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)

Nitrate reductase (NAD(P)H:nitrate oxidoreductase, EC 1.6.6.2) of the unicellular alga Cyanidium caldarium can exist in two interconvertible forms; one catalytically active and one inactive. The inactive nitrate reductase can be activated by mild treatment with denaturing agents of protein. By treatment with urea or mersalyl, activation of both the NADPH and benzyl viologen activities can be realized under mild conditions, whereas by treatment with heat, the activation of benzyl viologen activity is concomitant with loss of the NADPH activity. On the other hand, both activities are activated and destroyed concomitantly by ethylene glycol. In the present of FAD, either activation of benzyl viologen activity or loss of NADPH activity upon heating occur only at higher temperatures. The existence of a controlling region in the nitrate reductase molecule is postulated.
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PMID:Active and inactive nitrate reductase. Effects of mild treatment with denaturing agents of protein. 718 70

Assimilatory NAD(P)H-nitrate reductase (EC 1.6.6.2) from Ankistrodesmus braunii has been purified to homogeneity by affinity chromatography on blue Sepharose. The specific activity of the purified enzyme is in the range of 72 to 80 units/mg of protein. The electronic spectrum of the native enzyme shows absorption maxima at 278, 414 (Soret), 532 (beta), 562 (alpha), and 669 nm and shoulders at 455 and 484 nm, with an A278/A414 ratio of 2.56. The reduced enzyme shows absorption maxima at 424 (Soret), 528 (beta), 557 (alpha),and 669 n. The enzyme complex (Mr = 467,400) is composed of eight similar subunits (Mr = 58,750) and contains 4 molecules of FAD, 4 heme groups, and 2 atoms of molybdenum. Labile sulfide and nonheme iron were not detected. Electron micrographs show the eight subunits arranged alternately in two planes, and an 8-fold rotational symmetry was deduced from highly magnified images processed by optical superposition.
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PMID:Composition and structure of assimilatory nitrate reductase from Ankistrodesmus braunii. 719

Chlorella vulgaris was cultured on an ammonia-mineral salts medium until the nitrate reductase content reached a minimal level. These ammonia-grown cells were then induced by nitrate in the absence of molybdenum and of tungsten. A demolybdo nitrate reductase developed and reached high levels. This protein contained very little nitrate-reducing capacity, but had the full cytochrome c-reducing capacity of normal nitrate reductase. It was purified to homogeneity by the same procedures previously developed for the purification of nitrate reductase. The purified enzyme contained 1 molecule of heme and 1 molecule of FAD/subunit, but no detectable molybdenum or tungsten. This cytochrome c reductase was completely inhibited by antibodies raised against purified nitrate reductase of Chlorella. Mixtures prepared from normal nitrate reductase and the demolybdoenzyme could not be resolved by disc gel electrophoresis or by centrifugation in a density gradient. By a two-step enzyme induction (1, incubation with nitrate in absence of Mo; 2, incubation with Mo in absence of nitrate) the process of nitrate reductase synthesis could be cleanly separated from growth into two steps: Step 1, induction of cytochrome c reductase, was completely inhibited by cycloheximide. Step 2 was unaffected by cycloheximide, and most of the nitrate reductase synthesized accumulated in the form of the reversibly inactivated HCN complex of the enzyme.
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PMID:Purification and characterization of demolybdo nitrate reductase (NADH-cytochrome c oxidoreductase) of Chlorella vulgaris. 719 74

Desulfovibrio desulfuricans (ATCC 27774), a strictly anaerobic sulfate-reducing bacteria, is able to perform anaerobic nitrate respiration in which nitrate is first reduced to nitrite by the action of nitrate reductase, and nitrite reductase then catalyzes the six-electron reduction of nitrite to ammonia. The nitrite reductase was found to be a membrane-bound enzyme and has been purified to electrophoretic homogeneity. The purified enzyme has a minimal Mr = 66,000 as judged by sodium dodecyl sulfate gel electrophoresis and contains 6 c-type heme groups/molecule. Pure nitrite reductase exhibits a typical c-type cytochrome absorption spectrum with reduced alpha-band at 552.5 nm. NADH and NADPH do not function as direct electron donors for the nitrite reductase. Desulfovibrio vulgaris hydrogenase, however, is able to transfer electrons from H2 to the nitrite reductase using FAD as the electron transfer mediator. The dithionite-reduced nitrite reductase was demonstrated to be auto-oxidizable even in the presence of potassium cyanide. On addition of nitrite, the dithionite-reduced enzyme is re-oxidized immediately. Hydroxylamine, however, can only partially re-oxidize the reduced enzyme. Ascorbate reduces the enzyme to a limited extent and the partially reduced enzyme is neither auto-oxidizable nor re-oxidizable by nitrite or hydroxylamine. Purified nitrite reductase has a pH optimum in the range of 8.0-9.5 and optimal activity at 57 degrees C. Purified nitrite reductase also has hydroxylamine reductase activity, and the Km for nitrite was determined to be 1.14 mM and that for hydroxylamine is 113.5 mM. The difference in Km values seems to exclude the possibility of hydroxylamine being a free intermediate in the reduction of nitrite.
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PMID:The isolation of a hexaheme cytochrome from Desulfovibrio desulfuricans and its identification as a new type of nitrite reductase. 730 57

