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Sample records for nadph nitrate reductases

  1. Differential nitrate accumulation, nitrate reduction, nitrate reductase ...

    African Journals Online (AJOL)

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    2011-12-07

    Dec 7, 2011 ... reductase activity and nitrite accumulation depend on the exogenous nitrate. Nitrite itself is reduced to ammonium by palstidic nitrite reductase. Nitrite reductase is activated by both nitrate and nitrite ions by positive feed forward, whereas nitrate metabolites, most likely ammonium and glutamine; down.

  2. Differential nitrate accumulation, nitrate reduction, nitrate reductase ...

    African Journals Online (AJOL)

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    2011-12-07

    Dec 7, 2011 ... nitrate salts supply on nitrate accumulation, amino acid biosynthesis, total protein production, nitrate reductase activity and carbohydrate biosynthesis in the roots and leaves of the plants. The results indicate that both sodium and potassium nitrate supplementation had stimulatory effects on all of the.

  3. Differential nitrate accumulation, nitrate reduction, nitrate reductase ...

    African Journals Online (AJOL)

    For most of the cultivated crops, nitrate is the major source of nitrogen. Most steps in the nitrate assimilatory pathway are nitrate inducible. In this study, Cucurbita pepo were grown in washed sand per pot at three potassium and sodium nitrate supplies (25, 50 and 100 mM) to investigate the effects of nitrate salts supply on ...

  4. Comparative modelling and molecular docking of nitrate reductase from Bacillus weihenstephanensis (DS45

    Directory of Open Access Journals (Sweden)

    R. Seenivasagan

    2016-07-01

    Full Text Available Nitrate reductase catalyses the oxidation of NAD(PH and the reduction of nitrate to nitrite. NR serves as a central point for the integration of metabolic pathways by governing the flux of reduced nitrogen through several regulatory mechanisms in plants, algae and fungi. Bacteria express nitrate reductases that convert nitrate to nitrite, but mammals lack these specific enzymes. The microbial nitrate reductase reduces toxic compounds to nontoxic compounds with the help of NAD(PH. In the present study, our results revealed that Bacillus weihenstephanensis expresses a nitrate reductase enzyme, which was made to generate the 3D structure of the enzyme. Six different modelling servers, namely Phyre2, RaptorX, M4T Server, HHpred, SWISS MODEL and Mod Web, were used for comparative modelling of the structure. The model was validated with standard parameters (PROCHECK and Verify 3D. This study will be useful in the functional characterization of the nitrate reductase enzyme and its docking with nitrate molecules, as well as for use with autodocking.

  5. Structural Basis of Eukaryotic Nitrate Reduction: Crystal Structures of the Nitrate Reductase Active Site

    Science.gov (United States)

    Fischer, Katrin; Barbier, Guillaume G.; Hecht, Hans-Juergen; Mendel, Ralf R.; Campbell, Wilbur H.; Schwarz, Guenter

    2005-01-01

    Nitrate assimilation in autotrophs provides most of the reduced nitrogen on earth. In eukaryotes, reduction of nitrate to nitrite is catalyzed by the molybdenum-containing NAD(P)H:nitrate reductase (NR; EC 1.7.1.1-3). In addition to the molybdenum center, NR contains iron-heme and flavin adenine dinucleotide as redox cofactors involved in an internal electron transport chain from NAD(P)H to nitrate. Recombinant, catalytically active Pichia angusta nitrate-reducing, molybdenum-containing fragment (NR-Mo) was expressed in P. pastoris and purified. Crystal structures for NR-Mo were determined at 1.7 and 2.6 Å. These structures revealed a unique slot for binding nitrate in the active site and identified key Arg and Trp residues potentially involved in nitrate binding. Dimeric NR-Mo is similar in overall structure to sulfite oxidases, with significant differences in the active site. Sulfate bound in the active site caused conformational changes, as compared with the unbound enzyme. Four ordered water molecules located in close proximity to Mo define a nitrate binding site, a penta-coordinated reaction intermediate, and product release. Because yeast NAD(P)H:NR is representative of the family of eukaryotic NR, we propose a general mechanism for nitrate reduction catalysis. PMID:15772287

  6. Effect of tungsten and vanadium on the in vitro assembly of assimilatory nitrate reductase utilizing Neurospora mutant nit-1

    Energy Technology Data Exchange (ETDEWEB)

    Lee, K.Y.; Erickson, R.; Pan, S.S.; Jones, G.; May, F.; Nason, A.

    1974-06-01

    Tungsten or vanadium in a nutrient medium containing nitrate, but not ammonia, as the sole nitrogen source inhibited the formation of assimilatory NADPH-nitrate reductase (but not nitrate-inducible NADPH-cytochrome c reductase) and growth of wild type Neurospora crassa. Inhibition was prevented by high levels of molybdate in the nutrient medium but was not reversed by adding molybdate to subsequently prepared cell-free enzyme preparations. Neither tungsten nor vanadium was inhibitory when added to partially purified wild type Neurospora NADPH-nitrate reductase. Cell-free preparations of uninduced wild type grown in increasing concentrations of tungsten or vanadium showed progressive inhibition of ability to assemble nitrate reductase in vitro (with extracts of nitrate-induced Neurospora mutant nit-1) which was partially or entirely restored by subsequent addition of molybdate to the extracts of the tungsten- or vanadium-grown uninduced wild type. Tungsten or vanadium added to acid-treated bovine milk xanthine oxidase or to extracts of uninduced wild type also inhibited in vitro enzyme assembly, with added molybdate partially preventing inhibition. The tungsten or vanadium analogue of nitrate reductase formed in vivo or by in vitro assembly is considerably more labile than its molybdenum counterpart as indicated by the complete loss of /sup 185/W or /sup 48/V from the enzyme as a result of trichloroacetic acid precipitation or heat treatment.

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

    Science.gov (United States)

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

    2008-12-26

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

  8. Purification and Properties of an NADPH-Aldose Reductase (Aldehyde Reductase) from Euonymus japonica Leaves

    Science.gov (United States)

    Negm, Fayek B.

    1986-01-01

    The enzyme aldose (aldehyde) reductase was partially purified (142-fold) and characterized from Euonymus japonica leaves. The reductase, a dimer, had an average molecular weight of 67,000 as determined by gel filtration on Sephadex G-100. The enzyme was NADPH specific and reduced a broad range of substrates including aldoses, aliphatic aldehydes, and aromatic aldehydes. Maximum activity was observed at pH 8 in phosphate and Tris-HCl buffers and at pH 8.6 to 9.0 in glycine-NaOH buffer using dl-glyceraldehyde or 3-pyridinecarboxaldehyde as substrate. NADP was a competitive inhibitor with respect to NADPH with a Ki of 60 micromolar. Glycerol was an uncompetitive inhibitor to dl-glyceraldehyde (K′i = 460 millimolar). The Euonymus enzyme was inhibited by sulfhydryl inhibitor, phenobarbital, and high concentrations of Li2SO4. Pyrazol and metal chelating agents inhibited the enzyme slightly. Enzyme activity was detected in the leaves and berries of Celastrus orbiculatus and several species of Euonymus. Probable function of this enzyme is to reduce d-galactose to galactitol, a characteristic metabolite in phloem sap of members of the Celastraceae family. Images Fig. 1 PMID:16664750

  9. Effect of ammonium and nitrate on ferric chelate reductase and nitrate reductase in Vaccinium species.

    Science.gov (United States)

    Poonnachit, U; Darnell, R

    2004-04-01

    Most Vaccinium species have strict soil requirements for optimal growth, requiring low pH, high iron availability and nitrogen primarily in the ammonium form. These soils are limited and are often located near wetlands. Vaccinium arboreum is a wild species adapted to a wide range of soils, including high pH, low iron, and nitrate-containing soils. This broader soil adaptation in V. arboreum may be related to increased efficiency of iron or nitrate uptake compared with the cultivated Vaccinium species. Nitrate, ammonium and iron uptake, and nitrate reductase (NR) and ferric chelate reductase (FCR) activities were compared in two Vaccinium species grown hydroponically in either nitrate or ammonia, with or without iron. The species studied were the wild V. arboreum and the cultivated V. corymbosum interspecific hybrid, which exhibits the strict soil requirements of most Vaccinium species. Ammonium uptake was significantly greater than nitrate uptake in both species, while nitrate uptake was greater in the wild species, V. arboreum, compared with the cultivated species, V. corymbosum. The increased nitrate uptake in V. arboreum was correlated with increased root NR activity compared with V. corymbosum. The lower nitrate uptake in V. corymbosum was reflected in decreased plant dry weight in this species compared with V. arboreum. Root FCR activity increased significantly in V. corymbosum grown under iron-deficient conditions, compared with the same species grown under iron-sufficient conditions or with V. arboreum grown under either iron condition. V. arboreum appears to be more efficient in acquiring nitrate compared with V. corymbosum, possibly due to increased NR activity and this may partially explain the wider soil adaptation of V. arboreum.

  10. On The Regulation of Spinach Nitrate Reductase 1

    Science.gov (United States)

    Sanchez, Juan; Heldt, Hans W.

    1990-01-01

    A coupled assay has been worked out to study spinach (Spinacea oleracea L.) nitrate reductase under low, more physiological concentrations of NADH. In this assay the reduction of nitrate is coupled to the oxidation of malate catalyzed by spinach NAD-malate dehydrogenase. The use of this coupled system allows the assay of nitrate reductase activity at steady-state concentrations of NADH below micromolar. We have used this coupled assay to study the kinetic parameters of spinach nitrate reductase and to reinvestigate the putative regulatory role of adenine nucleotides, inorganic phosphate, amino acids, and calcium and calmodulin. PMID:16667335

  11. Optimum conditions for cotton nitrate reductase extraction and ...

    African Journals Online (AJOL)

    GREGO

    mM of glutamine in the extraction buffer stimulates significantly, in vitro, the reduction of nitrate. Enzyme activity is moreover optimal when 1 M of exogenous nitrate, as substrate, is added to the reaction medium. At these optimum conditions of nitrate reductase activity determination, the substrate was completely reduced ...

  12. Nitrate reductase assay using sodium nitrate for rapid detection of multidrug resistant tuberculosis

    OpenAIRE

    Macedo, Ma?ra Bidart; Groll, Andrea Von; Fissette, Krista; Palomino, Juan Carlos; da Silva, Pedro Eduardo Almeida; Martin, Anandi

    2012-01-01

    We validated the nitrate reductase assay (NRA) for the detection of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) using sodium nitrate (NaNO3) in replacement of potassium nitrate (KNO3) as nitrate source. NaNO3 is cheaper than KNO3 and has no restriction on use which facilitates the implementation of NRA to detect MDR-TB.

  13. Bioactivation of lapachol responsible for DNA scission by NADPH-cytochrome P450 reductase.

    Science.gov (United States)

    Kumagai, Y; Tsurutani, Y; Shinyashiki, M; Homma-Takeda, S; Nakai, Y; Yoshikawa, T; Shimojo, N

    1997-09-01

    The reduction of the naphthoquinone derivative, lapachol, which is responsible for its bioactivation was examined using microsomal preparations and NADPH-cytochrome P450 reductase (P450 reductase). Phenobarbital (PB) pretreatment resulted in an induction of enzyme activities for cytochrome c reduction (1.54 times) and lapachol reduction (1.20 times) by hepatic microsomal preparation of rats. The specific activity of lapachol reduction by purified P450 reductase showed 56-fold higher than that by untreated liver microsomes. Addition of antibody against P450 reductase (2 mg of IgG/mg of protein) to the microsomal incubation mixture caused an immunoinhibition of cytochrome c (32%) and lapachol (19%) reduction activities, suggesting that P450 reductase catalyzes lapachol reduction. Generation of superoxide anion radical (1321 nmol/mg per min) in approximately equivalent amounts of with NADPH consumption (941 nmol/mg per min) was detected during metabolism of lapachol by P450 reductase. Electron spin resonance (ESR) experiments confirmed generation of superoxide anion radical and hydroxyl radical as these 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) adducts. Incubation of lapachol with P450 reductase caused a cleavage of DNA which was reduced in the presence of Cu,Zn-superoxide dismutase (Cu,Zn-SOD), catalase(1), and hydroxyl radical scavengers such as dimethyl sulfoxide (DMSO) and thiourea. Taken together, these results indicate that lapachol is bioactivated by P450 reductase to reactive species, which promote DNA scission through the redox cycling based generation of superoxide anion radical.

  14. Compensatory periplasmic nitrate reductase activity supports anaerobic growth of Pseudomonas aeruginosa PAO1 in the absence of membrane nitrate reductase

    Science.gov (United States)

    Van Alst, Nadine E.; Sherrill, Lani A.; Iglewski, Barbara H.; Haidaris, Constantine G.

    2009-01-01

    Nitrate serves as a terminal electron acceptor under anaerobic conditions in Pseudomonas aeruginosa. Reduction of nitrate to nitrite generates a transmembrane proton motive force allowing ATP synthesis and anaerobic growth. Inner membrane-bound nitrate reductase NarGHI is encoded within the narK1K2GHJI operon and the periplasmic nitrate reductase NapAB is encoded within the napEFDABC operon. The role of the two dissimilatory nitrate reductases in anaerobic growth, and the regulation of their expression were examined by using a set of deletion mutants in P. aeruginosa PAO1. NarGHI mutants were unable to grow anaerobically, but plate cultures remained viable up to 120 hr. In contrast, nitrate sensor-response regulator mutant ΔnarXL displayed growth arrest initially, but resumed growth after 72 hr and reached early stationary phase in liquid culture after 120 hr. Genetic, transcriptional, and biochemical studies demonstrated that anaerobic growth recovery by the NarXL mutant was the result of NapAB periplasmic nitrate reductase expression. A novel transcriptional start site for napEFDABC expression was identified in the NarXL mutant grown anaerobically. Furthermore, mutagenesis of a consensus NarL-binding site monomer upstream of the novel transcriptional start site restored anaerobic growth recovery in the NarXL mutant. The data suggest that during anaerobic growth of wild type P. aeruginosa PAO1, nitrate response regulator NarL directly represses expression of periplasmic nitrate reductase, while inducing maximal expression of membrane nitrate reductase. PMID:19935885

  15. NADPH-dependent D-aldose reductases and xylose fermentation in Fusarium oxysporum

    DEFF Research Database (Denmark)

    Panagiotou, Gianni; Christakopoulos, P.

    2004-01-01

    Two aldose (xylose) reductases (ARI and ARII) from Fusarium oxysporum were purified and characterized. The native ARI was a monomer with M-r 41000, pI 5.2 and showed a 52-fold preference for NADPH over NADH, while ARII was homodimeric with a subunit of M-r 37000, pI 3.6 and a 60-fold preference...

  16. Analysis of nitrate reductase mRNA expression and nitrate reductase activity in response to nitrogen supply

    OpenAIRE

    Gholamreza Kavoosi; Sadegh Balotf; Homeira Eshghi; Hasan Hasani

    2014-01-01

    Nitrate is one of the major sources of nitrogen for the growth of plants. It is taken up by plant roots and transported to the leaves where it is reduced to nitrite in the. The main objective of this research was to investigate stimulatory effects of sodium nitrate, potassium nitrate, ammonia and urea on the production/generation of the nitrate reductase mRNA in Triticum aestivum plants. The plants were grown in standard nutrient solution for 21 days and then starved in a media without nitrat...

  17. Production and Characterization of Monoclonal Antibodies against NADPH-Cytochrome P-450 Reductases from Helianthus tuberosus1

    Science.gov (United States)

    Lesot, Agnès; Benveniste, Irène; Hasenfratz, Marie-Paule; Durst, Francis

    1992-01-01

    Monoclonal antibodies (mAbs) against a plant NADPH-cytochrome P-450 (Cyt P-450) reductase from Jerusalem artichoke (Helianthus tuberosus) tuber were prepared. These antibodies were produced by hybridoma resulting from the fusion of spleen cells from a rat immunized with a purified preparation of the reductase and mouse myeloma cells. The mAbs thus obtained were screened for their interaction with the reductases, first in western dots and then in blots, and for their ability to inhibit the NADPH-cytochrome c (Cyt c) reductase activity from Jerusalem artichoke microsomes. Among the 11 clones giving a positive response on western blots, only 6 were also able to inhibit microsomal NADPH-Cyt c reductase activity, and the microsomal Cyt P-450 monooxygenase activities dependent upon electrons transferred by the reductase. Thus, two families of mAbs were characterized: a family of mAbs that interact with epitopes of the reductase implicated in the reduction of Cyt P-450 by NADPH (binding sites for NADPH, flavin mononucleotide, flavin adenine dinucleotide, and Cyt P-450), and a structural family, whose members recognize epitopes outside the active site of the reductases. These mAbs specifically recognize the reductase, and all of them interact with all of the isoforms, indicating that important primary or secondary structural analogies exist between the isoforms, not only at the active site, but also at the level of epitopes not directly associated with catalytic activity. Images Figure 1 Figure 2 Figure 3 PMID:16653138

  18. Purification and properties of a NADPH-dependent erythrose reductase from the newly isolated Torula corallina.

    Science.gov (United States)

    Lee, Jung-Kul; Hong, Kwang-Won; Kim, Sang-Yong

    2003-01-01

    Torula corallina (KCCM-10171) is a yeast strain that is currently used for the industrial production of erythritol and has the highest erythritol yield ever reported for an erythritol-producing microorganism. Production of erythritol in T. corallina is catalyzed by erythrose reductase, an enzyme that converts erythrose to erythritol using NADPH as a cofactor. In this study, NADPH-dependent erythrose reductase was purified to homogeneity from the newly isolated T. corallina. The relative molecular weight of the erythrose reductase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography was 35.4 and 71.0 kDa, respectively, indicating that the enzyme is dimeric. This enzyme catalyzed both erythrose reduction and erythritol oxidation; both enzyme activities required NADP(H). The pH and temperature optima for erythrose reduction and erythritol oxidation were 6.0, 40 degrees C and 8.0, 45 degrees C, respectively. The sequence of the first 10 amino acids of this enzyme was N-V-K-N-F-Y-Q-P-N-D. The affinity (K(m)( )()= 7.12 mM) of the enzyme for erythrose was comparable to that of other known erythrose reductases, and the specificity for erythrose was very high, resulting in no production of other polyols, which may explain the high erythritol yield observed in this strain.

  19. The electron transfer reactions of NADPH: cytochrome P450 reductase with nonphysiological oxidants.

    Science.gov (United States)

    Cénas, N; Anusevicius, Z; Bironaité, D; Bachmanova, G I; Archakov, A I; Ollinger, K

    1994-12-01

    The steady-state kinetics of oxidation of rat liver NADPH: cytochrome P450 reductase (EC 1.6.2.4) by quinones, aromatic nitrocompounds, ferricyanide, Fe(EDTA)-, and cytochrome c has been studied. The logarithms of bimolecular rate constants of reduction (kcat/Km) of quinones and nitrocompounds increase with the increase in their single-electronreduction potential (E1(7)), reaching a maximum value at E1(7) > -0.15 V. The reactivities of nitroaromatics are about by an order of magnitude lower than the reactivities of quinones. For a series of nitroaromatics including the compounds with previously undetermined E1(7) values, an orthogonality was found between their reactivities toward cytochrome P450 reductase, flavocytochrome b2 (EC 1.1.2.3), and the NADPH: adrenodoxin reductase (EC 1.18.1.2)-adrenodoxin system. This indicates the absence of significant specific interactions during these reactions. The effects of ionic strength on reaction kinetics and the character of inhibition by a product of reaction, NADP+, are in accordance with the reduction of oxidants at the negatively charged site in the surroundings of FMN of P450 reductase. Quinones inactivate oxidized reductase modifying the NADP(H) binding site. The redox cycling of quinones markedly slows the inactivation. The kinetic data presented are consistent with an outer-sphere electron transfer mechanism. The analysis of kinetics of reduction of cytochrome c, ferricyanide, and Fe(EDTA)- using the model of Mauk et al. (A. G. Mauk, R. A. Scott, and H. B. Gray (1980) J. Am. Chem. Soc. 102, 4360-4363) gives calculated distances of FMN from the surface of protein globule, 0.33-0.63 nm. The data from nitroreductase reactions of cytochrome P450 reductase, flavocytochrome b2, and adrenodoxin were used for approximate evaluation of previously unknown E1(7) of nitrocompounds.

  20. Nitroreductase reactions of the NADPH: adrenodoxin reductase and the adrenodoxin complex.

    Science.gov (United States)

    Marcinkeviciene, J; Cenas, N; Kulys, J; Usanov, S A; Sukhova, N M; Selezneva, I S; Gryazev, V F

    1990-01-01

    NADPH: adrenodoxin reductase (E.C. 1.18.1.2) and its complex with adrenodoxin catalyze the aerobic oxidation of NADPH by a number of substituted 2-nitrofurans, 5-nitroimidazoles and p-derivatives of nitrobenzene. The nitrocompounds are reduced via an initial single-electron transfer. Under anaerobic conditions nitrofurans are reduced to the corresponding amines. The rate constants of adrenodoxin oxidation by nitrocompounds vary from 4 x 10(5) to 3 x 10(2) M-1 s-1. A linear correlation between the rate constant logarithm and the single-electron reduction potential at pH 7.0 (E7(1)) of nitrocompounds was observed. The relation between the reactivity and the polarographic half-wave potential (E7(1/2)) is distorted. The reactivity of adrenodoxin reductase is two orders of magnitude lower than that of adrenodoxin.

  1. Optimum conditions for cotton nitrate reductase extraction and ...

    African Journals Online (AJOL)

    Conditions of nitrate reductase extraction and activity measurement should be adapted to plant species, and to the organs of the same plant, because of extreme weaknesses and instabilities of the enzyme. Different extraction and reaction media have been compared in order to define the best conditions for cotton callus ...

  2. Transcriptional modulation of genes encoding nitrate reductase in ...

    African Journals Online (AJOL)

    2016-10-26

    Oct 26, 2016 ... The free aluminum (Al) content in soil can reach levels that are toxic to plants, and this has frequently limited increased productivity of cultures. Four genes encoding nitrate reductase (NR) were identified, named ZmNR1–4. With the aim of evaluating NR activity and the transcriptional modulation of the.

  3. Transcriptional modulation of genes encoding nitrate reductase in ...

    African Journals Online (AJOL)

    The free aluminum (Al) content in soil can reach levels that are toxic to plants, and this has frequently limited increased productivity of cultures. Four genes encoding nitrate reductase (NR) were identified, named ZmNR1–4. With the aim of evaluating NR activity and the transcriptional modulation of the ZmNR1, ZmNR2, ...

  4. Nitrate reductase gene involvement in hexachlorobiphenyl dechlorination by Phanerochaete chrysosporium

    International Nuclear Information System (INIS)

    De, Supriyo; Perkins, Michael; Dutta, Sisir K.

    2006-01-01

    Polychlorobiphenyl (PCB) degradation usually occurs through reductive dechlorination under anaerobic conditions and phenolic ring cleavage under aerobic conditions. In this paper, we provide evidence of nitrate reductase (NaR) mediated dechlorination of hexachlorobiphenyl (PCB-153) in Phanerochaete chrysosporium under non-ligninolytic condition and the gene involved. The NaR enzyme and its cofactor, molybdenum (Mo), were found to mediate reductive dechlorination of PCBs even in aerobic condition. Tungsten (W), a competitive inhibitor of this enzyme, was found to suppress this dechlorination. Chlorine release assay provided further evidence of this nitrate reductase mediated dechlorination. Commercially available pure NaR enzyme from Aspergillus was used to confirm these results. Through homology search using TBLASTN program, NaR gene was identified, primers were designed and the RT-PCR product was sequenced. The NaR gene was then annotated in the P. chrysosporium genome (GenBank accession no. AY700576). This is the first report regarding the presence of nitrate reductase gene in this fungus with the explanation why this fungus can dechlorinate PCBs even in aerobic condition. These fungal inoculums are used commercially as pellets in sawdust for enhanced bioremediation of PCBs at the risk of depleting soil nitrates. Hence, the addition of nitrates to the pellets will reduce this risk as well as enhance its activity

  5. Genomic and Bioinformatic Analysis of NADPH-Cytochrome P450 Reductase in Anopheles stephensi (Diptera: Culicidae)

    Science.gov (United States)

    Suwanchaichinda, C.; Brattsten, L. B.

    2014-01-01

    Abstract The cytochrome P450 monooxygenase (P450) enzyme system is a major mechanism of xenobiotic biotransformation. The nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CPR) is required for transfer of electrons from NADPH to P450. One CPR gene was identified in the genome of the malaria-transmitting mosquito Anopheles stephensi Liston (Diptera: Culicidae). The gene encodes a polypeptide containing highly conserved flavin mononucleotide-, flavin adenine dinucleotide-, and NADPH-binding domains, a unique characteristic of the reductase. Phylogenetic analysis revealed that the A. stephensi and other known mosquito CPRs belong to a monophyletic group distinctly separated from other insects in the same order, Diptera. Amino acid residues of CPRs involved in binding of P450 and cytochrome c are conserved between A. stephensi and the Norway rat Rattus norvegicus Berkenhout (Rodentia: Muridae). However, gene structure particularly within the coding region is evidently different between the two organisms. Such difference might arise during the evolution process as also seen in the difference of P450 families and isoforms found in these organisms. CPR in the mosquito A. stephensi is expected to be active and serve as an essential component of the P450 system. PMID:25368081

  6. Sucrose mimics the light induction of Arabidopsis nitrate reductase gene transcription

    DEFF Research Database (Denmark)

    Cheng, Chi-Lien; Acedo, Gregoria N; Kristensen, Michael

    1992-01-01

    can replace light in eliciting an increase of nitrate reductase mRNA accumulation in dark-adapted green Arabidopsis plants. We show further that sucrose alone is sufficient for the full expression of nitrate reductase genes in etiolated Arabidopsis plants. Finally, using a reporter gene, we show......Nitrate reductase, the first enzyme in nitrate assimilation, is located at the crossroad of two energy-consuming pathways: nitrate assimilation and carbon fixation. Light, which regulates the expression of many higher-plant carbon fixation genes, also regulates nitrate reductase gene expression....... Located in the cytosol, nitrate reductase obtains its reductant not from photosynthesis but from carbohydrate catabolism. This relationship prompted us to investigate the indirect role that light might play, via photosynthesis, in the regulation of nitrate reductase gene expression. We show that sucrose...

  7. Gamma-irradiation activates biochemical systems: induction of nitrate reductase activity in plant callus.

    OpenAIRE

    Pandey, K N; Sabharwal, P S

    1982-01-01

    Gamma-irradiation induced high levels of nitrate reductase activity (NADH:nitrate oxidoreductase, EC 1.6.6.1) in callus of Haworthia mirabilis Haworth. Subcultures of gamma-irradiated tissues showed autonomous growth on minimal medium. We were able to mimic the effects of gamma-irradiation by inducing nitrate reductase activity in unirradiated callus with exogenous auxin and kinetin. These results revealed that induction of nitrate reductase activity by gamma-irradiation is mediated through i...

  8. Immunological comparison of the NADH:nitrate reductase from different cucumber tissues

    Directory of Open Access Journals (Sweden)

    Jolanta Marciniak

    2014-01-01

    Full Text Available Soluble nitrate reductase from cucumber roots (Cucumis sativus L. was isolated and purified with blue-Sepharose 4B. Specific antibodies against the NR protein were raised by immunization of a goat. Using polyclonal antibodies anti-NR properties of the nitrate reductase from various cucumber tissues were examined. Experiments showed difference in immuno-logical properties of nitrate reductase (NR from cotyledon roots and leaves.

  9. Optimisation of nitrate reductase enzyme activity to synthesise silver nanoparticles.

    Science.gov (United States)

    Khodashenas, Bahareh; Ghorbani, Hamid Reza

    2016-06-01

    Today, the synthesis of silver nanoparticles (Ag NPs) is very common since it has many applications in different areas. The synthesis of these nanoparticles is done by means of physical, chemical, or biological methods. However, due to its inexpensive and environmentally friendly features, the biological method is more preferable. In the present study, using nitrate reductase enzyme available in the Escherichia coli (E. coli) bacterium, the biosynthesis of Ag NPs was investigated. In addition, the activity of the nitrate reductase enzyme was optimised by changing its cultural conditions, and the effects of silver nitrate (AgNO(3)) concentration and enzyme amount on nanoparticles synthesis were studied. Finally, the produced nanoparticles were studied using ultraviolet -visible (UV-Vis) spectrophotometer, dynamic light scattering technique, and transmission electron microscopy. UV-Visible spectrophotometric study showed the characteristic peak for Ag NPs at wavelength 405-420 nm for 1 mM metal precursor solution (AgNO(3)) with 1, 5, 10, and 20 cc supernatant and 435 nm for 0.01M AgNO(3) with 20 cc supernatant. In this study, it was found that there is a direct relationship between the AgNO(3) concentration and the size of produced Ag NPs.

  10. Determination of Nitrate Reductase Assay Depending on the Microbial Growth

    International Nuclear Information System (INIS)

    El-Kabbany, H.M.

    2012-01-01

    A rapid micro-dilution assay for determination of the antimicrobial susceptibility of different bacterial isolates was developed. This assay is based on the ability of the most of viable organisms to reduce nitrates. The MIC or MBC could be determined by nitrate reductase (NR) only after 30 to 90 min of incubation depending on the behaviour of microbial growth. Bacterial viability is detected by a positive nitrite reduction rather than visible turbidity. The nitrate reduction assay was compared with standard micro-assay using 250 isolates of different taxa against 10 antibiotics belonging to different classes. An excellent agreement of 82.5 % was found between the two methods and only 17.5 % of 1794 trials showed difference in the determined MIC by tow-dilution interval above or below the MIC determined by the turbidimetric method under the same test conditions. However, the nitrate reduction assay was more rapid and sensitive in detecting viable bacteria and so, established an accurate estimate of the minimal inhibitory concentration (MIC) or the minimal bacterial concentration (MBC). The nitrate reduction assay offers the additional advantage that it could be used to determine the MBC without having to subculture the broth. 232 cases of resistance were detected by NR and 4 different media were tested for susceptibility test. The bacterial isolates were exposed to ultra violet (UV) light for different period

  11. Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana

    Science.gov (United States)

    Tischner, R.; Ward, M. R.; Huffaker, R. C.

    1989-01-01

    Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrate uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.

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

    Science.gov (United States)

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

  13. Amperometric nitrate biosensor based on Carbon nanotube/Polypyrrole/Nitrate reductase biofilm electrode

    Energy Technology Data Exchange (ETDEWEB)

    Can, Faruk; Korkut Ozoner, Seyda; Ergenekon, Pinar; Erhan, Elif, E-mail: e.erhan@gyte.edu.tr

    2012-01-01

    This study describes the construction and characterization of an amperometric nitrate biosensor based on the Polypyrrole (PPy)/Carbon nanotubes (CNTs) film. Nitrate reductase (NR) was both entrapped into the growing PPy film and chemically immobilized via the carboxyl groups of CNTs to the CNT/PPy film electrode. The optimum amperometric response for nitrate was obtained in 0.1 M phosphate buffer solution (PBS), pH 7.5 including 0.1 M lithium chloride and 7 mM potassium ferricyanide with an applied potential of 0.13 V (vs. Ag/AgCl, 3 M NaCl). Sensitivity was found to be 300 nA/mM in a linear range of 0.44-1.45 mM with a regression coefficient of 0.97. The biosensor response showed a higher linear range in comparison to standard nitrate analysis methods which were tested in this study and NADH based nitrate biosensors. A minimum detectable concentration of 0.17 mM (S/N = 3) with a relative standard deviation (RSD) of 5.4% (n = 7) was obtained for the biosensor. Phenol and glucose inhibit the electrochemical reaction strictly at a concentration of 1 {mu}g/L and 20 mg/L, respectively. The biosensor response retained 70% of its initial response over 10 day usage period when used everyday. - Highlights: Black-Right-Pointing-Pointer K{sub 3}Fe(CN){sub 6} has been used for the first time as mediator for nitrate reductase. Black-Right-Pointing-Pointer Better performance was obtained in comparison to other nitrate biosensor studies operated with various mediators. Black-Right-Pointing-Pointer Analytical parameters were better than standard nitrate analysis methods.

  14. Amperometric nitrate biosensor based on Carbon nanotube/Polypyrrole/Nitrate reductase biofilm electrode

    International Nuclear Information System (INIS)

    Can, Faruk; Korkut Ozoner, Seyda; Ergenekon, Pinar; Erhan, Elif

    2012-01-01

    This study describes the construction and characterization of an amperometric nitrate biosensor based on the Polypyrrole (PPy)/Carbon nanotubes (CNTs) film. Nitrate reductase (NR) was both entrapped into the growing PPy film and chemically immobilized via the carboxyl groups of CNTs to the CNT/PPy film electrode. The optimum amperometric response for nitrate was obtained in 0.1 M phosphate buffer solution (PBS), pH 7.5 including 0.1 M lithium chloride and 7 mM potassium ferricyanide with an applied potential of 0.13 V (vs. Ag/AgCl, 3 M NaCl). Sensitivity was found to be 300 nA/mM in a linear range of 0.44–1.45 mM with a regression coefficient of 0.97. The biosensor response showed a higher linear range in comparison to standard nitrate analysis methods which were tested in this study and NADH based nitrate biosensors. A minimum detectable concentration of 0.17 mM (S/N = 3) with a relative standard deviation (RSD) of 5.4% (n = 7) was obtained for the biosensor. Phenol and glucose inhibit the electrochemical reaction strictly at a concentration of 1 μg/L and 20 mg/L, respectively. The biosensor response retained 70% of its initial response over 10 day usage period when used everyday. - Highlights: ► K 3 Fe(CN) 6 has been used for the first time as mediator for nitrate reductase. ► Better performance was obtained in comparison to other nitrate biosensor studies operated with various mediators. ► Analytical parameters were better than standard nitrate analysis methods.

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

    Science.gov (United States)

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

    2015-07-01

    At least four peroxiredoxins that are coupled with the thioredoxin (Trx) system have been shown to play a key role in redox metabolism in the unicellular phytoflagellate Euglena gracilis. In order to clarify Trx-mediated redox regulation in this alga, we herein identified three NADPH-dependent thioredoxin reductases (NTRs) using a homologous search and characterized their enzymatic properties and physiological roles. Each Euglena NTR protein belonged to the small, large, and NTRC types, and were named EgNTR1, EgNTR2, and EgNTRC, respectively. EgNTR2 was phylogenetically different from the known NTRs in eukaryotic algae. EgNTR1 was predicted to be localized in mitochondria, EgNTR2 in the cytosol, and EgNTRC in plastids. The catalytic efficiency of EgNTR2 for NADPH was 30-46-fold higher than those of EgNTR1 and truncated form of EgNTRC, suggested that large type EgNTR2 reduced Trx more efficiently. The silencing of EgNTR2 gene expression resulted in significant growth inhibition and cell hypertrophy in Euglena cells. These results suggest that EgNTRs function in each cellular compartment and are physiologically important, particularly in the cytosol. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2014-04-01

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

  17. Effect of Ammonium and Nitrate on Ferric Chelate Reductase and Nitrate Reductase in Vaccinium Species

    OpenAIRE

    POONNACHIT, U.; DARNELL, R.

    2004-01-01

    • Background and Aims Most Vaccinium species have strict soil requirements for optimal growth, requiring low pH, high iron availability and nitrogen primarily in the ammonium form. These soils are limited and are often located near wetlands. Vaccinium arboreum is a wild species adapted to a wide range of soils, including high pH, low iron, and nitrate‐containing soils. This broader soil adaptation in V. arboreum may be related to increased efficiency of iron or nitrate uptake compared with th...

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

    Science.gov (United States)

    Li, Meng; Guo, Wenbin; Chen, Xinhua

    2016-12-01

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

  19. Nitrate reductase activity in the diatom Biddulphia longicruris: characterization and daily oscillation

    Directory of Open Access Journals (Sweden)

    Cristina S Asano

    1995-01-01

    Full Text Available Nitrate reductase (NR activity was studied in the marine diatom Biddulphia longicruris. During 24 hours of sampling, NR activity was found during day time and in the transition day-night. Nitrite anions, the product of nitrate reduction, was released by the cells at the times NR was active, and accumulated in the culture medium. Whenever the cultures of B. longicmris were submitted to nitrogen deprivation, NR activity could not be detected. In vitro determination of KM values for NR using nitrate or NADH were respectively 50//M and 80 /*M. Temperature and pH dependence of NR activity were also determined for this organism.A atividade de nitrato redutase (NR foi estudada na diatomácea marinha Biddulphia longicruris. A NR é a enzima responsável pelo processo de assimilação de nitrato. O nitrato é reduzido no interior da célula a nitrito pela ação da NR. Esta enzima é normalmente oligomérica e utiliza o NADH como substrato doador de elétrons. Em alguns organismos também são encontradas NRs capazes de utilizarem o NADPH como doador eletrônico para a redução do nitrato a nitrito. Para a diatomácea B. longicmris foram apresentadas evidências de que a sua NR é específica para NADH e que a redução de nitrato em presença de NADPH não acontece. Determinações in vitro dos valores das constantes de Michaelis-Menten (KM usando nitrato e NADH como substratos, são respectivamente 50µ e 80µM. A temperatura ótima de reação enzimática e a sua dependência ao pH também foram estudadas. Cultivos de B. longicmris foram acompanhados por períodos de 24 horas e foi mostrado que a atividade de NR é encontrada em maiores níveis durante os períodos de transição de luz/escuro. Os anions nitrito, produtos da redução de nitrato, são eliminados pelas células nos períodos de maior atividade de NR e se acumulam no meio de cultura. Células submetidas à ausência de nitrato apresentam uma repressão da expressão de NR, sendo

  20. Characterization of two NADPH: cytochrome P450 reductases from cotton (Gossypium hirsutum).

    Science.gov (United States)

    Yang, Chang-Qing; Lu, Shan; Mao, Ying-Bo; Wang, Ling-Jian; Chen, Xiao-Ya

    2010-01-01

    Cytochrome P450 monooxygenases (P450s) are commonly involved in biosynthesis of endogenous compounds and catabolism of xenobiotics, and their activities rely on a partner enzyme, cytochrome P450 reductase (CPR, E.C.1.6.2.4). Two CPR cDNAs, GhCPR1 and GhCPR2, were isolated from cotton (Gossypium hirsutum). They are 71% identical to each other at the amino acid sequence level and belong to the Class I and II of dicotyledonous CPRs, respectively. The recombinant enzymes reduced cytochrome c, ferricyanide and dichlorophenolindophenol (DCPIP) in an NADPH-dependent manner, and supported the activity of CYP73A25, a cinnamate 4-hydroxylase of cotton. Both GhCPR genes were widely expressed in cotton tissues, with a reduced expression level of GhCPR2 in the glandless cotton cultivar. Expression of GhCPR2, but not GhCPR1, was inducible by mechanical wounding and elicitation, indicating that the GhCPR2 is more related to defense reactions, including biosynthesis of secondary metabolites. 2009 Elsevier Ltd. All rights reserved.

  1. Studies on the kinetic mechanism of nitrate reductase from spinach (Spinacea oleracea).

    Science.gov (United States)

    de la Rosa, F F; Palacián, E; Castillo, F

    1980-09-01

    Based on Lineweaver-Burk plots of the initial velocities, at different concentrations of NADH and nitrate, and product inhibition patterns, an Iso Ping Pong Bi Bi steady state kinetic mechanism is proposed for the spinach nitrate reductase. This mechanism incorporates the concept that the oxidized enzyme is present in two isomeric forms.

  2. Nitrate reductase from Spinacea oleracea. FAD and the reactivation of the enzyme treated with p-Hydroxymercuribenzoate.

    Science.gov (United States)

    Castillo, F; de la Rosa, F F; Palacián, E

    1977-12-01

    Spinach nitrate reductase complex previously inactivated by treatment with mercurials p-hydroxymercuribenzoate or p-hydroxymercuriphenyl sulphonate can be reactivated by incubation with dithioerythritol. The reactivation of NADH-diaphorase seems to be FAD-dependent, whereas that of FNH2-nitrate reductase is not. The requirement of FAD for NADH-inactivation of nitrate reductase treated with p-hydroxymercuribenzoate disappears after treatment with dithioerythritol.

  3. Overexpression of Nitrate Reductase in Tobacco Delays Drought-Induced Decreases in Nitrate Reductase Activity and mRNA1

    Science.gov (United States)

    Ferrario-Méry, Sylvie; Valadier, Marie-Hélène; Foyer, Christine H.

    1998-01-01

    Transformed (cauliflower mosaic virus 35S promoter [35S]) tobacco (Nicotiana plumbaginifolia L.) plants constitutively expressing nitrate reductase (NR) and untransformed controls were subjected to drought for 5 d. Drought-induced changes in biomass accumulation and photosynthesis were comparable in both lines of plants. After 4 d of water deprivation, a large increase in the ratio of shoot dry weight to fresh weight was observed, together with a decrease in the rate of photosynthetic CO2 assimilation. Foliar sucrose increased in both lines during water stress, but hexoses increased only in leaves from untransformed controls. Foliar NO3− decreased rapidly in both lines and was halved within 2 d of the onset of water deprivation. Total foliar amino acids decreased in leaves of both lines following water deprivation. After 4 d of water deprivation no NR activity could be detected in leaves of untransformed plants, whereas about 50% of the original activity remained in the leaves of the 35S-NR transformants. NR mRNA was much more stable than NR activity. NR mRNA abundance increased in the leaves of the 35S-NR plants and remained constant in controls for the first 3 d of drought. On the 4th d, however, NR mRNA suddenly decreased in both lines. Rehydration at d 3 caused rapid recovery (within 24 h) of 35S-NR transcripts, but no recovery was observed in the controls. The phosphorylation state of the protein was unchanged by long-term drought. There was a strong correlation between maximal extractable NR activity and ambient photosynthesis in both lines. We conclude that drought first causes increased NR protein turnover and then accelerates NR mRNA turnover. Constitutive NR expression temporarily delayed drought-induced losses in NR activity. 35S-NR expression may therefore allow more rapid recovery of N assimilation following short-term water deficit. PMID:9576799

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

    Science.gov (United States)

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

    1996-01-01

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

  5. NADH-Ferricyanide Reductase of Leaf Plasma Membranes : Partial Purification and Immunological Relation to Potato Tuber Microsomal NADH-Ferricyanide Reductase and Spinach Leaf NADH-Nitrate Reductase.

    Science.gov (United States)

    Askerlund, P; Laurent, P; Nakagawa, H; Kader, J C

    1991-01-01

    Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b(5) reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber

  6. Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species

    International Nuclear Information System (INIS)

    Gray, Joshua P.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2010-01-01

    Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and β-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from β-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury.

  7. NADPH-dependent thioredoxin reductase and 2-Cys peroxiredoxins are needed for the protection of Mg-protoporphyrin monomethyl ester cyclase

    DEFF Research Database (Denmark)

    Stenbæk, Anne; Hansson, Andreas; Wulff, Ragna Peterson

    2008-01-01

    Abstract: The chloroplast-localized NADPH-dependent thioredoxin reductase ( NTRC) has been found to be able to reduce hydrogen peroxide scavenging 2-Cys peroxiredoxins. We show that the Arabidopsis ntrc mutant is perturbed in chlorophyll biosynthesis and accumulate intermediates preceding...

  8. Inhibition of nitrate transport by anti-nitrate reductase IgG fragments and the identification of plasma membrane associated nitrate reductase in roots of barley seedlings

    Science.gov (United States)

    Ward, M. R.; Tischner, R.; Huffaker, R. C.

    1988-01-01

    Membrane associated nitrate reductase (NR) was detected in plasma membrane (PM) fractions isolated by aqueous two-phase partitioning from barley (Hordeum vulgare L. var CM 72) roots. The PM associated NR was not removed by washing vesicles with 500 millimolar NaCl and 1 millimolar EDTA and represented up to 4% of the total root NR activity. PM associated NR was stimulated up to 20-fold by Triton X-100 whereas soluble NR was only increased 1.7-fold. The latency was a function of the solubilization of NR from the membrane. NR, solubilized from the PM fraction by Triton X-100 was inactivated by antiserum to Chlorella sorokiniana NR. Anti-NR immunoglobulin G fragments purified from the anti-NR serum inhibited NO3- uptake by more than 90% but had no effect on NO2- uptake. The inhibitory effect was only partially reversible; uptake recovered to 50% of the control after thorough rinsing of roots. Preimmune serum immunoglobulin G fragments inhibited NO3- uptake 36% but the effect was completely reversible by rinsing. Intact NR antiserum had no effect on NO3- uptake. The results present the possibility that NO3- uptake and NO3- reduction in the PM of barley roots may be related.

  9. NADPH-thioredoxin reductase C mediates the response to oxidative stress and thermotolerance in the cyanobacterium Anabaena sp. PCC7120.

    Directory of Open Access Journals (Sweden)

    ANA MARÍA SÁNCHEZ-RIEGO

    2016-08-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Pandey, Amit V., E-mail: amit@pandeylab.org [Pediatric Endocrinology, Diabetology and Metabolism, Department of Clinical Research, University of Bern, Tiefenaustrasse 120c, CH-3004 Bern (Switzerland); Flueck, Christa E.; Mullis, Primus E. [Pediatric Endocrinology, Diabetology and Metabolism, Department of Clinical Research, University of Bern, Tiefenaustrasse 120c, CH-3004 Bern (Switzerland)

    2010-09-24

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

  11. EPR and redox properties of periplasmic nitrate reductase from Desulfovibrio desulfuricans ATCC 27774.

    Science.gov (United States)

    González, Pablo J; Rivas, María G; Brondino, Carlos D; Bursakov, Sergey A; Moura, Isabel; Moura, José J G

    2006-07-01

    Nitrate reductases are enzymes that catalyze the conversion of nitrate to nitrite. We report here electron paramagnetic resonance (EPR) studies in the periplasmic nitrate reductase isolated from the sulfate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774. This protein, belonging to the dimethyl sulfoxide reductase family of mononuclear Mo-containing enzymes, comprises a single 80-kDa subunit and contains a Mo bis(molybdopterin guanosine dinucleotide) cofactor and a [4Fe-4S] cluster. EPR-monitored redox titrations, carried out with and without nitrate in the potential range from 200 to -500 mV, and EPR studies of the enzyme, in both catalytic and inhibited conditions, reveal distinct types of Mo(V) EPR-active species, which indicates that the Mo site presents high coordination flexibility. These studies show that nitrate modulates the redox properties of the Mo active site, but not those of the [4Fe-4S] center. The possible structures and the role in catalysis of the distinct Mo(V) species detected by EPR are discussed.

  12. Mo and W bis-MGD enzymes: nitrate reductases and formate dehydrogenases.

    Science.gov (United States)

    Moura, José J G; Brondino, Carlos D; Trincão, José; Romão, Maria João

    2004-10-01

    Molybdenum and tungsten are second- and third-row transition elements, respectively, which are found in a mononuclear form in the active site of a diverse group of enzymes that generally catalyze oxygen atom transfer reactions. Mononuclear Mo-containing enzymes have been classified into three families: xanthine oxidase, DMSO reductase, and sulfite oxidase. The proteins of the DMSO reductase family present the widest diversity of properties among its members and our knowledge about this family was greatly broadened by the study of the enzymes nitrate reductase and formate dehydrogenase, obtained from different sources. We discuss in this review the information of the better characterized examples of these two types of Mo enzymes and W enzymes closely related to the members of the DMSO reductase family. We briefly summarize, also, the few cases reported so far for enzymes that can function either with Mo or W at their active site.

  13. The Effect of Nitrate Levels and Harvest Times on Fe, Zn, Cu, and K, Concentrations and Nitrate Reductase Activity in Lettuce and Spinach

    Directory of Open Access Journals (Sweden)

    Z. Gheshlaghi

    2015-09-01

    Full Text Available Leafy vegetables are considered as the main sources of nitrate in the human diet. In order to investigate the effect of nitrate levels and harvest times on nitrate accumulation, nitrate reductase activity, concentrations of Fe, Zn, Cu and K in Lettuce and Spinach and their relation to nitrate accumulation in these leafy vegetables, two harvest times (29 and 46 days after transplanting, two vegetable species of lettuce and spinach and two concentrations of nitrate (10 and 20 mM were used in a hydroponics greenhouse experiment with a completely randomized design and 3 replications. Modified Hoagland and Arnon nutrient solutions were used for the experiment. The results indicated that by increasing nitrate concentration of solution, nitrate accumulation in roots and shoots of lettuce and spinach increased significantly (P ≤ 0.05, and the same trend was observed for the nitrate reductase activity in the shoots of the two species. Increasing the nitrate concentrations of solution, reduced the shoot dry weight and the concentration of Fe and Cu in both species, where as it increased the K and Zn concentrations in the shoots of the two species in each both harvest times, the nitrate accumulation increased, but the nitrate reductase activity decreased in the shoots of the two species over the course of the growth. The Concentration of Fe, Cu and K decreased in the shoots of lettuce and the spinach with the time, despite the increase in Zn concentration in the shoots. The results also indicated that increasing nitrate concentrations of solution to the levels greater than the plant capacity for reduction and net uptake of nitrate, leads to the nitrate accumulation in the plants. Nitrate accumulation in plant tissue led to decreases in fresh shoot yield and Fe and Cu concentrations and nitrate reductase activities in both lettuce and spinach.

  14. Variant Cell Lines of Haplopappus gracilis with Disturbed Activities of Nitrate Reductase and Nitrite Reductase.

    Science.gov (United States)

    Gilissen, L J; Barneix, A J; van Staveren, M; Breteler, H

    1985-07-01

    Selected variant cell lines of Haplopappus gracilis (Nutt) Gray that showed disturbed growth after transfer from an alanine medium to NO(3) (-) medium were characterized. The in vivo NO(3) (-) reductase activity (NRA) was lower in these lines than in the wild type. In vitro NRA assays suggest that decreased in vivo NRA was not caused by a lower amount of active enzyme. Cells of the variant lines revealed up to 75% lower extractable activity of NO(2) (-) reductase as compared with the wild type. This coincided with higher accumulation of NO(2) (-) by the variant than by the wild type cells after transfer from alanine medium to NO(3) (-) medium. NO(2) (-) accumulation was transient or continuous, depending on cell line, metabolic state of the cells, and light conditions.

  15. Azospirillum Inoculation Alters Nitrate Reductase Activity and Nitrogen Uptake in Wheat Plant Under Water Deficit Conditions

    OpenAIRE

    N. Aliasgharzad, N. Aliasgharzad; Heydaryan, Zahra; Sarikhani, M.R

    2014-01-01

    Water deficit stress usually diminishes nitrogen uptake by plants. There are evidences that some nitrogen fixing bacteria can alleviate this stress by supplying nitrogen and improving its metabolism in plants. Four Azospirillum strains, A. lipoferum AC45-II, A. brasilense AC46-I, A. irakense AC49-VII and A. irakense AC51-VI were tested for nitrate reductase activity (NRA). In a pot culture experiment using a sandy loam soil, wheat plants (Triticum aestivum L. cv. Sardari) were inoculated with...

  16. The Flavin-Containing Reductase Domain of Cytochrome P450 BM3 Acts as a Surrogate for Mammalian NADPH-P450 Reductase.

    Science.gov (United States)

    Park, Seon-Ha; Kang, Ji-Yeon; Kim, Dong-Hyun; Ahn, Taeho; Yun, Chul-Ho

    2012-11-01

    Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium is a self-sufficient monooxygenase that consists of a heme domain and FAD/FMN-containing reductase domain (BMR). In this report, the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) by BMR was evaluated as a method for monitoring BMR activity. The electron transfer proceeds from NADPH to BMR and then to BMR substrates, MTT and CTC. MTT and CTC are monotetrazolium salts that form formazans upon reduction. The reduction of MTT and CTC followed classical Michaelis-Menten kinetics (kcat =4120 min(-1), Km =77 μM for MTT and kcat =6580 min(-1), Km =51 μM for CTC). Our continuous assay using MTT and CTC allows the simple, rapid measurement of BMR activity. The BMR was able to metabolize mitomycin C and doxorubicin, which are anticancer drug substrates for CPR, producing the same metabolites as those produced by CPR. Moreover, the BMR was able to interact with CYP1A2 and transfer electrons to promote the oxidation reactions of substrates by CYP1A2 and CYP2E1 in humans. The results of this study suggest the possibility of the utilization of BMR as a surrogate for mammalian CPR.

  17. Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617.

    Science.gov (United States)

    Correia, Cristina; Besson, Stéphane; Brondino, Carlos D; González, Pablo J; Fauque, Guy; Lampreia, Jorge; Moura, Isabel; Moura, José J G

    2008-11-01

    Membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617 can be solubilized in either of two ways that will ultimately determine the presence or absence of the small (Iota) subunit. The enzyme complex (NarGHI) is composed of three subunits with molecular masses of 130, 65, and 20 kDa. This enzyme contains approximately 14 Fe, 0.8 Mo, and 1.3 molybdopterin guanine dinucleotides per enzyme molecule. Curiously, one heme b and 0.4 heme c per enzyme molecule have been detected. These hemes were potentiometrically characterized by optical spectroscopy at pH 7.6 and two noninteracting species were identified with respective midpoint potentials at Em=+197 mV (heme c) and -4.5 mV (heme b). Variable-temperature (4-120 K) X-band electron paramagnetic resonance (EPR) studies performed on both as-isolated and dithionite-reduced nitrate reductase showed, respectively, an EPR signal characteristic of a [3Fe-4S]+ cluster and overlapping signals associated with at least three types of [4Fe-4S]+ centers. EPR of the as-isolated enzyme shows two distinct pH-dependent Mo(V) signals with hyperfine coupling to a solvent-exchangeable proton. These signals, called "low-pH" and "high-pH," changed to a pH-independent Mo(V) signal upon nitrate or nitrite addition. Nitrate addition to dithionite-reduced samples at pH 6 and 7.6 yields some of the EPR signals described above and a new rhombic signal that has no hyperfine structure. The relationship between the distinct EPR-active Mo(V) species and their plausible structures is discussed on the basis of the structural information available to date for closely related membrane-bound nitrate reductases.

  18. The effect of ionic and non-ionic surfactants on the growth, nitrate reductase and nitrite reductase activities of Spirodela polyrrhiza (L. Schleiden

    Directory of Open Access Journals (Sweden)

    Józef Buczek

    2014-01-01

    Full Text Available Inclusion into the medium of 5 mg•dm-3 of non-ionic (ENF or ionic (DBST surfactant caused 50-60% inhibition of nitrite reductase MR activity in S. polyrrhiza. At the same time, increased accumulation of NO2- in the plant tissues and lowering of the total and soluble protein contents were found. DBST also lowered the nitrate reductase (NR activity and the dry mass of the plants.

  19. Rapid induction of GFP expression by the nitrate reductase promoter in the diatom Phaeodactylum tricornutum

    Science.gov (United States)

    Ewe, Daniela; Río Bártulos, Carolina; Kroth, Peter G.; Gruber, Ansgar

    2016-01-01

    An essential prerequisite for a controlled transgene expression is the choice of a suitable promoter. In the model diatom Phaeodactylum tricornutum, the most commonly used promoters for trans-gene expression are the light dependent lhcf1 promoters (derived from two endogenous genes encoding fucoxanthin chlorophyll a/c binding proteins) and the nitrate dependent nr promoter (derived from the endogenous nitrate reductase gene). In this study, we investigated the time dependent expression of the green fluorescent protein (GFP) reporter under control of the nitrate reductase promoter in independently genetically transformed P. tricornutum cell lines following induction of expression by change of the nitrogen source in the medium via flow cytometry, microscopy and western blotting. In all investigated cell lines, GFP fluorescence started to increase 1 h after change of the medium, the fastest increase rates were observed between 2 and 3 h. Fluorescence continued to increase slightly for up to 7 h even after transfer of the cells to ammonium medium. The subsequent decrease of GFP fluorescence was much slower than the increase, probably due to the stability of GFP. The investigation of several cell lines transformed with nr based constructs revealed that, also in the absence of nitrate, the promoter may show residual activity. Furthermore, we observed a strong variation of gene expression between independent cell lines, emphasising the importance of a thorough characterisation of genetically modified cell lines and their individual expression patterns. PMID:27635322

  20. A novel NADPH-dependent aldehyde reductase gene from Vigna radiata confers resistance to the grapevine fungal toxin eutypine.

    Science.gov (United States)

    Guillén, P; Guis, M; Martínez-Reina, G; Colrat, S; Dalmayrac, S; Deswarte, C; Bouzayen, M; Roustan, J P; Fallot, J; Pech, J C; Latché, A

    1998-11-01

    Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzyl aldehyde, is a toxin produced by Eutypa lata, the causal agent of eutypa dieback of grapevines. It has previously been demonstrated that tolerance of some cultivars to this disease was correlated with their capacity to convert eutypine to the corresponding alcohol, eutypinol, which lacks phytotoxicity. We have thus purified to homogeneity a protein from Vigna radiata that exhibited eutypine-reducing activity and have isolated the corresponding cDNA. This encodes an NADPH-dependent reductase of 36 kDa that we have named Vigna radiata eutypine-reducing enzyme (VR-ERE), based on the capacity of a recombinant form of the protein to reduce eutypine into eutypinol. The strongest homologies (86.8%) of VR-ERE at the amino acid level were found with CPRD14, a drought-inducible gene of unknown function, isolated from Vigna unguiculata and with an aromatic alcohol dehydrogenase (71.7%) from Eucalyptus gunnii. Biochemical characterization of VR-ERE revealed that a variety of compounds containing an aldehyde group can act as substrates. However, the highest affinity was observed with 3-substituted benzaldehydes. Expression of a VR-ERE transgene in Vitis vinifera cells cultured in vitro conferred resistance to the toxin. This discovery opens up new biotechnological approaches for the generation of grapevines resistant to eutypa dieback.

  1. Colour formation in fermented sausages by meat-associated staphylococci with different nitrite- and nitrate-reductase activities

    DEFF Research Database (Denmark)

    Gøtterup, Jacob; Olsen, Karsten; Knøchel, Susanne

    2008-01-01

    nitrate depended on the specific Staphylococcus strain. Strains with high nitrate-reductase activity showed a significantly faster rate of pigment formation, but other factors were of influence as well. Product stability for the sliced, packaged sausage was evaluated as surface colour and oxidation...

  2. Inhibition of the Staphylococcus aureus NADPH-dependent enoyl-acyl carrier protein reductase by triclosan and hexachlorophene.

    Science.gov (United States)

    Heath, R J; Li, J; Roland, G E; Rock, C O

    2000-02-18

    Enoyl-acyl carrier protein reductase (FabI) plays a determinant role in completing cycles of elongation in type II fatty acid synthase systems and is an important target for antibacterial drugs. The FabI component of Staphylococcus aureus (saFabI) was identified, and its properties were compared with Escherichia coli FabI (ecFabI). ecFabI and saFabI had similar specific activities, and saFabI expression complemented the E. coli fabI(Ts) mutant, illustrating that the Gram-positive FabI was interchangeable with the Gram-negative FabI enzyme. However, ecFabI was specific for NADH, whereas saFabI exhibited specific and positive cooperative binding of NADPH. Triclosan and hexachlorophene inhibited both ecFabI and saFabI. The triclosan-resistant ecFabI(G93V) protein was also refractory to hexachlorophene inhibition, illustrating that both drugs bind at the FabI active site. Both the introduction of a plasmid expressing the safabI gene or a missense mutation in the chromosomal safabI gene led to triclosan resistance in S. aureus; however, these strains did not exhibit cross-resistance to hexachlorophene. The replacement of the ether linkage in triclosan by a carbon bridge in hexachlorophene prevented the formation of a stable FabI-NAD(P)(+)-drug ternary complex. Thus, the formation of this ternary complex is a key determinant of the antibacterial activity of FabI inhibitors.

  3. An inducible NADPH-cytochrome P450 reductase from Picrorhiza kurrooa - an imperative redox partner of cytochrome P450 enzymes.

    Science.gov (United States)

    Bhat, Wajid Waheed; Rana, Satiander; Dhar, Niha; Razdan, Sumeer; Pandith, Shahzad A; Vishwakarma, Ram; Lattoo, Surrinder K

    2014-06-01

    Picrorhiza kurrooa synthesizes a large array of pharmacologically important monoterpenoid iridoid glycosides called picrosides. Although chemical profile and pharmacological activities of P. kurrooa have been extensively studied, limited attempts have been made to decipher the biosynthetic route and to identify the key regulatory genes involved in picroside biosynthesis. In the present study, NADPH-cytochrome P450 reductase, a key enzyme involved in electron transfer to cytochrome P450s was identified from P. kurrooa. The full length cDNA (2679 bp) contained an open reading frame of 2133 bp, corresponding to 710 amino acids. PkCPR was heterologously expressed in Escherichia coli and the kinetic parameters of the recombinant enzyme were determined. Specific activity, V max and K m of PkCPR were found to be 5.8 ± 0.05 μmol min(-1) mg(-1), 8.1 ± 0.12 μmol min(-1) mg(-1) and 7.8 μM, respectively. PkCPR was found to be spatially regulated at transcript level, being maximally expressed in leaf tissues. Altitude was found to have a positive effect on the picroside concentration and the picroside content positively correlated with the PkCPR transcript levels in samples collected at varied altitudes. Further, transcript profiling under methyl jasmonate, salicylic acid, 2,4-dicholorophenoxy acetic acid and UV-B elicitations displayed differential transcriptional regulation of PkCPR that fully corroborated with the identified cis-elements within the PkCPR promoter. Expression of PkCPR was inducible by UV-B and phytohormone elicitation, indicating that the PkCPR is possibly related to defence reactions, including biosynthesis of secondary metabolites. Present study is so far the only report of identification and functional characterization of CPR ortholog from P. kurrooa.

  4. Mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli: pH and deuterium isotope effects with NADPH as the variable substrate

    International Nuclear Information System (INIS)

    Morrison, J.F.; Stone, S.R.

    1988-01-01

    The variations with pH of the kinetic parameters and primary deuterium isotope effects for the reaction of NADPH with dihydrofolate reductase from Escherichia coli have been determined. The aims of the investigations were to elucidate the chemical mechanism of the reaction and to obtain information about the location of the rate-limiting steps. The V and V/K/sub NADPH/ profiles indicate that a single ionizing group at the active center of the enzyme must be protonated for catalysis, whereas the K/sub i/ profiles show that the binding of NADPH to the free enzyme and of ATP-ribose to the enzyme-dihydrofolate complex is pH independent. From the results of deuterium isotope effects on V/K/sub NADPH/, it is concluded that NADPH behaves as a sticky substrate. It is this stickiness that raises artificially the intrinsic pK value of 6.4 for the Asp-27 residue of the enzyme-dihydrofolate complex to an observed value of 8.9. Thus, the binary enzyme complex is largely protonated at neutral pH. The elevation of the intrinsic pK value of 6.4 for the ternary enzyme-NADPH-dihydrofolate complex to 8.5 is not due to the kinetic effects of substrates. Rather, it is the consequence of the lower, pH-independent rate of product release and the faster pH-dependent catalytic step. The data for deuterium isotope and deuterium solvent isotope effects are consistent with the postulate that, for the reduction of dihydrofolate to tetrahydrofolate, protonation precedes hydride transfer. A scheme is proposed for the indirect transfer of a proton from the enzyme to dihydrofolate

  5. Purification and biochemical characterization of simplified eukaryotic nitrate reductase expressed in Pichia pastoris.

    Science.gov (United States)

    Barbier, Guillaume G; Joshi, Rama C; Campbell, Ellen R; Campbell, Wilbur H

    2004-09-01

    NAD(P)H:nitrate reductase (NaR, EC 1.7.1.1-3) is a useful enzyme in biotechnological applications, but it is very complex in structure and contains three cofactors-flavin adenine dinucleotide, heme-Fe, and molybdenum-molybdopterin (Mo-MPT). A simplified nitrate reductase (S-NaR1) consisting of Mo-MPT-binding site and nitrate-reducing active site was engineered from yeast Pichia angusta NaR cDNA (YNaR1). S-NaR1 was cytosolically expressed in high-density fermenter culture of methylotrophic yeast Pichia pastoris. Total amount of S-NaR1 protein produced was approximately 0.5 g per 10 L fermenter run, and methanol phase productivity was 5 microg protein/g wet cell weight/h. Gene copy number in genomic DNA of different clones showed direct correlation with the expression level. S-NaR1 was purified to homogeneity in one step by immobilized metal affinity chromatography (IMAC) and total amount of purified protein per run of fermentation was approximately 180 mg. Polypeptide size was approximately 55 kDa from electrophoretic analysis, and S-NaR1 was mainly homo-tetrameric in its active form, as shown by gel filtration. S-NaR1 accepted electrons efficiently from reduced bromphenol blue (kcat = 2081 s(-1)) and less so from reduced methyl viologen (kcat = 159 s(-1)). The nitrate KM for S-NaR1 was 30 +/- 3 microM, which is very similar to YNaR1. S-NaR1 is capable of specific nitrate reduction, and direct electric current, as shown by catalytic nitrate reduction using protein film cyclic voltammetry, can drive this reaction. Thus, S-NaR1 is an ideal form of this enzyme for commercial applications, such as an enzymatic nitrate biosensor formulated with S-NaR1 interfaced to an electrode system.

  6. The uptake and accumulation of phosphorous and nitrates and the activity of nitrate reductase in cucumber seedlings treated with PbCl2 or CdCl2

    Directory of Open Access Journals (Sweden)

    Marek Burzyński

    2014-01-01

    Full Text Available Treatment of 4-day-old cucumber (Cucumis sativus L. seedlings with PbCl2 or CdCl2 caused a significant increase in the accumulation of heavy metals by the plants, especially in the roots. The accumulated Pb initially enhanced the uptake of phosphorous after the plants had been transferred to a nutrient medium (6, 24 hrs, but after only 48 Ins the uptake had dropped to below control level. The plants treated with Cd exhibited a constant decreased phosphorous uptake level. The accumulated lead and cadmium also inhibited nitrate uptake and the activity of nitrate reductase. It is suggested that the reason for the decreased nitrate reductase activity lay rather in the lower nitrate uptake than in a direct effect of the heavy metals on the enzyme.

  7. Mechanism of inhibition of purified leaping mullet (Liza saliens) NADPH-cytochrome P450 reductase by toxic metals: aluminum and thallium.

    Science.gov (United States)

    Bozcaarmutlu, Azra

    2007-01-01

    Aluminum and thallium may reach life-threatening levels in aquatic systems in the near future because of their extensive use in various industrial fields. It is therefore important to study the mechanism of toxicity of aluminum and thallium on fish enzymes. To this aim, the effects of aluminum and thallium on the activity of purified leaping mullet (Liza saliens) cytochrome P450 reductase, an essential component of the important cytochrome P450 system, have been studied. Results indicated that both metal ions strongly inhibited the NADPH-cytochrome P450 reductase. The IC50 values of AlCl3 and TlCl3 were estimated to be 34 microM and 3 microM, respectively. The Lineweaver-Burk plot and Dixon plot revealed that both metal ions noncompetitively inhibited the purified mullet cytochrome P450 reductase. The K(i) values of Al3+ and Tl3+ were calculated from Dixon plots as 8.9 and 5.6 microM, respectively. The inhibitory effects of Al3+ and Tl3+ on purified cytochrome P450 reductase were partially recovered by 1 mM EDTA. Additionally, tin and magnesium were shown to have no apparent effect on purified mullet cytochrome P450 reductase.

  8. Acetaldehyde Oxime, A Product Formed during the In Vivo Nitrate Reductase Assay of Soybean Leaves.

    Science.gov (United States)

    Mulvaney, C S; Hageman, R H

    1984-09-01

    Evolution of nitrogen oxides (NO((x)), primarily as nitric oxide) from soybean (Glycine max [L.] Merr.) leaves during purged in vivo nitrate reductase assays had been reported; however, these reports were based on a method that had been used for determination of NO((x)) in air. This method also detects other N compounds. Preliminary work led us to doubt that the evolved N was nitric oxide. Studies were undertaken to identify the N compound evolved from the in vivo assay that had been reported as NO((x)). Material for identification was obtained by cryogenic trapping and fractional distillation, and by chemical trapping procedures. Mass spectrometry, ultraviolet spectroscopy, and (15)N-labeled nitrate were used to identify the compounds evolved and to determine whether these compounds were derived from nitrate. Acetaldehyde oxime was identified as the predominant N compound evolved and this compound is readily detected by the method for NO((x)) determination. Substantial quantities of acetaldehyde oxime (16.2 micromoles per gram fresh weight per hour) were evolved during the in vivo assay. Small amounts of nitrous oxide (0.63 micrograms N per gram fresh weight per hour) were evolved, but this compound is not detected as NO((x)). Acetaldehyde oxime and nitrous oxide were both produced as a result of nitrate ((15)NO(3) (-)) reduction during the assay.

  9. Acetaldehyde Oxime, A Product Formed during the In Vivo Nitrate Reductase Assay of Soybean Leaves 1

    Science.gov (United States)

    Mulvaney, Charlene S.; Hageman, Richard H.

    1984-01-01

    Evolution of nitrogen oxides (NO(x), primarily as nitric oxide) from soybean (Glycine max [L.] Merr.) leaves during purged in vivo nitrate reductase assays had been reported; however, these reports were based on a method that had been used for determination of NO(x) in air. This method also detects other N compounds. Preliminary work led us to doubt that the evolved N was nitric oxide. Studies were undertaken to identify the N compound evolved from the in vivo assay that had been reported as NO(x). Material for identification was obtained by cryogenic trapping and fractional distillation, and by chemical trapping procedures. Mass spectrometry, ultraviolet spectroscopy, and 15N-labeled nitrate were used to identify the compounds evolved and to determine whether these compounds were derived from nitrate. Acetaldehyde oxime was identified as the predominant N compound evolved and this compound is readily detected by the method for NO(x) determination. Substantial quantities of acetaldehyde oxime (16.2 micromoles per gram fresh weight per hour) were evolved during the in vivo assay. Small amounts of nitrous oxide (0.63 micrograms N per gram fresh weight per hour) were evolved, but this compound is not detected as NO(x). Acetaldehyde oxime and nitrous oxide were both produced as a result of nitrate (15NO3−) reduction during the assay. PMID:16663781

  10. Conformational changes of the NADPH-dependent cytochrome P450 reductase in the course of electron transfer to cytochromes P450.

    Science.gov (United States)

    Laursen, Tomas; Jensen, Kenneth; Møller, Birger Lindberg

    2011-01-01

    The NADPH-dependent cytochrome P450 reductase (CPR) is a key electron donor to eucaryotic cytochromes P450 (CYPs). CPR shuttles electrons from NADPH through the FAD and FMN-coenzymes into the iron of the prosthetic heme-group of the CYP. In the course of these electron transfer reactions, CPR undergoes large conformational changes. This mini-review discusses the new evidence provided for such conformational changes involving a combination of a "swinging" and "rotating" model and highlights the molecular mechanisms by which formation of these conformations are controlled and thereby enables CPR to serve as an effective electron transferring "nano-machine". Copyright © 2010 Elsevier B.V. All rights reserved.

  11. Seasonal Patterns of Nitrate Reductase and Nitrogenase Activities in Phaseolus vulgaris L. 1

    Science.gov (United States)

    Franco, Alvilio A.; Pereira, Joao C.; Neyra, Carlos A.

    1979-01-01

    The patterns of nitrate reductase activity (NRA) in the leaves (in vivo assay) and root nodule nitrogenase activity (C2H2 reduction) were investigated throughout the season in field-grown Phaseolus vulgaris plants. Maximal NRA (per g fresh weight) occurred at early stages of leaf development but total activity (per leaf) was maximal when the leaf reached full size. In mature plants, most NRA was associated with the upper leaves. Nitrogenase activity was initiated about 2 weeks after sowing, reached a maximum at flowering (5 weeks after sowing) and declined rapidly thereafter. Nitrogenase activity followed the pattern of nodule development. After flowering, P. vulgaris was apparently able to take up and assimilate NO−3 as evidenced by the increase in NO−3 content of the stem and the high levels of NRA in the leaves. Total plant NRA was maximal after flowering and addition of NH4NO3 to the soil at flowering resulted in even higher levels of NRA through most of the pod-filling period, thus resulting in higher seed yields (59% over control). It is proposed that P. vulgaris can benefit from both N2 fixation and NO−3 assimilation and that nitrate reductase plays an important role in the assimilation of nitrogen after flowering. PMID:16660740

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

    Science.gov (United States)

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

    2014-02-28

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

  13. Nitrogen nutrition of Canna indica: Effects of ammonium versus nitrate on growth, biomass allocation, photosynthesis, nitrate reductase activity and N uptake rates

    DEFF Research Database (Denmark)

    Konnerup, Dennis; Brix, Hans

    2010-01-01

    The effects of inorganic nitrogen (N) source (NH4+, NO3- or both) on growth, biomass allocation, photosynthesis, N uptake rate, nitrate reductase activity and mineral composition of Canna indica were studied in hydroponic culture. The relative growth rates (0.05-0.06 g g-1 d-1), biomass allocation...

