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)

Tobacco nitrate reductase (NR) produced in yeast retains cytochrome c reductase activity, but not NR activity. Biochemical data suggest that the haem and FAD domains are functional, and that the molybdenum cofactor (MoCo) domain is inactive owing to the absence of MoCo in yeast. The native form of the produced NR is dimeric. Thus MoCo is not involved in NR dimerization in higher plants, contrary to current assumptions.
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PMID:Characteristics of Nicotiana tabacum nitrate reductase protein produced in Saccharomyces cerevisiae. 189 32

The nitrate reductase (NADPH) (EC 1.6.6.3) from Aspergillus nidulans is influenced directly by mutations in the structural gene (niaD) for the major subunit of the enzyme and indirectly by mutation in any of several molybdenum cofactor loci (cnx). The cnxE-14 and the cnxH-3 mutants have been noted to contain the enzyme in two distinct forms following induction with nitrate. With the cnxH-3 as a prototype cnxH mutant, 10 other cnxH were found to be devoid of the assembled (dimeric) form of the enzyme. Two monoclonal antibodies specific for the native enzyme of the wild type (biA-1) recognized an epitope on the enzyme from the cnxE-14 and cnxH-3 mutants that was common to both and another that was unique to the cnxH gene specified protomer. Another monoclonal antibody recognized an epitope that occurs only in the assembled dimerio form of the enzyme from the wild type or the cnxE-14 mutant. The experiments further substantiate the cnxH phenotype as one involving unassembled protomers of the nitrate reductase in Aspergillus.
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PMID:Distinction of cnxH cofactor gene-specified protomers with monoclonal antibodies to Aspergillus nitrate reductase. 245 52

In Aspergillus nidulans, the nitrate assimilatory pathway is regulated by a variety of agents, one being the autogenous enzyme nitrate reductase. A major subunit of the enzyme which is specified by the niaD structural gene and is implicated in autogenous control exhibits both nitrate inducible diaphorase activity and ammonium repression. The former was used to test the extent to which alterations in the niaD specified protomer might affect its formation in selected niaD point and deletion mutants. Enzyme preparations from the wild type and mutant strains were compared on the basis of nitrate inducible co-activities and their reaction to specific monoclonal antibodies (MABS). Proteins in partially purified mycelial extracts were subjected to Western blot analyses with three MABs to functional native enzyme. Extracts of niaD point mutants exhibited nitrate induced co-activities which matched those of the wild type while those from deletion mutants were diminished or totally inactive. Nitrate reductase, from the wild type and specific cofactor mutants, shares an epitope common to both the monomeric and dimeric form in the case of one MAB, and exhibits epitopes unique to one or the other form in the case of the other two forms. Enzyme-antibody interaction occurs with or without inhibition of catalytic activity depending on the MAB involved.
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PMID:Monoclonal antibody probes for the niaD specified subunit in the NADPH-nitrate reductase from Aspergillus nidulans. 332 53

Assimilatory NADH:nitrate reductase from Chlorella is a homotetramer which contains one of each of the prosthetic groups FAD, heme, and Mo6+ per 100-kDa subunit. At low protein concentrations, this tetramer dissociates to a fully active dimer. To further elucidate the possible relationship between quaternary structure and activity, the functional size of nitrate reductase was determined by radiation inactivation analysis at high and low concentrations of enzyme where the principal physical species would be either tetrameric or dimeric, respectively. In both cases, the size obtained by this method was 100 kDa, suggesting that each subunit in the tetramer or dimer can function independently. These results confirm earlier results which indicated that the subunits are identical and that each contains a full complement of prosthetic groups. We also found that the functional sizes of the partial activities NADH:cytochrome c reductase, NADH:ferricyanide reductase, and reduced methyl viologen:nitrate reductase were fractions (approximately 58 kDa, 47 kDa, and 28 kDa, respectively) of the subunit molecular mass, suggesting that these domains are functionally independent.
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PMID:Radiation inactivation of assimilatory NADH:nitrate reductase from Chlorella. Catalytic and physical sizes of functional units. 351 Feb 7

