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Sample records for nitrite reductase activity

  1. Nitrate Reductase Regulates Expression of Nitrite Uptake and Nitrite Reductase Activities in Chlamydomonas reinhardtii 1

    PubMed Central

    Galván, Aurora; Cárdenas, Jacobo; Fernández, Emilio

    1992-01-01

    In Chlamydomonas reinhardtii mutants defective at the structural locus for nitrate reductase (nit-1) or at loci for biosynthesis of the molybdopterin cofactor (nit-3, nit-4, or nit-5 and nit-6), both nitrite uptake and nitrite reductase activities were repressed in ammonium-grown cells and expressed at high amounts in nitrogen-free media or in media containing nitrate or nitrite. In contrast, wild-type cells required nitrate induction for expression of high levels of both activities. In mutants defective at the regulatory locus for nitrate reductase (nit-2), very low levels of nitrite uptake and nitrite reductase activities were expressed even in the presence of nitrate or nitrite. Both restoration of nitrate reductase activity in mutants defective at nit-1, nit-3, and nit-4 by isolating diploid strains among them and transformation of a structural mutant upon integration of the wild-type nit-1 gene gave rise to the wild-type expression pattern for nitrite uptake and nitrite reductase activities. Conversely, inactivation of nitrate reductase by tungstate treatment in nitrate, nitrite, or nitrogen-free media made wild-type cells respond like nitrate reductase-deficient mutants with respect to the expression of nitrite uptake and nitrite reductase activities. Our results indicate that nit-2 is a regulatory locus for both the nitrite uptake system and nitrite reductase, and that the nitrate reductase enzyme plays an important role in the regulation of the expression of both enzyme activities. PMID:16668656

  2. Increased nitrite reductase activity of fetal versus adult ovine hemoglobin

    PubMed Central

    Blood, Arlin B.; Tiso, Mauro; Verma, Shilpa T.; Lo, Jennifer; Joshi, Mahesh S.; Azarov, Ivan; Longo, Lawrence D.; Gladwin, Mark T.; Kim-Shapiro, Daniel B.; Power, Gordon G.

    2009-01-01

    Growing evidence indicates that nitrite, NO2−, serves as a circulating reservoir of nitric oxide (NO) bioactivity that is activated during physiological and pathological hypoxia. One of the intravascular mechanisms for nitrite conversion to NO is a chemical nitrite reductase activity of deoxyhemoglobin. The rate of NO production from this reaction is increased when hemoglobin is in the R conformation. Because the mammalian fetus exists in a low-oxygen environment compared with the adult and is exposed to episodes of severe ischemia during the normal birthing process, and because fetal hemoglobin assumes the R conformation more readily than adult hemoglobin, we hypothesized that nitrite reduction to NO may be enhanced in the fetal circulation. We found that the reaction was faster for fetal than maternal hemoglobin or blood and that the reactions were fastest at 50–80% oxygen saturation, consistent with an R-state catalysis that is predominant for fetal hemoglobin. Nitrite concentrations were similar in blood taken from chronically instrumented normoxic ewes and their fetuses but were elevated in response to chronic hypoxia. The findings suggest an augmented nitrite reductase activity of fetal hemoglobin and that the production of nitrite may participate in the regulation of vascular NO homeostasis in the fetus. PMID:19028797

  3. A dissimilatory nitrite reductase in Paracoccus halodenitrificans

    NASA Technical Reports Server (NTRS)

    Grant, M. A.; Hochstein, L. I.

    1984-01-01

    Paracoccus halodenitrificans produced a membrane-associated nitrite reductase. Spectrophotometric analysis showed it to be associated with a cd-cytochrome and located on the inner side of the cytoplasmic membrane. When supplied with nitrite, membrane preparations produced nitrous oxide and nitric oxide in different ratios depending on the electron donor employed. The nitrite reductase was maximally active at relatively low concentrations of sodium chloride and remained attached to the membranes at 100 mM sodium chloride.

  4. Nitric Oxide (NO) Generation from Heme/Copper Assembly Mediated Nitrite Reductase Activity

    PubMed Central

    Hematian, Shabnam; Siegler, Maxime A.

    2014-01-01

    Nitric oxide (NO) as a cellular signaling molecule and vasodilator regulates a range of physiological and pathological processes. Nitrite (NO2−) is recycled in vivo to generate nitric oxide, particularly in physiologic hypoxia and ischemia. The cytochrome c oxidase (CcO) binuclear hemea3/CuB active site is one entity known to be responsible for cellular nitrite conversion to nitric oxide. We recently reported that a partially reduced heme/Cu assembly reduces nitrite ion, producing NO; the heme serves as the reductant and cupric ion provides a Lewis Acid interaction with nitrite, facilitating nitrite (N−O) bond cleavage (Hematian et al., J Am Chem Soc 134:18912–18915, 2012). To further investigate this nitrite reductase (NIR) chemistry, copper(II)-nitrito complexes with tri-and tetra-dentate ligands were used in this study, where either O,O'-bidentate or O-unidentate modes of nitrite binding to the cupric center are present. To study the role of the reducing ability of the ferrous heme center, two different tetraarylporphyrinate-iron(II) complexes, one with electron donating para-methoxy peripheral substituents, (TMPP)FeII, and the other with electron withdrawing 2,6-difluorophenyl substituents, (F8)FeII, were employed. The results show that differing nitrite coordination modes to copper(II) ion leads to varying kinetic behavior. Here, also, the ferrous heme is in all cases the source of the reducing equivalent required to take nitrite to nitric oxide, but the reduction ability of the heme center does not play a key role in the observed overall reaction rate. Based on our observations, reaction mechanisms are proposed and discussed in terms of heme/Cu heterobinuclear structures. PMID:24430198

  5. Nitric oxide generation from heme/copper assembly mediated nitrite reductase activity.

    PubMed

    Hematian, Shabnam; Siegler, Maxime A; Karlin, Kenneth D

    2014-06-01

    Nitric oxide (NO) as a cellular signaling molecule and vasodilator regulates a range of physiological and pathological processes. Nitrite (NO2 (-)) is recycled in vivo to generate nitric oxide, particularly in physiologic hypoxia and ischemia. The cytochrome c oxidase binuclear heme a 3/CuB active site is one entity known to be responsible for conversion of cellular nitrite to nitric oxide. We recently reported that a partially reduced heme/copper assembly reduces nitrite ion, producing nitric oxide; the heme serves as the reductant and the cupric ion provides a Lewis acid interaction with nitrite, facilitating nitrite (N-O) bond cleavage (Hematian et al., J. Am. Chem. Soc. 134:18912-18915, 2012). To further investigate this nitrite reductase chemistry, copper(II)-nitrito complexes with tridentate and tetradentate ligands were used in this study, where either O,O'-bidentate or O-unidentate modes of nitrite binding to the cupric center are present. To study the role of the reducing ability of the ferrous heme center, two different tetraarylporphyrinate-iron(II) complexes, one with electron-donating para-methoxy peripheral substituents and the other with electron-withdrawing 2,6-difluorophenyl substituents, were used. The results show that differing modes of nitrite coordination to the copper(II) ion lead to differing kinetic behavior. Here, also, the ferrous heme is in all cases the source of the reducing equivalent required to convert nitrite to nitric oxide, but the reduction ability of the heme center does not play a key role in the observed overall reaction rate. On the basis of our observations, reaction mechanisms are proposed and discussed in terms of heme/copper heterobinuclear structures.

  6. Enhanced nitrite reductase activity associated with the haptoglobin complexed hemoglobin dimer: functional and antioxidative implications.

    PubMed

    Roche, Camille J; Dantsker, David; Alayash, Abdu I; Friedman, Joel M

    2012-06-30

    The presence of acellular hemoglobin (Hb) within the circulation is generally viewed as a pathological state that can result in toxic consequences. Haptoglobin (Hp), a globular protein found in the plasma, binds with high avidity the αβ dimers derived from the dissociation of Hb tetramer and thus helps clear free Hb. More recently there have been compelling indications that the redox properties of the Hp bound dimer (Hb-Hp) may play a more active role in controlling toxicity by limiting the potential tissue damage caused by propagation of the free-radicals generated within the heme containing globin chains. The present study further examines the potential protective effect of Hp through its impact on the production of nitric oxide (NO) from nitrite through nitrite reductase activity of the Hp bound αβ Hb dimer. The presented results show that the Hb dimer in the Hb-Hp complex has oxygen binding, CO recombination and spectroscopic properties consistent with an Hb species having properties similar to but not exactly the same as the R quaternary state of the Hb tetramer. Consistent with these observations is the finding that the initial nitrite reductase rate for Hb-Hp is approximately ten times that of HbA under the same conditions. These results in conjunction with the earlier redox properties of the Hb-Hp are discussed in terms of limiting the pathophysiological consequences of acellular Hb in the circulation.

  7. Directing the mode of nitrite binding to a copper-containing nitrite reductase from Alcaligenes faecalis S-6: characterization of an active site isoleucine.

    PubMed

    Boulanger, Martin J; Murphy, Michael E P

    2003-02-01

    Unlike the heme cd(1)-based nitrite reductase enzymes, the molecular mechanism of copper-containing nitrite reductases remains controversial. A key source of controversy is the productive binding mode of nitrite in the active site. To identify and characterize the molecular determinants associated with nitrite binding, we applied a combinatorial mutagenesis approach to generate a small library of six variants at position 257 in nitrite reductase from Alcaligenes faecalis S-6. The activities of these six variants span nearly two orders of magnitude with one variant, I257V, the only observed natural substitution for Ile257, showing greater activity than the native enzyme. High-resolution (> 1.8 A) nitrite-soaked crystal structures of these variants display different modes of nitrite binding that correlate well with the altered activities. These studies identify for the first time that the highly conserved Ile257 in the native enzyme is a key molecular determinant in directing a catalytically competent mode of nitrite binding in the active site. The O-coordinate bidentate binding mode of nitrite observed in native and mutant forms with high activity supports a catalytic model distinct from the heme cd(1) NiRs. (The atomic coordinates for I257V[NO(2)(-)], I257L[NO(2)(-)], I257A[NO(2)(-)], I257T[NO(2)(-)], I257M[NO(2)(-)] and I257G[NO(2)(-)] AfNiR have been deposited in the Protein Data Bank [PDB identification codes are listed in Table 2].)

  8. Conserved active site residues limit inhibition of a copper-containing nitrite reductase by small molecules.

    PubMed

    Tocheva, Elitza I; Eltis, Lindsay D; Murphy, Michael E P

    2008-04-15

    The interaction of copper-containing dissimilatory nitrite reductase from Alcaligenes faecalis S-6 ( AfNiR) with each of five small molecules was studied using crystallography and steady-state kinetics. Structural studies revealed that each small molecule interacted with the oxidized catalytic type 2 copper of AfNiR. Three small molecules (formate, acetate and nitrate) mimic the substrate by having at least two oxygen atoms for bidentate coordination to the type 2 copper atom. These three anions bound to the copper ion in the same asymmetric, bidentate manner as nitrite. Consistent with their weak inhibition of the enzyme ( K i >50 mM), the Cu-O distances in these AfNiR-inhibitor complexes were approximately 0.15 A longer than that observed in the AfNiR-nitrite complex. The binding mode of each inhibitor is determined in part by steric interactions with the side chain of active site residue Ile257. Moreover, the side chain of Asp98, a conserved residue that hydrogen bonds to type 2 copper-bound nitrite and nitric oxide, was either disordered or pointed away from the inhibitors. Acetate and formate inhibited AfNiR in a mixed fashion, consistent with the occurrence of second acetate binding site in the AfNiR-acetate complex that occludes access to the type 2 copper. A fourth small molecule, nitrous oxide, bound to the oxidized metal in a side-on fashion reminiscent of nitric oxide to the reduced copper. Nevertheless, nitrous oxide bound at a farther distance from the metal. The fifth small molecule, azide, inhibited the reduction of nitrite by AfNiR most strongly ( K ic = 2.0 +/- 0.1 mM). This ligand bound to the type 2 copper center end-on with a Cu-N c distance of approximately 2 A, and was the only inhibitor to form a hydrogen bond with Asp98. Overall, the data substantiate the roles of Asp98 and Ile257 in discriminating substrate from other small anions.

  9. Residues in the Distal Heme Pocket of Arabidopsis Non-Symbiotic Hemoglobins: Implication for Nitrite Reductase Activity.

    PubMed

    Kumar, Nitin; Astegno, Alessandra; Chen, Jian; Giorgetti, Alejandro; Dominici, Paola

    2016-04-28

    It is well-established that plant hemoglobins (Hbs) are involved in nitric oxide (NO) metabolism via NO dioxygenase and/or nitrite reductase activity. The ferrous-deoxy Arabidopsis Hb1 and Hb2 (AHb1 and AHb2) have been shown to reduce nitrite to NO under hypoxia. Here, to test the hypothesis that a six- to five-coordinate heme iron transition might mediate the control of the nitrite reduction rate, we examined distal pocket mutants of AHb1 and AHb2 for nitrite reductase activity, NO production and spectroscopic features. Absorption spectra of AHbs distal histidine mutants showed that AHb1 mutant (H69L) is a stable pentacoordinate high-spin species in both ferrous and ferric states, whereas heme iron in AHb2 mutant (H66L) is hexacoordinated low-spin with Lys69 as the sixth ligand. The bimolecular rate constants for nitrite reduction to NO were 13.3 ± 0.40, 7.3 ± 0.5, 10.6 ± 0.8 and 171.90 ± 9.00 M(-1)·s(-1) for AHb1, AHb2, AHb1 H69L and AHb2 H66L, respectively, at pH 7.4 and 25 °C. Consistent with the reductase activity, the amount of NO detected by chemiluminescence was significantly higher in the AHb2 H66L mutant. Our data indicate that nitrite reductase activity is determined not only by heme coordination, but also by a unique distal heme pocket in each AHb.

  10. Residues in the Distal Heme Pocket of Arabidopsis Non-Symbiotic Hemoglobins: Implication for Nitrite Reductase Activity

    PubMed Central

    Kumar, Nitin; Astegno, Alessandra; Chen, Jian; Giorgetti, Alejandro; Dominici, Paola

    2016-01-01

    It is well-established that plant hemoglobins (Hbs) are involved in nitric oxide (NO) metabolism via NO dioxygenase and/or nitrite reductase activity. The ferrous-deoxy Arabidopsis Hb1 and Hb2 (AHb1 and AHb2) have been shown to reduce nitrite to NO under hypoxia. Here, to test the hypothesis that a six- to five-coordinate heme iron transition might mediate the control of the nitrite reduction rate, we examined distal pocket mutants of AHb1 and AHb2 for nitrite reductase activity, NO production and spectroscopic features. Absorption spectra of AHbs distal histidine mutants showed that AHb1 mutant (H69L) is a stable pentacoordinate high-spin species in both ferrous and ferric states, whereas heme iron in AHb2 mutant (H66L) is hexacoordinated low-spin with Lys69 as the sixth ligand. The bimolecular rate constants for nitrite reduction to NO were 13.3 ± 0.40, 7.3 ± 0.5, 10.6 ± 0.8 and 171.90 ± 9.00 M−1·s−1 for AHb1, AHb2, AHb1 H69L and AHb2 H66L, respectively, at pH 7.4 and 25 °C. Consistent with the reductase activity, the amount of NO detected by chemiluminescence was significantly higher in the AHb2 H66L mutant. Our data indicate that nitrite reductase activity is determined not only by heme coordination, but also by a unique distal heme pocket in each AHb. PMID:27136534

  11. NITRITE REDUCTASE ACTIVITY OF NON-SYMBIOTIC HEMOGLOBINS FROM ARABIDOPSIS THALIANA†

    PubMed Central

    Tiso, Mauro; Tejero, Jesús; Kenney, Claire; Frizzell, Sheila; Gladwin, Mark T.

    2013-01-01

    Plant non-symbiotic hemoglobins possess hexa-coordinate heme geometry similar to the heme protein neuroglobin. We recently discovered that deoxygenated neuroglobin converts nitrite to nitric oxide (NO), an important signaling molecule involved in many processes in plants. We sought to determine whether Arabidopsis thaliana non-symbiotic hemoglobins class 1 and 2 (AHb1 and AHb2) might function as nitrite reductases. We found that the reaction of nitrite with deoxygenated AHb1 and AHb2 generates NO gas and iron-nitrosyl-hemoglobin species. The bimolecular rate constants for nitrite reduction to NO are 19.8 ± 3.2 and 4.9 ± 0.2 M−1s−1, at pH = 7.4 and 25°C, respectively. We determined the pH dependence of these bimolecular rate constants and found a linear correlation with the concentration of protons, indicating the requirement for one proton in the reaction. Release of free NO gas during reaction in anoxic and hypoxic (2% oxygen) conditions was confirmed by chemiluminescence detection. These results demonstrate that deoxygenated AHb1 and AHb2 reduce nitrite to form NO via a mechanism analogous to that observed for hemoglobin, myoglobin and neuroglobin. Our findings suggest that during severe hypoxia and in the anaerobic plant roots, especially in water submerged species, non-symbiotic hemoglobins provide a viable pathway for NO generation via nitrite reduction. PMID:22620259

  12. Solubilization and Resolution of the Membrane-Bound Nitrite Reductase from Paracoccus Halodenitrificans into Nitrite and Nitric Oxide Reductases

    NASA Technical Reports Server (NTRS)

    Grant, Michael A.; Cronin, Sonja E.; Hochstein, Lawrence I.

    1984-01-01

    Membranes prepared from Paracoccus halodenitrificans reduced nitrite or nitric oxide to nitrous oxide. Extraction of these membranes with the detergent CHAPSO [3-(3-Chlolamidoporopyldimethylammonio)-1-(2- hydroxy-1-propanesulfonate)], followed by ammonium sulfate fractionation of the solubilized proteins, resulted in the separation of nitrite and nitric oxide reductase activities. The fraction containing nitrite reductase activity spectrally resembled a cd-type cytochrome. Several cytochromes were detected in the nitric oxide reductase fraction. Which, if any, of these cytochromes is associated with the reduction of nitric oxide is not clear at this time.

  13. Purification and characterization of assimilatory nitrite reductase from Candida utilis.

    PubMed

    Sengupta, S; Shaila, M S; Rao, G R

    1996-07-01

    Nitrate assimilation in many plants, algae, yeasts and bacteria is mediated by two enzymes, nitrate reductase (EC 1.6.6.2) and nitrite reductase (EC 1.7.7.1). They catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonia respectively. The nitrite reductase from an industrially important yeast, Candida utilis, has been purified to homogeneity. Purified nitrite reductase is a heterodimer and the molecular masses of the two subunits are 58 and 66 kDa. The native enzyme exhibits a molecular mass of 126 kDa as analysed by gel filtration. The identify of the two subunits of nitrite reductase was confirmed by immunoblotting using antibody for Cucurbita pepo leaf nitrite reductase. The presence of two different sized transcripts coding for the two subunits was confirmed by (a) in vitro translation of mRNA from nitrate-induced C. utilis followed by immunoprecipitation of the in vitro translated products with heterologous nitrite reductase antibody and (b) Northern-blot analysis. The 66 kDa subunit is acidic in nature which is probably due to its phosphorylated status. The enzyme is stable over a range of temperatures. Both subunits can catalyse nitrite reduction, and the reconstituted enzyme, at a higher protein concentration, shows an activity similar to that of the purified enzyme. Each of these subunits has been shown to contain a few unique peptides in addition to a large number of common peptides. Reduced Methyl Viologen has been found to be as effective an electron donor as NADPH in the catalytic process, a phenomenon not commonly seen for nitrite reductases from other systems.

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

    PubMed Central

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

    1998-01-01

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

  15. Boletus edulis Nitrite Reductase Reduces Nitrite Content of Pickles and Mitigates Intoxication in Nitrite-intoxicated Mice

    PubMed Central

    Zhang, Weiwei; Tian, Guoting; Feng, Shanshan; Wong, Jack Ho; Zhao, Yongchang; Chen, Xiao; Wang, Hexiang; Ng, Tzi Bun

    2015-01-01

    Pickles are popular in China and exhibits health-promoting effects. However, nitrite produced during fermentation adversely affects health due to formation of methemoglobin and conversion to carcinogenic nitrosamine. Fruiting bodies of the mushroom Boletus edulis were capable of inhibiting nitrite production during pickle fermentation. A 90-kDa nitrite reductase (NiR), demonstrating peptide sequence homology to fungal nitrite reductase, was isolated from B. edulis fruiting bodies. The optimum temperature and pH of the enzyme was 45 °C and 6.8, respectively. B. edulis NiR was capable of prolonging the lifespan of nitrite-intoxicated mice, indicating that it had the action of an antidote. The enzyme could also eliminate nitrite from blood after intragastric administration of sodium nitrite, and after packaging into capsule, this nitrite-eliminating activity could persist for at least 120 minutes thus avoiding immediate gastric degradation. B. edulis NiR represents the first nitrite reductase purified from mushrooms and may facilitate subsequent applications. PMID:26446494

  16. Boletus edulis Nitrite Reductase Reduces Nitrite Content of Pickles and Mitigates Intoxication in Nitrite-intoxicated Mice.

    PubMed

    Zhang, Weiwei; Tian, Guoting; Feng, Shanshan; Wong, Jack Ho; Zhao, Yongchang; Chen, Xiao; Wang, Hexiang; Ng, Tzi Bun

    2015-10-08

    Pickles are popular in China and exhibits health-promoting effects. However, nitrite produced during fermentation adversely affects health due to formation of methemoglobin and conversion to carcinogenic nitrosamine. Fruiting bodies of the mushroom Boletus edulis were capable of inhibiting nitrite production during pickle fermentation. A 90-kDa nitrite reductase (NiR), demonstrating peptide sequence homology to fungal nitrite reductase, was isolated from B. edulis fruiting bodies. The optimum temperature and pH of the enzyme was 45 °C and 6.8, respectively. B. edulis NiR was capable of prolonging the lifespan of nitrite-intoxicated mice, indicating that it had the action of an antidote. The enzyme could also eliminate nitrite from blood after intragastric administration of sodium nitrite, and after packaging into capsule, this nitrite-eliminating activity could persist for at least 120 minutes thus avoiding immediate gastric degradation. B. edulis NiR represents the first nitrite reductase purified from mushrooms and may facilitate subsequent applications.

  17. De novo-designed metallopeptides with type 2 copper centers: modulation of reduction potentials and nitrite reductase activities.

    PubMed

    Yu, Fangting; Penner-Hahn, James E; Pecoraro, Vincent L

    2013-12-04

    Enzymatic reactions involving redox processes are highly sensitive to the local electrostatic environment. Despite considerable effort, the complex interactions among different influential factors in native proteins impede progress toward complete understanding of the structure-function relationship. Of particular interest is the type 2 copper center Cu(His)3, which may act as an electron transfer center in peptidylglycine α-hydroxylating monooxygenase (PHM) or a catalytic center in copper nitrite reductase (CuNiR). A de novo design strategy is used to probe the effect of modifying charged amino acid residues around, but not directly bound to, a Cu(His)3 center embedded in three-stranded coiled coils (TRI-H)3 [TRI-H = Ac-G WKALEEK LKALEEK LKALEEK HKALEEK G-NH2]. Specifically, the peptide TRI-EH (=TRI-HK22E) alters an important lysine to glutamate just above the copper binding center. With a series of TRI-EH peptides mutated below the metal center, we use a variety of spectroscopies (EPR, UV-vis, XAS) to show a direct impact on the protonation equilibria, copper binding affinities, reduction potentials, and nitrite reductase activities of these copper-peptide complexes. The potentials at a specific pH vary by 100 mV, and the nitrite reductase activities range over a factor of 4 in rates. We also observe that the affinities, potentials, and catalytic activities are strongly influenced by the pH conditions (pH 5.8-7.4). In general, Cu(II) affinities for the peptides are diminished at low pH values. The interplay among these factors can lead to a 200 mV shift in reduction potential across these peptides, which is determined by the pH-dependent affinities of copper in both oxidation states. This study illustrates the strength of de novo protein design in elucidating the influence of ionizable residues on a particular redox system, an important step toward understanding the factors that govern the properties of this metalloenzyme with a goal of eventually improving the

  18. Nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase expression and activity in response to different nitrogen sources in nitrogen-starved wheat seedlings.

    PubMed

    Balotf, Sadegh; Kavoosi, Gholamreza; Kholdebarin, Bahman

    2016-01-01

    The objective of this study was to examine the expression and activity of nitrate reductase (NR, EC 1.7.1.1), nitrite reductase (NiR, EC 1.7.2.2), glutamine synthetase (GS, EC 6.3.1.2), and glutamate synthase (GOGAT, EC 1.4.7.1) in response to potassium nitrate, ammonium chloride, and ammonium nitrate in nitrogen-starved wheat seedlings. Plants were grown in standard nutrient solution for 17 days and then subjected to nitrogen starvation for 7 days. The starved plants were supplied with potassium nitrate ammonium nitrate and ammonium chloride (50 mM) for 4 days and the leaves were harvested. The relative expression of NR, NiR, GS, and GOGAT as well as the enzyme activities were investigated. Nitrogen starvation caused a significant decrease both in transcript levels and in NR, NiR, GS, and GOGAT activities. Potassium nitrate and ammonium nitrate treatments restored NR, NiR, GS, and GOGAT expressions and activities. Ammonium chloride increased only the expressions and activities of GS and GOGAT in a dose-dependent manner. The results of our study highlight the differential effects between the type and the amount of nitrogen salts on NR, NiR, GS, and GOGAT activities in wheat seedlings while potassium nitrate being more effective.

  19. The existence and significance of a mitochondrial nitrite reductase.

    PubMed

    Nohl, Hans; Staniek, Katrin; Kozlov, Andrey V

    2005-01-01

    The physiological functions of nitric oxide (NO) are well established. The finding that the endothelium-derived relaxing factor (EDRF) is NO was totally unexpected. It was shown that NO is a reaction product of an enzymatically catalyzed, overall, 5-electron oxidation of guanidinium nitrogen from L-arginine followed by the release of the free radical species NO. NO is synthesized by a single protein complex supported by cofactors, coenzymes (such as tetrahydrobiopterin) and cytochrome P450. The latter can uncouple from substrate oxidation producing O2*- radicals. The research groups of Richter [Ghafourifar P, Richter C. Nitric oxide synthase activity in mitochondria. FEBS Lett 1997; 418: 291-296.] and Boveris [Giulivi C, Poderoso JJ, Boveris A. Production of nitric oxide by mitochondria. J Biol Chem 1998; 273: 11038-11043.] identified a mitochondrial NO synthase (NOS). There are, however, increasing reports demonstrating that mitochondrial NO is derived from cytosolic NOS belonging to the Ca2+-dependent enzymes. NO was thought to control cytochrome oxidase. This assumption is controversial due to the life-time of NO in biological systems (millisecond range). We found a nitrite reductase in mitochondria which is of major interest. Any increase of nitrite in the tissue which is the first oxidation product of NO, for instance following NO donors, will stimulate NO-recycling via mitochondrial nitrite reductase. In this paper, we describe the identity and the function of mitochondrial nitrite reductase and the consequences of NO-recycling in the metabolic compartment of mitochondria.

  20. Human Neuroglobin Functions as a Redox-regulated Nitrite Reductase*

    PubMed Central

    Tiso, Mauro; Tejero, Jesús; Basu, Swati; Azarov, Ivan; Wang, Xunde; Simplaceanu, Virgil; Frizzell, Sheila; Jayaraman, Thottala; Geary, Lisa; Shapiro, Calli; Ho, Chien; Shiva, Sruti; Kim-Shapiro, Daniel B.; Gladwin, Mark T.

    2011-01-01

    Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved from a common ancestor to hemoglobin and myoglobin. It possesses a six-coordinate heme geometry with proximal and distal histidines directly bound to the heme iron, although coordination of the sixth ligand is reversible. We show that deoxygenated human neuroglobin reacts with nitrite to form nitric oxide (NO). This reaction is regulated by redox-sensitive surface thiols, cysteine 55 and 46, which regulate the fraction of the five-coordinated heme, nitrite binding, and NO formation. Replacement of the distal histidine by leucine or glutamine leads to a stable five-coordinated geometry; these neuroglobin mutants reduce nitrite to NO ∼2000 times faster than the wild type, whereas mutation of either Cys-55 or Cys-46 to alanine stabilizes the six-coordinate structure and slows the reaction. Using lentivirus expression systems, we show that the nitrite reductase activity of neuroglobin inhibits cellular respiration via NO binding to cytochrome c oxidase and confirm that the six-to-five-coordinate status of neuroglobin regulates intracellular hypoxic NO-signaling pathways. These studies suggest that neuroglobin may function as a physiological oxidative stress sensor and a post-translationally redox-regulated nitrite reductase that generates NO under six-to-five-coordinate heme pocket control. We hypothesize that the six-coordinate heme globin superfamily may subserve a function as primordial hypoxic and redox-regulated NO-signaling proteins. PMID:21296891

  1. [Properties of a nitrite reductase inhibitor protein from Pseudomonas aeruginosa].

    PubMed

    Karapetian, A V; Nalbandian, R M

    1993-08-01

    The amino acid composition and major physico-chemical properties of the "nonblue" copper protein isolated earlier from Pseudomonas aeruginosa have been determined. It has been found that the azurin oxidase, cytochrome c551 oxidase and superoxide dismutase activities of the enzyme are inhibited by this protein. The inhibition seems to be due to the protein interaction with the electron-accepting center of nitrite reductase.

  2. Nitrate metabolism in tobacco leaves overexpressing Arabidopsis nitrite reductase.

    PubMed

    Davenport, Susie; Le Lay, Pascaline; Sanchez-Tamburrrino, Juan Pablo

    2015-12-01

    Primary nitrogen assimilation in plants includes the reduction of nitrite to ammonium in the chloroplasts by the enzyme nitrite reductase (NiR EC:1.7.7.1) or in the plastids of non-photosynthetic organs. Here we report on a study overexpressing the Arabidopsis thaliana NiR (AtNiR) gene in tobacco plants under the control of a constitutive promoter (CERV - Carnation Etched Ring Virus). The aim was to overexpress AtNiR in an attempt to alter the level of residual nitrite in the leaf which can act as precursor to the formation of nitrosamines. The impact of increasing the activity of AtNiR produced an increase in leaf protein and a stay-green phenotype in the primary transformed AtNiR population. Investigation of the T1 homozygous population demonstrated elevated nitrate reductase (NR) activity, reductions in leaf nitrite and nitrate and the amino acids proline, glutamine and glutamate. Chlorophyl content of the transgenic lines was increased, as evidenced by the stay-green phenotype. This reveals the importance of NiR in primary nitrogen assimilation and how modification of this key enzyme affects both the nitrogen and carbon metabolism of tobacco plants.

  3. A cytochrome cd1-type nitrite reductase mediates the first step of denitrification in Alcaligenes eutrophus.

    PubMed

    Sann, R; Kostka, S; Friedrich, B

    1994-01-01

    Respiratory nitrite reductase (NIR) has been purified from the soluble extract of denitrifying cells of Alcaligenes eutrophus strain H16 to apparent electrophoretic homogeneity. The enzyme was induced under anoxic conditions in the presence of nitrite. Purified NIR showed typical features of a cytochrome cd1-type nitrite reductase. It appeared to be a dimer of kDa subunits, its activity was only weakly inhibited by the copper chelator diethyldithiocarbamate, and spectral analysis revealed absorption maxima which were characteristic for the presence of heme c and heme d1. The isoelectric point of 8.6 was considerably higher than the pI determined for cd1 nitrite reductases from pseudomonads. Eighteen amino acids at the N-terminus of the A. eutrophus NIR, obtained by protein sequencing, showed no significant homology to the N-terminal region of nitrite reductases from Pseudomonas stutzeri and Pseudomonas aeruginosa.

  4. Nitrite Reductase and Nitric-oxide Synthase Activity of the Mitochondrial Molybdopterin Enzymes mARC1 and mARC2*

    PubMed Central

    Sparacino-Watkins, Courtney E.; Tejero, Jesús; Sun, Bin; Gauthier, Marc C.; Thomas, John; Ragireddy, Venkata; Merchant, Bonnie A.; Wang, Jun; Azarov, Ivan; Basu, Partha; Gladwin, Mark T.

    2014-01-01

    Mitochondrial amidoxime reducing component (mARC) proteins are molybdopterin-containing enzymes of unclear physiological function. Both human isoforms mARC-1 and mARC-2 are able to catalyze the reduction of nitrite when they are in the reduced form. Moreover, our results indicate that mARC can generate nitric oxide (NO) from nitrite when forming an electron transfer chain with NADH, cytochrome b5, and NADH-dependent cytochrome b5 reductase. The rate of NO formation increases almost 3-fold when pH was lowered from 7.5 to 6.5. To determine if nitrite reduction is catalyzed by molybdenum in the active site of mARC-1, we mutated the putative active site cysteine residue (Cys-273), known to coordinate molybdenum binding. NO formation was abolished by the C273A mutation in mARC-1. Supplementation of transformed Escherichia coli with tungsten facilitated the replacement of molybdenum in recombinant mARC-1 and abolished NO formation. Therefore, we conclude that human mARC-1 and mARC-2 are capable of catalyzing reduction of nitrite to NO through reaction with its molybdenum cofactor. Finally, expression of mARC-1 in HEK cells using a lentivirus vector was used to confirm cellular nitrite reduction to NO. A comparison of NO formation profiles between mARC and xanthine oxidase reveals similar Kcat and Vmax values but more sustained NO formation from mARC, possibly because it is not vulnerable to autoinhibition via molybdenum desulfuration. The reduction of nitrite by mARC in the mitochondria may represent a new signaling pathway for NADH-dependent hypoxic NO production. PMID:24500710

  5. Chronic Exercise Downregulates Myocardial Myoglobin and Attenuates Nitrite Reductase Capacity During Ischemia-Reperfusion

    PubMed Central

    Nicholson, Chad K.; Lambert, Jonathan P.; Chow, Chi-Wing; Lefer, David J.; Calvert, John W.

    2013-01-01

    Background The infarct sparing effects of exercise are evident following both long-term and short-term training regimens. Here we compared the infarct-lowering effects of nitrite therapy, voluntary exercise, and the combination of both following myocardial ischemia-reperfusion (MI/R) injury. We also compared the degree to which each strategy increased cardiac nitrite levels, as well as the effects of each strategy on the nitrite reductase activity of the heart. Methods and Results Mice subjected to voluntary wheel running (VE) for 4 weeks displayed an 18% reduction in infarct size when compared to sedentary mice, whereas mice administered nitrite therapy (25 mg/L in drinking water) showed a 53% decrease. However, the combination of VE and nitrite exhibited no further protection than VE alone. Although the VE and nitrite therapy mice showed similar nitrite levels in the heart, cardiac nitrite reductase activity was significantly reduced in the VE mice. Additionally, the cardiac protein expression of myoglobin, a known nitrite reductase, was also reduced after VE. Further studies revealed that cardiac NFAT activity was lower after VE due to a decrease in calcineurin activity and an increase in GSK3β activity. Conclusion These data suggest that VE downregulates cardiac myoglobin levels by inhibiting calcineurin/NFAT signaling. Additionally, these results suggest that the modest infarct sparing effects of VE are the result of a decrease in the hearts ability to reduce nitrite to nitric oxide during MI/R. PMID:23962643

  6. Cardiac contractility in Antarctic teleost is modulated by nitrite through xanthine oxidase and cytochrome p-450 nitrite reductase.

    PubMed

    Garofalo, Filippo; Amelio, Daniela; Gattuso, Alfonsina; Cerra, Maria Carmela; Pellegrino, Daniela

    2015-09-15

    In mammalian and non-mammalian vertebrates, nitrite anion, the largest pool of intravascular and tissue nitric oxide storage, represents a key player of many biological processes, including cardiac modulation. As shown by our studies on Antarctic teleosts, nitrite-dependent cardiac regulation is of great relevance also in cold-blooded vertebrates. This study analysed the influence elicited by nitrite on the performance of the perfused beating heart of two Antarctic stenotherm teleosts, the haemoglobinless Chionodraco hamatus (icefish) and the red-blooded Trematomus bernacchii. Since haemoglobin is crucial in nitric oxide homeostasis, the icefish, a naturally occurring genetic knockout for this protein, provides exclusive opportunities to investigate nitric oxide/nitrite signaling. In vivo, nitrite conversion to nitric oxide requires the nitrite reductase activity of xanthine oxidase and cytochrome P-450, thus the involvement of these enzymes was also evaluated. We showed that, in C. hamatus and T. bernacchii, nitrite influenced cardiac performance by inducing a concentration-dependent positive inotropic effect which was unaffected by nitric oxide scavenging by PTIO in C. hamatus, while it was abolished in T. bernacchii. Specific inhibition of xanthine oxidase and cytochrome P-450 revealed, in the two teleosts, that the nitrite-dependent inotropism required the nitrite reductase activity of both enzymes. We also found that xanthine oxidase is more expressed in C. hamatus than in T. bernacchii, while the opposite was observed concerning cytochrome P-450. Results suggested that in the heart of C. hamatus and T. bernacchii, nitrite is an integral physiological source of nitric oxide with important signaling properties, which require the nitrite reductase activity of xanthine oxidase and cytochrome P-450.

  7. Nitrite controls the release of nitric oxide in Pseudomonas aeruginosa cd{sub 1} nitrite reductase

    SciTech Connect

    Rinaldo, Serena; Brunori, Maurizio; Cutruzzola, Francesca

    2007-11-23

    Nitrite reductase (cd{sub 1}NIR) from Pseudomonas aeruginosa, which catalyses the reduction of nitrite to nitric oxide (NO), contains a c-heme as the electron acceptor and a d{sub 1}-heme where catalysis occurs. Reduction involves binding of nitrite to the reduced d{sub 1}-heme, followed by dehydration to yield NO; release of NO and re-reduction of the enzyme close the cycle. Since NO is a powerful inhibitor of ferrous hemeproteins, enzymatic turnover demands the release of NO. We recently discovered that NO dissociation from the ferrous d{sub 1}-heme is fast, showing that cd{sub 1}NIR behaves differently from other hemeproteins. Here we demonstrate for the first time that the physiological substrate nitrite displaces NO from the ferrous enzyme, which enters a new catalytic cycle; this reaction depends on the conserved His369 whose role in substrate stabilization is crucial for catalysis. Thus we suggest that also in vivo the activity of cd{sub 1}NIR is controlled by nitrite.

  8. Comparative analysis of amino acid composition in the active site of nirk gene encoding copper-containing nitrite reductase (CuNiR) in bacterial spp.

    PubMed

    Adhikari, Utpal Kumar; Rahman, M Mizanur

    2017-04-01

    The nirk gene encoding the copper-containing nitrite reductase (CuNiR), a key catalytic enzyme in the environmental denitrification process that helps to produce nitric oxide from nitrite. The molecular mechanism of denitrification process is definitely complex and in this case a theoretical investigation has been conducted to know the sequence information and amino acid composition of the active site of CuNiR enzyme using various Bioinformatics tools. 10 Fasta formatted sequences were retrieved from the NCBI database and the domain and disordered regions identification and phylogenetic analyses were done on these sequences. The comparative modeling of protein was performed through Modeller 9v14 program and visualized by PyMOL tools. Validated protein models were deposited in the Protein Model Database (PMDB) (PMDB id: PM0080150 to PM0080159). Active sites of nirk encoding CuNiR enzyme were identified by Castp server. The PROCHECK showed significant scores for four protein models in the most favored regions of the Ramachandran plot. Active sites and cavities prediction exhibited that the amino acid, namely Glycine, Alanine, Histidine, Aspartic acid, Glutamic acid, Threonine, and Glutamine were common in four predicted protein models. The present in silico study anticipates that active site analyses result will pave the way for further research on the complex denitrification mechanism of the selected species in the experimental laboratory.

  9. DFT Study on Nitrite Reduction Mechanism in Copper-Containing Nitrite Reductase.

    PubMed

    Lintuluoto, Masami; Lintuluoto, Juha M

    2016-01-12

    Dissimilatory reduction of nitrite by copper-containing nitrite reductase (CuNiR) is an important step in the geobiochemical nitrogen cycle. The proposed mechanisms for the reduction of nitrite by CuNiRs include intramolecular electron and proton transfers, and these two events are understood to couple. Proton-coupled electron transfer is one of the key processes in enzyme reactions. We investigated the geometric structure of bound nitrite and the mechanism of nitrite reduction on CuNiR using density functional theory calculations. Also, the proton transfer pathway, the key residues, and their roles in the reaction mechanism were clarified in this study. In our results, the reduction of T2 Cu site promotes the proton transfer, and the hydrogen bond network around the binding site has an important role not only to stabilize the nitrite binding but also to promote the proton transfer to nitrite.

  10. Dissimilatory Nitrite Reductase Genes from Autotrophic Ammonia-Oxidizing Bacteria

    PubMed Central

    Casciotti, Karen L.; Ward, Bess B.

    2001-01-01

    The presence of a copper-containing dissimilatory nitrite reductase gene (nirK) was discovered in several isolates of β-subdivision ammonia-oxidizing bacteria using PCR and DNA sequencing. PCR primers Cunir3 and Cunir4 were designed based on published nirK sequences from denitrifying bacteria and used to amplify a 540-bp fragment of the nirK gene from Nitrosomonas marina and five additional isolates of ammonia-oxidizing bacteria. Amplification products of the expected size were cloned and sequenced. Alignment of the nucleic acid and deduced amino acid (AA) sequences shows significant similarity (62 to 75% DNA, 58 to 76% AA) between nitrite reductases present in these nitrifiers and the copper-containing nitrite reductase found in classic heterotrophic denitrifiers. While the presence of a nitrite reductase in Nitrosomonas europaea is known from early biochemical work, preliminary sequence data from its genome indicate a rather low similarity to the denitrifier nirKs. Phylogenetic analysis of the partial nitrifier nirK sequences indicates that the topology of the nirK tree corresponds to the 16S rRNA and amoA trees. While the role of nitrite reduction in the metabolism of nitrifying bacteria is still uncertain, these data show that the nirK gene is present in closely related nitrifying isolates from many oceanographic regions and suggest that nirK sequences retrieved from the environment may include sequences from ammonia-oxidizing bacteria. PMID:11319103

  11. Expression and purification of spinach nitrite reductase in E. coli

    SciTech Connect

    Bellissimo, D.; Privalle, L. )

    1991-03-11

    The study of structure-function relationships in nitrite reductase (NiR) by site-directed mutagenesis requires an expression system from which suitable quantities of active enzyme can be purified. Spinach NiR cDNA was cloned into pUC18 and expressed in E.coli JM109 as a beta-galactosidase fusion protein. The IPTG-induced fusion protein contains five additional amino acids at the N-terminus. The expressed NiR in aerobic cultures was mostly insoluble and inactive indicating the presence of inclusion bodies. By altering growth conditions, active NiR could represent 0.5-1.0% of the total E.coli protein, Effects of the addition of delta-aminolevulinic acid, a heme precursor, and anaerobic growth were also examined. Spinach NiR was purified approximately 200 fold to homogeneity. When subjected to electrophoresis on SDS polyacrylamide gels, the NiR migrated as a single band with similar mobility to pure spinach enzyme. The expressed enzyme also reacted with rabbit anti-spinach NiR antibody as visualized by Western blot analysis. The absorption spectrum of the E.coli-expressed enzyme was identical to spinach enzyme with a Soret and alpha band a 386 and 573 nm, respectively, and an A{sub 278}/A{sub 386} = 1.9. The addition of nitrite produced the characteristic shifts in the spectrum. The E. coli-expressed NiR catalyzed the methylviologen-dependent reduction of nitrite. The specific activity was 100 U/mg. The K{sub m} determined for nitrite was 0.3 mM which is in agreement with values reported for the enzyme. These results indicate that the E.coli-expressed NiR is fully comparable to spinach NiR in purity, catalytic activity and physical state. Site-directed mutants have been made using PCR to examine structure-function relationships in this enzyme.

  12. Expression of a fully functional cd1 nitrite reductase from Pseudomonas aeruginosa in Pseudomonas stutzeri.

    PubMed

    Arese, Marzia; Zumft, Walter G; Cutruzzolà, Francesca

    2003-01-01

    Nitrite reductases are redox enzymes catalysing the one electron reduction of nitrite to nitrogen monoxide (NO) within the bacterial denitrification process. We have cloned the gene for cd(1) nitrite reductase (Pa-nirS) from Pseudomonas aeruginosa into the NiRS(-) strain MK202 of Pseudomonas stutzeri and expressed the enzyme under denitrifying conditions. In the MK202 strain, denitrification is abolished by the disruption of the endogenous nitrite reductase gene; thus, cells can be grown only in the presence of oxygen. After complementation with Pa-nirS gene, cells supplemented with nitrate can be grown in the absence of oxygen. The presence of nitrite reductase was proven in vivo by the demonstration of NO production, showing that the enzyme was expressed in the active form, containing both heme c and d(1). A purification procedure for the recombinant PaNir has been developed, based on the P. aeruginosa purification protocol; spectroscopic analysis of the purified protein fully confirms the presence of the d(1) heme cofactor. Moreover, the functional characterisation of the recombinant NiR has been carried out by monitoring the production of NO by the purified NiR enzyme in the presence of nitrite by an NO electrode. The full recovery of the denitrification properties in the P. stutzeri MK202 strain by genetic complementation with Pa-NiR underlines the high homology between enzymes of nitrogen oxianion respiration. Our work provides an expression system for cd(1) nitrite reductase and its site-directed mutants in a non-pathogenic strain and is a starting point for the in vivo study of recombinant enzyme variants.

  13. The purification and properties of a cd-cytochrome nitrite reductase from Paracoccus halodenitrificans

    NASA Technical Reports Server (NTRS)

    Mancinelli, R. L.; Cronin, S.; Hochstein, L. I.

    1986-01-01

    Paracoccus halodenitrificans, grown anaerobically in the presence of nitrite, contained membrane and cytoplasmic nitrite reductases. When assayed in the presence of phenazine methosulfate and ascorbate, the membrane-bound enzyme produced nitrous oxide whereas the cytoplasmic enzyme produced nitric oxide. When both enzymes were assayed in the presence of methyl viologen and dithionite, the cytoplasmic enzyme produced ammonia. Following solubilization, the membrane-bound enzyme behaved like the cytoplasmic enzyme, producing nitric oxide in the presence of phenazine methosulfate and ascorbate, and ammonia when assayed in the presence of methyl viologen and dithionite. The cytoplasmic and membrane-bound enzymes were purified to essentially the same specific activity. Only a single nitrite-reductase activity was detected on electrophoretic gels and the electrophoretic behavior of both enzymes suggested they were identical. The spectral properties of both enzymes suggested they were cd-type cytochromes. These data suggest that the products of nitrite reduction by the cd-cytochrome nitrite reductase are determined by the location of the enzyme and the redox potential of the electron donor.

  14. The purification and properties of a cd-cytochrome nitrite reductase from Paracoccus halodenitrificans.

    PubMed

    Mancinelli, R L; Cronin, S; Hochstein, L I

    1986-01-01

    Paracoccus halodenitrificans, grown anaerobically in the presence of nitrite, contained membrane and cytoplasmic nitrite reductases. When assayed in the presence of phenazine methosulfate and ascorbate, the membrane-bound enzyme produced nitrous oxide whereas the cytoplasmic enzyme produced nitric oxide. When both enzymes were assayed in the presence of methyl viologen and dithionite, the cytoplasmic enzyme produced ammonia. Following solubilization, the membrane-bound enzyme behaved like the cytoplasmic enzyme, producing nitric oxide in the presence of phenazine methosulfate and ascorbate, and ammonia when assayed in the presence of methyl viologen and dithionite. The cytoplasmic and membrane-bound enzymes were purified to essentially the same specific activity. Only a single nitrite-reductase activity was detected on electrophoretic gels and the electrophoretic behavior of both enzymes suggested they were identical. The spectral properties of both enzymes suggested they were cd-type cytochromes. These data suggest that the products of nitrite reduction by the cd-cytochrome nitrite reductase are determined by the location of the enzyme and the redox potential of the electron donor.

  15. Structural study of the X-ray-induced enzymatic reaction of octahaem cytochrome C nitrite reductase.

    PubMed

    Trofimov, A A; Polyakov, K M; Lazarenko, V A; Popov, A N; Tikhonova, T V; Tikhonov, A V; Popov, V O

    2015-05-01

    Octahaem cytochrome c nitrite reductase from the bacterium Thioalkalivibrio nitratireducens catalyzes the reduction of nitrite to ammonium and of sulfite to sulfide. The reducing properties of X-ray radiation and the high quality of the enzyme crystals allow study of the catalytic reaction of cytochrome c nitrite reductase directly in a crystal of the enzyme, with the reaction being induced by X-rays. Series of diffraction data sets with increasing absorbed dose were collected from crystals of the free form of the enzyme and its complexes with nitrite and sulfite. The corresponding structures revealed gradual changes associated with the reduction of the catalytic haems by X-rays. In the case of the nitrite complex the conversion of the nitrite ions bound in the active sites to NO species was observed, which is the beginning of the catalytic reaction. For the free form, an increase in the distance between the oxygen ligand bound to the catalytic haem and the iron ion of the haem took place. In the case of the sulfite complex no enzymatic reaction was detected, but there were changes in the arrangement of the active-site water molecules that were presumably associated with a change in the protonation state of the sulfite ions.

  16. Correlations between the Electronic Properties of Shewanella oneidensis Cytochrome c Nitrite Reductase (ccNiR) and Its Structure: Effects of Heme Oxidation State and Active Site Ligation.

    PubMed

    Stein, Natalia; Love, Daniel; Judd, Evan T; Elliott, Sean J; Bennett, Brian; Pacheco, A Andrew

    2015-06-23

    The electrochemical properties of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), a homodimer that contains five hemes per protomer, were investigated by UV-visible and electron paramagnetic resonance (EPR) spectropotentiometries. Global analysis of the UV-vis spectropotentiometric results yielded highly reproducible values for the heme midpoint potentials. These midpoint potential values were then assigned to specific hemes in each protomer (as defined in previous X-ray diffraction studies) by comparing the EPR and UV-vis spectropotentiometric results, taking advantage of the high sensitivity of EPR spectra to the structural microenvironment of paramagnetic centers. Addition of the strong-field ligand cyanide led to a 70 mV positive shift of the active site's midpoint potential, as the cyanide bound to the initially five-coordinate high-spin heme and triggered a high-spin to low-spin transition. With cyanide present, three of the remaining hemes gave rise to distinctive and readily assignable EPR spectral changes upon reduction, while a fourth was EPR-silent. At high applied potentials, interpretation of the EPR spectra in the absence of cyanide was complicated by a magnetic interaction that appears to involve three of five hemes in each protomer. At lower applied potentials, the spectra recorded in the presence and absence of cyanide were similar, which aided global assignment of the signals. The midpoint potential of the EPR-silent heme could be assigned by default, but the assignment was also confirmed by UV-vis spectropotentiometric analysis of the H268M mutant of ccNiR, in which one of the EPR-silent heme's histidine axial ligands was replaced with a methionine.

  17. Enzymatic function of hemoglobin as a nitrite reductase that produces NO under allosteric control

    PubMed Central

    Huang, Zhi; Shiva, Sruti; Kim-Shapiro, Daniel B.; Patel, Rakesh P.; Ringwood, Lorna A.; Irby, Cynthia E.; Huang, Kris T.; Ho, Chien; Hogg, Neil; Schechter, Alan N.; Gladwin, Mark T.

    2005-01-01

    Hypoxic vasodilation is a fundamental, highly conserved physiological response that requires oxygen and/or pH sensing coupled to vasodilation. While this process was first characterized more than 80 years ago, the precise identity and mechanism of the oxygen sensor and mediators of vasodilation remain uncertain. In support of a possible role for hemoglobin (Hb) as a sensor and effector of hypoxic vasodilation, here we show biochemical evidence that Hb exhibits enzymatic behavior as a nitrite reductase, with maximal NO generation rates occurring near the oxy-to-deoxy (R-to-T) allosteric structural transition of the protein. The observed rate of nitrite reduction by Hb deviates from second-order kinetics, and sigmoidal reaction progress is determined by a balance between 2 opposing chemistries of the heme in the R (oxygenated conformation) and T (deoxygenated conformation) allosteric quaternary structures of the Hb tetramer — the greater reductive potential of deoxyheme in the R state tetramer and the number of unligated deoxyheme sites necessary for nitrite binding, which are more plentiful in the T state tetramer. These opposing chemistries result in a maximal nitrite reduction rate when Hb is 40–60% saturated with oxygen (near the Hb P50), an apparent ideal set point for hypoxia-responsive NO generation. These data suggest that the oxygen sensor for hypoxic vasodilation is determined by Hb oxygen saturation and quaternary structure and that the nitrite reductase activity of Hb generates NO gas under allosteric and pH control. PMID:16041407

  18. Molecular Cloning of Complementary DNA Encoding Maize Nitrite Reductase

    PubMed Central

    Lahners, Kristine; Kramer, Vance; Back, Eduard; Privalle, Laura; Rothstein, Steven

    1988-01-01

    Complementary DNA has been isolated that codes for maize nitrite reductase (NiR) by using the corresponding spinach gene (E Back et al. 1988 Mol Gen Genet 212:20-26) as a heterologous probe. The sequences of the complementary DNAs from the two species are 66% homologous while the deduced amino acid sequences are 86% similar when analogous amino acids are included. A high percentage of the differences in the DNA sequences is due to the extremely strong bias in the corn gene to have a G/C base in the third codon position with 559/569 codons ending in a G or C. Using a hydroponic system, maize seedlings grown in the absence of an exogenous nitrogen source were induced with nitrate or nitrite. Nitrate stimulated a rapid induction of the NiR mRNA in both roots and leaves. There is also a considerable induction of this gene in roots upon the addition of nitrite, although under the conditions used the final mRNA level was not as high as when nitrate was the inducer. There is a small but detectable level of NiR mRNA in leaves prior to induction, but no constitutive NiR mRNA can be seen in the roots. Analysis of genomic DNA supports the notion that there are at least two NiR genes in maize. Images Fig. 3 Fig. 4 Fig. 5 PMID:16666376

  19. Role of nitrate and nitrite in the induction of nitrite reductase in leaves of barley seedlings

    NASA Technical Reports Server (NTRS)

    Aslam, M.; Huffaker, R. C.

    1989-01-01

    The role of NO3- and NO2- in the induction of nitrite reductase (NiR) activity in detached leaves of 8-day-old barley (Hordeum vulgare L.) seedlings was investigated. Barley leaves contained 6 to 8 micromoles NO2-/gram fresh weight x hour of endogenous NiR activity when grown in N-free solutions. Supply of both NO2- and NO3- induced the enzyme activity above the endogenous levels (5 and 10 times, respectively at 10 millimolar NO2- and NO3- over a 24 hour period). In NO3(-)-supplied leaves, NiR induction occurred at an ambient NO3- concentration of as low as 0.05 millimolar; however, no NiR induction was found in leaves supplied with NO2- until the ambient NO2- concentration was 0.5 millimolar. Nitrate accumulated in NO2(-)-fed leaves. The amount of NO3- accumulating in NO2(-)-fed leaves induced similar levels of NiR as did equivalent amounts of NO3- accumulating in NO3(-)-fed leaves. Induction of NiR in NO2(-)-fed leaves was not seen until NO3- was detectable (30 nanomoles/gram fresh weight) in the leaves. The internal concentrations of NO3-, irrespective of N source, were highly correlated with the levels of NiR induced. When the reduction of NO3- to NO2- was inhibited by WO4(2-), the induction of NiR was inhibited only partially. The results indicate that in barley leaves in NiR is induced by NO3- directly, i.e. without being reduced to NO2-, and that absorbed NO2- induces the enzyme activity indirectly after being oxidized to NO3- within the leaf.

  20. The partial characterization of purified nitrite reductase and hydroxylamine oxidase from Nitrosomonas europaea

    PubMed Central

    Ritchie, G. A. F.; Nicholas, D. J. D.

    1974-01-01

    Nitrite reductase has been separated from cell-free extracts of Nitrosomonas and partially purified from hydroxylamine oxidase by polyacrylamide-gel electrophoresis. In its oxidized state the enzyme, which did not contain haem, had an extinction maximum at 590nm, which was abolished on reduction. Sodium diethyldithiocarbamate was a potent inhibitor of nitrite reductase. Enzyme activity was stimulated 2.5-fold when remixed with hydroxylamine oxidase, but was unaffected by mammalian cytochrome c. The enzyme also exhibited a low hydroxylamine-dependent nitrite reductase activity. The results suggest that this enzyme is similar to the copper-containing `denitrifying enzyme' of Pseudomonas denitrificans. A dithionite-reduced, 465nm-absorbing haemoprotein was associated with homogeneous preparations of hydroxylamine oxidase. The band at 465nm maximum was not reduced during the oxidation of hydroxylamine although the extinction was abolished on addition of hydroxylamine, NO2− or CO. These last-named compounds when added to the oxidized enzyme precluded the appearance of the 465nm-absorption band on addition of dithionite. Several properties of 465nm-absorbing haemoprotein are described. PMID:4154745

  1. Evaluation of nitrate reductase activity in Rhizobium japonicum

    SciTech Connect

    Streeter, J.G.; DeVine, P.J.

    1983-08-01

    Nitrate reductase activity was evaluated by four approaches, using four strains of Rhizobium japonicum and 11 chlorate-resistant mutants of the four strains. It was concluded that in vitro assays with bacteria or bacteroids provide the most simple and reliable assessment of the presence or absence of nitrate reductase. Nitrite reductase activity with methyl viologen and dithionite was found, but the enzyme activity does not confound the assay of nitrate reductase. 18 references

  2. Expression, and Molecular and Enzymatic Characterization of Cu-Containing Nitrite Reductase from a Marine Ammonia-Oxidizing Gammaproteobacterium, Nitrosococcus oceani

    PubMed Central

    Kondo, Keitaro; Yoshimatsu, Katsuhiko; Fujiwara, Taketomo

    2012-01-01

    Ammonia-oxidizing bacteria (AOB) remove intracellular nitrite to prevent its toxicity by a nitrifier denitrification pathway involving two denitrifying enzymes, nitrite reductase and nitric oxide reductase. Here, a Cu-containing nitrite reductase from Nitrosococcus oceani strain NS58, a gammaproteobacterial marine AOB, was expressed in Escherichia coli and purified to homogeneity. Sequence homology analysis indicated that the nitrite reductase from N. oceani was phylogenetically closer to its counterparts from denitrifying bacteria than that of the betaproteobacterium Nitrosomonas europaea. The recombinant enzyme was a homotrimer of a 32 kDa subunit molecule. The enzyme was green in the oxidized state with absorption peaks at 455 nm and 575 nm. EPR spectroscopy indicated the presence of type 2 Cu. Molecular activities and the affinity constant for the nitrite were determined to be 1.6×103 s−1 and 52 μM, respectively. PMID:22641151

  3. Nitrate reductase and nitrite as additional components of defense system in pigeonpea (Cajanus cajan L.) against Helicoverpa armigera herbivory.

    PubMed

    Kaur, Rimaljeet; Gupta, Anil Kumar; Taggar, Gaurav Kumar

    2014-10-01

    Amylase inhibitors serve as attractive candidates of defense mechanisms against insect attack. Therefore, the impediment of Helicoverpa armigera digestion can be the effective way of controlling this pest population. Nitrite was found to be a potent mixed non-competitive competitive inhibitor of partially purified α-amylase of H. armigera gut. This observation impelled us to determine the response of nitrite and nitrate reductase (NR) towards H. armigera infestation in nine pigeonpea genotypes (four moderately resistant, three intermediate and two moderately susceptible). The significant upregulation of NR in moderately resistant genotypes after pod borer infestation suggested NR as one of the factors that determine their resistance status against insect attack. The pod borer attack caused greater reduction of nitrate and significant accumulation of nitrite in moderately resistant genotypes. The activity of nitrite reductase (NiR) was also enhanced more in moderately resistant genotypes than moderately susceptible genotypes on account of H. armigera herbivory. Expression of resistance to H. armigera was further revealed when significant negative association between NR, NiR, nitrite and percent pod damage was observed. This is the first report that suggests nitrite to be a potent inhibitor of H. armigera α-amylase and also the involvement of nitrite and NR in providing resistance against H. armigera herbivory.

  4. Stable Copper-Nitrosyl Formation By Nitrite Reductase in Either Oxidation State

    SciTech Connect

    Tocheva, E.I.; Rosell, F.I.; Mauk, A.G.; Murphy, M.E.P.

    2009-06-04

    Nitrite reductase (NiR) is an enzyme that uses type 1 and type 2 copper sites to reduce nitrite to nitric oxide during bacterial denitrification. A copper-nitrosyl intermediate is a proposed, yet poorly characterized feature of the NiR catalytic cycle. This intermediate is formally described as Cu(I)-NO{sup +} and is proposed to be formed at the type 2 copper site after nitrite binding and electron transfer from the type 1 copper site. In this study, copper-nitrosyl complexes were formed by prolonged exposure of exogenous NO to crystals of wild-type and two variant forms of NiR from Alcaligenes faecalis (AfNiR), and the structures were determined to 1.8 {angstrom} or better resolution. Exposing oxidized wild-type crystals to NO results in the reverse reaction and formation of nitrite that remains bound at the active site. In a type 1 copper site mutant (H145A) that is incapable of electron transfer to the type 2 site, the reverse reaction is not observed. Instead, in both oxidized and reduced H145A crystals, NO is observed bound in a side-on manner to the type 2 copper. In AfNiR, Asp98 forms hydrogen bonds to both substrate and product bound to the type 2 Cu. In the D98N variant, NO is bound side-on but is more disordered when observed for the wild-type enzyme. The solution EPR spectra of the crystallographically characterized NiR-NO complexes indicate the presence of an oxidized type 2 copper site and thus are interpreted as resulting from stable copper-nitrosyls and formally assigned as Cu(II)-NO{sup -}. A reaction scheme in which a second NO molecule is oxidized to nitrite can account for the formation of a CuD-NO{sup -} species after exposure of the oxidized H145A variant to NO gas.

  5. The combined nitrate reductase and nitrite-dependent route of NO synthesis in potato immunity to Phytophthora infestans.

    PubMed

    Floryszak-Wieczorek, Jolanta; Arasimowicz-Jelonek, Magdalena; Izbiańska, Karolina

    2016-11-01

    In contrast to the in-depth knowledge concerning nitric oxide (NO) function, our understanding of NO synthesis in plants is still very limited. In view of the above, this paper provides a step by step presentation of the reductive pathway for endogenous NO generation involving nitrate reductase (NR) activity and nitrite implication in potato defense to Phytophthora infestans. A biphasic character of NO emission, peaking mainly at 3 and then at 24 hpi, was detected during the hypersensitive response (HR). In avr P. infestans potato leaves enhanced NR gene and protein expression was tuned with the depletion of nitrate contents and the increase in nitrite supply at 3 hpi. In the same time period a temporary down-regulation of nitrite reductase (NiR) and activity was found. The study for the link between NO signaling and HR revealed an up-regulation of used markers of effective defense, i.e. Nonexpressor of PR genes (NPR1), thioredoxins (Thx) and PR1, at early time-points (1-3 hpi) upon inoculation. In contrast to the resistant response, in the susceptible one a late overexpression (24-48 hpi) of NPR1 and PR1 mRNA levels was observed. Presented data confirmed the importance of nitrite processed by NR in NO generation in inoculated potato leaves. However, based on the pharmacological approach the potential formation of NO from nitrite bypassing the NR activity during HR response to P. infestans has also been discussed.

  6. Structure of octaheme cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens in a complex with phosphate

    SciTech Connect

    Trofimov, A. A.; Polyakov, K. M.; Boiko, K. M.; Filimonenkov, A. A.; Dorovatovskii, P. V.; Tikhonova, T. V.; Popov, V. O.; Koval'chuk, M. V.

    2010-01-15

    Octaheme cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens (TvNiR) catalyzes the reduction of nitrite and hydroxylamine to ammonia. The structures of the free enzyme and of the enzyme in complexes with the substrate (nitrite ion) and the inhibitor (azide ion) have been solved previously. In this study we report the structures of the oxidized complex of TvNiR with phosphate and of this complex reduced by europium(II) chloride (1.8- and 2.0-A resolution, the R factors are 15.9 and 16.7%, respectively) and the structure of the enzyme in the complex with cyanide (1.76-A resolution, the R factor is 16.5%), which was prepared by soaking a crystal of the oxidized phosphate complex of TvNiR. In the active site of the enzyme, the phosphate ion binds to the iron ion of the catalytic heme and to the side chains of the catalytic residues Arg131, Tyr303, and His361. The cyanide ion is coordinated to the heme-iron ion and is hydrogen bonded to the residue His361. In the structure of reduced TvNiR, the phosphate ion is bound in the same manner as in the structure of oxidized TvNiR, and the nine{sub c}oordinated europium ion is located on the surface of one of the crystallographically independent monomers of the enzyme.

  7. The anoxic plant mitochondrion as a nitrite: NO reductase.

    PubMed

    Gupta, Kapuganti J; Igamberdiev, Abir U

    2011-07-01

    Under the conditions of oxygen deprivation, accumulating nitrite can be reduced in the mitochondrial electron transport chain forming free radical nitric oxide (NO). By reducing nitrite to NO, plant mitochondria preserve the capacity to oxidize external NADH and NADPH and retain a limited power for ATP synthesis complementing glycolytic ATP production. NO participates in O(2) balance in mitochondria by competitively inhibiting cytochrome c oxidase which can oxidize it to nitrite when oxygen concentration increases. Some of the NO escapes to the cytosol, where the efficient scavenging system involving non-symbiotic hemoglobin oxygenates NO to nitrate and supports continuous anaerobic turnover of nitrogen species.

  8. Fine-tuned regulation of the dissimilatory nitrite reductase gene by oxygen and nitric oxide in Pseudomonas aeruginosa.

    PubMed

    Kuroki, Miho; Igarashi, Yasuo; Ishii, Masaharu; Arai, Hiroyuki

    2014-12-01

    Nitrite reductase (NIR) catalyses the reduction of nitrite to nitric oxide (NO) in the denitrification pathway. In Pseudomonas aeruginosa, expression of the gene encoding NIR (nirS) is induced by NO and is under control of the NO-sensing regulator DNR (dissimilatory nitrate respiration regulator). Because DNR is under control of the oxygen-sensing regulator ANR (anaerobic regulator of arginine deiminase and nitrate reductase), nirS is expressed only under low oxygen and anaerobic conditions. Both ANR and DNR are FNR (fumarate and nitrate reductase regulator)-type regulators and recognize the consensus FNR-binding motif. The motif of the nirS promoter is thought to be recognized only by DNR, and not by ANR. Here, mutant strains expressing either ANR or DNR were constructed and used to analyse the role of ANR and DNR in the activation of nirS expression. Analysis of transcriptional activity by microarray and quantitative reverse transcription polymerase chain reaction revealed that nirS is transcribed under low oxygen conditions in an ANR-dependent manner, although the expression level was 10-fold lower than that of the DNR-dependent expression. An artificial promoter containing the FNR-binding motif of the nirS promoter was also twofold upregulated by ANR. These results indicate that low-level expression of NIR in the presence of nitrite may provide NO as a trigger for the full expression of denitrification genes when oxygen is depleted.

  9. Expression of nitrite and nitric oxide reductases in free-living and plant-associated Agrobacterium tumefaciens C58 cells.

    PubMed

    Baek, Seung-Hun; Shapleigh, James P

    2005-08-01

    A number of the bacteria that form associations with plants are denitrifiers. To learn more about how the association with plants affects expression of denitrification genes, the regulation of nitrite and nitric oxide reductases was investigated in Agrobacterium tumefaciens. Analysis of free-living cells revealed that expression of the genes encoding nitrite and nitric oxide reductases, nirK and nor, respectively, requires low-oxygen conditions, nitric oxide, and the transcriptional regulator NnrR. Expression of nor was monitored in plant-associated bacteria using nor-gfp fusion expression. In root association experiments, only a small percentage of the attached cells were fluorescent, even when they were incubated under a nitrogen atmosphere. Inactivation of nirK had no significant effect on the ability of A. tumefaciens to bind to plant roots regardless of the oxygen tension, but it did decrease the occurrence of root-associated fluorescent cells. When wild-type cells containing the gfp fusion were infiltrated into leaves, most cells eventually became fluorescent. The same result was obtained when a nirK mutant was used, suggesting that nitric oxide activated nor expression in the endophytic bacteria. Addition of a nitric oxide synthase inhibitor to block nitric oxide generation by the plant prevented gfp expression in infiltrated nitrite reductase mutants, demonstrating that plant-derived nitric oxide can activate nor expression in infiltrated cells.

  10. Aminoglycoside-resistant mutants of Pseudomonas aeruginosa deficient in cytochrome d, nitrite reductase, and aerobic transport.

    PubMed Central

    Bryan, L E; Kwan, S

    1981-01-01

    Two gentamicin-resistant mutants of Pseudomonas aeruginosa PAO 503 were selected after ethyl methane sulfonate mutagenesis. Mutant PAO 2403 had significantly increased resistance to aminoglycoside but not to other antibiotics. Mutant PAO 2402 showed a similar spectrum of resistance but of lower magnitude. Both mutants showed no detectable cytochrome d and had a high frequency of reversion to a fully wild-type phenotype. PAO 2403 had a marked decrease and PAO 2402 had a moderate decrease in nitrite reductase activity. Both mutants had reduced uptake of gentamicin and dihydrostreptomycin. Mutant PAO 2403 showed a general decrease in transport rate of cationic compounds, whereas mutant PAO 2402 had only deficient glucose transport. Both mutants showed enhanced rates of glutamine transport and no change in glutamic acid transport. Other components of electron transport and oxidative phosphorylation were normal. These mutants involve ferrocytochrome C551 oxidoreductase formed only on anaerobic growth but illustrate transport defects in aerobically grown cells. PMID:6791588

  11. Functional analysis of a nitrite reductase promoter from birch in transgenic tobacco.

    PubMed

    Warning; Hachtel

    2000-06-29

    Nitrate assimilation is a highly regulated process in higher plants, and the regulatory cues governing gene expression in this pathway include both external and internal factors. In birch (Betula pendula Roth) the expression of nitrate reductase (NR) and nitrite reductase (NiR) genes is co-regulated by light and nitrate at the transcriptional level. In order to identify cis-acting DNA-elements involved in light and nitrate induction of the birch NiR gene, a 0.9 kb 5' flanking region of the NiR gene was isolated, analysed on the DNA level, and the transcription start site was determined. Deletion analysis of the birch NiR promoter region fused to the GUS reporter gene (uidA) in transgenic tobacco (Nicotiana tabacum) revealed the presence of light- and nitrate-responsive promoter fragments. The responsive fragments showed different activities in leaves and roots. Further, gel mobility shift assays using nuclear proteins from leaves detected a specific DNA-binding activity to the sequence between -146 and -267 bp that was induced in darkness and disappeared in the light. The deletion analysis has shown that this region is critical for light inducibility of the birch NiR gene in leaves.

  12. Serial crystallography captures enzyme catalysis in copper nitrite reductase at atomic resolution from one crystal

    PubMed Central

    Horrell, Sam; Antonyuk, Svetlana V.; Eady, Robert R.; Hasnain, S. Samar; Hough, Michael A.; Strange, Richard W.

    2016-01-01

    Relating individual protein crystal structures to an enzyme mechanism remains a major and challenging goal for structural biology. Serial crystallography using multiple crystals has recently been reported in both synchrotron-radiation and X-ray free-electron laser experiments. In this work, serial crystallography was used to obtain multiple structures serially from one crystal (MSOX) to study in crystallo enzyme catalysis. Rapid, shutterless X-ray detector technology on a synchrotron MX beamline was exploited to perform low-dose serial crystallography on a single copper nitrite reductase crystal, which survived long enough for 45 consecutive 100 K X-ray structures to be collected at 1.07–1.62 Å resolution, all sampled from the same crystal volume. This serial crystallography approach revealed the gradual conversion of the substrate bound at the catalytic type 2 Cu centre from nitrite to nitric oxide, following reduction of the type 1 Cu electron-transfer centre by X-ray-generated solvated electrons. Significant, well defined structural rearrangements in the active site are evident in the series as the enzyme moves through its catalytic cycle, namely nitrite reduction, which is a vital step in the global denitrification process. It is proposed that such a serial crystallography approach is widely applicable for studying any redox or electron-driven enzyme reactions from a single protein crystal. It can provide a ‘catalytic reaction movie’ highlighting the structural changes that occur during enzyme catalysis. The anticipated developments in the automation of data analysis and modelling are likely to allow seamless and near-real-time analysis of such data on-site at some of the powerful synchrotron crystallographic beamlines. PMID:27437114

  13. Role of Nitrate and Nitrite in the Induction of Nitrite Reductase in Leaves of Barley Seedlings 1

    PubMed Central

    Aslam, Muhammad; Huffaker, Ray C.

    1989-01-01

    The role of NO3− and NO2− in the induction of nitrite reductase (NiR) activity in detached leaves of 8-day-old barley (Hordeum vulgare L.) seedlings was investigated. Barley leaves contained 6 to 8 micromoles NO2−/gram fresh weight × hour of endogenous NiR activity when grown in N-free solutions. Supply of both NO2− and NO3− induced the enzyme activity above the endogenous levels (5 and 10 times, respectively at 10 millimolar NO2− and NO3− over a 24 hour period). In NO3−-supplied leaves, NiR induction occurred at an ambient NO3− concentration of as low as 0.05 millimolar; however, no NiR induction was found in leaves supplied with NO2− until the ambient NO2− concentration was 0.5 millimolar. Nitrate accumulated in NO2−-fed leaves. The amount of NO3− accumulating in NO2−-fed leaves induced similar levels of NiR as did equivalent amounts of NO3− accumulating in NO3−-fed leaves. Induction of NiR in NO2−-fed leaves was not seen until NO3− was detectable (30 nanomoles/gram fresh weight) in the leaves. The internal concentrations of NO3−, irrespective of N source, were highly correlated with the levels of NiR induced. When the reduction of NO3− to NO2− was inhibited by WO42−, the induction of NiR was inhibited only partially. The results indicate that in barley leaves NiR is induced by NO3− directly, i.e. without being reduced to NO2−, and that absorbed NO2− induces the enzyme activity indirectly after being oxidized to NO3− within the leaf. PMID:11537455

  14. Catalysis of nitrosyl transfer reactions by a dissimilatory nitrite reductase (cytochrome c,d1).

    PubMed

    Kim, C H; Hollocher, T C

    1984-02-25

    The dissimilatory nitrite reductase (cytochrome c,d1) from Pseudomonas aeruginosa was observed at pH 7.5 to catalyze nitrosyl transfer (nitrosation) between [15N]nitrite and several N-nucleophiles or H2 18O, with rate enhancement of the order of 10(8) relative to analogous chemical reactions. The reducing system (ascorbate, N,N,N',N'-tetramethylphenylenediamine) could reduce nitrite (but not NO) enzymatically and had essentially no direct chemical reactivity toward nitrite or NO. The N-nitrosations showed saturation kinetics with respect to the nucleophile and, while exhibiting Vmax values which varied by about 40-fold, nevertheless showed little or no dependence of Vmax on nucleophile pKa. The N-nitrosations and NO-2/H2O-18O exchange required the reducing system, whereas NO/H2O-18O exchange was inhibited by the reducing system. NO was not detected to serve as a nitrosyl donor to N-nucleophiles. These and other kinetic observations suggest that the enzymatic nitrosyl donor is an enzyme-bound species derived from reduced enzyme and one molecule of nitrite, possibly a heme-nitrosyl compound (E-FeII X NO+) for which there is precedence. Nitrosyl transfer to N-nucleophiles may occur within a ternary complex of enzyme, nitrite, and nucleophile. Catalysis of nitrosyl transfer by nitrite reductase represents a new class of enzymatic reactions and may present another example of electrophilic catalysis by a metal center. The nitrosyl donor trapped by these reactions is believed to represent an intermediate in the reduction of nitrite by cytochrome c,d1.

  15. Characterization of the nirK gene encoding the respiratory, Cu-containing nitrite reductase of Bradyrhizobium japonicum.

    PubMed

    Velasco, L; Mesa, S; Delgado, M J; Bedmar, E J

    2001-10-31

    The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from Bradyrhizobium japonicum USDA110 has been isolated and sequenced. The deduced amino acid sequence exhibited a high degree of similarity to other Cu-containing nitrite reductases from various sources. The full-length protein included a signal peptide for protein export. Analysis of the sequence upstream from the structural nirK gene revealed the presence of an anaerobox located 83 base pairs from the putative translational start codon. Cells of strain GRK308, a nitrite reductase-deficient derivative of strain USDA110, were unable to grow when cultured under microaerobic conditions (1% O(2)) in the presence of either nitrate or nitrite. Maximal expression of a nirK-lacZ fusion in strain USDA110 required simultaneously both low level oxygen conditions and the presence of nitrate. Expression of beta-galactosidase activity was not detected in the B. japonicum fixL 7403, fixJ 7360 and fixK(2) 9043 mutants transformed with the nirK-lacZ fusion after incubation of the cells under oxygen-limiting conditions either with or without nitrate. Complementation of B. japonicum 9043 with the fixK(2) gene restored beta-galactosidase activity to levels similar to those found in the parental strain. These results suggest that nirK expression depends on the low-oxygen-responsive two-component regulatory system FixLJ and on the Fnr/FixK-like DNA binding protein FixK(2).

  16. Nitrite Reductase NirBD Is Induced and Plays an Important Role during In Vitro Dormancy of Mycobacterium tuberculosis

    PubMed Central

    Akhtar, Shamim; Khan, Arshad; Sohaskey, Charles D.; Jagannath, Chinnaswamy

    2013-01-01

    Mycobacterium tuberculosis is one of the strongest reducers of nitrate among all mycobacteria. Reduction of nitrate to nitrite, mediated by nitrate reductase (NarGHJI) of M. tuberculosis, is induced during the dormant stage, and the enzyme has a respiratory function in the absence of oxygen. Nitrite reductase (NirBD) is also functional during aerobic growth when nitrite is the sole nitrogen source. However, the role of NirBD-mediated nitrite reduction during the dormancy is not yet characterized. Here, we analyzed nitrite reduction during aerobic growth as well as in a hypoxic dormancy model of M. tuberculosis in vitro. When nitrite was used as the sole nitrogen source in the medium, the organism grew and the reduction of nitrite was evident in both hypoxic and aerobic cultures of M. tuberculosis. Remarkably, the hypoxic culture of M. tuberculosis, compared to the aerobic culture, showed 32- and 4-fold-increased expression of nitrite reductase (NirBD) at the transcription and protein levels, respectively. More importantly, a nirBD mutant of M. tuberculosis was unable to reduce nitrite and compared to the wild-type (WT) strain had a >2-log reduction in viability after 240 h in the Wayne model of hypoxic dormancy. Dependence of M. tuberculosis on nitrite reductase (NirBD) was also seen in a human macrophage-based dormancy model where the nirBD mutant was impaired for survival compared to the WT strain. Overall, the increased expression and essentiality of nitrite reductase in the in vitro dormancy models suggested that NirBD-mediated nitrite reduction could be critical during the persistent stage of M. tuberculosis. PMID:23935045

  17. Patterns of product inhibition for bacterial nitrite reductase.

    PubMed

    Dhesi, R; Timkovich, R

    1984-09-28

    Product inhibition has been examined in the turnover kinetics of cytochrome cd1 from Pseudomonas aeruginosa (ATCC 19429) and from Paracoccus denitrificans1 (ATCC 13456). A common characteristic was a decrease in rate during the time course of assays that was not due to substrate depletion or irreversible inactivation. The product of nitrite reduction, nitric oxide (NO), acted as a product inhibitor in anaerobic assays with an apparent Ki of 0.2 microM, but only if the enzyme was first preincubated with NO for 15 min. The enzyme was inhibited by the oxidized form of electron donors and this could account for the decrease in rate during an assay. For the donors hydroquinone, ascorbate, TMPD, and azurin, measured values of the inhibition constant were at least ten fold lower than measured Km's. Cytochromes c as donors demonstrated a complex pattern of product inhibition by the ferric form. Although numerical values of Ki in these cases were not obtained, trends indicated that apparent values would be less than Km.