The nitrate reductase (NR) gene niaA of the oomycete Phytophthora infestans was selected from a gene library by heterologous hybridization. NiaA occurs as a single-copy gene ant its expression is regulated by the nitrogen source. The nucleotide sequence of niaA was determined and comparison of the deduced amino-acid sequence of 902 residues with NRs of higher fungi and plants revealed a significant homology, particularly within the three cofactor-binding domains for molybdenum, heme and FAD. The P. infestans niaA gene was used as a model gene to test whether oomycete genes are functional in the ascomycete Aspergillus nidulans, a fungus which is highly accessible for molecular genetic studies. The complete niaA gene was stably integrated into the genome of a nia- deletion mutant of A. nidulans. However, transformants containing one or more copies of the niaA gene were not able to complement the nia- mutant. This suggests that there is no functional expression of the introduced niaA gene in A. nidulans. In addition, the activity of two other oomycete gene promoters was analyzed in a transient expression assay. Plasmids containing chimaeric genes with the promoter of the P. infestans ubiquitin gene ubi3R, or the Bremia lactucae ham34 gene, fused to the coding sequence of the Escherichia coli beta-glucuronidase (GUS) reporter gene, were transferred to A. nidulans protoplasts. No significant GUS activity was detectable indicating that the ubi3R and ham34 promoters are not active in A. nidulans. Apparently, the regulatory sequences which are sufficient for gene activation in oomycetes are not functional in the ascomycete A. nidulans.
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PMID:NiaA, the structural nitrate reductase gene of Phytophthora infestans: isolation, characterization and expression analysis in Aspergillus nidulans. 761 59

We report a simple, enzymatic, end point method for determining nitrate in serum and urine with use of nitrate reductase from Aspergillus sp. (EC 1.6.6.2). The decrease in absorbance at 340 nm as a result of the oxidation of beta-NADPH is measured. We used FAD as a supplementary electron carrier and added an internal standard to avoid interference from possible inhibitors of the enzymatic reaction. The method was linear from 5 to 200 mumol/L nitrate in serum. Within-run (n = 12) and total (n = 14 days) imprecisions (CV) were 5.1-7.7% and 6.2-9.8%, respectively, at 20-90 mumol/L nitrate in serum. Recoveries of added nitrate were 80-106%. The median (range) concentration in serum of 20 healthy subjects was 16 (0-42) mumol/L.
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PMID:Nitrate determination in biological fluids by an enzymatic one-step assay with nitrate reductase. 776 10

Differences in the amino acid sequence between the bispecific NAD(P)H-nitrate reductase of birch (Betula pendula Roth) and the monospecific NADH-nitrate reductases of a variety of other higher plants have been found at the dinucleotide-binding site in the FAD domain. To pinpoint amino acid residues that determine the choice of reducing substrate, we introduced mutations into the cDNA coding for birch nitrate reductase. These mutations were aimed at replacing certain amino acids of the NAD(P)H-binding site by conserved amino acids located at identical positions in NADH-monospecific enzymes. The mutated cDNAs were integrated into the genome of tobacco by Agrobacterium-mediated transformation. Transgenic tobacco (Nicotiana tabacum) plants were grown on a medium containing ammonium as the sole nitrogen source to keep endogenous tobacco nitrate reductase activity low. Whereas some of the mutated enzymes showed a slight preference for NADPH, as does the nonmutated birch enzyme, the activity of some others greatly depended on the availability of NADH and was low with NADPH alone. Comparison of the mutations reveals that replacement of a single amino acid in the birch sequence (alanine871 by proline) is critical for the use of reducing substrate.
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PMID:The choice of reducing substrate is altered by replacement of an alanine by a proline in the FAD domain of a bispecific NAD(P)H-nitrate reductase from birch. 778 4

