Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.7.1.2 (nitrate reductase)
3,861 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The periplasmic nitrate reductase from Paracoccus denitrificans is a soluble two-subunit enzyme which binds two hemes (c-type), a [4Fe-4S] center, and a bis molybdopterin guanine dinucleotide cofactor (bis-MGD). A catalytic cycle for this enzyme is presented based on a study of these redox centers using electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) spectroscopies. The Mo(V) EPR signal of resting NAP (High g [resting]) has g(av) = 1.9898 is rhombic, exhibits low anisotropy, and is split by two weakly interacting protons which are not solvent-exchangeable. Addition of exogenous ligands to this resting state (e.g., nitrate, nitrite, azide) did not change the form of the signal. A distinct form of the High g Mo(V) signal, which has slightly lower anisotropy and higher rhombicity, was trapped during turnover of nitrate and may represent a catalytically relevant Mo(V) intermediate (High g [nitrate]). Mo K-edge EXAFS analysis was undertaken on the ferricyanide oxidized enzyme, a reduced sample frozen within 10 min of dithionite addition, and a nitrate-reoxidized form of the enzyme. The oxidized enzyme was fitted best as a di-oxo Mo(VI) species with 5 sulfur ligands (4 at 2. 43 A and 1 at 2.82 A), and the reduced form was fitted best as a mono-oxo Mo(IV) species with 3 sulfur ligands at 2.35 A. The addition of nitrate to the reduced enzyme resulted in reoxidation to a di-oxo Mo(VI) species similar to the resting enzyme. Prolonged incubation of NAP with dithionite in the absence of nitrate (i.e., nonturnover conditions) resulted in the formation of a species with a Mo(V) EPR signal that is quite distinct from the High g family and which has a g(av) = 1.973 (Low g [unsplit]). This signal resembles those of the mono-MGD xanthine oxidase family and is proposed to arise from an inactive form of the nitrate reductase in which the Mo(V) form is only coordinated by the dithiolene of one MGD. In samples of NAP that had been reduced with dithionite, treated with azide or cyanide, and then reoxidized with ferricyanide, two Mo(V) signals were detected with g(av) elevated compared to the High g signals. Kinetic analysis demonstrated that azide and cyanide displayed competitive and noncompetitive inhibition, respectively. EXAFS analysis of azide-treated samples show improvement to the fit when two nitrogens are included in the molybdenum coordination sphere at 2.52 A, suggesting that azide binds directly to Mo(IV). Based on these spectroscopic and kinetic data, models for Mo coordination during turnover have been proposed.
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PMID:Models for molybdenum coordination during the catalytic cycle of periplasmic nitrate reductase from Paracoccus denitrificans derived from EPR and EXAFS spectroscopy. 1041 73

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.
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PMID:A plasma membrane-bound enzyme of tobacco roots catalyses the formation of nitric oxide from nitrite. 1134 59

The nitrate reductase of the hyperthermophilic archaeon Pyrobaculum aerophilum was purified 137-fold from the cytoplasmic membrane. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the enzyme complex consists of three subunits with apparent molecular weights of 130,000, 52,000, and 32,000. The enzyme contained molybdenum (0.8-mol/mol complex), iron (15.4-mol/mol complex) and cytochrome b (0.49-mol/mol complex) as cofactors. The P. aerophilum nitrate reductase distinguishes itself from nitrate reductases of mesophilic bacteria and archaea by its very high specific activity using reduced benzyl viologen as the electron donor (V(max) with nitrate, 1,162 s(-1) (326 U/mg); V(max) with chlorate, 1,348 s(-1) (378 U/mg) [assayed at 75 degrees C]). The K(m) values for nitrate and chlorate were 58 and 140 microM, respectively. Azide was a competitive inhibitor and cyanide was a noncompetitive inhibitor of the nitrate reductase activity. The temperature optimum for activity was > 95 degrees C. When incubated at 100 degrees C, the purified nitrate reductase had a half-life of 1.5 h. This study constitutes the first description of a nitrate reductase from a hyperthermophilic archaeon.
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PMID:Properties of a thermostable nitrate reductase from the hyperthermophilic archaeon Pyrobaculum aerophilum. 1154 9