  18. Impact of residues remote from the catalytic centre on enzyme catalysis of copper nitrite reductase.

    PubMed

    Leferink, Nicole G H; Antonyuk, Svetlana V; Houwman, Joseline A; Scrutton, Nigel S; Eady, Robert R; Hasnain, S Samar

    2014-07-15

    Enzyme mechanisms are often probed by structure-informed point mutations and measurement of their effects on enzymatic properties to test mechanistic hypotheses. In many cases, the challenge is to report on complex, often inter-linked elements of catalysis. Evidence for long-range effects on enzyme mechanism resulting from mutations remains sparse, limiting the design/redesign of synthetic catalysts in a predictable way. Here we show that improving the accessibility of the active site pocket of copper nitrite reductase by mutation of a surface-exposed phenylalanine residue (Phe306), located 12 Å away from the catalytic site type-2 Cu (T2Cu), profoundly affects intra-molecular electron transfer, substrate-binding and catalytic activity. Structures and kinetic studies provide an explanation for the lower affinity for the substrate and the alteration of the rate-limiting step in the reaction. Our results demonstrate that distant residues remote from the active site can have marked effects on enzyme catalysis, by driving mechanistic change through relatively minor structural perturbations.

  19. Role of nitrite reductase in the ammonia-oxidizing pathway of Nitrosomonas europaea.

    PubMed

    Cantera, J Jason L; Stein, Lisa Y

    2007-10-01

    Metabolism of ammonia (NH(3)) and hydroxylamine (NH(2)OH) by wild-type and a nitrite reductase (nirK) deficient mutant of Nitrosomonas europaea was investigated to clarify the role of NirK in the NH(3) oxidation pathway. NirK-deficient N. europaea grew more slowly, consumed less NH(3), had a lower rate of nitrite (NO(2) (-)) production, and a significantly higher rate of nitrous oxide (N(2)O) production than the wild-type when incubated with NH(3) under high O(2) tension. In incubations with NH(3) under low O(2) tension, NirK-deficient N. europaea grew more slowly, but had only modest differences in NH(3) oxidation and product formation rates relative to the wild-type. In contrast, the nirK mutant oxidized NH(2)OH to NO(2) (-) at consistently slower rates than the wild-type, especially under low O(2) tension, and lost a significant pool of NH(2)OH-N to products other than NO(2) (-) and N(2)O. The rate of N(2)O production by the nirK mutant was ca. three times higher than the wild-type during hydrazine-dependent NO(2) (-) reduction under both high and low O(2) tension. Together, the results indicate that NirK activity supports growth of N. europaea by supporting the oxidation of NH(3) to NO(2) (-) via NH(2)OH, and stimulation of hydrazine-dependent NO(2) (-) reduction by NirK-deficient N. europaea indicated the presence of an alternative, enzymatic pathway for N(2)O production.

  20. Isoelectrophoretic characterization of Pseudomonas cytochrome oxidase/nitrite reductase and its heme d1-containing domain.

    PubMed

    Hull, H H; Wharton, D C

    1993-02-15

    The cytochrome oxidase/nitrite reductase of Pseudomonas aeruginosa has been purified to homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When this "homogeneous" protein is subjected to electrophoretic titration curve analysis in ampholines or to isoelectric focusing in immobilized pH gradient gels it is resolved into several bands, each of which possesses the olive-green color of the holoenzyme. Although the patterns of resolution replicate for a given enzyme preparation differences occur among different preparations. Furthermore, storage for several months at -20 degrees C leads to an increase in the number of isoelectrophoretic forms. All preparations, however, have two primary bands, one with a pI of 6.97 and the other of 7.02. Both these bands possess significant cytochrome oxidase activity after elution from the gels. When each of the primary bands is eluted and again subjected to isoelectric focusing under the same conditions as before, each band interconverts into two bands with pIs of 6.97 and 7.02. The addition of the ligand cyanide to the holoenzyme produces a shift in the pI of the two bands to pIs 7.04 and 7.12 while the addition of nitrite shifts some of the band at pI 6.97 into that at pI 7.02. The heme d1-containing dipeptide of the enzyme, produced by treatment with subtilisin, also exhibits considerable heterogeneity upon electrophoretic titration curve analysis and by isoelectric focusing in immobiline gels. Possible explanations for the observed isoelectrophoretic behavior in terms of protein conformation and heme chemistry are discussed.

  1. Dissecting the role of NtrC and RpoN in the expression of assimilatory nitrate and nitrite reductases in Bradyrhizobium diazoefficiens.

    PubMed

    López, María F; Cabrera, Juan J; Salas, Ana; Delgado, María J; López-García, Silvina L

    2017-04-01

    Bradyrhizobium diazoefficiens, a nitrogen-fixing endosymbiont of soybeans, is a model strain for studying rhizobial denitrification. This bacterium can also use nitrate as the sole nitrogen (N) source during aerobic growth by inducing an assimilatory nitrate reductase encoded by nasC located within the narK-bjgb-flp-nasC operon along with a nitrite reductase encoded by nirA at a different chromosomal locus. The global nitrogen two-component regulatory system NtrBC has been reported to coordinate the expression of key enzymes in nitrogen metabolism in several bacteria. In this study, we demonstrate that disruption of ntrC caused a growth defect in B. diazoefficiens cells in the presence of nitrate or nitrite as the sole N source and a decreased activity of the nitrate and nitrite reductase enzymes. Furthermore, the expression of narK-lacZ or nirA-lacZ transcriptional fusions was significantly reduced in the ntrC mutant after incubation under nitrate assimilation conditions. A B. diazoefficiens rpoN 1/2 mutant, lacking both copies of the gene encoding the alternative sigma factor σ(54), was also defective in aerobic growth with nitrate as the N source as well as in nitrate and nitrite reductase expression. These results demonstrate that the NtrC regulator is required for expression of the B. diazoefficiens nasC and nirA genes and that the sigma factor RpoN is also involved in this regulation.

  2. 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

    PubMed Central

    Frías, José E.

    2015-01-01

    ABSTRACT 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. IMPORTANCE 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

  3. ARM-microcontroller based portable nitrite electrochemical analyzer using cytochrome c reductase biofunctionalized onto screen printed carbon electrode.

    PubMed

    Santharaman, Paulraj; Venkatesh, Krishna Arun; Vairamani, Kanagavel; Benjamin, Alby Robson; Sethy, Niroj K; Bhargava, Kalpana; Karunakaran, Chandran

    2017-04-15

    Nitrite (NO2(-)) supplementation limits hypoxia-induced oxidative stress and activates the alternate NO pathway which may partially account for the nitrite-mediated cardioprotection. So, sensitive and selective biosensors with point-of-care devices need to be explored to detect the physiological nitrite level due to its important role in human pathophysiology. In this work, cytochrome c reductase (CcR) biofunctionalized self assembled monolayer (SAM) functionalized on gold nanoparticles (GNPs) in polypyrrole (PPy) nanocomposite onto the screen printed carbon electrode (SPCE) was investigated as a biosensor for the detection of nitrite based on its electrochemical and catalytic properties. CcR was covalently coupled with SAM layers on GNPs by using EDC and NHS. Direct electrochemical response of CcR biofunctionalized electrodes showed a couple of well-defined and nearly reversible cyclic voltammetric peaks at -0.34 and -0.45 vs. Ag/AgCl. Under optimal conditions, the biosensor could be used for the determination of NO2(-) with a linear range from 0.1-1600µm and a detection limit of 60nM with a sensitivity of 0.172µAµM(-1)cm(-2). Further, we have designed and developed a novel and cost effective portable electrochemical analyzer for the measurement of NO2(-) in hypoxia induced H9c2 cardiac cells using ARM microcontroller. The results obtained here using the developed portable electrochemical nitrite analyzer were also compared with the standard cyclic voltammetry instrument and found in agreement with each other.

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

    PubMed

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

    2013-05-01

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

  5. Nitrite reductase is critical for Pseudomonas aeruginosa survival during co-infection with the oral commensal Streptococcus parasanguinis.

    PubMed

    Scoffield, Jessica A; Wu, Hui

    2016-02-01

    Pseudomonas aeruginosa is the major aetiological agent of chronic pulmonary infections in cystic fibrosis (CF) patients. However, recent evidence suggests that the polymicrobial community of the CF lung may also harbour oral streptococci, and colonization by these micro-organisms may have a negative impact on P. aeruginosa within the CF lung. Our previous studies demonstrated that nitrite abundance plays an important role in P. aeruginosa survival during co-infection with oral streptococci. Nitrite reductase is a key enzyme involved in nitrite metabolism. Therefore, the objective of this study was to examine the role nitrite reductase (gene nirS) plays in P. aeruginosa survival during co-infection with an oral streptococcus, Streptococcus parasanguinis. Inactivation of nirS in both the chronic CF isolate FRD1 and acute wound isolate PAO1 reduced the survival rate of P. aeruginosa when co-cultured with S. parasanguinis. Growth of both mutants was restored when co-cultured with S. parasanguinis that was defective for H2O2 production. Furthermore, the nitrite reductase mutant was unable to kill Drosophila melanogaster during co-infection with S. parasanguinis. Taken together, these results suggest that nitrite reductase plays an important role for survival of P. aeruginosa during co-infection with S. parasanguinis.

  6. Inhibition of xanthine oxidase by the aldehyde oxidase inhibitor raloxifene: implications for identifying molybdopterin nitrite reductases.

    PubMed

    Weidert, E R; Schoenborn, S O; Cantu-Medellin, N; Choughule, K V; Jones, J P; Kelley, E E

    2014-02-15

    when choosing inhibition strategies as well as inhibitor concentrations when assigning relative NO2- reductase activity of AO and XOR.

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

    PubMed

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

    2017-04-04

    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+Na2S2O4+EDTA and MV+Na2S2O4, respectively. Kinetic parameters of Km and Vmax 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.

  8. Redox-coupled structural changes in nitrite reductase revealed by serial femtosecond and microfocus crystallography

    PubMed Central

    Fukuda, Yohta; Suzuki, Mamoru; Matsumura, Hiroyoshi; Inoue, Tsuyoshi; Iwata, So; Mizohata, Eiichi

    2016-01-01

    Serial femtosecond crystallography (SFX) has enabled the damage-free structural determination of metalloenzymes and filled the gaps of our knowledge between crystallographic and spectroscopic data. Crystallographers, however, scarcely know whether the rising technique provides truly new structural insights into mechanisms of metalloenzymes partly because of limited resolutions. Copper nitrite reductase (CuNiR), which converts nitrite to nitric oxide in denitrification, has been extensively studied by synchrotron radiation crystallography (SRX). Although catalytic Cu (Type 2 copper (T2Cu)) of CuNiR had been suspected to tolerate X-ray photoreduction, we here showed that T2Cu in the form free of nitrite is reduced and changes its coordination structure in SRX. Moreover, we determined the completely oxidized CuNiR structure at 1.43 Å resolution with SFX. Comparison between the high-resolution SFX and SRX data revealed the subtle structural change of a catalytic His residue by X-ray photoreduction. This finding, which SRX has failed to uncover, provides new insight into the reaction mechanism of CuNiR. PMID:26769972

  9. Isolation and characterization of a nitrite reductase gene and its use as a probe for denitrifying bacteria.

    PubMed Central

    Smith, G B; Tiedje, J M

    1992-01-01

    The dissimilatory nitrite reductase gene (nir) from denitrifying bacterium Pseudomonas stutzeri JM300 was isolated and sequenced. In agreement with recent sequence information from another strain of P. stutzeri (strain ZoBell), strain JM300 nir is the first gene in an operon and is followed immediately by a gene which codes for a tetraheme protein; 2.5 kb downstream from the nitrite reductase carboxyl terminus is the cytochrome c551 gene. P. stutzeri JM300 nir is 67% homologous to P. aeruginosa nir and 88% homologous to P. stutzeri ZoBell nir. Within the nitrite reductase promoter region is an fnr-like operator very similar to an operator upstream of a separate anaerobic pathway, that for arginine catabolism in P. aeruginosa. The denitrification genes in P. stutzeri thus may be under the same regulatory control as that found for other anaerobic pathways of pseudomonads. We have generated gene probes from restriction fragments within the nitrite reductase operon to evaluate their usefulness in ecology studies of denitrification. Probes generated from the carboxyl terminus region hybridized to denitrifying bacteria from five separate genera and did not cross-hybridize to any nondenitrifying bacteria among six genera tested. The denitrifier probes were successful in detecting denitrifying bacteria from samples such as a bioreactor consortium, aquifer microcosms, and denitrifying toluene-degrading enrichments. The probes also were used to reveal restriction fragment length polymorphism patterns indicating the diversity of denitrifiers present in these mixed communities. Images PMID:1539983

  10. Laue crystal structure of Shewanella oneidensis cytochrome c nitrite reductase from a high-yield expression system

    SciTech Connect

    Youngblut, Matthew; Judd, Evan T.; Srajer, Vukica; Sayyed, Bilal; Goelzer, Tyler; Elliott, Sean J.; Schmidt, Marius; Pacheco, A. Andrew

    2012-09-11

    The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) and its characterization by a variety of methods, notably Laue crystallography, are reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein 'small tetraheme c' replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated approximately 20 mg crude ccNiR per liter of culture, compared with 0.5-1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for Escherichia coli ccNiR, and is stable for over 2 weeks in pH 7 solution at 4 C. UV/vis spectropotentiometric titrations and protein film voltammetry identified five independent one-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the five reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed among the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good-quality crystals, with which the 2.59-{angstrom}-resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein).

  11. Laue Crystal Structure of Shewanella oneidensis Cytochrome c Nitrite Reductase from a High-yield Expression System

    PubMed Central

    Youngblut, Matthew; Judd, Evan T.; Srajer, Vukica; Sayyed, Bilal; Goelzer, Tyler; Elliott, Sean J.; Schmidt, Marius; Pacheco, A. Andrew

    2012-01-01

    The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), and its characterization by a variety of methods, notably Laue crystallography, is reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein “Small Tetra-heme c” replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated ~20 mg crude ccNiR/L culture, compared with 0.5–1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for E. coli ccNiR, and is stable for over two weeks in pH 7 solution at 4° C. UV/Vis spectropotentiometric titrations and protein film voltammetry identified 5 independent 1-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the 5 reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed amongst the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good quality crystals, with which the 2.59 Å resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein). PMID:22382353

  12. Nitrosylation of c heme in cd(1)-nitrite reductase is enhanced during catalysis.

    PubMed

    Rinaldo, Serena; Giardina, Giorgio; Cutruzzolà, Francesca

    2014-08-29

    The reduction of nitrite into nitric oxide (NO) in denitrifying bacteria is catalyzed by nitrite reductase. In several species, this enzyme is a heme-containing protein with one c heme and one d1 heme per monomer (cd1NiR), encoded by the nirS gene. For many years, the evidence of a link between NO and this hemeprotein represented a paradox, given that NO was known to tightly bind and, possibly, inhibit hemeproteins, including cd1NiRs. It is now established that, during catalysis, cd1NiRs diverge from "canonical" hemeproteins, since the product NO rapidly dissociates from the ferrous d1 heme, which, in turn, displays a peculiar "low" affinity for NO (KD=0.11 μM at pH 7.0). It has been also previously shown that the c heme reacts with NO at acidic pH but c heme nitrosylation was not extensively investigated, given that in cd1NiR it was considered a side reaction, rather than a genuine process controlling catalysis. The spectroscopic study of the reaction of cd1NiR and its semi-apo derivative (containing the sole c heme) with NO reported here shows that c heme nitrosylation is enhanced during catalysis; this evidence has been discussed in order to assess the potential of c heme nitrosylation as a regulatory process, as observed for cytochrome c nitrosylation in mammalian mitochondria.

  13. Electron transfer and docking between cytochrome cd1 nitrite reductase and different redox partners - A comparative study.

    PubMed

    Pedroso, Humberto A; Silveira, Célia M; Almeida, Rui M; Almeida, Ana; Besson, Stéphane; Moura, Isabel; Moura, José J G; Almeida, M Gabriela

    2016-09-01

    Cytochrome cd1 nitrite reductases (cd1NiRs) catalyze the reduction of nitrite to nitric oxide in denitrifying bacteria, such as Marinobacter hydrocarbonoclasticus. Previous work demonstrated that the enzymatic activity depends on a structural pre-activation triggered by the entry of electrons through the electron transfer (ET) domain, which houses a heme c center. The catalytic activity of M. hydrocarbonoclasticus cd1NiR (Mhcd1NiR) was tested by mediated electrochemistry, using small ET proteins and chemical redox mediators. The rate of enzymatic reaction depends on the nature of the redox partner, with cytochrome (cyt) c552 providing the highest value. In situations where cyt c552 is replaced by either a biological (cyt c from horse heart) or a chemical mediator the catalytic response was only observed at very low scan rates, suggesting that the intermolecular ET rate is much slower. Molecular docking simulations with the 3D model structure of Mhcd1NiR and cyt c552 or cyt c showed that hydrophobic interactions favor the formation of complexes where the heme c domain of the enzyme is the principal docking site. However, only in the case of cyt c552 the preferential areas of contact and Fe-Fe distances between heme c groups of the redox partners allow establishing competent ET pathways. The coupling of the enzyme with chemical redox mediators was also found not to be energetically favorable. These results indicate that although low activity functional complexes can be formed between Mhcd1NiR and different types of redox mediators, efficient ET is only observed with the putative physiological electron donor cyt c552.

  14. Light-regulated expression of the nitrate-reductase and nitrite-reductase genes in tomato and in the phytochrome-deficient aurea mutant of tomato.

    PubMed

    Becker, T W; Foyer, C; Caboche, M

    1992-08-01

    The phytochrome-deficient aurea mutant of tomato (Lycopersicon esculentum (L.) Mill) was used to investigate if phytochrome plays a role in the regulation of nitrate-reductase (NR, EC 1.6.6.1) and nitrite-reductase (NiR, EC 1.7.7.1) gene expression. We show that the expression of the tomato NR and NiR genes is stimulated by light and that this light response is mediated by the photoreceptor phytochrome. The red-light response of the NR and NiR genes was reduced in etiolated aurea seedlings when compared to isogenic wild-type cotyledons. The relative levels of NR mRNA and NiR transcripts and their diurnal fluctuations were identical in mature white-light-grown leaves of the wild-type and of the aurea mutant. The transcript levels for cab and RbcS (genes for the chlorophyll-a/b-binding protein of PSII and the small subunit of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively) in aurea leaves grown in white light were indistinguishable from the respective transcript levels in the leaves of the wildtype grown under the same conditions. Despite a severe reduction in the chlorophyll content, the rate of net CO2 uptake by leaves of the aurea mutant was only slightly reduced when compared to the rate of net photosynthesis of wild-type leaves. This difference in the photosynthetic performances of wild-type and aurea mutant plants disappeared during aging of the plants. The increase in zeaxanthin and the concomitant decrease in violaxanthin in leaves of the aurea mutant compared with the same pigment levels in leaves of the wild-type indicate that the activity of the xanthophyll cycle is increased in aurea leaves as a consequence of the reduced CO2-fixation capacity of the mutant leaves.

  15. Pseudomonas stutzeri Nitrite Reductase Gene Abundance in Environmental Samples Measured by Real-Time PCR

    PubMed Central

    Grüntzig, Verónica; Nold, Stephen C.; Zhou, Jizhong; Tiedje, James M.

    2001-01-01

    We used real-time PCR to quantify the denitrifying nitrite reductase gene (nirS), a functional gene of biogeochemical significance. The assay was tested in vitro and applied to environmental samples. The primer-probe set selected was specific for nirS sequences that corresponded approximately to the Pseudomonas stutzeri species. The assay was linear from 1 to 106 gene copies (r2 = 0.999). Variability at low gene concentrations did not allow detection of twofold differences in gene copy number at less than 100 copies. DNA spiking and cell-addition experiments gave predicted results, suggesting that this assay provides an accurate measure of P. stutzeri nirS abundance in environmental samples. Although P. stutzeri abundance was high in lake sediment and groundwater samples, we detected low or no abundance of this species in marine sediment samples from Puget Sound (Wash.) and from the Washington ocean margin. These results suggest that P. stutzeri may not be a dominant marine denitrifier. PMID:11157241

  16. Binding of NO and CO to the d(1) Heme of cd(1) nitrite reductase from Pseudomonas aeruginosa.

    PubMed

    Das, T K; Wilson, E K; Cutruzzolà, F; Brunori, M; Rousseau, D L

    2001-09-11

    The cd(1) nitrite reductase, a key enzyme in bacterial denitrification, catalyzes the one-electron reduction of nitrite to nitric oxide. The enzyme contains two redox centers, a c-type heme and a unique d(1) heme, which is a dioxoisobacteriochlorin. Nitric oxide, generated by this enzymatic pathway, if not removed from the medium, can bind to the ferrous d(1) cofactor with extremely high affinity and inhibit enzyme activity. In this paper, we report the resonance Raman investigation of the properties of nitric oxide and carbon monoxide binding to the d(1) site of the reduced enzyme. The Fe-ligand (Fe-NO and Fe-CO) stretching vibrational frequencies are unusually high in comparison to those of other ferrous heme complexes. The frequencies of the Fe-NO and N-O stretching modes appear at 585 and 1626 cm(-1), respectively, in the NO complex, while the frequencies of the Fe-CO and C-O stretching modes are at 563 and 1972 cm(-1), respectively, for the CO complex. Also, the widths (fwhm) of the Fe-CO and C-O stretching modes are smaller than those observed in the corresponding complexes of other heme proteins. The unusual spectroscopic characteristics of the d(1) cofactor are discussed in terms of both its unique electronic properties and the strongly polar distal environment around the iron-bound ligand. It is likely that the influence of a highly ruffled structure of heme d(1) on its electronic properties is the major factor causing anomalous Fe-ligand vibrational frequencies.

  17. Nitrite

    Integrated Risk Information System (IRIS)

    Nitrite ; CASRN 14797 - 65 - 0 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Effects

  18. Nitrate, nitrite and nitric oxide reductases: from the last universal common ancestor to modern bacterial pathogens.

    PubMed

    Vázquez-Torres, Andrés; Bäumler, Andreas J

    2016-02-01

    The electrochemical gradient that ensues from the enzymatic activity of cytochromes such as nitrate reductase, nitric oxide reductase, and quinol oxidase contributes to the bioenergetics of the bacterial cell. Reduction of nitrogen oxides by bacterial pathogens can, however, be uncoupled from proton translocation and biosynthesis of ATP or NH4(+), but still linked to quinol and NADH oxidation. Ancestral nitric oxide reductases, as well as cytochrome c oxidases and quinol bo oxidases evolved from the former, are capable of binding and detoxifying nitric oxide to nitrous oxide. The NO-metabolizing activity associated with these cytochromes can be a sizable source of antinitrosative defense in bacteria during their associations with host cells. Nitrosylation of terminal cytochromes arrests respiration, reprograms bacterial metabolism, stimulates antioxidant defenses and alters antibiotic cytotoxicity. Collectively, the bioenergetics and regulation of redox homeostasis that accompanies the utilization of nitrogen oxides and detoxification of nitric oxide by cytochromes of the electron transport chain increases fitness of many Gram-positive and -negative pathogens during their associations with invertebrate and vertebrate hosts.

  19. Diversity and abundance of nitrate reductase genes (narG and napA), nitrite reductase genes (nirS and nrfA), and their transcripts in estuarine sediments.

    PubMed

    Smith, Cindy J; Nedwell, David B; Dong, Liang F; Osborn, A Mark

    2007-06-01

    Estuarine systems are the major conduits for the transfer of nitrate from agricultural and other terrestrial-anthropogenic sources into marine ecosystems. Within estuarine sediments some microbially driven processes (denitrification and anammox) result in the net removal of nitrogen from the environment, while others (dissimilatory nitrate reduction to ammonium) do not. In this study, molecular approaches have been used to investigate the diversity, abundance, and activity of the nitrate-reducing communities in sediments from the hypernutrified Colne estuary, United Kingdom, via analysis of nitrate and nitrite reductase genes and transcripts. Sequence analysis of cloned PCR-amplified narG, napA, and nrfA gene sequences showed the indigenous nitrate-reducing communities to be both phylogenetically diverse and also divergent from previously characterized nitrate reduction sequences in soils and offshore marine sediments and from cultured nitrate reducers. In both the narG and nrfA libraries, the majority of clones (48% and 50%, respectively) were related to corresponding sequences from delta-proteobacteria. A suite of quantitative PCR primers and TaqMan probes was then developed to quantify phylotype-specific nitrate (narG and napA) and nitrite reductase (nirS and nrfA) gene and transcript numbers in sediments from three sites along the estuarine nitrate gradient. In general, both nitrate and nitrite reductase gene copy numbers were found to decline significantly (P < 0.05) from the estuary head towards the estuary mouth. The development and application, for the first time, of quantitative reverse transcription-PCR assays to quantify mRNA sequences in sediments revealed that transcript numbers for three of the five phylotypes quantified were greatest at the estuary head.

  20. Molecular interactions between multihaem cytochromes: probing the protein-protein interactions between pentahaem cytochromes of a nitrite reductase complex.

    PubMed

    Lockwood, Colin; Butt, Julea N; Clarke, Thomas A; Richardson, David J

    2011-01-01

    The cytochrome c nitrite reductase NrfA is a 53 kDa pentahaem enzyme that crystallizes as a decahaem homodimer. NrfA catalyses the reduction of NO2- to NH4+ through a six electron reduction pathway that is of major physiological significance to the anaerobic metabolism of enteric and sulfate reducing bacteria. NrfA receives electrons from the 21 kDa pentahaem NrfB donor protein. This requires that redox complexes form between the NrfA and NrfB pentahaem cytochromes. The formation of these complexes can be monitored using a range of methodologies for studying protein-protein interactions, including dynamic light scattering, gel filtration, analytical ultracentrifugation and visible spectroscopy. These methods have been used to show that oxidized NrfA exists in dynamic monomer-dimer equilibrium with a Kd (dissociation constant) of 4 μM. Significantly, the monomeric and dimeric forms of NrfA are equally active for either the six electron reduction of NO2- or HSO3-. When mixed together, NrfA and NrfB exist in equilibrium with NrfAB, which is described by a Kd of 50 nM. Thus, since NrfA and NrfB are present in micromolar concentrations in the periplasmic compartment, it is likely that NrfB remains tightly associated with its NrfA redox partner under physiological conditions.

  1. An electron transport system in maize roots for reactions of glutamate synthase and nitrite reductase : physiological and immunochemical properties of the electron carrier and pyridine nucleotide reductase.

    PubMed

    Suzuki, A; Oaks, A; Jacquot, J P; Vidal, J; Gadal, P

    1985-06-01

    A non-heme iron containing protein which bears an antigenic similarity to ferredoxin from spinach leaves (Spinacia oleracea L.) has been identified in extracts prepared from young roots of maize (Zea mays L., hybrid W64A x W182E). The ferredoxin-like root electron carrier could substitute for ferredoxin in a cytochrome c reduction system in which pyridine nucleotide (NADPH) reduces the root electron carrier in a reaction catalyzed by ferredoxin-NADP(+) reductase (EC 1.6.7.1) from spinach leaves. However, the root electron carrier did not mediate the photoreduction of NADP(+) in an illuminated reconstituted chloroplast system.A pyridine nucleotide reductase which shares identical immunological determinants with the ferredoxin-NADP(+) reductase from spinach leaves has also been characterized from maize roots. Root pyridine nucleotide reductase mediated the transfer of electrons from either NADPH or NADH to cytochrome c via ferredoxin or the root electron carrier. Under chemical reducing conditions with sodium dithionite and bicarbonate, the ferredoxin-like root electron carrier served as an electron carrier for the ferredoxin-requiring glutamate synthase (EC 1.4.7.1) and nitrite reductase (EC 1.7.7.1) obtained from maize roots or leaves. In the presence of root pyridine nucleotide reductase and root electron carrier, either NADPH or NADH served as the primary electron donor for glutamate synthesis in extracts from maize roots or leaves. The electron transport system originating with NADH or NADPH, was, however, not able to mediate the reduction of NO(2) (-) to NH(3).

  2. Regulation of Nitrate Reductase Activity in Corn (Zea mays L.) Seedlings by Endogenous Metabolites 1

    PubMed Central

    Schrader, L. E.; Hageman, R. H.

    1967-01-01

    Primary and secondary metabolites of inorganic nitrogen metabolism were evaluated as inhibitors of nitrate reductase (EC 1.6.6.1) induction in green leaf tissue of corn seedlings. Nitrite, nitropropionic acid, ammonium ions, and amino acids were not effective as inhibitors of nitrate reductase activity or synthesis. Increasing α-amino nitrogen and protein content of intact corn seedlings by culture techniques significantly enhanced rather than decreased the potential for induction of nitrate reductase activity in excised seedlings. Secondary metabolites, derived from phenylalanine and tyrosine, were tested as inhibitors of induction of nitrate reductase. Of the 9 different phenylpropanoid compounds tested, only coumarin, trans-cinnamic and trans-o-hydroxycinnamic acids inhibited induction of nitrate reductase. While coumarin alone exhibited a relatively greater inhibitory effect on enzyme induction than on general protein synthesis (the latter measured by incorporation of labeled amino acids), this differential effect may have been dependent upon unequal rates of synthesis and accumulation with respect to the initial levels of nitrate reductase and general proteins. Because of the short half-life of nitrate reductase, inhibitors of protein synthesis in general could still achieve differential regulation of nitrogen metabolism. Coumarin did not inhibit nitrate reductase activity when added directly to the assay mixture at 5 mm. Carbamyl phosphate and its chemical derivative, cyanate, were found to be competitive (with nitrate) inhibitors of nitrate reductase. The data suggest that cyanate is the active inhibitor in the carbamyl phosphate preparations. PMID:16656715

  3. Role of. pi. -cation radicals in the enzymatic cycles of peroxidases, catalases, and nitrite and sulfite reductases

    SciTech Connect

    Hanson, L K; Chang, C K; Davis, M S; Fajer, J

    1980-01-01

    Charge iterative extended Hueckel calculations, and magnetic and optical results on porphyrins, chlorins, and isobacteriochlorins (1) suggest that the catalytic cycles of the enzymes horseradish peroxidase, catalase, Neurospora crassa catalase, and nitrite and sulfite reductases proceed via ..pi..-cation radicals of their prosthetic groups; (2) offer distinguishing features for the optical and magnetic spectra of these radicals, pertinent to their detection as enzymatic intermediates; (3) reconcile the seemingly contradictory optical and NMR data on Compounds I of horseradish peroxidase; and (4) predict that the axial ligation of the heme differs for horseradish peroxidase and catalase.

  4. Copper, zinc superoxide dismutase and nitrate reductase coimmobilized bienzymatic biosensor for the simultaneous determination of nitrite and nitrate.

    PubMed

    Madasamy, Thangamuthu; Pandiaraj, Manickam; Balamurugan, Murugesan; Bhargava, Kalpana; Sethy, Niroj Kumar; Karunakaran, Chandran

    2014-02-15

    This work presents a novel bienzymatic biosensor for the simultaneous determination of nitrite (NO2(-)) and nitrate (NO3(-)) ions using copper, zinc superoxide dismutase (SOD1) and nitrate reductase (NaR) coimmobilized on carbon nanotubes (CNT)-polypyrrole (PPy) nanocomposite modified platinum electrode. Morphological changes of the PPy and CNT modified electrodes were investigated using scanning electron microscopy. The electrochemical behavior of the bienzymatic electrode (NaR-SOD1-CNT-PPy-Pt) was characterized by cyclic voltammetry exhibiting quasi-reversible redox peak at +0.06 V and reversible redox peaks at -0.76 and -0.62V vs. Ag/AgCl, for the immobilized SOD1 and NaR respectively. The electrocatalytic activity of SOD1 towards NO2(-) oxidation observed at +0.8 V was linear from 100 nM to 1mM with a detection limit of 50 nM and sensitivity of 98.5 ± 1.7 nA µM(-1)cm(-2). Similarly, the coimmobilized NaR showed its electrocatalytic activity towards NO3(-) reduction at -0.76 V exhibiting linear response from 500 nM to 10mM NO3(-) with a detection limit of 200 nM and sensitivity of 84.5 ± 1.56 nA µM(-1)cm(-2). Further, the present bienzymatic biosensor coated with cellulose acetate membrane for the removal of non-specific proteins was used for the sensitive and selective determinations of NO2(-) and NO3(-) present in human plasma, whole blood and saliva samples.

  5. Purification, Characterization, and Genetic Analysis of Cu-Containing Dissimilatory Nitrite Reductase from a Denitrifying Halophilic Archaeon, Haloarcula marismortui

    PubMed Central

    Ichiki, Hirotaka; Tanaka, Yoko; Mochizuki, Kiyotaka; Yoshimatsu, Katsuhiko; Sakurai, Takeshi; Fujiwara, Taketomo

    2001-01-01

    Cu-containing dissimilatory nitrite reductase (CuNiR) was purified from denitrifying cells of a halophilic archaeon, Haloarcula marismortui. The purified CuNiR appeared blue in the oxidized state, possessing absorption peaks at 600 and 465 nm in the visible region. Electron paramagnetic resonance spectroscopy suggested the presence of type 1 Cu (gII = 2.232; AII = 4.4 mT) and type 2 Cu centers (gII = 2.304; AII = 13.3 mT) in the enzyme. The enzyme contained two subunits, whose apparent molecular masses were 46 and 42 kDa, according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis indicated that the two subunits were identical, except that the 46-kDa subunit was 16 amino acid residues longer than the 42-kDa subunit in the N-terminal region. A nirK gene encoding the CuNiR was cloned and sequenced, and the deduced amino acid sequence with a residual length of 361 amino acids was homologous (30 to 41%) with bacterial counterparts. Cu-liganding residues His-133, Cys-174, His-182, and Met-187 (for type 1 Cu) and His-138, His-173, and His-332 (for type 2 Cu) were conserved in the enzyme. As generally observed in the halobacterial enzymes, the enzymatic activity of the purified CuNiR was enhanced during increasing salt concentration and reached its maximum in the presence of 2 M NaCl with the value of 960 μM NO2− · min−1 · mg−1. PMID:11418554

  6. Crystallization and preliminary structure determination of the membrane-bound complex cytochrome c nitrite reductase from Desulfovibrio vulgaris Hildenborough

    SciTech Connect

    Rodrigues, M. L.; Oliveira, T.; Matias, P. M.; Martins, I. C.; Valente, F. M. A.; Pereira, I. A. C.; Archer, M.

    2006-06-01

    The cytochrome c nitrite reductase complex from D. vulgaris Hildenborough has been crystallized. The preliminary crystallographic structure reveals a 2:1 NrfA:NrfH complex stoichiometry. The cytochrome c nitrite reductase (cNiR) isolated from Desulfovibrio vulgaris Hildenborough is a membrane-bound complex formed of NrfA and NrfH subunits. The catalytic subunit NrfA is a soluble pentahaem cytochrome c that forms a physiological dimer of about 120 kDa. The electron-donor subunit NrfH is a membrane-anchored tetrahaem cytochrome c of about 18 kDa molecular weight and belongs to the NapC/NirT family of quinol dehydrogenases, for which no structures are known. Crystals of the native cNiR membrane complex, solubilized with dodecylmaltoside detergent (DDM), were obtained using PEG 4K as precipitant. Anomalous diffraction data were measured at the Swiss Light Source to 2.3 Å resolution. Crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 79.5, b = 256.7, c = 578.2 Å. Molecular-replacement and MAD methods were combined to solve the structure. The data presented reveal that D. vulgaris cNiR contains one NrfH subunit per NrfA dimer.

  7. Frequencies, Timing, and Spatial Patterns of Co-Suppression of Nitrate Reductase and Nitrite Reductase in Transgenic Tobacco Plants.

    PubMed Central

    Palauqui, J. C.; Elmayan, T.; De Borne, F. D.; Crete, P.; Charles, C.; Vaucheret, H.

    1996-01-01

    Frequencies, timing, and spatial patterns of co-suppression of the nitrate (Nia) and nitrite (Nii) genes were analyzed in transgenic tobacco (Nicotiana tabacum) plants carrying either Nia or Nii cDNAs under the control of the 35S promoter, or a Nii gene with its own regulatory signals (promoter, introns, and terminator) cloned downstream of two copies of the enhancer of the 35S promoter. We show that (a) the frequencies of transgenic lines affected by co- suppression are similar for the three constructs, ranging from 19 to 25%; (b) Nia and Nii co-suppression are triggered stochastically during a phenocritical period of 2 weeks between germination and flowering; (c) the timing of co-suppression (i.e. the percentage of isogenic plants affected by co-suppression reported as a function of the number of days of culture) differs from one transgenic line to another; (d) the percentage of isogenic plants affected by co-suppression is increased by growing the plants in vitro prior to their transfer to the greenhouse and to the field; and (e) at the end of the culture period, plants are either unaffected, completely co-suppressed, or variegated. Suppressed and nonsuppressed parts of these variegated plants are separated by a vertical plane through the stem in Nia co-suppression, and separated by a horizontal plane in Nii co-suppression. PMID:12226457

  8. Fumarate Reductase Activity of Streptococcus faecalis

    PubMed Central

    Aue, B. J.; Diebel, R. H.

    1967-01-01

    Some characteristics of a fumarate reductase from Streptococcus faecalis are described. The enzyme had a pH optimum of 7.4; optimal activity was observed when the ionic strength of the phosphate buffer was adjusted to 0.088. The Km value of the enzyme for reduced flavin mononucleotide was 2 × 10−4 m as determined with a 26-fold preparation. In addition to fumarate, the enzyme reduced maleate and mesaconate. No succinate dehydrogenase activity was detected, but succinate did act as an inhibitor of the fumarate reductase activity. Other inhibitors were malonate, citraconate, and trans-, trans-muconate. Metal-chelating agents did not inhibit the enzyme. A limited inhibition by sulfhydryl-binding agents was observed, and the preparations were sensitive to air oxidation and storage. Glycine, alanine, histidine, and possibly lysine stimulated fumarate reductase activity in the cell-free extracts. However, growth in media supplemented with glycine did not enhance fumarate reductase activity. The enzymatic activity appears to be constitutive. PMID:4960892

  9. Comparative induction of nitrate reductase by nitrate and nitrite in barley leaves

    NASA Technical Reports Server (NTRS)

    Aslam, M.; Rosichan, J. L.; Huffaker, R. C.

    1987-01-01

    The comparative induction of nitrate reductase (NR) by ambient NO3- and NO2- as a function of influx, reduction (as NR was induced) and accumulation in detached leaves of 8-day-old barley (Hordeum vulgare L.) seedlings was determined. The dynamic interaction of NO3- influx, reduction and accumulation on NR induction was shown. The activity of NR, as it was induced, influenced its further induction by affecting the internal concentration of NO3-. As the ambient concentration of NO3- increased, the relative influences imposed by influx and reduction on NO3- accumulation changed with influx becoming a more predominant regulant. Significant levels of NO3- accumulated in NO2(-)-fed leaves. When the leaves were supplied cycloheximide or tungstate along with NO2-, about 60% more NO3- accumulated in the leaves than in the absence of the inhibitors. In NO3(-)-supplied leaves NR induction was observed at an ambient concentration of as low as 0.02 mM. No NR induction occurred in leaves supplied with NO2- until the ambient NO2- concentration was 0.5 mM. In fact, NR induction from NO2- solutions was not seen until NO3- was detected in the leaves. The amount of NO3- accumulating in NO2(-)-fed leaves induced similar levels of NR as did equivalent amounts of NO3- accumulating from NO3(-)-fed leaves. In all cases the internal concentration of NO3-, but not NO2-, was highly correlated with the amount of NR induced. The evidence indicated that NO3- was a more likely inducer of NR than was NO2-.

  10. Diversity, abundance, and distribution of NO-forming nitrite reductase-encoding genes in deep-sea subsurface sediments of the South China Sea.

    PubMed

    Li, M; Hong, Y; Cao, H; Klotz, M G; Gu, J-D

    2013-03-01

    In marine ecosystems, both nitrite-reducing bacteria and anaerobic ammonium-oxidizing (anammox) bacteria, containing different types of NO-forming nitrite reductase-encoding genes, contribute to the nitrogen cycle. The objectives of study were to reveal the diversity, abundance, and distribution of NO-forming nitrite reductase-encoding genes in deep-sea subsurface environments. Results showed that higher diversity and abundance of nirS gene than nirK and Scalindua-nirS genes were evident in the sediments of the South China Sea (SCS), indicating bacteria containing nirS gene dominated the NO-forming nitrite-reducing microbial community in this ecosystem. Similar diversity and abundance distribution patterns of both nirS and Scalindua-nirS genes were detected in this study sites, but different from nirK gene. Further statistical analyses also showed both nirS and Scalindua-nirS genes respond similarly to environmental factors, but differed from nirK gene. These results suggest that bacteria containing nirS and Scalindua-nirS genes share similar niche in deep-sea subsurface sediments of the SCS, but differed from those containing nirK gene, indicating that community structures of nitrite-reducing bacteria are segregated by the functional modules (NirS vs. NirK) rather than the competing processes (anammox vs. classical denitrification).

  11. Physiology and interaction of nitrate and nitrite reduction in Staphylococcus carnosus.

    PubMed Central

    Neubauer, H; Götz, F

    1996-01-01

    Staphylococcus carnosus reduces nitrate to ammonia in two steps. (i) Nitrate was taken up and reduced to nitrite, and nitrite was subsequently excreted. (ii) After depletion of nitrate, the accumulated nitrite was imported and reduced to ammonia, which again accumulated in the medium. The localization, energy gain, and induction of the nitrate and nitrite reductases in S. carnosus were characterized. Nitrate reductase seems to be a membrane-bound enzyme involved in respiratory energy conservation, whereas nitrite reductase seems to be a cytosolic enzyme involved in NADH reoxidation. Syntheses of both enzymes are inhibited by oxygen and induced to greater or lesser degrees by nitrate or nitrite, respectively. In whole cells, nitrite reduction is inhibited by nitrate and also by high concentrations of nitrite (> or = 10 mM). Nitrite did not influence nitrate reduction. Two possible mechanisms for the inhibition of nitrite reduction by nitrate that are not mutually exclusive are discussed. (i) Competition for NADH nitrate reductase is expected to oxidize the bulk of the NADH because of its higher specific activity. (ii) The high rate of nitrate reduction could lead to an internal accumulation of nitrite, possibly the result of a less efficient nitrite reduction or export. So far, we have no evidence for the presence of other dissimilatory or assimilatory nitrate or nitrite reductases in S. carnosus. PMID:8606176

  12. Physiology and interaction of nitrate and nitrite reduction in Staphylococcus carnosus.

    PubMed

    Neubauer, H; Götz, F

    1996-04-01

    Staphylococcus carnosus reduces nitrate to ammonia in two steps. (i) Nitrate was taken up and reduced to nitrite, and nitrite was subsequently excreted. (ii) After depletion of nitrate, the accumulated nitrite was imported and reduced to ammonia, which again accumulated in the medium. The localization, energy gain, and induction of the nitrate and nitrite reductases in S. carnosus were characterized. Nitrate reductase seems to be a membrane-bound enzyme involved in respiratory energy conservation, whereas nitrite reductase seems to be a cytosolic enzyme involved in NADH reoxidation. Syntheses of both enzymes are inhibited by oxygen and induced to greater or lesser degrees by nitrate or nitrite, respectively. In whole cells, nitrite reduction is inhibited by nitrate and also by high concentrations of nitrite (> or = 10 mM). Nitrite did not influence nitrate reduction. Two possible mechanisms for the inhibition of nitrite reduction by nitrate that are not mutually exclusive are discussed. (i) Competition for NADH nitrate reductase is expected to oxidize the bulk of the NADH because of its higher specific activity. (ii) The high rate of nitrate reduction could lead to an internal accumulation of nitrite, possibly the result of a less efficient nitrite reduction or export. So far, we have no evidence for the presence of other dissimilatory or assimilatory nitrate or nitrite reductases in S. carnosus.

  13. Effect of self-alkalization on nitrite accumulation in a high-rate denitrification system: Performance, microflora and enzymatic activities.

    PubMed

    Li, Wei; Shan, Xiao-Yu; Wang, Zhi-Yao; Lin, Xiao-Yu; Li, Chen-Xu; Cai, Chao-Yang; Abbas, Ghulam; Zhang, Meng; Shen, Li-Dong; Hu, Zhi-Qiang; Zhao, He-Ping; Zheng, Ping

    2016-01-01

    The self-alkalization of denitrifying automatic circulation (DAC) reactor resulted in a large increase of pH up to 9.20 and caused a tremendous accumulation of nitrite up to 451.1 ± 49.0 mgN L(-1) at nitrate loading rate (NLR) from 35 kgN m(-3) d(-1) to 55 kgN m(-3) d(-1). The nitrite accumulation was greatly relieved even at the same NLR once the pH was maintained at 7.6 ± 0.2 in the system. Enzymatic assays indicated that the long-term bacterial exposure to high pH significantly inhibited the activity of copper type nitrite reductase (NirK) rather than the cytochrome cd1 type nitrite reductase (NirS). The terminal restriction fragment length polymorphism (T-RFLP) analysis revealed that the dominant denitrifying bacteria shifted from the NirS-containing Thauear sp. 27 to the NirK-containing Hyphomicrobium nitrativorans strain NL23 during the self-alkalization. The significant nitrite accumulation in the high-rate denitrification system could be therefore, due to the inhibition of Cu-containing NirK by high pH from the self-alkalization. The results suggest that the NirK-containing H. nitrativorans strain NL23 could be an ideal functional bacterium for the conversion of nitrate to nitrite, i.e. denitritation, which could be combined with anaerobic ammonium oxidation (Anammox) to develop a new process for nitrogen removal from wastewater.

  14. Design and evaluation of primers targeting genes encoding NO-forming nitrite reductases: implications for ecological inference of denitrifying communities

    PubMed Central

    Bonilla-Rosso, Germán; Wittorf, Lea; Jones, Christopher M.; Hallin, Sara

    2016-01-01

    The detection of NO-forming nitrite reductase genes (nir) has become the standard when studying denitrifying communities in the environment, despite well-known amplification biases in available primers. We review the performance of 35 published and 121 newly designed primers targeting the nirS and nirK genes, against sequences from complete genomes and 47 metagenomes from three major habitats where denitrification is important. There were no optimal universal primer pairs for either gene, although published primers targeting nirS displayed up to 75% coverage. The alternative is clade-specific primers, which show a trade-off between coverage and specificity. The test against metagenomic datasets showed a distinct performance of primers across habitats. The implications of clade-specific nir primers choice and their performance for ecological inference when used for quantitative estimates and in sequenced-based community ecology studies are discussed and our phylogenomic primer evaluation can be used as a reference along with their environmental specificity as a guide for primer selection. Based on our results, we also propose a general framework for primer evaluation that emphasizes the testing of coverage and phylogenetic range using full-length sequences from complete genomes, as well as accounting for environmental range using metagenomes. This framework serves as a guideline to simplify primer performance comparisons while explicitly addressing the limitations and biases of the primers evaluated. PMID:27966627

  15. Novel genes for nitrite reductase and Amo-related proteins indicate a role of uncultivated mesophilic crenarchaeota in nitrogen cycling.

    PubMed

    Treusch, Alexander H; Leininger, Sven; Kletzin, Arnulf; Schuster, Stephan C; Klenk, Hans-Peter; Schleper, Christa

    2005-12-01

    Mesophilic crenarchaeota are frequently found in terrestrial and marine habitats worldwide, but despite their considerable abundance the physiology of these as yet uncultivated archaea has remained unknown. From a 1.2 Gb large-insert environmental fosmid library of a calcareous grassland soil, a 43 kb genomic fragment was isolated with a ribosomal RNA that shows its affiliation to group 1.1b of crenarchaeota repeatedly found in soils. The insert encoded a homologue of a copper-containing nitrite reductase with an unusual C-terminus that encoded a potential amicyanin-like electron transfer domain as well as two proteins related to subunits of ammonia monooxygenases or particulate methane monooxygenases (AmoAB/PmoAB) respectively. Expression of nirK and the amoA-like gene was shown by reverse transcription polymerase chain reaction (PCR) analyses in soil samples, the latter being found at higher levels when the soil was incubated with ammonia (measured by quantitative PCR). Further variants of both genes were amplified from soil samples and were found in the environmental database from the Sargasso Sea plankton. Taken together, our findings suggest that mesophilic terrestrial and marine crenarchaeota might be capable of ammonia oxidation under aerobic and potentially also under anaerobic conditions.

  16. Sequence of a cDNA encoding nitrite reductase from the tree Betula pendula and identification of conserved protein regions.

    PubMed

    Friemann, A; Brinkmann, K; Hachtel, W

    1992-02-01

    The sequence of an mRNA encoding nitrite reductase (NiR, EC 1.7.7.1.) from the tree Betula pendula was determined. A cDNA library constructed from leaf poly(A)+ mRNA was screened with an oligonucleotide probe deduced from NiR sequences from spinach and maize. A 2.5 kb cDNA was isolated that hybridized to an mRNA, the steady-state level of which increased markedly upon induction with nitrate. The nucleotide sequence of the cDNA contains a reading frame encoding a protein of 583 amino acids that reveals 79% identity with NiR from spinach. The transit peptide of the NiR precursor from birch was determined to be 22 amino acids in size by sequence comparison with NiR from spinach and maize and is the shortest transit peptide reported so far. A graphical evaluation of identities found in the NiR sequence alignment revealed nine well conserved sections each exceeding ten amino acids in size. Sequence comparisons with related redox proteins identified essential residues involved in cofactor binding. A putative binding site for ferredoxin was found in the N-terminal half of the protein.

  17. The effect of nitrite on aerobic phosphate uptake and denitrifying activity of phosphate-accumulating organisms.

    PubMed

    Yoshida, Y; Takahashi, K; Saito, T; Tanaka, K

    2006-01-01

    An anaerobic/aerobic/anoxic/aerobic sequencing batch reactor (SBR) was operated with municipal wastewater to investigate the effect of nitrite on biological phosphorus removal (BPR). When nitrite accumulated, aerobic phosphate uptake activity significantly decreased and, in case of hard exposure to nitrite, BPR severely deteriorated. The interesting observation was that the relative anoxic activity of phosphate accumulating organisms (PAOs) increased after nitrite exposure. Moreover batch tests of aerobic phosphate uptake in the presence/absence of nitrite indicated that PAOs with the higher relative anoxic activity are less sensitive to nitrite exposure. From these results, we concluded that BPR is sensitive to nitrite exposure, but BPR containing PAOs with the higher relative anoxic activity is possibly more stable against nitrite than BPR containing PAOs with the lower relative anoxic activity.

  18. HY5 regulates Nitrite Reductase 1 (NIR1) and Ammonium Transporter1;2 (AMT1;2) in Arabidopsis seedlings

    PubMed Central

    Huang, Lifen; Zhang, Hongcheng; Zhang, Huiyong; Deng, Xing Wang; Wei, Ning

    2016-01-01

    HY5 (Long Hypocotyles 5) is a key transcription factor in Arabidopsis thaliana that has a pivotal role in seedling development. Soil nitrogen is an essential macronutrient, and its uptake, assimilation and metabolism are influenced by nutrient availability and by lights. To understand the role of HY5 in nitrogen assimilation pathways, we examined the phenotype as well as the expression of selected nitrogen assimilation-related genes in hy5 mutant grown under various nitrogen limiting and nitrogen sufficient conditions, or different light conditions. We report that HY5 positively regulates nitrite reductase gene NIR1 and negatively regulates the ammonium transporter gene AMT1;2 under all nitrogen and light conditions tested, while it affects several other genes in a nitrogen supply-dependent manner. HY5 is not required for light induction of NIR1, AMT1;2 and NIA genes, but it is necessary for high level expression of NIR1 and NIA under optimal nutrient and light conditions. In addition, nitrogen deficiency exacerbates the abnormal root system of hy5. Together, our results suggest that HY5 exhibits the growth-promoting activity only when sufficient nutrients, including lights, are provided, and that HY5 has a complex involvement in nitrogen acquisition and metabolism in Arabidopsis seedlings. PMID:26259199

  19. Vertical distribution of nitrite reductase genes (nir S) in continental margin sediments of the Gulf of Mexico.

    PubMed

    Tiquia, Sonia M; Masson, Steven A; Devol, Allan

    2006-12-01

    Marine sediments account for up to 66% of the loss of nitrogen load to coastal areas. Sedimentary denitrification is the main sink for fixed nitrogen in the global nitrogen budget, and thus it is important to understand the structure and composition of denitrifying communities. To understand the structure and composition of denitrifying communities, the diversity of nitrite reductase (nirS) genes from sediments along the Gulf of Mexico was examined using a PCR-based cloning approach. Sediments were collected at three different depths (0-0.5, 4-5 and 19-21 cm). Geochemical analysis revealed decreasing nitrate and oxygen concentrations with increasing sediment depth. This trend coincided with the decrease in diversity of denitrifying bacteria. LIBSHUFF analysis indicated that the clone library in the shallowest sediment (depth, 0-0.5 cm) was significantly different from that in the deepest sediment (depth, 19-21 cm), and that the deeper sediments (depths of 4-5 and 19-21 cm) were significantly similar. Community structural shifts were evident between the shallowest (oxic zone) and deepest (anoxic zone) sediments. Community changes within the deepest sediments were more subtle, with the presence of different nirS clone sequences gradually becoming dominant or, alternatively, decreasing with depth. The changes in community structure at this depth are possibly driven by nutrient availability, with lower quality sources of carbon and energy leading to the disappearance of nirS sequences common in the top layer. The majority of recovered nirS sequences were phylogenetically divergent relative to known denitrifying bacteria in the database.

  20. Characterisation and expression analysis of a nitrate transporter and nitrite reductase genes, two members of a gene cluster for nitrate assimilation from the symbiotic basidiomycete Hebeloma cylindrosporum.

    PubMed

    Jargeat, Patricia; Rekangalt, David; Verner, Marie-Christine; Gay, Gilles; Debaud, Jean-Claude; Marmeisse, Roland; Fraissinet-Tachet, Laurence

    2003-06-01

    Symbiotic ectomycorrhizal fungi contribute to the nitrogen nutrition of their host-plants but little information is available on the molecular control of their nitrogen metabolism. We cloned and characterised genes encoding a nitrite reductase and a nitrate transporter in the ectomycorrhizal basidiomycete Hebeloma cylindrosporum. These two genes are divergently transcribed and linked to a previously cloned nitrate reductase gene, thus demonstrating that nitrate assimilation gene clusters occur in homobasidiomycetes. The nitrate transporter polypeptide (NRT2) is characterised by 12 transmembrane domains and presents both a long putative intracellular loop and a short C-terminal tail, two structural features which distinguish fungal high-affinity transporters from their plant homologues. In different wild-type genetic backgrounds, transcription of the two genes was repressed by ammonium and was strongly stimulated not only in the presence of nitrate but also in the presence of organic nitrogen sources or under nitrogen deficiency.

  1. Conserved Active Site Residues Limit Inhibition of a Copper-Containing Nitrite By Small Molecules

    SciTech Connect

    Tocheva, E.I.; Eltis, L.D.; Murphy, M.E.P.

    2009-05-26

    The interaction of copper-containing dissimilatory nitrite reductase from Alcaligenes faecalis S-6 ( AfNiR) with each of five small molecules was studied using crystallography and steady-state kinetics. Structural studies revealed that each small molecule interacted with the oxidized catalytic type 2 copper of AfNiR. Three small molecules (formate, acetate and nitrate) mimic the substrate by having at least two oxygen atoms for bidentate coordination to the type 2 copper atom. These three anions bound to the copper ion in the same asymmetric, bidentate manner as nitrite. Consistent with their weak inhibition of the enzyme ( K i >50 mM), the Cu-O distances in these AfNiR-inhibitor complexes were approximately 0.15 A longer than that observed in the AfNiR-nitrite complex. The binding mode of each inhibitor is determined in part by steric interactions with the side chain of active site residue Ile257. Moreover, the side chain of Asp98, a conserved residue that hydrogen bonds to type 2 copper-bound nitrite and nitric oxide, was either disordered or pointed away from the inhibitors. Acetate and formate inhibited AfNiR in a mixed fashion, consistent with the occurrence of second acetate binding site in the AfNiR-acetate complex that occludes access to the type 2 copper. A fourth small molecule, nitrous oxide, bound to the oxidized metal in a side-on fashion reminiscent of nitric oxide to the reduced copper. Nevertheless, nitrous oxide bound at a farther distance from the metal. The fifth small molecule, azide, inhibited the reduction of nitrite by AfNiR most strongly ( K ic = 2.0 +/- 0.1 mM). This ligand bound to the type 2 copper center end-on with a Cu-N c distance of approximately 2 A, and was the only inhibitor to form a hydrogen bond with Asp98. Overall, the data substantiate the roles of Asp98 and Ile257 in discriminating substrate from other small anions.

  2. Active sites of thioredoxin reductases: why selenoproteins?

    PubMed

    Gromer, Stephan; Johansson, Linda; Bauer, Holger; Arscott, L David; Rauch, Susanne; Ballou, David P; Williams, Charles H; Schirmer, R Heiner; Arnér, Elias S J

    2003-10-28

    Selenium, an essential trace element for mammals, is incorporated into a selected class of selenoproteins as selenocysteine. All known isoenzymes of mammalian thioredoxin (Trx) reductases (TrxRs) employ selenium in the C-terminal redox center -Gly-Cys-Sec-Gly-COOH for reduction of Trx and other substrates, whereas the corresponding sequence in Drosophila melanogaster TrxR is -Ser-Cys-Cys-Ser-COOH. Surprisingly, the catalytic competence of these orthologous enzymes is similar, whereas direct Sec-to-Cys substitution of mammalian TrxR, or other selenoenzymes, yields almost inactive enzyme. TrxRs are therefore ideal for studying the biology of selenocysteine by comparative enzymology. Here we show that the serine residues flanking the C-terminal Cys residues of Drosophila TrxRs are responsible for activating the cysteines to match the catalytic efficiency of a selenocysteine-cysteine pair as in mammalian TrxR, obviating the need for selenium. This finding suggests that the occurrence of selenoenzymes, which implies that the organism is selenium-dependent, is not necessarily associated with improved enzyme efficiency. Our data suggest that the selective advantage of selenoenzymes is a broader range of substrates and a broader range of microenvironmental conditions in which enzyme activity is possible.

  3. Enhanced silver nanoparticle synthesis by optimization of nitrate reductase activity.

    PubMed

    Vaidyanathan, Ramanathan; Gopalram, Shubaash; Kalishwaralal, Kalimuthu; Deepak, Venkataraman; Pandian, Sureshbabu Ram Kumar; Gurunathan, Sangiliyandi

    2010-01-01

    Nanostructure materials are attracting a great deal of attention because of their potential for achieving specific processes and selectivity, especially in biological and pharmaceutical applications. The generation of silver nanoparticles using optimized nitrate reductase for the reduction of Ag(+) with the retention of enzymatic activity in the complex is being reported. This report involves the optimization of enzyme activity to bring about enhanced nanoparticle synthesis. Response surface methodology and central composite rotary design (CCRD) were employed to optimize a fermentation medium for the production of nitrate reductase by Bacillus licheniformis at pH 8. The four variables involved in the study of nitrate reductase were Glucose, Peptone, Yeast extract and KNO(3). Glucose had a significant effect on nitrate reductase production. The optimized medium containing (%) Glucose: 1.5, Peptone: 1, Yeast extract: 0.35 and KNO(3): 0.35 resulted in a nitrate reductase activity of 452.206 U/ml which is same as that of the central level. The medium A (showing least nitrate reductase activity) and the medium B (showing maximum nitrate reductase activity) were compared for the synthesis. Spectrophotometric analysis revealed that the particles exhibited a peak at 431 nm and the A(431) for the medium B was 2-fold greater than that of the medium A. The particles were also characterized using TEM. The particles synthesized using the optimized enzyme activity ranged from 10 to 80 nm and therefore can be extended to various medicinal applications.

  4. Ammonia stimulates growth and nitrite-oxidizing activity of Nitrobacter winogradskyi

    PubMed Central

    Ma, Shouguang; Zhang, Demin; Zhang, Wenjun; Wang, Yinong

    2014-01-01

    The aim of this study was to obtain a nitrite-oxidizing bacterium with high nitrite oxidation activity for controlling nitrite levels. A nitrite-oxidizing bacterium, ZS-1, was isolated from the water of a coastal Pseudosciaena crocea-rearing pond. The strain was identified as Nitrobacter winogradskyi based on the phylogenetic analyses of the 16S ribosomal ribonucleic acid gene and nxrA sequence of ZS-1. Under aerobic condition, the nitrite-oxidizing activity of ZS-1 did not change considerably in the range of pH 7–9, but was strongly inhibited by lower (pH = 6) and higher (pH = 10) pH values. The optimum temperature range is 25–32 °C. Lower temperature made the adaptive phase of ZS-1 longer but did not affect its maximum nitrite oxidization rate. The nitrite-oxidizing activity of ZS-1 started to be inhibited by ammonia and nitrate when the concentrations of ammonia and nitrate reached 25 mg L−1 and 100 mg L−1, respectively. The inhibition was stronger with higher concentration of ammonia or nitrate. The nitrite-oxidizing activity of ZS-1, however, was not inhibited by high concentration of nitrite (500 mg L−1). The nitrite-oxidizing activity of ZS-1 was increased by low ammonia concentration (1 mg L−1 to 10 mg L−1). PMID:26019486

  5. Diversity and Abundance of Ammonia-Oxidizing Archaeal Nitrite Reductase (nirK) Genes in Estuarine Sediments of San Francisco Bay

    NASA Astrophysics Data System (ADS)

    Reji, L.; Lee, J. A.; Damashek, J.; Francis, C. A.

    2013-12-01

    Nitrification, the microbially-mediated aerobic oxidation of ammonia to nitrate via nitrite, is an integral component of the global biogeochemical nitrogen cycle. The first and rate-limiting step of nitrification, ammonia oxidation, is carried out by two distinct microbial groups: ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). Molecular ecological studies targeting the amoA gene have revealed the abundance and ubiquity of AOA in terrestrial as well as aquatic environments. In addition to the ammonia oxidation machinery that includes the amoA gene, AOA also encode a gene for copper-containing nitrite reductase (nirK). The distribution patterns and functional role of nirK in AOA remain mostly unknown; proposed functions include the indirect involvement in ammonia oxidation through the production of nitric oxide during nitrite reduction, and (2) nitrite detoxification. In the present study, the diversity and abundance of archaeal nirK genes in estuarine sediments were investigated using quantitative polymerase chain reaction, cloning and sequencing approaches. In sediment samples collected from the San Francisco Bay estuary, two archaeal nirK variants (AnirKa and AnirKb) were amplified using specific primer sets. Overall, AnirKa was observed to be significantly more abundant than AnirKb in the sediment samples, with variation in relative abundance spanning two to three orders of magnitude between sampling sites. Phylogenetic analysis revealed a number of unique archaeal nirK sequence types, as well as many that clustered with sequences from previous estuarine studies and cultured AOA isolates, such as Nitrosopumilus maritimus. This study yielded new insights into the diversity and abundance of archaeal nirK genes in estuarine sediments, and highlights the importance of further investigating the physiological role of this gene in AOA, as well as its suitability as a marker gene for studying AOA in the environment.

  6. Distribution of Prx-linked hydroperoxide reductase activity among microorganisms.

    PubMed

    Takeda, Kouji; Nishiyama, Yoshitaka; Yoda, Koji; Watanabe, Toshihiro; Nimura-Matsune, Kaori; Mura, Kiyoshi; Tokue, Chiyoko; Katoh, Tetzuya; Kawasaki, Shinji; Niimura, Youichi

    2004-01-01

    Peroxiredoxin (Prx) constitutes a large family of enzymes found in microorganisms, animals, and plants, but the detection of the activities of Prx-linked hydroperoxide reductases (peroxiredoxin reductases) in cell extracts, and the purification based on peroxide reductase activity, have only been done in bacteria and Trypanosomatidae. A peroxiredoxin reductase (NADH oxidase) from a bacterium, Amphibacillus, displayed only poor activities in the presence of purified Prx from Saccharomyces or Synechocystis, while it is highly active in the presence of bacterial Prx. These results suggested that an enzyme system different from that in bacteria might exist for the reduction of Prx in yeast and cyanobacteria. Prx-linked hydroperoxide reductase activities were detected in cell extracts of Saccharomyces, Synechocystis, and Chlorella, and the enzyme activities of Saccharomyces and Chlorella were induced under vigorously aerated culture conditions and intensive light exposure conditions, respectively. Partial purification of Prx-linked peroxidase from the induced yeast cells indicated that the Prx-linked peroxidase system consists of two protein components, namely, thioredoxin and thioredoxin reductase. This finding is consistent with the previous report on its purification based on its protein protection activity against oxidation [Chae et al., J. Biol. Chem., 269, 27670-27678 (1994)]. In this study we have confirmed that Prx-linked peroxidase activity are widely distributed, not only in bacteria species and Trypanosomatidae, but also in yeast and photosynthetic microorganisms, and showed reconstitution of the activity from partially purified interspecies components.

  7. Sodium nitrite blocks the activity of aminoglycosides against Pseudomonas aeruginosa biofilms.

    PubMed

    Zemke, Anna C; Gladwin, Mark T; Bomberger, Jennifer M

    2015-01-01

    Sodium nitrite has broad antimicrobial activity at pH 6.5, including the ability to prevent biofilm growth by Pseudomonas aeruginosa on the surfaces of airway epithelial cells. Because of its antimicrobial activity, nitrite is being investigated as an inhaled agent for chronic P. aeruginosa airway infections in cystic fibrosis patients. However, the interaction between nitrite and commonly used aminoglycosides is unknown. This paper investigates the interaction between nitrite and tobramycin in liquid culture, abiotic biofilms, and a biotic biofilm model simulating the conditions in the cystic fibrosis airway. The addition of nitrite prevented killing by aminoglycosides in liquid culture, with dose dependence between 1.5 and 15 mM. The effect was not blocked by the nitric oxide scavenger CPTIO or dependent on efflux pump activity. Nitrite shifted the biofilm minimal bactericidal concentration (MBC-biofilm) from 256 μg/ml to >1,024 μg/ml in an abiotic biofilm model. In a biotic biofilm model, the addition of 50 mM nitrite decreased the antibiofilm activity of tobramycin by up to 1.2 log. Respiratory chain inhibition recapitulated the inhibition of aminoglycoside activity by nitrite, suggesting a potential mechanism of inhibition of energy-dependent aminoglycoside uptake. In summary, sodium nitrite induces resistance to both gentamicin and tobramycin in P. aeruginosa grown in liquid culture, as an abiotic biofilm, or as a biotic biofilm.

  8. NO Reductase Activity of the Tetraheme Cytochrome c554 of Nitrosomonas europaea

    PubMed Central

    Upadhyay, Anup K.; Hooper, Alan B.; Hendrich, Michael P.

    2009-01-01

    The tetraheme cytochrome c554 (cyt c554) from Nitrosomonas europaea is believed to function as an electron-transfer protein from hydroxylamine oxidoreductase (HAO). We show here that cyt c554 also has significant NO reductase activity. The protein contains one high-spin and three low-spin c-type hemes. HAO catalyzed reduction of the cyt c554, ligand binding, intermolecular electron transfer, and kinetics of NO reduction by cyt c554 have been investigated. We detect the formation of a NO-bound ferrous heme species in cyt c554 by EPR and Mössbauer spectroscopies during the HAO catalyzed oxidation of hydroxylamine, indicating that N-oxide intermediates produced from HAO readily bind to cyt c554. In the half-reduced state of cyt c554, we detect a spin interaction between the [FeNO]7 state of heme 2 and the low-spin ferric state of heme 4. We find that ferrous cyt c554 will reduce NO at a rate greater than 16 s−1, which is comparable to rates of other known NO reductases. Carbon monoxide or nitrite are shown not to bind to the reduced protein, and previous results indicate the reactions with O2 are slow and that a variety of ligands will not bind in the oxidized state. Thus, the enzymatic site is highly selective for NO. The NO reductase activity of cyt c554 may be important during ammonia oxidation in N. europaea at low oxygen concentrations to detoxify NO produced by reduction of nitrite or incomplete oxidation of hydroxylamine. PMID:16569009

  9. Nitrite-dependent nitric oxide production pathway: implications for involvement of active nitrogen species in photoinhibition in vivo.

    PubMed Central

    Yamasaki, H

    2000-01-01

    Air pollution studies have shown that nitric oxide (NO), a gaseous free radical, is a potent photosynthetic inhibitor that reduces CO2 uptake activity in leaves. It is now recognized that NO is not only an air pollutant but also an endogenously produced metabolite, which may play a role in regulating plant cell functions. Although many studies have suggested the presence of mammalian-type NO synthase (NOS) in plants, the source of NO is still not clear. There has been a number of studies indicating that plant cells possess a nitrite-dependent NO production pathway which can be distinguished from the NOS-mediated reaction. Nitrate reductase (NR) has been recently found to be capable of producing NO through one-electron reduction of nitrite using NAD(P)H as an electron donor. This review focuses on current understanding of the mechanism for the nitrite-dependent NO production in plants. Impacts of NO produced by NR on photosynthesis are discussed in association with photo-oxidative stress in leaves. PMID:11128001

  10. The Effect of Low Osmotic Potential on Nitrite Reduction in Intact Spinach Chloroplasts 1

    PubMed Central

    Behrens, Paul W.; Xu, Fujuan; Werner, Marisa; Hoffman, Teresa; Marsho, Thomas V.; MacKay, A. Bryan

    1985-01-01

    The effect of water stress (reduced osmotic potential) on photosynthetic nitrite reduction was investigated using intact, isolated spinach (Spinacia oleracea) chloroplasts. Nitrite-dependent O2 evolution was inhibited 39% at −29.5 bars osmotic potential, relative to a control at −11 bars. In the presence of an uncoupler of photophosphorylation this inhibition was not seen. Reduced osmotic potential did not inhibit either methyl viologen reduction or photosynthetic O2 reduction. These results indicate that an inhibition of electron transport to ferredoxin cannot account for the observed inhibition of nitrite-dependent O2 evolution. In vitro assay of nitrite reductase activity showed that the interaction of the enzyme with nitrite was not affected by changes in the concentrations of ions or molecules that might be caused by water stress conditions. These results indicate that the most likely site for the effect of water stress on chloroplastic nitrite reduction is the interaction of ferredoxin with nitrite reductase. PMID:16664429

  11. Docking and molecular dynamics studies at trypanothione reductase and glutathione reductase active sites.

    PubMed

    Iribarne, Federico; Paulino, Margot; Aguilera, Sara; Murphy, Miguel; Tapia, Orlando

    2002-05-01

    A theoretical docking study on the active sites of trypanothione reductase (TR) and glutathione reductase (GR) with the corresponding natural substrates, trypanothione disulfide (T[S]2) and glutathione disulfide (GSSG), is reported. Molecular dynamics simulations were carried out in order to check the robustness of the docking results. The energetic results are in agreement with previous experimental findings and show the crossed complexes have lower stabilization energies than the natural ones. To test DOCK3.5, four nitro furanic compounds, previously designed as potentially active anti-chagasic molecules, were docked at the GR and TR active sites with the DOCK3.5 procedure. A good correlation was found between differential inhibitory activity and relative interaction energy (affinity). The results provide a validation test for the use of DOCK3.5 in connection with the design of anti-chagasic drugs.

  12. Pseudoazurin from Sinorhizobium meliloti as an electron donor to copper-containing nitrite reductase: influence of the redox partner on the reduction potentials of the enzyme copper centers.

    PubMed

    Ferroni, Félix M; Marangon, Jacopo; Neuman, Nicolás I; Cristaldi, Julio C; Brambilla, Silvina M; Guerrero, Sergio A; Rivas, María G; Rizzi, Alberto C; Brondino, Carlos D

    2014-08-01

    Pseudoazurin (Paz) is the physiological electron donor to copper-containing nitrite reductase (Nir), which catalyzes the reduction of NO2 (-) to NO. The Nir reaction mechanism involves the reduction of the type 1 (T1) copper electron transfer center by the external physiological electron donor, intramolecular electron transfer from the T1 copper center to the T2 copper center, and nitrite reduction at the type 2 (T2) copper catalytic center. We report the cloning, expression, and characterization of Paz from Sinorhizobium meliloti 2011 (SmPaz), the ability of SmPaz to act as an electron donor partner of S. meliloti 2011 Nir (SmNir), and the redox properties of the metal centers involved in the electron transfer chain. Gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis together with UV-vis and EPR spectroscopies revealed that as-purified SmPaz is a mononuclear copper-containing protein that has a T1 copper site in a highly distorted tetrahedral geometry. The SmPaz/SmNir interaction investigated electrochemically showed that SmPaz serves as an efficient electron donor to SmNir. The formal reduction potentials of the T1 copper center in SmPaz and the T1 and T2 copper centers in SmNir, evaluated by cyclic voltammetry and by UV-vis- and EPR-mediated potentiometric titrations, are against an efficient Paz T1 center to Nir T1 center to Nir T2 center electron transfer. EPR experiments proved that as a result of the SmPaz/SmNir interaction in the presence of nitrite, the order of the reduction potentials of SmNir reversed, in line with T1 center to T2 center electron transfer being thermodynamically more favorable.

  13. Uterine glutathione reductase activity: modulation by estrogens and progesterone.

    PubMed

    Díaz-Flores, M; Baiza-Gutman, L A; Pedrón, N N; Hicks, J J

    1999-10-29

    The aim of this study was to determine whether glutathione reductase activity in uterine tissue is regulated by sex hormones. In spayed rats uterine glutathione reductase was significantly increased by exogenous estrogen (P< 0.01), progesterone (P< 0.01) or estrogen plus progesterone (P<0.01). When enzyme activity is expressed per mg protein, daily administration of estrogen or progesterone induces a progressive increase of this enzyme between 24 to 48 h or 24 to 72 h of treatment, respectively. Whereas the combination of both steroids causes an earlier and higher increase in glutathione reductase activity at 24 h of treatment. Estradiol singly or in combination with progesterone induced the highest protein concentration in the uterus. Whereas uterine DNA concentration is only significantly affected by estradiol. Our results suggest that uterine glutathione reductase is regulated by estradiol and progesterone and may be involved in maintaining levels of reduced glutathione in the uterus. This compound may be required for control of the redox state of thiol groups and in detoxification reactions involving H2O2 and electrophylic substances. The antioxidant action of estrogens is partially due to the stimulation of glutathione reductase.

  14. [Ligand spectrum of hemoglobin activity of methemoglobin-reductase and hemolytic resistance of erythrocytes during chronic exposure to nitrates].

    PubMed

    Kiiza, D A; Artiukh, V P; Starodub, N F; Khmel'nitskiĭ, G A

    1992-01-01

    It is found that nitrite-ions formed as a result of biotransformation during long term feeding of calves with sodium and potassium nitrates induce changes in some biochemical parameters of blood, including HS-glutathione content in erythrocytes, acid hemolytic resistance of erythrocytes, activity of NAD-dependent methemoglobin-reductase, correlation of ligand forms of hemoglobin and its total content. It is supposed that the observed changes are of an adaptational character and, as a whole, provide for the optimization of both quantitative and qualitative composition of population of erythroid cells at the expense of erythropoiesis intensification.

  15. Enhanced activity and selectivity of carbon nanofiber supported Pd catalysts for nitrite reduction.

    PubMed

    Shuai, Danmeng; Choe, Jong Kwon; Shapley, John R; Werth, Charles J

    2012-03-06

    Pd-based catalyst treatment represents an emerging technology that shows promise to remove nitrate and nitrite from drinking water. In this work we use vapor-grown carbon nanofiber (CNF) supports in order to explore the effects of Pd nanoparticle size and interior versus exterior loading on nitrite reduction activity and selectivity (i.e., dinitrogen over ammonia production). Results show that nitrite reduction activity increases by 3.1-fold and selectivity decreases by 8.0-fold, with decreasing Pd nanoparticle size from 1.4 to 9.6 nm. Both activity and selectivity are not significantly influenced by Pd interior versus exterior CNF loading. Consequently, turnover frequencies (TOFs) among all CNF catalysts are similar, suggesting nitrite reduction is not sensitive to Pd location on CNFs nor Pd structure. CNF-based catalysts compare favorably to conventional Pd catalysts (i.e., Pd on activated carbon or alumina) with respect to nitrite reduction activity and selectivity, and they maintain activity over multiple reduction cycles. Hence, our results suggest new insights that an optimum Pd nanoparticle size on CNFs balances faster kinetics with lower ammonia production, that catalysts can be tailored at the nanoscale to improve catalytic performance for nitrite, and that CNFs hold promise as highly effective catalyst supports in drinking water treatment.

  16. Measurement of nitrous oxide reductase activity in aquatic sediments

    SciTech Connect

    Miller, L.G.; Oremland, R.S.; Paulsen, S.

    1986-01-01

    Denitrification in aquatic sediments was measured by an N/sub 2/O reductase assay. Sediments consumed small added quantities of N/sub 2/O over short periods (a few hours). In experiments with sediment slurries, N/sub 2/O reductase activity was inhibited by 0/sub 2/, C/sub 2/H/sub 2/, heat treatment, and by high levels of nitrate (1 mM) or sulfide (10 mM). However, ambient levels of nitrate (<100 ..mu..M) did not influence activity, and moderate levels (about 150 ..mu..M) induced only a short lag before reductase activity began. Moderate levels of sulfide (<1 mM) had no effect on N/sub 2/O reductase activity. Nitrous oxide reductase displayed Michaelis-Menten kinetics in sediments from freshwater, estuarine, and alkaline-saline environments. An in situ assay was devised in which a solution of N/sub 2/O was injected into sealed glass cores containing intact sediment. Two estimates of net rates of denitrification in San Francisco Bay under approximated in situ conditions were 0.009 and 0.041 mmol of N/sub 2/O per m/sup 2/ per h. Addition of chlorate to inhibit denitrification in these intact-core experiments (to estimate gross rates of N/sub 2/O consumption) resulted in approximately a 14% upward revision of estimates of net rates. These results were comparable to an in situ estimate of 0.022 mmol of N/sub 2/O per m/sup 2/ per h made with the acetylene block assay.