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

    Energy Technology Data Exchange (ETDEWEB)

    Wallen,J.; Paige, C.; Mallett, T.; Karplus, P.; Claiborne, A.

    2008-01-01

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

  15. The nitrate reductase activity of some root and stem parasites and their hosts

    International Nuclear Information System (INIS)

    Hunter, J.J.

    1984-12-01

    This investigation surveyed the nitrate reductase activity (NRA) of some South African root and stem parasites, as well as their hosts. Fourteen species - five stem and nine root parasites, representative of seven families - and eleven different hosts from eight families, were studied. Two methods were applied in the determination of the NRA of parasite and host, namely the in vivo and in vitro methods. Because of the limited literature on the NRA of parasitic flowering plants both the in vivo and in vitro methods were developed for the host species and subsequently applied to that specific species of parasite as well. Parasites and hosts were also investigated in their natural habitat. The NRA of the roots could, however, only be increased providing phorsynthetic products as a source of NADH, were available. By using [U- 14 C]-Sucrose it was confirmed that the parasite could have fulfilled this need. Generally, the investigation showed that the parasites that were studied, have not altogether lost their ability to reduce nitrate. However, it would appear that the host is used as a source of reduced nitrogen, rather than nitrate, under natural conditions

  16. Nitrate decreases xanthine oxidoreductase-mediated nitrite reductase activity and attenuates vascular and blood pressure responses to nitrite

    Directory of Open Access Journals (Sweden)

    Célio Damacena-Angelis

    2017-08-01

    Full Text Available Nitrite and nitrate restore deficient endogenous nitric oxide (NO production as they are converted back to NO, and therefore complement the classic enzymatic NO synthesis. Circulating nitrate and nitrite must cross membrane barriers to produce their effects and increased nitrate concentrations may attenuate the nitrite influx into cells, decreasing NO generation from nitrite. Moreover, xanthine oxidoreductase (XOR mediates NO formation from nitrite and nitrate. However, no study has examined whether nitrate attenuates XOR-mediated NO generation from nitrite. We hypothesized that nitrate attenuates the vascular and blood pressure responses to nitrite either by interfering with nitrite influx into vascular tissue, or by competing with nitrite for XOR, thus inhibiting XOR-mediated NO generation. We used two independent vascular function assays in rats (aortic ring preparations and isolated mesenteric arterial bed perfusion to examine the effects of sodium nitrate on the concentration-dependent responses to sodium nitrite. Both assays showed that nitrate attenuated the vascular responses to nitrite. Conversely, the aortic responses to the NO donor DETANONOate were not affected by sodium nitrate. Further confirming these results, we found that nitrate attenuated the acute blood pressure lowering effects of increasing doses of nitrite infused intravenously in freely moving rats. The possibility that nitrate could compete with nitrite and decrease nitrite influx into cells was tested by measuring the accumulation of nitrogen-15-labeled nitrite (15N-nitrite by aortic rings using ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS. Nitrate exerted no effect on aortic accumulation of 15N-nitrite. Next, we used chemiluminescence-based NO detection to examine whether nitrate attenuates XOR-mediated nitrite reductase activity. Nitrate significantly shifted the Michaelis Menten saturation curve to the right, with a 3-fold increase in

  17. Cold adaptation of the mononuclear molybdoenzyme periplasmic nitrate reductase from the Antarctic bacterium Shewanella gelidimarina

    Energy Technology Data Exchange (ETDEWEB)

    Simpson, Philippa J.L. [School of Chemistry, University of Sydney, New South Wales 2006 (Australia); Codd, Rachel, E-mail: rachel.codd@sydney.edu.au [School of Chemistry, University of Sydney, New South Wales 2006 (Australia); School of Medical Sciences (Pharmacology) and Bosch Institute, University of New South Wales, New South Wales 2006 (Australia)

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Cold-adapted phenotype of NapA from the Antarctic bacterium Shewanella gelidimarina. Black-Right-Pointing-Pointer Protein homology model of NapA from S. gelidimarina and mesophilic homologue. Black-Right-Pointing-Pointer Six amino acid residues identified as lead candidates governing NapA cold adaptation. Black-Right-Pointing-Pointer Molecular-level understanding of designing cool-temperature in situ oxyanion sensors. -- Abstract: The reduction of nitrate to nitrite is catalysed in bacteria by periplasmic nitrate reductase (Nap) which describes a system of variable protein subunits encoded by the nap operon. Nitrate reduction occurs in the NapA subunit, which contains a bis-molybdopterin guanine dinucleotide (Mo-MGD) cofactor and one [4Fe-4S] iron-sulfur cluster. The activity of periplasmic nitrate reductase (Nap) isolated as native protein from the cold-adapted (psychrophilic) Antarctic bacterium Shewanella gelidimarina (Nap{sub Sgel}) and middle-temperature adapted (mesophilic) Shewanella putrefaciens (Nap{sub Sput}) was examined at varied temperature. Irreversible deactivation of Nap{sub Sgel} and Nap{sub Sput} occurred at 54.5 and 65 Degree-Sign C, respectively. When Nap{sub Sgel} was preincubated at 21-70 Degree-Sign C for 30 min, the room-temperature nitrate reductase activity was maximal and invariant between 21 and 54 Degree-Sign C, which suggested that Nap{sub Sgel} was poised for optimal catalysis at modest temperatures and, unlike Nap{sub Sput}, did not benefit from thermally-induced refolding. At 20 Degree-Sign C, Nap{sub Sgel} reduced selenate at 16% of the rate of nitrate reduction. Nap{sub Sput} did not reduce selenate. Sequence alignment showed 46 amino acid residue substitutions in Nap{sub Sgel} that were conserved in NapA from mesophilic Shewanella, Rhodobacter and Escherichia species and could be associated with the Nap{sub Sgel} cold-adapted phenotype. Protein homology modeling of Nap{sub Sgel} using a

  18. Periplasmic nitrate reductase revisited: a sulfur atom completes the sixth coordination of the catalytic molybdenum.

    Science.gov (United States)

    Najmudin, Shabir; González, Pablo J; Trincão, José; Coelho, Catarina; Mukhopadhyay, Abhik; Cerqueira, Nuno M F S A; Romão, Carlos C; Moura, Isabel; Moura, José J G; Brondino, Carlos D; Romão, Maria J

    2008-06-01

    Nitrate reductase from Desulfovibrio desulfuricans ATCC 27774 (DdNapA) is a monomeric protein of 80 kDa harboring a bis(molybdopterin guanine dinucleotide) active site and a [4Fe-4S] cluster. Previous electron paramagnetic resonance (EPR) studies in both catalytic and inhibiting conditions showed that the molybdenum center has high coordination flexibility when reacted with reducing agents, substrates or inhibitors. As-prepared DdNapA samples, as well as those reacted with substrates and inhibitors, were crystallized and the corresponding structures were solved at resolutions ranging from 1.99 to 2.45 A. The good quality of the diffraction data allowed us to perform a detailed structural study of the active site and, on that basis, the sixth molybdenum ligand, originally proposed to be an OH/OH(2) ligand, was assigned as a sulfur atom after refinement and analysis of the B factors of all the structures. This unexpected result was confirmed by a single-wavelength anomalous diffraction experiment below the iron edge (lambda = 1.77 A) of the as-purified enzyme. Furthermore, for six of the seven datasets, the S-S distance between the sulfur ligand and the Sgamma atom of the molybdenum ligand Cys(A140) was substantially shorter than the van der Waals contact distance and varies between 2.2 and 2.85 A, indicating a partial disulfide bond. Preliminary EPR studies under catalytic conditions showed an EPR signal designated as a turnover signal (g values 1.999, 1.990, 1.982) showing hyperfine structure originating from a nucleus of unknown nature. Spectropotentiometric studies show that reduced methyl viologen, the electron donor used in the catalytic reaction, does not interact directly with the redox cofactors. The turnover signal can be obtained only in the presence of the reaction substrates. With use of the optimized conditions determined by spectropotentiometric titration, the turnover signal was developed with (15)N-labeled nitrate and in D(2)O-exchanged Dd

  19. In situ assay of nitrate reductase activity using portable water bath.

    Science.gov (United States)

    Rajsz, Adam; Wojtuń, Bronisław; Bytnerowicz, Andrzej

    2017-07-01

    In environmental research (i.e., plant ecophysiology, environmental microbiology, and environmental chemistry), some assays require incubation of samples at controlled temperature and darkness. Until now, due to a lack of equipment providing such possibility in situ, researchers had to move collected samples to the laboratory for incubation. Obviously, a delayed incubation and the ex situ conditions could seriously affect the assays' results. A good example of analysis where water bath use is needed is the nitrate reductase activity (NRA) in vivo assay where plant tissue samples are incubated in buffer solution at a predetermined temperature. We designed a transportable water bath with a temperature control which enables in situ measurements in many types of environmental studies. The presented device is small in size featuring a thermally insulated chamber and an electronically controlled thermostat system powered by a 12-V battery. Due to its modular design, it can be transported comfortably in difficult terrain. The incubation process can be carried out continuously in stable temperature and darkness. In order to examine the field usability of the presented device, we conducted measurements of plant nitrate reductase activity in difficult field conditions. The in situ assays were carried out at high altitudes in the Karkonosze mountains, SW Poland. The NRA was studied in two alpine species (Deschampsia caespitosa and Homogyne alpina). Our results showed low NR activity in H. alpina (mean 0.31 μM NO 2 g -1 DW h -1 ) and higher NRA in D. caespitosa (mean 2.7 μM NO 2 g -1 DW h -1 ). The obtained results were highly reproducible and had small variability (low standard error values).

  20. Exogenous auxin-induced NO synthesis is nitrate reductase-associated in Arabidopsis thaliana root primordia.

    Science.gov (United States)

    Kolbert, Zsuzsanna; Bartha, Bernadett; Erdei, László

    2008-06-16

    Nitric oxide (NO) functions in various physiological and developmental processes in plants. However, the source of this signaling molecule in the diversity of plant responses is not well understood. It is known that NO mediates auxin-induced adventitious and lateral root (LR) formation. In this paper, we provide genetic and pharmacological evidence that the production of NO is associated with the nitrate reductase (NR) enzyme during indole-3-butyric acid (IBA)-induced lateral root development in Arabidopsis thaliana L. NO production was detected using 4,5-diaminofluorescein diacetate (DAF-2DA) in the NR-deficient nia1, nia2 and Atnoa1 (former Atnos1) mutants of A. thaliana. An inhibitor for nitric oxide synthase (NOS) N(G)-monomethyl-l-arginine (l-NMMA) was applied. Our data clearly show that IBA increased LR frequency in the wild-type plant and the LR initials emitted intensive NO-dependent fluorescence of the triazol product of NO and DAF-2DA. Increased levels of NO were restricted only to the LR initials in contrast to primary root (PR) sections, where NO remained at the control level. The mutants had different NO levels in their control state (i.e. without IBA treatment): nia1, nia2 showed lower NO fluorescence than Atnoa1 or the wild-type plant. The role of NR in IBA-induced NO formation in the wild type was shown by the zero effects of the NOS inhibitors l-NMMA. Finally, it was clearly demonstrated that IBA was able to induce NO generation in both the wild-type and Atnoa1 plants, but failed to induce NO in the NR-deficient mutant. It is concluded that the IBA-induced NO production is nitrate reductase-associated during lateral root development in A. thaliana.

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

    Energy Technology Data Exchange (ETDEWEB)

    Frey, K.; Liu, J; Lombardo, M; Bolstad, D; Wright, D; Anderson, A

    2009-01-01

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

  2. Latent nitrate reductase activity is associated with the plasma membrane of corn roots

    Science.gov (United States)

    Ward, M. R.; Grimes, H. D.; Huffaker, R. C.

    1989-01-01

    Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U2). Analysis with marker enzymes indicated that the U2 fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.

  3. Molecular Cloning, Heterologous Expression, and Functional Characterization of an NADPH-Cytochrome P450 Reductase Gene from Camptotheca acuminata, a Camptothecin-Producing Plant.

    Directory of Open Access Journals (Sweden)

    Xixing Qu

    Full Text Available Camptothecin (CAM, a complex pentacyclic pyrroloqinoline alkaloid, is the starting material for CAM-type drugs that are well-known antitumor plant drugs. Although many chemical and biological research efforts have been performed to produce CAM, a few attempts have been made to uncover the enzymatic mechanism involved in the biosynthesis of CAM. Enzyme-catalyzed oxidoreduction reactions are ubiquitously presented in living organisms, especially in the biosynthetic pathway of most secondary metabolites such as CAM. Due to a lack of its reduction partner, most catalytic oxidation steps involved in the biosynthesis of CAM have not been established. In the present study, an NADPH-cytochrome P450 reductase (CPR encoding gene CamCPR was cloned from Camptotheca acuminata, a CAM-producing plant. The full length of CamCPR cDNA contained an open reading frame of 2127-bp nucleotides, corresponding to 708-amino acid residues. CamCPR showed 70 ~ 85% identities to other characterized plant CPRs and it was categorized to the group II of CPRs on the basis of the results of multiple sequence alignment of the N-terminal hydrophobic regions. The intact and truncate CamCPRs with N- or C-terminal His6-tag were heterologously overexpressed in Escherichia coli. The recombinant enzymes showed NADPH-dependent reductase activity toward a chemical substrate ferricyanide and a protein substrate cytochrome c. The N-terminal His6-tagged CamCPR showed 18- ~ 30-fold reduction activity higher than the C-terminal His6-tagged CamCPR, which supported a reported conclusion, i.e., the last C-terminal tryptophan of CPRs plays an important role in the discrimination between NADPH and NADH. Co-expression of CamCPR and a P450 monooxygenase, CYP73A25, a cinnamate 4-hydroxylase from cotton, and the following catalytic formation of p-coumaric acid suggested that CamCPR transforms electrons from NADPH to the heme center of P450 to support its oxidation reaction. Quantitative real-time PCR

  4. Asymmetric reduction of ketopantolactone using a strictly (R)-stereoselective carbonyl reductase through efficient NADPH regeneration and the substrate constant-feeding strategy.

    Science.gov (United States)

    Zhao, Man; Gao, Liang; Zhang, Li; Bai, Yanbin; Chen, Liang; Yu, Meilan; Cheng, Feng; Sun, Jie; Wang, Zhao; Ying, Xiangxian

    2017-11-01

    To characterize a recombinant carbonyl reductase from Saccharomyces cerevisiae (SceCPR1) and explore its use in asymmetric synthesis of (R)-pantolactone [(R)-PL]. The NADPH-dependent SceCPR1 exhibited strict (R)-enantioselectivity and high activity in the asymmetric reduction of ketopantolactone (KPL) to (R)-PL. Escherichia coli, coexpressing SceCPR1 and glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH), was constructed to fulfill efficient NADPH regeneration. During the whole-cell catalyzed asymmetric reduction of KPL, the spontaneous hydrolysis of KPL significantly affected the yield of (R)-PL, which was effectively alleviated by the employment of the substrate constant-feeding strategy. The established whole-cell bioreduction for 6 h afforded 458 mM (R)-PL with the enantiomeric excess value of >99.9% and the yield of 91.6%. Escherichia coli coexpressing SceCPR1 and EsGDH efficiently catalyzed the asymmetric synthesis of (R)-PL through the substrate constant-feeding strategy.

  5. Three spinach leaf nitrate reductase-3-hydroxy-3-methylglutaryl-CoA reductase kinases that are regulated by reversible phosphorylation and/or Ca2+ ions.

    Science.gov (United States)

    Douglas, P; Pigaglio, E; Ferrer, A; Halfords, N G; MacKintosh, C

    1997-07-01

    In spinach (Spinacea oleracea L.) leaf extracts, three protein kinases (PKI, PKII and PKIII) were identified each of which phosphorylated spinach nitrate reductase on serine-543, and inactivated the enzyme in the presence of nitrate reductase inhibitor, 14-3-3. PKIII was also very active in phosphorylating and inactivating Arabidopsis (Landsberg erecta) 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (HMGR1). PKI and PKII phosphorylated HMGR1 more slowly than PKIII, compared with their relative rates of phosphorylation of nitrate reductase. HMGR1 identical with those that are seen after phosphorylation of serine-577 by the sucrose non-fermenting (SNF1)-like PK, 3-hydroxy-3-methylglutaryl-Co A reductase kinase A (HRK-A), from cauliflower [Dale, Arró, Becerra, Morrice, Boronat, Hardie and Ferrer (1995) Eur. J. Biochem. 233, 506-513]. PKI was Ca2+-dependent when prepared in the absence of protein phosphatase (PP) inhibitors, and largely Ca2+-dependent when prepared in the presence of PP inhibitors (NaF and EGTA). The Ca2+-independent portion of PKI was inactivated by either PP2A or PP2C, while the Ca2+-dependent portion of PKI became increasingly activated during storage, which we presume was mimicking the effect of an unidentified PP. These findings indicate that PK1 is regulated by two functionally distinct phosphorylations. PKI had a molecular mass of 45 kDa on gel filtration and was active towards substrate peptides that terminated at the +2 residue from the phosphorylation site, whereas PKIII was inactive towards these peptides. PKII was Ca2+-stimulated under all conditions tested. PKIII was Ca2+-indepdented, inactivated by PP2A or PP2C, had a requirement for a hydrophobic residue in the +4 position of peptide substrates, had a molecular mass by gel filtration of approximately 140 kDa, and an antibody against the rye SNF1-related PK (RKIN1) recognized a 58 kDa subunit in fractions containing PKIII. These properties of PKIII are identical with those reported

  6. Three spinach leaf nitrate reductase-3-hydroxy-3-methylglutaryl-CoA reductase kinases that are required by reversible phosphorylation and/or Ca2+ ions.

    Science.gov (United States)

    Douglas, P; Pigaglio, E; Ferrer, A; Halfords, N G; MacKintosh, C

    1997-01-01

    In spinach (Spinacea oleracea L.) leaf extracts, three protein kinases (PKI, PKII and PKIII) were identified each of which phosphorylated spinach nitrate reductase on serine-543, and inactivated the enzyme in the presence of nitrate reductase inhibitor, 14-3-3. PKIII was also very active in phosphorylating and inactivating Arabidopsis (Landsberg erecta) 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (HMGR1). PKI and PKII phosphorylated HMGR1 more slowly than PKIII, compared with their relative rates of phosphorylation of nitrate reductase. HMGR1 identical with those that are seen after phosphorylation of serine-577 by the sucrose non-fermenting (SNF1)-like PK, 3-hydroxy-3-methylglutaryl-Co A reductase kinase A (HRK-A), from cauliflower [Dale, Arró, Becerra, Morrice, Boronat, Hardie and Ferrer (1995) Eur. J. Biochem. 233, 506-513]. PKI was Ca2+-dependent when prepared in the absence of protein phosphatase (PP) inhibitors, and largely Ca2+-dependent when prepared in the presence of PP inhibitors (NaF and EGTA). The Ca2+-independent portion of PKI was inactivated by either PP2A or PP2C, while the Ca2+-dependent portion of PKI became increasingly activated during storage, which we presume was mimicking the effect of an unidentified PP. These findings indicate that PK1 is regulated by two functionally distinct phosphorylations. PKI had a molecular mass of 45 kDa on gel filtration and was active towards substrate peptides that terminated at the +2 residue from the phosphorylation site, whereas PKIII was inactive towards these peptides. PKII was Ca2+-stimulated under all conditions tested. PKIII was Ca2+-indepdented, inactivated by PP2A or PP2C, had a requirement for a hydrophobic residue in the +4 position of peptide substrates, had a molecular mass by gel filtration of approximately 140 kDa, and an antibody against the rye SNF1-related PK (RKIN1) recognized a 58 kDa subunit in fractions containing PKIII. These properties of PKIII are identical with those reported

  7. Ammonium Inhibits Chromomethylase 3-Mediated Methylation of the Arabidopsis Nitrate Reductase Gene NIA2

    Science.gov (United States)

    Kim, Joo Yong; Kwon, Ye Jin; Kim, Sung-Il; Kim, Do Youn; Song, Jong Tae; Seo, Hak Soo

    2016-01-01

    Gene methylation is an important mechanism regulating gene expression and genome stability. Our previous work showed that methylation of the nitrate reductase (NR) gene NIA2 was dependent on chromomethylase 3 (CMT3). Here, we show that CMT3-mediated NIA2 methylation is regulated by ammonium in Arabidopsis thaliana. CHG sequences (where H can be A, T, or C) were methylated in NIA2 but not in NIA1, and ammonium [(NH4)2SO4] treatment completely blocked CHG methylation in NIA2. By contrast, ammonium had no effect on CMT3 methylation, indicating that ammonium negatively regulates CMT3-mediated NIA2 methylation without affecting CMT3 methylation. Ammonium upregulated NIA2 mRNA expression, which was consistent with the repression of NIA2 methylation by ammonium. Ammonium treatment also reduced the overall genome methylation level of wild-type Arabidopsis. Moreover, CMT3 bound to specific promoter and intragenic regions of NIA2. These combined results indicate that ammonium inhibits CMT3-mediated methylation of NIA2 and that of other target genes, and CMT3 selectively binds to target DNA sequences for methylation. PMID:26834755

  8. Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase

    Science.gov (United States)

    Bachmann, M.; Shiraishi, N.; Campbell, W. H.; Yoo, B. C.; Harmon, A. C.; Huber, S. C.; Davies, E. (Principal Investigator)

    1996-01-01

    Spinach leaf NADH:nitrate reductase (NR) responds to light/dark signals and photosynthetic activity in part as a result of rapid regulation by reversible protein phosphorylation. We have identified the major regulatory phosphorylation site as Ser-543, which is located in the hinge 1 region connecting the cytochrome b domain with the molybdenum-pterin cofactor binding domain of NR, using recombinant NR fragments containing or lacking the phosphorylation site sequence. Studies with NR partial reactions indicated that the block in electron flow caused by phosphorylation also could be localized to the hinge 1 region. A synthetic peptide (NR6) based on the phosphorylation site sequence was phosphorylated readily by NR kinase (NRk) in vitro. NR6 kinase activity tracked the ATP-dependent inactivation of NR during several chromatographic steps and completely inhibited inactivation/phosphorylation of native NR in vitro. Two forms of NRk were resolved by using anion exchange chromatography. Studies with synthetic peptide analogs indicated that both forms of NRk had similar specificity determinants, requiring a basic residue at P-3 (i.e., three amino acids N-terminal to the phosphorylated serine) and a hydrophobic residue at P-5. Both forms are strictly calcium dependent but belong to distinct families of protein kinases because they are distinct immunochemically.

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

    Science.gov (United States)

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

    2018-01-01

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

  10. Elevated CO2-induced production of nitric oxide (NO) by NO synthase differentially affects nitrate reductase activity in Arabidopsis plants under different nitrate supplies.

    Science.gov (United States)

    Du, Shaoting; Zhang, Ranran; Zhang, Peng; Liu, Huijun; Yan, Minggang; Chen, Ni; Xie, Huaqiang; Ke, Shouwei

    2016-02-01

    CO2 elevation often alters the plant's nitrate reductase (NR) activity, the first enzyme acting in the nitrate assimilation pathway. However, the mechanism underlying this process remains unknown. The association between elevated CO2-induced alterations of NR activity and nitric oxide (NO) was examined in Col-0 Arabidopsis fed with 0.2-10 mM nitrate, using NO donors, NO scavenger, and NO synthase (NOS) inhibitor. The noa1 mutant, in which most NOS activity was lost, and the NR activity-null mutant nia1 nia2 were also used to examine the above association. In response to CO2 elevation, NR activity increased in low-nitrate Col-0 plants but was inhibited in high-nitrate Col-0 plants. NO scavenger and NOS inhibitor could eliminate these two responses, whereas the application of NO donors mimicked these distinct responses in ambient CO2-grown Col-0 plants. Furthermore, in both low- and high-nitrate conditions, elevated CO2 increased NOS activity and NO levels in Col-0 and nia1 nia2 plants but had little effect on NO level and NR activity in noa1 plants. Considering all of these findings, this study concluded that, in response to CO2 elevation, either the NR activity induction in low-nitrate plants or the NR activity inhibition in high-nitrate plants is regulated by NOS-generated NO. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  11. Substrate-dependent modulation of the enzymatic catalytic activity: reduction of nitrate, chlorate and perchlorate by respiratory nitrate reductase from Marinobacter hydrocarbonoclasticus 617.

    Science.gov (United States)

    Marangon, Jacopo; Paes de Sousa, Patrícia M; Moura, Isabel; Brondino, Carlos D; Moura, José J G; González, Pablo J

    2012-07-01

    The respiratory nitrate reductase complex (NarGHI) from Marinobacter hydrocarbonoclasticus 617 (Mh, formerly Pseudomonas nautica 617) catalyzes the reduction of nitrate to nitrite. This reaction is the first step of the denitrification pathway and is coupled to the quinone pool oxidation and proton translocation to the periplasm, which generates the proton motive force needed for ATP synthesis. The Mh NarGH water-soluble heterodimer has been purified and the kinetic and redox properties have been studied through in-solution enzyme kinetics, protein film voltammetry and spectropotentiometric redox titration. The kinetic parameters of Mh NarGH toward substrates and inhibitors are consistent with those reported for other respiratory nitrate reductases. Protein film voltammetry showed that at least two catalytically distinct forms of the enzyme, which depend on the applied potential, are responsible for substrate reduction. These two forms are affected differentially by the oxidizing substrate, as well as by pH and inhibitors. A new model for the potential dependence of the catalytic efficiency of Nars is proposed. Copyright © 2012. Published by Elsevier B.V.

  12. Regeneration of NADPH Coupled with HMG-CoA Reductase Activity Increases Squalene Synthesis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Paramasivan, Kalaivani; Mutturi, Sarma

    2017-09-20

    Although overexpression of the tHMG1 gene is a well-known strategy for terpene synthesis in Saccharomyces cerevisiae, the optimal level for tHMG1p has not been established. In the present study, it was observed that two copies of the tHMG1 gene on a dual gene expression cassette improved squalene synthesis in laboratory strain by 16.8-fold in comparison to single-copy expression. It was also observed that tHMG1p is limited by its cofactor (NADPH), as the overexpression of NADPH regenerating genes', viz., ZWF1 and POS5 (full length and without mitochondrial presequence), has led to its increased enzyme activity. Further, it was demonstrated that overexpression of full-length POS5 has improved squalene synthesis in cytosol. Finally, when tHMG1 and full-length POS5 were co-overexpressed there was a net 27.5-fold increase in squalene when compared to control strain. These results suggest novel strategies to increase squalene accumulation in S. cerevisiae.

  13. Induction of the Nitrate Assimilation nirA Operon and Protein-Protein Interactions in the Maturation of Nitrate and Nitrite Reductases in the Cyanobacterium Anabaena sp. Strain PCC 7120.

    Science.gov (United States)

    Frías, José E; Flores, Enrique

    2015-07-01

    Nitrate is widely used as a nitrogen source by cyanobacteria, in which the nitrate assimilation structural genes frequently constitute the so-called nirA operon. This operon contains the genes encoding nitrite reductase (nirA), a nitrate/nitrite transporter (frequently an ABC-type transporter; nrtABCD), and nitrate reductase (narB). In the model filamentous cyanobacterium Anabaena sp. strain PCC 7120, which can fix N2 in specialized cells termed heterocysts, the nirA operon is expressed at high levels only in media containing nitrate or nitrite and lacking ammonium, a preferred nitrogen source. Here we examined the genes downstream of the nirA operon in Anabaena and found that a small open reading frame of unknown function, alr0613, can be cotranscribed with the operon. The next gene in the genome, alr0614 (narM), showed an expression pattern similar to that of the nirA operon, implying correlated expression of narM and the operon. A mutant of narM with an insertion mutation failed to produce nitrate reductase activity, consistent with the idea that NarM is required for the maturation of NarB. Both narM and narB mutants were impaired in the nitrate-dependent induction of the nirA operon, suggesting that nitrite is an inducer of the operon in Anabaena. It has previously been shown that the nitrite reductase protein NirA requires NirB, a protein likely involved in protein-protein interactions, to attain maximum activity. Bacterial two-hybrid analysis confirmed possible NirA-NirB and NarB-NarM interactions, suggesting that the development of both nitrite reductase and nitrate reductase activities in cyanobacteria involves physical interaction of the corresponding enzymes with their cognate partners, NirB and NarM, respectively. Nitrate is an important source of nitrogen for many microorganisms that is utilized through the nitrate assimilation system, which includes nitrate/nitrite membrane transporters and the nitrate and nitrite reductases. Many cyanobacteria

  14. Identification of a regulatory phosphorylation site in the hinge 1 region of nitrate reductase from spinach (Spinacea oleracea) leaves.

    Science.gov (United States)

    Douglas, P; Morrice, N; MacKintosh, C

    1995-12-18

    Purified nitrate reductase (NR) from spinach leaves was phosphorylated in vitro by NR-inactivating kinase on Ser-543 which is located in the hinge 1 region between the molybdenum-cofactor and haem-binding domains. Phosphorylation of Ser-543 allowed NR to be inhibited by the inhibitor, NIP. Degraded NR preparations in which a proportion of the subunits had lost 45 amino acids from the N-terminus during purification could be phosphorylated by NR kinase on Ser-543, but could not subsequently be fully inhibited by NIP, suggesting a role for the N-terminal tail of NR in NIP binding.

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

    Science.gov (United States)

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

    2016-04-01

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

  16. Purification and characterization of NAD(P)H quinone reductase from the latex of Hevea brasiliensis Müll.-Arg. (Euphorbiaceae).

    Science.gov (United States)

    Chareonthiphakorn, Nopphakaew; Wititsuwannakul, Dhirayos; Golan-Goldhirsh, Avi; Wititsuwannakul, Rapepun

    2002-09-01

    NAD(P)H quinone reductase [NAD(P)H-QR] present in the latex of Hevea brasiliensis Müll.-Arg. (Euphorbiaceae) was purified to homogeniety from the B-serum fraction obtained by freeze-thawing of the bottom fraction of ultracentrifuged fresh latex. The purification protocol involved acetone fractionation, heat treatment, ion exchange chromatography and affinity chromatography. The M(r) determined by SDS-PAGE for the protein subunit was 21 kDa, and the molecular mass of the native enzyme estimated by gel filtration was 83 kDa, indicating that the native enzyme is a homotetramer. The enzyme showed pH stability over a range of 6 to at least 10 (with an optimum at pH 8) and thermal stability up to 80 degrees C. High NAD(P)H-QR activity (70%) was still retained after 10 h of preincubation at 80 degrees C. A comparable substrate specificity for this enzyme was observed among menadione, p-benzoquinone, juglone, and plumbagin, with only duroquinone generating a lower activity. Positive correlations between latex NAD(P)H-QR activity and rubber yield per tapping [fresh latex (r=0.89, PPP<0.01) indicated that enzyme activity could possibly be used as a marker to predict the yield potential of selected clones.

  17. NADPH-dependent thioredoxin reductase C plays a role in nonhost disease resistance against Pseudomonas syringae pathogens by regulating chloroplast-generated reactive oxygen species

    Directory of Open Access Journals (Sweden)

    Yasuhiro Ishiga

    2016-04-01

    Full Text Available Chloroplasts are cytoplasmic organelles for photosynthesis in eukaryotic cells. In addition, recent studies have shown that chloroplasts have a critical role in plant innate immunity against invading pathogens. Hydrogen peroxide is a toxic by-product from photosynthesis, which also functions as a signaling compound in plant innate immunity. Therefore, it is important to regulate the level of hydrogen peroxide in response to pathogens. Chloroplasts maintain components of the redox detoxification system including enzymes such as 2-Cys peroxiredoxins (2-Cys Prxs, and NADPH-dependent thioredoxin reductase C (NTRC. However, the significance of 2-Cys Prxs and NTRC in the molecular basis of nonhost disease resistance is largely unknown. We evaluated the roles of Prxs and NTRC using knock-out mutants of Arabidopsis in response to nonhost Pseudomonas syringae pathogens. Plants lacking functional NTRC showed localized cell death (LCD accompanied by the elevated accumulation of hydrogen peroxide in response to nonhost pathogens. Interestingly, the Arabidopsis ntrc mutant showed enhanced bacterial growth and disease susceptibility of nonhost pathogens. Furthermore, the expression profiles of the salicylic acid (SA and jasmonic acid (JA-mediated signaling pathways and phytohormone analyses including SA and JA revealed that the Arabidopsis ntrc mutant shows elevated JA-mediated signaling pathways in response to nonhost pathogen. These results suggest the critical role of NTRC in plant innate immunity against nonhost P. syringae pathogens.

  18. NADPH-Cytochrome P450 Reductase: Molecular Cloning and Functional Characterization of Two Paralogs from Withania somnifera (L.) Dunal

    Science.gov (United States)

    Rana, Satiander; Lattoo, Surrinder K.; Dhar, Niha; Razdan, Sumeer; Bhat, Wajid Waheed; Dhar, Rekha S.; Vishwakarma, Ram

    2013-01-01

    Withania somnifera (L.) Dunal, a highly reputed medicinal plant, synthesizes a large array of steroidal lactone triterpenoids called withanolides. Although its chemical profile and pharmacological activities have been studied extensively during the last two decades, limited attempts have been made to decipher the biosynthetic route and identification of key regulatory genes involved in withanolide biosynthesis. Cytochrome P450 reductase is the most imperative redox partner of multiple P450s involved in primary and secondary metabolite biosynthesis. We describe here the cloning and characterization of two paralogs of cytochrome P450 reductase from W. somnifera. The full length paralogs of WsCPR1 and WsCPR2 have open reading frames of 2058 and 2142 bp encoding 685 and 713 amino acid residues, respectively. Phylogenetic analysis demonstrated that grouping of dual CPRs was in accordance with class I and class II of eudicotyledon CPRs. The corresponding coding sequences were expressed in Escherichia coli as glutathione-S-transferase fusion proteins, purified and characterized. Recombinant proteins of both the paralogs were purified with their intact membrane anchor regions and it is hitherto unreported for other CPRs which have been purified from microsomal fraction. Southern blot analysis suggested that two divergent isoforms of CPR exist independently in Withania genome. Quantitative real-time PCR analysis indicated that both genes were widely expressed in leaves, stalks, roots, flowers and berries with higher expression level of WsCPR2 in comparison to WsCPR1. Similar to CPRs of other plant species, WsCPR1 was un-inducible while WsCPR2 transcript level increased in a time-dependent manner after elicitor treatments. High performance liquid chromatography of withanolides extracted from elicitor-treated samples showed a significant increase in two of the key withanolides, withanolide A and withaferin A, possibly indicating the role of WsCPR2 in withanolide biosynthesis

  19. NADPH-cytochrome P450 reductase: molecular cloning and functional characterization of two paralogs from Withania somnifera (L. dunal.

    Directory of Open Access Journals (Sweden)

    Satiander Rana

    Full Text Available Withania somnifera (L. Dunal, a highly reputed medicinal plant, synthesizes a large array of steroidal lactone triterpenoids called withanolides. Although its chemical profile and pharmacological activities have been studied extensively during the last two decades, limited attempts have been made to decipher the biosynthetic route and identification of key regulatory genes involved in withanolide biosynthesis. Cytochrome P450 reductase is the most imperative redox partner of multiple P450s involved in primary and secondary metabolite biosynthesis. We describe here the cloning and characterization of two paralogs of cytochrome P450 reductase from W. somnifera. The full length paralogs of WsCPR1 and WsCPR2 have open reading frames of 2058 and 2142 bp encoding 685 and 713 amino acid residues, respectively. Phylogenetic analysis demonstrated that grouping of dual CPRs was in accordance with class I and class II of eudicotyledon CPRs. The corresponding coding sequences were expressed in Escherichia coli as glutathione-S-transferase fusion proteins, purified and characterized. Recombinant proteins of both the paralogs were purified with their intact membrane anchor regions and it is hitherto unreported for other CPRs which have been purified from microsomal fraction. Southern blot analysis suggested that two divergent isoforms of CPR exist independently in Withania genome. Quantitative real-time PCR analysis indicated that both genes were widely expressed in leaves, stalks, roots, flowers and berries with higher expression level of WsCPR2 in comparison to WsCPR1. Similar to CPRs of other plant species, WsCPR1 was un-inducible while WsCPR2 transcript level increased in a time-dependent manner after elicitor treatments. High performance liquid chromatography of withanolides extracted from elicitor-treated samples showed a significant increase in two of the key withanolides, withanolide A and withaferin A, possibly indicating the role of WsCPR2 in

  20. Nitrogenase and nitrate reductase activities in young Alnus glutinosa, relationship and effect of light-dark treatments

    International Nuclear Information System (INIS)

    Benamar, S.; Thiery, G.; Pizelle, G.

    1995-01-01

    Relations between in vivo nitrogenase (N-2-ase; EC 1.18.6.1) and nitrate reductase (NR; EC 1.6.6.1) activities were studied in young nodulated Alnus glutinosa (L. ) Gaertn. Positive correlations linked N-2-ase activity, constitutive (non-inducible by nitrate) leaf NR activity and plant growth. Light/dark treatments applied to the whole shoot or, separately, to the upper and lower part of shoot led to the finding that(a) the constitutive leaf NR activity depended on direct illumination of the leaf and did not appear subordinate to the N-2-ase activity; (b) the N-2-ase activity was much more efficiently supported by the illumination of the upper (young) leaves than by that of the lower (mature) leaves; (c) the maintenance of nitrate-inducible root NR activity required leaf illumination. The variations of both N-2-ase and root NR activities in response to light/dark treatments emphasize the importance of the photosynthesis for the reduction of dinitrogen and nitrate in nodulated roots of Alnus glutinosa

  1. Purification of a nitrate reductase kinase from Spinacea oleracea leaves, and its identification as a calmodulin-domain protein kinase.

    Science.gov (United States)

    Douglas, P; Moorhead, G; Hong, Y; Morrice, N; MacKintosh, C

    1998-10-01

    Spinach (Spinacea oleracea L.) nitrate reductase (NR) is inactivated by phosphorylation on serine-543, followed by binding of the phosphorylated enzyme to 14-3-3 proteins. We purified one of several chromatographically distinct NRserine-543 kinases from spinach leaf extracts, and established by Edman sequencing of 80 amino acid residues that it is a calcium-dependent (calmodulin-domain) protein kinase (CDPK), with peptide sequences very similar to Arabidopsis CDPK6 (accession no. U20623; also known as CPK3). The spinach CDPK was recognized by antibodies raised against Arabidopsis CDPK. Nitrate reductase was phosphorylated at serine-543 by bacterially expressed His-tagged CDPK6, and the phosphorylated NR was inhibited by 14-3-3 proteins. However, the bacterially expressed CDPK6 had a specific activity approx. 200-fold lower than that of the purified spinach enzyme. The physiological control of NR by CDPK is discussed, and the regulatory properties of the purified CDPK are considered with reference to current models for reversible intramolecular binding of the calmodulin-like domain to the autoinhibitory junction of CDPKs.

  2. Nitrate reductase and photosynthetic activities of wheat and their relationship with plant productivity under soil water deficit conditions (abstract)

    International Nuclear Information System (INIS)

    Ashraf, M.Y.; Sarwar, G.; Hussain, F.

    2005-01-01

    Experiments were conducted in lysimeters with wheat during two consecutive years. The first year experiment comprised of eight wheat genotypes with four water stress treatments, i.e. normal irrigation, pre-anthesis drought, post-anthesis drought and terminal drought, with four replications. The results showed that yield and yield parameters reduced with the severity of drought in all wheat lines. However, wheat lines Chakwal-86, DS-4 and Barani-83 had comparatively higher yield and yield components than others. The maximum reduction in all parameters was under terminal drought. The difference between pre- and post-anthesis drought was nonsignificant, particularly for grain yield. The second experiment was conducted with four wheat lines: two were tolerant (Chakwal-86 and DS-4) and two susceptible (Pavon and DS-17) under similar environments with same treatments to study the photosynthetic efficiency, nitrogen metabolism and their relationship with plant productivity (yield). The results showed that leaf area, transpiration, dry matter accumulation and nitrate reductase activity were reduced while diffusive resistance and total amino acids increased in all the wheat lines under water deficit conditions. The relationship between yield and leaf area, transpiration, dry matter accumulation and nitrate reductase activity was positive. The overall results showed that wheat lines Chakwal-86 and DS-4 showed better performance than others. (author)

  3. Heterologous expression and biochemical characterization of assimilatory nitrate and nitrite reductase reveals adaption and potential of Bacillus megaterium NCT-2 in secondary salinization soil.

    Science.gov (United States)

    Chu, Shaohua; Zhang, Dan; Wang, Daxin; Zhi, Yuee; Zhou, Pei

    2017-08-01

    Large accumulation of nitrate in soil has resulted in "salt stress" and soil secondary salinization. Bacillus megaterium NCT-2 which was isolated from secondary salinization soil showed high capability of nitrate reduction. The genes encoding assimilatory nitrate and nitrite reductase from NCT-2 were cloned and over-expressed in Escherichia coli. The optimum co-expression condition was obtained with E. coli BL21 (DE3) and 0.1mM IPTG for 10h when expression was carried out at 20°C and 120rpm in Luria-Bertani (LB) medium. The molecular mass of nitrate reductase was 87.3kDa and 80.5kDa for electron transfer and catalytic subunit, respectively. The large and small subunit of nitrite reductase was 88kDa and 11.7kDa, respectively. The purified recombinant enzymes showed broad activity range of temperature and pH. The maximum activities were obtained at 35°C and 30°C, pH 6.2 and 6.5, which was similar to the condition of greenhouse soils. Maximum stimulation of the enzymes occurred with addition of Fe 3+ , while Cu 2+ caused the maximum inhibition. The optimum electron donor was MV+Na 2 S 2 O 4 +EDTA and MV+Na 2 S 2 O 4 , respectively. Kinetic parameters of K m and V max were determined to be 670μM and 58U/mg for nitrate reductase, and 3100μM and 5.2U/mg for nitrite reductase. Results of quantitative real-time PCR showed that the maximum expression levels of nitrate and nitrite reductase were obtained at 50mM nitrate for 8h and 12h, respectively. These results provided information on novel assimilatory nitrate and nitrite reductase and their properties presumably revealed adaption of B. megaterium NCT-2 to secondary salinization condition. This study also shed light on the role played by the nitrate assimilatory pathway in B. megaterium NCT-2. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Nitric oxide generated by nitrate reductase increases nitrogen uptake capacity by inducing lateral root formation and inorganic nitrogen uptake under partial nitrate nutrition in rice.

    Science.gov (United States)

    Sun, Huwei; Li, Jiao; Song, Wenjing; Tao, Jinyuan; Huang, Shuangjie; Chen, Si; Hou, Mengmeng; Xu, Guohua; Zhang, Yali

    2015-05-01

    Increasing evidence shows that partial nitrate nutrition (PNN) can be attributed to improved plant growth and nitrogen-use efficiency (NUE) in rice. Nitric oxide (NO) is a signalling molecule involved in many physiological processes during plant development and nitrogen (N) assimilation. It remains unclear whether molecular NO improves NUE through PNN. Two rice cultivars (cvs Nanguang and Elio), with high and low NUE, respectively, were used in the analysis of NO production, nitrate reductase (NR) activity, lateral root (LR) density, and (15)N uptake under PNN, with or without NO production donor and inhibitors. PNN increased NO accumulation in cv. Nanguang possibly through the NIA2-dependent NR pathway. PNN-mediated NO increases contributed to LR initiation, (15)NH₄(+)/(15)NO₃(-) influx into the root, and levels of ammonium and nitrate transporters in cv. Nanguang but not cv. Elio. Further results revealed marked and specific induction of LR initiation and (15)NH₄(+)/(15)NO₃(-) influx into the roots of plants supplied with NH₄(+)+sodium nitroprusside (SNP) relative to those supplied with NH₄(+) alone, and considerable inhibition upon the application of cPTIO or tungstate (NR inhibitor) in addition to PNN, which is in agreement with the change in NO fluorescence in the two rice cultivars. The findings suggest that NO generated by the NR pathway plays a pivotal role in improving the N acquisition capacity by increasing LR initiation and the inorganic N uptake rate, which may represent a strategy for rice plants to adapt to a fluctuating nitrate supply and increase NUE. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  5. Developmental stage- and concentration-specific sodium nitroprusside application results in nitrate reductase regulation and the modification of nitrate metabolism in leaves of Medicago truncatula plants.

    Science.gov (United States)

    Antoniou, Chrystalla; Filippou, Panagiota; Mylona, Photini; Fasoula, Dionysia; Ioannides, Ioannis; Polidoros, Alexios; Fotopoulos, Vasileios

    2013-09-01

    Nitric oxide (NO) is a bioactive molecule involved in numerous biological events that has been reported to display both pro-oxidant and antioxidant properties in plants. Several reports exist which demonstrate the protective action of sodium nitroprusside (SNP), a widely used NO donor, which acts as a signal molecule in plants responsible for the expression regulation of many antioxidant enzymes. This study attempts to provide a novel insight into the effect of application of low (100 μΜ) and high (2.5 mM) concentrations of SNP on the nitrosative status and nitrate metabolism of mature (40 d) and senescing (65 d) Medicago truncatula plants. Higher concentrations of SNP resulted in increased NO content, cellular damage levels and reactive oxygen species (ROS) concentration, further induced in older tissues. Senescing M. truncatula plants demonstrated greater sensitivity to SNP-induced oxidative and nitrosative damage, suggesting a developmental stage-dependent suppression in the plant's capacity to cope with free oxygen and nitrogen radicals. In addition, measurements of the activity of nitrate reductase (NR), a key enzyme involved in the generation of NO in plants, indicated a differential regulation in a dose and time-dependent manner. Furthermore, expression levels of NO-responsive genes (NR, nitrate/nitrite transporters) involved in nitrogen assimilation and NO production revealed significant induction of NR and nitrate transporter during long-term 2.5 mM SNP application in mature plants and overall gene suppression in senescing plants, supporting the differential nitrosative response of M. truncatula plants treated with different concentrations of SNP.

  6. Rapid induction of GFP expression by the nitrate reductase promoter in the diatom Phaeodactylum tricornutum

    Czech Academy of Sciences Publication Activity Database

    Chu, L.; Ewe, Daniela; Bártulos, C.R.; Kroth, P. G.; Gruber, A.

    2016-01-01

    Roč. 4, AUG 25 (2016), e2344 ISSN 2167-8359 Institutional support: RVO:61388971 Keywords : Flow cytometry * Nitrogen source * Nitrate Subject RIV: EE - Microbiology, Virology Impact factor: 2.177, year: 2016

  7. Rapid induction of GFP expression by the nitrate reductase promoter in the diatom Phaeodactylum tricornutum

    Czech Academy of Sciences Publication Activity Database

    Chu, L.; Ewe, Daniela; Bártulos, C.R.; Kroth, P.G.; Gruber, A.

    2016-01-01

    Roč. 4, AUG 25 2016 (2016), e2344 ISSN 2167-8359 Institutional support: RVO:61388971 Keywords : Flow cytometry * Nitrogen source * Nitrate Subject RIV: EE - Microbiology, Virology Impact factor: 2.177, year: 2016

  8. Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in high nitrite excretion and NO emission from leaf and root tissue

    NARCIS (Netherlands)

    Lea, US; ten Hoopen, F; Provan, F; Kaiser, WM; Meyer, C; Lillo, C

    In wild-type Nicotiana plumbaginifolia Viv. and other higher plants, nitrate reductase (NR) is regulated at the post-translational level and is rapidly inactivated in response to, for example, a light-to-dark transition. This inactivation is caused by phosphorylation of a conserved regulatory serine

  9. NapGH components of the periplasmic nitrate reductase of Escherichia coli K-12: location, topology and physiological roles in quinol oxidation and redox balancing.

    OpenAIRE

    Brondijk, T Harma C; Nilavongse, Arjaree; Filenko, Nina; Richardson, David J; Cole, Jeffrey A

    2004-01-01

    Nap (periplasmic nitrate reductase) operons of many bacteria include four common, essential components, napD, napA, napB and napC (or a homologue of napC ). In Escherichia coli there are three additional genes, napF, napG and napH, none of which are essential for Nap activity. We now show that deletion of either napG or napH almost abolished Nap-dependent nitrate reduction by strains defective in naphthoquinone synthesis. The residual rate of nitrate reduction (approx. 1% of that of napG+ H+ ...

  10. The roles of tissue nitrate reductase activity and myoglobin in securing nitric oxide availability in deeply hypoxic crucian carp

    DEFF Research Database (Denmark)

    Hansen, Marie N; Lundberg, Jon O; Filice, Mariacristina

    2016-01-01

    . We also tested whether liver, muscle and heart tissue possess nitrate reductase activity that supplies nitrite to the tissues during severe hypoxia. Crucian carp exposed to deep hypoxia (1nitrite in red musculature to more than double the value in normoxic fish......In mammals, treatment with low doses of nitrite has a cytoprotective effect in ischemia/reperfusion events, as a result of nitric oxide formation and S-nitrosation of proteins. Interestingly, anoxia-tolerant lower vertebrates possess an intrinsic ability to increase intracellular nitrite...... concentration during anoxia in tissues with high myoglobin and mitochondria content, such as the heart. Here, we tested the hypothesis that red and white skeletal muscles develop different nitrite levels in crucian carp exposed to deep hypoxia and assessed whether this correlates with myoglobin concentration...

  11. Effect of NaCI and supplemental calcium on growth parameters and nitrate reductase activity in maize

    Directory of Open Access Journals (Sweden)

    Elżbieta Sacała

    2011-01-01

    Full Text Available In this study, investigated were the effects of NaCl (60 mmol/dm-3 and NaCl supplemented with different salts (5 mmol/dm-3 CaCl2, CaSO4, CaCO3, KCl, on growth of two maize varieties (Cyrkon and Limko. After 7 days of cultivation in nutrient solution the growth response to salinity of both maize varieties was similar. NaCl led to a dramatic decrease in growth of plants (approx. 50% reduction in fresh and dry weight of root, and 70% reduction in fresh weight of shoot. Addition of extra Ca2+ or K+ to nutrient solution containing NaCl did not definitely improve the growth parameters of maize. However, among the tested salts, CaCl2 had a beneficial visual effect on maize seedlings. In other cases the plants showed noticeable symptoms of salt damage. In long term exposure to salinity (two weeks growth of Cyrkon was more inhibited than Limko. Comparison of growth responses in short-term exposure to salinity (7 days with long-term (14 days showed that in Cyrkon variety the negative effects of NaCl were intensified and addition of CaCl2 to salinized solution had not positive effects on growth. On the contrary, in Limko variety, there was a significant improvement in growth (especially in root dry weight. This fact indicates that during longer exposure to salinity Limko was able to adapt to those conditions. Salinity caused a significant decrease in leaf nitrate reductase activity (60% and 30% reduction respectively in Limko and Cyrkon. Addition of CaCl2 to salinized nutrient solution resulted in greater enzyme inhibition in Cyrkon (50% decline in relation to plants grown under sole NaCl, and 30% increase in Limko. Inhibition of nitrate reductase activity did not cause a decrease in concentration of soluble protein in maize leaves.

  12. Nitrate reductase 15N discrimination in Arabidopsis thaliana, Zea mays, Aspergillus niger, Pichea angusta, and Escherichia coli

    Directory of Open Access Journals (Sweden)

    Eli eCarlisle

    2014-07-01

    Full Text Available Stable 15N isotopes have been used to examine movement of nitrogen (N through various pools of the global N cycle. A central reaction in the cycle involves nitrate (NO3– reduction to nitrite (NO2– catalyzed via nitrate reductase (NR. Discrimination against 15N by NR is a major determinant of isotopic differences among N pools. Here, we measured in vitro 15N discrimination by several NRs purified from plants, fungi, and a bacterium to determine the intrinsic 15N discrimination by the enzyme and to evaluate the validity of measurements made using 15N-enriched NO3–. Observed NR isotope discrimination ranged from 22‰ to 32‰ (kinetic isotope effects of 1.022 to 1.032 among the different isozymes at natural abundance 15N (0.37%. As the fractional 15N content of substrate NO3– increased from natural abundance, the product 15N fraction deviated significantly from that expected based on substrate enrichment and 15N discrimination measured at natural abundance. Additionally, isotopic discrimination by denitrifying bacteria used to reduce NO3– and NO2– in some protocols became a greater source of error as 15N enrichment increased. We briefly discuss potential causes of artifactual results with enriched 15N and recommend against the use of highly enriched 15N tracers to study N discrimination in plants or soils.

  13. Elevated CO2 levels affect the activity of nitrate reductase and carbonic anhydrase in the calcifying rhodophyte Corallina officinalis.

    Science.gov (United States)

    Hofmann, Laurie C; Straub, Sandra; Bischof, Kai

    2013-02-01

    The concentration of CO(2) in global surface ocean waters is increasing due to rising atmospheric CO(2) emissions, resulting in lower pH and a lower saturation state of carbonate ions. Such changes in seawater chemistry are expected to impact calcification in calcifying marine organisms. However, other physiological processes related to calcification might also be affected, including enzyme activity. In a mesocosm experiment, macroalgal communities were exposed to three CO(2) concentrations (380, 665, and 1486 µatm) to determine how the activity of two enzymes related to inorganic carbon uptake and nutrient assimilation in Corallina officinalis, an abundant calcifying rhodophyte, will be affected by elevated CO(2) concentrations. The activity of external carbonic anhydrase, an important enzyme functioning in macroalgal carbon-concentrating mechanisms, was inversely related to CO(2) concentration after long-term exposure (12 weeks). Nitrate reductase, the enzyme responsible for reduction of nitrate to nitrite, was stimulated by CO(2) and was highest in algae grown at 665 µatm CO(2). Nitrate and phosphate uptake rates were inversely related to CO(2), while ammonium uptake was unaffected, and the percentage of inorganic carbon in the algal skeleton decreased with increasing CO(2). The results indicate that the processes of inorganic carbon and nutrient uptake and assimilation are affected by elevated CO(2) due to changes in enzyme activity, which change the energy balance and physiological status of C. officinalis, therefore affecting its competitive interactions with other macroalgae. The ecological implications of the physiological changes in C. officinalis in response to elevated CO(2) are discussed.

  14. Loss of nitrate reductases NIA1 and NIA2 impairs stomatal closure by altering genes of core ABA signaling components in Arabidopsis.

    Science.gov (United States)

    Zhao, Chenchen; Cai, Shengguan; Wang, Yizhou; Chen, Zhong-Hua

    2016-06-02

    Nitrate reductases NIA1 and NIA2 determine NO production in plants and are critical to abscisic acid (ABA)-induced stomatal closure. However, the role for NIA1 and NIA2 in ABA signaling has not been paid much attention in nitrate reductase loss-of-function mutant nia1nia2. Recently, we have demonstrated that ABA-inhibited K(+)in current and ABA-enhanced slow anion current were absent in nia1nia2. Exogenous NO restored regulation of these channels for stomatal closure in nia1nia2. In this study, we found that mutating NIA1 and NIA2 impaired nearly all the key components of guard cell ABA signaling pathway in Arabidopsis. We also propose a simplified model for ABA signaling in the nia1nia2 mutant.

  15. Over-expression of a tobacco nitrate reductase gene in wheat (Triticum aestivum L. increases seed protein content and weight without augmenting nitrogen supplying.

    Directory of Open Access Journals (Sweden)

    Xiao-Qiang Zhao

    Full Text Available Heavy nitrogen (N application to gain higher yield of wheat (Triticum aestivum L. resulted in increased production cost and environment pollution. How to diminish the N supply without losing yield and/or quality remains a challenge. To meet the challenge, we integrated and expressed a tobacco nitrate reductase gene (NR in transgenic wheat. The 35S-NR gene was transferred into two winter cultivars, "Nongda146" and "Jimai6358", by Agrobacterium-mediation. Over-expression of the transgene remarkably enhanced T1 foliar NR activity and significantly augmented T2 seed protein content and 1000-grain weight in 63.8% and 68.1% of T1 offspring (total 67 individuals analyzed, respectively. Our results suggest that constitutive expression of foreign nitrate reductase gene(s in wheat might improve nitrogen use efficiency and thus make it possible to increase seed protein content and weight without augmenting N supplying.

  16. Escherichia coli MTC, a human NADPH P450 reductase competent mutagenicity tester strain for the expression of human cytochrome P450 isoforms 1A1, 1A2, 2A6, 3A4, or 3A5: catalytic activities and mutagenicity studies.

    NARCIS (Netherlands)

    Kranendonk, M.; Carreira, F.; Theisen, P.; Laires, A.; Fischer, C.W.; Rueff, J.; Estabrook, R.W.; Vermeulen, N.P.E.; Roda, R.

    1999-01-01

    We report here on the genetic engineering of four new Escherichia coli tester bacteria, coexpressing human CYP1A1, CYP2A6, CYP3A4 or CYP3A5 with human NADPH cytochrome P450 reductase (RED) by a biplasmid coexpression system, recently developed to express human CYP1A2 in the tester strain MTC. The

  17. Properties of nitrogen fertilization are decisive in determining the effects of elevated atmospheric CO2 on the activity of nitrate reductase in plants.

    Science.gov (United States)

    Zhang, Ranran; Du, Shaoting

    2016-01-01

    The concentration of atmospheric CO2 is predicted to double by the end of this century. The response of higher plants to an increase in atmospheric CO2 often includes a change in nitrate reductase (NR) activity. In a recent study, we showed that, under elevated CO2 levels, NR induction in low-nitrate plants and NR inhibition in high-nitrate plants are regulated by nitric oxide (NO) generated via nitric oxide synthases. This finding provides an explanation for the diverse responses of plants to elevated CO2 levels, and suggests that the use of nitrogen fertilizers on soil will have a major influence on the nitrogen assimilation capacity of plants in response to CO2 elevation.

  18. A web-based resource for the Arabidopsis P450, cytochromes b5, NADPH-cytochrome P450 reductases, and family 1 glycosyltransferases (http://www.P450.kvl.dk).

    Science.gov (United States)

    Paquette, Suzanne M; Jensen, Kenneth; Bak, Søren

    2009-12-01

    Gene and genome duplication is a key driving force in evolution of plant diversity. This has resulted in a number of large multi-gene families. Two of the largest multi-gene families in plants are the cytochromes P450 (P450s) and family 1 glycosyltransferases (UGTs). These two families are key players in evolution, especially of plant secondary metabolism, and in adaption to abiotic and biotic stress. In the model plant Arabidopsis thaliana there are 246 and 112 cytochromes P450 and UGTs, respectively. The Arabidopsis P450, cytochromes b(5), NADPH-cytochrome P450 reductases, and family 1 glycosyltransferases website (http://www.P450.kvl.dk) is a sequence repository of manually curated sequences, multiple sequence alignments, phylogenetic trees, sequence motif logos, 3D structures, intron-exon maps, and customized BLAST datasets.

  19. Resolution of two native monomeric 90 kDa nitrate reductase active proteins from Shewanella gelidimarina and the sequence of two napA genes

    International Nuclear Information System (INIS)

    Simpson, Philippa J.L.; McKinzie, Audra A.; Codd, Rachel

    2010-01-01

    Research highlights: → Two monomeric 90 kDa nitrate reductase active proteins from Shewanella gelidimarina. → Sequence of napA from napEDABC-type operon and napA from NapDAGHB-type operon. → Isolation of NAP as NapA or NapAB correlated with NapA P47E amino acid substitution. -- Abstract: The reduction of nitrate to nitrite in the bacterial periplasm occurs in the 90 kDa NapA subunit of the periplasmic nitrate reductase (NAP) system. Most Shewanella genomes contain two nap operons: napEDABC and napDAGHB, which is an unusual feature of this genus. Two native, monomeric, 90 kDa nitrate reductase active proteins were resolved by hydrophobic interaction chromatography from aerobic cultures of Shewanella gelidimarina replete with reduced nitrogen compounds. The 90 kDa protein obtained in higher yield was characterized as NapA by electronic absorption and electron paramagnetic resonance spectroscopies and was identified by LC/MS/MS and MALDI-TOF/TOF MS as NapA from the napEDABC-type operon. The other 90 kDa protein, which was unstable and produced in low yields, was posited as NapA from the napDAGHB-type operon. Two napA genes have been sequenced from the napEDABC-type and napDAGHB-type operons of S. gelidimarina. Native NAP from S. putrefaciens was resolved as one NapA monomer and one NapAB heterodimer. Two amino acid substitutions in NapA correlated with the isolation of NAP as a NapA monomer or a NapAB heterodimer. The resolution of native, redox-active NapA isoforms in Shewanella provides new insight into the respiratory versatility of this genus, which has implications in bioremediation and the assembly of microbial fuel cells.

  20. Involvement of nitrate reductase (NR) in osmotic stress-induced NO generation of Arabidopsis thaliana L. roots.

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    Kolbert, Zsuzsanna; Ortega, Leandro; Erdei, László

    2010-01-01

    Nitric oxide (NO) is undoubtedly a potential signal molecule in diverse developmental processes and stress responses. Despite our extensive knowledge about the role of NO in physiological and stress responses, the source of this gaseous molecule is still unresolved. The aim of this study was to investigate the potential role of nitrate reductase (NR) as the source of NO accumulation in the root system of wild-type and NR-deficient nia1, nia2 mutant Arabidopsis plants under osmotic stress conditions induced by a polyethylene glycol (PEG 6000) treatment. Reduction of primary root (PR) length was detected as the effect of osmotic stress in wild-type and NR-deficient plants. We found that osmotic stress-induced lateral root (LR) initiation in wild-type, but not in NR-mutant plants. High levels of NO formation occurred in roots of Col-1 plants as the effect of PEG treatment. The mammalian nitric oxide synthase (NOS) inhibitor N(G)-monomethyl-L-arginine (L-NMMA) had no effect on LR initiation or NO generation, while tungstate, an NR inhibitor, inhibited the later phase of osmotic stress-induced NO accumulation and slightly decreased the LR development. In nia1, nia2 roots, the PEG treatment induced the first phase of NO production, but later NO production was inhibited. We conclude that the first phase of PEG-induced NO generation is not dependent on NOS-like or NR activity. It is also suggested that the activity of NR in roots is required for the later phase of osmotic stress-induced NO formation.

  1. Direct nitrate reductase assay versus microscopic observation drug susceptibility test for rapid detection of MDR-TB in Uganda.

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    Freddie Bwanga

    Full Text Available The most common method for detection of drug resistant (DR TB in resource-limited settings (RLSs is indirect susceptibility testing on Lowenstein-Jensen medium (LJ which is very time consuming with results available only after 2-3 months. Effective therapy of DR TB is therefore markedly delayed and patients can transmit resistant strains. Rapid and accurate tests suitable for RLSs in the diagnosis of DR TB are thus highly needed. In this study we compared two direct techniques--Nitrate Reductase Assay (NRA and Microscopic Observation Drug Susceptibility (MODS for rapid detection of MDR-TB in a high burden RLS. The sensitivity, specificity, and proportion of interpretable results were studied. Smear positive sputum was collected from 245 consecutive re-treatment TB patients attending a TB clinic in Kampala, Uganda. Samples were processed at the national reference laboratory and tested for susceptibility to rifampicin and isoniazid with direct NRA, direct MODS and the indirect LJ proportion method as reference. A total of 229 specimens were confirmed as M. tuberculosis, of these interpretable results were obtained in 217 (95% with either the NRA or MODS. Sensitivity, specificity and kappa agreement for MDR-TB diagnosis was 97%, 98% and 0.93 with the NRA; and 87%, 95% and 0.78 with the MODS, respectively. The median time to results was 10, 7 and 64 days with NRA, MODS and the reference technique, respectively. The cost of laboratory supplies per sample was low, around 5 USD, for the rapid tests. The direct NRA and MODS offered rapid detection of resistance almost eight weeks earlier than with the reference method. In the study settings, the direct NRA was highly sensitive and specific. We consider it to have a strong potential for timely detection of MDR-TB in RLS.

  2. Analysis of the combined effects of lanthanum and acid rain, and their mechanisms, on nitrate reductase transcription in plants.

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    Xia, Binxin; Sun, Zhaoguo; Wang, Lihong; Zhou, Qing; Huang, Xiaohua

    2017-04-01

    Rare earth element (REE) pollution and acid rain are major global environmental concerns, and their spatial distributions overlap. Thus, both forms of pollution combine to act on plants. Nitrogen is important for plant growth, and nitrate reductase (NR) is a key plant enzyme that catalyzes nitrogen assimilation. Studying the combined effects of REEs and acid rain on plant nitrogen-based nutrients has important environmental significance. Here, soybean (Glycine max) plants, commonly used for toxicological studies, were exposed to lanthanum (La), a REE, and acid rain to study the NR activities and NR transcriptional levels in the roots. To explain how the pollution affected the NR transcriptional level, we simultaneously observed the contents of intracellular La and nutrient elements, protoplast morphology, membrane lipid peroxidation and intracellular pH. A combined treatment of 0.08mmol/L La and pH 4.5 acid rain increased the NR activity, decreased the NR transcriptional level, increased the intracellular nutrient elements' contents and caused deformations in membrane structures. Other combined treatments significantly decreased the aforementioned parameters and caused serious damage to the membrane structures. The variation in the amplitudes of combined treatments was greater than those of individual treatments. Compared with the control and individual treatments, combined treatments increased membrane permeability, the malondialdehyde content, and intracellular H + and La contents, and with an increasing La concentration or acid strength, the change in amplitude increased. Thus, the combined effects on NR gene transcription in soybean seedling roots were related to the intracellular nutrient elements' contents, protoplast morphology, membranous lipid peroxidation, intracellular pH and La content. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Impacts of some divalent cations on periplasmic nitrate reductase and dehydrogenase enzymes of Escherichia, Pseudomonas and Acinetobacter species

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    Christian E. Nwanyanwu

    2008-08-01

    Full Text Available The impacts of Hg2+, Cd2+ and Zn2+ on the activities of periplasmic nitrate reductase (NAP and dehydrogenase (DHA enzymes of three organisms isolated from soil and sediment-water interface were analysed in liquid culture studies. NAP and DHA activities were estimated from nitrite and triphenyl formazan were produced respectively after 4h incubation at 28 ± 2oC. Hg2+ completely inhibited NAP activity in Escherichia and Pseudomonas spp at all the concentrations (0.2 – 1mM while progressive inhibitions of NAP activity were observed in Escherichia and Pseudomonas spp with increasing concentrations of Zn2+ and Cd2+. Both metals were stimulatory to NAP of Acinetobacter sp at 0.2 – 1mM. Apart from stimulation of DHA activity by Zn2+ (0.2 – 1mM in Escherichia sp, Cd2+ (0.4 -1.0mM in Acinetobacter sp and (1.0mM in Pseudomonas sp, all the metals progressively inhibited DHA activities in the three organisms. In Escherichia sp, the activities of the two enzymes were negatively correlated on exposure to Zn2+ (r = -0.91 and positively correlated (r = >0.90 on exposure to Cd2+ and Hg2+. Based on IC50 values of the metals for the DHA and NAP enzymes, the most resistant of the three organisms were Escherichia sp and Acinetobacter sp respectively. Quantitatively, NAP with its lower IC50 values than DHA was a more sensitive toxicity measure for Hg2+ in all the organisms. The sensitivity of microbial metabolic enzymes to the toxic effects of metals varies with the type of enzyme, metal and the microorganism involved.

  4. [Effects of nitrogen application rate on nitrate reductase activity, nitric oxide content and gas exchange in winter wheat leaves].

    Science.gov (United States)

    Shangguan, Zhou-Ping

    2007-07-01

    In this paper, the effects of different nitrogen application rates on the nitrate reductase (NR) activity, nitric oxide (NO) content and gas exchange parameters in winter wheat (Triticum aestivum L.) leaves from tillering stage to heading stage and on grain yield were studied. The results showed that the photosynthetic rate (P(n)), transpiration rate (T(r)) and instantaneous water use efficiency (IWUE) of leaves as well as the grain yield were increased with increasing nitrogen application rate first but decreased then, with the values of all these parameters reached the highest in treatment N180. The NR activity increased with increasing nitrogen application rate, and there was a significant linear correlation between NR activity and NO content at tillering and jointing stages (R2 > or = 0.68, n = 15). NO content had a quadratic positive correlation with stomatal conductance (G(s)) (R2 > or = 0.43, n = 15). The lower NO content produced by lower NR activity under lower nitrogen application rate promoted the stoma opened, while the higher NO content produced by higher NR activity under higher nitrogen application rate induced the stoma closed. Although the leaf NO content had a quadratic positive correlation with stomatal conductance (R2 > or = 0.36, n = 15), no remarkable correlation was observed between NR activity and NO content at heading stage, suggesting that nitrogen fertilization could not affect leaf NO content through promoting NR activity, and further more, regulate the stomatal action. Under appropriate nitrogen application the leaf NR activity and NO content were lower, G(s), T(r) and IWUE were higher, and thus, the crop had a better drought-resistant ability, higher P(n), and higher grain yield.

  5. Process-driven bacterial community dynamics are key to cured meat colour formation by coagulase-negative staphylococci via nitrate reductase or nitric oxide synthase activities.

    Science.gov (United States)

    Sánchez Mainar, María; Leroy, Frédéric

    2015-11-06

    The cured colour of European raw fermented meats is usually achieved by nitrate-into-nitrite reduction by coagulase-negative staphylococci (CNS), subsequently generating nitric oxide to form the relatively stable nitrosomyoglobin pigment. The present study aimed at comparing this classical curing procedure, based on nitrate reductase activity, with a potential alternative colour formation mechanism, based on nitric oxide synthase (NOS) activity, under different acidification profiles. To this end, meat models with and without added nitrate were fermented with cultures of an acidifying strain (Lactobacillus sakei CTC 494) and either a nitrate-reducing Staphylococcus carnosus strain or a rare NOS-positive CNS strain (Staphylococcus haemolyticus G110), or by relying on the background microbiota. Satisfactory colour was obtained in the models prepared with added nitrate and S. carnosus. In the presence of nitrate but absence of added CNS, however, cured colour was only obtained when L. sakei CTC 494 was also omitted. This was ascribed to the pH dependency of the emerging CNS background microbiota, selecting for nitrate-reducing Staphylococcus equorum strains at mild acidification conditions but for Staphylococcus saprophyticus strains with poor colour formation capability when the pH decrease was more rapid. This reliance of colour formation on the composition of the background microbiota was further explored by a side experiment, demonstrating the heterogeneity in nitrate reduction of a set of 88 CNS strains from different species. Finally, in all batches prepared with S. haemolyticus G110, colour generation failed as the strain was systematically outcompeted by the background microbiota, even when imposing milder acidification profiles. Thus, when aiming at colour formation through CNS metabolism, technological processing can severely interfere with the composition and functionality of the meat-associated CNS communities, for both nitrate reductase and NOS activities

  6. The Effects of Dose Rhizoctonia Binucleat (BNR and Phosphorus to Nitrate Reductase Activity (NRA and Chlorophyll of Vanilla Seedling (Vanilla planifolia Andrews

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    Haryuni Haryuni

    2016-09-01

    Full Text Available Vanilla (Vanilla planifolia Andrews is one of the important exported commodities in Indonesia. Indonesia is one of top five major vanilla exporters in the world, that produce the high quality of Indonesian vanilla with high vanillin content (2.75%. The aims of this research were to determine the effects of dose binukleat Rhizoctonia (BNR and phosphorus as well as the interaction of the nitrate reductase activity (NRA and chlorophyll of the vanilla seedling (Vanilla planifolia Andrew. Method in this research used completely randomized factorial design, by involving two factors (dose of BNR inoculation and Phosphor. The first factor is without inoculation and inoculation BNR (M0, M1, M2, M3 wich consists of (0,5, 10, 15 g/polybag, the second factor is the dose of phosphorus fertilizer (P0, P1, P2, P3 which consists of (0, 3, 6, 9 g/polibag. The results showed that the inoculation dose of BNR and doses of phosphorus not significant and lower levels of NRA and chlorophyll while the interaction dose of BNR and phosphorus significantly and increase levels of NRA and chlorophyll of vanilla seedling. Nitrate Reductase Activity and chlorophyll has important role in metabolism process as a plant growth indicator.How to CiteHaryuni, H., & Dewi, T. S. K. (2016. The Effects of Dose Rhizoctonia Binucleat (BNR and Phosphorus to Nitrate Reductase Activity (NRA and Chlorophyll of Vanilla Seedling (Vanilla planifolia Andrews. Biosaintifika: Journal of Biology & Biology Education, 8(2, 141-147.