The amino acid modifier phenylglyoxal (PG) gradually inactivated the methyl viologen-coupled nitrate reductase activity of the anoxically grown whole cells of Paracoccus denitrificans. A double log plot of the pseudo-first-order inactivation rate constant versus PG concentration was linear with a mean slope of 1.4 (0.1M sodium phosphate) or 0.87 (0.1M sodium borate). Phenylglyoxalation of cells lowered the limiting velocity (V), while hardly affecting the apparent half-saturation concentration (K(m)) of nitrate. Nitrate afforded no protection against inactivation. The inhibition by PG could be removed by the detergent Triton X-100 or by the lipid-soluble tetraphenylphosphonium countercation, suggesting that PG exerts its effect at the level of nitrate transport. Based on studies with membrane potential- and pH-sensitive fluorescent probes, the inhibition was shown not to be due to changes in the electrochemical gradient of hydrogen ions. Both K(m) and V values for nitrate uptake increased in a hyperbolic fashion in response to exogenously added nitrite. Nitrite promoted a bypass of the inhibition caused by low concentrations of the proton-conducting agent carbonyl cyanide m-chlorophenylhydrazone (CCCP), but was almost ineffective in the case of the PG block. These results are rationalized in terms of two nitrate import pathways that are comparably inhibited by PG and differ in their sensitivities to CCCP. A simplified kinetic model for phenylglyoxalation is proposed to account for the observed nonintegral reaction orders.
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PMID:Inhibition by phenylglyoxal of nitrate transport in Paracoccus denitrificans: a comparison with the effect of a protonophorous uncoupler. 1250 99

Wild type (WT), and nitrate reductase (NR)- and nitrite-reductase (NiR)-deficient cells of Chlorella sorokiniana were used to characterize nitric oxide (NO) emission. The NO emission from nitrate-grown WT cells was very low in air, increased slightly after addition of nitrite (200 microM), but strongly under anoxia. Importantly, even completely NR-free mutants, as well as cells grown on tungstate, emitted NO when fed with nitrite under anoxia. Therefore, this NO production from nitrite was independent of NR and other molybdenum cofactor enzymes. Cyanide and inhibitors of mitochondrial complex III, myxothiazol or antimycin A, but not salicylhydroxamic acid (inhibitor of alternative oxidase) inhibited NO production by NR-free cells. In contrast, NiR-deficient cells growing on nitrate accumulated nitrite and emitted NO at very high equal rates in air and anoxia. This NO emission was 50% inhibited by salicylhydroxamic acid, indicating that in these cells the alternative oxidase pathway had been induced and reduced nitrite to NO.
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PMID:Mitochondrial electron transport as a source for nitric oxide in the unicellular green alga Chlorella sorokiniana. 1547 28

The salt-tolerant Rhodotorula glutinis yeast strain grew in medium containing nitrate, 1 mM tungsten, and trace amounts of molybdenum (as impurities from the reagents used). Isolation of electrophoretically homogenous preparation of nitrate reductase from the Rh. glutinis cells grown under these growth conditions is described. The isolated nitrate reductase is a molybdenum-containing homodimer with molecular mass of 130 kD, containing 0.177 mol of Mo per mol of the enzyme. The activity of the enzyme is maximal at pH 7.0 and 35-45 degrees C and is inhibited by low concentrations of azide and cyanide. The enzyme is almost insensitive to 1 mM tungsten.
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PMID:Isolation, purification, and characterization of nitrate reductase from a salt-tolerant Rhodotorula glutinis yeast strain grown in the presence of tungsten. 1609 46

Severely Ca-deficient Triticum aestivum L. seedlings accumulated high levels of nitrite and moderate levels of nitrate and organic nitrogen, but contained unaltered levels of hydroxylamine. Nitrite accumulation was not related to molybdenum deficiency, or altered cellular pH. Nitrate reductase was decreased by Ca deficiency, apparently by repression of enzyme synthesis from accumulated nitrite and not by inhibition of enzyme activity. Nitrite reductase and NADP diaphorase activities were not affected by Ca deficiency, and Ca did not restore activity to nitrite reductase inactivated by cyanide. The results indicated that the role of Ca is in intracellular transport of nitrite and not in induction or activity of enzymes.
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PMID:Evidence for a role of calcium in nitrate assimilation in wheat seedlings. 1665 39