  17. Measurement of nitrous oxide reductase activity in aquatic sediments

    USGS Publications Warehouse

    Miller, L.G.; Oremland, R.S.; Paulsen, S.

    1986-01-01

    Denitrification in aquatic sediments was measured by an N2O reductase assay. Sediments consumed small added quantities of N2O over short periods (a few hours). In experiments with sediment slurries, N2O reductase activity was inhibited by O2, C2H2, heat treatment, and by high levels of nitrate (1 mM) or sulfide (10 mM). However, ambient levels of nitrate (<100 μM) did not influence activity, and moderate levels (about 150 μM) induced only a short lag before reductase activity began. Moderate levels of sulfide (<1 mM) had no effect on N2O reductase activity. Nitrous oxide reductase displayed Michaelis-Menten kinetics in sediments from freshwater (Km = 2.17 μM), estuarine (Km = 14.5 μM), and alkaline-saline (Km = 501 μM) environments. An in situ assay was devised in which a solution of N2O was injected into sealed glass cores containing intact sediment. Two estimates of net rates of denitrification in San Francisco Bay under approximated in situ conditions were 0.009 and 0.041 mmol of N2O per m2 per h. Addition of chlorate to inhibit denitrification in these intact-core experiments (to estimate gross rates of N2O consumption) resulted in approximately a 14% upward revision of estimates of net rates. These results were comparable to an in situ estimate of 0.022 mmol of N2O per m2 per h made with the acetylene block assay.

  18. [Electrical activity and circulatory effects of nitrite in the rat cerebrum].

    PubMed

    Shumilova, T E; Smirnov, A G; Shereshkov, V I; Fedorova, M A; Nozdrachev, A D

    2015-01-01

    An association between the cerebrum electrical activity (CEA) in rats, blood supply of its cortex microregions (linear blood flow), and general cerebrum blood flow under acute nitrite hypoxia was studied. The phase character of the change in hemodynamic indices and the total capacity of electroencephalography (EEG) spectrum for 75 min after sodium nitrite introduction (30 mg/kg of body weight) was detected. The first phase (30 min) was associated with cerebrum adaptation to hypotension caused by nitrite and was completed by EEG normalization. The second phase was characterized by pathological EEG changes (in spite of restoration of hemodynamics in the cerebrum) caused by the growth of oxygen debt in the nervous tissue as a result of a decrease in the blood oxygen capacity by 60-75 min of the effect of nitrite.

  19. Molecular Components of Nitrate and Nitrite Efflux in Yeast

    PubMed Central

    Cabrera, Elisa; González-Montelongo, Rafaela; Giraldez, Teresa; de la Rosa, Diego Alvarez

    2014-01-01

    Some eukaryotes, such as plant and fungi, are capable of utilizing nitrate as the sole nitrogen source. Once transported into the cell, nitrate is reduced to ammonium by the consecutive action of nitrate and nitrite reductase. How nitrate assimilation is balanced with nitrate and nitrite efflux is unknown, as are the proteins involved. The nitrate assimilatory yeast Hansenula polymorpha was used as a model to dissect these efflux systems. We identified the sulfite transporters Ssu1 and Ssu2 as effective nitrate exporters, Ssu2 being quantitatively more important, and we characterize the Nar1 protein as a nitrate/nitrite exporter. The use of strains lacking either SSU2 or NAR1 along with the nitrate reductase gene YNR1 showed that nitrate reductase activity is not required for net nitrate uptake. Growth test experiments indicated that Ssu2 and Nar1 exporters allow yeast to cope with nitrite toxicity. We also have shown that the well-known Saccharomyces cerevisiae sulfite efflux permease Ssu1 is also able to excrete nitrite and nitrate. These results characterize for the first time essential components of the nitrate/nitrite efflux system and their impact on net nitrate uptake and its regulation. PMID:24363367

  20. Sequence homology requirements for transcriptional silencing of 35S transgenes and post-transcriptional silencing of nitrite reductase (trans)genes by the tobacco 271 locus.

    PubMed

    Thierry, D; Vaucheret, H

    1996-12-01

    The transgene locus of the tobacco plant 271 (271 locus) is located on a telomere and consists of multiple copies of a plasmid carrying an NptII marker gene driven by the cauliflower mosaic virus (CaMV) 19S promoter and the leaf-specific nitrite reductase Nii1 cDNA cloned in the antisense orientation under the control of the CaMV 35S promoter. Previous analysis of gene expression in leaves has shown that this locus triggers both post-transcriptional silencing of the host leaf-specific Nii genes and transcriptional silencing of transgenes driven by the 19S or 35S promoter irrespective of their coding sequence and of their location in the genome. In this paper we show that silencing of transgenes carrying Nii1 sequences occurs irrespective of the promoter driving their expression and of their location within the genome. This phenomenon occurs in roots as well as in leaves although root Nii genes share only 84% identity with leaf-specific Nii1 sequences carried by the 271 locus. Conversely, transgenes carrying the bean Nii gene (which shares 76% identity with the tobacco Nii1 gene) escape silencing by the 271 locus. We also show that transgenes driven by the figwort mosaic virus 34S promoter (which shares 63% identity with the 35S promoter) also escape silencing by the 271 locus. Taken together, these results indicate that a high degree of sequence similarity is required between the sequences of the silencing locus and of the target (trans)genes for both transcriptional and post-transcriptional silencing.

  1. Role of Bradyrhizobium japonicum cytochrome c550 in nitrite and nitrate respiration.

    PubMed

    Bueno, Emilio; Bedmar, Eulogio J; Richardson, David J; Delgado, María J

    2008-02-01

    Bradyrhizobium japonicum cytochrome c(550), encoded by cycA, has been previously suggested to play a role in denitrification, the respiratory reduction of nitrate to dinitrogen. However, the exact role of this cytochrome in the denitrification process is unknown. This study shows that cytochrome c(550) is involved in electron transfer to the copper-containing nitrite reductase of B. japonicum, as revealed by the inability of a cycA mutant strain to consume nitrite and, consequently, to grow under denitrifying conditions with nitrite as the electron acceptor. Mutation of cycA had no apparent effect on methylviologen-dependent nitrite reductase activity. However, succinate-dependent nitrite reduction was largely inhibited, suggesting that c(550) is the in vivo electron donor to copper-containing nitrite reductase. In addition, this study demonstrates that a cytochrome c(550) mutation has a negative effect on expression of the periplasmic nitrate reductase. This phenotype can be rescued by extending the growth period of the cells. A model is proposed whereby a mutation in cycA reduces expression of the cbb(3)-type oxidase, affecting oxygen consumption rate by the cells and consequently preventing maximal expression of the periplasmic nitrate reductase during the first days of the growth period.

  2. Oral Nitrate Reductase Activity Is Not Associated with Development of Non-Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH): A Pilot Study.

    PubMed

    Barzin, Gilda; Merat, Shahin; Nokhbeh-Zaeem, Habibeh; Saniee, Parastoo; Pedramnia, Shahrzad; Mostashfi Habibabadi, Ali; Nasseri-Moghaddam, Siavosh

    2014-01-01

    BACKGROUND NAFLD/NASH is a manifestation of metabolic syndrome and is associated with obesity/overweight. Not all obese/overweight individuals develop NASH. Gastro-esophageal reflux disease (GERD) is considered a gastrointestinal manifestation of the metabolic syndrome and is associated with obesity/overweight. Again not all obese/overweight individuals develop GERD. Recent data show association of dietary nitrate content and oral nitrate reductase activity (NRA) with GERD. Nitrates need to be converted to nitrite (done in human beings by nitrate reductase of oral bacteria exclusively) to be active in metabolic pathways. OBJECTIVE To assess the relation between NASH/NAFLD and oral NRA. METHODS Oral NRA was measured in individuals with NASH (compatible abdominal ultrasound and two elevated ALT/AST levels over six months) and was compared with that of those without NASH. Oral NRA was measured according to a previously reported protocol. RESULTS Eleven NASH patients and twelve controls were enrolled. Mean oral NRA activity were 2.82 vs. 3.51 μg nitrite-N formed per person per minute for cases and controls respectively (p=0.46). CONCLUSION According to our data, oral nitrite production is not different between individual swith and without NASH.

  3. Nitrate and periplasmic nitrate reductases

    PubMed Central

    Sparacino-Watkins, Courtney; Stolz, John F.; Basu, Partha

    2014-01-01

    The nitrate anion is a simple, abundant and relatively stable species, yet plays a significant role in global cycling of nitrogen, global climate change, and human health. Although it has been known for quite some time that nitrate is an important species environmentally, recent studies have identified potential medical applications. In this respect the nitrate anion remains an enigmatic species that promises to offer exciting science in years to come. Many bacteria readily reduce nitrate to nitrite via nitrate reductases. Classified into three distinct types – periplasmic nitrate reductase (Nap), respiratory nitrate reductase (Nar) and assimilatory nitrate reductase (Nas), they are defined by their cellular location, operon organization and active site structure. Of these, Nap proteins are the focus of this review. Despite similarities in the catalytic and spectroscopic properties Nap from different Proteobacteria are phylogenetically distinct. This review has two major sections: in the first section, nitrate in the nitrogen cycle and human health, taxonomy of nitrate reductases, assimilatory and dissimilatory nitrate reduction, cellular locations of nitrate reductases, structural and redox chemistry are discussed. The second section focuses on the features of periplasmic nitrate reductase where the catalytic subunit of the Nap and its kinetic properties, auxiliary Nap proteins, operon structure and phylogenetic relationships are discussed. PMID:24141308

  4. Terpenoids from Diplophyllum taxifolium with quinone reductase-inducing activity.

    PubMed

    Wang, Xiao; Zhang, Jiao-Zhen; Zhou, Jin-Chuan; Shen, Tao; Lou, Hong-Xiang

    2016-03-01

    Two new ent-prenylaromadendrane-type diterpenoids, diplotaxifols A (1) and B (2), a new ent-eudesmol, ent-eudesma-4(15),11(13)-dien-6α,12-diol (3), eight new eudesmanolides enantiomers (4-11) of the corresponding compounds from higher plants along with four known ent-eudesmanolides (12-15) were isolated from the 95% EtOH extract of Chinese liverwort Diplophyllum taxifolium. Their structures were elucidated on the basis of MS, NMR and IR spectral data, and confirmed by single-crystal X-ray diffraction analysis. The quinone reductase-inducing activity of the compounds was evaluated.

  5. Nitrite reductase genes (nirK and nirS) as functional markers to investigate diversity of denitrifying bacteria in Pacific northwest marine sediment communities

    SciTech Connect

    Braker, G.; Zhou, J.; Wu, L.; Devol, A.H.; Tiedje, J.M.

    2000-05-01

    Genetic heterogeneity of denitrifying bacteria in sediment samples from Puget Sound and two sites on the Washington continental margin was studied by PCR approaches amplifying nirK and nirS genes. These structurally different but functionally equivalent single-copy genes coding for nitrite reductases, a key enzyme of the denitrification process, were used as a molecular marker for denitrifying bacteria. nirS sequences could be amplified from samples of both sampling sites, whereas nirK sequences were detected only in samples from the Washington margin. To assess the underlying nir gene structure, PCR products of both genes were cloned and screened by restriction fragment length polymorphism (RFLP). Rarefraction analysis revealed a high level of diversity especially for nirS clones from Puget Sound and a slightly lower level of diversity for nirK and nirS clones from the Washington margin. One group dominated within nirK clones, but no dominance and only a few redundant clones were seen between sediment samples for nirS clones in both habitats. Hybridization and sequencing confirmed that all but one of the 228 putative nirS clones were nirS with levels of nucleotide identities as low as 45.3%. Phylogenetic analysis grouped nirS clones into three distinct subclusters within the nirS gene tree which corresponded to the two habitats from which they were obtained. These sequences had little relationship to any strain with known nirS sequences or to isolates (mostly close relatives of Pseudomonas stutzeri) from the Washington margin sediment samples. nirK clones were more closely related to each other than were the nirS clones, with 78.6% and higher nucleotide identities; clones showing only weak hybridization signals were not related to known nirK sequences. All nirK clones were also grouped into a distinct cluster which could not be placed with any strain with known nirK sequences. These findings show a very high diversity of nir sequences within small samples and that

  6. Vitamin K epoxide reductase: homology, active site and catalytic mechanism.

    PubMed

    Goodstadt, Leo; Ponting, Chris P

    2004-06-01

    Vitamin K epoxide reductase (VKOR) recycles reduced vitamin K, which is used subsequently as a co-factor in the gamma-carboxylation of glutamic acid residues in blood coagulation enzymes. VKORC1, a subunit of the VKOR complex, has recently been shown to possess this activity. Here, we show that VKORC1 is a member of a large family of predicted enzymes that are present in vertebrates, Drosophila, plants, bacteria and archaea. Four cysteine residues and one residue, which is either serine or threonine, are identified as likely active-site residues. In some plant and bacterial homologues the VKORC1 homologous domain is fused with domains of the thioredoxin family of oxidoreductases. These might reduce disulfide bonds of VKORC1-like enzymes as a prerequisite for their catalytic activities.

  7. Function of the Rhizobium etli CFN42 nirK gene in nitrite metabolism.

    PubMed

    Bueno, E; Gómez-Hernández, N; Girard, L; Bedmar, E J; Delgado, M J

    2005-02-01

    Rhizobium etli CFN42 is not capable of growing anaerobically with nitrate but it grows with nitrite as a terminal electron acceptor. This bacterium contains the nirK gene encoding the copper-containing Nir (nitrite reductase), which is located on the cryptic plasmid pCFN42f. Mutational analysis has demonstrated that a nirK deficient mutant was not capable of growing under nitrite-respiring conditions. Moreover, microaerobic growth of this mutant was inhibited by the presence of nitrite. Nir activity and nitrite uptake were highly diminished in a nirK mutant, compared with the wild-type levels after incubation under anaerobic conditions. Our results suggest that the copper-containing Nir may have both a respiratory and a nitrite-detoxifying role in R. etli.

  8. Erythrocyte aldose reductase activity and sorbitol levels in diabetic retinopathy

    PubMed Central

    Satyanarayana, A.; Balakrishna, N.; Ayyagari, Radha; Padma, M.; Viswanath, K.; Petrash, J. Mark

    2008-01-01

    Purpose Activation of polyol pathway due to increased aldose reductase (ALR2) activity has been implicated in the development of diabetic complications including diabetic retinopathy (DR), a leading cause of blindness. However, the relationship between hyperglycemia-induced activation of polyol pathway in retina and DR is still uncertain. We investigated the relationship between ALR2 levels and human DR by measuring ALR2 activity and its product, sorbitol, in erythrocytes. Methods We enrolled 362 type 2 diabetic subjects (T2D) with and without DR and 66 normal subjects in this clinical case-control study. Clinical evaluation of DR in T2D patients was done by fundus examination. ALR2 activity and sorbitol levels along with glucose and glycosylated hemoglobin (HbA1C) levels in erythrocytes were determined. Results T2D patients with DR showed significantly higher specific activity of ALR2 as compared to T2D patients without DR. Elevated levels of sorbitol in T2D patients with DR, as compared to T2D patients without DR, corroborated the increased ALR2 activity in erythrocytes of DR patients. However, the increased ALR2 activity was not significantly associated with diabetes duration, age, and HbA1C in both the DR group and total T2D subjects. Conclusions Levels of ALR2 activity as well as sorbitol in erythrocytes may have value as a quantitative trait to be included among other markers to establish a risk profile for development of DR. PMID:18385795

  9. QTL analysis of ferric reductase activity in the model legume lotus japonicus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Physiological and molecular studies have demonstrated that iron accumulation from the soil into Strategy I plants can be limited by ferric reductase activity. An initial study of Lotus japonicus ecotypes Miyakojima MG-20 and Gifu B-129 identified significant leaf chlorosis and ferric reductase activ...

  10. Inorganic nitrite attenuates NADPH oxidase-derived superoxide generation in activated macrophages via a nitric oxide-dependent mechanism.

    PubMed

    Yang, Ting; Peleli, Maria; Zollbrecht, Christa; Giulietti, Alessia; Terrando, Niccolo; Lundberg, Jon O; Weitzberg, Eddie; Carlström, Mattias

    2015-06-01

    Oxidative stress contributes to the pathogenesis of many disorders, including diabetes and cardiovascular disease. Immune cells are major sources of superoxide (O2(∙-)) as part of the innate host defense system, but exaggerated and sustained O2(∙-) generation may lead to progressive inflammation and organ injuries. Previous studies have proven organ-protective effects of inorganic nitrite, a precursor of nitric oxide (NO), in conditions manifested by oxidative stress and inflammation. However, the mechanisms are still not clear. This study aimed at investigating the potential role of nitrite in modulating NADPH oxidase (NOX) activity in immune cells. Mice peritoneal macrophages or human monocytes were activated by lipopolysaccharide (LPS), with or without coincubation with nitrite. O2(∙-) and peroxynitrite (ONOO(-)) formation were detected by lucigenin-based chemiluminescence and fluorescence techniques, respectively. The intracellular NO production was measured by DAF-FM DA fluorescence. NOX isoforms and inducible NO synthase (iNOS) expression were detected by qPCR. LPS increased both O2(∙-) and ONOO(-) production in macrophages, which was significantly reduced by nitrite (10µmol/L). Mechanistically, the effects of nitrite are (1) linked to increased NO generation, (2) similar to that observed with the NO donor DETA-NONOate, and (3) can be abolished by the NO scavenger carboxy-PTIO or by the xanthine oxidase (XO) inhibitor febuxostat. Nox2 expression was increased in activated macrophages, but was not influenced by nitrite. However, nitrite attenuated LPS-induced upregulation of iNOS expression. Similar to that observed in mice macrophages, nitrite also reduced O2(∙-) generation in LPS-activated human monocytes. In conclusion, XO-mediated reduction of nitrite attenuates NOX activity in activated macrophages, which may modulate the inflammatory response.

  11. Determination of the specific activities of methionine sulfoxide reductase A and B by capillary electrophoresis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A capillary electrophoresis (CE) method for the determination of methionine sulfoxide reductase A and methionine sulfoxide reductase B activities in mouse liver is described. The method is based on detection of the 4-(dimethylamino)azobenzene-4’-sulfonyl derivative of L-methionine (dabsyl Met), the ...

  12. Is there a role of inducible nitric oxide synthase activation in the delayed antiarrhythmic effect of sodium nitrite?

    PubMed

    Demeter-Haludka, Vivien; Juhász, László; Kovác, Mária; Gardi, János; Végh, Ágnes

    2017-04-01

    This study aimed to examine whether inducible nitric oxide synthase (iNOS) plays a role in the delayed antiarrhythmic effect of sodium nitrite. Twenty-one dogs were infused intravenously with sodium nitrite (0.2 μmol·kg(-1)·min(-1)) for 20 min, either in the absence (n = 12) or in the presence of the iNOS inhibitor S-(2-aminoethyl)-isothiourea (AEST) (total dose 2.0 mg·kg(-1) i.v., n = 9). Control dogs (n = 12) were given saline. Twenty-four hours later, all of the dogs were subjected to a 25 min period occlusion of the left anterior descending coronary artery followed by rapid reperfusion. Dogs treated with AEST and nitrite received again AEST prior to the occlusion. Compared with the controls, sodium nitrite markedly reduced the number of ectopic beats, the number and incidence of ventricular tachycardia, and the incidence of ventricular fibrillation during occlusion and increased survival (0% versus 50%) from the combined ischaemia and reperfusion insult. Although AEST completely inhibited iNOS activity, the nitrite-induced increase in NO bioavailability during occlusion was not substantially modified. Furthermore, AEST attenuated but did not completely abolish the antiarrhythmic effect of nitrite. The marked delayed antiarrhythmic effect of sodium nitrite is not entirely due to the activation of iNOS; other mechanisms may certainly play a role.

  13. Structural Basis for Activation of Class Ib Ribonucleotide Reductase

    SciTech Connect

    Boal, Amie K.; Cotruvo, Jr., Joseph A.; Stubbe, JoAnne; Rosenzweig, Amy C.

    2010-12-03

    The class Ib ribonucleotide reductase of Escherichia coli can initiate reduction of nucleotides to deoxynucleotides with either a Mn{sub 2}{sup III}-tyrosyl radical (Y{sm_bullet}) or a Fe{sub 2}{sup III}-Y{sm_bullet} cofactor in the NrdF subunit. Whereas Fe{sub 2}{sup III}-Y{sm_bullet} can self-assemble from Fe{sub 2}{sup II}-NrdF and O{sub 2}, activation of Mn{sub 2}{sup II}-NrdF requires a reduced flavoprotein, NrdI, proposed to form the oxidant for cofactor assembly by reduction of O{sub 2}. The crystal structures reported here of E. coli Mn{sub 2}{sup II}-NrdF and Fe{sub 2}{sup II}-NrdF reveal different coordination environments, suggesting distinct initial binding sites for the oxidants during cofactor activation. In the structures of Mn{sub 2}{sup II}-NrdF in complex with reduced and oxidized NrdI, a continuous channel connects the NrdI flavin cofactor to the NrdF Mn{sub 2}{sup II} active site. Crystallographic detection of a putative peroxide in this channel supports the proposed mechanism of Mn{sub 2}{sup III}-Y{sm_bullet} cofactor assembly.

  14. Identification of activators of methionine sulfoxide reductases A and B

    PubMed Central

    Cudic, Predrag; Joshi, Neelambari; Sagher, Daphna; Williams, Brandon T.; Stawikowski, Maciej J.; Weissbach, Herbert

    2016-01-01

    The methionine sulfoxide reductase (Msr) family of enzymes has been shown to protect cells against oxidative damage. The two major Msr enzymes, MsrA and MsrB, can repair oxidative damage to proteins due to reactive oxygen species, by reducing the methionine sulfoxide in proteins back to methionine. A role of MsrA in animal aging was first demonstrated in D. melanogaster where transgenic flies over-expressing recombinant bovine MsrA had a markedly extended life span. Subsequently, MsrA was also shown to be involved in the life span extension in C. elegans. These results supported other studies that indicated up-regulation, or activation, of the normal cellular protective mechanisms that cells use to defend against oxidative damage could be an approach to treat age related diseases and slow the aging process. In this study we have identified, for the first time, compounds structurally related to the natural products fusaricidins that markedly activate recombinant bovine and human MsrA and human MsrB. PMID:26718410

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

    PubMed

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

    2016-11-01

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

  16. Chromate reductase activity in Streptomyces sp. MC1.

    PubMed

    Polti, Marta A; Amoroso, María J; Abate, Carlos M

    2010-02-01

    Biological transformation of Cr(VI) to Cr(III) by enzymatic reduction may provide a less costly and more environmentally friendly approach to remediation. In a previous report a Cr(VI) resistant actinomycete strain, Streptomyces sp. MC1, was able to reduce Cr(VI) present in a synthetic medium, soil extract and soil samples. This is the first time optimal conditions such as pH, temperature, growth phase and electron donor have been elucidated in vitro for Cr(VI) reduction by a streptomycete. Chromate reductase of Streptomyces sp. MC1 is a constitutive enzyme which was mainly associated with biomass and required NAD(P)H as an electron donor. It was active over a broad temperature (19-39 degrees C) and pH (5-8) range, and optimum conditions were 30 degrees C and pH 7. The enzyme was present in supernatant, pellet and cell free extract. Bioremediation with the enzyme was observed in non-compatible cell reproduction systems, conditions frequently found in contaminated environments.

  17. Inhibition of carbonyl reductase activity in pig heart by alkyl phenyl ketones.

    PubMed

    Imamura, Yorishige; Narumi, Rika; Shimada, Hideaki

    2007-02-01

    The inhibitory effects of alkyl phenyl ketones on carbonyl reductase activity were examined in pig heart. In this study, carbonyl reductase activity was estimated as the ability to reduce 4-benzoylpyridine to S(-)-alpha-phenyl-4-pyridylmethanol in the cytosolic fraction from pig heart (pig heart cytosol). The order of their inhibitory potencies was hexanophenone > valerophenone > heptanophenone > butyrophenone > propiophenone. The inhibitory potencies of acetophenone and nonanophenone were much lower. A significant relationship was observed between Vmax/Km values for the reduction of alkyl phenyl ketones and their inhibitory potencies for carbonyl reductase activity in pig heart cytosol. Furthermore, hexanophenone was a competitive inhibitor for the enzyme activity. These results indicate that several alkyl phenyl ketones including hexanophenone inhibit carbonyl reductase activity in pig heart cytosol, by acting as substrate inhibitors.

  18. Human endothelial dihydrofolate reductase low activity limits vascular tetrahydrobiopterin recycling.

    PubMed

    Whitsett, Jennifer; Rangel Filho, Artur; Sethumadhavan, Savitha; Celinska, Joanna; Widlansky, Michael; Vasquez-Vivar, Jeannette

    2013-10-01

    Tetrahydrobiopterin (BH₄) is required for NO synthesis and inhibition of superoxide release from endothelial NO synthase. Clinical trials using BH₄ to treat endothelial dysfunction have produced mixed results. Poor outcomes may be explained by the rapid systemic and cellular oxidation of BH₄. One of the oxidation products of BH₄, 7,8-dihydrobiopterin (7,8-BH₂), is recycled back to BH₄ by dihydrofolate reductase (DHFR). This enzyme is ubiquitously distributed and shows a wide range of activity depending on species-specific factors and cell type. Information about the kinetics and efficiency of BH4 recycling in human endothelial cells receiving BH₄ treatment is lacking. To characterize this reaction, we applied a novel multielectrode coulometric HPLC method that enabled the direct quantification of 7,8-BH₂ and BH₄, which is not possible with fluorescence-based methodologies. We found that basal untreated BH₄ and 7,8-BH₂ concentrations in human endothelial cells (ECs) are lower than in bovine and murine endothelioma cells. Treatment of human ECs with BH₄ transiently increased intracellular BH₄ while accumulating the more stable 7,8-BH₂. This was different from bovine or murine ECs, which resulted in preferential BH₄ increase. Using BH₄ diastereomers, 6S-BH₄ and 6R-BH₄, the narrow contribution of enzymatic DHFR recycling to total intracellular BH₄ was demonstrated. Reduction of 7,8-BH₂ to BH₄ occurs at very slow rates in cells and needs supraphysiological levels of 7,8-BH₂, indicating this reaction is kinetically limited. Activity assays verified that human DHFR has very low affinity for 7,8-BH₂ (DHF7,8-BH₂) and folic acid inhibits 7,8-BH₂ recycling. We conclude that low activity of endothelial DHFR is an important factor limiting the benefits of BH4 therapies, which may be further aggravated by folate supplements.

  19. Increased 5. cap alpha. -reductase activity in idiopathic hirsutism

    SciTech Connect

    Serafini, P.; Lobo, R.A.

    1985-01-01

    In vitro, genital skin 5..cap alpha..-reductase activity (5..cap alpha..-RA) was measured in ten hirsute women with normal androgen levels (idiopathic hirsutism (IH)) and in ten hirsute women with elevated androgen levels (polycystic ovary syndrome (PCO)) in order to determine the influence of secreted androgens on 5..cap alpha..-RA. In vitro 5..cap alpha..-RA was assessed by incubations of skin with /sup 14/C-testosterone (T) for 2 hours, after which steroids were separated and the radioactivity of dihydrotestosterone (DHT) and 5..cap alpha..-androstane 3..cap alpha..-17..beta..-estradiol (3..cap alpha..-diol) in specific eluates were determined. All androgens were normal in IH with the exception of higher levels of 3..cap alpha..-diol glucuronide which were similar to the levels of PCO. The conversion ratio (CR) of T to DHT in IH and PCO were similar, yet significantly greater than the CR of control subjects. The CR of T to 3..cap alpha..-diol in IH and PCO were similar, yet higher than in control subjects. Serum androgens showed no correlation with 5..cap alpha..-RA, while the CR of T to DHT showed a significant positive correlation with the Ferriman and Gallwey score. The increased 5..cap alpha..-RA in IH appears to be independent of serum androgen levels and is, therefore, an inherent abnormality. The term idiopathic is a misnomer, because hirsutism in these patients may be explained on the basis of increased skin 5..cap alpha..-RA.

  20. Identification of promiscuous ene-reductase activity by mining structural databases using active site constellations

    PubMed Central

    Steinkellner, Georg; Gruber, Christian C.; Pavkov-Keller, Tea; Binter, Alexandra; Steiner, Kerstin; Winkler, Christoph; Łyskowski, Andrzej; Schwamberger, Orsolya; Oberer, Monika; Schwab, Helmut; Faber, Kurt; Macheroux, Peter; Gruber, Karl

    2014-01-01

    The exploitation of catalytic promiscuity and the application of de novo design have recently opened the access to novel, non-natural enzymatic activities. Here we describe a structural bioinformatic method for predicting catalytic activities of enzymes based on three-dimensional constellations of functional groups in active sites (‘catalophores’). As a proof-of-concept we identify two enzymes with predicted promiscuous ene-reductase activity (reduction of activated C–C double bonds) and compare them with known ene-reductases, that is, members of the Old Yellow Enzyme family. Despite completely different amino acid sequences, overall structures and protein folds, high-resolution crystal structures reveal equivalent binding modes of typical Old Yellow Enzyme substrates and ligands. Biochemical and biocatalytic data show that the two enzymes indeed possess ene-reductase activity and reveal an inverted stereopreference compared with Old Yellow Enzymes for some substrates. This method could thus be a tool for the identification of viable starting points for the development and engineering of novel biocatalysts. PMID:24954722

  1. Epigallocatechin-3-gallate potently inhibits the in vitro activity of hydroxy-3-methyl-glutaryl-CoA reductase[S

    PubMed Central

    Cuccioloni, Massimiliano; Mozzicafreddo, Matteo; Spina, Michele; Tran, Chi Nhan; Falconi, Maurizio; Eleuteri, Anna Maria; Angeletti, Mauro

    2011-01-01

    Hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) is the rate-controlling enzyme of cholesterol synthesis, and owing to its biological and pharmacological relevance, researchers have investigated several compounds capable of modulating its activity with the hope of developing new hypocholesterolemic drugs. In particular, polyphenol-rich extracts were extensively tested for their cholesterol-lowering effect as alternatives, or adjuvants, to the conventional statin therapies, but a full understanding of the mechanism of their action has yet to be reached. Our work reports on a detailed kinetic and equilibrium study on the modulation of HMGR by the most-abundant catechin in green tea, epigallocatechin-3-gallate (EGCG). Using a concerted approach involving spectrophotometric, optical biosensor, and chromatographic analyses, molecular docking, and site-directed mutagenesis on the cofactor site of HMGR, we have demonstrated that EGCG potently inhibits the in vitro activity of HMGR (Ki in the nanomolar range) by competitively binding to the cofactor site of the reductase. Finally, we evaluated the effect of combined EGCG-statin administration. PMID:21357570

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

    PubMed

    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.

  3. Endothelial human dihydrofolate reductase low activity limits vascular tetrahydrobiopterin recycling

    PubMed Central

    Whitsett, Jennifer; Filho, Artur Rangel; Sethumadhavan, Savitha; Celinska, Joanna; Widlansky, Michael; Vásquez-Vivar, Jeannette

    2013-01-01

    Tetrahydrobiopterin (BH4) is required for NO synthesis and inhibition of superoxide release from eNOS. Clinical trials using BH4 to treat endothelial dysfunction have produced mixed results. Poor outcomes may be explained by the rapid systemic and cellular oxidation of BH4. One of the oxidation products of BH4, 7,8-dihydrobiopterin (7,8-BH2), is recycled back to BH4 by dihydrofolate reductase (DHFR). This enzyme is ubiquitously distributed and shows a wide range of activity depending on species-specific factors and cell type. Information about the kinetics and efficiency of BH4 recycling in human endothelial cells receiving BH4 treatment is lacking. To characterize this reaction, we applied a novel multi-electrode coulometric HPLC method that enabled the direct quantification of 7,8-BH2 and BH4 which is not possible with fluorescent-based methodologies. We found that basal untreated BH4 and 7,8-BH2 concentrations in human ECs is lower than bovine and murine endothelioma cells. Treatment of human ECs with BH4 transiently increased intracellular BH4 while accumulating the more stable 7,8-BH2. This was different from bovine or murine ECs that resulted in preferential BH4 increase. Using BH4 diastereomers, 6S-BH4 and 6R-BH4, the narrow contribution of enzymatic DHFR recycling to total intracellular BH4 was demonstrated. Reduction of 7,8-BH2 to BH4 occurs at very slow rates in cells and needs supra-physiological levels of 7,8-BH2, indicating this reaction is kinetically limited. Activity assays verified that hDHFR has very low affinity for 7,8-BH2 (DHF7,8-BH2) and folic acid inhibits 7,8-BH2 recycling. We conclude that low activity of endothelial DHFR is an important factor limiting the benefits of BH4 therapies which may be further aggravated by folate supplements. PMID:23707606

  4. Immunological approach to the regulation of nitrate reductase in Monoraphidium braunii.

    PubMed

    Díez, J; López-Ruiz, A

    1989-02-01

    The effects of different culture conditions on nitrate reductase activity and nitrate reductase protein from Monoraphidium braunii have been studied, using two different immunological techniques, rocket immunoelectrophoresis and an enzyme-linked immunosorbent assay, to determine nitrate reductase protein. The nitrogen sources ammonium and glutamine repressed nitrate reductase synthesis, while nitrite, alanine, and glutamate acted as derepressors. There was a four- to eightfold increase of nitrate reductase activity and a twofold increase of nitrate reductase protein under conditions of nitrogen starvation versus growth on nitrate. Nitrate reductase synthesis was repressed in darkness. However, when Monoraphidium was grown under heterotrophic conditions with glucose as the carbon and energy source, the synthesis of nitrate reductase was maintained. With ammonium or darkness, changes in nitrate reductase activity correlated fairly well with changes in nitrate reductase protein, indicating that in both cases loss of activity was due to repression and not to inactivation of the enzyme. Experiments using methionine sulfoximine, to inhibit ammonium assimilation, showed that ammonium per se and not a product of its metabolism was the corepressor of the enzyme. The appearance of nitrate reductase activity after transferring the cells to induction media was prevented by cycloheximide and by 6-methylpurine, although in this latter case the effect was observed only in cells preincubated with the inhibitor for 1 h before the induction period.

  5. Nitrate reductase from Rhodopseudomonas sphaeroides.

    PubMed Central

    Kerber, N L; Cardenas, J

    1982-01-01

    The facultative phototroph Rhodopseudomonas sphaeroides DSM158 was incapable of either assimilating or dissimilating nitrate, although the organism could reduce it enzymatically to nitrite either anaerobically in the light or aerobically in the dark. Reduction of nitrate was mediated by a nitrate reductase bound to chromatophores that could be easily solubilized and functioned with chemically reduced viologens or photochemically reduced flavins as electron donors. The enzyme was solubilized, and some of its kinetic and molecular parameters were determined. It seemed to be nonadaptive, ammonia did not repress its synthesis, and its activity underwent a rapid decline when the cells entered the stationary growth phase. Studies with inhibitors and with metal antagonists indicated that molybdenum and possibly iron participate in the enzymatic reduction of nitrate. The conjectural significance of this nitrate reductase in phototrophic bacteria is discussed. PMID:6978883

  6. Circadian variation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in swine liver and ileum.

    PubMed

    Rogers, D H; Kim, D N; Lee, K T; Reiner, J M; Thomas, W A

    1981-07-01

    The temporal variation of HMG-CoA reductase activity in the liver and intestine of swine was investigated. The thin-layer chromatographic method widely used in the assay of the reductase was successfully applied to the porcine enzymes. Parallel circadian rhythms were demonstrated in both hepatic and ileal reductases from mash-fed animals. Peak activity occurred approximately 6 hr after feeding, 2.7-fold over the basal level in the liver, and 1.6-fold in the ileum. A milk-cholesterol diet caused a marked depression of both rhythms (90% in liver, 50% in ileum); however, the hourly variation in activity persisted in both organs. Cholestyramine was found to elevate hepatic activity (2.7-fold throughout the rhythm) without affecting that of the intestine. Clofibrate had no effect on either enzyme at any time during the cycle despite a 34% reduction in serum cholesterol concentrations.

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

    PubMed

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

    2008-09-01

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

  8. α-Glucosidase and aldose reductase inhibitory activities from the fruiting body of Phellinus merrillii.

    PubMed

    Huang, Guan-Jhong; Hsieh, Wen-Tsong; Chang, Heng-Yuan; Huang, Shyh-Shyun; Lin, Ying-Chih; Kuo, Yueh-Hsiung

    2011-05-25

    The inhibitory activity from the isolated component of the fruiting body Phellinus merrillii (PM) was evaluated against α-glucosidase and lens aldose reductase from Sprague-Dawley male rats and compared to the quercetin as an aldose reductase inhibitor and acarbose as an α-glucosidase inhibitor. The ethanol extracts of PM (EPM) showed the strong α-glucosidase and aldose reductase activities. α-Glucosidase and aldose reductase inhibitors were identified as hispidin (A), hispolon (B), and inotilone (C), which were isolated from EtOAc-soluble fractions of EPM. The above structures were elucidated by their spectra and comparison with the literatures. Among them, hispidin, hispolon, and inotilone exhibited potent against α-glucosidase inhibitor activity with IC(50) values of 297.06 ± 2.06, 12.38 ± 0.13, and 18.62 ± 0.23 μg/mL, respectively, and aldose reductase inhibitor activity with IC(50) values of 48.26 ± 2.48, 9.47 ± 0.52, and 15.37 ± 0.32 μg/mL, respectively. These findings demonstrated that PM may be a good source for lead compounds as alternatives for antidiabetic agents currently used. The importance of finding effective antidiabetic therapeutics led us to further investigate natural compounds.

  9. Color formation in nitrite-free dried hams as related to Zn-protoporphyrin IX and Zn-chelatase activity.

    PubMed

    Parolari, Giovanni; Benedini, Riccardo; Toscani, Tania

    2009-08-01

    The development of red pigment Zn-protoporphyrin IX (ZPP) in nitrite-free Parma hams was investigated in 5 leg muscles at several stages of processing and the activity of muscle Zn-chelatase was concurrently assayed for its potential role in ZPP formation. A steady increase of the pigment was observed throughout the manufacturing stages at mild temperatures while no development was observed during the prior cold resting phase. The enzyme was partly inactivated according to a muscle-dependent pattern, resulting in similar ZPP contents, hence color, in finished hams. It is concluded that enzyme-dependent synthesis of ZPP in nitrite-free dried hams contributes to color development, enabling muscles in dried hams to become more similar in redness than in green thighs. Therefore, checking raw meat for the enzyme content may be a means to control color formation in nitrite-free dry-cured meat derivatives.

  10. A diaper-embedded disposable nitrite sensor with integrated on-board urine-activated battery for UTI screening.

    PubMed

    Yu, W; Seo, W; Tan, T; Jung, B; Ziaie, B

    2016-08-01

    This paper reports a low-cost solution to the early detection of urinary nitrite, a common surrogate for urinary tract infection (UTI). We present a facile method to fabricate a disposable and flexible colorimetric [1] nitrite sensor and its urine-activated power source [2] on a hydrophobic (wax) paper through laser-assisted patterning and lamination. Such device, integrated with interface circuitry and a Bluetooth low energy (BLE) module can be embedded onto a diaper, and transmit semi-quantitative UTI monitoring information in a point-of-care and autonomous fashion. The proposed nitrite sensing platform achieves a sensitivity of 1.35 ms/(mg/L) and a detection limit of 4 mg/L.

  11. Inhibition of biomass activity in the via nitrite nitrogen removal processes by veterinary pharmaceuticals.

    PubMed

    Alvarino, Teresa; Katsou, Evina; Malamis, Simos; Suarez, Sonia; Omil, Francisco; Fatone, Francesco

    2014-01-01

    The inhibitory effect of two veterinary pharmaceuticals was studied for different types of biomass involved in via nitrite nitrogen removal processes. Batch tests were conducted to determine the inhibition level of acetaminophen (PAR) and doxycycline (DOX) on the activity of short-cut nitrifying, denitrifying and anoxic ammonium oxidation (anammox) biomass and phosphorus accumulating organisms (PAOs). All biomass types were affected by PAR and DOX, with anammox being the most sensitive bacteria. DOX inhibited more the biomass treating high strength nitrogenous effluents (HSNE) than low strength nitrogenous effluents (LSNE). The phosphorus uptake inhibition under anoxic conditions was lower than 25% in the presence of PAR up to 400 mg L(-1). The same DOX concentration inhibited anoxic phosphorus uptake more than 65% for biomass treating LSNE and HSNE. Heterotrophic denitrifying bacteria seem to be more robust at high DOX and PAR concentrations than anammox. Both veterinary products inactivated ammonium oxidizing, Accumulibacter phosphatis and denitrifying bacteria.

  12. HMG-CoA reductase activity in human liver microsomes: comparative inhibition by statins.

    PubMed

    Dansette, P M; Jaoen, M; Pons, C

    2000-05-01

    The aim of this study was to compare a number of vastatins, HMG-CoA reductase inhibitors, in human liver microsomes. HMG-CoA reductase activity was four times lower than the activity in untreated rat liver microsomes. Vastatins could be classified in this in vitro assay in three classes both in human and rat microsomes: the first one including cerivastatin with an IC50 of 6 nM, the second one with atorvastatin and fluvastatin (IC50) between 40 and 100 nM) and the third one containing pravastatin, simvastatin and lovastatin (IC50 between 100 and 300 nM).

  13. Aggregate Size and Architecture Determine Microbial Activity Balance for One-Stage Partial Nitritation and Anammox ▿

    PubMed Central

    Vlaeminck, Siegfried E.; Terada, Akihiko; Smets, Barth F.; De Clippeleir, Haydée; Schaubroeck, Thomas; Bolca, Selin; Demeestere, Lien; Mast, Jan; Boon, Nico; Carballa, Marta; Verstraete, Willy

    2010-01-01

    Aerobic ammonium-oxidizing bacteria (AerAOB) and anoxic ammonium-oxidizing bacteria (AnAOB) cooperate in partial nitritation/anammox systems to remove ammonium from wastewater. In this process, large granular microbial aggregates enhance the performance, but little is known about granulation so far. In this study, three suspended-growth oxygen-limited autotrophic nitrification-denitrification (OLAND) reactors with different inoculation and operation (mixing and aeration) conditions, designated reactors A, B, and C, were used. The test objectives were (i) to quantify the AerAOB and AnAOB abundance and the activity balance for the different aggregate sizes and (ii) to relate aggregate morphology, size distribution, and architecture putatively to the inoculation and operation of the three reactors. A nitrite accumulation rate ratio (NARR) was defined as the net aerobic nitrite production rate divided by the anoxic nitrite consumption rate. The smallest reactor A, B, and C aggregates were nitrite sources (NARR, >1.7). Large reactor A and C aggregates were granules capable of autonomous nitrogen removal (NARR, 0.6 to 1.1) with internal AnAOB zones surrounded by an AerAOB rim. Around 50% of the autotrophic space in these granules consisted of AerAOB- and AnAOB-specific extracellular polymeric substances. Large reactor B aggregates were thin film-like nitrite sinks (NARR, <0.5) in which AnAOB were not shielded by an AerAOB layer. Voids and channels occupied 13 to 17% of the anoxic zone of AnAOB-rich aggregates (reactors B and C). The hypothesized granulation pathways include granule replication by division and budding and are driven by growth and/or decay based on species-specific physiology and by hydrodynamic shear and mixing. PMID:19948857

  14. Gastric S-nitrosothiol formation drives the antihypertensive effects of oral sodium nitrite and nitrate in a rat model of renovascular hypertension.

    PubMed

    Pinheiro, Lucas C; Amaral, Jefferson H; Ferreira, Graziele C; Portella, Rafael L; Ceron, Carla S; Montenegro, Marcelo F; Toledo, Jose Carlos; Tanus-Santos, Jose E

    2015-10-01

    Many effects of nitrite and nitrate are attributed to increased circulating concentrations of nitrite, ultimately converted into nitric oxide (NO(•)) in the circulation or in tissues by mechanisms associated with nitrite reductase activity. However, nitrite generates NO(•) , nitrous anhydride, and other nitrosating species at low pH, and these reactions promote S-nitrosothiol formation when nitrites are in the stomach. We hypothesized that the antihypertensive effects of orally administered nitrite or nitrate involve the formation of S-nitrosothiols, and that those effects depend on gastric pH. The chronic effects of oral nitrite or nitrate were studied in two-kidney, one-clip (2K1C) hypertensive rats treated with omeprazole (or vehicle). Oral nitrite lowered blood pressure and increased plasma S-nitrosothiol concentrations independently of circulating nitrite levels. Increasing gastric pH with omeprazole did not affect the increases in plasma nitrite and nitrate levels found after treatment with nitrite. However, treatment with omeprazole severely attenuated the increases in plasma S-nitrosothiol concentrations and completely blunted the antihypertensive effects of nitrite. Confirming these findings, very similar results were found with oral nitrate. To further confirm the role of gastric S-nitrosothiol formation, we studied the effects of oral nitrite in hypertensive rats treated with the glutathione synthase inhibitor buthionine sulfoximine (BSO) to induce partial thiol depletion. BSO treatment attenuated the increases in S-nitrosothiol concentrations and antihypertensive effects of oral nitrite. These data show that gastric S-nitrosothiol formation drives the antihypertensive effects of oral nitrite or nitrate and has major implications, particularly to patients taking proton pump inhibitors.

  15. Mechanisms of Nitrite Bioactivation

    PubMed Central

    Kim-Shapiro, Daniel B.; Gladwin, Mark T.

    2014-01-01

    It is now accepted that the anion nitrite, once considered an inert oxidation product of nitric oxide (NO), contributes to hypoxic vasodilation, physiological blood pressure control, and redox signaling. As such, its application in therapeutics is being actively testing in pre-clinical models and in human phase I–II clinical trials. Major pathways for nitrite bioactivation involve its reduction to NO by members of the hemoglobin or molybdopterin family of proteins, or catalyzed dysproportionation. These conversions occur preferentially under hypoxic and acidic conditions. A number of enzymatic systems reduce nitrite to NO and their activity and importance are defined by oxygen tension, specific organ system and allosteric and redox effectors. In this work, we review different proposed mechanisms of nitrite bioactivation, focusing on analysis of kinetics and experimental evidence for the relevance of each mechanism under different conditions. PMID:24315961

  16. A substrate-bound structure of cyanobacterial biliverdin reductase identifies stacked substrates as critical for activity

    PubMed Central

    Takao, Haruna; Hirabayashi, Kei; Nishigaya, Yuki; Kouriki, Haruna; Nakaniwa, Tetsuko; Hagiwara, Yoshinori; Harada, Jiro; Sato, Hideaki; Yamazaki, Toshimasa; Sakakibara, Yoichi; Suiko, Masahito; Asada, Yujiro; Takahashi, Yasuhiro; Yamamoto, Ken; Fukuyama, Keiichi; Sugishima, Masakazu; Wada, Kei

    2017-01-01

    Biliverdin reductase catalyses the last step in haem degradation and produces the major lipophilic antioxidant bilirubin via reduction of biliverdin, using NAD(P)H as a cofactor. Despite the importance of biliverdin reductase in maintaining the redox balance, the molecular details of the reaction it catalyses remain unknown. Here we present the crystal structure of biliverdin reductase in complex with biliverdin and NADP+. Unexpectedly, two biliverdin molecules, which we designated the proximal and distal biliverdins, bind with stacked geometry in the active site. The nicotinamide ring of the NADP+ is located close to the reaction site on the proximal biliverdin, supporting that the hydride directly attacks this position of the proximal biliverdin. The results of mutagenesis studies suggest that a conserved Arg185 is essential for the catalysis. The distal biliverdin probably acts as a conduit to deliver the proton from Arg185 to the proximal biliverdin, thus yielding bilirubin. PMID:28169272

  17. Discovery and microassay of a nitrite-dependent carbonic anhydrase activity by stable-isotope dilution gas chromatography-mass spectrometry.

    PubMed

    Zinke, Maximilian; Hanff, Erik; Böhmer, Anke; Supuran, Claudiu T; Tsikas, Dimitrios

    2016-01-01

    The intrinsic activity of carbonic anhydrase (CA) is the hydration of CO2 to carbonic acid and its dehydration to CO2. CA may also function as esterase and phosphatase. Recently, we demonstrated that renal CA is mainly responsible for the reabsorption of nitrite (NO2(-)) which is the most abundant reservoir of the biologically highly potent nitric oxide (NO). By means of a stable-isotope dilution GC-MS method, we discovered a novel CA activity which strictly depends upon nitrite. We found that bovine erythrocytic CAII (beCAII) catalyses the incorporation of (18)O from H2 (18)O into nitrite at pH 7.4. After derivatization with pentafluorobenzyl bromide, gas chromatographic separation and mass spectrometric analysis, we detected ions at m/z 48 for singly (18)O-labelled nitrite ((16)O=N-(18)O(-)/(18)O=N-(16)O(-)) and at m/z 50 for doubly (18)O-labelled nitrite ((18)O=N-(18)O(-)) in addition to m/z 46 for unlabelled nitrite. Using (15)N-labelled nitrite ((15)NO2 (-), m/z 47) as an internal standard and selected-ion monitoring of m/z 46, m/z 48, m/z 50 and m/z 47, we developed a GC-MS microassay for the quantitative determination of the nitrite-dependent beCAII activity. The CA inhibitors acetazolamide and FC5 207A did not alter beCAII-catalysed formation of singly and doubly (18)O-labelled nitrite. Cysteine and the experimental CA inhibitor DIDS (a diisothiocyanate) increased several fold the beCAII-catalysed formation of the (18)O-labelled nitrite species. Cysteine, acetazolamide, FC5 207A, and DIDS by themselves had no effect on the incorporation of (18)O from H2 (18)O into nitrite. We conclude that erythrocytic CA possesses a nitrite-dependent activity which can only be detected when nitrite is used as the substrate and the reaction is performed in buffers of neutral pH values prepared in H2 (18)O. This novel CA activity, i.e., the nitrous acid anhydrase activity, represents a bioactivation of nitrite and may have both beneficial (via S-nitrosylation and subsequent

  18. Copper complexes relevant to the catalytic cycle of copper nitrite reductase: electrochemical detection of NO(g) evolution and flipping of NO2 binding mode upon Cu(II) → Cu(I) reduction.

    PubMed

    Maji, Ram Chandra; Barman, Suman Kumar; Roy, Suprakash; Chatterjee, Sudip K; Bowles, Faye L; Olmstead, Marilyn M; Patra, Apurba K

    2013-10-07

    Copper complexes of the deprotonated tridentate ligand, N-2-methylthiophenyl-2'-pyridinecarboxamide (HL1), were synthesized and characterized as part of our investigation into the reduction of copper(II) o-nitrito complexes into the related copper nitric oxide complexes and subsequent evolution of NO(g) such as occurs in the enzyme copper nitrite reductase. Our studies afforded the complexes [(L1)Cu(II)Cl]n (1), [(L1)Cu(II)(ONO)] (2), [(L1)Cu(II)(H2O)](ClO4)·H2O (3·H2O), [(L1)Cu(II)(CH3OH)](ClO4) (4), [(L1)Cu(II)(CH3CO2)]·H2O (5·H2O), and [Co(Cp)2][(L1)Cu(I)(NO2)(CH3CN)] (6). X-ray crystal structure determinations revealed distorted square-pyramidal coordination geometry around Cu(II) ion in 1-5. Substitution of the H2O of 3 by nitrite quantitatively forms 2, featuring the κ(2)-O,O binding mode of NO2(-) to Cu(II). Reduction of 2 generates two Cu(I) species, one with κ(1)-O and other with the κ(1)-N bonded NO2(-) group. The Cu(I) analogue of 2, compound 6, was synthesized. The FTIR spectrum of 6 reveals the presence of κ(1)-N bonded NO2(-). Constant potential electrolysis corresponding to Cu(II) → Cu(I) reduction of a CH3CN solution of 2 followed by reaction with acids, CH3CO2H or HClO4 generates 5 or 3, and NO(g), identified electrochemically. The isolated Cu(I) complex 6 independently evolves one equivalent of NO(g) upon reaction with acids. Production of NO(g) was confirmed by forming [Co(TPP)NO] in CH2Cl2 (λ(max) in CH2Cl2: 414 and 536 nm, ν(NO) = 1693 cm(-1)).

  19. Differential antioxidant and quinone reductase inducing activity of American, Asian, and Siberian ginseng

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The antioxidant and quinone reductase (QR) inducing activities of American, Asian, and Siberian ginseng have been reported using various plant materials, solvents, and assays. To directly establish their comparative bioactivity, the effects of extracts obtained from acidified methanol (MeOH), a gas...

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

    PubMed Central

    Hofmann, Laurie C.

    2013-01-01

    The concentration of CO2 in global surface ocean waters is increasing due to rising atmospheric CO2 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 CO2 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 CO2 concentrations. The activity of external carbonic anhydrase, an important enzyme functioning in macroalgal carbon-concentrating mechanisms, was inversely related to CO2 concentration after long-term exposure (12 weeks). Nitrate reductase, the enzyme responsible for reduction of nitrate to nitrite, was stimulated by CO2 and was highest in algae grown at 665 µatm CO2. Nitrate and phosphate uptake rates were inversely related to CO2, while ammonium uptake was unaffected, and the percentage of inorganic carbon in the algal skeleton decreased with increasing CO2. The results indicate that the processes of inorganic carbon and nutrient uptake and assimilation are affected by elevated CO2 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 CO2 are discussed. PMID:23314813

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

    PubMed

    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.

  2. Sequence diversity and enzyme activity of ferric-chelate reductase LeFRO1 in tomato.

    PubMed

    Kong, Danyu; Chen, Chunlin; Wu, Huilan; Li, Ye; Li, Junming; Ling, Hong-Qing

    2013-11-20

    Ferric-chelate reductase which functions in the reduction of ferric to ferrous iron on root surface is a critical protein for iron homeostasis in strategy I plants. LeFRO1 is a major ferric-chelate reductase involved in iron uptake in tomato. To identify the natural variations of LeFRO1 and to assess their effect on the ferric-chelate reductase activity, we cloned the coding sequences of LeFRO1 from 16 tomato varieties collected from different regions, and detected three types of LeFRO1 (LeFRO1(MM), LeFRO1(Ailsa) and LeFRO1(Monita)) with five amino acid variations at the positions 21, 24, 112, 195 and 582. Enzyme activity assay revealed that the three types of LeFRO1 possessed different ferric-chelate reductase activity (LeFRO1(Ailsa) > LeFRO1(MM) > LeFRO1(Monita)). The 112th amino acid residue Ala of LeFRO1 is critical for maintaining the high activity of ferric-chelate reductase, because modification of this amino acid resulted in a significant reduction of enzyme activity. Further, we showed that the combination of the amino acid residue Ile at the site 24 with Lys at the site 582 played a positive role in the enzyme activity of LeFRO1. In conclusion, the findings are helpful to understand the natural adaptation mechanisms of plants to iron-limiting stress, and may provide new knowledge to select and manipulate LeFRO1 for improving the iron deficiency tolerance in tomato.

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

    SciTech Connect

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

    2010-07-16

    Research highlights: {yields} Two monomeric 90 kDa nitrate reductase active proteins from Shewanella gelidimarina. {yields} Sequence of napA from napEDABC-type operon and napA from NapDAGHB-type operon. {yields} 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.

  4. 5 Alpha-reductase inhibitory and antiandrogenic activities of novel steroids in hamster seminal vesicles.

    PubMed

    Cabeza, Marisa; Bratoeff, Eugene; Flores, Eugenio; Ramírez, Elena; Calleros, Jorge; Montes, Diana; Quiroz, Alexandra; Heuze, Ivonne

    2002-11-01

    The pharmacological activity of several 16-bromosubstituted trienediones 4 and 5, 16-methyl substituted dienediones 6 and 7 and the 16-methyl substituted trienedione 8 was determined on gonadectomized hamster seminal vesicles by measuring the in vitro conversion of testosterone (T) to dihydrotestosterone (DHT) as 5alpha-reductase inhibitors and also the ability of these steroids to bind to the androgen receptor. Steroids 6 and 7 when injected together with T decreased the weight of the seminal vesicles thus showing an antiandrogenic effect. Compounds 5 and 6 reduced substantially the conversion of T to DHT and therefore can be considered good inhibitors for the enzyme 5alpha-reductase; however both steroids failed to form a complex with the androgen receptor. On the other hand compound 7 which showed a very small inhibitory activity for the enzyme 5alpha-reductase, exhibited a very high affinity for the androgen receptor and thus can be considered an effective antiandrogen. This compound also reduced substantially the weight of the seminal vesicles. Steroids 4 and 8 did not reduce the weight of the seminal vesicles and exhibited a low affinity for the androgen receptor; 8 showed a weak 5alpha-reductase inhibitory activity, whereas 4 exhibited a weak androgenic effect.

  5. Site-directed mutagenesis of azurin from Pseudomonas aeruginosa enhances the formation of an electron-transfer complex with a copper-containing nitrite reductase from Alcaligenes faecalis S-6.

    PubMed

    Kukimoto, M; Nishiyama, M; Tanokura, M; Murphy, M E; Adman, E T; Horinouchi, S

    1996-09-23

    Kinetic analysis of electron transfer between azurin from Pseudomonas aeruginosa and copper-containing nitrite reductase (NIR) from Akaligenes faecalis S-6 was carried out to investigate the specificity of electron transfer between copper-containing proteins. Apparent values of kcat and Km of NIR for azurin were 300-fold smaller and 172-fold larger than those for the physiological redox partner, pseudoazurin from A. faecalis S-6, respectively, suggesting that the electron transfer between azurin and NIR was less specific than that between pseudoazurin and NIR. One of the major differences in 3-D structure between these redox proteins, azurin and pseudoazurin, is the absence and presence of lysine residues near their type 1 copper sites, respectively. Three mutated azurins, D11K, P36K, and D11K/P36K, were constructed to evaluate the importance of lysine residues in the interaction with NIR. The redox potentials of D11K, P36K, and D11K/P36K azurins were higher than that of wild-type azurin by 48, 7, and 55 mV, respectively. As suggested by the increase in the redox potential, kinetic analysis of electron transfer revealed reduced ability of electron transfer in the mutated azurins. On the other hand, although each of the single mutations caused modest effects on the decrease in the Km value, the simultaneous mutations of D11K and P36K caused significant decrease in the Km value when compared to that for wild-type azurin. These results suggest that the introduction of two lysine residues into azurin facilitated docking to NIR.

  6. 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

    USGS Publications Warehouse

    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

  7. Type I and type II fatty acid biosynthesis in Eimeria tenella: enoyl reductase activity and structure.

    PubMed

    Lu, J Z; Muench, S P; Allary, M; Campbell, S; Roberts, C W; Mui, E; McLeod, R L; Rice, D W; Prigge, S T

    2007-12-01

    Apicomplexan parasites of the genus Eimeria are the major causative agent of avian coccidiosis, leading to high economic losses in the poultry industry. Recent results show that Eimeria tenella harbours an apicoplast organelle, and that a key biosynthetic enzyme, enoyl reductase, is located in this organelle. In related parasites, enoyl reductase is one component of a type II fatty acid synthase (FAS) and has proven to be an attractive target for antimicrobial compounds. We cloned and expressed the mature form of E. tenella enoyl reductase (EtENR) for biochemical and structural studies. Recombinant EtENR exhibits NADH-dependent enoyl reductase activity and is inhibited by triclosan with an IC50 value of 60 nm. The crystal structure of EtENR reveals overall similarity with other ENR enzymes; however, the active site of EtENR is unoccupied, a state rarely observed in other ENR structures. Furthermore, the position of the central beta-sheet appears to block NADH binding and would require significant movement to allow NADH binding, a feature not previously seen in the ENR family. We analysed the E. tenella genomic database for orthologues of well-characterized bacterial and apicomplexan FAS enzymes and identified 6 additional genes, suggesting that E. tenella contains a type II FAS capable of synthesizing saturated, but not unsaturated, fatty acids. Interestingly, we also identified sequences that appear to encode multifunctional type I FAS enzymes, a feature also observed in Toxoplasma gondii, highlighting the similarity between these apicomplexan parasites.

  8. Enhancement of N-nitrosamine formation on granular-activated carbon from N-methylaniline and nitrite

    SciTech Connect

    Dietrich, A.M.; Gallagher, D.L.; DeRosa, P.M.; Millington, D.S.; DiGiano, F.A.

    1986-10-01

    Sterile aqueous N-methylaniline solutions were allowed to equilibrate at various nitrite, F-400 granular-activated carbon, and pH levels for 1 week. The aqueous and activated carbon phases were extracted and analyzed for nitrosamines relative to an added internal standard. Selected ion monitoring GC/MS, utilizing continuous monitoring of the NO/sup +/ ion (m/z 29.9980) characteristic of nitrosamines, at medium resolution (R = 2500-3000) was applied to quantitatively measure nitrosamines at picograms per microliter concentrations. This method selected for nitrosamine products only and eliminated interferences from non-nitrosamine reaction products. Results indicate that the pressure of granular-activated carbon significantly enhanced the formation of nitrosamine from N-methyl-aniline (F = 145, P< 0.0001). The amount of N-nitrosomethylaniline formed in the presence of activated carbon was 75 times more than that formed in the absence of activated carbon under the same nitrite, pH, and precursor amine conditions. High nitrite concentrations and loss pH values significantly increased the conversion of secondary amine to nitrosamine. 25 references, 4 figures, 4 tables.

  9. Anion selective optodes: development of a fluorescent fiber optic sensor for the determination of nitrite activity

    NASA Astrophysics Data System (ADS)

    Barker, Susan L. R.; Shortreed, Michael R.; Kopelman, Raoul

    1996-12-01

    The response of state of the art anion optodes often cannot be described in a thermodynamically exact manner because the ionic strength within the membrane phase of such optodes changes during the course of a titration. Incorporating lipophilic charge sites in the anion optode membranes provides a constant ionic strength in the membrane phase, the ability to measure anion activities, and a more thermodynamically describable system. This configuration has been used to create a micrometer-sized nitrite-selective optode. Recent elucidation of the many biological roles of nitric oxide (NO) has spurred interest in sensitive and selective detection of this molecule. In biological systems NO is converted to NO2- within 30 sec and the biological concentration of NO2- is normally on the micromolar level. The optode we have prepared contains a selective vitamin B12 derivative ionophore, a fluorescent chromoionophore (ETH 2439 or ETH 5350), and lipophilic charge sites. These components are entrapped in a highly plasticized PVC matrix which is placed on the distal end of the fiber. Sensor characteristics such as limit of detection and reversibility are presented.

  10. Post-translational Regulation of Nitrate Reductase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nitrate reductase (NR) catalyzes the reduction of nitrate to nitrite, which is the first step in the nitrate assimilation pathway, but can also reduce nitrite to nitric oxide (NO), an important signaling molecule that is thought to mediate a wide array of of developmental and physiological processes...

  11. Impact of mitochondria on nitrite metabolism in HL-1 cardiomyocytes

    PubMed Central

    Dungel, Peter; Teuschl, Andreas H.; Banerjee, Asmita; Paier-Pourani, Jamile; Redl, Heinz; Kozlov, Andrey V.

    2013-01-01

    Apart from ATP synthesis mitochondria have many other functions, one being nitrite reductase activity. Nitric oxide (NO) released from nitrite has been shown to protect the heart from ischemia/reperfusion (I/R) injury in a cGMP-dependent manner. However, the exact impact of mitochondria on the release of NO from nitrite in cardiomyocytes is not completely understood. Besides mitochondria, a number of non-mitochondrial metalloproteins have been suggested to facilitate this process. The aim of this study was to investigate the impact of mitochondria on the bioactivation of nitrite in HL-1 cardiomyocytes. The levels of nitrosyl complexes of hemoglobin (NO-Hb) and cGMP levels were measured by electron spin resonance spectroscopy and enzyme immunoassay. In addition the formation of free NO was determined by confocal microscopy as well as intracellular nitrite and S-nitrosothiols by chemoluminescence analysis. NO was released from nitrite in cell culture in an oxygen-dependent manner. Application of specific inhibitors of the respiratory chain, p450, NO synthases (NOS) and xanthine oxidoreductase (XOR) showed that all four enzymatic systems are involved in the release of NO, but more than 50% of NO is released via the mitochondrial pathway. Only NO released by mitochondria activated cGMP synthesis. Cardiomyocytes co-cultured with red blood cells (RBC) competed with RBC for nitrite, but free NO was detected only in HL-1 cells suggesting that RBC are not a source of NO in this model. Apart from activation of cGMP synthesis, NO formed in HL-1 cells diffused out of the cells and formed NO-Hb complexes. In addition nitrite was converted by HL-1 cells to S-nitrosyl complexes. In HL-1 cardiomyocytes, several enzymatic systems are involved in nitrite reduction to NO but only the mitochondrial pathway of NO release activates cGMP synthesis. Our data suggest that this pathway may be a key regulator of myocardial contractility especially under hypoxic conditions. PMID:23730288

  12. Isolated and combined exposure to ammonia and nitrite in giant freshwater pawn (Macrobrachium rosenbergii): effects on the oxidative stress, antioxidant enzymatic activities and apoptosis in haemocytes.

    PubMed

    Zhang, Yufan; Ye, Chaoxia; Wang, Anli; Zhu, Xuan; Chen, Changhong; Xian, Jianan; Sun, Zhenzhu

    2015-10-01

    The residual contaminators such as ammonia and nitrite are widely considered as relevant sources of aquatic environmental pollutants, posing a great threat to shrimp survival. To study the toxicological effects of ammonia and nitrite exposure on the innate immune response in invertebrates, we investigated the oxidative stress and apoptosis in haemocytes of freshwater prawn (Macrobrachium rosenbergii) under isolated and combined exposure to ammonia and nitrite in order to provide useful information about adult prawn immune responses. M. rosenbergii (13.44 ± 2.75 g) were exposed to 0, 5, and 25 mg/L total ammonia-N (TAN) and 0, 5, and 20 mg/L nitrite-N for 24 h. All ammonia concentrations were combined with all nitrite concentrations, making a total of nine treatments studied. Following the exposure treatment, antioxidant enzyme activity, reactive oxygen species (ROS) generation, nitric oxide (NO) generation, and apoptotic cell ratio of haemocytes were measured using flow cytometry. Results indicated that ROS generation was sensitive to the combined effect of ammonia and nitrite, which subsequently affected the Cu-Zn SOD activity. In addition, CAT showed the highest activity at 5 mg/L TAN while GPx decreased at 5 mg/L TAN and returned towards baseline at 25 mg/L. NO generation synchronized with the apoptotic cell ratio in haemocytes, indicating that NO production was closely associated with programmed cell death. Both NO production and apoptotic ratios significantly decreased following 25 mg/L TAN, which may be due to the antagonistic regulation of NO and GPx. We hypothesized that the toxicological effect of nitrite exhibited less change in physiological changes compared to that of ammonia, because of the high tolerance to nitrite exposure in mature M. rosenbergii and/or the competitive effects of chloride ions. Taken together, these results showed that ammonia and nitrite caused a series of combined oxidative stress and apoptosis in M. rosenbergi, but further

  13. Sodium nitrite exerts an antihypertensive effect and improves endothelial function through activation of eNOS in the SHR

    PubMed Central

    Ling, Wei Chih; Murugan, Dharmani Devi; Lau, Yeh Siang; Vanhoutte, Paul M.; Mustafa, Mohd Rais

    2016-01-01

    Sodium nitrite (NaNO2) induces relaxation in isolated arteries partly through an endothelium-dependent mechanism involving NO-eNOS-sGC-cGMP pathway. The present study was designed to investigate the effect of chronic NaNO2 administration on arterial systolic blood pressure (SBP) and vascular function in hypertensive rats. NaNO2 (150 mg L−1) was given in drinking water for four weeks to spontaneously (SHR) and Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) treated hypertensive SD rats. Arterial SBP and vascular function in isolated aortae were studied. Total plasma nitrate/nitrite and vascular cyclic guanosine monophosphate (cGMP) levels were measured using commercially available assay kits. Vascular nitric oxide (NO) levels were evaluated by DAF-FM fluorescence while the proteins involved in endothelial nitric oxide synthase (eNOS) activation was determined by Western blotting. NaNO2 treatment reduced SBP, improved the impaired endothelium-dependent relaxation, increased plasma total nitrate/nitrite level and vascular tissue NO and cGMP levels in SHR. Furthermore, increased presence of phosphorylated eNOS and Hsp-90 was observed in NaNO2-treated SHR. The beneficial effect of nitrite treatment was not observed in L-NAME treated hypertensive SD rats. The present study provides evidence that chronic treatment of genetically hypertensive rats with NaNO2 improves endothelium-dependent relaxation in addition to its antihypertensive effect, partly through mechanisms involving activation of eNOS. PMID:27616322

  14. The retinaldehyde reductase activity of DHRS3 is reciprocally activated by retinol dehydrogenase 10 to control retinoid homeostasis.

    PubMed

    Adams, Mark K; Belyaeva, Olga V; Wu, Lizhi; Kedishvili, Natalia Y

    2014-05-23

    The retinoic acid-inducible dehydrogenase reductase 3 (DHRS3) is thought to function as a retinaldehyde reductase that controls the levels of all-trans-retinaldehyde, the immediate precursor for bioactive all-trans-retinoic acid. However, the weak catalytic activity of DHRS3 and the lack of changes in retinaldehyde conversion to retinol and retinoic acid in the cells overexpressing DHRS3 undermine its role as a physiologically important all-trans-retinaldehyde reductase. This study demonstrates that DHRS3 requires the presence of retinol dehydrogenase 10 (RDH10) to display its full catalytic activity. The RDH10-activated DHRS3 acts as a robust high affinity all-trans-retinaldehyde-specific reductase that effectively converts retinaldehyde back to retinol, decreasing the rate of retinoic acid biosynthesis. In turn, the retinol dehydrogenase activity of RDH10 is reciprocally activated by DHRS3. At E13.5, DHRS3-null embryos have ∼4-fold lower levels of retinol and retinyl esters, but only slightly elevated levels of retinoic acid. The membrane-associated retinaldehyde reductase and retinol dehydrogenase activities are decreased by ∼4- and ∼2-fold, respectively, in Dhrs3(-/-) embryos, and Dhrs3(-/-) mouse embryonic fibroblasts exhibit reduced metabolism of both retinaldehyde and retinol. Neither RDH10 nor DHRS3 has to be itself catalytically active to activate each other. The transcripts encoding DHRS3 and RDH10 are co-localized at least in some tissues during development. The mutually activating interaction between the two related proteins may represent a highly sensitive and conserved mechanism for precise control over the rate of retinoic acid biosynthesis.

  15. Photosystem I cyclic electron transport: Measurement of ferredoxin-plastoquinone reductase activity.

    PubMed

    Cleland, R E; Bendall, D S

    1992-12-01

    Absorbance changes of ferredoxin measured at 463 nm in isolated thylakoids were shown to arise from the activity of the enzyme ferredoxin-plastoquinone reductase (FQR) in cyclic electron transport. Under anaerobic conditions in the presence of DCMU and an appropriate concentration of reduced ferredoxin, a light-induced absorbance decrease due to further reduction of Fd was assigned to the oxidation of the other components in the cyclic pathway, primarily plastoquinone. When the light was turned off, Fd was reoxidised and this gave a direct quantitative measurement of the rate of cyclic electron transport due to the activity of FQR. This activity was sensitive to the classical inhibitor of cyclic electron transport, antimycin, and also to J820 and DBMIB. Antimycin had no effect on Fd reduction although this was inhibited by stigmatellin. This provides further evidence that there is a quinone reduction site outside the cytochrome bf complex. The effect of inhibitors of ferredoxin-NADP(+) reductase and experiments involving the modification of ferredoxin suggest that there may be some role for the reductase as a component of FQR. Contrary to expectations, NADPH2 inhibited FQR activity; ATP and ADP had no effect.

  16. In Situ Characterization of Nitrospira-Like Nitrite-Oxidizing Bacteria Active in Wastewater Treatment Plants

    PubMed Central

    Daims, Holger; Nielsen, Jeppe L.; Nielsen, Per H.; Schleifer, Karl-Heinz; Wagner, Michael

    2001-01-01

    Uncultivated Nitrospira-like bacteria in different biofilm and activated-sludge samples were investigated by cultivation-independent molecular approaches. Initially, the phylogenetic affiliation of Nitrospira-like bacteria in a nitrifying biofilm was determined by 16S rRNA gene sequence analysis. Subsequently, a phylogenetic consensus tree of the Nitrospira phylum including all publicly available sequences was constructed. This analysis revealed that the genus Nitrospira consists of at least four distinct sublineages. Based on these data, two 16S rRNA-directed oligonucleotide probes specific for the phylum and genus Nitrospira, respectively, were developed and evaluated for suitability for fluorescence in situ hybridization (FISH). The probes were used to investigate the in situ architecture of cell aggregates of Nitrospira-like nitrite oxidizers in wastewater treatment plants by FISH, confocal laser scanning microscopy, and computer-aided three-dimensional visualization. Cavities and a network of cell-free channels inside the Nitrospira microcolonies were detected that were water permeable, as demonstrated by fluorescein staining. The uptake of different carbon sources by Nitrospira-like bacteria within their natural habitat under different incubation conditions was studied by combined FISH and microautoradiography. Under aerobic conditions, the Nitrospira-like bacteria in bioreactor samples took up inorganic carbon (as HCO3− or as CO2) and pyruvate but not acetate, butyrate, and propionate, suggesting that these bacteria can grow mixotrophically in the presence of pyruvate. In contrast, no uptake by the Nitrospira-like bacteria of any of the carbon sources tested was observed under anoxic or anaerobic conditions. PMID:11679356

  17. Hydrolysis and volatile fatty acids accumulation of waste activated sludge enhanced by the combined use of nitrite and alkaline pH.

    PubMed

    Huang, Cheng; Liu, Congcong; Sun, Xiuyun; Sun, Yinglu; Li, Rui; Li, Jiansheng; Shen, Jinyou; Han, Weiqing; Liu, Xiaodong; Wang, Lianjun

    2015-12-01

    Volatile fatty acids (VFAs) production from anaerobic digestion of waste activated sludge (WAS) is often limited by the slow hydrolysis and/or poor substrate availability. Increased attention has been given to enhance the hydrolysis and acidification of WAS recently. This study presented an efficient and green strategy based on the combined use of nitrite pretreatment and alkaline pH to stimulate hydrolysis and VFA accumulation from WAS. Results showed that both proteins and polysaccharides increased in the presence of nitrite, indicating the enhancement of sludge solubilization and hydrolysis processes. Mechanism investigations showed that nitrite pretreatment could disintegrate the sludge particle and disperse extracellular polymeric substances (EPS). Then, anaerobic digestion tests demonstrated VFA production increased with nitrite treatment. The maximal VFA accumulation was achieved with 0.1 g N/L nitrite dosage and pH 10.0 at a sludge retention time (SRT) of 7 days, which was much higher VFA production in comparison with the blank, sole nitrite pretreatment, or sole pH 10. The potential analysis suggested that the combined nitrite pretreatment and alkaline pH is capable of enhancing WAS digestion with a great benefit for biological nutrient removal (BNR).

  18. Asymmetric reduction of activated alkenes using an enoate reductase from Gluconobacter oxydans.

    PubMed

    Richter, Nina; Gröger, Harald; Hummel, Werner

    2011-01-01

    A recombinant enoate reductase from Gluconobacter oxydans was heterologously expressed, purified, characterised and applied in the asymmetric reduction of activated alkenes. In addition to the determination of the kinetic properties, the major focus of this work was to utilise the enzyme in the biotransformation of different interesting compounds such as 3,5,5-trimethyl-2-cyclohexen-1,4-dione (ketoisophorone) and (E/Z)-3,7-dimethyl-2,6-octadienal (citral). The reaction proceeded with excellent stereoselectivities (>99% ee) as well as absolute chemo- and regioselectivity, only the activated C=C bond of citral was reduced by the enoate reductase, while non-activated C=C bond and carbonyl moiety remained untouched. The described strategy can be used for the production of enantiomerically pure building blocks, which are difficult to prepare by chemical means. In general, the results show that the investigated enoate reductase is a promising catalyst for the use in asymmetric C=C bond reductions.

  19. Major Peptides from Amaranth (Amaranthus cruentus) Protein Inhibit HMG-CoA Reductase Activity

    PubMed Central

    Soares, Rosana Aparecida Manólio; Mendonça, Simone; de Castro, Luíla Ívini Andrade; Menezes, Amanda Caroline Cardoso Corrêa Carlos; Arêas, José Alfredo Gomes

    2015-01-01

    The objective of this study was to identify the major peptides generated by the in vitro hydrolysis of Amaranthus cruentus protein and to verify the effect of these peptides on the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), a key enzyme in cholesterol biosynthesis. A protein isolate was prepared, and an enzymatic hydrolysis that simulated the in vivo digestion of the protein was performed. After hydrolysis, the peptide mixture was filtered through a 3 kDa membrane. The peptide profile of this mixture was determined by reversed phase high performance chromatography (RP-HPLC), and the peptide identification was performed by LC-ESI MS/MS. Three major peptides under 3 kDa were detected, corresponding to more than 90% of the peptides of similar size produced by enzymatic hydrolysis. The sequences identified were GGV, IVG or LVG and VGVI or VGVL. These peptides had not yet been described for amaranth protein nor are they present in known sequences of amaranth grain protein, except LVG, which can be found in amaranth α‑amylase. Their ability to inhibit the activity of HMG-CoA reductase was determined, and we found that the sequences GGV, IVG, and VGVL, significantly inhibited this enzyme, suggesting a possible hypocholesterolemic effect. PMID:25690031

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

    PubMed

    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

  1. Novel prenylated bichalcone and chalcone from Humulus lupulus and their quinone reductase induction activities.

    PubMed

    Yu, Liyan; Zhang, Fuxian; Hu, Zhijuan; Ding, Hui; Tang, Huifang; Ma, Zhongjun; Zhao, Xiaofeng

    2014-03-01

    A new prenylated chalcone xanthohumol M (1), a novel prenylated bichalcone humulusol (2) and six known chalcones (3-8) were found from Humulus lupulus. Their structures were determined by spectroscopic methods. All the chalcones' electrophilic abilities were assessed by GSH (glutathione) rapid screening, and their QR (quinone reductase) induction activities were evaluated using hepa 1c1c7 cells. The results of electrophilic assay and QR induction activity assay were quite well. New compounds 1 and 2, along with some known prenylated chalcones, displayed certain QR induction activity.