  7. Nitrate reductase, arginine deaminase, urease and dehydrogenase activities in natural soil (ridges with forest) and in cotton soil after acetamiprid treatments.

    Science.gov (United States)

    Singh, Dileep K; Kumar, Sunil

    2008-03-01

    Soil enzymes are indicators of microbial activities in soil and are often considered as an indicator of soil health and fertility. They are very sensitive to the agricultural practices, pH of the soil, nutrients, inhibitors and weather conditions. To understand the effect of an insecticide, acetamiprid (IUPAC Name: (E)-N1-[(6-chloro-3-pyridyl) methyl]-N2-cyano-N1-methyl acetamidine) on different soil enzyme activities, the experiments were conducted for three consecutive years (2003--2005) at control and cotton experimental fields of Indian Agricultural Research Institute (IARI) and natural area (ridges with forest) in Delhi. The combined results for all three years were presented here to understand the impact of acetamiprid on soil enzyme activities. Acetamiprid was applied three times in one crop season after 41, 48 and 73 days of sowing, to control the pest. Soil of treated fields was analyzed for insecticide residues immediately after first insecticide treatment and thereafter at definite period. The residues of acetamiprid in experimental soil was varied from 0.30+/-0.13 to 22.67+/-0.2 microg g(-1)d.wt. soil, during the crop period of 2003. The insecticide residues for 2004 ranged between 0.59+/-0.38 and 13.42+/-0.71 microg g(-1)d.wt. soil and for 2005 it ranged between 0.48+/-0.22 and 19.81+/-0.33 microg g(-1)d.wt. soil. An average half life of acetamiprid in our treated field was 11.2+/-1.7 days for all three years. Similarly, the soil from natural area and control were also tested for insecticide residues. No detectable insecticide residues had been found. Soil from three localities i.e. natural, control and experimental fields were tested for different enzyme activities. Nitrate reductase, arginine deaminase, urease and dehydrogenase activities were high in natural soil in comparison to control soil and insecticide treated soil in all three experimental years. At the same time, nitrate reductase activity was all time low in acetamiprid treated soil

  8. Heavy-metal-induced Inhibition of Aspergillus niger nitrate reductase: Applications for Rapid Contaminant Detection in Aqueous Samples

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William Arnold; Aiken, Abigail Marie; Peyton, Brent Michael; Petersen, James N.

    2003-03-01

    Enzyme inhibition assays have the potential to rapidly screen and identify heavy metals in environmental samples. Inhibition of nitrate reductase (NR) was examined as a method for detecting toxic metals. The activity of NR (EC 1.6.6.2) from Aspergillus niger was assayed as a function of metal concentration in the presence of Cd2+, Cr3+, Cr6+, Cu2+, Ni2+, Pb2+, and Zn2+. NR exhibited sensitivity to these metals at concentrations below 10 µM. Various buffers were screened for their ability to protect NR activity from metal inhibition, and 3-(N-morpholino) propanesulfonic acid (MOPS) was selected as the buffering system for the NR assays as it exhibited the least interference with metal inhibition, thus providing increased assay sensitivity. The hypothesis that chelating agents could prevent the inhibition of NR activity by metal ions was also tested. Results indicated that 10 mM ethylenediaminetetraacetic acid (EDTA) could protect NR activity from inhibition by Cr3+, Cu2+, Cd2+, Ni2+, and Zn2+ at concentrations below 100 µM, but that the EDTA had no effect on NR inhibition by Cr6+. An amount of 10 mM nitrilotriacetic acid (NTA) prevented NR inhibition by Cd2+, Cu2+, Ni2+, Pb2+, and Zn2+ at metal concentrations below 100 µM. However, 10 mM NTA was unable to protect the enzyme from inhibition by either Cr3+ or Cr6+. These results indicated that through specific metal chelation, a NR-based method for individually quantifying Cr3+ and Cr6+ species in aqueous solutions could be developed. The ability to restore activity to NR which been previously inhibited by exposure to 100 µM Pb2+, Cd2+, Zn2+, Cu2+, and Cr3+ was explored to determine whether NR activity could be recovered by EDTA additions for use in consecutive metal inhibition assays. The results showed NR activity could not be regained after exposure to Cr3+ or Cu2+, but did partially recover activity after Cd2+, Pb2+, and Zn2+ exposure.

  9. Structure and activity of NADPH-dependent reductase Q1EQE0 from Streptomyces kanamyceticus, which catalyses the R-selective reduction of an imine substrate.

    Science.gov (United States)

    Rodríguez-Mata, María; Frank, Annika; Wells, Elizabeth; Leipold, Friedemann; Turner, Nicholas J; Hart, Sam; Turkenburg, Johan P; Grogan, Gideon

    2013-07-22

    NADPH-dependent oxidoreductase Q1EQE0 from Streptomyces kanamyceticus catalyzes the asymmetric reduction of the prochiral monocyclic imine 2-methyl-1-pyrroline to the chiral amine (R)-2-methylpyrrolidine with >99% ee, and is thus of interest as a potential biocatalyst for the production of optically active amines. The structures of Q1EQE0 in native form, and in complex with the nicotinamide cofactor NADPH have been solved and refined to a resolution of 2.7 Å. Q1EQE0 functions as a dimer in which the monomer consists of an N-terminal Rossman-fold motif attached to a helical C-terminal domain through a helix of 28 amino acids. The dimer is formed through reciprocal domain sharing in which the C-terminal domains are swapped, with a substrate-binding cleft formed between the N-terminal subunit of monomer A and the C-terminal subunit of monomer B. The structure is related to those of known β-hydroxyacid dehydrogenases, except that the essential lysine, which serves as an acid/base in the (de)protonation of the nascent alcohol in those enzymes, is replaced by an aspartate residue, Asp187 in Q1EQE0. Mutation of Asp187 to either asparagine or alanine resulted in an inactive enzyme. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Enzyme kinetics, inhibitors, mutagenesis and electron paramagnetic resonance analysis of dual-affinity nitrate reductase in unicellular N(2)-fixing cyanobacterium Cyanothece sp. PCC 8801.

    Science.gov (United States)

    Wang, Tung-Hei; Chen, Yung-Han; Huang, Jine-Yung; Liu, Kang-Cheng; Ke, Shyue-Chu; Chu, Hsiu-An

    2011-11-01

    The assimilatory nitrate reductase (NarB) of N(2)-fixing cyanobacterium Cyanothece sp. PCC 8801 is a monomeric enzyme with dual affinity for substrate nitrate. We purified the recombinant NarB of Cyanothece sp. PCC 8801 and further investigated it by enzyme kinetics analysis, site-directed mutagenesis, inhibitor kinetics analysis, and electron paramagnetic resonance (EPR) spectroscopy. The NarB showed 2 kinetic regimes at pH 10.5 or 8 and electron-donor conditions methyl viologen or ferredoxin (Fd). Fd-dependent NR assay revealed NarB with very high affinity for nitrate (K(m)1, ∼1μM; K(m)2, ∼270μM). Metal analysis and EPR results showed that NarB contains a Mo cofactor and a [4Fe-4S] cluster. In addition, the R352A mutation on the proposed nitrate-binding site of NarB greatly altered both high- and low-affinity kinetic components. Furthermore, the effect of azide on the NarB of Cyanothece sp. PCC 8801 was more complex than that on the NarB of Synechococcus sp. PCC 7942 with its single kinetic regime. With 1mM azide, the kinetics of the wild-type NarB was transformed from 2 kinetic regimes to hyperbolic kinetics, and its activity was enhanced significantly under medium nitrate concentrations. Moreover, EPR results also suggested a structural difference between the two NarBs. Taken together, our results show that the NarB of Cyanothece sp. PCC 8801 contains only a single Mo-catalytic center, and we rule out that the enzyme has 2 independent, distinct catalytic sites. In addition, the NarB of Cyanothece sp. PCC 8801 may have a regulatory nitrate-binding site. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  11. Mechanism of biological denitrification inhibition: procyanidins induce an allosteric transition of the membrane-bound nitrate reductase through membrane alteration.

    Science.gov (United States)

    Bardon, Clément; Poly, Franck; Piola, Florence; Pancton, Muriel; Comte, Gilles; Meiffren, Guillaume; Haichar, Feth el Zahar

    2016-05-01

    Recently, it has been shown that procyanidins from Fallopia spp. inhibit bacterial denitrification, a phenomenon called biological denitrification inhibition (BDI). However, the mechanisms involved in such a process remain unknown. Here, we investigate the mechanisms of BDI involving procyanidins, using the model strain Pseudomonas brassicacearum NFM 421. The aerobic and anaerobic (denitrification) respiration, cell permeability and cell viability of P. brassicacearum were determined as a function of procyanidin concentration. The effect of procyanidins on the bacterial membrane was observed using transmission electronic microscopy. Bacterial growth, denitrification, NO3- and NO2-reductase activity, and the expression of subunits of NO3- (encoded by the gene narG) and NO2-reductase (encoded by the gene nirS) under NO3 or NO2 were measured with and without procyanidins. Procyanidins inhibited the denitrification process without affecting aerobic respiration at low concentrations. Procyanidins also disturbed cell membranes without affecting cell viability. They specifically inhibited NO3- but not NO2-reductase.Pseudomonas brassicacearum responded to procyanidins by over-expression of the membrane-bound NO3-reductase subunit (encoded by the gene narG). Our results suggest that procyanidins can specifically inhibit membrane-bound NO3-reductase inducing enzymatic conformational changes through membrane disturbance and that P. brassicacearum responds by over-expressing membrane-bound NO3-reductase. Our results lead the way to a better understanding of BDI. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  12. The reactivation of nitrate reductase from spinach (Spinacea oleracea L.) inactivated by NADH and cyanide: effects of peroxidase and associated systems.

    Science.gov (United States)

    Maldonado, J M; Notton, B A; Hewitt, E J

    1982-12-01

    Nitrate reductase of spinach (Spinacea oleracea L.) leaves which had been inactivated in vitro by treatment with NADH and cyanide, was reactivated by incubation with oxidant systems and measured as FMNH2-dependent activity. Ferricyanide, a purely chemical oxidant, produced rapid maximal reactivation (100%) which was 90% complete in less than 3 min. Reactivation occurred slowly and less completely (30-75% in 30 or 60 min) when the enzyme was incubated with pure horseradish peroxidase alone, depending on using one or 20 units and time. Addition of glucose and glucose oxidase to generate hydrogen peroxide increased reactivation slightly (10-15%) with 20 units of peroxidase but more (30-50%) with one unit and to 75-90% of ferricyanide values. Adding catalase decreased reactivation by more than half either with or without glucose oxidase. Glucose and glucose oxidase alone did not cause reactivation. Addition of superoxide dismutase increased reactivation from 50-75% of ferricyanide values with one unit of peroxidase alone but had no effect on greater reactivation obtained in the presence of glucose oxidase. The addition of p-cresol and manganese together increased reactivation with one unit of peroxidase and in the presence of glucose oxidase by about double, but omission of manganese had no effect. However, as shown previously, although trivalent manganese was formed, the residual presence of manganous ions inhibited reactivation. Nevertheless, peroxidase systems either alone or with additionally generated hydrogen peroxide can induce substantial reactivation of nitrate reductase in physiologically relevant conditions.

  13. Intensification of Doxorubicin-Related Oxidative Stress in the Heart by Hypothyroidism Is Not Related to the Expression of Cytochrome P450 NADPH-Reductase and Inducible Nitric Oxide Synthase, As Well As Activity of Xanthine Oxidase

    Directory of Open Access Journals (Sweden)

    Jaroslaw Dudka

    2012-01-01

    Full Text Available Cytochrome P450 NADPH-reductase (P450R, inducible synthase (iNOS and xanthine oxidase play an important role in the antracycline-related cardiotoxicity. The expression of P450R and iNOS is regulated by triiodothyronine. The aim of this study was to evaluate the effect of methimazole-induced hypothyreosis on oxidative stress secondary to doxorubicin administration. 48 hours after methimazole giving cessation, rats were exposed to doxorubicin (2.0, 5.0 and 15 mg/kg. Blood and heart were collected 4, 48 and 96 h after the drug administration. Animals exposed exclusively to doxorubicin or untreated ones were also assessed. The hypothyreosis (0.025% of methimazole significantly increased the doxorubicin effect on the cardiac carbonyl group and they may increase the glutathione level. An insignificant effect of methimazole was noticed in case of the cardiac lipid peroxidation product, the amount of DNA oxidative damages, iNOS and xanthine oxidase-enzymes responsible for red-ox activation of doxorubicin. However, the concentration of P450R was affected by a lower dose of methimazole in rats administered with doxorubicin. Since in rats receiving doxorubicin changes in oxidative stress caused by methimazole were not accompanied by elevation of bioreductive enzymes, it may be concluded that these changes in the oxidative stress were not related to the tested enzymes.

  14. Cloning of a nitrate reductase inactivator (NRI) cDNA from Spinacia oleracea L. and expression of mRNA and protein of NRI in cultured spinach cells.

    Science.gov (United States)

    Sonoda, Masatoshi; Ide, Hiroaki; Nakayama, Shinya; Sasaki, Asako; Kitazaki, Shinei; Sato, Takahide; Nakagawa, Hiroki

    2003-04-01

    The spinach ( Spinacia oleracea L. (cv. Hoyo) nitrate reductase inactivator (NRI) is a novel protein that irreversibly inactivates NR. Using degenerate primers based on an N-terminal amino acid sequence of NRI purified from spinach leaves and a cDNA library, we isolated a full-length NRI cDNA from spinach that contains an open reading frame encoding 479 amino acid residues. This protein shares 67.4% and 51.1-68.3% amino acid sequence similarities with a nucleotide pyrophosphatase (EC 3.6.1.9) from rice and three types of the nucleotide pyrophosphatase-like protein from Arabidopsis thaliana, respectively. Immunoblot analysis revealed that NRI was constitutively expressed in suspension-cultured spinach cells; however, its expression level is quite low in 1-day-subcultured cells. Moreover, northern blot analysis indicated that this expression was regulated at the mRNA level. These results suggest that NRI functions in mature cells.

  15. Overexpression of a GmCnx1 gene enhanced activity of nitrate reductase and aldehyde oxidase, and boosted mosaic virus resistance in soybean.

    Directory of Open Access Journals (Sweden)

    Zheng Zhou

    Full Text Available Molybdenum cofactor (Moco is required for the activities of Moco-dependant enzymes. Cofactor for nitrate reductase and xanthine dehydrogenase (Cnx1 is known to be involved in the biosynthesis of Moco in plants. In this work, a soybean (Glycine max L. Cnx1 gene (GmCnx1 was transferred into soybean using Agrobacterium tumefaciens-mediated transformation method. Twenty seven positive transgenic soybean plants were identified by coating leaves with phosphinothricin, bar protein quick dip stick and PCR analysis. Moreover, Southern blot analysis was carried out to confirm the insertion of GmCnx1 gene. Furthermore, expression of GmCnx1 gene in leaf and root of all transgenic lines increased 1.04-2.12 and 1.55-3.89 folds, respectively, as compared to wild type with GmCnx1 gene and in line 10 , 22 showing the highest expression. The activities of Moco-related enzymes viz nitrate reductase (NR and aldehydeoxidase (AO of T1 generation plants revealed that the best line among the GmCnx1 transgenic plants accumulated 4.25 μg g(-1 h(-1 and 30 pmol L(-1, respectively (approximately 2.6-fold and 3.9-fold higher than non-transgenic control plants.In addition, overexpression ofGmCnx1boosted the resistance to various strains of soybean mosaic virus (SMV. DAS-ELISA analysis further revealed that infection rate of GmCnx1 transgenic plants were generally lower than those of non-transgenic plants among two different virus strains tested. Taken together, this study showed that overexpression of a GmCnx1 gene enhanced NR and AO activities and SMV resistance, suggesting its important role in soybean genetic improvement.

  16. Role of cytochromes P450 1A1/2 in detoxication and activation of carcinogenic aristolochic acid I: studies with the hepatic NADPH:cytochrome P450 reductase null (HRN) mouse model.

    Science.gov (United States)

    Levová, Katerina; Moserová, Michaela; Kotrbová, Vera; Sulc, Miroslav; Henderson, Colin J; Wolf, C Roland; Phillips, David H; Frei, Eva; Schmeiser, Heinz H; Mares, Jaroslav; Arlt, Volker M; Stiborová, Marie

    2011-05-01

    Aristolochic acid (AA) causes aristolochic acid nephropathy, Balkan endemic nephropathy, and their urothelial malignancies. To identify enzymes involved in the metabolism of aristolochic acid I (AAI), the major toxic component of AA we used HRN (hepatic cytochrome P450 [Cyp] reductase null) mice, in which NADPH:Cyp oxidoreductase (Por) is deleted in hepatocytes. AAI was demethylated by hepatic Cyps in vitro to 8-hydroxy-aristolochic acid I (AAIa), indicating that less AAI is distributed to extrahepatic organs in wild-type (WT) mice. Indeed, AAI-DNA-adduct levels were significantly higher in organs of HRN mice, having low hepatic AAI demethylation capacity, than in WT mice. Absence of AAI demethylation in HRN mouse liver was confirmed in vitro; hepatic microsomes from WT, but not from HRN mice, oxidized AAI to AAIa. To define the role of hepatic Cyps in AAI demethylation, modulation of AAIa formation by CYP inducers was investigated. We conclude that AAI demethylation is attributable mainly to Cyp1a1/2. The higher AAI-DNA adduct levels in HRN than WT mice were the result of the lack of hepatic AAI demethylation concomitant with a higher activity of cytosolic NAD(P)H:quinone oxidoreductase (Nqo1), which activates AAI. Mouse hepatic Cyp1a1/2 also activated AAI to DNA adducts under hypoxic conditions in vitro, but in renal microsomes, Por and Cyp3a are more important than Cyp1a for AAI-DNA adduct formation. We propose that AAI activation and detoxication in mice are dictated mainly by AAI binding affinity to Cyp1a1/2 or Nqo1, by their turnover, and by the balance between oxidation and reduction of AAI by Cyp1a.

  17. Impact of elevated atmospheric CO2 on nitrate reductase transcription and activity in leaves and roots of Plantago major

    NARCIS (Netherlands)

    Fonseca, F; Bowsher, CG; Stulen, [No Value

    Vegetative plants of an inbred line, A4, of Plantage major ssp. pleiosperma (L.) Pilger were grown at 350 mu l 1(-1) or at elevated (700 mu l l(-1)) CO2 in non-limiting nutrient solution with nitrate. Both the relative growth rate (RGR) and the root to total plant weight ratio (RWR) were increased

  18. Tetrathionate reductase of Salmonella thyphimurium: a molybdenum containing enzyme

    International Nuclear Information System (INIS)

    Hinojosa-Leon, M.; Dubourdieu, M.; Sanchez-Crispin, J.A.; Chippaux, M.

    1986-01-01

    Use of radioactive molybdenum demonstrates that the tetrathionate reductase of Salmonella typhimurium is a molydenum containing enzyme. It is proposed that this enzyme shares with other molybdo-proteins, such as nitrate reductase, a common molybdenum containing cofactor the defect of which leads to the loss of the tetrathionate reductase and nitrate reductase activities

  19. Co-expression of human cytochrome P4501A1 (CYP1A1) variants and human NADPH-cytochrome P450 reductase in the baculovirus/insect cell system.

    Science.gov (United States)

    Schwarz, D; Kisselev, P; Honeck, H; Cascorbi, I; Schunck, W H; Roots, I

    2001-06-01

    1. Three human cytochrome P4501A1 (CYP1A1) variants, wild-type (CYP1A1.1), CYP1A1.2 (1462V) and CYP1A1.4 (T461N), were co-expressed with human NADPH-P450 reductase (OR) in Spodoptera frugiperda (Sf9) insect cells by baculovirus co-infection to elaborate a suitable system for studying the role of CYPA1 polymorphism in the metabolism of exogenous and endogenous substrates. 2. A wide range of conditions was examined to optimize co-expression with regard to such parameters as relative multiplicity of infection (MOI), time of harvest, haem precursor supplementation and post-translational stabilization. tinder optimized conditions, almost identical expression levels and molar OR/CYP1A1 ratios (20:1) were attained for all CYP1A1 variants. 3. Microsomes isolated from co-infected cells demonstrated ethoxyresorufin deethlylase activities (nmol/min(-1) nmol(-1) CYP1A1) of 16.0 (CYP1A1.1), 20.5 (CYP1A1.2) and 22.5 (CYP1A1.4). Pentoxyresorufin was dealkylated approximately 10-20 times slower with all enzyme variants. 4. All three CYP1A1 variants were active in metabolizing the precarcinogen benzo[a]pyrene (B[a]P), with wild-type enzyme showing the highest activity, followed by CYP1A1.4 (60%) and CYP1A1.2 (40%). Each variant produced all major metabolites including B[a]P-7,8-dihydrodiol, the precursor of the ultimate carcinogenic species. 5. These studies demonstrate that the baculovirus-mediated co-expression-by-co-infection approach all CYP1A1 variants yields functionally active enzyme systems with similar molar OR/CYP1A1 ratios, thus providing suitable preconditions to examine the metabolism of and environmental chemicals by the different CY1A1 variants.

  20. Nitrate reductase activity (NRA in the invasive alien Fallopia japonica: seasonal variation, differences among habitats types, and comparison with native species

    Directory of Open Access Journals (Sweden)

    Damian Chmura

    2016-09-01

    Full Text Available Nitrate reductase activity (NRA was studied in the invasive alien plant F. japonica (Japanese knotweed during the vegetation season and among natural, semi-natural, and human-made habitats and compared with NRA in selected native species. NRA was measured directly in the field from the beginning of May until the beginning of October. NRA was much higher than in the plant’s native range, i.e., East Asia, and showed a high degree of variation over time with the highest values being reached at the stage of fast vegetative growth and at the beginning of fruiting. NRA was highest on dumping sites probably due to the high nitrogen input into soils and near traffic and the emission of NOx by vehicles. A comparison of the enzyme activity in four selected native plant species indicated that NRA in F. japonica was the highest with the exception of Urtica dioica, which exhibited a similar activity of the enzyme. A detailed comparison with this species showed that differences between these species on particular dates were influenced by differences in the phenology of both plants. The initial results that were obtained suggest that nitrogen pollution in an environment can contribute to habitat invasibility and a high level of NRA, which in addition to the many plant traits that are commonly accepted as characteristic of invasiveness features, may be an important factor that enhances invasion success.

  1. Effects of elevated CO2 on the photosynthesis and nitrate reductase activity of Pyropia haitanensis (Bangiales, Rhodophyta) grown at different nutrient levels

    Science.gov (United States)

    Liu, Chunxiang; Zou, Dinghui

    2015-03-01

    Pyropia haitanensis, a commercially important species, was cultured at two CO2 concentrations (390×10-6 and 700×10-6 (parts per million)) and at low and high nutrient levels, to explore the effect of elevated CO2 on the species under nutrient enrichment. Results show that in CO2-enriched thalli, relative growth rate (RGR) was enhanced under nutrient enrichment. Elevated CO2 decreased phycobiliprotein (PB) contents, but increased the contents of soluble carbohydrates. Nutrient enrichment increased the contents of chlorophyll a (Chl a) and PB, while soluble carbohydrate content decreased. CO2 enrichment enhanced the relative maximum electronic transport rate and light saturation point. In nutrient-enriched thalli the activity of nitrate reductase (NRA) increased under elevated CO2. An instantaneous pH change in seawater (from 8.1 to 9.6) resulted in reduction of NRA, and the thalli grown under both elevated CO2 and nutrient enrichment exhibited less pronounced reduction than in algae grown at the ambient CO2. The thermal optima of NRA under elevated CO2 and/or nutrient enrichment shifted to a lower temperature (10-15°C) compared to that in ambient conditions (20°C). We propose that accelerated photosynthesis could result in growth increment. N assimilation remained high in acidified seawater and reflected increased temperature sensitivity in response to elevated CO2 and eutrophication.

  2. Nitrate reductase and nitrogenase activities in relation to N-uptake from soil, 15N-fertilizer and symbiotic fixation in soybean (Glycine max)

    International Nuclear Information System (INIS)

    Ruschel, A.P.; Saito, S.M.T.; Vose, P.B.

    1980-01-01

    Nitrate reductase (NRA) and nitrogenase (ARA) activities were evaluated in relation to nitrogen in the plant from soil (NFS), fertilizer (NFF) and symbiotic fixation (NFN 2 ) to study the pattern of utilization of nitrogen in nodulated and non nodulated soybean, 35, 55 and 75 days after planting. Three levels of ( 15 NH 4 ) 2 SO 4 - added to soil were used (0 - 25 and 50 kg N/ha), being the experiment conducted in the greenhouse, with a split plot statistical design and 4 replications. Maximum levels of RNA and ARA occurred 55 days after planting. Addition of 50 kg N/ha decreased NRA at all harvesting time studied; and nodule ARA only 75 days after planting. By that time the nodulated isoline showed higher NRA than the non nodulated one, the NFS and NFF of the isolines were not different 35 and 55 days after planting, but decreased at the last harvest, especially in nodulated soybean. Symbiotic N 2 -fixation increased plant-N after 55 days growth, contribution about 65% of plant-N in the period between 55 and 75 days after planting. Nodulated plant showed higher N than non nodulated, a sinergistic effect of the three sources of N studied on N increase of nodulated plants was observed. (Author) [pt

  3. Drought-Induced Effects on Nitrate Reductase Activity and mRNA and on the Coordination of Nitrogen and Carbon Metabolism in Maize Leaves1

    Science.gov (United States)

    Foyer, Christine H.; Valadier, Marie-Hélène; Migge, Andrea; Becker, Thomas W.

    1998-01-01

    Maize (Zea mays L.) plants were grown to the nine-leaf stage. Despite a saturating N supply, the youngest mature leaves (seventh position on the stem) contained little NO3− reserve. Droughted plants (deprived of nutrient solution) showed changes in foliar enzyme activities, mRNA accumulation, photosynthesis, and carbohydrate and amino acid contents. Total leaf water potential and CO2 assimilation rates, measured 3 h into the photoperiod, decreased 3 d after the onset of drought. Starch, glucose, fructose, and amino acids, but not sucrose (Suc), accumulated in the leaves of droughted plants. Maximal extractable phosphoenolpyruvate carboxylase activities increased slightly during water deficit, whereas the sensitivity of this enzyme to the inhibitor malate decreased. Maximal extractable Suc phosphate synthase activities decreased as a result of water stress, and there was an increase in the sensitivity to the inhibitor orthophosphate. A correlation between maximal extractable foliar nitrate reductase (NR) activity and the rate of CO2 assimilation was observed. The NR activation state and maximal extractable NR activity declined rapidly in response to drought. Photosynthesis and NR activity recovered rapidly when nutrient solution was restored at this point. The decrease in maximal extractable NR activity was accompanied by a decrease in NR transcripts, whereas Suc phosphate synthase and phosphoenolpyruvate carboxylase mRNAs were much less affected. The coordination of N and C metabolism is retained during drought conditions via modulation of the activities of Suc phosphate synthase and NR commensurate with the prevailing rate of photosynthesis. PMID:9576798

  4. Influence of the temporal and spatial variation of nitrate reductase, glutamine synthetase and soil composition in the N species content in lettuce (Lactuca sativa).

    Science.gov (United States)

    Pinto, Edgar; Fidalgo, Fernanda; Teixeira, Jorge; Aguiar, Ana A; Ferreira, Isabel M P L V O

    2014-04-01

    The variation of nitrate reductase (NR), glutamine synthetase (GS) and N content in lettuce was evaluated at 5 stages of lettuce growth. Soil physicochemical properties and its N content were also assessed to elucidate the soil-to-plant transfer of inorganic N and potential leaching to groundwater. A decrease of NR activity and an increase of NO3(-) and N-Kjeldahl content in lettuces were observed during plant growth, whereas GS activity and NH4(+) increased during the first few weeks of lettuce growth and then decreased. Although the temporal variation was similar in lettuces grown in different soils, quantitative differences were observed, indicating that high NO3(-) content in soil caused a higher NO3(-) accumulation in lettuce despite the higher NR activity during the initial stage of plant growth. Higher levels of NO3(-) and NH4(+) were correlated with higher levels of N-Kjeldahl in lettuce suggesting a positive effect of these N species in the biosynthesis of organic forms of N. Soil physicochemical properties influenced the mobility of inorganic N within the groundwater-soil-plant system. Sandy soils with low OM content allowed NO3(-) leaching, which was confirmed by higher NO3(-) levels in groundwater. Therefore, lettuces grown in those soils presented lower N content and the inputs of N to the environment were higher. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  5. Analytical properties of some commercially available nitrate reductase enzymes evaluated as replacements for cadmium in automated, semiautomated, and manual colorimetric methods for determination of nitrate plus nitrite in water

    Science.gov (United States)

    Patton, Charles J.; Kryskalla, Jennifer R.

    2013-01-01

    A multiyear research effort at the U.S. Geological Survey (USGS) National Water Quality Laboratory (NWQL) evaluated several commercially available nitrate reductase (NaR) enzymes as replacements for toxic cadmium in longstanding automated colorimetric air-segmented continuous-flow analyzer (CFA) methods for determining nitrate plus nitrite (NOx) in water. This research culminated in USGS approved standard- and low-level enzymatic reduction, colorimetric automated discrete analyzer NOx methods that have been in routine operation at the NWQL since October 2011. The enzyme used in these methods (AtNaR2) is a product of recombinant expression of NaR from Arabidopsis thaliana (L.) Heynh. (mouseear cress) in the yeast Pichia pastoris. Because the scope of the validation report for these new automated discrete analyzer methods, published as U.S. Geological Survey Techniques and Methods 5–B8, was limited to performance benchmarks and operational details, extensive foundational research with different enzymes—primarily YNaR1, a product of recombinant expression of NaR from Pichia angusta in the yeast Pichia pastoris—remained unpublished until now. This report documents research and development at the NWQL that was foundational to development and validation of the discrete analyzer methods. It includes: (1) details of instrumentation used to acquire kinetics data for several NaR enzymes in the presence and absence of known or suspected inhibitors in relation to reaction temperature and reaction pH; and (2) validation results—method detection limits, precision and bias estimates, spike recoveries, and interference studies—for standard- and low-level automated colorimetric CFA-YNaR1 reduction NOx methods in relation to corresponding USGS approved CFA cadmium-reduction (CdR) NOx methods. The cornerstone of this validation is paired sample statistical and graphical analysis of NOx concentrations from more than 3,800 geographically and seasonally diverse surface

  6. β-Subunits of the SnRK1 Complexes Share a Common Ancestral Function Together with Expression and Function Specificities; Physical Interaction with Nitrate Reductase Specifically Occurs via AKINβ1-Subunit1[C][OA

    Science.gov (United States)

    Polge, Cécile; Jossier, Mathieu; Crozet, Pierre; Gissot, Lionel; Thomas, Martine

    2008-01-01

    The SNF1/AMPK/SnRK1 kinases are evolutionary conserved kinases involved in yeast, mammals, and plants in the control of energy balance. These heterotrimeric enzymes are composed of one α-type catalytic subunit and two γ- and β-type regulatory subunits. In yeast it has been proposed that the β-type subunits regulate both the localization of the kinase complexes within the cell and the interaction of the kinases with their targets. In this work, we demonstrate that the three β-type subunits of Arabidopsis (Arabidopsis thaliana; AKINβ1, AKINβ2, and AKINβ3) restore the growth phenotype of the yeast sip1Δsip2Δgal83Δ triple mutant, thus suggesting the conservation of an ancestral function. Expression analyses, using AKINβ promoter∷β-glucuronidase transgenic lines, reveal different and specific patterns of expression for each subunit according to organs, developmental stages, and environmental conditions. Finally, our results show that the β-type subunits are involved in the specificity of interaction of the kinase with the cytosolic nitrate reductase. Together with previous cell-free phosphorylation data, they strongly support the proposal that nitrate reductase is a real target of SnRK1 in the physiological context. Altogether our data suggest the conservation of ancestral basic function(s) together with specialized functions for each β-type subunit in plants. PMID:18768910

  7. Xylose reductase from the thermophilic fungus Talaromyces ...

    Indian Academy of Sciences (India)

    Given the potential application of xylose reductase enzymes that preferentially utilize the reduced form of nicotinamide adenine dinucleotide (NADH) rather than NADPH in the fermentation of five carbon sugars by genetically engineered microorganisms, the coenzyme selectivity of TeXR was altered by site-directed ...

  8. ACCUMULATION OF NITROGEN COMPOUNDS AND NITRATE REDUCTASE ACTIVITY IN LETTUCE CULTIVATED IN DIFFERENT CROP SYSTEMS ACÚMULO DE COMPOSTOS NITROGENADOS E ATIVIDADE DA REDUTASE DO NITRATO EM ALFACE PRODUZIDA EM DIFERENTES SISTEMAS DE CULTIVO

    Directory of Open Access Journals (Sweden)

    Fernanda Nunes Ibrahim

    2008-09-01

    Full Text Available

    Nitrate content determination is important for food quality evaluation, therefore when ingested nitrate is reduced the nitrite, which can generate harmful compounds to the human organism. Ahead of this, the present work had as objective to study the transport and accumulation of nitrogen compounds and the nitrate reductase activity in lettuce cultivar 'Vera' produced in Registro (SP in different cropping systems. Were collected samples of the xylem sap, aerial part and root for quantification of nitrogen compounds and of the reductase activity in vivo. The nitrate concentration in the xylem sap, the nitrate and amino acids contents, as well as the nitrate reductase activity, demonstrated more intense transport, accumulation and assimilation in plants cultivated in in hydroponic solution, followed of the conventional system and finally, of the organic. The stem of the plant in the three systems of culture presented high capacity of accumulation and assimilation the nitrogen compounds. The nitrate reductase activity in leaves was superior of the root. The content nitrate, independent of the culture system, varied of 24.32 the 800.06 mg kg-1 of FW in the different parts of the plant. However, it did not exceed the maximum

  9. N-partitioning, nitrate reductase and glutamine synthetase activities in two contrasting varieties of maize Partição de nitrogênio e atividade das enzimas nitrato redutase e glutamina sintetase em duas cultivares contrastantes de milho

    Directory of Open Access Journals (Sweden)

    Altair Toledo Machado

    2001-02-01

    Full Text Available In order to identify useful parameters for maize genetic breeding programs aiming at a more efficient use of N, two maize varieties of contrasting N efficiency, Sol da Manhã NF (efficient and Catetão (inefficient were compared. Experiments were carried out under field and greenhouse conditions, at low and high N levels. The parameters analysed included total and relative plant and grain N content, biomass and the activities of nitrate reductase and glutamine synthetase in different parts of the plant. It was found that the translocation efficiency of N and photoassimilates to the developing seeds and the source-sink relations were significantly different for the two varieties. N content of the whole plant and grain, cob weight and the relative ear dry weight were useful parameters for characterizing the variety Sol da Manhã NF as to its efficient use of N. Enzymes activity of glutamine synthetase (transferase reaction and nitrate reductase did not differ among the varieties.Com o objetivo de identificar parâmetros que possam ser utilizados em programas de melhoramento genético em milho para uso eficiente de N, duas cultivares de milho contrastantes quanto ao uso deste nutriente, Sol da Manhã NF (eficiente e Catetão (não-eficiente, foram avaliadas em dois experimentos conduzidos no campo e em casa de vegetação, respectivamente, sob nível baixo e alto de N. Os caracteres avaliados foram: teor e conteúdo de N em diferentes partes da planta; massa seca; peso dos grãos e de diferentes partes da planta; biomassa, e atividade das enzimas nitrato redutase e glutamina sintetase. O mecanismo de translocação de N e de fotoassimilados para os grãos e a relação fonte/dreno foram importantes para diferenciar a cultivar eficiente da não-eficiente. Conteúdo de N nos grãos e total das plantas, peso do sabugo e relação peso de espiga/matéria seca foram importantes para caracterizar a cultivar Sol da Manhã NF eficiente no uso do N. A

  10. Effects of solar UV radiation on photosynthesis and enzyme activities (carbonic anhydrase and nitrate reductase) in marine macroalgae from southern Spain Efectos de la radiación solar UV sobre la fotosíntesis y actividades enzimáticas (anhidrasa carbónica y nitrato reductasa) en macralgas marinas del sur de España

    OpenAIRE

    FÉLIX L. FIGUEROA; BENJAMÍN VIÑEGLA

    2001-01-01

    The effects of solar ultraviolet (UV) radiation during daily cycles on photosynthesis and two key enzymes involved in carbon incorporation, the carbonic anhydrase, and in inorganic nitrogen reduction, the nitrate reductase, of macroalgae from southern Spain are presented. During daily cycles, photoinhibition in several intertidal macroalgae, expressed as decrease in the effective quantum yield from the morning to noon time, was linearly dependent on the daily integrated irradiance. However, r...

  11. The reduction of nitrate, nitrite and hydroxylamine to ammonia by enzymes from Cucurbita pepo L. in the presence of reduced benzyl viologen as electron donor

    Science.gov (United States)

    Cresswell, C. F.; Hageman, R. H.; Hewitt, E. J.; Hucklesby, D. P.

    1965-01-01

    1. Enzyme systems from Cucurbita pepo have been shown to catalyse the reduction of nitrite and hydroxylamine to ammonia in yields about 90–100%. 2. Reduced benzyl viologen serves as an efficient electron donor for both systems. Activity of the nitrite-reductase system is directly related to degree of dye reduction when expressed in terms of the function for oxidation–reduction potentials, but appears to decrease to negligible activity below about 9% dye reduction. 3. NADH and NADPH alone produce negligible nitrite loss, but NADPH can be linked to an endogenous diaphorase system to reduce nitrite to ammonia in the presence of catalytic amounts of benzyl viologen. 4. The NADH– or NADPH–nitrate-reductase system that is also present can accept electrons from reduced benzyl viologen, but shows relationships opposite to that for the nitrite-reductase system with regard to effect of degree of dye reduction on activity. The product of nitrate reduction may be nitrite alone, or nitrite and ammonia, or ammonia alone, according only to the degree of dye reduction. 5. The relative activities of nitrite-reductase and hydroxylamine-reductase systems show different relationships with degree of dye reduction and may become reversed in magnitude when effects of degree of dye reduction are tested over a suitable range. 6. Nitrite severely inhibits the rate of reduction of hydroxylamine without affecting the yield of ammonia as a percentage of total substrate loss, but hydroxylamine has a negligible effect on the activity of the nitrite-reductase system. 7. The apparent Km for nitrite (1 μm) is substantially less than that for hydroxylamine, for which variable values between 0·05 and 0·9mm (mean 0·51 mm) have been observed. 8. The apparent Km values for reduced benzyl viologen differ for the nitrite-reductase and hydroxylamine-reductase systems: 60 and 7·5 μm respectively. 9. It is concluded that free hydroxylamine may not be an intermediate in the reduction of nitrite

  12. Nitrate reductase and glutamine synthetase activity in coffee leaves during fruit development Atividade da redutase do nitrato e glutamina sintetase em folhas de cafeeiro durante o desenvolvimento dos frutos

    Directory of Open Access Journals (Sweden)

    Andre Rodrigues Reis

    2009-04-01

    Full Text Available Nitrate reductase is the first enzyme in the pathway of nitrate reduction by plants, followed by glutamine synthetase, which incorporates ammonia to glutamine. The purpose of this study was to evaluate the nitrate reductase and glutamine synthetase activity, total soluble protein content, N and Ni content in coffee leaves during fruit development under field conditions to establish new informations to help assess the N nutritional status and fertilizer management. The experimental design was in randomized complete blocks, arranged in a 3 x 6 factorial design, with five replications. The treatments consisted of 3 N rates (0 - control, 150 and 300 kg ha-1 and six evaluation periods (January, February, March, April, May, and June in six-year-old coffee (Coffea arabica L. plants of Catuaí Vermelho IAC 44 cv. The nitrate reductase and glutamine synthetase activities, leaf soluble protein, and N concentrations increased linearly with the N rates. During fruit development, the enzyme activity, leaf soluble protein and N content decreased, due to the leaf senescence process caused by nutrient mobilization to other organs, e.g, to the berries. Leaf Ni increased during fruit development. Beans and raisin-fruits of plants well-supplied with N had higher Ni contents. Enzyme activities, total leaf N and leaf soluble protein, evaluated during the green fruit stage in March, were significantly correlated with coffee yield. These variables can therefore be useful for an early assessment of the coffee N nutritional status as well as coffee yield and N fertilization management.A redutase do nitrato (RN é a primeira enzima na via de redução de nitrato pelas plantas, seguida da glutamina sintetase (GS, a qual incorpora amônia à glutamina. O objetivo deste trabalho foi avaliar a atividade dessas enzimas, o teor de proteína solúvel total e a concentração de N e de Ni em folhas e grãos de cafeeiro durante o desenvolvimento dos frutos, em condições de

  13. Nitrate biosensors and biological methods for nitrate determination.

    Science.gov (United States)

    Sohail, Manzar; Adeloju, Samuel B

    2016-06-01

    The inorganic nitrate (NO3‾) anion is present under a variety of both natural and artificial environmental conditions. Nitrate is ubiquitous within the environment, food, industrial and physiological systems and is mostly present as hydrated anion of a corresponding dissolved salt. Due to the significant environmental and toxicological effects of nitrate, its determination and monitoring in environmental and industrial waters are often necessary. A wide range of analytical techniques are available for nitrate determination in various sample matrices. This review discusses biosensors available for nitrate determination using the enzyme nitrate reductase (NaR). We conclude that nitrate determination using biosensors is an excellent non-toxic alternative to all other available analytical methods. Over the last fifteen years biosensing technology for nitrate analysis has progressed very well, however, there is a need to expedite the development of nitrate biosensors as a suitable alternative to non-enzymatic techniques through the use of different polymers, nanostructures, mediators and strategies to overcome oxygen interference. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Characterization of the reductase domain of rat neuronal nitric oxide synthase generated in the methylotrophic yeast Pichia pastoris. Calmodulin response is complete within the reductase domain itself.

    Science.gov (United States)

    Gachhui, R; Presta, A; Bentley, D F; Abu-Soud, H M; McArthur, R; Brudvig, G; Ghosh, D K; Stuehr, D J

    1996-08-23

    Rat neuronal NO synthase (nNOS) is comprised of a flavin-containing reductase domain and a heme-containing oxygenase domain. Calmodulin binding to nNOS increases the rate of electron transfer from NADPH into its flavins, triggers electron transfer from flavins to the heme, activates NO synthesis, and increases reduction of artificial electron acceptors such as cytochrome c. To investigate what role the reductase domain plays in calmodulin's activation of these functions, we overexpressed a form of the nNOS reductase domain (amino acids 724-1429) in the yeast Pichia pastoris that for the first time exhibits a complete calmodulin response. The reductase domain was purified by 2',5'-ADP affinity chromatography yielding 25 mg of pure protein per liter of culture. It contained 1 FAD and 0.8 FMN per molecule. Most of the protein as isolated contained an air-stable flavin semiquinone radical that was sensitive to FeCN6 oxidation. Anaerobic titration of the FeCN6-oxidized reductase domain with NADPH indicated the flavin semiquinone re-formed after addition of 1-electron equivalent and the flavins could accept up to 3 electrons from NADPH. Calmodulin binding to the recombinant reductase protein increased its rate of NADPH-dependent flavin reduction and its rate of electron transfer to cytochrome c, FeCN6, or dichlorophenolindophenol to fully match the rate increases achieved when calmodulin bound to native full-length nNOS. Calmodulin's activation of the reductase protein was associated with an increase in domain tryptophan and flavin fluorescence. We conclude that many of calmodulin's actions on native nNOS can be fully accounted for through its interaction with the nNOS reductase domain itself.

  15. Dicoumarol-sensitive NADPH: phenanthrenequinone oxidoreductase in channel catfish (Ictalurus punctatus).

    Science.gov (United States)

    Hasspieler, B M; Di Giulio, R T

    1994-04-01

    Phenanthrenequinone (PQ), which occurs widely as a pollutant and as a major metabolite of phenanthrene in a number of species, has been demonstrated to undergo futile redox cycling leading to oxidative stress. In the presence of cytosolic fractions of selected channel catfish tissues, PQ undergoes enzymatic reduction which is mediated by either NADH or NADPH and is composed of dicoumarol-sensitive and -insensitive components. Most notably, gastric cytosol catalyzed a disproportionately high level of NADPH-dependent, dicoumarol-sensitive PQ reduction as compared to gill, liver, and kidney cytosols. In the presence of stomach cytosol and NADPH, PQ facilitated production of superoxide anion at rates several fold higher than those mediated by menadione. The dicoumarol-sensitive PQ-reducing agent, which we have termed NADPH: phenanthrenequinone oxidoreductase (PQR), was purified by affinity chromatography and was demonstrated to be separable from DT diaphorase activity in gastric cytosol. Under aerobic conditions, purified PQR facilitates redox cycling of PQ as indicated by continued NADPH oxidation and hydrogen peroxide production. Under anaerobic conditions, NADPH oxidation is limited to a quantity indicative of PQ reduction to the hydroquinone. Substrate specificities, pH profiles, and kinetic characteristics combine to indicate that PQR represents a novel quinone reductase in this species.

  16. Characterization of mitochondrial thioredoxin reductase from C. elegans

    International Nuclear Information System (INIS)

    Lacey, Brian M.; Hondal, Robert J.

    2006-01-01

    Thioredoxin reductase catalyzes the NADPH-dependent reduction of the catalytic disulfide bond of thioredoxin. In mammals and other higher eukaryotes, thioredoxin reductases contain the rare amino acid selenocysteine at the active site. The mitochondrial enzyme from Caenorhabditis elegans, however, contains a cysteine residue in place of selenocysteine. The mitochondrial C. elegans thioredoxin reductase was cloned from an expressed sequence tag and then produced in Escherichia coli as an intein-fusion protein. The purified recombinant enzyme has a k cat of 610 min -1 and a K m of 610 μM using E. coli thioredoxin as substrate. The reported k cat is 25% of the k cat of the mammalian enzyme and is 43-fold higher than a cysteine mutant of mammalian thioredoxin reductase. The enzyme would reduce selenocysteine, but not hydrogen peroxide or insulin. The flanking glycine residues of the GCCG motif were mutated to serine. The mutants improved substrate binding, but decreased the catalytic rate

  17. Plasmid-encoded diacetyl (acetoin) reductase in Leuconostoc pseudomesenteroides

    DEFF Research Database (Denmark)

    Rattray, Fergal P; Myling-Petersen, Dorte; Larsen, Dianna

    2003-01-01

    A plasmid-borne diacetyl (acetoin) reductase (butA) from Leuconostoc pseudomesenteroides CHCC2114 was sequenced and cloned. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 257 amino acids which had high identity at the amino acid level to diacetyl (acetoin...... diacetyl (acetoin) reductase activity with NADH as coenzyme, but not with NADPH as coenzyme, suggesting the presence of another diacetyl (acetoin)-reducing activity in L. pseudomesenteroides. Plasmid-curing experiments demonstrated that the butA gene is carried on a 20-kb plasmid in L. pseudomesenteroides....

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

    Directory of Open Access Journals (Sweden)

    Alberto eGonzález

    2014-10-01

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

  19. Atividade da enzima nitrato redutase em milho cultivado sob diferentes níveis de adubação nitrogenada e potássica Activity of the enzyme reductase nitrate in corn cultivated under different levels of nitrogen and potassium fertilization

    Directory of Open Access Journals (Sweden)

    Sueli Maria da Silva

    2011-11-01

    Full Text Available Em função de seu uso na alimentação humana e animal e de um elevado potencial produtivo, o milho constitui-se num dos mais importantes cereais cultivados e comercializados no mundo. O objetivo deste trabalho foi avaliar o efeito de diferentes doses de nitrogênio e potássio na atividade da enzima redutase do nitrato na cultura do milho em casa de vegetação. Foram utilizadas cinco doses de nitrogênio (0; 50; 100; 150 e 200kg ha-1 e cinco doses de potássio (0; 40; 80; 120 e 160kg ha-1. A atividade da enzima foi estimada in vivo após as plantas atingirem quatro folhas totalmente desdobradas. Utilizou-se o método baseado no princípio de que a quantidade de nitrito liberada por fragmentos de tecidos vivos num tampão, na presença de uma agente permanente e do substrato, reflete a atividade potencial da enzima. A atividade da enzima nitrato redutase sofreu influência significativa da interação das doses de nitrogênio e potássio, já que a interação N=100kg ha-1 e K=40kg ha-1 proporcionou as melhores médias do experimento. A partir dessa dose, o aumento no fornecimento tanto de nitrogênio quanto de potássio promoveu redução da atividade enzimática.Because of the use in human feeding, animal, and high productive potential, corn is considered to be one of the most important cultivated and commercialized cereals in the world. The aim of this research was to evaluate the effect of different potassium doses and nitrogen in the activity of the enzyme nitrate reductase in the corn culture in a greenhouse environment. Five dosages of nitrogen (0; 50; 100; 150 and 200kg ha-1 and five potassium dosages (0; 40; 80; 120 and 160kg ha-1 were used. The activity of the nitrate reductase was esteemed, in vivo after the plants reached four leaves totally unfolded. The method that was used is based on the principle of the amount of nitrite liberated by fragments of alive tissue in a buffer in the presence of a permanent agent and substrate

  20. Determination of nitrite/nitrate in human biological material by the simple Griess reaction.

    Science.gov (United States)

    Guevara, I; Iwanejko, J; Dembińska-Kieć, A; Pankiewicz, J; Wanat, A; Anna, P; Gołabek, I; Bartuś, S; Malczewska-Malec, M; Szczudlik, A

    1998-06-22

    Since a number of pathological processes such as septic shock, inflammation, graft rejection, diabetes, etc. are associated with a release of nitric oxide (NO), rapid and accurate methods of monitoring of NO concentration are of interest. Various methods for measurement of nitrite and nitrate (NO2-, NO3- ) -- the stable metabolites of NO -- are commonly used for this purpose. In this paper we have shown that the proper Griess procedure for nitrite determination significantly increases the sensitivity of this method. This procedure, supplemented with deproteinization and reduction of nitrates to nitrites in the presence of NADPH-sensitive reductase, can be successfully applied for measurement of NOx levels in human body fluids (serum, urine and CSF). Deproteinization of samples with methanol/diethylether is required and does not influence the sensitivity of detection of NO metabolites. The recovery of the method is 88%+/-6% (n = 30). The NOx concentrations measured by this procedure ranged from 25.0 to 39.0 micromol/l in blood, 4.6 to 14.6 micromol/l in CSF and 0.37 to 2.52 mmol/l (adjusted to creatinine concentration) in urine. The coefficient of variation for this method was between 1.3-2.2%. This method can also be recommended for measurement of NOx produced by cells in tissue cell culture.

  1. Purification and characterization of (+)dihydroflavonol (3-hydroxyflavanone) 4-reductase from flowers of Dahlia variabilis.

    Science.gov (United States)

    Fischer, D; Stich, K; Britsch, L; Grisebach, H

    1988-07-01

    Individual flowers from inflorescences of Dahlia variabilis (cv Scarlet Star) in young developmental stages contained relatively high activity of (+)-dihydroflavonol (DHF) 4-reductase. The DHF reductase was purified from such flowers to apparent homogeneity by a five-step procedure. This included affinity adsorption on Blue Sepharose and elution of the enzyme with NADP+. By gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis it was shown that DHF reductase contains only one polypeptide chain with a Mr of about 41,000. The reductase required NADPH as cofactor and catalyzed transfer of the pro-S hydrogen of NADPH to the substrate. Flavanones and dihydroflavonols (3-hydroxyflavanones) were substrates for DHF reductase with pH optima of about 6.0 for flavanones and of about 6.8 for dihydroflavonols. Flavanones were reduced to the corresponding flavan-4-ols and (+)-dihydroflavonols to flavan-3,4-cis-diols. Apparent Michaelis constants determined for (2S)-naringenin, (2S)-eriodicytol, (+)-dihydrokaempferol, (+)-dihydroquercetin, and NADPH were, respectively, 2.3, 2, 10, 15, and 42 microM. V/Km values were higher for dihydroflavonols than for flavanones. Conversion of dihydromyricetin to leucodelphinidin was also catalyzed by the enzyme at a low rate, whereas flavones and flavonols were not accepted as substrates. DHF reductase was not inhibited by metal chelators.

  2. Respiratory arsenate reductase as a bidirectional enzyme

    Science.gov (United States)

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

    2009-01-01

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

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

    Science.gov (United States)

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

    2015-12-01

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

  4. Hydroxylated naphthoquinones as substrates for Escherichia coli anaerobic reductases.

    Science.gov (United States)

    Rothery, R A; Chatterjee, I; Kiema, G; McDermott, M T; Weiner, J H

    1998-01-01

    We have used two hydroxylated naphthoquinol menaquinol analogues, reduced plumbagin (PBH2, 5-hydroxy-2-methyl-1,4-naphthoquinol) and reduced lapachol [LPCH2, 2-hydroxy-3-(3-methyl-2-butenyl)-1, 4-naphthoquinol], as substrates for Escherichia coli anaerobic reductases. These compounds have optical, solubility and redox properties that make them suitable for use in studies of the enzymology of menaquinol oxidation. Oxidized plumbagin and oxidized lapachol have well resolved absorbances at 419 nm (epsilon=3.95 mM-1. cm-1) and 481 nm (epsilon=2.66 mM-1.cm-1) respectively (in Mops/KOH buffer, pH 7.0). PBH2 is a good substrate for nitrate reductase A (Km=282+/-28 microM, kcat=120+/-6 s-1) and fumarate reductase (Km=155+/-24 microM, kcat=30+/-2 s-1), but not for DMSO reductase. LPCH2 is a good substrate for nitrate reductase A (Km=57+/-35 microM, kcat=68+/-13 s-1), fumarate reductase (Km=85+/-27 microM, kcat=74+/-6 s-1) and DMSO reductase (Km=238+/-30 microM, kcat=191+/-21 s-1). The sensitivity of enzymic LPCH2 and PBH2 oxidation to 2-n-heptyl-4-hydroxyquinoline N-oxide inhibition is consistent with their oxidation occurring at sites of physiological quinol binding. PMID:9576848

  5. Avaliação do teste de nitrato redutase para a detecção rápida de resistência aos medicamentos de primeira linha em cepas de Mycobacterium tuberculosis isoladas de pacientes em um hospital geral Evaluation of the nitrate reductase assay for the rapid detection of resistance to first-line medications in Mycobacterium tuberculosis strains isolated from patients in a general hospital

    Directory of Open Access Journals (Sweden)

    Maria de Fátima Filardi Oliveira Mansur

    2012-04-01

    Full Text Available Comparamos o teste de nitrato redutase com o método de proporções, considerado como padrão ouro, em 57 cepas de Mycobacterium tuberculosis isoladas de pacientes atendidos no Hospital das Clínicas da Universidade Federal de Minas Gerais, em Belo Horizonte (MG. A sensibilidade, a especificidade e a acurácia para rifampicina e isoniazida foram de 100% para todas, enquanto essas foram, respectivamente, de 88,9%, 66,7% e 96,5% para estreptomicina e de 98,0%, 100% e 98,2% para etambutol. A média de tempo para a obtenção dos resultados foi de dez dias. Na amostra estudada, o teste de nitrato redutase mostrou grande acurácia e excelente concordância com o padrão ouro.We compared the nitrate reductase assay with the proportion method, which is considered the gold standard, in 57 Mycobacterium tuberculosis strains isolated from patients treated at the Federal University of Minas Gerais Hospital das Clínicas, located in the city of Belo Horizonte, Brazil. For rifampin and isoniazid, the sensitivity, specificity, and accuracy of the nitrate reductase assay were all 100%, whereas they were 100%, 88.9%, and 66.7%, respectively, for streptomycin and 98.0%, 100%, and 98.2%, respectively, for ethambutol. The mean time to results was ten days. In the study sample, the nitrate reductase assay proved highly accurate and showed excellent concordance with the gold standard.

  6. Evidence for a novel mechanism of time-resolved flavin fluorescence depolarization in glutathione reductase.

    NARCIS (Netherlands)

    Berg, van den P.A.W.; Hoek, van A.; Visser, A.J.W.G.

    2004-01-01

    Time-resolved flavin fluorescence anisotropy studies on glutathione reductase (GR) have revealed a remarkable new phenomenon: wild-type GR displays a rapid process of fluorescence depolarization, that is absent in mutant enzymes lacking a nearby tyrosine residue that blocks the NADPH-binding cleft.

  7. Kinetic properties and inhibition of Trypanosoma cruzi 3-hydroxy-3-methylglutaryl CoA reductase

    DEFF Research Database (Denmark)

    Hurtado-Guerrrero, Ramón; Pena Diaz, Javier; Montalvetti, Andrea

    2002-01-01

    A detailed kinetic analysis of the recombinant soluble enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) from Trypanosoma cruzi has been performed. The enzyme catalyzes the normal anabolic reaction and the reductant is NADPH. It also catalyzes the oxidation of mevalonate but at a lower...

  8. Inherited glutathione reductase deficiency and Plasmodium falciparum malaria--a case study

    NARCIS (Netherlands)

    Gallo, Valentina; Schwarzer, Evelin; Rahlfs, Stefan; Schirmer, R. Heiner; van Zwieten, Rob; Roos, Dirk; Arese, Paolo; Becker, Katja

    2009-01-01

    In Plasmodium falciparum-infected red blood cells (RBCs), the flavoenzyme glutathione reductase (GR) regenerates reduced glutathione, which is essential for antioxidant defense. GR utilizes NADPH produced in the pentose phosphate shunt by glucose-6-phosphate dehydrogenase (G6PD). Thus, conditions

  9. X-ray structural studies of quinone reductase 2 nanomolar range inhibitors

    OpenAIRE

    Pegan, Scott D; Sturdy, Megan; Ferry, Gilles; Delagrange, Philippe; Boutin, Jean A; Mesecar, Andrew D

    2011-01-01

    Quinone reductase 2 (QR2) is one of two members comprising the mammalian quinone reductase family of enzymes responsible for performing FAD mediated reductions of quinone substrates. In contrast to quinone reductase 1 (QR1) which uses NAD(P)H as its co-substrate, QR2 utilizes a rare group of hydride donors, N-methyl or N-ribosyl nicotinamide. Several studies have linked QR2 to the generation of quinone free radicals, several neuronal degenerative diseases, and cancer. QR2 has been also identi...

  10. Molecular Recognition in NADPH-Dependent Plant Thioredoxin Systems-Catalytic Mechanisms, Structural Snapshots and Target Identifications

    DEFF Research Database (Denmark)

    Hägglund, Per; Kirkensgaard, Kristine Groth; Maeda, Kenji

    2009-01-01

    NADPH-dependent thioredoxin Systems (NTS) control enzymatic activities and provide reducing equivalents to metabolic pathways in all types of organisms. from bacteria to mammals In these redox systems, thioredoxin reduces disulfide bonds in target proteins and receives electrons from NADPH via...... thioredoxin reductase (NTR). Plant NTS were first discovered in wheat seeds some 30 years ago and were demonstrated to play a key role in the seed germination process Since then, NTS have been identified in a large. variety of photosynthetic organisms. and,in organelle-specific pattern for their cellular...

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

    Science.gov (United States)

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

    2003-01-01

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

  12. Expression and site-directed mutagenesis of human dihydrofolate reductase

    Energy Technology Data Exchange (ETDEWEB)

    Prendergast, N.J.; Delcamp, T.J.; Smith, P.L.; Freisheim, J.H.

    1988-05-17

    A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-sate kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6 ..-->.. Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the k/sub cat/ for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the K/sub m/ values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by ..cap alpha..-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme.

  13. Thioredoxin Reductase and its Inhibitors

    Science.gov (United States)

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

    2014-01-01

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

  14. Structures of mammalian cytosolic quinone reductases.

    Science.gov (United States)

    Foster, C E; Bianchet, M A; Talalay, P; Faig, M; Amzel, L M

    2000-08-01

    The metabolism of quinone compounds presents one source of oxidative stress in mammals, as many pathways proceed by mechanisms that generate reactive oxygen species as by-products. One defense against quinone toxicity is the enzyme NAD(P)H:quinone oxidoreductase type 1 (QR1), which metabolizes quinones by a two-electron reduction mechanism, thus averting production of radicals. QR1 is expressed in the cytoplasm of many tissues, and is highly inducible. A closely related homologue, quinone reductase type 2 (QR2), has been identified in several mammalian species. QR2 is also capable of reducing quinones to hydroquinones, but unlike QR1, cannot use NAD(P)H. X-ray crystallographic studies of QR1 and QR2 illustrate that despite their different biochemical properties, these enzymes have very similar three-dimensional structures. In particular, conserved features of the active sites point to the close relationship between these two enzymes.

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

    Science.gov (United States)

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

    2016-11-25

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

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

    Science.gov (United States)

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

    2006-05-19

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

  17. Escherichia coli ferredoxin-NADP+ reductase and oxygen-insensitive nitroreductase are capable of functioning as ferric reductase and of driving the Fenton reaction.

    Science.gov (United States)

    Takeda, Kouji; Sato, Junichi; Goto, Kazuyuki; Fujita, Takanori; Watanabe, Toshihiro; Abo, Mitsuru; Yoshimura, Etsuro; Nakagawa, Junichi; Abe, Akira; Kawasaki, Shinji; Niimura, Youichi

    2010-08-01

    Two free flavin-independent enzymes were purified by detecting the NAD(P)H oxidation in the presence of Fe(III)-EDTA and t-butyl hydroperoxide from E. coli. The enzyme that requires NADH or NADPH as an electron donor was a 28 kDa protein, and N-terminal sequencing revealed it to be oxygen-insensitive nitroreductase (NfnB). The second enzyme that requires NADPH as an electron donor was a 30 kDa protein, and N-terminal sequencing revealed it to be ferredoxin-NADP(+) reductase (Fpr). The chemical stoichiometry of the Fenton activities of both NfnB and Fpr in the presence of Fe(III)-EDTA, NAD(P)H and hydrogen peroxide was investigated. Both enzymes showed a one-electron reduction in the reaction forming hydroxyl radical from hydrogen peroxide. Also, the observed Fenton activities of both enzymes in the presence of synthetic chelate iron compounds were higher than their activities in the presence of natural chelate iron compounds. When the Fenton reaction occurs, the ferric iron must be reduced to ferrous iron. The ferric reductase activities of both NfnB and Fpr occurred with synthetic chelate iron compounds. Unlike NfnB, Fpr also showed the ferric reductase activity on an iron storage protein, ferritin, and various natural iron chelate compounds including siderophore. The Fenton and ferric reductase reactions of both NfnB and Fpr occurred in the absence of free flavin. Although the k(cat)/K(m) value of NfnB for Fe(III)-EDTA was not affected by free flavin, the k(cat)/K(m) value of Fpr for Fe(III)-EDTA was 12-times greater in the presence of free FAD than in the absence of free FAD.

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

    Science.gov (United States)

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

    2017-08-01

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

  19. Nitroreductase reactions of Arabidopsis thaliana thioredoxin reductase.

    Science.gov (United States)

    Miskiniene, V; Sarlauskas, J; Jacquot, J P; Cenas, N

    1998-09-07

    Arabidopsis thaliana NADPH:thioredoxin reductase (TR, EC 1.6.4.5) catalyzed redox cycling of aromatic nitrocompounds, including the explosives 2,4,6-trinitrotoluene and tetryl, and the herbicide 3,5-dinitro-o-cresol. The yield of nitro anion radicals was equal to 70-90%. Redox cycling of tetryl was accompanied by formation of N-methylpicramide. Bimolecular rate constants of nitroaromatic reduction (kcat/Km) and reaction catalytic constants (kcat) increased upon an increase in oxidant single-electron reduction potential (E(1)7). Using compounds with an unknown E(1)7 value, the reactivity of TR increased parallelly to the increase in reactivity of ferredoxin:NADP+ reductase of Anabaena PCC 7119 (EC 1.18.1.2). This indicated that the main factor determining reactivity of nitroaromatics towards TR was their energetics of single-electron reduction. Incubation of reduced TR in the presence of tetryl or 2,4-dinitrochlorobenzene resulted in a loss of thioredoxin reductase activity, most probably due to modification of reduced catalytic disulfide, whereas nitroreductase reaction rates were unchanged. This means that on the analogy of quinone reduction by TR (D. Bironaite, Z. Anusevicius, J.-P. Jacquot, N. Cenas, Biochim. Biophys. Acta 1383 (1998) 82-92), FAD and not catalytic disulfide of TR was responsible for the reduction of nitroaromatics. Tetryl, 2,4,6-trinitrotoluene and thioredoxin increased the FAD fluorescence intensity of TR. This finding suggests that nitroaromatics may bind close to the thioredoxin-binding site at the catalytic disulfide domain of TR, and induce a conformational change of enzymes (S.B. Mulrooney, C.H. Williams Jr., Protein Sci. 6 (1997) 2188-2195). Our data indicate that certain nitroaromatic herbicides, explosives and other classes of xenobiotics may interfere with the reduction of thioredoxin by plant TR, and confer prooxidant properties to this antioxidant enzyme.

  20. Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans

    International Nuclear Information System (INIS)

    Nascimento, Alessandro S.; Ferrarezi, Thiago; Catalano-Dupuy, Daniela L.; Ceccarelli, Eduardo A.; Polikarpov, Igor

    2006-01-01

    Crystals adequate for X-ray diffraction analysis have been prepared from L. interrogans ferredoxin-NADP + reductase. Ferredoxin-NADP + reductase (FNR) is an FAD-containing enzyme that catalyzes electron transfer between NADP(H) and ferredoxin. Here, results are reported of the recombinant expression, purification and crystallization of FNR from Leptospira interrogans, a parasitic bacterium of animals and humans. The L. interrogans FNR crystals belong to a primitive monoclinic space group and diffract to 2.4 Å resolution at a synchrotron source

  1. Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans

    Energy Technology Data Exchange (ETDEWEB)

    Nascimento, Alessandro S.; Ferrarezi, Thiago [Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Saocarlense 400, São Carlos, SP, 13560-970 (Brazil); Catalano-Dupuy, Daniela L.; Ceccarelli, Eduardo A. [Facultad de Ciencias Bioquímicas y Farmacéuticas, Molecular Biology Division, Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario (Argentina); Polikarpov, Igor, E-mail: ipolikarpov@if.sc.usp.br [Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Saocarlense 400, São Carlos, SP, 13560-970 (Brazil)

    2006-07-01

    Crystals adequate for X-ray diffraction analysis have been prepared from L. interrogans ferredoxin-NADP{sup +} reductase. Ferredoxin-NADP{sup +} reductase (FNR) is an FAD-containing enzyme that catalyzes electron transfer between NADP(H) and ferredoxin. Here, results are reported of the recombinant expression, purification and crystallization of FNR from Leptospira interrogans, a parasitic bacterium of animals and humans. The L. interrogans FNR crystals belong to a primitive monoclinic space group and diffract to 2.4 Å resolution at a synchrotron source.

  2. Effects of solar UV radiation on photosynthesis and enzyme activities (carbonic anhydrase and nitrate reductase in marine macroalgae from southern Spain Efectos de la radiación solar UV sobre la fotosíntesis y actividades enzimáticas (anhidrasa carbónica y nitrato reductasa en macralgas marinas del sur de España

    Directory of Open Access Journals (Sweden)

    FÉLIX L. FIGUEROA

    2001-06-01

    Full Text Available The effects of solar ultraviolet (UV radiation during daily cycles on photosynthesis and two key enzymes involved in carbon incorporation, the carbonic anhydrase, and in inorganic nitrogen reduction, the nitrate reductase, of macroalgae from southern Spain are presented. During daily cycles, photoinhibition in several intertidal macroalgae, expressed as decrease in the effective quantum yield from the morning to noon time, was linearly dependent on the daily integrated irradiance. However, recovery, expressed as the increase in the effective quantum yield from noon to the afternoon, presented a different pattern; full recovery was found below daily integrated irradiance of 1.0 x10(4 kJ m-2. However, recovery reached only 50 % at higher irradiances. The existence of daily photoinhibition and full recovery in intertidal algae suggests that photoinhibition is a photoprotective mechanism against high solar radiation as in higher plants, and that patterns of photoinhibition and recovery are affected by accumulative doses. Activities of carbonic anhidrase and nitrate reductase were determined in three marine macroalgae (Plocamium cartilagineum, Ulva rigida and Fucus spiralis under full (PAR + UV-A + UV-B and excluded UV solar radiation (PAR. Under PAR + UV-A + UV-B, peaks of enzyme activity were found in P. cartilagineum during the evening, and accordingly to data previously published for other red macroalgae. This situation was modified by the absence of UV radiation since the increase in the activities was delayed several hours. In the three macroalgae and under full solar radiation, a significant and negative correlation was found only when data from nitrate reductase activity was shifted in time during at least four hours. This correlation is lost in Ulva rigida when UV radiation is excluded. The existence of these daily variations with a negative correlation of both enzyme activities could reflect a complex regulatory link between carbon and

  3. Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase

    DEFF Research Database (Denmark)

    Montalvetti, A; Pena Diaz, Javier; Hurtado, R

    2000-01-01

    reductase, obtaining K(m) values for the overall reaction of 40.3+/-5.8 microM for (R,S)-HMG-CoA and 81.4+/-5.3 microM for NADPH; V(max) was 33.55+/-1.8 units x mg(-1). Gel-filtration experiments suggested an apparent molecular mass of 184 kDa with subunits of 46 kDa. Finally, in order to achieve a better...

  4. Structure, function, and mechanism of cytosolic quinone reductases.

    Science.gov (United States)

    Bianchet, Mario A; Erdemli, Sabri Bora; Amzel, L Mario

    2008-01-01

    Quinone reductases type 1 (QR1) are FAD-containing enzymes that catalyze the reduction of many quinones, including menadione (Vit K3), to hydroquinones using reducing equivalents provided by NAD(P)H. The reaction proceeds with a ping-pong mechanism in which the NAD(P)H and the substrate occupy alternatively overlapping regions of the same binding site and participate in a double hydride transfer: one from NAD(P)H to the FAD of the enzyme, and one from the FADH(2) of the enzyme to the quinone substrate. The main function of QR1 is probably the detoxification of dietary quinones but it may also contribute to the reduction of vitamin K for its involvement in blood coagulation. In addition, the same reaction that QR1 uses in the detoxification of quinones, activates some compounds making them cytotoxic. Since QR1 is elevated in many tumors, this property has encouraged the development of chemotherapeutic compounds that become cytotoxic after reduction by QR1. The structures of QR1 alone, and in complexes with substrates, inhibitors, and chemotherapeutic prodrugs, combined with biochemical and mechanistic studies have provided invaluable insight into the mechanism of the enzyme as well as suggestions for the improvements of the chemotherapeutic prodrugs. Similar information is beginning to accumulate about another related enzyme, QR2.

  5. Disguised as a sulfate reducer: Growth of the Deltaproteobacterium Desulfurivibrio alkaliphilus by Sulfide Oxidation with Nitrate

    DEFF Research Database (Denmark)

    Thorup, Casper; Schramm, Andreas; Findlay, Alyssa Jean Lehsau

    2017-01-01

    This study demonstrates that the deltaproteobacterium Desulfurivibrio alkaliphilus can grow chemolithotrophically by coupling sulfide oxidation to the dissimilatory reduction of nitrate and nitrite to ammonium. Key genes of known sulfide oxidation pathways are absent from the genome of D...... of the sulfate reduction pathway. This is the first study providing evidence that a reductive-type DSR is involved in a sulfide oxidation pathway. Transcriptome sequencing further suggests that nitrate reduction to ammonium is performed by a novel type of periplasmic nitrate reductase and an unusual membrane......-anchored nitrite reductase....

