phosphate (GAP) is oxidized to 1,3 bisphophoglycerate (1,3-BPG). This reaction is catalyzed by NAD-dependent endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoded by the gapA gene. We constructed a recombinant E. coli strain by replacing the native NAD-dependent gapA gene with a NADP-dependent GAPDH from Clostridium acetobutylicum, encoded by the gene gapC. The beauty of this approach is that the recombinant E. coli strain produces 2 mol of NADPH, instead of NADH, per mole of glucose consumed. Metabolic flux analysis showed that the flux through the pentose phosphate (PP) pathway, one of the main pathways that produce NADPH, was reduced significantly in the recombinant strain when compared to that of the parent strain. The effectiveness of the NADPH enhancing system was tested using the production of lycopene and epsilon-caprolactone as model systems using two different background strains. The recombinant strains, with increased NADPH availability, consistently showed significant higher productivity than the parent strains. PMID:18852061

  11. Development of a substrate-coupled biocatalytic process driven by an NADPH-dependent sorbose reductase from Candida albicans for the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate.

    PubMed

    Cai, Ping; An, Mingdong; Xu, Lin; Xu, Sheng; Hao, Ning; Li, Yan; Guo, Kai; Yan, Ming

    2012-12-01

    A substrate-coupled biocatalytic process was developed based on the reactions catalyzed by an NADPH-dependent sorbose reductase (SOU1) from Candida albicans in which ethyl 4-chloro-3-oxobutanoate (COBE) was reduced to (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE], while NADPH was regenerated by the same enzyme via oxidation of sugar alcohols. (S)-CHBE yields of 1,140, 1,150, and 780 mM were obtained from 1,220 mM COBE when sorbitol, mannitol, and xylitol were used as co-substrates, respectively. Optimization of COBE and sorbitol proportions resulted in a maximum yield of (S)-CHBE (2,340 mM) from 2,500 mM COBE, and the enantiomeric excess was 99.6 %. The substrate-coupled system driven by SOU1 maintained a stable pH and a robust intracellular NADPH circulation; thus, pH adjustment and addition of extra coenzymes were unnecessary. PMID:22918792

  12. The P450 oxidoreductase, RedA, controls development beyond the mound stage in Dictyostelium discoideum

    PubMed Central

    Gonzalez-Kristeller, Daniela C; Farage, Layla; Fiorini, Leonardo C; Loomis, William F; da Silva, Aline M

    2008-01-01

    Background NADPH-cytochrome-P450 oxidoreductase (CPR) is a ubiquitous enzyme that belongs to a family of diflavin oxidoreductases and is required for activity of the microsomal cytochrome-P450 monooxygenase system. CPR gene-disruption experiments have demonstrated that absence of this enzyme causes developmental defects both in mouse and insect. Results Annotation of the sequenced genome of D. discoideum revealed the presence of three genes (redA, redB and redC) that encode putative members of the diflavin oxidoreductase protein family. redA transcripts are present during growth and early development but then decline, reaching undetectable levels after the mound stage. redB transcripts are present in the same levels during growth and development while redC expression was detected only in vegetative growing cells. We isolated a mutant strain of Dictyostelium discoideum following restriction enzyme-mediated integration (REMI) mutagenesis in which redA was disrupted. This mutant develops only to the mound stage and accumulates a bright yellow pigment. The mound-arrest phenotype is cell-autonomous suggesting that the defect occurs within the cells rather than in intercellular signaling. Conclusion The developmental arrest due to disruption of redA implicates CPR in the metabolism of compounds that control cell differentiation. PMID:18218133

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

    SciTech Connect

    Subramaniam, Sudhakar R.; Ellis, Elizabeth M., E-mail: elizabeth.ellis@strath.ac.uk

    2011-01-15

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

  14. Endoplasmic Reticulum Chaperones and Oxidoreductases: Critical Regulators of Tumor Cell Survival and Immunorecognition

    PubMed Central

    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

  15. Genetics Home Reference: Cytochrome P450 oxidoreductase deficiency

    MedlinePLUS

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

  16. MDR quinone oxidoreductases: the human and yeast zeta-crystallins.

    PubMed

    Porté, Sergio; Crosas, Eva; Yakovtseva, Evgenia; Biosca, Josep A; Farrés, Jaume; Fernández, M Rosario; Parés, Xavier

    2009-03-16

    The medium-chain dehydrogenase/reductase (MDR) superfamily can be divided into Zn-containing and Zn-lacking proteins. Zn-containing MDRs are generally well-known enzymes, mostly acting as dehydrogenases. The non-Zn MDR are much less studied, and classified in several families of NADP(H)-dependent reductases, including quinone oxidoreductases (QOR). zeta-Crystallins are the best studied group of QOR, have a structural function in the lens of several mammals, exhibit ortho-quinone reductase activity, and bind to specific adenine-uracil-rich elements (ARE) in RNA. In the present work, we have further characterized human zeta-crystallin and Saccharomyces cerevisiae Zta1p, the only QOR in yeast. Subcellular localization using a fluorescent protein tag indicates that zeta-crystallin is distributed in the cytoplasm but not in nucleus. The protein may also be present in mitochondria. Zta1p localizes in both cytoplasm and nucleus. NADPH, but not NADH, competitively prevents binding of zeta-crystallin to RNA, suggesting that the cofactor-binding site is involved in RNA binding. Interference of NADPH on Zta1p binding to RNA is much lower, consistent with a weaker binding of NADPH to the yeast enzyme. Disruption of the yeast ZTA1 gene does not affect cell growth under standard conditions but makes yeast more sensitive to oxidative stress agents. Sequence alignments, phylogenetic tree analysis and kinetic properties reveal a close relationship between zeta-crystallin and Zta1p. Amino acid conservation, between the substrate-binding sites of the two proteins and that of an E. coli QOR, indicates that zeta-crystallins maintained their kinetic function throughout evolution. Quinones are toxic compounds and a relevant step in their detoxification is reduction to their corresponding hydroquinones. Many enzymes of several superfamilies can reduce quinones, including NAD(P)H:quinone oxidoreductase 1 (NQO1 or DT-diaphorase), aldo-keto reductases and short-chain dehydrogenases/reductases. In this context, the physiological role of zeta-crystallins is discussed. PMID:19007762

  17. 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

  18. Novel SNPs in cytochrome P450 oxidoreductase.

    PubMed

    Hart, Steven N; Li, Ye; Nakamoto, Kaori; Wesselman, Chris; Zhong, Xiao-bo

    2007-08-01

    Cytochrome P450 oxidoreductase (POR) is the single flavoprotein which donates electrons to the microsomal cytochrome P450 enzymes for oxidation of their substrates. In this study, we sequenced all 15 exons and the surrounding intronic sequences of POR in 100 human liver samples to identify novel and confirm known genetic polymorphisms in POR. Thirty-four single nucleotide polymorphisms (SNPs) were identified including 9 in the coding exons (5 synonymous and 4 nonsynonymous), 20 in the intronic regions, and 5 in the 3'-UTR. Of these, 9 were novel SNPs, including three nonsynonymous SNPs, SNH313003 (817733G>C; K49N), SNH313020 (848661C>A; L420M), and SNH313029 (849577T>C; L577P) with minor allele frequencies of 0.005, 0.045, and 0.020, respectively. We also confirmed a previously reported non-synonymous SNP rs1057868 (A503V) as well as five synonymous SNPs (G5G, T29T, P129P, S485S, and S572S) all with allele frequencies similar to those previously reported. Structurally, these polymorphisms occur in different regions: SNH313003 (K49N) in the amino-terminal tail, SNH313020 (L420M) in the connecting domain, SNH313029 (L577P) in the NADPH-binding domain, and rs1057868 (A503V) in the FAD binding domain. PMID:17827787

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

    E-print Network

    Liao, Rongzhen

    Tungsten-dependent formaldehyde ferredoxin oxidoreductase: Reaction mechanism from quantum chemical theory Enzyme catalysis Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus is a tungsten the formaldehyde substrate binds directly to the tungsten ion. WVI =O then performs a nucleophilic attack

  20. Regulation of yeast replicative life span by thiol oxidoreductases

    PubMed Central

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

    2011-01-01

    Thiol-based redox reactions are involved in the regulation of a variety of biological functions, such as protection against oxidative stress, signal transduction and protein folding. Some proteins involved in redox regulation have been shown to modulate life span in organisms from yeast to mammals. To assess the role of thiol oxidoreductases in aging on a genome-wide scale, we analyzed the replicative life span of yeast cells lacking known and candidate thiol oxidoreductases. The data suggest the role of several pathways in regulation of yeast aging, including thioredoxin reduction, protein folding and degradation, peroxide reduction, PIP3 signaling, and ATP synthesis. PMID:20934449

  1. Mapping of the human guanosine monophosphate reductase gene (GMPR) to chromosome 6p23 by fluorescence in situ hybridization

    SciTech Connect

    Murano, Ichiro; Tsukahara, Masato; Kajii, Tadashi (Yamaguchi Univ. School of Medicine (Japan)); Yoshida, Akira (Beckman Research Institute of the City of Hope, Duarte, CA (United States))

    1994-01-01

    Guanosine monophosphate (GMP) reductase (EC 1.6.6.8) catalyzes the NADPH-dependent reductive deamination of guanosine monophosphate to inosine monophosphate: GMP + NADPH + 2H[sup +] [yields] IMP + NH[sub 4][sup +] + NADP[sup +]. The authors have previously cloned a human GMP reductase gene. They report here the precise localization of the human GMP reductase gene (GMPR) on human chromosomes. 2 refs.

  2. Uric acid and xanthine oxidoreductase in wound healing.

    PubMed

    Fernandez, Melissa L; Upton, Zee; Shooter, Gary K

    2014-02-01

    Chronic wounds are an important health problem because they are difficult to heal and treatment is often complicated, lengthy and expensive. For a majority of sufferers the most common outcomes are long-term immobility, infection and prolonged hospitalisation. There is therefore an urgent need for effective therapeutics that will enhance ulcer healing and patient quality of life, and will reduce healthcare costs. Studies in our laboratory have revealed elevated levels of purine catabolites in wound fluid from patients with venous leg ulcers. In particular, we have discovered that uric acid is elevated in wound fluid, with higher concentrations correlating with increased wound severity. We have also revealed a corresponding depletion in uric acid precursors, including adenosine. Further, we have revealed that xanthine oxidoreductase, the enzyme that catalyses the production of uric acid, is present at elevated levels in wound fluid. Taken together, these findings provide evidence that xanthine oxidoreductase may have a function in the formation or persistence of chronic wounds. Here we describe the potential function of xanthine oxidoreductase and uric acid accumulation in the wound site, and the effect of xanthine oxidoreductase in potentiating the inflammatory response. PMID:24357442

  3. Octomeric pyruvate-ferredoxin oxidoreductase from Desulfovibrio vulgaris

    E-print Network

    Hazen, Terry

    decarboxylation of pyruvate to produce acetyl-CoA is the gateway to the tricarboxylic acid (TCA) cycle. In aerobicOctomeric pyruvate-ferredoxin oxidoreductase from Desulfovibrio vulgaris Florian Garczarek a , Ming; accepted 19 January 2007 Available online 17 February 2007 Abstract Pyruvate-ferredoxin oxidoreductatse

  4. 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

  5. Hepatocyte circadian clock controls acetaminophen bioactivation through NADPH-cytochrome P450 oxidoreductase

    PubMed Central

    Johnson, Brian P.; Walisser, Jacqueline A.; Liu, Yan; Shen, Anna L.; McDearmon, Erin L.; Moran, Susan M.; McIntosh, Brian E.; Vollrath, Aaron L.; Schook, Andrew C.; Takahashi, Joseph S.; Bradfield, Christopher A.

    2014-01-01

    The diurnal variation in acetaminophen (APAP) hepatotoxicity (chronotoxicity) reportedly is driven by oscillations in metabolism that are influenced by the circadian phases of feeding and fasting. To determine the relative contributions of the central clock and the hepatocyte circadian clock in modulating the chronotoxicity of APAP, we used a conditional null allele of brain and muscle Arnt-like 1 (Bmal1, aka Mop3 or Arntl) allowing deletion of the clock from hepatocytes while keeping the central and other peripheral clocks (e.g., the clocks controlling food intake) intact. We show that deletion of the hepatocyte clock dramatically reduces APAP bioactivation and toxicity in vivo and in vitro because of a reduction in NADPH-cytochrome P450 oxidoreductase gene expression, protein, and activity. PMID:25512522

  6. Structure of a hyperthermophilic tungstopterin enzyme, aldehyde ferredoxin oxidoreductase

    Microsoft Academic Search

    M. K. Chan; D. C. Rees; S. Mukund; A. Kletzin; M. W. W. Adams

    1995-01-01

    The crystal structure of the tungsten-containing aldehyde ferrodoxin oxidoreductase (AOR) for Pyroccoccus furiosus, a hyperthermophilic archaoen (formerly archaebacterium) that grows optimally at 100°C, has been determined at 2.3 angstrom resolution by means of multiple isomorphous replacement and multiple crystal from averaging. AOR consists of two identical subunits, each containing an FeâSâ cluster and a molybdopterin-based tungsten cofactor that is analogous

  7. Cloning, overexpression, and mutagenesis of the Sporobolomyces salmonicolor AKU4429 gene encoding a new aldehyde reductase, which catalyzes the stereoselective reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (S)-4-chloro-3-hydroxybutanoate

    Microsoft Academic Search

    Keiko Kita; Takanobu Fukura; KOH-ICHI NAKASE; Kenji Okamoto; Hideshi Yanase; Michihiko Kataoka; Sakayu Shimizu

    1999-01-01

    The authors cloned and sequenced the gene encoding and NADPH-dependent aldehyde reductase (ARII) in Sporobolomyces salmonicolor AKU4429, which reduces ethyl 4-chloro-3-oxobutanoate (4-COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate. The ARII gene is 1,032 bp long, is interrupted by four introns, and encodes a 37,315-Da polypeptide. The deduced amino acid sequence exhibited significant levels of similarity to the amino acid sequences of members of

  8. An NAD(P)H-Nicotine Blue Oxidoreductase Is Part of the Nicotine Regulon and May Protect Arthrobacter nicotinovorans from Oxidative Stress during Nicotine Catabolism

    Microsoft Academic Search

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

    2007-01-01

    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

  9. Purification of NADH: hypothiocyanite oxidoreductase in Streptococcus sanguis.

    PubMed

    Courtois, P H; Pourtois, M

    1996-04-01

    NADH: hypothiocyanite oxidoreductase (NHOR) activity, found in some oral Streptococci, is postulated to protect these microorganisms against salivary peroxidase-produced hypothiocyanite. NHOR, however, has not been purified so far. The purification of NHOR from crude extracts of Streptococcus sanguis NCTC 7863 strain (by ultrafiltration and anion-exchange chromatography) revealed one fraction of 125 +/- kDa. However, SDS-PAGE electrophoresis provided a single protein of 21.1 +/- 1.2 kDa. This last discovery suggests that NHOR enzyme is a hexameric complex having six subunits. PMID:8733891

  10. WW domain-containing oxidoreductase in neuronal injury and neurological diseases

    PubMed Central

    Chang, Hsin-Tzu; Liu, Chan-Chuan; Chen, Shur-Tzu; Yap, Ye Vone; Chang, Nan-Shang; Sze, Chun-I

    2014-01-01

    The human and mouse WWOX/Wwox gene encodes a candidate tumor suppressor WW domain-containing oxidoreductase protein. This gene is located on a common fragile site FRA16D. WWOX participates in a variety of cellular events and acts as a transducer in the many signal pathways, including TNF, chemotherapeutic drugs, UV irradiation, Wnt, TGF-?, C1q, Hyal-2, sex steroid hormones, and others. While transiently overexpressed WWOX restricts relocation of transcription factors to the nucleus for suppressing cancer survival, physiological relevance of this regard in vivo has not been confirmed. Unlike many tumor suppressor genes, mutation of WWOX is rare, raising a question whether WWOX is a driver for cancer initiation. WWOX/Wwox was initially shown to play a crucial role in neural development and in the pathogenesis of Alzheimer's disease and neuronal injury. Later on, WWOX/Wwox was shown to participate in the development of epilepsy, mental retardation, and brain developmental defects in mice, rats and humans. Up to date, most of the research and review articles have focused on the involvement of WWOX in cancer. Here, we review the role of WWOX in neural injury and neurological diseases, and provide perspectives for the WWOX-regulated neurodegeneration. PMID:25537520

  11. 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.

  12. Radical reactions of thiamin pyrophosphate in 2-oxoacid oxidoreductases?

    PubMed Central

    Reed, George H.; Ragsdale, Stephen W.; Mansoorabadi, Steven O.

    2011-01-01

    Thiamin pyrophosphate (TPP) is essential in carbohydrate metabolism in all forms of life. TPP-dependent decarboxylation reactions of 2-oxo-acid substrates result in enamine adducts between the thiazolium moiety of the coenzyme and decarboxylated substrate. These central enamine intermediates experience different fates from protonation in pyruvate decarboxylase to oxidation by the 2-oxoacid dehydrogenase complexes, the pyruvate oxidases, and 2-oxoacid oxidoreductases. Virtually all of the TPP-dependent enzymes, including pyruvate decarboxylase, can be assayed by 1-electron redox reactions linked to ferricyanide. Oxidation of the enamines is thought to occur via a 2-electron process in the 2-oxoacid dehydrogenase complexes, wherein acyl group transfer is associated with reduction of the disulfide of the lipoamide moiety. However, discrete 1-electron steps occur in the oxidoreductases, where one or more [4Fe-4S] clusters mediate the electron transfer reactions to external electron acceptors. These radical intermediates can be detected in the absence of the acyl-group acceptor, coenzyme A (CoASH). The ?-electron system of the thiazolium ring stabilizes the radical. The extensively delocalized character of the radical is evidenced by quantitative analysis of nuclear hyperfine splitting tensors as detected by electron paramagnetic resonance (EPR) spectroscopy and by electronic structure calculations. The second electron transfer step is markedly accelerated by the presence of CoASH. While details of the second electron transfer step and its facilitation by CoASH remain elusive, expected redox properties of potential intermediates limit possible scenarios. PMID:22178227

  13. Impact of NAD(P)H:quinone oxidoreductase-1 on pigmentation.

    PubMed

    Choi, Tae-Young; Sohn, Kyung-Cheol; Kim, Jin-Hwa; Kim, Seong-Min; Kim, Cheol-Hee; Hwang, Jae-Sung; Lee, Jeung-Hoon; Kim, Chang Deok; Yoon, Tae-Jin

    2010-03-01

    We obtained metastasized melanoma tissue from a primary acral lentiginous melanoma (ALM) patient and established a melanoma cell line named primary culture of melanoma cell derived from lymph node (PML)-1. PML-1 cells had a light brown color and decreased the expression of melanogenesis markers, including tyrosinase (TYR), microphthalmia-associated transcription factor, and tyrosinase-related protein-1. To identify genes differentially regulated in PML-1 melanoma cells, we performed DNA microarray and two-dimensional matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses. Among the candidate genes identified, we chose NAD(P)H:quinone oxidoreductase-1 (NQO1) for further study. Reverse transcription-PCR and western blot analyses showed that NQO1 was markedly decreased in PML-1 cells and in several amelanotic melanoma cell lines. To investigate whether NQO1 affects the melanogenesis, we treated the cultured normal human melanocytes (NHMC) and zebrafish with NQO1 inhibitors, ES936 and dicoumarol. Interestingly, melanogenesis was significantly decreased by the addition of NQO1 inhibitors in both NHMC and zebrafish models. In contrast, overexpression of NQO1 using a recombinant adenovirus clearly induced melanogenesis, concomitantly with an increase of TYR protein level. These results suggest that NQO1 is a positive regulator of the pigmentation process. PMID:19759547

  14. 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

  15. Purification and characterization of a carbohydrate: acceptor oxidoreductase from Paraconiothyrium sp. that produces lactobionic acid efficiently.

    PubMed

    Kiryu, Takaaki; Nakano, Hirofumi; Kiso, Taro; Murakami, Hiromi

    2008-03-01

    A carbohydrate:acceptor oxidoreductase from Paraconiothyrium sp. was purified and characterized. The enzyme efficiently oxidized beta-(1-->4) linked sugars, such as lactose, xylobiose, and cellooligosaccharides. The enzyme also oxidized maltooligosaccharides, D-glucose, D-xylose, D-galactose, L-arabinose, and 6-deoxy-D-glucose. It specifically oxidized the beta-anomer of lactose. Molecular oxygen and 2,6-dichlorophenol indophenol were reduced by the enzyme as electron acceptors. The Paraconiothyrium enzyme was identified as a carbohydrate:acceptor oxidoreductase according to its specificity for electron donors and acceptors, and its molecular properties, as well as the N-terminal amino acid sequence. Further comparison of the amino acid sequences of lactose oxidizing enzymes indicated that carbohydrate:acceptor oxidoreductases belong to the same group as glucooligosaccharide oxidase, while they differ from cellobiose dehydrogenases and cellobiose:quinone oxidoreductases. PMID:18323642

  16. The respiratory molybdo-selenoprotein formate dehydrogenases of Escherichia coli have hydrogen: benzyl viologen oxidoreductase activity

    PubMed Central

    2011-01-01

    Background Escherichia coli synthesizes three membrane-bound molybdenum- and selenocysteine-containing formate dehydrogenases, as well as up to four membrane-bound [NiFe]-hydrogenases. Two of the formate dehydrogenases (Fdh-N and Fdh-O) and two of the hydrogenases (Hyd-1 and Hyd-2) have their respective catalytic subunits located in the periplasm and these enzymes have been shown previously to oxidize formate and hydrogen, respectively, and thus function in energy metabolism. Mutants unable to synthesize the [NiFe]-hydrogenases retain a H2: benzyl viologen oxidoreductase activity. The aim of this study was to identify the enzyme or enzymes responsible for this activity. Results Here we report the identification of a new H2: benzyl viologen oxidoreductase enzyme activity in E. coli that is independent of the [NiFe]-hydrogenases. This enzyme activity was originally identified after non-denaturing polyacrylamide gel electrophoresis and visualization of hydrogen-oxidizing activity by specific staining. Analysis of a crude extract derived from a variety of E. coli mutants unable to synthesize any [NiFe]-hydrogenase-associated enzyme activity revealed that the mutants retained this specific hydrogen-oxidizing activity. Enrichment of this enzyme activity from solubilised membrane fractions of the hydrogenase-negative mutant FTD147 by ion-exchange, hydrophobic interaction and size-exclusion chromatographies followed by mass spectrometric analysis identified the enzymes Fdh-N and Fdh-O. Analysis of defined mutants devoid of selenocysteine biosynthetic capacity or carrying deletions in the genes encoding the catalytic subunits of Fdh-N and Fdh-O demonstrated that both enzymes catalyze hydrogen activation. Fdh-N and Fdh-O can also transfer the electrons derived from oxidation of hydrogen to other redox dyes. Conclusions The related respiratory molybdo-selenoproteins Fdh-N and Fdh-O of Escherichia coli have hydrogen-oxidizing activity. These findings demonstrate that the energy-conserving selenium- and molybdenum-dependent formate dehydrogenases Fdh-N and Fdh-O exhibit a degree of promiscuity with respect to the electron donor they use and identify a new class of dihydrogen-oxidizing enzyme. PMID:21806784

  17. New Insights into Type II NAD(P)H:Quinone Oxidoreductases

    PubMed Central

    Melo, Ana M. P.; Bandeiras, Tiago M.; Teixeira, Miguel

    2004-01-01

    Type II NAD(P)H:quinone oxidoreductases (NDH-2) catalyze the two-electron transfer from NAD(P)H to quinones, without any energy-transducing site. NDH-2 accomplish the turnover of NAD(P)H, regenerating the NAD(P)+ pool, and may contribute to the generation of a membrane potential through complexes III and IV. These enzymes are usually constituted by a nontransmembrane polypeptide chain of ?50 kDa, containing a flavin moiety. There are a few compounds that can prevent their activity, but so far no general specific inhibitor has been assigned to these enzymes. However, they have the common feature of being resistant to the complex I classical inhibitors rotenone, capsaicin, and piericidin A. NDH-2 have particular relevance in yeasts like Saccharomyces cerevisiae and in several prokaryotes, whose respiratory chains are devoid of complex I, in which NDH-2 keep the [NADH]/[NAD+] balance and are the main entry point of electrons into the respiratory chains. Our knowledge of these proteins has expanded in the past decade, as a result of contributions at the biochemical level and the sequencing of the genomes from several organisms. The latter showed that most organisms contain genes that potentially encode NDH-2. An overview of this development is presented, with special emphasis on microbial enzymes and on the identification of three subfamilies of NDH-2. PMID:15590775

  18. Ã?Â?NADPH: Protochlorophyllide Oxidoreductase-Structure, Catalytic Function, and Role in Prolamellar Body Formation and Morphogenesis

    SciTech Connect

    Michael P. Timko

    2013-02-01

    The biosynthesis of chlorophyll is a critical biochemical step in the development of photosynthetic vascular plants and green algae. From photosynthetic bacteria (cyanobacteria) to algae, non-vascular plants, gymnosperms and vascular plants, mechanisms have evolved for protochlorophyllide reduction a key step in chlorophyll synthesis. Protochlorophyllide reduction is carried out by both a light-dependent (POR) and light-independent (LIPOR) mechanisms. NADPH: protochlorophyllide oxidoreductase (EC 1.3.1.33, abbreviated POR) catalyzes the light-dependent reduction of protochlorophyllide (PChlide) to chlorophyllide (Chlide). In contrast, a light-independent protochlorophyllide reductase (LIPOR) involves three plastid gene products (chlL, chlN, and chlB) and several nuclear factors. Our work focused on characterization of both the POR and LIPOR catalyzed processes.

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

    PubMed Central

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

    2014-01-01

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

  20. The Thiol:Disulfide Oxidoreductase DsbB Mediates the Oxidizing Effects of the Toxic Metalloid Tellurite (TeO32?) on the Plasma Membrane Redox System of the Facultative Phototroph Rhodobacter capsulatus?

    PubMed Central

    Borsetti, Francesca; Francia, Francesco; Turner, Raymond J.; Zannoni, Davide

    2007-01-01

    The highly toxic oxyanion tellurite (TeO32?) is a well known pro-oxidant in mammalian and bacterial cells. This work examines the effects of tellurite on the redox state of the electron transport chain of the facultative phototroph Rhodobacter capsulatus, in relation to the role of the thiol:disulfide oxidoreductase DsbB. Under steady-state respiration, the addition of tellurite (2.5 mM) to membrane fragments generated an extrareduction of the cytochrome pool (c- and b-type hemes); further, in plasma membranes exposed to tellurite (0.25 to 2.5 mM) and subjected to a series of flashes of light, the rate of the QH2:cytochrome c (Cyt c) oxidoreductase activity was enhanced. The effect of tellurite was blocked by the antibiotics antimycin A and/or myxothiazol, specific inhibitors of the QH2:Cyt c oxidoreductase, and, most interestingly, the membrane-associated thiol:disulfide oxidoreductase DsbB was required to mediate the redox unbalance produced by the oxyanion. Indeed, this phenomenon was absent from R. capsulatus MD22, a DsbB-deficient mutant, whereas the tellurite effect was present in membranes from MD22/pDsbBWT, in which the mutant gene was complemented to regain the wild-type DsbB phenotype. These findings were taken as evidence that the membrane-bound thiol:disulfide oxidoreductase DsbB acts as an “electron conduit” between the hydrophilic metalloid and the lipid-embedded Q pool, so that in habitats contaminated with subinhibitory amounts of TeIV, the metalloid is likely to function as a disposal for the excess reducing power at the Q-pool level of facultative phototrophic bacteria. PMID:17098900

  1. Structural and functional characterization of ferredoxin-NADP+-oxidoreductase using knock-out mutants of Arabidopsis.

    PubMed

    Lintala, Minna; Allahverdiyeva, Yagut; Kidron, Heidi; Piippo, Mirva; Battchikova, Natalia; Suorsa, Marjaana; Rintamäki, Eevi; Salminen, Tiina A; Aro, Eva-Mari; Mulo, Paula

    2007-03-01

    In Arabidopsis thaliana, the chloroplast-targeted enzyme ferredoxin-NADP+-oxidoreductase (FNR) exists as two isoforms, AtLFNR1 and AtLFNR2, encoded by the genes At5g66190 and At1g20020, respectively. Both isoforms are evenly distributed between the thylakoids and soluble stroma, and they are separated by two-dimensional electrophoresis in four distinct spots, suggesting post-translational modification of both isoforms. To reveal the functional specificity of AtLFNR1, we have characterized the T-DNA insertion mutants with an interrupted At5g66190 gene. Absence of AtLFNR1 resulted in a reduced size of the rosette with pale green leaves, which was accompanied by a low content of chlorophyll and light-harvesting complex proteins. Also the photosystem I/photosystem II (PSI/PSII) ratio was significantly lower in the mutant, but the PSII activity, measured as the F(V)/F(M) ratio, remained nearly unchanged and the excitation pressure of PSII was lower in the mutants than in the wild type. A slow re-reduction rate of P700 measured in the mutant plants suggested that AtLFNR1 is involved in PSI-dependent cyclic electron flow. Impaired function of FNR also resulted in decreased capacity for carbon fixation, whereas nitrogen metabolism was upregulated. In the absence of AtLFNR1, we found AtLFNR2 exclusively in the stroma, suggesting that AtLFNR1 is required for membrane attachment of FNR. Structural modeling supports the formation of a AtLFNR1-AtLFNR2 heterodimer that would mediate the membrane attachment of AtLFNR2. Dimer formation, in turn, might regulate the distribution of electrons between the cyclic and linear electron transfer pathways according to environmental cues. PMID:17335513

  2. 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

  3. Genetic variation in genes of the fatty acid synthesis pathway and breast cancer risk

    Microsoft Academic Search

    Daniele Campa; James McKay; Olga Sinilnikova; Anika Hüsing; Ulla Vogel; Rikke Dalgaard Hansen; Kim Overvad; Petra Mariann Witt; Françoise Clavel-Chapelon; Marie-Christine Boutron-Ruault; Veronique Chajes; Sabine Rohrmann; Jenny Chang-Claude; Heiner Boeing; Eva Fisher; Antonia Trichopoulou; Dimitrios Trichopoulos; Domenico Palli; Anna Villarini; Carlotta Sacerdote; Amalia Mattiello; Rosario Tumino; Petra H. M. Peeters; Carla H. van Gils; H. Bas Bueno-de-Mesquita; Eiliv Lund; María Dolores Chirlaque; Núria Sala; Laudina Rodriguez Suarez; Aurelio Barricarte; Miren Dorronsoro; Maria-José Sánchez; Per Lenner; Göran Hallmans; Kostas Tsilidis; Sheila Bingham; Kay-Tee Khaw; Valentina Gallo; Teresa Norat; Elio Riboli; Sabina Rinaldi; Gilbert Lenoir; Sean V. Tavtigian; Federico Canzian; Rudolf Kaaks

    2009-01-01

    Fatty acid synthase (FAS) is the major enzyme of lipogenesis. It catalyzes the NADPH-dependent condensation of acetyl-CoA\\u000a and malonyl-CoA to produce palmitic acid. Transcription of the FAS gene is controlled synergistically by the transcription\\u000a factors ChREBP (carbohydrate response element-binding protein), which is induced by glucose, and SREBP-1 (sterol response\\u000a element-binding protein-1), which is stimulated by insulin through the PI3K\\/Akt signal

  4. Association of Ferredoxin-NADP Oxidoreductase with the Chloroplastic Pyridine Nucleotide Dehydrogenase Complex in Barley Leaves1

    PubMed Central

    José Quiles, María; Cuello, Juan

    1998-01-01

    Barley (Hordeum vulgare L.) leaves were used to isolate and characterize the chloroplast NAD(P)H dehydrogenase complex. The stroma fraction and the thylakoid fraction solubilized with sodium deoxycholate were analyzed by native polyacrylamide gel electrophoresis, and the enzymes detected with NADH and nitroblue tetrazolium were electroeluted. The enzymes electroeluted from band S from the stroma fraction and from bands T1 (ET1) and T2 from the thylakoid fraction solubilized with sodium deoxycholate had ferredoxin-NADP oxidoreductase (FNR; EC 1.18.1.2) and NAD(P)H-FeCN oxidoreductase (NAD[P]H-FeCNR) activities. Their NADPH-FeCNR activities were inhibited by 2?-monophosphoadenosine-5?-diphosphoribose and by enzyme incubation with p-chloromercuriphenylsulfonic acid (p-CMPS), NADPH, and p-CMPS plus NADPH. They presented Michaelis constant NADPH values that were similar to those of FNRs from several sources. Their NADH-FeCNR activities, however, were not inhibited by 2?-monophosphoadenosine-5?-diphosphoribose but were weakly inhibited by enzyme incubation with NADH, p-CMPS, and p-CMPS plus NADH. We found that only ET1 contained two polypeptides of 29 and 35 kD, which reacted with the antibodies raised against the mitochondrial complex I TYKY subunit and the chloroplast ndhA gene product, respectively. However, all three enzymes contained two polypeptides of 35 and 53 kD, which reacted with the antibodies raised against barley FNR and the NADH-binding 51-kD polypeptide of the mitochondrial complex I, respectively. The results suggest that ET1 is the FNR-containing thylakoidal NAD(P)H dehydrogenase complex. PMID:9576793

  5. Ferredoxin:NADP+ Oxidoreductase Association with Phycocyanin Modulates Its Properties*

    PubMed Central

    Korn, Anja; Ajlani, Ghada; Lagoutte, Bernard; Gall, Andrew; Sétif, Pierre

    2009-01-01

    In photosynthetic organisms, ferredoxin:NADP+ oxidoreductase (FNR) is known to provide NADPH for CO2 assimilation, but it also utilizes NADPH to provide reduced ferredoxin. The cyanobacterium Synechocystis sp. strain PCC6803 produces two FNR isoforms, a small one (FNRS) similar to the one found in plant plastids and a large one (FNRL) that is associated with the phycobilisome, a light-harvesting complex. Here we show that a mutant lacking FNRL exhibits a higher NADP+/NADPH ratio. We also purified to homogeneity a phycobilisome subcomplex comprising FNRL, named FNRL-PC. The enzymatic activities of FNRL-PC were compared with those of FNRS. During NADPH oxidation, FNRL-PC exhibits a 30% decrease in the Michaelis constant Km(NADPH), and a 70% increase in Km(ferredoxin), which is in agreement with its predicted lower activity of ferredoxin reduction. During NADP+ reduction, the FNRL-PC shows a 29/43% decrease in the rate of single electron transfer from reduced ferredoxin in the presence/absence of NADP+. The increase in Km(ferredoxin) and the rate decrease of single reduction are attributed to steric hindrance by the phycocyanin moiety of FNRL-PC. Both isoforms are capable of catalyzing the NADP+ reduction under multiple turnover conditions. Furthermore, we obtained evidence that, under high ionic strength conditions, electron transfer from reduced ferredoxin is rate limiting during this process. The differences that we observe might not fully explain the in vivo properties of the Synechocystis mutants expressing only one of the isoforms. Therefore, we advocate that FNR localization and/or substrates availability are essential in vivo. PMID:19759024

  6. 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

  7. Glucose, lactate, and pyruvate biosensor arrays based on redox polymer/oxidoreductase nanocomposite thin-lms deposited on

    E-print Network

    Revzin, Alexander

    Glucose, lactate, and pyruvate biosensor arrays based on redox polymer/oxidoreductase nanocomposite, lactate, and pyruvate sensor arrays were fabricated by depositing electrostatically complexed monolayers-®lm of a cationic osmium redox polymer and anionic oxidoreductases, either glucose oxidase, lactate oxidase

  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. 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.

  10. P450 oxidoreductase deficiency: a disorder of steroidogenesis with multiple clinical manifestations.

    PubMed

    Miller, Walter L

    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. PMID:23092891

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

    SciTech Connect

    Xia, Chuanwu; Panda, Satya P.; Marohnic, Christopher C.; Martásek, Pavel; Masters, Bettie Sue; Kim, Jung-Ja P. (MCW); (Charles U); (UTSMC)

    2012-03-15

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

  12. NADPH:quinone oxidoreductase 1 regulates host susceptibility to ozone via isoprostane generation.

    PubMed

    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

    2013-02-15

    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 A(2)-isoprostane, a cyclopentenone isoprostane that blunts inflammation. Using GC-MS, we found that NQO1-null mice had greater lung tissue levels of D(2)- and E(2)-isoprostanes, the precursors of J(2)- and A(2)-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 A(2)-isoprostane inhibited ozone-induced NF-?B activation and IL-8 regulation. Furthermore, we determined that A(2)-isoprostane covalently modified the active Cys(179) domain in inhibitory ?B kinase in the presence of ozone in vitro, thus establishing the biochemical basis for A(2)-isoprostane inhibition of NF-?B. Our results demonstrate that host factors may regulate pulmonary susceptibility to ozone by regulating the generation of A(2)-isoprostanes in the lung. These observations provide the biochemical basis for the epidemiologic observation that NQO1 regulates pulmonary susceptibility to ozone. PMID:23275341

  13. 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

  14. 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

  15. Diflavin oxidoreductases activate the bioreductive prodrug PR-104A under hypoxia.

    PubMed

    Guise, Christopher P; Abbattista, Maria R; Tipparaju, Smitha R; Lambie, Neil K; Su, Jiechuang; Li, Dan; Wilson, William R; Dachs, Gabi U; Patterson, Adam V

    2012-01-01

    The clinical agent PR-104 is converted systemically to PR-104A, a nitrogen mustard prodrug designed to target tumor hypoxia. Reductive activation of PR-104A is initiated by one-electron oxidoreductases in a process reversed by oxygen. The identity of these oxidoreductases is unknown, with the exception of cytochrome P450 reductase (POR). To identify other hypoxia-selective PR-104A reductases, nine candidate oxidoreductases were expressed in HCT116 cells. Increased PR-104A-cytotoxicity was observed in cells expressing methionine synthase reductase (MTRR), novel diflavin oxidoreductase 1 (NDOR1), and inducible nitric-oxide synthase (NOS2A), in addition to POR. Plasmid-based expression of these diflavin oxidoreductases also enhanced bioreductive metabolism of PR-104A in an anoxia-specific manner. Diflavin oxidoreductase-dependent PR-104A metabolism was suppressed >90% by pan-flavoenzyme inhibition with diphenyliodonium chloride. Antibodies were used to quantify endogenous POR, MTRR, NDOR1, and NOS2A expression in 23 human tumor cell lines; however, only POR protein was detectable and its expression correlated with anoxic PR-104A reduction (r(2) = 0.712). An anti-POR monoclonal antibody was used to probe expression using human tissue microarrays; 13 of 19 cancer types expressed detectable POR with 21% of cores (185 of 874) staining positive; this heterogeneity suggests that POR is a useful biomarker for PR-104A activation. Immunostaining for carbonic anhydrase 9 (CAIX), reportedly an endogenous marker of hypoxia, revealed only moderate coexpression (9.6%) of both CAIX and POR across a subset of five cancer types. PMID:21984255

  16. 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

  17. Structure and Sequence Conservation of hao Cluster Genes of Autotrophic Ammonia-Oxidizing Bacteria: Evidence for Their Evolutionary History

    Microsoft Academic Search

    David J. Bergmann; Alan B. Hooper; Martin G. Klotz

    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

  18. [Control of buccal peroxidases by a bacterial NADH-hypothiocyanite oxidoreductase].

    PubMed

    Courtois, P

    1996-01-01

    Oral peroxidases (myeloperoxidase, sialoperoxidase) catalyze thiocyanate peroxidation into hypothiocyanite which is bacteriostatic or bactericidal against numerous bacterial species. NADH-hypothiocyanite-oxidoreductase is thought to protect bacteria which can express it; up to now, this enzyme activity was never purified. The present study analyzes, on one hand, the susceptibility of periodontal bacteria against hypothiocyanite and, on the other hand, proposes a purification design for the NADH-hypothiocyanite-oxidoreductase from Streptococcus sanguis, a commensal micro-organism of dental surfaces. The data suggest the importance of the bacterial biofilm on dental surfaces for production of antiseptic oxidants and for the control of their toxicity. PMID:9491629

  19. NAD(P)H : quinone oxidoreductase 1 inducer activity of some Saudi Arabian medicinal plants.

    PubMed

    Shahat, Abdelaaty A; Alsaid, Mansour S; Alyahya, Muhammad A; Higgins, Maureen; Dinkova-Kostova, Albena T

    2013-04-01

    Medicinal plants are a rich source of biologically-active phytochemicals and have been used in traditional medicine for centuries. Specific phytochemicals and extracts of their plant sources have the ability to reduce the risk for chronic degenerative diseases by induction of enzymes involved in xenobiotic metabolism, many of which also have antioxidant and anti-inflammatory functions. One such multifunctional cytoprotective enzyme is NAD(P)H : quinone oxidoreductase. In this study, we prepared extracts of 27 Saudi Arabian medicinal plants which belong to 18 different plant families and tested their ability to induce NAD(P)H : quinone oxidoreductase in murine hepatoma cells grown in microtiter plate wells. In addition to the Brassicaceae, a known source of NAD(P)H : quinone oxidoreductase inducer activity, we found substantial inducer activity in extracts from the Apiaceae, Apocynaceae, and the Asteraceae families. Five out of a total of eight active extracts are from plants which belong to the Asteraceae family. We further show that artemisinin, an agent which is used clinically for the treatment of malaria, contributes but does not fully account for the inducer activity of the extract of Artemisia monosperma. In contrast to artemisinin, deoxyartemisinin is inactive in this assay, demonstrating the critical role of the endoperoxide moiety of artemisinin for inducer activity. Thus, the NAD(P)H : quinone oxidoreductase inducer activity of extracts of some Saudi Arabian medicinal plants indicates the presence of specific phytochemicals which have the potential to protect against chronic degenerative diseases. PMID:23512501

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

    PubMed Central

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

    2013-01-01

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

  1. Glucose and Lactate Biosensors Based on Redox Polymer/Oxidoreductase Nanocomposite Thin

    E-print Network

    Revzin, Alexander

    Glucose and Lactate Biosensors Based on Redox Polymer/Oxidoreductase Nanocomposite Thin Films&M University, College Station, Texas 77843-3122 Glucose and lactate enzyme electrodes have been fabri- cated solutions and anionic glucose oxidase (GOX) or lactate oxidase (LAX) solutions to build the nanocomposite

  2. Continuous enzymatic production of lactobionic acid using glucose-fructose oxidoreductase in an ultrafiltration membrane reactor

    Microsoft Academic Search

    M. Satory; M. Fürlinger; D. Haltrich; K. D. Kulbe; F. Pittner; B. Nidetzky

    1997-01-01

    Glucose-fructose oxidoreductase from Zymomonas mobilis catalyzed the oxidation of various aldose sugars to the corresponding aldonic acids. The enzyme was used for the selective and high-yield conversion of lactose to lactobionic acid in batch, fed-batch and continous reaction mode. A productivity of 110 g L d was obtained in an ultrafiltration membrane reactor, operated for 70 h.

  3. Xanthine oxidoreductase is central to the evolution and function of the innate immune system

    Microsoft Academic Search

    Claudia Vorbach; Roger Harrison; Mario R. Capecchi

    2003-01-01

    The housekeeping enzyme xanthine oxidoreductase (XOR) has been studied intensively over the past 100 years, yet the complexity of its in vivo function is still poorly understood. A large body of literature focuses on the different catalytic forms of XOR and their importance in the synthesis of reactive oxygen and reactive nitrogen species, which are involved in many disease processes.

  4. 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

  5. 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

  6. NAD(P)H Cytochrome b5 Oxidoreductase Deficiency in Leishmania major Results in Impaired Linoleate Synthesis Followed by Increased Oxidative Stress and Cell Death*

    PubMed Central

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

    2012-01-01

    NAD(P)H cytochrome b5 oxidoreductase (Ncb5or), comprising cytochrome b5 and cytochrome b5 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 b5 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 O2 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 H2O2, 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

  7. Interaction of the molecular chaperone Hsp70 with human NAD(P)H:quinone oxidoreductase 1.

    PubMed

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

    2002-04-19

    NAD(P)H:quinone oxidoreductase 1 (EC; DT-Diaphorase, NQO1) is predominantly a cytosolic flavoenzyme that catalyzes a two-electron reduction. Using human tumor cell lines devoid of NQO1 enzymatic activity, we have previously identified a single nucleotide polymorphism (NQO1*2 allele) in the human NQO1 gene. This mutation has been characterized as a genetic polymorphism (NQO1*2), which leads to greatly diminished levels of protein due to rapid degradation of the NQO1*2 protein by the ubiquitin proteasomal pathway (UPP). In an attempt to decipher the mechanism responsible for the differential stability of wild-type NQO1*1 and mutant NQO1*2 proteins, we have investigated the interactions of these proteins with molecular chaperones of the Hsp family. Using co-immunoprecipitation studies (co-IPs), no association was observed between Hsp90 and either wild-type NQO1*1 or mutant NQO1*2 proteins. Hsp70, however, was found to associate with NQO1*1 protein in cells when co-IPs were performed with an anti-NQO1 antibody followed by immunoblotting with an anti-Hsp70 antibody or vice versa. Hsp40 could also be detected in the immunoprecipitated protein complex. Experiments were also performed using either the NQO1*1 or NQO1*2 coding regions in an in vitro transcription/translation system employing rabbit reticulocyte lysates (RRLs). Consistent with the cellular data, co-IP experiments in RRLs demonstrated an association of Hsp70 with wild-type NQO1*1 protein but not with NQO1*2 protein. To further elucidate the role of the association of Hsp70 with the NQO1*1 protein, site-directed mutagenesis was used to modify a proposed Hsp70 binding site near the N terminus of the NQO1 protein. We generated a plasmid containing an NQO1*1 coding region with a mutated Hsp70 binding site (isoleucine to aspartic acid at position 8, NQO1*1/I8D). In contrast to the NQO1*1 protein translated in RRLs, the NQO1*1/I8D protein did not associate with Hsp70, as demonstrated by co-IP, was catalytically inactive, and was degraded by the UPP. These data suggest that the association of Hsp70 with NQO1*1 may play an important role in the stability and functionality of the NQO1 protein. PMID:11821413

  8. NxrB encoding the beta subunit of nitrite oxidoreductase as functional and phylogenetic marker for nitrite-oxidizing Nitrospira.

    PubMed

    Pester, Michael; Maixner, Frank; Berry, David; Rattei, Thomas; Koch, Hanna; Lücker, Sebastian; Nowka, Boris; Richter, Andreas; Spieck, Eva; Lebedeva, Elena; Loy, Alexander; Wagner, Michael; Daims, Holger

    2014-10-01

    Nitrospira are the most widespread and diverse known nitrite-oxidizing bacteria and key nitrifiers in natural and engineered ecosystems. Nevertheless, their ecophysiology and environmental distribution are understudied because of the recalcitrance of Nitrospira to cultivation and the lack of a molecular functional marker, which would allow the detection of Nitrospira in the environment. Here we introduce nxrB, the gene encoding subunit beta of nitrite oxidoreductase, as a functional and phylogenetic marker for Nitrospira. Phylogenetic trees based on nxrB of Nitrospira were largely congruent to 16S ribosomal RNA-based phylogenies. By using new nxrB-selective polymerase chain reaction primers, we obtained almost full-length nxrB sequences from Nitrospira cultures, two activated sludge samples, and several geographically and climatically distinct soils. Amplicon pyrosequencing of nxrB fragments from 16 soils revealed a previously unrecognized diversity of terrestrial Nitrospira with 1801 detected species-level operational taxonomic units (OTUs) (using an inferred species threshold of 95% nxrB identity). Richness estimates ranged from 10 to 946 coexisting Nitrospira species per soil. Comparison with an archaeal amoA dataset obtained from the same soils [Environ. Microbiol. 14: 525-539 (2012)] uncovered that ammonia-oxidizing archaea and Nitrospira communities were highly correlated across the soil samples, possibly indicating shared habitat preferences or specific biological interactions among members of these nitrifier groups. PMID:24118804

  9. 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.

  10. CO oxidoreductase from Streptomyces strain G26 is a molybdenum hydroxylase.

    PubMed Central

    Bell, J M; Colby, J; Williams, E

    1988-01-01

    CO oxidoreductase was purified to 95% homogeneity from crude mycelial extracts of Streptomyces G26. The purified preparation has a specific activity of 25.7 units/mg, a 13-fold improvement on crude soluble mycelial extracts. The native enzyme (Mr 282,000) is composed of non-identical subunits of Mr 110,000 and 33,000. It is a molybdenum hydroxylase containing 1.6 mol of FAD, 7.3 mol of Fe, 8.3 mol of acid-labile sulphide and 1.3 mol of Mo per mol of enzyme. Purified CO oxidoreductase catalyses the reduction of benzyl viologen, confirming the previously reported ability of this enzyme to interact with low-potential acceptors. Cytochrome c reduction cannot be accounted for entirely by non-enzymic reduction by superoxide radicals. NAD+ and NADP+ are not reduced, nor is clostridial ferredoxin. PMID:3355539

  11. 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.