  2. Testosterone 5alpha-reductase inhibitory active constituents of Piper nigrum leaf.

    PubMed

    Hirata, Noriko; Tokunaga, Masashi; Naruto, Shunsuke; Iinuma, Munekazu; Matsuda, Hideaki

    2007-12-01

    Previously we reported that Piper nigrum leaf extract showed a potent stimulation effect on melanogenesis and that (-)-cubebin (1) and (-)-3,4-dimethoxy-3,4-desmethylenedioxycubebin (2) were isolated as active constituents. As a part of our continuous studies on Piper species for the development of cosmetic hair-care agents, testosterone 5alpha-reductase inhibitory activity of aqueous ethanolic extracts obtained from several different parts of six Piper species, namely Piper nigrum, P. methysticum, P. betle, P. kadsura, P. longum, and P. cubeba, were examined. Among them, the extracts of P. nigrum leaf, P. nigrum fruit and P. cubeba fruit showed potent inhibitory activity. Activity-guided fractionation of P. nigrum leaf extract led to the isolation of 1 and 2. Fruits of P. cubeba contain 1 as a major lignan, thus inhibitory activity of the fruit may be attributable to 1. As a result of further assay on other known constituents of the cited Piper species, it was found that piperine, a major alkaloid amide of P. nigrum fruit, showed potent inhibitory activity, thus a part of the inhibitory activity of P. nigrum fruit may depend on piperine. The 5alpha-reductase inhibitory activities of 1 and piperine were found for the first time. In addition, the P. nigrum leaf extract showed in vivo anti-androgenic activity using the hair regrowth assay in testosterone sensitive male C57Black/6CrSlc strain mice.

  3. Voltammetric characterization of the aerobic energy-dissipating nitrate reductase of Paracoccus pantotrophus: exploring the activity of a redox-balancing enzyme as a function of electrochemical potential.

    PubMed

    Gates, Andrew J; Richardson, David J; Butt, Julea N

    2008-01-01

    Paracoccus pantotrophus expresses two nitrate reductases associated with respiratory electron transport, termed NapABC and NarGHI. Both enzymes derive electrons from ubiquinol to reduce nitrate to nitrite. However, while NarGHI harnesses the energy of the quinol/nitrate couple to generate a transmembrane proton gradient, NapABC dissipates the energy associated with these reducing equivalents. In the present paper we explore the nitrate reductase activity of purified NapAB as a function of electrochemical potential, substrate concentration and pH using protein film voltammetry. Nitrate reduction by NapAB is shown to occur at potentials below approx. 0.1 V at pH 7. These are lower potentials than required for NarGH nitrate reduction. The potentials required for Nap nitrate reduction are also likely to require ubiquinol/ubiquinone ratios higher than are needed to activate the H(+)-pumping oxidases expressed during aerobic growth where Nap levels are maximal. Thus the operational potentials of P. pantotrophus NapAB are consistent with a productive role in redox balancing. A Michaelis constant (K(M)) of approx. 45 muM was determined for NapAB nitrate reduction at pH 7. This is in line with studies on intact cells where nitrate reduction by Nap was described by a Monod constant (K(S)) of less than 15 muM. The voltammetric studies also disclosed maximal NapAB activity in a narrow window of potential. This behaviour is resistant to change of pH, nitrate concentration and inhibitor concentration and its possible mechanistic origins are discussed.

  4. Molecular Underpinnings of Nitrite Effect on CymA-Dependent Respiration in Shewanella oneidensis

    PubMed Central

    Jin, Miao; Fu, Huihui; Yin, Jianhua; Yuan, Jie; Gao, Haichun

    2016-01-01

    Shewanella exhibit a remarkable versatility of respiration, with a diverse array of electron acceptors (EAs). In environments where these bacteria thrive, multiple EAs are usually present. However, we know little about strategies by which these EAs and their interaction affect ecophysiology of Shewanella. In this study, we demonstrate in the model strain, Shewanella oneidensis MR-1, that nitrite, not through nitric oxide to which it may convert, inhibits respiration of fumarate, and probably many other EAs whose reduction depends on quinol dehydrogenase CymA. This is achieved via the repression of cyclic adenosine monophosphate (cAMP) production, a second messenger required for activation of cAMP-receptor protein (Crp) which plays a primary role in regulation of respiration. If nitrite is not promptly removed, intracellular cAMP levels drop, and this impairs Crp activity. As a result, the production of nitrite reductase NrfA, CymA, and fumarate reductase FccA is substantially reduced. In contrast, nitrite can be simultaneously respired with trimethylamine N-oxide, resulting in enhanced biomass. PMID:27493647

  5. Taxis response of various denitrifying bacteria to nitrate and nitrite.

    PubMed

    Lee, Dong Yun; Ramos, Adela; Macomber, Lee; Shapleigh, James P

    2002-05-01

    The taxis response of Rhodobacter sphaeroides 2.4.1 and 2.4.3, Rhodopseudomonas palustris, and Agrobacterium tumefaciens to nitrate and nitrite was evaluated by observing the macroscopic behavior of cells suspended in soft agar and incubated under various conditions. R. sphaeroides 2.4.3, which is capable of both nitrate and nitrite reduction, showed a taxis response to both nitrate and nitrite. R. sphaeroides 2.4.1, which contains nitrate reductase but not nitrite reductase, did not show a taxis response towards either nitrogen oxide. Insertional inactivation of the nitrite reductase structural gene or its transcriptional regulator, NnrR, in strain 2.4.3 caused a loss of a taxis response towards both nitrate and nitrite. An isolate of 2.4.1 carrying a copy of the nitrite reductase gene from 2.4.3 showed a taxis response to both nitrogen oxides. The taxis response of 2.4.3 was observed under anaerobic conditions, suggesting that the taxis response was due to nitrate and nitrite respiration, not to inhibition of oxygen respiration by respiration of nitrogen oxides. Strain 2.4.3 showed a taxis response to nitrate and nitrite under photosynthetic and aerobic conditions. Changing the carbon source in the culture medium caused an unexpected subtle shift in the taxis response of 2.4.3 to nitrite. A taxis response to nitrogen oxides was also observed in R. palustris and A. tumefaciens. R. palustris exhibited a taxis response to nitrite but not to nitrate, while A. tumefaciens exhibited a response to both compounds.

  6. Taxis Response of Various Denitrifying Bacteria to Nitrate and Nitrite

    PubMed Central

    Lee, Dong Yun; Ramos, Adela; Macomber, Lee; Shapleigh, James P.

    2002-01-01

    The taxis response of Rhodobacter sphaeroides 2.4.1 and 2.4.3, Rhodopseudomonas palustris, and Agrobacterium tumefaciens to nitrate and nitrite was evaluated by observing the macroscopic behavior of cells suspended in soft agar and incubated under various conditions. R. sphaeroides 2.4.3, which is capable of both nitrate and nitrite reduction, showed a taxis response to both nitrate and nitrite. R. sphaeroides 2.4.1, which contains nitrate reductase but not nitrite reductase, did not show a taxis response towards either nitrogen oxide. Insertional inactivation of the nitrite reductase structural gene or its transcriptional regulator, NnrR, in strain 2.4.3 caused a loss of a taxis response towards both nitrate and nitrite. An isolate of 2.4.1 carrying a copy of the nitrite reductase gene from 2.4.3 showed a taxis response to both nitrogen oxides. The taxis response of 2.4.3 was observed under anaerobic conditions, suggesting that the taxis response was due to nitrate and nitrite respiration, not to inhibition of oxygen respiration by respiration of nitrogen oxides. Strain 2.4.3 showed a taxis response to nitrate and nitrite under photosynthetic and aerobic conditions. Changing the carbon source in the culture medium caused an unexpected subtle shift in the taxis response of 2.4.3 to nitrite. A taxis response to nitrogen oxides was also observed in R. palustris and A. tumefaciens. R. palustris exhibited a taxis response to nitrite but not to nitrate, while A. tumefaciens exhibited a response to both compounds. PMID:11976082

  7. Genistein inhibits activities of methylenetetrahydrofolate reductase and lactate dehydrogenase, enzymes which use NADH as a substrate.

    PubMed

    Grabowski, Michał; Banecki, Bogdan; Kadziński, Leszek; Jakóbkiewicz-Banecka, Joanna; Kaźmierkiewicz, Rajmund; Gabig-Cimińska, Magdalena; Węgrzyn, Grzegorz; Węgrzyn, Alicja; Banecka-Majkutewicz, Zyta

    2015-09-25

    Genistein (5, 7-dihydroxy-3- (4-hydroxyphenyl)-4H-1-benzopyran-4-one) is a natural isoflavone revealing many biological activities. Thus, it is considered as a therapeutic compound in as various disorders as cancer, infections and genetic diseases. Here, we demonstrate for the first time that genistein inhibits activities of bacterial methylenetetrahydrofolate reductase (MetF) and lactate dehydrogenase (LDH). Both enzymes use NADH as a substrate, and results of biochemical as well as molecular modeling studies with MetF suggest that genistein may interfere with binding of this dinucleotide to the enzyme. These results have implications for our understanding of biological functions of genistein and its effects on cellular metabolism.

  8. Differential cytochrome content and reductase activity in Geospirillum barnesii strain SeS3

    USGS Publications Warehouse

    Stolz, J.F.; Gugliuzza, T.; Switzer, Blum J.; Oremland, R.; Martinez, Murillo F.

    1997-01-01

    The protein composition, cytochrome content, and reductase activity in the dissimilatory selenate-reducing bacterium Geospirillum barnesii strain SeS3, grown with thiosulfate, nitrate, selenate, or fumarate as the terminal electron acceptor, was investigated. Comparison of seven high-molecular-mass membrane proteins (105.3, 90.3, 82.6, 70.2, 67.4, 61.1, and 57.3 kDa) by SDS-PAGE showed that their detection was dependent on the terminal electron acceptor used. Membrane fractions from cells grown on thiosulfate contained a 70.2-kDa c-type cytochrome with absorbance maxima at 552, 522, and 421 nm. A 61.1-kDa c-type cytochrome with absorption maxima at 552, 523, and 423 nm was seen in membrane fractions from cells grown on nitrate. No c-type cytochromes were detected in membrane fractions of either selenate- or fumarate-grown cells. Difference spectra, however, revealed the presence of a cytochrome b554 (absorption maxima at 554, 523, and 422 nm) in membrane fractions from selenate-grown cells and a cytochrome b556 (absorption maxima at 556, 520, and 416 nm) in membrane fractions from fumarate-grown cells. Analysis of reductase activity in the different membrane fractions showed variability in substrate specificity. However, enzyme activity was greatest for the substrate on which the cells had been grown (e.g., membranes from nitrate-grown cells exhibited the greatest activity with nitrate). These results show that protein composition, cytochrome content, and reductase activity are dependent on the terminal electron acceptor used for growth.

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

    PubMed

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

    2010-07-16

    The reduction of nitrate to nitrite in the bacterial periplasm occurs in the 90kDa 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, 90kDa nitrate reductase active proteins were resolved by hydrophobic interaction chromatography from aerobic cultures of Shewanella gelidimarina replete with reduced nitrogen compounds. The 90kDa 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 90kDa 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.

  10. THB1 regulates nitrate reductase activity and THB1 and THB2 transcription differentially respond to NO and the nitrate/ammonium balance in Chlamydomonas

    PubMed Central

    Sanz-Luque, Emanuel; Ocaña-Calahorro, Francisco; Galván, Aurora; Fernández, Emilio

    2015-01-01

    Nitric oxide (NO) has emerged as an important regulator of the nitrogen assimilation pathway in plants. Nevertheless, this free radical is a double-edged sword for cells due to its high reactivity and toxicity. Hemoglobins, which belong to a vast and ancestral family of proteins present in all kingdoms of life, have arisen as important NO scavengers, through their NO dioxygenase (NOD) activity. The green alga Chlamydomonas reinhardtii has 12 hemoglobins (THB1–12) belonging to the truncated hemoglobins family. THB1 and THB2 are regulated by the nitrogen source and respond differentially to NO and the nitrate/ammonium balance. THB1 expression is upregulated by NO in contrast to THB2, which is downregulated. THB1 has NOD activity and thus a role in nitrate assimilation. In fact, THB1 is upregulated by nitrate and is under the control of NIT2, the major transcription factor in nitrate assimilation. In Chlamydomonas, it has been reported that nitrate reductase (NR) has a redox regulation and is inhibited by NO through an unknown mechanism. Now, a model in which THB1 interacts with NR is proposed for its regulation. THB1 takes electrons from NR redirecting them to NO dioxygenation. Thus, when cells are assimilating nitrate and NO appears (i.e. as a consequence of nitrite accumulation), THB1 has a double role: 1) to scavenge NO avoiding its toxic effects and 2) to control the nitrate reduction activity. PMID:26252500

  11. SIRT3-AMPK Activation by Nitrite and Metformin Improves Hyperglycemia and Normalizes Pulmonary Hypertension Associated with Heart Failure with Preserved Ejection Fraction (PH-HFpEF)

    PubMed Central

    Lai, Yen-Chun; Tabima, Diana M.; Dube, John J.; Hughan, Kara S.; Vanderpool, Rebecca R.; Goncharov, Dmitry A.; St Croix, Claudette M.; Garcia-Ocaña, Adolfo; Goncharova, Elena A.; Tofovic, Stevan P.; Mora, Ana L.; Gladwin, Mark T.

    2016-01-01

    Background Pulmonary hypertension associated with heart failure with preserved ejection fraction (PH-HFpEF) is an increasingly recognized clinical complication of metabolic syndrome. No adequate animal model of PH-HFpEF is available and no effective therapies have been identified to date. A recent study suggested that dietary nitrate improves insulin resistance in eNOS null mice, and multiple studies have reported that both nitrate and its active metabolite, nitrite, have therapeutic activity in pre-clinical models of PH. Methods and Results In order to evaluate the efficacy and mechanism of nitrite in metabolic syndrome associated with PH-HFpEF, we developed a “two-hit” PH-HFpEF model in rats with multiple features of metabolic syndrome due to double leptin receptor defect (obese ZSF1) with the combined treatment of VEGF receptor blocker SU5416. Chronic oral nitrite treatment improved hyperglycemia in obese ZSF1 rats by a process that requires skeletal muscle SIRT3-AMPK-GLUT4 signaling. The glucose lowering effect of nitrite was abolished in SIRT3 deficient human skeletal muscle cells, as well as in SIRT3 knockout mice fed a high-fat diet. Skeletal muscle biopsies from humans with metabolic syndrome after 12 weeks of oral sodium nitrite and nitrate treatment (IND#115926) displayed increased activation of SIRT3 and AMPK. Finally, early treatments with nitrite and metformin at the time of SU5416 injection reduced pulmonary pressures and vascular remodeling in the PH-HFpEF model with robust activation of skeletal muscle SIRT3 and AMPK. Conclusions These studies validate a rodent model of metabolic syndrome and PH-HFpEF, suggesting a potential role of nitrite and metformin as a preventative treatment for this disease. PMID:26813102

  12. Quinone Reductase 2 Is a Catechol Quinone Reductase

    SciTech Connect

    Fu, Yue; Buryanovskyy, Leonid; Zhang, Zhongtao

    2008-09-05

    The functions of quinone reductase 2 have eluded researchers for decades even though a genetic polymorphism is associated with various neurological disorders. Employing enzymatic studies using adrenochrome as a substrate, we show that quinone reductase 2 is specific for the reduction of adrenochrome, whereas quinone reductase 1 shows no activity. We also solved the crystal structure of quinone reductase 2 in complexes with dopamine and adrenochrome, two compounds that are structurally related to catecholamine quinones. Detailed structural analyses delineate the mechanism of quinone reductase 2 specificity toward catechol quinones in comparison with quinone reductase 1; a side-chain rotational difference between quinone reductase 1 and quinone reductase 2 of a single residue, phenylalanine 106, determines the specificity of enzymatic activities. These results infer functional differences between two homologous enzymes and indicate that quinone reductase 2 could play important roles in the regulation of catecholamine oxidation processes that may be involved in the etiology of Parkinson disease.

  13. Glyphosate effect on shikimate, nitrate reductase activity, yield, and seed composition in corn.

    PubMed

    Reddy, Krishna N; Bellaloui, Nacer; Zablotowicz, Robert M

    2010-03-24

    When glyphosate is applied to glyphosate-resistant (GR) crops, drift to nonglyphosate-resistant (non-GR) crops may cause significant injury and reduce yields. Tools are needed to quantify injury and predict crop losses. In this study, glyphosate drift was simulated by direct application at 12.5% of the recommended label rate to non-GR corn (Zea mays L.) at 3 or 6 weeks after planting (WAP) during two field seasons in the Mississippi delta region of the southeastern USA. Visual plant injury, shikimate accumulation, nitrate reductase activity, leaf nitrogen, yield, and seed composition were evaluated. Effects were also evaluated in GR corn and GR corn with stacked glufosinate-resistant gene at the recommended label rate at 3 and 6 WAP. Glyphosate at 105 g ae/ha was applied once at 3 or 6 weeks after planting to non-GR corn. Glyphosate at 840 (lower label limit) or 1260 (upper label limit) g ae/ha was applied twice at 3 and 6 WAP to transgenic corn. Glyphosate caused injury (45-55%) and increased shikimate levels (24-86%) in non-GR compared to nontreated corn. In non-GR corn, glyphosate drift did not affect starch content but increased seed protein 8-21% while reducing leaf nitrogen reductase activity 46-64%, leaf nitrogen 7-16%, grain yield 49-54%, and seed oil 18-23%. In GR and GR stacked with glufosinate-resistant corn, glyphosate applied at label rates did not affect corn yield, leaf and seed nitrogen, or seed composition (protein, oil, and starch content). Yet, nitrate reductase activity was reduced 5-19% with glyphosate at 840 + 840 g/ha rate and 8-42% with glyphosate at 1260 + 1260 g/ha rate in both GR and GR stacked corn. These results demonstrate the potential for severe yield loss in non-GR corn exposed to glyphosate drift.

  14. Adventitious Arsenate Reductase Activity of the Catalytic Domain of the Human Cdc25B and Cdc25C Phosphatases†

    PubMed Central

    Bhattacharjee, Hiranmoy; Sheng, Ju; Ajees, A. Abdul; Mukhopadhyay, Rita; Rosen, Barry P.

    2013-01-01

    A number of eukaryotic enzymes that function as arsenate reductases are homologues of the catalytic domain of the human Cdc25 phosphatase. For example, the Leishmania major enzyme LmACR2 is both a phosphatase and an arsenate reductase, and its structure bears similarity to the structure of the catalytic domain of human Cdc25 phosphatase. These reductases contain an active site C-X5-R signature motif, where C is the catalytic cysteine, the five X residues form a phosphate binding loop, and R is a highly conserved arginine, which is also present in human Cdc25 phosphatases. We therefore investigated the possibility that the three human Cdc25 isoforms might have adventitious arsenate reductase activity. The sequences for the catalytic domains of Cdc25A, -B, and -C were cloned individually into a prokaryotic expression vector, and their gene products were purified from a bacterial host using nickel affinity chromatography. While each of the three Cdc25 catalytic domains exhibited phosphatase activity, arsenate reductase activity was observed only with Cdc25B and -C. These two enzymes reduced inorganic arsenate but not methylated pentavalent arsenicals. Alteration of either the cysteine and arginine residues of the Cys-X5-Arg motif led to the loss of both reductase and phosphatase activities. Our observations suggest that Cdc25B and -C may adventitiously reduce arsenate to the more toxic arsenite and may also provide a framework for identifying other human protein tyrosine phosphatases containing the active site Cys-X5-Arg loop that might moonlight as arsenate reductases. PMID:20025242

  15. Fatty acyl-CoA inhibition of beta-hydroxy-beta-methylglutaryl-CoA reductase activity.

    PubMed

    Faas, F H; Carter, W J; Wynn, J O

    1978-11-22

    The influence of the fatty acyl-CoA thioesters on rat liver microsomal hydroxymethylglutaryl-CoA reductase activity was tested in vitro to determine if the previously demonstrated inhibition of [14C]acetate incorporation into cholesterol is due to inhibition of this rate limiting step in cholesterol synthesis. The polyunsaturated fatty acyl-CoA thioesters caused the greatest inhibition of enzyme activity, 50 micron arachidonoyl-CoA inhibiting 67% and 5 micron inhibiting 22%. 50 micron linoleoyl-CoA inhibited 56% with the more saturated thioesters causing less inhibition. 50--100 micron free fatty acids, free CoA, cholesterol esters, phospholipids, carnitine derivatives, prostaglandins and non-specific detergents caused little or no inhibition of enzyme activity. Kinetic studies revealed the inhibition to be noncompetitive with respect to hydroxymethylglutaryl-CoA with a Ki for arachidonoyl CoA of 3.10 micron. Fatty acyl-CoA inhibition of in vitro cholesterol synthesis is due to inhibition of hydroxymethylglutaryl-CoA reductase activity. Variation in intracellular concentrations of fatty acyl-CoA thioesters may signficantly alter cholesterol synthesis.

  16. NADH-dependent decavanadate reductase, an alternative activity of NADP-specific isocitrate dehydrogenase protein.

    PubMed

    Rao, A V; Ramasarma, T

    2000-05-01

    The well known NADP-specific isocitrate dehydrogenase (IDH) obtained from pig heart was found to oxidize NADH with accompanying consumption of oxygen (NADH:O(2)=1:1) in presence of polyvanadate. This activity of the soluble IDH-protein has the following features common with the previously described membrane-enzymes: heat-sensitive, active only with NADH but not NADPH, increased rates in acidic pH, dependence on concentrations of the enzyme, NADH, decavanadate and metavanadate (the two constituents of polyvanadate), and sensitivity to SOD and EDTA. Utilizing NADH as the electron source the IDH protein was able to reduce decavanadate but not metavanadate. This reduced form of vanadyl (V(IV)) was similar in its eight-band electron spin resonance spectrum to vanadyl sulfate but had a 20-fold higher absorbance at its 700 nm peak. This decavanadate reductase activity of the protein was sensitive to heat and was not inhibited by SOD and EDTA. The IDH protein has the additional enzymic activity of NADH-dependent decavanadate reductase and is an example of "one protein--many functions".

  17. Dietary nitrates, nitrites, and cardiovascular disease.

    PubMed

    Hord, Norman G

    2011-12-01

    Dietary nitrate (NO(3)), nitrite (NO(2)), and arginine can serve as sources for production of NO(x) (a diverse group of metabolites including nitric oxide, nitrosothiols, and nitroalkenes) via ultraviolet light exposure to skin, mammalian nitrate/nitrite reductases in tissues, and nitric oxide synthase enzymes, respectively. NO(x) are responsible for the hypotensive, antiplatelet, and cytoprotective effects of dietary nitrates and nitrites. Current regulatory limits on nitrate intakes, based on concerns regarding potential risk of carcinogenicity and methemoglobinemia, are exceeded by normal daily intakes of single foods, such as soya milk and spinach, as well as by some recommended dietary patterns such as the Dietary Approaches to Stop Hypertension diet. This review includes a call for regulatory bodies to consider all available data on the beneficial physiologic roles of nitrate and nitrite in order to derive rational bases for dietary recommendations.

  18. Arabidopsis nitrate reductase activity is stimulated by the E3 SUMO ligase AtSIZ1

    PubMed Central

    Park, Bong Soo; Song, Jong Tae; Seo, Hak Soo

    2011-01-01

    Small ubiquitin-related modifier (SUMO) is a small polypeptide that modulates protein activity and regulates hormone signalling, abiotic and biotic responses in plants. Here we show that AtSIZ regulates nitrogen assimilation in Arabidopsis through its E3 SUMO ligase function. Dwarf plants of siz1-2 flower early, show abnormal seed development and have high salicylic acid content and enhanced resistance to bacterial pathogens. These mutant phenotypes are reverted to wild-type phenotypes by exogenous ammonium but not by nitrate, phosphate or potassium. Decreased nitrate reductase activity in siz1-2 plants resulted in low nitrogen concentrations, low nitric oxide production and high nitrate content in comparison with wild-type plants. The nitrate reductases, NIA1 and NIA2, are sumoylated by AtSIZ1, which dramatically increases their activity. Both sumoylated and non-sumoylated NIA1 and NIA2 can form dimers. Our results indicate that AtSIZ1 positively controls nitrogen assimilation by promoting sumoylation of NRs in Arabidopsis. PMID:21772271

  19. Copper removal ability by Streptomyces strains with dissimilar growth patterns and endowed with cupric reductase activity.

    PubMed

    Albarracín, Virginia Helena; Avila, Ana Lucía; Amoroso, María Julia; Abate, Carlos Mauricio

    2008-11-01

    Morphological, physiological and molecular characterization of three copper-resistant actinobacterial strains (AB2A, AB3 and AB5A) isolated from copper-polluted sediments of a drainage channel showed that they belonged to the genus Streptomyces. These characteristics plus their distinctive copper resistance phenotypes revealed considerable divergence among the isolates. Highly dissimilar growth patterns and copper removal efficiency were observed for the selected Streptomyces strains grown on minimal medium (MM) added with 0.5 mM of copper sulfate (MM(Cu)). Strain AB2A showed an early mechanism of copper uptake/retention (80% until day 3), followed by a drastic metal efflux process (days 5-7). In contrast, Streptomyces sp. AB3 and AB5A showed only copper retention phenotypes under the same culture conditions. Particularly, Streptomyces sp. AB5A showed a better efficiency in copper removal (94%), although a longer lag phase was observed for this microorganism grown for 7 days in MM(Cu). Cupric reductase activity was detected in both copper-adapted cells and nonadapted cells of all three strains but this activity was up to 100-fold higher in preadapted cells of Streptomyces sp. AB2A. To our knowledge, this is the first time that cupric reductase activity was demonstrated in Streptomyces strains.

  20. Triazine-benzimidazole hybrids: anticancer activity, DNA interaction and dihydrofolate reductase inhibitors.

    PubMed

    Singla, Prinka; Luxami, Vijay; Paul, Kamaldeep

    2015-04-15

    A new series of triazine-benzimidazole hybrids has been synthesized with different substitution of primary and secondary amines at one of the position of triazine in moderate to good yields. These compounds were evaluated for their inhibitory activities over 60 human tumor cell lines at one dose and five dose concentrations. Compounds 6b, 8 and 9 showed broad spectrum of antitumor activities with GI50 values of 9.79, 2.58 and 3.81μM, respectively. DNA binding studies also indicated strong interaction properties of these compounds. These synthesized compounds also showed inhibition of mammalian dihydrofolate reductase (DHFR). Compound 6b was depicted as the most active member of DHFR inhibitor with IC50 value of 1.05μM. Molecular modelling studies were used to identify the stabilized interactions of Compound 6b within the active site of enzyme for DHFR.

  1. Consequence of absence of nitrate reductase activity on photosynthesis in Nicotiana plumbaginifolia plants

    SciTech Connect

    Saux, C.; Lemoine, Y.; Marion-Poll, A.; Valadier, M.H.; Deng, M.; Morot-Gaudry, J.F.

    1987-05-01

    Chlorate-resistant Nicotiana plumbaginifolia (cv Viviani) mutants were found to be deficient in the nitrate reductase apoprotein (NR/sup -/ nia). Because they could not grow with nitrate as sole nitrogen source, they were cultivated as graftings on wild-type Nicotiana tabacum plants. The grafts of mutant plants were chlorotic compared to the grafts of wild type. Mutant leaves did not accumulate nitrogen but contained less malate and more glutamine than wild leaves. They exhibited a slight increase of the proportion of the light-harvesting chlorophyll a/b protein complexes and a lowering of the efficiency of energy transfer between these complexes and the active centers. After a 3 second /sup 14/CO/sub 2/ pulse, the total /sup 14/C incorporation of the mutant leaves was approximately 20/sup 5/ of that of the control. The /sup 14/C was essentially recovered in ribulose bisphosphate in these plants. It was consistent with a decline of ribulose bisphosphate carboxylase activity observed in the mutant. After a 3 second /sup 14/CO/sub 2/ pulse followed by a 60 second chase with normal CO/sub 2/, /sup 14/C was mainly accumulated in starch which was labeled more in the mutant than in the wild type. These results confirm the observation that in the nitrate reductase deficient leaves, chloroplasts were loaded with large starch inclusions preceding disorganization of the photosynthetic apparatus.

  2. Identification of a Noroxomaritidine Reductase with Amaryllidaceae Alkaloid Biosynthesis Related Activities.

    PubMed

    Kilgore, Matthew B; Holland, Cynthia K; Jez, Joseph M; Kutchan, Toni M

    2016-08-05

    Amaryllidaceae alkaloids are a large group of plant natural products with over 300 documented structures and diverse biological activities. Several groups of Amaryllidaceae alkaloids including the hemanthamine- and crinine-type alkaloids show promise as anticancer agents. Two reduction reactions are required for the production of these compounds: the reduction of norcraugsodine to norbelladine and the reduction of noroxomaritidine to normaritidine, with the enantiomer of noroxomaritidine dictating whether the derivatives will be the crinine-type or hemanthamine-type. It is also possible for the carbon-carbon double bond of noroxomaritidine to be reduced, forming the precursor for maritinamine or elwesine depending on the enantiomer reduced to an oxomaritinamine product. In this study, a short chain alcohol dehydrogenase/reductase that co-expresses with the previously discovered norbelladine 4'-O-methyltransferase from Narcissus sp. and Galanthus spp. was cloned and expressed in Escherichia coli Biochemical analyses and x-ray crystallography indicates that this protein functions as a noroxomaritidine reductase that forms oxomaritinamine from noroxomaritidine through a carbon-carbon double bond reduction. The enzyme also reduces norcraugsodine to norbelladine with a 400-fold lower specific activity. These studies identify a missing step in the biosynthesis of this pharmacologically important class of plant natural products.

  3. Ferric and cupric reductase activities by iron-limited cells of the green alga Chlorella kessleri: quantification via oxygen electrode.

    PubMed

    Weger, Harold G; Walker, Crystal N; Fink, Michael B

    2007-10-01

    The colorimetric Fe2+ indicators bathophenanthroline disulfonic acid (BPDS) and 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine (FZ) are routinely used to assay for plasma membrane ferric reductase activity in iron-limited algal cells and also in roots from iron-limited plants. Ferric reductase assays using these colorimetric indicators must take into account the fact that Fe3+ chelators (e.g. ethylenediaminetetraacetic acid) can also in general bind Fe2+ and may therefore compete with the colorimetric Fe2+ indicators, leading to the potential for underestimation of the ferric reduction rate. Conversely, the presence of BPDS or FZ may also facilitate the reduction of Fe3+ chelates, potentially leading to overestimation of ferric reduction rates. Last, both BPDS and FZ have non-negligible affinities for Fe3+ in addition to their well-known affinities for Fe2+; this leads to potential difficulties in ascertaining whether free and/or chelated Fe3+ are potential substrates for the ferric reductase. Similar issues arise when assaying for cupric reductase activity using the colorimetric Cu+ indicator bathocuproinedisulfonic acid (BCDS). In this paper, we describe an oxygen-electrode-based assay (conducted in darkness) for both ferric and cupric reductase activities that does not use colorimetric indicators. Using this assay system, we show that the plasma membrane metal reductase activity of iron-limited cells of the green alga Chlorella kessleri reduced complexed Fe3+ (i.e. Fe3+ chelates) but did not reduce free (non-chelated) Fe3+, and also reduced free Cu2+ to Cu+, but did not reduce Cu2+ that was part of Cu2+ chelates. We suggest that the potential for reduction of free Fe3+ cannot be adequately assayed using colorimetric assays. As well, the BPDS-based assay system consistently yielded similar estimates of ferric reductase activity compared with the O2-electrode-based assays at relatively low Fe3+ concentration, but higher estimates at higher Fe3

  4. A dual system formed by the ARC and NR molybdoenzymes mediates nitrite-dependent NO production in Chlamydomonas.

    PubMed

    Chamizo-Ampudia, Alejandro; Sanz-Luque, Emanuel; Llamas, Ángel; Ocaña-Calahorro, Francisco; Mariscal, Vicente; Carreras, Alfonso; Barroso, Juan B; Galván, Aurora; Fernández, Emilio

    2016-10-01

    Nitric oxide (NO) is a relevant signal molecule involved in many plant processes. However, the mechanisms and proteins responsible for its synthesis are scarcely known. In most photosynthetic organisms NO synthases have not been identified, and Nitrate Reductase (NR) has been proposed as the main enzymatic NO source, a process that in vitro is also catalysed by other molybdoenzymes. By studying transcriptional regulation, enzyme approaches, activity assays with in vitro purified proteins and in vivo and in vitro NO determinations, we have addressed the role of NR and Amidoxime Reducing Component (ARC) in the NO synthesis process. N\\R and ARC were intimately related both at transcriptional and activity level. Thus, arc mutants showed high NIA1 (NR gene) expression and NR activity. Conversely, mutants without active NR displayed an increased ARC expression in nitrite medium. Our results with nia1 and arc mutants and with purified enzymes support that ARC catalyses the NO production from nitrite taking electrons from NR and not from Cytb5-1/Cytb5-Reductase, the component partners previously described for ARC (proposed as NOFNiR, Nitric Oxide-Forming Nitrite Reductase). This NR-ARC dual system would be able to produce NO in the presence of nitrate, condition under which NR is unable to do it.

  5. Nitrite reduction in paracoccus halodenitrificans: Evidence for the role of a cd-type cytochrome in ammonia formation

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.; Cronin, S. E.

    1984-01-01

    Cell-free extracts prepared from Paracoccus halodenitrificans catalyzed the reduction of nitrate to ammonia in the presence of dithionite and methyl viologen. Enzyme activity was located in the soluble fraction and was associated with a cytochrome whose spectral properties resembled those of a cd-type cytochrome. Unlike the sissimilatory cd-cytochrome nitrate reductase associated with the membrane fraction of P. halodenitrificans, this soluble cd-cytochrome did not reduce nitrite to nitrous oxide.

  6. Discovery of potent and novel S-nitrosoglutathione reductase inhibitors devoid of cytochrome P450 activities.

    PubMed

    Sun, Xicheng; Qiu, Jian; Strong, Sarah A; Green, Louis S; Wasley, Jan W F; Blonder, Joan P; Colagiovanni, Dorothy B; Mutka, Sarah C; Stout, Adam M; Richards, Jane P; Rosenthal, Gary J

    2011-10-01

    The pyrrole based N6022 was recently identified as a potent, selective, reversible, and efficacious S-nitrosoglutathione reductase (GSNOR) inhibitor and is currently undergoing clinical development for the treatment of acute asthma. GSNOR is a member of the alcohol dehydrogenase family (ADH) and regulates the levels of S-nitrosothiols (SNOs) through catabolism of S-nitrosoglutathione (GSNO). Reduced levels of GSNO, as well as other nitrosothiols (SNOs), have been implicated in the pathogenesis of many diseases including those of the respiratory, cardiovascular, and gastrointestinal systems. Preservation of endogenous SNOs through GSNOR inhibition presents a novel therapeutic approach with broad applicability. We describe here the synthesis and structure-activity relationships (SAR) of novel pyrrole based analogues of N6022 focusing on removal of cytochrome P450 inhibition activities. We identified potent and novel GSNOR inhibitors having reduced CYP inhibition activities and demonstrated efficacy in a mouse ovalbumin (OVA) model of asthma.

  7. Two interacting binding sites for quinacrine derivatives in the active site of trypanothione reductase – a template for drug design

    PubMed Central

    Saravanamuthu, Ahilan; Vickers, Tim J.; Bond, Charles S.; Peterson, Mark R.; Hunter, William N.; Fairlamb, Alan H.

    2012-01-01

    SUMMARY Trypanothione reductase is a key enzyme in the trypanothione-based redox metabolism of pathogenic trypanosomes. Since this system is absent in humans, being replaced with glutathione and glutathione reductase, it offers a target for selective inhibition. The rational design of potent inhibitors requires accurate structures of enzyme-inhibitor complexes, but this is lacking for trypanothione reductase. We therefore used quinacrine mustard, an alkylating derivative of the competitive inhibitor quinacrine, to probe the active site of this dimeric flavoprotein. Quinacrine mustard irreversibly inactivates Trypanosoma cruzi trypanothione reductase, but not human glutathione reductase, in a time-dependent manner with a stoichiometry of two inhibitors bound per monomer. The rate of inactivation is dependent upon the oxidation state of trypanothione reductase, with the NADPH-reduced form being inactivated significantly faster than the oxidised form. Inactivation is slowed by clomipramine and a melarsen oxide-trypanothione adduct (both are competitive inhibitors) but accelerated by quinacrine. The structure of the trypanothione reductase-quinacrine mustard adduct was determined to 2.7 Å, revealing two molecules of inhibitor bound in the trypanothione-binding site. The acridine moieties interact with each other through π-stacking effects, and one acridine interacts in a similar fashion with a tryptophan residue. These interactions provide a molecular explanation for the differing effects of clomipramine and quinacrine on inactivation by quinacrine mustard. Synergism with quinacrine occurs as a result of these planar acridines being able to stack together in the active site cleft, thereby gaining an increased number of binding interactions, whereas antagonism occurs with non-planar molecules, such as clomipramine, where stacking is not possible. PMID:15102853

  8. Thioredoxin reductase.

    PubMed Central

    Mustacich, D; Powis, G

    2000-01-01

    The mammalian thioredoxin reductases (TrxRs) are a family of selenium-containing pyridine nucleotide-disulphide oxidoreductases with mechanistic and sequence identity, including a conserved -Cys-Val-Asn-Val-Gly-Cys- redox catalytic site, to glutathione reductases. TrxRs catalyse the NADPH-dependent reduction of the redox protein thioredoxin (Trx), as well as of other endogenous and exogenous compounds. The broad substrate specificity of mammalian TrxRs is due to a second redox-active site, a C-terminal -Cys-SeCys- (where SeCys is selenocysteine), that is not found in glutathione reductase or Escherichia coli TrxR. There are currently two confirmed forms of mammalian TrxRs, TrxR1 and TrxR2, and it is possible that other forms will be identified. The availability of Se is a key factor determining TrxR activity both in cell culture and in vivo, and the mechanism(s) for the incorporation of Se into TrxRs, as well as the regulation of TrxR activity, have only recently begun to be investigated. The importance of Trx to many aspects of cell function make it likely that TrxRs also play a role in protection against oxidant injury, cell growth and transformation, and the recycling of ascorbate from its oxidized form. Since TrxRs are able to reduce a number of substrates other than Trx, it is likely that additional biological effects will be discovered for TrxR. Furthermore, inhibiting TrxR with drugs may lead to new treatments for human diseases such as cancer, AIDS and autoimmune diseases. PMID:10657232

  9. Crystal structures of manganese- and cobalt-substituted myoglobin in complex with NO and nitrite reveal unusual ligand conformations.

    PubMed

    Zahran, Zaki N; Chooback, Lilian; Copeland, Daniel M; West, Ann H; Richter-Addo, George B

    2008-02-01

    Nitrite is now recognized as a storage pool of bioactive nitric oxide (NO). Hemoglobin (Hb) and myoglobin (Mb) convert, under certain conditions, nitrite to NO. This newly discovered nitrite reductase activity of Hb and Mb provides an attractive alternative to mammalian NO synthesis from the NO synthase pathway that requires dioxygen. We recently reported the X-ray crystal structure of the nitrite adduct of ferric horse heart Mb, and showed that the nitrite ligand binds in an unprecedented O-binding (nitrito) mode to the d(5) ferric center in Mb(III)(ONO) [D.M. Copeland, A. Soares, A.H. West, G.B. Richter-Addo, J. Inorg. Biochem. 100 (2006) 1413-1425]. We also showed that the distal pocket in Mb allows for different conformations of the NO ligand (120 degrees and 144 degrees ) in Mb(II)NO depending on the mode of preparation of the compound. In this article, we report the crystal structures of the nitrite and NO adducts of manganese-substituted hh Mb (a d(4) system) and of the nitrite adduct of cobalt-substituted hh Mb (a d(6) system). We show that the distal His64 residue directs the nitrite ligand towards the rare nitrito O-binding mode in Mn(III)Mb and Co(III)Mb. We also report that the distal pocket residues allow a stabilization of an unprecendented bent MnNO moiety in Mn(II)MbNO. These crystal structural data, when combined with the data for the aquo, methanol, and azide MnMb derivatives, provide information on the role of distal pocket residues in the observed binding modes of nitrite and NO ligands to wild-type and metal-substituted Mb.

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

    PubMed

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

    2014-04-01

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

  11. Evaluation of Giardia lamblia thioredoxin reductase as drug activating enzyme and as drug target.

    PubMed

    Leitsch, David; Müller, Joachim; Müller, Norbert

    2016-12-01

    The antioxidative enzyme thioredoxin reductase (TrxR) has been suggested to be a drug target in several pathogens, including the protist parasite Giardia lamblia. TrxR is also believed to catalyse the reduction of nitro drugs, e.g. metronidazole and furazolidone, a reaction required to render these compounds toxic to G. lamblia and other microaerophiles/anaerobes. It was the objective of this study to assess the potential of TrxR as a drug target in G. lamblia and to find direct evidence for the role of this enzyme in the activation of metronidazole and other nitro drugs. TrxR was overexpressed approximately 10-fold in G. lamblia WB C6 cells by placing the trxR gene behind the arginine deiminase (ADI) promoter on a plasmid. Likewise, a mutant TrxR with a defective disulphide reductase catalytic site was strongly expressed in another G. lamblia WB C6 cell line. Susceptibilities to five antigiardial drugs, i.e. metronidazole, furazolidone, nitazoxanide, albendazole and auranofin were determined in both transfectant cell lines and compared to wildtype. Further, the impact of all five drugs on TrxR activity in vivo was measured. Overexpression of TrxR rendered G. lamblia WB C6 more susceptible to metronidazole and furazolidone but not to nitazoxanide, albendazole, and auranofin. Of all five drugs tested, only auranofin had an appreciably negative effect on TrxR activity in vivo, albeit to a much smaller extent than expected. Overexpression of TrxR and mutant TrxR had hardly any impact on growth of G. lamblia WB C6, although the enzyme also exerts a strong NADPH oxidase activity which is a source of oxidative stress. Our results constitute first direct evidence for the notion that TrxR is an activator of metronidazole and furazolidone but rather question that it is a relevant drug target of presently used antigiardial drugs.

  12. Purification, properties, and sequence of glycerol trinitrate reductase from Agrobacterium radiobacter.

    PubMed Central

    Snape, J R; Walkley, N A; Morby, A P; Nicklin, S; White, G F

    1997-01-01

    Glycerol trinitrate (GTN) reductase, which enables Agrobacterium radiobacter to utilize GTN and related explosives as sources of nitrogen for growth, was purified and characterized, and its gene was cloned and sequenced. The enzyme was a 39-kDa monomeric protein which catalyzed the NADH-dependent reductive scission of GTN (Km = 23 microM) to glycerol dinitrates (mainly the 1,3-isomer) with a pH optimum of 6.5, a temperature optimum of 35 degrees C, and no dependence on metal ions for activity. It was also active on pentaerythritol tetranitrate (PETN), on isosorbide dinitrate, and, very weakly, on ethyleneglycol dinitrate, but it was inactive on isopropyl nitrate, hexahydro-1,3,5-trinitro-1,3,5-triazine, 2,4,6-trinitrotoluene, ammonium ions, nitrate, or nitrite. The amino acid sequence deduced from the DNA sequence was homologous (42 to 51% identity and 61 to 69% similarity) to those of PETN reductase from Enterobacter cloacae, N-ethylmaleimide reductase from Escherichia coli, morphinone reductase from Pseudomonas putida, and old yellow enzyme from Saccharomyces cerevisiae, placing the GTN reductase in the alpha/beta barrel flavoprotein group of proteins. GTN reductase and PETN reductase were very similar in many respects except in their distinct preferences for NADH and NADPH cofactors, respectively. PMID:9401040

  13. Esculetin, a Coumarin Derivative, Inhibits Aldose Reductase Activity in vitro and Cataractogenesis in Galactose-Fed Rats

    PubMed Central

    Kim, Chan-Sik; Kim, Junghyun; Lee, Yun Mi; Sohn, Eunjin; Kim, Jin Sook

    2016-01-01

    Naturally occurring coumarin compounds have received substantial attention due to their pharmaceutical effects. Esculetin is a coumarin derivative and a polyphenol compound that is used in a variety of therapeutic and pharmacological strategies. However, its effect on aldose reductase activity remains poorly understood. In this study, the potential beneficial effects of esculetin on lenticular aldose reductase were investigated in galactose-fed (GAL) rats, an animal model of sugar cataracts. Cataracts were induced in Sprague-Dawley (SD) rats via a 50% galactose diet for 2 weeks, and groups of GAL rats were orally treated with esculetin (10 or 50 mg/kg body weight). In vehicle-treated GAL rats, lens opacification was observed, and swelling and membrane rupture of the lens fiber cells were increased. Additionally, aldose reductase was highly expressed in the lens epithelium and superficial cortical fibers during cataract development in the GAL rats. Esculetin reduced rat lens aldose reductase (RLAR) activity in vitro, and esculetin treatment significantly inhibited lens opacity, as well as morphological alterations, such as swelling, vacuolation and liquefaction of lens fibers, via the inhibition of aldose reductase in the GAL rats. These results indicate that esculetin is a useful treatment for galactose-induced cataracts. PMID:26902086

  14. NADP(+)-dependent dehydrogenase activity of carbonyl reductase on glutathionylhydroxynonanal as a new pathway for hydroxynonenal detoxification.

    PubMed

    Moschini, Roberta; Peroni, Eleonora; Rotondo, Rossella; Renzone, Giovanni; Melck, Dominique; Cappiello, Mario; Srebot, Massimo; Napolitano, Elio; Motta, Andrea; Scaloni, Andrea; Mura, Umberto; Del-Corso, Antonella

    2015-06-01

    An NADP(+)-dependent dehydrogenase activity on 3-glutathionyl-4-hydroxynonanal (GSHNE) was purified to electrophoretic homogeneity from a line of human astrocytoma cells (ADF). Proteomic analysis identified this enzymatic activity as associated with carbonyl reductase 1 (EC 1.1.1.184). The enzyme is highly efficient at catalyzing the oxidation of GSHNE (KM 33 µM, kcat 405 min(-1)), as it is practically inactive toward trans-4-hydroxy-2-nonenal (HNE) and other HNE-adducted thiol-containing amino acid derivatives. Combined mass spectrometry and nuclear magnetic resonance spectroscopy analysis of the reaction products revealed that carbonyl reductase oxidizes the hydroxyl group of GSHNE in its hemiacetal form, with the formation of the corresponding 3-glutathionylnonanoic-δ-lactone. The relevance of this new reaction catalyzed by carbonyl reductase 1 is discussed in terms of HNE detoxification and the recovery of reducing power.

  15. Hexavalent Chromate Reductase Activity in Cell Free Extracts of Penicillium sp.

    PubMed Central

    Arévalo-Rangel, Damaris L.; Cárdenas-González, Juan F.; Martínez-Juárez, Víctor M.; Acosta-Rodríguez, Ismael

    2013-01-01

    A chromium-resistant fungus isolated from contaminated air with industrial vapors can be used for reducing toxic Cr(VI) to Cr(III). This study analyzes in vitro reduction of hexavalent chromium using cell free extract(s) of the fungus that was characterized based on optimal temperature, pH, use of electron donors, metal ions and initial Cr(VI) concentration in the reaction mixture. This showed the highest activity at 37°C and pH 7.0; there is an increase in Cr(VI) reductase activity with addition of NADH as an electron donor, and it was highly inhibited by Hg2+, Ca2+ and Mg2+, and azide, EDTA, and KCN. PMID:24027493

  16. Structural Mechanism of Allosteric Activity Regulation in a Ribonucleotide Reductase with Double ATP Cones.

    PubMed

    Johansson, Renzo; Jonna, Venkateswara Rao; Kumar, Rohit; Nayeri, Niloofar; Lundin, Daniel; Sjöberg, Britt-Marie; Hofer, Anders; Logan, Derek T

    2016-06-07

    Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides. Their overall activity is stimulated by ATP and downregulated by dATP via a genetically mobile ATP cone domain mediating the formation of oligomeric complexes with varying quaternary structures. The crystal structure and solution X-ray scattering data of a novel dATP-induced homotetramer of the Pseudomonas aeruginosa class I RNR reveal the structural bases for its unique properties, namely one ATP cone that binds two dATP molecules and a second one that is non-functional, binding no nucleotides. Mutations in the observed tetramer interface ablate oligomerization and dATP-induced inhibition but not the ability to bind dATP. Sequence analysis shows that the novel type of ATP cone may be widespread in RNRs. The present study supports a scenario in which diverse mechanisms for allosteric activity regulation are gained and lost through acquisition and evolutionary erosion of different types of ATP cone.

  17. A plasma membrane-bound enzyme of tobacco roots catalyses the formation of nitric oxide from nitrite.

    PubMed

    Stöhr, C; Strube, F; Marx, G; Ullrich, W R; Rockel, P

    2001-04-01

    Purified plasma membranes (PMs) of tobacco (Nicotiana tabacum L. cv. Samsun) roots exhibited a nitrite-reducing enzyme activity that resulted in nitric oxide (NO) formation. This enzyme activity was not detected in soluble protein fractions or in PM vesicles of leaves. At the pH optimum of pH 6.0, nitrite was reduced to NO with reduced cytochrome c as electron donor at a rate comparable to the nitrate-reducing activity of root-specific succinate-dependent PM-bound nitrate reductase (PM-NR). The hitherto unknown PM-bound nitrite: NO-reductase (NI-NOR) was insensitive to cyanide and anti-NR IgG and thereby proven to be different from PM-NR. Furthermore, PM-NR and NI-NOR were separated by gel-filtration chromatography and apparent molecular masses of 310 kDa for NI-NOR and 200 kDa for PM-NR were estimated. The PM-associated NI-NOR may reduce the apoplastic nitrite produced by PM-NR in vivo and may play a role in nitrate signalling via NO formation.

  18. The emerging role of nitrite as an endogenous modulator and therapeutic agent of cardiovascular function.

    PubMed

    Tota, B; Quintieri, A M; Angelone, T

    2010-01-01

    Recently, the circulating anion nitrite (NO2-), the largest physiological reservoir of nitric oxide (NO) in the body, has revealed itself as a signalling molecule mediating numerous biological responses. Since it was estimated that as much as 70% of plasma nitrite originates from nitric oxide synthases (NOSs), mainly in the endothelium by endothelial NOS, nitrite is considered an index of NOSs activity. Exogenous sources, principally environmental pollutants and intake of vegetables, also contribute to this NO reserve. In mammalian blood, nitrite, present at nanomolar concentrations, can be reduced to bioactive NO along a physiological oxygen and pH gradient either non-enzymatically (acidic disproportionation) or by a number of enzymes including xanthine oxidoreductase, NOS, mitochondrial cytochromes and deoxygenated haemoglobin and myoglobin. The various NO-dependent nitrite-induced biological responses include hypoxic vasodilation, inhibition of mitochondrial respiration, cytoprotection following ischemia/reperfusion, and regulation of protein and gene expression. Since NO is a major paracrine-autocrine cardiovascular modulator and nitrite acts mainly as an endocrine store of NO, it is not surprising that NO2 - exerts important cardiovascular actions both under normal and physio-pathological conditions. In the interdisciplinary framework of the NO cycle concept, this review illustrates the actions exerted by nitrite on the cardiovascular system. Since the majority of the NO2 - -oriented studies focused on the systemic and regional control of blood flow both under physiological and ischemia/reperfusion conditions, we will firstly consider this issue. Secondly, the nitrite- induced effects on myocardial contractile and relaxation processes will be discussed, emphasizing the biomedical interest of nitrite as a new therapeutic agent. The importance of cardiac myoglobin as nitrite-reductase able to exert cardioprotection through a novel function, in addition to its

  19. Structure-activity relationship for enantiomers of potent inhibitors of B. anthracis dihydrofolate reductase

    PubMed Central

    Bourne, Christina R.; Wakeham, Nancy; Nammalwar, Baskar; Tseitin, Vladimir; Bourne, Philip C.; Barrow, Esther W.; Mylvaganam, Shankari; Ramnarayan, Kal; Bunce, Richard A.; Berlin, K. Darrell; Barrow, William W.

    2012-01-01

    Background Bacterial resistance to antibiotic therapies is increasing and new treatment options are badly needed. There is an overlap between these resistant bacteria and organisms classified as likely bioterror weapons. For example, Bacillus anthracis is innately resistant to the anti-folate trimethoprim due to sequence changes found in the dihydrofolate reductase enzyme. Development of new inhibitors provides an opportunity to enhance the current arsenal of anti-folate antibiotics while also expanding the coverage of the anti-folate class. Methods We have characterized inhibitors of Bacillus anthracis dihydrofolate reductase by measuring the Ki and MIC values and calculating the energetics of binding. This series contains a core diaminopyrimidine ring, a central dimethoxybenzyl ring, and a dihydrophthalazine moiety. We have altered the chemical groups extended from a chiral center on the dihydropyridazine ring of the phthalazine moiety. The interactions for the most potent compounds were visualized by X-ray structure determination. Results We find that the potency of individual enantiomers is divergent with clear preference for the S-enantiomer, while maintaining a high conservation of contacts within the binding site. The preference for enantiomers seems to be predicated largely by differential interactions with protein residues Leu29, Gln30 and Arg53. Conclusions These studies have clarified the activity of modifications and of individual enantiomers, and highlighted the role of the less-active R-enantiomer in effectively diluting the more active S-enantiomer in racemic solutions. This directly contributes to the development of new antimicrobials, combating trimethoprim resistance, and treatment options for potential bioterrorism agents. PMID:22999981

  20. Nitrite in feed: From Animal health to human health

    SciTech Connect

    Cockburn, Andrew; Brambilla, Gianfranco; Fernández, Maria-Luisa; Arcella, Davide; Peteghem, Carlos van; Dorne, Jean-Lou

    2013-08-01

    Nitrite is widely consumed from the diet by animals and humans. However the largest contribution to exposure results from the in vivo conversion of exogenously derived nitrate to nitrite. Because of its potential to cause to methaemoglobin (MetHb) formation at excessive levels of intake, nitrite is regulated in feed and water as an undesirable substance. Forages and contaminated water have been shown to contain high levels of nitrate and represent the largest contributor to nitrite exposure for food-producing animals. Interspecies differences in sensitivity to nitrite intoxication principally result from physiological and anatomical differences in nitrite handling. In the case of livestock both pigs and cattle are relatively susceptible. With pigs this is due to a combination of low levels of bacterial nitrite reductase and hence potential to reduce nitrite to ammonia as well as reduced capacity to detoxify MetHb back to haemoglobin (Hb) due to intrinsically low levels of MetHb reductase. In cattle the sensitivity is due to the potential for high dietary intake and high levels of rumen conversion of nitrate to nitrite, and an adaptable gut flora which at normal loadings shunts nitrite to ammonia for biosynthesis. However when this escape mechanism gets overloaded, nitrite builds up and can enter the blood stream resulting in methemoglobinemia. Looking at livestock case histories reported in the literature no-observed-effect levels of 3.3 mg/kg body weight (b.w.) per day for nitrite in pigs and cattle were estimated and related to the total daily nitrite intake that would result from complete feed at the EU maximum permissible level. This resulted in margins of safety of 9-fold and 5-fold for pigs and cattle, respectively. Recognising that the bulkiness of animal feed limits their consumption, these margins in conjunction with good agricultural practise were considered satisfactory for the protection of livestock health. A human health risk assessment was also

  1. Methionine sulfoxide reductase regulates brain catechol-O-methyl transferase activity

    PubMed Central

    Moskovitz, Jackob; Walss-Bass, Consuelo; Cruz, Dianne A; Thompson, Peter M.; Bortolato, Marco

    2015-01-01

    Catechol-O-methyl transferase (COMT) plays a key role in the degradation of brain dopamine (DA). Specifically, low COMT activity results in higher DA levels in the prefrontal cortex (PFC), thereby reducing the vulnerability for attentional and cognitive deficits in both psychotic and healthy individuals. COMT activity is markedly reduced by a non-synonymous SNP that generates a valine-to-methionine substitution on the residue 108/158, by means of as-yet incompletely understood posttranslational mechanisms. One posttranslational modification is methionine sulfoxide, which can be reduced by the methionine sulfoxide reductase (Msr) A and B enzymes. We used recombinant COMT proteins (Val/Met108) and mice (wild-type (WT) and MsrA knockout) to determine the effect of methionine oxidation on COMT activity and COMT interaction with Msr, through a combination of enzymatic activity and Western blot assays. Recombinant COMT activity is positively regulated by MsrA, especially under oxidative conditions, while brains of MsrA knockout mice exhibited lower COMT activity (as compared with their WT counterparts). These results suggest that COMT activity may be reduced by methionine oxidation, and point to Msr as a key molecular determinant for the modulation of COMT activity in the brain. The role of Msr in modulating cognitive functions in healthy individuals and schizophrenia patients is yet to be determined. PMID:24735585

  2. Structure-activity relationship of pyrrole based S-nitrosoglutathione reductase inhibitors: carboxamide modification.

    PubMed

    Sun, Xicheng; Qiu, Jian; Strong, Sarah A; Green, Louis S; Wasley, Jan W F; Blonder, Joan P; Colagiovanni, Dorothy B; Stout, Adam M; Mutka, Sarah C; Richards, Jane P; Rosenthal, Gary J

    2012-03-15

    The enzyme S-nitrosoglutathione reductase (GSNOR) is a member of the alcohol dehydrogenase family (ADH) that regulates the levels of S-nitrosothiols (SNOs) through catabolism of S-nitrosoglutathione (GSNO). GSNO and SNOs are implicated in the pathogenesis of many diseases including those in respiratory, gastrointestinal, and cardiovascular systems. The pyrrole based N6022 was recently identified as a potent, selective, reversible, and efficacious GSNOR inhibitor which is currently in clinical development for acute asthma. We describe here the synthesis and structure-activity relationships (SAR) of novel pyrrole based analogs of N6022 focusing on carboxamide modifications on the pendant N-phenyl moiety. We have identified potent and novel GSNOR inhibitors that demonstrate efficacy in an ovalbumin (OVA) induced asthma model in mice.

  3. Utilization of iron-catecholamine complexes involving ferric reductase activity in Listeria monocytogenes.

    PubMed Central

    Coulanges, V; Andre, P; Ziegler, O; Buchheit, L; Vidon, D J

    1997-01-01

    Listeria monocytogenes is a ubiquitous potentially pathogenic organism requiring iron for growth and virulence. Although it does not produce siderophores, L. monocytogenes is able to obtain iron by using either exogenous siderophores produced by various microorganisms or natural catechol compounds widespread in the environment. In the presence of tropolone, an iron-chelating agent, growth of L. monocytogenes is completely inhibited. However, the growth inhibition can be relieved by the addition of dopamine or norepinephrine under their different isomeric forms, while the catecholamine derivatives 4-hydroxy-3-methoxyphenylglycol and normetanephrine did not relieve the inhibitory effect of tropolone. Preincubation of L. monocytogenes with chlorpromazine and yohimbine did not antagonize the growth-promoting effect of catecholamines in iron-complexed medium. In addition, norepinephrine stimulated the growth-promoting effect induced by human transferrin in iron-limited medium. Furthermore, dopamine and norepinephrine allowed 55Fe uptake by iron-deprived bacterial cells. The uptake of iron was energy dependent, as indicated by inhibition of 55Fe uptake at 0 degrees C as well as by preincubating the bacteria with KCN. Inhibition of 55Fe uptake by L. monocytogenes was also observed in the presence of Pt(II). Moreover, when assessed by a whole-cell ferric reductase assay, reductase activity of L. monocytogenes was inhibited by Pt(II). These data demonstrate that dopamine and norepinephrine can function as siderophore-like compounds in L. monocytogenes owing to their ortho-diphenol function and that catecholamine-mediated iron acquisition does not involve specific catecholamine receptors but acts through a cell-bound ferrireductase activity. PMID:9199450

  4. Disappearance of chloramines in the presence of bromide and nitrite. [Ammoniacal monochloramine, diethylchloramine, and chloramines produced by chlorinating a real and synthetic secondary (activated sludge) municipal waste effluent

    SciTech Connect

    Valentine, R.L.

    1982-01-01

    Batch experiments were used to study the reduction of chloramines in the presence of bromide and nitrite. Chloramines studies were ammoniacal monochloramine, diethylchloramine (DECA), and those produced by chlorinating a real and synthetic secondary (activated sludge) municipal waste effluent. Oxidant concentrations were measured using the DPD-FAS (N,N-diethyl-p-phenylenediamine, Ferrous Ammonium Sulfate) titrimetric procedure and/or spectrophotometrically. The degradation of NH/sub 2/Cl in the presence of bromide was found to occur via a mechanism consistent with a rate limiting step involving monochlorammonium ion (NH/sub 3/Cl/sup +/) and bromide ion. Experimental evidence suggests that the mixed haloamine, NHBrCl, was produced as an unstable intermediate. The oxidation of bromide by DECA did not occur by a mechanism similar to that describing the oxidation of bromide by NH/sub 2/Cl. The rate was not affected by added ammonia and was slower than that observed for comparable NH/sub 2/Cl-Br/sup -/ reactions. Chloramine loss in organic rich effluents was greatly accelerated by bromide addition. The reaction is not dependent on excess ammonia and is slower than that observed for a pure NH/sub 2/Cl-Br/sup -/ solution. Monochloramine can rapidly disappear in the presence of nitrite. The rates are too fast to be due solely to the hydrolysis of monochloramine. The presence of relatively small concentrations of nitrite can greatly accelerate the loss of NH/sub 2/Cl in the presence of bromide. Nitrite is not significantly consumed. Nitrite appears to increase the rate of bromide oxidation in a parallel acid catalyzed reaction mechanism which involves a rate limiting step described by a first order dependence on nitrite but no dependence on bromide. Empirical rate expressions and rate constants were determined for each reaction. 54 figures, 17 tables.

  5. Influence of Estimated Training Status on Anti and Pro-Oxidant Activity, Nitrite Concentration, and Blood Pressure in Middle-Aged and Older Women

    PubMed Central

    Jacomini, André M.; Dias, Danielle da Silva; Brito, Janaina de Oliveira; da Silva, Roberta F.; Monteiro, Henrique L.; Llesuy, Susana; De Angelis, Kátia; Amaral, Sandra L.; Zago, Anderson S.

    2017-01-01

    The purpose of this study was to compare the association between anti and pro-oxidant activity, nitrite concentration, and blood pressure (BP) in middle-aged and older women with different levels of estimated training status (TS). The sample consisted of 155 females (50–84 years) who were submitted to a physical examination to evaluate estimated TS through the “Functional Fitness Battery Test,” BP measurements, and plasma blood samples to evaluate pro-oxidant and antioxidant activity and nitrite concentrations. Participants were separated by age into a middle-aged group (<65 years) and an older (≥65 years) group and then subdivided in each group according to TS. Blood biochemistry was similar between groups. On the other hand, protein oxidation was lower in participants with higher TS, independent of age. Older females with higher TS presented higher nitrite concentrations, lower lipoperoxidation, and lower values of BP compared with those with lower TS. Lower GPx activity was observed in participants with higher TS compared with middle-aged with lower TS. Thus, our results suggest that good levels of TS may be associated with lower oxidative stress and higher nitrite concentration and may contribute to maintain normal or reduced blood pressure values. PMID:28326041

  6. Influence of long-term diesel fuel pollution on nitrite-oxidising activity and population size of nitrobacter spp in soil.

    PubMed

    Deni, Jamal; Penninckx, Michel J

    2004-01-01

    Previous investigations have shown that ammonia oxidation is not inhibited by diesel fuel in a soil with a long history of contamination contrary to a non-contaminated soil. As a consequence, ammonia oxidation does not constitute a Limited step in nitrification process (Appl. Environ. Microbiol. 65 (1999) 4008). Moreover, this type of soil also has had the opportunity to develop an abundant microbial population able to metabolise the diesel hydrocarbons. Whether the properties of soil with a long history of diesel fuel contamination may affect the activity of nitrite-oxidising bacteria was investigated. It was observed that re-exposure of soil to diesel fuel apparently stimulated the proliferation of nitrite-oxidising bacteria, as determined by most probable number (MPN) culture technique and MPN-polymerase chain reaction technique. The potential of nitrite-oxidising activity in soil treated with diesel fuel was about 4 times higher than in the control without addition. In the presence of diesel fuel and ammonium, the potential nitrite-oxidising activity was 40% higher than in presence of ammonium only. However, in the presence of hydrocarbon only, low proliferation of Nitrobacter was observed, probably because the heterotrophic bacteria were strongly limited by lack of nitrogen and did not produce sufficient organic metabolites that could be used by the Nitrobacter cells.

  7. Phytochemical Analysis of Agrimonia pilosa Ledeb, Its Antioxidant Activity and Aldose Reductase Inhibitory Potential

    PubMed Central

    Kim, Set Byeol; Hwang, Seung Hwan; Suh, Hong-Won; Lim, Soon Sung

    2017-01-01

    The aim of this study was to determine aldose reductase (AR) inhibitory activity and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity of compounds from Agrimonia pilosa Ledeb (AP). We isolated agrimoniin (AM), four flavonoid glucosides and two flavonoid glucuronides from the n-butanol fraction of AP 50% methanol extract. In addition to isolated compounds, the AR-inhibitory activity and the DPPH free radical scavenging activity of catechin, 5-flavonoids, and 4-flavonoid glucosides (known components of AP) against rat lens AR (RLAR) and DPPH assay were measured. AM showed IC50 values of 1.6 and 13.0 μM against RLAR and DPPH scavenging activity, respectively. Additionally, AM, luteolin-7-O-glucuronide (LGN), quercitrin (QU), luteolin (LT) and afzelin (AZ) showed high inhibitory activity against AR and were first observed to decrease sorbitol accumulation in the rat lens under high-sorbitol conditions ex vivo with inhibitory values of 47.6%, 91.8%, 76.9%, 91.8% and 93.2%, respectively. Inhibition of recombinant human AR by AM, LGN and AZ exhibited a noncompetitive inhibition pattern. Based on our results, AP and its constituents may play partial roles in RLAR and oxidative radical inhibition. Our results suggest that AM, LGN, QU, LT and AZ may potentially be used as natural drugs for treating diabetic complications. PMID:28208627

  8. Activities of nitrate reductase and glutamine synthetase in rice seedlings during cyanide metabolism.

    PubMed

    Yu, Xiao-Zhang; Zhang, Fu-Zhong

    2012-07-30

    A study was conducted to investigate activities of nitrate reductase (NR) and glutamine synthetase (GS) in plants during cyanide metabolism. Young rice seedlings (Oryza sativa L. cv. XZX 45) were grown in the nutrient solutions containing KNO(3) or NH(4)Cl and treated with free cyanide (KCN). Cyanide in solutions and in plant materials was analyzed to estimate the phyto-assimilation potential. Activities of NR and GS in different parts of rice seedlings were assayed in vivo. Seedlings grown on NH(4)(+) showed significantly higher relative growth rate than those on NO(3)(-) (p<0.05) in the presence of exogenous cyanide. The metabolic rates of cyanide by seedlings were all positively correlated to the concentrations supplied. A negligible difference was observed between the two treatments with nitrate and ammonium (p>0.05). Enzymatic assays showed that cyanide (≥0.97mg CN L(-1)) impaired NR activity significantly in both roots and shoots (p<0.05). The effect of cyanide on GS activity in roots was more evident at 1.93mg CN L(-1), suggesting that NR activity was more susceptible to change from cyanide application than GS activity. The results observed here suggest that the exogenous cyanide, which to a certain level has a beneficial role in plant nutrition.

  9. The Thiol Reductase Activity of YUCCA6 Mediates Delayed Leaf Senescence by Regulating Genes Involved in Auxin Redistribution

    PubMed Central

    Cha, Joon-Yung; Kim, Mi R.; Jung, In J.; Kang, Sun B.; Park, Hee J.; Kim, Min G.; Yun, Dae-Jin; Kim, Woe-Yeon

    2016-01-01

    Auxin, a phytohormone that affects almost every aspect of plant growth and development, is biosynthesized from tryptophan via the tryptamine, indole-3-acetamide, indole-3-pyruvic acid, and indole-3-acetaldoxime pathways. YUCCAs (YUCs), flavin monooxygenase enzymes, catalyze the conversion of indole-3-pyruvic acid (IPA) to the auxin (indole acetic acid). Arabidopsis thaliana YUC6 also exhibits thiol-reductase and chaperone activity in vitro; these activities require the highly conserved Cys-85 and are essential for scavenging of toxic reactive oxygen species (ROS) in the drought tolerance response. Here, we examined whether the YUC6 thiol reductase activity also participates in the delay in senescence observed in YUC6-overexpressing (YUC6-OX) plants. YUC6 overexpression delays leaf senescence in natural and dark-induced senescence conditions by reducing the expression of SENESCENCE-ASSOCIATED GENE 12 (SAG12). ROS accumulation normally occurs during senescence, but was not observed in the leaves of YUC6-OX plants; however, ROS accumulation was observed in YUC6-OXC85S plants, which overexpress a mutant YUC6 that lacks thiol reductase activity. We also found that YUC6-OX plants, but not YUC6-OXC85S plants, show upregulation of three genes encoding NADPH-dependent thioredoxin reductases (NTRA, NTRB, and NTRC), and GAMMA-GLUTAMYLCYSTEINE SYNTHETASE 1 (GSH1), encoding an enzyme involved in redox signaling. We further determined that excess ROS accumulation caused by methyl viologen treatment or decreased glutathione levels caused by buthionine sulfoximine treatment can decrease the levels of auxin efflux proteins such as PIN2-4. The expression of PINs is also reduced in YUC6-OX plants. These findings suggest that the thiol reductase activity of YUC6 may play an essential role in delaying senescence via the activation of genes involved in redox signaling and auxin availability. PMID:27242830

  10. Regulation of rat liver hydroxymethylglutaryl coenzyme A reductase by a new class of noncompetitive inhibitors. Effects of dichloroacetate and related carboxylic acids on enzyme activity.

    PubMed Central

    Stacpoole, P W; Harwood, H J; Varnado, C E

    1983-01-01

    Dichloroacetate (DCA) markedly reduces circulating cholesterol levels in animals and in patients with combined hyperlipoproteinemia or homozygous familial hypercholesterolemia (FH). To investigate the mechanism of its cholesterol-lowering action, we studied the effects of DCA and its hepatic metabolites, glyoxylate and oxalate, on the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) obtained from livers of healthy, reverse light-cycled rats. Oral administration of DCA for 4 d decreased HMG CoA reductase activity 46% at a dose of 50 mg/kg per d, and 82% at a dose of 100 mg/kg per d. A 24% decrease in reductase activity was observed as early as 1 h after a single dose of 50 mg/kg DCA. The inhibitory effect of the drug was due to a fall in both expressed enzyme activity and the total number of reductase molecules present. DCA also decreased reductase activity when added to suspensions of isolated hepatocytes. With chronic administration, DCA inhibited 3H2O incorporation into cholesterol by 38% and into triglycerides by 52%. When liver microsomes were incubated with DCA, the pattern of inhibition of reductase activity was noncompetitive for both HMG CoA (inhibition constant [Ki] 11.8 mM) and NADPH (Ki 11.6 mM). Inhibition by glyoxylate was also noncompetitive for both HMG CoA (Ki 1.2 mM) and NADPH (Ki 2.7 mM). Oxalate inhibited enzyme activity only at nonsaturating concentrations of NADPH (Ki 5.6 mM). Monochloroacetate, glycollate, and ethylene glycol, all of which can form glyoxylate, also inhibited reductase activity. Using solubilized and 60-fold purified HMG CoA reductase, we found that the inhibitory effect of glyoxylate was reversible. Furthermore, the inhibition by glyoxylate was an effect exerted on the reductase itself, rather than on its regulatory enzymes, reductase kinase and reductase phosphatase. We conclude that the cholesterol-lowering effect of DCA is mediated, at least in part, by inhibition of endogenous cholesterol

  11. An ene reductase from Clavispora lusitaniae for asymmetric reduction of activated alkenes.

    PubMed

    Ni, Yan; Yu, Hui-Lei; Lin, Guo-Qiang; Xu, Jian-He

    2014-03-05

    A putative ene reductase gene from Clavispora lusitaniae was heterologously overexpressed in Escherichia coli, and the encoded protein (ClER) was purified and characterized for its biocatalytic properties. This NADPH-dependent flavoprotein was identified with reduction activities toward a diverse range of activated alkenes including conjugated enones, enals, maleimide derivative and α,β-unsaturated carboxylic esters. The purified ClER exhibited a relatively high activity of 7.3 U mg(prot)⁻¹ for ketoisophorone while a remarkable catalytic efficiency (k(cat)/K(m)=810 s⁻¹ mM⁻¹) was obtained for 2-methyl-cinnamaldehyde due to the high affinity. A series of prochiral activated alkenes were stereoselectively reduced by ClER furnishing the corresponding saturated products in up to 99% ee. The practical applicability of ClER was further evaluated for the production of (R)-levodione, a valuable chiral compound, from ketoisophorone. Using the crude enzyme of ClER and glucose dehydrogenase (GDH), 500 mM of ketoisophorone was efficiently converted to (R)-levodione with excellent stereoselectivity (98% ee) within 1h. All these positive features demonstrate a high synthetic potential of ClER in the asymmetric reduction of activated alkenes.

  12. The contribution of bacteroidal nitrate and nitrite reduction to the formation of nitrosylleghaemoglobin complexes in soybean root nodules.

    PubMed

    Meakin, Georgina E; Bueno, Emilio; Jepson, Brian; Bedmar, Eulogio J; Richardson, David J; Delgado, María J

    2007-02-01

    It is becoming recognized that leghaemoglobin constitutes an important buffer for the cytotoxic nitric oxide radical (NO(*)) in root nodules, although the sources of this NO(*) within nodules are unclear. In Bradyrhizobium japonicum bacteroids, NO(*) can be produced through the denitrification process, during which nitrate is reduced to nitrite by the periplasmic nitrate reductase Nap, and nitrite is reduced to NO(*) by the respiratory nitrite reductase NirK. To assess the contribution of bacteroidal denitrification to the NO(*) within nitrate-treated soybean nodules, electron paramagnetic resonance and UV-visible spectroscopy were employed to study the presence of nitrosylleghaemoglobin (LbNO) within nodules from plants inoculated with wild-type, napA or nirK B. japonicum strains. Since it has been found that hypoxia induces NO(*) production in plant root tissue, and that plant roots can be subjected to hypoxic stress during drought and flooding, the effect of hypoxic stress on the formation of LbNO complexes within nodules was also investigated. Maximal levels of LbNO were observed in nodules from plants treated with nitrate and subjected to hypoxic conditions. It is shown that, in the presence of nitrate, all of the LbNO within normoxic nodules arises from nitrate reduction by the bacteroidal periplasmic nitrate reductase, whereas Nap activity is only responsible for half of the LbNO within hypoxic nodules. In contrast to Nap, NirK is not essential for LbNO formation under any condition tested.

  13. The haem-copper oxygen reductase of Desulfovibrio vulgaris contains a dihaem cytochrome c in subunit II.

    PubMed

    Lobo, Susana A L; Almeida, Claúdia C; Carita, João N; Teixeira, Miguel; Saraiva, Lígia M

    2008-12-01

    The genome of the sulphate reducing bacterium Desulfovibrio vulgaris Hildenborough, still considered a strict anaerobe, encodes two oxygen reductases of the bd and haem-copper types. The haem-copper oxygen reductase deduced amino acid sequence reveals that it is a Type A2 enzyme, which in its subunit II contains two c-type haem binding motifs. We have characterized the cytochrome c domain of subunit II and confirmed the binding of two haem groups, both with Met-His iron coordination. Hence, this enzyme constitutes the first example of a ccaa3 haem-copper oxygen reductase. The expression of D. vulgaris haem-copper oxygen reductase was found to be independent of the electron donor and acceptor source and is not altered by stress factors such as oxygen exposure, nitrite, nitrate, and iron; therefore the haem-copper oxygen reductase seems to be constitutive. The KCN sensitive oxygen reduction by D. vulgaris membranes demonstrated in this work indicates the presence of an active haem-copper oxygen reductase. D. vulgaris membranes perform oxygen reduction when accepting electrons from the monohaem cytochrome c553, thus revealing the first possible electron donor to the terminal oxygen reductase of D. vulgaris. The physiological implication of the presence of the oxygen reductase in this organism is discussed.

  14. Nitrite accumulation under constant temperature in anoxic denitrification process: The effects of carbon sources and COD/NO(3)-N.

    PubMed

    Ge, Shijian; Peng, Yongzhen; Wang, Shuying; Lu, Congcong; Cao, Xu; Zhu, Yunpeng

    2012-06-01

    Effects of external carbon sources and COD/NO(3)-N on nitrite accumulation through denitrification were studied at a temperature of 28±2.0 °C using mixed activated sludge. Nitrite accumulation was observed for each type of carbon source studied. Glucose resulted in the greatest nitrite accumulation and production rate, which were 14.51±2.41 mg/L and 0.121±0.013 g N/(g VSS d), respectively. Moreover, a higher COD/NO(3)-N ratio ranging from 1.0 to 15.0 increased accumulation to the maximum value of 0.34±0.03 g N/(g VSS d). It was assumed that the competition for electrons between nitrite reductase and nitrate reductase led to different reduction rates and finally caused the accumulation. In addition, it was reasonable to use the pH and ORP as proxies for monitoring the real endpoint of the denitrification process with the addition of carbon sources.

  15. Tomato fruit ascorbic acid content is linked with monodehydroascorbate reductase activity and tolerance to chilling stress.

    PubMed

    Stevens, R; Page, D; Gouble, B; Garchery, C; Zamir, D; Causse, M

    2008-08-01

    Quantitative trait loci (QTL) mapping is a step towards the identification of factors regulating traits such as fruit ascorbic acid content. A previously identified QTL controlling variations in tomato fruit ascorbic acid has been fine mapped and reveals that the QTL has a polygenic and epistatic architecture. A monodehydroascorbate reductase (MDHAR) allele is a candidate for a proportion of the increase in fruit ascorbic acid content. The MDHAR enzyme is active in different stages of fruit ripening, shows increased activity in the introgression lines containing the wild-type (Solanum pennellii) allele, and responds to chilling injury in tomato along with the reduced/oxidized ascorbate ratio. Low temperature storage of different tomato introgression lines with all or part of the QTL for ascorbic acid and with or without the wild MDHAR allele shows that enzyme activity explains 84% of the variation in the reduced ascorbic acid levels of tomato fruit following storage at 4 degrees C, compared with 38% at harvest under non-stress conditions. A role is indicated for MDHAR in the maintenance of ascorbate levels in fruit under stress conditions. Furthermore, an increased fruit MDHAR activity and a lower oxidation level of the fruit ascorbate pool are correlated with decreased loss of firmness because of chilling injury.

  16. Effect of dietary supplementation of vitamin C on growth, reactive oxygen species, and antioxidant enzyme activity of Apostichopus japonicus (Selenka) juveniles exposed to nitrite

    NASA Astrophysics Data System (ADS)

    Luo, Zuoyong; Wang, Baojie; Liu, Mei; Jiang, Keyong; Liu, Mingxing; Wang, Lei

    2014-07-01

    Different amounts of vitamin C were added to diets fed to juveniles (2.5 ± 0.15 g) of sea cucumber Apostichopus japonic u s (Selenka) in an attempt to reduce the stress response of specimens exposed to nitrite stress. A commercial feed was used as the control diet and three experimental diets were made by supplementing 1 000, 1 500, or 2 000 mg vitamin C/kg diet to control diet separately in a 45-day experiment. Sea cucumbers were exposed to three different levels (0.5, 1.0, and 1.5 mg/L) of nitrite stress for 4, 8, and 12 h at four time intervals (0, 15, 30, and 45 d). Growth of the animals was recorded during the experiment. Reactive oxygen species (ROS) (i.e. hydroxyl free radical (-OH), malondialdehyde (MDA) and total antioxidant capacity (T-AOC)) and antioxidant enzyme activities (i.e., superoxide dismutase (SOD) and catalase (CAT)) were measured. Response surface methodology (RSM) was used to analyze the effect of multiple factors on ROS indices and enzyme activities. Weight gain (WG) and special growth rate (SGR) of vitamin C supplementation groups were significantly higher than those of control group ( P < 0.05). The levels of -OH and MDA increased under exposure time extending and nitrite concentration increasing, whereas T-AOC level decreased. SOD and CAT activities increased at 4 h and 8 h and decreased at 12 h. During the days in which the animal consumed experimental diets, the levels of -OH and MDA decreased and that of T-AOC increased. This result suggests that diets containing vitamin C could reduce the nitrite stress response in the animals and increase their antioxidant capacity. The multifactor regression equation of growth performance, ROS indices, and duration of feeding results suggest that vitamin C supplementation of 1 400-2 000 mg/kg diet for 29-35 days could reduce effectively the effects of nitrite exposure.

