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 refined crystal structures of the recombinant cytochrome b reductase fragment of corn (Zea mays) nitrate reductase, its ADP complex and the active-site mutant Cys242Ser are reported here. The native structure has been refined at 2.5 A resolution to a crystallographic R-factor of 18.7% with root-mean-square (r.m.s) deviations from standard bond lengths and angles of 0.013 A and 2.0 degrees. The diffraction pattern of the crystals is highly anisotropic and correction of this effect lowered the crystallographic R-factor by 5% during the refinement. The structure of the enzyme co-crystallized with ADP has been solved at 2.7 A resolution and refined to an R-factor of 18.6% with r.m.s. deviations from standard bond lengths and angles of 0.014 A and 2.1 degrees. It revealed the binding site of the ADP moiety of the NADH cofactor, which is the electron donor for nitrate reduction. Based on this structure, a model of NADH at the active site of the enzyme was built and the implications for electron transfer from NADH to the flavin cofactor are discussed. The crystal structure of an active-site mutant enzyme, Cys242Ser, has been solved by difference Fourier synthesis and refined to an R-factor of 19.0% to 3.0 A resolution with standard deviations of bond lengths and angles of 0.017 A and 2.5 degrees. This structure analysis suggests that the observed decrease in catalytic activity of this mutant might be due to misalignment of the nicotinamide ring in its binding site. A model of the heme-containing domain of nitrate reductase has been built based on the X-ray structure of bovine cytochrome b5 and has been docked with the cytochrome b reductase fragment of nitrate reductase. The model of the complex contains six salt-bridges at the domain-domain interface and a hydrophobic core. In this model, His48, an invariant residue in the cytochrome b reductase family, forms an interaction with the propionic acid group of the D-ring of the heme cofactor. This group is in contact with the C-8 methyl group of the flavin ring. Residues that might influence the redox potential of the flavin cofactor are proposed and their possible role in electron transfer is discussed.
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PMID:Structural studies on corn nitrate reductase: refined structure of the cytochrome b reductase fragment at 2.5 A, its ADP complex and an active-site mutant and modeling of the cytochrome b domain. 776 Mar 34

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

Assimilatory nitrate reductase from Chlorella vulgaris catalyzes the rate-limiting step, the conversion of nitrate to nitrite, in nitrate assimilation. Initial rate studies of nitrate reductase activity, performed under optimum conditions of constant ionic strength (mu = 0.2) and pH (8.0) and using NADH as reductant, indicated the absence of substrate inhibition at NADH concentrations below 300 microM and NO3- concentrations less than 3 mM. Chlorella nitrate reductase exhibited a marked preference for NADH (Vmax = 9.2 mumol NADH/min/nmol heme and Km = 2.3 microM) as the physiological electron donor but could also utilize alpha-NADH (Vmax = 5.6 mumol NADH/min/nmol heme and Km = 131 microM) and NADPH (Vmax = 0.6 mumol NADPH/min/nmol heme and Km = 910 microM) though with significantly decreased efficiency. Examination of various NADH-analogs indicated that reduced nicotinamide hypoxanthine dinucleotide (NHDH) was used most efficiently (Vmax = 9.3 mumol NHDH/min/nmol heme and Km = 7.9 microM), while reduced nicotinamide mononucleotide (NMNH) was utilized least efficiently (Vmax = 0.07 mumol NMNH/min/nmol heme and Km = 676 microM). Overall, modifications to the nicotinamide moiety or the addition of a phosphate group were observed to result in the most significant decreases in Vmax, indicating poor reducing substrates. Product inhibition studies indicated both NAD+ (Ki = 2.2 mM) and NADP+ (Ki = 10.5 mM) to be competitive inhibitors of Chlorella NR. A variety of NAD+ analogs were also determined to act as competitive inhibitors with varying degrees of efficiency. 3-Pyridinealdehyde adenine dinucleotide was the most efficient inhibitor (Ki = 0.74 mM) while nicotinamide was the least efficient (Ki = 18.1 mM). Overall, changing substituents on the nicotinamide ring or its complete deletion produced the most effective inhibitors compared to NAD+. In contrast, changes in the adenine or ribose moieties produced less effective inhibitors when compared to NAD+. These results represent the most comprehensive analysis of the effect of modifications of the physiological reductant (NADH) and product (NAD+) on nitrate reductase activity.
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PMID:Assimilatory nitrate reductase: reduction and inhibition by NADH/NAD+ analogs. 797 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