  12. Up-regulation of NAD(P)H quinone oxidoreductase 1 during human liver injury

    Microsoft Academic Search

    Lauren M Aleksunes; Michael Goedken; José E Manautou

    AIM: To investigate the expression and activity of NAD(P)H quinone oxidoreductase 1 (NQO1) in human liver specimens obtained from patients with liver damage due to acetaminophen (APAP) overdose or primary biliary cirrhosis (PBC). METHODS: NQO1 activity was determined in cytosol from normal, APAP and PBC liver specimens. Western blot and immunohistochemical staining were used to determine patterns of NQO1 expression

  13. Purification and characterization of an (S)-3-hydroxycarboxylate oxidoreductase from Clostridium tyrobutyricum

    Microsoft Academic Search

    M. BayerH; H. Günther; H. Simon

    1994-01-01

    An NADP+ —dependent reversible 3-hydroxycarboxylate oxidoreductase present in Clostridium tyrobutyricum has been purified. As judged by gel electrophoresis the enzyme was pure after a 940-fold enrichment by four chromatographic steps. Its molecular mass was estimated to be 40–43 kDa. The enzyme was most active at pH 4.5 in the reduction of 3-oxobutyrate. Other substrates were 3-oxovalerate, 3-oxocaproate, 3-oxoisocaproate and 4-chloro-3-oxobutyrate.

  14. Expression of WW domain-containing oxidoreductase WOX1 in the developing murine nervous system

    Microsoft Academic Search

    S. T. CHEN; J. I. CHUANG; J. P. WANG; M. S. TSAI; H. LId; N.-S. CHANGe

    2004-01-01

    WW domain-containing oxidoreductase WOX1, also known as WWOX or FOR, is a proapoptotic protein and a putative tumor suppressor. Hyaluronidases such as PH-20, Hyal-1 and Hyal-2 induce the expression of WOX1, and hyaluronidases and hyaluronan are involved in the embryonic development. In the present study, we document the expression of WOX1 in the developing murine nervous system. Immunohistochemical analysis revealed

  15. Neutral 17?-hydroxysteroid oxidoreductase deficiency in testes causing male pseudohermaphroditism in an infant

    Microsoft Academic Search

    R. A. Harkness; D. Thistlethwaite; J. A. B. Darling; N. E. Skakkebaek; C. S. Corker

    1979-01-01

    A deficiency of neutral 17ß-hydroxysteroid oxidoreductase activity in tests has been diagnosed in an infant with male pseudohermaphroditism.In vivo stimulation tests of testicular endocrine function with human chorionic gonadotrophin provided an accurate diagnosis in contrast to estimates of enzymic activityin vitro in testes and other tissues. The discrepancy in testes may be due to the absence of gonadotrophin stimulation in

  16. AhpF and other NADH:peroxiredoxin oxidoreductases, homologues of low Mr thioredoxin reductase

    Microsoft Academic Search

    Leslie B. Poole; C. Michael Reynolds; Zachary A. Wood; P. Andrew Karplus; Holly R. Ellis; Marco Li Calzi

    2000-01-01

    A group of bacterial flavoproteins related to thioredoxin reductase contain an additional < 200-amino-acid domain including a redox-active disulfide center at their N-termini. These flavoproteins, designated NADH:peroxiredoxin oxidoreductases, catalyze the pyridine-nucleotide-dependent reduction of cysteine-based peroxidases (e.g. Salmonella typhimurium AhpC, a member of the peroxiredoxin family) which in turn reduce H2O2 or organic hydroperoxides. These enzymes catalyze rapid electron transfer (kcat

  17. 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

  18. Xanthine Oxidoreductase Reference Values in Platelet-Poor Plasma and Platelets in Healthy Volunteers

    PubMed Central

    Cecerska-Hery?, El?bieta; Klaudyna, Szupiluk; Dominika, M?czka; Dominika, Pawlak; Marta, Urba?ska; Do??gowska, Barbara

    2015-01-01

    Introduction. Xanthine oxidoreductase (XOR) is an enzyme belonging to the class of hydroxylases. XOR is stated, inter alia, in the kidneys, liver, and small intestine as well as in leukocytes and platelets and endothelial cells of capillaries. Its main role is to participate in the conversion of hypoxanthine to xanthine and the uric acid. It occurs in two isoforms: dehydrogenase (XD) and oxidase (XO), which is considered one of the sources of reactive oxygen species. Aim of the Study. Determination of reference values of xanthine oxidoreductase activity in PPP and platelets. Materials and Methods. Study group consisted of 70 healthy volunteers. The isoform activities of xanthine oxidoreductase were determined by kinetic spectrophotometry. Results. A statistically significant difference between the activity of the XOR in PPP and platelets (P < 0.001). The highest activity of XO was found in both PPP and blood platelets. Significant differences between the activity of the various isoforms in PPP (P = 0.0032) and platelets (P < 0.001) were also found. Conclusions. The healthy volunteers showed the highest activity XO (prooxidant) and the lowest XD (antioxidant), which indicates a slight oxidative stress and confirmed physiological effects of XOR. PMID:25688294

  19. Nuclear factor-E2-related factor 2 expression in liver is critical for induction of NAD(P)H:quinone oxidoreductase 1 during cholestasis

    PubMed Central

    Aleksunes, Lauren M.; Slitt, Angela L.; Maher, Jonathan M.; Dieter, Matthew Z.; Knight, Tamara R.; Goedken, Michael; Cherrington, Nathan J.; Chan, Jefferson Y.; Klaassen, Curtis D.; Manautou, José E.

    2006-01-01

    Bile duct ligation (BDL) causes hepatocellular oxidative stress and injury. The transcription factor nuclear factor-E2-related factor (Nrf2) induces expression of numerous genes including NAD(P)H:quinone oxidoreductase 1 (Nqo1) during periods of oxidative stress. Therefore, we hypothesized that BDL increases liver expression of mouse antioxidant genes in an Nrf2-dependent manner. BDL or sham surgeries were performed on male C57BL/6, Nrf2-null, and wild-type mice. Livers were collected at 1, 3, and 7 days after surgery for analysis of messenger ribonucleic acid (mRNA) levels of Nrf2-responsive genes as well as Nqo1 protein and activity. BDL increased mRNA expression of multiple Nrf2 genes in mouse liver, compared to sham-operated controls. Follow-up studies investigating protein expression, enzyme activity, and Nrf2 dependency were limited to Nqo1. Nqo1 protein expression and activity in mouse livers was increased 2- to 3-, and 4- to 5-fold at 3 and 7 days after BDL, respectively. Studies also showed that BDL increases Nqo1 mRNA, protein expression, and enzyme activity in livers from wild-type mice, but not in Nrf2-null mice. In conclusion, expression of Nrf2-dependent genes is increased during cholestasis. These studies also demonstrate that Nqo1 expression and activity in mouse liver are induced via an Nrf2-dependent mechanism. PMID:17278884

  20. Comparative Genomics of Thiol Oxidoreductases Reveals Widespread and Essential Functions of Thiol-based Redox Control of Cellular Processes

    PubMed Central

    2012-01-01

    Abstract Aims: Redox regulation of cellular processes is an important mechanism that operates in organisms from bacteria to mammals. Much of the redox control is provided by thiol oxidoreductases: proteins that employ cysteine residues for redox catalysis. We wanted to identify thiol oxidoreductases on a genome-wide scale and use this information to obtain insights into the general principles of thiol-based redox control. Results: Thiol oxidoreductases were identified by three independent methods that took advantage of the occurrence of selenocysteine homologs of these proteins and functional linkages among thiol oxidoreductases revealed by comparative genomics. Based on these searches, we describe thioredoxomes, which are sets of thiol oxidoreductases in organisms. Their analyses revealed that these proteins are present in all living organisms, generally account for 0.5%–1% of the proteome and that their use correlates with proteome size, distinguishing these proteins from those involved in core metabolic functions. We further describe thioredoxomes of Saccharomyces cerevisiae and humans, including proteins which have not been characterized previously. Thiol oxidoreductases occur in various cellular compartments and are enriched in the endoplasmic reticulum and cytosol. Innovation: We developed bioinformatics methods and used them to characterize thioredoxomes on a genome-wide scale, which in turn revealed properties of thioredoxomes. Conclusion: These data provide information about organization and properties of thiol-based redox control, whose use is increased with the increase in complexity of organisms. Our data also show an essential combined function of a set of thiol oxidoreductases, and of thiol-based redox regulation in general, in all living organisms. Antioxid. Redox Signal. 16, 193–201. PMID:21902454

  1. Allosteric nucleotide-binding site in the mitochondrial NADH:ubiquinone oxidoreductase (respiratory complex I).

    PubMed

    Grivennikova, Vera G; Gladyshev, Grigory V; Vinogradov, Andrei D

    2011-07-21

    The rotenone-insensitive NADH:hexaammineruthenium III (HAR) oxidoreductase reactions catalyzed by bovine heart and Yarrowia lipolytica submitochondrial particles or purified bovine complex I are stimulated by ATP and other purine nucleotides. The soluble fraction of mammalian complex I (FP) and prokaryotic complex I homolog NDH-1 in Paracoccus denitrificans plasma membrane lack stimulation of their activities by ATP. The stimulation appears as a decrease in apparent K(m) values for NADH and HAR. Thus, the "accessory" subunits of eukaryotic complex I bear an allosteric ATP-binding site. PMID:21624365

  2. Mechanistic insights into xanthine oxidoreductase from development studies of candidate drugs to treat hyperuricemia and gout.

    PubMed

    Nishino, Takeshi; Okamoto, Ken

    2015-03-01

    Xanthine oxidoreductase (XOR), which is widely distributed from humans to bacteria, has a key role in purine catabolism, catalyzing two steps of sequential hydroxylation from hypoxanthine to xanthine and from xanthine to urate at its molybdenum cofactor (Moco). Human XOR is considered to be a target of drugs not only for therapy of hyperuricemia and gout, but also potentially for a wide variety of other diseases. In this review, we focus on studies of XOR inhibitors and their implications for understanding the chemical nature and reaction mechanism of the Moco active site of XOR. We also discuss further experimental or clinical studies that would be helpful to clarify remaining issues. PMID:25501928

  3. A transcriptome-proteome integrated network identifies endoplasmic reticulum thiol oxidoreductase (ERp57) as a hub that mediates bone metastasis.

    PubMed

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

    2013-08-01

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

  4. The Role of Oxidoreductases in Determining the Function of the Neisserial Lipid A Phosphoethanolamine Transferase Required for Resistance to Polymyxin

    PubMed Central

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

    2014-01-01

    The decoration of the lipid A headgroups of the lipooligosaccharide (LOS) by the LOS phosphoethanolamine (PEA) transferase (LptA) in Neisseria spp. is central for resistance to polymyxin. The structure of the globular domain of LptA shows that the protein has five disulphide bonds, indicating that it is a potential substrate of the protein oxidation pathway in the bacterial periplasm. When neisserial LptA was expressed in Escherichia coli in the presence of the oxidoreductase, EcDsbA, polymyxin resistance increased 30-fold. LptA decorated one position of the E. coli lipid A headgroups with PEA. In the absence of the EcDsbA, LptA was degraded in E. coli. Neisseria spp. express three oxidoreductases, DsbA1, DsbA2 and DsbA3, each of which appear to donate disulphide bonds to different targets. Inactivation of each oxidoreductase in N. meningitidis enhanced sensitivity to polymyxin with combinatorial mutants displaying an additive increase in sensitivity to polymyxin, indicating that the oxidoreductases were required for multiple pathways leading to polymyxin resistance. Correlates were sought between polymyxin sensitivity, LptA stability or activity and the presence of each of the neisserial oxidoreductases. Only meningococcal mutants lacking DsbA3 had a measurable decrease in the amount of PEA decoration on lipid A headgroups implying that LptA stability was supported by the presence of DsbA3 but did not require DsbA1/2 even though these oxidoreductases could oxidise the protein. This is the first indication that DsbA3 acts as an oxidoreductase in vivo and that multiple oxidoreductases may be involved in oxidising the one target in N. meningitidis. In conclusion, LptA is stabilised by disulphide bonds within the protein. This effect was more pronounced when neisserial LptA was expressed in E. coli than in N. meningitidis and may reflect that other factors in the neisserial periplasm have a role in LptA stability. PMID:25215579

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

    PubMed

    Farooq, Amjad

    2015-03-01

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

  6. Genes

    NSDL National Science Digital Library

    BEGIN:VCARD VERSION:2.1 FN:Access Excellence N:Excellence; Access REV:2005-03-12 END:VCARD

    2005-03-12

    Illustration of the placement of genes in a chromosome. A gene can be defined as a region of DNA that controls a hereditary characteristic. It usually corresponds to a sequence used in the production of a specific protein or RNA. A gene carries biological information in a form that must be copied and transmitted from each cell to all its progeny. This includes the entire functional unit: coding DNA sequences, non-coding regulatory DNA sequences, and introns. Genes can be as short as 1000 base pairs or as long as several hundred thousand base pairs. It can even be carried by more than one chromosome. The estimate for the number of genes in humans has decreased as our knowledge has increased. As of 2001, humans are thought to have between 30,000 and 40,000 genes.

  7. 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...

  8. 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

  9. Survey of Human Oxidoreductases and Cytochrome P450 Enzymes Involved in the Metabolism of Chemicals.

    PubMed

    Rendic, Slobodan Petar; Guengerich, F Peter

    2014-12-01

    Analyzing the literature resources used in our previous reports, we calculated the fractions of the oxidoreductase enzymes FMO (microsomal flavin-containing monooxygenase), AKR (aldo-keto reductase), MAO (monoamine oxidase), and cytochrome P450 participating in metabolic reactions. The calculations show that the fractions of P450s involved in metabolism of all chemicals (general chemicals, natural and physiological compounds, and drugs) are rather consistent in the findings that > 90% of enzymatic reactions are catalyzed by P450s. Regarding drug metabolism, three-fourths of the human P450 reactions can be accounted for by a set of five P450s: 1A2, 2C9, 2C19, 2D6, and 3A4, and the largest fraction of the P450 reactions is catalyzed by P450 3A enzymes. P450 3A4 participation in metabolic reactions of drugs varied from 13% for general chemicals to 27% for drugs. PMID:25485457

  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 120min 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. Crystallization and preliminary X-ray analysis of NADH:rubredoxin oxidoreductase from Clostridium acetobutylicum

    PubMed Central

    Nishikawa, Koji; Shomura, Yasuhito; Kawasaki, Shinji; Niimura, Youichi; Higuchi, Yoshiki

    2010-01-01

    NADH:rubredoxin oxidoreductase (NROR), an O2-inducible protein, is a versatile electron donor for scavengers of O2 and reactive oxygen species (ROS) in Clostridium acetobutylicum. Recombinant NROR was overexpressed in Escherichia coli and purified to homogeneity; it was subsequently crystallized using the sitting-drop vapour-diffusion method at 293?K. Preliminary crystallo­graphic analysis revealed that the crystals belonged to space group P4122 or P4322, with unit-cell parameters a = b = 98.6, c = 88.3?Å, and diffracted to 2.1?Å resolution. Assuming that the crystals contained one molecule per asymmetric unit, the Matthews coefficient was calculated to be 2.7?Å3?Da?1 and the solvent content to be 54.1%. PMID:20057062

  12. NADPH quinone oxidoreductase 1 mediates breast cancer cell resistance to thymoquinone-induced apoptosis.

    PubMed

    Sutton, Kimberly M; Doucette, Carolyn D; Hoskin, David W

    2012-09-28

    Thymoquinone (TQ), a bioactive component of black caraway seed (Nigella sativa) oil, is reported to have antineoplastic properties. In this study we investigated the effect of TQ on a panel of human breast cancer cell lines. Cell viability assays showed that TQ killed T-47D, MDA-MB-231, and MDA-MB-468 cells via p53-independent induction of apoptosis; however, MCF-7 cells were refractory to the cytotoxic action of TQ. Western Blot analysis showed that MCF-7 cells expressed high levels of cytoprotective NADPH quinone oxidoreductase 1 (NQO1), which was responsible for TQ-resistance since inhibition of NQO1 with dicoumarol rendered MCF-7 cells TQ-sensitive. These findings may be clinically important when considering TQ as a possible adjunct treatment for breast cancer since a high percentage of breast tumors express NQO1. PMID:22960073

  13. Xanthine oxidoreductase-catalyzed reactive species generation: A process in critical need of reevaluation?

    PubMed Central

    Cantu-Medellin, Nadiezhda; Kelley, Eric E.

    2013-01-01

    Nearly 30 years have passed since the discovery of xanthine oxidoreductase (XOR) as a critical source of reactive species in ischemia/reperfusion injury. Since then, numerous inflammatory disease processes have been associated with elevated XOR activity and allied reactive species formation solidifying the ideology that enhancement of XOR activity equates to negative clinical outcomes. However, recent evidence may shatter this paradigm by describing a nitrate/nitrite reductase capacity for XOR whereby XOR may be considered a crucial source of beneficial •NO under ischemic/hypoxic/acidic conditions; settings similar to those that limit the functional capacity of nitric oxide synthase. Herein, we review XOR-catalyzed reactive species generation and identify key microenvironmental factors whose interplay impacts the identity of the reactive species (oxidants vs. •NO) produced. In doing so, we redefine existing dogma and shed new light on an enzyme that has weathered the evolutionary process not as gadfly but a crucial component in the maintenance of homeostasis. PMID:24024171

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

    PubMed Central

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

    2012-01-01

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

  15. 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

  16. 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.

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

    SciTech Connect

    Not Available

    1992-01-01

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

  18. 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

  19. 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

  20. A discovery study of daunorubicin induced cardiotoxicity in a sample of acute myeloid leukemia patients prioritizes P450 oxidoreductase polymorphisms as a potential risk factor

    PubMed Central

    Lubieniecka, Joanna M.; Graham, Jinko; Heffner, Daniel; Mottus, Randy; Reid, Ronald; Hogge, Donna; Grigliatti, Tom A.; Riggs, Wayne K.

    2013-01-01

    Anthracyclines are very effective chemotherapeutic agents; however, their use is hampered by the treatment-induced cardiotoxicity. Genetic variants that help define patient's sensitivity to anthracyclines will greatly improve the design of optimal chemotherapeutic regimens. However, identification of such variants is hampered by the lack of analytical approaches that address the complex, multi-genic character of anthracycline induced cardiotoxicity (AIC). Here, using a multi-SNP based approach, we examined 60 genes coding for proteins involved in drug metabolism and efflux and identified the P450 oxidoreductase (POR) gene to be most strongly associated with daunorubicin induced cardiotoxicity in a population of acute myeloid leukemia (AML) patients (FDR adjusted p-value of 0.15). In this sample of cancer patients, variation in the POR gene is estimated to account for some 11.6% of the variability in the drop of left ventricular ejection fraction (LVEF) after daunorubicin treatment, compared to the estimated 13.2% accounted for by the cumulative dose and ethnicity. In post-hoc analysis, this association was driven by 3 SNPs—the rs2868177, rs13240755, and rs4732513—through their linear interaction with cumulative daunorubicin dose. The unadjusted odds ratios (ORs) and confidence intervals (CIs) for rs2868177 and rs13240755 were estimated to be 1.89 (95% CI: 0.7435–4.819; p = 0.1756) and 3.18 (95% CI: 1.223–8.27; p = 0.01376), respectively. Although the contribution of POR variants is expected to be overestimated due to the multiple testing performed in this small pilot study, given that cumulative anthracycline dose is virtually the only factor used clinically to predict the risk of cardiotoxicity, the contribution that genetic analyses of POR can make to the assessment of this risk is worthy of follow up in future investigations. PMID:24273552

  1. 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 C2221, with two molecules in the asymmetric unit. PMID:25849509

  2. Novel Insights into the Enzymology, Regulation and Physiological Functions of Light-dependent Protochlorophyllide Oxidoreductase in Angiosperms

    Microsoft Academic Search

    Tatsuru Masuda; Ken-ichiro Takamiya

    2004-01-01

    The reduction of protochlorophyllide (Pchlide) is a key regulatory step in the biosynthesis of chlorophyll in phototrophic organisms. Two distinct enzymes catalyze this reduction; a light-dependent NADPH:protochlorophyllide oxidoreductase (POR) and light-independent Pchlide reductase (DPOR). Both enzymes are widely distributed among phototrophic organisms with the exception that only POR is found in angiosperms and only DPOR in anoxygenic photosynthetic bacteria. Consequently,

  3. The photoenzymatic cycle of NADPH: protochlorophyllide oxidoreductase in primary bean leaves ( Phaseolus vulgaris ) during the first days of photoperiodic growth

    Microsoft Academic Search

    Benoît Schoefs; Fabrice Franck

    2008-01-01

    The photoenzymatic cycle of the light-dependent NADPH:protochlorophyllide oxidoreductase (LPOR) was investigated in situ during\\u000a early stages of development of bean leaves under light-dark cycles (LDC). In the experimental system used in this study, prolamellar\\u000a bodies developed during night periods and disappeared during light periods. This was accompanied by changes in the photoactive\\u000a to non-photoactive Pchlide ratio, which was higher at the

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

    Microsoft Academic Search

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

    2005-01-01

    DsbA proteins, the primary catalysts of protein disulfide bond formation, are known to affect virulence and penicillin resistance\\u000a in Gram-negative bacteria. We identified a putative DsbA homologue in the Gram-positive pathogen Staphylococcus aureus that was able to restore the motility phenotype of an Escherichia coli dsbA mutant and thus demonstrated a functional thiol oxidoreductase activity. The staphylococcal DsbA (SaDsbA) had

  5. Substrate Specificity of the Oxidoreductase ERp57 Is Determined Primarily by Its Interaction with Calnexin and Calreticulin*

    PubMed Central

    Jessop, Catherine E.; Tavender, Timothy J.; Watkins, Rachel H.; Chambers, Joseph E.; Bulleid, Neil J.

    2009-01-01

    The formation of disulfides within proteins entering the secretory pathway is catalyzed by the protein disulfide isomerase family of endoplasmic reticulum localized oxidoreductases. One such enzyme, ERp57, is thought to catalyze the isomerization of non-native disulfide bonds formed in glycoproteins with unstructured disulfide-rich domains. Here we investigated the mechanism underlying ERp57 specificity toward glycoprotein substrates and the interdependence of ERp57 and the calnexin cycle for their correct folding. Our results clearly show that ERp57 must be physically associated with the calnexin cycle to catalyze isomerization reactions with most of its substrates. In addition, some glycoproteins only require ERp57 for correct disulfide formation if they enter the calnexin cycle. Hence, the specificity of ER oxidoreductases is not only determined by the physical association of enzyme and substrate but also by accessory factors, such as calnexin and calreticulin in the case of ERp57. These conclusions suggest that the calnexin cycle has evolved with a specialized oxidoreductase to facilitate native disulfide formation in complex glycoproteins. PMID:19054761

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

    PubMed

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

    2005-01-01

    In Gram-negative bacterial cells, disulfide bond formation occurs in the oxidative environment of the periplasm and is catalysed by Dsb (disulfide bond) proteins found in the periplasm and in the inner membrane. In this report the identification of a new subfamily of disulfide oxidoreductases encoded by a gene denoted dsbI, and functional characterization of DsbI proteins from Campylobacter jejuni and Helicobacter pylori, as well as DsbB from C. jejuni, are described. The N-terminal domain of DsbI is related to DsbB proteins and comprises five predicted transmembrane segments, while the C-terminal domain is predicted to locate to the periplasm and to fold into a beta-propeller structure. The dsbI gene is co-transcribed with a small ORF designated dba (dsbI-accessory). Based on a series of deletion and complementation experiments it is proposed that DsbB can complement the lack of DsbI but not the converse. In the presence of DsbB, the activity of DsbI was undetectable, hence it probably acts only on a subset of possible substrates of DsbB. To reconstruct the principal events in the evolution of DsbB and DsbI proteins, sequences of all their homologues identifiable in databases were analysed. In the course of this study, previously undetected variations on the common thiol-oxidoreductase theme were identified, such as development of an additional transmembrane helix and loss or migration of the second pair of Cys residues between two distinct periplasmic loops. In conjunction with the experimental characterization of two members of the DsbI lineage, this analysis has resulted in the first comprehensive classification of the DsbB/DsbI family based on structural, functional and evolutionary criteria. PMID:15632440

  7. A bacterial quercetin oxidoreductase QuoA-mediated perturbation in the phenylpropanoid metabolic network increases lignification with a concomitant decrease in phenolamides in Arabidopsis

    PubMed Central

    Swarup, Sanjay

    2013-01-01

    Metabolic perturbations by a gain-of-function approach provide a means to alter steady states of metabolites and query network properties, while keeping enzyme complexes intact. A combination of genetic and targeted metabolomics approach was used to understand the network properties of phenylpropanoid secondary metabolism pathways. A novel quercetin oxidoreductase, QuoA, from Pseudomonas putida, which converts quercetin to naringenin, thus effectively reversing the biosynthesis of quercetin through a de novo pathway, was expressed in Arabidopsis thaliana. QuoA transgenic lines selected for low, medium, and high expression levels of QuoA RNA had corresponding levels of QuoA activity and hypocotyl coloration resulting from increased anthocyanin accumulation. Stems of all three QuoA lines had increased tensile strength resulting from increased lignification. Sixteen metabolic intermediates from anthocyanin, lignin, and shikimate pathways had increased accumulation, of which 11 paralleled QuoA expression levels in the transgenic lines. The concomitant upregulation of the above pathways was explained by a significant downregulation of the phenolamide pathway and its precursor, spermidine. In a tt6 mutant line, lignifications as well as levels of the lignin pathway metabolites were much lower than those of QuoA transgenic lines. Unlike QuoA lines, phenolamides and spermidine were not affected in the tt6 line. Taken together, these results suggest that phenolamide pathway plays a major role in directing metabolic intermediates into the lignin pathway. Metabolic perturbations were accompanied by downregulation of five genes associated with branch-point enzymes and upregulation of their corresponding products. These results suggest that gene–metabolite pairs are likely to be co-ordinately regulated at critical branch points. Thus, these perturbations by a gain-of-function approach have uncovered novel properties of the phenylpropanoid metabolic network. PMID:24085580

  8. A bacterial quercetin oxidoreductase QuoA-mediated perturbation in the phenylpropanoid metabolic network increases lignification with a concomitant decrease in phenolamides in Arabidopsis.

    PubMed

    Reuben, Sheela; Rai, Amit; Pillai, Bhinu V S; Rodrigues, Amrith; Swarup, Sanjay

    2013-11-01

    Metabolic perturbations by a gain-of-function approach provide a means to alter steady states of metabolites and query network properties, while keeping enzyme complexes intact. A combination of genetic and targeted metabolomics approach was used to understand the network properties of phenylpropanoid secondary metabolism pathways. A novel quercetin oxidoreductase, QuoA, from Pseudomonas putida, which converts quercetin to naringenin, thus effectively reversing the biosynthesis of quercetin through a de novo pathway, was expressed in Arabidopsis thaliana. QuoA transgenic lines selected for low, medium, and high expression levels of QuoA RNA had corresponding levels of QuoA activity and hypocotyl coloration resulting from increased anthocyanin accumulation. Stems of all three QuoA lines had increased tensile strength resulting from increased lignification. Sixteen metabolic intermediates from anthocyanin, lignin, and shikimate pathways had increased accumulation, of which 11 paralleled QuoA expression levels in the transgenic lines. The concomitant upregulation of the above pathways was explained by a significant downregulation of the phenolamide pathway and its precursor, spermidine. In a tt6 mutant line, lignifications as well as levels of the lignin pathway metabolites were much lower than those of QuoA transgenic lines. Unlike QuoA lines, phenolamides and spermidine were not affected in the tt6 line. Taken together, these results suggest that phenolamide pathway plays a major role in directing metabolic intermediates into the lignin pathway. Metabolic perturbations were accompanied by downregulation of five genes associated with branch-point enzymes and upregulation of their corresponding products. These results suggest that gene-metabolite pairs are likely to be co-ordinately regulated at critical branch points. Thus, these perturbations by a gain-of-function approach have uncovered novel properties of the phenylpropanoid metabolic network. PMID:24085580

  9. Xanthine Oxidoreductase-Catalyzed Reduction of Nitrite to Nitric Oxide: Insights Regarding Where, When and How

    PubMed Central

    Cantu-Medellin, Nadiezhda; Kelley, Eric E.

    2013-01-01

    Numerous inflammatory disorders are associated with elevated levels of xanthine oxidoreductase (XOR) and allied enhancement of reactive species formation contributory to systemic pathology. Despite a long standing association between increased XOR activity and negative clinical outcomes, recent reports describe a paradigm shift where XOR mediates beneficial actions by catalyzing the reduction of NO2? to •NO. While provocative, these observations contradict reports of improved outcomes in similar models upon XOR inhibition as well as reports revealing strict anoxia as a requisite for XOR-mediated •NO formation. To garner a more clear understanding of conditions necessary for in vivo XOR-catalyzed •NO production, this review critically analyzes the impact of O2 tension, pH, substrate concentrations, glycoaminoglycan docking and inhibition strategies on the nitrite reductase activity of XOR and reveals a hypoxic milieu where this process may be operative. As such, information herein serves to link recent reports in which XOR activity has been identified as mediating the beneficial outcomes resulting from nitrite supplementation to a microenviromental setting where XOR can serve as substantial source of •NO. PMID:23454592

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

    SciTech Connect

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

    2009-09-02

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

  11. Evolving thermostability in mutant libraries of ligninolytic oxidoreductases expressed in yeast

    PubMed Central

    2010-01-01

    Background In the picture of a laboratory evolution experiment, to improve the thermostability whilst maintaining the activity requires of suitable procedures to generate diversity in combination with robust high-throughput protocols. The current work describes how to achieve this goal by engineering ligninolytic oxidoreductases (a high-redox potential laccase -HRPL- and a versatile peroxidase, -VP-) functionally expressed in Saccharomyces cerevisiae. Results Taking advantage of the eukaryotic machinery, complex mutant libraries were constructed by different in vivo recombination approaches and explored for improved stabilities and activities. A reliable high-throughput assay based on the analysis of T50 was employed for discovering thermostable oxidases from mutant libraries in yeast. Both VP and HRPL libraries contained variants with shifts in the T50 values. Stabilizing mutations were found at the surface of the protein establishing new interactions with the surrounding residues. Conclusions The existing tradeoff between activity and stability determined from many point mutations discovered by directed evolution and other protein engineering means can be circumvented combining different tools of in vitro evolution. PMID:20298573

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

    PubMed

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

    2014-12-01

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

  13. Mutations Associated with Functional Disorder of Xanthine Oxidoreductase and Hereditary Xanthinuria in Humans

    PubMed Central

    Ichida, Kimiyoshi; Amaya, Yoshihiro; Okamoto, Ken; Nishino, Takeshi

    2012-01-01

    Xanthine oxidoreductase (XOR) catalyzes the conversion of hypoxanthine to xanthine and xanthine to uric acid with concomitant reduction of either NAD+ or O2. The enzyme is a target of drugs to treat hyperuricemia, gout and reactive oxygen-related diseases. Human diseases associated with genetically determined dysfunction of XOR are termed xanthinuria, because of the excretion of xanthine in urine. Xanthinuria is classified into two subtypes, type I and type II. Type I xanthinuria involves XOR deficiency due to genetic defect of XOR, whereas type II xanthinuria involves dual deficiency of XOR and aldehyde oxidase (AO, a molybdoflavo enzyme similar to XOR) due to genetic defect in the molybdenum cofactor sulfurase. Molybdenum cofactor deficiency is associated with triple deficiency of XOR, AO and sulfite oxidase, due to defective synthesis of molybdopterin, which is a precursor of molybdenum cofactor for all three enzymes. The present review focuses on mutation or chemical modification studies of mammalian XOR, as well as on XOR mutations identified in humans, aimed at understanding the reaction mechanism of XOR and the relevance of mutated XORs as models to estimate the possible side effects of clinical application of XOR inhibitors. PMID:23203137

  14. Functional characterization of the FoxE iron oxidoreductase from the photoferrotroph Rhodobacter ferrooxidans SW2.

    PubMed

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

    2012-07-20

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

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

    PubMed Central

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

    2012-01-01

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

  16. Reduction of Clofazimine by Mycobacterial Type 2 NADH:Quinone Oxidoreductase

    PubMed Central

    Yano, Takahiro; Kassovska-Bratinova, Sacha; Teh, J. Shin; Winkler, Jeffrey; Sullivan, Kevin; Isaacs, Andre; Schechter, Norman M.; Rubin, Harvey

    2011-01-01

    The mechanism of action of clofazimine (CFZ), an antimycobacterial drug with a long history, is not well understood. The present study describes a redox cycling pathway that involves the enzymatic reduction of CFZ by NDH-2, the primary respiratory chain NADH:quinone oxidoreductase of mycobacteria and nonenzymatic oxidation of reduced CFZ by O2 yielding CFZ and reactive oxygen species (ROS). This pathway was demonstrated using isolated membranes and purified recombinant NDH-2. The reduction and oxidation of CFZ was measured spectrally, and the production of ROS was measured using a coupled assay system with Amplex Red. Supporting the ROS-based killing mechanism, bacteria grown in the presence of antioxidants are more resistant to CFZ. CFZ-mediated increase in NADH oxidation and ROS production were not observed in membranes from three different Gram-negative bacteria but was observed in Staphylococcus aureus and Saccharomyces cerevisiae, which is consistent with the known antimicrobial specificity of CFZ. A more soluble analog of CFZ, KS6, was synthesized and was shown to have the same activities as CFZ. These studies describe a pathway for a continuous and high rate of reactive oxygen species production in Mycobacterium smegmatis treated with CFZ and a CFZ analog as well as evidence that cell death produced by these agents are related to the production of these radical species. PMID:21193400

  17. Kinetics and regulation of mammalian NADH-ubiquinone oxidoreductase (Complex I).

    PubMed

    Chen, Xuewen; Qi, Feng; Dash, Ranjan K; Beard, Daniel A

    2010-09-01

    NADH-ubiquinone oxidoreductase (Complex I, European Commission No. 1.6.5.3) is one of the respiratory complexes that generate the proton-motive force required for the synthesis of ATP in mitochondria. The catalytic mechanism of Complex I has not been well understood, due to the complicated structure of this enzyme. Here, we develop a kinetic model for Complex I that accounts for electron transfer from NADH to ubiquinone through protein-bound prosthetic groups, which is coupled to the translocation of protons across the inner mitochondrial membrane. The model is derived based on the tri-bi enzyme mechanism combined with a simple model of the conformational changes associated with proton transport. To study the catalytic mechanism, parameter values are estimated by analyzing kinetic data. The model is further validated by independent data sets from additional experiments, effectively explaining the effect of pH on enzyme activity. Results imply that matrix pH significantly affects the enzyme turnover processes. The overall kinetic analysis demonstrates a hybrid ping-pong rapid-equilibrium random bi-bi mechanism, consolidating the characteristics from previously reported kinetic mechanisms and data. PMID:20816054

  18. Functional and structural characterization of protein disulfide oxidoreductase from Thermus thermophilus HB27.

    PubMed

    Pedone, Emilia; Fiorentino, Gabriella; Pirone, Luciano; Contursi, Patrizia; Bartolucci, Simonetta; Limauro, Danila

    2014-07-01

    The paper reports the characterization of a protein disulfide oxidoreductase (PDO) from the thermophilic Gram negative bacterium Thermus thermophilus HB27, identified as TTC0486 by genome analysis and named TtPDO. PDO members are involved in the oxidative folding, redox balance and detoxification of peroxides in thermophilic prokaryotes. Ttpdo was cloned and expressed in E. coli and the recombinant purified protein was assayed for the dithiol-reductase activity using insulin as substrate and compared with other PDOs characterized so far. In the thermophilic archaeon Sulfolobus solfataricus PDOs work as thiol-reductases constituting a peculiar redox couple with Thioredoxin reductase (SsTr). To get insight into the role of TtPDO, a hybrid redox couple with SsTr, homologous to putative Trs of T. thermophilus, was assayed. The results showed that SsTr was able to reduce TtPDO in a concentration dependent manner with a calculated K M of 34.72 ?M, suggesting the existence of a new redox system also in thermophilic bacteria. In addition, structural characterization of TtPDO by light scattering and circular dichroism revealed the monomeric structure and the high thermostability of the protein. The analysis of the genomic environment suggested a possible clustering of Ttpdo with TTC0487 and TTC0488 (tlpA). Accordingly, transcriptional analysis showed that Ttpdo is transcribed as polycistronic messenger. Primer extension analysis allowed the determination of its 5'end and the identification of the promoter region. PMID:24839097

  19. WW domain-containing oxidoreductase promotes neuronal differentiation via negative regulation of glycogen synthase kinase 3?

    PubMed Central

    Wang, H-Y; Juo, L-I; Lin, Y-T; Hsiao, M; Lin, J-T; Tsai, C-H; Tzeng, Y-H; Chuang, Y-C; Chang, N-S; Yang, C-N; Lu, P-J

    2012-01-01

    WW domain-containing oxidoreductase (WWOX), a putative tumour suppressor, is suggested to be involved in the hyperphosphorylation of Alzheimer's Tau. Tau is a microtubule-associated protein that has an important role in microtubule assembly and stability. Glycogen synthase kinase 3? (GSK3?) has a vital role in Tau hyperphosphorylation at its microtubule-binding domains. Hyperphosphorylated Tau has a low affinity for microtubules, thus disrupting microtubule stability. Bioinformatics analysis indicated that WWOX contains two potential GSK3?-binding FXXXLI/VXRLE motifs. Immunofluorescence, immunoprecipitation and molecular modelling showed that WWOX interacts physically with GSK3?. We demonstrated biochemically that WWOX can bind directly to GSK3? through its short-chain alcohol dehydrogenase/reductase domain. Moreover, the overexpression of WWOX inhibited GSK3?-stimulated S396 and S404 phosphorylation within the microtubule domains of Tau, indicating that WWOX is involved in regulating GSK3? activity in cells. WWOX repressed GSK3? activity, restored the microtubule assembly activity of Tau and promoted neurite outgrowth in SH-SY5Y cells. Conversely, RNAi-mediated knockdown of WWOX in retinoic acid (RA)-differentiated SH-SY5Y cells inhibited neurite outgrowth. These results suggest that WWOX is likely to be involved in regulating GSK3? activity, reducing the level of phosphorylated Tau, and subsequently promoting neurite outgrowth during neuron differentiation. In summary, our data reveal a novel mechanism by which WWOX promotes neuronal differentiation in response to RA. PMID:22193544

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

    PubMed Central

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

    2014-01-01

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

  1. Localization, Purification, and Characterization of Shikimate Oxidoreductase-Dehydroquinate Hydrolyase from Stroma of Spinach Chloroplasts 1

    PubMed Central

    Fiedler, Erich; Schultz, Gernot

    1985-01-01

    The stroma of chloroplasts is probably the sole site of the shikimate pathway enzymes shikimate oxidoreductase/dehydroquinate hydrolyase (SORase/DHQase) in spinach leaves. (a) The chromatographic behavior of the bifunctional protein SORase/DHQase on several separation materials with extracts from stroma compared with leaf extracts showed only one peak of enzymic activity originating from the stroma. (b) Polyacrylamide gel electrophoresis (PAGE) of these extracts followed by specific staining resulted in the same pattern without a band of extraplastidic enzyme. (c) In protoplast fractionation experiments it was shown that SORase/DHQase was present only in the soluble chloroplast protein fraction. An improved purification procedure for SORase/DHQase from stroma of chloroplasts, yield 40%, 1600 times as pure, gave essentially one protein band on sodium dodecyl sulfate-PAGE. Our results demonstrate that both enzyme functions are carried out by a single polypeptide. Nondenaturing PAGE exhibited a pattern of four bands with SORase/DHQase showing that they differ in charge but not in their molecular weight. Molecular weight was determined to be 67 kilodaltons (gel filtration) and 59 kilodaltons (PAGE) for all four forms. It was proven they were not due to artifacts. The four forms show similar kinetic properties, their Km and pH optima differing only very slightly. Response to some metabolites is reported. Images Fig. 3 Fig. 7 PMID:16664373

  2. Dietary nitrate ameliorates pulmonary hypertension: cytoprotective role for endothelial nitric oxide synthase and xanthine oxidoreductase

    PubMed Central

    Baliga, Reshma S; Milsom, Alexandra B; Ghosh, Suborno M; Trinder, Sarah L; MacAllister, Raymond J; Ahluwalia, Amrita; Hobbs, Adrian J

    2012-01-01

    Background Pulmonary hypertension (PH) is a multi-factorial disease characterized by increased pulmonary vascular resistance and right ventricular failure; morbidity and mortality remain unacceptably high. Loss of nitric oxide (NO) bioactivity is thought to contribute to the pathogenesis of PH and agents that augment pulmonary NO signaling are clinically effective in the disease. Inorganic nitrate (NO3?) and nitrite (NO2?) elicit a reduction in systemic blood pressure in healthy individuals; this effect is underpinned by endogenous and sequential reduction to NO. Herein, we determined whether dietary nitrate and nitrite might be preferentially reduced to NO by the hypoxia associated with PH, and thereby offer a convenient, inexpensive method of supplementing NO functionality to reduce disease severity. Methods & Results Dietary nitrate reduced the right ventricular pressure and hypertrophy, and pulmonary vascular re-modeling, in wild-type mice exposed to 3 weeks hypoxia; this beneficial activity was mirrored largely by dietary nitrite. The cytoprotective effects of dietary nitrate were associated with increased plasma & lung concentrations of nitrite and cGMP. The beneficial effects of dietary nitrate and nitrite were reduced in mice lacking endothelial NO synthase (eNOS) or treated with the xanthine oxidoreductase (XOR) inhibitor allopurinol. Conclusions These data demonstrate that dietary nitrate, and to a lesser extent dietary nitrite, elicit pulmonary dilatation, prevent pulmonary vascular remodeling, and reduce the RVH characteristic of PH. This favorable pharmacodynamic profile is dependent on eNOS and XOR -catalyzed reduction of nitrite to NO. Exploitation of this mechanism (i.e. dietary nitrate/nitrite supplementation) represents a viable, orally-active therapy for PH. PMID:22572914

  3. 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

  4. Migratory Activity of Human Breast Cancer Cells is Modulated by Differential Expression of Xanthine Oxidoreductase

    PubMed Central

    Fini, Mehdi A.; Orchard-Webb, David; Kosmider, Beata; Amon, Jeremy D.; Kelland, Robert; Shibao, Gayle; Wright, Richard M.

    2008-01-01

    Xanthine oxidoreductase (XOR) may exert an important, but poorly defined, role in the pathogenesis of breast cancer (BC). Loss of XOR expression was linked to aggressive BC, and recent clinical observations have suggested that decreasing XOR may be functionally linked to BC aggressiveness. The goal of the present investigation was to determine whether the decreased XOR observed in clinically aggressive BC was an intrinsic property of highly invasive mammary epithelial cells (MEC). Expression of XOR was investigated using HC11 mouse MEC, HB4a and MCF-10A normal human MEC, and several human mammary tumor cells including MCF-7 and MDA-MB-231. Consistent with clinical observations, data shown here revealed high levels of XOR in normal HC11 and MCF-10A cells that was markedly reduced in highly invasive mammary tumor cells. The contribution of XOR to tumor cell migration in vitro was investigated using MDA-MB-231 and MCF-7 cells and clonally selected derivatives of HC11 that exhibit either weak or strong migration in vitro. We observed that over-expression of an XOR cDNA in MDA-MB-231 and in HC11-C24, both possessing weak XOR expression and high migratory capacity, inhibited their migration in vitro. Conversely, pharmacological inhibition of XOR in MCF-7 and HC11-C4, both possessing high XOR expression and weak migratory capacity, stimulated their migration in vitro. Further experiments suggested that XOR derived ROS mediated this effect and also modulated COX-2 and MMP levels and function. These data demonstrate a functional link between XOR expression and MEC migration and suggest a potential role for XOR in suppressing BC pathogenesis. PMID:18767115

  5. Cyclin-Dependent Kinase Five Mediates Activation of Lung Xanthine Oxidoreductase in Response to Hypoxia

    PubMed Central

    Kim, Bo S.; Serebreni, Leonid; Fallica, Jonathan; Hamdan, Omar; Wang, Lan; Johnston, Laura; Kolb, Todd; Damarla, Mahendra; Damico, Rachel; Hassoun, Paul M.

    2015-01-01

    Background Xanthine oxidoreductase (XOR) is involved in oxidative metabolism of purines and is a source of reactive oxygen species (ROS). As such, XOR has been implicated in oxidant-mediated injury in multiple cardiopulmonary diseases. XOR enzyme activity is regulated, in part, via a phosphorylation-dependent, post-translational mechanism, although the kinase(s) responsible for such hyperactivation are unknown. Methods and Results Using an in silico approach, we identified a cyclin-dependent kinase 5 (CDK5) consensus motif adjacent to the XOR flavin adenine dinucleotide (FAD) binding domain. CDK5 is a proline-directed serine/threonine kinase historically linked to neural development and injury. We tested the hypothesis that CDK5 and its activators are mediators of hypoxia-induced hyperactivation of XOR in pulmonary microvascular endothelial cells (EC) and the intact murine lung. Using complementary molecular and pharmacologic approaches, we demonstrated that hypoxia significantly increased CDK5 activity in EC. This was coincident with increased expression of the CDK5 activators, cyclin-dependent kinase 5 activator 1 (CDK5r1 or p35/p25), and decreased expression of the CDK5 inhibitory peptide, p10. Expression of p35/p25 was necessary for XOR hyperactivation. Further, CDK5 physically associated with XOR and was necessary and sufficient for XOR phosphorylation and hyperactivation both in vitro and in vivo. XOR hyperactivation required the target threonine (T222) within the CDK5-consensus motif. Conclusions and Significance These results indicate that p35/CDK5-mediated phosphorylation of T222 is required for hypoxia-induced XOR hyperactivation in the lung. Recognizing the contribution of XOR to oxidative injury in cardiopulmonary disease, these observations identify p35/CDK5 as novel regulators of XOR and potential modifiers of ROS-mediated injury. PMID:25831123

  6. 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

  7. Identification of a Lactate-Quinone Oxidoreductase in Staphylococcus aureus that is Essential for Virulence

    PubMed Central

    Fuller, James R.; Vitko, Nicholas P.; Perkowski, Ellen F.; Scott, Eric; Khatri, Dal; Spontak, Jeffrey S.; Thurlow, Lance R.; Richardson, Anthony R.