  17. Nitrite, nitrite alternatives, and the control of Clostridium botulinum in cured meats.

    PubMed

    Pierson, M D; Smoot, L A

    1982-01-01

    Historically, nitrite has been a component of meat-curing additives for several centuries. In recent years the safety of nitrite as an additive in cured meats has been questioned mainly because of the possible formation of carcinogenic nitrosamines. Nitrite has many important functions in meat curing including its role in color development, flavor, antioxidant properties, and antimicrobial activity. The inhibition of Clostridium botulinum growth and toxin production is an especially important antimicrobial property of nitrite. This review discusses the effects of processing, curing ingredients (especially nitrite), and storage of cured meats in relation to the control of C. botulinum. If nitrite is eliminated from cured meats or the level of usage decreased, then alternatives for the antibotulinal function of nitrite need to be considered. Several potential alternatives including sorbates, parabens, and biological acidulants are discussed.

  18. Rapid suppression of 7-dehydrocholesterol reductase activity in keratinocytes by vitamin D.

    PubMed

    Zou, Ling; Porter, Todd D

    2015-04-01

    7-Dehydrocholesterol (7DHC) serves as the sterol substrate for both cholesterol and vitamin D3 (cholecalciferol) synthesis. The pivotal enzyme in these two pathways is 7-dehydrocholesterol reductase (DHCR7), which converts 7DHC to cholesterol. Treatment of adult human epidermal keratinocytes (HEKa) with 10μM cholecalciferol resulted in a rapid decrease in DHCR7 activity (19% of control activity at 2h). This loss of activity was observed only in HEKa cells, a primary cell line cultured from normal human skin, and not in an immortalized skin cell line (HaCaT cells) nor in two hepatoma cell lines. The decrease in DHCR7 activity was not due to direct inhibition or to dephosphorylation of the enzyme, and enzyme protein levels were not decreased. 25-Hydroxyvitamin D3 had a lesser effect on DHCR7 activity, while 1α,25-dihydroxyvitamin D3 had no effect on DHCR7, indicating that the vitamin D receptor is not involved. Treatment with cholecalciferol did not lead to the accumulation of 7-dehydrocholesterol, and a 50% decrease in lanosterol synthesis in these cells suggests that cholecalciferol down-regulates the entire cholesterolgenic pathway. As vitamin D has been reported to be an inhibitor of hedgehog (Hh) signaling through Smo, we tested the effect of cyclopamine, an established inhibitor of the Hh pathway, on DHCR7 activity. Cyclopamine (10μM) also rapidly decreased DHCR7 activity (50% of control activity at 3h), suggesting that vitamin D3 may modulate DHCR7 activity and cholesterol/vitamin D3 synthesis by inhibiting hedgehog signaling. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

  19. 21 CFR 181.34 - Sodium nitrite and potassium nitrite.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium nitrite and potassium nitrite. 181.34... nitrite and potassium nitrite. Sodium nitrite and potassium nitrite are subject to prior sanctions issued... without sodium or potassium nitrate, in the curing of red meat and poultry products....

  20. Bioactive fraction of Saraca indica prevents diabetes induced cataractogenesis: An aldose reductase inhibitory activity

    PubMed Central

    Somani, Gauresh; Sathaye, Sadhana

    2015-01-01

    Background: The present study was designed to investigate the effect of Saraca indica (SI) flowers extract and different bioactive fraction on in vitro aldose reductase (AR) inhibitory activity, high glucose-induced cataract in goat lens and in vivo streptozotocin (STZ; 45 mg/kg, i.p) induced cataract in rats. Methods: Extract of flowers of SI tested for inhibition against rat lens AR. Furthermore, bioactive fraction was investigated against high glucose-induced opacification of the lens in vitro lens culture and STZ induced diabetic cataract in rats. Identification of the bioactive component was attempted through high-performance thin-layer chromatography, high-performance liquid chromatography and liquid chromatography-mass spectrometry analysis. Results: Ethyl acetate fraction of S. indica (EASI) produced maximum inhibition that may be due to high phenolic content. Goat lenses in media containing glucose developed a distinctly opaque ring in 72 h and treatment with EASI fraction lowered lens opacity in 72 h. Prolonged treatment with EASI to STZ-induced diabetic rats inhibited the AR activity and delayed cataract progression in a dose dependent manner. Conclusion: Ethyl acetate fraction of S. indica fraction has potential to inhibit rat lens AR enzyme and prevent cataractogenesis not only in goat lens model (in vitro), but also in STZ induced diabetic rats (in vivo). This study is suggestive of the anticataract activity of EASI fraction that could be attributed to the phytoconstituents present in the same. PMID:25709218

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

    PubMed

    Rodriguez, Gabriel M; Atsumi, Shota

    2014-09-01

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

  2. Exogenous methyl jasmonate treatment increases glucosinolate biosynthesis and quinone reductase activity in kale leaf tissue.

    PubMed

    Ku, Kang-Mo; Jeffery, Elizabeth H; Juvik, John A

    2014-01-01

    Methyl jasmonate (MeJA) spray treatments were applied to the kale varieties 'Dwarf Blue Curled Vates' and 'Red Winter' in replicated field plantings in 2010 and 2011 to investigate alteration of glucosinolate (GS) composition in harvested leaf tissue. Aqueous solutions of 250 µM MeJA were sprayed to saturation on aerial plant tissues four days prior to harvest at commercial maturity. The MeJA treatment significantly increased gluconasturtiin (56%), glucobrassicin (98%), and neoglucobrassicin (150%) concentrations in the apical leaf tissue of these genotypes over two seasons. Induction of quinone reductase (QR) activity, a biomarker for anti-carcinogenesis, was significantly increased by the extracts from the leaf tissue of these two cultivars. Extracts of apical leaf tissues had greater MeJA mediated increases in phenolics, glucosinolate concentrations, GS hydrolysis products, and QR activity than extracts from basal leaf tissue samples. The concentration of the hydrolysis product of glucoraphanin, sulforphane was significantly increased in apical leaf tissue of the cultivar 'Red Winter' in both 2010 and 2011. There was interaction between exogenous MeJA treatment and environmental conditions to induce endogenous JA. Correlation analysis revealed that indole-3-carbanol (I3C) generated from the hydrolysis of glucobrassicin significantly correlated with QR activity (r = 0.800, P<0.001). Concentrations required to double the specific QR activity (CD values) of I3C was calculated at 230 µM, which is considerably weaker at induction than other isothiocyanates like sulforphane. To confirm relationships between GS hydrolysis products and QR activity, a range of concentrations of MeJA sprays were applied to kale leaf tissues of both cultivars in 2011. Correlation analysis of these results indicated that sulforaphane, NI3C, neoascorbigen, I3C, and diindolylmethane were all significantly correlated with QR activity. Thus, increased QR activity may be due to combined

  3. HMG-CoA reductase inhibitory activity and phytocomponent investigation of Basella alba leaf extract as a treatment for hypercholesterolemia

    PubMed Central

    Baskaran, Gunasekaran; Salvamani, Shamala; Ahmad, Siti Aqlima; Shaharuddin, Noor Azmi; Pattiram, Parveen Devi; Shukor, Mohd Yunus

    2015-01-01

    The enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase is the key enzyme of the mevalonate pathway that produces cholesterol. Inhibition of HMG-CoA reductase reduces cholesterol biosynthesis in the liver. Synthetic drugs, statins, are commonly used for the treatment of hypercholesterolemia. Due to the side effects of statins, natural HMG-CoA reductase inhibitors of plant origin are needed. In this study, 25 medicinal plant methanol extracts were screened for anti-HMG-CoA reductase activity. Basella alba leaf extract showed the highest inhibitory effect at about 74%. Thus, B. alba was examined in order to investigate its phytochemical components. Gas chromatography with tandem mass spectrometry and reversed phase high-performance liquid chromatography analysis revealed the presence of phenol 2,6-bis(1,1-dimethylethyl), 1-heptatriacotanol, oleic acid, eicosyl ester, naringin, apigenin, luteolin, ascorbic acid, and α-tocopherol, which have been reported to possess antihypercholesterolemic effects. Further investigation of in vivo models should be performed in order to confirm its potential as an alternative treatment for hypercholesterolemia and related cardiovascular diseases. PMID:25609924

  4. The Sulfur Oxygenase Reductase from the Mesophilic Bacterium Halothiobacillus neapolitanus Is a Highly Active Thermozyme

    PubMed Central

    Veith, Andreas; Botelho, Hugo M.; Kindinger, Florian; Gomes, Cláudio M.

    2012-01-01

    A biochemical, biophysical, and phylogenetic study of the sulfur oxygenase reductase (SOR) from the mesophilic gammaproteobacterium Halothiobacillus neapolitanus (HnSOR) was performed in order to determine the structural and biochemical properties of the enzyme. SOR proteins from 14 predominantly chemolithoautotrophic bacterial and archaeal species are currently available in public databases. Sequence alignment and phylogenetic analysis showed that they form a coherent protein family. The HnSOR purified from Escherichia coli after heterologous gene expression had a temperature range of activity of 10 to 99°C with an optimum at 80°C (42 U/mg protein). Sulfite, thiosulfate, and hydrogen sulfide were formed at various stoichiometries in a range between pH 5.4 and 11 (optimum pH 8.4). Circular dichroism (CD) spectroscopy and dynamic light scattering showed that the HnSOR adopts secondary and quaternary structures similar to those of the 24-subunit enzyme from the hyperthermophile Acidianus ambivalens (AaSOR). The melting point of the HnSOR was ≈20°C lower than that of the AaSOR, when analyzed with CD-monitored thermal unfolding. Homology modeling showed that the secondary structure elements of single subunits are conserved. Subtle changes in the pores of the outer shell and increased flexibility might contribute to activity at low temperature. We concluded that the thermostability was the result of a rigid protein core together with the stabilizing effect of the 24-subunit hollow sphere. PMID:22139503

  5. Expression and Enzyme Activity Detection of a Sepiapterin Reductase Gene from Musca domestica Larva.

    PubMed

    Tang, Yan; Pei, Zhihua; Liu, Lei; Wang, Dongfang; Kong, Lingcong; Liu, Shuming; Jiang, Xiuyun; Gao, Yunhang; Ma, Hongxia

    2017-02-01

    Tetrahydrobiopterin (BH4) is an essential cofactor for aromatic acid hydroxylases and nitric oxide synthase. Sepiapterin reductase (SPR) catalyzes the final steps of BH4 biosynthesis. Studies on SPR from several insects and other organisms have been reported. However, thus far, enzyme activity of SPR in Musca domestica is kept unknown. In this study, 186 differentially expressed genes including SPR gene from Musca domestica (MDSPR) were screened in subtractive cDNA library. The MDSPR gene was cloned, and the recombinant MDSPI16 protein was expressed as a 51-kDa protein in soluble form. The MDSPR exhibited strong activity to the substrate sepiapterin (SP). The values of Vmax and Km of the MDSPR for SP were 6.83 μM/min and 23.48 μM, and the optimum temperature and pH of MDSPR were 50 °C and 4.0, respectively. This study provides new hypotheses and methods for the production of BH4 using insect-derived SPR.

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

    NASA Technical Reports Server (NTRS)

    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.

  7. Trypanocidal Activity of Quinoxaline 1,4 Di-N-oxide Derivatives as Trypanothione Reductase Inhibitors.

    PubMed

    Chacón-Vargas, Karla Fabiola; Nogueda-Torres, Benjamin; Sánchez-Torres, Luvia E; Suarez-Contreras, Erick; Villalobos-Rocha, Juan Carlos; Torres-Martinez, Yuridia; Lara-Ramirez, Edgar E; Fiorani, Giulia; Krauth-Siegel, R Luise; Bolognesi, Maria Laura; Monge, Antonio; Rivera, Gildardo

    2017-02-01

    Chagas disease or American trypanosomiasis is a worldwide public health problem. In this work, we evaluated 26 new propyl and isopropyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives as potential trypanocidal agents. Additionally, molecular docking and enzymatic assays on trypanothione reductase (TR) were performed to provide a basis for their potential mechanism of action. Seven compounds showed better trypanocidal activity on epimastigotes than the reference drugs, and only four displayed activity on trypomastigotes; T-085 was the lead compound with an IC50 = 59.9 and 73.02 µM on NINOA and INC-5 strain, respectively. An in silico analysis proposed compound T-085 as a potential TR inhibitor with better affinity than the natural substrate. Enzymatic analysis revealed that T-085 inhibits parasite TR non-competitively. Compound T-085 carries a carbonyl, a CF3, and an isopropyl carboxylate group at 2-, 3- and 7-position, respectively. These results suggest the chemical structure of this compound as a good starting point for the design and synthesis of novel trypanocidal derivatives with higher TR inhibitory potency and lower toxicity.

  8. CIPK23 is involved in iron acquisition of Arabidopsis by affecting ferric chelate reductase activity.

    PubMed

    Tian, Qiuying; Zhang, Xinxin; Yang, An; Wang, Tianzuo; Zhang, Wen-Hao

    2016-05-01

    Iron deficiency is one of the major limiting factors affecting quality and production of crops in calcareous soils. Numerous signaling molecules and transcription factors have been demonstrated to play a regulatory role in adaptation of plants to iron deficiency. However, the mechanisms underlying the iron deficiency-induced physiological processes remain to be fully dissected. Here, we demonstrated that the protein kinase CIPK23 was involved in iron acquisition. Lesion of CIPK23 rendered Arabidopsis mutants hypersensitive to iron deficiency, as evidenced by stronger chlorosis in young leaves and lower iron concentration than wild-type plants under iron-deficient conditions by down-regulating ferric chelate reductase activity. We found that iron deficiency evoked an increase in cytosolic Ca(2+) concentration and the elevated Ca(2+) would bind to CBL1/CBL9, leading to activation of CIPK23. These novel findings highlight the involvement of calcium-dependent CBL-CIPK23 complexes in the regulation of iron acquisition. Moreover, mutation of CIPK23 led to changes in contents of mineral elements, suggesting that CBL-CIPK23 complexes could be as "nutritional sensors" to sense and regulate the mineral homeostasis in Arabisopsis.

  9. A Continuous Spectrophotometric Assay for APS Reductase Activity with Sulfite-Selective Probes

    PubMed Central

    Paritala, Hanumantharao; Carroll, Kate S.

    2013-01-01

    Mycobacterium tuberculosis (Mtb) adenosine 5′-phosphosulfate (APS) reductase (EC number 1.8.4.10), (APR) catalyzes the first committed step in sulfate reduction for the biosynthesis of essential reduced sulfur-containing biomolecules, such as cysteine, and is essential for survival in the latent phase of TB infection. Despite the importance of APR to Mtb, and other bacterial pathogens, current assay methods depend on use of [35S]-labeled APS or shunt AMP to a coupled-enzyme system. Both methods are cumbersome and require the use of expensive reagents. Here we report the development of a continuous spectrophotometric method for measuring APR activity by using novel sulfite-selective colorimetric or “off-on” fluorescent levulinate-based probes. The APR activity can thus be followed by monitoring the increase in absorbance or fluorescence of the resulting phenolate product. Using this assay, we determined Michelis-Menten kinetic constants (Km, kcat, kcat/Km) and apparent inhibition constant (Ki) for adenosine 5′-diphosphate (ADP), which compared favorably to values obtained in the gold-standard radioactive assay. The newly developed assay is robust and easy to perform with a simple spectrophotometer. PMID:23711725

  10. S-nitrosation of conserved cysteines modulates activity and stability of S-nitrosoglutathione reductase (GSNOR)

    PubMed Central

    Guerra, Damian; Ballard, Keith; Truebridge, Ian; Vierling, Elizabeth

    2016-01-01

    The free radical nitric oxide (NO•) regulates diverse physiological processes from vasodilation in humans to gas exchange in plants. S-nitrosoglutathione (GSNO) is considered a principal nitroso reservoir due to its chemical stability. GSNO accumulation is attenuated by GSNO reductase (GSNOR), a cysteine-rich cytosolic enzyme. Regulation of protein nitrosation is not well understood since NO•-dependent events proceed without discernible changes in GSNOR expression. Because GSNORs contain evolutionarily-conserved cysteines that could serve as nitrosation sites, we examined the effects of treating plant (Arabidopsis thaliana), mammalian (human), and yeast (Saccharomyces cerevisiae) GSNORs with nitrosating agents in vitro. Enzyme activity was sensitive to nitroso donors, while the reducing agent dithiothreitol (DTT) restored activity, suggesting catalytic impairment was due to S-nitrosation. Protein nitrosation was confirmed by mass spectrometry, by which mono-, di-, and tri-nitrosation were observed, and these signals were sensitive to DTT. GSNOR mutants in specific non-zinc coordinating cysteines were less sensitive to catalytic inhibition by nitroso donors and exhibited reduced nitrosation signals by mass spectrometry. Nitrosation also coincided with decreased tryptophan fluorescence, increased thermal aggregation propensity, and increased polydispersity—properties reflected by differential solvent accessibility of amino acids important for dimerization and the shape of the substrate and coenzyme binding pockets as assessed by hydrogen-deuterium exchange mass spectrometry. Collectively, these data suggest a mechanism for NO• signal transduction in which GSNOR nitrosation and inhibition transiently permit GSNO accumulation. PMID:27064847

  11. Targeted Mutations of Bacillus anthracis Dihydrofolate Reductase Condense Complex Structure-Activity Relationships

    SciTech Connect

    J Beierlein; N Karri; A Anderson

    2011-12-31

    Several antifolates, including trimethoprim (TMP) and a series of propargyl-linked analogues, bind dihydrofolate reductase from Bacillus anthracis (BaDHFR) with lower affinity than is typical in other bacterial species. To guide lead optimization for BaDHFR, we explored a new approach to determine structure-activity relationships whereby the enzyme is altered and the analogues remain constant, essentially reversing the standard experimental design. Active site mutants of the enzyme, Ba(F96I)DHFR and Ba(Y102F)DHFR, were created and evaluated with enzyme inhibition assays and crystal structures. The affinities of the antifolates increase up to 60-fold with the Y102F mutant, suggesting that interactions with Tyr 102 are critical for affinity. Crystal structures of the enzymes bound to TMP and propargyl-linked inhibitors reveal the basis of TMP resistance and illuminate the influence of Tyr 102 on the lipophilic linker between the pyrimidine and aryl rings. Two new inhibitors test and validate these conclusions and show the value of the technique for providing new directions during lead optimization.

  12. Potentiation of the reductase activity of protein disulphide isomerase (PDI) by 19-nortestosterone, bacitracin, fluoxetine, and ammonium sulphate.

    PubMed

    Hassan, Maya Haj; Alvarez, Eva; Cahoreau, Claire; Klett, Danièle; Lecompte, François; Combarnous, Yves

    2011-10-01

    Protein disulphide isomerase (PDI) in the endoplasmic reticulum catalyzes the rearrangement of disulphide bridges during folding of secreted proteins. It binds various molecules that inhibit its activity. But here, we looked for molecules that would potentiate its activity. PDI reductase activity was measured in vitro using di-eosin-oxidized glutathione as substrate. Its classical inhibitor bacitracin was found to exert a biphasic effect: stimulatory at low concentrations (∼10(-6) M) and inhibitory only at higher concentrations (∼10(-4)-10(-3) M). The weak oestrogenic molecule bisphenol A was found to exert a weak inhibitory effect on PDI reductase activity relative to the strong oestrogens, ethynylestradiol, and diethylstilbestrol. Like 19-nortestosterone, fluoxetine was found to exert a potentiating effect on PDI reductase activity and their potentiating effects could be reversed by increasing concentrations of oestrogens. In conclusion, this paper provides the first identification of potentiators of PDI activity that are potential pharmaceuticals against pathologies affecting protein folding such as Alzheimer's disease.

  13. Purification and properties of a dissimilatory nitrate reductase from Haloferax denitrificans

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.; Lang, F.

    1991-01-01

    A membrane-bound nitrate reductase (nitrite:(acceptor) oxidoreductase, EC 1.7.99.4) from the extremely halophilic bacterium Haloferax denitrificans was solubilized by incubating membranes in buffer lacking NaCl and purified by DEAE, hydroxylapatite, and Sepharose 6B gel filtration chromatography. The purified nitrate reductase reduced chlorate and was inhibited by azide and cyanide. Preincubating the enzyme with cyanide increased the extent of inhibition which in turn was intensified when dithionite was present. Although cyanide was a noncompetitive inhibitor with respect to nitrate, nitrate protected against inhibition. The enzyme, as isolated, was composed of two subunits (Mr 116,000 and 60,000) and behaved as a dimer during gel filtration (Mr 380,000). Unlike other halobacterial enzymes, this nitrate reductase was most active, as well as stable, in the absence of salt.

  14. Human Aldo-Keto Reductases and the Metabolic Activation of Polycyclic Aromatic Hydrocarbons

    PubMed Central

    2015-01-01

    Aldo-keto reductases (AKRs) are promiscuous NAD(P)(H) dependent oxidoreductases implicated in the metabolic activation of polycyclic aromatic hydrocarbons (PAH). These enzymes catalyze the oxidation of non-K-region trans-dihydrodiols to the corresponding o-quinones with the concomitant production of reactive oxygen species (ROS). The PAH o-quinones are Michael acceptors and can form adducts but are also redox-active and enter into futile redox cycles to amplify ROS formation. Evidence exists to support this metabolic pathway in humans. The human recombinant AKR1A1 and AKR1C1–AKR1C4 enzymes all catalyze the oxidation of PAH trans-dihydrodiols to PAH o-quinones. Many human AKRs also catalyze the NADPH-dependent reduction of the o-quinone products to air-sensitive catechols, exacerbating ROS formation. Moreover, this pathway of PAH activation occurs in a panel of human lung cell lines, resulting in the production of ROS and oxidative DNA damage in the form of 8-oxo-2′-deoxyguanosine. Using stable-isotope dilution liquid chromatography tandem mass spectrometry, this pathway of benzo[a]pyrene (B[a]P) metabolism was found to contribute equally with the diol-epoxide pathway to the activation of this human carcinogen in human lung cells. Evaluation of the mutagenicity of anti-B[a]P-diol epoxide with B[a]P-7,8-dione on p53 showed that the o-quinone produced by AKRs was the more potent mutagen, provided that it was permitted to redox cycle, and that the mutations observed were G to T transversions, reminiscent of those observed in human lung cancer. It is concluded that there is sufficient evidence to support the role of human AKRs in the metabolic activation of PAH in human lung cell lines and that they may contribute to the causation of human lung cancer. PMID:25279998

  15. Microbial activity balance in size fractionated suspended growth biomass from full-scale sidestream combined nitritation-anammox reactors.

    PubMed

    Shi, Yijing; Wells, George; Morgenroth, Eberhard

    2016-10-01

    The purpose of this study was to determine the abundance, distribution and activity of aerobic ammonia-oxidizing bacteria (AOB) and anammox in size fractionated aggregates from full-scale suspended growth combined nitritation-anammox sidestream reactors. Plants with or without a cyclone device were also studied to assess a purported enrichment of anammox granules. Specific aerobic ammonium oxidation rates (p=0.01) and specific oxygen uptake rates (p=0.02) were significantly greater in flocs than in granules. AOB abundance measured using quantitative FISH was significantly higher in flocs than in granules (p=0.01). Conversely, anammox abundance was significantly greater in granules (p=0.03). The average ratio of anammox/AOB in systems employing hydrocyclone separation devices was 2.4, significantly higher (p=0.02) than the average ratio (0.5) in a system without a hydrocyclone. Our results demonstrate substantial functional and population-level segregation between floccular and granular fractions, and provide a key corroboration that cyclone separation devices can increase anammox levels in such systems.

  16. Properties of some reductase enzymes in the nitrifying bacteria and their relationship to the oxidase systems

    PubMed Central

    Wallace, W.; Nicholas, D. J. D.

    1968-01-01

    The reductase enzymes in Nitrosomonas and Nitrobacter were studied under anaerobic conditions when the oxidase enzymes were inactive. The most effective electron-donor systems for nitrate reductase in Nitrobacter were reduced benzyl viologen alone, phenazine methosulphate with either NADH or NADPH, and FMN or FAD with NADH. Nitrite and hydroxylamine reductases were found in both nitrifying bacteria, and optimum activity for each enzyme was obtained with NADH or NADPH with either FMN or FAD. The product of both these enzymes was identified as ammonia. In extracts of Nitrosomonas the ammonia was further utilized by an NADPH-specific glutamate dehydrogenase. 15N-labelled nitrite, hydroxylamine and ammonia were rapidly incorporated into cell protein by Nitrosomonas, and Nitrobacter in addition incorporated [15N]nitrate. Relatively gentle methods of cell disruption were compared with ultrasonic treatment, to enable a more exact study to be undertaken of the intracellular distribution of the oxidase and reductase enzymes. The functional relationship of these opposing enzyme systems in the nitrifying bacteria is considered. PMID:4386932

  17. Both plant and bacterial nitrate reductases contribute to nitric oxide production in Medicago truncatula nitrogen-fixing nodules.

    PubMed

    Horchani, Faouzi; Prévot, Marianne; Boscari, Alexandre; Evangelisti, Edouard; Meilhoc, Eliane; Bruand, Claude; Raymond, Philippe; Boncompagni, Eric; Aschi-Smiti, Samira; Puppo, Alain; Brouquisse, Renaud

    2011-02-01

    Nitric oxide (NO) is a signaling and defense molecule of major importance in living organisms. In the model legume Medicago truncatula, NO production has been detected in the nitrogen fixation zone of the nodule, but the systems responsible for its synthesis are yet unknown and its role in symbiosis is far from being elucidated. In this work, using pharmacological and genetic approaches, we explored the enzymatic source of NO production in M. truncatula-Sinorhizobium meliloti nodules under normoxic and hypoxic conditions. When transferred from normoxia to hypoxia, nodule NO production was rapidly increased, indicating that NO production capacity is present in functioning nodules and may be promptly up-regulated in response to decreased oxygen availability. Contrary to roots and leaves, nodule NO production was stimulated by nitrate and nitrite and inhibited by tungstate, a nitrate reductase inhibitor. Nodules obtained with either plant nitrate reductase RNA interference double knockdown (MtNR1/2) or bacterial nitrate reductase-deficient (napA) and nitrite reductase-deficient (nirK) mutants, or both, exhibited reduced nitrate or nitrite reductase activities and NO production levels. Moreover, NO production in nodules was found to be inhibited by electron transfer chain inhibitors, and nodule energy state (ATP-ADP ratio) was significantly reduced when nodules were incubated in the presence of tungstate. Our data indicate that both plant and bacterial nitrate reductase and electron transfer chains are involved in NO synthesis. We propose the existence of a nitrate-NO respiration process in nodules that could play a role in the maintenance of the energy status required for nitrogen fixation under oxygen-limiting conditions.

  18. Independence of nitrate and nitrite inhibition of Desulfovibrio vulgaris Hildenborough and use of nitrite as a substrate for growth.

    PubMed

    Korte, Hannah L; Saini, Avneesh; Trotter, Valentine V; Butland, Gareth P; Arkin, Adam P; Wall, Judy D

    2015-01-20

    Sulfate-reducing microbes, such as Desulfovibrio vulgaris Hildenborough, cause “souring” of petroleum reservoirs through produced sulfide and precipitate heavy metals, either as sulfides or by alteration of the metal reduction state. Thus, inhibitors of these microbes, including nitrate and nitrite ions, are studied in order to limit their impact. Nitrite is a potent inhibitor of sulfate reducers, and it has been suggested that nitrate does not inhibit these microbes directly but by reduction to nitrite, which serves as the ultimate inhibitor. Here we provide evidence that nitrate inhibition of D. vulgaris can be independent of nitrite production. We also show that D. vulgaris can use nitrite as a nitrogen source or terminal electron acceptor for growth. Moreover, we report that use of nitrite as a terminal electron acceptor requires nitrite reductase (nrfA) as a D. vulgaris nrfA mutant cannot respire nitrite but remains capable of utilizing nitrite as a nitrogen source. These results illuminate previously uncharacterized metabolic abilities of D. vulgaris that may allow niche expansion in low-sulfate environments. Understanding these abilities may lead to better control of sulfate-reducing bacteria in industrial settings and more accurate prediction of their interactions in the environment.

  19. Ribonucleotide reductase activity is coupled to DNA synthesis via proliferating cell nuclear antigen.

    PubMed

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

    2012-04-24

    Synthesis of deoxynucleoside triphosphates (dNTPs) is required for both DNA replication and DNA repair and is catalyzed by ribonucleotide reductases (RNR), which convert ribonucleotides to their deoxy forms [1, 2]. Maintaining the correct levels of dNTPs for DNA synthesis is important for minimizing the mutation rate [3-7], and this is achieved by tight regulation of RNR [2, 8, 9]. In fission yeast, RNR is regulated in part by a small protein inhibitor, Spd1, which is degraded in S phase and after DNA damage to allow upregulation of dNTP supply [10-12]. Spd1 degradation is mediated by the activity of the CRL4(Cdt2) ubiquitin ligase complex [5, 13, 14]. This has been reported to be dependent on modulation of Cdt2 levels, which are cell cycle regulated, peaking in S phase, and which also increase after DNA damage in a checkpoint-dependent manner [7, 13]. We show here that Cdt2 level fluctuations are not sufficient to regulate Spd1 proteolysis and that the key step in this event is the interaction of Spd1 with the polymerase processivity factor proliferating cell nuclear antigen (PCNA), complexed onto DNA. This mechanism thus provides a direct link between DNA synthesis and RNR regulation.

  20. The transient catalytically competent coenzyme allocation into the active site of Anabaena ferredoxin NADP+ -reductase.

    PubMed

    Peregrina, José Ramón; Lans, Isaías; Medina, Milagros

    2012-01-01

    Ferredoxin-NADP(+) reductase (FNR) catalyses the electron transfer from ferredoxin to NADP(+) via its flavin FAD cofactor. A molecular dynamics theoretical approach is applied here to visualise the transient catalytically competent interaction of Anabaena FNR with its coenzyme, NADP(+). The particular role of some of the residues identified as key in binding and accommodating the 2'P-AMP moiety of the coenzyme is confirmed in molecular terms. Simulations also indicate that the architecture of the active site precisely contributes to the orientation of the N5 of the FAD isoalloxazine ring and the C4 of the coenzyme nicotinamide ring in the conformation of the catalytically competent hydride transfer complex and, therefore, contributes to the efficiency of the process. In particular, the side chain of the C-terminal Y303 in Anabaena FNR appears key to providing the optimum geometry by reducing the stacking probability between the isoalloxazine and nicotinamide rings, thus providing the required co-linearity and distance among the N5 of the flavin cofactor, the C4 of the coenzyme nicotinamide and the hydride that has to be transferred between them. All these factors are highly related to the reaction efficiency, mechanism and reversibility of the process.

  1. The epimerase activity of anthocyanidin reductase from Vitis vinifera and its regiospecific hydride transfers.

    PubMed

    Gargouri, Mahmoud; Chaudière, Jean; Manigand, Claude; Maugé, Chloé; Bathany, Katell; Schmitter, Jean-Marie; Gallois, Bernard

    2010-01-01

    Anthocyanidin reductase (ANR) from Vitis vinifera catalyzes an NADPH-dependent double reduction of anthocyanidins producing a mixture of (2S,3R)- and (2S,3S)-flavan-3-ols. At pH 7.5 and 30 degrees C, the first hydride transfer to anthocyanidin is irreversible, and no intermediate is released during catalysis. ANR reverse activity was assessed in the presence of excess NADP(+). Analysis of products by reverse phase and chiral phase HPLC demonstrates that ANR acts as a flavan-3-ol C(3)-epimerase under such conditions, but this is only observed with 2R-flavan-3-ols, not with 2S-flavan-3-ols produced by the enzyme in the forward reaction. In the presence of deuterated coenzyme 4S-NADPD, ANR transforms anthocyanidins into dideuterated flavan-3-ols. The regiospecificity of deuterium incorporation into catechin and afzelechin - derived from cyanidin and pelargonidin, respectively - was analyzed by liquid chromatography coupled with electro- spray ionization-tandem mass spectrometry (LC/ESI-MS/MS), and it was found that deuterium was always incorporated at C(2) and C(4). We conclude that C(3)-epimerization should be achieved by tautomerization between the two hydride transfers and that this produces a quinone methide intermediate which serves as C(4) target of the second hydride transfer, thereby avoiding any stereospecific modification of carbon 3. The inversion of C(2) stereochemistry required for 'reverse epimerization' suggests that the 2S configuration induces an irreversible product dissociation.

  2. A role for 5alpha-reductase activity in the development of male homosexuality?

    PubMed

    Alias, A G

    2004-12-01

    Higher body hair with lower mesmorphism ratings were observed in Caucasian homosexual men compared with the general male population, reflecting elevated 5alpha-reductase (5alphaR) activity, and higher dihydrotestosterone-to-testosterone (DHT-to-T) ratio, in sharp contrast to 46,XY 5alphaR 2 deficiency subjects, who are often born with ambiguous, or female genitalia, but tend to grow up to be muscular, heterosexual men with very little body hair, or beard. One study also showed them scoring around dull normal IQs. A greater prevalence of liberal body hair growth in men with higher IQs and/or educational levels was also observed in several samples. The exceptions to this statistical trend are too unsettling, however. Nevertheless, the results of a number of published studies, including one showing higher DHT-to-T ratio in homosexual men, done with different objectives over a span of 80 years, together strongly support these findings. Furthermore, in an animal model, "cognitive-enhancing effects" of "5alpha-reduced androgen [metabolites]" were recently demonstrated.

  3. Giardia, Entamoeba, and Trichomonas enzymes activate metronidazole (nitroreductases) and inactivate metronidazole (nitroimidazole reductases).

    PubMed

    Pal, Dibyarupa; Banerjee, Sulagna; Cui, Jike; Schwartz, Aaron; Ghosh, Sudip K; Samuelson, John

    2009-02-01

    Infections with Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis, which cause diarrhea, dysentery, and vaginitis, respectively, are each treated with metronidazole. Here we show that Giardia, Entamoeba, and Trichomonas have oxygen-insensitive nitroreductase (ntr) genes which are homologous to those genes that have nonsense mutations in metronidazole-resistant Helicobacter pylori isolates. Entamoeba and Trichomonas also have nim genes which are homologous to those genes expressed in metronidazole-resistant Bacteroides fragilis isolates. Recombinant Giardia, Entamoeba, and Trichomonas nitroreductases used NADH rather than the NADPH used by Helicobacter, and two recombinant Entamoeba nitroreductases increased the metronidazole sensitivity of transformed Escherichia coli strains. Conversely, the recombinant nitroimidazole reductases (NIMs) of Entamoeba and Trichmonas conferred very strong metronidazole resistance to transformed bacteria. The Ehntr1 gene of the genome project HM-1:IMSS strain of Entamoeba histolytica had a nonsense mutation, and the same nonsense mutation was present in 3 of 22 clinical isolates of Entamoeba. While ntr and nim mRNAs were variably expressed by cultured Entamoeba and Trichomonas isolates, there was no relationship to metronidazole sensitivity. We conclude that microaerophilic protists have bacterium-like enzymes capable of activating metronidazole (nitroreductases) and inactivating metronidazole (NIMs). While Entamoeba and Trichomonas displayed some of the changes (nonsense mutations and gene overexpression) associated with metronidazole resistance in bacteria, these changes did not confer metronidazole resistance to the microaerophilic protists examined here.

  4. Evidence for Increased 5α-Reductase Activity During Early Childhood in Daughters of Women With Polycystic Ovary Syndrome

    PubMed Central

    Torchen, Laura C.; Idkowiak, Jan; Fogel, Naomi R.; O'Neil, Donna M.; Shackleton, Cedric H. L.; Arlt, Wiebke

    2016-01-01

    Context: Polycystic ovary syndrome (PCOS) is a heritable, complex genetic disease. Animal models suggest that androgen exposure at critical developmental stages contributes to disease pathogenesis. We hypothesized that genetic variation resulting in increased androgen production produces the phenotypic features of PCOS by programming during critical developmental periods. Although we have not found evidence for increased in utero androgen levels in cord blood in the daughters of women with PCOS (PCOS-d), target tissue androgen production may be amplified by increased 5α-reductase activity analogous to findings in adult affected women. It is possible to noninvasively test this hypothesis by examining urinary steroid metabolites. Objective: We performed this study to investigate whether PCOS-d have altered androgen metabolism during early childhood. Design, Setting, and Participants: Twenty-one PCOS-d, 1–3 years old, and 36 control girls of comparable age were studied at an academic medical center. Main Outcome Measures: Urinary steroid metabolites were measured by gas chromatography/mass spectrometry. Twenty-four hour steroid excretion rates and precursor to product ratios suggestive of 5α-reductase and 11β-hydroxysteroid dehydrogenase activities were calculated. Results: Age did not differ but weight for length Z-scores were higher in PCOS-d compared to control girls (P = .02). PCOS-d had increased 5α-tetrahydrocortisol:tetrahydrocortisol ratios (P = .04), suggesting increased global 5α-reductase activity. There was no evidence for differences in 11β-hydroxysteroid dehydrogenase activity. Steroid metabolite excretion was not correlated with weight. Conclusions: Our findings suggest that differences in androgen metabolism are present in early childhood in PCOS-d. Increased 5α-reductase activity could contribute to the development of PCOS by amplifying target tissue androgen action. PMID:26990942

  5. Induction of Xylose Reductase and Xylitol Dehydrogenase Activities in Pachysolen tannophilus and Pichia stipitis on Mixed Sugars

    PubMed Central

    Bicho, Paul A.; Runnals, P. Lynn; Cunningham, J. Douglas; Lee, Hung

    1988-01-01

    The induction of xylose reductase and xylitol dehydrogenase activities on mixed sugars was investigated in the yeasts Pachysolen tannophilus and Pichia stipitis. Enzyme activities induced on d-xylose served as the controls. In both yeasts, d-glucose, d-mannose, and 2-deoxyglucose inhibited enzyme induction by d-xylose to various degrees. Cellobiose, l-arabinose, and d-galactose were not inhibitory. In liquid batch culture, P. tannophilus utilized d-glucose and d-mannose rapidly and preferentially over d-xylose, while d-galactose consumption was poor and lagged behind that of the pentose sugar. In P. stipitis, all three hexoses were used preferentially over d-xylose. The results showed that the repressibility of xylose reductase and xylitol dehydrogenase may limit the potential of yeast fermentation of pentose sugars in hydrolysates of lignocellulosic substrates. PMID:16347538

  6. Differences in nitric oxide steady states between arginine, hypoxanthine, uracil auxotrophs (AHU) and non-AHU strains of Neisseria gonorrhoeae during anaerobic respiration in the presence of nitrite.

    PubMed

    Barth, Kenneth; Clark, Virginia L

    2008-08-01

    Neisseria gonorrhoeae can grow by anaerobic respiration using nitrite as an alternative electron acceptor. Under these growth conditions, N. gonorrhoeae produces and degrades nitric oxide (NO), an important host defense molecule. Laboratory strain F62 has been shown to establish and maintain a NO steady-state level that is a function of the nitrite reductase/NO reductase ratio and is independent of cell number. The nitrite reductase activities (122-197 nmol NO2 reduced x min(-1) x OD600(-1)) and NO reductase activities (88-155 nmol NO reduced x min(-1) x OD600(-1)) in a variety of gonococcal clinical isolates were similar to the specific activities seen in F62 (241 nmol NO2 reduced x min(-1) x OD600(-1) and 88 nmol NO reduced x min(-1) x OD600(-1), respectively). In seven gonococcal strains, the NO steady-state levels established in the presence of nitrite were similar to that of F62 (801-2121 nmol x L-1 NO), while six of the strains, identified as arginine, hypoxanthine, and uracil auxotrophs (AHU), that cause asymptomatic infection in men had either two- to threefold (373-579 nmol x L-1 NO) or about 100-fold (13-24 nmol x L-1 NO) lower NO steady-state concentrations. All tested strains in the presence of a NO donor, 2,2'-(hydroxynitrosohydrazono)bis-ethanimine/NO, quickly lowered and maintained NO levels in the noninflammatory range of NO (<300 nmol x L-1). The generation of a NO steady-state concentration was directly affected by alterations in respiratory control in both F62 and an AHU strain, although differences in membrane function are suspected to be responsible for NO steady-state level differences in AHU strains.

  7. Response of AMP-activated protein kinase and energy metabolism to acute nitrite exposure in the Nile tilapia Oreochromis niloticus.

    PubMed

    Xu, Zhixin; Li, Erchao; Xu, Chang; Gan, Lei; Qin, Jian G; Chen, Liqiao

    2016-08-01

    Adenosine monophosphate-activated protein kinase (AMPK) is a prevalent mammalian energy metabolism sensor, but little is known about its role as an energy sensor in fish experiencing stress. We aimed to study AMPK in Oreochromis niloticus on both the molecular and the physical level. We found that the cDNAs encoding the AMPKα1 and AMPKα2 variants of the O. niloticus catalytic α subunit were 1753bp and 2563 bp long and encoded 571 and 557 amino acids, respectively. Both the AMPKα1 and the AMPKα2 isoform possess structural features similar to mammalian AMPKα, including a phosphorylation site at Thr172 in the N-terminus, and exhibit high homology with other fish and vertebrate AMPKα sequences (81.3%-98.1%). mRNA encoding the AMPKα isoforms was widely expressed in various tissues with distinctive patterns. AMPKα1 and AMPKα2 were primarily expressed in the intestines and brain, respectively. Under acute nitrite challenge, the mRNA encoding the AMPKα isoforms, as well as AMPK activity, changed over time. Its recovery period in freshwater, combined with the fact that it is highly conserved, suggests that fish AMPK, like its mammalian orthologues, acts as an energy metabolism sensor. Furthermore, subsequent decreases in AMPK mRNA levels and activity suggested that its action was transient but efficient. Physically, glucose, lactic acid and TGs in plasma, as well as energy materials in the hepatopancreas and muscle, were significantly altered over time, indicating changes in energy metabolism during the experimental period. These data have enabled us to characterize energy utilization in O. niloticus and further illustrate the role of fish AMPK as an energy sensor. This study provides new insight into energy metabolism and sensing by AMPK in teleost and necessitates further study of the multiple physiologic roles of AMPK in fish.

  8. Introducing a 2-His-1-Glu Nonheme Iron Center into Myoglobin Confers Nitric Oxide Reductase Activity

    SciTech Connect

    Lin, Y.W.; Robinson, H.; Yeung, N.; Gao, Y.-G.; Miner, K. D.; Lei, L.; Lu, Y.

    2010-07-28

    A conserved 2-His-1-Glu metal center, as found in natural nonheme iron-containing enzymes, was engineered into sperm whale myoglobin by replacing Leu29 and Phe43 with Glu and His, respectively (swMb L29E, F43H, H64, called Fe{sub B}Mb(-His)). A high resolution (1.65 {angstrom}) crystal structure of Cu(II)-CN?-Fe{sub B}Mb(-His) was determined, demonstrating that the unique 2-His-1-Glu metal center was successfully created within swMb. The Fe{sub B}Mb(-His) can bind Cu, Fe, or Zn ions, with both Cu(I)-Fe{sub B}Mb(-His) and Fe(II)-Fe{sub B}Mb(-His) exhibiting nitric oxide reductase (NOR) activities. Cu dependent NOR activity was significantly higher than that of Fe in the same metal binding site. EPR studies showed that the reduction of NO to N{sub 2}O catalyzed by these two enzymes resulted in different intermediates; a five-coordinate heme-NO species was observed for Cu(I)-Fe{sub B}Mb(-His) due to the cleavage of the proximal heme Fe-His bond, while Fe(II)-Fe{sub B}Mb(-His) remained six-coordinate. Therefore, both the metal ligand, Glu29, and the metal itself, Cu or Fe, play crucial roles in NOR activity. This study presents a novel protein model of NOR and provides insights into a newly discovered member of the NOR family, gNOR.

  9. Introducing a 2-His-1-Glu Nonheme Iron Center into Myoglobin Confers Nitric Oxide Reductase Activity

    SciTech Connect

    Y Lin; N Yeung; Y Gao; K Miner; L Lei; H Robinson; Y Lu

    2011-12-31

    A conserved 2-His-1-Glu metal center, as found in natural nonheme iron-containing enzymes, was engineered into sperm whale myoglobin by replacing Leu29 and Phe43 with Glu and His, respectively (swMb L29E, F43H, H64, called Fe{sub B}Mb(-His)). A high resolution (1.65 {angstrom}) crystal structure of Cu(II)-CN{sup -}-Fe{sub B}Mb(-His) was determined, demonstrating that the unique 2-His-1-Glu metal center was successfully created within swMb. The Fe{sub B}Mb(-His) can bind Cu, Fe, or Zn ions, with both Cu(I)-Fe{sub B}Mb(-His) and Fe(II)-Fe{sub B}Mb(-His) exhibiting nitric oxide reductase (NOR) activities. Cu dependent NOR activity was significantly higher than that of Fe in the same metal binding site. EPR studies showed that the reduction of NO to N{sub 2}O catalyzed by these two enzymes resulted in different intermediates; a five-coordinate heme-NO species was observed for Cu(I)-Fe{sub B}Mb(-His) due to the cleavage of the proximal heme Fe-His bond, while Fe(II)-Fe{sub B}Mb(-His) remained six-coordinate. Therefore, both the metal ligand, Glu29, and the metal itself, Cu or Fe, play crucial roles in NOR activity. This study presents a novel protein model of NOR and provides insights into a newly discovered member of the NOR family, gNOR.

  10. Kinetics of Hydrogen Atom Abstraction from Substrate by an Active Site Thiyl Radical in Ribonucleotide Reductase

    PubMed Central

    2015-01-01

    Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. Active E. coli class Ia RNR is an α2β2 complex that undergoes reversible, long-range proton-coupled electron transfer (PCET) over a pathway of redox active amino acids (β-Y122 → [β-W48] → β-Y356 → α-Y731 → α-Y730 → α-C439) that spans ∼35 Å. To unmask PCET kinetics from rate-limiting conformational changes, we prepared a photochemical RNR containing a [ReI] photooxidant site-specifically incorporated at position 355 ([Re]-β2), adjacent to PCET pathway residue Y356 in β. [Re]-β2 was further modified by replacing Y356 with 2,3,5-trifluorotyrosine to enable photochemical generation and spectroscopic observation of chemically competent tyrosyl radical(s). Using transient absorption spectroscopy, we compare the kinetics of Y· decay in the presence of substrate and wt-α2, Y731F-α2 ,or C439S-α2, as well as with 3′-[2H]-substrate and wt-α2. We find that only in the presence of wt-α2 and the unlabeled substrate do we observe an enhanced rate of radical decay indicative of forward radical propagation. This observation reveals that cleavage of the 3′-C–H bond of substrate by the transiently formed C439· thiyl radical is rate-limiting in forward PCET through α and has allowed calculation of a lower bound for the rate constant associated with this step of (1.4 ± 0.4) × 104 s–1. Prompting radical propagation with light has enabled observation of PCET events heretofore inaccessible, revealing active site chemistry at the heart of RNR catalysis. PMID:25353063

  11. The benzimidazole based drugs show good activity against T. gondii but poor activity against its proposed enoyl reductase enzyme target.

    PubMed

    Wilkinson, Craig; McPhillie, Martin J; Zhou, Ying; Woods, Stuart; Afanador, Gustavo A; Rawson, Shaun; Khaliq, Farzana; Prigge, Sean T; Roberts, Craig W; Rice, David W; McLeod, Rima; Fishwick, Colin W; Muench, Stephen P

    2014-02-01

    The enoyl acyl-carrier protein reductase (ENR) enzyme of the apicomplexan parasite family has been intensely studied for antiparasitic drug design for over a decade, with the most potent inhibitors targeting the NAD(+) bound form of the enzyme. However, the higher affinity for the NADH co-factor over NAD(+) and its availability in the natural environment makes the NADH complex form of ENR an attractive target. Herein, we have examined a benzimidazole family of inhibitors which target the NADH form of Francisella ENR, but despite good efficacy against Toxoplasma gondii, the IC50 for T. gondii ENR is poor, with no inhibitory activity at 1 μM. Moreover similar benzimidazole scaffolds are potent against fungi which lack the ENR enzyme and as such we believe that there may be significant off target effects for this family of inhibitors.

  12. Partial nitritation of stored source-separated urine by granular activated sludge in a sequencing batch reactor.

    PubMed

    Chen, Liping; Yang, Xiaoxiao; Tian, Xiujun; Yao, Song; Li, Jiuyi; Wang, Aimin; Yao, Qian; Peng, Dangcong

    2017-12-01

    The combination of partial nitritation (PN) and anaerobic ammonium oxidation (anammox) has been proposed as an ideal process for nitrogen removal from source-separated urine, while the high organic matters in urine cause instability of single-stage PN-anammox process. This study aims to remove the organic matters and partially nitrify the nitrogen in urine, producing an ammonium/nitrite solution suitable for anammox. The organic matters in stored urine were used as the electron donors to achieve 40% total nitrogen removal in nitritation-denitrification process in a sequencing batch reactor (SBR). Granular aggregates were observed and high mixed liquor suspended solids (9.5 g/L) were maintained in the SBR. Around 70-75% ammonium was oxidized to nitrite under the volumetric loading rates of 3.23 kg chemical oxygen demand (COD)/(m(3) d) and 1.86 kg N/(m(3) d), respectively. The SBR produced an ammonium/nitrite solution free of biodegradable organic matters, with a NO2(-)-N:NH4(+)-N of 1.24 ± 0.13. Fluorescence in situ hybridization images showed that Nitrosomonas-like ammonium-oxidizing bacteria, accounting for 7.2% of total bacteria, located in the outer layer (25 μm), while heterotrophs distributed homogeneously throughout the granular aggregates. High concentrations of free ammonia and nitrous acids in the reactor severely inhibited the growth of nitrite-oxidizing bacteria, resulting in their absence in the granular sludge. The microbial diversity analysis indicated Proteobacteria was the predominant phylum, in which Pseudomonas was the most abundant genus.

  13. The unique N terminus of the herpes simplex virus type 1 large subunit is not required for ribonucleotide reductase activity.

    PubMed

    Conner, J; Macfarlane, J; Lankinen, H; Marsden, H

    1992-01-01

    Using purified bacterially expressed herpes simplex virus type 1 ribonucleotide reductase large subunit (R1) and the proteolytic enzymes chymotrypsin and trypsin, we have generated stable N-terminal truncations. Chymotrypsin removes 246 amino acids from the amino terminus to produce a fragment (dN246R1) which retains full enzymic activity and affinity for the small subunit (R2). Treatment of R1 with trypsin produces a 120K protein and a cleavage at amino acid residue 305 to produce a fragment (dN305R1) which remains associated with a 33K N-terminal polypeptide. Although this 33K-dN305R1 complex retains full binding affinity for R2 its reductase activity is reduced by approximately 50%. Increasing the concentration of trypsin removes the 33K N-terminal polypeptide resulting in dN305R1 which, when bound to R2, has full ribonucleotide reductase activity. Like R1, dN246R1 and dN305R1 each exist as dimers showing that the first 305 amino acids of R1 are not necessary for dimer formation. These results indicate that, in structural studies of subunit interaction, dN246R1 or dN305R1 can be considered as suitable replacements for intact R1.

  14. Effect of dehydroepiandrosterone derivatives on the activity of 5α-reductase isoenzymes and on cancer cell line PC-3.

    PubMed

    Bratoeff, Eugene; Garrido, Mariana; Ramírez-Apan, Teresa; Heuze, Yvonne; Sánchez, Araceli; Soriano, Juan; Cabeza, Marisa

    2014-11-01

    It is well known that testosterone (T) under the influence of 5α-reductase enzyme is converted to dihydrotestosterone (DHT), which causes androgen-dependent diseases. The aim of this study was to synthesize new dehydroepiandrosterone derivatives (3a-e, 4a-i, 6 and 7) having potential inhibitory activity against the 5α-reductase enzyme. This paper also reports the in vivo pharmacological effect of these steroidal molecules. The results from this study showed that all compounds exhibited low inhibitory activity for 5α-reductase type 1 and 2 enzymes and they failed to bind to the androgen receptor. Furthermore, in the in vivo experiment, steroids 3b, 4f, and 4 g showed comparable antiandrogenic activity to that of finasteride; only derivatives 4d and 7 produced a considerable decrease in the weight of the prostate gland of gonadectomized hamsters treated with (T). On the other hand, compounds 4a, f and h showed 100% inhibition of the growth of prostate cancer cell line PC-3, with compound 4 g having a 98.2% antiproliferative effect at 50 μM. The overall data indicated that these steroidal molecules, having an aromatic ester moiety at C-3 (4f-h), could have anticancer properties.

  15. Inhibition of type 1 and type 2 5alpha-reductase activity by free fatty acids, active ingredients of Permixon.

    PubMed

    Raynaud, Jean Pierre; Cousse, Henri; Martin, Pierre Marie

    2002-10-01

    In different cell systems, the lipido-sterolic extract of Serenoa repens (LSESr, Permixon inhibits both type 1 and type 2 5alpha-reductase activity (5alphaR1 and 5alphaR2). LSESr is mainly constituted of fatty acids (90+/-5%) essentially as free fatty acids (80%). Among these free fatty acids, the main components are oleic and lauric acids which represent 65% and linoleic and myristic acids 15%. To evaluate the inhibitory effect of the different components of LSESr on 5alphaR1 or 5alphaR2 activity, the corresponding type 1 and type 2 human genes have been cloned and expressed in the baculovirus-directed insect cell expression system Sf9. The cells were incubated at pH 5.5 (5alphaR2) and pH 7.4 (5alphaR1) with 1 or 3nM testosterone in presence or absence of various concentrations of LSESr or of its different components. Dihydrotestosterone formation was measured with an automatic system combining HPLC and an on-line radiodetector. The inhibition of 5alphaR1 and 5alphaR2 activity was only observed with free fatty acids: esterified fatty acids, alcohols as well as sterols assayed were inactive. A specificity of the fatty acids in 5alphaR1 or 5alphaR2 inhibition has been found. Long unsaturated chains (oleic and linolenic) were active (IC(50)=4+/-2 and 13+/-3 microg/ml, respectively) on 5alphaR1 but to a much lesser extent (IC(50)>100 and 35+/-21 microg/ml, respectively) on 5alphaR2. Palmitic and stearic acids were inactive on the two isoforms. Lauric acid was active on 5alphaR1 (IC(50)=17+/-3 microg/ml) and 5alphaR2 (IC(50)=19+/-9 microg/ml). The inhibitory activity of myristic acid was evaluated on 5alphaR2 only and found active on this isoform (IC(50)=4+/-2 microg/ml). The dual inhibitory activity of LSESr on 5alpha-reductase type 1 and type 2 can be attributed to its high content in free fatty acids.

  16. Exogenous Methyl Jasmonate Treatment Increases Glucosinolate Biosynthesis and Quinone Reductase Activity in Kale Leaf Tissue

    PubMed Central

    Ku, Kang-Mo; Jeffery, Elizabeth H.; Juvik, John A.

    2014-01-01

    Methyl jasmonate (MeJA) spray treatments were applied to the kale varieties ‘Dwarf Blue Curled Vates’ and ‘Red Winter’ in replicated field plantings in 2010 and 2011 to investigate alteration of glucosinolate (GS) composition in harvested leaf tissue. Aqueous solutions of 250 µM MeJA were sprayed to saturation on aerial plant tissues four days prior to harvest at commercial maturity. The MeJA treatment significantly increased gluconasturtiin (56%), glucobrassicin (98%), and neoglucobrassicin (150%) concentrations in the apical leaf tissue of these genotypes over two seasons. Induction of quinone reductase (QR) activity, a biomarker for anti-carcinogenesis, was significantly increased by the extracts from the leaf tissue of these two cultivars. Extracts of apical leaf tissues had greater MeJA mediated increases in phenolics, glucosinolate concentrations, GS hydrolysis products, and QR activity than extracts from basal leaf tissue samples. The concentration of the hydrolysis product of glucoraphanin, sulforphane was significantly increased in apical leaf tissue of the cultivar ‘Red Winter’ in both 2010 and 2011. There was interaction between exogenous MeJA treatment and environmental conditions to induce endogenous JA. Correlation analysis revealed that indole-3-carbanol (I3C) generated from the hydrolysis of glucobrassicin significantly correlated with QR activity (r = 0.800, P<0.001). Concentrations required to double the specific QR activity (CD values) of I3C was calculated at 230 µM, which is considerably weaker at induction than other isothiocyanates like sulforphane. To confirm relationships between GS hydrolysis products and QR activity, a range of concentrations of MeJA sprays were applied to kale leaf tissues of both cultivars in 2011. Correlation analysis of these results indicated that sulforaphane, NI3C, neoascorbigen, I3C, and diindolylmethane were all significantly correlated with QR activity. Thus, increased QR activity may be due to

  17. Exposure to Silver Nanoparticles Inhibits Selenoprotein Synthesis and the Activity of Thioredoxin Reductase

    PubMed Central

    Srivastava, Milan; Singh, Sanjay

    2011-01-01

    Background: Silver nanoparticles (AgNPs) and silver (Ag)-based materials are increasingly being incorporated into consumer products, and although humans have been exposed to colloidal Ag in many forms for decades, this rise in the use of Ag materials has spurred interest into their toxicology. Recent reports have shown that exposure to AgNPs or Ag ions leads to oxidative stress, endoplasmic reticulum stress, and reduced cell proliferation. Previous studies have shown that Ag accumulates in tissues as silver sulfides (Ag2S) and silver selenide (Ag2Se). Objectives: In this study we investigated whether exposure of cells in culture to AgNPs or Ag ions at subtoxic doses would alter the effective metabolism of selenium, that is, the incorporation of selenium into selenoproteins. Methods: For these studies we used a keratinocyte cell model (HaCat) and a lung cell model (A549). We also tested (in vitro, both cellular and chemical) whether Ag ions could inhibit the activity of a key selenoenzyme, thioredoxin reductase (TrxR). Results: We found that exposure to AgNPs or far lower levels of Ag ions led to a dose-dependent inhibition of selenium metabolism in both cell models. The synthesis of protein was not altered under these conditions. Exposure to nanomolar levels of Ag ions effectively blocked selenium metabolism, suggesting that Ag ion leaching was likely the mechanism underlying observed changes during AgNP exposure. Exposure likewise inhibited TrxR activity in cultured cells, and Ag ions were potent inhibitors of purified rat TrxR isoform 1 (cytosolic) (TrxR1) enzyme. Conclusions: Exposure to AgNPs leads to the inhibition of selenoprotein synthesis and inhibition of TrxR1. Further, we propose these two sites of action comprise the likely mechanism underlying increases in oxidative stress, increases endoplasmic reticulum stress, and reduced cell proliferation during exposure to Ag. PMID:21965219

  18. Melatonin Reduces Cataract Formation and Aldose Reductase Activity in Lenses of Streptozotocin-induced Diabetic Rat

    PubMed Central

    Khorsand, Marjan; Akmali, Masoumeh; Sharzad, Sahab; Beheshtitabar, Mojtaba

    2016-01-01

    Background: The relationship between the high activity of aldose reductase (AR) and diabetic cataract formation has been previously investigated. The purpose of the present study was to determine the preventing effect of melatonin on streptozotocin (STZ)-induced diabetic cataract in rats. Methods: 34 adult healthy male Sprague-Dawely rats were divided into four groups. Diabetic control and diabetic+melatonin received a single dose of STZ (50 mg/kg, intraperitoneally), whereas the normal control and normal+melatonin received vehicle. The melatonin groups were gavaged with melatonin (5 mg/kg) daily for a period of 8 weeks, whereas the rats in the normal control and diabetic control groups received only the vehicle. The rats’ eyes were examined every week and cataract formation scores (0-4) were determined by slit-lamp microscope. At the end of the eighth week, the rats were sacrificed and markers of the polyol pathway and antioxidative (Glutathione, GSH) in their lens were determined. The levels of blood glucose, HbA1c and plasma malondialdhyde (MDA), as a marker of lipid peroxidation, were also measured. Results: Melatonin prevented STZ-induced hyperglycemia by decreased blood glucose and HbA1c levels. Slit lamp examination indicated that melatonin delayed cataract progression in diabetic rats. The results revealed that melatonin feeding increased the GSH levels, decreased the activities of AR and sorbitol dehydrogenase (SDH) and sorbitol formation in catractous lenses as well as plasma MDA content. Conclusion: In summary, for the first time we demonstrated that melatonin delayed the formation and progression of cataract in diabetic rat lenses. PMID:27365552

  19. Measurement of nitrite in urine by gas chromatography-mass spectrometry.

    PubMed

    Tsikas, Dimitrios; Suchy, Maria-Theresia; Mitschke, Anja; Beckmann, Bibiana; Gutzki, Frank-Mathias

    2012-01-01

    Nitric oxide (NO) is enzymatically produced from L-arginine and has a variety of biological functions. Autoxidation of NO in aqueous media yields nitrite (O = N-O(-)). NO and nitrite are oxidized in erythrocytes by oxyhemoglobin to nitrate (NO(3)(-)). Nitrate reductases from bacteria reduce nitrate to nitrite. Nitrite and nitrate are ubiquitous in nature, they are present throughout the body and they are excreted in the urine. Nitrite in urine has been used for several decades as an indicator and measure of bacteriuria. Since the identification of nitrite as a metabolite of NO, circulating nitrite is also used as an indicator of NO synthesis and is considered an NO storage form. In contrast to plasma nitrite, the significance of nitrite in the urine beyond bacteriuria is poorly investigated and understood. This chapter describes a gas chromatography-mass spectrometry (GC-MS) protocol for the quantitative determination of nitrite in urine of humans. Although the method is useful for detection and quantification of bacteriuria, the procedures described herein are optimum for urinary nitrite in conditions other than urinary tract infection. The method uses [(15)N]nitrite as internal standard and pentafluorobenzyl bromide as the derivatization agent. Derivatization is -performed on 100-μL aliquots and quantification of toluene extracts by selected-ion monitoring of m/z 46 for urinary nitrite and m/z 47 for the internal standard in the electron-capture negative-ion chemical ionization mode.

  20. 21 CFR 181.34 - Sodium nitrite and potassium nitrite.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium nitrite and potassium nitrite. 181.34...-Sanctioned Food Ingredients § 181.34 Sodium nitrite and potassium nitrite. Sodium nitrite and potassium... fixatives and preservative agents, with or without sodium or potassium nitrate, in the curing of red...

  1. 21 CFR 181.34 - Sodium nitrite and potassium nitrite.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium nitrite and potassium nitrite. 181.34...-Sanctioned Food Ingredients § 181.34 Sodium nitrite and potassium nitrite. Sodium nitrite and potassium... fixatives and preservative agents, with or without sodium or potassium nitrate, in the curing of red...

  2. 21 CFR 181.34 - Sodium nitrite and potassium nitrite.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium nitrite and potassium nitrite. 181.34...-Sanctioned Food Ingredients § 181.34 Sodium nitrite and potassium nitrite. Sodium nitrite and potassium... fixatives and preservative agents, with or without sodium or potassium nitrate, in the curing of red...

  3. 21 CFR 181.34 - Sodium nitrite and potassium nitrite.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium nitrite and potassium nitrite. 181.34...-Sanctioned Food Ingredients § 181.34 Sodium nitrite and potassium nitrite. Sodium nitrite and potassium... fixatives and preservative agents, with or without sodium or potassium nitrate, in the curing of red...

  4. Synthesis of 3-[(N-carboalkoxy)ethylamino]-indazole-dione derivatives and their biological activities on human liver carbonyl reductase.

    PubMed

    Berhe, Solomon; Slupe, Andrew; Luster, Choice; Charlier, Henry A; Warner, Don L; Zalkow, Leon H; Burgess, Edward M; Enwerem, Nkechi M; Bakare, Oladapo

    2010-01-01

    A series of indazole-dione derivatives were synthesized by the 1,3-dipolar cycloaddition reaction of appropriate substituted benzoquinones or naphthoquinones and N-carboalkoxyamino diazopropane derivatives. These compounds were evaluated for their effects on human carbonyl reductase. Several of the analogs were found to serve as substrates for carbonyl reductase with a wide range of catalytic efficiencies, while four analogs display inhibitory activities with IC(50) values ranging from 3-5 microM. Two of the inhibitors were studied in greater detail and were found to be noncompetitive inhibitors against both NADPH and menadione with K(I) values ranging between 2 and 11 microM. Computational studies suggest that conformation of the compounds may determine whether the indazole-diones bind productively to yield product or nonproductively to inhibit the enzyme.

  5. Interaction of Product Analogues With the Active Site of Rhodobacter Sphaeroides Dimethyl Sulfoxide Reductase

    SciTech Connect

    George, G.N.; Nelson, K.J.; Harris, H.H.; Doonan, C.J.; Rajagopalan, K.V.; /Saskatchewan U. /Duke U. /Sydney U.

    2007-07-09

    We report a structural characterization using X-ray absorption spectroscopy of Rhodobacter sphaeroides dimethylsulfoxide (DMSO) reductase reduced with trimethylarsine, and show that this is structurally analogous to the physiologically relevant dimethylsulfide-reduced DMSO reductase. Our data unambiguously indicate that these species should be regarded as formal MoIV species, and indicate a classical coordination complex of trimethylarsine oxide, with no special structural distortions. The similarity of the trimethylarsine and dimethylsulfide complexes suggests in turn that the dimethylsulfide reduced enzyme possesses a classical coordination of DMSO with no special elongation of the S-O bond, as previously suggested.

  6. A novel method of measuring reduction of nitrite-induced methemoglobin applied to fetal and adult blood of humans and sheep.

    PubMed

    Power, Gordon G; Bragg, Shannon L; Oshiro, Bryan T; Dejam, Andre; Hunter, Christian J; Blood, Arlin B

    2007-10-01

    The reaction of nitrite with deoxyhemoglobin results in the production of nitric oxide and methemoglobin, a reaction recently proposed as an important oxygen-sensitive source of vasoactive nitric oxide during hypoxic and anoxic stress, with several animal studies suggesting that nitrite may have therapeutic potential. Accumulation of toxic levels of methemoglobin is suppressed by reductase enzymes present within the erythrocyte. Using a novel method of measuring methemoglobin reductase activity in intact erythrocytes, we compared fetal and adult sheep and human blood. After nitrite-induced production of 20% methemoglobin, the blood was equilibrated with carbon monoxide, which effectively stopped further production. Methemoglobin disappearance was first order in nature with specific rate constants (k x 1,000) of 12.9 +/- 1.3 min(-1) for fetal sheep, 5.88 +/- 0.26 min(-1) for adult sheep, 4.27 +/- 0.34 for adult humans, and 3.30 +/- 0.15 for newborn cord blood, all statistically different from one another. The effects of oxygen tensions, pH, hemolysis, and methylene blue are reported. Studies of temperature dependence indicated an activation energy of 8,620 +/- 1,060 calories/mol (2.06 kJ/mol), appreciably higher than would be characteristic of processes limited by passive membrane diffusion. In conclusion, the novel methodology permits absolute quantification of the reduction of nitrite-induced methemoglobin in whole blood.

  7. Nitrite in organ protection

    PubMed Central

    Rassaf, Tienush; Ferdinandy, Peter; Schulz, Rainer

    2014-01-01

    In the last decade, the nitrate-nitrite-nitric oxide pathway has emerged to therapeutical importance. Modulation of endogenous nitrate and nitrite levels with the subsequent S-nitros(yl)ation of the downstream signalling cascade open the way for novel cytoprotective strategies. In the following, we summarize the actual literature and give a short overview on the potential of nitrite in organ protection. PMID:23826831

  8. Iridoid synthase activity is common among the plant progesterone 5β-reductase family.

    PubMed

    Munkert, Jennifer; Pollier, Jacob; Miettinen, Karel; Van Moerkercke, Alex; Payne, Richard; Müller-Uri, Frieder; Burlat, Vincent; O'Connor, Sarah E; Memelink, Johan; Kreis, Wolfgang; Goossens, Alain

    2015-01-01

    Catharanthus roseus, the Madagascar periwinkle, synthesizes bioactive monoterpenoid indole alkaloids, including the anti-cancer drugs vinblastine and vincristine. The monoterpenoid branch of the alkaloid pathway leads to the secoiridoid secologanin and involves the enzyme iridoid synthase (IS), a member of the progesterone 5β-reductase (P5βR) family. IS reduces 8-oxogeranial to iridodial. Through transcriptome mining, we show that IS belongs to a family of six C. roseus P5βR genes. Characterization of recombinant CrP5βR proteins demonstrates that all but CrP5βR3 can reduce progesterone and thus can be classified as P5βRs. Three of them, namely CrP5βR1, CrP5βR2, and CrP5βR4, can also reduce 8-oxogeranial, pointing to a possible redundancy with IS (corresponding to CrP5βR5) in secoiridoid synthesis. In-depth functional analysis by subcellular protein localization, gene expression analysis, in situ hybridization, and virus-induced gene silencing indicate that besides IS, CrP5βR4 may also participate in secoiridoid biosynthesis. We cloned a set of P5βR genes from angiosperm plant species not known to produce iridoids and demonstrate that the corresponding recombinant proteins are also capable of using 8-oxogeranial as a substrate. This suggests that IS activity is intrinsic to angiosperm P5βR proteins and has evolved early during evolution.

  9. Iridoid Synthase Activity Is Common among the Plant Progesterone 5β-Reductase Family.

    PubMed

    Munkert, Jennifer; Pollier, Jacob; Miettinen, Karel; Van Moerkercke, Alex; Payne, Richard; Müller-Uri, Frieder; Burlat, Vincent; O'Connor, Sarah E; Memelink, Johan; Kreis, Wolfgang; Goossens, Alain

    2014-09-19

    Catharanthus roseus, the Madagascar periwinkle, synthesizes bioactive monoterpenoid indole alkaloids, among which the anti-cancer drugs vinblastine and vincristine. The monoterpenoid branch of the alkaloid pathway leads to the secoiridoid secologanin and involves the enzyme iridoid synthase (IS), a member of the progesterone 5β-reductase (P5βR) family. IS reduces 8-oxogeranial to iridodial. Through transcriptome mining, we show that IS belongs to a family of six C. roseus P5βR genes. Characterisation of recombinant CrP5βR proteins demonstrates that all but CrP5βR3 can reduce progesterone, and thus can be classified as P5βRs. Three of them, namely CrP5βR1, CrP5βR2 and CrP5βR4, could also reduce 8-oxogeranial, pointing to a possible redundancy with IS (corresponding to CrP5βR5) in secoiridoid synthesis. In depth functional analysis by subcellular protein localisation, gene expression analysis, in situ hybridisation and virus-induced gene silencing, indicates that besides IS, CrP5βR4 may also participate in secoiridoid biosynthesis. Finally, we cloned a set of P5βR genes from angiosperm plant species not known to produce iridoids and demonstrate that the corresponding recombinant proteins are also capable of using 8-oxogeranial as a substrate. This suggests that 'IS activity' is intrinsic to angiosperm P5βR proteins and has evolved early during evolution.

  10. Pivotal role of dihydrofolate reductase knockdown in the anticancer activity of 2-hydroxyoleic acid

    PubMed Central

    Lladó, Victoria; Terés, Silvia; Higuera, Mónica; Álvarez, Rafael; Noguera-Salva, Maria Antònia; Halver, John E.; Escribá, Pablo V.; Busquets, Xavier

    2009-01-01

    α-Hydroxy-9-cis-octadecenoic acid, a synthetic fatty acid that modifies the composition and structure of lipid membranes. 2-Hydroxyoleic acid (HOA) generated interest due to its potent, yet nontoxic, anticancer activity. It induces cell cycle arrest in human lung cancer (A549) cells and apoptosis in human leukemia (Jurkat) cells. These two pathways may explain how HOA induces regression of a variety of cancers. We showed that HOA repressed the expression of dihydrofolate reductase (DHFR), the enzyme responsible for tetrahydrofolate (THF) synthesis. Folinic acid, which readily produces THF without the participation of DHFR, reverses the antitumor effects of HOA in A549 and Jurkat cells, as well as the inhibitory influence on cyclin D and cdk2 in A549 cells, and on DNA and PARP degradation in Jurkat cells. This effect was very specific, because either elaidic acid (an analog of HOA) or other lipids, failed to alter A549 or Jurkat cell growth. THF is a cofactor necessary for DNA synthesis. Thus, impairment of DNA synthesis appears to be a common mechanism involved in the different responses elicited by cancer cells following treatment with HOA, namely cell cycle arrest or apoptosis. Compared with other antifolates, such as methotrexate, HOA did not directly inhibit DHFR but rather, it repressed its expression, a mode of action that offers certain therapeutic advantages. These results not only demonstrate the effect of a fatty acid on the expression of DHFR, but also emphasize the potential of HOA to be used as a wide-spectrum drug against cancer. PMID:19666584

  11. Biosynthesis of catalytically active rat testosterone 5. alpha. -reductase in microinjected Xenopus oocytes: Evidence for tissue-specific differences in translatable mRNA

    SciTech Connect

    Farkash, Y.; Soreq, H.; Orly, J. )

    1988-08-01

    The enzyme 4-ene-3-ketosteroid-5{alpha}-oxidoreductase plays a key role in androgen-dependent target tissues, where it catalyzes the conversion of testosterone to the biologically active dihydrotestosterone. The regulation of 5{alpha}-reductase expression has not been studied at the molecular level as the enzyme is a membrane protein that is labile in cell-free homogenates. The authors developed a sensitive bioassay of the enzyme activity expressed in Xenopus oocytes microinjected with rat liver and prostate mRNA. After microinjection, incubation of intact oocytes in the presence of ({sup 3}H)testosterone revealed the in ovo appearance of active 5{alpha}-reductase. Polyandenylylated RNA was fractionated by sucrose gradient centrifugation, and the enzymatic activity was shown to be encoded by a 1,600- to 2,000-base-pair fraction of hepatic poly(A){sup +} RNA. 5{alpha}-Reductase mRNA was most efficiently translated when up to 80 ng of RNA was injected per oocyte. In the injected oocytes, 5{alpha}-reductase mRNA was found to be a short-lived molecule whereas its in ovo translatable 5{alpha}-reductase protein exhibited stable enzymatic activity for over 40 hr. Moreover, the levels of translatable tissue-specific 5{alpha}-reductase mRNAs as monitored in the Xenopus oocytes correlated with the variable 5{alpha}-reductase activities in female rat liver, male rat liver, and prostate homogenates. Altogether, these results provide supporting evidence in favor of the transcriptional control of 5{alpha}-reductase expression in rat tissues.

  12. Serenoa repens (Permixon) inhibits the 5alpha-reductase activity of human prostate cancer cell lines without interfering with PSA expression.

    PubMed

    Habib, Fouad K; Ross, Margaret; Ho, Clement K H; Lyons, Valerie; Chapman, Karen

    2005-03-20

    The phytotherapeutic agent Serenoa repens is an effective dual inhibitor of 5alpha-reductase isoenzyme activity in the prostate. Unlike other 5alpha-reductase inhibitors, Serenoa repens induces its effects without interfering with the cellular capacity to secrete PSA. Here, we focussed on the possible pathways that might differentiate the action of Permixon from that of synthetic 5alpha-reductase inhibitors. We demonstrate that Serenoa repens, unlike other 5alpha-reductase inhibitors, does not inhibit binding between activated AR and the steroid receptor-binding consensus in the promoter region of the PSA gene. This was shown by a combination of techniques: assessment of the effect of Permixon on androgen action in the LNCaP prostate cancer cell line revealed no suppression of AR and maintenance of PSA protein expression at control levels. This was consistent with reporter gene experiments showing that Permixon failed to interfere with AR-mediated transcriptional activation of PSA and that both testosterone and DHT were equally effective at maintaining this activity. Our results demonstrate that despite Serenoa repens effective inhibition of 5alpha-reductase activity in the prostate, it did not suppress PSA secretion. Therefore, we confirm the therapeutic advantage of Serenoa repens over other 5alpha-reductase inhibitors as treatment with the phytotherapeutic agent will permit the continuous use of PSA measurements as a useful biomarker for prostate cancer screening and for evaluating tumour progression.

  13. Thioredoxin reductase regulates AP-1 activity as well as thioredoxin nuclear localization via active cysteines in response to ionizing radiation.

    PubMed

    Karimpour, Shervin; Lou, Junyang; Lin, Lilie L; Rene, Luis M; Lagunas, Lucio; Ma, Xinrong; Karra, Sreenivasu; Bradbury, C Matthew; Markovina, Stephanie; Goswami, Prabhat C; Spitz, Douglas R; Hirota, Kiichi; Kalvakolanu, Dhananjaya V; Yodoi, Junji; Gius, David

    2002-09-12

    A recently identified class of signaling factors uses critical cysteine motif(s) that act as redox-sensitive 'sulfhydryl switches' to reversibly modulate specific signal transduction cascades regulating downstream proteins with similar redox-sensitive sites. For example, signaling factors such as redox factor-1 (Ref-1) and transcription factors such as the AP-1 complex both contain redox-sensitive cysteine motifs that regulate activity in response to oxidative stress. The mammalian thioredoxin reductase-1 (TR) is an oxidoreductase selenocysteine-containing flavoprotein that also appears to regulate multiple downstream intracellular redox-sensitive proteins. Since ionizing radiation (IR) induces oxidative stress as well as increases AP-1 DNA-binding activity via the activation of Ref-1, the potential roles of TR and thioredoxin (TRX) in the regulation of AP-1 activity in response to IR were investigated. Permanently transfected cell lines that overexpress wild type TR demonstrated constitutive increases in AP-1 DNA-binding activity as well as AP-1-dependent reporter gene expression, relative to vector control cells. In contrast, permanently transfected cell lines expressing a TR gene with the active site cysteine motif deleted were unable to induce AP-1 activity or reporter gene expression in response to IR. Transient genetic overexpression of either the TR wild type or dominant-negative genes demonstrated similar results using a transient assay system. One mechanism through which TR regulates AP-1 activity appears to involve TRX sub-cellular localization, with no change in the total TRX content of the cell. These results identify a novel function of the TR enzyme as a signaling factor in the regulation of AP-1 activity via a cysteine motif located in the protein.