  6. Reactions of lipoamide dehydrogenase and glutathione reductase with arsonic acids and arsonous acids.

    Science.gov (United States)

    Knowles, F C

    1985-10-01

    Lipoamide dehydrogenase reacts irreversibly with arsonous acids, RAs(OH)2, and arsonic acids, RAs(O)(OH)2, to form enzyme-inhibitor complexes. The formation of inactive enzyme requires NADH and is kinetically first order in the presence of excess arsonous acid. The second-order rate constant for formation of the enzyme-inhibitor complex was 545 min-1 M-1 for phenylarsonous acid, C6H5As(OH)2, and 5640 min-1 M-1 for methanearsonous acid, CH3As(OH)2. The kinetics of formation of inactive enzyme in the presence of arsonic acids was found to obey a rate law predicted by a two-step mechanism in which a rate-limiting reduction of an arsonic acid to the corresponding arsonous acid by reduced enzyme, E(SH)2, preceded formation of an inactive binary complex of reduced enzyme and arsonous acid: ES2 + NADH + H+ = E(SH)2 + NAD+; E(SH)2 + RAs(O)(OH)2 = ES2 + RAs(OH)2 + H2O; and E(SH)2 + RAs(OH)2 = ES2AsR + 2H2O. GSSG reductase reacts reversibly with C6H5As(OH)2 to form an inactive binary addition compound in the presence of NADPH. The value of the association constant for formation of enzyme inhibitor complex at pH 7.0 was 119 M-1. The initial rate of the GSSG reductase-catalyzed oxidation of NADPH by GSSG was insensitive to MeAs(OH)2. The kinetics of inhibition of GSSG reductase by arsenite and C6H5As(O)(OH)2 were found to obey the rate law described for lipoamide dehydrogenase and arsonic acids. GSSG reductase catalyzed the oxidation of NADPH by p-arsanilic acid. The initial rate of oxidation of NADPH was linearly dependent on enzyme concentration. The turnover number for GSSG reductase with p-arsanilic acid as an oxidant was 0.13 mol NADPH mol FAD-1 min-1.

  7. Role of Lysine-54 in determining cofactor specificity and binding in human dihydrofolate reductase

    International Nuclear Information System (INIS)

    Huang, Shaoming; Tan, Xuehai; Thompson, P.D.; Freisheim, J.H.; Appleman, J.R.; Blakley, R.L.; Sheridan, R.P.; Venkataraghavan, R.

    1990-01-01

    Lysine-54 of human dihydrofolate reductase (hDHFR) appears to be involved in the interaction with the 2'-phosphate of NADPH and is conserved as a basic residue in other species. Studies have suggested that in Lactobacillus casei dihydrofolate reductase Arg-43, the homologous residue at this position, plays an important role in the binding of NADPH and in the differentiation of K m values for NADPH and NADH. A Lys-54 to Gln-54 mutant (K54Q) of hDHFR has been constructed by oligodeoxynucleotide-directed mutagenesis in order to study the role of Lys-54 in differentiating K m and k cat values for NADPH and NADH as well as in other functions of hDHFR. The purpose of this paper is to delineate in quantitative terms the magnitude of the effect of the Lys-54 to Gln-54 replacement on the various kinetic parameters of hDHFR. Such quantitative effects cannot be predicted solely on the basis of X-ray structures. The ratio of K m (NADH)/K m (NADPH) decreases from 69 in the wild-type enzyme to 4.7 in the K54Q enzyme, suggesting that Lys-54, among other interactions between protein side-chain residues and the 2'-phosphate, makes a major contribution in terms of binding energy and differentiation of K m values for NADPH and NADH. Agents at concentrations that show activating effects on the wild-type enzyme such as potassium chloride and urea all inactivate the K54Q enzyme. There appear to be no gross conformational differences between wild-type and K54Q enzyme molecules as judged by competitive ELISA using peptide-specific antibodies against human dihydrofolate reductase and from protease susceptibility studies on both wild-type and K54Q mutant enzymes. The pH-rate profiles using NADPH for K54Q and wild-type enzymes show divergences at certain pH values, suggesting the possibility of alteration(s) in the steps of the catalytic pathway for the K54Q enzyme

  8. Does foliar application of salicylic acid protects nitrate reductase and ...

    African Journals Online (AJOL)

    The present study was conducted to assess whether exogenous applied salicylic acid (SA) as a foliar spray could ameliorate the adverse effects of virus infection in two maize cultivars (maize cv. sabaini and maize cv. Nab El-gamal). The plants were grown under normal field conditions for two weeks in sand clay soil, and ...

  9. Does foliar application of salicylic acid protects nitrate reductase and ...

    African Journals Online (AJOL)

    SAM

    2014-06-04

    Jun 4, 2014 ... interferes with tobacco mosaic virus replication via a novel salicylhydroxamic acid-sensitive mechanism. Plant Cell 9:547-557. Dat JF, Lopez DH, Foyer CH, Scott IM (1998). Parallel changes in H2O2 and catalase during thermotolerance induced by salicylic acid or heat acclimation in mustard seedlings.

  10. NADH-Ferricyanide Reductase of Leaf Plasma Membranes 1

    Science.gov (United States)

    Askerlund, Per; Laurent, Pascal; Nakagawa, Hiroki; Kader, Jean-Claude

    1991-01-01

    Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b5 reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber

  11. Altering coenzyme specificity of Pichia stipitis xylose reductase by the semi-rational approach CASTing

    Directory of Open Access Journals (Sweden)

    Zhang Jingqing

    2007-11-01

    Full Text Available Abstract Background The NAD(PH-dependent Pichia stipitis xylose reductase (PsXR is one of the key enzymes for xylose fermentation, and has been cloned into the commonly used ethanol-producing yeast Saccharomyces cerevisiae. In order to eliminate the redox imbalance resulting from the preference of this enzyme toward NADPH, efforts have been made to alter the coenzyme specificity of PsXR by site-directed mutagenesis, with limited success. Given the industrial importance of PsXR, it is of interest to investigate further ways to create mutants of PsXR that prefers NADH rather than NADPH, by the alternative directed evolution approach. Results Based on a homology model of PsXR, six residues were predicted to interact with the adenine ribose of NAD(PH in PsXR and altered using a semi-rational mutagenesis approach (CASTing. Three rounds of saturation mutagenesis were carried to randomize these residues, and a microplate-based assay was applied in the screening. A best mutant 2-2C12, which carried four mutations K270S, N272P, S271G and R276F, was obtained. The mutant showed a preference toward NADH over NADPH by a factor of about 13-fold, or an improvement of about 42-fold, as measured by the ratio of the specificity constant kcat/Kmcoenzyme. Compared with the wild-type, the kcatNADH for the best mutant was only slightly lower, while the kcatNADPH decreased by a factor of about 10. Furthermore, the specific activity of 2-2C12 in the presence of NADH was 20.6 U·mg-1, which is highest among PsXR mutants reported. Conclusion A seemingly simplistic and yet very effective mutagenesis approach, CASTing, was applied successfully to alter the NAD(PH preference for Pichia stipitis xylose reductase, an important enzyme for xylose-fermenting yeast. The observed change in the NAD(PH preference for this enzyme seems to have resulted from the altered active site that is more unfavorable for NADPH than NADH in terms of both Km and kcat. There are potentials for

  12. Interactions between inhibitors of dihydrofolate reductase.

    Science.gov (United States)

    Bowden, K; Hall, A D; Birdsall, B; Feeney, J; Roberts, G C

    1989-03-01

    The binding of substrates and inhibitors to dihydrofolate reductase was studied by steady-state kinetics and high-field 1H-n.m.r. spectroscopy. A series of 5-substituted 2,4-diaminopyrimidines were examined and were found to be 'tightly binding' inhibitors of the enzyme (Ki less than 10(-9) M). Studies on the binding of 4-substituted benzenesulphonamides and benzenesulphonic acids also established the existence of a 'sulphonamide-binding site' on the enzyme. Subsequent n.m.r. experiments showed that there are two binding sites for the sulphonamides on the enzyme, one of which overlaps the coenzyme (NADPH) adenine-ring-binding site. An examination of the pH-dependence of the binding of sulphonamides to the enzyme indicated the influence of an ionizable group on the enzyme that was not directly involved in the sulphonamide binding. The change in pKa value from 6.7 to 7.2 observed on sulphonamide binding suggests the involvement of a histidine residue, which could be histidine-28.

  13. NADPH oxidases in Microglia oxidant production

    DEFF Research Database (Denmark)

    Haslund-Vinding, J; McBean, G; Jaquet, V

    2017-01-01

    Microglia are the resident immune cells of the central nervous system (CNS) and constitute a self-sustaining population of CNS-adapted tissue macrophages. As mononuclear phagocytic cells, they express high levels of superoxide-producing NADPH oxidases (NOX). The sole function of members of the NOX...... excessive, badly-timed, or misplaced NOX activation in microglia may affect neuronal homeostasis in physiological or pathological conditions certainly merits further investigation. This article is protected by copyright. All rights reserved....

  14. External NAD(P)H dehydrogenases in Acanthamoeba castellanii mitochondria.

    Science.gov (United States)

    Antos-Krzeminska, Nina; Jarmuszkiewicz, Wieslawa

    2014-09-01

    The mitochondrial respiratory chain of plants and some fungi contains multiple rotenone-insensitive NAD(P)H dehydrogenases, of which at least two are located on the outer surface of the inner membrane (i.e., external NADH and external NADPH dehydrogenases). Annotated sequences of the putative alternative NAD(P)H dehydrogenases of the protozoan Acanthamoeba castellanii demonstrated similarity to plant and fungal sequences. We also studied activity of these dehydrogenases in isolated A. castellanii mitochondria. External NADPH oxidation was observed for the first time in protist mitochondria. The coupling parameters were similar for external NADH oxidation and external NADPH oxidation, indicating similar efficiencies of ATP synthesis. Both external NADH oxidation and external NADPH oxidation had an optimal pH of 6.8 independent of relevant ubiquinol-oxidizing pathways, the cytochrome pathway or a GMP-stimulated alternative oxidase. The maximal oxidizing activity with external NADH was almost double that with external NADPH. However, a lower Michaelis constant (K(M)) value for external NADPH oxidation was observed compared to that for external NADH oxidation. Stimulation by Ca(2+) was approximately 10 times higher for external NADPH oxidation, while NADH dehydrogenase(s) appeared to be slightly dependent on Ca(2+). Our results indicate that external NAD(P)H dehydrogenases similar to those in plant and fungal mitochondria function in mitochondria of A. castellanii. Copyright © 2014 Elsevier GmbH. All rights reserved.

  15. Crystallization and preliminary X-ray diffraction analysis of salutaridine reductase from the opium poppy Papaver somniferum

    International Nuclear Information System (INIS)

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

    2010-01-01

    Recombinant P. somniferum salutaridine reductase (SalR) was purified and crystallized with NADPH using the hanging-drop vapor-diffusion method. Crystals of the SalR–NADPH complex diffracted X-rays to a resolution of 1.9 Å. The opium poppy Papaver somniferum is the source of the narcotic analgesics morphine and codeine. Salutaridine reductase (SalR; EC 1.1.1.248) reduces the C-7 keto group of salutaridine to the C-7 (S)-hydroxyl group of salutaridinol in the biosynthetic pathway that leads to morphine in the opium poppy plant. P. somniferum SalR was overproduced in Escherichia coli and purified using cobalt-affinity and size-exclusion chromatography. Hexagonal crystals belonging to space group P6 4 22 or P6 2 22 were obtained using ammonium sulfate as precipitant and diffracted to a resolution of 1.9 Å

  16. NADPH-dependent D-aldose reductases and xylose fermentation in Fusarium oxysporum

    DEFF Research Database (Denmark)

    Panagiotou, Gianni; Christakopoulos, P.

    2004-01-01

    reactors. Oxygen limitation had considerable influence on xylose metabolism. Under anaerobic conditions (0 vvm), xylitol was the main product with a maximum yield of 0.34 mole of xylitol/mole of xylose while the maximum ethanol yield (1.02 moles of ethanol/mole of xylose) was obtained under aerobic...... conditions (0.3 vvm). When the artificial electron acceptor acetoin was added to an anaerobic batch fermentation of xylose by F. oxysporum, the ethanol yield increased while xylitol excretion was also decreased....

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

    Energy Technology Data Exchange (ETDEWEB)

    Timko, Michael P

    2013-02-01

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

  18. Structural and functional insights into Saccharomyces cerevisiae riboflavin biosynthesis reductase RIB7.

    Directory of Open Access Journals (Sweden)

    Zongyang Lv

    Full Text Available Saccharomyces cerevisiae RIB7 (ScRIB7 is a potent target for anti-fungal agents because of its involvement in the riboflavin biosynthesis pathway as a NADPH-dependent reductase. However, the catalytic mechanism of riboflavin biosynthesis reductase (RBSRs is controversial, and enzyme structure information is still lacking in eukaryotes. Here we report the crystal structure of Saccharomyces cerevisiae RIB7 at 2.10 Å resolution and its complex with NADPH at 2.35 Å resolution. ScRIB7 exists as a stable homodimer, and each subunit consists of nine central β-sheets flanked by five helices, resembling the structure of RIB7 homologues. A conserved G(76-X-G(78-Xn-G(181-G(182 motif is present at the NADPH pyrophosphate group binding site. Activity assays confirmed the necessity of Thr79, Asp83, Glu180 and Gly182 for the activity of ScRIB7. Substrate preference of ScRIB7 was altered by mutating one residue (Thr35 to a Lysine, implying that ScRIB7 Thr35 and its corresponding residue, a lysine in bacteria, are important in substrate-specific recognition.

  19. FQR1, a Novel Primary Auxin-Response Gene, Encodes a Flavin Mononucleotide-Binding Quinone Reductase1

    Science.gov (United States)

    Laskowski, Marta J.; Dreher, Kate A.; Gehring, Mary A.; Abel, Steffen; Gensler, Arminda L.; Sussex, Ian M.

    2002-01-01

    FQR1 is a novel primary auxin-response gene that codes for a flavin mononucleotide-binding flavodoxin-like quinone reductase. Accumulation of FQR1 mRNA begins within 10 min of indole-3-acetic acid application and reaches a maximum of approximately 10-fold induction 30 min after treatment. This increase in FQR1 mRNA abundance is not diminished by the protein synthesis inhibitor cycloheximide, demonstrating that FQR1 is a primary auxin-response gene. Sequence analysis reveals that FQR1 belongs to a family of flavin mononucleotide-binding quinone reductases. Partially purified His-tagged FQR1 isolated from Escherichia coli catalyzes the transfer of electrons from NADH and NADPH to several substrates and exhibits in vitro quinone reductase activity. Overexpression of FQR1 in plants leads to increased levels of FQR1 protein and quinone reductase activity, indicating that FQR1 functions as a quinone reductase in vivo. In mammalian systems, glutathione S-transferases and quinone reductases are classified as phase II detoxification enzymes. We hypothesize that the auxin-inducible glutathione S-transferases and quinone reductases found in plants also act as detoxification enzymes, possibly to protect against auxin-induced oxidative stress. PMID:11842161

  20. FQR1, a novel primary auxin-response gene, encodes a flavin mononucleotide-binding quinone reductase.

    Science.gov (United States)

    Laskowski, Marta J; Dreher, Kate A; Gehring, Mary A; Abel, Steffen; Gensler, Arminda L; Sussex, Ian M

    2002-02-01

    FQR1 is a novel primary auxin-response gene that codes for a flavin mononucleotide-binding flavodoxin-like quinone reductase. Accumulation of FQR1 mRNA begins within 10 min of indole-3-acetic acid application and reaches a maximum of approximately 10-fold induction 30 min after treatment. This increase in FQR1 mRNA abundance is not diminished by the protein synthesis inhibitor cycloheximide, demonstrating that FQR1 is a primary auxin-response gene. Sequence analysis reveals that FQR1 belongs to a family of flavin mononucleotide-binding quinone reductases. Partially purified His-tagged FQR1 isolated from Escherichia coli catalyzes the transfer of electrons from NADH and NADPH to several substrates and exhibits in vitro quinone reductase activity. Overexpression of FQR1 in plants leads to increased levels of FQR1 protein and quinone reductase activity, indicating that FQR1 functions as a quinone reductase in vivo. In mammalian systems, glutathione S-transferases and quinone reductases are classified as phase II detoxification enzymes. We hypothesize that the auxin-inducible glutathione S-transferases and quinone reductases found in plants also act as detoxification enzymes, possibly to protect against auxin-induced oxidative stress.

  1. A role for NADPH oxidase in antigen presentation

    Directory of Open Access Journals (Sweden)

    Gail J Gardiner

    2013-09-01

    Full Text Available The nicotinamide adenine dinucleotide phosphate (NADPH oxidase expressed in phagocytes is a multi-subunit enzyme complex that generates superoxide (O2.-. This radical is an important precursor of hydrogen peroxide (H2O2 and other reactive oxygen species (ROS needed for microbicidal activity during innate immune responses. Inherited defects in NADPH oxidase give rise to chronic granulomatous disease (CGD, a primary immunodeficiency characterized by recurrent infections and granulomatous inflammation. Interestingly, CGD, CGD carrier status, and oxidase gene polymorphisms have all been associated with autoinflammatory and autoimmune disorders, suggesting a potential role for NADPH oxidase in regulating adaptive immune responses. Here, NADPH oxidase function in antigen processing and presentation is reviewed. NADPH oxidase influences dendritic cell (DC crosspresentation by major histocompatibility complex class I molecules (MHC-I through regulation of the phagosomal microenvironment, while in B lymphocytes, NADPH oxidase alters epitope selection by major histocompatibility complex class II molecules (MHC-II.

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

    Energy Technology Data Exchange (ETDEWEB)

    Higashi, Yasuhiro; Kutchan, Toni M.; Smith, Thomas J. (Danforth)

    2011-11-18

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

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

    Science.gov (United States)

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

    2013-12-01

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

  4. Recent structural insights into the function of copper nitrite reductases.

    Science.gov (United States)

    Horrell, Sam; Kekilli, Demet; Strange, Richard W; Hough, Michael A

    2017-11-15

    Copper nitrite reductases (CuNiR) carry out the first committed step of the denitrification pathway of the global nitrogen cycle, the reduction of nitrite (NO 2 - ) to nitric oxide (NO). As such, they are of major agronomic and environmental importance. CuNiRs occur primarily in denitrifying soil bacteria which carry out the overall reduction of nitrate to dinitrogen. In this article, we review the insights gained into copper nitrite reductase (CuNiR) function from three dimensional structures. We particularly focus on developments over the last decade, including insights from serial femtosecond crystallography using X-ray free electron lasers (XFELs) and from the recently discovered 3-domain CuNiRs.

  5. A second target of benzamide riboside: dihydrofolate reductase.

    Science.gov (United States)

    Roussel, Breton; Johnson-Farley, Nadine; Kerrigan, John E; Scotto, Kathleen W; Banerjee, Debabrata; Felczak, Krzysztof; Pankiewicz, Krzysztof W; Gounder, Murugesan; Lin, HongXia; Abali, Emine Ercikan; Bertino, Joseph R

    2012-11-01

    Dihydrofolate reductase (DHFR) is an essential enzyme involved in de novo purine and thymidine biosynthesis. For several decades, selective inhibition of DHFR has proven to be a potent therapeutic approach in the treatment of various cancers including acute lymphoblastic leukemia, non-Hodgkin's lymphoma, osteogenic sarcoma, carcinoma of the breast, and head and neck cancer. Therapeutic success with DHFR inhibitor methotrexate (MTX) has been compromised in the clinic, which limits the success of MTX treatment by both acquired and intrinsic resistance mechanisms. We report that benzamide riboside (BR), via anabolism to benzamide adenine dinucleotide (BAD) known to potently inhibit inosine monophosphate dehydrogenase (IMPDH), also inhibits cell growth through a mechanism involving downregulation of DHFR protein. Evidence to support this second site of action of BR includes the finding that CCRF-CEM/R human T-cell lymphoblasic leukemia cells, resistant to MTX as a consequence of gene amplification and overexpression of DHFR, are more resistant to BR than are parental cells. Studies of the mechanism by which BR lowers DHFR showed that BR, through its metabolite BAD, reduced NADP and NADPH cellular levels by inhibiting nicotinamide adenine dinucleotide kinase (NADK). As consequence of the lack of NADPH, DHFR was shown to be destabilized. We suggest that, inhibition of NADK is a new approach to downregulate DHFR and to inhibit cell growth.

  6. Effects of arsenic on nitrate metabolism in arsenic hyperaccumulating and non-hyperaccumulating ferns

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Nandita [Soil and Water Science Department, University of Florida, Gainesville, Fl 32611-0290 (United States); Eco-Auditing group, National Botanical Research Institute, Rana Pratap Marg, Lucknow 226 001 (India); Ma, Lena Q., E-mail: lqma@ufl.ed [Soil and Water Science Department, University of Florida, Gainesville, Fl 32611-0290 (United States); Vu, Joseph C. [Chemistry Research Unit, CMAVE, USDA-ARS, Gainesville, FL 32608-1069 and Agronomy Department, University of Florida, Gainesville, FL 32611-0500 (United States); Raj, Anshita [Eco-Auditing group, National Botanical Research Institute, Rana Pratap Marg, Lucknow 226 001 (India)

    2009-08-15

    This study investigated the effects of arsenic on the in vitro activities of the enzymes (nitrate reductase and nitrite reductase) involved in nitrate metabolism in the roots, rhizomes, and fronds of four-month old Pteris vittata (arsenic - hyperaccumulator) and Pteris ensiformis (non-arsenic--hyperaccumulator) plants. The arsenic treatments (0, 150, and 300 muM as sodium arsenate) in hydroponics had adverse effects on the root and frond dry weights, and this effect was more evident in P. ensiformis than in P. vittata. Nitrate reductase and nitrite reductase activities of arsenate-treated plants were reduced more in P. ensiformis than in P. vittata. This effect was accompanied by similar decreases in tissue NO{sub 3}{sup -} concentrations. Therefore, this decrease is interpreted as being indirect, i.e., the consequence of the reduced NO{sub 3}{sup -} uptake and translocation in the plants. The study shows the difference in the tolerance level of the two Pteris species with varying sensitivity to arsenic. - Arsenic reduced the activity of nitrate and nitrite reductase more in Pteris ensiformis than Pteris vittata.

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

    Energy Technology Data Exchange (ETDEWEB)

    Rosenthal, Cindy [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Mueller, Uwe [Berliner Elektronenspeicherring-Gesellschaft für Synchrotronstrahlung mbH, Albert-Einstein-Strasse 15, D-12489 Berlin (Germany); Panjikar, Santosh [European Molecular Biology Laboratory Hamburg, Outstation Deutsches Elektronen-Synchrotron, Notkestrasse 85, D-22603 Hamburg (Germany); Sun, Lianli [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Department of TCM and Natural Drug Research, College of Pharmaceutical Sciences, 513 Zijingang Campus, Zhejiang University, 310058 Hangzhou (China); Ruppert, Martin [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Zhao, Yu [Department of TCM and Natural Drug Research, College of Pharmaceutical Sciences, 513 Zijingang Campus, Zhejiang University, 310058 Hangzhou (China); Stöckigt, Joachim, E-mail: stoeckig@mail.uni-mainz.de [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Department of TCM and Natural Drug Research, College of Pharmaceutical Sciences, 513 Zijingang Campus, Zhejiang University, 310058 Hangzhou (China)

    2006-12-01

    Perakine reductase, a novel member of the aldo-keto reductase enzyme superfamily of higher plants, is involved in the biosynthesis of monoterpenoid indole alkaloids in the Indian medicinal plant Rauvolfia serpentina. The enzyme has been crystallized in C-centered orthorhombic space group and diffracts to 2.0 Å resolution. Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C222{sub 1} and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å.

  8. Modeled Wet Nitrate Deposition

    Data.gov (United States)

    U.S. Environmental Protection Agency — Modeled data on nitrate wet deposition was obtained from Dr. Jeff Grimm at Penn State Univ. Nitrate wet depostion causes acidification and eutrophication of surface...

  9. Nitrate Enhanced Microbial Cr(VI) Reduction-Final Report

    Energy Technology Data Exchange (ETDEWEB)

    John F. Stolz

    2011-06-15

    A major challenge for the bioremediation of radionuclides (i.e., uranium, technetium) and metals (i.e., Cr(VI), Hg) is the co-occurrence of nitrate as it can inhibit metal transformation. Denitrification (nitrate reduction to dinitrogen gas) is considered the most important ecological process. For many metal and metalloid reducing bacteria, however, ammonia is the end product through respiratory nitrate reduction (RNRA). The focus of this work was to determine how RNRA impacts Cr(VI) transformation. The goal was to elucidate the specific mechanism(s) that limits Cr(VI) reduction in the presence of nitrate and to use this information to develop strategies that enhance Cr(VI) reduction (and thus detoxification). Our central hypothesis is that nitrate impacts the biotransformation of metals and metalloids in three ways 1) as a competitive alternative electron acceptor (inhibiting transformation), 2) as a co-metabolite (i.e., concomitant reduction, stimulating transformation), and 3) as an inducer of specific proteins and pathways involved in oxidation/reduction reactions (stimulating transformation). We have identified three model organisms, Geobacter metallireducens (mechanism 1), Sulfurospirillum barnesii, (mechasism 2), and Desulfovibrio desulfuricans (mechanisms 3). Our specific aims were to 1) investigate the role of Cr(VI) concentration on the kinetics of both growth and reduction of nitrate, nitrite, and Cr(VI) in these three organisms; 2) develop a profile of bacterial enzymes involved in nitrate transformation (e.g., oxidoreductases) using a proteomic approach; 3) investigate the function of periplasmic nitrite reductase (Nrf) as a chromate reductase; and 4) develop a strategy to maximize microbial chromium reduction in the presence of nitrate. We found that growth on nitrate by G. metallireducens was inhibited by Cr(VI). Over 240 proteins were identified by LC/MS-MS. Redox active proteins, outer membrane heavy metal efflux proteins, and chemotaxis sensory

  10. DROUGHT-INDUCED EFFECTS AND RECOVERY OF NITRATE ASSIMILATION AND NODULE ACTIVITY IN COWPEA PLANTS INOCULATED WITH BRADYRHIZOBIUM SPP. UNDER MODERATE NITRATE LEVEL

    Directory of Open Access Journals (Sweden)

    Silveira Joaquim Albenísio Gomes da

    2001-01-01

    Full Text Available This study was carried out to establish comparative effects of drought and recovery on the nitrate assimilation and nodule activity related to N2 fixation in cowpea plants [Vigna unguiculata L. (Walp.] previously inoculated with Bradyrhizobium spp. BR-3256 (CB-756 strain in the presence of 5 mol m-3 NO-3. Twenty-eight-day-old nodulated plants were submitted to water deprivation during 4 consecutive days and afterwards resupplied with nutrient solution during 2 days. The water deprivation caused a rapid increase in the nitrate content in root and a marked reduction in leaf nitrate reductase (NR activity. In contrast nodule NR activity was slightly increased by water deprivation. Concomitantly, in nodules of water stressed plants, leghemoglobin and glutamine synthetase (GS activity declined and a progressive reduction in ureide-N concentration in xylem sap was observed. Leaf-NR activity increased rapidly after rehydration while leaf nitrate content declined. In contrast both GS activity and soluble protein content in the nodule continued to decline in rewatered plants. In addition the concentration of leghemoglobin recovered well, while the xylem ureide-N content experienced a slight increase after rehydration. Despite the nitrate assimilation in leaves and the nodule activity had been both severely affected by water stress, the rapid recovery of nitrate reductase activity suggests that the nitrate assimilation process is less sensitive to drought/rehydration cycle when cowpea plants are nodulated in presence of moderate nitrate level.

  11. An 15N study of the effects of nitrate, ammonium, and nitrate + ammonium nutrition on nitrogen assimilation in Zea mays L

    International Nuclear Information System (INIS)

    Murphy, A.T.

    1984-10-01

    A brief review of the literature on the effects of nitrate and ammonium nitrogen sources on plant growth, and the assimilation of those nitrogen sources, has been presented. It was concluded that ammonium nutrition produces optimum growth, with nitrate + ammonium being a better nitrogen source than only nitrate. Leaf blade nitrate reductase activity exceeded that of the root in nitrate-fed plants, suggesting that the shoot is the major region of nitrate assimilation. This is further supported by the results of xylem exudate analysis, where 93% of the newly-absorbed nitrogen exported by the roots was detected as nitrate. Evidence in support of this hypothesis was also obtained by studying the distribution of 15 N in the various nitrogenous compounds. The effects of nitrogen source on plant growth, organic nitrogen and inorganic nitrogen contents, and the rates of incorporation into nitrogenous compounds were studied. The observed differences were explained with reference to the effects of the various nitrogen sources on the physiology of the plants. The experimental techniques included assays of the enzymes nitrate reductase and glutamine synthetase, whole plant growth studies, and the analysis of nitrogenous compounds of xylem exudate and those extracted from the leaf blade, leaf base, and root regions of maize plants after feeding with a nutrient solution containing nitrogen as 15 N

  12. Mechanistic studies with solubilized rat liver steroid 5 alpha-reductase: Elucidation of the kinetic mechanism

    International Nuclear Information System (INIS)

    Levy, M.A.; Brandt, M.; Greway, A.T.

    1990-01-01

    A solubilized preparation of steroid 5 alpha-reductase from rat liver has been used in studies focused toward an understanding of the kinetic mechanism associated with enzyme catalysis. From the results of analyses with product and dead-end inhibitors, a preferentially ordered binding of substrates and release of products from the surface of the enzyme is proposed. The observations from these experiments were identical with those using the steroid 5 alpha-reductase activity associated with rat liver microsomes. The primary isotope effects on steady-state kinetic parameters when [4S-2H]NADPH was used also were consistent with an ordered kinetic mechanism. Normal isotope effects were observed for all three kinetic parameters (Vm/Km for both testosterone and NADPH and Vm) at all substrate concentrations used experimentally. Upon extrapolation to infinite concentration of testosterone, the isotope effect on Vm/Km for NADPH approached unity, indicating that the nicotinamide dinucleotide phosphate is the first substrate binding to and the second product released from the enzyme. The isotope effects on Vm/Km for testosterone at infinite concentration of cofactor and on Vm were 3.8 +/- 0.5 and 3.3 +/- 0.4, respectively. Data from the pH profiles of these three steady-state parameters and the inhibition constants (1/Ki) of competitive inhibitors versus both substrates indicate that the binding of nicotinamide dinucleotide phosphate involves coordination of its anionic 2'-phosphate to a protonated enzyme-associated base with an apparent pK near 8.0. From these results, relative limits have been placed on several of the internal rate constants used to describe the ordered mechanism of the rat liver steroid 5 alpha-reductase

  13. Nitrate and ammonia as nitrogen sources for deep subsurface microorganisms

    Directory of Open Access Journals (Sweden)

    Heini eKutvonen

    2015-10-01

    Full Text Available We investigated the N-utilizing bacterial community in anoxic brackish groundwater of the low and intermediate level nuclear waste repository cave in Olkiluoto, Finland, at 100 m depth using 15N-based stable isotope probing (SIP and enrichment with 14/15N-ammonium or 14/15N-nitrate complemented with methane. 28 days of incubation at 12°C increased the concentration of bacterial 16S rRNA and nitrate reductase (narG gene copies in the substrate amended microcosms simultaneously with a radical drop in the overall bacterial diversity and OTU richness. Hydrogenophaga/Malikia were enriched in all substrate amended microcosms and Methylobacter in the ammonium and ammonium+methane supplemented microcosms. Sulfuricurvum was especially abundant in the nitrate+methane treatment and the unamended incubation control. Membrane-bound nitrate reductase genes (narG from Polarimonas sp. were detected in the original groundwater, while Burkholderia, Methylibium and Pseudomonas narG genes were enriched due to substrate supplements. Identified amoA genes belonged to Nitrosomonas sp. 15N-SIP revealed that Burkholderiales and Rhizobiales clades belonging to the minority groups in the original groundwater used 15N from ammonium and nitrate as N source indicating an important ecological function of these bacteria, despite their low number, in the groundwater N cycle in Olkiluoto bedrock system.

  14. 21 CFR 181.33 - Sodium nitrate and potassium nitrate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium nitrate and potassium nitrate. 181.33...-Sanctioned Food Ingredients § 181.33 Sodium nitrate and potassium nitrate. Sodium nitrate and potassium nitrate are subject to prior sanctions issued by the U.S. Department of Agriculture for use as sources of...

  15. Nitrate accumulation in spinach

    NARCIS (Netherlands)

    Steingröver, Eveliene Geertruda

    1986-01-01

    Leafy vegetables, like spinach, may contain high concentrations of nitrate. In the Netherlands, about 75% of mean daily intake of nitrate orginates from the consumption of vegatables. Hazards to human health are associated with the reduction of nitrate to nitrite. Acute nitrite poisoning causes

  16. Molecular Diagnosis of 5α-Reductase Type II Deficiency in Brazilian Siblings with 46,XY Disorder of Sex Development

    Directory of Open Access Journals (Sweden)

    Maricilda Palandi de Mello

    2011-12-01

    Full Text Available The steroid 5α-reductase type II enzyme catalyzes the conversion of testosterone (T to dihydrotestosterone (DHT, and its deficiency leads to undervirilization in 46,XY individuals, due to an impairment of this conversion in genital tissues. Molecular analysis in the steroid 5α-reductase type II gene (SRD5A2 was performed in two 46,XY female siblings. SRD5A2 gene sequencing revealed that the patients were homozygous for p.Gln126Arg missense mutation, which results from the CGA > CAA nucleotide substitution. The molecular result confirmed clinical diagnosis of 46,XY disorder of sex development (DSD for the older sister and directed the investigation to other family members. Studies on SRD5A2 protein structure showed severe changes at NADPH binding region indicating that structural modeling analysis can be useful to evaluate the deleterious role of a mutation as causing 5α-reductase type II enzyme deficiency.

  17. Interspecific variation for thermal dependence of glutathione reductase in sainfoin.

    Science.gov (United States)

    Kidambi, S P; Mahan, J R; Matches, A G

    1990-05-01

    Understanding the biochemical and physiological consequences of species variation would expedite improvement in agronomically useful genotypes of sainfoin (Onobrychis spp.) Information on variation among sainfoin species is lacking on thermal dependence of glutathione reductase (B.C. 1.6.4.2.), which plays an important role in the protection of plants from both high and low temperature stresses by preventing harmful oxidation of enzymes and membranes. Our objective was to investigate the interspecific variation for thermal dependency of glutathione reductase in sainfoin. Large variation among species was found for: (i) the minimum apparent Km (0.4-2.5 μM NADPH), (ii) the temperature at which the minimum apparent Km was observed (15°-5°C), and (iii) the thermal kinetic windows (2°-30°C width) over a 15°-45°C temperature gradient. In general, tetraploid species had narrower (≤17°C) thermal kinetic windows than did diploid species (∼30°C), with one exception among the diploids. Within the tetraploid species, the cultivars of O. viciifolia had a broader thermal kinetic window (≥7°C) than the plant introduction (PI 212241, >2 °C) itself.

  18. Modulation of NADPH oxidase activity by known uraemic retention solutes.

    Science.gov (United States)

    Schulz, Anna Marta; Terne, Cindy; Jankowski, Vera; Cohen, Gerald; Schaefer, Mandy; Boehringer, Falko; Tepel, Martin; Kunkel, Desiree; Zidek, Walter; Jankowski, Joachim

    2014-08-01

    Uraemia and cardiovascular disease appear to be associated with an increased oxidative burden. One of the key players in the genesis of reactive oxygen species (ROS) is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Based on initial experiments demonstrating a decreased inhibitory effect on NADPH oxidase activity in the presence of plasma from patients with CKD-5D after dialysis compared with before dialysis, we investigated the effect of 48 known and commercially available uraemic retention solutes on the enzymatic activity of NADPH oxidase. Mononuclear leucocytes isolated from buffy coats of healthy volunteers were isolated, lysed and incubated with NADH in the presence of plasma from healthy controls and patients with CKD-5D. Furthermore, the leucocytes were lysed and incubated in the presence of uraemic retention solute of interest and diphenyleneiodonium chloride (DPI), an inhibitor of NADPH oxidase. The effect on enzymatic activity of NADPH oxidase was quantified within an incubation time of 120 min. Thirty-nine of the 48 uraemic retention solutes tested had a significant decreasing effect on NADPH oxidase activity. Oxalate has been characterized as the strongest inhibitor of NADPH oxidase (90% of DPI inhibition). Surprisingly, none of the uraemic retention solutes we investigated was found to increase NADPH oxidase activity. Furthermore, plasma from patients with CKD-5D before dialysis caused significantly higher inhibitory effect on NADPH oxidase activity compared with plasma from healthy subjects. However, this effect was significantly decreased in plasma from patients with CKD-5D after dialysis. The results of this study show that uraemic retention solutes modulated the activity of the NADPH oxidase. The results of this study might be the basis for the development of inhibitors applicable as drug in the situation of increased oxidative stress. © 2014 Stichting European Society for Clinical Investigation Journal Foundation.

  19. Quantitative flux analysis reveals folate-dependent NADPH production

    Science.gov (United States)

    Fan, Jing; Ye, Jiangbin; Kamphorst, Jurre J.; Shlomi, Tomer; Thompson, Craig B.; Rabinowitz, Joshua D.

    2014-06-01

    ATP is the dominant energy source in animals for mechanical and electrical work (for example, muscle contraction or neuronal firing). For chemical work, there is an equally important role for NADPH, which powers redox defence and reductive biosynthesis. The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway, with malic enzyme sometimes also important. Although the relative contribution of glycolysis and oxidative phosphorylation to ATP production has been extensively analysed, similar analysis of NADPH metabolism has been lacking. Here we demonstrate the ability to directly track, by liquid chromatography-mass spectrometry, the passage of deuterium from labelled substrates into NADPH, and combine this approach with carbon labelling and mathematical modelling to measure NADPH fluxes. In proliferating cells, the largest contributor to cytosolic NADPH is the oxidative pentose phosphate pathway. Surprisingly, a nearly comparable contribution comes from serine-driven one-carbon metabolism, in which oxidation of methylene tetrahydrofolate to 10-formyl-tetrahydrofolate is coupled to reduction of NADP+ to NADPH. Moreover, tracing of mitochondrial one-carbon metabolism revealed complete oxidation of 10-formyl-tetrahydrofolate to make NADPH. As folate metabolism has not previously been considered an NADPH producer, confirmation of its functional significance was undertaken through knockdown of methylenetetrahydrofolate dehydrogenase (MTHFD) genes. Depletion of either the cytosolic or mitochondrial MTHFD isozyme resulted in decreased cellular NADPH/NADP+ and reduced/oxidized glutathione ratios (GSH/GSSG) and increased cell sensitivity to oxidative stress. Thus, although the importance of folate metabolism for proliferating cells has been long recognized and attributed to its function of producing one-carbon units for nucleic acid synthesis, another crucial function of this pathway is generating reducing power.

  20. Iron reductases from Pseudomonas aeruginosa.

    Science.gov (United States)

    Cox, C D

    1980-01-01

    Cell-free extracts of Pseudomonas aeruginosa contain enzyme activities which reduce Fe(III) to Fe(II) when iron is provided in certain chelates, but not when the iron is uncomplexed. Iron reductase activities for two substrates, ferripyochelin and ferric citrate, appear to be separate enzymes because of differences in heat stabilities, in locations in fractions of cell-free extracts, in reductant specificity, and in apparent sizes during gel filtration chromatography. Ferric citrate iron reductase is an extremely labile activity found in the cytoplasmic fraction, and ferripyochelin iron reductase is a more stable activity found in the periplasmic as well as cytoplasmic fraction of extracts. A small amount of activity detectable in the membrane fraction seemed to be loosely associated with the membranes. Although both enzymes have highest activity reduced nicotinamide adenine dinucleotide, reduced glutathione also worked with ferripyochelin iron reductase. In addition, oxygen caused an irreversible loss of a percentage of the ferripyochelin iron reductase following sparge of reaction mixtures, whereas the reductase for ferric citrate was not appreciably affected by oxygen. PMID:6766439

  1. NADPH Oxidases, Angiogenesis, and Peripheral Artery Disease

    Directory of Open Access Journals (Sweden)

    Pradeep Manuneedhi Cholan

    2017-07-01

    Full Text Available Peripheral artery disease (PAD is caused by narrowing of arteries in the limbs, normally occurring in the lower extremities, with severe cases resulting in amputation of the foot or leg. A potential approach for treatment is to stimulate the formation of new blood vessels to restore blood flow to limb tissues. This is a process called angiogenesis and involves the proliferation, migration, and differentiation of endothelial cells. Angiogenesis can be stimulated by reactive oxygen species (ROS, with NADPH oxidases (NOX being a major source of ROS in endothelial cells. This review summarizes the recent evidence implicating NOX isoforms in their ability to regulate angiogenesis in vascular endothelial cells in vitro, and in PAD in vivo. Increasing our understanding of the involvement of the NOX isoforms in promoting therapeutic angiogenesis may lead to new treatment options to slow or reverse PAD.

  2. Nitrate, NO and haemoglobin in plant adaptation to hypoxia: an alternative to classic fermentation pathways.

    Science.gov (United States)

    Igamberdiev, Abir U; Hill, Robert D

    2004-12-01

    The role of nitrate reduction to produce nitric oxide (NO) and its subsequent oxidation by oxyhaemoglobin as a mechanism to maintain plant cell energetics during hypoxia is examined. Nitrate reduction in hypoxic conditions can be considered as an alternative respiratory pathway, with nitrate as an intermediate electron acceptor, contributing to the oxidation of NADH. NO, produced in the reaction, does not accumulate due to the induction of hypoxia-induced (class 1) haemoglobins. These haemoglobins remain in the oxyhaemoglobin form, even at oxygen tensions two orders of magnitude lower than necessary to saturate cytochrome c oxidase. They act, probably in conjunction with a flavoprotein, as NO dioxygenases converting NO back to nitrate, consuming NAD(P)H in the process. The overall system oxidizes 2.5 moles of NADH per one mole of nitrate recycled during the reaction, leading to the maintenance of redox and energy status during hypoxia and resulting in the reduced production of ethanol and lactic acid.

  3. A new cotton SDR family gene encodes a polypeptide possessing aldehyde reductase and 3-ketoacyl-CoA reductase activities.

    Science.gov (United States)

    Pang, Yu; Song, Wen-Qiang; Chen, Fang-Yuan; Qin, Yong-Mei

    2010-03-01

    To understand regulatory mechanisms of cotton fiber development, microarray analysis has been performed for upland cotton (Gossypium hirsutum). Based on this, a cDNA (GhKCR3) encoding a polypeptide belonging to short-chain alcohol dehydrogenase/reductase family was isolated and cloned. It contains an open reading frame of 987 bp encoding a polypeptide of 328 amino acid residues. Following its overexpression in bacterial cells, the purified recombinant protein specifically uses NADPH to reduce a variety of short-chain aldehydes. A fragment between Gly180 and Gly191 was found to be essential for its catalytic activity. Though the GhKCR3 gene shares low sequence similarities to the ortholog of Saccharomyces cerevisiae YBR159w that encodes 3-ketoacyl-CoA reductase (KCR) catalyzing the second step of fatty acid elongation, it was surprisingly able to complement the yeast ybr159wDelta mutant. Gas chromatography-mass spectrometry analysis showed that very long-chain fatty acids, especially C26:0, were produced in the ybr159wDelta mutant cells expressing GhKCR3. Applying palmitoyl-CoA and malonyl-CoA as substrates, GhKCR3 showed KCR activity in vitro. Quantitative real time-PCR analysis indicated GhKCR3 transcripts accumulated in rapidly elongating fibers, roots, and stems. Our results suggest that GhKCR3 is probably a novel KCR contributing to very long-chain fatty acid biosynthesis in plants.

  4. Molecular cloning and biochemical characterization of a novel erythrose reductase from Candida magnoliae JH110

    Directory of Open Access Journals (Sweden)

    Ryu Yeon-Woo

    2010-06-01

    Full Text Available Abstract Background Erythrose reductase (ER catalyzes the final step of erythritol production, which is reducing erythrose to erythritol using NAD(PH as a cofactor. ER has gained interest because of its importance in the production of erythritol, which has extremely low digestibility and approved safety for diabetics. Although ERs were purified and characterized from microbial sources, the entire primary structure and the corresponding DNA for ER still remain unknown in most of erythritol-producing yeasts. Candida magnoliae JH110 isolated from honeycombs produces a significant amount of erythritol, suggesting the presence of erythrose metabolizing enzymes. Here we provide the genetic sequence and functional characteristics of a novel NADPH-dependent ER from C. magnoliae JH110. Results The gene encoding a novel ER was isolated from an osmophilic yeast C. magnoliae JH110. The ER gene composed of 849 nucleotides encodes a polypeptide with a calculated molecular mass of 31.4 kDa. The deduced amino acid sequence of ER showed a high degree of similarity to other members of the aldo-keto reductase superfamily including three ER isozymes from Trichosporonoides megachiliensis SNG-42. The intact coding region of ER from C. magnoliae JH110 was cloned, functionally expressed in Escherichia coli using a combined approach of gene fusion and molecular chaperone co-expression, and subsequently purified to homogeneity. The enzyme displayed a temperature and pH optimum at 42°C and 5.5, respectively. Among various aldoses, the C. magnoliae JH110 ER showed high specific activity for reduction of erythrose to the corresponding alcohol, erythritol. To explore the molecular basis of the catalysis of erythrose reduction with NADPH, homology structural modeling was performed. The result suggested that NADPH binding partners are completely conserved in the C. magnoliae JH110 ER. Furthermore, NADPH interacts with the side chains Lys252, Thr255, and Arg258, which could

  5. Nox NADPH oxidases and the endoplasmic reticulum.

    Science.gov (United States)

    Laurindo, Francisco R M; Araujo, Thaís L S; Abrahão, Thalita B

    2014-06-10

    Understanding isoform- and context-specific subcellular Nox reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase compartmentalization allows relevant functional inferences. This review addresses the interplay between Nox NADPH oxidases and the endoplasmic reticulum (ER), an increasingly evident player in redox pathophysiology given its role in redox protein folding and stress responses. Catalytic/regulatory transmembrane subunits are synthesized in the ER and their processing includes folding, N-glycosylation, heme insertion, p22phox heterodimerization, as shown for phagocyte Nox2. Dual oxidase (Duox) maturation also involves the regulation by ER-resident Duoxa2. The ER is the activation site for some isoforms, typically Nox4, but potentially other isoforms. Such location influences redox/Nox-mediated calcium signaling regulation via ER targets, such as sarcoendoplasmic reticulum calcium ATPase (SERCA). Growing evidence suggests that Noxes are integral signaling elements of the unfolded protein response during ER stress, with Nox4 playing a dual prosurvival/proapoptotic role in this setting, whereas Nox2 enhances proapoptotic signaling. ER chaperones such as protein disulfide isomerase (PDI) closely interact with Noxes. PDI supports growth factor-dependent Nox1 activation and mRNA expression, as well as migration in smooth muscle cells, and PDI overexpression induces acute spontaneous Nox activation. Mechanisms of PDI effects include possible support of complex formation and RhoGTPase activation. In phagocytes, PDI supports phagocytosis, Nox activation, and redox-dependent interactions with p47phox. Together, the results implicate PDI as possible Nox organizer. We propose that convergence between Noxes and ER may have evolutive roots given ER-related functional contexts, which paved Nox evolution, namely calcium signaling and pathogen killing. Overall, the interplay between Noxes and the ER may provide relevant insights in Nox-related (patho)physiology.

  6. The structure of Lactococcus lactis thioredoxin reductase reveals molecular features of photo-oxidative damage

    DEFF Research Database (Denmark)

    Skjoldager, Nicklas; Bang, Maria Blanner; Rykær, Martin

    2017-01-01

    The NADPH-dependent homodimeric flavoenzyme thioredoxin reductase (TrxR) provides reducing equivalents to thioredoxin, a key regulator of various cellular redox processes. Crystal structures of photo-inactivated thioredoxin reductase (TrxR) from the Gram-positive bacterium Lactococcus lactis have...... been determined. These structures reveal novel molecular features that provide further insight into the mechanisms behind the sensitivity of this enzyme toward visible light. We propose that a pocket on the si-face of the isoalloxazine ring accommodates oxygen that reacts with photo-excited FAD...... thus be a widespread feature among bacterial TrxR with the described characteristics, which affords applications in clinical photo-therapy of drug-resistant bacteria....

  7. Acute Ethanol Intake Induces NAD(PH Oxidase Activation and Rhoa Translocation in Resistance Arteries

    Directory of Open Access Journals (Sweden)

    Janaina A. Simplicio

    Full Text Available Abstract Background: The mechanism underlying the vascular dysfunction induced by ethanol is not totally understood. Identification of biochemical/molecular mechanisms that could explain such effects is warranted. Objective: To investigate whether acute ethanol intake activates the vascular RhoA/Rho kinase pathway in resistance arteries and the role of NAD(PH oxidase-derived reactive oxygen species (ROS on such response. We also evaluated the requirement of p47phox translocation for ethanol-induced NAD(PH oxidase activation. Methods: Male Wistar rats were orally treated with ethanol (1g/kg, p.o. gavage or water (control. Some rats were treated with vitamin C (250 mg/kg, p.o. gavage, 5 days before administration of water or ethanol. The mesenteric arterial bed (MAB was collected 30 min after ethanol administration. Results: Vitamin C prevented ethanol-induced increase in superoxide anion (O2- generation and lipoperoxidation in the MAB. Catalase and superoxide dismutase activities and the reduced glutathione, nitrate and hydrogen peroxide (H2O2 levels were not affected by ethanol. Vitamin C and 4-methylpyrazole prevented the increase on O2- generation induced by ethanol in cultured MAB vascular smooth muscle cells. Ethanol had no effect on phosphorylation levels of protein kinase B (Akt and eNOS (Ser1177 or Thr495 residues or MAB vascular reactivity. Vitamin C prevented ethanol-induced increase in the membrane: cytosol fraction ratio of p47phox and RhoA expression in the rat MAB. Conclusion: Acute ethanol intake induces activation of the RhoA/Rho kinase pathway by a mechanism that involves ROS generation. In resistance arteries, ethanol activates NAD(PH oxidase by inducing p47phox translocation by a redox-sensitive mechanism.

  8. Ammonium nitrate explosion hazards

    Directory of Open Access Journals (Sweden)

    Negovanović Milanka

    2015-01-01

    Full Text Available Ammonium nitrate (AN primarily is used as a fertilizer but it is also very important compound in the production of industrial explosives. The application of ammonium nitrate in the production of industrial explosives was related with the early era of Nobel dynamite and widely increased with the appearance of blasting agents such as ANFO and Slurry, in the middle of the last Century. Throughout the world millions of tons of ammonium nitrate are produced annually and handled without incident. Although ammonium nitrate generally is used safely, accidental explosions involving AN have high impact resulting in loss of lives and destruction of property. The paper presents the basic properties of ammonium nitrate as well as hazards in handling of ammonium nitrate in order to prevent accidents. Several accidents with explosions of ammonium nitrate resulted in catastrophic consequences are listed in the paper as examples of non-compliance with prescribed procedures.

  9. NADPH-generating systems in bacteria and archaea

    Science.gov (United States)

    Spaans, Sebastiaan K.; Weusthuis, Ruud A.; van der Oost, John; Kengen, Servé W. M.

    2015-01-01

    Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an essential electron donor in all organisms. It provides the reducing power that drives numerous anabolic reactions, including those responsible for the biosynthesis of all major cell components and many products in biotechnology. The efficient synthesis of many of these products, however, is limited by the rate of NADPH regeneration. Hence, a thorough understanding of the reactions involved in the generation of NADPH is required to increase its turnover through rational strain improvement. Traditionally, the main engineering targets for increasing NADPH availability have included the dehydrogenase reactions of the oxidative pentose phosphate pathway and the isocitrate dehydrogenase step of the tricarboxylic acid (TCA) cycle. However, the importance of alternative NADPH-generating reactions has recently become evident. In the current review, the major canonical and non-canonical reactions involved in the production and regeneration of NADPH in prokaryotes are described, and their key enzymes are discussed. In addition, an overview of how different enzymes have been applied to increase NADPH availability and thereby enhance productivity is provided. PMID:26284036

  10. NADPH-generating systems in bacteria and archaea

    Directory of Open Access Journals (Sweden)

    Sebastiaan K. Spaans

    2015-07-01

    Full Text Available Reduced nicotinamide adenine dinucleotide phosphate (NADPH is an essential electron donor in all organisms. It provides the reducing power that drives numerous anabolic reactions, including those responsible for the biosynthesis of all major cell components and many products in biotechnology. The efficient synthesis of many of these products, however, is limited by the rate of NADPH regeneration. Hence, a thorough understanding of the reactions involved in the generation of NADPH is required to increase its turnover through rational strain improvement. Traditionally, the main engineering targets for increasing NADPH availability have included the dehydrogenase reactions of the oxidative pentose phosphate pathway and the isocitrate dehydrogenase step of the tricarboxylic acid (TCA cycle. However, the importance of alternative NADPH-generating reactions has recently become evident. In the current review, the major canonical and non-canonical reactions involved in the production and regeneration of NADPH in prokaryotes are described, and their key enzymes are discussed. In addition, an overview of how different enzymes have been applied to increase NADPH availability and thereby enhance productivity is provided.

  11. Modulation of nitrate-nitrite conversion in the oral cavity.

    Science.gov (United States)

    van Maanen, J M; van Geel, A A; Kleinjans, J C

    1996-01-01

    The formation of nitrite from ingested nitrate can give rise to the induction of methemoglobinemia and endogenous nitrosation resulting in the formation of carcinogenic N-nitroso compounds. We investigated the possibility of modulation of the conversion of nitrate into nitrite in the oral cavity in order to seek ways of reducing the formation of the deleterious nitrite. We investigated the effectiveness of several mouthwash solutions with antibacterial constituents on the reduction of nitrate into nitrite in the oral cavity. In 15 studied subjects, the mean percentage of salivary nitrate reduced to nitrite after ingestion of 235 mg (3.8 mmol) nitrate was found to be 16.1 +/- 6.2%. The use of an antiseptic mouthwash with active antibacterial constituent chlorhexidine resulted in an almost complete decrease of the mean percentage of reduced nitrate, to 0.9 +/- 0.8%. Mouthwash solutions with antibacterial component triclosan or antimicrobial enzymes amyloglucosidase and glucose oxidase did not affect the reduction of nitrate into nitrite. A toothpaste with active components triclosan and zinc citrate with synergistic antiplaque activity was also without effect. Use of a pH-regulating chewing gum resulted in a rise in the pH in the oral cavity from 6.8 to 7.3. At 30 min after nitrate ingestion, this rise was accompanied by a significant increase in the salivary nitrite concentration, which might be explained by the pH being close to the optimal pH for nitrate reductase of 8. In conclusion, a limited number of possibilities of modulation of the conversion of nitrate into nitrite in the oral cavity are available.

  12. Nitrate Sensing and Metabolism Modulate Motility, Biofilm Formation, and Virulence in Pseudomonas aeruginosa▿

    Science.gov (United States)

    Van Alst, Nadine E.; Picardo, Kristin F.; Iglewski, Barbara H.; Haidaris, Constantine G.

    2007-01-01

    Infection by the bacterial opportunist Pseudomonas aeruginosa frequently assumes the form of a biofilm, requiring motility for biofilm formation and dispersal and an ability to grow in nutrient- and oxygen-limited environments. Anaerobic growth by P. aeruginosa is accomplished through the denitrification enzyme pathway that catalyzes the sequential reduction of nitrate to nitrogen gas. Mutants mutated in the two-component nitrate sensor-response regulator and in membrane nitrate reductase displayed altered motility and biofilm formation compared to wild-type P. aeruginosa PAO1. Analysis of additional nitrate dissimilation mutants demonstrated a second level of regulation in P. aeruginosa motility that is independent of nitrate sensor-response regulator function and is associated with nitric oxide production. Because motility and biofilm formation are important for P. aeruginosa pathogenicity, we examined the virulence of selected regulatory and structural gene mutants in the surrogate model host Caenorhabditis elegans. Interestingly, the membrane nitrate reductase mutant was avirulent in C. elegans, while nitrate sensor-response regulator mutants were fully virulent. The data demonstrate that nitrate sensing, response regulation, and metabolism are linked directly to factors important in P. aeruginosa pathogenesis. PMID:17526746

  13. Isolated menthone reductase and nucleic acid molecules encoding same

    Science.gov (United States)

    Croteau, Rodney B; Davis, Edward M; Ringer, Kerry L

    2013-04-23

    The present invention provides isolated menthone reductase proteins, isolated nucleic acid molecules encoding menthone reductase proteins, methods for expressing and isolating menthone reductase proteins, and transgenic plants expressing elevated levels of menthone reductase protein.

  14. Diversity of Nitrate-Reducing and Denitrifying Bacteria in a Marine Aquaculture Biofilter and their Response to Sulfide

    DEFF Research Database (Denmark)

    Krieger, Bärbel; Schwermer, Carsten U.; Rezakhani, Nastaran

    2006-01-01

    was developed containing a 3-stage biofilter for nitrification, denitrification/anaerobic sludge digestion, and sulfide oxidation. Sulfate reduction in the anaerobic part of the system leads to sulfide concentrations exceeding 5 mM, which may affect nitrate reduction and denitrification. Sulfide can inhibit...... nitrous oxide reductase, trigger a shift from denitrification to dissimilatory nitrate reduction to ammonium (DNRA), or be used as electron donor for nitrate reduction. The goal of this study was to identify and isolate nitrate-reducing and denitrifying bacteria from the biofilter and to investigate...

  15. Biomass accumulation, photochemical efficiency of photosystem II, nutrient contents and nitrate reductase activity in young rosewood plants (Aniba rosaeodora Ducke submitted to different NO3-:NH4+ ratios Acúmulo de biomassa, eficiência fotoquímica do fotossistema II, conteúdo de nutrientes e atividade da redutase do nitrato em plantas jovens de pau-rosa (Aniba rosaeodora Ducke submetidas a diferentes relações NO3-:NH4+

    Directory of Open Access Journals (Sweden)

    Denize Caranhas de Sousa Barreto

    2007-01-01

    Full Text Available The rosewood (Aniba rosaeodora Ducke is a native tree species of Amazon rainforest growing naturally in acidic forest soils with reduced redox potential. However, this species can also been found growing in forest gaps containing oxide soils. Variations in the forms of mineral nitrogen (NO3- or NH4+ may be predicted in these different edaphic conditions. Considering that possibility, an experiment was carried out to analyze the effects of different NO3-:NH4+ ratios on the growth performance, mineral composition, chloroplastid pigment contents, photochemical efficiency photosystem II (PSII, and nitrate redutase activity (RN, E.C.1.6.6.1 on A. rosaeodora seedlings. Nine-month-old seedlings were grown in pots with a washed sand capacity of 7.5 kg and submitted to different NO3-:NH4+ ratios (T1 = 0:100%, T2 = 25:75%, T3 = 50:50%, T4 = 75:25%, and T5 = 100:0%. The lowest relative growth rate was observed when the NO3-:NH4+ ratio was equal to 0:100%. In general, high concentrations of NO3- rather than NH4+ favored a greater nutrient accumulation in different parts of the plant. For the chloroplastid pigment, the highest Chl a, Chl b, Chl tot, Chl a/b and Chl tot/Cx+c contents were found in the treatment with 75:25% of NO3-:NH4+, and for Chl b and Cx+c it was observed no difference. In addition, there was a higher photochemical efficiency of PSII (Fv/Fm when high NO3- concentrations were used. A linear and positive response for the nitrate reductase activity was recorded when the nitrate content increased on the culture substrate. Our results suggest that A. rosaeodora seedlings have a better growth performance when the NO3- concentrations in the culture substrate were higher than the NH4+ concentrations.O pau-rosa (Aniba rosaeodora Ducke habita, naturalmente, solos florestais ácidos com potencial redox reduzido. No entanto, estas espécies têm sido encontradas também em clareiras que, teoricamente, apresentam solos mais oxidados. Nestas diferentes

  16. The unique glutathione reductase from Xanthomonas campestris: Gene expression and enzyme characterization

    International Nuclear Information System (INIS)

    Loprasert, Suvit; Whangsuk, Wirongrong; Sallabhan, Ratiboot; Mongkolsuk, Skorn

    2005-01-01

    The glutathione reductase gene, gor, was cloned from the plant pathogen Xanthomonas campestris pv. phaseoli. Its gene expression and enzyme characteristics were found to be different from those of previously studied homologues. Northern blot hybridization, promoter-lacZ fusion, and enzyme assay experiments revealed that its expression, unlike in Escherichia coli, is OxyR-independent and constitutive upon oxidative stress conditions. The deduced amino acid sequence shows a unique NADPH binding motif where the most highly conserved arginine residue, which is critical for NADPH binding, is replaced by glutamine. Interestingly, a search of the available Gor amino acid sequences from various sources, including other Xanthomonas species, revealed that this replacement is specific to the genus Xanthomonas. Recombinant Gor enzyme was purified and characterized, and was found to have a novel ability to use both, NADPH and NADH, as electron donor. A gor knockout mutant was constructed and shown to have increased expression of the organic peroxide-inducible regulator gene, ohrR

  17. Characterization of recombinant glyoxylate reductase from thermophile Thermus thermophilus HB27.

    Science.gov (United States)

    Ogino, Hiroyasu; Nakayama, Hitoshi; China, Hideyasu; Kawata, Takuya; Doukyu, Noriyuki; Yasuda, Masahiro

    2008-01-01

    A glyoxylate reductase gene from the thermophilic bacterium Thermus thermophilus HB27 (TthGR) was cloned and expressed in Escherichia coli cells. The recombinant enzyme was highly purified to homogeneity and characterized. The purified TthGR showed thermostability up to 70 degrees C. In contrast, the maximum reaction condition was relatively mild (45 degrees C and pH 6.7). Although the kcat values against co-enzyme NADH and NADPH were similar, the Km value against co-enzyme NADH was approximately 18 times higher than that against NADPH. TthGR prefers NADPH rather than NADH as an electron donor. These results indicate that a phosphate group of a co-enzyme affects the binding affinity rather than the reaction efficiency, and TthGR demands appropriate amount of phosphate for a high activity. Furthermore, it was found that the half-lives of TthGR in the presence of 25% dimethyl sulfoxide and diethylene glycol were significantly longer than that in the absence of an organic solvent.

  18. Potential testicular toxicity of sodium nitrate in adult rats.

    Science.gov (United States)

    Aly, Hamdy A A; Mansour, Ahmed M; Abo-Salem, Osama M; Abd-Ellah, Hala F; Abdel-Naim, Ashraf B

    2010-02-01

    Nitrate is a common contaminant in groundwater aquifers. Current study aimed at evaluating the potential testicular toxicity of sodium nitrate in rats. Sodium nitrate was given orally to rats at doses of 50, 100 or 200 mg/kg/day for 60 consecutive days. Sperm count and motility, daily sperm production and testis weight were significantly decreased specially at high doses. Testicular activity of lactate dehydrogenase-X, glucose-6-phosphate dehydrogenase, and acid phosphatase were inhibited in a dose-related manner. Lipid peroxides and hydrogen peroxide production were significantly increased in all treated animals. This was accompanied by inhibition of testicular activities of superoxide dismutase and glutathione peroxidase. Fifty mg/kg of sodium nitrate did not significantly alter catalase or glutathione reductase activity. Glutathione was significantly decreased by sodium nitrate in a dose-related manner. The decrease in sperm count and motility and daily sperm production was confirmed by histopathological studies which indicated chromatolysis, pyknosis and necrosis in spermatocytes. In conclusion, subchronic exposure of rats to sodium nitrate results in testicular toxicity as evidenced by decreased sperm count and motility, daily sperm production and testis weight, inhibited activity of enzyme markers of spermatogenesis and induction of histopathological changes. These effects are attributed, at least partly, to testicular oxidative stress. Copyright 2009. Published by Elsevier Ltd.

  19. Methylenetetrahydrofolate reductase (MTHFR) gene polymorphism ...

    African Journals Online (AJOL)

    Administrator

    2011-10-19

    Oct 19, 2011 ... Polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) gene are associated with abortion, early embryo loss and recurrent spontaneous abortion in human. However, information on the association between MTHFR polymorphism and cow abortion is scarce. In the present study, the effects.

  20. Association between methylenetetrahydrofolate reductase (MTHFR ...

    African Journals Online (AJOL)

    Association between methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism and risk of ischemic stroke in North Indian population: A hospital based case–control study. Amit Kumar, Shubham Misra, Anjali Hazarika, Pradeep Kumar, Ram Sagar, Abhishek Pathak, Kamalesh Chakravarty, Kameshwar ...

  1. Methylenetetrahydrofolate reductase (MTHFR) gene polymorphism ...

    African Journals Online (AJOL)

    Polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) gene are associated with abortion, early embryo loss and recurrent spontaneous abortion in human. However, information on the association between MTHFR polymorphism and cow abortion is scarce. In the present study, the effects of MTHFR ...

  2. Trametes versicolor carboxylate reductase uncovered.

    Science.gov (United States)

    Winkler, Margit; Winkler, Christoph K

    The first carboxylate reductase from Trametes versicolor was identified, cloned, and expressed in Escherichia coli . The enzyme reduces aromatic acids such as benzoic acid and derivatives, cinnamic acid, and 3-phenylpropanoic acid, but also aliphatic acids such as octanoic acid are reduced.

  3. Rhodobacter capsulatus gains a competitive advantage from respiratory nitrate reduction during light-dark transitions.

    Science.gov (United States)

    Ellington, M J K; Richardson, D J; Ferguson, S J

    2003-04-01

    Rhodobacter capsulatus N22DNAR(+) possesses a periplasmic nitrate reductase and is capable of reducing nitrate to nitrite under anaerobic conditions. In the absence of light this ability cannot support chemoheterotrophic growth in batch cultures. This study investigated the effect of nitrate reduction on the growth of R. capsulatus N22DNAR(+) during multiple light-dark cycles of anaerobic photoheterotrophic/dark chemoheterotrophic growth conditions in carbon-limited continuous cultures. The reduction of nitrate did not affect the photoheterotrophic growth yield of R. capsulatus N22DNAR(+). After a transition from photoheterotrophic to dark chemoheterotrophic growth conditions, the reduction of nitrate slowed the initial washout of a R. capsulatus N22DNAR(+) culture. Towards the end of a period of darkness nitrate-reducing cultures maintained higher viable cell counts than non-nitrate-reducing cultures. During light-dark cycling of a mixed culture, the strain able to reduce nitrate (N22DNAR(+)) outcompeted the strain which was unable to reduce nitrate (N22). The evidence indicates that the periplasmic nitrate reductase activity supports slow growth that retards the washout of a culture during anaerobic chemoheterotrophic conditions, and provides a protonmotive force for cell maintenance during the dark period before reillumination. This translates into a selective advantage during repeated light-dark cycles, such that in mixed culture N22DNAR(+) outcompetes N22. Exposure to light-dark cycles will be a common feature for R. capsulatus in its natural habitats, and this study shows that nitrate respiration may provide a selective advantage under such conditions.

  4. Nitrite Reductase NirS Is Required for Type III Secretion System Expression and Virulence in the Human Monocyte Cell Line THP-1 by Pseudomonas aeruginosa▿

    Science.gov (United States)

    Van Alst, Nadine E.; Wellington, Melanie; Clark, Virginia L.; Haidaris, Constantine G.; Iglewski, Barbara H.

    2009-01-01

    The nitrate dissimilation pathway is important for anaerobic growth in Pseudomonas aeruginosa. In addition, this pathway contributes to P. aeruginosa virulence by using the nematode Caenorhabditis elegans as a model host, as well as biofilm formation and motility. We used a set of nitrate dissimilation pathway mutants to evaluate the virulence of P. aeruginosa PA14 in a model of P. aeruginosa-phagocyte interaction by using the human monocytic cell line THP-1. Both membrane nitrate reductase and nitrite reductase enzyme complexes were important for cytotoxicity during the interaction of P. aeruginosa PA14 with THP-1 cells. Furthermore, deletion mutations in genes encoding membrane nitrate reductase (ΔnarGH) and nitrite reductase (ΔnirS) produced defects in the expression of type III secretion system (T3SS) components, extracellular protease, and elastase. Interestingly, exotoxin A expression was unaffected in these mutants. Addition of exogenous nitric oxide (NO)-generating compounds to ΔnirS mutant cultures restored the production of T3SS phospholipase ExoU, whereas nitrite addition had no effect. These data suggest that NO generated via nitrite reductase NirS contributes to the regulation of expression of selected virulence factors in P. aeruginosa PA14. PMID:19651860

  5. Nitrate leaching index

    Science.gov (United States)

    The Nitrate Leaching Index is a rapid assessment tool that evaluates nitrate (NO3) leaching potential based on basic soil and climate information. It is the basis for many nutrient management planning efforts, but it has considerable limitations because of : 1) an oversimplification of the processes...

  6. Agricultural nitrate pollution

    DEFF Research Database (Denmark)

    Anker, Helle Tegner

    2015-01-01

    Despite the passing of almost 25 years since the adoption of the EU Nitrates Directive, agricultural nitrate pollution remains a major concern in most EU Member States. This is also the case in Denmark, although a fairly strict regulatory regime has resulted in almost a 50 per cent reduction...

  7. Sistema NADPH oxidasa: nuevos retos y perspectivas = NADPH oxidase system: new challenges and perspectives

    Directory of Open Access Journals (Sweden)

    Arango Rincón, Julián Camilo

    2010-12-01

    Full Text Available El sistema NADPH oxidasa es un complejo multiproteico encargado de producir especies reactivas del oxígeno (ROS, por reactive oxygen species en diferentes células y tejidos. Es de gran importancia en las células fagocíticas (principalmente neutrófilos y macrófagos porque participa en la destrucción de microorganismos patógenos, mediante la fagocitosis y la formación de las trampas extracelulares de neutrófilos (NET, por neutrophils extracelular traps, así como en la activación de procesos inflamatorios. Las alteraciones en la producción de ROS por parte de las células fagocíticas a causa de defectos genéticos en los componentes del sistema generan la inmunodeficiencia primaria denominada enfermedad granulomatosa crónica (EGC. Este es un artículo de revisión sobre los componentes del sistema NADPH oxidasa, su distribución celular, mecanismo de activación y acción, así como de las funciones que desempeña en otros tejidos. Además, se revisan los defectos moleculares que llevan a la EGC y el tratamiento de esta, incluyendo la terapia con IFNγ, y finalmente las perspectivas para el estudio del sistema.

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

    Science.gov (United States)

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

    2017-08-16

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

  9. Initial kinetics of 15N-nitrate labelling of root and shoot N fractions of barley cultured at different relative addition rates of nitrate-N

    International Nuclear Information System (INIS)

    Agrell, D.; Larsson, C.M.; Larsson, M.; MacKown, C.T.; Rufty, T.W.

    1997-01-01

    The reduction of absorbed nitrate in the root and N transport to the shoot were studied in young barley (Hordeum vulgare L.) plants growing at low external nitrate levels. Plants were grown at three relative addition rates (RA) of nitrate: 0.04, 0.09, and 0.14 d -1 , which represent different degrees of growth limiting nitrate supply. Root nitrate reduction and N transport in situ were estimated using 15 N labelled nitrate exposures ranging from 5 to 60 min. With increasing RA, nitrate uptake in absolute terms increased, but the proportion of absorbed 15 N-nitrate that was reduced in the root decreased markedly. After 10-20 min of exposure to the label, 75, 49 and 27 % of the 15 N taken up was recovered as reduced 15 N in the root at RAs 0.04, 0.09, and 0.14 d -1 , respectively. The response pattern was supported by root nitrate reductase activities and xylem sap nitrate measurements. The decreasing proportion of reduced 15 N in the root with higher nitrate supply rates was matched by a relative increase in 15 N-nitrate storage and, to some extent, an increase in N transport to the shoot. Although small amounts of 15 N were rapidly transported to the shoot, the accumulation of label in the shoot at the end of the 60 min period remained a relatively small proportion of total 15 N uptake, which indicates delayed movement of 15 N out of the root. The results clearly indicate that differing degrees of adjustment occur in important nitrate assimilation processes throughout the N deficiency range. (author)

  10. Efficient one-step production of (S-1-phenyl-1,2-ethanediol from (R-enantiomer plus NAD+–NADPH in-situ regeneration using engineered Escherichia coli

    Directory of Open Access Journals (Sweden)

    Zhang Rongzhen

    2012-12-01

    Full Text Available Abstract Background Candida parapsilosis CCTCC M203011 catalyzes the stereoinversion of (R-1-phenyl-1,2-ethanediol (PED through oxidation and reduction. Its NAD+-linked (R-carbonyl reductase (RCR catalyzes the oxidization of (R-PED to 2-hydroxyacetophenone (HAP, and its NADPH-dependent (S-carbonyl reductase (SCR catalyzes the reduction of HAP to (S-PED. The reactions require NAD+ and NADPH as cofactors. However, even if NAD+ and NADPH are added, the biotransformation of (S-PED from the (R-enantiomer by an Escherichia coli strain co-expressing RCR and SCR is slow and gives low yields, probably as a result of insufficient or imbalanced redox cofactors. To prepare (S-PED from the (R-enantiomer in one-step efficiently, plus redox cofactor regeneration, we introduced pyridine nucleotide transhydrogenases (PNTs from E. coli to the metabolic pathway of (S-PED. Results The PNTs were successfully introduced into the E. coli strain RSAB. Most of the PNT activities occurred in the cell membrane of E. coli. The introduction of PNTs increased intracellular NAD+ and NADH concentrations and decreased the NADPH pool without affecting the total nucleotide concentration and cell growth properties. The presence of PNTs increased the NADH/NAD+ ratio slightly and reduced the NADPH/NADP+ ratio about two-fold; the ratio of NADPH/NADP+ to NADH/NAD+ was reduced from 36 to 17. So, the PNTs rebalanced the cofactor pathways: the rate of RCR was increased, while the rate of SCR was decreased. When the ratio of NAD+/NADPH was 3.0 or higher, the RSAB strain produced (S-PED with the highest optical purity, 97.4%, and a yield of 95.2% at 6 h. The introduction of PNTs stimulated increases of 51.5% and 80.6%, respectively, in optical purity and yield, and simultaneously reduced the reaction time seven-fold. Conclusions In this work, PNTs were introduced into E. coli to rebalance the cofactor pools within the engineered (S-PED pathways. The efficient one-step production of (S

  11. Seasonal variation in denitrification and dissimilatory nitrate reduction to ammonia process rates and corresponding key functional genes along an estuarine nitrate gradient

    Science.gov (United States)

    Smith, Cindy J.; Dong, Liang F.; Wilson, John; Stott, Andrew; Osborn, A. Mark; Nedwell, David B.