Five cysteine residues in the recombinant cytochrome b reductase domain of corn leaf NADH:nitrate reductase (EC 1.6.6.1) were modified by site-directed mutagenesis. At least two amino acid replacement mutants were generated for each of the 5 cysteines of this domain. Characteristics of the amino acid replacement mutants correlated well with the structural location of the cysteine residues in the preliminary three-dimensional model of the cytochrome b reductase domain: somewhat exposed cysteines could be replaced by hydrophilic amino acid residues, while more buried cysteines by hydrophobic residues. An exception was found for the invariant cysteine near the C terminus, which is found in all nitrate reductases and also in the closely related NADH: cytochrome b5 reductase, as well as, most other members of this flavoenzyme family. No substitution for the invariant cysteine yielded highly active enzyme, although these mutants had normal visible spectra. When the invariant cysteine was mutated to serine, the cytochrome b reductase domain was resistant to inhibition by pchloromercuribenzoate, an inhibitor of nitrate reductases. Kinetic analysis suggested that the catalytic efficiency of the mutant was markedly reduced. We concluded, the invariant cysteine plays an important role in catalysis and may be essential for high catalytic efficiency of nitrate reductases.
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PMID:Identification of an "essential" cysteine of nitrate reductase via mutagenesis of its recombinant cytochrome b reductase domain. 818 55

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

A recombinant protein corresponding to the putative heme-binding domain of assimilatory NADH:nitrate reductase from Chlorella vulgaris has been expressed and purified from transformed Escherichia coli BL21 cells. The recombinant protein, exhibited a subunit molecular mass of approximately 10 kDa with a N-terminal sequence beginning with the residues PAGA in agreement with that predicted by cDNA analysis. The UV-visible spectrum of the protein confirmed the incorporation of heme with maxima at 413 nm and 423, 528, and 557 nm for the oxidized and reduced forms, respectively. Circular dichroism spectra indicated the environment of the heme chromophore was very similar to that of the native enzyme. Potentiometric titrations of the recombinant heme domain yielded a midpoint potential of +16 mV (n = 1, pH 7), substantially higher than the values of -160 mV obtained for the native enzyme and -28 mV obtained for a previously expressed recombinant heme domain that contained part of the Mo-pterin domain. These results indicate that portions of the amino acid sequence that are involved in the formation of the Mo-pterin domain of Chlorella nitrate reductase influence the redox potential of the heme prosthetic group.
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PMID:Expression and characterization of the heme-binding domain of Chlorella nitrate reductase. 842 4

In this paper, we describe the gene (nia) coding for the apoenzyme of the nitrate reductase (NR) of petunia. A full-size genomic clone was isolated from a genomic library, using the tobacco nia2 cDNA as a probe, and sequenced. The open reading frame is interrupted by three introns and encodes a protein of 909 amino acids which reveals between 92% and 68% identity to the NADH NR apoenzyme from other higher plants. Southern analyses indicated that the NR apoenzyme is encoded by a single-copy gene, although another region homologous to part of nia was also identified. The analysis of the steady-state level of nia mRNA showed that the petunia nia is regulated by the nitrogen source and is under the control of the circadian rhythm.
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PMID:Analysis of the petunia nitrate reductase apoenzyme-encoding gene: a first step for sequence modification analysis. 851 83

In order to study the variation of nitrate reductase (NR) genes among grass species, gene number, intron size and number, and the heme-hinge fragment sequence of 25 grass species were compared. Genomic DNA cut with six restriction enzymes and hybridized with the barley NAD(P)H and NADH NR gene probes revealed a single NAD(P)H NR gene copy and two or more NADH NR gene copies per haploid genome in most of the species examined. Major exceptions were Hordeum vulgare, H. vulgare ssp. spontaneum, and Avena strigosa, which appeared to have a single NADH NR gene copy. The NADH NR gene intron number and lengths were examined by polymerase chain reaction amplification. Introns I and III appeared to be absent in at least one of the NADH NR genes in the grass species, while intron II varied from 0.8 to 2.4 kilobases in length. The NADH NR gene heme-hinge regions were amplified and sequenced. The estimated average overall nucleotide substitution rate in the sequenced region was 7.8 x 10(-10) substitutions/site per year. The synonymous substitution rate was 2.11 x 10(-9) substitutions/synonymous site per year and the nonsynonymous substitution rate was 4.10 x 10(-10) substitutions/nonsynonymous site per year. Phylogenetic analyses showed that all of the wild Hordeum species examined clustered in a group separate from H. vulgare and H. vulgare ssp. spontaneum.
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PMID:Variation of nitrate reductase genes in selected grass species. 853 1

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.
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PMID:Physiology and interaction of nitrate and nitrite reduction in Staphylococcus carnosus. 860 76


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