THE ASSIMILATORY NITRATE REDUCTASE (NADH: nitrate oxidoreductase, E.C. 1.6.6.2.) from the marine diatom Thalassiosira pseudonana, Hasle and Heimdal, has been purified 200-fold and characterized. The regulation of nitrate reductase in response to various conditions of nitrogen nutrition has been investigated.Nitrate reductase activity is repressed by the presence of ammonium in vivo, and its synthesis is derepressed when ammonium is absent. The derepression process is sensitive to cycloheximide and apparently requires protein synthesis. Repression of enzyme activity by ammonium is neither inhibited nor delayed by the presence of cycloheximide. In vitro, ammonium does not inhibit enzyme activity.NADH is the physiological electron donor for the enzyme in a flavin-dependent reaction. Spectral studies have indicated the presence of a b-type cytochrome associated with the enzyme. It is possible to observe enzymatic oxidation-reduction reactions which represent partial functions of the over-all electron transport capacity of this enzyme. Nitrate reductase will accept electrons from artificial electron donors such as reduced methyl viologen in a flavin-independent reaction. Further, dithionitereduced flavin adenine dinucleotide can donate electrons to the enzyme to reduce nitrate to nitrite. Finally, the nitrate reductase will exhibit a diaphorase activity and reduce the artificial electron acceptor mammalian cytochrome c in flavin-adeninedinucleotide-dependent reaction.Inhibition studies with potassium cyanide, sodium azide, and o-phenanthroline have yielded indirect evidence for metal component (s) of the enzyme.The inhibition of the NADH-requiring enzyme activities by p-hydroxymercuribenzoate has shown that an essential sulfhydryl group is involved in the initial portion of the electron transport. Heat treatment exerts an effect similar to the p-hydroxymercuribenzoate inhibition; namely, the NADH-requiring activities are rapidly inactivated, whereas the terminal nitrate-reducing activities are relatively stable to heat.The T. pseudonana nitrate reductase molecule has the hydrodynamic properties of an ellipsoid with a frictional coefficient of 1.69 and a molecular weight of 330,000.
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PMID:Purification and Characterization of the Nitrate Reductase from the Diatom Thalassiosira pseudonana. 1665 41

The 4S cytochrome c (Cyt c) reductase activity of several plant species was markedly stimulated by cyanide and ferrocyanide but those of the 8S nitrate reductase component and other particulate components of the maize (Zea mays L.) scutellum by comparison, were increased only slightly. The effect of cyanide and ferrocyanide was not due to elimination of cytochrome oxidase interference but resulted from the stimulation of NADH-dependent reduction of Cyt c. A 4S Cyt c reductase component which could be isolated by ammonium sulfate fractionation and diethyl-aminoethyl-cellulose chromatography was found to be stimulated markedly by cyanide and ferrocyanide. The remaining 4S Cyt c reductase, which was insensitive to cyanide and ferrocyanide, was also fractionated with ammonium sulfate into two components. One of these, like the 8S Cyt c reductase, was sensitive to a protease from the maize roots which is relatively specific for nitrate reductase. This 4S Cyt c reductase species could be a subunit of nitrate reductase.
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PMID:Nitrate Reductase and Soluble Cytochrome c Reductase(s) in Higher Plants. 1666 Mar 77

Nitrate reductase from wheat (Triticum aestivum L. cv Bindawarra) leaves is inactivated by pretreatment with NADH, in the absence of nitrate, a 50% loss of activity occurring in 30 minutes at 25 degrees C with 10 micromolar NADH. Nitrate (50 micromolar) prevented inactivation by 10 micromolar NADH while cyanide (1 micromolar) markedly enhanced the degree of inactivation.A rapid reactivation of NADH-inactivated nitrate reductase occurred after treatment with 0.3 millimolar ferricyanide or exposure to light (230 milliwatts per square centimeter) plus 20 micromolar flavin adenine dinucleotide. When excess NADH was removed, the enzyme was also reactivated by autoxidation. Nitrate did not influence the rate of reactivation.Leaf nitrate reductase, from plants grown for 12 days on 1 millimolar nitrate, isolated in the late photoperiod or dark period, was activated by ferricyanide or light treatment. This suggests that, at these times of the day, the nitrate reductase in the leaves of the low nitrate plants is in a partially inactive state (NADH-inactivated). The nitrate reductase from moisture-stressed plants showed a greater degree of activation after light treatment, and inactive enzyme in them was detected earlier in the photoperiod.
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PMID:Reversible Inactivation of Nitrate Reductase by NADH and the Occurrence of Partially Inactive Enzyme in the Wheat Leaf. 1666 70


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