The carbon monoxide oxidases (COXs) purified from the carboxydotrophic bacteria Pseudomonas carboxydohydrogena and Pseudomonas carboxydoflava were found to be molybdenum hydroxylases, identical in cofactor composition and spectral properties to the recently characterized enzyme from Pseudomonas carboxydovorans (O. Meyer, J. Biol. Chem. 257:1333-1341, 1982). All three enzymes exhibited a cofactor composition of two flavin adenine dinucleotides, two molybdenums, eight irons and eight labile sulfides per dimeric molecule, typical for molybdenum-containing iron-sulfur flavoproteins. The millimolar extinction coefficient of the COXs at 450 nm was 72 (per two flavin adenine dinucleotides), a value similar to that of milk xanthine oxidase and chicken liver xanthine dehydrogenase at 450 nm. That molybdopterin, the novel prosthetic group of the molybdenum cofactor of a variety of molybdoenzymes (J. Johnson and K. V. Rajagopalan, Proc. Natl. Acad. Sci. U.S.A. 79:6856-6860, 1982) is also a constituent of COXs from carboxydotrophic bacteria is indicated by the formation of identical fluorescent cofactor derivatives, by complementation of the nitrate reductase activity in extracts of Neurospora crassa nit-l, and by the presence of organic phosphate additional to flavin adenine dinucleotides. Molybdopterin is tightly but noncovalently bound to the protein. COX, sulfite oxidase, xanthine oxidase, and xanthine dehydrogenase each contains 2 mol of molybdopterin per mol of enzyme. The presence of a trichloroacetic acid-releasable, so-far-unidentified, phosphorous-containing moiety in COX is suggested by the results of phosphate analysis.
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PMID:Molybdopterin in carbon monoxide oxidase from carboxydotrophic bacteria. 658 59

Nitrate reductase of Neurospora crassa is a dimeric protein composed of two identical subunits, each possessing three separate domains, with flavin, heme, and molybdenum-containing cofactors. A number of mutants of nit-3, the structural gene that encodes Neurospora nitrate reductase, have been characterized at the molecular level. Amber nonsense mutants of nit-3 were found to possess a truncated protein detected by a specific antibody, whereas Ssu-1-suppressed nonsense mutants showed restoration of the wild-type, full-length nitrate reductase monomer. The mutants show constitutive expression of the truncated nitrate reductase protein; however normal control, which requires nitrate induction, was restored in the suppressed mutant strains. Three conventional nit-3 mutants were isolated by the polymerase chain reaction and sequenced; two of these mutants were due to the deletion of a single base in the coding region for the flavin domain, the third mutant was a nonsense mutation within the amino-terminal molybdenum-containing domain. Homologous recombination was shown to occur when a deleted nit-3 gene was introduced by transformation into a host strain with a single point mutation in the resident nit-3 gene. New, severely damaged, null nit-3 mutants were created by repeat-induced point mutation and demonstrated to be useful as host strains for transformation experiments.
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PMID:Molecular characterization of conventional and new repeat-induced mutants of nit-3, the structural gene that encodes nitrate reductase in Neurospora crassa. 847 43

The phototrophic bacterium Rhodobacter sphaeroides DSM 158 has a periplasmic nitrate reductase which is induced by nitrate and it is not repressed by ammonium or oxygen. In a Tn5 mutant lacking nitrate reductase activity, transposon insertion is localized in a 1.2 kb EcoRI fragment. A 0.6 kb BamHI-EcoRI segment of this region was used as a probe to isolate, from the wild-type strain, a 6.8 kb PstI fragment carrying the putative genes coding for the periplasmic nitrate reductase. In vivo protein expression and DNA sequence analysis reveal the presence in this region of three genes, napABC, probably organized in an operon. These genes are required for nitrate reduction, as deduced by mutational and complementation studies. The napA gene codes for a protein with a high homology to the periplasmic nitrate reductase from Alcaligenes eutrophus and, to a lesser extent, to other prokaryotic nitrate reductases and molybdenum-containing enzymes. The napB gene product has two haem c-binding sites and shows a high homology with the cytochrome c-type subunit of the periplasmic nitrate reductase from A. eutrophus. NAPA and NAPB proteins appear to be translated with signal peptides of 29 and 24 residues, respectively, indicating that mature proteins are located in the periplasm. The napC gene codes for a 25 kDa protein with a transmembrane sequence of 17 hydrophobic residues. NAPC has four haem c-binding sites and is homologous to the membrane-bound c-type cytochromes encoded by Pseudomonas stutzeri nirT and Escherichia coli torC genes. The phenotypes of defined insertion mutants constructed for each gene also indicate that periplasmic nitrate reductase from R. sphaeroides DSM 158 is a dimeric complex of a 90 kDa catalytic subunit (NAPA) and a 15 kDa cytochrome c (NAPB), which receives electrons from a membrane-anchored tetrahaem protein (NAPC), thus allowing electron flow between membrane and periplasm. This nitrate-reducing system differs from the assimilatory and respiratory bacterial nitrate reductases at the level of cellular localization, regulatory properties, biochemical characteristics and gene organization.
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PMID:Isolation of periplasmic nitrate reductase genes from Rhodobacter sphaeroides DSM 158: structural and functional differences among prokaryotic nitrate reductases. 873 Aug 72