    2011-01-01

    Staphylococcus aureus is an important human pathogen commonly infecting nearly every host tissue. The ability of S. aureus to resist innate immunity is critical to its success as a pathogen, including its propensity to grow in the presence of host nitric oxide (NO·). Upon exogenous NO· exposure, S. aureus immediately excretes copious amounts of L-lactate to maintain redox balance. However, after prolonged NO·-exposure, S. aureus reassimilates L-lactate specifically and in this work, we identify the enzyme responsible for this L-lactate-consumption as a L-lactate-quinone oxidoreductase (Lqo, SACOL2623). Originally annotated as Mqo2 and thought to oxidize malate, we show that this enzyme exhibits no affinity for malate but reacts specifically with L-lactate (KM?=??330??M). In addition to its requirement for reassimilation of L-lactate during NO·-stress, Lqo is also critical to respiratory growth on L-lactate as a sole carbon source. Moreover, ?lqo mutants exhibit attenuation in a murine model of sepsis, particularly in their ability to cause myocarditis. Interestingly, this cardiac-specific attenuation is completely abrogated in mice unable to synthesize inflammatory NO· (iNOS?/?). We demonstrate that S. aureus NO·-resistance is highly dependent on the availability of a glycolytic carbon sources. However, S. aureus can utilize the combination of peptides and L-lactate as carbon sources during NO·-stress in an Lqo-dependent fashion. Murine cardiac tissue has markedly high levels of L-lactate in comparison to renal or hepatic tissue consistent with the NO·-dependent requirement for Lqo in S. aureus myocarditis. Thus, Lqo provides S. aureus with yet another means of replicating in the presence of host NO·. PMID:22919585

  8. Purification, characterization, and properties of an aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646.

    PubMed Central

    Li, T; Rosazza, J P

    1997-01-01

    An aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646 was purified 196-fold by a combination of Mono-Q, Reactive Green 19 agarose affinity, and hydroxyapatite chromatographies. The purified enzyme runs as a single band of 140 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass was estimated to be 163 +/- 3.8 kDa by gel filtration, indicating that this enzyme is a monomeric protein. The binding of the enzyme to Reactive Green 19 agarose was Mg2+ dependent. The binding capacity was estimated to be about 0.2 mg of Reactive Green agarose per ml in the presence of 10 mM MgCl2. This enzyme can catalyze the reduction of a wide range of aryl carboxylic acids, including substituted benzoic acids, phenyl-substituted aliphatic acids, heterocyclic carboxylic acids, and polyaromatic ring carboxylic acids, to produce the corresponding aldehydes. The Km values for benzoate, ATP, and NADPH were determined to be 645 +/- 75, 29.3 +/- 3.1, and 57.3 +/- 12.5 microM, respectively. The Vmax was determined to be 0.902 +/- 0.04 micromol/min/mg of protein. Km values for (S)-(+)-alpha-methyl-4-(2-methylpropyl)-benzeneacetic acid (ibuprofen) and its (R)-(-) isomer were determined to be 155 +/- 18 and 34.5 +/- 2.5 microM, respectively. The Vmax for the (S)-(+) and (R)-(-) isomers were 1.33 and 0.15 micromol/min/mg of protein, respectively. Anthranilic acid is a competitive inhibitor with benzoic acid as a substrate, with a Ki of 261 +/- 30 microM. The N-terminal and internal amino acid sequences of a 76-kDa peptide from limited alpha-chymotrypsin digestion were determined. PMID:9171390

  9. 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

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

    PubMed

    Szczepaniak, Krzysztof; Worch, Remigiusz; Grzyb, Joanna

    2013-05-15

    Ferredoxin:NADP(+) oxidoreductase (FNR) is a plant and cyanobacterial photosynthetic enzyme, also found in non-photosynthetic tissues, where it is involved in redox reactions of biosynthetic pathways. In vivo it transfers electrons to nicotinamide adenine dinucleotide phosphate (NADP(+)), forming its reduced version, NADPH, while in vitro it can also use NADPH to reduce several substrates, such as ferricyanide, various quinones and nitriles. As an oxidoreductase catalyzing reaction of a broad range of substrates, FNR may be used in biotechnological processes. Quantum dots are semiconductor nanocrystals of a few to several nanometers diameter, having very useful luminescent properties. We present the spectroscopic and functional characteristics of a covalent conjugation of FNR and CdSe/ZnS quantum dots. Two types of quantum dots, of different diameter and emission maximum (550 and 650 nm), were used for comparison. Steady-state fluorescence and gel electrophoresis confirmed efficient conjugation, while fluorescence correlation spectroscopy (FCS) allowed for determination of the conjugates' radii. The nanohybrids sustained enzymatic activity; however, changes in maximal reaction rates and Michaelis constant were found. Detailed analysis of the kinetic parameters showed that the changes in the enzyme activity depend on the substrate used for activity measurement but also on the size of the quantum dots. The presented nanohybrids, as the first example using plant and photosynthetic enzyme as a protein partner, may became a tool to study photosynthesis as well as other biosynthetic and biotechnological processes, involving enzymatically catalyzed electron transfer. PMID:23611948

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

    PubMed

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

    2011-01-01

    Several microorganisms were isolated from soil/sediment samples of Antarctic Peninsula. The enrichment technique using (RS)-1-(phenyl)ethanol as a carbon source allowed us to isolate 232 psychrophile/psychrotroph microorganisms. We also evaluated the enzyme activity (oxidoreductases) for enantioselective oxidation reactions, by using derivatives of (RS)-1-(phenyl)ethanol as substrates. Among the studied microorganisms, 15 psychrophile/psychrotroph strains contain oxidoreductases that catalyze the (S)-enantiomer oxidation from racemic alcohols to their corresponding ketones. Among the identified microorganisms, Flavobacterium sp. and Arthrobacter sp. showed excellent enzymatic activity. These new bacteria strains were selected for optimization study, in which the (RS)-1-(4-methyl-phenyl)ethanol oxidation was evaluated in several reaction conditions. From these studies, it was observed that Flavobacterium sp. has an excellent enzymatic activity at 10 °C and Arthrobacter sp. at 15 and 25 °C. We have also determined the growth curves of these bacteria, and both strains showed optimum growth at 25 °C, indicating that these bacteria are psychrotroph. PMID:21673897

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-04-01

    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

  14. 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

  15. T. Wydrzynski and K. Satoh (eds): Photosystem II: The Water/Plastoquinone Oxido-Reductase in Photosynthesis, pp. 000000. 2005 Kluwer Academic Publishers. Printed in The Netherlands.

    E-print Network

    Crofts, Antony R.

    T. Wydrzynski and K. Satoh (eds): Photosystem II: The Water/Plastoquinone Oxido-Reductase in Photosynthesis, pp. 000­000. © 2005 Kluwer Academic Publishers. Printed in The Netherlands. FIRST PROOF Chapter 8-crofts@life.uiuc.edu The Quinone Iron Acceptor Complex Vasili Petrouleas Institute of Materials Science, NCSR `Demokritos', 153 10

  16. Low NAD(P)H:quinone oxidoreductase activity is associated with increased risk of leukemia with MLL translocations in infants and children

    E-print Network

    California at Berkeley, University of

    NEOPLASIA Low NAD(P)H:quinone oxidoreductase activity is associated with increased risk of leukemia with an increased risk of adult leukemia. A small British study suggested that NQO1 C609T was associated with an in- creased risk of infant leukemias with MLL translocations, especially infant acute lymphoblastic leukemia

  17. The hybrid-cluster protein ('prismane protein') from Escherichia coli. Characterization of the hybrid-cluster protein, redox properties of the [2Fe-2S] and [4Fe-2S-2O] clusters and identification of an associated NADH oxidoreductase containing FAD and [2Fe-2S].

    PubMed

    van den Berg, W A; Hagen, W R; van Dongen, W M

    2000-02-01

    Hybrid-cluster proteins ('prismane proteins') have previously been isolated and characterized from strictly anaerobic sulfate-reducing bacteria. These proteins contain two types of Fe/S clusters unique in biological systems: a [4Fe-4S] cubane cluster with spin-admixed S = 3/2 ground-state paramagnetism and a novel type of hybrid [4Fe-2S-2O] cluster, which can attain four redox states. Genomic sequencing reveals that genes encoding putative hybrid-cluster proteins are present in a range of bacterial and archaeal species. In this paper we describe the isolation and spectroscopic characterization of the hybrid-cluster protein from Escherichia coli. EPR spectroscopy shows the presence of a hybrid cluster in the E. coli protein with characteristics similar to those in the proteins of anaerobic sulfate reducers. EPR spectra of the reduced E. coli hybrid-cluster protein, however, give evidence for the presence of a [2Fe-2S] cluster instead of a [4Fe-4S] cluster. The hcp gene encoding the hybrid-cluster protein in E. coli and other facultative anaerobes occurs, in contrast with hcp genes in obligate anaerobic bacteria and archaea, in a small operon with a gene encoding a putative NADH oxidoreductase. This NADH oxidoreductase was also isolated and shown to contain FAD and a [2Fe-2S] cluster as cofactors. It catalysed the reduction of the hybrid-cluster protein with NADH as an electron donor. Midpoint potentials (25 degrees C, pH 7.5) for the Fe/S clusters in both proteins indicate that electrons derived from the oxidation of NADH (Em NADH/NAD+ couple: -320 mV) are transferred along the [2Fe-2S] cluster of the NADH oxidoreductase (Em = -220 mV) and the [2Fe-2S] cluster of the hybrid-cluster protein (Em = -35 mV) to the hybrid cluster (Em = -50, +85 and +365 mV for the three redox transitions). The physiological function of the hybrid-cluster protein has not yet been elucidated. The protein is only detected in the facultative anaerobes E. coli and Morganella morganii after cultivation under anaerobic conditions in the presence of nitrate or nitrite, suggesting a role in nitrate-and/or nitrite respiration. PMID:10651802

  18. Diversity and Spatial Distribution of Hydrazine Oxidoreductase (hzo) Gene in the Oxygen Minimum Zone Off Costa Rica

    E-print Network

    Kong, Liangliang

    Anaerobic ammonia oxidation (anammox) as an important nitrogen loss pathway has been reported in marine oxygen minimum zones (OMZs), but the community composition and spatial distribution of anammox bacteria in the eastern ...

  19. Factor XI is a substrate for oxidoreductases: enhanced activation of reduced FXI and its role in antiphospholipid syndrome thrombosis.

    PubMed

    Giannakopoulos, Bill; Gao, Lu; Qi, Miao; Wong, Jason W; Yu, Demin M; Vlachoyiannopoulos, Panayiotis G; Moutsopoulos, Harry M; Atsumi, Tatsuya; Koike, Takao; Hogg, Philip; Qi, Jian C; Krilis, Steven A

    2012-09-01

    Factor XI (FXI), a disulfide-linked covalent homodimer, circulates in plasma, and upon activation initiates the intrinsic/consolidation phase of coagulation. We present evidence that disulfide bonds in FXI are reduced to free thiols by oxidoreductases thioredoxin-1 (TRX-1) and protein disulfide isomerase (PDI). We identified that Cys362-Cys482 and Cys118-Cys147 disulfide bonds are reduced by TRX-1. The activation of TRX-1-treated FXI by thrombin, FXIIa or FXIa was significantly increased compared to non-reduced FXI, indicating that the reduced factor is more efficiently activated than the oxidized protein. Using a novel ELISA system, we compared the amount of reduced FXI in antiphospholipid syndrome (APS) thrombosis patients with levels in healthy controls, and found that APS patients have higher levels of reduced FXI. This may have implication for understanding the contribution of FXI to APS thrombosis, and the predisposition to thrombosis in patients with elevated plasma levels of reduced FXI. PMID:22704541

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

    PubMed

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

    2009-03-01

    The aim of this study was to investigate the effect of tart cherry juice on serum uric acid levels, hepatic xanthine oxidoreductase activity and two non-invasive biomarkers of oxidative stress (total antioxidant capacity and malondialdehyde concentration), in normal and hyperuricemic rats. Tart cherry juice (5 ml/kg) was given by oral gavage to rats for 2 weeks. Allopurinol (5 mg/kg) was used as a positive control and was also given by oral gavage. Data showed that tart cherry juice treatment did not cause any significant reduction in the serum uric acid levels in normal rats, but significantly reduced (P<0.05) the serum uric acid levels of hyperuricemic rats in a time-dependent manner. Tart cherry juice treatment also inhibited hepatic xanthine oxidase/dehydrogenase activity. Moreover, a significant increase (P<0.05) in serum total antioxidant capacity was observed in tart cherry juice treated-rats in both normal and hyperuricemic groups. The oral administration of tart cherry juice also led to a significant reduction (P<0.05) in MDA concentration in the hyperuricemic rats. Although the hypouricemic effect of allopurinol, as a putative inhibitor of xanthine oxidoreductase, was much higher than that of tart cherry, it could not significantly change anti-oxidative parameters. These features of tart cherry make it an attractive candidate for the prophylactic treatment of hyperuricaemia, particularly if it is to be taken on a long-term basis. Further investigations to define its clinical efficacy would be highly desirable. PMID:22691805

  1. 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

  2. [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.

  3. 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

  4. Prenatal diagnosis of P450 oxidoreductase deficiency (ORD): a disorder causing low pregnancy estriol, maternal and fetal virilization, and the Antley-Bixler syndrome phenotype.

    PubMed

    Shackleton, Cedric; Marcos, Josep; Arlt, Wiebke; Hauffa, Berthold P

    2004-08-30

    We report studies on the second pregnancy of a woman who had previously given birth to a virilized female infant. The cause of the virilization had not been established, but common forms of congenital adrenal hyperplasia (CAH) were excluded. Longitudinal monitoring of the second pregnancy revealed that estriol excretion failed to increase normally, reaching a maximum 0.7 mg/24 hr at the end of pregnancy (normal mean 30 mg/24 hr). The mother showed signs of virilization by the 23rd week of gestation and aromatase deficiency was suspected. However, predicted urinary metabolites for diagnosis of aromatase deficiency (for example, 16alpha-hydroxyandrosterone) were not increased significantly during the pregnancy. Interestingly, excretion of the androgen metabolite androsterone increased rapidly at the beginning of pregnancy and peaked around the 20th week, suggesting increased production of testosterone and 5alphaDHT, probably the cause of maternal virilization. Urine steroid analysis by GC/MS showed gradually increasing excretion (9 mg/24 hr) of the normally minor metabolite 5alpha-pregnane-3beta,20alpha-diol (epiallopregnanediol), an epimer of the dominant progesterone metabolite pregnanediol (5beta-pregnane-3alpha,20alpha-diol). We believe epiallopregnanediol is largely the maternal urinary excretion product of fetal 5-pregnene-3beta,20alpha-diol, the principal metabolite of pregnenolone, implying a build-up of the latter steroid in the fetal adrenal. These findings suggested that the 'block' in the estriol biosynthetic pathway occurs at an early stage with 17-hydroxylation of pregnenolone being affected. The male baby born of this pregnancy had normal genitalia but showed a urinary steroid profile indicating partial deficiencies of P450c17 and P450c21. However, no mutations in the corresponding CYP17 and CYP21 genes were identified. Urinary steroid analysis carried out on his virilized older sibling showed the same pattern of metabolites. Recently, we determined that this disorder is caused by mutations in P450 oxidoreductase (OR), the essential redox partner for CYP17 and CYP21 hydroxylases. The novel metabolic profile has now been seen in many patients, most diagnosed with the skeletal dysplasia Antley-Bixler syndrome. We propose that excessive excretion of epiallopregnanediol together with low estriol may be prenatally diagnostic for OR deficiency (ORD). PMID:15316970

  5. Differential expression of genes encoding the light-dependent and light-independent enzymes for protochlorophyllide reduction during development in loblolly pine

    Microsoft Academic Search

    Jeffrey S. Skinner; Michael P. Timko

    1999-01-01

    The expression patterns of the two distinct subfamilies of genes (designated porA and porB) encoding the light- dependent NADPH:protochlorophyllide oxidoreductases (PORs) in loblolly pine (Pinus taeda L.) were examined. Transcripts arising from both gene subfamilies were shown to be present at high levels in the cotyledons of dark-grown pine seedlings and to a lesser extent in their stems. Exposure of

  6. Identification of the NF-E2-related Factor2-dependent Genes Conferring Protection against Oxidative Stress in Primary Cortical Astrocytes Using Oligonucleotide Microarray Analysis

    Microsoft Academic Search

    Jong-Min Lee; Marcus J. Calkins; Kaimin Chan; Yuet Wai Kan; Jeffrey A. Johnson

    2003-01-01

    The antioxidant responsive element (ARE) mediates transcriptional regulation of phase II detoxification en- zymes and antioxidant proteins such as NAD(P)H:qui- none oxidoreductase (NQO1), glutathione S-trans- ferases, and glutamate-cysteine ligase. In this study, we demonstrate that NF-E2-related factor-2 (Nrf2) plays a major role in transcriptional activation of ARE-driven genes and identify Nrf2-dependent genes by oligonu- cleotide microarray analysis using primary cortical

  7. The Oxidoreductase DsbA Plays a Key Role in the Ability of the Crohn's Disease-Associated Adherent-Invasive Escherichia coli Strain LF82 To Resist Macrophage Killing?

    PubMed Central

    Bringer, Marie-Agnès; Rolhion, Nathalie; Glasser, Anne-Lise; Darfeuille-Michaud, Arlette

    2007-01-01

    Adherent-invasive Escherichia coli (AIEC) isolated from Crohn's disease patients is able to adhere to and invade intestinal epithelial cells and to replicate in mature phagolysosomes within macrophages. Here, we show that the dsbA gene, encoding a periplasmic oxidoreductase, was required for AIEC strain LF82 to adhere to intestinal epithelial cells and to survive within macrophages. The LF82-?dsbA mutant did not express flagella and, probably as a consequence of this, did not express type 1 pili. The role of DsbA in adhesion is restricted to the loss of flagella and type 1 pili, as forced contact between bacteria and cells and induced expression of type 1 pili restored the wild-type phenotype. In contrast, the dsbA gene is essential for AIEC LF82 bacteria to survive within macrophages, irrespective of the loss of flagella and type 1 pilus expression, and the survival ability of LF82-?dsbA was as low as that of the nonpathogenic E. coli K-12, which was efficiently killed by macrophages. We also provide evidence that the dsbA gene is needed for LF82 bacteria to grow and survive in an acidic and nutrient-poor medium that partly mimics the harsh environment of the phagocytic vacuole. In addition, under such stress conditions dsbA transcription is highly up-regulated. Finally, the CpxRA signaling pathway does not play a role in regulation of dsbA expression in AIEC LF82 bacteria under conditions similar to those of mature phagolysosomes. PMID:17449627

  8. The oxidoreductase DsbA plays a key role in the ability of the Crohn's disease-associated adherent-invasive Escherichia coli strain LF82 to resist macrophage killing.

    PubMed

    Bringer, Marie-Agnès; Rolhion, Nathalie; Glasser, Anne-Lise; Darfeuille-Michaud, Arlette

    2007-07-01

    Adherent-invasive Escherichia coli (AIEC) isolated from Crohn's disease patients is able to adhere to and invade intestinal epithelial cells and to replicate in mature phagolysosomes within macrophages. Here, we show that the dsbA gene, encoding a periplasmic oxidoreductase, was required for AIEC strain LF82 to adhere to intestinal epithelial cells and to survive within macrophages. The LF82-DeltadsbA mutant did not express flagella and, probably as a consequence of this, did not express type 1 pili. The role of DsbA in adhesion is restricted to the loss of flagella and type 1 pili, as forced contact between bacteria and cells and induced expression of type 1 pili restored the wild-type phenotype. In contrast, the dsbA gene is essential for AIEC LF82 bacteria to survive within macrophages, irrespective of the loss of flagella and type 1 pilus expression, and the survival ability of LF82-DeltadsbA was as low as that of the nonpathogenic E. coli K-12, which was efficiently killed by macrophages. We also provide evidence that the dsbA gene is needed for LF82 bacteria to grow and survive in an acidic and nutrient-poor medium that partly mimics the harsh environment of the phagocytic vacuole. In addition, under such stress conditions dsbA transcription is highly up-regulated. Finally, the CpxRA signaling pathway does not play a role in regulation of dsbA expression in AIEC LF82 bacteria under conditions similar to those of mature phagolysosomes. PMID:17449627

  9. Stable Expression of Human Cytochrome P450 3A4 in Conjunction with Human NADPH-Cytochrome P450 Oxidoreductase in V79 Chinese Hamster Cells

    Microsoft Academic Search

    Anneliese Schneider; Wolfgang A. Schmalix; Vasanthi Siruguri; Els M. de Groene; G. Jean Horbach; Britta Kleingeist; Dieter Lang; Ronald Böcker; Claire Belloc; Philippe Beaune; Helmut Greim; Johannes Doehmer

    1996-01-01

    V79 Chinese hamster cells were constructed for stable expression of human cytochrome P450 3A4 with and without coexpression of human NADPH-cytochrome P450 oxidoreductase. Expression of the cDNAs was shown by Northern and Western analyses. Activity was tested by 6?-hydroxylation of testosterone for cytochrome P450 3A4 and by cytochrome c reduction for NADPH-cytochrome P450 reductase. Five V79 cell lines were obtained

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

    PubMed

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

    2015-04-01

    Oxidoreductases are an important family of enzymes that are used in many biotechnological processes. An experimental design was applied to optimize partition and purification of two recombinant oxidoreductases, glucose dehydrogenase (GDH) from Bacillus subtilis and d-galactose dehydrogenase (GalDH) from Pseudomonas fluorescens AK92 in aqueous two-phase systems (ATPS). Response surface methodology (RSM) with a central composite rotatable design (CCRD) was performed to optimize critical factors like polyethylene glycol (PEG) concentration, concentration of salt and pH value. The best partitioning conditions was achieved in an ATPS composed of 12% PEG-6000, 15% K2HPO4 with pH 7.5 at 25°C, which ensured partition coefficient (KE) of 66.6 and 45.7 for GDH and GalDH, respectively. Under these experimental conditions, the activity of GDH and GalDH was 569.5U/ml and 673.7U/ml, respectively. It was found that these enzymes preferentially partitioned into the top PEG-rich phase and appeared as single bands on SDS-PAGE gel. Meanwhile the validity of the response model was confirmed by a good agreement between predicted and experimental results. Collectively, according to the obtained data it can be inferred that the ATPS optimization using RSM approach can be applied for recovery and purification of any enzyme from oxidoreductase family. PMID:25591389

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

    E-print Network

    Mashalidis, Ellene H.

    2013-10-08

    22 Figure 2.2. Gene cluster comparisons of PNPOx-like proteins in Mtb and M. smegmatis 23 Figure 2.3. Structural superimposition of E. coli PNPOx and Mtb PNPOx-like proteins (protomers) 25 xiv Table 2.3. Summary of Dali results... for PNPOx-like proteins Rv2607, Rv1155, Rv2991, and Rv2074 26 Figure 2.4. Sequence alignment of E. coli PNPOx, H. sapiens PNPOx, and Mtb PNPOx-like proteins 27 Figure 2.5. Structure of the E. coli PNPOx active site 28 Figure 2...

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

    SciTech Connect

    Dong, G.; Wearsch, P.A.; Peaper, D.R.; Cresswell, P.; Reinisch, K.M.; (Yale-MED)

    2009-03-02

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

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

    PubMed Central

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

    2014-01-01

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

  14. Overproduction of stromal ferredoxin:NADPH oxidoreductase in H2O 2-accumulating Brassica napus leaf protoplasts.

    PubMed

    Tewari, Rajesh Kumar; Satoh, Mamoru; Kado, Sayaka; Mishina, Kohei; Anma, Misato; Enami, Kazuhiko; Hanaoka, Mitsumasa; Watanabe, Masami

    2014-12-01

    The isolation of Brassica napus leaf protoplasts induces reactive oxygen species generation and accumulation in the chloroplasts. An activated isoform of NADPH oxidase-like protein was detected in the protoplasts and the protoplast chloroplasts. The purpose of this study is to define the NADH oxidase-like activities in the H2O2-accumulating protoplast chloroplasts. Proteomic analysis of this protein revealed an isoform of ferredoxin:NADPH oxidoreductase (FNR1). While leaves highly expressed the LFNR1 transcript, protoplasts decreased the expression significantly. The protoplast chloroplasts predominantly expressed soluble FNR1 proteins. While the albino leaves of white kale (Brassica oleracea var. acephala f. tricolor cv. white pigeon) expressed FNR1 protein at the same level as B. napus leaves, the protoplasts of albino leaves displayed reduced FNR1 expression. The albino leaf protoplasts of white kale generated and accumulated H2O2 in the cytoplasm and on the plasma membrane. Intracellular pH showed that the chloroplasts were acidic, which suggest that excess H(+) was generated in chloroplast stroma. NADPH content of the protoplast chloroplasts increased by over sixfold during the isolation of protoplasts. This study reports a possibility of mediating electrons to oxygen by an overproduced soluble FNR, and suggests that the FNR has a function in utilizing any excess reducing power of NADPH. PMID:25255860

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

    PubMed

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

    2014-10-17

    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 21kDa 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 (13)C/(15)N-resolved NOESY, CBCA(CO)NH, and 2D [(13)C/(15)N,(1)H]-HSQC were used, and 86.8% if exclusively 3D (13)C/(15)N-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 20kDa. PMID:25462951

  16. Why is the molybdenum-substituted tungsten-dependent formaldehyde ferredoxin oxidoreductase not active? A quantum chemical study.

    PubMed

    Liao, Rong-Zhen

    2013-02-01

    Formaldehyde ferredoxin oxidoreductase is a tungsten-dependent enzyme that catalyzes the oxidative degradation of formaldehyde to formic acid. The molybdenum ion can be incorporated into the active site to displace the tungsten ion, but is without activity. Density functional calculations have been employed to understand the incapacitation of the enzyme caused by molybdenum substitution. The calculations show that the enzyme with molybdenum (Mo-FOR) has higher redox potential than that with tungsten, which makes the formation of the Mo(VI)=O complex endothermic by 14 kcal/mol. Following our previously suggested mechanism for this enzyme, the formaldehyde substrate oxidation was also investigated for Mo-FOR using the same quantum-mechanics-only model, except for the displacement of tungsten by molybdenum. The calculations demonstrate that formaldehyde oxidation occurs via a sequential two-step mechanism. Similarly to the tungsten-catalyzed reaction, the Mo(VI)=O species performs the nucleophilic attack on the formaldehyde carbon, followed by proton transfer in concert with two-electron reduction of the metal center. The first step is rate-limiting, with a total barrier of 28.2 kcal/mol. The higher barrier is mainly due to the large energy penalty for the formation of the Mo(VI)=O species. PMID:23183892

  17. 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

  18. Gelatin and starch as stabilizers of the coupled enzyme system of luminous bacteria NADH:FMN-oxidoreductase-luciferase.

    PubMed

    Bezrukikh, Anna; Esimbekova, Elena; Nemtseva, Elena; Kratasyuk, Valentina; Shimomura, Osamu

    2014-09-01

    We have studied the effects of a gel-like environment on the characteristics of enzyme preparations based on the coupled enzyme system of luminous bacteria, NADH:FMN-oxidoreductase-luciferase, to design a stable immobilizing reagent for bioluminescent analysis. Natural polymers, gelatin and starch, were used to create a viscous, structured microenvironment. The stability of the coupled enzyme system to such physical and chemical environmental factors as temperature, pH, and ionic strength in gelatin and starch-containing media was examined. It was shown that both gelatin and starch have a stabilizing effect on the enzymes of luminous bacteria under specific conditions. In particular, the enzymes' activity is increased twofold in the presence of 1 and 5% of gelatin at 20 °C and 25 °C, respectively (temperatures lower than the gel point). Also, the acceptable pH range of the coupled enzyme system expands into the alkaline region and becomes 6.8-8.1. Stabilization at low ionic strength (0.01-0.06 mol L(-1)) is observed. At the same time, microenvironments based on either gelatin or starch do not change the enzymes' thermal inactivation rate constants in the temperature range from 25 to 43 °C. Finally, gelatin and starch are suitable for development of a reagent for immobilization of enzymes which would be stable and resistant to physical and chemical environmental conditions. PMID:25002335

  19. 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

  20. 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.

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

    PubMed Central

    Dinkova-Kostova, Albena T.; Talalay, Paul

    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 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

  2. 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.

  3. 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

  4. The crystal structure of NAD(P)H quinone oxidoreductase 1 in complex with its potent inhibitor dicoumarol.

    PubMed

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

    2006-05-23

    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 for binding to NQO1. Dicoumarol also disrupts the binding of NQO1 to p53, p73, and ODC and induces their ubiquitin-independent proteasomal degradation. We report here the crystal structure of human NQO1 in complex with dicoumarol at 2.75 A resolution. We have identified the interactions of dicoumarol with the different residues of NQO1 and the conformational changes imposed upon dicoumarol binding. The most prominent conformational changes that occur in the presence of dicoumarol involve Tyr 128 and Phe 232 that are present on the surface of the NQO1 catalytic pocket. On the basis of the comparison of the NQO1 structure in complex with different NQO1 inhibitors and our previous analysis of NQO1 mutants, we propose that the specific conformation of Tyr 128 and Phe 232 is important for NQO1 interaction with p53 and other client proteins. PMID:16700548

  5. Excited-state charge separation in the photochemical mechanism of the light-driven enzyme protochlorophyllide oxidoreductase.

    PubMed

    Heyes, Derren J; Hardman, Samantha J O; Hedison, Tobias M; Hoeven, Robin; Greetham, Greg M; Towrie, Michael; Scrutton, Nigel S

    2015-01-26

    The unique light-driven enzyme protochlorophyllide oxidoreductase (POR) is an important model system for understanding how light energy can be harnessed to power enzyme reactions. The ultrafast photochemical processes, essential for capturing the excitation energy to drive the subsequent hydride- and proton-transfer chemistry, have so far proven difficult to detect. We have used a combination of time-resolved visible and IR spectroscopy, providing complete temporal resolution over the picosecond-microsecond time range, to propose a new mechanism for the photochemistry. Excited-state interactions between active site residues and a carboxyl group on the Pchlide molecule result in a polarized and highly reactive double bond. This so-called "reactive" intramolecular charge-transfer state creates an electron-deficient site across the double bond to trigger the subsequent nucleophilic attack of NADPH, by the negatively charged hydride from nicotinamide adenine dinucleotide phosphate. This work provides the crucial, missing link between excited-state processes and chemistry in POR. Moreover, it provides important insight into how light energy can be harnessed to drive enzyme catalysis with implications for the design of light-activated chemical and biological catalysts. PMID:25488797

  6. c-Maf negatively regulates ARE-mediated detoxifying enzyme genes expression and anti-oxidant induction

    Microsoft Academic Search

    Saravanakumar Dhakshinamoorthy; Anil K Jaiswal

    2002-01-01

    Anti-oxidant response element (ARE) and nuclear factors including Nrf2 and small Maf (MafG and MafK) proteins are known to regulate expression and induction of detoxifying enzyme genes including quinone oxidoreductase1 (NQO1). Nrf2 upregulates and small Maf proteins lacking the transcriptional activation domain down regulates ARE-mediated expression and induction. In this report, we have investigated the role of c-Maf (large Maf)

  7. The Structural and Functional Basis of Catalysis Mediated by NAD(P)H:acceptor Oxidoreductase (FerB) of Paracoccus denitrificans

    PubMed Central

    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

  8. Chromogenic Identification of Genetic Regulatory Signals in Bacillus subtilis Based on Expression of a Cloned Pseudomonas Gene

    Microsoft Academic Search

    Mark M. Zukowski; Dairena F. Gaffney; Denis Speck; Muriel Kauffmann; Annie Findeli; Anne Wisecup; Jean-Pierre Lecocq

    1983-01-01

    A method to isolate fragments of DNA that promote gene expression in Bacillus subtilis is described. The system is based on production of catechol 2,3-dioxygenase [CatO2ase; catechol:oxygen 2,3-oxidoreductase (decyclizing), EC 1.13.11.2] encoded by the Pseudomonas putida TOL plasmid gene xylE. The gene was transferred to a B. subtilis\\/Escherichia coli plasmid vector to construct pTG402. Although xylE is functionally expressed in

  9. NADH: flavin oxidoreductase/NADH oxidase and ROS regulate microsclerotium development in Nomuraea rileyi.

    PubMed

    Liu, Juanjuan; Yin, Youping; Song, Zhangyong; Li, Yan; Jiang, Shasha; Shao, Changwen; Wang, Zhongkang

    2014-07-01

    Based on transcriptome library, an NADH: flavinoxidore ductase/NADH oxidase gene (Nox) was cloned from Nomuraea rileyi. The 1,663-bp full-length cDNA contains an open reading frame of 1,233 bp coding 410 amino acids. The expression level of Nox was up-regulated and co-related to the intracellular H2O2 concentration during microsclerotium (MS) initiation. Rotenone inhibition showed that inhibition of Nox could cause a noticeable decrease in the MS yields. Silencing of Nox resulted in the MS yields, H2O2 and virulence decreased by 98.5, 38 and 21.5%, respectively. On the other hand, MS yields increased by 24.8-61% when induced by H2O2 or menadione. Furthermore, the reactive oxygen species (ROS) scavenger, ascorbic acid (up to 0.03 g ascorbic acid l(-1)), completely inhibited the formation of MS. In conclusion, the results obtained suggested that ROS promoted MS development, and that Nox was required for MS differentiation through regulation of intracellular H2O2 concentration. Besides, Nox had a great impact on the virulence in N. rileyi. PMID:24497186

  10. Characterization of NADPH-cytochrome P450 reductase gene from the cotton bollworm, Helicoverpa armigera.

    PubMed

    Liu, Dong; Zhou, Xiaojie; Li, Mei; Zhu, Shunyi; Qiu, Xinghui

    2014-07-25

    A complete cDNA encoding the NADPH-cytochrome P450 reductase (haCPR) and its genomic sequence from the cotton bollworm Helicoverpa armigera were cloned and sequenced. The open reading frame of haCPR codes for a protein of 687 amino acid residues with a calculated molecular mass of 77.4kDa. The haCPR gene spans over 11 kb and its coding region is interrupted by 11 introns. haCPR is ubiquitously expressed in various tissues and at various stages of development. Escherichia coli produced haCPR enzyme exhibited catalytic activity for NADPH-dependent reduction of cytochrome c, following Michaelis-Menten kinetics. The functionality of CPR was further demonstrated by its capacity to support cytochrome P450 (e.g. haCYP9A14 and chicken CYP1A5) mediated O-dealkylation activity of alkoxyresorufins. The flavoprotein-specific inhibitor (diphenyleneiodonium chloride, DPI) showed a potent inhibition to haCPR activity (IC50=1.69 ?M). Inhibitory effect of secondary metabolites in the host plants (tannic acid, quercetin and gossypol) on CPR activity (with an IC50 value ranged from 15 to 90 ?M) was also observed. PMID:24768738

  11. Tropine Forming Tropinone Reductase Gene from Withania somnifera (Ashwagandha): Biochemical Characteristics of the Recombinant Enzyme and Novel Physiological Overtones of Tissue-Wide Gene Expression Patterns

    PubMed Central

    Kushwaha, Amit Kumar; Sangwan, Neelam Singh; Trivedi, Prabodh Kumar; Negi, Arvind Singh; Misra, Laxminarain; Sangwan, Rajender Singh

    2013-01-01

    Withania somnifera is one of the most reputed medicinal plants of Indian systems of medicine synthesizing diverse types of secondary metabolites such as withanolides, alkaloids, withanamides etc. Present study comprises cloning and E. coli over-expression of a tropinone reductase gene (WsTR-I) from W. somnifera, and elucidation of biochemical characteristics and physiological role of tropinone reductase enzyme in tropane alkaloid biosynthesis in aerial tissues of the plant. The recombinant enzyme was demonstrated to catalyze NADPH-dependent tropinone to tropine conversion step in tropane metabolism, through TLC, GC and GC-MS-MS analyses of the reaction product. The functionally active homodimeric ?60 kDa enzyme catalyzed the reaction in reversible manner at optimum pH 6.7. Catalytic kinetics of the enzyme favoured its forward reaction (tropine formation). Comparative 3-D models of landscape of the enzyme active site contours and tropinone binding site were also developed. Tissue-wide and ontogenic stage-wise assessment of WsTR-I transcript levels revealed constitutive expression of the gene with relatively lower abundance in berries and young leaves. The tissue profiles of WsTR-I expression matched those of tropine levels. The data suggest that, in W. somnifera, aerial tissues as well possess tropane alkaloid biosynthetic competence. In vivo feeding of U-[14C]-sucrose to orphan shoot (twigs) and [14C]-chasing revealed substantial radiolabel incorporation in tropinone and tropine, confirming the de novo synthesizing ability of the aerial tissues. This inherent independent ability heralds a conceptual novelty in the backdrop of classical view that these tissues acquire the alkaloids through transportation from roots rather than synthesis. The TR-I gene expression was found to be up-regulated on exposure to signal molecules (methyl jasmonate and salicylic acid) and on mechanical injury. The enzyme's catalytic and structural properties as well as gene expression profiles are discussed with respect to their physiological overtones. PMID:24086372

  12. Substrate-specific modulation of CYP3A4 activity by genetic variants of cytochrome P450 oxidoreductase (POR)

    PubMed Central

    Agrawal, Vishal; Choi, Ji Ha; Giacomini, Kathleen M.; Miller, Walter L.

    2010-01-01

    Objectives CYP3A4 receives electrons from P450 oxidoreductase (POR) to metabolize about 50% of clinically used drugs. There is substantial inter-individual variation in CYP3A4 catalytic activity that is not explained by CYP3A4 genetic variants. CYP3A4 is flexible and distensible, permitting it to accommodate substrates varying in shape and size. To elucidate mechanisms of variability in CYP3A4 catalysis, we examined the effects of genetic variants of POR, and explored the possibility that substrate-induced conformational changes in CYP3A4 differentially affect the ability of POR variants to support catalysis. Methods We expressed human CYP3A4 and four POR variants (Q153R, A287P, R457H, A503V) in bacteria, reconstituted them in vitro and measured the Michaelis constant and maximum velocity with testosterone, midazolam, quinidine and erythromycin as substrates. Results POR A287P and R457H had low activity with all substrates; Q153R had 76–94% of wild type (WT) activity with midazolam and erythromycin, but 129–150% activity with testosterone and quinidine. The A503V polymorphism reduced CYP3A4 activity to 61–77% of wild type with testosterone and midazolam, but had nearly wild type activity with quinidine and erythromycin. Conclusion POR variants affect CYP3A4 activities. The impact of a POR variant on catalysis by CYP3A4 is substrate-specific, probably due to substrate-induced conformational changes in CYP3A4. PMID:20697309

  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. Quinol-cytochrome c Oxidoreductase and Cytochrome c4 Mediate Electron Transfer during Selenate Respiration in Thauera selenatis*

    PubMed Central

    Lowe, Elisabeth C.; Bydder, Sarah; Hartshorne, Robert S.; Tape, Hannah L. U.; Dridge, Elizabeth J.; Debieux, Charles M.; Paszkiewicz, Konrad; Singleton, Ian; Lewis, Richard J.; Santini, Joanne M.; Richardson, David J.; Butler, Clive S.

    2010-01-01

    Selenate reductase (SER) from Thauera selenatis is a periplasmic enzyme that has been classified as a type II molybdoenzyme. The enzyme comprises three subunits SerABC, where SerC is an unusual b-heme cytochrome. In the present work the spectropotentiometric characterization of the SerC component and the identification of redox partners to SER are reported. The mid-point redox potential of the b-heme was determined by optical titration (Em + 234 ± 10 mV). A profile of periplasmic c-type cytochromes expressed in T. selenatis under selenate respiring conditions was undertaken. Two c-type cytochromes were purified (?24 and ?6 kDa), and the 24-kDa protein (cytc-Ts4) was shown to donate electrons to SerABC in vitro. Protein sequence of cytc-Ts4 was obtained by N-terminal sequencing and liquid chromatography-tandem mass spectrometry analysis, and based upon sequence similarities, was assigned as a member of cytochrome c4 family. Redox potentiometry, combined with UV-visible spectroscopy, showed that cytc-Ts4 is a diheme cytochrome with a redox potential of +282 ± 10 mV, and both hemes are predicted to have His-Met ligation. To identify the membrane-bound electron donors to cytc-Ts4, growth of T. selenatis in the presence of respiratory inhibitors was monitored. The specific quinol-cytochrome c oxidoreductase (QCR) inhibitors myxothiazol and antimycin A partially inhibited selenate respiration, demonstrating that some electron flux is via the QCR. Electron transfer via a QCR and a diheme cytochrome c4 is a novel route for a member of the DMSO reductase family of molybdoenzymes. PMID:20388716

  15. Placental 17 beta-hydroxysteroid oxidoreductase, lactate dehydrogenase and malate dehydrogenase during the latter half of pregnancy in the mouse.

    PubMed

    Blomquist, C H; Hensleigh, H C; Block, D L; Feeney, L A

    1993-07-01

    The specific activity of 17 beta-hydroxysteroid oxidoreductase (17-HOR) with estradiol-17 beta (E2), estrone (E1) and testosterone (T), as well as that of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) were measured in homogenates of CF-1 mouse placenta during the latter half of pregnancy. 17-HOR activity with E2 and T increased over 100-fold between days 9 and 12, and 3- to 4-fold between days 15 and 19, with no further change to day 21. In contrast, activity with E1 increased 39-fold between days 9 and 12, 3.8-fold between days 15 and 19 but then decreased between days 19 and 21. The E2/T activity ratio was constant while the E2/E1 ratio increased between days 9 and 21. LDH increased 2-fold between days 9 and 12 with no further increase to day 19. MDH was constant from day 9 to 19. Activity with E2 was inhibited by T, 5 alpha-dihydrotestosterone (5 alpha-DHT) and DHA but not by E1, androstenedione (A) or 20 alpha-dihydroprogesterone. Activity with T was inhibited by E2, 5 alpha-DHT and DHA, but not by A. In contrast, activity with E1 was inhibited by A and DHA but not by E2, T or 5 alpha-DHT. The results suggest placental 17-HOR is developmentally regulated. Although the results are also suggestive of multiple forms of 17-HOR, a single enzyme with an ordered kinetic mechanism cannot be ruled out. PMID:8338791

  16. Reevaluating the relationship between EPR spectra and enzyme structure for the iron–sulfur clusters in NADH:quinone oxidoreductase

    PubMed Central

    Yakovlev, Gregory; Reda, Torsten; Hirst, Judy

    2007-01-01

    NADH:quinone oxidoreductase (complex I) plays a pivotal role in cellular energy production. It employs a series of redox cofactors to couple electron transfer to the generation of a proton-motive force across the inner mitochondrial or bacterial cytoplasmic membrane. Complex I contains a noncovalently bound flavin mononucleotide at the active site for NADH oxidation and eight or nine iron–sulfur clusters to transfer electrons between the flavin and a quinone-binding site. Understanding the mechanism of complex I requires the properties of these clusters to be defined, both individually and as an ensemble. Most functional information on the clusters has been gained from EPR spectroscopy, but some clusters are not observed by EPR and attributing the observed signals to the structurally defined clusters is difficult. The current consensus picture relies on correlating the spectra from overexpressed subunits (containing one to four clusters) with those from intact complexes I. Here, we analyze spectra from the overexpressed NuoG subunit from Escherichia coli complex I and compare them with spectra from the intact enzyme. Consequently, we propose that EPR signals N4 and N5 have been misassigned: signal N4 is from NuoI (not NuoG) and signal N5 is from the conserved cysteine-ligated [4Fe-4S] cluster in NuoG (not from the cluster with a histidine ligand). The consequences of reassigning the EPR signals and their associated functional information on the free energy profile for electron transfer through complex I are discussed. PMID:17640900

  17. The short-chain oxidoreductase Q9HYA2 from Pseudomonas aeruginosa PAO1 contains an atypical catalytic center

    PubMed Central

    Huether, Robert; Mao, Qilong; Duax, William L; Umland, Timothy C

    2010-01-01

    The characteristic oxidation or reduction reaction mechanisms of short-chain oxidoreductase (SCOR) enzymes involve a highly conserved Asp-Ser-Tyr-Lys catalytic tetrad. The SCOR enzyme Q9HYA2 from the pathogenic bacterium Pseudomonas aeruginosa was recognized to possess an atypical catalytic tetrad composed of Lys118-Ser146-Thr159-Arg163. Orthologs of Q9HYA2 containing the unusual catalytic tetrad along with conserved substrate and cofactor recognition residues were identified in 27 additional species, the majority of which are bacterial pathogens. However, this atypical catalytic tetrad was not represented within the Protein Data Bank. The crystal structures of unligated and NADPH-complexed Q9HYA2 were determined at 2.3 Å resolution. Structural alignment to a polyketide ketoreductase (KR), a typical SCOR, demonstrated that Q9HYA2's Lys118, Ser146, and Arg163 superimposed upon the KR's catalytic Asp114, Ser144, and Lys161, respectively. However, only the backbone of Q9HYA2's Thr159 overlapped KR's catalytic Tyr157. The Thr159 hydroxyl in apo Q9HYA2 is poorly positioned for participating in catalysis. In the Q9HYA2–NADPH complex, the Thr159 side chain was modeled in two alternate rotamers, one of which is positioned to interact with other members of the tetrad and the bound cofactor. A chloride ion is bound at the position normally occupied by the catalytic tyrosine hydroxyl. The putative active site of Q9HYA2 contains a chemical moiety at each catalytically important position of a typical SCOR enzyme. This is the first observation of a SCOR protein with this alternate catalytic center that includes threonine replacing the catalytic tyrosine and an ion replacing the hydroxyl moiety of the catalytic tyrosine. PMID:20340135

  18. 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

  19. 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

  20. WrbA from Escherichia coli and Archaeoglobus fulgidus Is an NAD(P)H:Quinone Oxidoreductase

    PubMed Central

    Patridge, Eric V.; Ferry, James G.

    2006-01-01

    WrbA (tryptophan [W] repressor-binding protein) was discovered in Escherichia coli, where it was proposed to play a role in regulation of the tryptophan operon; however, this has been put in question, leaving the function unknown. Here we report a phylogenetic analysis of 30 sequences which indicated that WrbA is the prototype of a distinct family of flavoproteins which exists in a diversity of cell types across all three domains of life and includes documented NAD(P)H:quinone oxidoreductases (NQOs) from the Fungi and Viridiplantae kingdoms. Biochemical characterization of the prototypic WrbA protein from E. coli and WrbA from Archaeoglobus fulgidus, a hyperthermophilic species from the Archaea domain, shows that these enzymes have NQO activity, suggesting that this activity is a defining characteristic of the WrbA family that we designate a new type of NQO (type IV). For E. coli WrbA, the KmNADH was 14 ± 0.43 ?M and the Kmbenzoquinone was 5.8 ± 0.12 ?M. For A. fulgidus WrbA, the KmNADH was 19 ± 1.7 ?M and the Kmbenzoquinone was 37 ± 3.6 ?M. Both enzymes were found to be homodimeric by gel filtration chromatography and homotetrameric by dynamic light scattering and to contain one flavin mononucleotide molecule per monomer. The NQO activity of each enzyme is retained over a broad pH range, and apparent initial velocities indicate that maximal activities are comparable to the optimum growth temperature for the respective organisms. The results are discussed and implicate WrbA in the two-electron reduction of quinones, protecting against oxidative stress. PMID:16672604

  1. 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

  2. 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

  3. Reference genes for gene expression studies in wheat flag leaves grown under different farming conditions

    PubMed Central

    2011-01-01

    Background Internal control genes with highly uniform expression throughout the experimental conditions are required for accurate gene expression analysis as no universal reference genes exists. In this study, the expression stability of 24 candidate genes from Triticum aestivum cv. Cubus flag leaves grown under organic and conventional farming systems was evaluated in two locations in order to select suitable genes that can be used for normalization of real-time quantitative reverse-transcription PCR (RT-qPCR) reactions. The genes were selected among the most common used reference genes as well as genes encoding proteins involved in several metabolic pathways. Findings Individual genes displayed different expression rates across all samples assayed. Applying geNorm, a set of three potential reference genes were suitable for normalization of RT-qPCR reactions in winter wheat flag leaves cv. Cubus: TaFNRII (ferredoxin-NADP(H) oxidoreductase; AJ457980.1), ACT2 (actin 2; TC234027), and rrn26 (a putative homologue to RNA 26S gene; AL827977.1). In addition of these three genes that were also top-ranked by NormFinder, two extra genes: CYP18-2 (Cyclophilin A, AY456122.1) and TaWIN1 (14-3-3 like protein, AB042193) were most consistently stably expressed. Furthermore, we showed that TaFNRII, ACT2, and CYP18-2 are suitable for gene expression normalization in other two winter wheat varieties (Tommi and Centenaire) grown under three treatments (organic, conventional and no nitrogen) and a different environment than the one tested with cv. Cubus. Conclusions This study provides a new set of reference genes which should improve the accuracy of gene expression analyses when using wheat flag leaves as those related to the improvement of nitrogen use efficiency for cereal production. PMID:21951810

  4. FaQR, Required for the Biosynthesis of the Strawberry Flavor Compound 4-Hydroxy-2,5-Dimethyl-3(2H)-Furanone, Encodes an Enone Oxidoreductase

    PubMed Central

    Raab, Thomas; López-Ráez, Juan Antonio; Klein, Dorothée; Caballero, Jose Luis; Moyano, Enriqueta; Schwab, Wilfried; Muñoz-Blanco, Juan

    2006-01-01

    The flavor of strawberry (Fragaria × ananassa) fruit is dominated by an uncommon group of aroma compounds with a 2,5-dimethyl-3(H)-furanone structure. We report the characterization of an enzyme involved in the biosynthesis of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF; Furaneol), the key flavor compound in strawberries. Protein extracts were partially purified, and the observed distribution of enzymatic activity correlated with the presence of a single polypeptide of ?37 kD. Sequence analysis of two peptide fragments showed total identity with the protein sequence of a strongly ripening-induced, auxin-dependent putative quinone oxidoreductase, Fragaria × ananassa quinone oxidoreductase (FaQR). The open reading frame of the FaQR cDNA consists of 969 bp encoding a 322–amino acid protein with a calculated molecular mass of 34.3 kD. Laser capture microdissection followed by RNA extraction and amplification demonstrated the presence of FaQR mRNA in parenchyma tissue of the strawberry fruit. The FaQR protein was functionally expressed in Escherichia coli, and the monomer catalyzed the formation of HDMF. After chemical synthesis and liquid chromatography–tandem mass spectrometry analysis, 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone was confirmed as a substrate of FaQR and the natural precursor of HDMF. This study demonstrates the function of the FaQR enzyme in the biosynthesis of HDMF as enone oxidoreductase and provides a foundation for the improvement of strawberry flavor and the biotechnological production of HDMF. PMID:16517758

  5. Identification of rice genes associated with cosmic-ray response via co-expression gene network analysis.

    PubMed

    Hwang, Sun-Goo; Kim, Dong Sub; Hwang, Jung Eun; Han, A-Reum; Jang, Cheol Seong

    2014-05-15

    In order to better understand the biological systems that are affected in response to cosmic ray (CR), we conducted weighted gene co-expression network analysis using the module detection method. By using the Pearson's correlation coefficient (PCC) value, we evaluated complex gene-gene functional interactions between 680 CR-responsive probes from integrated microarray data sets, which included large-scale transcriptional profiling of 1000 microarray samples. These probes were divided into 6 distinct modules that contained 20 enriched gene ontology (GO) functions, such as oxidoreductase activity, hydrolase activity, and response to stimulus and stress. In particular, modules 1 and 2 commonly showed enriched annotation categories such as oxidoreductase activity, including enriched cis-regulatory elements known as ROS-specific regulators. These results suggest that the ROS-mediated irradiation response pathway is affected by CR in modules 1 and 2. We found 243 ionizing radiation (IR)-responsive probes that exhibited similarities in expression patterns in various irradiation microarray data sets. The expression patterns of 6 randomly selected IR-responsive genes were evaluated by quantitative reverse transcription polymerase chain reaction following treatment with CR, gamma rays (GR), and ion beam (IB); similar patterns were observed among these genes under these 3 treatments. Moreover, we constructed subnetworks of IR-responsive genes and evaluated the expression levels of their neighboring genes following GR treatment; similar patterns were observed among them. These results of network-based analyses might provide a clue to understanding the complex biological system related to the CR response in plants. PMID:24631263

  6. Pivotal Roles of Three Conserved Carboxyl Residues of NuoC (30k) Segment in the Structural Integrity of Proton-translocating NADH-Quinone Oxidoreductase (NDH-1) from Escherichia coli#

    PubMed Central

    Castro-Guerrero, Norma; Sinha, Prem Kumar; Torres-Bacete, Jesus; Matsuno-Yagi, Akemi; Yagi, Takao

    2010-01-01

    The prokaryotic proton-translocating NADH-quinone oxidoreductase (NDH-1) is an L-shaped membrane-bound enzyme that contains 14 subunits (NuoA-NuoN/Nqo1-Nqo14). All subunits have their counterparts in the eukaryotic enzyme (complex I). NDH-1 consists of two domains: the peripheral arm (NuoB,C,D,E,F,G, and I) and the membrane arm (NuoA,H,J,K,L,M, and N). In Escherichia coli NDH-1 the hydrophilic subunits NuoC/Nqo5/30k and NuoD/Nqo4/49k are fused together in a single polypeptide as the NuoCD subunit. The NuoCD subunit is the only subunit that does not bear a cofactor in the peripheral arm. While some roles for inhibitor- and quinone-association have been reported for the NuoD segment, structural and functional roles of the NuoC segment remain mostly elusive. In the current work, 14 highly conserved residues of the NuoC segment were mutated and 21 mutants were constructed using the chromosomal gene manipulation technique. From the enzymatic assays and immunochemical and blue-native gel analyses, it was found that residues Glu-138, Glu-140, and Asp-143 that are anticipated to be in the third ?-helix are absolutely required for the energy-transducing NDH-1 activities and the assembly of the whole enzyme. Together with available information for the hydrophobic subunits, it is proposed that Glu-138, Glu-140, and Asp-143 of the NuoC segment may have a pivotal role in structural stability of NDH-1. PMID:20979355

  7. Nrf2 and c-Jun regulation of antioxidant response element (ARE)-mediated expression and induction of ?-glutamylcysteine synthetase heavy subunit gene

    Microsoft Academic Search

    Janet Jeyapaul; Anil K Jaiswal

    2000-01-01

    ?-Glutamylcysteine synthetase (?-GCS) is a rate-limiting enzyme in the de novo synthesis of glutathione, a known scavenger of electrophiles and reactive oxygen species (ROS). The ?-GCS gene is expressed ubiquitously and induced coordinately with NAD(P)H:quinone oxidoreductase1 (NQO1) and glutathione S-transferase Ya (GST Ya) in response to xenobiotics and antioxidants. The antioxidant response element (ARE) is required for expression and induction

  8. Site-directed mutagenesis studies of the metal-binding center of the iron-dependent propanediol oxidoreductase from Escherichia coli.