  14. Overexpression and enhanced specific activity of aldoketo reductases (AKR1B1 & AKR1B10) in human breast cancers.

    PubMed

    Reddy, K Ashok; Kumar, P Uday; Srinivasulu, M; Triveni, B; Sharada, K; Ismail, Ayesha; Reddy, G Bhanuprakash

    2017-02-01

    The incidence of breast cancer in India is on the rise and is rapidly becoming the primary cancer in Indian women. The aldoketo reductase (AKR) family has more than 190 proteins including aldose reductase (AKR1B1) and aldose reductase like protein (AKR1B10). Apart from liver cancer, the status of AKR1B1 and AKR1B10 with respect to their expression and activity has not been reported in other human cancers. We studied the specific activity and expression of AKR1B1 and AKR1B10 in breast non tumor and tumor tissues and in the blood. Fresh post-surgical breast cancer and non-cancer tissues and blood were collected from the subjects who were admitted for surgical therapy. Malignant, benign and pre-surgical chemotherapy samples were evaluated by histopathology scoring. Expression of AKR1B1 and AKR1B10 was carried out by immunoblotting and immunohistochemistry (IHC) while specific activity was determined spectrophotometrically. The specific activity of AKR1B1 was significantly higher in red blood cells (RBC) in all three grades of primary surgical and post-chemotherapy samples. Specific activity of both AKR1B1 and AKR1B10 increased in tumor samples compared to their corresponding non tumor samples (primary surgical and post-chemotherapy). Immunoblotting and IHC data also indicated overexpression of AKR1B1 in all grades of tumors compared to their corresponding non tumor samples. There was no change in the specific activity of AKR1B1 in benign samples compared to all grades of tumor and non-tumors.

  15. Ethylene could influence ferric reductase, iron transporter, and H+-ATPase gene expression by affecting FER (or FER-like) gene activity.

    PubMed

    Lucena, Carlos; Waters, Brian M; Romera, F Javier; García, María José; Morales, María; Alcántara, Esteban; Pérez-Vicente, Rafael

    2006-01-01

    In previous works, it has been shown, by using ethylene inhibitors and precursors, that ethylene could participate in the regulation of the enhanced ferric reductase activity of Fe-deficient Strategy I plants. However, it was not known whether ethylene regulates the ferric reductase gene expression or other aspects related to this activity. This paper is a study of the effects of ethylene inhibitors and precursors on the expression of the genes encoding the ferric reductases and iron transporters of Arabidopsis thaliana (FRO2 and IRT1) and Lycopersicon esculentum (=Solanum lycopersicum) (FRO1 and IRT1) plants. The effects of ethylene inhibitors and precursors on the activity of the iron reductase and the iron transporter have been examined in parallel. Also studied were the effects of ethylene inhibitors and precursors on the expression of the H(+)-ATPase genes of cucumber (CsHA1 and CsHA2) and the transcription factor genes of tomato (LeFER) and Arabidopsis (AtFRU or AtFIT1, an LeFER homologue) that regulate ferric reductase, iron transporter, and H(+)-ATPse activity. The results obtained suggest that ethylene participates in the regulation of ferric reductase, the iron transporter, and H(+)-ATPase gene expression by affecting the FER (or FER-like) levels.

  16. Growth of Campylobacter jejuni on nitrate and nitrite: electron transport to NapA and NrfA via NrfH and distinct roles for NrfA and the globin Cgb in protection against nitrosative stress.

    PubMed

    Pittman, Marc S; Elvers, Karen T; Lee, Lucy; Jones, Michael A; Poole, Robert K; Park, Simon F; Kelly, David J

    2007-01-01

    Pathways of electron transport to periplasmic nitrate (NapA) and nitrite (NrfA) reductases have been investigated in Campylobacter jejuni, a microaerophilic food-borne pathogen. The nap operon is unusual in lacking napC (encoding a tetra-haem c-type cytochrome) and napF, but contains a novel gene of unknown function, napL. The iron-sulphur protein NapG has a major role in electron transfer to the NapAB complex, but we show that slow nitrate-dependent growth of a napG mutant can be sustained by electron transfer from NrfH, the electron donor to the nitrite reductase NrfA. A napL mutant possessed approximately 50% lower NapA activity than the wild type but showed normal growth with nitrate as the electron acceptor. NrfA was constitutive and was shown to play a role in protection against nitrosative stress in addition to the previously identified NO-inducible single domain globin, Cgb. However, nitrite also induced cgb expression in an NssR-dependent manner, suggesting that growth of C. jejuni with nitrite causes nitrosative stress. This was confirmed by lack of growth of cgb and nssR mutants, and slow growth of the nrfA mutant, in media containing nitrite. Thus, NrfA and Cgb together provide C. jejuni with constitutive and inducible components of a robust defence against nitrosative stress.

  17. The effect of melatonin on lipid peroxidation and nitrite/nitrate levels, and on superoxide dismutase and catalase activities in kainic acid-induced injury.

    PubMed

    Akcay, Yasemin Delen; Yalcin, Ayfer; Sozmen, Eser Yildirim

    2005-01-01

    Kainic acid (KA) initiates neuronal injury and death by inducing oxidative stress and nitric oxide release from various regions of the brain. It was recently shown that melatonin has free radical-scavenging action and may protect against kainate-induced toxicity. In order to assess the possible supportive effect of melatonin treatment in KA-induced injury in the rat brain cortex, we determined malondialdehyde (MDA) levels as an index of lipid peroxidation, and assessed the activities of catalase (CAT) and superoxide dismutase (SOD) and the levels of nitrite/nitrate 35 male rats were divided into five groups, each receiving a different intraperitoneal treatment: saline solution (0.2 ml), kainic acid (15 mg/kg), melatonin (20 mg/kg), KA then melatonin (each as above, 15 min apart), or melatonin then KA (each as above, 30 min apart). Administration of KA caused an about five-fold increase in the catalase activity and an increase in the SOD activity in the cortex relative to the activities for the controls. Treatment with melatonin 15 min after KA injection kept malondialdehyde levels and catalase and superoxide dismutase activities at the normal levels, and led to an increase in the levels of nitrite/nitrate. Our data suggests that melatonin treatment following KA administration has a protective effect on antioxidant enzyme activities and thus supports the role of melatonin and oxidative stress in the regulation of antioxidative enzyme activity.

  18. An arsenate reductase homologue possessing phosphatase activity from sweet potato (Ipomoea batatas [L.] Lam): kinetic studies and characterization.

    PubMed

    Chan, Ya-Hui; Lin, Chao-Yi; Pai, Shou-Hsiung; Huang, Jenq-Kuen; Lin, Chi-Tsai

    2011-04-13

    A cDNA encoding a putative arsenate reductase homologue (IbArsR) was cloned from sweet potato (Ib). The deduced protein showed a high level of sequence homology (16-66%) with ArsRs from other organisms. A 3-D homology structure was created based on AtArsR (PDB code 1T3K ) from Arabidopsis thaliana. The putative active site of protein tyrosine phosphatase (HC(X)(5)R) is conserved in all reported ArsRs. IbArsR was overexpressed and purified. The monomeric nature of the enzyme was confirmed by 15% SDS-PAGE and molecular mass determination of the native enzyme via ESI Q-TOF. The IbArsR lacks arsenate reductase activity but possesses phosphatase activity. The Michaelis constant (K(M)) value for p-nitrophenyl phosphate (pNPP) was 11.11 mM. The phosphatase activity was inhibited by 0.5 mM sodium arsenate [As(V)]. The protein's half-life of deactivation at 25 °C was 6.1 min, and its inactivation rate constant K(d) was 1.1 × 10(-1) min(-1). The enzyme was active in a broad pH range from 4.0 to 11.0 with optimum activity at pH 10.0. Phosphatase would remove phosphate group from nucleic acid or dephosphorylation of other enzymes as regulation signaling.

  19. Inhibition of vacuolation toxin activity of Helicobacter pylori by iodine, nitrite and potentiation by sodium chloride, sterigmatocystin and fluoride.

    PubMed

    Ma, Fengjuan; Zhao, Wenyuan; Kudo, Masanobu; Aoki, Kazuo; Misumi, Junichi

    2002-10-01

    The toxin VacA produced by Helicobacter pylori is an important determinant of virulence. VacA causes vacuolation of cultured cells such as HeLa cells. Iodine, nitrite, sodium chloride, thiocyanate and fungus toxin sterigmatocystin are universally present in nature and could possibly be related to carcinogenesis of the stomach. The present study was designed to examine the effects of the above-mentioned compound on VacA-induced vacuolation of HeLa cells, which was quantitated using the neutral red uptake assay. VacA-induced vacuolation was inhibited by BafA1 and NPPB. Formation of large vacuoles was inhibited in the presence of iodine, nitrite, but enhanced by sodium chloride, thiocyanate, fluoride and sterigmatocystin. Our results indicate that VacA toxin may interact with other gastric cancer risk factors present naturally in the environment, and suggest that those compounds may modulate the development of gastric cancer induced by H. pylori.

  20. Diurnal variation in the fraction of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in the active form in the mammary gland of the lactating rat.

    PubMed Central

    Smith, R A; Middleton, B; West, D W

    1986-01-01

    'Expressed' and 'total' activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) were measured in freeze-clamped samples of mammary glands from lactating rats at intervals throughout the 24 h light/dark cycle. 'Expressed' activities were measured in microsomal fractions isolated and assayed in the presence of 100 mM-KF. 'Total' activities were determined in microsomal preparations from the same homogenates but washed free of KF and incubated with exogenously added sheep liver phosphoprotein phosphatase before assay. Both 'expressed' and 'total' activities of HMG-CoA reductase underwent a diurnal cycle, which had a major peak 6 h into the light phase and a nadir 15 h later, i.e. 9 h into the dark period. Both activities showed a secondary peak of activity (around 68% of the maximum activity) at the time of changeover from dark to light, with a trough in the value of the 'expressed' activity that was close to the nadir value. 'Expressed' activity was lower than 'total' at all time points, indicating the presence of enzyme molecules inactivated by covalent phosphorylation. Nevertheless the 'expressed'/'total' activity ratio was comparatively constant and varied only between 43% and 75%. Immunotitration of enzyme activity, with antiserum raised in sheep against purified rat liver HMG-CoA reductase, confirmed the presence of both active and inactive forms of the enzyme and indicated that at the peak and nadir the variation in 'expressed' HMG-CoA reductase activity resulted from changes in the total number of enzyme molecules rather than from covalent modification. The sample obtained after 3 h of the light phase exhibited an anomalously low 'total' HMG-CoA reductase activity, which could be increased when Cl- replaced F- in the homogenization medium. The result suggests that at that time the activity of the enzyme could be regulated by mechanisms other than covalent phosphorylation or degradation. PMID:3814075

  1. Nitrite inhalants: history, epidemiology, and possible links to AIDS.

    PubMed Central

    Haverkos, H W; Kopstein, A N; Wilson, H; Drotman, P

    1994-01-01

    Nitrite inhalants have been commonly abused substances in the United States. Nitrite inhalants and AIDS was a popular topic in the early 1980s, when the cause of AIDS was not known. With the discovery of HIV, concern about nitrite use in the USA waned. However, nitrite inhalant use is associated with behavioral relapse and HIV transmission among gay men, with decreased lymphocyte counts and natural killer cell activity in a few laboratory studies, and it remains a candidate cofactor in the pathogenesis of AIDS-related Kaposi's sarcoma. Discouraging nitrite use continues to be a worthwhile public health goal. PMID:9644194

  2. Modification of membrane sulfhydryl groups in bacteriostatic action of nitrite

    SciTech Connect

    Buchman, G.W. III; Hansen, J.N.

    1987-01-01

    The mechanism by which nitrite inhibits outgrowing spores of bacillus cereus T was examined by using techniques developed earlier for nitrite analogs. The morphological stage of inhibition, cooperativity effects, effect of pH on inhibition, kinetics of protection against tritiated iodoacetate incorporation into membrane sulfhydryl groups, and protection against the bacteriocidal effect of carboxymethylation of iodoacetate indicate that nitrite acts as a membrane-directed sulfhydryl agent. The mechanism by which nitrite modifies the chemical reactivity of the sulfhyrdyl group could be either direct covalent modification or inactivation through communication with another modified membrane component. Profiles of pH effects suggest that the active agent is the protonated form of nitrite. The nitrite concentrations which modify membrane sulfhydryl activity coincide with those which have a bacteriostatic effect. These results are consistent with membrane sulfhydryl modification as a component of the mechanism of nitrite-induced bacteriostasis in this aerobic sporeformer.

  3. HMG-CoA reductase inhibitor rosuvastatin improves abnormal brain electrical activity via mechanisms involving eNOS.

    PubMed

    Seker, F B; Kilic, U; Caglayan, B; Ethemoglu, M S; Caglayan, A B; Ekimci, N; Demirci, S; Dogan, A; Oztezcan, S; Sahin, F; Yilmaz, B; Kilic, E

    2015-01-22

    Apart from its repressing effect on plasma lipid levels, 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors exert neuroprotective functions in animal models of neurodegenerative disorders. In view of these promising observations, we were interested in whether HMG-CoA reductase inhibition would affect epileptiform activity in the brain. To elucidate this issue, atorvastatin, simvastatin and rosuvastatin were administered orally at a dose of 20 mg/kg each for 3 days and their anti-epileptic activities were tested and compared in rats. Epileptiform activity in the brain was induced by an intracortical penicillin G injection. Among HMG-CoA reductase inhibitors, simvastatin-treatment was less effective in terms of spike frequency as compared with atorvastatin- and rosuvastatin-treated animals. Atorvastatin treatment reduced spike frequencies and amplitudes significantly throughout the experiment. However, the most pronounced anti-epileptic effect was observed in rosuvastatin-treated animals, which was associated with improved blood-brain barrier (BBB) integrity, increased expression of endothelial nitric oxide synthase (eNOS) mRNA and decreased expressions of pro-apoptotic p53, Bax and caspase-3 mRNAs. Inhibition of eNOS activity with L-NG-Nitroarginine Methyl Ester (L-NAME) reversed the anti-epileptic effect of rosuvastatin significantly. However, L-NAME did not alter the effect of rosuvastatin on the levels of p53, Bax and caspase-3 mRNA expression. Here, we provide evidence that among HMG-CoA reductase inhibitors, rosuvastatin was the most effective statin on the reduction of epileptiform activity, which was associated with improved BBB permeability, increased expression of eNOS and decreased expressions of pro-apoptotic p53, Bax and caspase-3. Our observation also revealed that the anti-epileptic effect of rosuvastatin was dependent on the increased expression level of eNOS. The robust anti-epileptic effect encourages proof-of-concept studies with

  4. Stereospecific micellar electrokinetic chromatography assay of methionine sulfoxide reductase activity employing a multiple layer coated capillary.

    PubMed

    Zhu, Qingfu; El-Mergawy, Rabab G; Heinemann, Stefan H; Schönherr, Roland; Jáč, Pavel; Scriba, Gerhard K E

    2013-09-01

    A micellar electrokinetic chromatography method for the analysis of the l-methionine sulfoxide diastereomers employing a successive multiple ionic-polymer layer coated fused-silica capillary was developed and validated in order to investigate the stereospecificity of methionine sulfoxide reductases. The capillary coating consisted of a first layer of hexadimethrine and a second layer of dextran sulfate providing a stable strong cathodic EOF and consequently highly repeatable analyte migration times. The methionine sulfoxide diastereomers, methionine as product as well as β-alanine as internal standard were derivatized by dabsyl chloride and separated using a 35 mM sodium phosphate buffer, pH 8.0, containing 25 mM SDS as BGE and a separation voltage of 25 kV. The method was validated in the range of 0.15-2.0 mM with respect to linearity and precision. The LODs of the analytes ranged between 0.04 and 0.10 mM. The assay was subsequently applied to determine the stereospecificity of methionine sulfoxide reductases as well as the enzyme kinetics of human methionine sulfoxide reductase A. Monitoring the decrease of the l-methionine-(S)-sulfoxide Km = 411.8 ± 33.8 μM and Vmax = 307.5 ± 10.8 μM/min were determined.

  5. Genome sequence of the chemolithoautotrophic nitrite-oxidizing bacterium Nitrobacter winogradskyi Nb-255

    SciTech Connect

    Hauser, Loren John; Land, Miriam L; Larimer, Frank W; Arp, D J; Hickey, W J

    2006-03-01

    The alphaproteobacterium Nitrobacter winogradskyi (ATCC 25391) is a gram-negative facultative chemolithoautotroph capable of extracting energy from the oxidation of nitrite to nitrate. Sequencing and analysis of its genome revealed a single circular chromosome of 3,402,093 bp encoding 3,143 predicted proteins. There were extensive similarities to genes in two alphaproteobacteria, Bradyrhizobium japonicum USDA110 (1,300 genes) and Rhodopseudomonas palustris CGA009 CG (815 genes). Genes encoding pathways for known modes of chemolithotrophic and chemoorganotrophic growth were identified. Genes encoding multiple enzymes involved in anapleurotic reactions centered on C2 to C4 metabolism, including a glyoxylate bypass, were annotated. The inability of N. winogradskyi to grow on C6 molecules is consistent with the genome sequence, which lacks genes for complete Embden-Meyerhof and Entner-Doudoroff pathways, and active uptake of sugars. Two gene copies of the nitrite oxidoreductase, type I ribulose-1,5-bisphosphate carboxylase/oxygenase, cytochrome c oxidase, and gene homologs encoding an aerobic-type carbon monoxide dehydrogenase were present. Similarity of nitrite oxidoreductases to respiratory nitrate reductases was confirmed. Approximately 10% of the N. winogradskyi genome codes for genes involved in transport and secretion, including the presence of transporters for various organic-nitrogen molecules. The N. winogradskyi genome provides new insight into the phylogenetic identity and physiological capabilities of nitrite-oxidizing bacteria. The genome will serve as a model to study the cellular and molecular processes that control nitrite oxidation and its interaction with other nitrogen-cycling processes.

  6. Genome Sequence of the Chemolithoautotrophic Nitrite-Oxidizing Bacterium Nitrobacter winogradskyi Nb-255

    PubMed Central

    Starkenburg, Shawn R.; Chain, Patrick S. G.; Sayavedra-Soto, Luis A.; Hauser, Loren; Land, Miriam L.; Larimer, Frank W.; Malfatti, Stephanie A.; Klotz, Martin G.; Bottomley, Peter J.; Arp, Daniel J.; Hickey, William J.

    2006-01-01

    The alphaproteobacterium Nitrobacter winogradskyi (ATCC 25391) is a gram-negative facultative chemolithoautotroph capable of extracting energy from the oxidation of nitrite to nitrate. Sequencing and analysis of its genome revealed a single circular chromosome of 3,402,093 bp encoding 3,143 predicted proteins. There were extensive similarities to genes in two alphaproteobacteria, Bradyrhizobium japonicum USDA110 (1,300 genes) and Rhodopseudomonas palustris CGA009 CG (815 genes). Genes encoding pathways for known modes of chemolithotrophic and chemoorganotrophic growth were identified. Genes encoding multiple enzymes involved in anapleurotic reactions centered on C2 to C4 metabolism, including a glyoxylate bypass, were annotated. The inability of N. winogradskyi to grow on C6 molecules is consistent with the genome sequence, which lacks genes for complete Embden-Meyerhof and Entner-Doudoroff pathways, and active uptake of sugars. Two gene copies of the nitrite oxidoreductase, type I ribulose-1,5-bisphosphate carboxylase/oxygenase, cytochrome c oxidase, and gene homologs encoding an aerobic-type carbon monoxide dehydrogenase were present. Similarity of nitrite oxidoreductases to respiratory nitrate reductases was confirmed. Approximately 10% of the N. winogradskyi genome codes for genes involved in transport and secretion, including the presence of transporters for various organic-nitrogen molecules. The N. winogradskyi genome provides new insight into the phylogenetic identity and physiological capabilities of nitrite-oxidizing bacteria. The genome will serve as a model to study the cellular and molecular processes that control nitrite oxidation and its interaction with other nitrogen-cycling processes. PMID:16517654

  7. Characterization of quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus: enzymatic activity and active site structure.

    PubMed

    Terasaka, Erina; Okada, Norihiro; Sato, Nozomi; Sako, Yoshihiko; Shiro, Yoshitsugu; Tosha, Takehiko

    2014-07-01

    Nitric oxide reductase (NOR) catalyzes the reduction of nitric oxide to generate nitrous oxide. We recently reported on the crystal structure of a quinol-dependent NOR (qNOR) from Geobacillus stearothermophilus [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238-246], and suggested that a water channel from the cytoplasm, which is not observed in cytochrome c-dependent NOR (cNOR), functions as a pathway transferring catalytic protons. Here, we further investigated the functional and structural properties of qNOR, and compared the findings with those for cNOR. The pH optimum for the enzymatic reaction of qNOR was in the alkaline range, whereas Pseudomonas aeruginosa cNOR showed a higher activity at an acidic pH. The considerably slower reduction rate, and a correlation of the pH dependence for enzymatic activity and the reduction rate suggest that the reduction process is the rate-determining step for the NO reduction by qNOR, while the reduction rate for cNOR was very fast and therefore is unlikely to be the rate-determining step. A close examination of the heme/non-heme iron binuclear center by resonance Raman spectroscopy indicated that qNOR has a more polar environment at the binuclear center compared with cNOR. It is plausible that a water channel enhances the accessibility of the active site to solvent water, creating a more polar environment in qNOR. This structural feature could control certain properties of the active site, such as redox potential, which could explain the different catalytic properties of the two NORs. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

  8. Effects of SRT and DO on N2O reductase activity in an anoxic-oxic activated sludge system.

    PubMed

    Noda, N; Kaneko, N; Mikami, M; Kimochi, Y; Tsuneda, S; Hirata, A; Mizuochi, M; Inamori, Y

    2003-01-01

    Nitrous oxide (N2O) is emitted from wastewater treatment processes, and is known to be a green house gas contributing to global warming. It is thus important to develop technology that can suppress N2O emission. The effects of sludge retention time (SRT) and dissolved oxygen (DO) on N2O emission in an anoxic-oxic activated sludge system were estimated. Moreover, the microbial community structure in the sludge, which plays an important role in N2O suppression, was clarified based on nitrous oxide reductase (nosZ) gene analysis by molecular biological techniques. The results showed that under low SRT conditions, nitrification efficiency was reduced and the N2O emission rate in the oxic reactors was increased. It was also observed that N2O emission was enhanced under low DO conditions, where the available oxygen is insufficient for nitrification. Moreover, molecular analysis revealed that the clones identified in this study were closely related to Ralstonia eutropha and Paracoccus denitrificans. The fact that the identified sequences are not closely related to known culturable denitrifier nosZ sequences indicates a substantial in situ diversity of denitrifiers contributing to N2O suppression, which are not reflected in the cultivatable fraction of the population. The further application of these new molecular techniques should serve to enhance our knowledge of the microbial community of denitrifying bacteria contributing to N2O suppression in wastewater treatment systems.

  9. Mercuric reductase activity and evidence of broad-spectrum mercury resistance among clinical isolates of rapidly growing mycobacteria

    SciTech Connect

    Steingrube, V.A.; Wallace, R.J. Jr.; Steele, L.C.; Pang, Y.J. )

    1991-05-01

    Resistance to mercury was evaluated in 356 rapidly growing mycobacteria belonging to eight taxonomic groups. Resistance to inorganic Hg2+ ranged from 0% among the unnamed third biovariant complex of Mycobacterium fortuitum to 83% among M. chelonae-like organisms. With cell extracts and 203Hg(NO3)2 as the substrate, mercuric reductase (HgRe) activity was demonstrable in six of eight taxonomic groups. HgRe activity was inducible and required NADPH or NADH and a thiol donor for optimai activity. Species with HgRe activity were also resistant to organomercurial compounds, including phenylmercuric acetate. Attempts at intraspecies and intragenus transfer of HgRe activity by conjugation or transformation were unsuccessful. Mercury resistance is common in rapidly growing mycobacteria and appears to function via the same inducible enzyme systems already defined in other bacterial species. This system offers potential as a strain marker for epidemiologic investigations and for studying genetic systems in rapidly growing mycobacteria.

  10. Effect of pH and HNO2 concentration on the activity of ammonia-oxidizing bacteria in a partial nitritation reactor.

    PubMed

    Claros, J; Jiménez, E; Aguado, D; Ferrer, J; Seco, A; Serralta, J

    2013-01-01

    Ammonia-oxidizing bacteria (AOB) are very sensitive to environmental conditions and wastewater treatment plant operational parameters. One of the most important factors affecting their activity is pH. Its effect is associated with: NH3/NH4(+) and HNO2/NO2(-) chemical equilibriums and biological reaction rates. The aim of this study was to quantify and model the effect of pH and free nitrous acid (FNA) concentration on the activity of AOB present in a lab-scale partial nitritation reactor. For this purpose, two sets of batch experiments were carried out using biomass from this reactor. Fluorescent in situ hybridization analysis showed that Nitrosomona eutropha and Nitrosomona europaea species were dominant in the partial nitritation reactor (>94%). The experimental results showed that FNA inhibits the AOB activity. This inhibition was properly modelled by the non-competitive inhibition function and the half inhibition constant value was determined as 1.32 mg HNO2-N L(-1). The optimal pH for these AOB was found to be in the range 7.4-7.8. The pH inhibitory effect was stronger at high pH values than at low pH values. Therefore, an asymmetric inhibition function was proposed to represent the pH effect on these bacteria. A combination of two sigmoidal functions was able to reproduce the experimental results obtained.

  11. Inhabitancy of active Nitrosopumilus-like ammonia-oxidizing archaea and Nitrospira nitrite-oxidizing bacteria in the sponge Theonella swinhoei

    PubMed Central

    Feng, Guofang; Sun, Wei; Zhang, Fengli; Karthik, Loganathan; Li, Zhiyong

    2016-01-01

    Nitrification directly contributes to the ammonia removal in sponges, and it plays an indispensable role in sponge-mediated nitrogen cycle. Previous studies have demonstrated genomic evidences of nitrifying lineages in the sponge Theonella swinhoei. However, little is known about the transcriptional activity of nitrifying community in this sponge. In this study, combined DNA- and transcript-based analyses were performed to reveal the composition and transcriptional activity of the nitrifiers in T. swinhoei from the South China Sea. Transcriptional activity of ammonia-oxidizing archaea (AOA) and nitrite-oxidizing bacteria (NOB) in this sponge were confirmed by targeting their nitrifying genes,16S rRNA genes and their transcripts. Phylogenetic analysis coupled with RDP rRNA classification indicated that archaeal 16S rRNA genes, amoA (the subunit of ammonia monooxygenase) genes and their transcripts were closely related to Nitrosopumilus-like AOA; whereas nitrifying bacterial 16S rRNA genes, nxrB (the subunit of nitrite oxidoreductase) genes and their transcripts were closely related to Nitrospira NOB. Quantitative assessment demonstrated relative higher abundances of nitrifying genes and transcripts of Nitrosopumilus-like AOA than those of Nitrospira NOB in this sponge. This study illustrated the transcriptional potentials of Nitrosopumilus-like archaea and Nitrospira bacteria that would predominantly contribute to the nitrification functionality in the South China Sea T. swinhoei. PMID:27113140

  12. Protein kinase activity associated with the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10).

    PubMed Central

    Chung, T D; Wymer, J P; Smith, C C; Kulka, M; Aurelian, L

    1989-01-01

    The large subunit of the herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (RR1) is demonstrated to possess serine/threonine-specific kinase activity. Computer-assisted sequence analysis identified regions within the amino terminus of ICP10 that are homologous to the catalytic domain of known protein kinases (PKs). An in vitro kinase assay confirmed the ability of ICP10, immunoprecipitated from either HSV-2-infected cells or from cells transfected with an ICP10 expression vector, to autophosphorylate and transphosphorylate exogenous substrates in the presence of ATP and Mg2+. The HSV-1 RR1 was shown to be negative for PK activity under these conditions. PK activity was localized to a 57-kDa amino-terminal region within ICP10 that lacked RR activity. Images PMID:2545912

  13. Design, synthesis, and biological activity of diaryl ether inhibitors of Toxoplasma gondii enoyl reductase.

    PubMed

    Cheng, Gang; Muench, Stephen P; Zhou, Ying; Afanador, Gustavo A; Mui, Ernest J; Fomovska, Alina; Lai, Bo Shiun; Prigge, Sean T; Woods, Stuart; Roberts, Craig W; Hickman, Mark R; Lee, Patty J; Leed, Susan E; Auschwitz, Jennifer M; Rice, David W; McLeod, Rima

    2013-04-01

    Triclosan is a potent inhibitor of Toxoplasma gondii enoyl reductase (TgENR), which is an essential enzyme for parasite survival. In view of triclosan's poor druggability, which limits its therapeutic use, a new set of B-ring modified analogs were designed to optimize its physico-chemical properties. These derivatives were synthesized and evaluated by in vitro assay and TgENR enzyme assay. Some analogs display improved solubility, permeability and a comparable MIC50 value to that of triclosan. Modeling of these inhibitors revealed the same overall binding mode with the enzyme as triclosan, but the B-ring modifications have additional interactions with the strongly conserved Asn130.

  14. Nitrite-catalase interaction as an important element of nitrite toxicity.

    PubMed

    Titov, V Yu; Petrenko, Yu M

    2003-06-01

    It was established that nitrite in the presence of chloride, bromide, and thiocyanate decreases the rate of hydrogen peroxide decomposition by catalase. The decrease was recorded by the permanganatometric method and by a method of dynamic calorimetry. Nitrite was not destroyed in the course of the reaction and the total value of heat produced in the process was not changed by its presence. These facts suggest that nitrite induces inhibition of catalase with no change in the essence of the enzymatic process. Even micromolar nitrite concentrations induced a considerable decrease in catalase activity. However, in the absence of chloride, bromide, and thiocyanate inhibition was not observed. In contrast, fluoride protected catalase from nitrite inhibition in the presence of the above-mentioned halides and pseudohalide. As hydrogen peroxide is a necessary factor for triggering a number of important toxic effects of nitrite, the latter increases its toxicity by inhibiting catalase. This was shown by the example of nitrite-induced hemoglobin oxidation. The naturally existing gradient of chloride and other anion concentrations between intra- and extracellular media appears to be the most important mechanism of cell protection from inhibition of intracellular catalase by nitrite. Possible mechanisms of this inhibition are discussed.

  15. The L-arginine/NO pathway, homoarginine, and nitrite-dependent renal carbonic anhydrase activity in young people with type 1 diabetes mellitus.

    PubMed

    Carmann, Christina; Lilienthal, Eggert; Weigt-Usinger, Katharina; Schmidt-Choudhury, Anjona; Hörster, Irina; Kayacelebi, Arslan Arinc; Beckmann, Bibiana; Chobanyan-Jürgens, Kristine; Tsikas, Dimitrios; Lücke, Thomas

    2015-09-01

    High circulating levels of asymmetric dimethylarginine (ADMA) and low circulating levels of homoarginine (hArg) are known cardiovascular risk factors in adults. While in adults with type 1 diabetes mellitus (T1DM) circulating ADMA is significantly elevated, in children and adolescents the reported ADMA data are contradictory. In 102 children with T1DM and 95 healthy controls (HC) serving as controls, we investigated the L-arginine (Arg)/nitric oxide (NO) pathway. Children with T1DM were divided into two groups, i.e., in children with newly diagnosed diabetes mellitus [T1DM-ND; n = 10; age, 8.8 (4.4-11.2) years; HbA1c, 13 (8.9-13.9) %] and in those with long-term treatment [T1DM-T; n = 92; age, 12.5 (10.5-15.4) years; HbA1c, 8.0 (7.2-8.6) %]. The age of the HC was 11.3 (8-13.3) years. Amino acids and NO metabolites of the Arg/NO pathway, creatinine and the oxidative stress biomarker malondialdehyde (MDA) were measured by GC-MS or GC-MS/MS. Plasma hArg, ADMA and the hArg/ADMA molar ratio did not differ between the T1DM and HC groups. There was a significant difference between T1DM-T and HC with regard to plasma nitrite [0.53 (0.48-0.61) vs 2.05 (0.86-2.36) µM, P < 0.0001] as well as to urinary nitrite [0.09 (0.06-0.17) vs 0.22 (0.13-0.37) μmol/mmol creatinine, P < 0.0001]. Plasma, but not urinary nitrite, differed between T1DM-ND and HC [0.55 (0.50-0.66) vs 2.05 (0.86-2.36) µM, P < 0.0001]. Plasma MDA did not differ between the groups. The urinary nitrate-to-nitrite molar ratio (UNOXR), a measure of nitrite-dependent renal carbonic anhydrase (CA) activity, was higher in T1DM-T [1173 (738-1481), P < 0.0001] and T1DM-ND [1341 (1117-1615), P = 0.0007] compared to HC [540 (324-962)], but did not differ between T1DM-T and T1DM-ND (P = 0.272). The lower nitrite excretion in the children with T1DM may indicate enhanced renal CA-dependent nitrite reabsorption compared with healthy children. Yet, lower plasma nitrite concentration in the T1DM patients may have also

  16. [Illumination's effect on the growth and nitrate reductase activity of typical red-tide algae in the East China Sea].

    PubMed

    Li, Hong-mei; Shi, Xiao-yong; Ding, Yan-yan; Tang, Hong-jie

    2013-09-01

    Two typical red-tide algae, Skeletonema costatum and Prorocentrum donghaiense were selected as studied objects. The nitrate reductase activity (NRA) and the growth of the two algae under different illuminations through incubation experiment were studied. The illumination condition was consistent with in situ. Results showed that P. donghaiense and S. costatum could grow normally in the solar radiation ranged from 30-60 W x m(-2), and the growth curve was "S" type. However, when solar radiation was below 9 W x m(-2), the two alga could hardly grow. In the range of 0-60 W x m(-2), three parameters (NRAmax, micro(max), Bf) increased with the increasing of light intensity, indicating that the light intensity can influence the grow of alga indirectly through influencing the nitrate reductase activity. The micro(max) and NRAmax in unite volume of Skeletonema costatum were higher than those of Prorocentrum donghaiense, indicating that Skeletonema costatum can better utilize the nitrate than Prorocentrum donghaiense.

  17. Compensating for the absence of selenocysteine in high-molecular weight thioredoxin reductases: the electrophilic activation hypothesis.

    PubMed

    Lothrop, Adam P; Snider, Gregg W; Flemer, Stevenson; Ruggles, Erik L; Davidson, Ronald S; Lamb, Audrey L; Hondal, Robert J

    2014-02-04

    Mammalian thioredoxin reductase (TR) is a pyridine disulfide oxidoreductase that uses the rare amino acid selenocysteine (Sec) in place of the more commonly used amino acid cysteine (Cys). Selenium is a Janus-faced element because it is both highly nucleophilic and highly electrophilic. Cys orthologs of Sec-containing enzymes may compensate for the absence of a Sec residue by making the active site Cys residue more (i) nucleophilic, (ii) electrophilic, or (iii) reactive by increasing both S-nucleophilicity and S-electrophilicity. It has already been shown that the Cys ortholog TR from Drosophila melanogaster (DmTR) has increased S-nucleophilicity [Gromer, S., Johansson, L., Bauer, H., Arscott, L. D., Rauch, S., Ballou, D. P., Williams, C. H., Jr., Schrimer, R. H., and Arnér, E. S (2003) Active sites of thioredoxin reductases: Why selenoproteins? Proc. Natl. Acad. Sci. U.S.A. 100, 12618-12623]. Here we present evidence that DmTR also enhances the electrophilicity of Cys490 through the use of an "electrophilic activation" mechanism. This mechanism is proposed to work by polarizing the disulfide bond that occurs between Cys489 and Cys490 in the C-terminal redox center by the placement of a positive charge near Cys489. This polarization renders the sulfur atom of Cys490 electron deficient and enhances the rate of thiol/disulfide exchange that occurs between the N- and C-terminal redox centers. Our hypothesis was developed by using a strategy of homocysteine (hCys) for Cys substitution in the Cys-Cys redox dyad of DmTR to differentiate the function of each Cys residue. The results show that hCys could substitute for Cys490 with little loss of thioredoxin reductase activity, but that substitution of hCys for Cys489 resulted in a 238-fold reduction in activity. We hypothesize that replacement of Cys489 with hCys destroys an interaction between the sulfur atom of Cys489 and His464 crucial for the proposed electrophilic activation mechanism. This electrophilic activation

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

    PubMed

    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.

  19. Variation of glucosinolates and quinone reductase activity among different varieties of Chinese kale and improvement of glucoraphanin by metabolic engineering.

    PubMed

    Qian, Hongmei; Sun, Bo; Miao, Huiying; Cai, Congxi; Xu, Chaojiong; Wang, Qiaomei

    2015-02-01

    The variation of glucosinolates and quinone reductase (QR) activity in fourteen varieties of Chinese kale (Brassica oleracea var. alboglabra Bailey) was investigated in the present study. Results showed that gluconapin (GNA), instead of glucoraphanin (GRA), was the most predominant glucosinolate in all varieties, and QR activity was remarkably positively correlated with the glucoraphanin level. AOP2, a tandem 2-oxoglutarate-dependent dioxygenase, catalyzes the conversion of glucoraphanin to gluconapin in glucosinolate biosynthesis. Here, antisense AOP2 was transformed into Gailan-04, the variety with the highest gluconapin content and ratio of GNA/GRA. The glucoraphanin content and corresponding QR activity were notably increased in transgenic plants, while no significant difference at the level of other main nutritional compounds (total phenolics, vitamin C, carotenoids and chlorophyll) was observed between the transgenic lines and the wide-type plants. Taken together, metabolic engineering is a good practice for improvement of glucoraphanin in Chinese kale.

  20. Evidence that the amino acid residue Cys117 of chloroplastic monodehydroascorbate reductase is involved in its activity and structural stability.

    PubMed

    Li, Feng; Wu, Qing-Yun; Sun, Yan-Li; Ma, Na-Na; Wang, Xiao-Yun; Meng, Qing-Wei

    2010-04-01

    Monodehydroascorbate reductase (MDAR; EC 1.6.5.4) is crucial for AsA regeneration and essential for maintaining the reduced pool of AsA. And the amino acid residue C117 of chloroplastic MDAR is the conserved cysteine residue in MDAR isoforms. A series mutation of conserved amino acid residue cysteine117 (C117) was constructed to investigate its role in MDAR structural stability and activity. Our study revealed that mutation in this conserved residue could cause pronounced loss of activity and conformational changes. Spectroscopic experiments indicated that these mutations influenced transition from the molten globule intermediate to the native state in folding process. These results suggested that amino acid residue C117 played a relatively important role in keeping MDAR structural stability and activity.

  1. Compensating for the Absence of Selenocysteine in High-Molecular Weight Thioredoxin Reductases: The Electrophilic Activation Hypothesis

    PubMed Central

    2015-01-01

    Mammalian thioredoxin reductase (TR) is a pyridine disulfide oxidoreductase that uses the rare amino acid selenocysteine (Sec) in place of the more commonly used amino acid cysteine (Cys). Selenium is a Janus-faced element because it is both highly nucleophilic and highly electrophilic. Cys orthologs of Sec-containing enzymes may compensate for the absence of a Sec residue by making the active site Cys residue more (i) nucleophilic, (ii) electrophilic, or (iii) reactive by increasing both S-nucleophilicity and S-electrophilicity. It has already been shown that the Cys ortholog TR from Drosophila melanogaster (DmTR) has increased S-nucleophilicity [Gromer, S., Johansson, L., Bauer, H., Arscott, L. D., Rauch, S., Ballou, D. P., Williams, C. H., Jr., Schrimer, R. H., and Arnér, E. S (2003) Active sites of thioredoxin reductases: Why selenoproteins? Proc. Natl. Acad. Sci. U.S.A. 100, 12618–12623]. Here we present evidence that DmTR also enhances the electrophilicity of Cys490 through the use of an “electrophilic activation” mechanism. This mechanism is proposed to work by polarizing the disulfide bond that occurs between Cys489 and Cys490 in the C-terminal redox center by the placement of a positive charge near Cys489. This polarization renders the sulfur atom of Cys490 electron deficient and enhances the rate of thiol/disulfide exchange that occurs between the N- and C-terminal redox centers. Our hypothesis was developed by using a strategy of homocysteine (hCys) for Cys substitution in the Cys-Cys redox dyad of DmTR to differentiate the function of each Cys residue. The results show that hCys could substitute for Cys490 with little loss of thioredoxin reductase activity, but that substitution of hCys for Cys489 resulted in a 238-fold reduction in activity. We hypothesize that replacement of Cys489 with hCys destroys an interaction between the sulfur atom of Cys489 and His464 crucial for the proposed electrophilic activation mechanism. This electrophilic

  2. Evidence for the participation of Cys sub 558 and Cys sub 559 at the active site of mercuric reductase

    SciTech Connect

    Miller, S.M.; Moore, M.J.; Massey, V.; Williams, C.H. Jr.; Distefano, M.D.; Ballou, D.P.; Walsh, C.T. )

    1989-02-07

    Mercuric reductase, with FAD and a reducible disulfide at the active site, catalyzes the two-electron reduction of Hg(II) by NADPH. Addition of reducing equivalents rapidly produces a spectrally distinct EH{sub 2} form of the enzyme containing oxidized FAD and reduced active site thiols. Formation of EH{sub 2} has previously been reported to require only 2 electrons for reduction of the active site disulfide. The authors present results of anaerobic titrations of mercuric reductase with NADPH and dithionite showing that the equilibrium conversion of oxidized enzyme to EH{sub 2} actually requires 2 equiv of reducing agent or 4 electrons. Kinetic studies conducted both at 4{degree}C and at 25{degree}C indicate that reduction of the active site occurs rapidly, as previously reported; this is followed by a slower reduction of another redox group via reaction with the active site. ({sup 14}C)Iodoacetamide labeling experiments demonstrate that the C-terminal residues, Cys{sub 558} and Cys{sub 559}, are involved in this disulfide. The fluorescence, but not the absorbance, of the enzyme-bound FAD was found to be highly dependent on the redox state of the C-terminal thiols. Thus, E{sub ox} with Cys{sub 558} and Cys{sub 559} as thiols exhibits less than 50% of the fluorescence of E{sub ox} where these residues are present as a disulfide, indicating that the thiols remain intimately associated with the active site. Initial velocity measurements show that the auxiliary disulfide must be reduced before catalytic Hg(II) reduction can occur, consistent with the report of a preactivation phenomenon with NADPH or cysteine. A modified minimal catalytic mechanism is proposed as well as several chemical mechanisms for the Hg(II) reduction step.

  3. Chemical Constituents of Smilax china L. Stems and Their Inhibitory Activities against Glycation, Aldose Reductase, α-Glucosidase, and Lipase.

    PubMed

    Lee, Hee Eun; Kim, Jin Ah; Whang, Wan Kyunn

    2017-03-11

    The search for natural inhibitors with anti-diabetes properties has gained increasing attention. Among four selected Smilacaceae family plants, Smilax china L. stems (SCS) showed significant in vitro anti-glycation and rat lens aldose reductase inhibitory activities. Bioactivity-guided isolation was performed with SCS and four solvent fractions were obtained, which in turn yielded 10 compounds, including one phenolic acid, three chlorogenic acids, four flavonoids, one stilbene, and one phenylpropanoid glycoside; their structures were elucidated using nuclear magnetic resonance and mass spectrometry. All solvent fractions, isolated compounds, and stem extracts from plants sourced from six different provinces of South Korea were next tested for their inhibitory effects against advanced glycation end products, as well as aldose reductase. α-Glucosidase, and lipase assays were also performed on the fractions and compounds. Since compounds 3, 4, 6, and 8 appeared to be the superior inhibitors among the tested compounds, a comparative study was performed via high-performance liquid chromatography with photodiode array detection using a self-developed analysis method to confirm the relationship between the quantity and bioactivity of the compounds in each extract. The findings of this study demonstrate the potent therapeutic efficacy of SCS and its potential use as a cost-effective natural alternative medicine against type 2 diabetes and its complications.

  4. Mutagenesis of the redox-active disulfide in mercuric ion reductase: Catalysis by mutant enzymes restricted to flavin redox chemistry

    SciTech Connect

    Distefano, M.D.; Au, K.G.; Walsh, C.T. )

    1989-02-07

    Mercuric reductase, a flavoenzyme that possesses a redox-active cystine, Cys{sub 135}Cys{sub 140}, catalyzes the reduction of Hg(II) to Hg(0) by NADPH. As a probe of mechanism, the authors have constructed mutants lacking a redox-active disulfide by eliminating Cys{sub 135} (Ala{sub 135}Cys{sub 140}), Cys{sub 14} (Cys{sub 135}Ala{sub 140}), or both (Ala{sub 135}Ala{sub 140}). Additionally, they have made double mutants that lack Cys{sub 135} (Ala{sub 135}Cys{sub 139}Cys{sub 140}) or Cys{sub 140} (Cys{sub 135}Cys{sub 139}Ala{sub 140}) but introduce a new Cys in place of Gly{sub 139} with the aim of constructing dithiol pairs in the active site that do not form a redox-active disulfide. The resulting mutant enzymes all lack redox-active disulfides and are hence restricted to FAD/FADH{sub 2} redox chemistry. Each mutant enzyme possesses unique physical and spectroscopic properties that reflect subtle differences in the FAD microenvironment. Preliminary evidence for the Ala{sub 135}Cys{sub 139}Cys{sub 14} mutant enzyme suggests that this protein forms a disulfide between the two adjacent Cys residues. Hg(II) titration experiments that correlate the extent of charge-transfer quenching with Hg(II) binding indicate that the Ala{sub 135}Cys{sub 140} protein binds Hg(II) with substantially less avidity than does the wild-type enzyme. All mutant mercuric reductases catalyze transhydrogenation and oxygen reduction reactions through obligatory reduced flavin intermediates at rates comparable to or greater than that of the wild-type enzyme. In multiple-turnover assays which monitored the production of Hg(0), two of the mutant enzymes were observed to proceed through at least 30 turnovers at rates ca. 1000-fold slower than that of wild-type mercuric reductase. They conclude that the Cys{sub 135} and Cys{sub 140} thiols serve as Hg(II) ligands that orient the Hg(II) for subsequent reduction by a reduced flavin intermediate.

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

    PubMed Central

    Salvamani, Shamala; Gunasekaran, Baskaran; Shukor, Mohd Yunus; Shaharuddin, Noor Azmi; Sabullah, Mohd Khalizan

    2016-01-01

    Inflammation and oxidative stress are believed to contribute to the pathology of several chronic diseases including hypercholesterolemia (elevated levels of cholesterol in blood) and atherosclerosis. HMG-CoA reductase inhibitors of plant origin are needed as synthetic drugs, such as statins, which are known to cause adverse effects on the liver and muscles. Amaranthus viridis (A. viridis) has been used from ancient times for its supposedly medically beneficial properties. In the current study, different parts of A. viridis (leaf, stem, and seed) were evaluated for potential anti-HMG-CoA reductase, antioxidant, and anti-inflammatory activities. The putative HMG-CoA reductase inhibitory activity of A. viridis extracts at different concentrations was determined spectrophotometrically by NADPH oxidation, using HMG-CoA as substrate. A. viridis leaf extract revealed the highest HMG-CoA reductase inhibitory effect at about 71%, with noncompetitive inhibition in Lineweaver-Burk plot analysis. The leaf extract showed good inhibition of hydroperoxides, 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO), and ferric ion radicals in various concentrations. A. viridis leaf extract was proven to be an effective inhibitor of hyaluronidase, lipoxygenase, and xanthine oxidase enzymes. The experimental data suggest that A. viridis leaf extract is a source of potent antioxidant and anti-inflammatory agent and may modulate cholesterol metabolism by inhibition of HMG-CoA reductase. PMID:27051453

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

    PubMed

    Salvamani, Shamala; Gunasekaran, Baskaran; Shukor, Mohd Yunus; Shaharuddin, Noor Azmi; Sabullah, Mohd Khalizan; Ahmad, Siti Aqlima

    2016-01-01

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

  7. Chemical constituents from the aerial parts of Aster koraiensis with protein glycation and aldose reductase inhibitory activities.

    PubMed

    Lee, Jun; Lee, Yun Mi; Lee, Byong Won; Kim, Joo-Hwan; Kim, Jin Sook

    2012-02-24

    Two new eudesmane-type sesquiterpene glucosides, 9β-O-(E-p-hydroxycinnamoyl)-1β,6β-dihydroxy-trans-eudesm-3-en-6-O-β-D-glucopyranoside (1) and 9α-O-(E-p-hydroxycinnamoyl)-1α,6α-11-trihydroxy-trans-eudesm-3-en-6-O-β-D-glucopyranoside (2), were isolated by the activity-guidedfractionation of an EtOAc-soluble fraction from the aerial parts of Aster koraiensis. A new dihydrobenzofuran glucoside, (2R,3S)-6-acetyl-2-[1-O-(β-D-glucopyranosyl)-2-propenyl]-5-hydroxy-3-methoxy-2,3-dihydrobenzofuran (3), was also isolated, in addition to 15 known compounds. The structures of 1-3 were determined by spectroscopic data interpretation. All of the isolates were evaluated for in vitro inhibitory activity against the formation of advanced glycation end-products and rat lens aldose reductase.

  8. Activity-guided isolation of constituents of Tephrosia purpurea with the potential to induce the phase II enzyme, quinone reductase.

    PubMed

    Chang, L C; Gerhäuser, C; Song, L; Farnsworth, N R; Pezzuto, J M; Kinghorn, A D

    1997-09-01

    An isoflavone, 7,4'-dihydroxy-3',5'-dimethoxyisoflavone (1), and a chalcone, (+)-tephropurpurin (2), both novel compounds, as well as six constituents of known structure, (+)-purpurin (3), pongamol (4), lanceolatin B (5), (-)-maackiain (6), (-)-3-hydroxy-4-methoxy-8,9-methylene-dioxypterocarpan (7), and (-)-medicarpin (8), were obtained as active compounds from Tephrosia purpurea, using a bioassay based on the induction of quinone reductase (QR) activity with cultured Hepa 1c1c7 mouse hepatoma cells. Additionally, three inactive compounds of known structure, 3'-methoxydaidzein, desmoxyphyllin B, and 3,9-dihydroxy-8-methoxycoumestan, were isolated and identified. The structure elucidation of compounds 1 and 2 was carried out by spectral data interpretation.

  9. Sequence and properties of pentaerythritol tetranitrate reductase from Enterobacter cloacae PB2.

    PubMed

    French, C E; Nicklin, S; Bruce, N C

    1996-11-01

    Pentaerythritol tetranitrate reductase, which reductively liberates nitrite from nitrate esters, is related to old yellow enzyme. Pentaerythritol tetranitrate reductase follows a ping-pong mechanism with competitive substrate inhibition by NADPH, is strongly inhibited by steroids, and is capable of reducing the unsaturated bond of 2-cyclohexen-1-one.

  10. Characterizing and predicting carboxylic acid reductase activity for diversifying bioaldehyde production.

    PubMed

    Moura, Matthew; Pertusi, Dante; Lenzini, Stephen; Bhan, Namita; Broadbelt, Linda J; Tyo, Keith E J

    2016-05-01

    Chemicals with aldehyde moieties are useful in the synthesis of polymerization reagents, pharmaceuticals, pesticides, flavors, and fragrances because of their high reactivity. However, chemical synthesis of aldehydes from carboxylic acids has unfavorable thermodynamics and limited specificity. Enzymatically catalyzed reductive bioaldehyde synthesis is an attractive route that overcomes unfavorable thermodynamics by ATP hydrolysis in ambient, aqueous conditions. Carboxylic acid reductases (Cars) are particularly attractive, as only one enzyme is required. We sought to increase the knowledge base of permitted substrates for four Cars. Additionally, the Lys2 enzyme family was found to be mechanistically the same as Cars and two isozymes were also tested. Our results show that Cars prefer molecules where the carboxylic acid is the only polar/charged group. Using this data and other published data, we develop a support vector classifier (SVC) for predicting Car reactivity and make predictions on all carboxylic acid metabolites in iAF1260 and Model SEED.

  11. Inhibition of glycation and aldose reductase activity using dietary flavonoids: A lens organ culture studies.

    PubMed

    Patil, Kapil K; Gacche, Rajesh N

    2017-05-01

    On the eve of increasing incidence of diabetes mellitus and related complications, the search for novel, safe and alternatives therapeutic approaches are evolving. In the present investigation, a panel of ten dietary flavonoids such as 4'-methoxyflavanone, formononetin, hesperetin, hesperidin, naringenin, naringin, rutin, diadzin, silibinin and silymarin was evaluated as possible inhibitors of sugar induced cataractogenesis using bovine lens organ culture studies. The effect of selected flavonoids was observed on glycation induced lens opacity, AGE fluorescence, carbonyl group formation (a biomarker of glycation), protein aggregation and aldose reductase (AR) inhibition. The results obtained clearly demonstrate the efficacy of rutin and silibinin as promising leads for inhibition of glycation reaction and amelioration of sugar induced cataractogenesis. The findings of the present study may be useful for designing and development of the novel lead molecules for the management of diabetic cataract.

  12. Design, synthesis, and biological activity of diaryl ether inhibitors of Toxoplasma gondii enoyl reductase

    PubMed Central

    Cheng, Gang; Muench, Stephen P.; Zhou, Ying; Afanador, Gustavo A.; Mui, Ernest J.; Fomovska, Alina; Lai, Bo Shiun; Prigge, Sean T.; Woods, Stuart; Roberts, Craig W.; Hickman, Mark R.; Lee, Patty J.; Leed, Susan E.; Auschwitz, Jennifer M.; Rice, David W.; McLeod, Rima

    2013-01-01

    Triclosan is a potent inhibitor of Toxoplasma gondii enoyl reductase (TgENR), which is an essential enzyme for parasite survival. In view of triclosan’s poor druggability, which limits its therapeutic use, a new set of B-ring modified analogs were designed to optimize its physico-chemical properties. These derivatives were synthesized and evaluated by in vitro assay and TgENR enzyme assay. Some analogs display improved solubility, permeability and a comparable MIC50 value to that of triclosan. Modeling of these inhibitors revealed the same overall binding mode with the enzyme as triclosan, but the Bring modifications have additional interactions with the strongly conserved Asn130. PMID:23453069

  13. FAD binding, cobinamide binding and active site communication in the corrin reductase (CobR)

    PubMed Central

    Lawrence, Andrew D.; Taylor, Samantha L.; Scott, Alan; Rowe, Michelle L.; Johnson, Christopher M.; Rigby, Stephen E. J.; Geeves, Michael A.; Pickersgill, Richard W.; Howard, Mark J.; Warren, Martin J.

    2014-01-01

    Adenosylcobalamin, the coenzyme form of vitamin B12, is one Nature's most complex coenzyme whose de novo biogenesis proceeds along either an anaerobic or aerobic metabolic pathway. The aerobic synthesis involves reduction of the centrally chelated cobalt metal ion of the corrin ring from Co(II) to Co(I) before adenosylation can take place. A corrin reductase (CobR) enzyme has been identified as the likely agent to catalyse this reduction of the metal ion. Herein, we reveal how Brucella melitensis CobR binds its coenzyme FAD (flavin dinucleotide) and we also show that the enzyme can bind a corrin substrate consistent with its role in reduction of the cobalt of the corrin ring. Stopped-flow kinetics and EPR reveal a mechanistic asymmetry in CobR dimer that provides a potential link between the two electron reduction by NADH to the single electron reduction of Co(II) to Co(I). PMID:24909839

  14. Construction of effective disposable biosensors for point of care testing of nitrite.

    PubMed

    Monteiro, Tiago; Rodrigues, Patrícia R; Gonçalves, Ana Luisa; Moura, José J G; Jubete, Elena; Añorga, Larraitz; Piknova, Barbora; Schechter, Alan N; Silveira, Célia M; Almeida, M Gabriela

    2015-09-01

    In this paper we aim to demonstrate, as a proof-of-concept, the feasibility of the mass production of effective point of care tests for nitrite quantification in environmental, food and clinical samples. Following our previous work on the development of third generation electrochemical biosensors based on the ammonia forming nitrite reductase (ccNiR), herein we reduced the size of the electrodes' system to a miniaturized format, solved the problem of oxygen interference and performed simple quantification assays in real samples. In particular, carbon paste screen printed electrodes (SPE) were coated with a ccNiR/carbon ink composite homogenized in organic solvents and cured at low temperatures. The biocompatibility of these chemical and thermal treatments was evaluated by cyclic voltammetry showing that the catalytic performance was higher with the combination acetone and a 40°C curing temperature. The successful incorporation of the protein in the carbon ink/solvent composite, while remaining catalytically competent, attests for ccNiR's robustness and suitability for application in screen printed based biosensors. Because the direct electrochemical reduction of molecular oxygen occurs when electroanalytical measurements are performed at the negative potentials required to activate ccNiR (ca.-0.4V vs Ag/AgCl), an oxygen scavenging system based on the coupling of glucose oxidase and catalase activities was successfully used. This enabled the quantification of nitrite in different samples (milk, water, plasma and urine) in a straightforward way and with small error (1-6%). The sensitivity of the biosensor towards nitrite reduction under optimized conditions was 0.55 A M(-1) cm(-2) with a linear response range 0.7-370 μM.

  15. Nitrate reduction in Haloferax alexandrinus: the case of assimilatory nitrate reductase.

    PubMed

    Kilic, Volkan; Kilic, Gözde Aydoğan; Kutlu, Hatice Mehtap; Martínez-Espinosa, Rosa María

    2017-03-21

    Haloferax alexandrinus Strain TM JCM 10717(T) = IFO 16590(T) is an extreme halophilic archaeon able to produce significant amounts of canthaxanthin. Its genome sequence has been analysed in this work using bioinformatics tools available at Expasy in order to look for genes encoding nitrate reductase-like proteins: respiratory nitrate reductase (Nar) and/or assimilatory nitrate reductase (Nas). The ability of the cells to reduce nitrate under aerobic conditions was tested. The enzyme in charge of nitrate reduction under aerobic conditions (Nas) has been purified and characterised. It is a monomeric enzyme (72 ± 1.8 kDa) that requires high salt concentration for stability and activity. The optimum pH value for activity was 9.5. Effectiveness of different substrates, electron donors, cofactors and inhibitors was also reported. High nitrite concentrations were detected within the culture media during aerobic/microaerobic cells growth. The main conclusion from the results is that this haloarchaeon reduces nitrate aerobically thanks to Nas and may induce denitrification under anaerobic/microaerobic conditions using nitrate as electron acceptor. The study sheds light on the role played by haloarchaea in the biogeochemical cycle of nitrogen, paying special attention to nitrate reduction processes. Besides, it provides useful information for future attempts on microecological and biotechnological implications of haloarchaeal nitrate reductases.

  16. Relationship of changing delta 4-steroid 5 alpha-reductase activity to (125I)iododeoxyuridine uptake during regeneration of involuted rat prostates

    SciTech Connect

    Kitahara, S.; Higashi, Y.; Takeuchi, S.; Oshima, H. )

    1989-04-01

    To elucidate the phenotypic expression of proliferating prostatic cells, rats were castrated, and the regenerating process of involuted ventral prostates during testosterone propionate (TP) administration was investigated by examining morphology, (5-{sup 125}I)iododeoxyuridine ({sup 125}I-UdR) uptake, DNA content, weight, acid phosphatase, and delta 4-steroid 5 alpha-reductase (5 alpha-reductase) activities. Morphologically, TP treatment initially increased the number of epithelial cells lining glandular lobules and subsequently restored the shape of epithelial cells. {sup 125}I-UdR uptake peaked on Day 3 of TP treatment and stayed at higher levels than for uncastrated controls until Day 14 of treatment. Prostatic weight, protein content, acid phosphatase, and DNA content returned to uncastrated control levels by Day 14 of TP treatment. TP administration markedly stimulated prostatic 5 alpha-reductase activity, which peaked on the Day 5 of treatment and decreased to uncastrated control levels by Day 14 of treatment. It is concluded that TP administration to castrated rats initially induced active mitotic division of the remaining stem cells, followed by formation of differentiated functional epithelial cells. Prostatic 5 alpha-reductase was highly active at the initial phase of active mitotic cell division. The major portion of the increased enzyme activity can be regarded as a phenotypic expression of stem or transient cells of prostatic epithelium.

  17. Transformation of heavy metal fractions on soil urease and nitrate reductase activities in copper and selenium co-contaminated soil.

    PubMed

    Hu, Bin; Liang, Dongli; Liu, Juanjuan; Lei, Lingming; Yu, Dasong

    2014-12-01

    This study aims to explore the effects of the distribution, transformation and bioavailability of different fractions of copper (Cu) and selenium (Se) in co-contaminated soils on soil enzymes, providing references for the phytoremediation of contaminated areas and agriculture environmental protection. Pot experiments and laboratory analysis were used to investigate the transformation and bioavailability of additional Cu and Se for pakchoi (Brassica chinensis) in co-contaminated soil. In the uncontaminated soil, Cu mainly existed in residual form, whereas Se was present in residual form and in elemental and organic-sulfide matter-bound form. In the contaminated soil, Cu mainly bound to Fe-Mn oxidates, whereas Se was in exchangeable and carbonates forms. After a month of pakchoi growth, Cu tended to transfer into organic matter-bound fractions, whereas Se tended to bound to Fe-Mn oxidates. The IR (reduced partition index) value of Cu decreased as the concentrations of Cu and Se gradually increased, whereas the IR value of Se decreased as the concentration of Se increased. The IR value before pakchoi planting and after it was harvested was not affected by the concentration of exogenous Cu. Soil urease and nitrate reductase activities were inhibited by Cu and Se pollution either individually or combined in different degrees, following the order nitrate reductase>urease. The significant correlation between the IR value and soil enzyme activities suggests that this value could be used to evaluate the bioavailability of heavy metals in soil. Path analysis showed that the variations in exchangeable Cu and organic-sulfide matter-bound and elemental Se had direct effects on the activities of the two enzymes, suggesting their high bioavailability. Therefore, the IR value and the transformation of metals in soil could be used as indicators in evaluating the bioavailability of heavy metals.

  18. Targeting Ribonucleotide Reductase M2 and NF-κB Activation with Didox to Circumvent Tamoxifen Resistance in Breast Cancer.

    PubMed

    Shah, Khyati N; Wilson, Elizabeth A; Malla, Ritu; Elford, Howard L; Faridi, Jesika S

    2015-11-01

    Tamoxifen is widely used as an adjuvant therapy for patients with estrogen receptor (ERα)-positive tumors. However, the clinical benefit is often limited because of the emergence of drug resistance. In this study, overexpression of ribonucleotide reductase M2 (RRM2) in MCF-7 breast cancer cells resulted in a reduction in the effectiveness of tamoxifen, through downregulation of ERα66 and upregulation of the 36-kDa variant of ER (ERα36). We identified that NF-κB, HIF1α, and MAPK/JNK are the major pathways that are affected by RRM2 overexpression and result in increased NF-κB activity and increased protein levels of EGFR, HER2, IKKs, Bcl-2, RelB, and p50. RRM2-overexpressing cells also exhibited higher migratory and invasive properties. Through time-lapse microscopy and protein profiling studies of tamoxifen-treated MCF-7 and T-47D cells, we have identified that RRM2, along with other key proteins, is altered during the emergence of acquired tamoxifen resistance. Inhibition of RRM2 using siRRM2 or the ribonucleotide reductase (RR) inhibitor didox not only eradicated and effectively prevented the emergence of tamoxifen-resistant populations but also led to the reversal of many of the proteins altered during the process of acquired tamoxifen resistance. Because didox also appears to be a potent inhibitor of NF-κB activation, combining didox with tamoxifen treatment cooperatively reverses ER-α alterations and inhibits NF-κB activation. Finally, inhibition of RRM2 by didox reversed tamoxifen-resistant in vivo tumor growth and decreased in vitro migratory and invasive properties, revealing a beneficial effect of combination therapy that includes RRM2 inhibition to delay or abrogate tamoxifen resistance.

  19. Synergistic Inhibition of Microbial Sulfide Production by Combinations of the Metabolic Inhibitor Nitrite and Biocides▿

    PubMed Central

    Greene, E. Anne; Brunelle, Veronique; Jenneman, Gary E.; Voordouw, Gerrit

    2006-01-01

    MICs of six broad-spectrum biocides and two specific metabolic inhibitors and fractional inhibitory concentration indexes (FICIs) for controlling a sulfide-producing consortium were determined. Nitrite was synergistic (FICI < 1) with all but one biocide due to its specific inhibition of dissimilatory sulfite reductase. Hence, combining nitrite with biocides allows more efficient and cost-effective control of sulfate-reducing bacteria. PMID:16997976

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

    PubMed

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

    2015-06-05

    Short-chain dehydrogenases/reductases (SDRs) constitute a large, functionally diverse branch of enzymes within the class of NAD(P)(H) dependent oxidoreductases. In humans, over 80 genes have been identified with distinct metabolic roles in carbohydrate, amino acid, lipid, retinoid and steroid hormone metabolism, frequently associated with inherited genetic defects. Besides metabolic functions, a subset of atypical SDR proteins appears to play critical roles in adapting to redox status or RNA processing, and thereby controlling metabolic pathways. Here we present an update on the human SDR superfamily and a ligand identification strategy using differential scanning fluorimetry (DSF) with a focused library of oxidoreductase and metabolic ligands to identify substrate classes and inhibitor chemotypes. This method is applicable to investigate structure-activity relationships of oxidoreductases and ultimately to better understand their physiological roles.

  1. Implications and problems in analysing cytotoxic activity of hydroxyurea in combination with a potential inhibitor of ribonucleotide reductase.

    PubMed

    Nocentini, G; Barzi, A; Franchetti, P

    1990-01-01

    The cytotoxicity of hydroxyurea in combination with 2.2'-bipyridyl-6-carbothioamide (a potential inhibitor of ribonucleotide reductase) on P388 murine leukemia is reported. Synergistic activity was studied using various interpretations of the isobologram method and the combination index method. We evaluated the pros and cons of these methods and their overall usefulness. In our opinion, to obtain all possible information from a compound association, it is important to choose a formally correct method that (a) can quantitatively evaluate synergism or antagonism, (b) may offer the possibility of averaging final results, (c) needs a minimal amount of experimental data, and (d) is rapid. Moreover, we emphasize both the utility of testing at least three molar ratios of compound association and the importance of carefully choosing the fractional inhibition used in calculating the combination effect. Such evaluation of drug combinations gives information essential to the preparation of new anticancer drug regimens and to the early assessment of biochemical interactions.

  2. Purification and characterization of a yeast carbonyl reductase for synthesis of optically active (R)-styrene oxide derivatives.

    PubMed

    Kizaki, Noriyuki; Sawa, Ikuo; Yano, Miho; Yasohara, Yoshihiko; Hasegawa, Junzo

    2005-01-01

    Optically active styrene oxide derivatives are versatile chiral building blocks. Stereoselective reduction of phenacyl halide to chiral 2-halo-1-phenylethanol is the key reaction of the most economical synthetic route. Rhodotorula glutinis var. dairenensis IFO415 was discovered on screening as a potent microorganism reducing a phenacyl halide to the (R)-form of the corresponding alcohol. An NADPH-dependent carbonyl reductase was purified to homogeneity through four steps from this strain. The relative molecular mass of the enzyme was estimated to be 40,000 on gel filtration and 30,000 on SDS-polyacrylamide gel electrophoresis. This enzyme reduced a broad range of carbonyl compounds in addition to phenacyl halides. Some properties of the enzyme and preparation of a chiral styrene oxide using the crude enzyme are reported herein.

  3. Structure-activity relationships of pyrrole based S-nitrosoglutathione reductase inhibitors: pyrrole regioisomers and propionic acid replacement.

    PubMed

    Sun, Xicheng; Qiu, Jian; Strong, Sarah A; Green, Louis S; Wasley, Jan W F; Colagiovanni, Dorothy B; Mutka, Sarah C; Blonder, Joan P; Stout, Adam M; Richards, Jane P; Chun, Lawrence; Rosenthal, Gary J

    2011-06-15

    S-Nitrosoglutathione reductase (GSNOR) is a member of the alcohol dehydrogenase family (ADH) that regulates the levels of S-nitrosothiols (SNOs) through catabolism of S-nitrosoglutathione (GSNO). GSNO and SNOs are implicated in the pathogenesis of many diseases including those in respiratory, cardiovascular, and gastrointestinal systems. The pyrrole based N6022 was recently identified as a potent, selective, reversible, and efficacious GSNOR inhibitor which is currently undergoing clinical development. We describe here the synthesis and structure-activity relationships (SAR) of novel pyrrole based analogues of N6022 focusing on scaffold modification and propionic acid replacement. We identified equally potent and novel GSNOR inhibitors having pyrrole regioisomers as scaffolds using a structure based approach.

  4. Nitrate reductase activity in some subarctic species and UV influence in the foliage of Betula pendula Roth. seedlings.

    PubMed

    Krywult, Marek; Turunen, Minna; Sutinen, Marja-Liisa; Derome, Kirsti; Norokorpi, Yrjö

    2002-02-04

    Nitrate reductase (NR) activity was studied in the foliage of five subarctic species: mature trees of European white birch (Betula pubescens Erch. S.S.), Scots pine (Pinus sylvestris L.), Norway spruce (Picea abies L. Karst), Ericaceous shrub bilberry (Vaccinium myrtillus L.), naturally growing in a forest, and seed-grown silver birch (Betula pendula Roth.) seedlings in an ultraviolet (UV) exclusion field experiment at the Pallas-Ounastunturi National Park in Finnish Lapland (68 degrees N). Mean NR activity ranged from 0 in bilberry to 1477 (S.D. = 277.7) and 1910 (S.D. = 785.4) nmol g(-1) DW h(-1) in mature trees of European white birch and silver birch seedlings, respectively. Significant differences due to UV exclosure treatments were determined for the NR activity of silver birch seedlings (F = 3.62, P= 0.025*) after three growing seasons (191 days) of UV exclusion. The ambient and control silver birch seedlings had or tended to have higher NR activity than those grown under UV exclusion. No relationship was found between the foliage NR activity and total nitrogen content, which ranged from 0.61 to 1.35% per seedling. The present study suggests large differences in NR activity between the species and the induction of NR activity in silver birch seedlings due to ambient UV radiation.

  5. Dimerization and enzymatic activity of fungal 17β-hydroxysteroid dehydrogenase from the short-chain dehydrogenase/reductase superfamily

    PubMed Central

    Kristan, Katja; Deluca, Dominga; Adamski, Jerzy; Stojan, Jure; Rižner, Tea Lanišnik

    2005-01-01

    Background 17β-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17β-HSDcl) is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. SDR proteins usually function as dimers or tetramers and 17β-HSDcl is also a homodimer under native conditions. Results We have investigated here which secondary structure elements are involved in the dimerization of 17β-HSDcl and examined the importance of dimerization for the enzyme activity. Sequence similarity with trihydroxynaphthalene reductase from Magnaporthe grisea indicated that Arg129 and His111 from the αE-helices interact with the Asp121, Glu117 and Asp187 residues from the αE and αF-helices of the neighbouring subunit. The Arg129Asp and His111Leu mutations both rendered 17β-HSDcl monomeric, while the mutant 17β-HSDcl-His111Ala was dimeric. Circular dichroism spectroscopy analysis confirmed the conservation of the secondary structure in both monomers. The three mutant proteins all bound coenzyme, as shown by fluorescence quenching in the presence of NADP+, but both monomers showed no enzymatic activity. Conclusion We have shown by site-directed mutagenesis and structure/function analysis that 17β-HSDcl dimerization involves the αE and αF helices of both subunits. Neighbouring subunits are connected through hydrophobic interactions, H-bonds and salt bridges involving amino acid residues His111 and Arg129. Since the substitutions of these two amino acid residues lead to inactive monomers with conserved secondary structure, we suggest dimerization is a prerequisite for catalysis. A detailed understanding of this dimerization could lead to the development of compounds that will specifically prevent dimerization, thereby serving as a new type of inhibitor. PMID:16359545

  6. Effects of nitrate addition to a diet on fermentation and microbial populations in the rumen of goats, with special reference to Selenomonas ruminantium having the ability to reduce nitrate and nitrite.

    PubMed

    Asanuma, Narito; Yokoyama, Shota; Hino, Tsuneo

    2015-04-01

    This study investigated the effects of dietary nitrate addition on ruminal fermentation characteristics and microbial populations in goats. The involvement of Selenomonas ruminantium in nitrate and nitrite reduction in the rumen was also examined. As the result of nitrate feeding, the total concentration of ruminal volatile fatty acids decreased, whereas the acetate : propionate ratio and the concentrations of ammonia and lactate increased. Populations of methanogens, protozoa and fungi, as estimated by real-time PCR, were greatly decreased as a result of nitrate inclusion in the diet. There was modest or little impact of nitrate on the populations of prevailing species or genus of bacteria in the rumen, whereas Streptococcus bovis and S. ruminantium significantly increased. Both the activities of nitrate reductase (NaR) and nitrite reductase (NiR) per total mass of ruminal bacteria were increased by nitrate feeding. Quantification of the genes encoding NaR and NiR by real-time PCR with primers specific for S. ruminantium showed that these genes were increased by feeding nitrate, suggesting that the growth of nitrate- and nitrite-reducing S. ruminantium is stimulated by nitrate addition. Thus, S. ruminantium is likely to play a major role in nitrate and nitrite reduction in the rumen.

  7. Adaptation of cytochrome-b5 reductase activity and methaemoglobinaemia in areas with a high nitrate concentration in drinking-water.

    PubMed Central

    Gupta, S. K.; Gupta, R. C.; Seth, A. K.; Gupta, A. B.; Bassin, J. K.; Gupta, A.

    1999-01-01

    An epidemiological investigation was undertaken in India to assess the prevalence of methaemoglobinaemia in areas with high nitrate concentration in drinking-water and the possible association with an adaptation of cytochrome-b5 reductase. Five areas were selected, with average nitrate ion concentrations in drinking-water of 26, 45, 95, 222 and 459 mg/l. These areas were visited and house schedules were prepared in accordance with a statistically designed protocol. A sample of 10% of the total population was selected in each of the areas, matched for age and weight, giving a total of 178 persons in five age groups. For each subject, a detailed history was documented, a medical examination was conducted and blood samples were taken to determine methaemoglobin level and cytochrome-b5 reductase activity. Collected data were subjected to statistical analysis to test for a possible relationship between nitrate concentration, cytochrome-b5 reductase activity and methaemoglobinaemia. High nitrate concentrations caused methaemoglobinaemia in infants and adults. The reserve of cytochrome-b5 reductase activity (i.e. the enzyme activity not currently being used, but which is available when needed; for example, under conditions of increased nitrate ingestion) and its adaptation with increasing water nitrate concentration to reduce methaemoglobin were more pronounced in children and adolescents. PMID:10534899

  8. Adaptation of cytochrome-b5 reductase activity and methaemoglobinaemia in areas with a high nitrate concentration in drinking-water.

    PubMed

    Gupta, S K; Gupta, R C; Seth, A K; Gupta, A B; Bassin, J K; Gupta, A

    1999-01-01

    An epidemiological investigation was undertaken in India to assess the prevalence of methaemoglobinaemia in areas with high nitrate concentration in drinking-water and the possible association with an adaptation of cytochrome-b5 reductase. Five areas were selected, with average nitrate ion concentrations in drinking-water of 26, 45, 95, 222 and 459 mg/l. These areas were visited and house schedules were prepared in accordance with a statistically designed protocol. A sample of 10% of the total population was selected in each of the areas, matched for age and weight, giving a total of 178 persons in five age groups. For each subject, a detailed history was documented, a medical examination was conducted and blood samples were taken to determine methaemoglobin level and cytochrome-b5 reductase activity. Collected data were subjected to statistical analysis to test for a possible relationship between nitrate concentration, cytochrome-b5 reductase activity and methaemoglobinaemia. High nitrate concentrations caused methaemoglobinaemia in infants and adults. The reserve of cytochrome-b5 reductase activity (i.e. the enzyme activity not currently being used, but which is available when needed; for example, under conditions of increased nitrate ingestion) and its adaptation with increasing water nitrate concentration to reduce methaemoglobin were more pronounced in children and adolescents.

  9. Ferric reductase activity of low molecular weight human milk fraction is associated with enhanced iron solubility and uptake in Caco-2 cells.

    PubMed

    Pullakhandam, Raghu; Nair, Madhavan Krishnapillai; Kasula, Sunanda; Kilari, Sreenivasulu; Thippande, Tippeswamy Gowda

    2008-09-19

    It is known that the fractional absorption of extrinsic iron from human milk is higher in infants and adults. A low molecular weight milk fraction has been proposed to increase the bioavailability of iron from human milk. Nevertheless, the mechanisms remained elusive. Here in we demonstrate ferric reductase activity (Km7.73x10(-6)M) in low molecular weight human milk fraction (10kF, filtrate derived from ultra filtration of milk whey through 10kDa cutoff membrane), which increased ferric iron solubility and iron uptake in Caco-2 cells. The 10kF fraction was as effective as ascorbic acid (1:20 iron to ascorbic acid) in increasing the ferric iron solubility and uptake in Caco-2 cells. Further, gel filtration chromatography on peptide column led to co-elution of ferric reductase and iron solubilization activities at an apparent molecular mass of <1500Da. Interestingly, only these fractions containing ferric reductase activity also stimulated the uptake of iron in Caco-2 cells. Thus, it is concluded that human milk possesses ferric reductase activity and is associated with ferric iron solubilization and enhanced absorption.

  10. Exploring the inhibitory activity of Withaferin-A against Pteridine reductase-1 of L. donovani.

    PubMed

    Chandrasekaran, Sambamurthy; Veronica, Jalaja; Gundampati, Ravi Kumar; Sundar, Shyam; Maurya, Radheshyam

    2016-12-01

    Withaferin A is an abundant withanolide present in Withania somnifera leaves and to some extent in roots. It has been known for its profound anti-cancer properties, but its role in counteracting the Leishmania donovani infection has to be explored. Pteridine reductase 1 (PTR1) is involved in pteridine salvage and an important enzyme for the parasite growth, which could be targeted for the development of an efficient antileishmanial drug. We employed molecular docking studies to identify the binding mode of withaferin A with PTR1 in silico. We further cloned, expressed, and purified PTR1 of L. donovani and performed the enzyme kinetics using the Michaelis-Menten equation and enzyme inhibition studies with withaferin A by plotting the Lineweaver-Burk graph, which followed an uncompetitive mode of inhibition. We also showed the inhibition of the enzyme in the crude lysate of treated parasites. Thus, our study contributes towards understanding the mode of action of withaferin A against L. donovani parasite.

  11. Bioactive profiles, antioxidant activities, nitrite scavenging capacities and protective effects on H2O2-injured PC12 cells of Glycyrrhiza glabra L. leaf and root extracts.

    PubMed

    Dong, Yi; Zhao, Mouming; Zhao, Tiantian; Feng, Mengying; Chen, Huiping; Zhuang, Mingzhu; Lin, Lianzhu

    2014-06-30

    This study compared the total flavonoid content of Glycyrrhiza glabra L. leaf and root extracts. Results suggested that the total flavonoid content in the leaf extract was obviously higher than that in the root extract. Pinocembrin, the main compound in the leaf extract after purification by column chromatography, showed good antioxidant activity and nitrite scavenging capacity, but moderate inhibitory effect on mushroom tyrosinase. Liquiritin was the main compound in root extract and possessed strong inhibitory effect on mushroom tyrosinase. Both compounds exhibited significant protection effect on H2O2-injured PC12 cells at a low concentration. These results indicate that Glycyrrhiza glabra L. leaf is potential as an important raw material for functional food.