    2015-01-01

    This research investigated spatial-temporal variation in benthic bacterial community structure, rates of denitrification and dissimilatory nitrate reduction to ammonium (DNRA) processes and abundances of corresponding genes and transcripts at three sites—the estuary-head, mid-estuary and the estuary mouth (EM) along the nitrate gradient of the Colne estuary over an annual cycle. Denitrification rates declined down the estuary, while DNRA rates were higher at the estuary head and middle than the EM. In four out of the six 2-monthly time-points, rates of DNRA were greater than denitrification at each site. Abundance of gene markers for nitrate-reduction (nitrate reductase narG and napA), denitrification (nitrite reductase nirS) and DNRA (DNRA nitrite reductase nrfA) declined along the estuary with significant relationships between denitrification and nirS abundance, and DNRA and nrfA abundance. Spatially, rates of denitrification, DNRA and corresponding functional gene abundances decreased along the estuary. However, temporal correlations between rate processes and functional gene and transcript abundances were not observed. PMID:26082763

  12. NADPH oxidase-dependent H2O2 production is required for salt-induced antioxidant defense in Arabidopsis thaliana.

    Science.gov (United States)

    Ben Rejeb, Kilani; Benzarti, Maâli; Debez, Ahmed; Bailly, Christophe; Savouré, Arnould; Abdelly, Chedly

    2015-02-01

    The involvement of hydrogen peroxide (H2O2) generated by nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) in the antioxidant defense system was assessed in salt-challenged Arabidopsis thaliana seedlings. In the wild-type, short-term salt exposure led to a transient and significant increase of H2O2 concentration, followed by a marked increase in catalase (CAT, EC 1.11.16), ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) activities. Pre-treatment with either a chemical trap for H2O2 (dimethylthiourea) or two widely used NADPH oxidase inhibitors (imidazol and diphenylene iodonium) significantly decreased the above-mentioned enzyme activities under salinity. Double mutant atrbohd/f plants failed to induce the antioxidant response under the culture conditions. Under long-term salinity, the wild-type was more salt-tolerant than the mutant based on the plant biomass production. The better performance of the wild-type was related to a significantly higher photosynthetic activity, a more efficient K(+) selective uptake, and to the plants' ability to deal with the salt-induced oxidative stress as compared to atrbohd/f. Altogether, these data suggest that the early H2O2 generation by NADPH oxidase under salt stress could be the beginning of a reaction cascade that triggers the antioxidant response in A. thaliana in order to overcome the subsequent reactive oxygen species (ROS) production, thereby mitigating the salt stress-derived injuries. Copyright © 2014 Elsevier GmbH. All rights reserved.

  13. Inhibition of steroid 5 alpha-reductase by specific aliphatic unsaturated fatty acids.

    Science.gov (United States)

    Liang, T; Liao, S

    1992-01-01

    Human or rat microsomal 5 alpha-reductase activity, as measured by enzymic conversion of testosterone into 5 alpha-dihydrotestosterone or by binding of a competitive inhibitor, [3H]17 beta-NN-diethulcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([3H]4-MA) to the reductase, is inhibited by low concentrations (less than 10 microM) of certain polyunsaturated fatty acids. The relative inhibitory potencies of unsaturated fatty acids are, in decreasing order: gamma-linolenic acid greater than cis-4,7,10,13,16,19-docosahexaenoic acid = cis-6,9,12,15-octatetraenoic acid = arachidonic acid = alpha-linolenic acid greater than linoleic acid greater than palmitoleic acid greater than oleic acid greater than myristoleic acid. Other unsaturated fatty acids such as undecylenic acid, erucic acid and nervonic acid, are inactive. The methyl esters and alcohol analogues of these compounds, glycerols, phospholipids, saturated fatty acids, retinoids and carotenes were inactive even at 0.2 mM. The results of the binding assay and the enzymic assay correlated well except for elaidic acid and linolelaidic acid, the trans isomers of oleic acid and linoleic acid respectively, which were much less active than their cis isomers in the binding assay but were as potent in the enzymic assay. gamma-Linolenic acid had no effect on the activities of two other rat liver microsomal enzymes: NADH:menadione reductase and glucuronosyl transferase. gamma-Linolenic acid, the most potent inhibitor tested, decreased the Vmax. and increased Km values of substrates, NADPH and testosterone, and promoted dissociation of [3H]4-MA from the microsomal reductase. gamma-Linolenic acid, but not the corresponding saturated fatty acid (stearic acid), inhibited the 5 alpha-reductase activity, but not the 17 beta-dehydrogenase activity, of human prostate cancer cells in culture. These results suggest that unsaturated fatty acids may play an important role in regulating androgen action in target cells. PMID:1637346

  14. Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae.

    Science.gov (United States)

    Zhang, Min; Jiang, Shao-tong; Zheng, Zhi; Li, Xing-jiang; Luo, Shui-zhong; Wu, Xue-feng

    2015-07-01

    Rhizopus oryzae is valuable as a producer of organic acids via lignocellulose catalysis. R. oryzae metabolizes xylose, which is one component of lignocellulose hydrolysate. In this study, a novel NADPH-dependent xylose reductase gene from R. oryzae AS 3.819 (Roxr) was cloned and expressed in Pichia pastoris GS115. Homology alignment suggested that the 320-residue protein contained domains and active sites belonging to the aldo/keto reductase family. SDS-PAGE demonstrated that the recombinant xylose reductase has a molecular weight of approximately 37 kDa. The optimal catalytic pH and temperature of the purified recombinant protein were 5.8 and 50 °C, respectively. The recombinant protein was stable from pH 4.4 to 6.5 and at temperatures below 42 °C. The recombinant enzyme has bias for D-xylose and L-arabinose as substrates and NADPH as its coenzyme. Real-time quantitative reverse transcription PCR tests suggested that native Roxr expression is regulated by a carbon catabolite repression mechanism. Site-directed mutagenesis at two possible key sites involved in coenzyme binding, Thr(226)  → Glu(226) and Val(274)  → Asn(274), were performed, respectively. The coenzyme specificity constants of the resulted RoXR(T226E) and RoXR(V274N) for NADH increased 18.2-fold and 2.4-fold, which suggested possibility to improve the NADH preference of this enzyme through genetic modification. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Crystal structures and atomic model of NADPH oxidase.

    Science.gov (United States)

    Magnani, Francesca; Nenci, Simone; Millana Fananas, Elisa; Ceccon, Marta; Romero, Elvira; Fraaije, Marco W; Mattevi, Andrea

    2017-06-27

    NADPH oxidases (NOXs) are the only enzymes exclusively dedicated to reactive oxygen species (ROS) generation. Dysregulation of these polytopic membrane proteins impacts the redox signaling cascades that control cell proliferation and death. We describe the atomic crystal structures of the catalytic flavin adenine dinucleotide (FAD)- and heme-binding domains of Cylindrospermum stagnale NOX5. The two domains form the core subunit that is common to all seven members of the NOX family. The domain structures were then docked in silico to provide a generic model for the NOX family. A linear arrangement of cofactors (NADPH, FAD, and two membrane-embedded heme moieties) injects electrons from the intracellular side across the membrane to a specific oxygen-binding cavity on the extracytoplasmic side. The overall spatial organization of critical interactions is revealed between the intracellular loops on the transmembrane domain and the NADPH-oxidizing dehydrogenase domain. In particular, the C terminus functions as a toggle switch, which affects access of the NADPH substrate to the enzyme. The essence of this mechanistic model is that the regulatory cues conformationally gate NADPH-binding, implicitly providing a handle for activating/deactivating the very first step in the redox chain. Such insight provides a framework to the discovery of much needed drugs that selectively target the distinct members of the NOX family and interfere with ROS signaling.

  16. Fatty acyl-CoA reductases of birds

    Directory of Open Access Journals (Sweden)

    Hellenbrand Janine

    2011-12-01

    Full Text Available Abstract Background Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters. Results cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba, domestic chicken (Gallus gallus domesticus and domestic goose (Anser anser domesticus. Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland. Conclusion The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis.

  17. VT Nitrate Leaching Index

    Data.gov (United States)

    Vermont Center for Geographic Information — (Link to Metadata) Nitrate Leaching Index data for the state of Vermont. This is a derivative product based on the SSURGO soils data for all counties except Essex...

  18. NADPH Oxidases and Their Roles in Skin Homeostasis and Carcinogenesis.

    Science.gov (United States)

    Rudolf, Jana; Raad, Houssam; Taieb, Alain; Rezvani, Hamid Reza

    2017-11-17

    Skin protects the body from dehydration, pathogens, and external mutagens. NADPH oxidases are central components for regulating the cellular redox balance. There is increasing evidence indicating that reactive oxygen species (ROS) generated by members of this enzyme family play important roles in the physiology and pathophysiology of the skin. Recent Advances: NADPH oxidases are active producers of ROS such as superoxide and hydrogen peroxide. Different isoforms are found in virtually all tissues. They play pivotal roles in normal cell homeostasis and in the cellular responses to various stressors. In particular, these enzymes are integral parts of redox-sensitive prosurvival and proapoptotic signaling pathways, in which they act both as effectors and as modulators. However, continuous (re)activation of NADPH oxidases can disturb the redox balance of cells, in the worst-case scenario in a permanent manner. Abnormal NADPH oxidase activity has been associated with a wide spectrum of diseases, as well as with aging and carcinogenesis. Sunlight with its beneficial and deleterious effects induces the activation of NADPH oxidases in the skin. Evidence for the important roles of this enzyme family in skin cancer and skin aging, as well as in many chronic skin diseases, is now emerging. Understanding the precise roles of NADPH oxidases in normal skin homeostasis, in the cellular responses to solar radiation, and during carcinogenesis will pave the way for their validation as therapeutic targets not only for the prevention and treatment of skin cancers but also for many other skin-related disorders. Antioxid. Redox Signal. 00, 000-000.

  19. ECTO-NOX (ENOX) proteins of the cell surface lack thioredoxin reductase activity.

    Science.gov (United States)

    Bosneaga, Elena; Kim, Chinpal; Shen, Bernard; Watanabe, Takahiro; Morre, Dorothy M; Morré, D James

    2008-01-01

    This study was to determine if ENOX proteins of the cell surface act as cell surface thioredoxin reductases. To measure formation of thiols a turbimetric insulin assay was used. No turbidity was observed with insulin alone or with insulin plus DTT. However, the combination of insulin +DTT + recombinant his-tagged ENOX2 (tNOX) did result in increased turbidity. An ENOX1 (CNOX) preparation also resulted in turbidity changes. In contrast, we were unable to demonstrate ENOX2-dependent insulin reduction by high density SDS-PAGE. Inclusion of reduced serum albumin as a source of free thiols for the protein disulfide interchange activity catalyzed by ENOX2 failed to result in insulin reduction in the presence of ENOX2. A direct effect of ENOX2 on thioredoxin reduction in the presence of NADPH also was not observed. The DTNB assay for thioredoxin reductase activity also failed to reveal activity. Thus, ENOX proteins appear not to function as thioredoxin reductases at the cell surface nor do they appear to recognize reduced insulin as a substrate for protein disulfide-thiol interchange. The enhanced turbidity of insulin solutions resulting from ENOX presence was traced to ENOX-catalyzed insulin fibrillation either through nucleation enhancement or some other mechanism. Fibrillation was determined using Thioflavin T fluorescence which paralleled the turbimetric results and the formation of multimers (polymerization) observed on SDS-PAGE.

  20. Leishmania chagasi: uptake of iron bound to lactoferrin or transferrin requires an iron reductase.

    Science.gov (United States)

    Wilson, Mary E; Lewis, Troy S; Miller, Melissa A; McCormick, Michael L; Britigan, Bradley E

    2002-03-01

    Leishmania chagasi can utilize iron bound to transferrin, lactoferrin, or other chelates. We investigated the mechanism of iron uptake. Promastigotes preferentially took up iron in a reduced rather than an oxidized form, suggesting that extracellular iron must be reduced prior to internalization. Similar to literature reports, a 70-kDa protein in promastigote membrane-containing microsomes bound to [125I]-labeled transferrin. However, [125I]lactoferrin and [125I]albumin also bound a similar 70-kDa protein, suggesting that binding might not be specific. Both total and fractionated promastigotes exhibited an NADPH-dependent iron reductase activity. In contrast to trypanosomes, which take up transferrin through a specific receptor, these data support a model in which a parasite-associated or secreted reductase reduces ferric to ferrous iron, decreasing its affinity for the extracellular chelate and allowing it to be readily internalized by the parasite. This could account for the ability of the parasite to utilize iron from multiple sources in diverse host environments. Index Descriptors and Abbreviations. Index descriptors: Cryptococcus neoformans, Histoplasma capsulatum, iron, iron reductase, lactoferrin, L. chagasi, leishmaniasis, nutrient acquisition, protozoan, Saccharomyces cerevisiae, Trypanosoma brucei, Trypanosoma cruzi, transferrin; Abbreviations used: DNA, deoxyribonucleic acid; DTT, dithiothreitol; HBSS, Hanks' balanced salt solution; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NEM, N-ethylmaleimide; RNA, ribonucleic acid.

  1. Differential regulation of wheat quinone reductases in response to powdery mildew infection.

    Science.gov (United States)

    Greenshields, David L; Liu, Guosheng; Selvaraj, Gopalan; Wei, Yangdou

    2005-11-01

    At least two types of quinone reductases are present in plants: (1) the zeta-crystallin-like quinone reductases (QR1, EC 1.6.5.5) that catalyze the univalent reduction of quinones to semiquinone radicals, and (2) the DT-diaphorase-like quinone reductases (QR2, EC 1.6.99.2) that catalyze the divalent reduction of quinones to hydroquinones. QR2s protect cells from oxidative stress by making the quinones available for conjugation, thereby releasing them from the superoxide-generating one electron redox cycling, catalyzed by QR1s. Two genes, putatively encoding a QR1 and a QR2, respectively, were isolated from an expressed sequence tag collection derived from the epidermis of a diploid wheat Triticum monococcum L. 24 h after inoculation with the powdery mildew fungus Blumeria graminis (DC) EO Speer f. sp. tritici Em. Marchal. Northern analysis and tissue-specific RT-PCR showed that TmQR1 was repressed while TmQR2 was induced in the epidermis during powdery mildew infection. Heterologous expression of TmQR2 in Escherichia coli confirmed that the gene encoded a functional, dicumarol-inhibitable QR2 that could use either NADH or NADPH as an electron donor. The localization of dicumarol-inhibitable QR2 activity around powdery mildew infection sites was accomplished using a histochemical technique, based on tetrazolium dye reduction.

  2. Research progress on the roles of aldose reductase in diabetic retinopathy

    Directory of Open Access Journals (Sweden)

    Hong-Zhe Li

    2015-07-01

    Full Text Available Aldose reductase(ARbelonging to nicotinamide-adenine dinucleotide phosphate(NADPH-dependent aldehyde-keto reductase superfamily, is the key rate-limiting enzyme in the polyol pathway which plays an important role in the body's high-sugar metabolism. AR is widely present in the kidneys, blood vessels, lens, retina, heart, skeletal muscle and other tissues and organs, converts glucose to sorbitol which easy permeability of cell membranes, cause cell swelling, degeneration, necrosis, and have a close relationship with the development of chronic complications of diabetes mellitus. Diabetic retinopathy(DRis a multifactorial disease, the exact cause is currently unknown, but polyol pathway has been demonstrated to play an important role in the pathogenesis of DR. Clinical risk factors such as blood sugar control, blood pressure and other treatments for DR only play a part effect of remission or invalid, if we can find out DR genes associated with the disease, this will contribute to a better understanding of the pathological mechanisms and contribute to the development of new treatments and drugs. The current research progress of AR, AR gene polymorphism, Aldose reductase inhibitors to DR was reviewed in this article.

  3. Targeting the Thioredoxin Reductase-Thioredoxin System from Staphylococcus aureus by Silver Ions.

    Science.gov (United States)

    Liao, Xiangwen; Yang, Fang; Li, Hongyan; So, Pui-Kin; Yao, Zhongping; Xia, Wei; Sun, Hongzhe

    2017-12-18

    The thioredoxin system, which is composed of NADPH, thioredoxin reductase (TrxR), and thioredoxin (Trx), is one of the major disulfide reductase systems used by bacteria against oxidative stress. In particular, this reductase system is crucial for the survival of the pathogenic bacterium Staphylococcus aureus, which lacks a natural glutathione/glutaredoxin (Grx) system. Although silver ions and silver-containing materials have been used as antibacterial agents for centuries, the antibacterial mechanism of silver is not well-understood. Herein, we demonstrate that silver ions bind to the active sites of S. aureus TrxR and Trx with dissociation constants of 1.4 ± 0.1 μM and 15.0 ± 5.0 μM and stoichiometries of 1 and 2 Ag + ions per protein, respectively. Importantly, silver ion binding leads to oligomerization and functional disruption of TrxR as well as Trx. Silver also depleted intracellular thiol levels in S. aureus, disrupting bacterial thiol-redox homeostasis. Our study provides new insights into the antibacterial mechanism of silver ions. Moreover, the Trx and TrxR system might serve as a feasible target for the design of antibacterial drugs.

  4. Signal transduction and activation of the NADPH oxidase in eosinophils

    Directory of Open Access Journals (Sweden)

    Mark A Lindsay

    1997-12-01

    Full Text Available Activation of the eosinophil NADPH oxidase and the subsequent release of toxic oxygen radicals has been implicated in the mechanism of parasite killing and inflammation. At present, little is known of the signal transduction pathway that govern agonist-induced activation of the respiratory burst and is the subject of this review. In particular, we focus on the ability of leukotrine B4 to activate the NADPH oxidase in guinea-pig peritoneal eosinophils which can be obtained in sufficient number and purity for detailed biochemical experiments to be performed.

  5. Mutational analysis of the respiratory nitrate transporter NarK2 of Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Michelle M Giffin

    Full Text Available Mycobacterium tuberculosis induces nitrate reductase activity in response to decreasing oxygen levels. This is due to regulation of both the transcription and the activity of the nitrate transporter NarK2. A model of NarK2 structure is proposed containing 12 membrane spanning regions consistent with other members of the major facilitator superfamily. The role of the proton gradient was determined by exposing M. tuberculosis to uncouplers. Nitrite production decreased indicating that the importation of nitrate involved an H(+/nitrate symporter. The addition of nitrite before nitrate had no effect, suggesting no role for a nitrate/nitrite antiporter. In addition the NarK2 knockout mutant showed no defect in nitrite export. NarK2 is proposed to be a Type I H(+/nitrate symporter. Site directed mutagenesis was performed changing 23 amino acids of NarK2. This allowed the identification of important regions and amino acids of this transporter. Five of these mutants were inactive for nitrate transport, seven produced reduced activity and eleven mutants retained wild type activity. NarK2 is inactivated in the presence of oxygen by an unknown mechanism. However none of the mutants, including those with mutated cysteines, were altered in their response to oxygen levels. The assimilatory nitrate transporter NasA of Bacillus subtilis was expressed in the M. tuberculosis NarK2 mutant. It remained active during aerobic incubation showing that the point of oxygen control is NarK2.

  6. Immunocytochemical localization of APS reductase and bisulfite reductase in three Desulfovibrio species

    NARCIS (Netherlands)

    Kremer, D.R.; Veenhuis, M.; Fauque, G.; Peck Jr., H.D.; LeGall, J.; Lampreia, J.; Moura, J.J.G.; Hansen, T.A.

    1988-01-01

    The localization of APS reductase and bisulfite reductase in Desulfovibrio gigas, D. vulgaris Hildenborough and D. thermophilus was studied by immunoelectron microscopy. Polyclonal antibodies were raised against the purified enzymes from each strain. Cells fixed with formaldehyde/glutaraldehyde were

  7. Fatty acyl-CoA reductase

    Energy Technology Data Exchange (ETDEWEB)

    Reiser, Steven E.; Somerville, Chris R.

    1998-12-01

    The present invention relates to bacterial enzymes, in particular to an acyl-CoA reductase and a gene encoding an acyl-CoA reductase, the amino acid and nucleic acid sequences corresponding to the reductase polypeptide and gene, respectively, and to methods of obtaining such enzymes, amino acid sequences and nucleic acid sequences. The invention also relates to the use of such sequences to provide transgenic host cells capable of producing fatty alcohols and fatty aldehydes.

  8. Two X-linked chronic granulomatous disease patients with unusual NADPH oxidase properties

    NARCIS (Netherlands)

    Wolach, Baruch; Broides, Arnon; Zeeli, Tal; Gavrieli, Ronit; de Boer, Martin; van Leeuwen, Karin; Levy, Jacov; Roos, Dirk

    2011-01-01

    Chronic granulomatous disease (CGD) is an immune deficiency syndrome caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the enzyme that generates reactive oxygen species (ROS) in phagocytizing leukocytes. This study evaluates the NADPH oxidase capacity in two

  9. Structural and mutational studies on an aldo-keto reductase AKR5C3 from Gluconobacter oxydans

    Science.gov (United States)

    Liu, Xu; Wang, Chao; Zhang, Lujia; Yao, Zhiqiang; Cui, Dongbing; Wu, Liang; Lin, Jinping; Yuan, Yu-Ren Adam; Wei, Dongzhi

    2014-01-01

    An aldo-keto reductase AKR5C3 from Gluconobacter oxydans (designated as Gox0644) is a useful enzyme with various substrates, including aldehydes, diacetyl, keto esters, and α-ketocarbonyl compounds. The crystal structures of AKR5C3 in apoform in complex with NADPH and the D53A mutant (AKR5C3-D53A) in complex with NADPH are presented herein. Structure comparison and site-directed mutagenesis combined with biochemical kinetics analysis reveal that the conserved Asp53 in the AKR5C3 catalytic tetrad has a crucial role in securing active pocket conformation. The gain-of-function Asp53 to Ala mutation triggers conformational changes on the Trp30 and Trp191 side chains, improving NADPH affinity to AKR5C3, which helps increase catalytic efficiency. The highly conserved Trp30 and Trp191 residues interact with the nicotinamide moiety of NADPH and help form the NADPH-binding pocket. The AKR5C3-W30A and AKR5C3-W191Y mutants show decreased activities, confirming that both residues facilitate catalysis. Residue Trp191 is in the loop structure, and the AKR5C3-W191Y mutant does not react with benzaldehyde, which might also determine substrate recognition. Arg192, which is involved in the substrate binding, is another important residue. The introduction of R192G increases substrate-binding affinity by improving hydrophobicity in the substrate-binding pocket. These results not only supplement the AKRs superfamily with crystal structures but also provide useful information for understanding the catalytic properties of AKR5C3 and guiding further engineering of this enzyme. PMID:25131535

  10. Identification and functional evaluation of the reductases and dehydrogenases from Saccharomyces cerevisiae involved in vanillin resistance.

    Science.gov (United States)

    Wang, Xinning; Liang, Zhenzhen; Hou, Jin; Bao, Xiaoming; Shen, Yu

    2016-04-01

    Vanillin, a type of phenolic released during the pre-treatment of lignocellulosic materials, is toxic to microorganisms and therefore its presence inhibits the fermentation. The vanillin can be reduced to vanillyl alcohol, which is much less toxic, by the ethanol producer Saccharomyces cerevisiae. The reducing capacity of S. cerevisiae and its vanillin resistance are strongly correlated. However, the specific enzymes and their contribution to the vanillin reduction are not extensively studied. In our previous work, an evolved vanillin-resistant strain showed an increased vanillin reduction capacity compared with its parent strain. The transcriptome analysis suggested the reductases and dehydrogenases of this vanillin resistant strain were up-regulated. Using this as a starting point, 11 significantly regulated reductases and dehydrogenases were selected in the present work for further study. The roles of these reductases and dehydrogenases in the vanillin tolerance and detoxification abilities of S. cerevisiae are described. Among the candidate genes, the overexpression of the alcohol dehydrogenase gene ADH6, acetaldehyde dehydrogenase gene ALD6, glucose-6-phosphate 1-dehydrogenase gene ZWF1, NADH-dependent aldehyde reductase gene YNL134C, and aldo-keto reductase gene YJR096W increased 177, 25, 6, 15, and 18 % of the strain μmax in the medium containing 1 g L(-1) vanillin. The in vitro detected vanillin reductase activities of strain overexpressing ADH6, YNL134C and YJR096W were notably higher than control. The vanillin specific reduction rate increased by 8 times in ADH6 overexpressed strain but not in YNL134C and YJR096W overexpressed strain. This suggested that the enzymes encoded by YNL134C and YJR096W might prefer other substrate and/or could not show their effects on vanillin on the high background of Adh6p in vivo. Overexpressing ALD6 and ZWF1 mainly increased the [NADPH]/[NADP(+)] and [GSH]/[GSSG] ratios but not the vanillin reductase activities. Their

  11. Functional properties and differential mode of regulation of the nitrate transporter from a plant symbiotic ascomycete

    Science.gov (United States)

    Montanini, Barbara; Viscomi, Arturo R.; Bolchi, Angelo; Martin, Yusé; Siverio, José M.; Balestrini, Raffaella; Bonfante, Paola; Ottonello, Simone

    2005-01-01

    Nitrogen assimilation by plant symbiotic fungi plays a central role in the mutualistic interaction established by these organisms, as well as in nitrogen flux in a variety of soils. In the present study, we report on the functional properties, structural organization and distinctive mode of regulation of TbNrt2 (Tuber borchii NRT2 family transporter), the nitrate transporter of the mycorrhizal ascomycete T. borchii. As revealed by experiments conducted in a nitrate-uptake-defective mutant of the yeast Hansenula polymorpha, TbNrt2 is a high-affinity transporter (Km=4.7 μM nitrate) that is bispecific for nitrate and nitrite. It is expressed in free-living mycelia and in mycorrhizae, where it preferentially accumulates in the plasma membrane of root-contacting hyphae. The TbNrt2 mRNA, which is transcribed from a single-copy gene clustered with the nitrate reductase gene in the T. borchii genome, was specifically up-regulated following transfer of mycelia to nitrate- (or nitrite)-containing medium. However, at variance with the strict nitrate-dependent induction commonly observed in other organisms, TbNrt2 was also up-regulated (at both the mRNA and the protein level) following transfer to a nitrogen-free medium. This unusual mode of regulation differs from that of the adjacent nitrate reductase gene, which was expressed at basal levels under nitrogen deprivation conditions and required nitrate for induction. The functional and expression properties, described in the present study, delineate TbNrt2 as a versatile transporter that may be especially suited to cope with the fluctuating (and often low) mineral nitrogen concentrations found in most natural, especially forest, soils. PMID:16201972

  12. Ammonium nitrate and iron nutrition effects on some nitrogen assimilation enzymes and metabolites in Spirulina platensis.

    Science.gov (United States)

    Esen, Merve; Ozturk Urek, Raziye

    2015-01-01

    The effect of various concentrations of ammonium nitrate (5-60 mM), an economical nitrogen source, on the growth, nitrate-ammonium uptake rates, production of some pigments and metabolites, and some nitrogen assimilation enzymes such as nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthase (GOGAT) in Spirulina platensis (Gamont) Geitler was investigated. Ten millimolars of ammonium nitrate stimulated the growth, production of pigments and the other metabolites, and enzyme activities, whereas 30 and 60 mM ammonium nitrate caused inhibition. In the presence of 10 mM ammonium nitrate, different concentrations of iron were tried in the growth media of S. platensis. After achieving the best growth, levels of metabolite and pigment production, and enzyme activities in the presence of 10 mM ammonium nitrate as a nitrogen source, different iron concentrations (10-100 µM) were tried in the growth medium of S. platensis. The highest growth, pigment and metabolite levels, and enzyme activities were determined in the medium containing 50 µM iron and 10 mM ammonium nitrate. In this optimum condition, the highest dry biomass level, chlorophyll a, and pyruvate contents were obtained as 55.42 ± 3.8 mg mL(-1) , 93.114 ± 7.9 µg g(-1) , and 212.5 ± 18.7 µg g(-1) , respectively. The highest NR, NiR, GS, and GOGAT activities were 67.16 ± 5.1, 777.92 ± 52, 0.141 ± 0.01, and 44.45 ± 3.6, respectively. Additionally, 10 mM ammonium nitrate is an economical and efficient nitrogen source for nitrogen assimilation of S. platensis, and 50 µM iron is optimum for the growth of S. platensis. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

  13. Comparison of nitrate accumulation, nitrogen uptake and utilization efficiency among different spinach (Spinacia oleracea L. genotypes

    Directory of Open Access Journals (Sweden)

    Zhou Jianjian

    2017-10-01

    Full Text Available A hydroponic experiment was conducted to study the difference of nitrate accumulation,nitrogen uptake and utilization efficiency between four spinach (Spinacia oleracea L. genotypes (So10,So13,So18 and So57. Results showed that So13 had the highest nitrate contents under two nitrate (NO3--N level (0.5 mmol·L-1,15 mmol·L-1 conditions,whereas So10 had the lowest nitrate contents. So18 had the similar nitrate contents with So13 under low NO3- level,while it showed no significant difference of nitrate contents with So57 under high NO3- treatment. The 15NO3--N uptake rates of So13 were the highest one among four genotypes,while the N utilization efficiency (NutE and N utilization ratio (NUR of So13 were significantly lower than those of So18 and So57. The shoot dry mass,nitrate reductase activity,NutE,NUR of So18 and So57 were higher than those of So13 and So10,while their 15NO3--N uptake rates were lower than those of So13. The shoot dry mass,nitrate reductase activity,NutE,N utilization ratio of So10 were significantly lower than those of So18 and So57,and its 15NO3--N uptake rate was significantly lower than those of So13. Among the four spinach genotypes,the So57 can be selected as elite germplasm using for spinach production for its relatively lower nitrate content and higher N efficiency.

  14. Production of (R)-Ethyl-4-Chloro-3-Hydroxybutanoate Using Saccharomyces cerevisiae YOL151W Reductase Immobilized onto Magnetic Microparticles.

    Science.gov (United States)

    Choo, Jin Woo; Kim, Hyung Kwoun

    2015-11-01

    For the synthesis of various pharmaceuticals, chiral alcohols are useful intermediates. Among them, (R)-ethyl-4-chloro-3-hydroxybutanoate ((R)-ECHB) is an important building block for the synthesis of L-carnitine. (R)-ECHB is produced from ethyl-4-chloro-3-oxobutanoate (ECOB) by a reductase-mediated, enantioselective reduction reaction. The Saccharomyces cerevisiae YOL151W reductase that is expressed in Escherichia coli cells exhibited an enantioselective reduction reaction toward ECOB. By virtue of the C-terminal His-tag, the YOL151W reductase was purified from the cell-free extract using Ni(2+)-NTA column chromatography and immobilized onto Ni(2+)-magnetic microparticles. The physical properties of the immobilized reductase (Imm-Red) were measured using electron microscopy, a magnetic property measurement system, and a zeta potential system; the average size of the particles was approximately 1 μm and the saturated magnetic value was 31.76 emu/g. A neodymium magnet was used to recover the immobilized enzyme within 2 min. The Imm-Red showed an optimum temperature at 45°C and an optimum pH at 6.0. In addition, Bacillus megaterium glucose dehydrogenase (GDH) was produced in the E. coli cells and was used in the coupling reaction to regenerate the NADPH cofactor. The reduction/oxidation coupling reaction composed of the Imm-Red and GDH converted 20 mM ECOB exclusively into (R)- ECHB with an e.e.p value of 98%.

  15. Oxygen and xenobiotic reductase activities of cytochrome P450.

    NARCIS (Netherlands)

    Goeptar, A.R.; Scheerens, H.; Vermeulen, N.P.E.

    1995-01-01

    The oxygen reductase and xenobiotic reductase activities of cytochrome P450 (P450) are reviewed. During the oxygen reductase activity of P450, molecular oxygen is reduced to superoxide anion radicals (O

  16. Crystal structures and atomic model of NADPH oxidase

    NARCIS (Netherlands)

    Magnani, Francesca; Nenci, Simone; Fananas, Elisa Millana; Ceccon, Marta; Romero, Elvira; Fraaije, Marco W.; Mattevi, Andrea

    2017-01-01

    NADPH oxidases (NOXs) are the only enzymes exclusively dedicated to reactive oxygen species (ROS) generation. Dysregulation of these polytopic membrane proteins impacts the redox signaling cascades that control cell proliferation and death. We describe the atomic crystal structures of the catalytic

  17. NADPH-generating systems in bacteria and archaea

    NARCIS (Netherlands)

    Spaans, S.K.; Weusthuis, R.A.; Oost, van der J.; Kengen, S.W.M.

    2015-01-01

    Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an essential electron donor in all organisms. It provides the reducing power that drives numerous anabolic reactions, including those responsible for the biosynthesis of all major cell components and many products in biotechnology. The

  18. Modulation of NADPH oxidase activity by known uraemic retention solutes

    DEFF Research Database (Denmark)

    Schulz, Anna Marta; Terne, Cindy; Jankowski, Vera

    2014-01-01

    BACKGROUND: Uraemia and cardiovascular disease appear to be associated with an increased oxidative burden. One of the key players in the genesis of reactive oxygen species (ROS) is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Based on initial experiments demonstrating a decreased ...

  19. Pinus taeda phenylpropenal double-bond reductase: purification, cDNA cloning, heterologous expression in Escherichia coli, and subcellular localization in P. taeda.

    Science.gov (United States)

    Kasahara, Hiroyuki; Jiao, Ying; Bedgar, Diana L; Kim, Sung-Jin; Patten, Ann M; Xia, Zhi-Qiang; Davin, Laurence B; Lewis, Norman G

    2006-08-01

    A phenylpropenal double-bond reductase (PPDBR) was obtained from cell suspension cultures of loblolly pine (Pinus taeda L.). Following trypsin digestion and amino acid sequencing, the cDNA encoding this protein was subsequently cloned, with the functional recombinant protein expressed in Escherichia coli and characterized. PPDBR readily converted both dehydrodiconiferyl and coniferyl aldehydes into dihydrodehydrodiconiferyl and dihydroconiferyl aldehydes, when NADPH was added as cofactor. However, it was unable to reduce directly either the double bond of dehydrodiconiferyl or coniferyl alcohols in the presence of NADPH. During this reductive step, the corresponding 4-proR hydrogen was abstracted from [4R-3H]-NADPH during hydride transfer. This is thus the first report of a double-bond reductase involved in phenylpropanoid metabolism, and which is presumed to be involved in plant defense. In situ mRNA hybridization indicated that the PPDBR transcripts in P. taeda stem sections were localized to the vascular cambium, as well as to radial and axial parenchyma cell types. Additionally, using P. taeda cell suspension culture crude protein extracts, dehydrodiconiferyl and coniferyl alcohols could be dehydrogenated to afford dehydrodiconiferyl and coniferyl aldehydes. Furthermore, these same extracts were able to convert dihydrodehydrodiconiferyl and dihydroconiferyl aldehydes into the corresponding alcohols. Taken together, these results indicate that in the crude extracts dehydrodiconiferyl and coniferyl alcohols can be converted to dihydrodehydrodiconiferyl and dihydroconiferyl alcohols through a three-step process, i.e. by initial phenylpropenol oxidation, then sequential PPDBR and phenylpropanal reductions, respectively.

  20. Characterization of xylose reductase from Candida tropicalis ...

    African Journals Online (AJOL)

    USER

    2010-08-02

    Aug 2, 2010 ... Xylose reductase gene, enzyme cofactors and plasmids. E.coli BL21(DE3) was used as host strains for ... C. tropicalis xylose reductase gene was isolated from plasmid. pMD18-T (TaKaRa, Japan). Enzyme ..... the gels is instable, soft and even dissolve in the solution containing multivalent anions or high ...

  1. Isolation and characterization of mercuric reductase by newly isolated halophilic bacterium, Bacillus firmus MN8

    Directory of Open Access Journals (Sweden)

    M. Noroozi

    2017-12-01

    Full Text Available The current study was aimed at isolating and identifying the halophilic and halotolerant bacteria which can produce mercuric reductase in Gavkhuni wetland in Iran. Moreover, tracking and sequencing merA gene and kinetic properties of mercuric reductase in the selected strain were performed in this study. Soil samples were taken from Gavkhuni wetland and cultured in nutrient agar medium with 5% NaCl. To examine the tolerance of purified colonies to mercury, agar dilution method was administered. Similarly, the phylogenetic analysis based on 16SrRNA gene sequencing was conducted. To investigate enzyme activity of kinetic parameters, a spectrophotometer was used to measure the NADPH oxidation decrease at 340 n.m. The results showed that among the 21 halophilic and halotolerant strains isolated from Gavkhuni wetland, 4 were resistant to mercuric chloride. A strain designated MN8 was selected for further studies because it showed the highest resistance to mercury. According to phylogenetic sequencing of 16S rRNA gene and phenotypic characteristics, the strain was categorized in the Bacillus genus and nearly related to Bacillus firmus. This strain had merA gene. The mercuric reductase showed Vmax and Km values of 0.106 U/mg and 24.051 µM, respectively. Evaluation of different concentrations of NaCl at 37°C and pH=7.5 in mercuric reductase enzyme activity indicated that the enzyme shows 50% activity in concentration of 1.5 M. Optimum pH and temperature of  enzyme activity were 7.5 and 35 °C, respectively. The results suggested that MN8 strain could be a proper candidate for bioremediation of mercury-contaminated environments such as industrial wastewaters.

  2. Serum nitrate levels as an index of endothelial function in pre-eclamsia and normal pregnancy.

    Science.gov (United States)

    Gupta, Rani; Maruthy, K N; Mhaskar, Arun M; Padmanabhan, Laxmi Devi

    2003-04-01

    The study was conducted in St. John's Medical College Hospital and Department of Physiology, with the aim of studying the serum nitrate levels in pre-eclampsia and normal pregnancy. The total number of subjects studied in various groups were 159, control (n = 55), first trimester (n = 13), second trimester normal (n = 42), second trimester pre-eclampsia/PET (n = 5), third trimester normal (n = 32), third trimester pre-eclampsia/PET (n = 12). The serum nitrate was measured by one step enzymatic assay using Nitrate reductase from Aspergillus species. The nitrate levels in the third trimester pre-eclamptic group was found to be significant lower (P = 0.02), as compared to normal subjects, however the renal functions were normal in all the subjects.

  3. Yttrium Nitrate mediated Nitration of Phenols at room temperature in ...

    Indian Academy of Sciences (India)

    Rapid nitration of electron rich phenols using Y(NO₃)₃.6H₂O in glacial acetic acid at room temperature was observed with good yield. The method allows nitration of phenols without oxidation, and isolation of nitration product in a rapid and simple way. The described method is selective for phenols.

  4. Yttrium Nitrate mediated Nitration of Phenols at room temperature in ...

    Indian Academy of Sciences (India)

    which resulted in many neurological disorders.1 Thus, the mechanism of nitration in biological system explains how environment and genetic factors induce neurological disorder. Conventional nitration involves use of concentrated H2SO4 and HNO3 mixture as the nitrating agent. Such a method suffers from drawbacks.

  5. Nitrate ammonification by Nautilia profundicola AmH: experimental evidence consistent with a free hydroxylamine intermediate.

    Directory of Open Access Journals (Sweden)

    Thomas E Hanson

    2013-07-01

    Full Text Available The process of nitrate reduction via nitrite controls the fate and bioavailability of mineral nitrogen within ecosystems; i.e. whether it is retained as ammonium (ammonification or lost as nitrous oxide or dinitrogen (denitrification. Here, we present experimental evidence for a novel pathway of microbial nitrate reduction, the reverse hydroxylamine:ubiquinone reductase module (reverse-HURM pathway. Instead of a classical ammonia-forming nitrite reductase that performs a 6 electron-transfer process, the pathway is thought to employ two catalytic redox modules operating in sequence: the reverse-HURM reducing nitrite to hydroxylamine followed by a hydroxylamine reductase that converts hydroxylamine to ammonium. Experiments were performed on Nautilia profundicola strain AmH, whose genome sequence led to the reverse-HURM pathway proposal. N. profundicola produced ammonium from nitrate, which was assimilated into biomass. Furthermore, genes encoding the catalysts of the reverse-HURM pathway were preferentially expressed during growth of N. profundicola on nitrate as an electron acceptor relative to cultures grown on polysulfide as an electron acceptor. Finally, nitrate-grown cells of N. profundicola were able to rapidly and stoichiometrically convert high concentrations of hydroxylamine to ammonium in resting cell assays. These experiments are consistent with the reverse-HURM pathway and a free hydroxylamine intermediate, but could not definitively exclude direct nitrite reduction to ammonium by the reverse-HURM with hydroxylamine as an off-pathway product. N. profundicola and related organisms are models for a new pathway of nitrate ammonification that may have global impact due to the wide distribution of these organisms in hypoxic environments and symbiotic or pathogenic associations with animal hosts.

  6. Nitrate Leaching Management

    Science.gov (United States)

    Nitrate (NO3) leaching is a significant nitrogen (N) loss process for agriculture that must be managed to minimize NO3 enrichment of groundwater and surface waters. Managing NO3 leaching should involve the application of basic principles of understanding the site’s hydrologic cycle, avoiding excess ...

  7. The human short-chain dehydrogenase/reductase (SDR) superfamily: a bioinformatics summary.

    Science.gov (United States)

    Bray, James E; Marsden, Brian D; Oppermann, Udo

    2009-03-16

    The short-chain dehydrogenase/reductase (SDR) superfamily represents one of the largest protein superfamilies known to date. Enzymes of this family usually catalyse NAD(P)(H) dependent reactions with a substrate spectrum ranging from polyols, retinoids, steroids and fatty acid derivatives to xenobiotics. We have currently identified 73 SDR superfamily members within the human genome. A status report of the human SDR superfamily is provided in terms of 3D structure determination, co-factor preferences, subcellular localisation and functional annotation. A simple scoring system for measuring structural and functional information (SFS score) has also been introduced to monitor the status of 5 key metrics. Currently there are 17 SDR members with an SFS score of zero indicating that almost a quarter of the human SDR superfamily lacks substantial functional annotation.

  8. Transmembrane topology of FRO2, a ferric chelate reductase from Arabidopsis thaliana.

    Science.gov (United States)

    Schagerlöf, Ulrika; Wilson, Greer; Hebert, Hans; Al-Karadaghi, Salam; Hägerhäll, Cecilia

    2006-09-01

    Iron uptake in Arabidopsis thaliana is mediated by ferric chelate reductase FRO2, a transmembrane protein belonging to the flavocytochrome b family. There is no high resolution structural information available for any member of this family. We have determined the transmembrane topology of FRO2 experimentally using the alkaline phosphatase fusion method. The resulting topology is different from that obtained by theoretical predictions and contains 8 transmembrane helices, 4 of which build up the highly conserved core of the protein. This core is present in the entire flavocytochrome b family. The large water soluble domain of FRO2, which contains NADPH, FAD and oxidoreductase sequence motifs, was located on the inside of the membrane.

  9. Oxalate synthesis in leaves is associated with root uptake of nitrate and its assimilation in spinach (Spinacia oleracea L.) plants.

    Science.gov (United States)

    Liu, Xiao Xia; Zhou, Kai; Hu, Yan; Jin, Rong; Lu, Ling Li; Jin, Chong Wei; Lin, Xian Yong

    2015-08-15

    Excessive accumulation of oxalate in numerous vegetables adversely affects their quality as food. While it is known that nitrate could effectively stimulate oxalate accumulation in many vegetables, little information is available about the mechanism of nitrate-induced oxalate accumulation. In this study, we examined the association of oxalate synthesis with nitrate uptake and assimilation in two genotypes of spinach (Spinacia oleracea L.), Heizhenzhu and Weilv. Increasing nitrate levels resulted in enhanced synthesis of oxalate, as well as increased root uptake of nitrate and leaf activities of nitrate reductase (NR) and glutamine synthetase (GS) for both genotypes. Correlation analysis revealed that oxalate accumulation in spinach leaves was positively related with rate of nitrate uptake by roots, as well as leaf activities of NR and GS. Addition of plasmalemma H(+)-ATPase inhibitor sodium vanadate (Na3VO4) significantly decreased leaf oxalate accumulation in both genotypes. Presence of NR or GS inhibitors led to reduction of leaf oxalate contents, GS/NR activities and decreased nitrate uptake rate. Significantly higher levels of nitrate root uptake, leaf NR and GS activities were observed in the high-oxalate genotype Heizhenzhu than in Weilv. Oxalate synthesis in leaves of spinach is not only positively associated with root uptake of nitrate, but also with its assimilation within the plants. © 2014 Society of Chemical Industry.

  10. Functioning of nitric oxide cycle in gastric mucosa of rats under excessive combined intake of sodium nitrate and fluoride.

    Science.gov (United States)

    Akimov, O Ye; Kostenko, V O

    2016-01-01

    In the article the function of nitric oxide (·NO) cycle in rat’s gastric mucosa was assessed under excessive combined chronic fluoride and nitrate intake during 30 days. It was estimated that general nitric oxide synthase activity (NOS) was increased during excessive sodium fluoride intake meanwhile influence on gene­ral nitrate reduction was not statistically significant, but general nitrite reduction was increased. General arginase activity decreased. Excessive sodium nitrate intake decreased NOS activity, but increased nitrate, nitrite reduction and general arginase activity. Combined sodium nitrate and fluoride intake increased NOS by 18.9%, nitrate reduction by 71.7%, nitrite by 161.5%, arginase activity increase by 61.4%. The highest amounts of peroxynitrite were obtained from excessive sodium fluoride intake group, excessive sodium nitrate intake showed the lowest levels and combined excessive sodium nitrate and fluoride intake levels had intermediate results. Summarizing the data obtained, the authors have made a conclusion that combined excessive sodium nitrate and fluoride intake creates optimal conditions for functioning of nitrate-nitrite reductases.

  11. Properties of latent and thiol-activated rat hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase and regulation of enzyme activity.

    Science.gov (United States)

    Dotan, I; Shechter, I

    1983-10-15

    The effect of the thiols glutathione (GSH), dithiothreitol (DTT), and dithioerythritol (DTE) on the conversion of an inactive, latent form (El) of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) to a catalyticaly active form (Ea) is examined. Latent hepatic microsomal HMG-CoA reductase is activated to a similar degree of activation by DTT and DTE and to a lower extent by GSH. All three thiols affect both Km and Vmax values of the enzyme toward HMG-CoA and NADPH. Studies of the effect of DTT on the affinity binding of HMG-CoA reductase to agarose-hexane-HMG-CoA (AG-HMG-CoA) resin shows that thiols are necessary for the binding of the enzyme to the resin. Removal of DTT from AG-HMG-CoA-bound soluble Ea (active enzyme) does not cause dissociation of the enzyme from the resin at low salt concentrations. Substitution of DTT by NADPH does not promote binding of soluble El (latent enzyme) to AG-HMG-CoA. The enzymatic activity of Ea in the presence of DTT and GSH indicates that these thiols compete for the same binding site on the enzyme. Diethylene glycol disulfide (ESSE) and glutathione disulfide (GSSG) inhibit the activity of Ea. ESSE is more effective for the inhibition of Ea than GSSG, causing a higher degree of maximal inhibition and affecting the enzymatic activity at lower concentrations. A method is described for the rapid conversion of soluble purified Ea to El using gel-filtration chromatography on Bio-Gel P-4 columns. These combined results point to the importance of the thiol/disulfide ratio for the modulation of hepatic HMG-CoA reductase activity.

  12. Horizontal transfer of a nitrate assimilation gene cluster and ecological transitions in fungi: a phylogenetic study.

    Directory of Open Access Journals (Sweden)

    Jason C Slot

    Full Text Available High affinity nitrate assimilation genes in fungi occur in a cluster (fHANT-AC that can be coordinately regulated. The clustered genes include nrt2, which codes for a high affinity nitrate transporter; euknr, which codes for nitrate reductase; and NAD(PH-nir, which codes for nitrite reductase. Homologs of genes in the fHANT-AC occur in other eukaryotes and prokaryotes, but they have only been found clustered in the oomycete Phytophthora (heterokonts. We performed independent and concatenated phylogenetic analyses of homologs of all three genes in the fHANT-AC. Phylogenetic analyses limited to fungal sequences suggest that the fHANT-AC has been transferred horizontally from a basidiomycete (mushrooms and smuts to an ancestor of the ascomycetous mold Trichoderma reesei. Phylogenetic analyses of sequences from diverse eukaryotes and eubacteria, and cluster structure, are consistent with a hypothesis that the fHANT-AC was assembled in a lineage leading to the oomycetes and was subsequently transferred to the Dikarya (Ascomycota+Basidiomycota, which is a derived fungal clade that includes the vast majority of terrestrial fungi. We propose that the acquisition of high affinity nitrate assimilation contributed to the success of Dikarya on land by allowing exploitation of nitrate in aerobic soils, and the subsequent transfer of a complete assimilation cluster improved the fitness of T. reesei in a new niche. Horizontal transmission of this cluster of functionally integrated genes supports the "selfish operon" hypothesis for maintenance of gene clusters.

  13. Anti-HMG-CoA Reductase, Antioxidant, and Anti-Inflammatory Activities of Amaranthus viridis Leaf Extract as a Potential Treatment for Hypercholesterolemia

    Directory of Open Access Journals (Sweden)

    Shamala Salvamani

    2016-01-01

    Full Text Available Inflammation and oxidative stress are believed to contribute to the pathology of several chronic diseases including hypercholesterolemia (elevated levels of cholesterol in blood and atherosclerosis. HMG-CoA reductase inhibitors of plant origin are needed as synthetic drugs, such as statins, which are known to cause adverse effects on the liver and muscles. Amaranthus viridis (A. viridis has been used from ancient times for its supposedly medically beneficial properties. In the current study, different parts of A. viridis (leaf, stem, and seed were evaluated for potential anti-HMG-CoA reductase, antioxidant, and anti-inflammatory activities. The putative HMG-CoA reductase inhibitory activity of A. viridis extracts at different concentrations was determined spectrophotometrically by NADPH oxidation, using HMG-CoA as substrate. A. viridis leaf extract revealed the highest HMG-CoA reductase inhibitory effect at about 71%, with noncompetitive inhibition in Lineweaver-Burk plot analysis. The leaf extract showed good inhibition of hydroperoxides, 2,2-diphenyl-1-picrylhydrazyl (DPPH, nitric oxide (NO, and ferric ion radicals in various concentrations. A. viridis leaf extract was proven to be an effective inhibitor of hyaluronidase, lipoxygenase, and xanthine oxidase enzymes. The experimental data suggest that A. viridis leaf extract is a source of potent antioxidant and anti-inflammatory agent and may modulate cholesterol metabolism by inhibition of HMG-CoA reductase.

  14. Role of a novel dual flavin reductase (NR1) and an associated histidine triad protein (DCS-1) in menadione-induced cytotoxicity

    International Nuclear Information System (INIS)

    Kwasnicka-Crawford, Dorota A.; Vincent, Steven R.

    2005-01-01

    Microsomal cytochrome P450 reductase catalyzes the one-electron transfer from NADPH via FAD and FMN to various electron acceptors, such as cytochrome P450s or to some anti-cancer quinone drugs. This results in generation of free radicals and toxic oxygen metabolites, which can contribute to the cytotoxicity of these compounds. Recently, a cytosolic NADPH-dependent flavin reductase, NR1, has been described which is highly homologous to the microsomal cytochrome P450 reductase. In this study, we show that over-expression of NR1 in human embryonic kidney cells enhances the cytotoxic action of the model quinone, menadione. Furthermore, we show that a novel human histidine triad protein DCS-1, which is expressed together with NR1 in many tissues, can significantly reduce menadione-induced cytotoxicity in these cells. We also show that DCS-1 binds NF1 and directly modulates its activity. These results suggest that NR1 may play a role in carcinogenicity and cell death associated with one-electron reductions

  15. Expression, purification and characterization of enoyl-ACP reductase II, FabK, from Porphyromonas gingivalis

    Energy Technology Data Exchange (ETDEWEB)

    Hevener, Kirk E.; Mehboob, Shahila; Boci, Teuta; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E. (UIC)

    2012-10-25

    The rapid rise in bacterial drug resistance coupled with the low number of novel antimicrobial compounds in the discovery pipeline has led to a critical situation requiring the expedient discovery and characterization of new antimicrobial drug targets. Enzymes in the bacterial fatty acid synthesis pathway, FAS-II, are distinct from their mammalian counterparts, FAS-I, in terms of both structure and mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. Enoyl-acyl carrier protein reductase II, FabK, is a key, rate-limiting enzyme in the FAS-II pathway for several bacterial pathogens. The organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the US population and incurs a high national burden in terms of cost of treatment. P. gingivalis expresses FabK as the sole enoyl reductase enzyme in its FAS-II cycle, which makes this a particularly appealing target with potential for selective antimicrobial therapy. Herein we report the molecular cloning, expression, purification and characterization of the FabK enzyme from P. gingivalis, only the second organism from which this enzyme has been isolated. Characterization studies have shown that the enzyme is a flavoprotein, the reaction dependent upon FMN and NADPH and proceeding via a Ping-Pong Bi-Bi mechanism to reduce the enoyl substrate. A sensitive assay measuring the fluorescence decrease of NADPH as it is converted to NADP{sup +} during the reaction has been optimized for high-throughput screening. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x-rays to high resolution.

  16. Carboxylic acid reductase enzymes (CARs).

    Science.gov (United States)

    Winkler, Margit

    2018-04-01

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

  17. Ferrisiderophore reductase activity in Bacillus megaterium.

    Science.gov (United States)

    Arceneaux, J E; Byers, B R

    1980-01-01

    The release of iron from ferrisiderophores (microbial ferric-chelating iron transport cofactors) by cell-free extracts of Bacillus megaterium was demonstrated. Reductive transfer of iron from ferrisiderophores to the ferrous-chelating agent ferrozine was measured spectrophotometrically. This ferrisiderophore reductase activity (reduced nicotinamide adenine dinucleotide phosphate:ferrisiderophore oxidoreductase) was associated primarily with the cell soluble rather than particulate (membrane) fraction. Ferrisiderophore reductase was inhibited by oxygen and required the addition of a reductant (reduced nicotinamide adenine dinucleotide phosphate was most effective) for maximal activity. The activity was destroyed by both heat and protease treatments and was inhibited by iodoacetamide treatment. Ferrisiderophore reductase activity for several microbial ferrisiderophores was measured; highest activity was displayed for ferrischizokinen, the ferrisiderophore produced by this organism. The Km and Vmax values of the reductase for ferrischizokinen were 2.5 x 10(-4) M and 35.7 nmol/min per mg of the ferrisiderophore reductase reaction. Preliminary fractionation of the cell soluble material by gel filtration chromatography resulted in the demonstration of ferrisiderophore reductase activity in three peaks of different molecular weight. Ferrisiderophore reductase probably mediates entrance of iron into cellular metabolism. PMID:6444944

  18. Evidence for two interconverting protein isomers in the methotrexate complex of dihydrofolate reductase from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Falzone, C.J.; Benkovic, S.J. (Pennsylvania State Univ., University Park (United States)); Wright, P.E. (Research Inst. of Scripps Clinic, La Jolla, CA (United States))

    1991-02-26

    Two-dimensional {sup 1}H NMR methods and a knowledge of the X-ray crystal structure have been used to make resonance assignments for the amino acid side chains of dihydrofolate reductase from Escherichia coli complexed with methotrexate. The H7 proton on the pteridine ring of methotrexate was found to have NOEs to the methyl protons of Leu-28 which were assigned by using the L28F mutant. These NOEs indicated that the orientation of the methotrexate pteridine ring is similar in both solution and crystal structures. During the initial assignment process, it became evident that many of the resonances in this complex, unlike those of the folate complex, are severally broadened or doubled. The observation of two distinct sets of resonances in a ratio of approximately 2:1 was attributed to the presence of two protein isomers. Many of the side chains with clearly doubled resonances were located in the {beta}-sheet and the active site. Preliminary studies on the apoprotein also revealed doubled resonances in the absence of the inhibitor, indicating the existence of the protein isomers prior to methotrexate binding. In contrast to the methotrexate complex, the binary complex with folate and the ternary MTX-NADPH-DHFR complex presented a single enzyme form. These results are proposed to reflect the ability of folate and NADPH to bind predominantly to one protein isomer.

  19. Plant Glyoxylate/Succinic Semialdehyde Reductases: Comparative Biochemical Properties, Function during Chilling Stress, and Subcellular Localization

    Directory of Open Access Journals (Sweden)

    Adel Zarei

    2017-08-01

    Full Text Available Plant NADPH-dependent glyoxylate/succinic semialdehyde reductases 1 and 2 (cytosolic GLYR1 and plastidial/mitochondrial GLYR2 are considered to be of particular importance under abiotic stress conditions. Here, the apple (Malus × domestica Borkh. and rice (Oryza sativa L. GLYR1s and GLYR2s were characterized and their kinetic properties were compared to those of previously characterized GLYRs from Arabidopsis thaliana [L.] Heynh. The purified recombinant GLYRs had an affinity for glyoxylate and succinic semialdehyde, respectively, in the low micromolar and millimolar ranges, and were inhibited by NADP+. Comparison of the GLYR activity in cell-free extracts from wild-type Arabidopsis and a glyr1 knockout mutant revealed that approximately 85 and 15% of the cellular GLYR activity is cytosolic and plastidial/mitochondrial, respectively. Recovery of GLYR activity in purified mitochondria from the Arabidopsis glyr1 mutant, free from cytosolic GLYR1 or plastidial GLYR2 contamination, provided additional support for the targeting of GLYR2 to mitochondria, as well as plastids. The growth of plantlets or roots of various Arabidopsis lines with altered GLYR activity responded differentially to succinic semialdehyde or glyoxylate under chilling conditions. Taken together, these findings highlight the potential regulation of highly conserved plant GLYRs by NADPH/NADP+ ratios in planta, and their roles in the reduction of toxic aldehydes in plants subjected to chilling stress.

  20. Nuclear magnetic resonance studies on bacterial dihydrofolate reductase containing (methyl-/sup 13/C)methionine

    Energy Technology Data Exchange (ETDEWEB)

    Blakley, R.L.; Cocco, L.; London, R.E.; Walker, T.E.; Matwiyoff, N.A.

    1978-06-13

    (methyl-/sup 13/C)Methionine has been incorporated with high efficiency by Streptococcus faecium var. Durans strain A into dihydrofolate reductase isoenzyme 2. In the /sup 13/C NMR spectrum of the purified enzyme the resonances corresponding to the seven methionine residues are partially resolved into three composite peaks. Denaturation with urea collapses these into a single peak centered at 15.32 ppm, whereas the resonance of free methionine is at 15.04 ppm. Spectra of the free enzyme, its complex with methotrexate, and its complex with methotrexate and reduced nicotinamide adenine dinucleotide phosphate (NADPH) have been simulated, permitting more accurate estimates of line widths and nuclear Overhauser enhancement (NOE) values. These, together with the T/sub 1/ values, cannot be explained solely by the effects of macromolecular tumbling and very rapid rotation of the methionine methyl group about its axis. A model assuming, in addition, the occurrence of free rotation about the methionine CH/sub 2/-S bond is also unsatisfactory, and it is concluded that internal rotation about the CH/sub 2/-S bond is highly restricted so that the methyl group oscillates through a relatively narrow angular range. Complex formation with NADPH produced rather small changes in the spectrum of the native enzyme, probably due to conformational transitions in the enzyme. However, NADP/sup +/ produced several changes,including movement of one resonance downfield by at least 1.7 ppM.

  1. Refining method for bismuth nitrate

    International Nuclear Information System (INIS)

    Shibata, Shigeyuki.

    1997-01-01

    The present invention concerns a method of separating and removing α ray emitting nuclides present in an aqueous solution of bismuth nitrate by an industrially convenient method. A nitric acid concentration in the aqueous solution of bismuth nitrate in which α ray emitting nuclides are dissolved is lowered to coprecipitate the bismuth oxynitrate and the α ray emitting nuclides. The coprecipitation materials are separated from the aqueous solution of bismuth nitrate to separate the α ray emitting nuclides dissolved in the aqueous solution of bismuth nitrate thereby refining the aqueous solution of bismuth nitrate. (T.M.)

  2. Mechanisms for suppressing NADPH oxidase in the vascular wall

    Directory of Open Access Journals (Sweden)

    Gregory J Dusting

    2005-03-01

    Full Text Available Oxidative stress underlies many forms of vascular disease as well as tissue injury following ischemia and reperfusion. The major source of oxidative stress in the artery wall is an NADPH oxidase. This enzyme complex as expressed in vascular cells differs from that in phagocytic leucocytes both in biochemical structure and functions. The crucial flavin-containing catalytic subunits, Nox1 and Nox4, are not found in leucocytes, but are highly expressed in vascular cells and upregulated with vascular remodeling, such as that found in hypertension and atherosclerosis. The difference in catalytic subunits offers the opportunity to develop "vascular specific" NADPH oxidase inhibitors that do not compromise the essential physiological signaling and phagocytic functions carried out by reactive oxygen and nitrogen species. Nitric oxide and targeted inhibitors of NADPH oxidase that block the source of oxidative stress in the vasculature are more likely to prevent the deterioration of vascular function that leads to stroke and heart attack, than are conventional antioxidants. The roles of Nox isoforms in other inflammatory conditions are yet to be explored.

  3. Nitrate in drinking water

    DEFF Research Database (Denmark)

    Schullehner, Jörg

    Annual nationwide exposure maps for nitrate in drinking water in Denmark from the 1970s until today will be presented based on the findings in Schullehner & Hansen (2014) and additional work on addressing the issue of private well users and estimating missing data. Drinking water supply in Denmark...... is highly decentralized and fully relying on simple treated groundwater. At the same time, Denmark has an intensive agriculture, making groundwater resources prone to nitrate pollution. Drinking water quality data covering the entire country for over 35 years are registered in the public database Jupiter....... In order to create annual maps of drinking water quality, these data had to be linked to 2,852 water supply areas, which were for the first time digitized, collected in one dataset and connected to the Jupiter database. Analyses of the drinking water quality maps showed that public water supplies...

  4. Nitrate in drinking water

    DEFF Research Database (Denmark)

    Schullehner, Jörg; Hansen, Birgitte; Sigsgaard, Torben

    is highly decentralized and fully relying on simple treated groundwater. At the same time, Denmark has an intensive agriculture, making groundwater resources prone to nitrate pollution. Drinking water quality data covering the entire country for over 35 years are registered in the public database Jupiter......Annual nationwide exposure maps for nitrate in drinking water in Denmark from the 1970s until today will be presented based on the findings in Schullehner & Hansen (2014) and additional work on addressing the issue of private well users and estimating missing data. Drinking water supply in Denmark....... In order to create annual maps of drinking water quality, these data had to be linked to 2,852 water supply areas, which were for the first time digitized, collected in one dataset and connected to the Jupiter database. Analyses of the drinking water quality maps showed that public water supplies...

  5. A novel NAD(P)H-dependent carbonyl reductase specifically expressed in the thyroidectomized chicken fatty liver: catalytic properties and crystal structure.

    Science.gov (United States)

    Fukuda, Yudai; Sone, Takeki; Sakuraba, Haruhiko; Araki, Tomohiro; Ohshima, Toshihisa; Shibata, Takeshi; Yoneda, Kazunari

    2015-10-01

    A gene encoding a functionally unknown protein that is specifically expressed in the thyroidectomized chicken fatty liver and has a predicted amino acid sequence similar to that of NAD(P)H-dependent carbonyl reductase was overexpressed in Escherichia coli; its product was purified and characterized. The expressed enzyme was an NAD(P)H-dependent broad substrate specificity carbonyl reductase and was inhibited by arachidonic acid at 1.5 μm. Enzymological characterization indicated that the enzyme could be classified as a cytosolic-type carbonyl reductase. The enzyme's 3D structure was determined using the molecular replacement method at 1.98 Å resolution in the presence of NADPH and ethylene glycol. The asymmetric unit consisted of two subunits, and a noncrystallographic twofold axis generated the functional dimer. The structures of the subunits, A and B, differed from each other. In subunit A, the active site contained an ethylene glycol molecule absent in subunit B. Consequently, Tyr172 in subunit A rotated by 103.7° in comparison with subunit B, which leads to active site closure in subunit A. In Y172A mutant, the Km value for 9,10-phenanthrenequinone (model substrate) was 12.5 times higher than that for the wild-type enzyme, indicating that Tyr172 plays a key role in substrate binding in this carbonyl reductase. Because the Tyr172-containing active site lid structure (Ile164-Gln174) is not conserved in all known carbonyl reductases, our results provide new insights into substrate binding of carbonyl reductase. The catalytic properties and crystal structure revealed that thyroidectomized chicken fatty liver carbonyl reductase is a novel enzyme. © 2015 FEBS.

  6. Disguised as a Sulfate Reducer: Growth of the Deltaproteobacterium Desulfurivibrio alkaliphilus by Sulfide Oxidation with Nitrate

    Directory of Open Access Journals (Sweden)

    Casper Thorup

    2017-07-01

    Full Text Available This study demonstrates that the deltaproteobacterium Desulfurivibrio alkaliphilus can grow chemolithotrophically by coupling sulfide oxidation to the dissimilatory reduction of nitrate and nitrite to ammonium. Key genes of known sulfide oxidation pathways are absent from the genome of D. alkaliphilus. Instead, the genome contains all of the genes necessary for sulfate reduction, including a gene for a reductive-type dissimilatory bisulfite reductase (DSR. Despite this, growth by sulfate reduction was not observed. Transcriptomic analysis revealed a very high expression level of sulfate-reduction genes during growth by sulfide oxidation, while inhibition experiments with molybdate pointed to elemental sulfur/polysulfides as intermediates. Consequently, we propose that D. alkaliphilus initially oxidizes sulfide to elemental sulfur, which is then either disproportionated, or oxidized by a reversal of the sulfate reduction pathway. This is the first study providing evidence that a reductive-type DSR is involved in a sulfide oxidation pathway. Transcriptome sequencing further suggests that nitrate reduction to ammonium is performed by a novel type of periplasmic nitrate reductase and an unusual membrane-anchored nitrite reductase.

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

    Science.gov (United States)

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

    2013-01-01

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

  8. Acute Ethanol Intake Induces NAD(P)H Oxidase Activation and Rhoa Translocation in Resistance Arteries.

    Science.gov (United States)

    Simplicio, Janaina A; Hipólito, Ulisses Vilela; Vale, Gabriel Tavares do; Callera, Glaucia Elena; Pereira, Camila André; Touyz, Rhian M; Tostes, Rita de Cássia; Tirapelli, Carlos R

    2016-11-01

    The mechanism underlying the vascular dysfunction induced by ethanol is not totally understood. Identification of biochemical/molecular mechanisms that could explain such effects is warranted. To investigate whether acute ethanol intake activates the vascular RhoA/Rho kinase pathway in resistance arteries and the role of NAD(P)H oxidase-derived reactive oxygen species (ROS) on such response. We also evaluated the requirement of p47phox translocation for ethanol-induced NAD(P)H oxidase activation. Male Wistar rats were orally treated with ethanol (1g/kg, p.o. gavage) or water (control). Some rats were treated with vitamin C (250 mg/kg, p.o. gavage, 5 days) before administration of water or ethanol. The mesenteric arterial bed (MAB) was collected 30 min after ethanol administration. Vitamin C prevented ethanol-induced increase in superoxide anion (O2-) generation and lipoperoxidation in the MAB. Catalase and superoxide dismutase activities and the reduced glutathione, nitrate and hydrogen peroxide (H2O2) levels were not affected by ethanol. Vitamin C and 4-methylpyrazole prevented the increase on O2- generation induced by ethanol in cultured MAB vascular smooth muscle cells. Ethanol had no effect on phosphorylation levels of protein kinase B (Akt) and eNOS (Ser1177 or Thr495 residues) or MAB vascular reactivity. Vitamin C prevented ethanol-induced increase in the membrane: cytosol fraction ratio of p47phox and RhoA expression in the rat MAB. Acute ethanol intake induces activation of the RhoA/Rho kinase pathway by a mechanism that involves ROS generation. In resistance arteries, ethanol activates NAD(P)H oxidase by inducing p47phox translocation by a redox-sensitive mechanism. O mecanismo da disfunção vascular induzido pelo consumo de etanol não é totalmente compreendido. Justifica-se, assim a identificação de mecanismos bioquímicos e moleculares que poderiam explicar tais efeitos. Investigar se a ingestão aguda de etanol ativa a via vascular RhoA/Rho quinase

  9. Enterosalivary nitrate metabolism and the microbiome: Intersection of microbial metabolism, nitric oxide and diet in cardiac and pulmonary vascular health.

    Science.gov (United States)

    Koch, Carl D; Gladwin, Mark T; Freeman, Bruce A; Lundberg, Jon O; Weitzberg, Eddie; Morris, Alison

    2017-04-01

    Recent insights into the bioactivation and signaling actions of inorganic, dietary nitrate and nitrite now suggest a critical role for the microbiome in the development of cardiac and pulmonary vascular diseases. Once thought to be the inert, end-products of endothelial-derived nitric oxide (NO) heme-oxidation, nitrate and nitrite are now considered major sources of exogenous NO that exhibit enhanced vasoactive signaling activity under conditions of hypoxia and stress. The bioavailability of nitrate and nitrite depend on the enzymatic reduction of nitrate to nitrite by a unique set of bacterial nitrate reductase enzymes possessed by specific bacterial populations in the mammalian mouth and gut. The pathogenesis of pulmonary hypertension (PH), obesity, hypertension and CVD are linked to defects in NO signaling, suggesting a role for commensal oral bacteria to shape the development of PH through the formation of nitrite, NO and other bioactive nitrogen oxides. Oral supplementation with inorganic nitrate or nitrate-containing foods exert pleiotropic, beneficial vascular effects in the setting of inflammation, endothelial dysfunction, ischemia-reperfusion injury and in pre-clinical models of PH, while traditional high-nitrate dietary patterns are associated with beneficial outcomes in hypertension, obesity and CVD. These observations highlight the potential of the microbiome in the development of novel nitrate- and nitrite-based therapeutics for PH, CVD and their risk factors. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Purification and characterization of an NADH-hexacyanoferrate(III) reductase from spinach leaf plasma membrane.

    Science.gov (United States)

    Bérczi, A; Fredlund, K M; Møller, I M

    1995-06-20

    Plasma membranes were purified from spinach (Spinacea oleracea L.) leaves by aqueous two-phase partitioning. The NADH-hexacyanoferrate(III) reductase was released from the membrane by Chaps solubilization and purified 360-fold by ion-exchange chromatography followed by affinity chromatography and size-exclusion chromatography on FPLC. A major band of 45 kDa and a minor contaminant of 66 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The band at 45 kDa cross-reacted with antibodies raised against an NADH-hexacyanoferrate(III) reductase from potato tuber microsomes. The native size of the enzyme was 160 kDa as determined by size-exclusion chromatography indicating that it is a tetramer. Two-dimensional gel electrophoresis, isoelectric focusing, followed by SDS-PAGE revealed three main bands of identical molecular weight with pI of 5.3-5.6. The enzyme contained about one flavin adenine dinucleotide (FAD) per 45-kDa subunit as determined by fluorescence spectroscopy, was specific for the beta-hydrogen of NADH, preferred NADH over NADPH as electron donor, and preferred hexacyanoferrate(III) as electron acceptor, e.g., it reduced Fe3+-EDTA, cytochrome c, oxygen, and duroquinone at 70% whereas FAD, flavin mononucleotide, duroquinone, and ubiquinone0 did not affect the activity.