    PubMed

    Obradors, N; Cabiscol, E; Aguilar, J; Ros, J

    1998-11-15

    The amino acid residues involved in the metal-binding site in the iron-containing dehydrogenase family were characterized by the site-directed mutagenesis of selected candidate residues of propanediol oxidoreductase from Escherichia coli. Based on the findings that mutations H263R, H267A and H277A resulted in iron-deficient propanediol oxidoreductases without catalytic activity, we identified three conserved His residues as iron ligands, which also bind zinc. The Cys362, a residue highly conserved among these dehydrogenases, was considered another possible ligand by comparison with the sequences of the medium-chain dehydrogenases. Mutation of Cys362 to Ile, resulted in an active enzyme that was still able to bind iron, with minor changes in the Km values and decreased thermal stability. Furthermore, in an attempt to produce an enzyme specific only for the zinc ion, three mutations were designed to mimic the catalytic zinc-binding site of the medium-chain dehydrogenases: (1) V262C produced an enzyme with altered kinetic parameters which nevertheless retained a significant ability to bind both metals, (2) the double mutant V262C-M265D was inactive and too unstable to allow purification, and (3) the insertion of a cysteine at position 263 resulted in a catalytically inactive enzyme without iron-binding capacity, while retaining the ability to bind zinc. This mutation could represent a conceivable model of one of the steps in the evolution from iron to zinc-dependent dehydrogenases. PMID:9851711

  9. Investigation of protein FTT1103 electroactivity using carbon and mercury electrodes. Surface-inhibition approach for disulfide oxidoreductases using silver amalgam powder.

    PubMed

    Ve?erková, Renata; Hernychová, Lenka; Dobeš, Petr; Vrba, Ji?í; Josyp?uk, Bohdan; Bartošík, Martin; Vacek, Jan

    2014-06-01

    Recently, it was shown that electrochemical methods can be used for analysis of poorly water-soluble proteins and for study of their structural changes and intermolecular (protein-ligand) interactions. In this study, we focused on complex electrochemical investigation of recombinant protein FTT1103, a disulfide oxidoreductase with structural similarity to well described DsbA proteins. This thioredoxin-like periplasmic lipoprotein plays an important role in virulence of bacteria Francisella tularensis. For electrochemical analyses, adsorptive transfer (ex situ) square-wave voltammetry with pyrolytic graphite electrode, and alternating-current voltammetry and constant-current chronopotentiometric stripping analysis with mercury electrodes, including silver solid amalgam electrode (AgSAE) were used. AgSAE was used in poorly water-soluble protein analysis for the first time. In addition to basic redox, electrocatalytic and adsorption/desorption characterization of FTT1103, electrochemical methods were also used for sensitive determination of the protein at nanomolar level and study of its interaction with surface of AgSA microparticles. Proposed electrochemical protocol and AgSA surface-inhibition approach presented here could be used in future for biochemical studies focused on proteins associated with membranes as well as on those with disulfide oxidoreductase activity. PMID:24856508

  10. Molecular characterization of an NADPH-dependent acetoin reductase/2,3-butanediol dehydrogenase from Clostridium beijerinckii NCIMB 8052.

    PubMed

    Raedts, John; Siemerink, Marco A J; Levisson, Mark; van der Oost, John; Kengen, Servé W M

    2014-03-01

    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 Zn(2+) 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

  11. 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

  12. Genes and Gene Therapy

    MedlinePLUS

    ... correctly, a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... or prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

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

    PubMed Central

    Grabowska, Anna D.; Wywia?, Ewa; Dunin-Horkawicz, Stanislaw; ?asica, Anna M.; Wösten, Marc M. S. M.; Nagy-Staro?, Anna; Godlewska, Renata; Bocian-Ostrzycka, Katarzyna; Pie?kowska, Katarzyna; ?aniewski, Pawe?; Bujnicki, Janusz M.; van Putten, Jos P. M.; Jagusztyn-Krynicka, E. Katarzyna

    2014-01-01

    Background Bacterial Dsb enzymes are involved in the oxidative folding of many proteins, through the formation of disulfide bonds between their cysteine residues. The Dsb protein network has been well characterized in cells of the model microorganism Escherichia coli. To gain insight into the functioning of the Dsb system in epsilon-Proteobacteria, where it plays an important role in the colonization process, we studied two homologs of the main Escherichia coli Dsb oxidase (EcDsbA) that are present in the cells of the enteric pathogen Campylobacter jejuni, the most frequently reported bacterial cause of human enteritis in the world. Methods and Results Phylogenetic analysis suggests the horizontal transfer of the epsilon-Proteobacterial DsbAs from a common ancestor to gamma-Proteobacteria, which then gave rise to the DsbL lineage. Phenotype and enzymatic assays suggest that the two C. jejuni DsbAs play different roles in bacterial cells and have divergent substrate spectra. CjDsbA1 is essential for the motility and autoagglutination phenotypes, while CjDsbA2 has no impact on those processes. CjDsbA1 plays a critical role in the oxidative folding that ensures the activity of alkaline phosphatase CjPhoX, whereas CjDsbA2 is crucial for the activity of arylsulfotransferase CjAstA, encoded within the dsbA2-dsbB-astA operon. Conclusions Our results show that CjDsbA1 is the primary thiol-oxidoreductase affecting life processes associated with bacterial spread and host colonization, as well as ensuring the oxidative folding of particular protein substrates. In contrast, CjDsbA2 activity does not affect the same processes and so far its oxidative folding activity has been demonstrated for one substrate, arylsulfotransferase CjAstA. The results suggest the cooperation between CjDsbA2 and CjDsbB. In the case of the CjDsbA1, this cooperation is not exclusive and there is probably another protein to be identified in C. jejuni cells that acts to re-oxidize CjDsbA1. Altogether the data presented here constitute the considerable insight to the Epsilonproteobacterial Dsb systems, which have been poorly understood so far. PMID:25181355

  14. Structural and Functional Insights into the Catalytic Inactivity of the Major Fraction of Buffalo Milk Xanthine Oxidoreductase

    PubMed Central

    Gadave, Kaustubh S.; Panda, Santanu; Singh, Surender; Kalra, Shalini; Malakar, Dhruba; Mohanty, Ashok K.; Kaushik, Jai K.

    2014-01-01

    Background Xanthine oxidoreductase (XOR) existing in two interconvertible forms, xanthine dehydrogenase (XDH) and xanthine oxidase (XO), catabolises xanthine to uric acid that is further broken down to antioxidative agent allantoin. XOR also produces free radicals serving as second messenger and microbicidal agent. Large variation in the XO activity has been observed among various species. Both hypo and hyper activity of XOR leads to pathophysiological conditions. Given the important nutritional role of buffalo milk in human health especially in south Asia, it is crucial to understand the functional properties of buffalo XOR and the underlying structural basis of variations in comparison to other species. Methods and Findings Buffalo XO activity of 0.75 U/mg was almost half of cattle XO activity. Enzymatic efficiency (kcat/Km) of 0.11 sec?1 µM?1 of buffalo XO was 8–10 times smaller than that of cattle XO. Buffalo XOR also showed lower antibacterial activity than cattle XOR. A CD value (??430 nm) of 46,000 M?1 cm?1 suggested occupancy of 77.4% at Fe/S I centre. Buffalo XOR contained 0.31 molybdenum atom/subunit of which 48% existed in active sulfo form. The active form of XO in buffalo was only 16% in comparison to ?30% in cattle. Sequencing revealed 97.4% similarity between buffalo and cattle XOR. FAD domain was least conserved, while metal binding domains (Fe/S and Molybdenum) were highly conserved. Homology modelling of buffalo XOR showed several variations occurring in clusters, especially close to FAD binding pocket which could affect NAD+ entry in the FAD centre. The difference in XO activity seems to be originating from cofactor deficiency, especially molybdenum. Conclusion A major fraction of buffalo milk XOR exists in a catalytically inactive form due to high content of demolybdo and desulfo forms. Lower Fe/S content and structural factors might be contributing to lower enzymatic efficiency of buffalo XOR in a minor way. PMID:24498153

  15. 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.

  16. Carbohydrate metabolism genes and pathways in insects: insights from the honey bee genome

    PubMed Central

    Kunieda, T; Fujiyuki, T; Kucharski, R; Foret, S; Ament, S A; Toth, A L; Ohashi, K; Takeuchi, H; Kamikouchi, A; Kage, E; Morioka, M; Beye, M; Kubo, T; Robinson, G E; Maleszka, R

    2006-01-01

    Carbohydrate-metabolizing enzymes may have particularly interesting roles in the honey bee, Apis mellifera, because this social insect has an extremely carbohydrate-rich diet, and nutrition plays important roles in caste determination and socially mediated behavioural plasticity. We annotated a total of 174 genes encoding carbohydrate-metabolizing enzymes and 28 genes encoding lipid-metabolizing enzymes, based on orthology to their counterparts in the fly, Drosophila melanogaster, and the mosquito, Anopheles gambiae. We found that the number of genes for carbohydrate metabolism appears to be more evolutionarily labile than for lipid metabolism. In particular, we identified striking changes in gene number or genomic organization for genes encoding glycolytic enzymes, cellulase, glucose oxidase and glucose dehydrogenases, glucose-methanol-choline (GMC) oxidoreductases, fucosyltransferases, and lysozymes. PMID:17069632

  17. [Detection and analysis of sulfur metabolism genes in Sphaerotilus natans subsp. sulfidivorans representatives].

    PubMed

    Belousova, E V; Chernousova, E Iu; Dubinina, G A; Turova, T P; Grabovich, M Iu

    2013-01-01

    The lithotrophic capacity of the betaproteobacteria Sphaerotilus natans subsp. sulfidivorans was confirmed at genetic level: functional genes of sulfur metabolism were detected (aprBA, soxB, and sqr, coding for adenylyl phosphosulfate reductase, thiosulfate-cleaving enzyme, and sulfide:quinone oxidoreductase, respectively), and the expression of aprA and soxB genes was demonstrated. An evolutionary scenario for soxB genes in Sphaerotilus representatives is suggested based on comparative analysis of codon occurrence frequency, DNA base composition (G + C content), and topology of phylogenetic trees. The ancestor bacterium of the Sphaerotilus-Leptothrix group was capable of lithotrophic growth in the presence of reduced sulfur compounds. However, in the course of further evolution, the sulfur metabolism genes, including the soxB gene, were lost by some Sphaerotilus strains. As a result, the lithotrophic Sphaerotilus-Leptothrix group split into two phylogenetic lineages, lithotrophic and organotrophic ones. PMID:25509396

  18. 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

  19. Influence of Populus genotype on gene expression by the wood decay fungus Phanerochaete chrysosporium.

    PubMed

    Gaskell, Jill; Marty, Amber; Mozuch, Michael; Kersten, Philip J; Splinter BonDurant, Sandra; Sabat, Grzegorz; Azarpira, Ali; Ralph, John; Skyba, Oleksandr; Mansfield, Shawn D; Blanchette, Robert A; Cullen, Dan

    2014-09-01

    We examined gene expression patterns in the lignin-degrading fungus Phanerochaete chrysosporium when it colonizes hybrid poplar (Populus alba × tremula) and syringyl (S)-rich transgenic derivatives. A combination of microarrays and liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowed detection of a total of 9,959 transcripts and 793 proteins. Comparisons of P. chrysosporium transcript abundance in medium containing poplar or glucose as a sole carbon source showed 113 regulated genes, 11 of which were significantly higher (>2-fold, P < 0.05) in transgenic line 64 relative to the parental line. Possibly related to the very large amounts of syringyl (S) units in this transgenic tree (94 mol% S), several oxidoreductases were among the upregulated genes. Peptides corresponding to a total of 18 oxidoreductases were identified in medium consisting of biomass from line 64 or 82 (85 mol% S) but not in the parental clone (65 mol% S). These results demonstrate that P. chrysosporium gene expression patterns are substantially influenced by lignin composition. PMID:25015893

  20. Influence of Populus Genotype on Gene Expression by the Wood Decay Fungus Phanerochaete chrysosporium

    PubMed Central

    Gaskell, Jill; Marty, Amber; Mozuch, Michael; Kersten, Philip J.; Splinter BonDurant, Sandra; Sabat, Grzegorz; Azarpira, Ali; Ralph, John; Skyba, Oleksandr; Mansfield, Shawn D.; Blanchette, Robert A.

    2014-01-01

    We examined gene expression patterns in the lignin-degrading fungus Phanerochaete chrysosporium when it colonizes hybrid poplar (Populus alba × tremula) and syringyl (S)-rich transgenic derivatives. A combination of microarrays and liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowed detection of a total of 9,959 transcripts and 793 proteins. Comparisons of P. chrysosporium transcript abundance in medium containing poplar or glucose as a sole carbon source showed 113 regulated genes, 11 of which were significantly higher (>2-fold, P < 0.05) in transgenic line 64 relative to the parental line. Possibly related to the very large amounts of syringyl (S) units in this transgenic tree (94 mol% S), several oxidoreductases were among the upregulated genes. Peptides corresponding to a total of 18 oxidoreductases were identified in medium consisting of biomass from line 64 or 82 (85 mol% S) but not in the parental clone (65 mol% S). These results demonstrate that P. chrysosporium gene expression patterns are substantially influenced by lignin composition. PMID:25015893

  1. Promoter Polymorphism With Higher Gene Expression and Increased Susceptibility to Parkinson’s Disease

    E-print Network

    Wei Wang; Wei-dong Le; Tianhong Pan; Janet L. Stringer; Anil K. Jaiswal

    The N-ribosyldihydronicotinamide (NRH):quinone oxidoreductase 2 (NQO2) gene encodes an enzyme that catalyzes activation of quinones. Blood DNA from 80 control individuals and 118 age-matched Parkinson’s disease patients were analyzed for NQO2 gene promoter polymorphisms. The results revealed three allelic variants, designated I-29, I-16, and D. These results were confirmed in fibroblast cell lines. In patients with Parkinson’s disease, there was a significant increase in the frequency of the D allele, but there was no difference in the frequency of the alleles in familial compared to sporadic Parkinson’s disease. The D and I-16 promoters direct higher NQO2 gene expression that results in higher enzyme activity. Overexpression of NQO2 in the catecholaminergic neuroblastoma SH-SY5Y cells resulted in increased production of reactive oxygen species when exposed to exogenous dopamine. The results suggest that the association of the D promoter with Parkinson’s disease may be due to an increase in expression of the NQO2 gene. Key Words: NRH:quinone oxidoreductase 2—Promoter polymorphism—Parkinson’s disease. PARKINSON’S disease is a neurodegenerative disease characterized by tremor, slowness of movement,

  2. Comparative Studies on Ferredoxin-NADP+ Oxidoreductase Isoenzymes Derived from Different Organs by Antibodies Specific for the Radish Root- and Leaf-Enzymes.

    PubMed

    Morigasaki, S.; Jin, T.; Wada, K.

    1993-10-01

    Determination of the prosthetic group and titration of sulfhydryl group of ferredoxin-NADP+ oxidoreductase (FNR) from roots of radish (Raphanus sativus var acanthiformis cv Miyashige) confirmed its similarity to leaf-FNR. Antisera directed against radish root-FNR and leaf-FNR distinguished the enzyme forms from roots and leaves of radish as well as other flowering plants. The FNR isoenzymes showed organ-specific distributions. In horsetail (Equisetum arvense L.) and cultured liverwort cells (Marchantia polymorpha), at least two FNR isoenzymes were distinguished by the antisera. FNR from Chlorella vulgaris reacted only with the anti-root-FNR antiserum. FNR from a cyanobacterium, Spirulina spp., failed to react with either antiserum. PMID:12231952

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

    PubMed Central

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

    2013-01-01

    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 E. 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 inhibition of vital respiratory enzymes in the bacterial inner membrane represents one of the secondary modes of action for polymyxins. PMID:24169795

  4. Time-lapse anomalous X-ray diffraction shows how Fe(2+) substrate ions move through ferritin protein nanocages to oxidoreductase sites.

    PubMed

    Pozzi, Cecilia; Di Pisa, Flavio; Lalli, Daniela; Rosa, Camilla; Theil, Elizabeth; Turano, Paola; Mangani, Stefano

    2015-04-01

    Ferritin superfamily protein cages reversibly synthesize internal biominerals, Fe2O3·H2O. Fe(2+) and O2 (or H2O2) substrates bind at oxidoreductase sites in the cage, initiating biomineral synthesis to concentrate iron and prevent potentially toxic reactions products from Fe(2+)and O2 or H2O2 chemistry. By freezing ferritin crystals of Rana catesbeiana ferritin M (RcMf) at different time intervals after exposure to a ferrous salt, a series of high-resolution anomalous X-ray diffraction data sets were obtained that led to crystal structures that allowed the direct observation of ferrous ions entering, moving along and binding at enzyme sites in the protein cages. The ensemble of crystal structures from both aerobic and anaerobic conditions provides snapshots of the iron substrate bound at different cage locations that vary with time. The observed differential occupation of the two iron sites in the enzyme oxidoreductase centre (with Glu23 and Glu58, and with Glu58, His61 and Glu103 as ligands, respectively) and other iron-binding sites (with Glu53, His54, Glu57, Glu136 and Asp140 as ligands) reflects the approach of the Fe(2+) substrate and its progression before the enzymatic cycle 2Fe(2+) + O2 ? Fe(3+)-O-O-Fe(3+) ? Fe(3+)-O(H)-Fe(3+) and turnover. The crystal structures also revealed different Fe(2+) coordination compounds bound to the ion channels located at the threefold and fourfold symmetry axes of the cage. PMID:25849404

  5. FeS/S/FeS(2) redox system and its oxidoreductase-like chemistry in the iron-sulfur world.

    PubMed

    Wang, Wei; Yang, Bin; Qu, Youpeng; Liu, Xiaoyang; Su, Wenhui

    2011-06-01

    The iron-sulfur world (ISW) theory is an intriguing prediction regarding the origin of life on early Earth. It hypothesizes that life arose as a geochemical process from inorganic starting materials on the surface of sulfide minerals in the vicinity of deep-sea hot springs. During the last two decades, many experimental studies have been carried out on this topic, and some interesting results have been achieved. Among them, however, the processes of carbon/nitrogen fixation and biomolecular assembly on the mineral surface have received an inordinate amount of attention. To the present, an abiotic model for the oxidation-reduction of intermediates participating in metabolic pathways has been ignored. We examined the oxidation-reduction effect of a prebiotic FeS/S/FeS(2) redox system on the interconversion between several pairs of ?-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/FeS(2) redox system suggests that it can determine the redox homeostasis of metabolic intermediates in the early evolutionary phase of life. The results provide a possible pathway for the development of primordial redox biochemistry in the iron-sulfur world. Key Words: Iron-sulfur world-FeS/S/FeS(2) redox system-Oxidoreductase-like chemistry. Astrobiology 11, 471-476. PMID:21707387

  6. Transcriptional analysis of Pleurotus ostreatus laccase genes.

    PubMed

    Pezzella, Cinzia; Lettera, Vincenzo; Piscitelli, Alessandra; Giardina, Paola; Sannia, Giovanni

    2013-01-01

    Fungal laccases (p-diphenol:oxygen oxidoreductase; EC 1.10.3.2) are multi-copper-containing oxidases that catalyse the oxidation of a great variety of phenolic compounds and aromatic amines through simultaneous reduction of molecular oxygen to water. Fungi generally produce several laccase isoenzymes encoded by complex multi-gene families. The Pleurotus ostreatus genome encodes 11 putative laccase coding genes, and only six different laccase isoenzymes have been isolated and characterised so far. Laccase expression was found to be regulated by culture conditions and developmental stages even if the redundancy of these genes still raises the question about their respective functions in vivo. In this context, laccase transcript profiling analysis has been used to unravel the physiological role played by the different isoforms produced by P. ostreatus. Even if reported results depict a complex picture of the transcriptional responses exhibited by the analysed laccase genes, they were allowed to speculate on the isoform role in vivo. Among the produced laccases, LACC10 (POXC) seems to play a major role during vegetative growth, since its transcription is downregulated when the fungus starts the fructification process. Furthermore, a new tessera has been added to the puzzling mosaic of the heterodimeric laccase LACC2 (POXA3). LACC2 small subunit seems to play an additional physiological role during fructification, beside that of LACC2 complex activation/stabilisation. PMID:22395908

  7. Characterization of an Azospirillum brasilense Sp7 gene homologous to Alcaligenes eutrophus phbB and to Rhizobium meliloti nodG.

    PubMed

    Vieille, C; Elmerich, C

    1992-02-01

    A 4 kb SalI fragment from Azospirillum brasilense Sp7 that shares homology with a 6.8 kb EcoRI fragment carrying nodGEFH and part of nodP of Rhizobium meliloti 41 was cloned in pUC18 to yield pAB503. The nucleotide sequence of a 2 kb SalI-SmaI fragment of the pAB503 insert revealed an open reading frame, named ORF3, encoding a polypeptide sharing 40% identity with R. meliloti NodG. The deduced polypeptide also shared 60% identity with the Alcaligenes eutrophus NADPH-dependent acetoacetyl-CoA (AA-CoA) reductase, encoded by the phbB gene and involved in poly-beta-hydroxybutyrate (PHB) synthesis. Northern blot analysis and promoter extension mapping indicated that ORF3 is expressed as a monocistronic operon from a promoter that resembles the Escherichia coli sigma 70 consensus promoter. An ORF3-lacZ translational fusion was constructed and was very poorly expressed in E. coli, but was functional and constitutively expressed in Azospirillum. Tn5-Mob insertions in ORF3 did not affect growth, nitrogen fixation, PHB synthesis or NAD(P)H-linked AA-CoA reductase activity. An ORF3 DNA sequence was used to probe total DNA of several Azospirillum strains. No ORF3 homologues were found in A. irakense, A. amazonense, A. halopraeferens or in several A. lipoferum strains. PMID:1538694

  8. Cloning and sequence analysis demonstrate the chromate reduction ability of a novel chromate reductase gene from Serratia sp

    PubMed Central

    DENG, PENG; TAN, XIAOQING; WU, YING; BAI, QUNHUA; JIA, YAN; XIAO, HONG

    2015-01-01

    The ChrT gene encodes a chromate reductase enzyme which catalyzes the reduction of Cr(VI). The chromate reductase is also known as flavin mononucleotide (FMN) reductase (FMN_red). The aim of the present study was to clone the full-length ChrT DNA from Serratia sp. CQMUS2 and analyze the deduced amino acid sequence and three-dimensional structure. The putative ChrT gene fragment of Serratia sp. CQMUS2 was isolated by polymerase chain reaction (PCR), according to the known FMN_red gene sequence from Serratia sp. AS13. The flanking sequences of the ChrT gene were obtained by high efficiency TAIL-PCR, while the full-length gene of ChrT was cloned in Escherichia coli for subsequent sequencing. The nucleotide sequence of ChrT was submitted onto GenBank under the accession number, KF211434. Sequence analysis of the gene and amino acids was conducted using the Basic Local Alignment Search Tool, and open reading frame (ORF) analysis was performed using ORF Finder software. The ChrT gene was found to be an ORF of 567 bp that encodes a 188-amino acid enzyme with a calculated molecular weight of 20.4 kDa. In addition, the ChrT protein was hypothesized to be an NADPH-dependent FMN_red and a member of the flavodoxin-2 superfamily. The amino acid sequence of ChrT showed high sequence similarity to the FMN reductase genes of Klebsiella pneumonia and Raoultella ornithinolytica, which belong to the flavodoxin-2 superfamily. Furthermore, ChrT was shown to have a 85.6% similarity to the three-dimensional structure of Escherichia coli ChrR, sharing four common enzyme active sites for chromate reduction. Therefore, ChrT gene cloning and protein structure determination demonstrated the ability of the gene for chromate reduction. The results of the present study provide a basis for further studies on ChrT gene expression and protein function. PMID:25667630

  9. Identification of pathways, gene networks and paralogous gene families in Daphnia pulex responding to exposure to the toxic cyanobacterium Microcystis aeruginosa

    PubMed Central

    Asselman, Jana; De Coninck, Dieter IM; Glaholt, Stephen; Colbourne, John K; Janssen, Colin R; Shaw, Joseph R; De Schamphelaere, Karel AC

    2013-01-01

    Although cyanobacteria produce a wide range of natural toxins that impact aquatic organisms, food webs and water quality, the mechanisms of toxicity are still insufficiently understood. Here, we implemented a whole-genome expression microarray to identify pathways, gene networks and paralogous gene families responsive to Microcystis stress in Daphnia pulex. Therefore, neonates of a sensitive isolate were given a diet contaminated with Microcystis to contrast with those given a control diet for sixteen days. The microarray revealed 2247 differentially expressed (DE) genes (7.6% of the array) in response to Microcystis, of which 17% are lineage specific( i.e., these genes have no detectable homology to any other gene in currently available databases) and 49% are gene duplicates (paralogs). We identified four pathways/gene networks and eight paralogous gene families affected by Microcystis. Differential regulation of the ribosome, including 3 paralogous gene families encoding 40S, 60S and mitochondrial ribosomal proteins, suggests an impact of Microcystis on protein synthesis of D. pulex. In addition, differential regulation of the oxidative phosphorylation pathway (including the NADH ubquinone oxidoreductase gene family) and the trypsin paralogous gene family (a major component of the digestive system in D. pulex) could explain why fitness is reduced based on energy budget considerations. PMID:22799445

  10. 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

  11. Genetic alterations of the WWOX gene in breast cancer.

    PubMed

    Ekizoglu, Seda; Muslumanoglu, Mahmut; Dalay, Nejat; Buyru, Nur

    2012-09-01

    FRA3B and FRA16D are the most sensitive common chromosomal fragile site loci in the human genome and two tumor suppressor genes FHIT (Fragile Histidine Triad) and WWOX (WW domain-containing oxidoreductase gene) map to this sites. The WWOX gene is composed of 9 exons and encodes a 46-kD protein that contains 414 amino acids. Loss of heterozygosity, homozygous deletions, and chromosomal translocations affecting WWOX has been reported in several types of cancer, including ovarian, esophageal, lung and stomach carcinoma, and multiple myeloma. The aim of this study was to determine the role of WWOX as a tumor suppressor gene in patients with breast cancer. Tumor and adjacent non-cancerous tissue samples were obtained from 81 patients with breast cancer. DNA was isolated from all tissue samples, and all exons and flanking intronic sequences of the WWOX gene were analyzed by PCR amplification and direct sequencing. We detected 14 different alterations in the coding sequence and one base substitution at the intron 6 splice site (+1 G-A). In addition to exonic and splice-site alterations, we detected 23 different alterations in the non-coding region of the gene. All coding region mutations identified in this study were in the exons between 4 and 9. We did not observe any alterations in exons 1-3. We conclude that mutations in critical region of the WWOX gene are frequent and may have an important role in breast carcinogenesis. PMID:21983861

  12. 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...

  13. 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

  14. The thioredoxin system of Penicillium chrysogenum and its possible role in penicillin biosynthesis.

    PubMed Central

    Cohen, G; Argaman, A; Schreiber, R; Mislovati, M; Aharonowitz, Y

    1994-01-01

    Penicillium chrysogenum is an important producer of penicillin antibiotics. A key step in their biosynthesis is the oxidative cyclization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N by the enzyme isopenicillin N synthase (IPNS). bis-ACV, the oxidized disulfide form of ACV is, however, not a substrate for IPNS. We report here the characterization of a broad-range disulfide reductase from P. chrysogenum that efficiently reduces bis-ACV to the thiol monomer. When coupled in vitro with IPNS, it converts bis-ACV to isopenicillin N and may therefore play a role in penicillin biosynthesis. The disulfide reductase consists of two protein components, a 72-kDa NADPH-dependent reductase, containing two identical subunits, and a 12-kDa general disulfide reductant. The latter reduces disulfide bonds in low-molecular-weight compounds and in proteins. The genes coding for the reductase system were cloned and sequenced. Both possess introns. A comparative analysis of their predicted amino acid sequences showed that the 12-kDa protein shares 26 to 60% sequence identity with thioredoxins and that the 36-kDa protein subunit shares 44 to 49% sequence identity with the two known bacterial thioredoxin reductases. In addition, the P. chrysogenum NADPH-dependent reductase is able to accept thioredoxin as a substrate. These results establish that the P. chrysogenum broad-range disulfide reductase is a member of the thioredoxin family of oxidoreductases. This is the first example of the cloning of a eucaryotic thioredoxin reductase gene. Images PMID:8106340

  15. Genetic variants in metabolizing genes NQO1, NQO2, MTHFR and risk of prostate cancer: a study from North India.

    PubMed

    Mandal, Raju K; Nissar, Kamran; Mittal, Rama D

    2012-12-01

    Quinone oxidoreductases (NAD(P)H): quinone oxidoreductase 1 (NQO1) and NRH: quinone oxidoreductase 2 (NQO2) are an antioxidant enzyme, important in the detoxification of environmental carcinogens. Methylene-tetra-hydrofolate reductase (MTHFR), plays a role in folate metabolism and may have oncogenic role through disruption of normal DNA methylation pattern, synthesis, and impaired DNA repair. In a case-control study, genotyping was done in 195 PCa and 250 age matched unrelated healthy controls of similar ethnicity to determine variants in NQO1 exon 4 (C > T, rs4986998), exon 6 (C > T, rs1800566), NQO2 -3423 (G > A, rs2070999) and MTHFR exon 4 (C > T, rs1801133) by PCR-RFLP methods. Heterozygous genotype CT and variant allele career genotype (CT + TT) of NQO1 exon 4 showed increased risk of PCa (OR = 2.06, p = 0.033; OR = 2.02, p = 0.027). Variant allele T also revealed increased risk (OR = 1.87, p = 0.029). Similarly variant genotype TT (OR = 2.71, p = 0.009), combined genotype (CT + TT) (OR = 1.59, p = 0.019) and T allele (OR = 1.63, p = 0.002) of NQO1 exon 6 demonstrated significant risk for PCa. Diplotypes of NQO1 (exon 4 and 6), C-T (OR = 1.56, Pc = 0.007) and T-T (OR = 0.011, Pc = 3.86) was associated with an increased risk for PCa. NQO2 and MTHFR did not show any risk with PCa. Our results strongly support that common sequence variants and diplotypes of NQO1 exon 4 and 6 genes may have role in PCa risk in the North Indian population, indicating the importance of genes involved in metabolism with respect to PCa risk. Additional studies on larger populations are needed to clarify the role of variation in these genes in PCa carcinogenesis. PMID:23054000

  16. The mammalian aldehyde oxidase gene family

    PubMed Central

    2009-01-01

    Aldehyde oxidases (EC 1.2.3.1) are a small group of structurally conserved cytosolic proteins represented in both the animal and plant kingdoms. In vertebrates, aldehyde oxidases constitute the small sub-family of molybdo-flavoenzymes, along with the evolutionarily and structurally related protein, xanthine oxidoreductase. These enzymes require a molybdo-pterin cofactor (molybdenum cofactor, MoCo) and flavin adenine dinucleotide for their catalytic activity. Aldehyde oxidases have broad substrate specificity and catalyse the hydroxylation of N-heterocycles and the oxidation of aldehydes to the corresponding acid. In humans, a single aldehyde oxidase gene (AOX1) and two pseudogenes clustering on a short stretch of chromosome 2q are known. In other mammals, a variable number of structurally conserved aldehyde oxidase genes has been described. Four genes (Aox1, Aox3, Aox4 and Aox3l1), coding for an equivalent number of catalytically active enzymes, are present in the mouse and rat genomes. Although human AOX1 and its homologous proteins are best known as drug metabolising enzymes, the physiological substrate(s) and function(s) are as yet unknown. The present paper provides an update of the available information on the evolutionary history, tissue- and cell-specific distribution and function of mammalian aldehyde oxidases. PMID:20038499

  17. Electron microscopic analysis and structural characterization of novel NADP(H)-containing methanol: N,N'-dimethyl-4-nitrosoaniline oxidoreductases from the gram-positive methylotrophic bacteria Amycolatopsis methanolica and Mycobacterium gastri MB19.

    PubMed Central

    Bystrykh, L V; Vonck, J; van Bruggen, E F; van Beeumen, J; Samyn, B; Govorukhina, N I; Arfman, N; Duine, J A; Dijkhuizen, L

    1993-01-01

    The quaternary protein structure of two methanol:N,N'-dimethyl-4-nitrosoaniline (NDMA) oxidoreductases purified from Amycolatopsis methanolica and Mycobacterium gastri MB19 was analyzed by electron microscopy and image processing. The enzymes are decameric proteins (displaying fivefold symmetry) with estimated molecular masses of 490 to 500 kDa based on their subunit molecular masses of 49 to 50 kDa. Both methanol:NDMA oxidoreductases possess a tightly but noncovalently bound NADP(H) cofactor at an NADPH-to-subunit molar ratio of 0.7. These cofactors are redox active toward alcohol and aldehyde substrates. Both enzymes contain significant amounts of Zn2+ and Mg2+ ions. The primary amino acid sequences of the A. methanolica and M. gastri MB19 methanol:NDMA oxidoreductases share a high degree of identity, as indicated by N-terminal sequence analysis (63% identity among the first 27 N-terminal amino acids), internal peptide sequence analysis, and overall amino acid composition. The amino acid sequence analysis also revealed significant similarity to a decameric methanol dehydrogenase of Bacillus methanolicus C1. Images PMID:8449887

  18. 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

  19. 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

  20. 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

  1. 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

  2. Steady-State Kinetic Mechanism of the Proline:Ubiquinone Oxidoreductase Activity of Proline Utilization A (PutA) from Escherichia coli

    PubMed Central

    Moxley, Michael A.; Tanner, John J.; Becker, Donald F.

    2011-01-01

    The multifunctional proline utilization A (PutA) flavoenzyme from Escherichia coli performs the oxidation of proline to glutamate in two catalytic steps using separate proline dehydrogenase (PRODH) and ?1-pyrroline-5-carboxylate (P5C) dehydrogenase domains. In the first reaction, the oxidation of proline is coupled to the reduction of ubiquinone (CoQ) by the PRODH domain, which has a ?8?8-barrel structure that is conserved in bacterial and eukaryotic PRODH enzymes. The structural requirements of the benzoquinone moiety were examined by steady-state kinetics using CoQ analogs. PutA displayed activity with all the analogs tested; the highest kcat/Km was obtained with CoQ2. The kinetic mechanism of the PRODH reaction was investigated use a variety of steady-state approaches. Initial velocity patterns measured using proline and CoQ1, combined with dead-end and product inhibition studies, suggested a two-site ping-pong mechanism for PutA. The kinetic parameters for PutA were not strongly influenced by solvent viscosity suggesting that diffusive steps do not significantly limit the overall reaction rate. In summary, the kinetic data reported here, along with analysis of the crystal structure data for the PRODH domain, suggest that the proline:ubiquinone oxidoreductase reaction of PutA occurs via a rapid equilibrium ping-pong mechanism with proline and ubiquinone binding at two distinct sites. PMID:22040654

  3. Expression of rat liver NAD(P)H:quinone-acceptor oxidoreductase in Escherichia coli and mutagenesis in vitro at Arg-177.

    PubMed

    Chen, H H; Ma, J X; Forrest, G L; Deng, P S; Martino, P A; Lee, T D; Chen, S

    1992-06-15

    A prokaryotic expression plasmid, pKK-DT2, containing the cDNA of rat liver NAD(P)H:quinone-acceptor oxidoreductase (EC 1.6.99.2; DT-diaphorase) was constructed and used to transform Escherichia coli strain JM109. The rat liver quinone reductase was expressed in strain in JM109 and was inducible with isopropyl beta-D-thiogalactopyranoside (IPTG). The expressed rat protein was purified by affinity chromatography and had kinetic and physical properties identical with the protein purified from rat liver in that it could utilize either NADH or NADPH as the electron donor and its activity was inhibited by dicoumarol. In addition, we have generated four mutants, Arg-177----His (R177H), Arg-177----Ala (R177A), Arg-177----Cys (R177C) and Arg-177----Leu (R177L), using this expression system. Several of the mutants behaved anomalously on SDS/PAGE, but all of the mutant proteins had the expected M(r) as determined by electrospray m.s. These results and those obtained from enzyme kinetic analysis, u.v./visible absorption spectral analysis, and flavin and tryptophan fluorescence analysis of the wild-type enzyme and four mutants indicated that mutations at Arg-177 changed the conformation of the enzyme, resulting in a decrease in enzyme activity. Replacing Arg-177 with leucine altered the protein conformation and decreased FAD incorporation. PMID:1622401

  4. Detection of functional hydrogen-bonded water molecules with protonated/deprotonated key carboxyl side chains in the respiratory enzyme ba3-oxidoreductase.

    PubMed

    Nicolaides, Antonis; Soulimane, Tewfik; Varotsis, Constantinos

    2015-03-11

    The protonation/deprotonation of active carboxyl side chains by water networks forming the proton loading and exit sites in proteins are important steps in protein catalysis. An excellent system to study such basic principles is the heme-copper ba3 from T. thermophilus because it utilizes one proton input channel and it delivers protons to the active site for both O2 chemistry and proton pumping. We report the interaction of the heme a3 Fe propionate-A and the Asp372-His376 pair which forms the valve for the exit pathway for the protons with internal water molecules in ba3 oxidoreductase by light minus dark FTIR spectroscopy in conjunction with H2O/H2(18)O/D2O exchange. The proton loading site consists of several water molecules including w941/w946 which are H-bonded to propionate-A-H(+), acting as the Zundel cation. The detection of two H2(18)O sensitive bands at 3640 and 3634 cm(-1) shows the existence of weakly H-bonded water molecules. PMID:25728291

  5. Analysis of experimental errors in bioprocesses. 1. Production of lactobionic acid and sorbitol using the GFOR (glucose-fructose oxidoreductase) enzyme from permeabilized cells of Zymomonas mobilis.

    PubMed

    Severo, João B; Pinto, José C; Ferraz, Helen C; Alves, Tito L M

    2011-09-01

    The proper determination of experimental errors in bioprocesses can be very important because experimental errors can exert a major impact on the analysis of experimental results. Despite this, the effect of experimental errors on the analysis of bioprocess data has been largely overlooked in the literature. For this reason, we performed detailed statistical analyses of experimental errors obtained during the production of lactobionic acid and sorbitol in a system utilizing as catalyst the GFOR (glucose-fructose oxidoreductase) enzyme from permeabilized cells of the bacteria Zymomonas mobilis. The magnitude of the experimental errors thus obtained were then correlated with the process operation conditions and with the composition of the culture media used for bacterial growth. It is shown that experimental errors can depend very significantly on the operation conditions and affect the interpretation of available experimental data. More specifically, in this study, experimental errors depended on the nutritional supplements added to the cultivation medium, the inoculation process, and the reaction time, which may be of fundamental importance for actual process development. The results obtained also indicate, for the first time, that GFOR activity can be affected by the composition of the medium in which cells are cultivated. PMID:21328074

  6. 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

  7. GmcA Is a Putative Glucose-Methanol-Choline Oxidoreductase Required for the Induction of Asexual Development in Aspergillus nidulans

    PubMed Central

    Etxebeste, Oier; Herrero-García, Erika; Cortese, Marc S.; Garzia, Aitor; Oiartzabal-Arano, Elixabet; de los Ríos, Vivian; Ugalde, Unai; Espeso, Eduardo A.

    2012-01-01

    Aspergillus nidulans asexual differentiation is induced by Upstream Developmental Activators (UDAs) that include the bZIP-type Transcription Factor (TF) FlbB. A 2D-PAGE/MS-MS-coupled screen for proteins differentially expressed in the presence and absence of FlbB identified 18 candidates. Most candidates belong to GO term classes involved in osmotic and/or oxidative stress response. Among these, we focused on GmcA, a putative glucose-methanol-choline oxidoreductase which is upregulated in a ?flbB background. GmcA is not required for growth since no differences were detected in the radial extension upon deletion of gmcA. However, its activity is required to induce conidiation under specific culture conditions. A ?gmcA strain conidiates profusely under acid conditions but displays a characteristic fluffy aconidial phenotype in alkaline medium. The absence of asexual development in a ?gmcA strain can be suppressed, on one hand, using high concentrations of non-fermentable carbon sources like glycerol, and on the other hand, when the cMyb-type UDA TF flbD is overexpressed. Overall, the results obtained in this work support a role for GmcA at early stages of conidiophore initiation. PMID:22792266

  8. Structure-based computational study of two disease resistance gene homologues (Hm1 and Hm2) in maize (Zea mays L.) with implications in plant-pathogen interactions.

    PubMed

    Dehury, Budheswar; Patra, Mahesh Chandra; Maharana, Jitendra; Sahu, Jagajjit; Sen, Priyabrata; Modi, Mahendra Kumar; Choudhury, Manabendra Dutta; Barooah, Madhumita

    2014-01-01

    The NADPH-dependent HC-toxin reductases (HCTR1 and 2) encoded by enzymatic class of disease resistance homologous genes (Hm1 and Hm2) protect maize by detoxifying a cyclic tetrapeptide, HC-toxin, secreted by the fungus Cochliobolus carbonum race 1(CCR1). Unlike the other classes' resistance (R) genes, HCTR-mediated disease resistance is an inimitable mechanism where the avirulence (Avr) component from CCR1 is not involved in toxin degradation. In this study, we attempted to decipher cofactor (NADPH) recognition and mode of HC-toxin binding to HCTRs through molecular docking, molecular dynamics (MD) simulations and binding free energy calculation methods. The rationality and the stability of docked complexes were validated by 30-ns MD simulation. The binding free energy decomposition of enzyme-cofactor complex was calculated to find the driving force behind cofactor recognition. The overall binding free energies of HCTR1-NADPH and HCTR2-NADPH were found to be -616.989 and -16.9749 kJ mol-1 respectively. The binding free energy decomposition revealed that the binding of NADPH to the HCTR1 is mainly governed by van der Waals and nonpolar interactions, whereas electrostatic terms play dominant role in stabilizing the binding mode between HCTR2 and NADPH. Further, docking analysis of HC-toxin with HCTR-NADPH complexes showed a distinct mode of binding and the complexes were stabilized by a strong network of hydrogen bond and hydrophobic interactions. This study is the first in silico attempt to unravel the biophysical and biochemical basis of cofactor recognition in enzymatic class of R genes in cereal crop maize. PMID:24847713

  9. 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

  10. Chromosomal organization of the human dihydrofolate reductase genes: dispersion, selective amplification, and a novel form of polymorphism.

    PubMed Central

    Anagnou, N P; O'Brien, S J; Shimada, T; Nash, W G; Chen, M J; Nienhuis, A W

    1984-01-01

    The human dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) gene family includes a functional gene (hDHFR) and at least four intronless genes. Three intronless genes (hDHFR-psi 2, hDHFR-psi 3, and hDHFR-psi 4) are identifiable as pseudogenes because of DNA sequence divergence from the functional gene with introns, while one intronless gene (hDHFR-psi 1) is completely homologous to the coding sequences of the functional gene. Analysis of genomic DNA from two panels of somatic human-rodent cell hybrids with specific molecular probes provide insight into the chromosomal organization and assignment of these genes. The five genes are dispersed in that each one is found on a different chromosome. The functional gene hDHFR has been assigned to chromosome 5, and one pseudogene (hDHFR-psi 4), to chromosome 3. In a human cell line (HeLa) that was selected for methotrexate resistance, the functional locus became amplified, while there was no amplification of the four intronless pseudogenes. hDHFR-psi 1 was found to be present in DNA of some individuals and absent from DNA of others, consistent with a recent evolutionary origin of this gene originally suggested by its sequence identity to the coding portions of the functional gene. The presence or absence of this intronless pseudogene represents a previously unreported form of DNA polymorphism. Images PMID:6089182

  11. 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

  12. Improved biocatalysts from a synthetic circular permutation library of the flavin-dependent oxidoreductase old yellow enzyme.

    PubMed

    Daugherty, Ashley B; Govindarajan, Sridhar; Lutz, Stefan

    2013-09-25

    Members of the old yellow enzyme (OYE) family are widely used, effective biocatalysts for the stereoselective trans-hydrogenation of activated alkenes. To further expand their substrate scope and improve catalytic performance, we have applied a protein engineering strategy called circular permutation (CP) to enhance the function of OYE1 from Saccharomyces pastorianus. CP can influence a biocatalyst's function by altering protein backbone flexibility and active site accessibility, both critical performance features because the catalytic cycle for OYE1 is thought to involve rate-limiting conformational changes. To explore the impact of CP throughout the OYE1 protein sequence, we implemented a highly efficient approach for cell-free cpOYE library preparation by combining whole-gene synthesis with in vitro transcription/translation. The versatility of such an ex vivo system was further demonstrated by the rapid and reliable functional evaluation of library members under variable environmental conditions with three reference substrates ketoisophorone, cinnamaldehyde, and (S)-carvone. Library analysis identified over 70 functional OYE1 variants with several biocatalysts exhibiting over an order of magnitude improved catalytic activity. Although catalytic gains of individual cpOYE library members vary by substrate, the locations of new protein termini in functional variants for all tested substates fall within the same four distinct loop/lid regions near the active site. Our findings demonstrate the importance of these structural elements in enzyme function and support the hypothesis of conformational flexibility as a limiting factor for catalysis in wild type OYE. PMID:23987134

  13. Kinetic and Structural Studies of Aldehyde Oxidoreductase from Desulfovibrio gigas Reveal a Dithiolene-Based Chemistry for Enzyme Activation and Inhibition by H2O2

    PubMed Central

    Brondino, Carlos D.; Moura, José J. G.; Romão, Maria J.; González, Pablo J.; Santos-Silva, Teresa

    2013-01-01

    Mononuclear Mo-containing enzymes of the xanthine oxidase (XO) family catalyze the oxidative hydroxylation of aldehydes and heterocyclic compounds. The molybdenum active site shows a distorted square-pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. The XO family member aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is an exception as presents in its catalytically competent form an equatorial oxo ligand instead of the sulfido ligand. Despite this structural difference, inactive samples of DgAOR can be activated upon incubation with dithionite plus sulfide, a procedure similar to that used for activation of desulfo-XO. The fact that DgAOR does not need a sulfido ligand for catalysis indicates that the process leading to the activation of inactive DgAOR samples is different to that of desulfo-XO. We now report a combined kinetic and X-ray crystallographic study to unveil the enzyme modification responsible for the inactivation and the chemistry that occurs at the Mo site when DgAOR is activated. In contrast to XO, which is activated by resulfuration of the Mo site, DgAOR activation/inactivation is governed by the oxidation state of the dithiolene moiety of the pyranopterin cofactor, which demonstrates the non-innocent behavior of the pyranopterin in enzyme activity. We also showed that DgAOR incubation with dithionite plus sulfide in the presence of dioxygen produces hydrogen peroxide not associated with the enzyme activation. The peroxide molecule coordinates to molybdenum in a ?2 fashion inhibiting the enzyme activity. PMID:24391748

  14. 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.