  12. Nitrates and Nitrites TNC Presentation

    EPA Pesticide Factsheets

    The Nitrates and Nitrites Presentation gives an overview of nitrates and nitrites in drinking water, why it is important to monitor them and what to do in cases where the results exceed the maximum contaminant level (MCL).

  13. YNL134C from Saccharomyces cerevisiae encodes a novel protein with aldehyde reductase activity for detoxification of furfural derived from lignocellulosic biomass.

    PubMed

    Zhao, Xianxian; Tang, Juan; Wang, Xu; Yang, Ruoheng; Zhang, Xiaoping; Gu, Yunfu; Li, Xi; Ma, Menggen

    2015-05-01

    Furfural and 5-hydroxymethylfurfural (HMF) are the two main aldehyde compounds derived from pentoses and hexoses, respectively, during lignocellulosic biomass pretreatment. These two compounds inhibit microbial growth and interfere with subsequent alcohol fermentation. Saccharomyces cerevisiae has the in situ ability to detoxify furfural and HMF to the less toxic 2-furanmethanol (FM) and furan-2,5-dimethanol (FDM), respectively. Herein, we report that an uncharacterized gene, YNL134C, was highly up-regulated under furfural or HMF stress and Yap1p and Msn2/4p transcription factors likely controlled its up-regulated expression. Enzyme activity assays showed that YNL134C is an NADH-dependent aldehyde reductase, which plays a role in detoxification of furfural to FM. However, no NADH- or NADPH-dependent enzyme activity was observed for detoxification of HMF to FDM. This enzyme did not catalyse the reverse reaction of FM to furfural or FDM to HMF. Further studies showed that YNL134C is a broad-substrate aldehyde reductase, which can reduce multiple aldehydes to their corresponding alcohols. Although YNL134C is grouped into the quinone oxidoreductase family, no quinone reductase activity was observed using 1,2-naphthoquinone or 9,10-phenanthrenequinone as a substrate, and phylogenetic analysis indicates that it is genetically distant to quinone reductases. Proteins similar to YNL134C in sequence from S. cerevisiae and other microorganisms were phylogenetically analysed.

  14. Probing the active site of cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala.

    PubMed

    Sonawane, Prashant; Patel, Krunal; Vishwakarma, Rishi Kishore; Srivastava, Sameer; Singh, Somesh; Gaikwad, Sushama; Khan, Bashir M

    2013-09-01

    Lack of three dimensional crystal structure of cinnamoyl CoA reductase (CCR) limits its detailed active site characterization studies. Putative active site residues involved in the substrate/NADPH binding and catalysis for Leucaena leucocephala CCR (Ll-CCRH1; GenBank: DQ986907) were identified by amino acid sequence alignment and homology modeling. Putative active site residues and proximal H215 were subjected for site directed mutagenesis, and mutated enzymes were expressed, purified and assayed to confirm their functional roles. Mutagenesis of S136, Y170 and K174 showed complete loss of activity, indicating their pivotal roles in catalysis. Mutant S212G exhibited the catalytic efficiencies less than 10% of wild type, showing its indirect involvement in substrate binding or catalysis. R51G, D77G, F30V and I31N double mutants showed significant changes in Km values, specifying their roles in substrate binding. Finally, chemical modification and substrate protection studies corroborated the presence Ser, Tyr, Lys, Arg and carboxylate group at the active site of Ll-CCRH1.

  15. Profiles of Glucosinolates, Their Hydrolysis Products, and Quinone Reductase Inducing Activity from 39 Arugula (Eruca sativa Mill.) Accessions.

    PubMed

    Ku, Kang-Mo; Kim, Moo Jung; Jeffery, Elizabeth H; Kang, Young-Hwa; Juvik, John A

    2016-08-31

    Glucosinolates, their hydrolysis product concentrations, and the quinone reductase (QR) inducing activity of extracts of leaf tissue were assayed from 39 arugula (Eruca sativa Mill.) accessions. Arugula accessions from Mediterranean countries (n = 16; Egypt, Greece, Italy, Libya, Spain, and Turkey) and Northern Europe (n = 2; Poland and United Kingdom) were higher in glucosinolates and their hydrolysis products, especially glucoraphanin and sulforaphane, compared to those from Asia (n = 13; China, India, and Pakistan) and Middle East Asia (n = 8; Afghanistan, Iran, and Israel). The QR inducing activity was also the highest in Mediterranean and Northern European arugula accessions, possibly due to a significant positive correlation between sulforaphane and QR inducing activity (r = 0.54). No nitrile hydrolysis products were found, suggesting very low or no epithiospecifier protein activity from these arugula accessions. Broad sense heritability (H(2)) was estimated to be 0.91-0.98 for glucoinolates, 0.55-0.83 for their hydrolysis products, and 0.90 for QR inducing activity.

  16. Antitumor Indolequinones Induced Apoptosis in Human Pancreatic Cancer Cells via Inhibition of Thioredoxin Reductase and Activation of Redox Signaling

    PubMed Central

    Yan, Chao; Siegel, David; Newsome, Jeffery; Chilloux, Aurelie; Moody, Christopher J.

    2012-01-01

    Indolequinones (IQs) were developed as potential antitumor agents against human pancreatic cancer. IQs exhibited potent antitumor activity against the human pancreatic cancer cell line MIA PaCa-2 with growth inhibitory IC50 values in the low nanomolar range. IQs were found to induce time- and concentration-dependent apoptosis and to be potent inhibitors of thioredoxin reductase 1 (TR1) in MIA PaCa-2 cells at concentrations equivalent to those inducing growth-inhibitory effects. The mechanism of inhibition of TR1 by the IQs was studied in detail in cell-free systems using purified enzyme. The C-terminal selenocysteine of TR1 was characterized as the primary adduction site of the IQ-derived reactive iminium using liquid chromatography-tandem mass spectrometry analysis. Inhibition of TR1 by IQs in MIA PaCa-2 cells resulted in a shift of thioredoxin-1 redox state to the oxidized form and activation of the p38/c-Jun NH2-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) signaling pathway. Oxidized thioredoxin is known to activate apoptosis signal-regulating kinase 1, an upstream activator of p38/JNK in the MAPK signaling cascade and this was confirmed in our study providing a potential mechanism for IQ-induced apoptosis. These data describe the redox and signaling events involved in the mechanism of growth inhibition induced by novel inhibitors of TR1 in human pancreatic cancer cells. PMID:22147753

  17. HMG-CoA reductase regulates CCL17-induced colon cancer cell migration via geranylgeranylation and RhoA activation

    SciTech Connect

    Al-Haidari, Amr A.; Syk, Ingvar; Thorlacius, Henrik

    2014-03-28

    Highlights: • Simvastatin blocked CCL17-induced and CCR4-dependent RhoA activation in HT29 cells. • CCL17/CCR4-mediated migration of colon cancer cells was antagonised by simvastatin. • Cell migration recovered by adding Mevalonate and geranylgeranyl pyrophosphate. • Targeting HMG-CoA reductase might be useful to inhibit colon cancer metastasis. - Abstract: Background: Simvastatin is widely used to lower cholesterol levels in patients with cardiovascular diseases, although accumulating evidence suggests that statins, such as simvastatin, also exert numerous anti-tumoral effects. Aim: The aim of this study was to examine the effect of simvastatin on colon cancer cell migration. Methods: Migration assays were performed to evaluate CCL17-induced colon cancer cell (HT-29) chemotaxis. In vitro tumor growth and apoptosis were assessed using a proliferation assay and annexin V assay, respectively. Active RhoA protein levels in CCL17-stimulated colon cancer cells were quantified using a G-LISA assay. Results: We found that simvastatin dose-dependently decreased CCL17-induced colon cancer cell migration. Simvastatin had no effect on colon cancer cell proliferation or apoptosis. Inhibition of beta chemokine receptor 4, CCR4, reduced CCL17-evoked activation of RhoA in colon cancer cells. Moreover, administration of mevalonate reversed the inhibitory effect of simvastatin on CCL17-induced colon cancer cell migration. Interestingly, co-incubation with geranylgeranyl pyrophosphate (GGPP) antagonized the inhibitory impact of simvastatin on colon cancer cell migration triggered by CCL17. Moreover, we observed that simvastatin decreased CCL17-induced activation of RhoA in colon cancer cells. Administration of mevalonate and GGPP reversed the inhibitory effect of simvastatin on CCL17-provoked RhoA activation in colon cancer cells. Conclusions: Taken together, our findings show for the first time that HMG-CoA reductase regulates CCL17-induced colon cancer cell migration via

  18. Peptide methionine sulfoxide reductase from Escherichia coli and Mycobacterium tuberculosis protects bacteria against oxidative damage from reactive nitrogen intermediates.

    PubMed

    St John, G; Brot, N; Ruan, J; Erdjument-Bromage, H; Tempst, P; Weissbach, H; Nathan, C

    2001-08-14

    Inducible nitric oxide synthase (iNOS) plays an important role in host defense. Macrophages expressing iNOS release the reactive nitrogen intermediates (RNI) nitrite and S-nitrosoglutathione (GSNO), which are bactericidal in vitro at a pH characteristic of the phagosome of activated macrophages. We sought to characterize the active intrabacterial forms of these RNI and their molecular targets. Peptide methionine sulfoxide reductase (MsrA; EC ) catalyzes the reduction of methionine sulfoxide (Met-O) in proteins to methionine (Met). E. coli lacking MsrA were hypersensitive to killing not only by hydrogen peroxide, but also by nitrite and GSNO. The wild-type phenotype was restored by transformation with plasmids encoding msrA from E. coli or M. tuberculosis, but not by an enzymatically inactive mutant msrA, indicating that Met oxidation was involved in the death of these cells. It seemed paradoxical that nitrite and GSNO kill bacteria by oxidizing Met residues when these RNI cannot themselves oxidize Met. However, under anaerobic conditions, neither nitrite nor GSNO was bactericidal. Nitrite and GSNO can both give rise to NO, which may react with superoxide produced by bacteria during aerobic metabolism, forming peroxynitrite, a known oxidant of Met to Met-O. Thus, the findings are consistent with the hypotheses that nitrite and GSNO kill E. coli by intracellular conversion to peroxynitrite, that intracellular Met residues in proteins constitute a critical target for peroxynitrite, and that MsrA can be essential for the repair of peroxynitrite-mediated intracellular damage.

  19. New evidence of similarity between human and plant steroid metabolism: 5alpha-reductase activity in Solanum malacoxylon.

    PubMed

    Rosati, Fabiana; Danza, Giovanna; Guarna, Antonio; Cini, Nicoletta; Racchi, Milvia Luisa; Serio, Mario

    2003-01-01

    The physiological role of steroid hormones in humans is well known, and the metabolic pathway and mechanisms of action are almost completely elucidated. The role of plant steroid hormones, brassinosteroids, is less known, but an increasing amount of data on brassinosteroid biosynthesis is showing unexpected similarities between human and plant steroid metabolic pathways. Here we focus our attention on the enzyme 5alpha-reductase (5alphaR) for which a plant ortholog of the mammalian system, DET2, was recently described in Arabidopsis thaliana. We demonstrate that campestenone, the natural substrate of DET2, is reduced to 5alpha-campestanone by both human 5alphaR isozymes but with different affinities. Solanum malacoxylon, which is a calcinogenic plant very active in the biosynthesis of vitamin D-like molecules and sterols, was used to study 5alphaR activity. Leaves and calli were chosen as examples of differentiated and undifferentiated tissues, respectively. Two separate 5alphaR activities were found in calli and leaves of Solanum using campestenone as substrate. The use of progesterone allowed the detection of both activities in calli. Support for the existence of two 5alphaR isozymes in S. malacoxylon was provided by the differential actions of inhibitors of the human 5alphaR in calli and leaves. The evidence for the presence of two isozymes in different plant tissues extends the analogies between plant and mammalian steroid metabolic pathways.

  20. Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

    SciTech Connect

    Droux, M.; Miginiac-Maslow, M.; Jacquot, J.P.; Gadal, P.; Crawford, N.A.; Kosower, N.S.; Buchanan, B.B.

    1987-07-01

    The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with (/sup 14/C)iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.

  1. Effect of acetic acid present in bagasse hydrolysate on the activities of xylose reductase and xylitol dehydrogenase in Candida guilliermondii.

    PubMed

    Lima, Luanne Helena Augusto; das Graças de Almeida Felipe, Maria; Vitolo, Michele; Torres, Fernando Araripe Gonçalves

    2004-11-01

    The first two steps in xylose metabolism are catalyzed by NAD(P)H-dependent xylose reductase (XR) (EC 1.1.1.21) and NAD(P)-dependent xylitol dehydrogenase (XDH) (EC 1.1.1.9), which lead to xylose-->xylitol-->xylulose conversion. Xylitol has high commercial value, due to its sweetening and anticariogenic properties, as well as several clinical applications. The acid hydrolysis of sugarcane bagasse allows the separation of a xylose-rich hemicellulosic fraction that can be used as a substrate for Candida guilliermondii to produce xylitol. However, the hydrolysate contains acetic acid, an inhibitor of microbial metabolism. In this study, the effect of acetic acid on the activities of XR and XDH and on xylitol formation by C. guilliermondii were studied. For this purpose, fermentations were carried out in bagasse hydrolysate and in synthetic medium. The activities of XR and XDH were higher in the medium containing acetic acid than in control medium. Moreover, none of the fermentative parameters were significantly altered during cell culture. It was concluded that acetic acid does not interfere with xylitol formation since the increase in XR activity is proportional to XDH activity, leading to a greater production of xylitol and its subsequent conversion to xylulose.

  2. Cytochrome cb-type nitric oxide reductase with cytochrome c oxidase activity from Paracoccus denitrificans ATCC 35512.

    PubMed

    Fujiwara, T; Fukumori, Y

    1996-04-01

    A highly active nitric oxide reductase was purified from Paracoccus denitrificans ATCC 35512, formerly named Thiosphaera pantotropha, which was anaerobically cultivated in the presence of nitrate. The enzyme was composed of two subunits with molecular masses of 34 and 15 kDa and contained two hemes b and one heme c per molecule. Copper was not found in the enzyme. The spectral properties suggested that one of the two hemes b and heme c were in six-coordinated low-spin states and another heme b was in a five-coordinated high-spin state and reacted with carbon monoxide. The enzyme showed high cytochrome c-nitric oxide oxidoreductase activity and formed nitrous oxide from nitric oxide with the expected stoichiometry when P. denitrificans ATCC 35512 ferrocytochrome c-550 was used as the electron donor. The V max and Km values for nitric oxide were 84 micromol of nitric oxide per min/mg of protein and 0.25 microM, respectively. Furthermore, the enzyme showed ferrocytochrome c-550-O2 oxidoreductase activity with a V max of 8.4 micromol of O2 per min/mg of protein and a Km value of 0.9 mM. Both activities were 50% inhibited by about 0.3 mM KCN.

  3. S-nitrosoglutathione reductase (GSNOR) activity is down-regulated during pepper (Capsicum annuum L.) fruit ripening.

    PubMed

    Rodríguez-Ruiz, Marta; Mioto, Paulo; Palma, José M; Corpas, Francisco J

    2016-12-27

    Pepper (Capsicum annuum L.) is an annual plant species of great agronomic importance whose fruits undergo major metabolic changes through development and ripening. These changes include emission of volatile organic compounds associated with respiration, destruction of chlorophylls and synthesis of new pigments (red/yellow carotenoids plus xanthophylls and anthocyans) responsible for color shift, protein degradation/synthesis and changes in total soluble reducing equivalents. Previous data have shown that, during the ripening of pepper fruit, an enhancement of protein tyrosine nitration takes place. On the other hand, it is well known that S-nitrosoglutathione reductase (GSNOR) activity can modulate the transnitrosylation equilibrium between GSNO and S-nitrosylated proteins and, consequently, regulate cellular NO homeostasis. In this study, GSNOR activity, protein content and gene expression were analyzed in green and red pepper fruits. The content of S-nitrosylated proteins on diaminofluorescein (DAF) gels was also studied. The data show that, while GSNOR activity and protein expression diminished during fruit ripening, S-nitrosylated protein content increased. Some of the protein candidates for S-nitrosylation identified, such as cytochorme c oxidase and peroxiredoxin II E, have previously been described as targets of this posttranslational modification in other plant species. These findings corroborate the important role played by GSNOR activity in the NO metabolism during the process of pepper fruit ripening.

  4. Identification of flavonoids and flavonoid rhamnosides from Rhododendron mucronulatum for. albiflorum and their inhibitory activities against aldose reductase.

    PubMed

    Mok, So-Youn; Lee, Sanghyun

    2013-01-15

    To investigate the therapeutic potential of compounds from natural sources, Rhododendron mucronulatum for. albiflorum flowers (RMAF) and R. mucronulatum flowers (RMF) were tested for inhibition of aldose reductase (AR). The methanol extracts of RMAF and RMF exhibited AR inhibitory activities (IC(50) values 1.07 and 1.29 μg/mL, respectively). The stepwise polarity fractions of RMAF were tested for in vitro inhibition of AR from rat lenses. Of these, the ethyl acetate (EtOAc) fraction exhibited AR inhibitory activity (IC(50) 0.15 μg/mL). A chromatography of the active EtOAc fraction of RMAF led to the isolation of six flavonoids, which were identified by spectroscopic analysis as kaempferol (1), afzelin (2), quercetin (3), quercitrin (4), myricetin (5) and myricitrin (6). Compounds 1-6 exhibited high AR inhibitory activity, with IC(50) values of 0.79, 0.31, 0.48, 0.13, 11.92 and 2.67 μg/mL, respectively. HPLC/UV analysis revealed that the major flavonoids of RMAF and RMF are quercitrin (4) and myricitrin (6). Our results suggest that RMAF containing these six flavonoids could be a useful natural source in the development of a novel AR inhibitory agent against diabetic complications.

  5. Effects of Metmyoglobin Reducing Activity and Thermal Stability of NADH-Dependent Reductase and Lactate Dehydrogenase on Premature Browning in Ground Beef.

    PubMed

    Djimsa, Blanchefort A; Abraham, Anupam; Mafi, Gretchen G; VanOverbeke, Deborah L; Ramanathan, Ranjith

    2017-02-01

    Premature browning is a condition wherein ground beef exhibits a well-done appearance before reaching the USDA recommended internal cooked meat temperature of 71.1 °C; however, the mechanism is unclear. The objectives of this study were: (1) to determine the effects of packaging and temperature on metmyoglobin reducing activity (MRA) of cooked ground beef patties and (2) to assess the effects of temperature and pH on thermal stability of NADH-dependent reductase, lactate dehydrogenase (LDH), and oxymyoglobin (OxyMb) in-vitro. Beef patties (lean: fat = 85:15) were packaged in high-oxygen modified atmosphere (HiOX-MAP) or vacuum (VP) and cooked to either 65 or 71 °C. Internal meat color and MRA of both raw and cooked patties were determined. Purified NADH-dependent reductase and LDH were used to determine the effects of pH and temperature on enzyme activity. MRA of cooked patties was temperature and packaging dependent (P < 0.05). Vacuum packaged patties cooked to 71 °C had greater (P < 0.05) MRA than HiOX-MAP counterparts. Thermal stability of OxyMb, NADH-dependent reductase, and LDH were different and pH-dependent. LDH was able to generate NADH at 84 °C; whereas NADH-dependent reductase was least stable to heat. The results suggest that patties have MRA at cooking temperatures, which can influence cooked meat color.

  6. E2F1 promote the aggressiveness of human colorectal cancer by activating the ribonucleotide reductase small subunit M2

    SciTech Connect

    Fang, Zejun; Gong, Chaoju; Liu, Hong; Zhang, Xiaomin; Mei, Lingming; Song, Mintao; Qiu, Lanlan; Luo, Shuchai; Zhu, Zhihua; Zhang, Ronghui; Gu, Hongqian; Chen, Xiang

    2015-08-21

    As the ribonucleotide reductase small subunit, the high expression of ribonucleotide reductase small subunit M2 (RRM2) induces cancer and contributes to tumor growth and invasion. In several colorectal cancer (CRC) cell lines, we found that the expression levels of RRM2 were closely related to the transcription factor E2F1. Mechanistic studies were conducted to determine the molecular basis. Ectopic overexpression of E2F1 promoted RRM2 transactivation while knockdown of E2F1 reduced the levels of RRM2 mRNA and protein. To further investigate the roles of RRM2 which was activated by E2F1 in CRC, CCK-8 assay and EdU incorporation assay were performed. Overexpression of E2F1 promoted cell proliferation in CRC cells, which was blocked by RRM2 knockdown attenuation. In the migration and invasion tests, overexpression of E2F1 enhanced the migration and invasion of CRC cells which was abrogated by silencing RRM2. Besides, overexpression of RRM2 reversed the effects of E2F1 knockdown partially in CRC cells. Examination of clinical CRC specimens demonstrated that both RRM2 and E2F1 were elevated in most cancer tissues compared to the paired normal tissues. Further analysis showed that the protein expression levels of E2F1 and RRM2 were parallel with each other and positively correlated with lymph node metastasis (LNM), TNM stage and distant metastasis. Consistently, the patients with low E2F1 and RRM2 levels have a better prognosis than those with high levels. Therefore, we suggest that E2F1 can promote CRC proliferation, migration, invasion and metastasis by regulating RRM2 transactivation. Understanding the role of E2F1 in activating RRM2 transcription will help to explain the relationship between E2F1 and RRM2 in CRC and provide a novel predictive marker for diagnosis and prognosis of the disease. - Highlights: • E2F1 promotes RRM2 transactivation in CRC cells. • E2F1 promotes the proliferation of CRC cells by activating RRM2. • E2F1 promotes the migration and

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

    PubMed

    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.

  8. Rational design of broad spectrum antibacterial activity based on a clinically relevant enoyl-acyl carrier protein (ACP) reductase inhibitor.

    PubMed

    Schiebel, Johannes; Chang, Andrew; Shah, Sonam; Lu, Yang; Liu, Li; Pan, Pan; Hirschbeck, Maria W; Tareilus, Mona; Eltschkner, Sandra; Yu, Weixuan; Cummings, Jason E; Knudson, Susan E; Bommineni, Gopal R; Walker, Stephen G; Slayden, Richard A; Sotriffer, Christoph A; Tonge, Peter J; Kisker, Caroline

    2014-06-06

    Determining the molecular basis for target selectivity is of particular importance in drug discovery. The ideal antibiotic should be active against a broad spectrum of pathogenic organisms with a minimal effect on human targets. CG400549, a Staphylococcus-specific 2-pyridone compound that inhibits the enoyl-acyl carrier protein reductase (FabI), has recently been shown to possess human efficacy for the treatment of methicillin-resistant Staphylococcus aureus infections, which constitute a serious threat to human health. In this study, we solved the structures of three different FabI homologues in complex with several pyridone inhibitors, including CG400549. Based on these structures, we rationalize the 65-fold reduced affinity of CG400549 toward Escherichia coli versus S. aureus FabI and implement concepts to improve the spectrum of antibacterial activity. The identification of different conformational states along the reaction coordinate of the enzymatic hydride transfer provides an elegant visual depiction of the relationship between catalysis and inhibition, which facilitates rational inhibitor design. Ultimately, we developed the novel 4-pyridone-based FabI inhibitor PT166 that retained favorable pharmacokinetics and efficacy in a mouse model of S. aureus infection with extended activity against Gram-negative and mycobacterial organisms.

  9. A nickel hydride complex in the active site of methyl-coenzyme m reductase: implications for the catalytic cycle.

    PubMed

    Harmer, Jeffrey; Finazzo, Cinzia; Piskorski, Rafal; Ebner, Sieglinde; Duin, Evert C; Goenrich, Meike; Thauer, Rudolf K; Reiher, Markus; Schweiger, Arthur; Hinderberger, Dariush; Jaun, Bernhard

    2008-08-20

    Methanogenic archaea utilize a specific pathway in their metabolism, converting C1 substrates (i.e., CO2) or acetate to methane and thereby providing energy for the cell. Methyl-coenzyme M reductase (MCR) catalyzes the key step in the process, namely methyl-coenzyme M (CH3-S-CoM) plus coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. The active site of MCR contains the nickel porphinoid F430. We report here on the coordinated ligands of the two paramagnetic MCR red2 states, induced when HS-CoM (a reversible competitive inhibitor) and the second substrate HS-CoB or its analogue CH3-S-CoB are added to the enzyme in the active MCR red1 state (Ni(I)F430). Continuous wave and pulse EPR spectroscopy are used to show that the MCR red2a state exhibits a very large proton hyperfine interaction with principal values A((1)H) = [-43,-42,-5] MHz and thus represents formally a Ni(III)F430 hydride complex formed by oxidative addition to Ni(I). In view of the known ability of nickel hydrides to activate methane, and the growing body of evidence for the involvement of MCR in "reverse" methanogenesis (anaerobic oxidation of methane), we believe that the nickel hydride complex reported here could play a key role in helping to understand both the mechanism of "reverse" and "forward" methanogenesis.

  10. Slow-Onset Inhibition of the FabI Enoyl Reductase from Francisella tularensis: Residence Time and in Vivo Activity

    SciTech Connect

    Lu, H.; England, K; Ende, C; Truglio, J; Luckner, S; Reddy, B; Marlenee, N; Knudson, S; Knudson, D; et. al.

    2009-01-01

    Francisella tularensis is a highly virulent and contagious Gram-negative intracellular bacterium that causes the disease tularemia in mammals. The high infectivity and the ability of the bacterium to survive for weeks in a cool, moist environment have raised the possibility that this organism could be exploited deliberately as a potential biological weapon. Fatty acid biosynthesis (FAS-II) is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterials. The FAS-II enoyl reductase ftuFabI has been cloned and expressed, and a series of diphenyl ethers have been identified that are subnanomolar inhibitors of the enzyme with MIC90 values as low as 0.00018 ?g mL-1. The existence of a linear correlation between the Ki and MIC values strongly suggests that the antibacterial activity of the diphenyl ethers results from direct inhibition of ftuFabI within the cell. The compounds are slow-onset inhibitors of ftuFabI, and the residence time of the inhibitors on the enzyme correlates with their in vivo activity in a mouse model of tularemia infection. Significantly, the rate of breakdown of the enzyme-inhibitor complex is a better predictor of in vivo activity than the overall thermodynamic stability of the complex, a concept that has important implications for the discovery of novel chemotherapeutics that normally rely on equilibrium measurements of potency.

  11. Red-cell GSH regeneration and glutathione reductase activity in G6PD variants in the Ferrara area.

    PubMed

    Anderson, B B; Carandina, G; Lucci, M; Perry, G M; Vullo, C

    1987-12-01

    Red-cell studies were carried out on three groups of G6PD-deficient subjects with different G6PD variants from the Ferrara area of Northern Italy. Red-cell GSH and activities of G6PD, glutathione reductase (GR), glutathione peroxidase (GPx) and superoxide dismutase (SOD) were measured. A method was developed to measure red-cell GSH regeneration after oxidation of endogenous GSH in whole blood by diamide and only this clearly distinguished the variants from each other and from normal. Regeneration by 1 h was lowest in the Mediterranean variant, 0-10.2% in contrast to 93-98% in normal. A predisposition to a haemolytic crisis after ingestion of fava beans was not clearcut, but subjects appeared to be at risk if GSH regeneration at 1 h was less than 30% of the endogenous level, and red-cell FAD+ was very high indicated by high in vitro GR activity and inhibition by added FAD+. It is suggested that the most informative tests in G6PD deficiency are measurements of GSH regeneration in intact red cells plus GR activity and/or red-cell flavin compounds.

  12. A nanotherapy strategy significantly enhances anticryptosporidial activity of an inhibitor of bifunctional thymidylate synthase-dihydrofolate reductase from Cryptosporidium.

    PubMed

    Mukerjee, Anindita; Iyidogan, Pinar; Castellanos-Gonzalez, Alejandro; Cisneros, José A; Czyzyk, Daniel; Ranjan, Amalendu Prakash; Jorgensen, William L; White, A Clinton; Vishwanatha, Jamboor K; Anderson, Karen S

    2015-01-01

    Cryptosporidiosis, a gastrointestinal disease caused by protozoans of the genus Cryptosporidium, is a common cause of diarrheal diseases and often fatal in immunocompromised individuals. Bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from Cryptosporidium hominis (C. hominis) has been a molecular target for inhibitor design. C. hominis TS-DHFR inhibitors with nM potency at a biochemical level have been developed however drug delivery to achieve comparable antiparasitic activity in Cryptosporidium infected cell culture has been a major hurdle for designing effective therapies. Previous mechanistic and structural studies have identified compound 906 as a nM C. hominis TS-DHFR inhibitor in vitro, having μM antiparasitic activity in cell culture. In this work, proof of concept studies are presented using a nanotherapy approach to improve drug delivery and the antiparasitic activity of 906 in cell culture. We utilized PLGA nanoparticles that were loaded with 906 (NP-906) and conjugated with antibodies to the Cryptosporidium specific protein, CP2, on the nanoparticle surface in order to specifically target the parasite. Our results indicate that CP2 labeled NP-906 (CP2-NP-906) reduces the level of parasites by 200-fold in cell culture, while NP-906 resulted in 4.4-fold decrease. Moreover, the anticryptosporidial potency of 906 improved 15 to 78-fold confirming the utility of the antibody conjugated nanoparticles as an effective drug delivery strategy.

  13. Non-covalent forces tune the electron transfer complex between ferredoxin and sulfite reductase to optimize enzymatic activity.

    PubMed

    Kim, Ju Yaen; Kinoshita, Misaki; Kume, Satoshi; Gt, Hanke; Sugiki, Toshihiko; Ladbury, John E; Kojima, Chojiro; Ikegami, Takahisa; Kurisu, Genji; Goto, Yuji; Hase, Toshiharu; Lee, Young-Ho

    2016-11-01

    Although electrostatic interactions between negatively charged ferredoxin (Fd) and positively charged sulfite reductase (SiR) have been predominantly highlighted to characterize complex formation, the detailed nature of intermolecular forces remains to be fully elucidated. We investigated interprotein forces for the formation of an electron transfer complex between Fd and SiR and their relationship to SiR activity using various approaches over NaCl concentrations between 0 and 400 mM. Fd-dependent SiR activity assays revealed a bell-shaped activity curve with a maximum ∼40-70 mM NaCl and a reverse bell-shaped dependence of interprotein affinity. Meanwhile, intrinsic SiR activity, as measured in a methyl viologen-dependent assay, exhibited saturation above 100 mM NaCl. Thus, two assays suggested that interprotein interaction is crucial in controlling Fd-dependent SiR activity. Calorimetric analyses showed the monotonic decrease in interprotein affinity on increasing NaCl concentrations, distinguished from a reverse bell-shaped interprotein affinity observed from Fd-dependent SiR activity assay. Furthermore, Fd:SiR complex formation and interprotein affinity were thermodynamically adjusted by both enthalpy and entropy through electrostatic and non-electrostatic interactions. A residue-based NMR investigation on the addition of SiR to (15)N-labeled Fd at the various NaCl concentrations also demonstrated that a combination of electrostatic and non-electrostatic forces stabilized the complex with similar interfaces and modulated the binding affinity and mode. Our findings elucidate that non-electrostatic forces are also essential for the formation and modulation of the Fd:SiR complex. We suggest that a complex configuration optimized for maximum enzymatic activity near physiological salt conditions is achieved by structural rearrangement through controlled non-covalent interprotein interactions.

  14. The Nitric Oxide Reductase Mechanism of a Flavo-Diiron Protein: Identification of Active-Site Intermediates and Products

    PubMed Central

    2015-01-01

    The unique active site of flavo-diiron proteins (FDPs) consists of a nonheme diiron-carboxylate site proximal to a flavin mononucleotide (FMN) cofactor. FDPs serve as the terminal components for reductive scavenging of dioxygen or nitric oxide to combat oxidative or nitrosative stress in bacteria, archaea, and some protozoan parasites. Nitric oxide is reduced to nitrous oxide by the four-electron reduced (FMNH2–FeIIFeII) active site. In order to clarify the nitric oxide reductase mechanism, we undertook a multispectroscopic presteady-state investigation, including the first Mössbauer spectroscopic characterization of diiron redox intermediates in FDPs. A new transient intermediate was detected and determined to be an antiferromagnetically coupled diferrous-dinitrosyl (S = 0, [{FeNO}7]2) species. This species has an exchange energy, J ≥ 40 cm–1 (JS1 ° S2), which is consistent with a hydroxo or oxo bridge between the two irons. The results show that the nitric oxide reductase reaction proceeds through successive formation of diferrous-mononitrosyl (S = 1/2, FeII{FeNO}7) and the S = 0 diferrous-dinitrosyl species. In the rate-determining process, the diferrous-dinitrosyl converts to diferric (FeIIIFeIII) and by inference N2O. The proximal FMNH2 then rapidly rereduces the diferric site to diferrous (FeIIFeII), which can undergo a second 2NO → N2O turnover. This pathway is consistent with previous results on the same deflavinated and flavinated FDP, which detected N2O as a product (HayashiBiochemistry2010, 49, 704020669924). Our results do not support other proposed mechanisms, which proceed either via “super-reduction” of [{FeNO}7]2 by FMNH2 or through FeII{FeNO}7 directly to a diferric-hyponitrite intermediate. The results indicate that an S = 0 [{FeNO}7}]2 complex is a proximal precursor to N–N bond formation and N–O bond cleavage to give N2O and that this conversion can occur without redox participation of the FMN cofactor. PMID:24828196

  15. Involvement of tristetraprolin in transcriptional activation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin

    SciTech Connect

    Ness, Gene C.; Edelman, Jeffrey L.; Brooks, Patricia A.

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer siRNAs to tristetraprolin blocks transcription of HMGR in vivo in rat liver. Black-Right-Pointing-Pointer siRNAs to tristetraprolin inhibits insulin activation of HMGR transcription. Black-Right-Pointing-Pointer Insulin acts to rapidly increase tristetraprolin in liver nuclear extracts. -- Abstract: Several AU-rich RNA binding element (ARE) proteins were investigated for their possible effects on transcription of hepatic 3-hydroxy-3-methyglutaryl coenzyme A reductase (HMGR) in normal rats. Using in vivo electroporation, four different siRNAs to each ARE protein were introduced together with HMGR promoter (-325 to +20) luciferase construct and compared to saline controls. All four siRNAs to tristetraprolin (TTP) completely eliminated transcription from the HMGR promoter construct. Since insulin acts to rapidly increase hepatic HMGR transcription, the effect of TTP siRNA on induction by insulin was tested. The 3-fold stimulation by insulin was eliminated by this treatment. In comparison, siRNA to AU RNA binding protein/enoyl coenzyme A hydratase (AUH) had no effect. These findings indicate a role for TTP in the insulin-mediated activation of hepatic HMGR transcription.

  16. Dissimilatory arsenate reductase activity and arsenate-respiring bacteria in bovine rumen fluid, hamster feces, and the termite hindgut

    USGS Publications Warehouse

    Herbel, M.J.; Switzer, Blum J.; Hoeft, S.E.; Cohen, S.M.; Arnold, L.L.; Lisak, J.; Stolz, J.F.; Oremland, R.S.

    2002-01-01

    Bovine rumen fluid and slurried hamster feces completely reduced millimolar levels of arsenate to arsenite upon incubation under anoxic conditions. This activity was strongly inhibited by autoclaving or aerobic conditions, and partially inhibited by tungstate or chloramphenicol. The rate of arsenate reduction was faster in feces from a population of arsenate-watered (100 ppm) hamsters compared to a control group watered without arsenate. Using radioisotope methods, arsenate reductase activity in hamster feces was also detected at very low concentrations of added arsenate (???10 ??M). Bacterial cultures were isolated from these materials, as well as from the termite hindgut, that grew using H2 as their electron donor, acetate as their carbon source, and arsenate as their respiratory electron acceptor. The three cultures aligned phylogenetically either with well-established enteric bacteria, or with an organism associated with feedlot fecal wastes. Because arsenite is transported across the gut epithelium more readily than arsenate, microbial dissimilatory reduction of arsenate in the gut may promote the body's absorption of arsenic and hence potentiate its toxicity. ?? 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

  17. Low red blood cell glutathione reductase and pyridoxine phosphate oxidase activities not related to dietary riboflavin: selection by malaria?

    PubMed

    Anderson, B B; Giuberti, M; Perry, G M; Salsini, G; Casadio, I; Vullo, C

    1993-05-01

    This study was designed to confirm that low dietary riboflavin does not contribute to the flavin-deficient red blood cells commonly found in subjects in Ferrara Province, northern Italy. In this area it is primarily an inherited characteristic believed to have been selected for by malaria, which was endemic from the 12th century. In parallel with assessment of daily riboflavin intake (DRI), flavin adenine dinucleotide-dependent glutathione reductase (EGR) and flavin mononucleotide-dependent pyridoxine phosphate oxidase (PPO) were measured in beta-thalassemic heterozygotes, their normal relatives, and normal spouses (representative of the normal population). In all of these groups there is a high incidence of deficiency of these flavin enzymes. We found that the majority had an adequate riboflavin intake and there was no significant correlation of EGR and PPO activities with DRI. Thus, interpretation of low EGR activity is discussed with reference to studies of EGR done to detect nutritional riboflavin deficiency in countries where there is malnutrition and endemic malaria.

  18. Pre-exposure to nitrite in the absence of ammonium strongly inhibits anammox.

    PubMed

    Carvajal-Arroyo, José M; Puyol, Daniel; Li, Guangbin; Lucero-Acuña, Armando; Sierra-Álvarez, Reyes; Field, Jim A

    2014-01-01

    Anaerobic ammonium oxidizing bacteria (Anammox) are known to be inhibited by their substrate, nitrite. However, the mechanism of inhibition and the physiological conditions under which nitrite impacts the performance of anammox bioreactors are still unknown. This study investigates the role of pre-exposing anammox bacteria to nitrite alone on their subsequent activity and metabolism after ammonium has been added. Batch experiments were carried out with anammox granular biofilm pre-exposed to nitrite over a range of concentrations and durations in the absence of ammonium. The effect of pre-exposure to nitrite alone compared to nitrite simultaneously fed with ammonium was evaluated by measuring the anammox activity and the accumulation of the intermediate, nitric oxide. The results show that the inhibitory effect was more dramatic when bacteria were pre-exposed to nitrite in absence of ammonium, as revealed by the lower activity and the higher accumulation of nitric oxide. The nitrite concentration causing 50% inhibition was 53 and 384 mg N L(-1) in the absence or the presence of ammonium, respectively. The nitrite inhibition was thus 7.2-fold more severe in the absence of ammonium. Biomass exposure to nitrite (25 mg N L(-1)), in absence of ammonium, led to accumulation of nitric oxide. On the other hand when the biomass was exposed to nitrite in presence of ammonium, accumulation of nitric oxide was only observed at much higher nitrite concentrations (500 mg N L(-1)). The inhibitory effect of nitrite in the absence of ammonium was very rapid. The rate of decay of the anammox activity was equivalent to the diffusion rate of nitrite up to 46% of activity loss. The results taken as a whole suggest that nitrite inhibition is more acute when anammox cells are not actively metabolizing. Accumulation of nitric oxide in the headspace most likely indicates disruption of the anammox biochemistry by nitrite inhibition, caused by an interruption of the hydrazine synthesis step.

  19. Chromate/Nitrite Interactions in Shewanella Oneidensis MR-1: Evidence for Multiple Cr(VI) Reduction Mechanisms Dependent on Physiological Growth Conditions

    SciTech Connect

    Apel, William Arnold; Viamajala, S.; Peyton, Brent Michael; Petersen, J. N.

    2002-06-01

    Inhibition of hexavalent chromium [Cr(VI)] reduction due to nitrate and nitrite was observed during tests with Shewanella oneidensis MR-1 (previously named Shewanella putrefaciens MR-1 and henceforth referred to as MR-1). Initial Cr(VI) reduction rates were measured at various nitrite concentrations, and a mixed inhibition kinetic model was used to determine the kinetic parameters-maximum Cr(VI) reduction rate and inhibition constant [V(max,Cr(VI)) and K(i,Cr(VI))]. Values of V(max,Cr(VI)) and K(i,Cr(VI)) obtained with MR-1 cultures grown under denitrifying conditions were observed to be significantly different from the values obtained when the cultures were grown with fumarate as the terminal electron acceptor. It was also observed that a single V(max,Cr(VI)) and K(i,Cr(VI)) did not adequately describe the inhibition kinetics of either nitrate-grown or fumarate-grown cultures. The inhibition patterns indicate that Cr(VI) reduction in MR-1 is likely not limited to a single pathway, but occurs via different mechanisms some of which are dependent on growth conditions. Inhibition of nitrite reduction due to the presence of Cr(VI) was also studied, and the kinetic parameters V(max,NO2) and K(i,NO2) were determined. It was observed that these coefficients also differed significantly between MR-1 grown under denitrifying conditions and fumarate reducing conditions. The inhibition studies suggest the involvement of nitrite reductase in Cr(VI) reduction. Because nitrite reduction is part of the anaerobic respiration process, inhibition due to Cr(VI) might be a result of interaction with the components of the anaerobic respiration pathway such as nitrite reductase. Also, differences in the degree of inhibition of nitrite reduction activity by chromate at different growth conditions suggest that the toxicity mechanism of Cr(VI) might also be dependent on the conditions of growth. Cr(VI) reduction has been shown to occur via different pathways, but to our knowledge, multiple

  20. In vitro effect of D-004, a lipid extract of the fruit of the cuban royal palm (Roystonea regia), on prostate steroid 5α-reductase activity

    PubMed Central

    Pérez, L. Yohani; Menéndez, Roberto; Má, Rosa; González, Rosa M.

    2006-01-01

    Background: D-004, a lipid extract of the fruit of the Cuban royal palm (Roystonea regia), has been found to reduce prostatic hyperplasia (PH) induced with testosterone (T), but not PH induced with dihydrotestosterone (DHT), in rodents, suggesting the inhibition of prostate 5α-reductase activity. Objectives: The aims of this study were to assess whether D-004 inhibits prostate 5α-reductase activity in vitro and to examine the effects of D-004 on enzyme kinetics. Methods: This experimental study was conducted at the Pharmacology Department, Center of Natural Products, National Center for Scientific Research, Havana, Cuba. Soluble rat prostate preparations were used as the source of 5α-reductase, and (3H)-DHT production was measured to determine prostate 5α-reductase activity. Cell-free rat prostate homogenates were pre-incubated with carboxymethyl cellulose 2% alone (control tubes) or D-004 (0.24–125 μg/mL) suspended in the vehicle (treated tubes) for 10 minutes prior to adding the labeled substrate (3H)-T Once the reaction was stopped, sterols were extracted with chloroform and aliquots were applied on silica gel plates developed in benzene-acetone (4:1, v/v). Areas containing DHT were scraped and radioactivity was counted. The median inhibitory concentration (IC50) was determined by measuring the conversion of T to DHT The apparent Michaelis-Menten constant (Km) and Vmax values before and after adding D-004 were determined in kinetic studies using labeled T (0.5-25 μmol/L). Results: Compared with controls, D-004 significantly and dose-dependently inhibited the enzymatic reaction at doses of 1.95 to 125.0 μg/mL) (all, P < 0.05). The IC50 of D-004 required to inhibit 5a-reductase activity was 2.25 μg/mL. Enzyme inhibition was noncompetitive, since D-004 lowered the Vmax from 15.3 to 10.0 nmol DHT/min · mg−1 protein, while the Km (4.54 μmol/L) was almost unaffected. Conclusions: D-004 dose-dependently and noncompetitively inhibited in vitro 5α-reductase

  1. Post-translational control of nitrate reductase activity responding to light and photosynthesis evolved already in the early vascular plants.

    PubMed

    Nemie-Feyissa, Dugassa; Królicka, Adriana; Førland, Nina; Hansen, Margarita; Heidari, Behzad; Lillo, Cathrine

    2013-05-01

    Regulation of nitrate reductase (NR) by reversible phosphorylation at a conserved motif is well established in higher plants, and enables regulation of NR in response to rapid fluctuations in light intensity. This regulation is not conserved in algae NR, and we wished to test the evolutionary origin of the regulatory mechanism by physiological examination of ancient land plants. Especially a member of the lycophytes is of interest since their NR is candidate for regulation by reversible phosphorylation based on sequence analysis. We compared Selaginella kraussiana, a member of the lycophytes and earliest vascular plants, with the angiosperm Arabidopsis thaliana, and also tested the moss Physcomitrella patens. Interestingly, optimization of assay conditions revealed that S. kraussiana NR used NADH as an electron donor like A. thaliana, whereas P. patens NR activity depended on NADPH. Examination of light/darkness effects showed that S. kraussiana NR was rapidly regulated similar to A. thaliana NR when a differential (Mg(2+) contra EDTA) assay was used to reveal activity state of NR. This implies that already existing NR enzyme was post-translationally activated by light in both species. Light had a positive effect also on de novo synthesis of NR in S. kraussiana, which could be shown after the plants had been exposed to a prolonged dark period (7 days). Daily variations in NR activity were mainly caused by post-translational modifications. As for angiosperms, the post-translational light activation of NR in S. kraussiana was inhibited by 3-(3,4-dichlorophenyl)-1*1-dimethylurea (DCMU), an inhibitor of photosynthesis and stomata opening. Evolutionary, a post-translational control mechanism for NR have occurred before or in parallel with development of vascular tissue in land plants, and appears to be part of a complex mechanisms for coordination of CO2 and nitrogen metabolism in these plants.

  2. Effect of pH and nitrite concentration on nitrite oxidation rate.

    PubMed

    Jiménez, E; Giménez, J B; Ruano, M V; Ferrer, J; Serralta, J

    2011-10-01

    The effect of pH and nitrite concentration on the activity of the nitrite oxidizing bacteria (NOB) in an activated sludge reactor has been determined by means of laboratory batch experiments based on respirometric techniques. The bacterial activity was measured at different pH and at different total nitrite concentrations (TNO₂). The experimental results showed that the nitrite oxidation rate (NOR) depends on the TNO₂ concentration independently of the free nitrous acid (FNA) concentration, so FNA cannot be considered as the real substrate for NOB. NOB were strongly affected by low pH values (no activity was detected at pH 6.5) but no inhibition was observed at high pH values (activity was nearly the same for the pH range 7.5-9.95). A kinetic expression for nitrite oxidation process including switch functions to model the effect of TNO₂ concentration and pH inhibition is proposed. Substrate half saturation constant and pH inhibition constants have been obtained.

  3. Reduction of nitrite to NO in an organised triphasic medium by platinum carbonyl clusters and redox active dyes as electron carriers.

    PubMed

    Sen Gupta, Nalinava; Basu, Susmit; Payra, Pramatha; Mathur, Pradeep; Bhaduri, Sumit; Lahiri, Goutam Kumar

    2007-06-28

    Artificial electron donors such as leuco methylene blue and leuco safranin O reduce nitrite ion to nitric oxide. The reaction is effected in a U-tube where nitrite ion and dye in two aqueous layers are separated by a layer of dichloromethane (a close model for a biological liquid membrane) that contains the platinum carbonyl cluster ([Bu(4)N]2[Pt12(CO)24], Chini cluster). On passing dihydrogen an electron transfer chain involving dihydrogen, the dye, the clusters and the nitrite ion is initiated. The cluster catalytically reduces the dye in the presence of dihydrogen, the reduced dye migrates across the phase boundaries and in turn reduces the nitrite ions. The resultant nitric oxide in the effluent gas has been identified by its reactions with cobalamine and myoglobin. When safranin O is the dye, an adduct is formed between the reduced dye and NO. It has been identified by spectroscopic techniques and its probable structure investigated by DFT calculations.

  4. Synthesis of Superparamagnetic Core-Shell Structure Supported Pd Nanocatalysts for Catalytic Nitrite Reduction with Enhanced Activity, No Detection of Undesirable Product of Ammonium, and Easy Magnetic Separation Capability.

    PubMed

    Sun, Wuzhu; Yang, Weiyi; Xu, Zhengchao; Li, Qi; Shang, Jian Ku

    2016-01-27

    Superparamagnetic nanocatalysts could minimize both the external and internal mass transport limitations and neutralize OH(-) produced in the reaction more effectively to enhance the catalytic nitrite reduction efficiency with the depressed product selectivity to undesirable ammonium, while possess an easy magnetic separation capability. However, commonly used qusi-monodispersed superparamagnetic Fe3O4 nanosphere is not suitable as catalyst support for nitrite reduction because it could reduce the catalytic reaction efficiency and the product selectivity to N2, and the iron leakage could bring secondary contamination to the treated water. In this study, protective shells of SiO2, polymethylacrylic acid, and carbon were introduced to synthesize Fe3O4@SiO2/Pd, Fe3O4@PMAA/Pd, and Fe3O4@C/Pd catalysts for catalytic nitrite reduction. It was found that SiO2 shell could provide the complete protection to Fe3O4 nanosphere core among these shells. Because of its good dispersion, dense structure, and complete protection to Fe3O4, the Fe3O4@SiO2/Pd catalyst demonstrated the highest catalytic nitrite reduction activity without the detection of NH4(+) produced. Due to this unique structure, the activity of Fe3O4@SiO2/Pd catalysts for nitrite reduction was found to be independent of the Pd nanoparticle size or shape, and their product selectivity was independent of the Pd nanoparticle size, shape, and content. Furthermore, their superparamagnetic nature and high saturation magnetization allowed their easy magnetic separation from treated water, and they also demonstrated a good stability during the subsequent recycling experiment.

  5. Electrostatic Fields Near the Active Site of Human Aldose Reductase: 2. New Inhibitors and Complications due to Hydrogen Bonds†

    PubMed Central

    Xu, Lin; Cohen, Aina E.; Boxer, Steven G.

    2011-01-01

    Vibrational Stark effect spectroscopy was used to measure electrostatic fields in the hydrophobic region of the active site of human aldose reductase (hALR2). A new nitrile-containing inhibitor was designed and synthesized, and the x-ray structure of its complex, along with cofactor NADP+, with wild-type hALR2 was determined at 1.3 Å resolution. The nitrile is found to be in close proximity to T113, consistent with a hydrogen bond interaction. Two vibrational absorption peaks were observed at room temperature in the nitrile region when the inhibitor binds to wild-type hALR2, indicating that the nitrile probe experiences two different microenvironments, and these could be empirically separated into a hydrogen bonded and non-hydrogen bonded population by comparison with the mutant T113A, where a hydrogen bond to the nitrile is not present. Classical molecular dynamics simulations based on the structure predict a double-peaked distribution in protein electric fields projected along the nitrile probe. The interpretation of these two peaks as a hydrogen bond formation-dissociation process between the probe nitrile group and a nearby amino acid side chain is used to explain the observation of two IR bands, and the simulations were used to investigate the molecular details of this conformational change. Hydrogen bonding complicates the simplest analysis of vibrational frequency shifts as being due solely to electrostatic interactions through the vibrational Stark effect, and the consequences of this complication are discussed. PMID:21859105

  6. Enantioselective Reduction of Citral Isomers in NCR Ene Reductase: Analysis of an Active-Site Mutant Library.

    PubMed

    Kress, Nico; Rapp, Johanna; Hauer, Bernhard

    2017-02-08

    A deeper understanding of the >99 % S-selective reduction of both isomers of citral catalyzed by NCR ene reductase was achieved by active-site mutational studies and docking simulation. Though structurally similar, the E/Z isomers of citral showed a significantly varying selectivity response to introduced mutations. Although it was possible to invert (E)-citral reduction enantioselectivity to ee 46 % (R) by introducing mutation W66A, for (Z)-citral it remained ≥88 % (S) for all single-residue variants. Residue 66 seems to act as a lever for opposite binding modes. This was underlined by a W66A-based double-mutant library that enhanced the (E)-citral derived enantioselectivity to 63 % (R) and significantly lowered the S selectivity for (Z)-citral to 44 % (S). Formation of (R)-citronellal from an (E/Z)-citral mixture is a desire in industrial (-)-menthol synthesis. Our findings pave the way for a rational enzyme engineering solution.

  7. Biological Nitrogen Removal through Nitritation Coupled with Thiosulfate-Driven Denitritation

    NASA Astrophysics Data System (ADS)

    Qian, Jin; Zhou, Junmei; Zhang, Zhen; Liu, Rulong; Wang, Qilin

    2016-06-01

    A novel biological nitrogen removal system based on nitritation coupled with thiosulfate-driven denitritation (Nitritation-TDD) was developed to achieve a high nitrogen removal rate and low sludge production. A nitritation sequential batch reactor (nitritation SBR) and an anoxic up-flow sludge bed (AnUSB) reactor were applied for effective nitritation and denitritation, respectively. Above 75% nitrite was accumulated in the nitritation SBR with an influent ammonia loading rate of 0.43 kg N/d/m3. During Nitritation-TDD operation, particle sizes (d50) of the sludge decreased from 406 to 225 um in nitritation SBR and from 327–183 um in AnUSB reactor. Pyrosequencing tests revealed that ammonium-oxidizing bacteria (AOB) population was stabilized at approximately 7.0% (calculated as population of AOB-related genus divided by the total microbial population) in the nitritation SBR. In contrast, nitrite-oxidizing bacteria (NOB) population decreased from 6.5–0.6% over the same time, indicating the effective nitrite accumulation in the nitritation SBR. Thiobacillus, accounting for 34.2% in the AnUSB reactor, was mainly responsible for nitrogen removal via autotrophic denitritation, using an external source of thiosulfate as electron donor. Also, it was found that free nitrous acid could directly affect the denitritation activity.

  8. Biological Nitrogen Removal through Nitritation Coupled with Thiosulfate-Driven Denitritation

    PubMed Central

    Qian, Jin; Zhou, Junmei; Zhang, Zhen; Liu, Rulong; Wang, Qilin

    2016-01-01

    A novel biological nitrogen removal system based on nitritation coupled with thiosulfate-driven denitritation (Nitritation-TDD) was developed to achieve a high nitrogen removal rate and low sludge production. A nitritation sequential batch reactor (nitritation SBR) and an anoxic up-flow sludge bed (AnUSB) reactor were applied for effective nitritation and denitritation, respectively. Above 75% nitrite was accumulated in the nitritation SBR with an influent ammonia loading rate of 0.43 kg N/d/m3. During Nitritation-TDD operation, particle sizes (d50) of the sludge decreased from 406 to 225 um in nitritation SBR and from 327–183 um in AnUSB reactor. Pyrosequencing tests revealed that ammonium-oxidizing bacteria (AOB) population was stabilized at approximately 7.0% (calculated as population of AOB-related genus divided by the total microbial population) in the nitritation SBR. In contrast, nitrite-oxidizing bacteria (NOB) population decreased from 6.5–0.6% over the same time, indicating the effective nitrite accumulation in the nitritation SBR. Thiobacillus, accounting for 34.2% in the AnUSB reactor, was mainly responsible for nitrogen removal via autotrophic denitritation, using an external source of thiosulfate as electron donor. Also, it was found that free nitrous acid could directly affect the denitritation activity. PMID:27272192

  9. Nitrate Reduction to Nitrite, a Possible Source of Nitrite for Growth of Nitrite-Oxidizing Bacteria

    PubMed Central

    Belser, L. W.

    1977-01-01

    Growth yields and other parameters characterizing the kinetics of growth of nitrite-oxidizing bacteria are presented. These parameters were measured during laboratory enrichments of soil samples with added nitrite. They were then used to reanalyze data for nitrite oxidizer growth in a previously reported field study (M. G. Volz, L. W. Belser, M. S. Ardakani, and A. D. McLaren, J. Environ. Qual. 4:179-182, 1975), where nitrate, but not nitrite or ammonium, was added. In that report, analysis of the field data indicated that in unsaturated soils, the reduction of nitrate to nitrite may be a significant source of nitrite for the growth of nitrite oxidizers. A yield of 1.23 × 104 cells per μg of N was determined to be most appropriate for application to the field. It was determined that if nitrite came only from mineralized organic nitrogen via ammonium oxidation, 35 to 90% of the organic nitrogen would have had to have been mineralized to produce the growth observed. However, it is estimated that only about 2% of the organic nitrogen could have been mineralized during the growth period. Thus, it appears that another source of nitrite is required, the most likely being the reduction of nitrate to nitrite coupled to the oxidation of organic matter. PMID:921264

  10. The cytochrome ba3 oxygen reductase from Thermus thermophilus uses a single input channel for proton delivery to the active site and for proton pumping.

    PubMed

    Chang, Hsin-Yang; Hemp, James; Chen, Ying; Fee, James A; Gennis, Robert B

    2009-09-22

    The heme-copper oxygen reductases are redox-driven proton pumps that generate a proton motive force in both prokaryotes and mitochondria. These enzymes have been divided into 3 evolutionarily related groups: the A-, B- and C-families. Most experimental work on proton-pumping mechanisms has been performed with members of the A-family. These enzymes require 2 proton input pathways (D- and K-channels) to transfer protons used for oxygen reduction chemistry and for proton pumping, with the D-channel transporting all pumped protons. In this work we use site-directed mutagenesis to demonstrate that the ba(3) oxygen reductase from Thermus thermophilus, a representative of the B-family, does not contain a D-channel. Rather, it utilizes only 1 proton input channel, analogous to that of the A-family K-channel, and it delivers protons to the active site for both O2 chemistry and proton pumping. Comparison of available subunit I sequences reveals that the only structural elements conserved within the oxygen reductase families that could perform these functions are active-site components, namely the covalently linked histidine-tyrosine, the Cu(B) and its ligands, and the active-site heme and its ligands. Therefore, our data suggest that all oxygen reductases perform the same chemical reactions for oxygen reduction and comprise the essential elements of the proton-pumping mechanism (e.g., the proton-loading and kinetic-gating sites). These sites, however, cannot be located within the D-channel. These results along with structural considerations point to the A-propionate region of the active-site heme and surrounding water molecules as the proton-loading site.

  11. Regulative roles of glutathione reductase and four glutaredoxins in glutathione redox, antioxidant activity, and iron homeostasis of Beauveria bassiana.

    PubMed

    Zhang, Long-Bin; Tang, Li; Ying, Sheng-Hua; Feng, Ming-Guang

    2016-07-01

    Multiple glutaredoxins (Grx) and glutathione reductase (Glr) are vital for the thiol-disulfide redox system in budding yeast but generally unexplored in filamentous fungi. Here we characterized the Beauveria bassiana redox system comprising dithiol Grx1, monothiol Grx2-4, Grx-like Grx5, and Glr orthologue. Each grx or glr deletion was compensated by increased transcripts of some other grx genes in normal cultures. Particularly, grx3 compensated the absence of grx1, grx2, grx5, or glr under oxidative stress while its absence was compensated only by undeletable grx4 under normal conditions but by most of other undeleted grx and glr genes in response to menadione. Consequently, the redox state was disturbed in Δglr more than in Δgrx3 but not in Δgrx1/2/5. Superoxide dismutases were more active in normal Δgrx1-3 cultures but less in Δgrx5 or Δglr response to menadione. Total catalase activity increased differentially in all the mutant cultures stressed with or without H2O2 while total peroxidase activity decreased more in the normal or H2O2-stressed culture of Δglr than of Δgrx3. Among the mutants, Δgrx3 showed slightly increased sensitivity to menadione or H2O2; Δglr exhibited greater sensitivity to thiol-oxidizing diamide than thiol-reducing 1-chloro-2,4-dinitrobenzene as well as increased sensitivity to the two oxidants. Intriguingly, all the mutants grew slower in a Fe(3+)-inclusive medium perhaps due to elevated transcripts of two Fe(3+) transporter genes. More or fewer phenotypes linked with biocontrol potential were altered in four deletion mutants excluding Δgrx5. All the changes were restored by targeted gene complementation. Overall, Grx3 played more critical role than other Grx homologues in the Glr-dependent redox system of the fungal entomopathogen.

  12. ROS-Mediated Inhibition of S-nitrosoglutathione Reductase Contributes to the Activation of Anti-oxidative Mechanisms

    PubMed Central

    Kovacs, Izabella; Holzmeister, Christian; Wirtz, Markus; Geerlof, Arie; Fröhlich, Thomas; Römling, Gaby; Kuruthukulangarakoola, Gitto T.; Linster, Eric; Hell, Rüdiger; Arnold, Georg J.; Durner, Jörg; Lindermayr, Christian

    2016-01-01

    Nitric oxide (NO) has emerged as a signaling molecule in plants being involved in diverse physiological processes like germination, root growth, stomata closing and response to biotic and abiotic stress. S-nitrosoglutathione (GSNO) as a biological NO donor has a very important function in NO signaling since it can transfer its NO moiety to other proteins (trans-nitrosylation). Such trans-nitrosylation reactions are equilibrium reactions and depend on GSNO level. The breakdown of GSNO and thus the level of S-nitrosylated proteins are regulated by GSNO-reductase (GSNOR). In this way, this enzyme controls S-nitrosothiol levels and regulates NO signaling. Here we report that Arabidopsis thaliana GSNOR activity is reversibly inhibited by H2O2 in vitro and by paraquat-induced oxidative stress in vivo. Light scattering analyses of reduced and oxidized recombinant GSNOR demonstrated that GSNOR proteins form dimers under both reducing and oxidizing conditions. Moreover, mass spectrometric analyses revealed that H2O2-treatment increased the amount of oxidative modifications on Zn2+-coordinating Cys47 and Cys177. Inhibition of GSNOR results in enhanced levels of S-nitrosothiols followed by accumulation of glutathione. Moreover, transcript levels of redox-regulated genes and activities of glutathione-dependent enzymes are increased in gsnor-ko plants, which may contribute to the enhanced resistance against oxidative stress. In sum, our results demonstrate that reactive oxygen species (ROS)-dependent inhibition of GSNOR is playing an important role in activation of anti-oxidative mechanisms to damping oxidative damage and imply a direct crosstalk between ROS- and NO-signaling. PMID:27891135

  13. Nitrite inhibition of denitrification by Pseudomonas fluorescens

    SciTech Connect

    Almeida, J.S.; Julio, S.M.; Reis, M.A.M. |

    1995-05-05

    Using a pure culture of Pseudomonas fluorescens as a model system nitrite inhibition of denitrification was studied. A mineral media with acetate and nitrate as sole electron donor and acceptor, respectively, was used. Results obtained in continuous stirred-tank reactors (CSTR) operated at pH values between 6.6 and 7.8 showed that growth inhibition depended only on the nitrite undissociated fraction concentration (nitrous acid). A mathematical model to describe this dependence is put forward. The maximum nitrous acid concentration compatible with cell growth and denitrification activity was found to be 66 {mu}g N/L. Denitrification activity was partially associated with growth, as described by the Luedeking-Piret equation. However, when the freshly inoculated reactor was operated discontinuously, nitrite accumulation caused growth uncoupling from denitrification activity. The authors suggest that these results can be interpreted considering that (a) nitrous acid acts as a proton uncoupler; and (b) cultures continuously exposed to nitrous acid prevent the uncoupling effect but not the growth inhibition. Examination of the growth dependence on nitrite concentration at pH 7.0 showed that adapted cultures (growth on CSTR) are less sensitive to nitrous acid inhibition than the ones cultivated in batch.

  14. Inhibition of Rat 5α-Reductase Activity and Testosterone-Induced Sebum Synthesis in Hamster Sebocytes by an Extract of Quercus acutissima Cortex

    PubMed Central

    Koseki, Junichi; Matsumoto, Takashi; Matsubara, Yosuke; Tsuchiya, Kazuaki; Mizuhara, Yasuharu; Sekiguchi, Kyoji; Nishimura, Hiroaki; Watanabe, Junko; Kaneko, Atsushi; Hattori, Tomohisa; Maemura, Kazuya; Kase, Yoshio

    2015-01-01

    Objective. Bokusoku (BK) is an extract from the Quercus cortex used in folk medicine for treatment of skin disorders and convergence, and is present in jumihaidokuto, a traditional Japanese medicine that is prescribed for purulent skin diseases like acne vulgaris. The excess of sebum production induced by androgen is involved in the development of acne. Our aim is to examine whether BK and its constituents inhibit testosterone metabolism and testosterone-induced sebum synthesis. Methods. Measurements of 5α-reductase activity and lipogenesis were performed using rat liver microsomes and hamster sebocytes, respectively. Results. BK dose-dependently reduced the conversion of testosterone to a more active androgen, dihydrotestosterone in a 5α-reductase enzymatic reaction. Twenty polyphenols in BK categorized as gallotannin, ellagitannin, and flavonoid were identified by LC-MS/MS. Nine polyphenols with gallate group, tetragalloyl glucose, pentagalloyl glucose, eugeniin, 1-desgalloyl eugeniin, casuarinin, castalagin, stenophyllanin C, (−)-epicatechin gallate, and (−)-epigallocatechin gallate, inhibited testosterone metabolism. In particular, pentagalloyl glucose showed the strongest activity. BK and pentagalloyl glucose suppressed testosterone-induced lipogenesis, whereas they weakly inhibited the lipogenic action of insulin. Conclusions. BK inhibited androgen-related pathogenesis of acne, testosterone conversion, and sebum synthesis, partially through 5α-reductase inhibition, and has potential to be a useful agent in the therapeutic strategy of acne. PMID:25709710

  15. Differential regulatory role of nitric oxide in mediating nitrate reductase activity in roots of tomato (Solanum lycocarpum)

    PubMed Central

    Jin, Chong Wei; Du, Shao Ting; Zhang, Yong Song; Lin, Xian Yong; Tang, Cai Xian

    2009-01-01

    Background and Aims Nitric oxide (NO) has been demonstrated to stimulate the activity of nitrate reductase (NR) in plant roots supplied with a low level of nitrate, and to affect proteins differently, depending on the ratio of NO to the level of protein. Nitrate has been suggested to regulate the level of NO in plants. This present study examined interactive effects of NO and nitrate level on NR activity in roots of tomato (Solanum lycocarpum). Methods NR activity, mRNA level of NR gene and concentration of NR protein in roots fed with 0·5 mm or 5 mm nitrate and treated with the NO donors, sodium nitroprusside (SNP) and diethylamine NONOate sodium (NONOate), and the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO), were measured in 25-d-old seedlings. Key Results Addition of SNP and NONOate enhanced but cPTIO decreased NR activity in the roots fed with 0·5 mm nitrate. The opposite was true for the roots fed with 5 mm nitrate. However, the mRNA level of the NR gene and the protein concentration of NR enzyme in the roots were not affected by SNP treatment, irrespective of nitrate pre-treatment. Nevertheless, a low rate of NO gas increased while cPTIO decreased the NR activities of the enzyme extracts from the roots at both nitrate levels. Increasing the rate of NO gas further increased NR activity in the enzyme extracts of the roots fed with 0·5 mm nitrate but decreased it when 5 mm nitrate was supplied. Interestingly, the stimulative effect of NO gas on NR activity could be reversed by NO removal through N2 flushing in the enzyme extracts from the roots fed with 0·5 mm nitrate but not from those with 5 mm nitrate. Conclusions The effects of NO on NR activity in tomato roots depend on levels of nitrate supply, and probably result from direct interactions between NO and NR protein. PMID:19376780

  16. Mechanism of reaction of myeloperoxidase with nitrite.

    PubMed

    Burner, U; Furtmuller, P G; Kettle, A J; Koppenol, W H; Obinger, C

    2000-07-07

    Myeloperoxidase (MPO) is a major neutrophil protein and may be involved in the nitration of tyrosine residues observed in a wide range of inflammatory diseases that involve neutrophils and macrophage activation. In order to clarify if nitrite could be a physiological substrate of myeloperoxidase, we investigated the reactions of the ferric enzyme and its redox intermediates, compound I and compound II, with nitrite under pre-steady state conditions by using sequential mixing stopped-flow analysis in the pH range 4-8. At 15 degrees C the rate of formation of the low spin MPO-nitrite complex is (2.5 +/- 0.2) x 10(4) m(-1) s(-1) at pH 7 and (2.2 +/- 0.7) x 10(6) m(-1) s(-1) at pH 5. The dissociation constant of nitrite bound to the native enzyme is 2.3 +/- 0.1 mm at pH 7 and 31.3 +/- 0.5 micrometer at pH 5. Nitrite is oxidized by two one-electron steps in the MPO peroxidase cycle. The second-order rate constant of reduction of compound I to compound II at 15 degrees C is (2.0 +/- 0.2) x 10(6) m(-1) s(-1) at pH 7 and (1.1 +/- 0.2) x 10(7) m(-1) s(-1) at pH 5. The rate constant of reduction of compound II to the ferric native enzyme at 15 degrees C is (5.5 +/- 0.1) x 10(2) m(-1) s(-1) at pH 7 and (8.9 +/- 1.6) x 10(4) m(-1) s(-1) at pH 5. pH dependence studies suggest that both complex formation between the ferric enzyme and nitrite and nitrite oxidation by compounds I and II are controlled by a residue with a pK(a) of (4.3 +/- 0.3). Protonation of this group (which is most likely the distal histidine) is necessary for optimum nitrite binding and oxidation.

  17. Detection and diversity of fungal nitric oxide reductase genes (p450nor) in agricultural soils

    DOE PAGES

    Higgins, Steven A.; Welsh, Allana; Orellana, Luis H.; ...

    2016-03-11

    Members of the Fungi convert nitrate (NO3-) and nitrite (NO2-) to gaseous nitrous oxide (N2O) (denitrification), but the fungal contributions to N-loss from soil remain uncertain. Cultivation-based methodologies that include antibiotics to selectively assess fungal activities have limitations and complementary molecular approaches to assign denitrification potential to fungi are desirable. Microcosms established with soils from two representative U.S. Midwest agricultural regions produced N2O from added NO3- or NO2- in the presence of antibiotics to inhibit bacteria. Cultivation efforts yielded 214 fungal isolates belonging to at least 15 distinct morphological groups, of which 151 produced N2O from NO2-. Novel PCR primersmore » targeting the p450nor gene that encodes the nitric oxide (NO) reductase responsible for N2O production in fungi yielded 26 novel p450nor amplicons from DNA of 37 isolates and 23 amplicons from environmental DNA obtained from two agricultural soils. The sequences shared 54-98% amino acid identity to reference P450nor sequences within the phylum Ascomycota, and expand the known fungal P450nor sequence diversity. p450nor was detected in all fungal isolates that produced N2O from nitrite, whereas nirK (encoding the NO-forming nitrite reductase) was amplified in only 13-74% of the N2O-forming isolates using two separate nirK primer sets. Altogether, our findings demonstrate the value of p450nor-targeted PCR to complement existing approaches to assess the fungal contributions to denitrification and N2O formation.« less

  18. Density functional theory study of model complexes for the revised nitrate reductase active site in Desulfovibrio desulfuricans NapA.

    PubMed

    Hofmann, Matthias

    2009-09-01

    [Mo(SSCH3)(S2C2(CH3)2)2](x) complexes with charges x between -3 and +3 were investigated by density functional theory computations as minimal nitrate reductase active-site models. The strongly reduced species (x = -2, -3) exist preferentially as pentacoordinate sulfo complexes separated from a thiolate anion. The oxidized extremes (x > 0) clearly prefer hexacoordinate complexes with an eta(2)-MeSS ligand. Among the neutral and especially for the singly negatively charged species structures with eta(2)-MeSS and eta(1)-MeSS ligands are energetically close to the sulfo methyl sulfide complex without SS bonding. For x = -1 the three isomers lie in a 1.5 kcal mol(-1) energy range. Putative mechanistic pathways for nitrate reduction from the literature were investigated computationally: (1) reduction at a pentacoordinate sulfo complex, (2) reduction at the ligand, and (3) reduction at the molybdenum center with an R-S-S ligand. All three pathways could be traced at least for some overall charges but no definite conclusion can be drawn about the mechanism. Complexes with larger dithiolato ligands were also computed in order to model the tricyclic metallopterin framework more accurately: the first heterocyclus (5,6-dihydro-2H-pyran) stabilizes the nitrate complex and the molybdenum oxo product complex by approximately 10 kcal mol(-1) and also reduces the activation barrier (by approximately 5 kcal mol(-1)). The effect of the second (1,2,3,4-tetrahydropyrazin) and third heterocyclus (2-amino-3H-pyrimidin-4-one) on the relative energies is relatively small. For bigger models derived from an experimental protein structure, nitrate reduction at a persulfo molybdenum(IV) complex fragment (mechanism 3) is clearly favored over the oxidation of a molybdenum-bound sulfur atom (mechanism 2). Mechanism 1 could not be investigated for the big models but seems the least favorable on the basis of the results from smaller models.

  19. Methyl jasmonate and 1-methylcyclopropene treatment effects on quinone reductase inducing activity and post-harvest quality of broccoli.

    PubMed

    Ku, Kang Mo; Choi, Jeong Hee; Kim, Hyoung Seok; Kushad, Mosbah M; Jeffery, Elizabeth H; Juvik, John A

    2013-01-01

    Effect of pre-harvest methyl jasmonate (MeJA) and post-harvest 1-methylcyclopropene (1-MCP) treatments on broccoli floret glucosinolate (GS) concentrations and quinone reductase (QR, an in vitro anti-cancer biomarker) inducing activity were evaluated two days prior to harvest, at harvest and at 10, 20, and 30 days of post-harvest storage at 4 °C. MeJA treatments four days prior to harvest of broccoli heads was observed to significantly increase floret ethylene biosynthesis resulting in chlorophyll catabolism during post-harvest storage and reduced product quality. Post-harvest treatment with 1-methylcyclopropene (1-MCP), which competitively binds to protein ethylene receptors, maintained post-harvest floret chlorophyll concentrations and product visual quality in both control and MeJA-treated broccoli. Transcript abundance of BoPPH, a gene which is responsible for the synthesis of pheophytinase, the primary enzyme associated with chlorophyll catabolism in broccoli, was reduced by 1-MCP treatment and showed a significant, negative correlation with floret chlorophyll concentrations. The GS, glucobrassicin, neoglucobrassicin, and gluconasturtiin were significantly increased by MeJA treatments. The products of some of the GS from endogenous myrosinase hydrolysis [sulforaphane (SF), neoascorbigen (NeoASG), N-methoxyindole-3-carbinol (NI3C), and phenethyl isothiocyanate (PEITC)] were also quantified and found to be significantly correlated with QR. Sulforaphane, the isothiocyanate hydrolysis product of the GS glucoraphanin, was found to be the most potent QR induction agent. Increased sulforaphane formation from the hydrolysis of glucoraphanin was associated with up-regulated gene expression of myrosinase (BoMyo) and the myrosinase enzyme co-factor gene, epithiospecifier modifier1 (BoESM1). This study demonstrates the combined treatment of MeJA and 1-MCP increased QR activity without post-harvest quality loss.

  20. Detoxification of superoxide without production of H2O2: Antioxidant activity of superoxide reductase complexed with ferrocyanide

    PubMed Central

    Molina-Heredia, Fernando P.; Houée-Levin, Chantal; Berthomieu, Catherine; Touati, Danièle; Tremey, Emilie; Favaudon, Vincent; Adam, Virgile; Nivière, Vincent

    2006-01-01

    The superoxide radical O2·̅ is a toxic by-product of oxygen metabolism. Two O2·̅ detoxifying enzymes have been described so far, superoxide dismutase and superoxide reductase (SOR), both forming H2O2 as a reaction product. Recently, the SOR active site, a ferrous iron in a [Fe2+ (N-His)4 (S-Cys)] pentacoordination, was shown to have the ability to form a complex with the organometallic compound ferrocyanide. Here, we have investigated in detail the reactivity of the SOR–ferrocyanide complex with O2·̅ by pulse and γ-ray radiolysis, infrared, and UV-visible spectroscopies. The complex reacts very efficiently with O2·̅. However, the presence of the ferrocyanide adduct markedly modifies the reaction mechanism of SOR, with the formation of transient intermediates different from those observed for SOR alone. A one-electron redox chemistry appears to be carried out by the ferrocyanide moiety of the complex, whereas the SOR iron site remains in the reduced state. Surprisingly, the toxic H2O2 species is no longer the reaction product. Accordingly, in vivoexperiments showed that formation of the SOR–ferrocyanide complex increased the antioxidant capabilities of SOR expressed in an Escherichia coli sodA sodB recA mutant strain. Altogether, these data describe an unprecedented O2·̅ detoxification activity, catalyzed by the SOR–ferrocyanide complex, which does not conduct to the production of the toxic H2O2 species. PMID:17001016

  1. The P aracoccus denitrificans NarK‐like nitrate and nitrite transporters—probing nitrate uptake and nitrate/nitrite exchange mechanisms

    PubMed Central

    Goddard, Alan D.; Bali, Shilpa; Mavridou, Despoina A.I.; Luque‐Almagro, Victor M.; Gates, Andrew J.; Dolores Roldán, M.; Newstead, Simon; Richardson, David J.

    2016-01-01

    Summary Nitrate and nitrite transport across biological membranes is often facilitated by protein transporters that are members of the major facilitator superfamily. Paracoccus denitrificans contains an unusual arrangement whereby two of these transporters, NarK1 and NarK2, are fused into a single protein, NarK, which delivers nitrate to the respiratory nitrate reductase and transfers the product, nitrite, to the periplasm. Our complementation studies, using a mutant lacking the nitrate/proton symporter NasA from the assimilatory nitrate reductase pathway, support that NarK1 functions as a nitrate/proton symporter while NarK2 is a nitrate/nitrite antiporter. Through the same experimental system, we find that Escherichia coli NarK and NarU can complement deletions in both narK and nasA in P. denitrificans, suggesting that, while these proteins are most likely nitrate/nitrite antiporters, they can also act in the net uptake of nitrate. Finally, we argue that primary sequence analysis and structural modelling do not readily explain why NasA, NarK1 and NarK2, as well as other transporters from this protein family, have such different functions, ranging from net nitrate uptake to nitrate/nitrite exchange. PMID:27696579

  2. In Vitro Screening for β-Hydroxy-β-methylglutaryl-CoA Reductase Inhibitory and Antioxidant Activity of Sequentially Extracted Fractions of Ficus palmata Forsk

    PubMed Central

    Iqbal, Danish; Khan, M. Salman; Khan, Amir; Khan, Mohd. Sajid; Srivastava, Ashwani K.; Bagga, Paramdeep

    2014-01-01

    Hypercholesterolemia-induced oxidative stress has been strongly implicated in the pathogenesis of atherosclerosis, which is one of the major causes of mortality worldwide. The current work, for the first time, accounts the antioxidant, genoprotective, antilipoperoxidative, and HMG-CoA reductase (EC 1.1.1.34) inhibitory properties of traditional medicinal plant, Ficus palmata Forsk. Our result showed that among sequentially extracted fractions of Ficus palmata Forsk, FPBA (F. palmata bark aqueous extract) and FPLM (F. palmata leaves methanolic extract) extracts have higher phenolic content and also exhibited significantly more radical scavenging (DPPH and Superoxide) and antioxidant (FRAP) capacity. Moreover, FPBA extract also exhibited significantly higher inhibition of lipid peroxidation assay. Additionally, results showed almost complete and partial protection of oxidatively damaged DNA by these plant extracts when compared to mannitol. Furthermore, our results showed that FPBA extract (IC50 = 9.1 ± 0.61 µg/mL) exhibited noteworthy inhibition of HMG-CoA reductase activity as compared to other extracts, which might suggest its role as cardioprotective agent. In conclusion, results showed that FPBA extract not only possess significant antioxidant and genoprotective property but also is able to attenuate the enzymatic activity of HMG-CoA reductase, which might suggest its role in combating various oxidative stress-related diseases, including atherosclerosis. PMID:24883325

  3. Enhancing activity and selectivity in a series of pyrrol-1-yl-1-hydroxypyrazole-based aldose reductase inhibitors: The case of trifluoroacetylation.