  11. Crystallization and preliminary X-ray crystallographic studies of the alkanesulfonate FMN reductase from Escherichia coli

    International Nuclear Information System (INIS)

    Gao, Benlian; Bertrand, Adam; Boles, William H.; Ellis, Holly R.; Mallett, T. Conn

    2005-01-01

    Crystallization of the native and SeMet FMN reductase protein of the E. coli alkanesulfonate monooxygenase two-component enzyme system is reported. The alkanesulfonate FMN reductase (SsuE) from Escherichia coli catalyzes the reduction of FMN by NADPH to provide reduced flavin for the monooxygenase (SsuD) enzyme. The vapor-diffusion technique yielded single crystals that grow as hexagonal rods and diffract to 2.9 Å resolution using synchrotron X-ray radiation. The protein crystallizes in the primitive hexagonal space group P622. The SsuE protein lacks any cysteine or methionine residues owing to the role of the SsuE enzyme in the acquisition of sulfur during sulfate starvation. Therefore, substitution of two leucine residues (Leu114 and Leu165) to methionine was performed to obtain selenomethionine-containing SsuE for MAD phasing. The selenomethionine derivative of SsuE has been expressed and purified and crystals of the protein have been obtained with and without bound FMN. These preliminary studies should lead to the structure solution of SsuE. It is anticipated that this new protein structure will provide detailed structural information on specific active-site regions of the protein and insight into the mechanism of flavin reduction and transfer of reduced flavin

  12. Nitrite reductase activity and inhibition of H₂S biogenesis by human cystathionine ß-synthase.

    Directory of Open Access Journals (Sweden)

    Carmen Gherasim

    Full Text Available Nitrite was recognized as a potent vasodilator >130 years and has more recently emerged as an endogenous signaling molecule and modulator of gene expression. Understanding the molecular mechanisms that regulate nitrite metabolism is essential for its use as a potential diagnostic marker as well as therapeutic agent for cardiovascular diseases. In this study, we have identified human cystathionine ß-synthase (CBS as a new player in nitrite reduction with implications for the nitrite-dependent control of H₂S production. This novel activity of CBS exploits the catalytic property of its unusual heme cofactor to reduce nitrite and generate NO. Evidence for the possible physiological relevance of this reaction is provided by the formation of ferrous-nitrosyl (Fe(II-NO CBS in the presence of NADPH, the human diflavin methionine synthase reductase (MSR and nitrite. Formation of Fe(II-NO CBS via its nitrite reductase activity inhibits CBS, providing an avenue for regulating biogenesis of H₂S and cysteine, the limiting reagent for synthesis of glutathione, a major antioxidant. Our results also suggest a possible role for CBS in intracellular NO biogenesis particularly under hypoxic conditions. The participation of a regulatory heme cofactor in CBS in nitrite reduction is unexpected and expands the repertoire of proteins that can liberate NO from the intracellular nitrite pool. Our results reveal a potential molecular mechanism for cross-talk between nitrite, NO and H₂S biology.

  13. Ebselen: A thioredoxin reductase-dependent catalyst for α-tocopherol quinone reduction

    International Nuclear Information System (INIS)

    Fang Jianguo; Zhong Liangwei; Zhao Rong; Holmgren, Arne

    2005-01-01

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

  14. Cloning and expression of koala (Phascolarctos cinereus) liver cytochrome P450 reductase.

    Science.gov (United States)

    Kong, Sandra; Ngo, Suong N T; McKinnon, Ross A; Stupans, Ieva

    2009-07-01

    The cloning, expression and characterization of hepatic NADPH-cytochrome P450 reductase (CPR) from koala (Phascolarctos cinereus) is described. Two 2059 bp koala liver CPR cDNAs, designated CPR1 and CPR2, were cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The koala CPR cDNAs encode proteins of 678 amino acids and share 85% amino acid sequence identity to human CPR. Transfection of the koala CPR cDNAs into Cos-7 cells resulted in the expression of proteins, which were recognized by a goat-antihuman CPR antibody. The koala CPR1 and 2 cDNA-expressed enzymes catalysed cytochrome c reductase at the rates of 4.9 +/- 0.5 and 2.6 +/- 0.4 nmol/min/mg protein (mean +/- SD, n = 3), respectively which were comparable to that of rat CPR cDNA-expressed enzyme. The apparent Km value for CPR activity in koala liver microsomes was 11.61 +/- 6.01 microM, which is consistent with that reported for rat CPR enzyme. Northern analysis detected a CPR mRNA band of approximately 2.6 kb. Southern analysis suggested a single PCR gene across species. The present study provides primary molecular data regarding koala CPR1 and CPR2 genes in this unique marsupial species.

  15. Clone-Specific Response in Leaf Nitrate Reductase Activity among Unrelated Hybrid Poplars in relation to Soil Nitrate Availability

    Directory of Open Access Journals (Sweden)

    Julien Fortier

    2012-01-01

    Full Text Available In this field study, we used in vivo NRA activity in hybrid poplar leaves as an indicator of NO3- assimilation for five unrelated hybrid poplar clones. We also examined if leaf NRA of these clones is influenced to the same extent by different levels of soil NO3- availability in two riparian agroforestry systems located in pastures. Leaf NRA differences of more than one order of magnitude were observed between the clones, clearly showing their different abilities to reduce NO3- in leaves. Clone DxN-3570, a P. deltoides x P. nigra hybrid (Aigeiros intrasectional hybrid, always had the highest leaf NRA during the field assays. This clone was also the only one to increase its leaf NRA with increasing NO3- soil availability, which resulted in a significant Site x Clone interaction and a positive relationship between soil NO3- concentration and NRA. All of the four other clones studied had one or both parental species from the Tacamahaca section. They had relatively low leaf NRA and they did not increase their leaf NRA when grown on the NO3- rich site. These results provide evidence that NO3- assimilation in leaves varies widely among hybrid poplars of different parentages, suggesting potential preferences for N forms.

  16. Reduction of the explosive 2,4,6-trinitrophenylmethylnitramine (tetryl) catalyzed by oxygen sensitive nitro reductase enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Shah, M.M.; Spain, J.C. [Tyndall Air Force Base, FL (United States)

    1995-12-01

    Reduction of nitroaromatic compounds by nitroreductase enzymes generally leads to the formation of the corresponding amines. However, we recently found that the incubation of the explosive 2,4,6-trinitrophenylmethylnitramine (tetryl) with ferredoxin-NADP oxidoreductase, an oxygen sensitive nitroreductase from spinach in the presence of NADPH led to the elimination of the nitramine nitro group from tetryl and the formation of N-methylpicramide (NMP). Other oxygen sensitive nitroreductase enzymes including glutathione reductase, xanthine oxidase, and cytochrome c reductase were also able to release nitrite from tetryl. Nitrite was not eliminated from tetryl by an oxygen insensitive nitrobenzene reductase. For every mole of tetryl reduced, one mole each of nitrite and NMP were produced. The rate of nitrite elimination was inhibited under aerobic conditions. Subsequent oxygen uptake studies suggested that under aerobic conditions, molecular oxygen was reduced by FNR and tetryl served as the redox mediator. Our results suggest that under aerobic conditions; tetryl is reduced to the nitroanion radical by the enzyme and this radical is involved in the reduction of molecular oxygen.

  17. Inhibition of Human Steroid 5-Reductase (AKR1D1) by Finasteride and Structure of the Enzyme-Inhibitor Complex

    Energy Technology Data Exchange (ETDEWEB)

    Drury, J.; Di Costanzo, L; Penning, T; Christianson, D

    2009-01-01

    The {Delta}{sup 4}-3-ketosteroid functionality is present in nearly all steroid hormones apart from estrogens. The first step in functionalization of the A-ring is mediated in humans by steroid 5{alpha}- or 5{beta}-reductase. Finasteride is a mechanism-based inactivator of 5{alpha}-reductase type 2 with subnanomolar affinity and is widely used as a therapeutic for the treatment of benign prostatic hyperplasia. It is also used for androgen deprivation in hormone-dependent prostate carcinoma, and it has been examined as a chemopreventive agent in prostate cancer. The effect of finasteride on steroid 5{beta}-reductase (AKR1D1) has not been previously reported. We show that finasteride competitively inhibits AKR1D1 with low micromolar affinity but does not act as a mechanism-based inactivator. The structure of the AKR1D1 {center_dot} NADP{sup +} {center_dot} finasteride complex determined at 1.7 {angstrom} resolution shows that it is not possible for NADPH to reduce the {Delta}{sup 1-2}-ene of finasteride because the cofactor and steroid are not proximal to each other. The C3-ketone of finasteride accepts hydrogen bonds from the catalytic residues Tyr-58 and Glu-120 in the active site of AKR1D1, providing an explanation for the competitive inhibition observed. This is the first reported structure of finasteride bound to an enzyme involved in steroid hormone metabolism.

  18. Characterization of the osmotic response element of the human aldose reductase gene promoter.

    Science.gov (United States)

    Ruepp, B; Bohren, K M; Gabbay, K H

    1996-08-06

    Aldose reductase (EC 1.1.1.21) catalyzes the NADPH-mediated conversion of glucose to sorbitol. The hyperglycemia of diabetes increases sorbitol production primarily through substrate availability and is thought to contribute to the pathogenesis of many diabetic complications. Increased sorbitol production can also occur at normoglycemic levels via rapid increases in aldose reductase transcription and expression, which have been shown to occur upon exposure of many cell types to hyperosmotic conditions. The induction of aldose reductase transcription and the accumulation of sorbitol, an organic osmolyte, have been shown to be part of the physiological osmoregulatory mechanism whereby renal tubular cells adjust to the intraluminal hyperosmolality during urinary concentration. Previously, to explore the mechanism regulating aldose reductase levels, we partially characterized the human aldose reductase gene promoter present in a 4.2-kb fragment upstream of the transcription initiation start site. A fragment (-192 to +31 bp) was shown to contain several elements that control the basal expression of the enzyme. In this study, we examined the entire 4.2-kb human AR gene promoter fragment by deletion mutagenesis and transfection studies for the presence of osmotic response enhancer elements. An 11-bp nucleotide sequence (TGGAAAATTAC) was located 3.7 kb upstream of the transcription initiation site that mediates hypertonicity-responsive enhancer activity. This osmotic response element (ORE) increased the expression of the chloramphenicol acetyltransferase reporter gene product 2-fold in transfected HepG2 cells exposed to hypertonic NaCl media as compared with isoosmotic media. A more distal homologous sequence is also described; however, this sequence has no osmotic enhancer activity in transfected cells. Specific ORE mutant constructs, gel shift, and DNA fragment competition studies confirm the nature of the element and identify specific nucleotides essential for enhancer

  19. 8-Methoxy-naphtho[2,3-b]thiophen-4,9-quinone, a non-competitive inhibitor of trypanothione reductase

    Directory of Open Access Journals (Sweden)

    Zani Carlos L

    2003-01-01

    Full Text Available The enzyme trypanothione reductase is a recognised drug target in trypanosomatids and has been used in the search of new compounds with potential activity against diseases such as leishmaniasis, Chagas disease and African trypanosomiasis. 8-Methoxy-naphtho [2,3-b] thiophen-4,9-quinone was selected in a screening of natural and synthetic compounds using an in vitro assay with the recombinant enzyme from Trypanosoma cruzi. Its mode of inhibition fits a non-competitive model with respect to the substrate (trypanothione and to the co-factor (NADPH, with Ki-values of 5 and 3.6 µM, respectively. When tested against human glutathione reductase, this compound did not display any significant inhibition at 100 µM, indicating a good selectivity against the parasite enzyme.

  20. NADPH Supply and Mannitol Biosynthesis. Characterization, Cloning, and Regulation of the Non-Reversible Glyceraldehyde-3-Phosphate Dehydrogenase in Celery Leaves1

    Science.gov (United States)

    Gao, Zhifang; Loescher, Wayne H.

    2000-01-01

    Mannitol, a sugar alcohol, is a major primary photosynthetic product in celery (Apium graveolens L. cv Giant Pascal). We report here on purification, characterization, and cDNA cloning of cytosolic non-reversible glyceraldehyde-3-P dehydrogenase (nr-G3PDH, EC 1.2.1.9), the apparent key contributor of the NADPH required for mannitol biosynthesis in celery leaves. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, purified nr-G3PDH showed a molecular mass of 53 kD. A 1,734-bp full-length cDNA clone (accession no. AF196292) encoding nr-G3PDH was identified using polymerase chain reaction and rapid amplification of cDNA ends techniques. The cDNA clone has an open reading frame of 1,491 bp encoding 496 amino acid residues with a calculated molecular weight of 53,172. Km values for the celery nr-G3PDH were low (6.8 μm for NADP+ and 29 μm for d-glyceraldehyde-3-P). NADPH, 3-phosphoglycerate, and ATP were competitive inhibitors, and cytosolic levels of these three metabolites (as determined by nonaqueous fractionation) were all above the concentrations necessary to inhibit activity in vitro, suggesting that nr-G3PDH may be regulated through feedback inhibition by one or more metabolites. We also determined a tight association between activities of nr-G3PDH and mannose-6-P reductase and mRNA expression levels in response to both leaf development and salt treatment. Collectively, our data clearly show metabolic, developmental, and environmental regulation of nr-G3PDH, and also suggest that the supply of NADPH necessary for mannitol biosynthesis is under tight metabolic control. PMID:10982446

  1. X-ray structural studies of quinone reductase 2 nanomolar range inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Pegan, Scott D.; Sturdy, Megan; Ferry, Gilles; Delagrange, Philippe; Boutin, Jean A.; Mesecar, Andrew D. (IdRS); (Purdue); (Colorado); (UIC)

    2011-09-06

    Quinone reductase 2 (QR2) is one of two members comprising the mammalian quinone reductase family of enzymes responsible for performing FAD mediated reductions of quinone substrates. In contrast to quinone reductase 1 (QR1) which uses NAD(P)H as its co-substrate, QR2 utilizes a rare group of hydride donors, N-methyl or N-ribosyl nicotinamide. Several studies have linked QR2 to the generation of quinone free radicals, several neuronal degenerative diseases, and cancer. QR2 has been also identified as the third melatonin receptor (MT3) through in cellulo and in vitro inhibition of QR2 by traditional MT3 ligands, and through recent X-ray structures of human QR2 (hQR2) in complex with melatonin and 2-iodomelatonin. Several MT3 specific ligands have been developed that exhibit both potent in cellulo inhibition of hQR2 nanomolar, affinity for MT3. The potency of these ligands suggest their use as molecular probes for hQR2. However, no definitive correlation between traditionally obtained MT3 ligand affinity and hQR2 inhibition exists limiting our understanding of how these ligands are accommodated in the hQR2 active site. To obtain a clearer relationship between the structures of developed MT3 ligands and their inhibitory properties, in cellulo and in vitro IC{sub 50} values were determined for a representative set of MT3 ligands (MCA-NAT, 2-I-MCANAT, prazosin, S26695, S32797, and S29434). Furthermore, X-ray structures for each of these ligands in complex with hQR2 were determined allowing for a structural evaluation of the binding modes of these ligands in relation to the potency of MT3 ligands.

  2. X-ray structural studies of quinone reductase 2 nanomolar range inhibitors.

    Science.gov (United States)

    Pegan, Scott D; Sturdy, Megan; Ferry, Gilles; Delagrange, Philippe; Boutin, Jean A; Mesecar, Andrew D

    2011-07-01

    Quinone reductase 2 (QR2) is one of two members comprising the mammalian quinone reductase family of enzymes responsible for performing FAD mediated reductions of quinone substrates. In contrast to quinone reductase 1 (QR1) which uses NAD(P)H as its co-substrate, QR2 utilizes a rare group of hydride donors, N-methyl or N-ribosyl nicotinamide. Several studies have linked QR2 to the generation of quinone free radicals, several neuronal degenerative diseases, and cancer. QR2 has been also identified as the third melatonin receptor (MT3) through in cellulo and in vitro inhibition of QR2 by traditional MT3 ligands, and through recent X-ray structures of human QR2 (hQR2) in complex with melatonin and 2-iodomelatonin. Several MT3 specific ligands have been developed that exhibit both potent in cellulo inhibition of hQR2 nanomolar, affinity for MT3. The potency of these ligands suggest their use as molecular probes for hQR2. However, no definitive correlation between traditionally obtained MT3 ligand affinity and hQR2 inhibition exists limiting our understanding of how these ligands are accommodated in the hQR2 active site. To obtain a clearer relationship between the structures of developed MT3 ligands and their inhibitory properties, in cellulo and in vitro IC₅₀ values were determined for a representative set of MT3 ligands (MCA-NAT, 2-I-MCANAT, prazosin, S26695, S32797, and S29434). Furthermore, X-ray structures for each of these ligands in complex with hQR2 were determined allowing for a structural evaluation of the binding modes of these ligands in relation to the potency of MT3 ligands. Copyright © 2011 The Protein Society.

  3. Detoxification of hexavalent chromium by Leucobacter sp. uses a reductase with specificity for dihydrolipoamide.

    Science.gov (United States)

    Sarangi, Abhipsa; Krishnan, Chandraraj

    2016-02-01

    Leucobacter sp. belongs to the metal stressed community and possesses higher tolerance to metals including chromium and can detoxify toxic hexavalent chromium by reduction to less toxic trivalent chromium. But, the mechanism of reduction of hexavalent chromium by Leucobacter sp. has not been studied. Understanding the enzyme catalyzing reduction of chromium is important to improve the species for application in bioremediation. Hence, a soluble reductase catalyzing the reduction of hexavalent chromium was purified from a Leucobacter sp. and characterized. The pure chromate reductase was obtained from the cell-free extract through hydrophobic interaction and gel filtration column chromatographic methods. It was a monomeric enzyme and showed similar molecular weights in both gel filtration (∼68 KDa) and SDS-PAGE (64 KDa). It reduced Cr(VI) using both NADH and NADPH as the electron donor, but exhibited higher activity with NADH. The optimal activity was found at pH 5.5 and 30 °C. The K(m) and V(max) for Cr(VI) reduction with NADH were 46.57 μM and 0.37 μmol min(-1) (mg protein) (-1), respectively. The activity was inhibited by p-hydroxy mercury benzoate, Ag(2+) and Hg(2+) indicating the role of thiol groups in the catalysis. The spectrophotometric analysis of the purified enzyme showed the absence of bound flavin in the enzyme. The N-terminal amino acid sequence and LC/MS analysis of trypsin digested purified enzyme showed similarity to dihydrolipoyl dehydrogenase. The purified enzyme had dihydrolipoyl dehydrogenase activity with dihydrolipoamide as the substrate, which suggested that Leucobacter sp. uses reductase with multiple substrate specificity for reduction of Cr(VI) detoxification. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Inhibition of aldo-keto reductase family 1 member B10 by unsaturated fatty acids.

    Science.gov (United States)

    Hara, Akira; Endo, Satoshi; Matsunaga, Toshiyuki; Soda, Midori; El-Kabbani, Ossama; Yashiro, Koji

    2016-11-01

    A human member of the aldo-keto reductase (AKR) superfamily, AKR1B10, is a cytosolic NADPH-dependent reductase toward various carbonyl compounds including reactive aldehydes, and is normally expressed in intestines. The enzyme is overexpressed in several extraintestinal cancers, and suggested as a potential target for cancer treatment. We found that saturated and cis-unsaturated fatty acids inhibit AKR1B10. Among the saturated fatty acids, myristic acid was the most potent, showing the IC 50 value of 4.2 μM cis-Unsaturated fatty acids inhibited AKR1B10 more potently, and linoleic, arachidonic, and docosahexaenoic acids showed the lowest IC 50 values of 1.1 μM. The inhibition by these fatty acids was reversible and kinetically competitive with respect to the substrate, showing the K i values of 0.24-1.1 μM. These fatty acids, except for α-linoleic acid, were much less inhibitory to structurally similar aldose reductase. Site-directed mutagenesis study suggested that the fatty acids interact with several active site residues of AKR1B10, of which Gln114, Val301 and Gln303 are responsible for the inhibitory selectivity. Linoleic and arachidonic acids also effectively inhibited AKR1B10-mediated 4-oxo-2-nonenal metabolism in HCT-15 cells. Thus, the cis-unsaturated fatty acids may be used as an adjuvant therapy for treatment of cancers that up-regulate AKR1B10. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Sunflower (Helianthus annuus) fatty acid synthase complex: enoyl-[acyl carrier protein]-reductase genes.

    Science.gov (United States)

    González-Thuillier, Irene; Venegas-Calerón, Mónica; Garcés, Rafael; von Wettstein-Knowles, Penny; Martínez-Force, Enrique

    2015-01-01

    Enoyl-[acyl carrier protein]-reductases from sunflower. A major factor contributing to the amount of fatty acids in plant oils are the first steps of their synthesis. The intraplastidic fatty acid biosynthetic pathway in plants is catalysed by type II fatty acid synthase (FAS). The last step in each elongation cycle is carried out by the enoyl-[ACP]-reductase, which reduces the dehydrated product of β-hydroxyacyl-[ACP] dehydrase using NADPH or NADH. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus) seeds, two enoyl-[ACP]-reductase genes have been identified and cloned from developing seeds with 75 % identity: HaENR1 (GenBank HM021137) and HaENR2 (HM021138). The two genes belong to the ENRA and ENRB families in dicotyledons, respectively. The genetic duplication most likely originated after the separation of di- and monocotyledons. RT-qPCR revealed distinct tissue-specific expression patterns. Highest expression of HaENR1 was in roots, stems and developing cotyledons whereas that of H a ENR2 was in leaves and early stages of seed development. Genomic DNA gel blot analyses suggest that both are single-copy genes. In vivo activity of the ENR enzymes was tested by complementation experiments with the JP1111 fabI(ts) E. coli strain. Both enzymes were functional demonstrating that they interacted with the bacterial FAS components. That different fatty acid profiles resulted infers that the two Helianthus proteins have different structures, substrate specificities and/or reaction rates. The latter possibility was confirmed by in vitro analysis with affinity-purified heterologous-expressed enzymes that reduced the crotonyl-CoA substrate using NADH with different V max.

  6. Direct antioxidant properties of bilirubin andbiliverdin. Is there a role for biliverdin reductase?

    Directory of Open Access Journals (Sweden)

    Thomas eJansen

    2012-03-01

    Full Text Available Reactive oxygen species (ROS and signaling events are involved in the pathogenesis of endothelial dysfunction and represent a major contribution to vascular regulation. Molecular signaling is highly dependent on reactive oxygen species. But depending on the amount of ROS production it might have toxic or protective effects. Despite a large number of negative outcomes in large clinical trials (e.g. HOPE, HOPE-TOO, antioxidant molecules and agents are important players to influence the critical balance between production and elimination of RONS. However, chronic systemic antioxidant therapy lacks clinical efficacy, probably by interfering with important physiological redox signaling pathways. Therefore, it may be a much more promising attempt to induce intrinsic antioxidant pathways in order to increase the antioxidants not systemically but at the place of oxidative stress and complications. Among others, heme oxygenase (HO has been shown to be important for attenuating the overall production of ROS in a broad range of disease states through its ability to degrade heme and to produce carbon monoxide (CO, biliverdin/bilirubin, and the release of free iron with subsequent ferritin induction. With the present review we would like to highlight the important antioxidant role of the heme oxygenase system and especially discuss the contribution of the biliverdin, bilirubin and biliverdin reductase to these beneficial effects. The bilierdin reductase was reported to confer an antioxidant redox amplification cycle by which low, physiological bilirubin concentrations confer potent antioxidant protection via recycling of biliverdin from oxidized bilirubin by the biliverdin reductase, linking this sink for oxidants to the NADPH pool. To date the existence and role of this antioxidant redox cycle is still under debate and we present and discuss the pros and cons as well as our own findings on this topic.

  7. fHANT-AC genes of the ectomycorrhizal fungus Laccaria bicolor are not repressed by l-glutamine allowing simultaneous utilization of nitrate and organic nitrogen sources.

    Science.gov (United States)

    Kemppainen, Minna J; Alvarez Crespo, Maria C; Pardo, Alejandro G

    2010-08-01

    In boreal and temperate forest ectomycorrhizal fungi play a crucial role in nitrogen cycling by assimilating nitrogenous compounds from soil and transferring them to tree hosts. The expression profile of fHANT-AC genes, nitrate transporter (Lbnrt), nitrate reductase (Lbnr) and nitrite reductase (Lbnir), responsible for nitrate utilization in the ectomycorrhizal fungus Laccaria bicolor, was studied on variable N regimens. The three genes were shown to be under a common regulation: repressed in the presence of ammonium while growth on nitrate resulted in high transcripts accumulation. The presence of nitrate was shown not to be indispensable for activation of Laccaria fHANT-AC as also N starvation and growth on urea and l-asparagine resulted in high transcript levels. Equally high expression of Laccaria fHANT-AC genes was detected in mycelia grown on variable concentrations of l-glutamine. This finding shows that in L. bicolor N metabolite repression of fHANT-AC is not signalled via l-glutamine like described in ascomycetes. The expression patterns of Lbnrt and Lbnir were also studied in an Lbnr RNA-silenced Laccaria strain. No differences were observed on the N source regulation or the degree of transcript accumulation of these genes, indicating that the presence of high nitrate reductase activity is not a core regulator of L. bicolor fHANT-AC expression. The simultaneous utilization of nitrate and organic N sources, already suggested by high transcript levels of Laccaria fHANT-AC genes on organic N, was supported by the increase of culture medium pH as a result of nitrate transporter activity. The possible ecological and evolutionary significance of the herein reported high regulatory flexibility of Laccaria nitrate utilization pathway for ectomycorrizal fungi and the ectomycorrhizal symbiosis is discussed. © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd.

  8. dl-Asparaginium nitrate

    Directory of Open Access Journals (Sweden)

    Nabila Moussa Slimane

    2009-09-01

    Full Text Available In the title compound, C4H9N2O3+·NO3−, alternatively called (1RS-2-carbamoyl-1-carboxyethanaminium nitrate, the asymmetric unit comprises one asparaginium cation and one nitrate anion. The strongest cation–cation O—H...O hydrogen bond in the structure, together with other strong cation–cation N—H...O hydrogen bonds, generates a succession of infinite chains of R22(8 rings along the b axis. Additional cation–cation C—H...O hydrogen bonds link these chains into two-dimensional layers formed by alternating R44(24 and R42(12 rings. Connections between these layers are provided by the strong cation–anion N—H...O hydrogen bonds, as well as by one weak C—H...O interaction, thus forming a three-dimensional network. Some of the cation–anion N—H...O hydrogen bonds are bifurcated of the type D—H...(A1,A2.

  9. Dicumarol inhibition of NADPH:quinone oxidoreductase induces growth inhibition of pancreatic cancer via a superoxide-mediated mechanism.

    Science.gov (United States)

    Cullen, Joseph J; Hinkhouse, Marilyn M; Grady, Matthew; Gaut, Andrew W; Liu, Jingru; Zhang, Yu Ping; Weydert, Christine J Darby; Domann, Frederick E; Oberley, Larry W

    2003-09-01

    NADPH:quinone oxidoreductase (NQO(1)), a homodimeric, ubiquitous, flavoprotein, catalyzes the two-electron reduction of quinones to hydroquinones. This reaction prevents the one-electron reduction of quinones by cytochrome P450 reductase and other flavoproteins that would result in oxidative cycling with generation of superoxide (O(2)(.-)). NQO(1) gene regulation may be up-regulated in some tumors to accommodate the needs of rapidly metabolizing cells to regenerate NAD(+). We hypothesized that pancreatic cancer cells would exhibit high levels of this enzyme, and inhibiting it would suppress the malignant phenotype. Reverse transcription-PCR, Western blots, and activity assays demonstrated that NQO(1) was up-regulated in the pancreatic cancer cell lines tested but present in very low amounts in the normal human pancreas. To determine whether inhibition of NQO(1) would alter the malignant phenotype, MIA PaCa-2 pancreatic cancer cells were treated with a selective inhibitor of NQO(1), dicumarol. Dicumarol increased intracellular production of O(2)(.-), as measured by hydroethidine staining, and inhibited cell growth. Both of these effects were blunted with infection of an adenoviral vector containing the cDNA for manganese superoxide dismutase. Dicumarol also inhibited cell growth, plating efficiency, and growth in soft agar. We conclude that inhibition of NQO(1) increases intracellular O(2)(.-) production and inhibits the in vitro malignant phenotype of pancreatic cancer. These mechanisms suggest that altering the intracellular redox environment of pancreatic cancer cells may inhibit growth and delineate a potential strategy directed against pancreatic cancer.

  10. Disguised as a Sulfate Reducer: Growth of the Deltaproteobacterium Desulfurivibrio alkaliphilus by Sulfide Oxidation with Nitrate.

    Science.gov (United States)

    Thorup, Casper; Schramm, Andreas; Findlay, Alyssa J; Finster, Kai W; Schreiber, Lars

    2017-07-18

    This study demonstrates that the deltaproteobacterium Desulfurivibrio alkaliphilus can grow chemolithotrophically by coupling sulfide oxidation to the dissimilatory reduction of nitrate and nitrite to ammonium. Key genes of known sulfide oxidation pathways are absent from the genome of D. alkaliphilus Instead, the genome contains all of the genes necessary for sulfate reduction, including a gene for a reductive-type dissimilatory bisulfite reductase (DSR). Despite this, growth by sulfate reduction was not observed. Transcriptomic analysis revealed a very high expression level of sulfate-reduction genes during growth by sulfide oxidation, while inhibition experiments with molybdate pointed to elemental sulfur/polysulfides as intermediates. Consequently, we propose that D. alkaliphilus initially oxidizes sulfide to elemental sulfur, which is then either disproportionated, or oxidized by a reversal of the sulfate reduction pathway. This is the first study providing evidence that a reductive-type DSR is involved in a sulfide oxidation pathway. Transcriptome sequencing further suggests that nitrate reduction to ammonium is performed by a novel type of periplasmic nitrate reductase and an unusual membrane-anchored nitrite reductase. IMPORTANCE Sulfide oxidation and sulfate reduction, the two major branches of the sulfur cycle, are usually ascribed to distinct sets of microbes with distinct diagnostic genes. Here we show a more complex picture, as D. alkaliphilus , with the genomic setup of a sulfate reducer, grows by sulfide oxidation. The high expression of genes typically involved in the sulfate reduction pathway suggests that these genes, including the reductive-type dissimilatory bisulfite reductases, are also involved in as-yet-unresolved sulfide oxidation pathways. Finally, D. alkaliphilus is closely related to cable bacteria, which grow by electrogenic sulfide oxidation. Since there are no pure cultures of cable bacteria, D. alkaliphilus may represent an

  11. Nitrate storage and dissimilatory nitrate reduction by eukaryotic microbes

    DEFF Research Database (Denmark)

    Kamp, Anja; Høgslund, Signe; Risgaard-Petersen, Nils

    2015-01-01

    The microbial nitrogen cycle is one of the most complex and environmentally important element cycles on Earth and has long been thought to be mediated exclusively by prokaryotic microbes. Rather recently, it was discovered that certain eukaryotic microbes are able to store nitrate intracellularly...... and use it for dissimilatory nitrate reduction in the absence of oxygen. The paradigm shift that this entailed is ecologically significant because the eukaryotes in question comprise global players like diatoms, foraminifers, and fungi. This review article provides an unprecedented overview of nitrate...... storage and dissimilatory nitrate reduction by diverse marine eukaryotes placed into an eco-physiological context. The advantage of intracellular nitrate storage for anaerobic energy conservation in oxygen-depleted habitats is explained and the life style enabled by this metabolic trait is described...

  12. Thermodynamic and NMR analyses of NADPH binding to lipocalin-type prostaglandin D synthase

    Energy Technology Data Exchange (ETDEWEB)

    Qin, Shubin [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Shimamoto, Shigeru [Faculty of Science and Engineering, Kinki University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502 (Japan); Maruno, Takahiro; Kobayashi, Yuji [Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Kawahara, Kazuki; Yoshida, Takuya [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Ohkubo, Tadayasu, E-mail: ohkubo@phs.osaka-u.ac.jp [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2015-12-04

    Lipocalin-type prostaglandin D synthase (L-PGDS) is one of the most abundant proteins in human cerebrospinal fluid (CSF) with dual functions as a prostaglandin D{sub 2} (PGD{sub 2}) synthase and a transporter of lipophilic ligands. Recent studies revealed that L-PGDS plays important roles in protecting against various neuronal diseases induced by reactive oxygen species (ROS). However, the molecular mechanisms of such protective actions of L-PGDS remain unknown. In this study, we conducted thermodynamic and nuclear magnetic resonance (NMR) analyses, and demonstrated that L-PGDS binds to nicotinamide coenzymes, including NADPH, NADP{sup +}, and NADH. Although a hydrophilic ligand is not common for L-PGDS, these ligands, especially NADPH showed specific interaction with L-PGDS at the upper pocket of its ligand-binding cavity with an unusually bifurcated shape. The binding affinity of L-PGDS for NADPH was comparable to that previously reported for NADPH oxidases and NADPH in vitro. These results suggested that L-PGDS potentially attenuates the activities of NADPH oxidases through interaction with NADPH. Given that NADPH is the substrate for NADPH oxidases that play key roles in neuronal cell death by generating excessive ROS, these results imply a novel linkage between L-PGDS and ROS. - Highlights: • Interactions of L-PGDS with nicotinamide coenzymes were studied by ITC and NMR. • The binding affinity of L-PGDS was strongest to NADPH among nicotinamide coenzymes. • NADPH binds to the upper part of L-PGDS ligand-binding cavity. • L-PGDS binds to both lipophilic and hydrophilic ligands. • This study implies a novel linkage between L-PGDS and reactive oxygen species.

  13. 33 CFR 126.28 - Ammonium nitrate, ammonium nitrate fertilizers, fertilizer mixtures, or nitro carbo nitrate...

    Science.gov (United States)

    2010-07-01

    ..., combustible liquids, corrosive liquids, chlorates, permanganates, finely divided metals, caustic soda... molten ammonium nitrate if a fire occurred (and thus become potential detonators for the storage piles...

  14. YNL134C from Saccharomyces cerevisiae encodes a novel protein with aldehyde reductase activity for detoxification of furfural derived from lignocellulosic biomass.

    Science.gov (United States)

    Zhao, Xianxian; Tang, Juan; Wang, Xu; Yang, Ruoheng; Zhang, Xiaoping; Gu, Yunfu; Li, Xi; Ma, Menggen

    2015-05-01

    Furfural and 5-hydroxymethylfurfural (HMF) are the two main aldehyde compounds derived from pentoses and hexoses, respectively, during lignocellulosic biomass pretreatment. These two compounds inhibit microbial growth and interfere with subsequent alcohol fermentation. Saccharomyces cerevisiae has the in situ ability to detoxify furfural and HMF to the less toxic 2-furanmethanol (FM) and furan-2,5-dimethanol (FDM), respectively. Herein, we report that an uncharacterized gene, YNL134C, was highly up-regulated under furfural or HMF stress and Yap1p and Msn2/4p transcription factors likely controlled its up-regulated expression. Enzyme activity assays showed that YNL134C is an NADH-dependent aldehyde reductase, which plays a role in detoxification of furfural to FM. However, no NADH- or NADPH-dependent enzyme activity was observed for detoxification of HMF to FDM. This enzyme did not catalyse the reverse reaction of FM to furfural or FDM to HMF. Further studies showed that YNL134C is a broad-substrate aldehyde reductase, which can reduce multiple aldehydes to their corresponding alcohols. Although YNL134C is grouped into the quinone oxidoreductase family, no quinone reductase activity was observed using 1,2-naphthoquinone or 9,10-phenanthrenequinone as a substrate, and phylogenetic analysis indicates that it is genetically distant to quinone reductases. Proteins similar to YNL134C in sequence from S. cerevisiae and other microorganisms were phylogenetically analysed. Copyright © 2015 John Wiley & Sons, Ltd.

  15. Engineering an NADPH/NADP+ Redox Biosensor in Yeast

    DEFF Research Database (Denmark)

    Zhang, Jie; Sonnenschein, Nikolaus; Pihl, Thomas Peter Boye

    2016-01-01

    Genetically encoded biosensors have emerged as powerful tools for timely and precise in vivo evaluation of cellular metabolism. In particular, biosensors that can couple intercellular cues with downstream signaling responses are currently attracting major attention within health science and biote......Genetically encoded biosensors have emerged as powerful tools for timely and precise in vivo evaluation of cellular metabolism. In particular, biosensors that can couple intercellular cues with downstream signaling responses are currently attracting major attention within health science...... and biotechnology. Still, there is a need for bioprospecting and engineering of more biosensors to enable real-time monitoring of specific cellular states and controlling downstream actuation. In this study, we report the engineering and application of a transcription factor-based NADPH/NADP+ redox biosensor...... in the budding yeast Saccharomyces cerevisiae. Using the biosensor, we are able to monitor the cause of oxidative stress by chemical induction, and changes in NADPH/NADP+ ratios caused by genetic manipulations. Because of the regulatory potential of the biosensor, we also show that the biosensor can actuate upon...

  16. Engineered P450 biocatalysts show improved activity and regio-promiscuity in aromatic nitration.

    Science.gov (United States)

    Zuo, Ran; Zhang, Yi; Jiang, Chao; Hackett, John C; Loria, Rosemary; Bruner, Steven D; Ding, Yousong

    2017-04-12

    Nitroaromatics are among the most important and commonly used chemicals but their production often suffers from multiple unsolved challenges. We have previously described the development of biocatalytic nitration processes driven by an engineered P450 TxtE fusion construct. Herein we report the creation of improved nitration biocatalysts through constructing and characterizing fusion proteins of TxtE with the reductase domain of CYP102A1 (P450BM3, BM3R). The majority of constructs contained variable linker length while one was rationally designed for optimizing protein-protein interactions. Detailed biochemical characterization identified multiple active chimeras that showed improved nitration activity, increased coupling efficiency and higher total turnover numbers compared with TxtE. Substrate promiscuity of the most active chimera was further assessed with a substrate library. Finally, a biocatalytic nitration process was developed to nitrate 4-Me-DL-Trp. The production of both 4-Me-5-NO 2 -L-Trp and 4-Me-7-NO 2 -L-Trp uncovered remarkable regio-promiscuity of nitration biocatalysts.

  17. Function of Ubiquinone in Electron Transport from Reduced Nicotinamide Adenine Dinucleotide to Nitrate and Oxygen in Aerobacter aerogenes

    Science.gov (United States)

    Knook, D. L.; Planta, R. J.

    1971-01-01

    The possible role of quinones in the electron transport system of Aerobacter aerogenes was investigated. The only quinone found in measurable amounts in bacteria grown in minimal media under both aerobic and anaerobic conditions was ubiquinone-8. Membrane-bound ubiquinone-8 could be removed by extraction with pentane, or destroyed by ultraviolet irradiation, with a concomitant loss of both reduced nicotinamide adenine dinucleotide (NADH) oxidase and NADH-linked respiratory nitrate reductase activity. In the extracted membrane preparations, these enzymatic activities could be restored, both to the same degree, by incorporation of ubiquinone-6, -8, or -10, but not by incorporation of menaquinones. The NADH oxidation and the nitrate reduction were sensitive to the respiratory inhibitors dicoumarol, lapachol, and cyanide. The results obtained indicate that ubiquinone-8 mediates the electron transport between NADH and oxygen as well as between NADH and nitrate. Branching of the electron transport chain to oxygen and nitrate occurs after an initial common pathway. PMID:4100202

  18. Decomposition of metal nitrate solutions

    International Nuclear Information System (INIS)

    Haas, P.A.; Stines, W.B.

    1982-01-01

    Oxides in powder form are obtained from aqueous solutions of one or more heavy metal nitrates (e.g. U, Pu, Th, Ce) by thermal decomposition at 300 to 800 deg C in the presence of about 50 to 500% molar concentration of ammonium nitrate to total metal. (author)

  19. Evaluation of nitrate destruction methods

    International Nuclear Information System (INIS)

    Taylor, P.A.; Kurath, D.E.; Guenther, R.

    1993-01-01

    A wide variety of high nitrate-concentration aqueous mixed [radioactive and Resource Conservation and Recovery Act (RCRA) hazardous] wastes are stored at various US Department of Energy (DOE) facilities. These wastes will ultimately be solidified for final disposal, although the waste acceptance criteria for the final waste form is still being determined. Because the nitrates in the wastes will normally increase the volume or reduce the integrity of all of the waste forms under consideration for final disposal, nitrate destruction before solidification of the waste will generally be beneficial. This report describes and evaluates various technologies that could be used to destroy the nitrates in the stored wastes. This work was funded by the Department of Energy's Office of Technology Development, through the Chemical/Physical Technology Support Group of the Mixed Waste Integrated Program. All the nitrate destruction technologies will require further development work before a facility could be designed and built to treat the majority of the stored wastes. Several of the technologies have particularly attractive features: the nitrate to ammonia and ceramic (NAC) process produces an insoluble waste form with a significant volume reduction, electrochemical reduction destroys nitrates without any chemical addition, and the hydrothermal process can simultaneously treat nitrates and organics in both acidic and alkaline wastes. These three technologies have been tested using lab-scale equipment and surrogate solutions. At their current state of development, it is not possible to predict which process will be the most beneficial for a particular waste stream

  20. Malic enzyme tracers reveal hypoxia-induced switch in adipocyte NADPH pathway usage.

    Science.gov (United States)

    Liu, Ling; Shah, Supriya; Fan, Jing; Park, Junyoung O; Wellen, Kathryn E; Rabinowitz, Joshua D

    2016-05-01

    The critical cellular hydride donor NADPH is produced through various means, including the oxidative pentose phosphate pathway (oxPPP), folate metabolism and malic enzyme. In growing cells, it is efficient to produce NADPH via the oxPPP and folate metabolism, which also make nucleotide precursors. In nonproliferating adipocytes, a metabolic cycle involving malic enzyme holds the potential to make both NADPH and two-carbon units for fat synthesis. Recently developed deuterium ((2)H) tracer methods have enabled direct measurement of NADPH production by the oxPPP and folate metabolism. Here we enable tracking of NADPH production by malic enzyme with [2,2,3,3-(2)H]dimethyl-succinate and [4-(2)H]glucose. Using these tracers, we show that most NADPH in differentiating 3T3-L1 mouse adipocytes is made by malic enzyme. The associated metabolic cycle is disrupted by hypoxia, which switches the main adipocyte NADPH source to the oxPPP. Thus, (2)H-labeled tracers enable dissection of NADPH production routes across cell types and environmental conditions.

  1. Dietary nitrate improves age-related hypertension and metabolic abnormalities in rats via modulation of angiotensin II receptor signaling and inhibition of superoxide generation

    DEFF Research Database (Denmark)

    Hezel, M.; Peleli, Maria; Liu, M.

    2016-01-01

    . Finally, nitrate treatment in aged rats normalized the gene expression profile of ANG II receptors (AT1A, AT2, AT1A/AT2 ratio) in the renal and cardiovascular systems without altering plasma levels of renin or ANG II. Our results show that boosting the nitrate-nitrite-NO pathway can partly compensate...... that increased angiotensin II (ANG II) signaling is also implicated in the pathogenesis of endothelial dysfunction and hypertension by accelerating formation of reactive oxygen species. This study was designed to test the hypothesis that dietary nitrate supplementation could reduce blood pressure and improve...... glucose tolerance in aged rats, via attenuation of NADPH oxidase activity and ANG II receptor signaling. Dietary nitrate supplementation for two weeks reduced blood pressure (10–15 mmHg) and improved glucose clearance in old, but not in young rats. These favorable effects were associated with increased...

  2. NADPH Oxidase Activation Contributes to Heavy Ion Irradiation–Induced Cell Death

    Directory of Open Access Journals (Sweden)

    Yupei Wang

    2017-03-01

    Full Text Available Increased oxidative stress plays an important role in heavy ion radiation–induced cell death. The mechanism involved in the generation of elevated reactive oxygen species (ROS is not fully illustrated. Here we show that NADPH oxidase activation is closely related to heavy ion radiation–induced cell death via excessive ROS generation. Cell death and cellular ROS can be greatly reduced in irradiated cancer cells with the preincubation of diphenyleneiodium, an inhibitor of NADPH oxidase. Most of the NADPH oxidase (NOX family proteins (NOX1, NOX2, NOX3, NOX4, and NOX5 showed increased expression after heavy ion irradiation. Meanwhile, the cytoplasmic subunit p47phox was translocated to the cell membrane and localized with NOX2 to form reactive NADPH oxidase. Our data suggest for the first time that ROS generation, as mediated by NADPH oxidase activation, could be an important contributor to heavy ion irradiation–induced cell death.

  3. Redox engineering by ectopic expression of glutamate dehydrogenase genes links NADPH availability and NADH oxidation with cold growth in Saccharomyces cerevisiae.

    Science.gov (United States)

    Ballester-Tomás, Lidia; Randez-Gil, Francisca; Pérez-Torrado, Roberto; Prieto, Jose Antonio

    2015-07-09

    Cold stress reduces microbial growth and metabolism being relevant in industrial processes like wine making and brewing. Knowledge on the cold transcriptional response of Saccharomyces cerevisiae suggests the need of a proper redox balance. Nevertheless, there are no direct evidence of the links between NAD(P) levels and cold growth and how engineering of enzymatic reactions requiring NAD(P) may be used to modify the performance of industrial strains at low temperature. Recombinant strains of S. cerevisiae modified for increased NADPH- and NADH-dependent Gdh1 and Gdh2 activity were tested for growth at low temperature. A high-copy number of the GDH2-encoded glutamate dehydrogenase gene stimulated growth at 15°C, while overexpression of GDH1 had detrimental effects, a difference likely caused by cofactor preferences. Indeed, neither the Trp(-) character of the tested strains, which could affect the synthesis of NAD(P), nor changes in oxidative stress susceptibility by overexpression of GDH1 and GDH2 account for the observed phenotypes. However, increased or reduced NADPH availability by knock-out or overexpression of GRE3, the NADPH-dependent aldose reductase gene, eliminated or exacerbated the cold-growth defect observed in YEpGDH1 cells. We also demonstrated that decreased capacity of glycerol production impairs growth at 15 but not at 30°C and that 15°C-grown baker's yeast cells display higher fermentative capacity than those cultivated at 30°C. Thus, increasing NADH oxidation by overexpression of GDH2 would help to avoid perturbations in the redox metabolism induced by a higher fermentative/oxidative balance at low temperature. Finally, it is shown that overexpression of GDH2 increases notably the cold growth in the wine yeast strain QA23 in both standard growth medium and synthetic grape must. Redox constraints limit the growth of S. cerevisiae at temperatures below the optimal. An adequate supply of NAD(P) precursors as well as a proper level of reducing

  4. Studying solubility of praseodymium(3) nitrate with nitrates of aniline, guanidine in aqueous solution

    International Nuclear Information System (INIS)

    Kuznetsova, L.S.; Zhuravlev, E.F.

    1977-01-01

    The solubility in the systems praseodymium nitrate-aniline nitrate-water(1) and praseodymium nitrate-guanidine nitrate-water(2) has been studied at 20 and 40 deg C. In the given temperature range the state diagrams of the systems correspond to the isotherms of the simple eutonic type. Aniline nitrate and hexahydrate of praseodymium nitrate in the first system and guanidine nitrate and hexahydrate of praseodymium nitrate in the second system are equilibrium solid phases. The most developed crystallization fields are those of amine nitrates; eutonics are enriched with praseodymium nitrate

  5. Low level nitrate or nitroethane preconditioning enhances the bactericidal effect of suboptimal experimental chlorate treatment against Escherichia coli and Salmonella Typhimurium but not Campylobacter in swine.

    Science.gov (United States)

    Anderson, Robin C; Jung, Yong Soo; Genovese, Kenneth J; McReynolds, Jackson L; Callaway, Todd R; Edrington, Thomas S; Harvey, Roger B; Nisbet, David J

    2006-01-01

    An experimental chlorate product that targets the respiratory nitrate reductase enzyme of bacteria such as Salmonella and Escherichia coli has shown promising results in reducing concentrations of these bacteria in the gut of food animals. Because expression of the target enzyme is induced by nitrate, we administered short-duration, low level nitrate or nitroethane preconditioning treatments to finishing swine to see if these would enhance the ability of an experimental chlorate product to kill these bacteria. Results from these studies showed that preconditioning the gut microflora of swine with low levels of nitrate or nitrocompounds enhanced (more than tenfold) the ability of the chlorate product to kill Salmonella and E. coli, but not Campylobacter. Further studies are needed before these compounds can be fed as feed additives to animals, although it is likely that nitrate preconditioning may be more near to market than the nitrocompounds, which may require more comprehensive review by regulatory authorities.

  6. A new strategy to improve the efficiency and sustainability of Candida parapsilosis catalyzing deracemization of (R,S)-1-phenyl-1,2-ethanediol under non-growing conditions: increase of NADPH availability.

    Science.gov (United States)

    Nie, Yao; Xu, Yan; Hu, Qing Sen; Xiao, Rong

    2009-01-01

    Microbial oxidoreductive systems have been widely used in asymmetric syntheses of optically active alcohols. However, when reused in multi-batch reaction, the catalytic efficiency and sustainability of non-growing cells usually decreased because of continuous consumption of required cofactors during the reaction process. A novel method for NADPH regeneration in cells was proposed by using pentose metabolism in microorganisms. Addition of D-xylose, L-arabinose, or D-ribose to the reaction significantly improved the conversion efficiency of deracemization of racemic 1-phenyl-1,2-ethanediol to (S)-isomer by Candida parapsilosis cells already used once, which afforded the product with high optical purity over 97%e.e. in high yield over 85% under an increased substrate concentration of 15 g/l. Compared with reactions without xylose, xylose added to multi-batch reactions had no influence on the activity of the enzyme catalyzing the key step in deracemization, but performed a promoting effect on the recovery of the metabolic activity of the non-growing cells with its consumption in each batch. The detection of activities of xylose reductase and xylitol dehydrogenase from cell-free extract of C. parapsilosis made xylose metabolism feasible in cells, and the depression of the pentose phosphate pathway inhibitor to this reaction further indicated that xylose facilitated the NADPH-required deracemization through the pentose phosphate pathway in C. parapsilosis. moreover, by investigating the cofactor pool, the xylose addition in reaction batches giving more NADPH, compared with those without xylose, suggested that the higher catalytic efficiency and sustainability of C. parapsilosis non-growing cells had resulted from xylose metabolism recycling NADPH for the deracemization.

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

    Science.gov (United States)

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

    2015-06-05

    Short-chain dehydrogenases/reductases (SDRs) constitute a large, functionally diverse branch of enzymes within the class of NAD(P)(H) dependent oxidoreductases. In humans, over 80 genes have been identified with distinct metabolic roles in carbohydrate, amino acid, lipid, retinoid and steroid hormone metabolism, frequently associated with inherited genetic defects. Besides metabolic functions, a subset of atypical SDR proteins appears to play critical roles in adapting to redox status or RNA processing, and thereby controlling metabolic pathways. Here we present an update on the human SDR superfamily and a ligand identification strategy using differential scanning fluorimetry (DSF) with a focused library of oxidoreductase and metabolic ligands to identify substrate classes and inhibitor chemotypes. This method is applicable to investigate structure-activity relationships of oxidoreductases and ultimately to better understand their physiological roles. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  8. Differentially regulated NADPH: cytochrome p450 oxidoreductases in parsely

    International Nuclear Information System (INIS)

    Koopmann, E.; Hahlbrock, K.

    1997-01-01

    Two NADPH:cytochrome P450 oxidoreductases (CPRs) from parsley (Petroselinum crispum) were cloned, and the complete proteins were expressed and functionally identified in yeast. The two enzymes, designated CPR1 and CPR2, are 80% identical in amino acid sequence with one another and about 75% identical with CPRs from several other plant species. The mRNA accumulation patterns for CPR1 and CPR2 in fungal elicitor-treated or UV-irradiated cultured parsley cells and in developing or infected parsley plants were compared with those for cinnamate 4-hydroxylase (C4H), one of the most abundant CPR-dependent P450 enzymes in plants. All treatments strongly induced the mRNAs for C4H and CPR1 but not for CPR2, suggesting distinct metabolic roles of CPR1 and CPR2 and a functional relationship between CPR1 and C4H

  9. Traumatic Brain Injury and NADPH Oxidase: A Deep Relationship

    Directory of Open Access Journals (Sweden)

    Cristina Angeloni

    2015-01-01

    Full Text Available Traumatic brain injury (TBI represents one of the major causes of mortality and disability in the world. TBI is characterized by primary damage resulting from the mechanical forces applied to the head as a direct result of the trauma and by the subsequent secondary injury due to a complex cascade of biochemical events that eventually lead to neuronal cell death. Oxidative stress plays a pivotal role in the genesis of the delayed harmful effects contributing to permanent damage. NADPH oxidases (Nox, ubiquitary membrane multisubunit enzymes whose unique function is the production of reactive oxygen species (ROS, have been shown to be a major source of ROS in the brain and to be involved in several neurological diseases. Emerging evidence demonstrates that Nox is upregulated after TBI, suggesting Nox critical role in the onset and development of this pathology. In this review, we summarize the current evidence about the role of Nox enzymes in the pathophysiology of TBI.

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

    International Nuclear Information System (INIS)

    Moon, Jeong Chan; Jang, Ho Hee; Chae, Ho Byoung; Lee, Jung Ro; Lee, Sun Yong; Jung, Young Jun; Shin, Mi Rim; Lim, Hye Song; Chung, Woo Sik; Yun, Dae-Jin; Lee, Kyun Oh; Lee, Sang Yeol

    2006-01-01

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

  11. Engineering Cofactor Preference of Ketone Reducing Biocatalysts: A Mutagenesis Study on a γ-Diketone Reductase from the Yeast Saccharomyces cerevisiae Serving as an Example

    Directory of Open Access Journals (Sweden)

    Michael Katzberg

    2010-04-01

    Full Text Available The synthesis of pharmaceuticals and catalysts more and more relies on enantiopure chiral building blocks. These can be produced in an environmentally benign and efficient way via bioreduction of prochiral ketones catalyzed by dehydrogenases. A productive source of these biocatalysts is the yeast Saccharomyces cerevisiae, whose genome also encodes a reductase catalyzing the sequential reduction of the γ-diketone 2,5-hexanedione furnishing the diol (2S,5S-hexanediol and the γ-hydroxyketone (5S-hydroxy-2-hexanone in high enantio- as well as diastereoselectivity (ee and de >99.5%. This enzyme prefers NADPH as the hydrogen donating cofactor. As NADH is more stable and cheaper than NADPH it would be more effective if NADH could be used in cell-free bioreduction systems. To achieve this, the cofactor binding site of the dehydrogenase was altered by site-directed mutagenesis. The results show that the rational approach based on a homology model of the enzyme allowed us to generate a mutant enzyme having a relaxed cofactor preference and thus is able to use both NADPH and NADH. Results obtained from other mutants are discussed and point towards the limits of rationally designed mutants.

  12. Variability of nitrate and phosphate

    Digital Repository Service at National Institute of Oceanography (India)

    Sardessai, S.; Sundar, D.

    and the high-nitrate waters farther upstream led to a sharp increase in nitrates in the first few kilometres from the mouth of the Mandovi. Thus, at the end of the summer monsoon, the channels of the Mandovi and Zuari have nitrate concentrations of the order... in June. Throughout this period, the properties of the waters at the mouth migrate upstream owing to horizontal mixing. One consequence of this mixing is a steady rise in salinity in the channels of the Mandovi and Zuari. Since the concentration June 13...

  13. Methylenetetrahydrofolate reductase A1298C polymorphism and ...

    African Journals Online (AJOL)

    Methylenetetrahydrofolate reductase A1298C polymorphism and breast cancer risk: A meta analysis of 33 studies. ... were searched for case‑control studies relating the association between MTHFR A1298C polymorphism and BC risk and estimated summary odds ratios (ORs) with confidence intervals (CIs) for assessment.

  14. Methylenetetrahydrofolate reductase (MTHFR) C677T gene ...

    Indian Academy of Sciences (India)

    vitamin B12 and riboflavin that are required in Hcy metabolic pathway. Gene that encodes the methylenete- trahydrofolate reductase (MTHFR) enzyme that .... tors like climate, food habits, lifestyle and genetic makeup are common. Validation of the results of the present study in different ethnic groups with larger sample ...

  15. phenotype correlation of methylene tetrahydrofolate reductase ...

    African Journals Online (AJOL)

    Rabah M. Shawky

    2014-06-21

    Jun 21, 2014 ... ORIGINAL ARTICLE. Study of genotype–phenotype correlation of methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in a sample of Egyptian autistic children. Rabah M. Shawky a,. *, Farida El-baz b. , Tarek M. Kamal c. , Reham M. Elhossiny b. ,. Mona A. Ahmed b. , Ghada H. El Nady d.

  16. Scandium extraction by methyltrialkylammonium nitrate from nitrate solutions

    International Nuclear Information System (INIS)

    Stepanov, S.I.; Kiyatkina, N.G.; Fedotov, O.N.

    1987-01-01

    Chemistry of scandium extraction by methyltrialkylammonium (MTAA) nitrate in toluene from nitrate solutions is studied. Methods of saturation, equilibrium shift, physicochemical analysis, isomolar series and UV-spectroscopy are used to determine the composition of extracted complexes. It is shown that with low saturation degrees of extractant (R 4 N) 6 Sc(NO 3 ) 9 complex is formed in organic phase and with saturation - (R 4 N) 3 xSc(NO 3 ) 6 complex

  17. Rare earth(3) nitrates extraction with trialkylmethylammonium nitrate in toluene

    International Nuclear Information System (INIS)

    Pyartman, A.K.; Kovalev, S.V.; Keskinov, V.A.; Khokhlova, N.V.

    1997-01-01

    Extraction of rare earth(3) nitrates [praseodymium(3)-lutetium(3)] with trialkylmethylammonium nitrate in toluene at T = 298.15 K and pH 2 is studied. Extraction isotherms are described with regard to formation of compounds of (R 4 N) i [Ln(NO 3 ) 3+i ] composition (i = 2, 3) in organic phase. Values of extraction constants are calculated, they are decreasing in the praseodymium(3) - lutetium(3) series

  18. Nebivolol prevents ethanol-induced reactive oxygen species generation and lipoperoxidation in the rat kidney by regulating NADPH oxidase activation and expression.

    Science.gov (United States)

    do Vale, Gabriel T; Gonzaga, Natália A; Simplicio, Janaina A; Tirapelli, Carlos R

    2017-03-15

    We studied whether the β 1 -adrenergic antagonist nebivolol would prevent ethanol-induced reactive oxygen species generation and lipoperoxidation in the rat renal cortex. Male Wistar rats were treated with ethanol (20% v/v) for 2 weeks. Nebivolol (10mg/kg/day; p.o. gavage) prevented both the increase in superoxide anion (O 2 - ) generation and thiobarbituric acid reactive substances (TBARS) concentration induced by ethanol in the renal cortex. Ethanol decreased nitrate/nitrite (NOx) concentration in the renal cortex, and nebivolol prevented this response. Nebivolol did not affect the reduction of hydrogen peroxide (H 2 O 2 ) concentration induced by ethanol. Nebivolol prevented the ethanol-induced increase of catalase (CAT) activity. Both SOD activity and the levels of reduced glutathione (GSH) were not affected by treatment with nebivolol or ethanol. Neither ethanol nor nebivolol affected the expression of Nox1, Nox4, eNOS, nNOS, CAT, Nox organizer 1 (Noxo1), c-Src, p47 phox or superoxide dismutase (SOD) isoforms in the renal cortex. On the other hand, treatment with ethanol increased Nox2 expression, and nebivolol prevented this response. Finally, nebivolol reduced the expression of protein kinase (PK) Cδ and Rac1. The major finding of our study is that nebivolol prevented ethanol-induced reactive oxygen species generation and lipoperoxidation in the kidney by a mechanism that involves reduction on the expression of Nox2, a catalytic subunit of NADPH oxidase. Additionally, we demonstrated that nebivolol reduces NADPH oxidase-derived reactive oxygen species by decreasing the expression of PKCδ and Rac1, which are important activators of NADPH oxidase. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Biochemical Function, Molecular Structure and Evolution of an Atypical Thioredoxin Reductase from Desulfovibrio vulgaris

    Directory of Open Access Journals (Sweden)

    Odile Valette

    2017-09-01

    Full Text Available Thioredoxin reductase (TR regulates the intracellular redox environment by reducing thioredoxin (Trx. In anaerobes, recent findings indicate that the Trx redox network is implicated in the global redox regulation of metabolism but also actively participates in protecting cells against O2. In the anaerobe Desulfovibrio vulgaris Hildenborough (DvH, there is an intriguing redundancy of the Trx system which includes a classical system using NADPH as electron source, a non-canonical system using NADH and an isolated TR (DvTRi. The functionality of DvTRi was questioned due to its lack of reactivity with DvTrxs. Structural analysis shows that DvTRi is a NAD(PH-independent TR but its reducer needs still to be identified. Moreover, DvTRi reduced by an artificial electron source is able to reduce in turn DvTrx1 and complexation experiments demonstrate a direct interaction between DvTRi and DvTrx1. The deletion mutant tri exhibits a higher sensitivity to disulfide stress and the gene tri is upregulated by O2 exposure. Having DvTRi in addition to DvTR1 as electron source for reducing DvTrx1 must be an asset to combat oxidative stress. Large-scale phylogenomics analyses show that TRi homologs are confined within the anaerobes. All TRi proteins displayed a conserved TQ/NGK motif instead of the HRRD motif, which is selective for the binding of the 2′-phosphate group of NADPH. The evolutionary history of TRs indicates that tr1 is the common gene ancestor in prokaryotes, affected by both gene duplications and horizontal gene events, therefore leading to the appearance of TRi through subfunctionalization over the evolutionary time.

  20. The nitrate-reduction gene cluster components exert lineage-dependent contributions to optimization of Sinorhizobium symbiosis with soybeans.

    Science.gov (United States)

    Liu, Li Xue; Li, Qin Qin; Zhang, Yun Zeng; Hu, Yue; Jiao, Jian; Guo, Hui Juan; Zhang, Xing Xing; Zhang, Biliang; Chen, Wen Xin; Tian, Chang Fu

    2017-12-01

    Receiving nodulation and nitrogen fixation genes does not guarantee rhizobia an effective symbiosis with legumes. Here, variations in gene content were determined for three Sinorhizobium species showing contrasting symbiotic efficiency on soybeans. A nitrate-reduction gene cluster absent in S. sojae was found to be essential for symbiotic adaptations of S. fredii and S. sp. III. In S. fredii, the deletion mutation of the nap (nitrate reductase), instead of nir (nitrite reductase) and nor (nitric oxide reductase), led to defects in nitrogen-fixation (Fix - ). By contrast, none of these core nitrate-reduction genes were required for the symbiosis of S. sp. III. However, within the same gene cluster, the deletion of hemN1 (encoding oxygen-independent coproporphyrinogen III oxidase) in both S. fredii and S. sp. III led to the formation of nitrogen-fixing (Fix + ) but ineffective (Eff - ) nodules. These Fix + /Eff - nodules were characterized by significantly lower enzyme activity of glutamine synthetase indicating rhizobial modulation of nitrogen-assimilation by plants. A distant homologue of HemN1 from S. sojae can complement this defect in S. fredii and S. sp. III, but exhibited a more pleotropic role in symbiosis establishment. These findings highlighted the lineage-dependent optimization of symbiotic functions in different rhizobial species associated with the same host. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  1. Histidine hydrogen-deuterium exchange mass spectrometry for probing the microenvironment of histidine residues in dihydrofolate reductase.

    Directory of Open Access Journals (Sweden)

    Masaru Miyagi

    2011-02-01

    Full Text Available Histidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS determines the HDX rates at the imidazole C(2-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK(a values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR, an enzyme proposed to undergo multiple conformational changes during catalysis.Using His-HDX-MS, the pK(a values and the half-lives (t(1/2 of HDX reactions of five histidine residues of apo-DHFR, DHFR in complex with methotrexate (DHFR-MTX, DHFR in complex with MTX and NADPH (DHFR-MTX-NADPH, and DHFR in complex with folate and NADP+ (DHFR-folate-NADP+ were determined. The results showed that the two parameters (pK(a and t(1/2 are sensitive to the changes of the microenvironment around the histidine residues. Although four of the five histidine residues are located far from the active site, ligand binding affected their pK(a, t(1/2 or both. This is consistent with previous observations of ligand binding-induced distal conformational changes on DHFR. Most of the observed pK(a and t(1/2 changes could be rationalized using the X-ray structures of apo-DHFR, DHFR-MTX-NADPH, and DHFR-folate-NADP+. The availability of the neutron diffraction structure of DHFR-MTX enabled us to compare the protonation states of histidine imidazole rings.Our results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins.

  2. Histidine hydrogen-deuterium exchange mass spectrometry for probing the microenvironment of histidine residues in dihydrofolate reductase.

    Science.gov (United States)

    Miyagi, Masaru; Wan, Qun; Ahmad, Md Faiz; Gokulrangan, Giridharan; Tomechko, Sara E; Bennett, Brad; Dealwis, Chris

    2011-02-16

    Histidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C(2)-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK(a) values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR), an enzyme proposed to undergo multiple conformational changes during catalysis. Using His-HDX-MS, the pK(a) values and the half-lives (t(1/2)) of HDX reactions of five histidine residues of apo-DHFR, DHFR in complex with methotrexate (DHFR-MTX), DHFR in complex with MTX and NADPH (DHFR-MTX-NADPH), and DHFR in complex with folate and NADP+ (DHFR-folate-NADP+) were determined. The results showed that the two parameters (pK(a) and t(1/2)) are sensitive to the changes of the microenvironment around the histidine residues. Although four of the five histidine residues are located far from the active site, ligand binding affected their pK(a), t(1/2) or both. This is consistent with previous observations of ligand binding-induced distal conformational changes on DHFR. Most of the observed pK(a) and t(1/2) changes could be rationalized using the X-ray structures of apo-DHFR, DHFR-MTX-NADPH, and DHFR-folate-NADP+. The availability of the neutron diffraction structure of DHFR-MTX enabled us to compare the protonation states of histidine imidazole rings. Our results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins.

  3. Biochemical, Physiological and Transcriptomic Comparison between Burley and Flue-Cured Tobacco Seedlings in Relation to Carbohydrates and Nitrate Content

    Directory of Open Access Journals (Sweden)

    Yafei Li

    2017-12-01

    Full Text Available Burley tobacco is a genotype of chloroplast-deficient mutant with accumulates high levels of tobacco-specific nitrosamines (TSNAs which would induce malignant tumors in animals. Nitrate is a principle precursor of tobacco-specific nitrosamines. Nitrate content in burley tobacco was significantly higher than that in flue-cured tobacco. The present study investigated differences between the two tobacco types to explore the mechanisms of nitrate accumulation in burley tobacco. transcripts (3079 related to the nitrogen and carbon metabolism were observed. Expression of genes involved in carbon fixation, glucose and starch biosynthesis, nitrate translocation and assimilation were significantly low in burley tobacco than flue-cured tobacco. Being relative to flue-cured tobacco, burley tobacco was significantly lower at total nitrogen and carbohydrate content, nitrate reductase and glutamine synthetase activities, chlorophyll content and photosynthetic rate (Pn, but higher nitrate content. Burley tobacco required six-fold more nitrogen fertilizers than flue-cured tobacco, but both tobaccos had a similar leaf biomass. Reduced chlorophyll content and photosynthetic rate (Pn might result in low carbohydrate formation, and low capacity of nitrogen assimilation and translocation might lead to nitrate accumulation in burley tobacco.

  4. Nitrate augmented myocardial viability assessment

    International Nuclear Information System (INIS)

    Wadhwa, S.; Mansberb, R.; Fernandes, V.B.

    1997-01-01

    Full text: 24 hour 201 TI reinjection imaging improves myocardial viability detection when compared to standard 3-4 hour redistribution imaging, however, it is a time-consuming approach and some images do not provide adequate quality due to high background activity on the delayed scan. We tested whether sensitivity of redistribution and same day reinjection imaging could be improved by giving short-acting nitrates immediately prior to redistribution and reinjection imaging. Eighteen patients underwent a stress test (exercise or pharmacological) and 4 hour redistribution 201 TI SPECT study. Immediately after redistribution imaging, each patient was given 600 μg of sublingual glyceryl trinitrate and reimaged 15-20 minutes later (nitrate augmented image). Immediately following nitrate augmentation imaging, each patient was reinjected with 30 MBq of 201 TI and reimaged 20 minutes later (nitrate augmented reinjection images). Each patient returned 24 hours later and was reinjected with 40 MBq of 201 TI and imaged 20 minutes later (24 hour reinjection images). In all, each patient had five SPECT images as follows: stress/redistribution/ nitrate augmented redistribution/nitrate augmented same day reinjection/ 24 hour reinjection. The myocardium was divided into 11 segments and perfusion to each segment was scored by consensus method (2 blinded assessors) on a 4 point graded scale (0 = no perfusion, 1 = minimal perfusion, 2 = moderate perfusion, 3 = normal perfusion). Perfusion scores were analysed on a segment by segment basis; as well, each patient was given an overall perfusion score equal to the sum of the perfusion score for each segment. 150 segments with reduced perfusion were identified, of these 23 (15.3%) showed improvement in the redistribution images, 60 (40%) segments improved in the nitrate augmented images, 49 (32.7%) improved in the nitrate augmented reinjection images and 52 (34.7%) improved in the 24 hour reinjection images. To assess overall cardiac

  5. Angiotensin II inhibits the Na+-K+ pump via PKC-dependent activation of NADPH oxidase.

    Science.gov (United States)

    White, Caroline N; Figtree, Gemma A; Liu, Chia-Chi; Garcia, Alvaro; Hamilton, Elisha J; Chia, Karin K M; Rasmussen, Helge H

    2009-04-01

    The sarcolemmal Na(+)-K(+) pump, pivotal in cardiac myocyte function, is inhibited by angiotensin II (ANG II). Since ANG II activates NADPH oxidase, we tested the hypothesis that NADPH oxidase mediates the pump inhibition. Exposure to 100 nmol/l ANG II increased superoxide-sensitive fluorescence of isolated rabbit ventricular myocytes. The increase was abolished by pegylated superoxide dismutase (SOD), by the NADPH oxidase inhibitor apocynin, and by myristolated inhibitory peptide to epsilon-protein kinase C (epsilonPKC), previously implicated in ANG II-induced Na(+)-K(+) pump inhibition. A role for epsilonPKC was also supported by an ANG II-induced increase in coimmunoprecipitation of epsilonPKC with the receptor for the activated kinase and with the cytosolic p47(phox) subunit of NADPH oxidase. ANG II decreased electrogenic Na(+)-K(+) pump current in voltage-clamped myocytes. The decrease was abolished by SOD, by the gp91ds inhibitory peptide that blocks assembly and activation of NADPH oxidase, and by epsilonPKC inhibitory peptide. Since colocalization should facilitate NADPH oxidase-dependent regulation of the Na(+)-K(+) pump, we examined whether there is physical association between the pump subunits and NADPH oxidase. The alpha(1)-subunit coimmunoprecipitated with caveolin 3 and with membrane-associated p22(phox) and cytosolic p47(phox) NADPH oxidase subunits at baseline. ANG II had no effect on alpha(1)/caveolin 3 or alpha(1)/p22(phox) interaction, but it increased alpha(1)/p47(phox) coimmunoprecipitation. We conclude that ANG II inhibits the Na(+)-K(+) pump via PKC-dependent NADPH oxidase activation.