  15. 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

  16. 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

  17. Hepatic microsomal enzyme activity in the koala and tammar wallaby: high 17beta-hydroxysteroid oxidoreductase activity in koala liver microsomes.

    PubMed

    Stupans, I; Kong, S; Kirlich, A; Murray, M; Bailey, E L; Jones, B R; McKinnon, R A

    1999-05-01

    We have studied the hepatic microsomal xenobiotic metabolising capacity of koala (Phascolarctos cinereus) and tammar wallaby (Macropus eugenii). Total cytochrome P450 content in hepatic microsomes from koala (0.87 +/- 0.18 nmol/mg protein, n = 4, mean (S.D.) and rat were comparable while tammar wallaby displayed reduced P450 content (0.24 +/- 0.04 nmol/mg protein). Associated microsomal activities (NADPH cytochrome P450 reductase, aminopyrine N-demethylation, aniline hydroxylation, and androstenedione 6beta- and 16alpha-hydroxylation) in koala liver were similar to or reduced relative to rat. Hepatic microsomal NADPH-supported 17beta-hydroxysteroid oxidoreductase (17beta-HSOR) activity was significantly higher in koala (9.99+/-3.08 nmol/mg protein/min) than in tammar wallaby liver (0.86 +/- 0.16 nmol/mg protein/min). However, when NADH was utilised as cofactor the activity was similar in both marsupial species (koala, 1.44 +/- 0.84 nmol/mg protein/min; tammar wallaby, 1.52 +/- 0.44 nmol/mg protein/min). Michaelis-Menten parameters for the kinetics of 17beta-HSOR androstenedione reduction by NADPH and NADH were determined in the koala. The Km for androstenedione was of the order of 1.9-4 microM (n = 4) irrespective of the cofactor used, whilst the Km for NADPH was 0.04-0.05 microM (n = 2) and for NADH was 134-430 microM (n = 2). Potential inhibitors were evaluated for their effects on NADPH-mediated 17beta-HSOR activity with menadione and, to lesser extents, menthone, benzaldehyde and metyrapone eliciting significant inhibition. From detailed kinetic studies menthone was found to be an uncompetitive inhibitor of the activity in koala liver (Ki 220 microM). PMID:10390058

  18. Arsenite pretreatment enhances the cytotoxicity of mitomycin C in human cancer cell lines via increased NAD(P)H quinone oxidoreductase 1 expression.

    PubMed

    Lin, Yi-Ling; Ho, I-Ching; Su, Pei-Fen; Lee, Te-Chang

    2006-08-01

    Arsenic is an effective therapeutic agent for the treatment of patients with refractory or relapsed acute promyelocytic leukemia. The use of arsenic for treating solid tumors, particularly in combination with other chemotherapeutic agents, has been extensively studied. Here, we report that arsenite-resistant human lung cancer CL3R15 cells constitutively overexpress NAD(P)H quinone oxidoreductase 1 (NQO1), an enzyme responsible for activation of mitomycin C (MMC), and are more susceptible to MMC cytotoxicity than parental CL3 cells. The effects of arsenite pretreatment on NQO1 induction were examined in CL3, H1299, H460, and MC-T2 cells. Arsenite pretreatment significantly enhanced the expression of NQO1 and susceptibility to MMC in CL3, H1299, and MC-T2 cells, but not in H460 cells that express high endogenous levels of NQO1. Alternatively, arsenic pretreatment reduced adriamycin sensitivity of CL3 cells. Arsenite-mediated MMC susceptibility was abrogated by dicumarol (DIC), an NQO1 inhibitor, indicating that NQO1 is one of the key regulators of arsenite-mediated MMC susceptibility. Various cancer cell lines showed different basal levels of NQO1 activity and a different capacity for NQO1 induction in response to arsenite treatment. However, overall, there was a positive correlation between induced NQO1 activity and MMC susceptibility in cells pretreated with various doses of arsenite. These results suggest that arsenite may increase NQO1 activity and thus enhance the antineoplastic activity of MMC. In addition, our results also showed that inhibition of NQO1 activity by DIC reversed the arsenite resistance of CL3R15 cells. PMID:16494910

  19. The reaction of NADPH with bovine mitochondrial NADH:ubiquinone oxidoreductase revisited: I. Proposed consequences for electron transfer in the enzyme.

    PubMed

    Albracht, Simon P J

    2010-08-01

    Bovine NADH:ubiquinone oxidoreductase (Complex I) is the first complex in the mitochondrial respiratory chain. It has long been assumed that it contained only one FMN group. However, as demonstrated in 2003, the intact enzyme contains two FMN groups. The second FMN was proposed to be located in a conserved flavodoxin fold predicted to be present in the PSST subunit. The long-known reaction of Complex I with NADPH differs in many aspects from that with NADH. It was proposed that the second flavin group was specifically involved in the reaction with NADPH. The X-ray structure of the hydrophilic domain of Complex I from Thermus thermophilus (Sazanov and Hinchliffe 2006, Science 311, 1430-1436) disclosed the positions of all redox groups of that enzyme and of the subunits holding them. The PSST subunit indeed contains the predicted flavodoxin fold although it did not contain FMN. Inspired by this structure, the present paper describes a re-evaluation of the enigmatic reactions of the bovine enzyme with NADPH. Published data, as well as new freeze-quench kinetic data presented here, are incompatible with the general opinion that NADPH and NADH react at the same site. Instead, it is proposed that these pyridine nucleotides react at opposite ends of the 90 A long chain of prosthetic groups in Complex I. Ubiquinone is proposed to react with the Fe-S clusters in the TYKY subunit deep inside the hydrophilic domain. A new model for electron transfer in Complex I is proposed. In the accompanying paper this model is compared with the one advocated in current literature. PMID:20628895

  20. 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

  1. Semiquinone and cluster N6 signals in His-tagged proton-translocating NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli.

    PubMed

    Narayanan, Madhavan; Gabrieli, David J; Leung, Steven A; Elguindy, Mahmoud M; Glaser, Carl A; Saju, Nitha; Sinha, Subhash C; Nakamaru-Ogiso, Eiko

    2013-05-17

    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

  2. Structural and Functional Investigation of Flavin Binding Center of the NqrC Subunit of Sodium-Translocating NADH:Quinone Oxidoreductase from Vibrio harveyi.

    PubMed

    Borshchevskiy, Valentin; Round, Ekaterina; Bertsova, Yulia; Polovinkin, Vitaly; Gushchin, Ivan; Ishchenko, Andrii; Kovalev, Kirill; Mishin, Alexey; Kachalova, Galina; Popov, Alexander; Bogachev, Alexander; Gordeliy, Valentin

    2015-01-01

    Na+-translocating NADH:quinone oxidoreductase (NQR) is a redox-driven sodium pump operating in the respiratory chain of various bacteria, including pathogenic species. The enzyme has a unique set of redox active prosthetic groups, which includes two covalently bound flavin mononucleotide (FMN) residues attached to threonine residues in subunits NqrB and NqrC. The reason of FMN covalent bonding in the subunits has not been established yet. In the current work, binding of free FMN to the apo-form of NqrC from Vibrio harveyi was studied showing very low affinity of NqrC to FMN in the absence of its covalent bonding. To study structural aspects of flavin binding in NqrC, its holo-form was crystallized and its 3D structure was solved at 1.56 Å resolution. It was found that the isoalloxazine moiety of the FMN residue is buried in a hydrophobic cavity and that its pyrimidine ring is squeezed between hydrophobic amino acid residues while its benzene ring is extended from the protein surroundings. This structure of the flavin-binding pocket appears to provide flexibility of the benzene ring, which can help the FMN residue to take the bended conformation and thus to stabilize the one-electron reduced form of the prosthetic group. These properties may also lead to relatively weak noncovalent binding of the flavin. This fact along with periplasmic location of the FMN-binding domains in the vast majority of NqrC-like proteins may explain the necessity of the covalent bonding of this prosthetic group to prevent its loss to the external medium. PMID:25734798

  3. Functional analysis of two isoforms of leaf-type ferredoxin-NADP(+)-oxidoreductase in rice using the heterologous expression system of Arabidopsis.

    PubMed

    Higuchi-Takeuchi, Mieko; Ichikawa, Takanari; Kondou, Youichi; Matsui, Keiko; Hasegawa, Yukako; Kawashima, Mika; Sonoike, Kintake; Mori, Masaki; Hirochika, Hirohiko; Matsui, Minami

    2011-09-01

    Ferredoxin-NADP(+)-oxidoreductase (FNR) mediates electron transfer between ferredoxin (Fd) and NADP(+); therefore, it is a key enzyme that provides the reducing power used in the Calvin cycle. Other than FNR, nitrite reductase, sulfite reductase, glutamate synthase, and Fd-thioredoxin reductase also accept electrons from Fd, an electron carrier protein in the stroma. Therefore, the regulation of electron partitioning in the chloroplast is important for photosynthesis and other metabolic pathways. The regulatory mechanism of electron partitioning, however, remains to be elucidated. We found, by taking advantage of a gain-of-function approach, that expression of two rice (Oryza sativa) full-length cDNAs of leaf-type FNRs (OsLFNR1 and OsLFNR2) led to altered chlorophyll fluorescence and growth in Arabidopsis (Arabidopsis thaliana) and rice. We revealed that overexpression of the OsLFNR1 and OsLFNR2 full-length cDNAs resulted in distinct phenotypes despite the high sequence similarity between them. Expression of OsLFNR1 affected the nitrogen assimilation pathway without inhibition of photosynthesis under normal conditions. On the other hand, OsLFNR2 expression led to the impairment of photosynthetic linear electron transport as well as Fd-dependent cyclic electron flow around photosystem I. The endogenous protein level of OsLFNR was found to be suppressed in both OsLFNR1- and OsLFNR2-overexpressing rice plants, leading to changes in the stoichiometry of the two LFNR isoforms within the thylakoid and soluble fractions. Thus, we propose that the stoichiometry of two LFNR isoforms plays an important role in electron partitioning between carbon fixation and nitrogen assimilation. PMID:21734114

  4. Characterization of Chlorophenol 4-Monooxygenase (TftD) and NADH:FAD Oxidoreductase (TftC) of Burkholderia cepacia AC1100*

    PubMed Central

    Webb, Brian N.; Ballinger, Jordan W.; Kim, Eunjung; Belchik, Sara M.; Lam, Ka-Sum; Youn, Buhyun; Nissen, Mark S.; Xun, Luying; Kang, ChulHee

    2010-01-01

    Burkholderia cepacia AC1100 completely degrades 2,4,5-trichlorophenol, in which an FADH2-dependent monooxygenase (TftD) and an NADH:FAD oxidoreductase (TftC) catalyze the initial steps. TftD oxidizes 2,4,5-trichlorophenol (2,4,5-TCP) to 2,5-dichloro-p-benzoquinone, which is chemically reduced to 2,5-dichloro-p-hydroquinone (2,5-DiCHQ). Then, TftD oxidizes the latter to 5-chloro-2-hydroxy-p-benzoquinone. In those processes, TftC provides all the required FADH2. We have determined the crystal structures of dimeric TftC and tetrameric TftD at 2.0 and 2.5 Å resolution, respectively. The structure of TftC was similar to those of related flavin reductases. The stacked nicotinamide:isoalloxazine rings in TftC and sequential reaction kinetics suggest that the reduced FAD leaves TftC after NADH oxidation. The structure of TftD was also similar to the known structures of FADH2-dependent monooxygenases. Its His-289 residue in the re-side of the isoalloxazine ring is within hydrogen bonding distance with a hydroxyl group of 2,5-DiCHQ. An H289A mutation resulted in the complete loss of activity toward 2,5-DiCHQ and a significant decrease in catalytic efficiency toward 2,4,5-TCP. Thus, His-289 plays different roles in the catalysis of 2,4,5-TCP and 2,5-DiCHQ. The results support that free FADH2 is generated by TftC, and TftD uses FADH2 to separately transform 2,4,5-TCP and 2,5-DiCHQ. Additional experimental data also support the diffusion of FADH2 between TftC and TftD without direct physical interaction between the two enzymes. PMID:19915006

  5. Electrical Wiring of the Aldehyde Oxidoreductase PaoABC with a Polymer Containing Osmium Redox Centers: Biosensors for Benzaldehyde and GABA

    PubMed Central

    Badalyan, Artavazd; Dierich, Marlen; Stiba, Konstanze; Schwuchow, Viola; Leimkühler, Silke; Wollenberger, Ulla

    2014-01-01

    Biosensors for the detection of benzaldehyde and ??aminobutyric acid (GABA) are reported using aldehyde oxidoreductase PaoABC from Escherichia coli immobilized in a polymer containing bound low potential osmium redox complexes. The electrically connected enzyme already electrooxidizes benzaldehyde at potentials below ?0.15 V (vs. Ag|AgCl, 1 M KCl). The pH-dependence of benzaldehyde oxidation can be strongly influenced by the ionic strength. The effect is similar with the soluble osmium redox complex and therefore indicates a clear electrostatic effect on the bioelectrocatalytic efficiency of PaoABC in the osmium containing redox polymer. At lower ionic strength, the pH-optimum is high and can be switched to low pH-values at high ionic strength. This offers biosensing at high and low pH-values. A “reagentless” biosensor has been formed with enzyme wired onto a screen-printed electrode in a flow cell device. The response time to addition of benzaldehyde is 30 s, and the measuring range is between 10–150 µM and the detection limit of 5 µM (signal to noise ratio 3:1) of benzaldehyde. The relative standard deviation in a series (n = 13) for 200 µM benzaldehyde is 1.9%. For the biosensor, a response to succinic semialdehyde was also identified. Based on this response and the ability to work at high pH a biosensor for GABA is proposed by coimmobilizing GABA-aminotransferase (GABA-T) and PaoABC in the osmium containing redox polymer. PMID:25587431

  6. Structural and Functional Investigation of Flavin Binding Center of the NqrC Subunit of Sodium-Translocating NADH:Quinone Oxidoreductase from Vibrio harveyi

    PubMed Central

    Bertsova, Yulia; Polovinkin, Vitaly; Gushchin, Ivan; Ishchenko, Andrii; Kovalev, Kirill; Mishin, Alexey; Kachalova, Galina; Popov, Alexander; Bogachev, Alexander; Gordeliy, Valentin

    2015-01-01

    Na+-translocating NADH:quinone oxidoreductase (NQR) is a redox-driven sodium pump operating in the respiratory chain of various bacteria, including pathogenic species. The enzyme has a unique set of redox active prosthetic groups, which includes two covalently bound flavin mononucleotide (FMN) residues attached to threonine residues in subunits NqrB and NqrC. The reason of FMN covalent bonding in the subunits has not been established yet. In the current work, binding of free FMN to the apo-form of NqrC from Vibrio harveyi was studied showing very low affinity of NqrC to FMN in the absence of its covalent bonding. To study structural aspects of flavin binding in NqrC, its holo-form was crystallized and its 3D structure was solved at 1.56 Å resolution. It was found that the isoalloxazine moiety of the FMN residue is buried in a hydrophobic cavity and that its pyrimidine ring is squeezed between hydrophobic amino acid residues while its benzene ring is extended from the protein surroundings. This structure of the flavin-binding pocket appears to provide flexibility of the benzene ring, which can help the FMN residue to take the bended conformation and thus to stabilize the one-electron reduced form of the prosthetic group. These properties may also lead to relatively weak noncovalent binding of the flavin. This fact along with periplasmic location of the FMN-binding domains in the vast majority of NqrC-like proteins may explain the necessity of the covalent bonding of this prosthetic group to prevent its loss to the external medium. PMID:25734798

  7. 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

  8. Reduction of hydrophilic ubiquinones by the flavin in mitochondrial NADH:ubiquinone oxidoreductase (Complex I) and production of reactive oxygen species.

    PubMed

    King, Martin S; Sharpley, Mark S; Hirst, Judy

    2009-03-10

    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

  9. 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

  10. Role of Ser457 of NADPH-cytochrome P450 oxidoreductase in catalysis and control of FAD oxidation-reduction potential.

    PubMed

    Shen, A L; Kasper, C B

    1996-07-23

    Site-directed mutagenesis of Ser457 of NADPH-cytochrome P450 oxidoreductase demonstrates that this residue plays a major role in both hydride transfer from NADPH to FAD and modulation of FAD redox potential. Substitution of Ser457 with alanine or cysteine decreases the rates of reduction of the substrates cytochrome c and potassium ferricyanide approximately 100-fold, while substitution with threonine produces a 20-fold decrease in activity. No changes are observed in k(m)NADPH, KiNADP+, or flavin content, indicating that these substitutions have no effect on cofactor binding but affect catalysis only. k(m)cyt c values are decreased in parallel with the observed decreases in the rates of the reductive half-reaction. Stopped-flow studies with the S457A mutant show a 100-fold decrease in the rate of flavin reduction. The primary deuterium isotope effect on Kcat for cytochrome c reduction increases from 2.7 for the wild-type enzyme to 9.0 for the S457A mutant, consistent with a change in the rate-determining step from NADP+ release in the wild-type enzyme to hydride transfer in the S457A mutant. The primary deuterium isotope effect on K1 for flavin reduction at high ionic strength (I = 535 mM) increases from 12.2 for the wild-type enzyme to > 20 for the S457A mutant, consistent again with an increase in the relative rate limitation of hydride transfer. Furthermore, anaerobic titration of S457A indicates that the redox potential of the FAD semiquinone has been decreased. Data presented in this study support the hypothesis that Ser457 is involved in hydrogen bonding interactions which stabilize both the transition state for hydride transfer and the reduced FAD. PMID:8755724

  11. Aromatic aldehydes at the active site of aldehyde oxidoreductase from Desulfovibrio gigas: reactivity and molecular details of the enzyme-substrate and enzyme-product interaction.

    PubMed

    Correia, Hugo D; Marangon, Jacopo; Brondino, Carlos D; Moura, Jose J G; Romão, Maria J; González, Pablo J; Santos-Silva, Teresa

    2015-03-01

    Desulfovibrio gigas aldehyde oxidoreductase (DgAOR) is a mononuclear molybdenum-containing enzyme from the xanthine oxidase (XO) family, a group of enzymes capable of catalyzing the oxidative hydroxylation of aldehydes and heterocyclic compounds. The kinetic studies reported in this work showed that DgAOR catalyzes the oxidative hydroxylation of aromatic aldehydes, but not heterocyclic compounds. NMR spectroscopy studies using (13)C-labeled benzaldehyde confirmed that DgAOR catalyzes the conversion of aldehydes to the respective carboxylic acids. Steady-state kinetics in solution showed that high concentrations of the aromatic aldehydes produce substrate inhibition and in the case of 3-phenyl propionaldehyde a suicide substrate behavior. Hydroxyl-substituted aromatic aldehydes present none of these behaviors but the kinetic parameters are largely affected by the position of the OH group. High-resolution crystallographic structures obtained from single crystals of active-DgAOR soaked with benzaldehyde showed that the side chains of Phe425 and Tyr535 are important for the stabilization of the substrate in the active site. On the other hand, the X-ray data of DgAOR soaked with trans-cinnamaldehyde showed a cinnamic acid molecule in the substrate channel. The X-ray data of DgAOR soaked with 3-phenyl propionaldehyde showed clearly how high substrate concentrations inactivate the enzyme by binding covalently at the surface of the enzyme and blocking the substrate channel. The different reactivity of DgAOR versus aldehyde oxidase and XO towards aromatic aldehydes and N-heterocyclic compounds is explained on the basis of the present kinetic and structural data. PMID:25261288

  12. Defining redox centers in human electron transfer flavoprotein: ubiquinone oxidoreductase (ETF:QO) by expression in Saccharomyces cerevisiae

    SciTech Connect

    Frerman, F.E.; Beard, S.; Goodman, S.I. [Univ. of Colorado School of Medicine, Denver, CO (United States)

    1994-09-01

    Mutations in ETF or ETC:QO cause glutaric acidemia type II (GA2). ETF:QO is an iron-sulfur flavoprotein in the inner mitochondrial membrane which transfers electrons from ETF in the mitochondrial matrix to ubiquinone (Q). The human ETF:QO gene is on chromosome 4q32{r_arrow}qter, and encodes a 617 amino acid precursor which is processed to the 64 kDa mature form in the mitochondrion. One ETF:QO mutation in GA2 is a G{r_arrow}T transversion in a donor splice site, deleting the 222 bp upstream exon from the transcript. The deleted 74 amino acids are near the carboxyl terminus just beyond a predicted membrane helix, and include C561, one of four cysteine residues predicted to ligate the 4Fe4S cluster. The mutant protein is not stable in patient fibroblasts. We have expressed cDNAs encoding wild type (wt) ETF:QO, ETF:QO with the 74 amino acid deletion, and ETFF:QO with only a C561A mutation, in S cerevisiae. In all instances, precursor and mature ETF:QOs were stably inserted into the mitochondrial membrane. ETF:QO (C561A) is extracted from the membrane under the same conditions as wt ETF:QO, but ETF:QO with the deletion is much more difficult to extract. Wt ETF:QO accepts electrons from ETF and reduces Q but, while both mutant proteins accept electrons from ETF, neither of them reduces Q. This work demonstrates that C561 in human ETF:QO is essential for Q reduction (probably because it ligands the 4Fe4S cluster), that mutant proteins that are unstable in man may be stable in other systems, that cleavage of signal peptide from precursor proteins can occur within the inner mitochondrial membrane, and the general usefulness of expressing human mitochondrial proteins in yeast.

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. Cell Growth Defect Factor1/CHAPERONE-LIKE PROTEIN OF POR1 Plays a Role in Stabilization of Light-Dependent Protochlorophyllide Oxidoreductase in Nicotiana benthamiana and Arabidopsis[C][W

    PubMed Central

    Lee, Jae-Yong; Lee, Ho-Seok; Song, Ji-Young; Jung, Young Jun; Reinbothe, Steffen; Park, Youn-Il; Lee, Sang Yeol; Pai, Hyun-Sook

    2013-01-01

    Angiosperms require light for chlorophyll biosynthesis because one reaction in the pathway, the reduction of protochlorophyllide (Pchlide) to chlorophyllide, is catalyzed by the light-dependent protochlorophyllide oxidoreductase (POR). Here, we report that Cell growth defect factor1 (Cdf1), renamed here as CHAPERONE-LIKE PROTEIN OF POR1 (CPP1), an essential protein for chloroplast development, plays a role in the regulation of POR stability and function. Cdf1/CPP1 contains a J-like domain and three transmembrane domains, is localized in the thylakoid and envelope membranes, and interacts with POR isoforms in chloroplasts. CPP1 can stabilize POR proteins with its holdase chaperone activity. CPP1 deficiency results in diminished POR protein accumulation and defective chlorophyll synthesis, leading to photobleaching and growth inhibition of plants under light conditions. CPP1 depletion also causes reduced POR accumulation in etioplasts of dark-grown plants and as a result impairs the formation of prolamellar bodies, which subsequently affects chloroplast biogenesis upon illumination. Furthermore, in cyanobacteria, the CPP1 homolog critically regulates POR accumulation and chlorophyll synthesis under high-light conditions, in which the dark-operative Pchlide oxidoreductase is repressed by its oxygen sensitivity. These findings and the ubiquitous presence of CPP1 in oxygenic photosynthetic organisms suggest the conserved nature of CPP1 function in the regulation of POR. PMID:24151298

  18. Human NADPH-P450 oxidoreductase modulates the level of cytochrome P450 CYP2D6 holoprotein via haem oxygenase-dependent and -independent pathways.

    PubMed Central

    Ding, S; Yao, D; Deeni, Y Y; Burchell, B; Wolf, C R; Friedberg, T

    2001-01-01

    NADPH-P450 oxidoreductase (CPR) is essential for the activity of cytochrome P450 (P450). Previous studies demonstrated that CPR regulates the levels of various P450 isoforms in vitro. We investigated the mechanistic basis for this regulation. By transfection of Chinese hamster ovary DUKXB11 cells we obtained the cell line DUKX/2D6, which expressed human CYP2D6, a P450 isoform. Subsequently, DUKX/2D6 cells were transfected with human CPR cDNA to generate the cell line DUKX/2D6/CPR-3. Expression of recombinant CPR decreased the level of spectrally detectable CYP2D6 holoprotein in DUKX/2D6/CPR-3 cells by 70%, whereas the level of immunodetectable apoprotein remained unchanged. Addition of the radical scavenger DMSO increased levels of CYP2D6 holoenzyme in DUKX/2D6/CPR-3 cells but not in DUKX/2D6 cells. A similar effect was noted when cells were grown in the presence of hemin. Importantly, combined treatment with DMSO and hemin increased levels of CYP2D6 holoenzyme in DUKX/2D6/CPR-3 but not in DUKX/2D6 cells even further than either treatment alone. None of these treatments affected the level of immunodetectable CYP2D6. This demonstrates that expression of CPR increases production of damaging radicals but also that CPR may alter haem homoeostasis. In agreement with this, the activity of haem oxygenase, a rate-limiting enzyme in haem metabolism, was compared with that in DUKX/DHFR control cells (expressing dihydrofolate reductase), and was 3-fold higher in DUKX/2D6/CPR-3 but similar in DUKX/2D6 cells. Furthermore, treatment of cells with sodium arsenite increased levels of haem oxygenase concomitant with a marked decrease of spectrally detectable CYP2D6 and a rise in levels of ferritin, which sequesters free iron released from the destruction of haem. These data demonstrate that CPR regulates P450 activity by supplying electrons and also by altering P450 levels via radical-and haem oxygenase-mediated pathways. PMID:11368792

  19. Human NADPH-P450 oxidoreductase modulates the level of cytochrome P450 CYP2D6 holoprotein via haem oxygenase-dependent and -independent pathways.

    PubMed

    Ding, S; Yao, D; Deeni, Y Y; Burchell, B; Wolf, C R; Friedberg, T

    2001-06-01

    NADPH-P450 oxidoreductase (CPR) is essential for the activity of cytochrome P450 (P450). Previous studies demonstrated that CPR regulates the levels of various P450 isoforms in vitro. We investigated the mechanistic basis for this regulation. By transfection of Chinese hamster ovary DUKXB11 cells we obtained the cell line DUKX/2D6, which expressed human CYP2D6, a P450 isoform. Subsequently, DUKX/2D6 cells were transfected with human CPR cDNA to generate the cell line DUKX/2D6/CPR-3. Expression of recombinant CPR decreased the level of spectrally detectable CYP2D6 holoprotein in DUKX/2D6/CPR-3 cells by 70%, whereas the level of immunodetectable apoprotein remained unchanged. Addition of the radical scavenger DMSO increased levels of CYP2D6 holoenzyme in DUKX/2D6/CPR-3 cells but not in DUKX/2D6 cells. A similar effect was noted when cells were grown in the presence of hemin. Importantly, combined treatment with DMSO and hemin increased levels of CYP2D6 holoenzyme in DUKX/2D6/CPR-3 but not in DUKX/2D6 cells even further than either treatment alone. None of these treatments affected the level of immunodetectable CYP2D6. This demonstrates that expression of CPR increases production of damaging radicals but also that CPR may alter haem homoeostasis. In agreement with this, the activity of haem oxygenase, a rate-limiting enzyme in haem metabolism, was compared with that in DUKX/DHFR control cells (expressing dihydrofolate reductase), and was 3-fold higher in DUKX/2D6/CPR-3 but similar in DUKX/2D6 cells. Furthermore, treatment of cells with sodium arsenite increased levels of haem oxygenase concomitant with a marked decrease of spectrally detectable CYP2D6 and a rise in levels of ferritin, which sequesters free iron released from the destruction of haem. These data demonstrate that CPR regulates P450 activity by supplying electrons and also by altering P450 levels via radical-and haem oxygenase-mediated pathways. PMID:11368792

  20. Influence of Various Polymorphic Variants of Cytochrome P450 Oxidoreductase (POR) on Drug Metabolic Activity of CYP3A4 and CYP2B6

    PubMed Central

    Naranmandura, Hua; Zeng, Su; Chen, Shu Qing

    2012-01-01

    Cytochrome P450 oxidoreductase (POR) is known as the sole electron donor in the metabolism of drugs by cytochrome P450 (CYP) enzymes in human. However, little is known about the effect of polymorphic variants of POR on drug metabolic activities of CYP3A4 and CYP2B6. In order to better understand the mechanism of the activity of CYPs affected by polymorphic variants of POR, six full-length mutants of POR (e.g., Y181D, A287P, K49N, A115V, S244C and G413S) were designed and then co-expressed with CYP3A4 and CYP2B6 in the baculovirus-Sf9 insect cells to determine their kinetic parameters. Surprisingly, both mutants, Y181D and A287P in POR completely inhibited the CYP3A4 activity with testosterone, while the catalytic activity of CYP2B6 with bupropion was reduced to approximately ?70% of wild-type activity by Y181D and A287P mutations. In addition, the mutant K49N of POR increased the CLint (Vmax/Km) of CYP3A4 up to more than 31% of wild-type, while it reduced the catalytic efficiency of CYP2B6 to 74% of wild-type. Moreover, CLint values of CYP3A4-POR (A115V, G413S) were increased up to 36% and 65% of wild-type respectively. However, there were no appreciable effects observed by the remaining two mutants of POR (i.e., A115V and G413S) on activities of CYP2B6. In conclusion, the extent to which the catalytic activities of CYP were altered did not only depend on the specific POR mutations but also on the isoforms of different CYP redox partners. Thereby, we proposed that the POR-mutant patients should be carefully monitored for the activity of CYP3A4 and CYP2B6 on the prescribed medication. PMID:22719896

  1. Cloning, Sequencing, and Characterization of a Gene Cluster Involved in EDTA Degradation from the Bacterium BNC1

    PubMed Central

    Bohuslavek, Jan; Payne, Jason W.; Liu, Yong; Bolton, Harvey; Xun, Luying

    2001-01-01

    EDTA is a chelating agent, widely used in many industries. Because of its ability to mobilize heavy metals and radionuclides, it can be an environmental pollutant. The EDTA monooxygenases that initiate EDTA degradation have been purified and characterized in bacterial strains BNC1 and DSM 9103. However, the genes encoding the enzymes have not been reported. The EDTA monooxygenase gene was cloned by probing a genomic library of strain BNC1 with a probe generated from the N-terminal amino acid sequence of the monooxygenase. Sequencing of the cloned DNA fragment revealed a gene cluster containing eight genes. Two of the genes, emoA and emoB, were expressed in Escherichia coli, and the gene products, EmoA and EmoB, were purified and characterized. Both experimental data and sequence analysis showed that EmoA is a reduced flavin mononucleotide-utilizing monooxygenase and that EmoB is an NADH:flavin mononucleotide oxidoreductase. The two-enzyme system oxidized EDTA to ethylenediaminediacetate (EDDA) and nitrilotriacetate (NTA) to iminodiacetate (IDA) with the production of glyoxylate. The emoA and emoB genes were cotranscribed when BNC1 cells were grown on EDTA. Other genes in the cluster encoded a hypothetical transport system, a putative regulatory protein, and IDA oxidase that oxidizes IDA and EDDA. We concluded that this gene cluster is responsible for the initial steps of EDTA and NTA degradation. PMID:11157232

  2. Sodium arsenite-induced stress-related gene expression in normal human epidermal, HaCaT, and HEL30 keratinocytes.

    PubMed Central

    Trouba, Kevin J; Geisenhoffer, Kristen M; Germolec, Dori R

    2002-01-01

    Arsenic is a carcinogen that poses a significant health risk in humans. Based on evidence that arsenic has differential effects on human, rodent, normal, and transformed cells, these studies addressed the relative merits of using normal human epidermal keratinocytes (NHEK) and immortalized human (HaCaT) and mouse (HEL30) keratinocytes when examining stress-induced gene expression that may contribute to carcinogenesis. We hypothesize that redox-related gene expression is differentially modulated by arsenic in normal versus immortalized keratinocytes. To test the hypothesis, we exposed keratinocytes to sodium arsenite for 4 or 24 hr, at which time serine threonine kinase-25 (stk25) and nicotine adenine dinucleotide phosphate [nad(p)h] quinone oxidoreductase gene expression were measured. The effect of glutathione reduction on arsenite-induced cytotoxicity and gene expression in NHEK also was evaluated by addition of l-buthionine-[S,R]-sulfoximine (BSO) to culture media. Results indicate the term LC(50) for arsenite is approximately 10-15 microM in NHEK and HEL30 keratinocytes and 30 microM in HaCaT keratinocytes. Compared with HaCaT and HEL30 keratinocytes, a nontoxic concentration of arsenite (2.5 microM) increases stk25 and nad(p)h quinone oxidoreductase gene expression in NHEK, an effect partially attenuated by BSO. These data indicate that NHEK and HaCaT/HEL30 keratinocytes have similar sensitivities toward arsenite-induced cytotoxicity but unique gene expression responses. They also suggest that arsenite modulates gene expression in NHEK involved in cellular signaling and other aspects of intermediary metabolism that may contribute to the carcinogenic process. PMID:12426128

  3. 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

  4. Identification of the Genes Involved in the Fruiting Body Production and Cordycepin Formation of Cordyceps militaris Fungus

    PubMed Central

    Zheng, Zhuang-li; Qiu, Xue-hong

    2015-01-01

    A mutant library of Cordyceps militaris was constructed by improved Agrobacterium tumefaciens-mediated transformation and screened for degradation features. Six mutants with altered characters in in vitro and in vivo fruiting body production, and cordycepin formation were found to contain a single copy T-DNA. T-DNA flanking sequences of these mutants were identified by thermal asymmetric interlaced-PCR approach. ATP-dependent helicase, cytochrome oxidase subunit I and ubiquitin-like activating enzyme were involved in in vitro fruiting body production, serine/threonine phosphatase involved in in vivo fruiting body production, while glucose-methanol-choline oxidoreductase and telomerase reverse transcriptase involved in cordycepin formation. These genes were analyzed by bioinformatics methods, and their molecular function and biology process were speculated by Gene Ontology (GO) analysis. The results provided useful information for the control of culture degeneration in commercial production of C. militaris.

  5. The Drosophila Molybdenum Cofactor Gene Cinnamon Is Homologous to Three Escherichia Coli Cofactor Proteins and to the Rat Protein Gephyrin

    PubMed Central

    Kamdar, K. P.; Shelton, M. E.; Finnerty, V.

    1994-01-01

    Essentially all organisms depend upon molybdenum oxidoreductases which require a molybdopterin cofactor for catalytic activity. Mutations resulting in a lack of the cofactor show a pleiotropic loss of molybdoenzyme activities and thereby define genes involved in cofactor biosynthesis or utilization. In prokaryotes, two operons are directly associated with biosynthesis of the pterin moiety and its side chain while additional loci play a role in the acquisition of molybdenum and/or activation of the cofactor. Here we report the cloning of cinnamon, a Drosophila molybdenum cofactor gene encoding a protein with sequence similarity to three of the prokaryotic cofactor proteins. In addition, the Drosophila cinnamon protein is homologous to gephyrin, a protein isolated from the rat central nervous system. Our results suggest that some portions of the prokaryotic cofactor biosynthetic pathway composed of monofunctional proteins have evolved into a multifunctional protein in higher eukaryotes. PMID:8088525

  6. Cloning and characterization of a gene from Bacillus stearothermophilus var. non-diastaticus encoding a glycerol dehydrogenase.

    PubMed

    Mallinder, P R; Pritchard, A; Moir, A

    1992-01-01

    A 4.1-kb EcoRI fragment which includes the gene (gldA) encoding a glycerol dehydrogenase (G1DH; EC 1.1.1.6; glycerol:NAD oxidoreductase) from Bacillus stearothermophilus var. non-diastaticus has been cloned by virtue of its ability to restore glycerol utilisation to Escherichia coli glycerol kinase (glpK) and glycerol-3-phosphate dehydrogenase (glpD) mutants. Sequencing suggests that the gldA gene is likely to be monocistronic and encodes a protein of 39450 Da. The deduced amino acid composition and sequence of G1DH reveals that the protein is extremely similar to a characterized metal-dependent NAD-dependent G1DH from B. stearothermophilus RS93. The enzyme has limited homology to the iron-activated alcohol dehydrogenase of Zymomonas mobilis and the butanol dehydrogenase of Clostridium acetobutylicum. PMID:1339360

  7. Cloning, overexpression, and mutagenesis of the Sporobolomyces salmonicolor AKU4429 gene encoding a new aldehyde reductase, which catalyzes the stereoselective reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (S)-4-chloro-3-hydroxybutanoate

    SciTech Connect

    Kita, Keiko; Fukura, Takanobu; Nakase, Kohichi; Okamoto, Kenji; Yanase, Hideshi; Kataoka, Michihiko; Shimizu, Sakayu

    1999-12-01

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

  8. Purification and Characterization of a NADPH-Dependent Aldehyde Reductase from Mung Bean That Detoxifies Eutypine, a Toxin from Eutypa lata1

    PubMed Central

    Colrat, Ségolène; Latché, Alain; Guis, Monique; Pech, Jean-Claude; Bouzayen, Mondher; Fallot, Jean; Roustan, Jean-Paul

    1999-01-01

    Eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin produced by Eutypa lata, the causal agent of eutypa dieback in the grapevine (Vitis vinifera). Eutypine is enzymatically converted by numerous plant tissues into eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), a metabolite that is nontoxic to grapevine. We report a four-step procedure for the purification to apparent electrophoretic homogeneity of a eutypine-reducing enzyme (ERE) from etiolated mung bean (Vigna radiata) hypocotyls. The purified protein is a monomer of 36 kD, uses NADPH as a cofactor, and exhibits a Km value of 6.3 ?m for eutypine and a high affinity for 3- and 4-nitro-benzaldehyde. The enzyme failed to catalyze the reverse reaction using eutypinol as a substrate. ERE detoxifies eutypine efficiently over a pH range from 6.2 to 7.5. These data strongly suggest that ERE is an aldehyde reductase that could probably be classified into the aldo-keto reductase superfamily. We discuss the possible role of this enzyme in eutypine detoxification. PMID:9952458

  9. 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

  10. 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

  11. 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 Central

    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

  12. Flavodoxin:quinone reductase (FqrB): a redox partner of pyruvate:ferredoxin oxidoreductase that reversibly couples pyruvate oxidation to NADPH production in Helicobacter pylori and Campylobacter jejuni.

    PubMed

    St Maurice, Martin; Cremades, Nunilo; Croxen, Matthew A; Sisson, Gary; Sancho, Javier; Hoffman, Paul S

    2007-07-01

    Pyruvate-dependent reduction of NADP has been demonstrated in cell extracts of the human gastric pathogen Helicobacter pylori. However, NADP is not a substrate of purified pyruvate:ferredoxin oxidoreductase (PFOR), suggesting that other redox active enzymes mediate this reaction. Here we show that fqrB (HP1164), which is essential and highly conserved among the epsilonproteobacteria, exhibits NADPH oxidoreductase activity. FqrB was purified by nickel interaction chromatography following overexpression in Escherichia coli. The protein contained flavin adenine dinucleotide and exhibited NADPH quinone reductase activity with menadione or benzoquinone and weak activity with cytochrome c, molecular oxygen, and 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB). FqrB exhibited a ping-pong catalytic mechanism, a k(cat) of 122 s(-1), and an apparent K(m) of 14 muM for menadione and 26 muM for NADPH. FqrB also reduced flavodoxin (FldA), the electron carrier of PFOR. In coupled enzyme assays with purified PFOR and FldA, FqrB reduced NADP in a pyruvate- and reduced coenzyme A (CoA)-dependent manner. Moreover, in the presence of NADPH, CO(2), and acetyl-CoA, the PFOR:FldA:FqrB complex generated pyruvate via CO(2) fixation. PFOR was the rate-limiting enzyme in the complex, and nitazoxanide, a specific inhibitor of PFOR of H. pylori and Campylobacter jejuni, also inhibited NADP reduction in cell-free lysates. These capnophilic (CO(2)-requiring) organisms contain gaps in pathways of central metabolism that would benefit substantially from pyruvate formation via CO(2) fixation. Thus, FqrB provides a novel function in pyruvate metabolism and, together with production of superoxide anions via quinone reduction under high oxygen tensions, contributes to the unique microaerobic lifestyle that defines the epsilonproteobacterial group. PMID:17468253

  13. 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

  14. Characterization of the tumor suppressor gene WWOX in primary human oral squamous cell carcinomas

    PubMed Central

    Pimenta, Flávio J.; Gomes, Dawidson A.; Perdigão, Paolla F.; Barbosa, Alvimar A.; Romano-Silva, Marco A.; Gomez, Marcus V.; Aldaz, C. Marcelo; De Marco, Luiz; Gomez, Ricardo S.

    2014-01-01

    Oral squamous cell carcinoma (OSCC) is the most common malignant neoplasm of the oral cavity, representing ~90% of all oral carcinomas and accounting for 3–5% of all malignancies. The WWOX gene (WW-domain containing oxidoreductase) is a candidate tumor suppressor gene located at 16q23.3–24.1, spanning the second most common fragile site, FRA16D. In this report, the role of the WWOX gene was investigated in 20 tumors and 10 normal oral mucosas, and we demonstrated an altered WWOX gene in 50% (10/20) of OSCCs. Using nested RT-PCR, mRNA transcription was altered in 35% of the tumors, with the complete absence of transcripts in 2 samples as well as absence of exons 6–8 (2 tumors), exon 7 (1 tumor), exon 7 and exon 6–8 (1 tumor) and partial loss of exons 8 and 9 (1 tumor). To determine if the aberrant transcripts were translated, Western blots were performed in all samples; however, only the normal protein was detected. By immunohistochemistry, a reduction in Wwox protein expression was observed, affecting 40% of the tumors when compared with normal mucosa. In addition, a novel somatic mutation (S329F) was found. The presence of alterations in mRNA transcription correlated with the reduced expression of Wwox protein in the tumors. These results show that the WWOX gene is frequently altered in OSCC and may contribute to the carcinogenesis processes in oral cancer. PMID:16152610

  15. Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae.

    PubMed

    Gonzalez-Perez, David; Alcalde, Miguel

    2014-01-01

    The ligninolytic enzymatic consortium produced by white-rot fungi is one of the most efficient oxidative systems found in nature, with many potential applications that range from the production of 2nd generation biofuels to chemicals synthesis. In the current study, two high redox potential oxidoreductase fusion genes (laccase -Lac- and versatile peroxidase -Vp-) that had been evolved in the laboratory were re-assembled in Saccharomyces cerevisiae. First, cell viability and secretion were assessed after co-transforming the Lac and Vp genes into yeast. Several expression cassettes were inserted in vivo into episomal bi-directional vectors in order to evaluate inducible promoter and/or terminator pairs of different strengths in an individual and combined manner. The synthetic white-rot yeast model harboring Vp(GAL1/CYC1)-Lac(GAL10/ADH1) displayed up to 1000 and 100 Units per L of peroxidase and laccase activity, respectively, representing a suitable point of departure for future synthetic biology studies. PMID:24830983

  16. 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

  17. 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

  18. 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

    2015-03-15

    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

  19. Genome-Wide Survey and Expression Analysis Suggest Diverse Roles of Glutaredoxin Gene Family Members During Development and Response to Various Stimuli in Rice

    PubMed Central

    Garg, Rohini; Jhanwar, Shalu; Tyagi, Akhilesh K.; Jain, Mukesh

    2010-01-01

    Glutaredoxins (GRXs) are glutathione-dependent oxidoreductase enzymes involved in a variety of cellular processes. In this study, our analysis revealed the presence of 48 genes encoding GRX proteins in the rice genome. GRX proteins could be classified into four classes, namely CC-, CGFS-, CPYC- and GRL-type, based on phylogenetic analysis. The classification was supported with organization of predicted conserved putative motifs in GRX proteins. We found that expansion of this gene family has occurred largely via whole genome duplication events in a species-specific manner. We explored rice oligonucleotide array data to gain insights into the function of GRX gene family members during various stages of development and in response to environmental stimuli. The comprehensive expression analysis suggested diverse roles of GRX genes during growth and development in rice. Some of the GRX genes were expressed in specific organs/developmental stages only. The expression of many of rice GRX genes was influenced by various phytohormones, abiotic and biotic stress conditions, suggesting an important role of GRX proteins in response to these stimuli. The identification of GRX genes showing differential expression in specific tissues or in response to environmental stimuli provide a new avenue for in-depth characterization of selected genes of importance. PMID:21044985

  20. Gene Modifications

    NSDL National Science Digital Library

    This animation shows how a gene is constructed to eventually produce a protein in a Bt corn plant. This is the fifth of a series of seven animations that detail the process of crop genetic engineering. To begin at the beginning, see Overview of Crop Genetic Engineering. (To return to the animation previous to this, go to Gene Regions. To go to the next animation, go to Gene Gun.)

  1. 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. © 2015 Wiley Periodicals, Inc. PMID:25622915

  2. Molecular alterations in the tumor suppressor gene WWOX in oral leukoplakias

    PubMed Central

    Pimenta, Flávio Juliano; Cordeiro, Gabriela Tavares; Pimenta, Luiz Gustavo Garcia Santos; Viana, Michelle Beatriz; Lopes, Joyce; Gomez, Marcus Vinícius; Aldaz, C. Marcelo; De Marco, Luiz; Gomez, Ricardo Santiago

    2014-01-01

    Summary Oral leukoplakia is the most prevalent and potentially malignant disorder of the oral mucosa. Previous studies have demonstrated that molecular changes of the WWOX gene (WW-domain containing oxidoreductase), a candidate tumor suppressor gene located at 16q23.3–24.1 that spans FRA16D, the second most common fragile site, are present in several malignant neoplasias, including oral squamous cell carcinoma. In this report, the role of the WWOX gene was investigated in 23 cases of oral leukoplakias. Using nested RT-PCR and immunohistochemistry, altered mRNA transcription and/or reduced Wwox protein expression was observed in 35% of the lesions when compared with normal mucosa. The majority of lesions (4/6) with altered transcripts had a reduction in the expression of Wwox protein. Although normal WWOX expression was found in some lesions with dysplasia, all lesions with WWOX mRNA and/or protein expression showed histological evidence of dysplasia and none of the cases without dysplasia presented this alteration. These results show that the WWOX gene alteration is an early genetic alteration and may contribute to oral carcinogenesis. PMID:18061530

  3. 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

  4. Identification of genes specifically required for the anaerobic metabolism of benzene in Geobacter metallireducens

    PubMed Central

    Zhang, Tian; Tremblay, Pier-Luc; Chaurasia, Akhilesh K.; Smith, Jessica A.; Bain, Timothy S.; Lovley, Derek R.

    2014-01-01

    Although the biochemical pathways for the anaerobic degradation of many of the hydrocarbon constituents in petroleum reservoirs have been elucidated, the mechanisms for anaerobic activation of benzene, a very stable molecule, are not known. Previous studies have demonstrated that Geobacter metallireducens can anaerobically oxidize benzene to carbon dioxide with Fe(III) as the sole electron acceptor and that phenol is an intermediate in benzene oxidation. In an attempt to identify enzymes that might be involved in the conversion of benzene to phenol, whole-genome gene transcript abundance was compared in cells metabolizing benzene and cells metabolizing phenol. Eleven genes had significantly higher transcript abundance in benzene-metabolizing cells. Five of these genes had annotations suggesting that they did not encode proteins that could be involved in benzene metabolism and were not further studied. Strains were constructed in which one of the remaining six genes was deleted. The strain in which the monocistronic gene Gmet 0232 was deleted metabolized phenol, but not benzene. Transcript abundance of the adjacent monocistronic gene, Gmet 0231, predicted to encode a zinc-containing oxidoreductase, was elevated in cells metabolizing benzene, although not at a statistically significant level. However, deleting Gmet 0231 also yielded a strain that could metabolize phenol, but not benzene. Although homologs of Gmet 0231 and Gmet 0232 are found in microorganisms not known to anaerobically metabolize benzene, the adjacent localization of these genes is unique to G. metallireducens. The discovery of genes that are specifically required for the metabolism of benzene, but not phenol in G. metallireducens is an important step in potentially identifying the mechanisms for anaerobic benzene activation. PMID:24904558

  5. Effect of simulated microgravity on oxidation-sensitive gene expression in PC12 cells

    NASA Astrophysics Data System (ADS)

    Kwon, Ohwon; Sartor, Maureen; Tomlinson, Craig R.; Millard, Ronald W.; Olah, Mark E.; Sankovic, John M.; Banerjee, Rupak K.