The mob mutants of Escherichia coli are pleiotropically defective in molybdoenzyme activities because they are unable to catalyse the conversion of molybdopterin guanine dinucleotide, the active form of the molybdenum cofactor. The mob locus comprises two genes. The product of mobA, protein FA, has previously been purified to homogeneity and is able to restore molybdoenzyme activities following incubation with cell extracts of mob strains. The mobB gene, although not essential for the biosynthesis of active molybdoenzymes, encodes a protein which, sequence analysis strongly suggests, contains a nucleotide-binding site. We have overproduced the products of both the mobA and mobB genes in engineered E. coli strains and purified each to homogeneity. The preparation of protein FA (MobA) is simpler than that previously published and produces a much greater yield of active protein. The isolated MobB protein, which is dimeric in solution, acts in the presence of protein FA, to enhance the level of nitrate reductase activation achieved on incubation with mob cell extracts. Equilibrium dialysis experiments show that purified MobB binds 0.83 mol GTP/mol protein with a Kd of 2.0 microM. Isolated MobB also catalyses a low GTPase activity (turnover number of 3 x 10(-3) min-1) with a K(m) for GTP to GDP of 7.5 microM. Under the conditions tested, protein FA did not affect the GTP-binding or GTPase activity of MobB. Intrinsic (tryptophan) protein fluorescence measurements show that MobB also binds the nucleotides ATP, TTP and GDP, but with lower affinity than GTP. These results are consistent with a model whereby MobB binds the guanine nucleotide which is attached to molybdopterin during the biosynthesis of the molybdenum cofactor.
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PMID:The product of the molybdenum cofactor gene mobB of Escherichia coli is a GTP-binding protein. 921 27

We report the identification of a number of mutations that result in amino acid replacements (and their phenotypic characterization) in either the MogA-like domain or domains 2 and 3 of the MoeA-like region of the Aspergillus nidulans cnxE gene. These domains are functionally required since mutations that result in amino acid substitutions in any one domain lead to the loss or to a substantial reduction in all three identified molybdoenzyme activities (i.e., nitrate reductase, xanthine dehydrogenase, and nicotinate hydroxylase). Certain cnxE mutants that show partial growth with nitrate as the nitrogen source in contrast do not grow on hypoxanthine or nicotinate. Complementation between mutants carrying lesions in the MogA-like domain or the MoeA-like region, respectively, most likely occurs at the protein level. A homology model of CnxE based on the dimeric structure of E. coli MoeA is presented and the position of inactivating mutations (due to amino acid replacements) in the MoeA-like functional region of the CnxE protein is mapped to this model. Finally, the activity of nicotinate hydroxylase, unlike that of nitrate reductase and xanthine dehydrogenase, is not restored in cnxE mutants grown in the presence of excess molybdate.
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PMID:Mutational analysis of the gephyrin-related molybdenum cofactor biosynthetic gene cnxE from the lower eukaryote Aspergillus nidulans. 1207 59

Nitrate reductase (NR; EC 1.6.6.1-3) catalyzes NAD(P)H reduction of nitrate to nitrite. NR serves plants, algae, and fungi as a central point for integration of metabolism by governing flux of reduced nitrogen by several regulatory mechanisms. The NR monomer is composed of a ~100-kD polypeptide and one each of FAD, heme-iron, and molybdenum-molybdopterin (Mo-MPT). NR has eight sequence segments: (a) N-terminal "acidic" region; (b) Mo-MPT domain with nitrate-reducing active site; (c) interface domain; (d) Hinge 1 containing serine phosphorylated in reversible activity regulation with inhibition by 14-3-3 binding protein; (e) cytochrome b domain; (f) Hinge 2; (g) FAD domain; and (h) NAD(P)H domain. The cytochrome b reductase fragment contains the active site where NAD(P)H transfers electrons to FAD. A complete three-dimensional dimeric NR structure model was built from structures of sulfite oxidase and cytochrome b reductase. Key active site residues have been investigated. NR structure, function, and regulation are now becoming understood.
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PMID:NITRATE REDUCTASE STRUCTURE, FUNCTION AND REGULATION: Bridging the Gap between Biochemistry and Physiology. 1501 11


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