  6. Sulforaphane-induced transcription of thioredoxin reductase in lens: possible significance against cataract formation

    Directory of Open Access Journals (Sweden)

    Varma SD

    2013-10-01

    Full Text Available Shambhu D Varma, Krish Chandrasekaran, Svitlana Kovtun Department of Ophthalmology and Visual Sciences, University of Maryland, Baltimore, MD, USA Purpose: Sulforaphane is a phytochemically derived organic isothiocyanate 1-isothiocyanato-4-methylsulfinyl-butane present naturally in crucifers, including broccoli and cauliflower. Biochemically, it has been reported to induce the transcription of several antioxidant enzymes. Since such enzymes have been implicated in preventing cataract formation triggered by the intraocular generation of oxy-radical species, the purpose of this investigation was to examine whether it could induce the formation of antioxidant enzymes in the eye lens. Thioredoxin reductase (TrxR was used as the target of such induction. Methods: Mice lenses were cultured for an overnight period of 17 hours in medium 199 fortified with 10% fetal calf serum. Incubation was conducted in the absence and presence of sulforaphane (5 µM. Subsequently, the lenses were homogenized in phosphate-buffered saline (PBS, followed by centrifugation. TrxR activity was determined in the supernatant by measuring the nicotinamide adenine dinucleotide phosphate (reduced (NADPH-dependent reduction of 5,5´-dithiobis-2-nitrobenzoic acid (DTNB. Non-specific reduction of DTNB was corrected for by conducting parallel determinations in the presence of aurothiomalate. The reduction of DTNB was followed spectrophotometrically at 410 nm. Results: The activity of TrxR in the lenses incubated with sulforaphane was found to be elevated to 18 times of that observed in lenses incubated without sulforaphane. It was also noticeably higher in the lenses incubated without sulforaphane than in the un-incubated fresh lenses. However, this increase was much lower than that observed for lenses incubated with sulforaphane. Conclusion: Sulforaphane has been found to enhance TrxR activity in the mouse lens in culture. In view of the protective effect of the antioxidant enzymes

  7. In vivo induction of phase II detoxifying enzymes, glutathione transferase and quinone reductase by citrus triterpenoids

    Directory of Open Access Journals (Sweden)

    Ahmad Hassan

    2010-09-01

    Full Text Available Abstract Background Several cell culture and animal studies demonstrated that citrus bioactive compounds have protective effects against certain types of cancer. Among several classes of citrus bioactive compounds, limonoids were reported to prevent different types of cancer. Furthermore, the structures of citrus limonoids were reported to influence the activity of phase II detoxifying enzymes. The purpose of the study was to evaluate how variations in the structures of citrus limonoids (namely nomilin, deacetyl nomilin, and isoobacunoic acid and a mixture of limonoids would influence phase II enzyme activity in excised tissues from a mouse model. Methods In the current study, defatted sour orange seed powder was extracted with ethyl acetate and subjected to silica gel chromatography. The HPLC, NMR and mass spectra were used to elucidate the purity and structure of compounds. Female A/J mice were treated with three limonoids and a mixture in order to evaluate their effect on phase II enzymes in four different tissues. Assays for glutathione S-transferase and NAD(PH: quinone reductase (QR were used to evaluate induction of phase II enzymatic activity. Results The highest induction of GST against 1-chloro-2,4-dinitrobenzene (CDNB was observed in stomach (whole, 58% by nomilin, followed by 25% isoobacunoic acid and 19% deacetyl nomilin. Deacetyl nomilin in intestine (small as well as liver significantly reduced GST activity against CDNB. Additionally isoobacunoic acid and the limonoid mixture in liver demonstrated a significant reduction of GST activity against CDNB. Nomilin significantly induced GST activity against 4-nitroquinoline 1-oxide (4NQO, intestine (280% and stomach (75% while deacetyl nomilin showed significant induction only in intestine (73%. Induction of GST activity was also observed in intestine (93% and stomach (45% treated with the limonoid mixture. Finally, a significant induction of NAD(PH: quinone reductase (QR activity was

  8. Disentangling the rhizosphere effect on nitrate reducers and denitrifiers: insight into the role of root exudates.

    Science.gov (United States)

    Henry, S; Texier, S; Hallet, S; Bru, D; Dambreville, C; Chèneby, D; Bizouard, F; Germon, J C; Philippot, L

    2008-11-01

    To determine to which extent root-derived carbon contributes to the effects of plants on nitrate reducers and denitrifiers, four solutions containing different proportions of sugar, organic acids and amino acids mimicking maize root exudates were added daily to soil microcosms at a concentration of 150 microg C g(-1) of soil. Water-amended soils were used as controls. After 1 month, the size and structure of the nitrate reducer and denitrifier communities were analysed using the narG and napA, and the nirK, nirS and nosZ genes as molecular markers respectively. Addition of artificial root exudates (ARE) did not strongly affect the structure or the density of nitrate reducer and denitrifier communities whereas potential nitrate reductase and denitrification activities were stimulated by the addition of root exudates. An effect of ARE composition was also observed on N(2)O production with an N(2)O:(N(2)O + N(2)) ratio of 0.3 in microcosms amended with ARE containing 80% of sugar and of 1 in microcosms amended with ARE containing 40% of sugar. Our study indicated that ARE stimulated nitrate reduction or denitrification activity with increases in the range of those observed with the whole plant. Furthermore, we demonstrated that the composition of the ARE affected the nature of the end-product of denitrification and could thus have a putative impact on greenhouse gas emissions.

  9. EXTRACTION OF URANYL NITRATE FROM AQUEOUS SOLUTIONS

    Science.gov (United States)

    Furman, N.H.; Mundy, R.J.

    1957-12-10

    An improvement in the process is described for extracting aqueous uranyl nitrate solutions with an organic solvent such as ether. It has been found that the organic phase will extract a larger quantity of uranyl nitrate if the aqueous phase contains in addition to the uranyl nitrate, a quantity of some other soluble nitrate to act as a salting out agent. Mentioned as suitable are the nitrates of lithium, calcium, zinc, bivalent copper, and trivalent iron.

  10. Sodium nitrate ingestion increases skeletal muscle nitrate content in humans.

    Science.gov (United States)

    Nyakayiru, Jean; Kouw, Imre W K; Cermak, Naomi M; Senden, Joan M; van Loon, Luc J C; Verdijk, Lex B

    2017-09-01

    Nitrate ([Formula: see text]) ingestion has been shown to have vasoactive and ergogenic effects that have been attributed to increased nitric oxide (NO) production. Recent observations in rodents suggest that skeletal muscle tissue serves as an endogenous [Formula: see text] "reservoir." The present study determined [Formula: see text] contents in human skeletal muscle tissue in a postabsorptive state and following ingestion of a sodium nitrate bolus (NaNO 3 ). Seventeen male, type 2 diabetes patients (age 72 ± 1 yr; body mass index 26.5 ± 0.5 kg/m 2 ; means ± SE) were randomized to ingest a dose of NaNO 3 (NIT; 9.3 mg [Formula: see text]/kg body wt) or placebo (PLA; 8.8 mg NaCl/kg body wt). Blood and muscle biopsy samples were taken before and up to 7 h following [Formula: see text] or placebo ingestion to assess [Formula: see text] [and plasma nitrite ([Formula: see text])] concentrations. Additionally, basal plasma and muscle [Formula: see text] concentrations were assessed in 10 healthy young (CON-Y; age 21 ± 1 yr) and 10 healthy older (CON-O; age 75 ± 1 yr) control subjects. In all groups, baseline [Formula: see text] concentrations were higher in muscle (NIT, 57 ± 7; PLA, 61 ± 7; CON-Y, 80 ± 10; CON-O, 54 ± 6 µmol/l) than in plasma (NIT, 35 ± 3; PLA, 32 ± 3; CON-Y, 38 ± 3; CON-O, 33 ± 3 µmol/l; P ≤ 0.011). Ingestion of NaNO 3 resulted in a sustained increase in plasma [Formula: see text], plasma [Formula: see text], and muscle [Formula: see text] concentrations (up to 185 ± 25 µmol/l) in the NIT group (time effect P nitrate ingestion is usually limited to the changes observed in plasma nitrate and nitrite concentrations. The present investigation assessed the skeletal muscle nitrate content in humans during the postabsorptive state, as well as following dietary nitrate ingestion. We show that basal nitrate content is higher in skeletal muscle tissue than in plasma and that ingestion of a dietary nitrate bolus strongly increases both plasma

  11. NADPH phagocyte oxidase knockout mice control Trypanosoma cruzi proliferation, but develop circulatory collapse and succumb to infection.

    Directory of Open Access Journals (Sweden)

    Helton C Santiago

    Full Text Available (•NO is considered to be a key macrophage-derived cytotoxic effector during Trypanosoma cruzi infection. On the other hand, the microbicidal properties of reactive oxygen species (ROS are well recognized, but little importance has been attributed to them during in vivo infection with T. cruzi. In order to investigate the role of ROS in T. cruzi infection, mice deficient in NADPH phagocyte oxidase (gp91(phox (-/- or phox KO were infected with Y strain of T. cruzi and the course of infection was followed. phox KO mice had similar parasitemia, similar tissue parasitism and similar levels of IFN-γ and TNF in serum and spleen cell culture supernatants, when compared to wild-type controls. However, all phox KO mice succumbed to infection between day 15 and 21 after inoculation with the parasite, while 60% of wild-type mice were alive 50 days after infection. Further investigation demonstrated increased serum levels of nitrite and nitrate (NOx at day 15 of infection in phox KO animals, associated with a drop in blood pressure. Treatment with a NOS2 inhibitor corrected the blood pressure, implicating NOS2 in this phenomenon. We postulate that superoxide reacts with (•NO in vivo, preventing blood pressure drops in wild type mice. Hence, whilst superoxide from phagocytes did not play a critical role in parasite control in the phox KO animals, its production would have an important protective effect against blood pressure decline during infection with T. cruzi.

  12. Analysis and prediction of the physiological effects of altered coenzyme specificity in xylose reductase and xylitol dehydrogenase during xylose fermentation by Saccharomyces cerevisiae

    Science.gov (United States)

    Krahulec, Stefan; Klimacek, Mario; Nidetzky, Bernd

    2012-01-01

    An advanced strategy of Saccharomyces cerevisiae strain development for fermentation of xylose applies tailored enzymes in the process of metabolic engineering. The coenzyme specificities of the NADPH-preferring xylose reductase (XR) and the NAD+-dependent xylitol dehydrogenase (XDH) have been targeted in previous studies by protein design or evolution with the aim of improving the recycling of NADH or NADPH in their two-step pathway, converting xylose to xylulose. Yeast strains expressing variant pairs of XR and XDH that according to in vitro kinetic data were suggested to be much better matched in coenzyme usage than the corresponding pair of wild-type enzymes, exhibit widely varying capabilities for xylose fermentation. To achieve coherence between enzyme properties and the observed strain performance during fermentation, we explored the published kinetic parameters for wild-type and engineered forms of XR and XDH as possible predictors of xylitol by-product formation (Yxylitol) in yeast physiology. We found that the ratio of enzymatic reaction rates using NADP(H) and NAD(H) that was calculated by applying intracellular reactant concentrations to rate equations derived from bi-substrate kinetic analysis, succeeded in giving a statistically reliable forecast of the trend effect on Yxylitol. Prediction based solely on catalytic efficiencies with or without binding affinities for NADP(H) and NAD(H) were not dependable, and we define a minimum demand on the enzyme kinetic characterization to be performed for this purpose. An immediate explanation is provided for the typically lower Yxylitol in the current strains harboring XR engineered for utilization of NADH as compared to strains harboring XDH engineered for utilization of NADP+. The known XDH enzymes all exhibit a relatively high Km for NADP+ so that physiological boundary conditions are somewhat unfavorable for xylitol oxidation by NADP+. A criterion of physiological fitness is developed for engineered XR

  13. Two modes of regulation of the fatty acid elongase ELOVL6 by the 3-ketoacyl-CoA reductase KAR in the fatty acid elongation cycle.

    Directory of Open Access Journals (Sweden)

    Tatsuro Naganuma

    Full Text Available Fatty acids (FAs are diverse molecules, and such diversity is important for lipids to exert their functions under several environmental conditions. FA elongation occurs at the endoplasmic reticulum and produces a variety of FA species; the FA elongation cycle consists of four distinct enzyme reactions. For this cycle to be driven efficiently, there must exist coordinated regulation of protein components of the FA elongation machinery. However, such regulation is poorly understood. In the present study, we performed biochemical analyses using the FA elongase ELOVL6 and the 3-ketoacyl-CoA reductase KAR, which catalyze the first and second steps of the FA elongation cycle, respectively. In vitro FA elongation assays using membrane fractions demonstrated that ELOVL6 activity was enhanced ∼10-fold in the presence of NADPH, although ELOVL6 itself did not require NADPH for its catalysis. On the other hand, KAR does use NADPH as a reductant in its enzyme reaction. Activity of purified ELOVL6 was enhanced by ∼3-fold in the presence of KAR. This effect was KAR enzyme activity-independent, since it was observed in the absence of NADPH and in the KAR mutant. However, ELOVL6 enzyme activity was further enhanced in a KAR enzyme activity-dependent manner. Therefore, KAR regulates ELOVL6 via two modes. In the first mode, KAR may induce conformational changes in ELOVL6 to become structure that can undergo catalysis. In the second mode, conversion of 3-ketoacyl-CoA to 3-hydroxyacyl-CoA by KAR may facilitate release of the product from the presumed ELOVL6-KAR complex.

  14. Functional characterization of an anthocyanidin reductase gene from the fibers of upland cotton (Gossypium hirsutum).

    Science.gov (United States)

    Zhu, Yue; Wang, Haiyun; Peng, Qingzhong; Tang, Yuntao; Xia, Guixian; Wu, Jiahe; Xie, De-Yu

    2015-05-01

    Metabolic profiling, gene cloning, enzymatic analysis, ectopic expression, and gene silencing experiments demonstrate that the anthocyanidin reductase (ANR) pathway is involved in the biosynthesis of proanthocyanidins in upland cotton. Proanthocyanidins (PAs) are oligomeric or polymeric flavan-3-ols, however, the biosynthetic pathway of PAs in cotton remains to be elucidated. Here, we report on an anthocyanidin reductase (ANR) gene from cotton fibers and the ANR pathway of PAs. Phytochemical analysis demonstrated that leaves, stems, roots, and early developing fibers produced PAs and their monomers, including (-)-epicatechin, (-)-catechin, (-)-epigallocatechin, and (-)-gallocatechin. Crude PA extractions from different tissues were boiled in Butanol:HCl. Cyanidin, delphinidin, and pelargonidin were produced, indicating that cotton PAs include diverse extension unit structures. An ANR cDNA homolog (named GhANR1) was cloned from developing fibers. The open reading frame, composed of 1,011 bp nucleotides, was expressed in E. coli to obtain a recombinant protein. In the presence of NADPH, the recombinant enzyme catalyzed cyanidin, delphinidin, and pelargonidin to (-)-epicatechin and (-)-catechin, (-)-epigallocatechin and (-)-gallocatechin, and (-)-epiafzelechin and (-)-afzelechin, respectively. The ectopic expression of GhANR11 in an Arabidopsis ban mutant allowed for the reconstruction of the ANR pathway and PA biosynthesis in the seed coat. Virus-induced gene silencing (VIGS) of GhANR11 led to a significant increase in anthocyanins and a decrease in the PAs, (-)-epicatechin, and (-)-catechin in the stems and leaves of VIGS-infected plants. Taken together, these data demonstrate that the ANR pathway contributes to the biosynthesis of flavan-3-ols and PAs in cotton.

  15. Drug-enhanced carbon monoxide production from heme by cytochrome P450 reductase

    Directory of Open Access Journals (Sweden)

    Dragic Vukomanovic

    2017-01-01

    Full Text Available Carbon monoxide (CO formed endogenously is considered to be cytoprotective, and the vast majority of CO formation is attributed to the degradation of heme by heme oxygenases-1 and -2 (HO-1, HO-2. Previously, we observed that brain microsomes containing HO-2 produced many-fold more CO in the presence of menadione and its congeners; herein we explored these observations further. We determined the effects of various drugs on CO production of rat brain microsomes and recombinant human cytochrome P450 reductase (CPR; CO was measured by gas chromatography with reductive detection. Brain microsomes of Sprague-Dawley rats or recombinant human cytochrome P450 reductase (CPR were incubated with NADPH and various drugs in closed vials in phosphate buffer at pH 7.4 and 37°C. After 15 minutes, the reaction was stopped by cooling in dry ice, and the headspace gas was analyzed for CO production using gas chromatography with reductive (mercuric oxide detection. We observed drug-enhanced CO production in the presence of both microsomes and recombinant CPR alone; the presence of HO was not required. A range of structurally diverse drugs were capable of amplifying this CO formation; these molecules had structures consistent with redox cycling capability. The addition of catalase to a reaction mixture, that contained activating drugs, inhibited the production of CO. Drug-enhanced CO formation can be catalyzed by CPR. The mechanism of CPR activation was not through classical drug-receptor mediation. Redox cycling may be involved in the drug-induced amplification of CO production by CPR through the production of reactive oxygen species.

  16. Biochemical properties of human dehydrogenase/reductase (SDR family) member 7.

    Science.gov (United States)

    Stambergova, Hana; Skarydova, Lucie; Dunford, James E; Wsol, Vladimir

    2014-01-25

    Dehydrogenase/reductase (SDR family) member 7 (DHRS7, retSDR4, SDR34C1) is a previously uncharacterized member of the short-chain dehydrogenase/reductase (SDR) superfamily. While human SDR members are known to play an important role in various (patho)biochemical pathways including intermediary metabolism and biotransformation of xenobiotics, only 20% of them are considered to be well characterized. Based on phylogenetic tree and SDR sequence clusters analysis DHRS7 is a close relative to well-known SDR member 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) that participates in metabolism of endogenous and xenobiotic substances with carbonyl group. The aim of present study is to determine the basic biochemical properties of DHRS7 and its possible involvement in metabolism of substrates with carbonyl group. For the first time the computational predictions of this membrane protein and membrane topology were experimentally confirmed. DHRS7 has been demonstrated to be an integral protein facing the lumen of the endoplasmic reticulum with lack of posttranscriptional glycosylation modification. Subsequently, NADP(H) cofactor preference and enzymatic reducing activity of DHRS7 was determined towards endogenous substrates with a steroid structure (cortisone, 4-androstene-3,17-dion) and also toward relevant exogenous substances bearing a carbonyl group harmful to human health (1,2-naphtoquinone, 9,10-phenantrenequinone). In addition to 11β-HSD1, DHRS7 is another enzyme from SDR superfamily that have been proved, at least in vitro, to contribute to the metabolism of xenobiotics with carbonyl group. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2018-01-13

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

  18. Effects of 3G cell phone exposure on the structure and function of the human cytochrome P450 reductase.

    Science.gov (United States)

    Tanvir, Shazia; Thuróczy, György; Selmaoui, Brahim; Silva Pires Antonietti, Viviane; Sonnet, Pascal; Arnaud-Cormos, Delia; Lévêque, Philippe; Pulvin, Sylviane; de Seze, René

    2016-10-01

    Cell phones increase exposure to radiofrequency (RF) electromagnetic fields (EMFs). Whether EMFs exert specific effects on biological systems remains debatable. This study investigated the effect of cell phone exposure on the structure and function of human NADPH-cytochrome P450 reductase (CPR). CPR plays a key role in the electron transfer to cytochrome P450, which takes part in a wide range of oxidative metabolic reactions in various organisms from microbes to humans. Human CPR was exposed for 60min to 1966-MHz RF inside a transverse electromagnetic cell (TEM-cell) placed in an incubator. The specific absorption rate (SAR) was 5W·kg(-1). Conformation changes have been detected through fluorescent spectroscopy of flavin and tryptophan residues, and investigated through circular dichroism, dynamic light scattering and microelectrophoresis. These showed that CPR was narrowed. By using cytochrome C reductase activity to assess the electron flux through the CPR, the Michaelis Menten constant (Km) and the maximum initial velocity (Vmax) decreased by 22% as compared with controls. This change was due to small changes in the tertiary and secondary structures of the protein at 37°C. The relevance of these findings to an actual RF exposure scenario demands further biochemical and in-vivo confirmation. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Functional characterization and stability improvement of a ‘thermophilic-like’ ene-reductase from Rhodococcus opacus 1CP

    Directory of Open Access Journals (Sweden)

    Anika eRiedel

    2015-10-01

    Full Text Available Ene-reductases are widely applied for the asymmetric synthesis of relevant industrial chemicals. A novel ene-reductase OYERo2 was found within a set of 14 putative Old Yellow Enzymes (OYEs obtained by genome mining of the actinobacterium Rhodococcus opacus 1CP. Multiple sequence alignment suggested that the enzyme belongs to the group of ‘thermophilic-like’ OYEs. OYERo2 was produced in Escherichia coli and biochemically characterized. The enzyme is strongly NADPH dependent and uses non-covalently bound FMNH2 for the reduction of activated α,β-unsaturated alkenes. In the active form OYERo2 is a dimer. Optimal catalysis occurs at pH 7.3 and 37 °C. OYERo2 showed highest specific activities (4550 U mg-1 on maleimides, which are efficiently converted to the corresponding succinimides. The OYERo2-mediated reduction of prochiral alkenes afforded the (R-products with excellent optical purity (ee > 99%. OYERo2 is not as thermo-resistant as related OYEs. Introduction of a characteristic intermolecular salt bridge by site-specific mutagenesis raised the half-life of enzyme inactivation at 32 °C from 28 min to 87 min and improved the tolerance towards organic co-solvents. The suitability of OYERo2 for application in industrial biocatalysis is discussed.

  20. Structural studies provide clues for analog design of specific inhibitors of Cryptosporidium hominis thymidylate synthase-dihydrofolate reductase.

    Science.gov (United States)

    Kumar, Vidya P; Cisneros, Jose A; Frey, Kathleen M; Castellanos-Gonzalez, Alejandro; Wang, Yiqiang; Gangjee, Aleem; White, A Clinton; Jorgensen, William L; Anderson, Karen S

    2014-09-01

    Cryptosporidium is the causative agent of a gastrointestinal disease, cryptosporidiosis, which is often fatal in immunocompromised individuals and children. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer, bacterial infections, and malaria. Cryptosporidium hominis has a bifunctional thymidylate synthase and dihydrofolate reductase enzyme, compared to separate enzymes in the host. We evaluated lead compound 1 from a novel series of antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines as an inhibitor of Cryptosporidium hominis thymidylate synthase with selectivity over the human enzyme. Complementing the enzyme inhibition compound 1 also has anti-cryptosporidial activity in cell culture. A crystal structure with compound 1 bound to the TS active site is discussed in terms of several van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate (TS), cofactor NADPH and inhibitor methotrexate (DHFR). Another crystal structure in complex with compound 1 bound in both the TS and DHFR active sites is also reported here. The crystal structures provide clues for analog design and for the design of ChTS-DHFR specific inhibitors. Copyright © 2014. Published by Elsevier Ltd.

  1. Lapachol inhibition of vitamin K epoxide reductase and vitamin K quinone reductase.

    Science.gov (United States)

    Preusch, P C; Suttie, J W

    1984-11-01

    Lapachol [2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone] has been shown to be a potent inhibitor of both vitamin K epoxide reductase and the dithiothreitol-dependent vitamin K quinone reductase of rat liver microsomes in vitro. These observations explain the anticoagulant activity of lapachol previously observed in both rats and humans. Lapachol inhibition of the vitamin K epoxide and quinone reductases resembled coumarin anticoagulant inhibition, and was observed in normal strain but not in warfarin-resistant strain rat liver microsomes. This similarity of action suggests that the lactone functionality of the coumarins is not critical for their activity. The initial-velocity steady-state inhibition patterns for lapachol inhibition of the solubilized vitamin K epoxide reductase were consistent with tight binding of lapachol to the oxidized form of the enzyme, and somewhat lower affinity for the reduced form. It is proposed that lapachol assumes a 4-enol tautomeric structure similar to that of the 4-hydroxy coumarins. These structures are analogs of the postulated hydroxyvitamin K enolate intermediate bound to the oxidized form of the enzyme in the chemical reaction mechanism of vitamin K epoxide reductase, thus explaining their high affinity.

  2. Mitochondrial type II NAD(PH dehydrogenases in fungal cell death

    Directory of Open Access Journals (Sweden)

    A. Pedro Gonçalves

    2015-03-01

    Full Text Available During aerobic respiration, cells produce energy through oxidative phosphorylation, which includes a specialized group of multi-subunit complexes in the inner mitochondrial membrane known as the electron transport chain. However, this canonical pathway is branched into single polypeptide alternative routes in some fungi, plants, protists and bacteria. They confer metabolic plasticity, allowing cells to adapt to different environmental conditions and stresses. Type II NAD(PH dehydrogenases (also called alternative NAD(PH dehydrogenases are non-proton pumping enzymes that bypass complex I. Recent evidence points to the involvement of fungal alternative NAD(PH dehydrogenases in the process of programmed cell death, in addition to their action as overflow systems upon oxidative stress. Consistent with this, alternative NAD(PH dehydrogenases are phylogenetically related to cell death - promoting proteins of the apoptosis-inducing factor (AIF-family.

  3. Interferon gamma/NADPH oxidase defence system in immunity and cancer

    Czech Academy of Sciences Publication Activity Database

    Hodný, Zdeněk; Reiniš, Milan; Hubáčková, Soňa; Vašicová, Pavla; Bartek, Jiří

    -, 01 Sep (2015) ISSN 2162-4011 Institutional support: RVO:68378050 ; RVO:61388971 Keywords : IFNγ * NADPH oxidase * immunity * cancer Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.266, year: 2014

  4. Interacting TCP and NLP transcription factors control plant responses to nitrate availability.

    Science.gov (United States)

    Guan, Peizhu; Ripoll, Juan-José; Wang, Renhou; Vuong, Lam; Bailey-Steinitz, Lindsay J; Ye, Dening; Crawford, Nigel M

    2017-02-28

    Plants have evolved adaptive strategies that involve transcriptional networks to cope with and survive environmental challenges. Key transcriptional regulators that mediate responses to environmental fluctuations in nitrate have been identified; however, little is known about how these regulators interact to orchestrate nitrogen (N) responses and cell-cycle regulation. Here we report that teosinte branched1/cycloidea/proliferating cell factor1-20 (TCP20) and NIN-like protein (NLP) transcription factors NLP6 and NLP7, which act as activators of nitrate assimilatory genes, bind to adjacent sites in the upstream promoter region of the nitrate reductase gene, NIA1 , and physically interact under continuous nitrate and N-starvation conditions. Regions of these proteins necessary for these interactions were found to include the type I/II Phox and Bem1p (PB1) domains of NLP6&7, a protein-interaction module conserved in animals for nutrient signaling, and the histidine- and glutamine-rich domain of TCP20, which is conserved across plant species. Under N starvation, TCP20-NLP6&7 heterodimers accumulate in the nucleus, and this coincides with TCP20 and NLP6&7-dependent up-regulation of nitrate assimilation and signaling genes and down-regulation of the G 2 /M cell-cycle marker gene, CYCB1;1 TCP20 and NLP6&7 also support root meristem growth under N starvation. These findings provide insights into how plants coordinate responses to nitrate availability, linking nitrate assimilation and signaling with cell-cycle progression.

  5. Respiratory and dissimilatory nitrate-reducing communities from an extreme saline alkaline soil of the former lake Texcoco (Mexico).

    Science.gov (United States)

    Alcántara-Hernández, Rocio J; Valenzuela-Encinas, César; Marsch, Rodolfo; Dendooven, Luc

    2009-01-01

    The diversity of the dissimilatory and respiratory nitrate-reducing communities was studied in two soils of the former lake Texcoco (Mexico). Genes encoding the membrane-bound nitrate reductase (narG) and the periplasmic nitrate reductase (napA) were used as functional markers. To investigate bacterial communities containing napA and narG in saline alkaline soils of the former lake Texcoco, libraries of the two sites were constructed (soil T3 with pH 11 and electrolytic conductivity in saturated extract (EC(SE)) 160 dS m(-1) and soil T1 with pH 8.5 and EC(SE) 0.8 dS m(-1)). Phylogenetic analysis of napA sequences separated the clone families into two main groups: dependent or independent of NapB. Most of napA sequences from site T1 were grouped in the NapB-dependent clade, meanwhile most of the napA sequences from the extreme soil T3 were affiliated to the NapB-independent group. For both sites, partial narG sequences were associated with representatives of the Proteobacteria, Firmicutes and Actinobacteria phyla, but the proportions of the clones were different. Our results support the concept of a specific and complex nitrate-reducing community for each soil of the former lake Texcoco.

  6. Radiation chemistry of nitrate ices

    International Nuclear Information System (INIS)

    Kishore, K.; Moorthy, P.N.; Rao, K.N.

    1978-01-01

    The yields of various products formed in γ-irradiated nitrate ices, viz NO 2 - , H 2 O 2 , O 2 and H 2 , have been measured at different nitrate concentrations. Daniel's method of partition of yields has been employed to evaluate the direct and indirect effect contributions to the yields of the first three products. G(H 2 ) is close to zero at all nitrate concentrations above approximately 0.5 mol dm -3 . The mechanism of product formation has been discussed in the light of various reactions put forth to explain the radiolysis of fluid aqueous nitrate solutions. The effect of various scavengers for the primary species on the product yields has also been investigated. From this it is inferred that nitrate is able to scavenge both the reducing species, viz. H and e - sub(m) giving nitrite and possibly also the holes (h + sub(m)) giving O 2 , and excited water molecules (H 2 O*) giving H 2 O 2 and additional nitrite. (author)

  7. Nox family NADPH oxidases: Molecular mechanisms of activation.

    Science.gov (United States)

    Brandes, Ralf P; Weissmann, Norbert; Schröder, Katrin

    2014-11-01

    NADPH oxidases of the Nox family are important enzymatic sources of reactive oxygen species (ROS). Numerous homologue-specific mechanisms control the activity of this enzyme family involving calcium, free fatty acids, protein-protein interactions, intracellular trafficking, and posttranslational modifications such as phosphorylation, acetylation, or sumoylation. After a brief review on the classic pathways of Nox activation, this article will focus on novel mechanisms of homologue-specific activity control and on cell-specific aspects which govern Nox activity. From these findings of the recent years it must be concluded that the activity control of Nox enzymes is much more complex than anticipated. Moreover, depending on the cellular activity state, Nox enzymes are selectively activated or inactivated. The complex upstream signaling aspects of these events make the development of "intelligent" Nox inhibitors plausible, which selectively attenuate disease-related Nox-mediated ROS formation without altering physiological signaling ROS. This approach might be of relevance for Nox-mediated tissue injury in ischemia-reperfusion and inflammation and also for chronic Nox overactivation as present in cancer initiation and cardiovascular disease. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. NADPH oxidase deficiency underlies dysfunction of aged CD8+ Tregs

    Science.gov (United States)

    Wen, Zhenke; Shimojima, Yasuhiro; Shirai, Tsuyoshi; Li, Yinyin; Ju, Jihang; Yang, Zhen; Tian, Lu; Goronzy, Jörg J.

    2016-01-01

    Immune aging results in progressive loss of both protective immunity and T cell–mediated suppression, thereby conferring susceptibility to a combination of immunodeficiency and chronic inflammatory disease. Here, we determined that older individuals fail to generate immunosuppressive CD8+CCR7+ Tregs, a defect that is even more pronounced in the age-related vasculitic syndrome giant cell arteritis. In young, healthy individuals, CD8+CCR7+ Tregs are localized in T cell zones of secondary lymphoid organs, suppress activation and expansion of CD4 T cells by inhibiting the phosphorylation of membrane-proximal signaling molecules, and effectively inhibit proliferative expansion of CD4 T cells in vitro and in vivo. We identified deficiency of NADPH oxidase 2 (NOX2) as the molecular underpinning of CD8 Treg failure in the older individuals and in patients with giant cell arteritis. CD8 Tregs suppress by releasing exosomes that carry preassembled NOX2 membrane clusters and are taken up by CD4 T cells. Overexpression of NOX2 in aged CD8 Tregs promptly restored suppressive function. Together, our data support NOX2 as a critical component of the suppressive machinery of CD8 Tregs and suggest that repairing NOX2 deficiency in these cells may protect older individuals from tissue-destructive inflammatory disease, such as large-vessel vasculitis. PMID:27088800

  9. Metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, in mouse liver by alcohol dehydrogenase Adh1 and aldehyde reductase AKR1A4

    International Nuclear Information System (INIS)

    Short, Duncan M.; Lyon, Robert; Watson, David G.; Barski, Oleg A.; McGarvie, Gail; Ellis, Elizabeth M.

    2006-01-01

    The reductive metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, was studied in mouse liver. Using an HPLC-based stopped assay, the primary reduced metabolite was identified as 6-hydroxy-trans, trans-2,4-hexadienal (OH/CHO) and the secondary metabolite as 1,6-dihydroxy-trans, trans-2,4-hexadiene (OH/OH). The main enzymes responsible for the highest levels of reductase activity towards trans, trans-muconaldehyde were purified from mouse liver soluble fraction first by Q-sepharose chromatography followed by either blue or red dye affinity chromatography. In mouse liver, trans, trans-muconaldehyde is predominantly reduced by an NADH-dependent enzyme, which was identified as alcohol dehydrogenase (Adh1). Kinetic constants obtained for trans, trans-muconaldehyde with the native Adh1 enzyme showed a V max of 2141 ± 500 nmol/min/mg and a K m of 11 ± 4 μM. This enzyme was inhibited by pyrazole with a K I of 3.1 ± 0.57 μM. Other fractions were found to contain muconaldehyde reductase activity independent of Adh1, and one enzyme was identified as the NADPH-dependent aldehyde reductase AKR1A4. This showed a V max of 115 nmol/min/mg and a K m of 15 ± 2 μM and was not inhibited by pyrazole

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

    Science.gov (United States)

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

    2017-04-01

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

  11. Young and Especially Senescent Endothelial Microvesicles Produce NADPH: The Fuel for Their Antioxidant Machinery

    Directory of Open Access Journals (Sweden)

    Guillermo Bodega

    2018-01-01

    Full Text Available In a previous study, we demonstrated that endothelial microvesicles (eMVs have a well-developed enzymatic team involved in reactive oxygen species detoxification. In the present paper, we demonstrate that eMVs can synthesize the reducing power (NAD(PH that nourishes this enzymatic team, especially those eMVs derived from senescent human umbilical vein endothelial cells. Moreover, we have demonstrated that the molecules that nourish the enzymatic machinery involved in NAD(PH synthesis are blood plasma metabolites: lactate, pyruvate, glucose, glycerol, and branched-chain amino acids. Drastic biochemical changes are observed in senescent eMVs to optimize the synthesis of reducing power. Mitochondrial activity is diminished and the glycolytic pathway is modified to increase the activity of the pentose phosphate pathway. Different dehydrogenases involved in NADPH synthesis are also increased. Functional experiments have demonstrated that eMVs can synthesize NADPH. In addition, the existence of NADPH in eMVs was confirmed by mass spectrometry. Multiphoton confocal microscopy images corroborate the synthesis of reducing power in eMVs. In conclusion, our present and previous results demonstrate that eMVs can act as autonomous reactive oxygen species scavengers: they use blood metabolites to synthesize the NADPH that fuels their antioxidant machinery. Moreover, senescent eMVs have a stronger reactive oxygen species scavenging capacity than young eMVs.

  12. Blockade of TGF-β 1 Signalling Inhibits Cardiac NADPH Oxidase Overactivity in Hypertensive Rats

    Directory of Open Access Journals (Sweden)

    José Luis Miguel-Carrasco

    2012-01-01

    Full Text Available NADPH oxidases constitute a major source of superoxide anion (⋅O2 - in hypertension. Several studies suggest an important role of NADPH oxidases in different effects mediated by TGF-β 1. In this study we show that chronic administration of P144, a peptide synthesized from type III TGF-β 1 receptor, significantly reduced the cardiac NADPH oxidase expression and activity as well as in the nitrotyrosine levels observed in control spontaneously hypertensive rats (V-SHR to levels similar to control normotensive Wistar Kyoto rats. In addition, P144 was also able to reduce the significant increases in the expression of collagen type I protein and mRNA observed in hearts from V-SHR. In addition, positive correlations between collagen expression, NADPH oxidase activity, and nitrotyrosine levels were found in all animals. Finally, TGF-β 1-stimulated Rat-2 exhibited significant increases in NADPH oxidase activity that was inhibited in the presence of P144. It could be concluded that the blockade of TGF-β 1 with P144 inhibited cardiac NADPH oxidase in SHR, thus adding new data to elucidate the involvement of this enzyme in the profibrotic actions of TGF-β 1.

  13. Introducing extra NADPH consumption ability significantly increases the photosynthetic efficiency and biomass production of cyanobacteria.

    Science.gov (United States)

    Zhou, Jie; Zhang, Fuliang; Meng, Hengkai; Zhang, Yanping; Li, Yin

    2016-11-01

    Increasing photosynthetic efficiency is crucial to increasing biomass production to meet the growing demands for food and energy. Previous theoretical arithmetic analysis suggests that the light reactions and dark reactions are imperfectly coupled due to shortage of ATP supply, or accumulation of NADPH. Here we hypothesized that solely increasing NADPH consumption might improve the coupling of light reactions and dark reactions, thereby increasing the photosynthetic efficiency and biomass production. To test this hypothesis, an NADPH consumption pathway was constructed in cyanobacterium Synechocystis sp. PCC 6803. The resulting extra NADPH-consuming mutant grew much faster and achieved a higher biomass concentration. Analyses of photosynthesis characteristics showed the activities of photosystem II and photosystem I and the light saturation point of the NADPH-consuming mutant all significantly increased. Thus, we demonstrated that introducing extra NADPH consumption ability is a promising strategy to increase photosynthetic efficiency and to enable utilization of high-intensity lights. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  14. Ethanol-induced erectile dysfunction and increased expression of pro-inflammatory proteins in the rat cavernosal smooth muscle are mediated by NADPH oxidase-derived reactive oxygen species.

    Science.gov (United States)

    Leite, Letícia N; do Vale, Gabriel T; Simplicio, Janaina A; De Martinis, Bruno S; Carneiro, Fernando S; Tirapelli, Carlos R

    2017-06-05

    Ethanol consumption is associated with an increased risk of erectile dysfunction (ED), but the molecular mechanisms through which ethanol causes ED remain elusive. Reactive oxygen species are described as mediators of ethanol-induced cell toxicity/damage in distinctive tissues. The enzyme NADPH oxidase is the main source of reactive oxygen species in the endothelium and vascular smooth muscle cells and ethanol is described to increase NADPH oxidase activation and reactive oxygen species generation. This study evaluated the contribution of NADPH oxidase-derived reactive oxygen species to ethanol-induced ED, endothelial dysfunction and production of pro-inflammatory and redox-sensitive proteins in the rat cavernosal smooth muscle (CSM). Male Wistar rats were treated with ethanol (20% v/v) or ethanol plus apocynin (30mg/kg/day; p.o. gavage) for six weeks. Apocynin prevented both the decreased in acetylcholine-induced relaxation and intracavernosal pressure induced by ethanol. Ethanol increased superoxide anion (O 2 - ) generation and catalase activity in CSM, and treatment with apocynin prevented these responses. Similarly, apocynin prevented the ethanol-induced decreased of nitrate/nitrite (NOx), hydrogen peroxide (H 2 O 2 ) and SOD activity. Treatment with ethanol increased p47phox translocation to the membrane as well as the expression of Nox2, COX-1, catalase, iNOS, ICAM-1 and p65. Apocynin prevented the effects of ethanol on protein expression and p47phox translocation. Finally, treatment with ethanol increased both TNF-α production and neutrophil migration in CSM. The major new finding of this study is that NADPH oxidase-derived reactive oxygen species play a role on chronic ethanol consumption-induced ED and endothelial dysfunction in the rat CSM. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Dataset generated for Dissection of mechanisms of Trypanothione Reductase and Tryparedoxin Peroxidase through dynamic network analysis and simulations in leishmaniasis

    Directory of Open Access Journals (Sweden)

    Anurag Kumar

    2017-12-01

    Full Text Available Leishmaniasis is the second largest parasitic killer disease caused by the protozoan parasite Leishmania, transmitted by the bite of sand flies. It's endemic in the eastern India with 165.4 million populations at risk with the current drug regimen. Three forms of leishmaniasis exist in which cutaneous is the most common form caused by Leishmania major. Trypanothione Reductase (TryR, a flavoprotein oxidoreductase, unique to thiol redox system, is considered as a potential target for chemotherapy for trypanosomatids infection. It is involved in the NADPH dependent reduction of Trypanothione disulphide to Trypanothione. Similarly, is Tryparedoxin Peroxidase (Txnpx, for detoxification of peroxides, an event pivotal for survival of Leishmania in two disparate biological environment. Fe-S plays a major role in regulating redox balance. To check for the closeness between human homologs of these proteins, we have carried the molecular clock analysis followed by molecular modeling of 3D structure of this protein, enabling us to design and test the novel drug like molecules. Molecular clock analysis suggests that human homologs of TryR i.e. Glutathione Reductase and Txnpx respectively are highly diverged in phylogenetic tree, thus, they serve as good candidates for chemotherapy of leishmaniasis. Furthermore, we have done the homology modeling of TryR using template of same protein from Leishmania infantum (PDB ID: 2JK6. This was done using Modeller 9.18 and the resultant models were validated. To inhibit this target, molecular docking was done with various screened inhibitors in which we found Taxifolin acts as common inhibitors for both TryR and Txnpx. We constructed the protein-protein interaction network for the proteins that are involved in the redox metabolism from various Interaction databases and the network was statistically analysed. Keywords: Trypanothione Reductase, Tryparedoxin Peroxidase, L.major, Homology modeling, Molecular clock analysis

  16. Changes of sodium nitrate, nitrite, and N-nitrosodiethylamine during in vitro human digestion.

    Science.gov (United States)

    Kim, Hyeong Sang; Hur, Sun Jin

    2017-06-15

    This study aimed to determine the changes in sodium nitrate, sodium nitrite, and N-nitrosodiethylamine (NDEA) during in vitro human digestion, and the effect of enterobacteria on the changes in these compounds. The concentrations of nitrate, nitrite, and NDEA were significantly reduced from 150, 150, and 1ppm to 42.8, 63.2, and 0.85ppm, respectively, during in vitro human digestion (pdigestion. This study is the first to report that E. coli can dramatically reduce the amount of nitrite during in vitro human digestion and this may be due to the effect of nitrite reductase present in E. coli. We therefore conclude that the amounts of potentially harmful substances and their toxicity can be decreased during human digestion. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Molecular Cloning and Functional Characterization of a Dihydroflavonol 4-Reductase from Vitis bellula

    Directory of Open Access Journals (Sweden)

    Yue Zhu

    2018-04-01

    Full Text Available Vitis bellula is a new grape crop in southern China. Berries of this species are rich in antioxidative anthocyanins and proanthocyanidins. This study reports cloning and functional characterization of a cDNA encoding a V. bellula dihydroflavonol reductase (VbDFR involved in the biosynthesis of anthocyanins and proanthocyanidins. A cDNA including 1014 bp was cloned from young leaves and its open reading frame (ORF was deduced encoding 337 amino acids, highly similar to V. vinifera DFR (VvDFR. Green florescence protein fusion and confocal microscopy analysis determined the cytosolic localization of VbDFR in plant cells. A soluble recombinant VbDFR was induced and purified from E. coli for enzyme assay. In the presence of NADPH, the recombinant enzyme catalyzed dihydrokaempferol (DHK and dihydroquercetin (DHQ to their corresponding leucoanthocyanidins. The VbDFR cDNA was introduced into tobacco plants via Agrobacterium-mediated transformation. The overexpression of VbDFR increased anthocyanin production in flowers. Anthocyanin hydrolysis and chromatographic analysis revealed that transgenic flowers produced pelargonidin and delphinidin, which were not detected in control flowers. These data demonstrated that the overexpression of VbDFR produced new tobacco anthocyanidins. In summary, all data demonstrate that VbDFR is a useful gene to provide three types of substrates for metabolic engineering of anthocyanins and proanthocyanidins in grape crops and other crops.

  18. The relationship between the nitrogen and nitrate content and nitrate ...

    African Journals Online (AJOL)

    The interrelationships between the nitrate-N and nitrogen content and dry matter yield of Midmar ryegrass (Lolium multiflorum cv. Midmar) were investigated. Data were collected from N fertility trials, from two seasons (1985 and 1987) on four soil sites (Metz, Griffin, Clovelly and Katspruit) with N fertilizer rates ranging from 0 ...

  19. Efficiency of nitrate uptake in spinach : impact of external nitrate concentration and relative growth rate on nitrate influx and efflux

    NARCIS (Netherlands)

    Ter Steege, MW; Stulen, [No Value; Wiersema, PK; Posthumus, F; Vaalburg, W

    1999-01-01

    Regulation of nitrate influx and efflux in spinach (Spinacia oleracea L., cv. Subito), was studied in short-term label experiments with N-13- and N-15-nitrate. Nitrate fluxes were examined in relation to the N demand for growth, defined as relative growth rate (RGR) times plant N concentration.

  20. Regulation of NADPH oxidase 5 by protein kinase C isoforms.

    Directory of Open Access Journals (Sweden)

    Feng Chen

    Full Text Available NADPH oxidase5 (Nox5 is a novel Nox isoform which has recently been recognized as having important roles in the pathogenesis of coronary artery disease, acute myocardial infarction, fetal ventricular septal defect and cancer. The activity of Nox5 and production of reactive oxygen species is regulated by intracellular calcium levels and phosphorylation. However, the kinases that phosphorylate Nox5 remain poorly understood. Previous studies have shown that the phosphorylation of Nox5 is PKC dependent, but this contention was based on the use of pharmacological inhibitors and the isoforms of PKC involved remain unknown. Thus, the major goals of this study were to determine whether PKC can directly regulate Nox5 phosphorylation and activity, to identify which isoforms are involved in the process, and to understand the functional significance of this pathway in disease. We found that a relatively specific PKCα inhibitor, Ro-32-0432, dose-dependently inhibited PMA-induced superoxide production from Nox5. PMA-stimulated Nox5 activity was significantly reduced in cells with genetic silencing of PKCα and PKCε, enhanced by loss of PKCδ and the silencing of PKCθ expression was without effect. A constitutively active form of PKCα robustly increased basal and PMA-stimulated Nox5 activity and promoted the phosphorylation of Nox5 on Ser490, Thr494, and Ser498. In contrast, constitutively active PKCε potently inhibited both basal and PMA-dependent Nox5 activity. Co-IP and in vitro kinase assay experiments demonstrated that PKCα directly binds to Nox5 and modifies Nox5 phosphorylation and activity. Exposure of endothelial cells to high glucose significantly increased PKCα activation, and enhanced Nox5 derived superoxide in a manner that was in prevented by a PKCα inhibitor, Go 6976. In summary, our study reveals that PKCα is the primary isoform mediating the activation of Nox5 and this maybe of significance in our understanding of the vascular

  1. Decoding NADPH oxidase 4 expression in human tumors

    Directory of Open Access Journals (Sweden)

    Jennifer L. Meitzler

    2017-10-01

    Full Text Available NADPH oxidase 4 (NOX4 is a redox active, membrane-associated protein that contributes to genomic instability, redox signaling, and radiation sensitivity in human cancers based on its capacity to generate H2O2 constitutively. Most studies of NOX4 in malignancy have focused on the evaluation of a small number of tumor cell lines and not on human tumor specimens themselves; furthermore, these studies have often employed immunological tools that have not been well characterized. To determine the prevalence of NOX4 expression across a broad range of solid tumors, we developed a novel monoclonal antibody that recognizes a specific extracellular region of the human NOX4 protein, and that does not cross-react with any of the other six members of the NOX gene family. Evaluation of 20 sets of epithelial tumors revealed, for the first time, high levels of NOX4 expression in carcinomas of the head and neck (15/19 patients, esophagus (12/18 patients, bladder (10/19 patients, ovary (6/17 patients, and prostate (7/19 patients, as well as malignant melanoma (7/15 patients when these tumors were compared to histologically-uninvolved specimens from the same organs. Detection of NOX4 protein upregulation by low levels of TGF-β1 demonstrated the sensitivity of this new probe; and immunofluorescence experiments found that high levels of endogenous NOX4 expression in ovarian cancer cells were only demonstrable associated with perinuclear membranes. These studies suggest that NOX4 expression is upregulated, compared to normal tissues, in a well-defined, and specific group of human carcinomas, and that its expression is localized on intracellular membranes in a fashion that could modulate oxidative DNA damage.

  2. Nitrate and bicarbonate selective CHEMFETs

    NARCIS (Netherlands)

    Antonisse, M.M.G.; Engbersen, Johannes F.J.; Reinhoudt, David

    1995-01-01

    The development of durable anion selective CHEMFET micro sensors is described. Selectivity in these sensors is either obtained from differences in hydration energy of the anions (the Hlofmeister series, giving nitrate selectivity) or by introduction of a new class of uranyl salophene ionophores

  3. Extreme nitrite tolerance in the clown knifefish Chitala ornata is linked to up-regulation of methaemoglobin reductase activity.

    Science.gov (United States)

    Gam, Le Thi Hong; Jensen, Frank Bo; Damsgaard, Christian; Huong, Do Thi Thanh; Phuong, Nguyen Thanh; Bayley, Mark

    2017-06-01

    The clown knifefish is a facultative air breather, which is widely farmed in freshwater ponds in Vietnam. Here we report a very high nitrite tolerance (96h LC 50 of 7.82mM) in this species and examine the effects of 1mM (LC 5 ) and 2.5mM (LC 10 ) ambient nitrite on haemoglobin (Hb) derivatives, electrolyte levels, acid-base status, and total body water content during 7days of exposure. Furthermore, we tested the hypothesis that erythrocyte methaemoglobin (metHb) reductase activity is upregulated by nitrite exposure. Plasma nitrite levels increased for 2-3days but stayed below environmental levels and fell towards control values during the last half of the exposure period. Plasma nitrate, in contrast, rose continuously, reflecting detoxification of nitrite to nitrate. MetHb generated from the reaction between nitrite and erythrocyte Hb reached 38% at day 2, but then decreased to 17% by the end of experiment. The first order rate constant for metHb reduction by erythrocyte metHb reductase increased from 0.01 in controls to 0.046min -1 after 6days of nitrite exposure, showing up-regulation of this enzyme. While such upregulation has been suggested in nitrite-exposed fish species, this study provides the first experimental evidence. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. The protein inhibitor of nNOS (PIN/DLC1/LC8) binding does not inhibit the NADPH-dependent heme reduction in nNOS, a key step in NO synthesis

    International Nuclear Information System (INIS)

    Parhad, Swapnil S.; Jaiswal, Deepa; Ray, Krishanu; Mazumdar, Shyamalava

    2016-01-01

    The neuronal nitric oxide synthase (nNOS) is an essential enzyme involved in the synthesis of nitric oxide (NO), a potent neurotransmitter. Although previous studies have indicated that the dynein light chain 1 (DLC1) binding to nNOS could inhibit the NO synthesis, the claim is challenged by contradicting reports. Thus, the mechanism of nNOS regulation remained unclear. nNOS has a heme-bearing, Cytochrome P450 core, and the functional enzyme is a dimer. The electron flow from NADPH to Flavin, and finally to the heme of the paired nNOS subunit within a dimer, is facilitated upon calmodulin (CaM) binding. Here, we show that DLC1 binding to nNOS-CaM complex does not affect the electron transport from the reductase to the oxygenase domain. Therefore, it cannot inhibit the rate of NADPH-dependent heme reduction in nNOS, which results in L-Arginine oxidation. Also, the NO release activity does not decrease with increasing DLC1 concentration in the reaction mix, which further confirmed that DLC1 does not inhibit nNOS activity. These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell. - Highlights: • The effect of interaction of nNOS with DLC1 has been debatable with contradicting reports in literature. • Purified DLC1 has no effect on electron transport between reductase and oxygenase domain of purified nNOS-CaM. • The NO release activity of nNOS was not altered by DLC1, supporting that DLC1 does not inhibit the enzyme. • These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell.

  5. The protein inhibitor of nNOS (PIN/DLC1/LC8) binding does not inhibit the NADPH-dependent heme reduction in nNOS, a key step in NO synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Parhad, Swapnil S. [Tata Institute of Fundamental Research (TIFR), Homi Bhabha Road, Mumbai 400 005 (India); Jaiswal, Deepa [Tata Institute of Fundamental Research (TIFR), Homi Bhabha Road, Mumbai 400 005 (India); TIFR Centre for Interdisciplinary Sciences, 21 Brundavan Colony, Narsingi, Hyderabad 500075 (India); Ray, Krishanu, E-mail: krishanu@tifr.res.in [Tata Institute of Fundamental Research (TIFR), Homi Bhabha Road, Mumbai 400 005 (India); Mazumdar, Shyamalava, E-mail: shyamal@tifr.res.in [Tata Institute of Fundamental Research (TIFR), Homi Bhabha Road, Mumbai 400 005 (India)

    2016-03-25

    The neuronal nitric oxide synthase (nNOS) is an essential enzyme involved in the synthesis of nitric oxide (NO), a potent neurotransmitter. Although previous studies have indicated that the dynein light chain 1 (DLC1) binding to nNOS could inhibit the NO synthesis, the claim is challenged by contradicting reports. Thus, the mechanism of nNOS regulation remained unclear. nNOS has a heme-bearing, Cytochrome P450 core, and the functional enzyme is a dimer. The electron flow from NADPH to Flavin, and finally to the heme of the paired nNOS subunit within a dimer, is facilitated upon calmodulin (CaM) binding. Here, we show that DLC1 binding to nNOS-CaM complex does not affect the electron transport from the reductase to the oxygenase domain. Therefore, it cannot inhibit the rate of NADPH-dependent heme reduction in nNOS, which results in L-Arginine oxidation. Also, the NO release activity does not decrease with increasing DLC1 concentration in the reaction mix, which further confirmed that DLC1 does not inhibit nNOS activity. These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell. - Highlights: • The effect of interaction of nNOS with DLC1 has been debatable with contradicting reports in literature. • Purified DLC1 has no effect on electron transport between reductase and oxygenase domain of purified nNOS-CaM. • The NO release activity of nNOS was not altered by DLC1, supporting that DLC1 does not inhibit the enzyme. • These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell.

  6. Targeting NADPH oxidase decreases oxidative stress in the transgenic sickle cell mouse penis.

    Science.gov (United States)

    Musicki, Biljana; Liu, Tongyun; Sezen, Sena F; Burnett, Arthur L

    2012-08-01

    Sickle cell disease (SCD) is a state of chronic vasculopathy characterized by endothelial dysfunction and increased oxidative stress, but the sources and mechanisms responsible for reactive oxygen species (ROS) production in the penis are unknown. We evaluated whether SCD activates NADPH oxidase, induces endothelial nitric oxide synthase (eNOS) uncoupling, and decreases antioxidants in the SCD mouse penis. We further tested the hypothesis that targeting NADPH oxidase decreases oxidative stress in the SCD mouse penis. SCD transgenic (sickle) mice were used as an animal model of SCD. Hemizygous (hemi) mice served as controls. Mice received an NADPH oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Penes were excised at baseline for molecular studies. Markers of oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NADPH oxidase subunits p67(phox) , p47(phox) , and gp91(phox) ), and enzymatic antioxidants (superoxide dismutase [SOD]1, SOD2, catalase, and glutathione peroxidase-1 [GPx1]) were measured by Western blot in penes. Sources of ROS, oxidative stress, and enzymatic antioxidants in the SCD penis. Relative to hemi mice, SCD increased (Ppenis. Apocynin treatment of sickle mice reversed (P0.05) prevented eNOS uncoupling in the penis. Apocynin treatment of hemi mice did not affect any of these parameters. NADPH oxidase and eNOS uncoupling are sources of oxidative stress in the SCD penis; decreased GPx1 further contributes to oxidative stress. Inhibition of NADPH oxidase upregulation decreases oxidative stress, implying a major role for NADPH oxidase as a ROS source and a potential target for improving vascular function in the SCD mouse penis. © 2012 International Society for Sexual Medicine.

  7. Perturbation of human coronary artery endothelial cell redox state and NADPH generation by methylglyoxal.

    Directory of Open Access Journals (Sweden)

    Philip E Morgan

    Full Text Available Diabetes is associated with elevated plasma glucose, increased reactive aldehyde formation, oxidative damage, and glycation/glycoxidation of biomolecules. Cellular detoxification of, or protection against, such modifications commonly requires NADPH-dependent reducing equivalents (e.g. GSH. We hypothesised that reactive aldehydes may modulate cellular redox status via the inhibition of NADPH-generating enzymes, resulting in decreased thiol and NADPH levels. Primary human coronary artery endothelial cells (HCAEC were incubated with high glucose (25 mM, 24 h, 37°C, or methylglyoxal (MGO, glyoxal, or glycolaldehyde (100-500 µM, 1 h, 37°C, before quantification of intracellular thiols and NADPH-generating enzyme activities. Exposure to MGO, but not the other species examined, significantly (P<0.05 decreased total thiols (∼35%, further experiments with MGO showed significant losses of GSH (∼40% and NADPH (∼10%; these changes did not result in an immediate loss of cell viability. Significantly decreased (∼10% NADPH-producing enzyme activity was observed for HCAEC when glucose-6-phosphate or 2-deoxyglucose-6-phosphate were used as substrates. Cell lysate experiments showed significant MGO-dose dependent inhibition of glucose-6-phosphate-dependent enzymes and isocitrate dehydrogenase, but not malic enzyme. Analysis of intact cell or lysate proteins showed that arginine-derived hydroimidazolones were the predominant advanced glycation end-product (AGE formed; lower levels of N(ε-(carboxyethyllysine (CEL and N(ε-(carboxymethyllysine (CML were also detected. These data support a novel mechanism by which MGO exposure results in changes in redox status in human coronary artery endothelial cells, via inhibition of NADPH-generating enzymes, with resultant changes in reduced protein thiol and GSH levels. These changes may contribute to the endothelial cell dysfunction observed in diabetes-associated atherosclerosis.

  8. Structure and mechanism of dimethylsulfoxide reductase, a molybdopterin-containing enzyme of DMSO reductase family

    International Nuclear Information System (INIS)

    McEwan, A.G.; Ridge, J.P.; McDevitt, C.A.; Hanson, G.R.

    2001-01-01

    Full text: Apart from nitrogenase, enzymes containing molybdenum are members of a superfamily, the molybdopterin-containing enzymes. Most of these enzymes catalyse an oxygen atom transfer and two electron transfer reaction. During catalysis the Mo at the active site cycles between the Mo(VI) and Mo(IV) states. The DMSO reductase family of molybdopterin-containing enzymes all contain a bis(molybdopterin guanine dinucleotide)Mo cofactor and over thirty examples have now been described. Over the last five years crystal structures of dimethylsulfoxide (DMSO) reductase and four other enzymes of the DMSO reductase family have revealed that enzymes of this family have a similar tertiary structure. The Mo atom at the active site is coordinated by four thiolate ligands provided by the dithiolene side chains of the two MGD molecules of the bis(MGD)Mo cofactor as well as a ligand provided by an amino acid side chain. In addition, an oxygen atom in the form of an oxo, hydroxo or aqua group is also coordinated to the Mo atom. In the case of dimethylsulfoxide reductase X-ray crystallography of the product-reduced species and Raman spectroscopy has demonstrated that the enzyme contains a single exchangeable oxo group that is H-bonded to W116

  9. Genetics of nitrate accumulation in lettuce

    OpenAIRE

    Reinink, K.

    1993-01-01

    This study evaluated the prospects of breeding for low nitrate content in lettuce ( Lactuca sativa L.). A lettuce collection was screened and accessions with low nitrate content were identified. These were used to study the genetics of nitrate accumulation. Nitrate accumulation inherited quantitatively, in a mainly additive fashion with only minor effects of dominance. No important maternal effects were detected. Estimates of the additive genotypic variance and t...

  10. A Novel Aldo-Keto Reductase, HdRed, from the Pacific Abalone Haliotis discus hannai, Which Reduces Alginate-derived 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid to 2-Keto-3-deoxy-d-gluconate*

    Science.gov (United States)

    Mochizuki, Shogo; Nishiyama, Ryuji; Inoue, Akira; Ojima, Takao

    2015-01-01

    Abalone feeds on brown seaweeds and digests seaweeds' alginate with alginate lyases (EC 4.2.2.3). However, it has been unclear whether the end product of alginate lyases (i.e. unsaturated monouronate-derived 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH)) is assimilated by abalone itself, because DEH cannot be metabolized via the Embden-Meyerhof pathway of animals. Under these circumstances, we recently noticed the occurrence of an NADPH-dependent reductase, which reduced DEH to 2-keto-3-deoxy-d-gluconate, in hepatopancreas extract of the pacific abalone Haliotis discus hannai. In the present study, we characterized this enzyme to some extent. The DEH reductase, named HdRed in the present study, could be purified from the acetone-dried powder of hepatopancreas by ammonium sulfate fractionation followed by conventional column chromatographies. HdRed showed a single band of ∼40 kDa on SDS-PAGE and reduced DEH to 2-keto-3-deoxy-d-gluconate with an optimal temperature and pH at around 50 °C and 7.0, respectively. HdRed exhibited no appreciable activity toward 28 authentic compounds, including aldehyde, aldose, ketose, α-keto-acid, uronic acid, deoxy sugar, sugar alcohol, carboxylic acid, ketone, and ester. The amino acid sequence of 371 residues of HdRed deduced from the cDNA showed 18–60% identities to those of aldo-keto reductase (AKR) superfamily enzymes, such as human aldose reductase, halophilic bacterium reductase, and sea hare norsolorinic acid (a polyketide derivative) reductase-like protein. Catalytic residues and cofactor binding residues known in AKR superfamily enzymes were fairly well conserved in HdRed. Phylogenetic analysis for HdRed and AKR superfamily enzymes indicated that HdRed is an AKR belonging to a novel family. PMID:26555267

  11. A Novel Aldo-Keto Reductase, HdRed, from the Pacific Abalone Haliotis discus hannai, Which Reduces Alginate-derived 4-Deoxy-L-erythro-5-hexoseulose Uronic Acid to 2-Keto-3-deoxy-D-gluconate.

    Science.gov (United States)

    Mochizuki, Shogo; Nishiyama, Ryuji; Inoue, Akira; Ojima, Takao

    2015-12-25

    Abalone feeds on brown seaweeds and digests seaweeds' alginate with alginate lyases (EC 4.2.2.3). However, it has been unclear whether the end product of alginate lyases (i.e. unsaturated monouronate-derived 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH)) is assimilated by abalone itself, because DEH cannot be metabolized via the Embden-Meyerhof pathway of animals. Under these circumstances, we recently noticed the occurrence of an NADPH-dependent reductase, which reduced DEH to 2-keto-3-deoxy-D-gluconate, in hepatopancreas extract of the pacific abalone Haliotis discus hannai. In the present study, we characterized this enzyme to some extent. The DEH reductase, named HdRed in the present study, could be purified from the acetone-dried powder of hepatopancreas by ammonium sulfate fractionation followed by conventional column chromatographies. HdRed showed a single band of ∼ 40 kDa on SDS-PAGE and reduced DEH to 2-keto-3-deoxy-D-gluconate with an optimal temperature and pH at around 50 °C and 7.0, respectively. HdRed exhibited no appreciable activity toward 28 authentic compounds, including aldehyde, aldose, ketose, α-keto-acid, uronic acid, deoxy sugar, sugar alcohol, carboxylic acid, ketone, and ester. The amino acid sequence of 371 residues of HdRed deduced from the cDNA showed 18-60% identities to those of aldo-keto reductase (AKR) superfamily enzymes, such as human aldose reductase, halophilic bacterium reductase, and sea hare norsolorinic acid (a polyketide derivative) reductase-like protein. Catalytic residues and cofactor binding residues known in AKR superfamily enzymes were fairly well conserved in HdRed. Phylogenetic analysis for HdRed and AKR superfamily enzymes indicated that HdRed is an AKR belonging to a novel family. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Nitrate tolerance impairs nitric oxide-mediated vasodilation in vivo

    DEFF Research Database (Denmark)

    Laursen, Jørn Bech; Boesgaard, Søren; Poulsen, Henrik E.

    1996-01-01

    Nitrates, Nitrate tolerence, Nitric oxide, acetylcholine, N-acetylcholine, N-acetylcysteine, L-NAME, Rat, Anesthetized......Nitrates, Nitrate tolerence, Nitric oxide, acetylcholine, N-acetylcholine, N-acetylcysteine, L-NAME, Rat, Anesthetized...

  13. 21 CFR 172.160 - Potassium nitrate.

    Science.gov (United States)

    2010-04-01

    ... Preservatives § 172.160 Potassium nitrate. The food additive potassium nitrate may be safely used as a curing... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Potassium nitrate. 172.160 Section 172.160 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN...

  14. 21 CFR 172.170 - Sodium nitrate.

    Science.gov (United States)

    2010-04-01

    ... Preservatives § 172.170 Sodium nitrate. The food additive sodium nitrate may be safely used in or on specified... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium nitrate. 172.170 Section 172.170 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN...

  15. 76 FR 47238 - Ammonium Nitrate From Russia

    Science.gov (United States)

    2011-08-04

    ... COMMISSION Ammonium Nitrate From Russia Determination On the basis of the record \\1\\ developed in the subject... order on ammonium nitrate from Russia would be likely to lead to continuation or recurrence of material... Commission are contained in USITC Publication 4249 (August 2011), entitled Ammonium Nitrate from Russia...

  16. Method of producing thin cellulose nitrate film

    International Nuclear Information System (INIS)

    Lupica, S.B.

    1975-01-01

    An improved method for forming a thin nitrocellulose film of reproducible thickness is described. The film is a cellulose nitrate film, 10 to 20 microns in thickness, cast from a solution of cellulose nitrate in tetrahydrofuran, said solution containing from 7 to 15 percent, by weight, of dioctyl phthalate, said cellulose nitrate having a nitrogen content of from 10 to 13 percent

  17. The protein inhibitor of nNOS (PIN/DLC1/LC8) binding does not inhibit the NADPH-dependent heme reduction in nNOS, a key step in NO synthesis.

    Science.gov (United States)

    Parhad, Swapnil S; Jaiswal, Deepa; Ray, Krishanu; Mazumdar, Shyamalava

    2016-03-25

    The neuronal nitric oxide synthase (nNOS) is an essential enzyme involved in the synthesis of nitric oxide (NO), a potent neurotransmitter. Although previous studies have indicated that the dynein light chain 1 (DLC1) binding to nNOS could inhibit the NO synthesis, the claim is challenged by contradicting reports. Thus, the mechanism of nNOS regulation remained unclear. nNOS has a heme-bearing, Cytochrome P450 core, and the functional enzyme is a dimer. The electron flow from NADPH to Flavin, and finally to the heme of the paired nNOS subunit within a dimer, is facilitated upon calmodulin (CaM) binding. Here, we show that DLC1 binding to nNOS-CaM complex does not affect the electron transport from the reductase to the oxygenase domain. Therefore, it cannot inhibit the rate of NADPH-dependent heme reduction in nNOS, which results in l-Arginine oxidation. Also, the NO release activity does not decrease with increasing DLC1 concentration in the reaction mix, which further confirmed that DLC1 does not inhibit nNOS activity. These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. High temperature interaction studies on equimolar nitrate mixture of uranyl nitrate hexahydrate and gadolinium nitrate hexahydrate

    International Nuclear Information System (INIS)

    Kalekar, Bhupesh B.; Raje, Naina; Reddy, A.V.R.

    2015-01-01

    Rare earths including gadolinium form a sizeable fraction of the fission products in the nuclear fission of fissile material in the reactor. These fission products can interact with uranium dioxide fuel and can form various compounds which can alter the thermal behavior of the fuel. The mixed oxide formed due to the high temperature interactions of mixture of uranyl nitrate hexahydrate (UNH) and gadolinium nitrate hexahydrate (GdNH) has been studied using thermal and X- ray diffraction techniques. The equimolar mixture of UNH and GdNH was prepared by mixing the weighed amount of individual nitrates and grinding gently with mortar and pestle. Thermogravimetry (TG) measurements were carried out by separately heating 100 mg of mixture and individual nitrates at heating rate of 10°C min -1 using Netzsch thermal analyzer (Model No.: STA 409 PC Luxx) in high purity nitrogen atmosphere with a flow rate of 120 mL min -1 . The XRD measurement was carried out on a Philips X-ray diffractometer (Model PW1710) using nickel-filtered Cu-Kα radiation

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

    International Nuclear Information System (INIS)

    Wang, Yinxi; Liu, Dan; Zhang, Huifeng; Wang, Yixin; Wei, Ling; Liu, Yutong; Liao, Jieying; Gao, Hui-Ming; Zhou, Hui

    2017-01-01

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

  20. Yttrium Nitrate mediated Nitration of Phenols at room temperature in ...

    Indian Academy of Sciences (India)

    chromatographic purification. 2.1 Procedure for Nitration of Phenol. Phenol (94 mg, 1 mmol) dissolved in 3 mL glacial acetic acid in a 50 mL test tube was treated with solid Y(NO3)3.6H2O. (383 mg, 1 mmol) with constant shaking at RT for 10 min. The reaction was monitored by TLC at 10% EtOAc in. Petroleum benzene.

  1. Methemoglobin reductase activity in intact fish red blood cells

    DEFF Research Database (Denmark)

    Jensen, Frank B; Nielsen, Karsten

    2018-01-01

    Red blood cells (RBCs) possess methemoglobin reductase activity that counters the ongoing oxidation of hemoglobin (Hb) to methemoglobin (metHb), which in circulating blood is caused by Hb autoxidation or reactions with nitrite. We describe an assay for determining metHb reductase activity in intact...

  2. 21 CFR 864.7375 - Glutathione reductase assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... reductase deficiency, or riboflavin deficiency. (b) Classification. Class II (performance standards). [45 FR...

  3. Nitration of naphthalene and remarks on the mechanism of electrophilic aromatic nitration*

    OpenAIRE

    Olah, George A.; Narang, Subhash C.; Olah, Judith A.

    1981-01-01

    Naphthalene was nitrated with a variety of nitrating agents. Comparison of data with Perrin's electrochemical nitration [Perrin, C. L. (1977) J. Am. Chem. Soc. 99, 5516-5518] shows that nitration of naphthalene gives an α-nitronaphthalene to β-nitronaphthalene ratio that varies between 9 and 29 and is thus not constant. Perrin's data, therefore, are considered to be inconclusive evidence for the proposed one-electron transfer mechanism for the nitration of naphthalene and other reactive aroma...

  4. Siroheme- and [Fe4-S4]-dependent NirA from Mycobacterium tuberculosis is a sulfite reductase with a covalent Cys-Tyr bond in the active site.

    Science.gov (United States)

    Schnell, Robert; Sandalova, Tatyana; Hellman, Ulf; Lindqvist, Ylva; Schneider, Gunter

    2005-07-22

    The nirA gene of Mycobacterium tuberculosis is up-regulated in the persistent state of the bacteria, suggesting that it is a potential target for the development of antituberculosis agents particularly active against the pathogen in its dormant phase. This gene encodes a ferredoxin-dependent sulfite reductase, and the structure of the enzyme has been determined using x-ray crystallography. The enzyme is a monomer comprising 555 amino acids and contains a [Fe4-S4] cluster and a siroheme cofactor. The molecule is built up of three domains with an alpha/beta fold. The first domain consists of two ferredoxin-like subdomains, related by a pseudo-2-fold symmetry axis passing through the whole molecule. The other two domains, which provide much of the binding interactions with the cofactors, have a common fold that is unique to the sulfite/nitrite reductase family. The domains form a trilobal structure, with the cofactors and the active site located at the interface of all three domains in the center of the molecule. NirA contains an unusual covalent bond between the side chains of Tyr69 and Cys161 in the active site, in close proximity to the siroheme cofactor. Removal of this covalent bond by site-directed mutagenesis impairs catalytic activity, suggesting that it is important for the enzymatic reaction. These residues are part of a sequence fingerprint, able to distinguish between ferredoxin-dependent sulfite and nitrite reductases. Comparison of NirA with the structure of the truncated NADPH-dependent sulfite reductase from Escherichia coli suggests a binding site for the external electron donor ferredoxin close to the [Fe4-S4] cluster.

  5. Nitrate Removal from Ground Water: A Review

    Directory of Open Access Journals (Sweden)

    Archna

    2012-01-01

    Full Text Available Nitrate contamination of ground water resources has increased in Asia, Europe, United States, and various other parts of the world. This trend has raised concern as nitrates cause methemoglobinemia and cancer. Several treatment processes can remove nitrates from water with varying degrees of efficiency, cost, and ease of operation. Available technical data, experience, and economics indicate that biological denitrification is more acceptable for nitrate removal than reverse osmosis and ion exchange. This paper reviews the developments in the field of nitrate removal processes which can be effectively used for denitrifying ground water as well as industrial water.