    2006-01-01

    Oxygen utilization by and oxygen dependence of cellular processes may be different in biological systems that are exposed to microgravity (micro-g). A baseline in which cellular changes in oxygen sensitive molecular processes occur during micro-g conditions would be important to pursue this question. The objective of this research is to analyze oxidation-sensitive gene expression in a model cell line [rat pheochromocytoma (PC12)] under simulated micro-g conditions. The PC12 cell line is well characterized in its response to oxygen, and is widely recognized as a sensitive model for studying the responses of oxygen-sensitive molecular and cellular processes. This study uses the rotating wall vessel bioreactor (RWV) designed at NASA to simulate micro-g. Gene expression in PC12 cells in response to micro-g was analyzed by DNA microarray technology. The microarray analysis of PC12 cells cultured for 4 days under simulated micro-g under standardized oxygen environment conditions revealed more than 100 genes whose expression levels were changed at least twofold (up-regulation of 65 genes and down-regulation of 39 genes) compared with those from cells in the unit gravity (unit-g) control. This study observed that genes involved in the oxidoreductase activity category were most significantly differentially expressed under micro-g conditions. Also, known oxidation-sensitive transcription factors such as hypoxia-inducible factor-2?, c-myc, and the peroxisome proliferator-activated receptor-? were changed significantly. Our initial results from the gene expression microarray studies may provide a context in which to evaluate the effect of varying oxygen environments on the background of differential gene regulation of biological processes under variable gravity conditions.

  6. Effect of simulated microgravity on oxidation-sensitive gene expression in PC12 cells.

    PubMed

    Kwon, Ohwon; Sartor, Maureen; Tomlinson, Craig R; Millard, Ronald W; Olah, Mark E; Sankovic, John M; Banerjee, Rupak K

    2006-01-01

    Oxygen utilization by and oxygen dependence of cellular processes may be different in biological systems that are exposed to microgravity (micro-g). A baseline in which cellular changes in oxygen sensitive molecular processes occur during micro-g conditions would be important to pursue this question. The objective of this research is to analyze oxidation-sensitive gene expression in a model cell line [rat pheochromocytoma (PC12)] under simulated micro-g conditions. The PC12 cell line is well characterized in its response to oxygen, and is widely recognized as a sensitive model for studying the responses of oxygen-sensitive molecular and cellular processes. This study uses the rotating wall vessel bioreactor (RWV) designed at NASA to simulate micro-g. Gene expression in PC12 cells in response to micro-g was analyzed by DNA microarray technology. The microarray analysis of PC12 cells cultured for 4 days under simulated micro-g under standardized oxygen environment conditions revealed more than 100 genes whose expression levels were changed at least twofold (up-regulation of 65 genes and down-regulation of 39 genes) compared with those from cells in the unit gravity (unit-g) control. This study observed that genes involved in the oxidoreductase activity category were most significantly differentially expressed under micro-g conditions. Also, known oxidation-sensitive transcription factors such as hypoxia-inducible factor-2alpha, c-myc, and the peroxisome proliferator-activated receptor-gamma were changed significantly. Our initial results from the gene expression microarray studies may provide a context in which to evaluate the effect of varying oxygen environments on the background of differential gene regulation of biological processes under variable gravity conditions. PMID:19081771

  7. 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

  8. Gene Control

    NSDL National Science Digital Library

    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.

  9. Gene Gun

    NSDL National Science Digital Library

    How the gene gun works to transform cells with new DNA. This is thesixth of a series of seven animations that detail the process of cropgenetic engineering. To begin at the beginning, see Overview of Crop Genetic Engineering. (To return to the animation previous to this, go to Gene Modification. To go to the next animation, go to Backcross Breeding.)

  10. 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).

  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 MAPPING

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gene mapping is the science of determining the location of a gene in a species' genome. The genome of most mammalian species is comprised of approximately three billion bases of deoxyribonucleic acid (DNA) contained in 18-35 separate linear molecules (chromosomes). Mammals are diploid organisms so e...

  13. Gene Puzzles

    NSDL National Science Digital Library

    Science Netlinks

    2001-10-20

    In this Science NetLinks lesson, students will examine a fictional pedigree and determine which gene is responsible for a given trait. The genetic information for individuals is depicted as a jigsaw puzzle. Without delving into a complicated explanation of the process, the activity in this lesson will help students build an understanding of how offspring inherit genes from their parents.

  14. Pathogen-origin horizontally transferred genes contribute to the evolution of Lepidopteran insects

    PubMed Central

    2011-01-01

    Background Horizontal gene transfer (HGT), a source of genetic variation, is generally considered to facilitate hosts' adaptability to environments. However, convincing evidence supporting the significant contribution of the transferred genes to the evolution of metazoan recipients is rare. Results In this study, based on sequence data accumulated to date, we used a unified method consisting of similarity search and phylogenetic analysis to detect horizontally transferred genes (HTGs) between prokaryotes and five insect species including Drosophila melanogaster, Anopheles gambiae, Bombyx mori, Tribolium castaneum and Apis mellifera. Unexpectedly, the candidate HTGs were not detected in D. melanogaster, An. gambiae and T. castaneum, and 79 genes in Ap. mellifera sieved by the same method were considered as contamination based on other information. Consequently, 14 types of 22 HTGs were detected only in the silkworm. Additionally, 13 types of the detected silkworm HTGs share homologous sequences in species of other Lepidopteran superfamilies, suggesting that the majority of these HTGs were derived from ancient transfer events before the radiation of Ditrysia clade. On the basis of phylogenetic topologies and BLAST search results, donor bacteria of these genes were inferred, respectively. At least half of the predicted donor organisms may be entomopathogenic bacteria. The predicted biochemical functions of these genes include four categories: glycosyl hydrolase family, oxidoreductase family, amino acid metabolism, and others. Conclusions The products of HTGs detected in this study may take part in comprehensive physiological metabolism. These genes potentially contributed to functional innovation and adaptability of Lepidopteran hosts in their ancient lineages associated with the diversification of angiosperms. Importantly, our results imply that pathogens may be advantageous to the subsistence and prosperity of hosts through effective HGT events at a large evolutionary scale. PMID:22151541

  15. Gene Cloning

    NSDL National Science Digital Library

    2009-09-08

    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.

  16. Role of the C-terminal extension stacked on the re-face of the isoalloxazine ring moiety of the flavin adenine dinucleotide prosthetic group in ferredoxin-NADP(+) oxidoreductase from Bacillus subtilis.

    PubMed

    Seo, Daisuke; Asano, Tomoya; Komori, Hirofumi; Sakurai, Takeshi

    2014-08-01

    Ferredoxin-NADP(+) oxidoreductase [EC 1.18.1.2] from Bacillus subtilis (BsFNR) is homologous to the bacterial NADPH-thioredoxin reductase, but possesses a unique C-terminal extension that covers the re-face of the isoalloxazine ring moiety of the flavin adenine dinucleotide (FAD) prosthetic group. In this report, we utilize BsFNR mutants depleted of their C-terminal residues to examine the importance of the C-terminal extension in reactions with NADPH and ferredoxin (Fd) from B. subtilis by spectroscopic and steady-state reaction analyses. The depletions of residues Y313 to K332 (whole C-terminal extension region) and S325 to K332 (His324 intact) resulted in significant increases in the catalytic efficiency with NADPH in diaphorase assay with ferricyanide, whereas Km values for ferricyanide were increased. In the cytochrome c reduction assay in the presence of B. subtilis ferredoxin, the S325-K332 depleted mutant displayed a significant decrease in the turnover rate with an Fd concentration range of 1-10 ?M. The Y313-K332 depleted mutant demonstrated an increase in the rate of the direct reduction of horse heart cytochrome c in the absence of Fd. These data indicated that depletion of the C-terminal extension plays an important role in the reaction of BsFNR with ferredoxin. PMID:24529496

  17. ?-Lapachone, a substrate of NAD(P)H:quinone oxidoreductase, induces anti-inflammatory heme oxygenase-1 via AMP-activated protein kinase activation in RAW264.7 macrophages

    PubMed Central

    Byun, Seung Jae; Son, Young; Hwan Cho, Baik; Chung, Hun-Taeg; Pae, Hyun-Ock

    2013-01-01

    AMP-activated protein kinase (AMPK), a crucial regulator of energy metabolic homeostasis, is suggested to regulate inflammatory responses, but its precise mechanisms are not fully understood. It has been reported that pharmacological activation of AMPK induces heme oxygenase-1 (HO-1) expression. ?-Lapachone (BL), a well-known substrate of NAD(P)H:quinone oxidoreductase (NQO1), has been demonstrated to stimulate AMPK activation via NQO1 activation, and to exert anti-inflammatory effects in macrophages. Here we examined whether AMPK activation by BL would be linked to HO-1 expression in RAW264.7 macrophages and whether HO-1 expression could mediate the anti-inflammatory effects of BL. BL treatment induced concentration- and time-dependent AMPK phosphorylation and HO-1 expression. 5-Aminoimidazole-4-carboxamide-1-?-D-ribofuranoside, an AMPK activator, also induced HO-1 expression. In contrast, compound C (CC), an inhibitor of AMPK activation, prevented the increase in BL-induced HO-1 expression. BL pretreatment reduced lipopolysaccharide-induced production of tumor necrosis factor-?, a pro-inflammatory cytokine, and expression of inducible nitric oxide synthase, a pro-inflammatory enzyme. These inhibitory effects BL were almost completely abolished by CC and partly by tin protoporphyrin-IX, a competitive inhibitor of HO-1. Accordingly, the present results indicate that BL induces anti-inflammatory HO-1 expression in macrophages via AMPK activation, providing one of possible mechanisms by which BL can exert anti-inflammatory effects. PMID:23525626

  18. Calcium and calcium sensing receptor modulates the expression of thymidylate synthase, NAD(P)H:quinone oxidoreductase 1 and survivin in human colon carcinoma cells: promotion of cytotoxic response to mitomycin C and fluorouracil

    PubMed Central

    Liu, Guangming; Hu, Xin; Varani, James; Chakrabarty, Subhas

    2008-01-01

    Ca2+ and the cell-surface calcium sensing receptor (CaSR) constitute a novel and robust ligand/receptor system in regulating the proliferation and differentiation of colonic epithelial cells. Here we show that activation of CaSR by extracellular Ca2+ (or CaSR agonists) enhanced the sensitivity of human colon carcinoma cells to mitomycin C (MMC) and fluorouracil (5-FU). Activation of CaSR up-regulated the expression of MMC activating enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO-1) and down-regulated the expression of 5-FU target, thymidylate synthase (TS) and the anti-apoptotic protein survivin. Cells that were resistant to drugs expressed little or no CaSR but abundant amount of survivin. Disruption of CaSR expression by shRNA targeting the CaSR abrogated these modulating effects of CaSR activation on the expression of NQO1, TS, survivin and cytotoxic response to drugs. It is concluded that activation of CaSR can enhance colon cancer cell sensitivity to MMC and 5-FU and can modulate the expression of molecules involved in the cellular responses to these cytotoxic drugs. PMID:18618519

  19. Variation in P450 oxidoreductase (POR) A503V and flavin-containing monooxygenase (FMO)-3 E158K is associated with minor alterations in nicotine metabolism, but does not alter cigarette consumption.

    PubMed

    Chenoweth, Meghan J; Zhu, Andy Z X; Sanderson Cox, Lisa; Ahluwalia, Jasjit S; Benowitz, Neal L; Tyndale, Rachel F

    2014-03-01

    The rates of nicotine metabolism differ widely, even after controlling for genetic variation in the major nicotine-metabolizing enzyme, CYP2A6. Genetic variants in an additional nicotine-metabolizing enzyme, flavin-containing monooxygenase (FMO)-3, and an obligate microsomal CYP-supportive enzyme, cytochrome P450 oxidoreductase (POR), were investigated. We examined the impact of FMO3 E158K and POR A503V before and after stratifying by CYP2A6 metabolism group. In 130 nonsmokers of African descent who received 4 mg oral nicotine, FMO3 158K trended toward slower nicotine metabolism in reduced CYP2A6 metabolizers (P=0.07) only, whereas POR 503V was associated with faster CYP2A6 activity (nicotine metabolite ratio) in normal (P=0.03), but not reduced, CYP2A6 metabolizers. Neither FMO3 158K nor POR 503V significantly altered the nicotine metabolic ratio (N=659), cigarette consumption (N=667), or urine total nicotine equivalents (N=418) in smokers of African descent. Thus, FMO3 E158K and POR A503V are minor sources of nicotine metabolism variation, insufficient to appreciably alter smoking. PMID:24448396

  20. Genes V.

    SciTech Connect

    Lewin, B.

    1994-12-31

    This fifth edition book encompasses a wide range of topics covering 1,272 pages. The book is arranged into nine parts with a total of 36 chapters. These nine parts include Introduction; DNA as a Store of Information; Translation; Constructing Cells; Control of Prokaryotypic Gene Expression; Perpetuation of DNA; Organization of the Eukaryotypic Genome; Eukaryotypic Transcription and RNA Processing; The Dynamic Genome; and Genes in Development.

  1. 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

  2. IruO is a reductase for heme degradation by IsdI and IsdG proteins in Staphylococcus aureus.

    PubMed

    Loutet, Slade A; Kobylarz, Marek J; Chau, Crystal H T; Murphy, Michael E P

    2013-09-01

    Staphylococcus aureus is a common hospital- and community-acquired bacterium that can cause devastating infections and is often multidrug-resistant. Iron acquisition is required by S. aureus during an infection, and iron acquisition pathways are potential targets for therapies. The gene NWMN2274 in S. aureus strain Newman is annotated as an oxidoreductase of the diverse pyridine nucleotide-disulfide oxidoreductase (PNDO) family. We show that NWMN2274 is an electron donor to IsdG and IsdI catalyzing the degradation of heme, and we have renamed this protein IruO. Recombinant IruO is a FAD-containing NADPH-dependent reductase. In the presence of NADPH and IruO, either IsdI or IsdG degraded bound heme 10-fold more rapidly than with the chemical reductant ascorbic acid. Varying IsdI-heme substrate and monitoring loss of the heme Soret band gave a K(m) of 15 ± 4 ?M, a k(cat) of 5.2 ± 0.7 min(-1), and a k(cat)/K(m) of 5.8 × 10(3) M(-1) s(-1). From HPLC and electronic spectra, the major heme degradation products are 5-oxo-?-bilirubin and 15-oxo-?-bilirubin (staphylobilins), as observed with ascorbic acid. Although heme degradation by IsdI or IsdG can occur in the presence of H2O2, the addition of catalase and superoxide dismutase did not disrupt NADPH/IruO heme degradation reactions. The degree of electron coupling between IruO and IsdI or IsdG remains to be determined. Homologs of IruO were identified by sequence similarity in the genomes of Gram-positive bacteria that possess IsdG-family heme oxygenases. A phylogeny of these homologs identifies a distinct clade of pyridine nucleotide-disulfide oxidoreductases likely involved in iron uptake systems. IruO is the likely in vivo reductant required for heme degradation by S. aureus. PMID:23893407

  3. IruO Is a Reductase for Heme Degradation by IsdI and IsdG Proteins in Staphylococcus aureus*

    PubMed Central

    Loutet, Slade A.; Kobylarz, Marek J.; Chau, Crystal H. T.; Murphy, Michael E. P.

    2013-01-01

    Staphylococcus aureus is a common hospital- and community-acquired bacterium that can cause devastating infections and is often multidrug-resistant. Iron acquisition is required by S. aureus during an infection, and iron acquisition pathways are potential targets for therapies. The gene NWMN2274 in S. aureus strain Newman is annotated as an oxidoreductase of the diverse pyridine nucleotide-disulfide oxidoreductase (PNDO) family. We show that NWMN2274 is an electron donor to IsdG and IsdI catalyzing the degradation of heme, and we have renamed this protein IruO. Recombinant IruO is a FAD-containing NADPH-dependent reductase. In the presence of NADPH and IruO, either IsdI or IsdG degraded bound heme 10-fold more rapidly than with the chemical reductant ascorbic acid. Varying IsdI-heme substrate and monitoring loss of the heme Soret band gave a Km of 15 ± 4 ?m, a kcat of 5.2 ± 0.7 min?1, and a kcat/Km of 5.8 × 103 m?1 s?1. From HPLC and electronic spectra, the major heme degradation products are 5-oxo-?-bilirubin and 15-oxo-?-bilirubin (staphylobilins), as observed with ascorbic acid. Although heme degradation by IsdI or IsdG can occur in the presence of H2O2, the addition of catalase and superoxide dismutase did not disrupt NADPH/IruO heme degradation reactions. The degree of electron coupling between IruO and IsdI or IsdG remains to be determined. Homologs of IruO were identified by sequence similarity in the genomes of Gram-positive bacteria that possess IsdG-family heme oxygenases. A phylogeny of these homologs identifies a distinct clade of pyridine nucleotide-disulfide oxidoreductases likely involved in iron uptake systems. IruO is the likely in vivo reductant required for heme degradation by S. aureus. PMID:23893407

  4. Evolutionary diversification and characterization of the eubacterial gene family encoding DXR type II, an alternative isoprenoid biosynthetic enzyme

    PubMed Central

    2013-01-01

    Background Isoprenoids constitute a vast family of natural compounds performing diverse and essential functions in all domains of life. In most eubacteria, isoprenoids are synthesized through the methylerythritol 4-phosphate (MEP) pathway. The production of MEP is usually catalyzed by deoxyxylulose 5-phosphate reductoisomerase (DXR-I) but a few organisms use an alternative DXR-like enzyme (DXR-II). Results Searches through 1498 bacterial complete proteomes detected 130 sequences with similarity to DXR-II. Phylogenetic analysis identified three well-resolved clades: the DXR-II family (clustering 53 sequences including eleven experimentally verified as functional enzymes able to produce MEP), and two previously uncharacterized NAD(P)-dependent oxidoreductase families (designated DLO1 and DLO2 for DXR-II-like oxidoreductases 1 and 2). Our analyses identified amino acid changes critical for the acquisition of DXR-II biochemical function through type-I functional divergence, two of them mapping onto key residues for DXR-II activity. DXR-II showed a markedly discontinuous distribution, which was verified at several levels: taxonomic (being predominantly found in Alphaproteobacteria and Firmicutes), metabolic (being mostly found in bacteria with complete functional MEP pathways with or without DXR-I), and phenotypic (as no biological/phenotypic property was found to be preferentially distributed among DXR-II-containing strains, apart from pathogenicity in animals). By performing a thorough comparative sequence analysis of GC content, 3:1 dinucleotide frequencies, codon usage and codon adaptation indexes (CAI) between DXR-II sequences and their corresponding genomes, we examined the role of horizontal gene transfer (HGT), as opposed to an scenario of massive gene loss, in the evolutionary origin and diversification of the DXR-II subfamily in bacteria. Conclusions Our analyses support a single origin of the DXR-II family through functional divergence, in which constitutes an exceptional model of acquisition and maintenance of redundant gene functions between non-homologous genes as a result of convergent evolution. Subsequently, although old episodic events of HGT could not be excluded, the results supported a prevalent role of gene loss in explaining the distribution of DXR-II in specific pathogenic eubacteria. Our results highlight the importance of the functional characterization of evolutionary shortcuts in isoprenoid biosynthesis for screening specific antibacterial drugs and for regulating the production of isoprenoids of human interest. PMID:24004839

  5. Genetical and Biochemical Characterization of QA-3 Mutants and Revertants in the QA Gene Cluster of NEUROSPORA CRASSA

    PubMed Central

    Case, Mary E.; Pueyo, Carmen; Barea, J. Lopez; Giles, Norman H.

    1978-01-01

    The qa-3 gene, one of the four genes in the qa gene cluster, encodes quinate (shikimate) dehydrogenase (quinate: NAD oxidoreductase, ER 1.1.1.24), the first enzyme in the inducible quinic acid catabolic pathway in Neurospora crassa. Genetic analyses have localized 26 qa-3 mutants at 11 sites on the qa-3 genetic map on the basis of prototroph frequencies. Certain mutants, e.g., 336–3–10 and 336–3–3, are located at opposite ends of the qa-3 gene. Data from four-point crosses (qa-1S mutant 124 x five different qa-3 mutants in triple mutants qa-3, qa-4, qa-2) indicate the following orientation of the qa-3 gene within the qa cluster: qa-1, qa-3 mutant 336–3–10 ("left" end) qa-3 mutant 336–3–3 ("right" end), qa-4, qa-2. Ultraviolet-induced revertants have been obtained from 14 of the qa-3 mutants. The revertable mutants fall into two major classes: those that revert by changes either at the same site or at a second site within the qa-3 gene, and those that revert by unlinked suppressor mutations. The intragenic revertants can be further distinguished by quantative and/or qualitative differences in their quinate dehydrogenase activities. Some revertants with activities either equivalent to or less than wild type produce a thermostable enzyme, and others an enzyme which is thermolabile in vitro at 35°. A concentration of quinic acid or shikimic acid as low as 50 µm protects the enzyme markedly from heat inactivation. The genetic organization and the orientation of the qa-3 gene are discussed with respect to its direction of transcription and to the possible localization of a promoter (initiator) region(s) within the qa gene cluster. PMID:151647

  6. cDNA cloning of the human homologues of the mouse Ke4 and Ke6 genes at the centromeric end of the human MHC region

    SciTech Connect

    Ando, Asako; Kikuti, Yara Yukie; Shigenari, Atsuko [Tokai Univ. School of Medicine, Isehara, Kanagawa (Japan)] [and others] [Tokai Univ. School of Medicine, Isehara, Kanagawa (Japan); and others

    1996-08-01

    cDNA clones corresponding to the KHE4 and HKE6 genes at the centromeric end of the HLA region on human chromosome 6p21.3 were isolated and characterized. The predicted amino acid sequences of HKE4 and HKE6 exhibited 81.5 and 85.6% identity to the mouse homologues, Ke4 and Ke6, respectively. HKE4 may encode a membrane protein with histidine-rich charge clusters. HKE6 possesses remarkable amino acid sequence conservation with several bacterial proteins with oxidoreductase function and also shows significant homology with the two unique functional domains containing the nucleotide cofactor binding site and the consensus motif characteristic of the members of the superfamily of short-chain alcohol dehydrogenases such as human and rat steroid and prostaglandin dehydrogenases. 13 refs., 2 figs.

  7. The supposed tumor suppressor gene WWOX is mutated in an early lethal microcephaly syndrome with epilepsy, growth retardation and retinal degeneration

    PubMed Central

    2014-01-01

    Background WWOX, encoding WW domain-containing oxidoreductase, spans FRA16D, the second most common chromosomal fragile site frequently altered in cancers. It is therefore considered a tumor suppressor gene, but its direct implication in cancerogenesis remains controversial. Methods and results By whole-exome sequencing, we identified a homozygous WWOX nonsense mutation, p.Arg54*, in a girl from a consanguineous family with a severe syndrome of growth retardation, microcephaly, epileptic seizures, retinopathy and early death, a phenotype highly similar to the abormalities reported in lde/lde rats with a spontaneous functional null mutation of Wwox. As in rats, no tumors were observed in the patient or heterozygous mutation carriers. Conclusions Our finding, a homozygous loss-of-function germline mutation in WWOX in a patient with a lethal autosomal recessive syndrome, supports an alternative role of WWOX and indicates its importance for human viability. PMID:24456803

  8. Gene Ontology Driven Classification of Gene

    E-print Network

    Spang, Rainer

    Gene Ontology Driven Classification of Gene Expression Patterns Claudio Lottaz and Rainer Spang Genetics #12;Overview 23-Jul-02 2 / 17Claudio Lottaz: GO driven classification of gene expression patterns Overview · Introduction · Gene Ontology driven classification of gene expression patterns · Preliminary

  9. Identification of a new gene, tmoF, in the Pseudomonas mendocina KR1 gene cluster encoding toluene-4-monooxygenase.

    PubMed

    Yen, K M; Karl, M R

    1992-11-01

    Five genes, tmoABCDE, encoding toluene-4-monooxygenase (T4MO) were previously mapped to a 3.6-kb region of a 10.2-kb SacI DNA fragment isolated from Pseudomonas mendocina KR1 (K.-M. Yen, M. R. Karl, L. M. Blatt, M. J. Simon, R. B. Winter, P. R. Fausset, H. S. Lu, A. A. Harcourt, and K. K. Chen, J. Bacteriol. 173:5315-5327, 1991). In this report, we describe the identification and characterization of a DNA region in the SacI fragment whose expression enhances the T4MO activity determined by the tmoABCDE gene cluster. This region was mapped immediately downstream of the putative transcription termination sequence previously located at the end of the tmoABCDE gene cluster (Yen et al., J. Bacteriol., 1991) and was found to stimulate T4MO activity two- to threefold when expressed in Escherichia coli or Pseudomonas putida. Determination of the nucleotide sequence of this region revealed an open reading frame (ORF) of 978 bp. Expression of the ORF resulted in the synthesis of an approximately 37-kDa polypeptide whose N-terminal amino acid sequence completely matched that of the product predicted from the ORF. The ORF thus defines a gene, which has now been designated tmoF. The TmoF protein shares amino acid sequence homology with the reductases of several mono- and dioxygenase systems. In addition, the reductase component of the naphthalene dioxygenase system, encoded by the nahAa gene of plasmid NAH7 from P. putida G7, could largely replace the TmoF protein in stimulating T4MO activity, and TmoF could partially replace the NahAa protein in forming active naphthalene dioxygenase. The overall properties of tmoF suggest that it is a member of the T4mo gene cluster and encodes the NADH:ferredoxin oxidoreductase of the T4MO system. PMID:1429451

  10. Characterization of a putative stereoselective oxidoreductase from Gluconobacter oxydans and its application in producing ethyl (R)-4-chloro-3-hydroxybutanoate ester.

    PubMed

    Liu, Xu; Chen, Rong; Yang, Zhongwei; Wang, Jiale; Lin, Jinping; Wei, Dongzhi

    2014-04-01

    A gene encoding an NADH-dependent short-chain dehydrogenase/reductase (gox2036) from Gluconobacter oxydans 621H was cloned and heterogeneously expressed in Escherichia coli. The protein (Gox2036) was purified to homogeneity and biochemically characterized. Gox2036 was a homotetramer with a subunit size of approximately 28 kDa. Gox2036 had a strict requirement for NAD?/NADH as the cofactor. Gox2036 displayed preference for oxidation of secondary alcohols and 2,3-diols as well as for reduction of ?-diketones, hydroxy ketones, ?-ketoesters, and ?-ketoesters. However, Gox2036 was poorly active on 1,2-diols and acetoin and showed no activity on primary alcohols, polyols, and aldehydes. The optimum pH values for the oxidation and reduction reactions were 9 and 6, respectively. Gox2036 was highly selective in the reduction of various ?-ketones and ?-ketoesters. Among the substrates tested, ethyl 4-chloro acetoacetate was reduced to ethyl (R)-4-chloro-3-hydroxybutanoate ester with an excellent conversion yield of 96.9 % and optical purity of >99 % e.e. using an efficient in situ NADH-recycling system involving glucose and a glucose dehydrogenase from Bacillus subtilis (BsGDH). PMID:24113812

  11. Attention Genes

    ERIC Educational Resources Information Center

    Posner, Michael I.; Rothbart, Mary K.; Sheese, Brad E.

    2007-01-01

    A major problem for developmental science is understanding how the cognitive and emotional networks important in carrying out mental processes can be related to individual differences. The last five years have seen major advances in establishing links between alleles of specific genes and the neural networks underlying aspects of attention. These…

  12. Designer Genes.

    ERIC Educational Resources Information Center

    Miller, Judith; Miller, Mark

    1983-01-01

    Genetic technologies may soon help fill some of the most important needs of humanity from food to energy to health care. The research of major designer genes companies and reasons why the initial mad rush for biotechnology has slowed are reviewed. (SR)

  13. The protective role of NAD(P)H:quinone oxidoreductase 1 on acetaminophen-induced liver injury is associated with prevention of adenosine triphosphate depletion and improvement of mitochondrial dysfunction.

    PubMed

    Hwang, Jung Hwan; Kim, Yong-Hoon; Noh, Jung-Ran; Gang, Gil-Tae; Kim, Kyoung-Shim; Chung, Hyo Kyun; Tadi, Surendar; Yim, Yong-Hyeon; Shong, Minho; Lee, Chul-Ho

    2014-09-17

    An overdose of acetaminophen (APAP) causes hepatotoxicity due to its metabolite, N-acetyl-p-benzoquinone imine. NAD(P)H:quinone oxidoreductase 1 (NQO1) is an important enzyme for detoxification, because it catabolizes endogenous/exogenous quinone to hydroquinone. Although various studies have suggested the possible involvement of NQO1 in APAP-induced hepatotoxicity, its precise role in this remains unclear. We investigated the role of NQO1 against APAP-induced hepatotoxicity using a genetically modified rodent model. NQO1 wild-type (WT) and knockout (KO) mice were treated with different doses of APAP, and we evaluated the mortality and toxicity markers for cell death caused by APAP. NQO1 KO mice showed high sensitivity to APAP-mediated hepatotoxicity (as indicated by a large necrotic region) as well as increased levels of nitrotyrosine adducts and reactive oxygen species. APAP-induced cell death in the livers and primary hepatocytes of NQO1 KO mice, which was accompanied by an extensive reduction in adenosine triphosphate (ATP) levels. In accordance with this ATP depletion, cytosolic increases in mitochondrial proteins such as apoptosis-inducing factor, second mitochondria-derived activator of caspases/DIABLO, endonuclease G, and cytochrome c, which indicate severe mitochondrial dysfunction, were observed in NQO1 KO mice but not in WT mice after APAP exposure. Severe mitochondrial depolarization was also greater in hepatocytes isolated from NQO1 KO mice. Collectively, our data suggest that NQO1 plays a critical role in protection against energy depletion caused by APAP, and NQO1 may be useful in the development of therapeutic approaches to effectively diminish the hepatotoxicity caused by an APAP overdose. PMID:25224400

  14. 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

  15. Hypoxia inducible factor-1 is activated by transcriptional co-activator with PDZ-binding motif (TAZ) versus WWdomain-containing oxidoreductase (WWOX) in hypoxic microenvironment of bone metastasis from breast cancer.

    PubMed

    Bendinelli, Paola; Maroni, Paola; Matteucci, Emanuela; Luzzati, Alessandro; Perrucchini, Giuseppe; Desiderio, Maria Alfonsina

    2013-07-01

    The hypoxic microenvironment of bone marrow favours the bone metastasis process. Hypoxia inducible factor (HIF)-1? is hallmark for hypoxia, correlating with poor prognosis and radio/chemotherapy resistance of primary-breast carcinoma. For bone metastasis, the molecular mechanisms involved in HIF-1? expression and HIF-1 (?/? heterodimer)-transcription factor activity are scarcely known. We studied the role played by HIF-1 in the cross-talk between neoplastic and supportive-microenvironmental cells. Also, WWdomain-containing oxidoreductase (Wwox) and transcriptional co-activator with PDZ-binding motif (TAZ) were taken into consideration evaluating whether these Hippo-pathway effectors affect bone-metastatic phenotype through HIF-1 activity. Considering bone-metastasis specimens, nuclear HIF-1?-TAZ co-localisation occurred in neoplastic and supportive cells, such as fibroblasts and endotheliocytes. Based on these data, the functional importance was verified using 1833-bone metastatic clone under hypoxia: nuclear HIF-1? and TAZ expression increased and co-immunoprecipitated, activating HIF-1-DNA binding and transactivation. In contrast, Wwox localised at perinuclear level in neoplastic cells of bone metastasis, being almost absent in supportive cells, and Wwox-protein expression diminished in hypoxic-1833 cells. Thus, TAZ regulation of HIF-1 activity might be important for bone-secondary growth, participating in metastasis-stroma cross-talk. Further, TAZ and HIF-1?-protein levels seemed correlated. In fact, blocking cyclooxygenase-2 with NS398 in hypoxic-1833 cells, not only HIF-1? decreased but also molecular-mechanism(s) upstream of the Hippo pathway were triggered: LATS-dependent TAZ phosphorylation seemed responsible for TAZ nucleus/cytoplasm translocation and degradation. In the 1833-xenograft model, NS398 largely prevented the outgrowth of bone-metastatic cells, probably related to remarkable-extracellular matrix assembly. We gained clinical insight into HIF-1? and TAZ as candidate biomarkers for bone avidity, relevant for early-therapeutic intervention against bone metastasis. PMID:23566416

  16. 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.

  17. Isotropic exchange interaction between Mo and the proximal FeS center in the xanthine oxidase family member aldehyde oxidoreductase from Desulfovibrio gigas on native and polyalcohol inhibited samples: an EPR and QM/MM study.

    PubMed

    Gómez, María C; Neuman, Nicolás I; Dalosto, Sergio D; González, Pablo J; Moura, José J G; Rizzi, Alberto C; Brondino, Carlos D

    2015-03-01

    Aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is a homodimeric molybdenum-containing protein that catalyzes the hydroxylation of aldehydes to carboxylic acids and contains a Mo-pyranopterin active site and two FeS centers called FeS 1 and FeS 2. The electron transfer reaction inside DgAOR is proposed to be performed through a chemical pathway linking Mo and the two FeS clusters involving the pyranopterin ligand. EPR studies performed on reduced as-prepared DgAOR showed that this pathway is able to transmit very weak exchange interactions between Mo(V) and reduced FeS 1. Similar EPR studies but performed on DgAOR samples inhibited with glycerol and ethylene glycol showed that the value of the exchange coupling constant J increases ~2 times upon alcohol inhibition. Structural studies in these DgAOR samples have demonstrated that the Mo-FeS 1 bridging pathway does not show significant differences, confirming that the changes in J observed upon inhibition cannot be ascribed to structural changes associated neither with pyranopterin and FeS 1 nor with changes in the electronic structure of FeS 1, as its EPR properties remain unchanged. Theoretical calculations indicate that the changes in J detected by EPR are related to changes in the electronic structure of Mo(V) determined by the replacement of the OHx labile ligand for an alcohol molecule. Since the relationship between electron transfer rate and isotropic exchange interaction, the present results suggest that the intraenzyme electron transfer process mediated by the pyranopterin moiety is governed by a Mo ligand-based regulatory mechanism. PMID:25344343

  18. 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.

  19. Role of NQO1 609C>T and NQO2 -3423G>A gene polymorphisms in esophageal cancer risk in Kashmir valley and meta analysis.

    PubMed

    Malik, Manzoor Ahmad; Zargar, Showkat Ali; Mittal, Balraj

    2012-09-01

    Esophageal cancer (EC) is a complex multifactorial disorder, where environmental and genetic factors play major role. NADPH:quinone oxidoreductase 1 (NQO1) and NRH:quinone oxidoreductase 2 (NQO2) are phase II cytosolic enzymes that catalyze metabolism of quinones, important in the detoxification of environmental carcinogens. A case-control study was performed to investigated the associations of NQO1 609C>T and NQO2 -3423G>A polymorphisms with susceptibility to EC in a high-risk Kashmiri population of India in 135 EC patients and 195 unrelated healthy controls using PCR-RFLP. We also performed a meta analysis of nine published studies (1,224 cases and 1,740 controls) on NQO1 609C>T and evaluated the association between the NQO1 609C>T polymorphisms and esophageal cancer risk. A significant difference in NQO1 609C>T and NQO2 -3423G>A genotype distribution between EC cases and corresponding controls groups was observed (OR = 2.65; 95 % CI = 1.29-5.42 and OR = 1.88; 95 % CI = 1.02-3.49 respectively). Further, gene-gene interaction showed significantly increased risk for esophageal adenocarcinoma with variant genotypes of NQO1 609C>T and NQO2 -3423G>A polymorphisms and interaction with environmental risk factors revealed pronounced risk of EC with NQO1 609C>T TT genotype in high salted tea users of Kashmir valley (OR = 3.72, 95 % CI = 0.98-14.19). Meta analysis of NQO 609C>T polymorphism also suggested association of the polymorphism with EC in Asians as well as Europeans. In conclusion, NQO1 609C>T and NQO2 -3423G>A genetic variations modulate risk of EC in high-risk Kashmir population. PMID:22736108

  20. 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.

  1. 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

  2. Induction of erythroid differentiation and modulation of gene expression by tiazofurin in K-562 leukemia cells.

    PubMed Central

    Olah, E; Natsumeda, Y; Ikegami, T; Kote, Z; Horanyi, M; Szelenyi, J; Paulik, E; Kremmer, T; Hollan, S R; Sugar, J

    1988-01-01

    Tiazofurin (2-beta-D-ribofuranosyl-4-thiazole-carboxamide; NSC 286193), an antitumor carbon-linked nucleoside that inhibits IMP dehydrogenase (IMP:NAD+ oxidoreductase; EC 1.1.1.205) and depletes guanylate levels, can activate the erythroid differentiation program of K-562 human leukemia cells. Tiazofurin-mediated cell differentiation is a multistep process. The inducer initiates early (less than 6 hr) metabolic changes that precede commitment to differentiation; among these early changes are decreases in IMP dehydrogenase activity and in GTP concentration, as well as alterations in the expression of certain protooncogenes (c-Ki-ras). K-562 cells do express commitment-i.e., cells exhibit differentiation without tiazofurin. Guanosine was effective in preventing the action of tiazofurin, thus providing evidence that the guanine nucleotides are critically involved in tiazofurin-initiated differentiation. Activation of transcription of the erythroid-specific gene that encodes A gamma-globin is a late (48 hr) but striking effect of tiazofurin. Down-regulation of the c-ras gene appears to be part of the complex process associated with tiazofurin-induced erythroid differentiation and relates to the perturbations of GTP metabolism. Images PMID:2901100

  3. Sandro Rusconi Gene transfer

    E-print Network

    Málaga, Universidad de

    Program 37 'Somatic Gene Therapy' 2001-today Swiss Natl. Res. Program 50 'Endocrine disruptors' 2002 with other doping, detection Techniques of gene transfer (Gene Therapy) problems and solutions, vectors Somatic Gene Therapy (somatic gene transfer) UNIFR Rusconi 2003 Definition of GT: 'Use genes as drugs

  4. Evidence for repeated gene duplications in Tritrichomonas foetus supported by EST analysis and comparison with the Trichomonas vaginalis genome.

    PubMed

    Oyhenart, Jorge; Breccia, Javier D

    2014-12-15

    Tritrichomonas foetus causes a venereal infection in cattle; the disease has mild or no clinical manifestation in bulls, while cows may present vaginitis, placentitis, pyometra and abortion in the more severe cases. T. foetus has one of the largest known genomes among trichomonads. However molecular data are fragmentary and have minimally contributed to the understanding of the biology and pathogenesis of this protozoan. In a search of new T. foetus genes, a detailed exploration was performed using recently available expressed sequences. Genes involved in the central carbon metabolism (phosphoenol pyruvate carboxykinase, glyceraldehyde-3-phosphate dehydrogenase, fructose-1,6-bisphosphate aldolase, thioredoxin peroxidase, alpha and beta chains of succinyl CoA synthetase, malate dehydrogenase, malate oxidoreductase and enolase) as well as in cell structure and motility (actin, ?-tubulin and ?-tubulin) were found duplicated and, in many cases, repeatedly duplicated. Homology analysis suggested that massive expansions might have occurred in the T. foetus genome in a similar way it was also predicted for Trichomonas vaginalis, while conservation assessment showed that duplications have been acquired after differentiation of the two species. Therefore, gene duplications might be common among these parasitic protozoans. PMID:25458117

  5. Loss of Nuclear Gene Expression during the Phytochrome A-Mediated Far-Red Block of Greening Response1

    PubMed Central

    McCormac, Alex C.; Terry, Matthew J.

    2002-01-01

    We have examined the expression of the HEMA1 gene, which encodes the key chlorophyll synthesis enzyme glutamyl-tRNA reductase, during the phytochrome A-mediated far-red light (FR) block of greening response in Arabidopsis. Our results demonstrate that the FR block of greening comprises two separate responses: a white light (WL) intensity-independent response that requires 3 d of FR and is associated with a loss of expression of the nuclear genes HEMA1 and Lhcb following the transfer to WL (transcriptionally coupled response) and a WL intensity-dependent response that is induced by 1 d of FR and is transcriptionally uncoupled. Both responses required phytochrome A. The transcriptionally uncoupled response correlated with a deregulation of tetrapyrrole synthesis and potential photooxidative damage and was inhibited by cytokinin. The transcriptionally coupled FR response was additive with the loss of expression following Norflurazon-induced photobleaching and was absent in the presence of sucrose or after lower fluence rate (1 ?mol m?2 s?1) FR treatments. Both pathways leading to the loss of nuclear gene expression were inhibited by overexpression of NADPH:protochlorophyllide oxidoreductase, indicating a role for plastid signaling in the FR-mediated pathway. The significance of identifying a distinct phytochrome A-mediated plastid signaling pathway is discussed. PMID:12226519

  6. 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

  7. Increased Nrf2 Activation in Livers from Keap1-Knockdown Mice Increases Expression of Cytoprotective Genes that Detoxify Electrophiles more than those that Detoxify Reactive Oxygen Species

    PubMed Central

    Reisman, Scott A.; Yeager, Ronnie L.; Yamamoto, Masayuki; Klaassen, Curtis D.

    2009-01-01

    Nuclear factor erythroid 2–related factor 2 (Nrf2) is a transcription factor critical for protection against electrophilic and oxidative stress. In a recently engineered mouse with knockdown of kelch-like ECH associated protein 1 (Keap1-kd mice), the cytosolic repressor of Nrf2, there is a 55% decrease in Keap1 mRNA and a 200% increase in Nrf2 protein in liver. Experiments with Nrf2-null mice have demonstrated the effects of a lack of Nrf2. However, little is known about the biological effects of more Nrf2 activation. Accordingly, the hepatic phenotype of Keap1-kd mice, as well as the hepatic mRNA expression of cytoprotective genes were compared among wild-type, Nrf2-null, and Keap1-kd mice. Three distinct patterns of hepatic gene expression were identified among wild-type, Nrf2-null, and Keap1-kd mice. The first pattern encompassed genes that were lower in Nrf2-null mice and considerably higher in Keap1-kd mice than wild-type mice, which included genes mainly responsible for the detoxification and elimination of electrophiles, such as NAD(P)H:quinone oxidoreductase 1 and glutathione-S-transferases (Gst), and multidrug resistance–associated proteins. The second pattern encompassed genes that were lower in Nrf2-null mice but not increased in Keap1-kd mice, and included genes, such as epoxide hydrolase-1, UDP-glucuronosyltransferases, aldehyde dehydrogenases, as well as genes important in the detoxification of reactive oxygen species, such as superoxide dismutase 1 and 2, catalase, and peroxiredoxin 1. The third pattern encompassed genes that were not different among wild-type, Nrf2-null, and Keap1-kd mice and included genes such as glutathione peroxidase, microsomal Gsts, and uptake transporters. In conclusion, the present study suggests that increased activation of hepatic Nrf2 is more important for the detoxification and elimination of electrophiles than reactive oxygen species. PMID:19129213

  8. The aryl hydrocarbon receptor and estrogen receptor alpha differentially modulate nuclear factor erythroid-2-related factor 2 transactivation in MCF-7 breast cancer cells.

    PubMed

    Lo, Raymond; Matthews, Jason

    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. PMID:23583297

  9. 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.

  10. Induced expression of cytochrome P450 1A and NAD(P)H:quinone oxidoreductase determined at mRNA, protein, and enzyme activity levels in rats exposed to the carcinogenic azo dye 1-phenylazo-2-naphthol (Sudan I).

    PubMed

    Stiborová, Marie; Dra?ínská, Helena; Martínek, Václav; Svášková, Dagmar; Hodek, Petr; Milichovský, Jan; Hejduková, Žaneta; Brotánek, Jaroslav; Schmeiser, Heinz H; Frei, Eva

    2013-02-18

    Sudan I (1-phenylazo-2-hydroxynaphthol) is a suspected human carcinogen causing tumors in the livers and urinary bladders of rats, mice, and rabbits. Here, we investigated for the first time the influence of Sudan I exposure on the expression of several biotransformation enzymes in the livers, kidneys, and lungs of rats concomitantly at the mRNA and protein levels and assayed their enzymatic activities. We also studied its effect on the formation of Sudan I-derived DNA adducts in vitro. Sudan I increased the total amounts of cytochrome P450 (P450) in all organs tested. Western blots using antibodies raised against various P450s, NADPH:P450 reductase, and NAD(P)H:quinone oxidoreductase 1 (NQO1) showed that the expression of P450 1A1 and NQO1 was induced in the liver, kidney, and lung of rats treated with Sudan I. The higher protein levels correlated with increased enzyme activities of P450 1A1/2 and NQO1. Furthermore, 9.9-, 5.9-, and 2.8-fold increases in the formation of Sudan I oxidative metabolites catalyzed by microsomes isolated from the liver, kidney, and lung, respectively, of rats treated with Sudan I were found. The relative amounts of P450 1A and NQO1 mRNA, measured by real-time polymerase chain reaction (RT-PCR) analysis, demonstrated that Sudan I induced the expression of P450 1A1 and NQO1 mRNA in the liver, kidney, and lung, and of P450 1A2 mRNA in kidney and lung. Finally, microsomes isolated from livers, kidneys, and lungs of Sudan I exposed rats more effectively catalyzed the formation of Sudan I-DNA adducts than microsomes from organs of control rats. This was attributable to the higher P450 1A1 expression. Because P450 1A1 is playing a major role in the bioactivation of Sudan I in rat and human systems, its induction by Sudan I may have a profound effect on cancer risk by this azo dye. In addition, the induction of P450 1A1/2 and NQO1 enzymes can influence individual human susceptibility to other environmental carcinogens and have an effect on cancer risk. PMID:23289503

  11. Gene - Gene Interactions Among MCP Genes Polymorphisms in Asthma

    PubMed Central

    Lee, June-Hyuk

    2014-01-01

    Purpose Monocyte chemoattractant proteins (MCPs) are important cytokines that involved in cellular activation and releasing of inflammatoy mediators by basophils and eosinophils in allergic disease. Some MCP gene variants implicate in asthma and monoclonal antibody for MCP-3 blocks allergic inflammations in the patients with asthma. Detection of interactions between gene and environment or between genes for complex disease such as asthma is important. We searched for an evidence of genetic effect of single nucleotide polymorphisms (SNPs) of MCP genes as well as gene - gene interactions involved in asthma. Methods Four hundreds asthmatics and four hundreds normal controls were enrolled. Asthma was defined as a positive bronchodilator response or positive methacholine provocation test with compatible clinical symptoms. Seven MCP gene SNPs (2 SNPs in MCP-1, 1 in MCP-2, and 4 in MCP-3) were included. Association analyses between SNP and asthma, and the tests for gene - gene interaction were performed. Results Strong linkage disequilibria were found among 7 MCP gene polymorphisms. There was no SNP that showed a significant association with asthma among 7 SNPs of 3 MCP genes. No haplotype was associated with asthma, either. The combination of MCP1-2518G>A, MCP2+46A>C, and MCP3+563C>T was the best predictive model for asthma as compared to the control in tests for gene - gene interaction. The MCP1-2518G>A and MCP2+46A>C was the second best predictive combination and this had the highest synergistic interaction effect on the subject's status than any other combination of polymorphisms. Complete linkages were not associated with the gene - gene interactions models. Conclusions MCP gene polymorphisms probably interact with each other; thus, these findings may help in developing a possible genetic marker to predict asthma. PMID:24991457

  12. Gene expression profiles in testis of pigs with extreme high and low levels of androstenone

    PubMed Central

    Moe, Maren; Meuwissen, Theo; Lien, Sigbjørn; Bendixen, Christian; Wang, Xuefei; Conley, Lene Nagstrup; Berget, Ingunn; Tajet, Håvard; Grindflek, Eli

    2007-01-01

    Background: Boar taint is a major obstacle when using uncastrated male pigs for swine production. One of the main compounds causing this taint is androstenone, a pheromone produced in porcine testis. Here we use microarrays to study the expression of thousands of genes simultaneously in testis of high and low androstenone boars. The study allows identification of genes and pathways associated with elevated androstenone levels, which is essential for recognising potential molecular markers for breeding purposes. Results: Testicular tissue was collected from 60 boars, 30 with extreme high and 30 with extreme low levels of androstenone, from each of the two breeds Duroc and Norwegian Landrace. The samples were hybridised to porcine arrays containing 26,877 cDNA clones, detecting 563 and 160 genes that were differentially expressed (p < 0.01) in Duroc and Norwegian Landrace, respectively. Of these significantly up- and down-regulated clones, 72 were found to be common for the two breeds, suggesting the possibility of both general and breed specific mechanisms in regulation of, or response to androstenone levels in boars. Ten genes were chosen for verification of expression patterns by quantitative real competitive PCR and real-time PCR. As expected, our results point towards steroid hormone metabolism and biosynthesis as important biological processes for the androstenone levels, but other potential pathways were identified as well. Among these were oxidoreductase activity, ferric iron binding, iron ion binding and electron transport activities. Genes belonging to the cytochrome P450 and hydroxysteroid dehydrogenase families were highly up-regulated, in addition to several genes encoding different families of conjugation enzymes. Furthermore, a number of genes encoding transcription factors were found both up- and down-regulated. The high number of clones belonging to ferric iron and iron ion binding suggests an importance of these genes, and the association between these pathways and androstenone levels is not previously described. Conclusion: This study contributes to the understanding of the complex genetic system controlling and responding to androstenone levels in pig testis. The identification of new pathways and genes involved in the biosynthesis and metabolism of androstenone is an important first step towards finding molecular markers to reduce boar taint. PMID:17988377

  13. Steroidogenesis of the testis -- new genes and pathways.

    PubMed

    Flück, Christa E; Pandey, Amit V

    2014-05-01

    Defects of androgen biosynthesis cause 46,XY disorder of sexual development (DSD). All steroids are produced from cholesterol and the early steps of steroidogenesis are common to mineralocorticoid, glucocorticoid and sex steroid production. Genetic mutations in enzymes and proteins supporting the early biosynthesis pathways cause adrenal insufficiency (AI), DSD and gonadal insufficiency. The classic androgen biosynthesis defects with AI are lipoid CAH, CYP11A1 and HSD3B2 deficiencies. Deficiency of CYP17A1 rarely causes AI, and HSD17B3 or SRD5A2 deficiencies only cause 46,XY DSD and gonadal insufficiency. All androgen biosynthesis depends on 17,20 lyase activity of CYP17A1 which is supported by P450 oxidoreductase (POR) and cytochrome b5 (CYB5). Therefore 46,XY DSD with apparent 17,20 lyase deficiency may be due to mutations in CYP17A1, POR or CYB5. Illustrated by patients harboring mutations in SRD5A2, normal development of the male external genitalia depends largely on dihydrotestosterone (DHT) which is converted from circulating testicular testosterone (T) through SRD5A2 in the genital skin. In the classic androgen biosynthetic pathway, T is produced from DHEA and androstenedione/-diol in the testis. However, recently found mutations in AKR1C2/4 genes in undervirilized 46,XY individuals have established a role for a novel, alternative, backdoor pathway for fetal testicular DHT synthesis. In this pathway, which has been first elucidated for the tammar wallaby pouch young, 17-hydroxyprogesterone is converted directly to DHT by 5?-3? reductive steps without going through the androgens of the classic pathway. Enzymes AKR1C2/4 catalyse the critical 3?HSD reductive reaction which feeds 17OH-DHP into the backdoor pathway. In conclusion, androgen production in the fetal testis seems to utilize two pathways but their exact interplay remains to be elucidated. PMID:24793988

  14. Sandro Rusconi Gene transfer

    E-print Network

    Málaga, Universidad de

    Research Program 37 'Somatic Gene Therapy' 2001-today Swiss Natl. Res. Program 50 'Endocrine disruptors, comparison with other doping, detection Techniques of gene transfer (Gene Therapy) problems and solutions getting oldcomp2.mov movie clip deleted Now, let's talk about Somatic Gene Therapy (somatic gene transfer

  15. Computational Gene Finding

    E-print Network

    Cheng, Jianlin Jack

    Computational Gene Finding Dong Xu Digital Biology Laboratory Computer Science Department://digbio.missouri.edu #12; Protein-encoding genes and gene structures Computational models for coding regions;What Is a Gene? Definition: A gene is the nucleotide sequence that stores the information which

  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

    ... Health Plans For Your Patients 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. Insert or gene Laboratory Gene transfer vectora

    E-print Network

    Kay, Mark A.