Nitrate reductase is a multiredox enzyme possessing three functional domains associated with the prosthetic groups FAD, heme iron, and molybdopterin. In Aspergillus nidulans, it is encoded by the niaD gene. A homologous transformation system has been used whereby a major deletion at the niiAniaD locus of the host was repaired by gene replacement. Employing site-directed mutagenesis and this transformation system, nine niaD mutants were generated carrying specific amino acid substitutions. Mutants in which alanine replaced cysteine 150, which is thought to bind the molybdenum atom of the molybdenum-pterin, and in which alanine replaced histidine 547, which putatively binds heme iron, had no detectable nitrate reductase (NAR) activity. This clearly establishes an essential catalytic role for these residues. Of the remaining mutants, all altered in the NADPH/FAD domain, two were temperature-sensitive for NAR activity, two had reduced NAR activity levels, and three had normal levels. Since some of these mutants change residues conserved between homologous nitrate reductases from a wide range of species, it is clear that such amino acid identities do not necessarily signify essential roles for the activity of the enzyme. These findings are considered in the light of predicted structural/functional roles for the altered amino acids.
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PMID:Site-directed mutagenesis of nitrate reductase from Aspergillus nidulans. Identification of some essential and some nonessential amino acids among conserved residues. 789 4

A gene has been constructed coding for a chimeric flavocytochrome b5 protein that comprises the soluble domain of rat hepatic cytochrome b5 as the NH2-terminal portion of the chimera and the flavin-containing domain of spinach assimilatory NADH:nitrate reductase as the C terminus. The chimeric protein has been expressed in Escherichia coli and purified to homogeneity using a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP-agarose, anion-exchange chromatography, and fast protein liquid chromatography gel filtration with an estimated molecular mass of 43 kDa from polyacrylamide gel electrophoresis. Visible and fluorescence spectroscopy indicated the purified protein contained both a b-type cytochrome and FAD prosthetic groups. The chimeric hemoflavoprotein immunologically cross-reacted with both anti-rat cytochrome b5 and anti-spinach nitrate reductase polyclonal antibodies, indicating the conservation of antigenic determinants from both native domains. NH2-terminal and internal amino acid sequencing of the native and CNBr-digested protein confirmed the presence of peptides derived from both the heme- and flavin-binding portions of the sequence which were identical to the deduced amino acid sequence. The chimera exhibited both NADH: ferricyanide reductase and NADH:cytochrome c reductase activities with Vmax values of 88 and 37 mumol of NADH consumed per min/nmol of heme (mu = 0.05 and pH 7.0) and Km values of 2.1, 32, and 1.4 microM for NADH, ferricyanide, and cytochrome c, respectively. This work represents the first successful bacterial expression of a mammalian-plant chimeric metalloflavoprotein. The chimera exhibited properties extremely similar to those of the native cytochrome b5 heme and spinach nitrate reductase FAD components.
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PMID:Construction and expression of a flavocytochrome b5 chimera. 817 67

1. A soluble reduced Methyl Viologen-dependent assimilatory nitrate reductase from Azotobacter vinelandii strain UW136 grown aerobically on nitrate was purified to homogeneity by the criteria of nitrate reductase activity staining, and coincidence of a Coomassie Blue-staining protein band on polyacrylamide gels run under non-denaturing conditions. The specific activity was 3 mumol of NO2- formed/min per mg of protein. 2. Gel filtration on Superose-12 and SDS/PAGE showed that the enzyme had an M(r) of 105,000 and was monomeric. The enzyme contained 1 Mo atom, 4 Fe atoms and 4 acid-labile sulphide atoms per molecule; no evidence for the presence of cytochrome or FAD was found. 3. Mo was present in a molybdenum cofactor, which on extraction was capable of activating apo-(nit-1) nitrate reductase present in crude extracts of nit-1 mutants of Neurospora crassa. 4. As isolated, the enzyme had e.p.r. signals assigned to Mo(V) with g-values g1 = 2.023; g2 = 1.998; g3 = 1.993 and with gav. = 2.004 indicating an unusual environment of Mo in this enzyme. 5. Reduction with S2O4(2-) bleached the e.p.r. signals which, on reoxidation after the addition of NO3(2-) to initiate enzyme turnover, exhibited at short times Mo(V) signals similar to those of dissimilatory nitrate reductases, with g1 = 1.998; g2 = 1.989; g3 = 1.981 and gav. = 1.989. Prolonged incubation subsequently gave a mixture of both e.p.r. species. 6. Neither NADH nor NADPH was effective as an electron donor, but reduced Methyl Viologen (apparent Km 998 microM) and reduced Bromophenol Blue (apparent Km 158 microM) were effective. With these donors the apparent Km values for nitrate were 70 microM and 217 microM respectively.
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PMID:Purification and characterization of the assimilatory nitrate reductase of Azotobacter vinelandii. 838 Sep 91


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