  6. Identification of a 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid Reductase, FlRed, in an Alginolytic Bacterium Flavobacterium sp. Strain UMI-01

    Directory of Open Access Journals (Sweden)

    Akira Inoue

    2015-01-01

    Full Text Available In alginate-assimilating bacteria, alginate is depolymerized to unsaturated monosaccharide by the actions of endolytic and exolytic alginate lyases (EC 4.2.2.3 and EC 4.2.2.11. The monosaccharide is non-enzymatically converted to 4-deoxy-l-ery thro-5-hexoseulose uronic acid (DEH, then reduced to 2-keto-3-deoxy-d-gluconate (KDG by a specific reductase, and metabolized through the Entner–Doudoroff pathway. Recently, the NADPH-dependent reductase A1-R that belongs to short-chain dehydrogenases/reductases (SDR superfamily was identified as the DEH-reductase in Sphingomonas sp. A1. We have subsequently noticed that an SDR-like enzyme gene, flred, occurred in the genome of an alginolytic bacterium Flavobacterium sp. strain UMI-01. In the present study, we report on the deduced amino-acid sequence of flred and DEH-reducing activity of recombinant FlRed. The deduced amino-acid sequence of flred comprised 254 residues and showed 34% amino-acid identities to that of A1-R from Sphingomonas sp. A1 and 80%–88% to those of SDR-like enzymes from several alginolytic bacteria. Common sequence motifs of SDR-superfamily enzymes, e.g., the catalytic tetrad Asn-Lys-Tyr-Ser and the cofactor-binding sequence Thr-Gly-x-x-x-Gly-x-Gly in Rossmann fold, were completely conserved in FlRed. On the other hand, an Arg residue that determined the NADPH-specificity of Sphingomonas A1-R was replaced by Glu in FlRed. Thus, we investigated cofactor-preference of FlRed using a recombinant enzyme. As a result, the recombinant FlRed (recFlRed was found to show high specificity to NADH. recFlRed exhibited practically no activity toward variety of aldehyde, ketone, keto ester, keto acid and aldose substrates except for DEH. On the basis of these results, we conclude that FlRed is the NADH-dependent DEH-specific SDR of Flavobacterium sp. strain UMI-01.

  7. Nitrate metabolism in the gromiid microbial universe

    DEFF Research Database (Denmark)

    Høgslund, Signe; Risgaard-Petersen, Nils; Cedhagen, Tomas

    Eukaryotic nitrate respiration supported by intracellular nitrate storages contributes substantially to the nitrogen cycle. Research focus is currently directed towards two phyla: Foraminifera and diatoms, but the widespread Gromia in the Rhizaria may be another key organism. These giant protists...... enclose and regulate a small biogeochemical universe within their cell. Their transparent proteinaceous cell wall surrounds a complex matrix consisting of sediment, bacteria and nitrate which is concentrated to hundreds of mM in the gromiid cell. The nitrate is respired to dinitrogen, but in contrast...... to the findings of eukaryotic mediated nitrate reduction in some foraminifera and diatoms, nitrate respiration in gromiids seems to be mediated by bacterial endosymbionts. The role of endobionts in nitrate accumulating eukaryotes is of fundamental importance for understanding the evolutionary path...

  8. Denitrification of nitrate waste solutions

    International Nuclear Information System (INIS)

    Michaels, S.L.; Michel, R.C.; Terpandjian, P.D.; Vora, J.N.

    1976-01-01

    Bacterial denitrification by Pseudomonas Stutzeri has been chosen as the method for removing nitrate from the effluent stream of the Y-12 uranium purification process. A model was developed to predict bacterial growth and carbon and nitrate depletion during the induction period and steady state operation. Modification of analytical procedures and automatic control of the pH in the reactor are recommended to improve agreement between the prediction of the model and experimental data. An initial carbon-to-nitrogen (C/N) mass ratio of 1.4-1.5 insures adequate population growth during the induction period. Further experiments in batch reactors and in steady state flow reactors are recommended to obtain more reliable kinetic rate constants

  9. 2-Amino-5-chloropyridinium nitrate

    Directory of Open Access Journals (Sweden)

    Donia Zaouali Zgolli

    2009-11-01

    Full Text Available The title structure, C5H6ClN2+·NO3−, is held together by extensive hydrogen bonding between the NO3− ions and 2-amino-5-chloropyridinium H atoms. The cation–anion N—H...O hydrogen bonds link the ions into a zigzag- chain which develops parallel to the b axis. The structure may be compared with that of the related 2-amino-5-cyanopyridinium nitrate.

  10. NADPH-d activity in rat thymus after the application of retinoid acid

    Directory of Open Access Journals (Sweden)

    F. Dorko

    2012-02-01

    Full Text Available The aim of this work was to determine the localization of nicotinamide-adenine dinucleotide phosphate-diaphorase (NADPH-d activity as the marker for synthesis of nitric oxide synthase (NOS in the rat thymus after the application of retinoid acid (RA on 1st, 7th, 14th and 21st days of gestation. The given results can build the basis for understanding of the role of NOS in rat thymus. NADPH-d positive cells were represented with dark-blue color and were localized on corticomedullar junction of the thymus. These cells were of different intensity of coloring and were shaped in oval, circle or irregular forms. NADPH-d positive nerve fibers were observed in perivascular topography. They were marked more strongly in the case of control group. The result of application of RA to gravid rats was that the birth weights of newborn rats and their thymuses were smaller, but without statistically significance.

  11. Activated barrier crossing dynamics in the non-radiative decay of NADH and NADPH

    Science.gov (United States)

    Blacker, Thomas S.; Marsh, Richard J.; Duchen, Michael R.; Bain, Angus J.

    2013-08-01

    In live tissue, alterations in metabolism induce changes in the fluorescence decay of the biological coenzyme NAD(P)H, the mechanism of which is not well understood. In this work, the fluorescence and anisotropy decay dynamics of NADH and NADPH were investigated as a function of viscosity in a range of water-glycerol solutions. The viscosity dependence of the non-radiative decay is well described by Kramers and Kramers-Hubbard models of activated barrier crossing over a wide viscosity range. Our combined lifetime and anisotropy analysis indicates common mechanisms of non-radiative relaxation in the two emitting states (conformations) of both molecules. The low frequencies associated with barrier crossing suggest that non-radiative decay is mediated by small scale motion (e.g. puckering) of the nicotinamide ring. Variations in the fluorescence lifetimes of NADH and NADPH when bound to different enzymes may therefore be attributed to differing levels of conformational restriction upon binding.

  12. Ancient Plant Glyoxylate/Succinic Semialdehyde Reductases: GLYR1s Are Cytosolic, Whereas GLYR2s Are Localized to Both Mitochondria and Plastids

    Directory of Open Access Journals (Sweden)

    Barry J. Shelp

    2017-04-01

    Full Text Available Plant NADPH-dependent glyoxylate/succinic semialdehyde reductases 1 and 2 (GLYR1 and GLYR2 are considered to be involved in detoxifying harmful aldehydes, thereby preserving plant health during exposure to various abiotic stresses. Phylogenetic analysis revealed that the two GLYR isoforms appeared in the plant lineage prior to the divergence of the Chlorophyta and Streptophyta, which occurred approximately 750 million years ago. Green fluorescent protein fusions of apple (Malus x domestica Borkh., rice (Oryza sativa L. and Arabidopsis thaliana [L.] Heynh GLYRs were transiently expressed in tobacco (Nicotiana tabaccum L. suspension cells or Arabidopsis protoplasts, as well in methoxyfenozide-induced, stably transformed Arabidopsis seedlings. The localization of apple GLYR1 confirmed that this isoform is cytosolic, whereas apple, rice and Arabidopsis GLYR2s were localized to both mitochondria and plastids. These findings highlight the potential involvement of GLYRs within distinct compartments of the plant cell.

  13. Photochemical reduction of uranyl nitrate

    Energy Technology Data Exchange (ETDEWEB)

    Duerksen, W.K.

    1993-10-20

    The photochemical reduction of uranyl nitrate solutions to tetravalent uranium was investigated as a means of producing uranium dioxide feed for the saltless direct oxide reduction (SDOR) process. At high uranium concentrations, reoxidation of U{sup +4} occurs rapidly. The kinetics of the nitric oxidation of tetravalent uranium depend on the concentrations of hydrogen ion, nitrate ion, nitrous acid, and tetravalent uranium in the same manner as was reported elsewhere for the nitrate oxidation of PU{sup +3}. Reaction rate data were successfully correlated with a mechanism in which nitrogen dioxide is the reactive intermediate. Addition of a nitrous acid scavenger suppresses the reoxidation reaction. An immersion reactor employing a mercury vapor lamp gave reduction times fast enough for routine production usage. Precipitation techniques for conversion of aqueous U(NO{sub 3}){sub 4} to hydrous UO{sub 2} were evaluated. Prolonged dewatering times tended to make the process time consuming. Use of 3- to 4-M aqueous NaOH gave the best dewatering times observed. Reoxidation of the UO{sub 2} by water of hydration was encountered, which required the drying process to be carried out under a reducing atmosphere.

  14. Functional heterogeneity of NADPH oxidase-mediated contractions to endothelin with vascular aging.

    Science.gov (United States)

    Meyer, Matthias R; Barton, Matthias; Prossnitz, Eric R

    2014-11-24

    Aging, a physiological process and main risk factor for cardiovascular and renal diseases, is associated with endothelial cell dysfunction partly resulting from NADPH oxidase-dependent oxidative stress. Because increased formation of endothelium-derived endothelin-1 (ET-1) may contribute to vascular aging, we studied the role of NADPH oxidase function in age-dependent contractions to ET-1. Renal arteries and abdominal aortas from young and old C57BL6 mice (4 and 24 months of age) were prepared for isometric force measurements. Contractions to ET-1 (0.1-100 nmol/L) were determined in the presence and absence of the NADPH oxidase-selective inhibitor gp91ds-tat (3 μmol/L). To exclude age-dependent differential effects of NO bioactivity between vascular beds, all experiments were conducted in the presence of the NO synthase inhibitor L-NAME (300 μmol/L). In young animals, ET-1-induced contractions were 6-fold stronger in the renal artery than in the aorta (prenal artery and aorta, respectively (pAging had no effect on NADPH oxidase-dependent and -independent contractions to ET-1 in the renal artery. In contrast, contractions to ET-1 were markedly reduced in the aged aorta (5-fold, page-dependent heterogeneity of NADPH oxidase-mediated vascular contractions to ET-1, demonstrating an inherent resistance to functional changes in the renal artery but not in the aorta with aging. Thus, local activity of NADPH oxidase differentially modulates responses to ET-1 with aging in distinct vascular beds. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Mitochondrial regulation of NADPH oxidase in hindlimb unweighting rat cerebral arteries.

    Directory of Open Access Journals (Sweden)

    Ran Zhang

    Full Text Available Exposure to microgravity results in post-flight cardiovascular deconditioning and orthostatic intolerance in astronauts. Vascular oxidative stress injury and mitochondrial dysfunction have been indicated in this process. To elucidate the mechanism for this condition, we investigated whether mitochondria regulated NADPH oxidase in hindlimb unweighting (HU rat cerebral and mesenteric arteries. Four-week HU was used to simulate microgravity in rats. Vascular superoxide generation, protein and mRNA levels of Nox2/Nox4, and the activity of NADPH oxidase were examined in the present study. Compared with control rats, the levels of superoxide increased in cerebral (P<0.001 but not in mesenteric vascular smooth muscle cells. The protein and mRNA levels of Nox2 and Nox4 were upregulated significantly (P<0.001 and P<0.001 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively in HU rat cerebral arteries but not in mesenteric arteries. NADPH oxidases were activated significantly by HU (P<0.001 in cerebral arteries but not in mesenteric arteries. Chronic treatment with mitochondria-targeted antioxidant mitoTEMPO attenuated superoxide levels (P<0.001, decreased the protein and mRNA expression levels of Nox2/Nox4 (P<0.01 and P<0.05 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively and the activity of NADPH oxidase (P<0.001 in HU rat cerebral arteries, but exerted no effects on HU rat mesenteric arteries. Therefore, mitochondria regulated the expression and activity of NADPH oxidases during simulated microgravity. Both mitochondria and NADPH oxidase participated in vascular redox status regulation.

  16. Crosstalk of mitochondria with NADPH oxidase via reactive oxygen and nitrogen species signalling and its role for vascular function.

    Science.gov (United States)

    Daiber, Andreas; Di Lisa, Fabio; Oelze, Matthias; Kröller-Schön, Swenja; Steven, Sebastian; Schulz, Eberhard; Münzel, Thomas

    2017-06-01

    Cardiovascular diseases are associated with and/or caused by oxidative stress. This concept has been proven by using the approach of genetic deletion of reactive species producing (pro-oxidant) enzymes as well as by the overexpression of reactive species detoxifying (antioxidant) enzymes leading to a marked reduction of reactive oxygen and nitrogen species (RONS) and in parallel to an amelioration of the severity of diseases. Likewise, the development and progression of cardiovascular diseases is aggravated by overexpression of RONS producing enzymes as well as deletion of antioxidant RONS detoxifying enzymes. Thus, the consequences of the interaction (redox crosstalk) of superoxide/hydrogen peroxide produced by mitochondria with other ROS producing enzymes such as NADPH oxidases (Nox) are of outstanding importance and will be discussed including the consequences for endothelial nitric oxide synthase (eNOS) uncoupling as well as the redox regulation of the vascular function/tone in general (soluble guanylyl cyclase, endothelin-1, prostanoid synthesis). Pathways and potential mechanisms leading to this crosstalk will be analysed in detail and highlighted by selected examples from the current literature including hypoxia, angiotensin II-induced hypertension, nitrate tolerance, aging and others. The general concept of redox-based activation of RONS sources via "kindling radicals" and enzyme-specific "redox switches" will be discussed providing evidence that mitochondria represent key players and amplifiers of the burden of oxidative stress. This article is part of a themed section on Redox Biology and Oxidative Stress in Health and Disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.12/issuetoc. © 2015 The British Pharmacological Society.

  17. Correlation of denitrification-accepted fraction of electrons with NAD(P)H fluorescence for Pseudomonas aeruginosa performing simultaneous denitrification and respiration at extremely low dissolved oxygen conditions.

    Science.gov (United States)

    Chen, Fan; Xia, Qing; Ju, Lu-Kwang

    2004-01-01

    In cystic fibrosis airway infection, Pseudomonas aeruginosa forms a microaerobic biofilm and undergoes significant physiological changes. It is important to understand the bacterium's metabolism at microaerobic conditions. In this work, the culture properties and two indicators (the denitrification-accepted e- fraction and an NAD(P)H fluorescence fraction) for the culture's "fractional approach" to a fully anaerobic denitrifying state were examined in continuous cultures with practically zero DO but different aeration rates. With decreasing aeration, specific OUR decreased while specific NAR and NIR increased and kept Y(ATP/S) relatively constant. P. aeruginosa thus appeared to effectively compensate for energy generation at microaerobic conditions with denitrification. At the studied dilution rate of 0.06 h(-1), the maximum specific OUR was 2.8 mmol O2/g cells-h and the Monod constant for DO, in the presence of nitrate, was extremely low (Y(X/S) increased significantly (from 0.24 to 0.34) with increasing aeration, attributed to a roughly opposite trend of Y(ATP/X) (ATP generation required for cell growth). As for the denitrification-accepted e- fraction and the fluorescence fraction, both decreased with increasing aeration as expected. The two fractions, however, were not directly proportional. The fluorescence fraction changed more rapidly than the e- fraction at very low aeration rates, whereas the opposite was true at higher aeration. The results demonstrated the feasibility of using online NAD(P)H fluorescence to monitor sensitive changes of cellular physiology and provided insights to the shift of e- -accepting mechanisms of P. aeruginosa under microaerobic conditions.

  18. Investigation into common solubility of dysprosium nitrate with nitrates of aniline, pyridine in aqueous solutions

    International Nuclear Information System (INIS)

    Katamanov, V.L.; Zhuravlev, E.F.

    1976-01-01

    Data are given on solubility at 20 and 40 deg C in the systems: dysprosium nitrate - aniline nitrate -water and dysprosium nitrate - pyridine nitrate - water. Solubilities are determined by the method of sections. It is found that isotherms of the first system are of eutonic type, equilibrium solid phases of dysprosium hexahydrate nitrate and aniline nitrate correspond to non-variant points of intersection of lines of saturated solutions. The solubility of aniline nitrate is much lower than that of dysprosium hexahydrate nitrate. An increase in the concentration of Dy 3+ ions in a binary solution results in salting-out of aniline nitrate and in absence of any interaction in the system. The compound Dy(NO 3 ) 3 x2PyxHNO 3 is formed in a congruent way in the second system considered

  19. Engineering of xylose reductase and overexpression of xylitol dehydrogenase and xylulokinase improves xylose alcoholic fermentation in the thermotolerant yeast Hansenula polymorpha

    Directory of Open Access Journals (Sweden)

    Voronovsky Andriy Y

    2008-07-01

    Full Text Available Abstract Background The thermotolerant methylotrophic yeast Hansenula polymorpha is capable of alcoholic fermentation of xylose at elevated temperatures (45 – 48°C. Such property of this yeast defines it as a good candidate for the development of an efficient process for simultaneous saccharification and fermentation. However, to be economically viable, the main characteristics of xylose fermentation of H. polymorpha have to be improved. Results Site-specific mutagenesis of H. polymorpha XYL1 gene encoding xylose reductase was carried out to decrease affinity of this enzyme toward NADPH. The modified version of XYL1 gene under control of the strong constitutive HpGAP promoter was overexpressed on a Δxyl1 background. This resulted in significant increase in the KM for NADPH in the mutated xylose reductase (K341 → R N343 → D, while KM for NADH remained nearly unchanged. The recombinant H. polymorpha strain overexpressing the mutated enzyme together with native xylitol dehydrogenase and xylulokinase on Δxyl1 background was constructed. Xylose consumption, ethanol and xylitol production by the constructed strain were determined for high-temperature xylose fermentation at 48°C. A significant increase in ethanol productivity (up to 7.3 times was shown in this recombinant strain as compared with the wild type strain. Moreover, the xylitol production by the recombinant strain was reduced considerably to 0.9 mg × (L × h-1 as compared to 4.2 mg × (L × h-1 for the wild type strain. Conclusion Recombinant strains of H. polymorpha engineered for improved xylose utilization are described in the present work. These strains show a significant increase in ethanol productivity with simultaneous reduction in the production of xylitol during high-temperature xylose fermentation.

  20. NO signaling is a key component of the root growth response to nitrate in Zea mays L.

    Science.gov (United States)

    Trevisan, Sara; Manoli, Alessandro; Quaggiotti, Silvia

    2014-01-01

    Roots are considered to be a vital organ system of plants due to their involvement in water and nutrient uptake, anchorage, propagation, storage functions, secondary metabolite (including hormones) biosynthesis, and accumulation. Crops are strongly dependent on the availability of nitrogen in soil and on the efficiency of nitrogen utilization for biomass production and yield. However, knowledge about molecular responses to nitrogen fluctuations mainly derives from the study of model species. Nitric oxide (NO) has been proposed to be implicated in plant adaptation to environment, but its exact role in the response of plants to nutritional stress is still under evaluation. Recently a novel role for NO production and scavenging, thanks to the coordinate spatio-temporal expression of nitrate reductase and non-symbiotic hemoglobins, in the maize root response to nitrate has been postulated. This control of NO homeostasis is preferentially accomplished by the cells of the root transition zone (TZ) which seem to represent the most nitrate responsive portion of maize root. The TZ is already known to function as a sensory center able to gather information from the external environment and to re-elaborate them in an adequate response. These results indicate that it could play a central role also for nitrate sensing by roots. A lot of work is still needed to identify and characterize other upstream and downstream signals involved in the "nitrate-NO" pathway, leading to root architecture adjustments and finally to stress adaptation.

  1. The Tail Wagging the Dog: Insights into Catalysis in R67 Dihydrofolate Reductase

    Energy Technology Data Exchange (ETDEWEB)

    Kamath, Ganesh K [ORNL; Agarwal, Pratul K [ORNL

    2010-01-01

    Plasmid-encoded R67 dihydrofolate reductase (DHFR) catalyzes a hydride transfer reaction between substrate dihydrofolate (DHF) and its cofactor, nicotinamide adenine dinucleotide phosphate (NADPH). R67 DHFR is a homotetramer that exhibits numerous characteristics of a primitive enzyme, including promiscuity in binding of substrate and cofactor, formation of nonproductive complexes, and the absence of a conserved acid in its active site. Furthermore, R67's active site is a pore, which is mostly accessible by bulk solvent. This study uses a computational approach to characterize the mechanism of hydride transfer. Not surprisingly, NADPH remains fixed in one-half of the active site pore using numerous interactions with R67. Also, stacking between the nicotinamide ring of the cofactor and the pteridine ring of the substrate, DHF, at the hourglass center of the pore, holds the reactants in place. However, large movements of the p-aminobenzoylglutamate tail of DHF occur in the other half of the pore because of ion pair switching between symmetry-related K32 residues from two subunits. This computational result is supported by experimental results that the loss of these ion pair interactions (located >13 {angstrom} from the center of the pore) by addition of salt or in asymmetric K32M mutants leads to altered enzyme kinetics [Hicks, S. N., et al. (2003) Biochemistry 42, 10569-10578; Hicks, S. N., et al. (2004) J. Biol. Chem. 279, 46995?47002]. The tail movement at the edge of the active site, coupled with the fixed position of the pteridine ring in the center of the pore, leads to puckering of the pteridine ring and promotes formation of the transition state. Flexibility coupled to R67 function is unusual as it contrasts with the paradigm that enzymes use increased rigidity to facilitate attainment of their transition states. A comparison with chromosomal DHFR indicates a number of similarities, including puckering of the nicotinamide ring and changes in the DHF tail

  2. Identification of a mitochondrial external NADPH dehydrogenase by overexpression in transgenic ¤Nicotiana sylvestris¤

    DEFF Research Database (Denmark)

    Michalecka, A.M.; Agius, S.C.; Møller, I.M.

    2004-01-01

    (P)H dehydrogenases, was introduced into Nicotiana sylvestris. Transgenic lines with high transcript and protein levels for St-NDB1 had up to threefold increased activity of external NADPH dehydrogenase in isolated mitochondria as compared to the wild type (WT). In two lines, the external NADPH dehydrogenase activity...

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

    Energy Technology Data Exchange (ETDEWEB)

    Heaslet, Holly; Harris, Melissa; Fahnoe, Kelly; Sarver, Ronald; Putz, Henry; Chang, Jeanne; Subramanyam, Chakrapani; Barreiro, Gabriela; Miller, J. Richard; Pfizer

    2010-09-02

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

  4. The cytochrome bd respiratory oxygen reductases.

    Science.gov (United States)

    Borisov, Vitaliy B; Gennis, Robert B; Hemp, James; Verkhovsky, Michael I

    2011-11-01

    Cytochrome bd is a respiratory quinol: O₂ oxidoreductase found in many prokaryotes, including a number of pathogens. The main bioenergetic function of the enzyme is the production of a proton motive force by the vectorial charge transfer of protons. The sequences of cytochromes bd are not homologous to those of the other respiratory oxygen reductases, i.e., the heme-copper oxygen reductases or alternative oxidases (AOX). Generally, cytochromes bd are noteworthy for their high affinity for O₂ and resistance to inhibition by cyanide. In E. coli, for example, cytochrome bd (specifically, cytochrome bd-I) is expressed under O₂-limited conditions. Among the members of the bd-family are the so-called cyanide-insensitive quinol oxidases (CIO) which often have a low content of the eponymous heme d but, instead, have heme b in place of heme d in at least a majority of the enzyme population. However, at this point, no sequence motif has been identified to distinguish cytochrome bd (with a stoichiometric complement of heme d) from an enzyme designated as CIO. Members of the bd-family can be subdivided into those which contain either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I, designated as the Q-loop. However, it is not clear whether there is a functional consequence of this difference. This review summarizes current knowledge on the physiological functions, genetics, structural and catalytic properties of cytochromes bd. Included in this review are descriptions of the intermediates of the catalytic cycle, the proposed site for the reduction of O₂, evidence for a proton channel connecting this active site to the bacterial cytoplasm, and the molecular mechanism by which a membrane potential is generated. 2011 Elsevier B.V. All rights reserved.

  5. Cucurbitacin delta 23-reductase from the fruit of Cucurbita maxima var. Green Hubbard. Physicochemical and fluorescence properties and enzyme-ligand interactions.

    Science.gov (United States)

    Dirr, H W; Schabort, J C; Weitz, C

    1986-02-01

    Cucurbitacin delta 23-reductase from Cucurbita maxima var. Green Hubbard fruit displays an apparent Mr of 32,000, a Stokes radius of 263 nm and a diffusion coefficient of 8.93 X 10(-7) cm2 X s-1. The enzyme appears to possess a homogeneous dimeric quaternary structure with a subunit Mr of 15,000. Two tryptophan and fourteen tyrosine residues per dimer were found. Emission spectral properties of the enzyme and fluorescence quenching by iodide indicate the tryptophan residues to be buried within the protein molecule. In the pH range 5-7, where no conformational changes were detected, protonation of a sterically related ionizable group with a pK of approx. 6.0 markedly influenced the fluorescence of the tryptophan residues. Protein fluorescence quenching was employed to determine the dissociation constants for binding of NADPH (Kd 17 microM), NADP+ (Kd 30 microM) and elaterinide (Kd 227 microM). Fluorescence energy transfer between the tryptophan residues and enzyme-bound NADPH was observed.

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

    Science.gov (United States)

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

    2017-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Kenneth R. Olson

    2017-08-01

    Full Text Available Catalase is well-known as an antioxidant dismutating H2O2 to O2 and H2O. However, catalases evolved when metabolism was largely sulfur-based, long before O2 and reactive oxygen species (ROS became abundant, suggesting catalase metabolizes reactive sulfide species (RSS. Here we examine catalase metabolism of H2Sn, the sulfur analog of H2O2, hydrogen sulfide (H2S and other sulfur-bearing molecules using H2S-specific amperometric electrodes and fluorophores to measure polysulfides (H2Sn; SSP4 and ROS (dichlorofluorescein, DCF. Catalase eliminated H2Sn, but did not anaerobically generate H2S, the expected product of dismutation. Instead, catalase concentration- and oxygen-dependently metabolized H2S and in so doing acted as a sulfide oxidase with a P50 of 20 mmHg. H2O2 had little effect on catalase-mediated H2S metabolism but in the presence of the catalase inhibitor, sodium azide (Az, H2O2 rapidly and efficiently expedited H2S metabolism in both normoxia and hypoxia suggesting H2O2 is an effective electron acceptor in this reaction. Unexpectedly, catalase concentration-dependently generated H2S from dithiothreitol (DTT in both normoxia and hypoxia, concomitantly oxidizing H2S in the presence of O2. H2S production from DTT was inhibited by carbon monoxide and augmented by NADPH suggesting that catalase heme-iron is the catalytic site and that NADPH provides reducing equivalents. Catalase also generated H2S from garlic oil, diallyltrisulfide, thioredoxin and sulfur dioxide, but not from sulfite, metabisulfite, carbonyl sulfide, cysteine, cystine, glutathione or oxidized glutathione. Oxidase activity was also present in catalase from Aspergillus niger. These results show that catalase can act as either a sulfide oxidase or sulfur reductase and they suggest that these activities likely played a prominent role in sulfur metabolism during evolution and may continue do so in modern cells as well. This also appears to be the first observation of catalase

  8. Nitrous oxide emission by the non-denitrifying, nitrate ammonifier Bacillus licheniformis.

    Science.gov (United States)

    Sun, Yihua; De Vos, Paul; Heylen, Kim

    2016-01-19

    Firmicutes have the capacity to remove excess nitrate from the environment via either denitrification, dissimilatory nitrate reduction to ammonium or both. The recent renewed interest in their nitrogen metabolism has revealed many interesting features, the most striking being their wide variety of dissimilatory nitrate reduction pathways. In the present study, nitrous oxide production from Bacillus licheniformis, a ubiquitous Gram-positive, spore-forming species with many industrial applications, is investigated. B. licheniformis has long been considered a denitrifier but physiological experiments on three different strains demonstrated that nitrous oxide is not produced from nitrate in stoichiometric amounts, rather ammonium is the most important end-product, produced during fermentation. Significant strain dependency in end-product ratios, attributed to nitrite and ammonium, and medium dependency in nitrous oxide production were also observed. Genome analyses confirmed the lack of a nitrite reductase to nitric oxide, the key enzyme of denitrification. Based on the gene inventory and building on knowledge from other non-denitrifying nitrous oxide emitters, hypothetical pathways for nitrous oxide production, involving NarG, NirB, qNor and Hmp, are proposed. In addition, all publically available genomes of B. licheniformis demonstrated similar gene inventories, with specific duplications of the nar operon, narK and hmp genes as well as NarG phylogeny supporting the evolutionary separation of previously described distinct BALI1 and BALI2 lineages. Using physiological and genomic data we have demonstrated that the common soil bacterium B. licheniformis does not denitrify but is capable of fermentative dissimilatory nitrate/nitrite reduction to ammonium (DNRA) with concomitant production of N2O. Considering its ubiquitous nature and non-fastidious growth in the lab, B. licheniformis is a suitable candidate for further exploration of the actual mechanism of N2O

  9. Nitrate reduction associated with respiration in Sinorhizobium meliloti 2011 is performed by a membrane-bound molybdoenzyme.

    Science.gov (United States)

    Ferroni, Felix M; Rivas, María G; Rizzi, Alberto C; Lucca, María E; Perotti, Nora I; Brondino, Carlos D

    2011-10-01

    The purification and biochemical characterization of the respiratory membrane-bound nitrate reductase from Sinorhizobium meliloti 2011 (Sm NR) is reported together with the optimal conditions for cell growth and enzyme production. The best biomass yield was obtained under aerobic conditions in a fed-batch system using Luria-Bertani medium with glucose as carbon source. The highest level of Sm NR production was achieved using microaerobic conditions with the medium supplemented with both nitrate and nitrite. Sm NR is a mononuclear Mo-protein belonging to the DMSO reductase family isolated as a heterodimeric enzyme containing two subunits of 118 and 45 kDa. Protein characterization by mass spectrometry showed homology with respiratory nitrate reductases. UV-Vis spectra of as-isolated and dithionite reduced Sm NR showed characteristic absorption bands of iron-sulfur and heme centers. Kinetic studies indicate that Sm NR follows a Michaelis-Menten mechanism (K (m) = 97 ± 11 μM, V = 9.4 ± 0.5 μM min(-1), and k (cat) = 12.1 ± 0.6 s(-1)) and is inhibited by azide, chlorate, and cyanide with mixed inhibition patterns. Physiological and kinetic studies indicate that molybdenum is essential for NR activity and that replacement of this metal for tungsten inhibits the enzyme. Although no narGHI gene cluster has been annotated in the genome of rhizobia, the biochemical characterization indicates that Sm NR is a Mo-containing NR enzyme with molecular organization similar to NarGHI.

  10. Nitrate contamination of groundwater and its countermeasures

    Energy Technology Data Exchange (ETDEWEB)

    Mitamura, Hisayoshi [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

    2003-03-01

    The inevitable increases of food production and energy consumption with an increase in world population become main causes of an increase of nitrate load to the environment. Although nitrogen is essential for the growth of animal and plant as a constituent element of protein, excessive nitrate load to the environment contaminates groundwater resources used as drinking water and leads to seriously adverse effects on the health of man and livestock. In order to clarify the problem of nitrate contamination of groundwater and search a new trend of technology development from the viewpoint of environment remediation and protection, the present paper has reviewed adverse effects of nitrate on human health, the actual state of nitrogen cycle, several kinds of nitrate sources, measures for reducing nitrate level, etc. (author)

  11. Alpha autoradiography by cellulose nitrate layer

    International Nuclear Information System (INIS)

    Simonovic, J.; Vukovic, J.; Antanasijevic, R.

    1977-01-01

    From domestic cellulose nitrate bulk material thin layers for α-particle autoradiography were prepared. An artificial test specimen of a uniformly alpha labelled grid source was used. The efficiency of autoradiography by cellulose nitrate was calculated comparing with data from an Ilford K2 nuclear emulsion exposed under the same conditions as the cellulose nitrate film. The resolution was determined as the distance from grid pitch edge at which the track density fell considerably. (Auth.)

  12. Alpha autoradiography by cellulose nitrate layer

    International Nuclear Information System (INIS)

    Simonovic, J.; Vukovic, J.; Antanasijevic, R.

    1976-01-01

    From domestic cellulose nitrate bulk material thin layers for α-particle autoradiography were prepared. An artifical test specimen of a uniformly alpha labelled grid source was used. The efficiency of autoradiographs by cellulose nitrate was calculated comparing with data from an Ilford K2 nuclear emulsion exposed under the same conditions as the cellulose nitrate film. The resolution was determined as the distance from grid pitch edge at which the track density fell considerably. (orig.) [de

  13. Nitrat i drikkevandet og vores sundhed

    DEFF Research Database (Denmark)

    Hansen, Birgitte; Schullehner, Jörg; Sigsgaard, Torben

    2014-01-01

    Nitrat i drikkevandet er uønsket, da det kan påvirke vores sundhed negativt. Den øvre grænse for hvor meget nitrat der tillades i drikkevandet er fastsat i forhold til risikoen for akut forgiftning med nitrit og blå børn-syndromet. Men nitrat i drikkevandet mistænkes også for at være medvirkende...

  14. Vitamin E--a selective inhibitor of the NADPH oxidoreductase enzyme system in human granulocytes

    International Nuclear Information System (INIS)

    Butterick, C.J.; Baehner, R.L.; Boxer, L.A.; Jersild, R.A. Jr.

    1983-01-01

    The cellular sites of H 2 O 2 formation in phagocytizing granulocytes have been identified with cerium chloride. A precipitate was visible in phagosomes and on plasma membranes from intact normal cells in the presence of either 0.71 mM NADH or NADPH. X-ray microanalysis permitted identification of cerium deposition within the phagosomes even in the absence of reduced pyridine nucleotides. Catalase ablated the formation of the reaction product. Intact granulocytes obtained from subjects receiving 1600 units of vitamin E daily for 2 weeks exhibited reaction product in the presence of NADH but not NADPH. Intact cells from subjects treated with vitamin E demonstrated diminished numbers of phagocytic vesicles containing reaction product. During phagocytosis the granulocytes treated with vitamin E consumed oxygen but exhibited significantly reduced rates of hydrogen-peroxide-dependent glucose-1- 14 C oxidation to 14 CO 2 . Isolated phagocytic vesicles obtained from granulocytes after ingestion of opsonized lipopolysaccharide-paraffin oil droplets contained reaction product when exposed to 0.71 mM NADPH. No reaction product was evident at 0.71 mM NADH but was evident at 2.0 mM NADH. Isolated phagocytic vesicles from the granulocytes of subjects receiving vitamin E exhibited reaction product only in the presence of NADH. These observations suggest that vitamin E interferes with the electron transport chain apparently required for the oxidation of NADPH to form H 2 O 2 in the phagocytizing granulocyte

  15. Alcohol-induced bone loss is blocked in p47phox -/- mice lacking functional nadph oxidases

    Science.gov (United States)

    Chronic ethanol (EtOH) consumption produces bone loss. Previous data suggest a role for NADPH oxidase enzymes (Nox) since the pan-Nox inhibitor diphenylene iodonium (DPI) blocks EtOH-induced bone loss in rats. The current study utilized mice in which Nox enzymes 1,2,3 and 5 are inactivated as a resu...

  16. Inhibition of NADPH oxidases prevents chronic ethanol-induced bone loss in female rats

    Science.gov (United States)

    Previous in vitro data suggest that ethanol (EtOH) activates NADPH (nicotinamide adenine dinucleotide phosphate) oxidase (Nox) in osteoblasts leading to accumulation of reactive oxygen species (ROS). This might be a mechanism underlying inhibition of bone formation and increased bone resorption obse...

  17. Localization of NADPH-diaphorase in the brain of the chicken.

    Science.gov (United States)

    Brüning, G

    1993-08-08

    NADPH-diaphorase, an enzyme catalyzed reaction thought to reflect the activity of nitric oxide synthase in the mammalian nervous system, was mapped in the brain of the chicken. Intensely stained neurons and fibers were found in most parts of the telencephalon, in particular in the neostriatum, paleostriatum augmentatum, olfactory tubercle, lobus parolfactorius, hyperstriatum accessorium, and hyperstriatum ventrale. Medial to the nucleus taeniae, an accumulation of stained cells was observed that appeared to merge with a band of stained neurons located dorsal to the occipitomesencephalic tract. These are considered to belong to the nucleus interstitialis of the dorsal olfactory projection. Further caudally, neurons with different staining intensities were found in the lateral hypothalamic area, lateral mammillary nucleus, periventricular organ, ventral tegmental area, medial spiriform nucleus, optic tectum, isthmooptic nucleus, mesencephalic trigeminal nucleus, interpeduncular nucleus, and central gray of the mesencephalon. A particularly dense cluster of NADPH-diaphorase positive neurons was located in the locus coeruleus. It is proposed that these might represent cholinergic cells intermingled with catecholaminergic neurons, thus forming the avian counterpart of the tegmental cholinergic nuclei of mammals. Several NADPH-diaphorase reactive neurons were seen in the parabrachial nucleus and medial and dorsal vestibular nucleus, as well as scattered in the reticular formation. In the caudal medulla, intensely stained cells were grouped around the central canal. Therefore the pattern of expression of NADPH-diaphorase, and thus possibly of nitric oxide synthase, within the avian and mammalian brain might be largely conserved.

  18. Pancreatic Beta-Cell Purification by Altering FAD and NAD(PH Metabolism

    Directory of Open Access Journals (Sweden)

    P. de Vos

    2008-07-01

    Full Text Available Isolation of primary beta cells from other cells within in the pancreatic islets is of importance for many fields of islet research. However, up to now, no satisfactory method has been developed that gained high numbers of viable beta cells, without considerable alpha-cell contamination. In this study, we investigated whether rat beta cells can be isolated from nonbeta endocrine cells by manipulating the flavin adenine dinucleotide (FAD and nicotinamide-adenine dinucleotide phosphate (NAD(PH autofluorescence. Beta cells were isolated from dispersed islets by flow cytometry, based on their high FAD and NAD(PH fluorescence. To improve beta cell yield and purity, the cellular FAD and NAD(PH contents were altered by preincubation in culture media containing varying amounts of D-glucose and amino acids. Manipulation of the cellular FAD and NAD(PH fluorescence improves beta cell yield and purity after sorting. This method is also a fast and reliable method to measure beta cell functional viability. A conceivable application is assessing beta cell viability before transplantation.

  19. The microglial NADPH oxidase complex as a source of oxidative stress in Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Landreth Gary E

    2006-11-01

    Full Text Available Abstract Alzheimer's disease is the most common cause of dementia in the elderly, and manifests as progressive cognitive decline and profound neuronal loss. The principal neuropathological hallmarks of Alzheimer's disease are the senile plaques and the neurofibrillary tangles. The senile plaques are surrounded by activated microglia, which are largely responsible for the proinflammatory environment within the diseased brain. Microglia are the resident innate immune cells in the brain. In response to contact with fibrillar beta-amyloid, microglia secrete a diverse array of proinflammatory molecules. Evidence suggests that oxidative stress emanating from activated microglia contribute to the neuronal loss characteristic of this disease. The source of fibrillar beta-amyloid induced reactive oxygen species is primarily the microglial nicotinamide adenine dinucleotide phosphate (NADPH oxidase. The NADPH oxidase is a multicomponent enzyme complex that, upon activation, produces the highly reactive free radical superoxide. The cascade of intracellular signaling events leading to NADPH oxidase assembly and the subsequent release of superoxide in fibrillar beta-amyloid stimulated microglia has recently been elucidated. The induction of reactive oxygen species, as well as nitric oxide, from activated microglia can enhance the production of more potent free radicals such as peroxynitrite. The formation of peroxynitrite causes protein oxidation, lipid peroxidation and DNA damage, which ultimately lead to neuronal cell death. The elimination of beta-amyloid-induced oxidative damage through the inhibition of the NADPH oxidase represents an attractive therapeutic target for the treatment of Alzheimer's disease.

  20. Abnormal microsomal detoxification implicated in Fanconi anemia group C by interaction of the FAC protein with NADPH cytochrome P450 reductase

    NARCIS (Netherlands)

    Kruyt, FAE; Hoshino, T; Liu, JM; Joseph, P; Jaiswal, AK; Youssoufian, H

    1998-01-01

    The FAC protein encoded by the Fanconi anemia (FA) complementation group C gene is thought to function in the cytoplasm at a step before DNA repair. Because FA cells are susceptible to mitomycin C, we considered the possibility that FAC might interact with enzymes involved in the bioreductive

  1. One-electron reduction of mitomycin c by rat liver : role of cytochrome P-450 and NADPH-cytochrome P-450 reductase

    NARCIS (Netherlands)

    Vromans, R M; Van de Straat, R; Groeneveld, M.; Vermeulen, N P

    1. The role of cytochrome P-450 in the one-electron reduction of mitomycin c was studied in rat hepatic microsomal systems and in reconstituted systems of purified cytochrome P-450. Formation of H2O2 from redox cycling of the reduced mitomycin c in the presence of O2 and the alkylation of

  2. Structure and mechanism of a bacterial haloalcohol dehalogenase : a new variation of the short-chain dehydrogenase/reductase fold without an NAD(P)H binding site

    NARCIS (Netherlands)

    Jong, R.M.de; Tiesinga, J.J.W.; Rozeboom, H.J.; Kalk, K.H.; Janssen, D.B.; Dijkstra, B.W.

    2003-01-01

    Haloalcohol dehalogenases are bacterial enzymes that catalyze the cofactor-independent dehalogenation of vicinal haloalcohols such as the genotoxic environmental pollutant 1,3-dichloro-2-propanol, thereby producing an epoxide, a chloride ion and a proton. Here we present X-ray structures of the

  3. Synthesis of cerous ammonium nitrate using ceric ammonium nitrate and anhydrous ammonia as a reducing agent

    International Nuclear Information System (INIS)

    Bourleaux, G.; Colombet, P.; Rouxel, J.; Gradeff, P.S.; Mauermann, H.

    1988-01-01

    The reduction of ceric ammonium nitrate by anhydrous ammonia has been studied. The reaction yields cerous ammonium nitrate complex. This is an easy method to prepare an anhydrous Ce (III) salt, suitable for synthesis of organo cerium (III) derivatives [fr

  4. Nuclear magnetic resonance study of interaction of ligands with Streptococcus faecium dihydrofolate reductase labeled with [#betta#-13C]tryptophan

    International Nuclear Information System (INIS)

    London, R.E.; Groff, J.P.; Cocco, L.; Blakley, R.L.

    1982-01-01

    Dihydrofolate reductase from Streptococcus faecium has been labeled with [#betta#- 13 C]tryptophan. We have determined changes occurring in the chemical shifts and line widths of the four resonances of the 13 C NMR spectrum of the labeled enzyme, due to its interaction with various ligands. These include the coenzyme, NPDPH and related nucleotides, folate and its polyglutamate derivatives, and many inhibitors including methotrexate and trimethoprim. In addition, paramagnetic relaxation effects produced by a bound spin-labeled analogue of 2'-phosphoadenosine-5'-diphosphoribose on the tryptophan C/sup #betta#/ carbons have been measured. Distances calculated from the relaxation data have been compared with corresponding distances in the crystallographic model of the NADPH-methotrexate ternary complex of Lactobacillus casei reductase. The paramagnetic relaxation data indicate that the two downfield resonances (1 and 2) correspond to tryptophans (W/sub A/ and W/sub B/) that are more remote from the catalytic site, and from the crystallographic model these are seen to be Trp-115 and Trp-160. The upfield resonances (3 and 4) that show broadening due to chemical exchange correspond to closer residues (W/sub C/ and W/sub D/), and these are identified with Trp-6 and Trp-22. However, the relaxation data do not permit specific assignments within the nearer and farther pairs. Although resonance 3, which is split due to chemical exchange, was formerly assigned to Trp-6, data obtained for the enzyme in the presence of various ligands are better interpreted if resonance 3 is assigned to Trp-22, which is located on a loop that joins elements of secondary structure and forms one side of the ligand-binding cavity

  5. Regulation of ribonucleotide reductase by Spd1 involves multiple mechanisms

    DEFF Research Database (Denmark)

    Nestoras, Konstantinos; Mohammed, Asma Hadi; Schreurs, Ann-Sofie

    2010-01-01

    The correct levels of deoxyribonucleotide triphosphates and their relative abundance are important to maintain genomic integrity. Ribonucleotide reductase (RNR) regulation is complex and multifaceted. RNR is regulated allosterically by two nucleotide-binding sites, by transcriptional control, and...

  6. Reduction of Folate by Dihydrofolate Reductase from Thermotoga maritima

    NARCIS (Netherlands)

    Loveridge, E Joel; Hroch, Lukas; Hughes, Robert L; Williams, Thomas; Davies, Rhidian L; Angelastro, Antonio; Luk, Louis Y P; Maglia, Giovanni; Allemann, Rudolf K

    2017-01-01

    Mammalian dihydrofolate reductases (DHFR) catalyse the reduction of folate more efficiently than the equivalent bacterial enzymes, despite typically having similar efficiencies for the reduction of their natural substrate dihydrofolate. In contrast, we show here that DHFR from the hyperthermophilic

  7. Ozone affects pollen viability and NAD(P)H oxidase release from Ambrosia artemisiifolia pollen

    Energy Technology Data Exchange (ETDEWEB)

    Pasqualini, Stefania, E-mail: spas@unipg.it [Department of Applied Biology, University of Perugia, Perugia (Italy); Tedeschini, Emma; Frenguelli, Giuseppe [Department of Applied Biology, University of Perugia, Perugia (Italy); Wopfner, Nicole; Ferreira, Fatima [Department of Molecular Biology, CD Laboratory for Allergy Diagnosis and Therapy, University of Salzburg, Salzburg (Austria); D' Amato, Gennaro [Division of Respiratory and Allergic Diseases, ' A. Cardarelli' High Speciality Hospital, Naples (Italy); Ederli, Luisa [Department of Applied Biology, University of Perugia, Perugia (Italy)

    2011-10-15

    Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O{sub 3}) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O{sub 3} fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O{sub 3} fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O{sub 3}, determined from the mRNA levels of the major allergens. We conclude that O{sub 3} can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase. - Highlights: > O{sub 3} reduces the viability of ragweed pollen. > ROS and allergens of ragweed pollen were not affected by O{sub 3} exposure. > O{sub 3} enhances the activity of the ROS-generating enzyme NAD(P)H oxidase. > O{sub 3} increases ragweed pollen allergenicity through NAD(P)H-oxidase stimulation. - This study focuses on the effects of the atmospheric pollutant ozone on ROS content and NAD(P)H oxidase activity of ragweed pollen grains.

  8. Critical role of NADPH oxidase in neuronal oxidative damage and microglia activation following traumatic brain injury.

    Directory of Open Access Journals (Sweden)

    Quan-Guang Zhang

    Full Text Available BACKGROUND: Oxidative stress is known to play an important role in the pathology of traumatic brain injury. Mitochondria are thought to be the major source of the damaging reactive oxygen species (ROS following TBI. However, recent work has revealed that the membrane, via the enzyme NADPH oxidase can also generate the superoxide radical (O(2(-, and thereby potentially contribute to the oxidative stress following TBI. The current study thus addressed the potential role of NADPH oxidase in TBI. METHODOLOGY/PRINCIPAL FINDINGS: The results revealed that NADPH oxidase activity in the cerebral cortex and hippocampal CA1 region increases rapidly following controlled cortical impact in male mice, with an early peak at 1 h, followed by a secondary peak from 24-96 h after TBI. In situ localization using oxidized hydroethidine and the neuronal marker, NeuN, revealed that the O(2(- induction occurred in neurons at 1 h after TBI. Pre- or post-treatment with the NADPH oxidase inhibitor, apocynin markedly inhibited microglial activation and oxidative stress damage. Apocynin also attenuated TBI-induction of the Alzheimer's disease proteins β-amyloid and amyloid precursor protein. Finally, both pre- and post-treatment of apocynin was also shown to induce significant neuroprotection against TBI. In addition, a NOX2-specific inhibitor, gp91ds-tat was also shown to exert neuroprotection against TBI. CONCLUSIONS/SIGNIFICANCE: As a whole, the study demonstrates that NADPH oxidase activity and superoxide production exhibit a biphasic elevation in the hippocampus and cortex following TBI, which contributes significantly to the pathology of TBI via mediation of oxidative stress damage, microglial activation, and AD protein induction in the brain following TBI.

  9. NADPH oxidase 4 regulates homocysteine metabolism and protects against acetaminophen-induced liver damage in mice.

    Science.gov (United States)

    Murray, Thomas V A; Dong, Xuebin; Sawyer, Greta J; Caldwell, Anna; Halket, John; Sherwood, Roy; Quaglia, Alberto; Dew, Tracy; Anilkumar, Narayana; Burr, Simon; Mistry, Rajesh K; Martin, Daniel; Schröder, Katrin; Brandes, Ralf P; Hughes, Robin D; Shah, Ajay M; Brewer, Alison C

    2015-12-01

    Glutathione is the major intracellular redox buffer in the liver and is critical for hepatic detoxification of xenobiotics and other environmental toxins. Hepatic glutathione is also a major systemic store for other organs and thus impacts on pathologies such as Alzheimer's disease, Sickle Cell Anaemia and chronic diseases associated with aging. Glutathione levels are determined in part by the availability of cysteine, generated from homocysteine through the transsulfuration pathway. The partitioning of homocysteine between remethylation and transsulfuration pathways is known to be subject to redox-dependent regulation, but the underlying mechanisms are not known. An association between plasma Hcy and a single nucleotide polymorphism within the NADPH oxidase 4 locus led us to investigate the involvement of this reactive oxygen species- generating enzyme in homocysteine metabolism. Here we demonstrate that NADPH oxidase 4 ablation in mice results in increased flux of homocysteine through the betaine-dependent remethylation pathway to methionine, catalysed by betaine-homocysteine-methyltransferase within the liver. As a consequence NADPH oxidase 4-null mice display significantly lowered plasma homocysteine and the flux of homocysteine through the transsulfuration pathway is reduced, resulting in lower hepatic cysteine and glutathione levels. Mice deficient in NADPH oxidase 4 had markedly increased susceptibility to acetaminophen-induced hepatic injury which could be corrected by administration of N-acetyl cysteine. We thus conclude that under physiological conditions, NADPH oxidase 4-derived reactive oxygen species is a regulator of the partitioning of the metabolic flux of homocysteine, which impacts upon hepatic cysteine and glutathione levels and thereby upon defence against environmental toxins. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  10. Increasing anaerobic acetate consumption and ethanol yields in Saccharomyces cerevisiae with NADPH-specific alcohol dehydrogenase.

    Science.gov (United States)

    Henningsen, Brooks M; Hon, Shuen; Covalla, Sean F; Sonu, Carolina; Argyros, D Aaron; Barrett, Trisha F; Wiswall, Erin; Froehlich, Allan C; Zelle, Rintze M

    2015-12-01

    Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter(-1) acetate during fermentation of 114 g liter(-1) glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter(-1), this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter(-1) and raised the ethanol yield to 7% above the wild-type level. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  11. Unexpected genetic and structural relationships of a long-forgotten flavoenzyme to NAD(P)H:quinone reductase (DT-diaphorase)

    Science.gov (United States)

    Zhao, Q; Yang, X L; Holtzclaw, W D; Talalay, P

    1997-03-04

    A mammalian cytosolic FAD-dependent enzyme that catalyzes the reduction of quinones by N-ribosyl- and N-alkyldihydronicotinamides, but not by NADH, NADPH, or NMNH (reduced nicotinamide mononucleotide), was isolated from bovine kidney more than 30 years ago [S. Liao, J. T. Dulaney and H. G. Williams-Ashman (1962) J. Biol. Chem. 237, 2981-2987]. This enzyme is designated here as quinone reductase type 2 (QR2). Bovine QR2 is a homodimer that migrates on SDS/PAGE at approximately 22 kDa. Three tryptic peptides of bovine QR2 (representing 39 amino acids) showed 43% identity to human NAD(P)H:quinone reductase (DT-diaphorase; EC 1.6.99.2), here designated QR1 and 82% identity to a related human cDNA clone [called hNQO2 by A. K. Jaiswal, P. Burnett, M. Adesnik and O. W. McBride (1990) Biochemistry 29, 1899-1906], and designated here as hQR2. The protein encoded by the latter cDNA did not show QR activity when tested with conventional nicotinamide nucleotides. The unexpected high homology between the old flavoenzyme and hQR2 prompted us to clone and overexpress hQR2. The properties of hQR2 were identical to those of the flavoenzyme described by S. Liao and H. G. Williams-Ashman, thus establishing their genetic identity. Recombinant human QR2: (i) reacts with N-ribosyl- and N-alkyldihydronicotinamides, but not with NADH, NADPH, or NMNH; (ii) is very weakly inhibited by dicumarol or Cibacron blue; (iii) is very potently inhibited by benzo[a]pyrene. The x-ray crystal structure of rat QR1 shows that the 43 amino acid C-terminal tail of QR1 provides the binding site for the hydrophilic portions of NADH and NADPH. In the absence of this binding site in QR2, the enzyme retains the essential catalytic machinery, including affinity for FAD, but cannot bind phosphorylated hydride donors.

  12. Mercury resistance and mercuric reductase activities and expression among chemotrophic thermophilic Aquificae.

    Science.gov (United States)

    Freedman, Zachary; Zhu, Chengsheng; Barkay, Tamar

    2012-09-01

    Mercury (Hg) resistance (mer) by the reduction of mercuric to elemental Hg is broadly distributed among the Bacteria and Archaea and plays an important role in Hg detoxification and biogeochemical cycling. MerA is the protein subunit of the homodimeric mercuric reductase (MR) enzyme, the central function of the mer system. MerA sequences in the phylum Aquificae form the deepest-branching lineage in Bayesian phylogenetic reconstructions of all known MerA homologs. We therefore hypothesized that the merA homologs in two thermophilic Aquificae, Hydrogenobaculum sp. strain Y04AAS1 (AAS1) and Hydrogenivirga sp. strain 128-5-R1-1 (R1-1), specified Hg resistance. Results supported this hypothesis, because strains AAS1 and R1-1 (i) were resistant to >10 μM Hg(II), (ii) transformed Hg(II) to Hg(0) during cellular growth, and (iii) possessed Hg-dependent NAD(P)H oxidation activities in crude cell extracts that were optimal at temperatures corresponding with the strains' optimal growth temperatures, 55°C for AAS1 and 70°C for R1-1. While these characteristics all conformed with the mer system paradigm, expression of the Aquificae mer operons was not induced by exposure to Hg(II) as indicated by unity ratios of merA transcripts, normalized to gyrA transcripts for hydrogen-grown AAS1 cultures, and by similar MR specific activities in thiosulfate-grown cultures with and without Hg(II). The Hg(II)-independent expression of mer in the deepest-branching lineage of MerA from bacteria whose natural habitats are Hg-rich geothermal environments suggests that regulated expression of mer was a later innovation likely in environments where microorganisms were intermittently exposed to toxic concentrations of Hg.

  13. Dissection of malonyl-coenzyme A reductase of Chloroflexus aurantiacus results in enzyme activity improvement.

    Directory of Open Access Journals (Sweden)

    Changshui Liu

    Full Text Available The formation of fusion protein in biosynthetic pathways usually improves metabolic efficiency either channeling intermediates and/or colocalizing enzymes. In the metabolic engineering of biochemical pathways, generating unnatural protein fusions between sequential biosynthetic enzymes is a useful method to increase system efficiency and product yield. Here, we reported a special case. The malonyl-CoA reductase (MCR of Chloroflexus aurantiacus catalyzes the conversion of malonyl-CoA to 3-hydroxypropionate (3HP, and is a key enzyme in microbial production of 3HP, an important platform chemical. Functional domain analysis revealed that the N-terminal region of MCR (MCR-N; amino acids 1-549 and the C-terminal region of MCR (MCR-C; amino acids 550-1219 were functionally distinct. The malonyl-CoA was reduced into free intermediate malonate semialdehyde with NADPH by MCR-C fragment, and further reduced to 3HP by MCR-N fragment. In this process, the initial reduction of malonyl-CoA was rate limiting. Site-directed mutagenesis demonstrated that the TGXXXG(AX(1-2G and YXXXK motifs were important for enzyme activities of both MCR-N and MCR-C fragments. Moreover, the enzyme activity increased when MCR was separated into two individual fragments. Kinetic analysis showed that MCR-C fragment had higher affinity for malonyl-CoA and 4-time higher K cat/K m value than MCR. Dissecting MCR into MCR-N and MCR-C fragments also had a positive effect on the 3HP production in a recombinant Escherichia coli strain. Our study showed the feasibility of protein dissection as a new strategy in biosynthetic systems.

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

    Science.gov (United States)

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

    2014-10-01

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

  15. Cyclophosphamide as a potent inhibitor of tumor thioredoxin reductase in vivo

    International Nuclear Information System (INIS)

    Wang Xufang; Zhang Jinsong; Xu Tongwen

    2007-01-01

    Cyclophosphamide (CTX) is in the nitrogen mustard group of alkylating antineoplastic chemotherapeutic agents. It is one of the most frequently used antitumor agents for the treatment of a broad spectrum of human cancers. Thioredoxin reductase (TrxR) catalyze the NADPH-dependent reduction of thioredoxin and play an important role in multiple cellular events related to carcinogenesis including cell proliferation, apoptosis, and cell signaling. This enzyme represents a promising target for the development of cytostatic agents. The purpose of this study is to determine whether CTX could target TrxR in vivo. Lewis lung carcinoma and solid H22 hepatoma treated with 50-250 mg/kg CTX for 3 h lost TrxR activity in a dose-dependent fashion. Over 75% and 95% of TrxR activity was lost at the dose of 250 mg/kg. There was, however, a recovery of TrxR activity such that it attained normal levels by 120 h after a dose of 250 mg/kg. In addition, we found that CTX caused a preferential TrxR inhibition over other antioxidant enzymes, such as glutathione peroxidase, catalase, and superoxide dismutase. We also used ascites H22 cells to investigate cancer cells response after TrxR was inhibited by CTX in vivo since CTX is needed to be activated by liver cytochrome P450 enzymes. The time course and dose-dependent changes of cellular TrxR activity were similar with those in tumor tissue. CTX caused a dose-dependent cellular proliferation inhibition which was positively correlated with TrxR inhibition at 3 h. Furthermore, when 3 h CTX-treated cells with various TrxR backgrounds, harvested from ascites-bearing mice, were implanted into mice, the proliferations of these cells were again proportionally dependent on TrxR activity. The TrxR inhibition could thereby be considered as a crucial mechanism contributing to anticancer effect seen upon clinical use of CTX

  16. Increased ethanol productivity in xylose-utilizing Saccharomyces cerevisiae via a randomly mutagenized xylose reductase.

    Science.gov (United States)

    Runquist, David; Hahn-Hägerdal, Bärbel; Bettiga, Maurizio

    2010-12-01

    Baker's yeast (Saccharomyces cerevisiae) has been genetically engineered to ferment the pentose sugar xylose present in lignocellulose biomass. One of the reactions controlling the rate of xylose utilization is catalyzed by xylose reductase (XR). In particular, the cofactor specificity of XR is not optimized with respect to the downstream pathway, and the reaction rate is insufficient for high xylose utilization in S. cerevisiae. The current study describes a novel approach to improve XR for ethanol production in S. cerevisiae. The cofactor binding region of XR was mutated by error-prone PCR, and the resulting library was expressed in S. cerevisiae. The S. cerevisiae library expressing the mutant XR was selected in sequential anaerobic batch cultivation. At the end of the selection process, a strain (TMB 3420) harboring the XR mutations N272D and P275Q was enriched from the library. The V(max) of the mutated enzyme was increased by an order of magnitude compared to that of the native enzyme, and the NADH/NADPH utilization ratio was increased significantly. The ethanol productivity from xylose in TMB 3420 was increased ∼40 times compared to that of the parent strain (0.32 g/g [dry weight {DW}] × h versus 0.007 g/g [DW] × h), and the anaerobic growth rate was increased from ∼0 h(-1) to 0.08 h(-1). The improved traits of TMB 3420 were readily transferred to the parent strain by reverse engineering of the mutated XR gene. Since integrative vectors were employed in the construction of the library, transfer of the improved phenotype does not require multicopy expression from episomal plasmids.

  17. Isethionate formation from taurine in Chromohalobacter salexigens: purification of sulfoacetaldehyde reductase.

    Science.gov (United States)

    Krejcík, Zdenek; Hollemeyer, Klaus; Smits, Theo H M; Cook, Alasdair M

    2010-05-01

    Bacterial generation of isethionate (2-hydroxyethanesulfonate) from taurine (2-aminoethanesulfonate) by anaerobic gut bacteria was established in 1980. That phenomenon in pure culture was recognized as a pathway of assimilation of taurine-nitrogen. Based on the latter work, we predicted from genome-sequence data that the marine gammaproteobacterium Chromohalobacter salexigens DSM 3043 would exhibit this trait. Quantitative conversion of taurine to isethionate, identified by mass spectrometry, was confirmed, and the taurine-nitrogen was recovered as cell material. An eight-gene cluster was predicted to encode the inducible vectorial, scalar and regulatory enzymes involved, some of which were known from other taurine pathways. The genes (Csal_0153-Csal_0156) encoding a putative ATP-binding-cassette (ABC) transporter for taurine (TauAB(1)B(2)C) were shown to be inducibly transcribed by reverse transcription (RT-) PCR. An inducible taurine : 2-oxoglutarate aminotransferase [EC 2.6.1.55] was found (Csal_0158); the reaction yielded glutamate and sulfoacetaldehyde. The sulfoacetaldehyde was reduced to isethionate by NADPH-dependent sulfoacetaldehyde reductase (IsfD), a member of the short-chain alcohol dehydrogenase superfamily. The 27 kDa protein (SDS-PAGE) was identified by peptide-mass fingerprinting as the gene product of Csal_0161. The putative exporter of isethionate (IsfE) is encoded by Csal_0160; isfE was inducibly transcribed (RT-PCR). The presumed transcriptional regulator, TauR (Csal_0157), may autoregulate its own expression, typical of GntR-type regulators. Similar gene clusters were found in several marine and terrestrial gammaproteobacteria, which, in the gut canal, could be the source of not only mammalian, but also arachnid and cephalopod isethionate.

  18. 49 CFR 176.410 - Division 1.5 materials, ammonium nitrate and ammonium nitrate mixtures.

    Science.gov (United States)

    2010-10-01

    ... ammonium nitrate mixtures. 176.410 Section 176.410 Transportation Other Regulations Relating to... nitrate and ammonium nitrate mixtures. (a) This section prescribes requirements to be observed with..., blasting, type B, and Explosives, blasting, type E, Division 1.5 compatibility group D. (f) No mixture...

  19. Aldose reductase inhibitory activity and antioxidant capacity of pomegranate extracts

    OpenAIRE

    Karasu, Çimen; Cumaoğlu, Ahmet; Gürpinar, Ali Rifat; Kartal, Murat; Kovacikova, Lucia; Milackova, Ivana; Stefek, Milan

    2012-01-01

    The pomegranate, Punica granatum L., has been the subject of current interest as a medicinal agent with wide-ranging therapeutic indications. In the present study, pomegranate ethanolic seed and hull extracts were tested, in comparison with a commercial sample, for the inhibition of aldose reductase, an enzyme involved in the etiology of diabetic complications. In vitro inhibition of rat lens aldose reductase was determined by a conventional method. Pomegranate ethanolic hull extract and comm...

  20. Safety in the Chemical Laboratory: Nitric Acid, Nitrates, and Nitro Compounds.

    Science.gov (United States)

    Bretherick, Leslie

    1989-01-01

    Discussed are the potential hazards associated with nitric acid, inorganic and organic nitrate salts, alkyl nitrates, acyl nitrates, aliphatic nitro compounds, aromatic nitro compounds, and nitration reactions. (CW)

  1. Engineering Styrene Monooxygenase for Biocatalysis: Reductase-Epoxidase Fusion Proteins.

    Science.gov (United States)

    Heine, Thomas; Tucker, Kathryn; Okonkwo, Nonye; Assefa, Berhanegebriel; Conrad, Catleen; Scholtissek, Anika; Schlömann, Michael; Gassner, George; Tischler, Dirk

    2017-04-01

    The enantioselective epoxidation of styrene and related compounds by two-component styrene monooxygenases (SMOs) has targeted these enzymes for development as biocatalysts. In the present work, we prepare genetically engineered fusion proteins that join the C-terminus of the epoxidase (StyA) to the N-terminus of the reductase (StyB) through a linker peptide and demonstrate their utility as biocatalysts in the synthesis of Tyrain purple and other indigoid dyes. A single-vector expression system offers a simplified platform for transformation and expansion of the catalytic function of styrene monooxygenases, and the resulting fusion proteins are self-regulated and couple efficiently NADH oxidation to styrene epoxidation. We find that the reductase domain proceeds through a sequential ternary-complex mechanism at low FAD concentration and a double-displacement mechanism at higher concentrations of FAD. Single-turnover studies indicate an observed rate constant for FAD-to-FAD hydride transfer of ~8 s -1 . This step is rate limiting in the styrene epoxidation reaction and helps to ensure that flavin reduction and styrene epoxidation reactions proceed without wasteful side reactions. Comparison of the reductase activity of the fusion proteins with the naturally occurring reductase, SMOB, and N-terminally histidine-tagged reductase, NSMOB, suggests that the observed changes in catalytic mechanism are due in part to an increase in flavin-binding affinity associated with the N-terminal extension of the reductase.

  2. Nitration Study of Cyclic Ladder Polyphenylsilsesquioxane

    Directory of Open Access Journals (Sweden)

    LIANG Jia-xiang

    2017-05-01

    Full Text Available Several nitration reagents including fuming nitric acid, HNO3-H2SO4, KNO3-H2SO4, HNO3-KNO3, CH3COOH-KNO3, (CH3CO2O-HNO3 were used to nitrate cyclic ladder polyphenylsilsesquioxane (CL-PPSQ in different conditions in order to enhance the compatibility of the CL-PPSQ in polymers, the NO2-PPSQ was obtained. FTIR, element analysis, GPC, TGA and 1H NMR were used to characterize the structures of the nitrated products. The results show that the nitrating abilities of the fuming nitric acid, HNO3-H2SO4 and KNO3-H2SO4 are very strong. Many nitro groups can be linked with phenyl groups in CL-PPSQ, but with low molecular mass, fracture occurs in siloxane segment. However, the Mn of the product NO2-PPSQ sharply drops by 50% compared with that of CL-PPSQ, so the nitration reagents can break the cyclic structure of CL-PPSQ. The nitrating reagents of HNO3-KNO3 and CH3COOH-KNO3 have no nitration effects on CL-PPSQ. At last, NO2-CL-PPSQ was prepared using (CH3CO2O-HNO3 because of the moderate nitration process and ability. The cyclic structure of PPSQ is remained, although the number of —NO2 group is not too much. At the same time, the nitration mechanism using different nitration reagents was analyzed. A certain amount of NO2+, which is a kind of activator owning strong nitration ability, can be found in the fuming nitric acid and H2SO4-HNO3(KNO3 systems. As to the (CH3CO2O-HNO3 system, the main activator is CH3COONO2.

  3. Legacy Nitrate Impacts on Groundwater and Streams

    Science.gov (United States)

    Tesoriero, A. J.; Juckem, P. F.; Miller, M. P.

    2017-12-01

    Decades of recharge of high-nitrate groundwater have created a legacy—a mass of high-nitrate groundwater—that has implications for future nitrate concentrations in groundwater and in streams. In the United States, inorganic nitrogen fertilizer applications to the land surface have increased ten-fold since 1950, resulting in sharp increases in nitrate concentrations in recharging groundwater, which pose a risk to deeper groundwater and streams. This study assesses the factors that control time lags and eventual concentrations of legacy nitrate in groundwater and streams. Results from the USGS National Water-Quality Assessment Project are presented which elucidate nitrate trends in recharging groundwater, delineate redox zones and assess groundwater and stream vulnerability to legacy nitrate sources on a regional scale. This study evaluated trends and transformations of agricultural chemicals based on groundwater age and water chemistry data along flow paths from recharge areas to streams at 20 study sites across the United States. Median nitrate recharge concentrations in these agricultural areas have increased markedly over the last 50 years, from 4 to 7.5 mg N/L. The effect that nitrate accumulation in shallow aquifers will have on drinking water quality and stream ecosystems is dependent on the redox zones encountered along flow paths and on the age distribution of nitrate discharging to supply wells and streams. Delineating redox zones on a regional scale is complicated by the spatial variability of reaction rates. To overcome this limitation, we applied logistic regression and machine learning techniques to predict the probability of a specific redox condition in groundwater in the Chesapeake Bay watershed and the Fox-Wolf-Peshtigo study area in Wisconsin. By relating redox-active constituent concentrations in groundwater samples to indicators of residence time and/or electron donor availability, we were able to delineate redox zones on a regional scale

  4. Consumption of NADPH for 2-HG Synthesis Increases Pentose Phosphate Pathway Flux and Sensitizes Cells to Oxidative Stress

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    Susan J. Gelman

    2018-01-01

    Full Text Available Summary: Gain-of-function mutations in isocitrate dehydrogenase 1 (IDH1 occur in multiple types of human cancer. Here, we show that these mutations significantly disrupt NADPH homeostasis by consuming NADPH for 2-hydroxyglutarate (2-HG synthesis. Cells respond to 2-HG synthesis, but not exogenous administration of 2-HG, by increasing pentose phosphate pathway (PPP flux. We show that 2-HG production competes with reductive biosynthesis and the buffering of oxidative stress, processes that also require NADPH. IDH1 mutants have a decreased capacity to synthesize palmitate and an increased sensitivity to oxidative stress. Our results demonstrate that, even when NADPH is limiting, IDH1 mutants continue to synthesize 2-HG at the expense of other NADPH-requiring pathways that are essential for cell viability. Thus, rather than attempting to decrease 2-HG synthesis in the clinic, the consumption of NADPH by mutant IDH1 may be exploited as a metabolic weakness that sensitizes tumor cells to ionizing radiation, a commonly used anti-cancer therapy. : Using liquid chromatography/mass spectrometry (LC/MS and stable isotope tracing, Gelman et al. find that 2-HG production in cells with IDH1 mutations leads to increased pentose phosphate pathway activity to generate NADPH. Production of 2-HG competes with other NADPH-dependent pathways and sensitizes cells to redox stress. Keywords: 2-hydroxyglutarate, cancer metabolism, LC/MS, metabolomcis, pentose phosphate pathway, redox regulation

  5. Characterization of a heme c nitrite reductase from a non-ammonifying microorganism, Desulfovibrio vulgaris Hildenborough.

    Science.gov (United States)

    Pereira, I A; LeGall, J; Xavier, A V; Teixeira, M

    2000-08-31

    A cytochrome c nitrite reductase (NiR) was purified for the first time from a microorganism not capable of growing on nitrate, the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough. It was isolated from the membranes as a large heterooligomeric complex of 760 kDa, containing two cytochrome c subunits of 56 and 18 kDa. This complex has nitrite and sulfite reductase activities of 685 micromol NH(4)(+)/min/mg and 1.0 micromol H(2)/min/mg. The enzyme was studied by UV-visible and electron paramagnetic resonance (EPR) spectroscopies. The overall redox behavior was determined through a visible redox titration. The data were analyzed with a set of four redox transitions, with an E(0)' of +160 mV (12% of total absorption), -5 mV (38% of total absorption), -110 mV (38% of total absorption) and -210 mV (12% of total absorption) at pH 7.6. The EPR spectra of oxidized and partially reduced NiR show a complex pattern, indicative of multiple heme-heme magnetic interactions. It was found that D. vulgaris Hildenborough is not capable of using nitrite as a terminal electron acceptor. These results indicate that in this organism the NiR is not involved in the dissimilative reduction of nitrite, as is the case with the other similar enzymes isolated so far. The possible role of this enzyme in the detoxification of nitrite and/or in the reduction of sulfite is discussed.

  6. Methylenetetrahydrofolate Reductase Activity and Folate Metabolism

    Directory of Open Access Journals (Sweden)

    Nursen Keser

    2014-04-01

    Full Text Available Folate is a vital B vitamin which is easily water-soluble. It is a natural source which is found in the herbal and animal foods. Folate has important duties in the human metabolism, one of them is the adjustment of the level of plasma homocysteine. Reduction in MTHFR (methylenetetrahydrofolate reductase,which is in charge of the metabolism of homocysteine activity affects the level of homocysteine. Therefore MTHFR is an important enzyme in folate metabolism. Some of the mutations occurring in the MTHFR gene is a risk factor for various diseases and may be caused the hyperhomocysteinemia or the homocystinuria, and they also may lead to metabolic problems. MTHFR is effective in the important pathways such as DNA synthesis, methylation reactions and synthesis of RNA. C677T and A1298C are the most commonly occurring polymorphisms in the gene of MTHFR. The frequency of these polymorphisms show differences in the populations. MTHFR, folate distribution, metabolism of homocysteine and S-adenosylmethionine, by the MTHFR methylation the genetic defects have the potential of affecting the risk of disease in the negative or positive way.