    Insert or gene Laboratory Gene transfer vectora Host rangeb functionc containment leveld MMLV based, CC BSL-2 including neurons TX BSL-2+/BSL-3 Poxvirus based- Broad host range S, E, M, T, DR, MP BSL-2 cells. c General categories of cellular genes and functions: S, structural proteins: actin, myosin, etc

  19. 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

  20. Aspirin may promote mitochondrial biogenesis via the production of hydrogen peroxide and the induction of Sirtuin1/PGC-1? genes

    PubMed Central

    Kamble, Pratibha; Selvarajan, Krithika; Narasimhulu, Chandrakala Aluganti; Nandave, Mukesh; Parthasarathy, Sampath

    2013-01-01

    Based on the rapid hydrolysis of acetyl salicylic acid (ASA, Aspirin) to salicylic acid (SA), the ability of SA to form dihydroxy benzoic acid (DBA), and the latter’s redox reactions to yield hydrogen peroxide (H2O2), we predicted that ASA may have the potential to induce Sirtuin1 (Sirt1) and its downstream effects. We observed that treatment of cultured liver cells with ASA resulted in the induction of Sirt1, peroxisome proliferator-activated receptor-gamma co-activator-1? (PGC-1?), and NAD(P)H quinone oxidoreductase 1 (Nqo1) genes. Paraoxonase 1 (PON1) and Aryl hydrocarbon receptor (AhR) siRNA transfections inhibited the induction of gene expressions by ASA suggesting the need for the acetyl ester hydrolysis and hydroxylation to DHBA. The latter also induced Sirt1, confirming the proposed pathway. As predicted, ASA and SA treatment resulted in the production of H2O2, a known inducer of Sirt1 and confirmed in the current studies. More importantly, ASA treatment resulted in an increase in mitochondria as seen by tracking dyes. We suggest that DHBA, generated from ASA, via its oxidation/reduction reactions mediated by Nqo1 might be involved in the production of O2-. and H2O2. As Sirt1 and PGC-1? profoundly affect mitochondrial metabolism and energy utilization, ASA may have therapeutic potential beyond its ability to inhibit cyclooxygenases. PMID:23228932

  1. Aspirin may promote mitochondrial biogenesis via the production of hydrogen peroxide and the induction of Sirtuin1/PGC-1? genes.

    PubMed

    Kamble, Pratibha; Selvarajan, Krithika; Aluganti Narasimhulu, Chandrakala; Nandave, Mukesh; Parthasarathy, Sampath

    2013-01-15

    Based on the rapid hydrolysis of acetyl salicylic acid (ASA, Aspirin) to salicylic acid (SA), the ability of SA to form dihydroxy benzoic acid (DBA), and the latter's redox reactions to yield hydrogen peroxide (H(2)O(2)), we predicted that ASA may have the potential to induce Sirtuin1 (Sirt1) and its downstream effects. We observed that treatment of cultured liver cells with ASA resulted in the induction of Sirt1, peroxisome proliferator-activated receptor-gamma co-activator-1? (PGC-1?), and NAD(P)H quinone oxidoreductase 1 (Nqo1) genes. Paraoxonase 1 (PON1) and Aryl hydrocarbon receptor (AhR) siRNA transfections inhibited the induction of gene expressions by ASA suggesting the need for the acetyl ester hydrolysis and hydroxylation to DHBA. The latter also induced Sirt1, confirming the proposed pathway. As predicted, ASA and SA treatment resulted in the production of H(2)O(2), a known inducer of Sirt1 and confirmed in the current studies. More importantly, ASA treatment resulted in an increase in mitochondria as seen by tracking dyes. We suggest that DHBA, generated from ASA, via its oxidation/reduction reactions mediated by Nqo1 might be involved in the production of O(2)(-.) and H(2)O(2). As Sirt1 and PGC-1? profoundly affect mitochondrial metabolism and energy utilization, ASA may have therapeutic potential beyond its ability to inhibit cyclooxygenases. PMID:23228932

  2. Lactate and succinate oxidoreductases in marine invertebrates

    Microsoft Academic Search

    C. S. Hammen

    1969-01-01

    Nineteen species of littoral marine invertebrates, representing Porifera, Cnidaria, Ctenophora, Brachiopoda, Mollusca, and Arthropoda, were studied with respect to the ability of tissue extracts to catalyze the lactate and succinate dehydrogenase reactions in both directions. Pyruvate reductase (PR) activity varied tremendously with species, from 0.014 µmole\\/min\\/g of tissue in the etenophore Mnemiopsis leidyi to 145 µ-moles\\/min in leg muscle of

  3. Gene gymnastics

    PubMed Central

    Vijayachandran, Lakshmi S; Thimiri Govinda Raj, Deepak B; Edelweiss, Evelina; Gupta, Kapil; Maier, Josef; Gordeliy, Valentin; Fitzgerald, Daniel J; Berger, Imre

    2013-01-01

    Most essential activities in eukaryotic cells are catalyzed by large multiprotein assemblies containing up to ten or more interlocking subunits. The vast majority of these protein complexes are not easily accessible for high resolution studies aimed at unlocking their mechanisms, due to their low cellular abundance and high heterogeneity. Recombinant overproduction can resolve this bottleneck and baculovirus expression vector systems (BEVS) have emerged as particularly powerful tools for the provision of eukaryotic multiprotein complexes in high quality and quantity. Recently, synthetic biology approaches have begun to make their mark in improving existing BEVS reagents by de novo design of streamlined transfer plasmids and by engineering the baculovirus genome. Here we present OmniBac, comprising new custom designed reagents that further facilitate the integration of heterologous genes into the baculovirus genome for multiprotein expression. Based on comparative genome analysis and data mining, we herein present a blueprint to custom design and engineer the entire baculovirus genome for optimized production properties using a bottom-up synthetic biology approach. PMID:23328086

  4. 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

  5. New patentable use of an old neuroleptic compound thioridazine to combat tuberculosis: a gene regulation perspective.

    PubMed

    Dutta, Noton K; Mazumdar, Kaushiki; Dastidar, Sujata G; Karakousis, Petros C; Amaral, Leonard

    2011-05-01

    Use of the old antipsychotic phenothiazine thioridazine (THZ) for therapy of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) infection is now being seriously considered. It is reported that THZ primarily acts on enzymes involved in fatty acid metabolism and membrane proteins, particularly efflux pumps, as well as oxidoreductases and proteins involved in aerobic respiration that overlap with a number of conventional antituberculous drugs. It targets the products of the Rv3160c-Rv3161c operon, which are perhaps required for the detoxification of THZ, members of the sigma factor SigB regulon that play a crucial role in protecting the pathogen against cell envelope damage, and Rv2745c, a transcription factor that regulates ATP-dependent proteolysis. Some of these genes have been shown to be essential for the survival or persistence of Mycobacterium tuberculosis in the infected host. Since THZ targets multiple pathways, including those involved in cell wall processes and respiratory chain components, it may serve as a model for multi-target drug development, as well as constitute a highly potent addition to a combination of antituberculous drug regimens. The discussion of some of the patents relevant to thioridazine to combat tuberculosis is also included in the present manuscript. PMID:21517741

  6. 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

  7. 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

  8. The nit1C gene cluster of Pseudomonas pseudoalcaligenes CECT5344 involved in assimilation of nitriles is essential for growth on cyanide.

    PubMed

    Estepa, Jessica; Luque-Almagro, Victor M; Manso, Isabel; Escribano, M Paz; Martínez-Luque, Manuel; Castillo, Francisco; Moreno-Vivián, Conrado; Roldán, M Dolores

    2012-06-01

    A proteomic approach was used to identify several proteins induced by cyanide in the alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344, two of them, NitB and NitG, encoded by genes that belong to the nit1C gene cluster. The predicted products of the nit1C gene cluster are a Fis-like ?(54) -dependent transcriptional activator (NitA), a nitrilase (NitC), an S-adenosylmethionine superfamily member (NitD), an N-acyltransferase superfamily member (NitE), a trifunctional polypeptide of the AIRS/GARS family (NitF), an NADH-dependent oxidoreductase (NitH) and two hypothetical proteins of unknown function (NitB and NitG). RT-PCR analysis suggested that nitBCDEFGH genes were co-transcribed, whereas the regulatory nitA gene was divergently transcribed. Real-time RT-PCR revealed that expression of the nitBCDEFGH genes was induced by cyanide and repressed by ammonium. The P.?pseudoalcaligenes CECT5344 nit1C gene cluster was found to be involved in assimilation of free and organic cyanides (nitriles) as deduced for the inability to grow with cyanides showed by the NitA, NitB and NitC mutant strains. The wild-type strain CECT5344 showed a nitrilase activity that allows growth on cyanide or hydroxynitriles. The NitB and NitC mutants only presented low basal levels of nitrilase activity that were not enough to support growth on either free cyanide or aliphatic nitriles, suggesting that nitrilase NitC is specific and essential for cyanide and aliphatic nitriles assimilation. PMID:23760796

  9. Gene doping: gene delivery for olympic victory

    PubMed Central

    Gould, David

    2013-01-01

    With one recently recommended gene therapy in Europe and a number of other gene therapy treatments now proving effective in clinical trials it is feasible that the same technologies will soon be adopted in the world of sport by unscrupulous athletes and their trainers in so called ‘gene doping’. In this article an overview of the successful gene therapy clinical trials is provided and the potential targets for gene doping are highlighted. Depending on whether a doping gene product is secreted from the engineered cells or is retained locally to, or inside engineered cells will, to some extent, determine the likelihood of detection. It is clear that effective gene delivery technologies now exist and it is important that detection and prevention plans are in place. PMID:23082866

  10. Comparative transcript profiling of gene expression between seedless Ponkan mandarin and its seedy wild type during floral organ development by suppression subtractive hybridization and cDNA microarray

    PubMed Central

    2012-01-01

    Background Seedlessness is an important agronomic trait for citrus, and male sterility (MS) is one main cause of seedless citrus fruit. However, the molecular mechanism of citrus seedlessness remained not well explored. Results An integrative strategy combining suppression subtractive hybridization (SSH) library with cDNA microarray was employed to study the underlying mechanism of seedlessness of a Ponkan mandarin seedless mutant (Citrus reticulata Blanco). Screening with custom microarray, a total of 279 differentially expressed clones were identified, and 133 unigenes (43 contigs and 90 singletons) were obtained after sequencing. Gene Ontology (GO) distribution based on biological process suggested that the majority of differential genes are involved in metabolic process and respond to stimulus and regulation of biology process; based on molecular function they function as DNA/RNA binding or have catalytic activity and oxidoreductase activity. A gene encoding male sterility-like protein was highly up-regulated in the seedless mutant compared with the wild type, while several transcription factors (TFs) such as AP2/EREBP, MYB, WRKY, NAC and C2C2-GATA zinc-finger domain TFs were down-regulated. Conclusion Our research highlighted some candidate pathways that participated in the citrus male gametophyte development and could be beneficial for seedless citrus breeding in the future. PMID:22897898

  11. Isolation and partial nucleotide sequence of the laccase gene from Neurospora crassa: amino acid sequence homology of the protein to human ceruloplasmin.

    PubMed

    Germann, U A; Lerch, K

    1986-12-01

    The laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) gene from Neurospora crassa was cloned and part of its nucleotide sequence corresponding to the carboxyl-terminal region of the protein has been determined. The gene was cloned by cDNA synthesis with a laccase-specific synthetic deoxyundecanucleotide as primer and poly(A) RNA isolated from cycloheximide-treated N. crassa cultures as template. Based on the nucleotide sequence of the cDNA obtained, a unique 21-mer was synthesized and used to screen a genomic DNA library from N. crassa. Five different positive clones were isolated and shown to share an overlapping DNA region with the same pattern of restriction sites. Sequence analysis of the common 1.36-kilobase Sal I fragment revealed an open reading frame of 726 nucleotides. The amino acid sequence deduced is in complete agreement with the primary structures of several tryptic peptides isolated previously from N. crassa laccase. The analyzed carboxyl-terminal region of laccase exhibits a striking sequence homology to the carboxyl-terminal part of the third homology unit of the multicopper oxidase ceruloplasmin and to a smaller extent, to the low molecular weight blue copper proteins plastocyanin and azurin. Based on amino acid sequence comparison between these proteins, putative copper ligands of N. crassa laccase are proposed. Moreover, these data further support the hypothesis that the small blue copper proteins and the multicopper oxidases have evolved from the same ancestral gene. PMID:2947240

  12. 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

  13. Discovery of genes related to diabetic nephropathy in various animal models by current techniques.

    PubMed

    Wada, Jun; Sun, Lin; Kanwar, Yashpal S

    2011-01-01

    One of the major problems facing clinical nephrology currently throughout the world is an exponential increase in patients with end-stage renal disease (ESRD), which is largely related to a high incidence of diabetic nephropathy. The latter is characterized by a multitude of metabolic and signaling events following excessive channeling of glucose, which leads to an increased synthesis of extracellular matrix (ECM) glycoproteins resulting in glomerulosclerosis, interstitial fibrosis and ultimately ESRD. With the incidence of nephropathy at pandemic levels and a high rate of ESRD, physicians around the world must treat a disproportionately large number of diabetic patients with upto-date innovative measures. In this regard, identification of genes that are crucially involved in the progression of diabetic nephropathy would enhance the discovery of new biomarkers and could also promote the development of novel therapeutic strategies. Over the last decade, we focused on the recent methodologies of high-throughput and genome-wide screening for identification of relevant genes in various animal models, which included the following: (1) single nucleotide polymorphism-based genome- wide screening; (2) the transcriptome approach, such as differential display reverse transcription polymerase chain reaction (DDRT-PCR), representational difference analysis of cDNA (cDNA-RDA)/suppressive subtractive hybridization, SAGE (serial analysis of gene expression) and DNA Microarray; and (3) the proteomic approach and 2- dimensional polyacrylamide gel electrophoresis (2D- PAGE) coupled with mass spectroscopic analysis. Several genes, such as Tim44 (translocase of inner mitochondrial membrane- 44), RSOR/MIOX (renal specific oxidoreductase/myo-inositol oxygenase), UbA52, Rap1b (Ras-related GTPase), gremlin, osteopontin, hydroxysteroid dehydrogenase- 3? isotype 4 and those of the Wnt signaling pathway, were identified as differentially expressed genes in kidneys of diabetic rodents. Functional analysis of these genes and the subsequent translational research in the clinical settings would be very valuable in the prevention and treatment of diabetic nephropathy. Future trends for identification of the biomarkers and therapeutic target genes should also include genome scale DNA/histonemethylation profiling, metabolomic approaches (e.g. metabolic phenotyping by 1H spectroscopy) and lectin microarray for glycan profiling along with the development of robust data-mining strategies. PMID:21252517

  14. 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

  15. Tests for gene clustering

    Microsoft Academic Search

    Dannie Durand; David Sankoff

    2002-01-01

    Comparing chromosomal gene order in two or more related species is an important approach to studying the forces that guide genome organization and evolution. Linked clusters of similar genes found in related genomes are often used to support arguments of evolutionary relatedness or functional selection. However, as the gene order and the gene complement of sister genomes diverge progressively due

  16. Corneal gene therapy

    Microsoft Academic Search

    Eytan A. Klausner; Dan Peer; Robert L. Chapman; Richard F. Multack; Shridhar V. Andurkar

    2007-01-01

    Gene therapy to the cornea can potentially correct inherited and acquired diseases of the cornea. Factors that facilitate corneal gene delivery are the accessibility and transparency of the cornea, its stability ex vivo and the immune privilege of the eye. Initial corneal gene delivery studies characterized the relationship between intraocular modes of administration and location of reporter gene expression. The

  17. 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, "Raw Data" conditions genes Entries of the Raw Data matrix: · Ratio values · Absolute values · ... · Row = gene's expression pattern · Column = experiment/condition's profile #12;CG 3 Data Preprocessing

  18. Differential Gene Rainer Spang

    E-print Network

    Spang, Rainer

    Differential Gene Expression Rainer Spang Courses in Practical DNA Microarray Analysis #12;Two cell. etc.... For every sample (cell line/patient) we have the expression levels of thousands of genes and the information whether it is A or B #12;Differential gene expression: Which genes are differentially expressed

  19. 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…

  20. Life with 6000 Genes

    Microsoft Academic Search

    A. Gofieau; B. G. Barrell; H. Bussey; R. W. Davis; B. Dujon; H. Feldmann; F. Galibert; J. D. Hoheisel; C. Jacq; M. Johnston; E. J. Louis; H. W. Mewes; Y. Murakami; P. Philippsen; H. Tettelin; S. G. Oliver

    1996-01-01

    The genome of the yeast Saccharomyces cerevisiae has been completely sequenced through a worldwide collaboration. The sequence of 12,068 kilobases defines 5885 potential protein-encoding genes, approximately 140 genes specifying ribosomal RNA, 40 genes for small nuclear RNA molecules, and 275 transfer RNA genes. In addition, the complete sequence provides information about the higher order organization of yeast's 16 chromosomes and

  1. Regulation of aldo-keto reductase AKR1B10 gene expression: involvement of transcription factor Nrf2.

    PubMed

    Nishinaka, Toru; Miura, Takeshi; Okumura, Manami; Nakao, Fumika; Nakamura, Haruka; Terada, Tomoyuki

    2011-05-30

    Aldo-keto reductase 1B10 (AKR1B10) is an aldose reductase-like oxidoreductase of human origin. The expression of AKR1B10 is highly induced in the cells of various cancers such as lung non-small-cell carcinoma and hepatocellular carcinoma. Since the enzyme exhibits broad substrate specificities toward various xenobiotics such as anti-tumor drugs or various endogenous compounds such as retinaldehyde, AKR1B10 may play an important role in tumor progression or drug resistance. However, very little is known about its gene regulation. In this study, we investigated the regulation of AKR1B10 expression. A -3282bp of the 5'-flanking fragment of AKR1B10 gene was isolated from A549 lung carcinoma cells. This region contains several putative regulatory motifs such as AP-1, NF-?B and antioxidant response element. In addition, a complex polymorphic microsatellite with repetitive sequences enriched with C and T was found. However, luciferase reporter assay revealed that the microsatellite polymorphism did not influence the basal promoter activity. We found that an antioxidant ethoxyquin induced the AKR1B10 expression based on RT-PCR analysis and luciferase reporter assay. Since ethoxyquin is known to activate the gene expression mediated through transcription factor Nrf2, the involvement of Nrf2 was examined. Forced expression of dominant-negative Nrf2 mutant suppressed the ethoxyquin-induced AKR1B10 expression, and co-introduction of Nrf2 expression plasmid into the cells significantly augmented the luciferase reporter activity. Deletion analysis revealed that Nrf2-regulating cis-element(s) lay within -539bp of the 5'-flanking region. These results suggest that Nrf2 is one of the major factors involved in the AKR1B10 gene regulation. PMID:21277289

  2. Role of the WWOX gene, encompassing fragile region FRA16D, in suppression of pancreatic carcinoma cells.

    PubMed

    Nakayama, Shunji; Semba, Shuho; Maeda, Naoko; Aqeilan, Rami I; Huebner, Kay; Yokozaki, Hiroshi

    2008-07-01

    The WW-domain-containing oxidoreductase (WWOX) gene spans the common chromosomal fragile site FRA16D (16q23.2) and is believed to be a tumor suppressor in various human malignancies. We have previously shown frequent down-modulation of Wwox expression in pancreatic carcinoma (PC); however, biological function of Wwox in pancreatic duct carcinogenesis remains unknown. In PANC-1 (Wwox-negative) PC-derived cells, restoration of recombinant WWOX gene expression with adenoviral gene delivery (Ad-WWOX) effectively increased the number of cells with subG(1) DNA contents in a multiplicity of infection-dependent manners: Ad-WWOX infection up-regulated caspase-3 activity and reduced procaspase-3 and procaspase-8 levels. We also confirmed that restoration of WWOX gene suppressed cell growth in vitro and tumorigenicity in vivo. In addition, transduction of wild-type WWOX-expressing vector inhibited PANC-1 colony formation; however, substitution of Y33 of Wwox with arginine did not lead to inhibition of colony formation, suggesting the biological significance of the WW1 domain of Wwox for its tumor-suppressing activity. In PC tissue samples, abundant cytoplasmic Wwox expression was detected in the normal pancreatic duct epithelium, whereas Wwox expression was frequently reduced not only in a large fraction of PC but also in precancerous lesions in accord with the pancreatic intraepithelial neoplasia (PanIN) grade, which was closely correlated with patients' poorer outcome. Interestingly, the existence of Wwox expression was associated with elevated mothers against decapentaplegic homolog 4 (Smad4) protein levels in vitro and in vivo. These findings suggest that down-modulation of Wwox expression is an early event and may be associated with the down-regulation of Smad4 protein levels during pancreatic duct carcinogenesis. PMID:18460020

  3. 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

  4. Reference Genes for Real-Time PCR Quantification of Messenger RNAs and MicroRNAs in Mouse Model of Obesity

    PubMed Central

    Matoušková, Petra; Bártíková, Hana; Boušová, Iva; Hanušová, Veronika; Szotáková, Barbora; Skálová, Lenka

    2014-01-01

    Obesity and metabolic syndrome is increasing health problem worldwide. Among other ways, nutritional intervention using phytochemicals is important method for treatment and prevention of this disease. Recent studies have shown that certain phytochemicals could alter the expression of specific genes and microRNAs (miRNAs) that play a fundamental role in the pathogenesis of obesity. For study of the obesity and its treatment, monosodium glutamate (MSG)-injected mice with developed central obesity, insulin resistance and liver lipid accumulation are frequently used animal models. To understand the mechanism of phytochemicals action in obese animals, the study of selected genes expression together with miRNA quantification is extremely important. For this purpose, real-time quantitative PCR is a sensitive and reproducible method, but it depends on proper normalization entirely. The aim of present study was to identify the appropriate reference genes for mRNA and miRNA quantification in MSG mice treated with green tea catechins, potential anti-obesity phytochemicals. Two sets of reference genes were tested: first set contained seven commonly used genes for normalization of messenger RNA, the second set of candidate reference genes included ten small RNAs for normalization of miRNA. The expression stability of these reference genes were tested upon treatment of mice with catechins using geNorm, NormFinder and BestKeeper algorithms. Selected normalizers for mRNA quantification were tested and validated on expression of NAD(P)H:quinone oxidoreductase, biotransformation enzyme known to be modified by catechins. The effect of selected normalizers for miRNA quantification was tested on two obesity- and diabetes- related miRNAs, miR-221 and miR-29b, respectively. Finally, the combinations of B2M/18S/HPRT1 and miR-16/sno234 were validated as optimal reference genes for mRNA and miRNA quantification in liver and 18S/RPlP0/HPRT1 and sno234/miR-186 in small intestine of MSG mice. These reference genes will be used for mRNA and miRNA normalization in further study of green tea catechins action in obese mice. PMID:24465854

  5. Gene doping in sports.

    PubMed

    Unal, Mehmet; Ozer Unal, Durisehvar

    2004-01-01

    Gene or cell doping is defined by the World Anti-Doping Agency (WADA) as "the non-therapeutic use of genes, genetic elements and/or cells that have the capacity to enhance athletic performance". New research in genetics and genomics will be used not only to diagnose and treat disease, but also to attempt to enhance human performance. In recent years, gene therapy has shown progress and positive results that have highlighted the potential misuse of this technology and the debate of 'gene doping'. Gene therapies developed for the treatment of diseases such as anaemia (the gene for erythropoietin), muscular dystrophy (the gene for insulin-like growth factor-1) and peripheral vascular diseases (the gene for vascular endothelial growth factor) are potential doping methods. With progress in gene technology, many other genes with this potential will be discovered. For this reason, it is important to develop timely legal regulations and to research the field of gene doping in order to develop methods of detection. To protect the health of athletes and to ensure equal competitive conditions, the International Olympic Committee, WADA and International Sports Federations have accepted performance-enhancing substances and methods as being doping, and have forbidden them. Nevertheless, the desire to win causes athletes to misuse these drugs and methods. This paper reviews the current status of gene doping and candidate performance enhancement genes, and also the use of gene therapy in sports medicine and ethics of genetic enhancement. PMID:15157120

  6. 1,3-Propanediol production by new recombinant Escherichia coli containing genes from pathogenic bacteria.

    PubMed

    Przysta?owska, Hanna; Zeyland, Joanna; Szymanowska-Powa?owska, Daria; Szalata, Marlena; S?omski, Ryszard; Lipi?ski, Daniel

    2015-02-01

    1,3-Propanediol (1,3-PDO) is an organic compound, which is a valuable intermediate product, widely used as a monomer for synthesizing biodegradable polymers, increasing their strength; as well as an ingredient of textile, cosmetic and medical products. 1,3-PDO is mostly synthesized chemically. Global companies have developed technologies for 1,3-PDO synthesis from petroleum products such as acrolein and ethylene oxide. A potentially viable alternative is offered by biotechnological processes using microorganisms capable of synthesizing 1,3-PDO from renewable substrates (waste glycerol, a by-product of biofuel production, or glucose). In the present study, genes from Citrobacter freundii and Klebsiella pneumoniae were introduced into Escherichia coli bacteria to enable the synthesis of 1,3-PDO from waste glycerol. These strains belong to the best 1,3-PDO producers, but they are pathogenic, which restricts their application in industrial processes. The present study involved the construction of two gene expression constructs, containing a total of six heterologous glycerol catabolism pathway genes from C. freundii ATCC 8090 and K. pneumoniae ATCC 700721. Heterologous genes encoding glycerol dehydratase (dhaBCE) and the glycerol dehydratase reactivation factor (dhaF, dhaG) from C. freundii and gene encoding 1,3-PDO oxidoreductase (dhaT) from K. pneumoniae were expressed in E. coli under the control of the T7lac promoter. An RT-PCR analysis and overexpression confirmed that 1,3-PDO synthesis pathway genes were expressed on the RNA and protein levels. In batch fermentation, recombinant E. coli bacteria used 32.6gl(-1) of glycerol to produce 10.6 gl(-1) of 1,3-PDO, attaining the efficiency of 0.4 (mol?,?-PDO molglycerol(-1)). The recombinant E. coli created is capable of metabolizing glycerol to produce 1,3-PDO, and the efficiency achieved provides a significant research potential of the bacterium. In the face of shortage of fossil fuel supplies and climate warming there is an increasing industrial need to exchange the chemical way of chemicals synthesis for biotechnological - more ecological manner. The 1,3-PDO production from glycerol is an desirable alternative to the traditional production from non-renewable resources. This work is a part of project, which opens a way to development of innovative "green chemistry" and new perspectives to chemical industry. PMID:25644946

  7. Human Gene Therapy: Genes without Frontiers?

    ERIC Educational Resources Information Center

    Simon, Eric J.

    2002-01-01

    Describes the latest advancements and setbacks in human gene therapy to provide reference material for biology teachers to use in their science classes. Focuses on basic concepts such as recombinant DNA technology, and provides examples of human gene therapy such as severe combined immunodeficiency syndrome, familial hypercholesterolemia, and…

  8. 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

  9. Your Genes, Your Choices

    MedlinePLUS

    Your Genes, Your Choices describes the Human Genome Project, the science behind it, and the ethical, legal, and social ... Nothing could be further from the truth. Your Genes, Your Choices points out how the progress of ...

  10. Proto-genes and de novo gene birth

    E-print Network

    Carvunis, Anne-Ruxandra

    Novel protein-coding genes can arise either through re-organization of pre-existing genes or de novo. Processes involving re-organization of pre-existing genes, notably after gene duplication, have been extensively described. ...

  11. Gene therapies for osteoarthritis

    Microsoft Academic Search

    Christopher H. Evans

    2004-01-01

    Osteoarthritis (OA) is a major health problem in urgent need of better treatment. Gene therapy offers to meet this need. Of\\u000a the different strategies for using gene therapy in OA, local gene transfer to synovium is in the most advanced stage of development.\\u000a Local gene transfer brings several advantages, including a focused, local therapy that promises greater efficacy with reduced

  12. Reading and Generalist Genes

    ERIC Educational Resources Information Center

    Haworth, Claire M. A.; Meaburn, Emma L.; Harlaar, Nicole; Plomin, Robert

    2007-01-01

    Twin-study research suggests that many (but not all) of the same genes contribute to genetic influence on diverse learning abilities and disabilities, a hypothesis called "generalist genes". This generalist genes hypothesis was tested using a set of 10 DNA markers (single nucleotide polymorphisms [SNPs]) found to be associated with early reading…

  13. Dopamine genes and ADHD

    Microsoft Academic Search

    J. M Swanson; Pamela Flodman; James Kennedy; M. Anne Spence; Robert Moyzis; Sabrina Schuck; Michael Murias; Joan Moriarity; Cathy Barr; Moyra Smith; Michael Posner

    2000-01-01

    Family, twin, and adoption studies have documented a strong genetic basis for ADHD\\/HKD, but these studies do not identify specific genes linked to the disorder. Molecular genetic studies can identify allelic variations of specific genes that are functionally associated with ADHD\\/HKD, and dopamine genes have been the initial candidates based on the site of action of the stimulants drugs, which

  14. Gene Regulation by Methylation

    Microsoft Academic Search

    Wolf C. Mueller; Andreas von Deimling

    Epigenetic gene regulation of specific genes strongly affects clinical outcome of malignant glioma. MGMT is the best studied gene for the connection of promoter methylation and clinical course in glioblastoma. While MGMT promoter methylation analysis currently does not alter treatment of glioblastoma patients, mainly because of a lack of convincing\\u000a therapy to radiotherapy and concomitant administration of alkylating drugs, there

  15. Introduction The COMT gene

    E-print Network

    Maddox, W. Todd

    Introduction The COMT gene References Dopamine-Related Genetic Influences on Exploratory Decision: The goal of the present work is to explore the role of the prefrontal dopamine gene (COMT) in exploratory.D., Oas-Terpstra, J., Moreno, F. (2009). Prefrontal and Striatal Dopaminergic Genes Predict Individual

  16. Gene expression heterogeneous

    E-print Network

    Khaitovich, Philipp

    Magazine R359 Gene expression becomes heterogeneous with age Mehmet Somel1*, Philipp Khaitovich1 for an age-dependent increase in variation in gene expression has yet been found [6­9]. Using eight microarray data sets from different studies in humans and rats, we find that gene expression becomes more

  17. Optimization of Heterologous Gene

    E-print Network

    Matsumura, Ichiro

    Optimization of Heterologous Gene Expression for In Vitro Evolution BioTechniques 30:474-476 (March 2001) Proteins can be evolved in vitro by randomly mutating the corresponding genes, expressing is the development of the high-throughput as- say or "screen". In particular, the wild- type gene should initially

  18. Effect of the combined probiotics with aflatoxin B?-degrading enzyme on aflatoxin detoxification, broiler production performance and hepatic enzyme gene expression.

    PubMed

    Zuo, Rui-yu; Chang, Juan; Yin, Qing-qiang; Wang, Ping; Yang, Yu-rong; Wang, Xiao; Wang, Guo-qiang; Zheng, Qiu-hong

    2013-09-01

    In order to degrade aflatoxin B? (AFB?), AFB?-degrading microbes (probiotics) such as Lactobacillus casei, Bacillus subtilis and Pichia anomala, and the AFB?-degrading enzyme from Aspergillus oryzae were selected and combined to make feed additive. Seventy-five 43-day-old male Arbor Acres broilers were randomly divided into 5 groups, 15 broilers for each group. The broilers were given with 5 kinds of diets such as the basal diet, 400 ?g/kg AFB? supplement without feed additive, and 200, 400, 800 ?g/kg AFB? supplement with 0.15% feed additive. The feeding experimental period was 30 d, which was used to determine production performance of broilers. In addition, serum, liver and chest muscle were selected for measuring AFB? residues, gene expressions, microscopic and antioxidant analyses. The results showed that adding 0.15% feed additive in broiler diets could significantly relieve the negative effect of AFB? on chicken's production performance and nutrient metabolic rates (P<0.05). It could also improve AFB? metabolism, hepatic cell structure, antioxidant activity, and many hepatic enzyme gene expressions involved in oxidoreductase, apoptosis, cell growth, immune system and metabolic process (P<0.05). It could be concluded that the feed additive was able to degrade AFB? and improve animal production. PMID:23831311

  19. Cloning, characterization, and expression in Escherichia coli of a gene encoding Listeria seeligeri catalase, a bacterial enzyme highly homologous to mammalian catalases.

    PubMed Central

    Haas, A; Brehm, K; Kreft, J; Goebel, W

    1991-01-01

    A gene coding for catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase; EC 1.11.1.6) of the gram-positive bacterium Listeria seeligeri was cloned from a plasmid library of EcoRI-digested chromosomal DNA, with Escherichia coli DH5 alpha as a host. The recombinant catalase was expressed in E. coli to an enzymatic activity approximately 50 times that of the combined E. coli catalases. The nucleotide sequence was determined, and the deduced amino acid sequence revealed 43.2% amino acid sequence identity between bovine liver catalase and L. seeligeri catalase. Most of the amino acid residues which are involved in catalytic activity, the formation of the active center accession channel, and heme binding in bovine liver catalase were also present in L. seeligeri catalase at the corresponding positions. The recombinant protein contained 488 amino acid residues and had a calculated molecular weight of 55,869. The predicted isoelectric point was 5.0. Enzymatic and genetic analyses showed that there is most probably a single catalase of this type in L. seeligeri. A perfect 21-bp inverted repeat, which was highly homologous to previously reported binding sequences of the Fur (ferric uptake regulon) protein of E. coli, was detected next to the putative promoter region of the L. seeligeri catalase gene. Images PMID:1860824

  20. The tumour suppressor gene WWOX is mutated in autosomal recessive cerebellar ataxia with epilepsy and mental retardation

    PubMed Central

    Mallaret, Martial; Synofzik, Matthis; Lee, Jaeho; Sagum, Cari A.; Mahajnah, Muhammad; Sharkia, Rajech; Drouot, Nathalie; Renaud, Mathilde; Klein, Fabrice A. C.; Anheim, Mathieu; Tranchant, Christine; Mignot, Cyril; Mandel, Jean-Louis; Bedford, Mark; Bauer, Peter; Salih, Mustafa A.; Schüle, Rebecca; Schöls, Ludger; Aldaz, C. Marcelo

    2014-01-01

    We previously localized a new form of recessive ataxia with generalized tonic-clonic epilepsy and mental retardation to a 19 Mb interval in 16q21-q23 by homozygosity mapping of a large consanguineous Saudi Arabian family. We now report the identification by whole exome sequencing of the missense mutation changing proline 47 into threonine in the first WW domain of the WW domain containing oxidoreductase gene, WWOX, located in the linkage interval. Proline 47 is a highly conserved residue that is part of the WW motif consensus sequence and is part of the hydrophobic core that stabilizes the WW fold. We demonstrate that proline 47 is a key amino acid essential for maintaining the WWOX protein fully functional, with its mutation into a threonine resulting in a loss of peptide interaction for the first WW domain. We also identified another highly conserved homozygous WWOX mutation changing glycine 372 to arginine in a second consanguineous family. The phenotype closely resembled the index family, presenting with generalized tonic-clonic epilepsy, mental retardation and ataxia, but also included prominent upper motor neuron disease. Moreover, we observed that the short-lived Wwox knock-out mouse display spontaneous and audiogenic seizures, a phenotype previously observed in the spontaneous Wwox mutant rat presenting with ataxia and epilepsy, indicating that homozygous WWOX mutations in different species causes cerebellar ataxia associated with epilepsy. PMID:24369382

  1. Connectionist Approaches for Predicting Mouse Gene Function from Gene Expression

    E-print Network

    Bonner, Anthony

    Connectionist Approaches for Predicting Mouse Gene Function from Gene Expression Emad Andrews. emad@cs.toronto.edu Abstract. Identifying gene function has many useful applications especially in Gene Therapy. Identifying gene function based on gene expression data is much easier in prokaryotes than

  2. 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.

  3. UniGene

    NSDL National Science Digital Library

    Created by the National Center for Biotechnology Information, UniGene is "an experimental system for automatically partitioning GenBank sequences into a non-redundant set of gene-oriented clusters." In addition to gene sequences, this Web site also offers thousands of novel expressed sequence tag (EST) sequences, a useful gene discovery resource. Organisms currently cataloged include human, rat, mouse, cow, zebrafish, clawed frog, fruitfly, mosquito, wheat, rice, barley, maize, and cress. Users may also access the Digital Differential Display to compare gene expression fingerprints for cancer cells and their normal counterparts. Other Web site features include query tips, FAQs, and relevant external links.

  4. [Imprinted genes in plants].

    PubMed

    Zhang, Li-Geng; Yang, Ruo-Fei; Fu, Feng-Ling; Li, Wan-Chen

    2010-12-01

    The expression of imprinted genes is regulated by epigenetic mechanism. In plant endosperm, the allele of imprinted genes is expressed in a pattern of parent-of-origin-dependent. The expression of imprinted genes plays essential roles in the development of embryos and their annexe structures, as well as seed size, reproductive barriers and apomixis. Along with the progress of plant epigenetic research, the exploration of imprinted genes is becoming hotspot in epigenetic research. This review focused on the parental conflict theory about the origin of imprinted genes, and the latest research advances in expression regulation mechanism of plant imprinted genes, using the examples of the important imprinted genes MEA, FIS2, FWA, MPC, and PHE1 in Arabidopsis, and FIEI and FIE2 in maize. PMID:21513148

  5. Oncogenes, genes, and growth factors

    SciTech Connect

    Guroff, G.

    1989-01-01

    This book contains 12 chapters. Some of the chapter titles are: The Epidermal Growth Factor Receptor Gene; Structure and Expression of the Nerve Growth Factor Gene; The Erythropoietin Gene; The Interleukin-2 Gene; The Transferrin Gene; and The Transferrin Receptor Gene.

  6. 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

  7. Visualizing differentially expressed genes

    NASA Astrophysics Data System (ADS)

    Islam, Atiq U.; Iftekharuddin, Khan M.; Russomanno, David J.

    2006-08-01

    Identification of significantly differentially expressed genes (marker genes) among sample groups is a central issue in microarray analysis. This identification is important to understand the molecular pathway of diseases. Many statistical methods have been proposed to locate marker genes. These methods depend on a cutoff value for selection. A tightfisted cutoff may omit some of the important marker genes, whereas a generous threshold increases the number of false positives. Although robust models for identifying marker genes more accurately is an area of intense research, effective tools for the evaluation of results is often ignored in the literature. Despite the robustness of many of these methods, there is always some probability of false positives. In this paper, we propose a novel approach that exploits parallel coordinates to visualize the gene expression patterns so that one can compare the expression level changes of the marker genes between sample groups and determine whether the selected marker genes are valid. Such visualization is useful to measure the validity of the marker gene selection process as well as to fine tune the parameters of a particular method. A prediction method based on the selected marker genes is used to measure the reliability of our process.

  8. Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis

    Microsoft Academic Search

    Sung-Jin Kim; Mi-Ran Kim; Diana L. Bedgar; Syed G. A. Moinuddin; Claudia L. Cardenas; Laurence B. Davin; Chulhee Kang; Norman G. Lewis

    2004-01-01

    Of 17 genes annotated in the Arabidopsis genome database as cinnamyl alcohol dehydrogenase (CAD) homologues, an in silico analysis revealed that 8 genes were misannotated. Of the remaining nine, six were catalytically competent for NADPH-dependent reduction of p-coumaryl, caffeyl, coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes, whereas three displayed very low activity and only at very high substrate concentrations. Of the nine

  9. Genes and Disease

    NSDL National Science Digital Library

    1998-01-01

    The National Center for Biotechnology Information of the National Library of Medicine (part of the National Institutes of Health) has posted this webpage, Genes and Disease, which provides information "for some 60 diseases associated with specific genes, and has links to the 1998 Gene Map as well as to PubMed, protein sequences, Online Mendelian Inheritance in Man, and associations related to each disease."

  10. Gene therapy for restenosis

    Microsoft Academic Search

    Roy C. Smith; Kenneth Walsh

    2000-01-01

    This review provides an overview of candidate genes that are currently being evaluated for genetic strategies in vascular\\u000a gene therapy. We discuss treatment strategies that have proven efficacious in limiting postinterventional restenosis through\\u000a evaluation with in vivo model systems. The candidate strategies utilize genes that are either cytotoxic, regulate vascular\\u000a smooth muscle cell differentiation or proliferation. In addition, we review

  11. DNA, Genes and Chromosomes

    NSDL National Science Digital Library

    Mrs. Fomby

    2007-11-07

    Today you will learn about the parts of DNA and what DNA, genes and chromosomes are. Today you will learn what DNA, genes and chromosomes are and the parts of the DNA molecule. Look at all of the websites, take whatever notes you need to. At the end of the assignment, be able to describle DNA, the parts of DNA, genes and chromosomes. Covers Biology Core Curriculum, ...

  12. A Diet Rich in Olive Oil Phenolics Reduces Oxidative Stress in the Heart of SAMP8 Mice by Induction of Nrf2-Dependent Gene Expression

    PubMed Central

    Bayram, Banu; Ozcelik, Beraat; Grimm, Stefanie; Roeder, Thomas; Schrader, Charlotte; Ernst, Insa M.A.; Wagner, Anika E.; Grune, Tilman; Frank, Jan

    2012-01-01

    Abstract A Mediterranean diet rich in olive oil has been associated with health benefits in humans. It is unclear if and to what extent olive oil phenolics may mediate these health benefits. In this study, we fed senescence-accelerated mouse-prone 8 (SAMP8, n=11 per group) semisynthetic diets with 10% olive oil containing either high (HP) or low amounts of olive oil phenolics (LP) for 4.5 months. Mice consuming the HP diet had significantly lower concentrations of the oxidative damage markers thiobarbituric acid–reactive substances and protein carbonyls in the heart, whereas proteasomal activity was similar in both groups. Nrf2-dependent gene expression may be impaired during the aging process. Therefore, we measured Nrf2 and its target genes glutathione-S-transferase (GST), ?-glutamyl cysteine synthetase (?-GCS), nicotinamide adenine dinucleotide phosphate [NAD(P)H]:quinone oxidoreductase (NQO1), and paraoxonase-2 (PON2) in the hearts of these mice. Nrf2 as well as GST, ?-GCS, NQO1, and PON2 mRNA levels were significantly higher in heart tissue of the HP as compared to the LP group. The HP-fed mice had significantly higher PON1 activity in serum compared to those receiving the LP diet. Furthermore, HP feeding increased relative SIRT1 mRNA levels. Additional mechanistic cell culture experiments were performed, and they suggest that the olive oil phenolic hydroxytyrosol present in the HP oil may be responsible for the induction of Nrf2-dependent gene expression and the increase in PON activity. In conclusion, a diet rich in olive oil phenolics may prevent oxidative stress in the heart of SAMP8 mice by modulating Nrf2-dependent gene expression. PMID:22236145

  13. History of gene therapy.

    PubMed

    Wirth, Thomas; Parker, Nigel; Ylä-Herttuala, Seppo

    2013-08-10

    Two decades after the initial gene therapy trials and more than 1700 approved clinical trials worldwide we not only have gained much new information and knowledge regarding gene therapy in general, but also learned to understand the concern that has persisted in society. Despite the setbacks gene therapy has faced, success stories have increasingly emerged. Examples for these are the positive recommendation for a gene therapy product (Glybera) by the EMA for approval in the European Union and the positive trials for the treatment of ADA deficiency, SCID-X1 and adrenoleukodystrophy. Nevertheless, our knowledge continues to grow and during the course of time more safety data has become available that helps us to develop better gene therapy approaches. Also, with the increased understanding of molecular medicine, we have been able to develop more specific and efficient gene transfer vectors which are now producing clinical results. In this review, we will take a historical view and highlight some of the milestones that had an important impact on the development of gene therapy. We will also discuss briefly the safety and ethical aspects of gene therapy and address some concerns that have been connected with gene therapy as an important therapeutic modality. PMID:23618815

  14. Refining discordant gene trees

    PubMed Central

    2014-01-01

    Background Evolutionary studies are complicated by discordance between gene trees and the species tree in which they evolved. Dealing with discordant trees often relies on comparison costs between gene and species trees, including the well-established Robinson-Foulds, gene duplication, and deep coalescence costs. While these costs have provided credible results for binary rooted gene trees, corresponding cost definitions for non-binary unrooted gene trees, which are frequently occurring in practice, are challenged by biological realism. Result We propose a natural extension of the well-established costs for comparing unrooted and non-binary gene trees with rooted binary species trees using a binary refinement model. For the duplication cost we describe an efficient algorithm that is based on a linear time reduction and also computes an optimal rooted binary refinement of the given gene tree. Finally, we show that similar reductions lead to solutions for computing the deep coalescence and the Robinson-Foulds costs. Conclusion Our binary refinement of Robinson-Foulds, gene duplication, and deep coalescence costs for unrooted and non-binary gene trees together with the linear time reductions provided here for computing these costs significantly extends the range of trees that can be incorporated into approaches dealing with discordance. PMID:25434729

  15. 'COGNITIVE GENES 'R EVEAL HIGHER CODON COMPLEXITY THAN 'SOMATIC GENES

    Microsoft Academic Search

    Christoph S. Herrmann; Wolfgang S. Herrmann

    In this article we want to apply the concept of complexity to the analysis and com- parison of genes. A multitude of genes has been identified coding somatic function. Recently the analysis of mental disorders yielded insights about genes coding cogni- tive functions. According to the theory of evolution they evolved from other genes through mutation. Therefore, 'cognitive genes' and

  16. Testing Groups of Genes Part II: Scoring Gene Ontology Terms

    E-print Network

    Spang, Rainer

    ­ Decorrelating the GO (elim, weight), Alexa et al. (2006) ­ Parent-child approach, Grossmann et al. (2007 the genes of its child ­ a node can contain genes that are not found in the children · a subset of genes that we call significant genes (differentially expressed genes) Goal: · find the nodes from the graph

  17. Gene 254 (2000) 253263 www.elsevier.com/locate/gene

    E-print Network

    Horvitz, H. Robert

    Gene 254 (2000) 253­263 www.elsevier.com/locate/gene The C. elegans gene lin-9,which acts in an Rb The Caenorhabditis elegans gene lin-9 functions in an Rb-related pathway that acts antagonistically to a receptor are multipotent and can adopt either vulval or non- one gene in each pathway is mutated do the cells P3.p

  18. Using Gene Expression Noise to Understand Gene Regulation

    E-print Network

    Munsky, Brian

    REVIEW Using Gene Expression Noise to Understand Gene Regulation Brian Munsky,1 * Gregor Neuert,2 environments display variable phenotypes. Stochastic gene expression, or gene expression "noise," has been and messenger RNA levels, and they have discovered strong connections between noise and gene regulation

  19. Gene 256 (2000) 245252 www.elsevier.com/locate/gene

    E-print Network

    Schnaufer, Achim

    Gene 256 (2000) 245­252 www.elsevier.com/locate/gene Redundant and non-functional guide RNA genes in Trypanosoma brucei are a consequence of multiple genes per minicircle Nicholas J. Savill *, Paul G. Higgs classes that contain several functional gRNA genes can be lost from the population via drift and replaced

  20. Gene 259 (2000) 129138 www.elsevier.com/locate/gene

    E-print Network

    Hedges, Blair

    Gene 259 (2000) 129­138 www.elsevier.com/locate/gene Evolutionary history of the enolase gene tissue-specific isozymes encoded by three genes: alpha (a), beta (b), and gamma (c) enolase. Limited taxonomic sampling of enolase has obscured the timing of gene duplication events. To help clarify

  1. The Gene Ontology (GO) database and informatics Gene Ontology Consortium*

    E-print Network

    Botstein, David

    The Gene Ontology (GO) database and informatics resource Gene Ontology Consortium* GO-EBI, EMBL and Accepted September 12, 2003 ABSTRACT The Gene Ontology (GO) project (http://www. geneontology and cellular biology and are freely available for community use in the annotation of genes, gene products

  2. Gene 252 (2000) 8393 www.elsevier.com/locate/gene

    E-print Network

    Aksoy, Serap

    Gene 252 (2000) 83­93 www.elsevier.com/locate/gene A family of genes with growth factor 45% amino acid identity and 61% similarity to one another. Both genes are preferentially expressed of adults as well as in puparia, while neither gene is expressed during the larval developmental stages

  3. Differentially expressed genes between drought-tolerant and drought-sensitive barley genotypes in response to drought stress during the reproductive stage.