    PubMed

    Papastavrou, Nikolaos; Chatzopoulou, Maria; Ballekova, Jana; Cappiello, Mario; Moschini, Roberta; Balestri, Francesco; Patsilinakos, Alexandros; Ragno, Rino; Stefek, Milan; Nicolaou, Ioannis

    2017-04-21

    Aldose reductase (ALR2) has been the target of therapeutic intervention for over 40 years; first, for its role in long-term diabetic complications and more recently as a key mediator in inflammation and cancer. However, efforts to prepare small-molecule aldose reductase inhibitors (ARIs) have mostly yielded carboxylic acids with rather poor pharmacokinetics. To address this limitation, the 1-hydroxypyrazole moiety has been previously established as a bioisostere of acetic acid in a group of aroyl-substituted pyrrolyl derivatives. In the present work, optimization of this new class of ARIs was achieved by the addition of a trifluoroacetyl group on the pyrrole ring. Eight novel compounds were synthesized and tested for their inhibitory activity towards ALR2 and selectivity against aldehyde reductase (ALR1). All compounds proved potent and selective inhibitors of ALR2 (IC50/ALR2 = 0.043-0.242 μΜ, Selectivity index = 190-858), whilst retaining a favorable physicochemical profile. The most active (4g) and selective (4d) compounds were further evaluated for their ability to inhibit sorbitol formation in rat lenses ex vivo and to exhibit substrate-specific inhibition.

  4. Dissecting the Structural Elements for the Activation of β-Ketoacyl-(Acyl Carrier Protein) Reductase from Vibrio cholerae

    PubMed Central

    Hou, Jing; Zheng, Heping; Chruszcz, Maksymilian; Zimmerman, Matthew D.; Shumilin, Igor A.; Osinski, Tomasz; Demas, Matt; Grimshaw, Sarah

    2015-01-01

    ABSTRACT β-Ketoacyl-(acyl carrier protein) reductase (FabG) catalyzes the key reductive reaction in the elongation cycle of fatty acid synthesis (FAS), which is a vital metabolic pathway in bacteria and a promising target for new antibiotic development. The activation of the enzyme is usually linked to the formation of a catalytic triad and cofactor binding, and crystal structures of FabG from different organisms have been captured in either the active or inactive conformation. However, the structural elements which enable activation of FabG require further exploration. Here we report the findings of structural, enzymatic, and binding studies of the FabG protein found in the causative agent of cholera, Vibrio cholerae (vcFabG). vcFabG exists predominantly as a dimer in solution and is able to self-associate to form tetramers, which is the state seen in the crystal structure. The formation of the tetramer may be promoted by the presence of the cofactor NADP(H). The transition between the dimeric and tetrameric states of vcFabG is related to changes in the conformations of the α4/α5 helices on the dimer-dimer interface. Two glycine residues adjacent to the dimer interface (G92 and G141) are identified to be the hinge for the conformational changes, while the catalytic tyrosine (Y155) and a glutamine residue that forms hydrogen bonds to both loop β4-α4 and loop β5-α5 (Q152) stabilize the active conformation. The functions of the aforementioned residues were confirmed by binding and enzymatic assays for the corresponding mutants. IMPORTANCE This paper describes the results of structural, enzymatic, and binding studies of FabG from Vibrio cholerae (vcFabG). In this work, we dissected the structural elements responsible for the activation of vcFabG. The structural information provided here is essential for the development of antibiotics specifically targeting bacterial FabG, especially for the multidrug-resistant strains of V. cholerae. PMID:26553852

  5. Enzymatic activity of coenzyme B(12) derivatives with altered axial nucleotides: probing the mechanochemical triggering hypothesis in ribonucleotide reductase.

    PubMed

    Brown, K L; Zou, X; Li, J; Chen, G

    2001-11-05

    Theoretical studies (J. Inorg. Biochem. 2001, 83, 121) of the involvement of the bulky 5,6-dimethylbenzimidazole (Dmbz) ligand of coenzyme B(12) (5'-deoxyadenosylcobalamin, AdoCbl) in the mechanism of activation of the carbon-cobalt bond of the coenzyme for homolytic cleavage by AdoCbl-dependent enzymes (the "mechanochemical triggering" mechanisms) have shown that a purely steric, ground-state mechanism can supply only a few kilocalories per mole (of the observed 13-16 kcal mol(-1)) of activation, but that an electronic mechanism, operating to stabilize the transition state, can explain all of the observed catalytic effect. To address these mechanisms experimentally, analogues of AdoCbl in which the Dmbz ligand is replaced by benzimidazole (Ado(Bzim)Cbl) or by imidazole (Ado(Im)Cbl) have been prepared and characterized. Both of these analogues support turnover in the AdoCbl-dependent ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii at 100% of the activity of AdoCbl itself, but the Ado(Im)Cbl analogue has a significantly higher K(m). 5'-Deoxyadenosylcobinamide, the analogue in which the axial nucleotide has been chemically removed, in contrast, is inactive in the spectrophotometric assay, which indicates that it has at most 1% of the activity of AdoCbl. Stopped-flow spectrophotometric measurements of the formation of cob(II)alamin at the enzyme active site show that RTPR binds Ado(Bzim)Cbl slightly more weakly than it does AdoCbl, but binds Ado(Im)Cbl 8-fold more weakly. While the equilibrium constant for cob(II)alamin formation is nearly the same for Ado(Bzim)Cbl and AdoCbl, it is 5-fold smaller for Ado(Im)Cbl. Finally, the forward rate constant for enzyme-induced Co-C bond homolysis was about the same for Ado(Bzim)Cbl and for AdoCbl but was 17-fold smaller for Ado(Im)Cbl. These results are consistent with a small contribution from ground-state mechanochemical triggering, but they do not in themselves rule out transition-state mechanical

  6. Syringic Acid Extracted from Herba dendrobii Prevents Diabetic Cataract Pathogenesis by Inhibiting Aldose Reductase Activity

    PubMed Central

    Wei, Xiaoyong; Chen, Dan; Yi, Yanchun; Qi, Hui; Gao, Xinxin; Fang, Hua; Gu, Qiong; Wang, Ling; Gu, Lianquan

    2012-01-01

    Objective. Effects of Syringic acid (SA) extracted from dendrobii on diabetic cataract (DC) pathogenesis were explored. Methods. Both in vitro and in vivo DC lens models were established using D-gal, and proliferation of HLEC exposed to SA was determined by MMT assay. After 60-day treatment with SA, rat lens transparency was observed by anatomical microscopy using a slit lamp. SA protein targets were extracted and isolated using 2-DE and MALDI TOF/TOF. AR gene expression was investigated using qRT-PCR. Interaction sites and binding characteristics were determined by molecule-docking techniques and dynamic models. Results. Targeting AR, SA provided protection from D-gal-induced damage by consistently maintaining lens transparency and delaying lens turbidity development. Inhibition of AR gene expression by SA was confirmed by qRT-PCR. IC50 of SA for inhibition of AR activity was 213.17 μg/mL. AR-SA binding sites were Trp111, His110, Tyr48, Trp20, Trp79, Leu300, and Phe122. The main binding modes involved hydrophobic interactions and hydrogen bonding. The stoichiometric ratio of non-covalent bonding between SA and AR was 1.0 to 13.3. Conclusion. SA acts to prevent DC in rat lenses by inhibiting AR activity and gene expression, which has potential to be developed into a novel drug for therapeutic management of DC. PMID:23365598

  7. The peroxidase and peroxynitrite reductase activity of human erythrocyte peroxiredoxin 2.

    PubMed

    Manta, Bruno; Hugo, Martín; Ortiz, Cecilia; Ferrer-Sueta, Gerardo; Trujillo, Madia; Denicola, Ana

    2009-04-15

    Peroxiredoxin 2 (Prx2) is a 2-Cys peroxiredoxin extremely abundant in the erythrocyte. The peroxidase activity was studied in a steady-state approach yielding an apparent K(M) of 2.4 microM for human thioredoxin and a very low K(M) for H2O2 (0.7 microM). Rate constants for the reaction of peroxidatic cysteine with the peroxide substrate, H2O2 or peroxynitrite, were determined by competition kinetics, k(2) = 1.0 x 10(8) and 1.4 x 10(7) M(-1) s(-1) at 25 degrees C and pH 7.4, respectively. Excess of both oxidants inactivated the enzyme by overoxidation and also tyrosine nitration and dityrosine were observed with peroxynitrite treatment. Prx2 associates into decamers (5 homodimers) and we estimated a dissociation constant K(d) < 10(-23) M(4) which confirms the enzyme exists as a decamer in vivo. Our kinetic results indicate Prx2 is a key antioxidant enzyme for the erythrocyte and reveal red blood cells as active oxidant scrubbers in the bloodstream.

  8. Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1.

    PubMed

    Randall, Matthew J; Spiess, Page C; Hristova, Milena; Hondal, Robert J; van der Vliet, Albert

    2013-01-01

    Cigarette smoking remains a major health concern worldwide, and many of the adverse effects of cigarette smoke (CS) can be attributed to its abundant electrophilic aldehydes, such as acrolein (2-propenal). Previous studies indicate that acrolein readily reacts with thioredoxin reductase 1 (TrxR1), a critical enzyme involved in regulation of thioredoxin (Trx)-mediated redox signaling, by alkylation at its selenocysteine (Sec) residue. Because alkylation of Sec within TrxR1 has significant implications for its enzymatic function, we explored the potential importance of TrxR1 alkylation in acrolein-induced activation or injury of bronchial epithelial cells. Exposure of human bronchial epithelial HBE1 cells to acrolein (1-30 μM) resulted in dose-dependent loss of TrxR thioredoxin reductase activity, which coincided with its alkylation, as determined by biotin hydrazide labeling, and was independent of initial GSH status. To test the involvement of TrxR1 in acrolein responses in HBE1 cells, we suppressed TrxR1 using siRNA silencing or augmented TrxR1 by cell supplementation with sodium selenite. Acrolein exposure of HBE1 cells induced dose-dependent activation of the MAP kinases, extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, and activation of JNK was markedly enhanced after selenite-mediated induction of TrxR1, and was associated with increased alkylation of TrxR1. Conversely, siRNA silencing of TrxR1 significantly suppressed the ability of acrolein to activate JNK, and also appeared to attenuate acrolein-dependent activation of ERK and p38. Alteration of initial TrxR1 levels by siRNA or selenite supplementation also affected initial Trx1 redox status and acrolein-mediated alkylation of Trx1, but did not significantly affect acrolein-mediated activation of HO-1 or cytotoxicity. Collectively, our findings indicate that alkylation of TrxR1 and/or Trx1 may contribute directly to acrolein-mediated activation of MAP kinases such as JNK, and

  9. Electron transport to periplasmic nitrate reductase (NapA) of Wolinella succinogenes is independent of a NapC protein.

    PubMed

    Simon, Jörg; Sänger, Monica; Schuster, Stephan C; Gross, Roland

    2003-07-01

    The rumen bacterium Wolinella succinogenes grows by respiratory nitrate ammonification with formate as electron donor. Whereas the enzymology and coupling mechanism of nitrite respiration is well known, nitrate reduction to nitrite has not yet been examined. We report here that intact cells and cell fractions catalyse nitrate and chlorate reduction by reduced viologen dyes with high specific activities. A gene cluster encoding components of a putative periplasmic nitrate reductase system (napA, G, H, B, F, L, D) was sequenced. The napA gene was inactivated by inserting a kanamycin resistance gene cassette. The resulting mutant did not grow by nitrate respiration and did not reduce nitrate during growth by fumarate respiration, in contrast to the wild type. An antigen was detected in wild-type cells using an antiserum raised against the periplasmic nitrate reductase (NapA) from Paracoccus pantotrophus. This antigen was absent in the W. succinogenes napA mutant. It is concluded that the periplasmic nitrate reductase NapA is the only respiratory nitrate reductase in W. succinogenes, although a second nitrate-reducing enzyme is apparently induced in the napA mutant. The nap cluster of W. succinogenes lacks a napC gene whose product is thought to function in quinol oxidation and electron transfer to NapA in other bacteria. The W. succinogenes genome encodes two members of the NapC/NirT family, NrfH and FccC. Characterization of corresponding deletion mutants indicates that neither of these two proteins is required for nitrate respiration. A mutant lacking the genes encoding respiratory nitrite reductase (nrfHA) had wild-type properties with respect to nitrate respiration. A model of the electron transport chain of nitrate respiration is proposed in which one or more of the napF, G, H and L gene products mediate electron transport from menaquinol to the periplasmic NapAB complex. Inspection of the W. succinogenes genome sequence suggests that ammonia formation from

  10. Molybdate-dependent expression of the periplasmic nitrate reductase in Bradyrhizobium japonicum.

    PubMed

    Bonnard, N; Tresierra-Ayala, A; Bedmar, E J; Delgado, M J

    2005-02-01

    The napEDABC genes of Bradyrhizobium japonicum encode the periplasmic nitrate reductase, an Mo-containing enzyme which catalyses the reduction of nitrate to nitrite when oxygen concentrations are limiting. In this bacterium, another set of genes, modABC, code for a high affinity ABC-type Mo transport system. A B. japonicum modA mutant has been obtained that is not capable of growing anaerobically with nitrate and lacks nitrate reductase activity. Under nitrate respiring conditions, when Mo concentrations are limiting, the B. japonicum modA mutant lacked both the 90 kDa protein corresponding to the NapA component of the periplasmic nitrate reductase, and the membrane-bound 25 kDa c-type cytochrome NapC. Regulatory studies using a napE-lacZ fusion indicated that napE expression was highly reduced in the modA mutant background when the cells were incubated anaerobically with nitrate under Mo-deficient conditions.

  11. Convenient synthesis of deazaflavin cofactor FO and its activity in F(420)-dependent NADP reductase.

    PubMed

    Hossain, Mohammad S; Le, Cuong Q; Joseph, Ebenezer; Nguyen, Toan Q; Johnson-Winters, Kayunta; Foss, Frank W

    2015-05-14

    F420 and FO are phenolic 5-deazaflavin cofactors that complement nicotinamide and flavin redox coenzymes in biochemical oxidoreductases and photocatalytic systems. Specifically, these 5-deazaflavins lack the single electron reactivity with O2 of riboflavin-derived coenzymes (FMN and FAD), and, in general, have a more negative redox potential than NAD(P)(+). For example, F420-dependent NADP(+) oxidoreductase (Fno) is critical to the conversion of CO2 to CH4 by methanogenic archaea, while FO functions as a light-harvesting agent in DNA repair. The preparation of these cofactors is an obstacle to their use in biochemical studies and biotechnology. Here, a convenient synthesis of FO was achieved by improving the redox stability of synthetic intermediates containing a polar, electron-rich aminophenol fragment. Improved yields and simplified purification techniques for FO are described. Additionally, Fno activity was restored with FO in the absence of F420. Investigating the FO-dependent NADP(+)/NADPH redox process by stopped-flow spectrophotometry, steady state kinetics were defined as having a Km of 4.00 ± 0.39 μM and a kcat of 5.27 ± 0.14 s(-1). The preparation of FO should enable future biochemical studies and novel uses of F420 mimics.

  12. Comparative bioavailability of ammonium, nitrate, nitrite and urea to typically harmful cyanobacterium Microcystis aeruginosa.

    PubMed

    Li, Jihua; Zhang, Jibiao; Huang, Wei; Kong, Fanlong; Li, Yue; Xi, Min; Zheng, Zheng

    2016-09-15

    Phosphorus is generally considered as the prime limiting nutrient responsible for cyanobacterial blooms. However, recent research is drawing attention to the importance of bioavailable nitrogen (N) in freshwater eutrophication. This study investigated the bioavailability of NO3(-)-N, NO2(-)-N, NH4(+)-N and Urea-N under different concentrations of 1.2, 3.6 and 6.0mgL(-1) to Microcystis aeruginosa. Overall, Urea-N ranked the first in promoting M. aeruginosa growth, followed by NO3(-)-N and NO2(-)-N. However, the algal growth cultured in NH4(+)-N was depressed under test N levels. The bioavailability of N to M. aeruginosa was seriously influenced by both N forms and N concentrations (p<0.01). Total N concentrations in Urea-N treatment decreased the fastest, which were corresponding with the μ values of M. aeruginosa. The high enzymic activities of nitrate reductase, nitrite reductase and glutamine synthetase indicated that the decomposition process for urea is effective, which contributed in N assimilation and utilization in M. aeruginosa cells.

  13. The Structure of Glycerol Trinitrate Reductase NerA from Agrobacterium radiobacter Reveals the Molecular Reason for Nitro- and Ene-Reductase Activity in OYE Homologues

    PubMed Central

    Oberdorfer, Gustav; Binter, Alexandra; Wallner, Silvia; Durchschein, Katharina; Hall, Mélanie; Faber, Kurt; Macheroux, Peter; Gruber, Karl

    2013-01-01

    In recent years, Old Yellow Enzymes (OYEs) and their homologues have found broad application in the efficient asymmetric hydrogenation of activated C=C bonds with high selectivities and yields. Members of this class of enzymes have been found in many different organisms and are rather diverse on the sequence level, with pairwise identities as low as 20 %, but they exhibit significant structural similarities with the adoption of a conserved (αβ)8-barrel fold. Some OYEs have been shown not only to reduce C=C double bonds, but also to be capable of reducing nitro groups in both saturated and unsaturated substrates. In order to understand this dual activity we determined and analyzed X-ray crystal structures of NerA from Agrobacterium radiobacter, both in its apo form and in complex with 4-hydroxybenzaldehyde and with 1-nitro-2-phenylpropene. These structures, together with spectroscopic studies of substrate binding to several OYEs, indicate that nitro-containing substrates can bind to OYEs in different binding modes, one of which leads to C=C double bond reduction and the other to nitro group reduction. PMID:23606302

  14. Zeatin reductase in Phaseolus embryos

    SciTech Connect

    Martin, R.C.; Mok, David, W.S.; Mok, M.C. )

    1989-04-01

    Zeatin was converted to O-xylosylzeatin in embryos of Phaseolus vulgaris . O-xylosyldihydrozeatin was also identified as a zeatin metabolite. Incubation of embryo extracts with {sup 14}C-zeatin and {sup 14}C-O-xylosylzeatin revealed that reduction preceeds the O-xylosylation of zeatin. An enzyme responsible for reducing the N{sup 6}-side chain was isolated and partially purified using ammonium sulfate fractionation and affinity, gel filtration and anion exchange chromatography. The NADPH dependent reductase was zeatin specific and did not recognize cis-zeatin, ribosylzeatin, i{sup 6}Ade or i{sup 6}Ado. Two forms of the reductase could be separated by either gel filtration or anion exchange HPLC. The HMW isozyme (Mr. 55,000) eluted from the anion exchange column later than the LMW isozyme (Mr. 25,000). Interspecific differences in zeatin reductase activity were also detected.

  15. Synthesis and activity of novel 16-dehydropregnenolone acetate derivatives as inhibitors of type 1 5α-reductase and on cancer cell line SK-LU-1.

    PubMed

    Silva-Ortiz, Aylin Viviana; Bratoeff, Eugene; Ramírez-Apan, Teresa; Heuze, Yvonne; Sánchez, Araceli; Soriano, Juan; Cabeza, Marisa

    2015-12-15

    Testosterone (T) plays a crucial role in prostate growth. In androgen-dependent tissues T is reduced to dihydrotestosterone (DHT) because of the presence of the 5α-reductase enzyme. This androgen is more active than T, since it has a higher affinity for the androgen receptor (AR). When this mechanism is altered, androgen-dependent diseases, including prostate cancer, could result. The aim of this study was to synthesize several 16-dehydropregnenolone acetate derivatives containing a triazole ring at C-21 and a linear or alicyclic ester moiety at C-3 of the steroidal skeleton. These steroids were designed as potential inhibitors of the activity of both types (1 and 2) of 5α-reductase. The cytotoxic activity of these compounds was also evaluated on a panel of PC-3, MCF7, and SK-LU-1 human cancer cell lines. The results from this study showed that with the exception of steroids 20-oxo-21-(1H-1,2,4-triazole-1-yl)pregna-5,16-dien-3β-yl-propionate and 20-oxo-21-(1H-1,2,4-triazole-1-yl)pregna-5,16-dien-3β-yl-pentanoate, the compounds exhibit a lower inhibitory activity for both isoenzymes of 5α-reductase than finasteride. Furthermore the 3β-hydroxy-21-(1H-1,2,4-triazole-1-yl)pregna-5,16-dien-20-one and 20-oxo-21-(1H-1,2,4-triazole-1-yl)pregna-5,16-dien-3β-yl-acetate derivatives display 80% cytotoxic activity on the SK-LU-1 cell line. These results also indicated that the triazole derivatives, which have a hydroxyl or acetoxy group at C-3, could have an anticancer effect, whereas the derivatives with a alicyclic ester group at C-3 do not show biological activity.

  16. Mercury (II) removal by resistant bacterial isolates and mercuric (II) reductase activity in a new strain of Pseudomonas sp. B50A.

    PubMed

    Giovanella, Patricia; Cabral, Lucélia; Bento, Fátima Menezes; Gianello, Clesio; Camargo, Flávio Anastácio Oliveira

    2016-01-25

    This study aimed to isolate mercury resistant bacteria, determine the minimum inhibitory concentration for Hg, estimate mercury removal by selected isolates, explore the mer genes, and detect and characterize the activity of the enzyme mercuric (II) reductase produced by a new strain of Pseudomonas sp. B50A. The Hg removal capacity of the isolates was determined by incubating the isolates in Luria Bertani broth and the remaining mercury quantified by atomic absorption spectrophotometry. A PCR reaction was carried out to detect the merA gene and the mercury (II) reductase activity was determined in a spectrophotometer at 340 nm. Eight Gram-negative bacterial isolates were resistant to high mercury concentrations and capable of removing mercury, and of these, five were positive for the gene merA. The isolate Pseudomonas sp. B50A removed 86% of the mercury present in the culture medium and was chosen for further analysis of its enzyme activity. Mercuric (II) reductase activity was detected in the crude extract of this strain. This enzyme showed optimal activity at pH 8 and at temperatures between 37 °C and 45 °C. The ions NH4(+), Ba(2+), Sn(2+), Ni(2+) and Cd(2+) neither inhibited nor stimulated the enzyme activity but it decreased in the presence of the ions Ca(2+), Cu(+) and K(+). The isolate and the enzyme detected were effective in reducing Hg(II) to Hg(0), showing the potential to develop bioremediation technologies and processes to clean-up the environment and waste contaminated with mercury.

  17. Crosstalk between nitrite, myoglobin and reactive oxygen species to regulate vasodilation under hypoxia.

    PubMed

    Totzeck, Matthias; Hendgen-Cotta, Ulrike B; Kelm, Malte; Rassaf, Tienush

    2014-01-01

    The systemic response to decreasing oxygen levels is hypoxic vasodilation. While this mechanism has been known for more than a century, the underlying cellular events have remained incompletely understood. Nitrite signaling is critically involved in vessel relaxation under hypoxia. This can be attributed to the presence of myoglobin in the vessel wall together with other potential nitrite reductases, which generate nitric oxide, one of the most potent vasodilatory signaling molecules. Questions remain relating to the precise concentration of nitrite and the exact dose-response relations between nitrite and myoglobin under hypoxia. It is furthermore unclear whether regulatory mechanisms exist which balance this interaction. Nitrite tissue levels were similar across all species investigated. We then investigated the exact fractional myoglobin desaturation in an ex vivo approach when gassing with 1% oxygen. Within a short time frame myoglobin desaturated to 58±12%. Given that myoglobin significantly contributes to nitrite reduction under hypoxia, dose-response experiments using physiological to pharmacological nitrite concentrations were conducted. Along all concentrations, abrogation of myoglobin in mice impaired vasodilation. As reactive oxygen species may counteract the vasodilatory response, we used superoxide dismutase and its mimic tempol as well as catalase and ebselen to reduce the levels of reactive oxygen species during hypoxic vasodilation. Incubation of tempol in conjunction with catalase alone and catalase/ebselen increased the vasodilatory response to nitrite. Our study shows that modest hypoxia leads to a significant nitrite-dependent vessel relaxation. This requires the presence of vascular myoglobin for both physiological and pharmacological nitrite levels. Reactive oxygen species, in turn, modulate this vasodilation response.

  18. Novel synthetic inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity that inhibit tumor cell proliferation and are structurally unrelated to existing statins.

    PubMed

    Perchellet, Jean-Pierre H; Perchellet, Elisabeth M; Crow, Kyle R; Buszek, Keith R; Brown, Neil; Ellappan, Sampathkumar; Gao, Ge; Luo, Diheng; Minatoya, Machiko; Lushington, Gerald H

    2009-11-01

    Pilot-scale libraries of eight-membered medium ring lactams (MRLs) and related tricyclic compounds (either seven-membered lactams, thiolactams or amines) were screened for their ability to inhibit the catalytic activity of human recombinant 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase in vitro. A dozen of the synthetic compounds mimic the inhibition of purified HMG-CoA reductase activity caused by pravastatin, fluvastatin and sodium salts of lovastatin, mevastatin and simvastatin in this cell-free assay, suggesting direct interaction with the rate-limiting enzyme of cholesterol biosynthesis. Moreover, several MRLs inhibit the metabolic activity of L1210 tumor cells in vitro to a greater degree than fluvastatin, lovastatin, mevastatin and simvastatin, whereas pravastatin is inactive. Although the correlation between the concentration-dependent inhibitions of HMG-CoA reductase activity over 10 min in the cell-free assay and L1210 tumor cell proliferation over 4 days in culture is unclear, some bioactive MRLs elicit interesting combinations of statin-like (IC50: 7.4-8.0 microM) and anti-tumor (IC50: 1.4-2.3 microM) activities. The HMG-CoA reductase-inhibiting activities of pravastatin and an MRL persist in the presence of increasing concentrations of NADPH. But increasing concentrations of HMG-CoA block the HMG-CoA reductase-inhibiting activity of pravastatin without altering that of an MRL, suggesting that MRLs and existing statins may have different mechanisms of enzyme interaction and inhibition. When tested together, suboptimal concentrations of synthetic MRLs and existing statins have additive inhibitory effects on HMG-CoA reductase activity. Preliminary molecular docking studies with MRL-based inhibitors indicate that these ligands fit sterically well into the HMG-CoA reductase statin-binding receptor model and, in contrast to mevastatin, may occupy a narrow channel housing the pyridinium moiety on NADP+.

  19. [Nitrogenase, hydrogenase and nitrate reductase activities, oxygen consumption, and ATP content in nodules formed by strains of Rhizobium leguminosarum 128C53 and 300 in symbiosis with pea plants].

    PubMed

    Bedmar, E J; Olivares, J

    1986-10-01

    The nitrogenase activity, nitrate reductase activity and oxygen uptake as well as the hydrogen incorporation and ATP content were examined in the root nodules and bacteroids, respectively, formed by Rhizobium leguminosarum strains 128C53 (hydrogenase positive) and 300 (hydrogenase negative) in symbiosis with Pisum sativum plants grown in the presence of 2 mM KNO3. The strain 128C53 showed the greatest values for all parameters analyzed, except for the nitrate reductase activity, which was higher for the strain 300. Similarly, nodule nitrate reductase activity in strain 300 was greater than that in strain 128C53 when plants grew in the absence of combined nitrogen. In general, the highest values were obtained when determinations were made after 7 hours of plant illumination. However, the hydrogenase activity of strain 128C53 and the nitrate reductase activities of both strains increased with the light period, reaching a maximum after 14 hours of illumination. These results suggest that the benefits derived from the superior symbiotic properties and from the presence of hydrogenase activity in strain 128C53 could be counteracted by the higher rates of the nodule nitrate reductase activity in strain 300.

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

    PubMed

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

    1996-01-01

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

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

    PubMed Central

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

    1996-01-01

    we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions. PMID:8597660

  2. Pharmacologically relevant receptor binding characteristics and 5alpha-reductase inhibitory activity of free Fatty acids contained in saw palmetto extract.

    PubMed

    Abe, Masayuki; Ito, Yoshihiko; Oyunzul, Luvsandorj; Oki-Fujino, Tomomi; Yamada, Shizuo

    2009-04-01

    Saw palmetto extract (SPE), used widely for the treatment of benign prostatic hyperplasia (BPH) has been shown to bind alpha(1)-adrenergic, muscarinic and 1,4-dihydropyridine (1,4-DHP) calcium channel antagonist receptors. Major constituents of SPE are lauric acid, oleic acid, myristic acid, palmitic acid and linoleic acid. The aim of this study was to investigate binding affinities of these fatty acids for pharmacologically relevant (alpha(1)-adrenergic, muscarinic and 1,4-DHP) receptors. The fatty acids inhibited specific [(3)H]prazosin binding in rat brain in a concentration-dependent manner with IC(50) values of 23.8 to 136 microg/ml, and specific (+)-[(3)H]PN 200-110 binding with IC(50) values of 24.5 to 79.5 microg/ml. Also, lauric acid, oleic acid, myristic acid and linoleic acid inhibited specific [(3)H]N-methylscopolamine ([(3)H]NMS) binding in rat brain with IC(50) values of 56.4 to 169 microg/ml. Palmitic acid had no effect on specific [(3)H]NMS binding. The affinity of oleic acid, myristic acid and linoleic acid for each receptor was greater than the affinity of SPE. Scatchard analysis revealed that oleic acid and lauric acid caused a significant decrease in the maximal number of binding sites (B(max)) for [(3)H]prazosin, [(3)H]NMS and (+)-[(3)H]PN 200-110. The results suggest that lauric acid and oleic acid bind noncompetitively to alpha(1)-adrenergic, muscarinic and 1,4-DHP calcium channel antagonist receptors. We developed a novel and convenient method of determining 5alpha-reductase activity using LC/MS. With this method, SPE was shown to inhibit 5alpha-reductase activity in rat liver with an IC(50) of 101 microg/ml. Similarly, all the fatty acids except palmitic acid inhibited 5alpha-reductase activity, with IC(50) values of 42.1 to 67.6 microg/ml. In conclusion, lauric acid, oleic acid, myristic acid, and linoleic acid, major constituents of SPE, exerted binding activities of alpha(1)-adrenergic, muscarinic and 1,4-DHP receptors and inhibited 5

  3. Microbial community and metabolism activity in a bioelectrochemical denitrification system under long-term presence of p-nitrophenol.

    PubMed

    Chen, Dan; Yang, Kai; Wei, Li; Wang, Hongyu

    2016-10-01

    Bioelectrochemical denitrification system (BEDS) is a promising technology for nitrate removal from wastewaters. The hazards and effects concerning p-nitrophenol (PNP) towards BEDS lack enough investigations and possess great research prospects. This study investigated how PNP affected the nitrate removal efficiency, microbial communities, functional denitrifying genes abundances, nitrate and nitrite reductase activities, diffusible signal factors (DSF) release, and extracellular polymeric substances (EPS) production in the BEDS. Results indicated that nitrate removal efficiency decreased with initial PNP concentration increased from 0 to 100mg/L. Phylum Firmicutes and class Clostridia were the main contributors for denitrification process in this BEDS. The abundances of the denitrifying genes nirS, nirK, napA, and narG all presented decreased trends with increasing PNP. In addition, the concentrations of nitrate reductase (NR), nitrite reductase (NIR), and EPS obviously decreased, while the concentration of DSF increased with increasing PNP, which demonstrated that higher PNP would inhibit the biofilm formation.

  4. Nitrates and nitrites in the treatment of ischemic cardiac disease.

    PubMed

    Nossaman, Vaughn E; Nossaman, Bobby D; Kadowitz, Philip J

    2010-01-01

    The organic nitrite, amyl of nitrite, was initially used as a therapeutic agent in the treatment of angina pectoris, but was replaced over a decade later by the organic nitrate, nitroglycerin (NTG), due to the ease of administration and longer duration of action. The administration of organic nitrate esters, such as NTG, continues to be used in the treatment of angina pectoris and heart failure since the birth of modern pharmacology. Their clinical effectiveness is due to vasodilator activity in large veins and arteries through an as yet unidentified method of delivering nitric oxide (NO), or a NO-like compound. The major drawback is the development of tolerance with NTG, and the duration and route of administration with amyl of nitrite. Although the nitrites are no longer used in the treatment of hypertension or ischemic heart disease, the nitrite anion has recently been discovered to possess novel pharmacologic actions, such as modulating hypoxic vasodilation, and providing cytoprotection in ischemia-reperfusion injury. Although the actions of these 2 similar chemical classes (nitrites and organic nitrates) have often been considered to be alike, we still do not understand their mechanism of action. Finally, the nitrite anion, either from sodium nitrite or an intermediate NTG form, may act as a storage form for NO and provide support for investigating the use of these agents in the treatment of ischemic cardiovascular states. We review what is presently known about the use of nitrates and nitrites including the historical, current, and potential uses of these agents, and their mechanisms of action.

  5. [Protonation of nitrite is an obligatory stage in the generation of nitric oxide from nitrite in biological systems].

    PubMed

    Mikoian, V D; Kubrina, L N; Khachatrian, G N; Vanin, A F

    2006-01-01

    The yield of nitric oxide from 1 mM sodium nitrite differs 200 times when the process was initiated by 10 mM sodium dithionite in the solution of 5 or 150 mM HEPES-buffer (pH 7.4). Dithionite acted both as a strong reductant and an agent that induced a local acidification of solutions without notable change in pH value. The amount of nitric oxide was estimated by the EPR method by measuring the incorporation of nitric oxide to water-soluble complexes of Fe with N-methyl-D-glucamine dithiocarbamate (MGD), which led to the formation of EPR-detectable mononitrosyl iron complexes with MGD (MNIC-MGD). Ten seconds after dithionite addition, the concentration of MNIC - MGD complexes reached 2 microM in 5 mM HEPES-buffer in contrast to 0.01 microM in 150 mM HEPES-buffer. The difference was suggested to be due to a higher life-time of zones with decreased pH values in a weaker weak buffer solution. The life-time was high enough to ensure the protonation of a part of nitrite. The resulting nitrous acid was decomposed to form nitric oxide. The difference in the formation of nitric oxide from nitrite was also observed in weak and strong buffer solutions in the presence of hemoglobin (0.3 mM) or serum albumin (0.5 mM). However, the ratios of nitric oxide yields in weak and strong buffer did not exceed 3-4 times. The increase in the formation of nitric oxide from nitrite was characteristic for the solutions containing both proteins. Large amounts of nitric oxide formed from nitrite was observed in mouse liver preparation subjected to freezing-thawing procedure followed by incubation in 150 mM HEPES-buffer (pH 7.4) and addition of dithionite. The proposition was made that the presence of zones with low pH value in cells and tissues can ensure the predominant operation of the acid mechanism formation of nitric oxide from nitrite. The contribution of the formation of nitric oxide from nitrite catalyzing with heme-containing proteins nitrite reductases can be minor one under these

  6. Involvement of the narJ or narW gene product in the formation of active nitrate reductase in Escherichia coli.

    PubMed

    Blasco, F; Pommier, J; Augier, V; Chippaux, M; Giordano, G

    1992-01-01

    Two membrane-bound nitrate reductases, NRA and NRZ, exist in Escherichia coli. Both isoenzymes are composed of three structural subunits, alpha, beta, and gamma encoded by narG/narZ, narH/narY and narI/narV, respectively. The genes are in transcription units which also contain a fourth gene encoding a polypeptide, delta, which is not part of the final enzyme. A strain which is devoid of, or does not express, the nar genes, was used to investigate the role of the delta and gamma polypeptides in the formation and/or processing of the nitrate reductase. When only the alpha and beta polypeptides are produced, an (alpha beta) complex exists which is inactive and soluble. When the alpha, beta and delta polypeptides are produced, the (alpha beta) complex is active with artificial donors such as benzyl viologen but is soluble. When the alpha, beta and gamma polypeptides are produced, the (alpha beta) complex is inactive but partially binds the membrane. It was concluded that the gamma polypeptide is involved in the binding of the (alpha beta) complex to the membrane while the delta polypeptide is indispensable for the (alpha beta) nitrate reductase activity. The activation by the delta polypeptide does not seem to involve the insertion of the redox centres of the enzyme since the purified inactive (alpha beta) complex was shown to contain the four iron-sulphur centres and the molybdenum cofactor, which are normally present in the native purified enzyme. The extreme sensitivity of this inactive complex to thermal denaturation or tryptic treatment favours the idea that the delta polypeptide promotes the correct assembly of the alpha and beta subunits. Although this corresponds to the definition of a chaperone protein this possibility has been rejected. In this study we have also demonstrated that the delta or gamma polypeptide encoded by one nar operon can be substituted successfully for by its respective counterpart from the other nar operon to give an active membrane bound

  7. Renal carbonic anhydrases are involved in the reabsorption of endogenous nitrite.

    PubMed

    Chobanyan-Jürgens, Kristine; Schwarz, Alexandra; Böhmer, Anke; Beckmann, Bibiana; Gutzki, Frank-Mathias; Michaelsen, Jan T; Stichtenoth, Dirk O; Tsikas, Dimitrios

    2012-02-15

    Nitrite (ONO(-)) exerts nitric oxide (NO)-related biological actions and its concentration in the circulation may be of particular importance. Nitrite is excreted in the urine. Hence, the kidney may play an important role in nitrite/NO homeostasis in the vasculature. We investigated a possible involvement of renal carbonic anhydrases (CAs) in endogenous nitrite reabsorption in the proximal tubule. The potent CA inhibitor acetazolamide was administered orally to six healthy volunteers (5 mg/kg) and nitrite was measured in spot urine samples before and after administration. Acetazolamide increased abruptly nitrite excretion in the urine, strongly suggesting that renal CAs are involved in nitrite reabsorption in healthy humans. Additional in vitro experiments support our hypothesis that nitrite reacts with CO(2), analogous to the reaction of peroxynitrite (ONOO(-)) with CO(2), to form acid-labile nitrito carbonate [ONOC(O)O(-)]. We assume that this reaction is catalyzed by CAs and that nitrito carbonate represents the nitrite form that is actively transported into the kidney. The significance of nitrite reabsorption in the kidney and the underlying mechanisms, notably a direct involvement of CAs in the reaction between nitrite and CO(2), remain to be elucidated.

  8. Retention and leaching of nitrite by municipal solid waste incinerator bottom ash under the landfill circumstance.

    PubMed

    Yao, Jun; Kong, Qingna; Zhu, Huayue; Long, Yuyang; Shen, Dongsheng

    2015-01-01

    The retention and leaching of nitrite by municipal solid waste incinerator (MSWI) bottom ash could affect its migration in the landfill. In this study, the effect of the dosage of MSWI bottom ash as well as the variation of the landfill environmental parameters including pH, anions and organic matter on the nitrite retention and leaching behavior was investigated by batch experiments. The highest removal percentage (73.0%) of nitrite was observed when the dosage of MSWI bottom ash was 10 g L(-1) in 2 mg L(-1) nitrite solution. Further increase of the dosage would retard the retention, as the nitrite leaching from MSWI bottom ash was enhanced. The optimum retention of nitrite was observed when the pH was 5.0, while the leaching of nitrite showed a consistent reduction with the increase of pH. Besides, the presence of Cl(-), SO4(2)(-) and acetic acid could enhance the leaching of nitrite and mitigate the retention process. However, the retention of nitrite was enhanced by PO4(3)(-), which was probably due to the formation of the apatite, an active material for the adsorption of the nitrite. These results suggested that MSWI bottom ash could affect the migration of nitrite in the landfill, which was related to the variation of the landfill circumstance.

  9. 21 CFR 573.700 - Sodium nitrite.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Sodium nitrite. 573.700 Section 573.700 Food and... Listing § 573.700 Sodium nitrite. Sodium nitrite may be safely used in canned pet food containing meat and... byproducts so that the level of sodium nitrite does not exceed 20 parts per million. (b) To assure safe...

  10. 21 CFR 573.700 - Sodium nitrite.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Sodium nitrite. 573.700 Section 573.700 Food and... Listing § 573.700 Sodium nitrite. Sodium nitrite may be safely used in canned pet food containing meat and... byproducts so that the level of sodium nitrite does not exceed 20 parts per million. (b) To assure safe...

  11. 21 CFR 573.700 - Sodium nitrite.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Sodium nitrite. 573.700 Section 573.700 Food and... Listing § 573.700 Sodium nitrite. Sodium nitrite may be safely used in canned pet food containing meat and... byproducts so that the level of sodium nitrite does not exceed 20 parts per million. (b) To assure safe...

  12. 21 CFR 573.700 - Sodium nitrite.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Sodium nitrite. 573.700 Section 573.700 Food and... Listing § 573.700 Sodium nitrite. Sodium nitrite may be safely used in canned pet food containing meat and... byproducts so that the level of sodium nitrite does not exceed 20 parts per million. (b) To assure safe...

  13. 21 CFR 573.700 - Sodium nitrite.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Sodium nitrite. 573.700 Section 573.700 Food and... Listing § 573.700 Sodium nitrite. Sodium nitrite may be safely used in canned pet food containing meat and... byproducts so that the level of sodium nitrite does not exceed 20 parts per million. (b) To assure safe...

  14. Oxygen as Intermediate in Anoxic Environments: Nitrite-Dependent Methane Oxidation and Beyond

    NASA Astrophysics Data System (ADS)

    Ettwig, K. F.

    2014-12-01

    In recent years the known diversity of hydrocarbon activation mechanisms under anaerobic conditions has been extended by intra-aerobic denitrification, a process in which oxygen is derived from NO and used for substrate activation. For two phylogenetically unrelated bacterial species, the freshwater NC10 phylum bacterium Methylomirabilis oxyfera [1] and the marine γ-proteobacterial strain HdN1 [2] it has been shown that, under anoxic conditions with nitrate and/or nitrite, mono-oxygenases are used for methane and hexadecane oxidation, respectively. No degradation was observed with nitrous oxide (N2O) only. In the anaerobic methanotroph M. oxyfera, which lacks apparent nitrous oxide reductase in its genome, substrate activation in the presence of nitrite was directly associated with both O2 and N2 formation. These findings strongly argue for the role of nitric oxide (NO), or an oxygen species derived from it, in the activation reaction of methane. Although intracellular oxygen generation has been experimentally documented and elegantly explains the utilization of 'aerobic' pathways under anoxic conditions, research about the underlying molecular mechanism has just started. The proposed candidate enzymes for oxygen (or possibly another another reactive intermediate) production from NO, an NO dismutase (NOD) [3], related to quinol-dependent NO reductases (qNORs), is present and highly expressed in both M. oxyfera and strain HdN1. Besides that, several recently sequenced species from the Cytophaga-Flavobacterium-Bacteroides group harbor Nod/Nor genes, but experimential evidence is needed to show if these have NOD activity, are unusual but functional qNORs, or represent transition states between the two. Additionally, for several anaerobic hydrocarbon-degrading organisms the biochemical mechanism of substrate activation has not been elucidated yet: whereas signature genes of anaerobic degradation are missing, monooxygenase genes are present. Also these microorganisms

  15. HMG-CoA reductase inhibitor improves endothelial dysfunction in spontaneous hypertensive rats via down-regulation of caveolin-1 and activation of endothelial nitric oxide synthase.

    PubMed

    Suh, Jung-Won; Choi, Dong-Ju; Chang, Hyuk-Jae; Cho, Young-Seok; Youn, Tae-Jin; Chae, In-Ho; Kim, Kwang-Il; Kim, Cheol-Ho; Kim, Hyo-Soo; Oh, Buyng-Hee; Park, Young-Bae

    2010-01-01

    Hypertension is associated with endothelial dysfunction and increased cardiovascular risk. Caveolin-1 regulates nitric oxide (NO) signaling by modulating endothelial nitric oxide synthase (eNOS). The purpose of this study was to examine whether HMG-CoA reductase inhibitor improves impaired endothelial function of the aorta in spontaneous hypertensive rat (SHR) and to determine the underlying mechanisms involved. Eight-week-old male SHR were assigned to either a control group (CON, n=11) or a rosuvastatin group (ROS, n=12), rosuvastatin (10 mg/kg/day) administered for eight weeks. Abdominal aortic rings were prepared and responses to acetylcholine (10(-9)-10(-4) M) were determined in vitro. To evaluate the potential role of NO and caveolin-1, we examined the plasma activity of NOx, eNOS, phosphorylated-eNOS and expression of caveolin-1. The relaxation in response to acetylcholine was significantly enhanced in ROS compared to CON. Expression of eNOS RNA was unchanged, whereas NOx level and phosphorylated-eNOS at serine-1177 was increased accompanied with depressed level of caveolin-1 in ROS. We conclude that 3-Hydroxy-3-methylglutaryl Coenzyme-A (HMG-CoA) reductase inhibitor can improve impaired endothelial dysfunction in SHR, and its underlying mechanisms are associated with increased NO production. Furthermore, HMG-CoA reductase inhibitor can activate the eNOS by phosphorylation related to decreased caveolin-1 abundance. These results imply the therapeutic strategies for the high blood pressure-associated endothelial dysfunction through modifying caveolin status.

  16. Camphene, a Plant-Derived Monoterpene, Reduces Plasma Cholesterol and Triglycerides in Hyperlipidemic Rats Independently of HMG-CoA Reductase Activity

    PubMed Central

    Vallianou, Ioanna; Peroulis, Nikolaos; Pantazis, Panayotis; Hadzopoulou-Cladaras, Margarita

    2011-01-01

    Background Central to the pathology of coronary heart disease is the accumulation of lipids, cholesterol and triglycerides, within the intima of arterial blood vessels. The search for drugs to treat dislipidemia, remains a major pharmaceutical focus. In this study, we evaluated the hypolipidemic properties of the essential oil from Chios mastic gum (MGO). Methodology/Principal Findings The hypolipidemic effect of MGO was investigated in naïve as well as in rats susceptible to detergent-induced hyperlipidemia. Serum cholesterol and triglycerides were determined using commercial kits. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase activity was measured in HepG2 cell extracts using a radioactive assay; cellular cholesterol and cholesterol esters were assessed using gas chromatography. MGO administration into naïve rats resulted in a dose-dependent reduction in the constitutive synthesis of serum cholesterol and triglycerides. In hyperlipidemic rats, MGO treatment had also a strong hypolipidemic effect. By testing various components of MGO, we show for the first time that the hypolipidemic action is associated with camphene. Administration of camphene at a dose of 30 µg/gr of body weight in hyperlipidemic rats resulted in a 54.5% reduction of total cholesterol (p<0.001), 54% of Low Density Lipoprotein (LDL)-cholesterol (p<0.001) and 34.5% of triglycerides (p<0.001). Treatment of HepG2 cells with camphene led to a decrease in cellular cholesterol content to the same extend as mevinolin, a known HMG-CoA reductase inhibitor. The hypolipidemic action of camphene is independent of HMG-CoA reductase activity, suggesting that its hypocholesterolemic and hypotriglyceridemic effects are associated with a mechanism of action different than that of statins. Conclusions Given the critical role that the control of hyperlipidemia plays in cardiovascular disease, the results of our study provide insights into the use of camphene as an alternative lipid lowering agent

  17. [Effect of chloditan on the changes of activity of glutathione transferase, glutathione reductase and glutathione content in the adrenal glands and liver in rats].

    PubMed

    Zorich, P A; Tronko, N D; Mikosha, A S

    1994-01-01

    The chloditan (o.p-DDD, mitotane), which causes the destruction of the human and dog adrenal cortex, on the most essential system of xenobiotic metabolism: glutathione-S-transferase--glutathione has been studied. The effect of o,p-DDD on GSH level and activity of glutathione-S-transferase and glutathione reductase which maintain the level of reduced glutathione was analyzed in the adrenal and liver tissue of rats. This species is resistant to adrenocorticolytic action of o,p-DDD. It was shown that feeding of rats weighting 200-240 g with oil solution of o,p-DDD (75 mg daily) for 3 days causes the decrease in activity of glutathione-S-transferase and content of oxidazed glutathione in the adrenals with simultaneous increase of the content of reduced glutathione. The glutathione-S-transferase and glutathione reductase activity in the liver rises under the effect of o,p-DDD, the decrease of the GSH level being observed. The revealed changes may explain the species sensitivity of animals to o,p-DDD.

  18. Severe Methemoglobinemia due to Sodium Nitrite Poisoning

    PubMed Central

    Hayakawa, Mineji; Gando, Satoshi

    2016-01-01

    Case. We report a case of severe methemoglobinemia due to sodium nitrite poisoning. A 28-year-old man was brought to our emergency department because of transient loss of consciousness and cyanosis. He was immediately intubated and ventilated with 100% oxygen. A blood test revealed a methemoglobin level of 92.5%. Outcome. We treated the patient with gastric lavage, activated charcoal, and methylene blue (2 mg/kg) administered intravenously. Soon after receiving methylene blue, his cyanosis resolved and the methemoglobin level began to decrease. After relocation to the intensive care unit, his consciousness improved and he could recall ingesting approximately 15 g sodium nitrite about 1 hour before he was brought to our hospital. The patient was discharged on day 7 without neurologic impairment. Conclusion. Severe methemoglobinemia may be fatal. Therefore, accurate diagnosis of methemoglobinemia is very important so that treatment can be started as soon as possible. PMID:27563472

  19. Achievement of high nitrite accumulation via endogenous partial denitrification (EPD).

    PubMed

    Ji, Jiantao; Peng, Yongzhen; Wang, Bo; Wang, Shuying

    2017-01-01

    This study proposed a novel strategy for achievement of partial denitrification driven by endogenous carbon sources in an anaerobic/anoxic/aerobic activated sludge system. Results showed that in the steady-stage, the nitrate-to-nitrite transformation ratio (NTR) was kept at around 87% without nitrate in the effluent. During the anaerobic period, exogenous carbon sources was completely taken up, accompanied by the consumption of glycogen and production of polyhydroxyalkanoates (PHAs). During the anoxic period, nitrate was reduced to nitrite by using PHAs as carbon sources, followed by the replenishment of glycogen. Thus, the phenotype of denitrifying GAOs was clearly observed and endogenous partial denitrification (EPD) occurred. Furthermore, results showed the nitrate reduction was prior to the nitrite reduction in the presence of nitrate, which led to the high nitrite accumulation.

  20. Human biliverdin reductase-based peptides activate and inhibit glucose uptake through direct interaction with the kinase domain of insulin receptor

    PubMed Central

    Gibbs, Peter E. M.; Lerner-Marmarosh, Nicole; Poulin, Amelia; Farah, Elie; Maines, Mahin D.

    2014-01-01

    Insulin binding changes conformation of the insulin receptor kinase (IRK) domain and initiates glucose uptake through the insulin, IGF-1, phosphatidyl inositol 3-kinase (PI3K), and MAPK pathways; human biliverdin reductase (hBVR) is an IRK substrate and pathway effector. This is the first report on hBVR peptide-mediated IRK activation and conformational change. 290KYCCSRK, which increased IRK Vmax without changing Km, stimulated glucose uptake and potentiated insulin and IGF-1 stimulation in 4 cell lines. KYCCSRK in native hBVR was necessary for the hBVR and IRK cross-activation. Peptide treatment also activated PI3K downstream effectors, Akt and ERK, phosphorylation, and Elk transcriptional activity. In cells transfected with CMV-regulated EGFP-VP-peptide plasmid, C292→A mutant did not stimulate glucose uptake; K296→A decreased uptake and kinase activity. KEDQYMKMTV, corresponding to hBVR's SH2-binding domain, was a potent inhibitor of glucose uptake and IRK. The mechanism of action of peptides was examined using cells expressing IRK (aa 988–1263) activated by coexpressed KYCCSRK. Three active cys-mutants of IRK, with fluorophore coupled to cysteines, C1056, C1138, or C1234, were examined for changes in fluorescence emission spectra in the presence of peptides. KYCCSRK and KEDQYMKMTV bound to different sites in IRK. The findings identify novel agents for activating or inhibiting insulin signaling and offer a new approach for treatment of type 2 diabetes and hypoglycemia.—Gibbs, P. E. M., Lerner-Marmarosh, N., Poulin, A., Farah, E., Maines, M. D. Human biliverdin reductase-based peptides activate and inhibit glucose uptake through direct interaction with the kinase domain of insulin receptor. PMID:24568842

  1. Low-dose intravenous nitrite improves hemodynamics in a canine model of acute pulmonary thromboembolism.

    PubMed

    Dias-Junior, Carlos A C; Gladwin, Mark T; Tanus-Santos, Jose E

    2006-12-15

    Acute pulmonary thomboembolism (APT)-induced pulmonary hypertension can be counteracted by activating the nitric oxide (NO)-cGMP pathway. Recent studies have demonstrated that the naturally occurring anion nitrite (NO(2)(-)) is a bioactive storage reservoir for NO, and is reduced to NO under conditions of hypoxia and acidosis. We hypothesized that nitrite infused intravenously could attenuate the hemodynamic changes associated with APT. APT was induced with autologous blood clots injected into the right atrium in mongrel dogs. After APT (or saline), the dogs received an intravenous nitrite (or saline) infusion (6.75 micromol/kg over 15 min and then 0.28 micromol/kg/min) and hemodynamic evaluations were carried out for 2 h. Plasma nitrite concentrations were measured using ozone-based reductive chemiluminescence methodologies. APT decreased cardiac index (CI) and increased pulmonary vascular resistance index (PVRI); these effects were improved during infusions of sodium nitrite. Accordingly, nitrite infusion increased cardiac index by 28%, reduced the PVRI by 48%, and the systemic vascular resistance index (SVRI) by 21% in embolized dogs, suggesting a greater effect on the ischemic embolized vascular system than the systemic circulation following embolization. Interestingly, in nonembolized control dogs the same nitrite infusion decreased MAP and CI (all P<0.05). The nitrite infusion increased plasma nitrite concentrations by approximately 2 microM, and produced dose-dependent effects on PVRI, MAP, and SVRI. Remarkably, blood levels of nitrite as low as 500 nM decreased PVRI and SVRI in this model, suggesting a potential role of nitrite in physiological blood flow regulation. These results suggest that a low-dose nitrite infusion produces beneficial hemodynamic effects in a dog model of APT. These findings suggest a new therapeutic application for nitrite and support emerging evidence for a surprisingly potent and potentially physiological vasoactivity of nitrite.

  2. Nitrite Assimilation and Amino Nitrogen Synthesis in Isolated Spinach Chloroplasts 1

    PubMed Central

    Magalhaes, A. C.; Neyra, C. A.; Hageman, R. H.

    1974-01-01

    The assimilation of nitrite leading to de novo synthesis of amino nitrogen in a chloroplast-enriched fraction isolated from freshly harvested young spinach (Spinacia oleracea L.) leaves was demonstrated. The preparations showed approximately 55% intact chloroplasts as determined by light scattering properties and fixed CO2 at rates of approximately 100 μmoles hr−1 mg chlorophyll−1. The chloroplast-enriched fraction contained the enzymes, nitrite reductase and NADPH-glutamate dehydrogenase, needed for the reduction of nitrite and incorporation of ammonia into glutamate. Kinetic studies showed that the reduction of nitrite by the chloroplast-enriched fraction is light-dependent, and the process proceeds at rates of 6 to 12 μmoles hr−1 mg chlorophyll−1. The addition of nitrite to the chloroplast preparation caused a 3-fold increase in the production of α-amino nitrogen when compared with the control without nitrite. There was a stoichiometric relation between amino-nitrogen synthesis and nitrite disappearance from the medium. The ratio of amino-nitrogen: NO2− ranged from 0.6 to 0.9. The initial rate of amino-nitrogen production was faster when α-ketoglutarate was added to the nitrite reducing chloroplast medium than when it was omitted. However, these high rates were not sustained and the total amino-nitrogen production at the end of a 30-minute period was only slightly higher. These data show that chloroplasts are functionally able and contain the enzyme complement necessary to utilize light energy for the reduction of nitrite to amino nitrogen. Thus, chloroplasts should be considered as a major site for in vivo amino-nitrogen synthesis in green plants. PMID:16658715

  3. Mutagenicity of some alkyl nitrites used as recreational drugs

    SciTech Connect

    Dunkel, V.C.; Cameron, T.P. ); Rogers-Back, A.M.; Lawlor, T.E.; Harbell, J.W. )

    1989-01-01

    When the AIDS epidemic was in its earliest stages, and prior to identification of HIV as the etiological factor, the use of volatile nitrites by the male homosexual community to enhance sexual activities appeared to have a significant role in this disease. Preliminary observations indicated that that portion of the male homosexual community which developed Kaposi's sarcoma were also heavy nitrite users. These nitrites had been demonstrated to be mutagenic in bacteria and thus it was postulated that they could be responsible for the appearance of the sarcoma. To evaluate further the genotoxic activity of these chemicals, six nitrites, including those most commonly used by homosexuals for sexual gratification, were selected for testing in the mouse lymphoma TK {plus minus} and Salmonell typhimurium mutagenicity assays. One chemical, n-amyl nitrite, was negative in the mouse lymphoma assay, while the other five chemicals, n-butyl, isobutyl, iso-amyl, sec-butyl, and n-propyl nitrite, were positive. All six compounds were positive in the Salmonella assay. The mutagenic and known toxic effects of these chemicals remain a concern because a large population of teenagers and young adults continue to abuse these substances.

  4. Production of a highly active, soluble form of the cytochrome P450 reductase (CPR A) from Candida tropicalis

    DOEpatents

    Donnelly, Mark

    2006-08-01

    The present invention provides soluble cytochrome p450 reductase (CPR) proteins from Candida sp. having an altered N-terminal region which results in reduced hydrophobicity of the N-terminal region. Also provided are host cells comprising the subject soluble CPR proteins. In addition, the present invention provides nucleotide and corresponding amino acid sequences for soluble CPR proteins and vectors comprising the nucleotide sequences. Methods for producing a soluble CPR, for increasing production of a dicarboxylic acid, and for detecting a cytochrome P450 are also provided.

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

    PubMed

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

    2013-09-01

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

  6. Kinetic Characterization of Nitrite Uptake and Reduction by Chlamydomonas reinhardtii1

    PubMed Central

    Córdoba, Francisco; Cárdenas, Jacobo; Fernández, Emilio

    1986-01-01

    Kinetics of nitrite uptake and reduction by Chlamydomonas reinhardtii cells growing phototrophically has been studied by means of progress curves and the Michaelis-Menten integrated equation. Both uptake and reduction processes exhibited hyperbolic saturation kinetics, the nitrite uptake system lacking a diffusion component. Nitrite uptake and reduction showed significant differences in Ks for nitrite at pH 7.5 (1.6 versus 20 micromolar, respectively), optimal pH, activation energy values, and sensitivity toward reagents of sulfhydryl groups. Ks values for nitrite uptake were halved in cells subjected to darkness or to nitrogen-starvation. Nitrate inhibited nitrite uptake by a partially competitive mechanism. The same inhibition pattern was found for nitrite uptake by C. reinhardtii mutant 305 cells incapable of nitrate assimilation. The results demonstrate that C. reinhardtii cells take up nitrite via a highly specific carrier, probably energy-dependent, kinetically responsive to environmental changes, distinguishable from the enzymic nitrite reduction and endowed with an active site for nitrite not usable for nitrate transport. PMID:16665164

  7. Augmentation of CFTR maturation by S-nitrosoglutathione reductase

    PubMed Central

    Sawczak, Victoria; Zaidi, Atiya; Butler, Maya; Bennett, Deric; Getsy, Paulina; Zeinomar, Maryam; Greenberg, Zivi; Forbes, Michael; Rehman, Shagufta; Jyothikumar, Vinod; DeRonde, Kim; Sattar, Abdus; Smith, Laura; Corey, Deborah; Straub, Adam; Sun, Fei; Palmer, Lisa; Periasamy, Ammasi; Randell, Scott; Kelley, Thomas J.; Lewis, Stephen J.

    2015-01-01

    S-nitrosoglutathione (GSNO) reductase regulates novel endogenous S-nitrosothiol signaling pathways, and mice deficient in GSNO reductase are protected from airways hyperreactivity. S-nitrosothiols are present in the airway, and patients with cystic fibrosis (CF) tend to have low S-nitrosothiol levels that may be attributed to upregulation of GSNO reductase activity. The present study demonstrates that 1) GSNO reductase activity is increased in the cystic fibrosis bronchial epithelial (CFBE41o−) cells expressing mutant F508del-cystic fibrosis transmembrane regulator (CFTR) compared with the wild-type CFBE41o− cells, 2) GSNO reductase expression level is increased in the primary human bronchial epithelial cells expressing mutant F508del-CFTR compared with the wild-type cells, 3) GSNO reductase colocalizes with cochaperone Hsp70/Hsp90 organizing protein (Hop; Stip1) in human airway epithelial cells, 4) GSNO reductase knockdown with siRNA increases the expression and maturation of CFTR and decreases Stip1 expression in human airway epithelial cells, 5) increased levels of GSNO reductase cause a decrease in maturation of CFTR, and 6) a GSNO reductase inhibitor effectively reverses the effects of GSNO reductase on CFTR maturation. These studies provide a novel approach to define the subcellular location of the interactions between Stip1 and GSNO reductase and the role of S-nitrosothiols in these interactions. PMID:26637637

  8. The effect of aluminium-stress and exogenous spermidine on chlorophyll degradation, glutathione reductase activity and the photosystem II D1 protein gene (psbA) transcript level in lichen Xanthoria parietina.

    PubMed

    Sen, Gulseren; Eryilmaz, Isil Ezgi; Ozakca, Dilek

    2014-02-01

    In this study, the effects of short-term aluminium toxicity and the application of spermidine on the lichen Xanthoria parietina were investigated at the physiological and transcriptional levels. Our results suggest that aluminium stress leads to physiological processes in a dose-dependent manner through differences in lipid peroxidation rate, chlorophyll content and glutathione reductase (EC 1.6.4.2) activity in aluminium and spermidine treated samples. The expression of the photosystem II D1 protein (psbA) gene was quantified using semi-quantitative RT-PCR. Increased glutathione reductase activity and psbA mRNA transcript levels were observed in the X. parietina thalli that were treated with spermidine before aluminium-stress. The results showed that the application of spermidine could mitigate aluminium-induced lipid peroxidation and chlorophyll degradation on lichen X. parietina thalli through an increase in psbA transcript levels and activity of glutathione reductase (GR) enzymes.

  9. The Role of Human Aldo-Keto Reductases in the Metabolic Activation and Detoxication of Polycyclic Aromatic Hydrocarbons: Interconversion of PAH Catechols and PAH o-Quinones.

    PubMed

    Zhang, Li; Jin, Yi; Huang, Meng; Penning, Trevor M

    2012-01-01

    Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental pollutants. They are procarcinogens requiring metabolic activation to elicit their deleterious effects. Aldo-keto reductases (AKR) catalyze the oxidation of proximate carcinogenic PAH trans-dihydrodiols to yield electrophilic and redox-active PAH o-quinones. AKRs are also found to be capable of reducing PAH o-quinones to form PAH catechols. The interconversion of o-quinones and catechols results in the redox-cycling of PAH o-quinones to give rise to the generation of reactive oxygen species and subsequent oxidative DNA damage. On the other hand, PAH catechols can be intercepted through phase II metabolism by which PAH o-quinones could be detoxified and eliminated. The aim of the present review is to summarize the role of human AKRs in the metabolic activation/detoxication of PAH and the relevance of phase II conjugation reactions to human lung carcinogenesis.

  10. Metabolic fates and effects of nitrite in brown trout under normoxic and hypoxic conditions: blood and tissue nitrite metabolism and interactions with branchial NOS, Na+/K+-ATPase and hsp70 expression.

    PubMed

    Jensen, Frank B; Gerber, Lucie; Hansen, Marie N; Madsen, Steffen S

    2015-07-01

    Nitrite secures essential nitric oxide (NO) bioavailability in hypoxia at low endogenous concentrations, whereas it becomes toxic at high concentrations. We exposed brown trout to normoxic and hypoxic water in the absence and presence of added ambient nitrite to decipher the cellular metabolism and effects of nitrite at basal and elevated concentrations under different oxygen regimes. We also tested hypotheses concerning the influence of nitrite on branchial nitric oxide synthase (NOS), Na(+)/K(+)-ATPase (nka) and heat shock protein (hsp70) mRNA expression. Basal plasma and erythrocyte nitrite levels were higher in hypoxia than normoxia, suggesting increased NOS activity. Nitrite exposure strongly elevated nitrite concentrations in plasma, erythrocytes, heart tissue and white muscle, which was associated with an extensive metabolism of nitrite to nitrate and to iron-nitrosylated and S-nitrosated compounds. Nitrite uptake was slightly higher in hypoxia than normoxia, and high internal nitrite levels extensively converted blood hemoglobin to methemoglobin and nitrosylhemoglobin. Hypoxia increased inducible NOS (iNOS) mRNA levels in the gills, which was overruled by a strong inhibition of iNOS expression by nitrite in both normoxia and hypoxia, suggesting negative-feedback regulation of iNOS gene expression by nitrite. A similar inhibition was absent for neuronal NOS. Branchial NKA activity stayed unchanged, but mRNA levels of the nkaα1a subunit increased with hypoxia and nitrite, which may have countered an initial NKA inhibition. Nitrite also increased hsp70 gene expression, probably contributing to the cytoprotective effects of nitrite at low concentrations. Nitrite displays a concentration-dependent switch between positive and negative effects similar to other signaling molecules.

  11. Nitrate/Nitrite as Critical Mediators to Limit Oxidative Injury and Inflammation

    PubMed Central

    Waltz, Paul; Escobar, Daniel; Botero, Ana Maria

    2015-01-01

    Abstract Significance: Nitric oxide (NO) is a critical signaling molecule marked by complex chemistry and varied biological responses depending on the context of the redox environment. In the setting of inflammation, NO can not only contribute to tissue injury and be causative of oxidative damage but can also signal as an adaptive molecule to limit inflammatory signaling in multiple cell types and tissues. Recent Advances: An advance in our understanding of NO biology was the recognition of the nitrate-nitrite-NO axis, whereby nitrate (predominantly from dietary sources) could be converted to nitrite and nitrite could be reduced to NO. Critical Issues: Intriguingly, the recognition of multiple enzymes that serve as nitrite reductases in the setting of hypoxia or ischemia established the concept of nitrite as a circulating endocrine reservoir of NO, with the selective release of NO at sites that were primed for this reaction. This review highlights the anti-inflammatory roles of nitrite in numerous clinical conditions, including ischemia/reperfusion, transplant, cardiac arrest, and vascular injury, and in gastrointestinal inflammation. Future Directions: These preclinical and clinical investigations set up further clinical trials and studies that elucidate the endogenous role this pathway plays in protection against inflammatory signaling. Antioxid. Redox Signal. 23, 328–339. PMID:26140517

  12. Inhibitory effect of nitrite on coagulation processes demonstrated by thrombelastography.

    PubMed

    Park, J W; Piknova, B; Nghiem, K; Lozier, J N; Schechter, A N

    2014-08-31

    Nitric oxide (NO) can be generated by two-step reduction pathway in which nitrate is converted first into nitrite and then into NO via several mechanisms, as well as from arginine by endogenous nitric oxide synthase (NOS). We have recently shown that nitrite ions in the presence of erythrocytes inhibit platelet aggregation and activation, as measured by aggregometry and flow cytometric analysis of P-selectin, through its reduction to NO under partially deoxygenated conditions. In the current study, we investigated how nitrite may affect overall clotting processes via modulating platelet function using thrombelastography (TEG). We measured three major TEG parameters, reaction time (R, time to initial fibrin formation), α angle (velocity of clot growth) and maximum amplitude (MA, maximum clot strength) using blood from healthy volunteers. An NO donor (DEANONOate) showed inhibitory effects on all TEG parameters in platelet rich plasma (PRP) and whole blood, resulting in delayed R, decreased angle, and reduced MA in a dose dependent manner. Nitrite ions also exhibited inhibitory effects in whole blood at 20% hematocrit, and this was greatly enhanced under hypoxic conditions, being demonstrable at 0.1 μM concentration. Neither compound changed any TEG parameters in plasma. Our results suggest that nitrite affects overall blood clotting and that TEG may be used to follow this process. Further the physiological effects of factors which determine NO bioavailability, such as endogenous levels of blood and tissue nitrite, may be useful as biomarkers for predicting hemostatic potential.

  13. Inhibitory effect of nitrite on coagulation processes demonstrated by thrombelastography

    PubMed Central

    Park, J. W.; Piknova, B.; Nghiem, K.; Lozier, J. N.; Schechter, A. N.

    2014-01-01

    Nitric oxide (NO) can be generated by two-step reduction pathway in which nitrate is converted first into nitrite and then into NO via several mechanisms, as well as from arginine by endogenous nitric oxide synthase (NOS). We have recently shown that nitrite ions in the presence of erythrocytes inhibit platelet aggregation and activation, as measured by aggregometry and flow cytometric analysis of P-selectin, through its reduction to NO under partially deoxygenated conditions. In the current study, we investigated how nitrite may affect overall clotting processes via modulating platelet function using thrombelastography (TEG). We measured three major TEG parameters, reaction time (R, time to initial fibrin formation), α angle (velocity of clot growth) and maximum amplitude (MA, maximum clot strength) using blood from healthy volunteers. An NO donor (DEANONOate) showed inhibitory effects on all TEG parameters in platelet rich plasma (PRP) and whole blood, resulting in delayed R, decreased angle, and reduced MA in a dose dependent manner. Nitrite ions also exhibited inhibitory effects in whole blood at 20% hematocrit, and this was greatly enhanced under hypoxic conditions, being demonstrable at 0.1 μM concentration. Neither compound changed any TEG parameters in plasma. Our results suggest that nitrite affects overall blood clotting and that TEG may be used to follow this process. Further the physiological effects of factors which determine NO bioavailability, such as endogenous levels of blood and tissue nitrite, may be useful as biomarkers for predicting hemostatic potential. PMID:24858214

  14. The NsrR Regulon of Escherichia coli K-12 Includes Genes Encoding the Hybrid Cluster Protein and the Periplasmic, Respiratory Nitrite Reductase▿

    PubMed Central

    Filenko, Nina; Spiro, Stephen; Browning, Douglas F.; Squire, Derrick; Overton, Tim W.; Cole, Jeff; Constantinidou, Chrystala

    2007-01-01

    Successful pathogens must be able to protect themselves against reactive nitrogen species generated either as part of host defense mechanisms or as products of their own metabolism. The regulatory protein NsrR (a member of the Rrf2 family of transcription factors) plays key roles in this stress response. Microarray analysis revealed that NsrR represses nine operons encoding 20 genes in Escherichia coli MG1655, including the hmpA, ytfE, and ygbA genes that were previously shown to be regulated by NsrR. Novel NsrR targets revealed by this study include hcp-hcr (which were predicted in a recent bioinformatic study to be NsrR regulated) and the well-studied nrfA promoter that directs the expression of the periplasmic respiratory nitrite reductase. Conversely, transcription from the ydbC promoter is strongly activated by NsrR. Regulation of the nrf operon by NsrR is consistent with the ability of the periplasmic nitrite reductase to reduce nitric oxide and hence protect against reactive nitrogen species. Gel retardation assays were used to show that both FNR and NarL bind to the hcp promoter. The expression of hcp and the contiguous gene hcr is not induced by hydroxylamine. As hmpA and ytfE encode a nitric oxide reductase and a mechanism to repair iron-sulfur centers damaged by nitric oxide, the demonstration that hcp-hcr, hmpA, and ytfE are the three transcripts most tightly regulated by NsrR highlights the possibility that the hybrid cluster protein, HCP, might also be part of a defense mechanism against reactive nitrogen stress. PMID:17449618

  15. The metabolic impact of extracellular nitrite on aerobic metabolism of Paracoccus denitrificans.

    PubMed

    Hartop, K R; Sullivan, M J; Giannopoulos, G; Gates, A J; Bond, P L; Yuan, Z; Clarke, T A; Rowley, G; Richardson, D J

    2017-02-07

    Nitrite, in equilibrium with free nitrous acid (FNA), can inhibit both aerobic and anaerobic growth of microbial communities through bactericidal activities that have considerable potential for control of microbial growth in a range of water systems. There has been much focus on the effect of nitrite/FNA on anaerobic metabolism and so, to enhance understanding of the metabolic impact of nitrite/FNA on aerobic metabolism, a study was undertaken with a model denitrifying bacterium Paracoccus denitrificans PD1222. Extracellular nitrite inhibits aerobic growth of P. denitrificans in a pH-dependent manner that is likely to be a result of both nitrite and free nitrous acid (pKa = 3.25) and subsequent reactive nitrogen oxides generated from the intracellular passage of FNA into P. denitrificans. Increased expression of a gene encoding a flavohemoglobin protein (Fhp) (Pden_1689) was observed in response to extracellular nitrite. Construction and analysis of a deletion mutant established Fhp to be involved in endowing nitrite/FNA resistance at high extracellular nitrite concentrations. Global transcriptional analysis confirmed nitrite-dependent expression of fhp and indicated that P. denitrificans expressed a number of stress response systems associated with protein, DNA and lipid repair. It is therefore suggested that nitrite causes a pH-dependent stress response that is due to the production of associated reactive nitrogen species, such as nitric oxide from the internalisation of FNA.

  16. Purification and characterization of a highly active chromate reductase from endophytic Bacillus sp. DGV19 of Albizzia lebbeck (L.) Benth. actively involved in phytoremediation of tannery effluent-contaminated sites.

    PubMed

    Manikandan, Muthu; Gopal, Judy; Kumaran, Rangarajulu Senthil; Kannan, Vijayaraghavan; Chun, Sechul

    2016-01-01

    Phytoremediation using timber-yielding tree species is considered to be the most efficient method for chromium/tannery effluent-contaminated sites. In this study, we have chosen Albizzia lebbeck, a chromium hyperaccumulator plant, and studied one of its chromium detoxification processes operated by its endophytic bacterial assemblage. Out of the four different groups of endophytic bacteria comprising Pseudomonas, Rhizobium, Bacillus, and Salinicoccus identified from A. lebbeck employed in phytoremediation of tannery effluent-contaminated soil, Bacillus predominated with three species, which exhibited not only remarkable chromium accumulation ability but also high chromium reductase activity. A chromate reductase was purified to homogeneity from the most efficient chromium accumulator, Bacillus sp. DGV 019, and the purified 34.2-kD enzyme was observed to be stable at temperatures from 20°C to 60°C. The enzyme was active over a wide range of pH values (4.0-9.0). Furthermore, the enzyme activity was enhanced with the electron donors NADH, followed by NADPH, not affected by glutathione and ascorbic acid. Cu(2+) enhanced the activity of the purified enzyme but was inhibited by Zn(2+) and etheylenediamine tetraacetic acid (EDTA). In conclusion, due to its versatile adaptability the chromate reductase can be used for chromium remediation.

  17. Nitrite toxicity to the crayfish Procambarus clarkii

    SciTech Connect

    Gutzmer, M.P.; Tomasso, J.R.

    1985-03-01

    The purpose of this study was to determine the effects of acute nitrite exposure to the crayfish Procambarus clarkii (Decapoda). Specific objectives of this study included (1) determining the 24-, 48-, 72- and 96-h LC-50's of nitrite to crayfish of different weights and genders in freshwater, (2) determining the LC-50's of nitrite to crayfish in water with elevated chloride concentrations, and (3), in order to gain insight into the mechanisms of nitrite toxicity in crayfish, determining hemolymph nitrite concentrations in crayfish exposed to nitrite in freshwater and water with elevated chloride concentrations.

  18. Synthesis and highly potent hypolipidemic activity of alpha-asarone- and fibrate-based 2-acyl and 2-alkyl phenols as HMG-CoA reductase inhibitors.

    PubMed

    Mendieta, Aarón; Jiménez, Fabiola; Garduño-Siciliano, Leticia; Mojica-Villegas, Angélica; Rosales-Acosta, Blanca; Villa-Tanaca, Lourdes; Chamorro-Cevallos, Germán; Medina-Franco, José L; Meurice, Nathalie; Gutiérrez, Rsuini U; Montiel, Luisa E; Cruz, María Del Carmen; Tamariz, Joaquín

    2014-11-01

    In the search for new potential hypolipidemic agents, the present study focused on the synthesis of 2-acyl phenols (6a-c and 7a-c) and their saturated side-chain alkyl phenols (4a-c and 5a-c), and on the evaluation of their hypolipidemic activity using a murine Tyloxapol-induced hyperlipidemic protocol. The whole series of compounds 4-7 greatly and significantly reduced elevated serum levels of total cholesterol, LDL-cholesterol, and triglycerides, with series 6 and 7 showing the greatest potency ever found in our laboratory. At the minimum dose (25mg/kg/day), the latter compounds lowered cholesterol by 68-81%, LDL by 72-86%, and triglycerides by 59-80%. This represents a comparable performance than that shown by simvastatin. Experimental evidence and docking studies suggest that the activity of these derivatives is associated with the inhibition of HMG-CoA reductase.

  19. Implications of Limited Thermophilicity of Nitrite Reduction for Control of Sulfide Production in Oil Reservoirs

    PubMed Central

    Fida, Tekle Tafese; Chen, Chuan; Okpala, Gloria

    2016-01-01

    ABSTRACT Nitrate reduction to nitrite in oil fields appears to be more thermophilic than the subsequent reduction of nitrite. Concentrated microbial consortia from oil fields reduced both nitrate and nitrite at 40 and 45°C but only nitrate at and above 50°C. The abundance of the nirS gene correlated with mesophilic nitrite reduction activity. Thauera and Pseudomonas were the dominant mesophilic nitrate-reducing bacteria (mNRB), whereas Petrobacter and Geobacillus were the dominant thermophilic NRB (tNRB) in these consortia. The mNRB Thauera sp. strain TK001, isolated in this study, reduced nitrate and nitrite at 40 and 45°C but not at 50°C, whereas the tNRB Petrobacter sp. strain TK002 and Geobacillus sp. strain TK003 reduced nitrate to nitrite but did not reduce nitrite further from 50 to 70°C. Testing of 12 deposited pure cultures of tNRB with 4 electron donors indicated reduction of nitrate in 40 of 48 and reduction of nitrite in only 9 of 48 incubations. Nitrate is injected into high-temperature oil fields to prevent sulfide formation (souring) by sulfate-reducing bacteria (SRB), which are strongly inhibited by nitrite. Injection of cold seawater to produce oil creates mesothermic zones. Our results suggest that preventing the temperature of these zones from dropping below 50°C will limit the reduction of nitrite, allowing more effective souring control. IMPORTANCE Nitrite can accumulate at temperatures of 50 to 70°C, because nitrate reduction extends to higher temperatures than the subsequent reduction of nitrite. This is important for understanding the fundamentals of thermophilicity and for the control of souring in oil fields catalyzed by SRB, which are strongly inhibited by nitrite. PMID:27208132

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

    PubMed

    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 muta