    PubMed

    Guo, Peiguo; Baum, Michael; Grando, Stefania; Ceccarelli, Salvatore; Bai, Guihua; Li, Ronghua; von Korff, Maria; Varshney, Rajeev K; Graner, Andreas; Valkoun, Jan

    2009-01-01

    Drought tolerance is a key trait for increasing and stabilizing barley productivity in dry areas worldwide. Identification of the genes responsible for drought tolerance in barley (Hordeum vulgare L.) will facilitate understanding of the molecular mechanisms of drought tolerance, and also facilitate the genetic improvement of barley through marker-assisted selection or gene transformation. To monitor the changes in gene expression at the transcriptional level in barley leaves during the reproductive stage under drought conditions, the 22K Affymetrix Barley 1 microarray was used to screen two drought-tolerant barley genotypes, Martin and Hordeum spontaneum 41-1 (HS41-1), and one drought-sensitive genotype Moroc9-75. Seventeen genes were expressed exclusively in the two drought-tolerant genotypes under drought stress, and their encoded proteins may play significant roles in enhancing drought tolerance through controlling stomatal closure via carbon metabolism (NADP malic enzyme, NADP-ME, and pyruvate dehydrogenase, PDH), synthesizing the osmoprotectant glycine-betaine (C-4 sterol methyl oxidase, CSMO), generating protectants against reactive-oxygen-species scavenging (aldehyde dehydrogenase,ALDH, ascorbate-dependent oxidoreductase, ADOR), and stabilizing membranes and proteins (heat-shock protein 17.8, HSP17.8, and dehydrin 3, DHN3). Moreover, 17 genes were abundantly expressed in Martin and HS41-1 compared with Moroc9-75 under both drought and control conditions. These genes were possibly constitutively expressed in drought-tolerant genotypes. Among them, seven known annotated genes might enhance drought tolerance through signalling [such as calcium-dependent protein kinase (CDPK) and membrane steroid binding protein (MSBP)], anti-senescence (G2 pea dark accumulated protein, GDA2), and detoxification (glutathione S-transferase, GST) pathways. In addition, 18 genes, including those encoding Delta(l)-pyrroline-5-carboxylate synthetase (P5CS), protein phosphatase 2C-like protein (PP2C), and several chaperones, were differentially expressed in all genotypes under drought; thus they were more likely to be general drought-responsive genes in barley. These results could provide new insights into further understanding of drought-tolerance mechanisms in barley. PMID:19561048

  4. Protection by the NDI1 Gene against Neurodegeneration in a Rotenone Rat Model of Parkinson's Disease

    PubMed Central

    Marella, Mathieu; Seo, Byoung Boo; Nakamaru-Ogiso, Eiko; Greenamyre, J. Timothy; Matsuno-Yagi, Akemi; Yagi, Takao

    2008-01-01

    It is widely recognized that mitochondrial dysfunction, most notably defects in the NADH-quinone oxidoreductase (complex I), is closely related to the etiology of sporadic Parkinson's disease (PD). In fact, rotenone, a complex I inhibitor, has been used for establishing PD models both in vitro and in vivo. A rat model with chronic rotenone exposure seems to reproduce pathophysiological conditions of PD more closely than acute mouse models as manifested by neuronal cell death in the substantia nigra and Lewy body-like cytosolic aggregations. Using the rotenone rat model, we investigated the protective effects of alternative NADH dehydrogenase (Ndi1) which we previously demonstrated to act as a replacement for complex I both in vitro and in vivo. A single, unilateral injection of recombinant adeno-associated virus carrying the NDI1 gene into the vicinity of the substantia nigra resulted in expression of the Ndi1 protein in the entire substantia nigra of that side. It was clear that the introduction of the Ndi1 protein in the substantia nigra rendered resistance to the deleterious effects caused by rotenone exposure as assessed by the levels of tyrosine hydroxylase and dopamine. The presence of the Ndi1 protein also prevented cell death and oxidative damage to DNA in dopaminergic neurons observed in rotenone-treated rats. Unilateral protection also led to uni-directional rotation of the rotenone-exposed rats in the behavioral test. The present study shows, for the first time, the powerful neuroprotective effect offered by the Ndi1 enzyme in a rotenone rat model of PD. PMID:18197244

  5. Evolutionary Origin of Orphan Genes

    E-print Network

    Evolutionary Origin of Orphan Genes Diethard Tautz, Max-Planck Institute for Evolutionary Biology they turn out to be useful. Orphan genes may have played key roles in generating lineage- specific with a gene family that was known before. Such genes were originally called `orphan' genes (Dujon, 1996

  6. 4. AERIAL VIEW OF GENE WASH RESERVOIR AND GENE CAMP ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    4. AERIAL VIEW OF GENE WASH RESERVOIR AND GENE CAMP LOOKING SOUTHWEST. DAM AND SPILLWAY VISIBLE IN BOTTOM OF PHOTO. - Gene Wash Reservoir & Dam, 2 miles west of Parker Dam, Parker Dam, San Bernardino County, CA

  7. GENE EXPRESSION NETWORKS

    EPA Science Inventory

    "Gene expression network" is the term used to describe the interplay, simple or complex, between two or more gene products in performing a specific cellular function. Although the delineation of such networks is complicated by the existence of multiple and subtle types of intera...

  8. The Gene Ontology Consortium

    Microsoft Academic Search

    Michael Ashburner; Catherine A. Ball; Judith A. Blake; David Botstein; Heather Butler; J. Michael Cherry; Allan P. Davis; Kara Dolinski; Selina S. Dwight; Janan T. Eppig; Midori A. Harris; David P. Hill; Laurie Issel-Tarver; Andrew Kasarskis; Suzanna Lewis; John C. Matese; Joel E. Richardson; Martin Ringwald; Gerald M. Rubin; Gavin Sherlock

    2000-01-01

    Genomic sequencing has made it clear that a large fraction of the genes specifying the core biological functions are shared by all eukaryotes. Knowledge of the biological role of such shared proteins in one organism can often be transferred to other organisms. The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied

  9. Antitumor gene therapy

    Microsoft Academic Search

    C. Cirielli; M. C. Capogrossi; A. Passaniti

    1997-01-01

    Gene therapy as an anti-tumor strategy is becoming a powerful tool for cytokine delivery to inhibit the growth of many tumors. Several delivery systems are being utilized and designed for the expression of specific genes to achieve a therapeutic result. Liposomes, retroviral vectors, and adenoviral vectors have all been used and eventual clinical application may depend on the type of

  10. What Is a Gene?

    MedlinePLUS

    ... medicine — so new that scientists are still doing experiments to see if it works. It uses the technology of genetic engineering to treat a disease caused by a gene that has changed in some way. One method being tested is replacing sick genes with healthy ...

  11. Gene therapy for newborns

    Microsoft Academic Search

    DONALD B. KOHN; ROBERTSON PARKMAN

    Application of gene therapy to treat genetic and infectious diseases may have several ad- vantages if performed in newborns. Because of the minimal adverse effect of the underlying disease on cells of the newborn, the relatively small size of in- fants, and the large amount of future growth, gene therapy may be more successful in newborns than in older children

  12. Transcriptome analysis of candidate genes and signaling pathways associated with light-induced brown film formation in Lentinula edodes.

    PubMed

    Tang, Li-Hua; Jian, Hua-Hua; Song, Chun-Yan; Bao, Da-Peng; Shang, Xiao-Dong; Wu, Da-Qiang; Tan, Qi; Zhang, Xue-Hong

    2013-06-01

    High-throughput Illumina RNA-seq was used for deep sequencing analysis of the transcriptome of poly(A)+ RNA from mycelium grown under three different conditions: 30 days darkness (sample 118), 80 days darkness (313W), and 30 days darkness followed by 50 days in the light (313C), in order to gain insight into the molecular mechanisms underlying the process of light-induced brown film (BF) formation in the edible mushroom, Lentinula edodes. Of the three growth conditions, BF formation occurred in 313C samples only. Approximately 159.23 million reads were obtained, trimmed, and de novo assembled into 31,511 contigs with an average length of 1,746 bp and an N 50 of 2,480 bp. Based on sequence orientations determined by a BLASTX search against the NR, Swiss-Prot, COG, and KEGG databases, 24,246 (76.9 %) contigs were assigned putative descriptions. Comparison of 313C/118 and 313C/313W expression profiles revealed 3,958 and 5,651 significantly differentially expressed contigs (DECs), respectively. Annotation using the COG database revealed that candidate genes for light-induced BF formation encoded proteins linked to light reception (e.g., WC-1, WC-2, phytochrome), light signal transduction pathways (e.g., two-component phosphorelay system, mitogen-activated protein kinase pathway), and pigment formation (e.g., polyketide synthase, O-methyltransferase, laccase, P450 monooxygenase, oxidoreductase). Several DECs were validated using quantitative real-time polymerase chain reaction. Our report is the first to identify genes associated with light-induced BF formation in L. edodes and represents a valuable resource for future genomic studies on this commercially important mushroom. PMID:23624682

  13. Entrez Gene: gene-centered information at NCBI

    Microsoft Academic Search

    Donna R. Maglott; James Ostell; Kim D. Pruitt; Tatiana A. Tatusova

    2005-01-01

    Entrez Gene (www.ncbi.nlm.nih.gov\\/entrez\\/query. fcgi?db=gene) is NCBI's database for gene-specific information. Entrez Gene includes records from genomes that have been completely sequenced, that have an active research community to con- tribute gene-specific information or that are sched- uled for intense sequence analysis. The content of Entrez Gene represents the result of both curation and automated integration of data from NCBI's Reference

  14. GeneEd: Genetics, Education, Discovery

    NSDL National Science Digital Library

    The GeneEd website was created by the National Library of Medicine (NLM), the National Human Genome Research Institute (NHGRI), and the National Institutes of Health (NIH) as a helpful resource for the teaching and learning of genetics. On the site, visitors can find labs and experiments, fact sheets, and teacher resources on topics including DNA forensics, genetic conditions, evolution, and biostatistics. First-time visitors will want to start their journey by looking over the Topics tab at the top of the page. There are 40 different thematic areas here consisting of articles, video clips, webcasts, and links to additional quality resources vetted by the GeneEd web team. The Labs & Experiments section includes virtual labs that explore the genetics of different organisms as well as links to resources provided by the Howard Hughes Medical Institute and Cold Spring Harbor Laboratory. Young people may also wish to take a look at the Careers in Genetics section as it features interviews with scientists that will inspire and delight.

  15. Genes from scratch – the evolutionary fate of de novo genes

    PubMed Central

    Schlötterer, Christian

    2015-01-01

    Although considered an extremely unlikely event, many genes emerge from previously noncoding genomic regions. This review covers the entire life cycle of such de novo genes. Two competing hypotheses about the process of de novo gene birth are discussed as well as the high death rate of de novo genes. Despite the high death rate, some de novo genes are retained and remain functional, even in distantly related species, through their integration into gene networks. Further studies combining gene expression with ribosome profiling in multiple populations across different species will be instrumental for an improved understanding of the evolutionary processes operating on de novo genes. PMID:25773713

  16. Molecular characterization of berberine bridge enzyme genes from opium poppy

    Microsoft Academic Search

    Peter J. Facchini; Catherine Penzes; Alison C. Johnson; Dean Bull

    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) ((SI-reticu1ine:oxygen oxidoreductase (methylene bridge forming), EC 1.5.3.9) catalyzes the stereospecific conversion of the N-methyl moiety of (9-reticuline into the berberine bridge carbon of (9-scoulerine and represents the first committed step in the pathway

  17. Gene expression changes linked to antimicrobial resistance, oxidative stress, iron depletion and retained motility are observed when Burkholderia cenocepacia grows in cystic fibrosis sputum

    PubMed Central

    Drevinek, Pavel; Holden, Matthew TG; Ge, Zhaoping; Jones, Andrew M; Ketchell, Ian; Gill, Ryan T; Mahenthiralingam, Eshwar

    2008-01-01

    Background Bacteria from the Burkholderia cepacia complex (Bcc) are the only group of cystic fibrosis (CF) respiratory pathogens that may cause death by an invasive infection known as cepacia syndrome. Their large genome (> 7000 genes) and multiple pathways encoding the same putative functions make virulence factor identification difficult in these bacteria. Methods A novel microarray was designed to the genome of Burkholderia cenocepacia J2315 and transcriptomics used to identify genes that were differentially regulated when the pathogen was grown in a CF sputum-based infection model. Sputum samples from CF individuals infected with the same B. cenocepacia strain as genome isolate were used, hence, other than a dilution into a minimal growth medium (used as the control condition), no further treatment of the sputum was carried out. Results A total of 723 coding sequences were significantly altered, with 287 upregulated and 436 downregulated; the microarray-observed expression was validated by quantitative PCR on five selected genes. B. cenocepacia genes with putative functions in antimicrobial resistance, iron uptake, protection against reactive oxygen and nitrogen species, secretion and motility were among the most altered in sputum. Novel upregulated genes included: a transmembrane ferric reductase (BCAL0270) implicated in iron metabolism, a novel protease (BCAL0849) that may play a role in host tissue destruction, an organic hydroperoxide resistance gene (BCAM2753), an oxidoreductase (BCAL1107) and a nitrite/sulfite reductase (BCAM1676) that may play roles in resistance to the host defenses. The assumptions of growth under iron-depletion and oxidative stress formulated from the microarray data were tested and confirmed by independent growth of B. cenocepacia under each respective environmental condition. Conclusion Overall, our first full transcriptomic analysis of B. cenocepacia demonstrated the pathogen alters expression of over 10% of the 7176 genes within its genome when it grows in CF sputum. Novel genetic pathways involved in responses to antimicrobial resistance, oxidative stress, and iron metabolism were revealed by the microarray analysis. Virulence factors such as the cable pilus and Cenocepacia Pathogenicity Island were unaltered in expression. However, B. cenocepacia sustained or increased expression of motility-associated genes in sputum, maintaining a potentially invasive phenotype associated with cepacia syndrome. PMID:18801206

  18. Ectopic expression of the WWOX gene suppresses stemness of human ovarian cancer stem cells

    PubMed Central

    YAN, HONG CHAO; XU, JUN; FANG, LI SHA; QIU, YING YING; LIN, XIAO MAN; HUANG, HONG XIANG; HAN, QIU YU

    2015-01-01

    The present study aimed to investigate the effects of the WW domain-containing oxidoreductase (WWOX) gene on the stem cell properties of human ovarian cancer stem cells. A eukaryotic expression vector containing the WWOX gene was transfected into human ovarian cancer stem cells and Western blotting was used to assess the expression of WWOX protein in the transfected cells compared with the control cells (untransfected cells and cells transfected with the empty vector). The self-renewal abilities of these three types of stem cells was also assessed in vitro. To monitor changes in their differentiation potential, cells were cultured in medium supplemented with serum, and the expression of specific stem cell markers was determined. Drug-sensitivity tests were used to measure the sensitivity of the stem cells to cisplatin, doxorubicin, and mitoxantrone. The cells were also transplanted into non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice to determine the changes in their tumorigenicity in vivo. Cells transfected with the WWOX-expressing plasmid stably expressed WWOX protein, while no WWOX protein was detected in control cells. Compared with the two types of control cells, WWOX-expressing stem cells manifested significantly reduced self-renewal ability. Compared with control cells, the expression levels of stem cell markers, including CD133, CD117, ATP-binding cassette sub-family G member 2, Nanog, octamer-binding transcription factor 4 and breast cancer resistance protein, were significantly lower in WWOX-expressing cells, while the level of the differentiation marker E-cadherin was significantly higher in WWOX-expressing cells. Furthermore, WWOX-expressing cells were more sensitive to treatment with cisplatin, doxorubicin and mitoxantrone. In NOD/SCID mice, the tumorigenicity of WWOX-expressing cells was significantly lower compared with that of control cells. The results indicate that the tumor suppressor WWOX suppresses stem cell properties in cancer stem cells, including self-renewal ability, differentiation potential, in vivo tumorigenic capability, high-level expression of stem cell genes and multidrug resistance. PMID:25789010

  19. Dopamine genes and ADHD.

    PubMed

    Swanson, J M; Flodman, P; Kennedy, J; Spence, M A; Moyzis, R; Schuck, S; Murias, M; Moriarity, J; Barr, C; Smith, M; Posner, M

    2000-01-01

    Family, twin, and adoption studies have documented a strong genetic basis for ADHD/HKD, but these studies do not identify specific genes linked to the disorder. Molecular genetic studies can identify allelic variations of specific genes that are functionally associated with ADHD/HKD, and dopamine genes have been the initial candidates based on the site of action of the stimulants drugs, which for a half century have provided the primary pharmacological treatment for ADHD/HKD. Two candidate dopamine genes have been investigated and reported to be associated with ADHD/HKD: the dopamine transporter (DAT1) gene [Cook et al., American Journal of Human Genetics 1995;56:993-998, Gill et al., Molecular Psychiatry 1997;2:311-313] and the dopamine receptor D4 (DRD4) gene [LaHoste et al., Molecular Psychiatry 1996;1:121-124: Smalley et al., 1998;3:427-430; Swanson et al., Molecular Psychiatry 1998;3:38-41]. Speculative hypotheses [Swanson and Castellanos, NIH Consensus Development Conference: Diagnosis and Treatment of Attention Deficit Hyperactivity Disorder, November 1998. p. 37-42] have suggested that specific alleles of these dopamine genes may alter dopamine transmission in the neural networks implicated in ADHD/HKD (e.g. that the 10-repeat allele of the DAT1 gene may be associated with hyperactive re-uptake of dopamine or that the 7-repeat allele of the DRD4 gene may be associated with a subsensitive postsynaptic receptor). These and other variants of the dopamine hypothesis of ADHD will be discussed. PMID:10654656

  20. TIGR Drosophila Gene Index

    NSDL National Science Digital Library

    1999-01-01

    TIGR, The Institute for Genomic Research, has announced the release of the Drosophila Gene Index (DGI). The Drosophila Gene Index, which may be searched by Nucleotide or Protein Sequence, Identifier (TC, ET, EST, GB) Tissue, cDNA Library Name or cDNA Library Identifier(cat#), or Gene Product Name, contains some 50,500 total sequences (ET, EST, TC, and singletons). Data may be requested free of charge by "researchers at non-profit institutions using it for non-commercial purposes;" instructions are provided on-site.

  1. Genes and Social Behavior

    PubMed Central

    Robinson, Gene E.; Fernald, Russell D.; Clayton, David F.

    2011-01-01

    What specific genes and regulatory sequences contribute to the organization and functioning of brain circuits that support social behavior? How does social experience interact with information in the genome to modulate these brain circuits? Here we address these questions by highlighting progress that has been made in identifying and understanding two key “vectors of influence” that link genes, brain, and social behavior: 1) social information alters gene readout in the brain to influence behavior; and 2) genetic variation influences brain function and social behavior. We also briefly discuss how evolutionary changes in genomic elements influence social behavior and outline prospects for a systems biology of social behavior. PMID:18988841

  2. Oxygen at Nanomolar Levels Reversibly Suppresses Process Rates and Gene Expression in Anammox and Denitrification in the Oxygen Minimum Zone off Northern Chile

    PubMed Central

    Stewart, Frank J.; Thamdrup, Bo; De Brabandere, Loreto; Revsbech, Niels Peter; Ulloa, Osvaldo; Canfield, Don E.; DeLong, Edward F.

    2014-01-01

    ABSTRACT A major percentage (20 to 40%) of global marine fixed-nitrogen loss occurs in oxygen minimum zones (OMZs). Concentrations of O2 and the sensitivity of the anaerobic N2-producing processes of anammox and denitrification determine where this loss occurs. We studied experimentally how O2 at nanomolar levels affects anammox and denitrification rates and the transcription of nitrogen cycle genes in the anoxic OMZ off Chile. Rates of anammox and denitrification were reversibly suppressed, most likely at the enzyme level. Fifty percent inhibition of N2 and N2O production by denitrification was achieved at 205 and 297 nM O2, respectively, whereas anammox was 50% inhibited at 886 nM O2. Coupled metatranscriptomic analysis revealed that transcripts encoding nitrous oxide reductase (nosZ), nitrite reductase (nirS), and nitric oxide reductase (norB) decreased in relative abundance above 200 nM O2. This O2 concentration did not suppress the transcription of other dissimilatory nitrogen cycle genes, including nitrate reductase (narG), hydrazine oxidoreductase (hzo), and nitrite reductase (nirK). However, taxonomic characterization of transcripts suggested inhibition of narG transcription in gammaproteobacteria, whereas the transcription of anammox narG, whose gene product is likely used to oxidatively replenish electrons for carbon fixation, was not inhibited. The taxonomic composition of transcripts differed among denitrification enzymes, suggesting that distinct groups of microorganisms mediate different steps of denitrification. Sulfide addition (1 µM) did not affect anammox or O2 inhibition kinetics but strongly stimulated N2O production by denitrification. These results identify new O2 thresholds for delimiting marine nitrogen loss and highlight the utility of integrating biogeochemical and metatranscriptomic analyses. PMID:25352619

  3. Temporal and spatial dynamics of nrf2-antioxidant response elements mediated gene targets in cortex and hippocampus after controlled cortical impact traumatic brain injury in mice.

    PubMed

    Miller, Darren M; Wang, Juan A; Buchanan, Ashley K; Hall, Edward D

    2014-07-01

    The pathophysiological importance of oxidative damage after traumatic brain injury (TBI) has been extensively demonstrated. The transcription factor nuclear factor erythoid related factor 2 (Nrf2) mediates antioxidant and cytoprotective genes by binding to antioxidant response elements (ARE) present in nuclear DNA. In this study, we characterized the time course of Nrf2-ARE-mediated expression in the cortex and hippocampus using a unilateral controlled cortical impact model of focal TBI. Ipsilateral hippocampal and cortical tissue was collected for Western-blot protein analysis (n=6/group) or quantitative reverse transcription-polymerase chain reaction for mRNA (n=3/group) at 3, 6, 12, 24, 48, and 72?h or 1 week post-injury. Multiple genes mediated by Nrf2-ARE were altered post-TBI. Specifically, Nrf2 mRNA increased significantly post-TBI at 48 and 72?h in the cortex and at 48 and 72?h and 1 week in the hippocampus with a coincident increase in glial fibrillary acidic protein mRNA, thereby implying this response is likely occurring in astrocytes. Presumably linked to Nrf2 activation, heme-oxygenase-1, nicotinamide adenine dinucleotide phosphate-quinone-oxidoreductase 1, glutathione reductase, and catalase mRNA overlap throughout the post-injury time course. This study demonstrates the first evidence of such changes during the first week after focal TBI and that increases in expression of some Nrf2-ARE-mediated cytoprotective genes are not observed until 24-48?h post-injury. Unfortunately, this does not precede, but rather coincides with, the occurrence of lipid peroxidative damage. This is the first known comparison between the time course of peroxidative damage and that of Nrf2-ARE activation during the first week post-TBI. These results underscore the necessity to discover pharmacological agents to accelerate and amplify Nrf2-ARE-mediated expression early post-TBI. PMID:24628668

  4. Positive Darwinian Selection after Gene Duplication in Primate Ribonuclease Genes

    Microsoft Academic Search

    Jianzhi Zhang; Helene F. Rosenberg; Masatoshi Nei

    1998-01-01

    Evolutionary mechanisms of origins of new gene function have been a subject of long-standing debate. Here we report a convincing case in which positive Darwinian selection operated at the molecular level during the evolution of novel function by gene duplication. The genes for eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) in primates belong to the ribonuclease gene family, and

  5. Harnessing Gene Expression Networks to Prioritize Candidate Epileptic Encephalopathy Genes

    PubMed Central

    Oliver, Karen L.; Lukic, Vesna; Thorne, Natalie P.; Berkovic, Samuel F.; Scheffer, Ingrid E.; Bahlo, Melanie

    2014-01-01

    We apply a novel gene expression network analysis to a cohort of 182 recently reported candidate Epileptic Encephalopathy genes to identify those most likely to be true Epileptic Encephalopathy genes. These candidate genes were identified as having single variants of likely pathogenic significance discovered in a large-scale massively parallel sequencing study. Candidate Epileptic Encephalopathy genes were prioritized according to their co-expression with 29 known Epileptic Encephalopathy genes. We utilized developing brain and adult brain gene expression data from the Allen Human Brain Atlas (AHBA) and compared this to data from Celsius: a large, heterogeneous gene expression data warehouse. We show replicable prioritization results using these three independent gene expression resources, two of which are brain-specific, with small sample size, and the third derived from a heterogeneous collection of tissues with large sample size. Of the nineteen genes that we predicted with the highest likelihood to be true Epileptic Encephalopathy genes, two (GNAO1 and GRIN2B) have recently been independently reported and confirmed. We compare our results to those produced by an established in silico prioritization approach called Endeavour, and finally present gene expression networks for the known and candidate Epileptic Encephalopathy genes. This highlights sub-networks of gene expression, particularly in the network derived from the adult AHBA gene expression dataset. These networks give clues to the likely biological interactions between Epileptic Encephalopathy genes, potentially highlighting underlying mechanisms and avenues for therapeutic targets. PMID:25014031

  6. Gene 237 (1999) 403411 www.elsevier.com/locate/gene

    E-print Network

    Handelsman, Jo

    Gene 237 (1999) 403­411 www.elsevier.com/locate/gene Zwittermicin A biosynthetic cluster Elizabeth cereus. The nucleotide sequence of 2.7 kb of DNA flanking the zwittermicin A self-resistance gene, zma of genes that is involved in zwittermicin A biosynthesis, representing the first biosynthetic pathway

  7. A Gene Scrapbook A Tribute to Gene Loh

    E-print Network

    A Gene Scrapbook A Tribute to Gene Loh on the Occasion of His Retirement Feb 22, 2003 #12;The Early of Technology, 1961 #12;The Missing Years Not much is known about Gene's whereabouts between 1961 until his (probably kelp) for transport by sea. #12;Why did Gene leave Cornell? He got tired of shoveling all

  8. 5. OVERHEAD VIEW OF GENE CAMP LOOKING SOUTH. GENE PUMP ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. OVERHEAD VIEW OF GENE CAMP LOOKING SOUTH. GENE PUMP PLANT IS AT CENTER WITH ADMINISTRATIVE COMPLEX IN FOREGROUND AND RESIDENTIAL AREA BEYOND PLANT. - Gene Pump Plant, South of Gene Wash Reservoir, 2 miles west of Whitsett Pump Plant, Parker Dam, San Bernardino County, CA

  9. Gene 252 (2000) 137145 www.elsevier.com/locate/gene

    E-print Network

    Kimble, Judith

    Gene 252 (2000) 137­145 www.elsevier.com/locate/gene A HECT domain ubiquitin ligase closely related, and a catalytic HECT domain. A Caenorhabditis elegans gene was cloned encoding a HECT domain protein (CeWWP1 an embryonic lethal phenotype, despite the presence of at least six additional C. elegans genes encoding HECT

  10. Inferring gene orders from gene maps using the breakpoint distance

    E-print Network

    Paris-Sud XI, Université de

    Inferring gene orders from gene maps using the breakpoint distance Guillaume Blin1 , Eric Blais2- notation of chromosomes as ordered sequences of genes. Unfortunately, different genetic mapping techniques usually give rise to different maps with unequal gene content, and often containing sets of unordered

  11. Identifying Gene Regulatory Networks from Gene Expression Data

    E-print Network

    Filkov, Vladimir

    27 Identifying Gene Regulatory Networks from Gene Expression Data Vladimir Filkov University of California, Davis 27.1 Introduction................................ ........... 27-1 27.2 Gene Networks ............................ ........... 27-2 Definition · Biological Properties · Utility 27.3 Gene Expression: Data and Analysis

  12. Gene 240 (1999) 4555 www.elsevier.com/locate/gene

    E-print Network

    Trinkle-Mulcahy, Laura

    Gene 240 (1999) 45­55 www.elsevier.com/locate/gene Alternative splicing regulates the production by the same gene Annie C.Y. Chang a,1, Bjo¨rn Sohlberg a,1, Laura Trinkle-Mulcahy b, Felix Claverie-Martin a,2 mutations in rne, a gene that encodes Escherichia coli ribonuclease E. NIPP-1 was identified in bovine

  13. GENE4200/GENE6200 Fall Semester Every Year

    E-print Network

    Arnold, Jonathan

    Syllabus GENE4200/GENE6200 Fall Semester Every Year Instructors Richard Meagher and Jeff Bennetzen how much writing 300 words is. Anything longer than this will not be graded in your answer. #12;GENE4200 Honor Credit students and GENE6200 students will be expected to turn in extra written assignments

  14. Gene 241 (2000) 101105 www.elsevier.com/locate/gene

    E-print Network

    Purugganan, Michael D.

    Gene 241 (2000) 101­105 www.elsevier.com/locate/gene Interspecific evolution in plant Abstract Several intragenically linked microsatellites have been identified in the floral regulatory genes in the introns of these two genes, suggesting that they are hotspots for microsatellite formation. The sequences

  15. Some Genes Are Dominant

    NSDL National Science Digital Library

    WGBH Educational Foundation

    2007-04-19

    This interactive activity, adapted from the Dolan DNA Learning Center, illustrates how Gregor Mendel used pure-bred yellow and green peas to show that some genes are dominant and others are recessive.

  16. Gene structure and expression

    SciTech Connect

    Hawkins, J. (London Univ. (United Kingdom))

    1990-01-01

    This book describes the structure of genes in molecular terms and summarizes present knowledge about how their activity is regulated. It covers a range of topics, including a review of the structure and replication of DNA, transcription and translation, prokaryotic and eukaryotic gene organization and expression, retroviruses and oncogenes. The book also includes a chapter on the methodology of DNA manipulation including sections on site-directed mutagenesis, the polymerase chain reaction, reporter genes and restriction fragment length polymorphisms. The hemoglobin gene system and the genetics of the proteins of the immune system are presented in the latter half of the book to show the structure and expression of the most well-studied systems in higher eukaryotes. The final chapter reviews the differences between prokaryotic and the eukaryotic genomes.

  17. Microfluidic gene synthesis

    E-print Network

    Kong, David Sun, 1979-

    2008-01-01

    The ability to synthesize custom de novo DNA constructs rapidly, accurately, and inexpensively is highly desired by researchers, as synthetic genes and longer DNA constructs are enabling to numerous powerful applications ...

  18. Evolutionary Fingerprinting of Genes

    PubMed Central

    Kosakovsky Pond, Sergei L.; Scheffler, Konrad; Gravenor, Michael B.; Poon, Art F.Y.; Frost, Simon D.W.

    2010-01-01

    Over time, natural selection molds every gene into a unique mosaic of sites evolving rapidly or resisting change—an “evolutionary fingerprint” of the gene. Aspects of this evolutionary fingerprint, such as the site-specific ratio of nonsynonymous to synonymous substitution rates (dN/dS), are commonly used to identify genetic features of potential biological interest; however, no framework exists for comparing evolutionary fingerprints between genes. We hypothesize that protein-coding genes with similar protein structure and/or function tend to have similar evolutionary fingerprints and that comparing evolutionary fingerprints can be useful for discovering similarities between genes in a way that is analogous to, but independent of, discovery of similarity via sequence-based comparison tools such as Blast. To test this hypothesis, we develop a novel model of coding sequence evolution that uses a general bivariate discrete parameterization of the evolutionary rates. We show that this approach provides a better fit to the data using a smaller number of parameters than existing models. Next, we use the model to represent evolutionary fingerprints as probability distributions and present a methodology for comparing these distributions in a way that is robust against variations in data set size and divergence. Finally, using sequences of three rapidly evolving RNA viruses (HIV-1, hepatitis C virus, and influenza A virus), we demonstrate that genes within the same functional group tend to have similar evolutionary fingerprints. Our framework provides a sound statistical foundation for efficient inference and comparison of evolutionary rate patterns in arbitrary collections of gene alignments, clustering homologous and nonhomologous genes, and investigation of biological and functional correlates of evolutionary rates. PMID:19864470

  19. Cystic fibrosis modifier genes.

    PubMed Central

    Davies, Jane; Alton, Eric; Griesenbach, Uta

    2005-01-01

    Since the recognition that CFTR genotype was not a good predictor of pulmonary disease severity in CF, several candidate modifier genes have been identified. It is unlikely that a single modifier gene will be found, but more probable that several haplotypes in combination may contribute, which in itself presents a major methodological challenge. The aims of such studies are to increase our understanding of disease pathogenesis, to aid prognosis and ultimately to lead to the development of novel treatments. PMID:16025767

  20. Cell and Gene Therapy

    Microsoft Academic Search

    Robbie Norville

    \\u000a Cell and Gene therapies are novel additions to the current multimodal approach of chemotherapy, radiation, surgery, and hematopoietic\\u000a stem cell transplant (HSCT) for the treatment of pediatric cancers. The manipulation of genes and the immune system can treat\\u000a cancer and prevent genetic diseases as well as increase the body's ability to receive intense treatment that would be impossible\\u000a otherwise. Humans

  1. Gene Therapy for Autoimmune Disorders

    Microsoft Academic Search

    C. H. Evans; S. C. Ghivizzani; T. J. Oligino; P. D. Robbins

    2000-01-01

    Although many autoimmune disorders do not have a strong genetic basis, their treatment may nevertheless be improved by gene therapies. Most strategies seek to transfer genes encoding immunomodulatory products that will alter host immune responses in a beneficial manner. Used in this fashion, genes serve as biological delivery vehicles for the products they encode. By this means gene therapy overcomes

  2. Gene Center Munich Genzentrum Mnchen

    E-print Network

    Frey, Erwin

    Gene Center Munich Genzentrum München Symposium December 1, 2010 'From Genes to Networks ­ Systems:50 ­ 12:50 Prof. Dr. Ulrike Gaul (Alexander von Humboldt Professor, LMU Munich) "Decoding regulatory gene. Patrick Cramer (LMU Munich) "Global mechanisms of gene transcription" 14:30 ­ 15:10 Prof. Dr. Gertrud

  3. Gene finding with Hidden Markov

    E-print Network

    Pachter, Lior

    Gene finding with Hidden Markov models Rachel Brem, Mike Eisen, Lior Pachter #12;Gene Structure 5 to Gene recognition · Homology ­ BLAST, Procrustes, Exonerate · De Novo ­ GRAIL, FGENESH, GENSCAN, Genie, Glimmer, SNAP · Hybrids ­ GenomeScan, Genie · Comparative ­ Rosetta, SLAM, Twinscan #12;Ab-initio gene

  4. 5____________________________________________________________________________ Gene Regulation in Spermatogenesis

    E-print Network

    Wilkinson, Miles F.

    5____________________________________________________________________________ Gene Regulation Nuclear Receptors C. HeatShock Factors D. Sox E. Plzf F. Dmrt1 G. CAF1 H. Homeobox Genes I. Perspective IV. TestisSpecific Gene Expression and DNA Methylation A. Male Germ Cell­Specific Genes B. SomaticCell Testis

  5. Identifying The Most Significant Genes From Gene Expression Profiles For Sample Classification

    E-print Network

    Al-Mubaid, Hisham

    Identifying The Most Significant Genes From Gene Expression Profiles For Sample Classification of gene expression profiles. This generated gene data include complex variations of expression levels with the existing techniques. Keywords: Bioinformatics, Gene Selection, Gene Classification. I. INTRODUCTION The DNA

  6. FunGene: the functional gene pipeline and repository

    PubMed Central

    Fish, Jordan A.; Chai, Benli; Wang, Qiong; Sun, Yanni; Brown, C. Titus; Tiedje, James M.; Cole, James R.

    2013-01-01

    Ribosomal RNA genes have become the standard molecular markers for microbial community analysis for good reasons, including universal occurrence in cellular organisms, availability of large databases, and ease of rRNA gene region amplification and analysis. As markers, however, rRNA genes have some significant limitations. The rRNA genes are often present in multiple copies, unlike most protein-coding genes. The slow rate of change in rRNA genes means that multiple species sometimes share identical 16S rRNA gene sequences, while many more species share identical sequences in the short 16S rRNA regions commonly analyzed. In addition, the genes involved in many important processes are not distributed in a phylogenetically coherent manner, potentially due to gene loss or horizontal gene transfer. While rRNA genes remain the most commonly used markers, key genes in ecologically important pathways, e.g., those involved in carbon and nitrogen cycling, can provide important insights into community composition and function not obtainable through rRNA analysis. However, working with ecofunctional gene data requires some tools beyond those required for rRNA analysis. To address this, our Functional Gene Pipeline and Repository (FunGene; http://fungene.cme.msu.edu/) offers databases of many common ecofunctional genes and proteins, as well as integrated tools that allow researchers to browse these collections and choose subsets for further analysis, build phylogenetic trees, test primers and probes for coverage, and download aligned sequences. Additional FunGene tools are specialized to process coding gene amplicon data. For example, FrameBot produces frameshift-corrected protein and DNA sequences from raw reads while finding the most closely related protein reference sequence. These tools can help provide better insight into microbial communities by directly studying key genes involved in important ecological processes. PMID:24101916

  7. GoGene: gene annotation in the fast lane.

    PubMed

    Plake, Conrad; Royer, Loic; Winnenburg, Rainer; Hakenberg, Jörg; Schroeder, Michael

    2009-07-01

    High-throughput screens such as microarrays and RNAi screens produce huge amounts of data. They typically result in hundreds of genes, which are often further explored and clustered via enriched GeneOntology terms. The strength of such analyses is that they build on high-quality manual annotations provided with the GeneOntology. However, the weakness is that annotations are restricted to process, function and location and that they do not cover all known genes in model organisms. GoGene addresses this weakness by complementing high-quality manual annotation with high-throughput text mining extracting co-occurrences of genes and ontology terms from literature. GoGene contains over 4,000,000 associations between genes and gene-related terms for 10 model organisms extracted from more than 18,000,000 PubMed entries. It does not cover only process, function and location of genes, but also biomedical categories such as diseases, compounds, techniques and mutations. By bringing it all together, GoGene provides the most recent and most complete facts about genes and can rank them according to novelty and importance. GoGene accepts keywords, gene lists, gene sequences and protein sequences as input and supports search for genes in PubMed, EntrezGene and via BLAST. Since all associations of genes to terms are supported by evidence in the literature, the results are transparent and can be verified by the user. GoGene is available at http://gopubmed.org/gogene. PMID:19465383

  8. Characterization of two cotton cDNAs encoding trans-2-enoyl-CoA reductase reveals a putative novel NADPH-binding motif

    Microsoft Academic Search

    Wen-Qiang Song; Yong-Mei Qin; Mihoko Saito; Tsuyoshi Shirai; F. M. Pujol; Alexander J. Kastaniotis; J. Kalervo Hiltunen; Yu-Xian Zhu

    2009-01-01

    Very long chain fatty acids are important components of plant lipids, suberins, and cuticular waxes. Trans-2-enoyl- CoA reductase (ECR) catalyses the fourth reaction of fatty acid elongation, which is NADPH dependent. In the present study, the expression of two cotton ECR (GhECR) genes revealed by quantitative RT-PCR analysis was up- regulated during cotton fibre elongation. GhECR1 and 2 each contain

  9. Analysis of a range of catabolic mutants provides evidence that phytanoyl-coenzyme A does not act as a substrate of the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase complex in Arabidopsis during dark-induced senescence.

    PubMed

    Araújo, Wagner L; Ishizaki, Kimitsune; Nunes-Nesi, Adriano; Tohge, Takayuki; Larson, Tony R; Krahnert, Ina; Balbo, Ilse; Witt, Sandra; Dörmann, Peter; Graham, Ian A; Leaver, Christopher J; Fernie, Alisdair R

    2011-09-01

    The process of dark-induced senescence in plants is not fully understood, however, the functional involvement of an electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO), has been demonstrated. Recent studies have revealed that the enzymes isovaleryl-coenzyme A (CoA) dehydrogenase and 2-hydroxyglutarate dehydrogenase act as important electron donors to this complex. In addition both enzymes play a role in the breakdown of cellular carbon storage reserves with isovaleryl-CoA dehydrogenase being involved in degradation of the branched-chain amino acids, phytol, and lysine while 2-hydroxyglutarate dehydrogenase is exclusively involved in lysine degradation. Given that the chlorophyll breakdown intermediate phytanoyl-CoA accumulates dramatically both in knockout mutants of the ETF/ETFQO complex and of isovaleryl-CoA dehydrogenase following growth in extended dark periods we have investigated the direct importance of chlorophyll breakdown for the supply of carbon and electrons during this process. For this purpose we isolated three independent Arabidopsis (Arabidopsis thaliana) knockout mutants of phytanoyl-CoA 2-hydroxylase and grew them under the same extended darkness regime as previously used. Despite the fact that these mutants accumulated phytanoyl-CoA and also 2-hydroxyglutarate they exhibited no morphological changes in comparison to the other mutants previously characterized. These results are consistent with a single entry point of phytol breakdown into the ETF/ETFQO system and furthermore suggest that phytol is not primarily metabolized by this pathway. Furthermore analysis of isovaleryl-CoA dehydrogenase/2-hydroxyglutarate dehydrogenase double mutants generated here suggest that these two enzymes essentially account for the entire electron input via the ETF complex. PMID:21788362

  10. Molecular cloning and functional expression of codeinone reductase: the penultimate enzyme in morphine biosynthesis in the opium poppy Papaver somniferum.

    PubMed

    Unterlinner, B; Lenz, R; Kutchan, T M

    1999-06-01

    The narcotic analgesic morphine is the major alkaloid of the opium poppy Papaver somniferum. Its biosynthetic precursor codeine is currently the most widely used and effective antitussive agent. Along the morphine biosynthetic pathway in opium poppy, codeinone reductase catalyzes the NADPH-dependent reduction of codeinone to codeine. In this study, we have isolated and characterized four cDNAs encoding codeinone reductase isoforms and have functionally expressed them in Escherichia coli. Heterologously expressed codeinone reductase-calmodulin-binding peptide fusion protein was purified from E. coli using calmodulin affinity column chromatography in a yield of 10 mg enzyme l-1. These four isoforms demonstrated very similar physical properties and substrate specificity. As least six alleles appear to be present in the poppy genome. A comparison of the translations of the nucleotide sequences indicate that the codeinone reductase isoforms are 53% identical to 6'-deoxychalcone synthase from soybean suggesting an evolutionary although not a functional link between enzymes of phenylpropanoid and alkaloid biosynthesis. By sequence comparison, both codeinone reductase and 6'-deoxy- chalcone synthase belong to the aldo/keto reductase family, a group of structurally and functionally related NADPH-dependent oxidoreductases, and thereby possibly arise from primary metabolism. PMID:10417697

  11. Biliverdin amides reveal roles for propionate side chains in bilin reductase recognition and in holophytochrome assembly and photoconversion†

    PubMed Central

    Shang, Lixia; Rockwell, Nathan C.; Martin, Shelley S.; Lagarias, J. Clark

    2010-01-01

    Linear tetrapyrroles (bilins) perform important antioxidant and light harvesting functions in cells from bacteria to humans. To explore the role of the propionate moieties in bilin metabolism, we report the semisynthesis of mono- and di-amides of biliverdin IX? and those of its non-natural XIII? isomer. Initially, these were examined as substrates of two types of NADPH-dependent biliverdin reductase, BVR and BvdR, and of the representative ferredoxin-dependent bilin reductase, phycocyanobilin:ferredoxin oxidoreductase (PcyA). Our studies indicate that the NADPH-dependent biliverdin reductases are less accommodating to amidation of the propionic acid sidechains of biliverdin IX? than PcyA, which does not require free carboxylic acid sidechains to yield its phytobilin product, phycocyanobilin. Bilin amides were also assembled with BV-type and phytobilin-type apophytochromes, demonstrating a role for the 8-propionate in formation of the spectroscopically native Pr dark states of these biliprotein photosensors. Neither ionizable propionate sidechain proved essential to primary photoisomerization for both classes of phytochromes, but an unsubstituted 12-propionate was required for full photointerconversion of phytobilin-type phytochrome Cph1. Taken together, these studies provide insight into the roles of the ionizable propionate sidechains in substrate discrimination by two bilin reductase families while further underscoring the mechanistic differences between the photoconversions of BV-type and phytobilin-type phytochromes. PMID:20565135

  12. Fusion genes in breast cancer

    E-print Network

    Batty, Elizabeth

    2012-02-07

    in breast cancer support the model that there are few commonly mutated genes, and many genes which are mutated much less frequently. Two genes stand out as often mutated in breast cancer across all subtypes: T P53 and PIK3C A. PIK3CA has been reported... distinguish between histologically similar tumours which are molecularly different (Rouzier et al., 2005). Gene expression profiling suggests that the different subtypes of breast cancer vary widely, harbouring different gene alterations and responding...

  13. Virus induced gene silencing of Arabidopsis gene homologues in wheat identify genes conferring improved drought tolerance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In a non-model staple crop like wheat, functional validation of potential drought stress responsive genes identified in Arabidopsis could provide gene targets for wheat breeding. Virus induced gene silencing (VIGS) of genes of interest can overcome the inherent problems of polyploidy and limited tra...

  14. Gene finding in metatranscriptomic sequences

    PubMed Central

    2014-01-01

    Background Metatranscriptomic sequencing is a highly sensitive bioassay of functional activity in a microbial community, providing complementary information to the metagenomic sequencing of the community. The acquisition of the metatranscriptomic sequences will enable us to refine the annotations of the metagenomes, and to study the gene activities and their regulation in complex microbial communities and their dynamics. Results In this paper, we present TransGeneScan, a software tool for finding genes in assembled transcripts from metatranscriptomic sequences. By incorporating several features of metatranscriptomic sequencing, including strand-specificity, short intergenic regions, and putativ