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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)

Reconstitution of the apoprotein of the molybdoenzyme nitrate reductase in extracts of the Neurospora crassa mutant nit-1 with molybdenum cofactor released by denaturation of purified molybdoenzymes is efficient in the absence of exogenous MoO2-4 under defined conditions. Evidence is presented that this molybdate-independent reconstitution is due to transfer of intact Mo cofactor, a complex of Mo and molybdopterin (MPT), the organic constituent of the cofactor. This complex can be separated from denatured protein by gel filtration, and from excess MoO2-4 by reverse-phase HPLC. Sulfite oxidase, native xanthine dehydrogenase, and cyanolyzed xanthine dehydrogenase are equipotent Mo cofactor donors. Other well-studied inactive forms of xanthine dehydrogenase are also shown to be good cofactor sources. Using xanthine dehydrogenase specifically radiolabeled in the cyanolyzable sulfur, it is shown that this terminal ligand of Mo is rapidly removed from Mo cofactor under the conditions used for reconstitution.
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PMID:The relationship of Mo, molybdopterin, and the cyanolyzable sulfur in the Mo cofactor. 623 87

The reduced, metal-free pterin of the molybdenum cofactor has been termed molybdopterin. Oxidation of any molybdopterin-containing protein in the presence or absence of iodine yields oxidized molybdopterin derivatives termed Form A and Form B, respectively. Application of these procedures to whole cells and cell extracts has demonstrated the presence of molybdopterin in wild-type Neurospora crassa, and its absence in the cofactor-deficient mutant nit-1. In order to demonstrate that the reconstitution of nitrate reductase activity in nit-1 extracts results from the incorporation of molybdopterin into the apoprotein, active molybdopterin, free of contaminating amino acids or peptides, was isolated from chicken liver sulfite oxidase and used in the reconstitution system. The results show that, during reconstitution, exogenous molybdopterin is specifically incorporated into the nitrate reductase protein, confirming the role of molybdopterin as the organic moiety of the molybdenum cofactor.
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PMID:In vitro reconstitution of nitrate reductase activity of the Neurospora crassa mutant nit-1: specific incorporation of molybdopterin. 623 11

The regulation of synthesis of the hydrogenase which is a component of the formate hydrogen-lyase complex was studied by means of a strain of Escherichia coli possessing a transcriptional fusion of the hydrogenase gene (hyd) with the lacZ gene (hyd::lac fusion). Formation of active hydrogenase in the wild strain requires the presence of nickel in the medium; transcription of the hyd gene, however, is independent from the presence of Ni2+. Ni2+ addition to Ni2+-prestarved cells did not lead to any activation of presumptive hydrogenase apoprotein. Regulatory mutants were isolated in which nitrate repression of hyd::lac expression was relieved. Two main classes of regulatory mutants were identified: (i) Mutants with a defect in nitrate reductase; (ii) mutants with a cis-dominant regulatory mutation closely linked to the hyd::lac fusion. In the presence of formate which acts as an inducer, the hyd::lac fusion was also expressed under aerobic conditions. The results infer that nitrate repression of transcription of the hydrogenase structural gene is not effected by nitrate itself but requires the function of the electron transport chain leading to nitrate and that mutations in the promoter/operator region of the hyd cistron may confer insensitivity to redox control both by oxygen and nitrate.
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PMID:Regulation of the synthesis of hydrogenase (formate hydrogen-lyase linked) of E. coli. 644 May 7

The isolation and characterization of mutants altered for nitrate assimilation in Neurospora crassa is described. The mutants isolated can be subdivided into five classes on the basis of growth test that correspond to the growth patterns of existing mutants at six distinct loci. Mutants with growth characteristics like those of nit-2, nit-3 and nit-6 are assigned to those loci on the basis of noncomplementation and lack of recombination. Mutants that, from their growth patterns, appear to lack the molybdenum-containing cofactor for both nitrate reductase and xanthine dehydrogenase subdivide into three loci (nit-y, nit-8 and nit-9), all of which are gentically distinct from nit-1. nit-9 is a complex locus consisting of three complementation groups and thus appears similar ao the cnxABC locus of Asperillus nidulans. Extensive complementational and recombinational analyses reveal that nit-4 and nit-5 are alleles of the same locus, and two new alleles of that locus have been isolated. The results indicate that, as in A. nidulans, nitrate assimilation in N. crassa requires at least four loci (nit-1, 7, 8 and 9) to produce the molybdenum co-factor for nitrate reductase (and xanthine dehydrogenase), one locus (nit-3) to code for the nitrate reductase apoprotein, one locus (nit-6) to code for the nitrite reductase approtein and only one lous (nit-4/5) for the regulation of induction of the pathway by nitrate and nitrite.
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PMID:The isolation and characterization of mutants defective in nitrate assimilation in Neurospora crassa. 644 99

A mutant of Escherichia coli K12 is described which is unable to reduce nitrate with a variety of physiological electron donors but which retains nitrate reductase activity with the artificial electron donor benzyl viologen. It is suggested that the affected gene, chlI, located close to chlC, encodes the cytochrome bNR apoprotein.
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PMID:A mutant defective in electron transfer to nitrate in Escherichia coli K12. 699 26

Demolybdo-nitrate reductase (cytochrome c reductase) (NADH: acceptor oxidoreductase, EC 1.6.99.3) of Chlorella vulgaris can be activated in vitro to nitrate reductase by insertion of Mo from molybdate into the apoprotein. Evidence is here presented that reduction of the enzyme by reduced pyridine nucleotides inhibits the process of molybdenum insertion. This report also describes the effect of molybdate and tungstate concentration on the activation process. The activation is sigmoidally related to molybdate concentration with a calculated Hill coefficient of NH = 3. At suboptimal molybdate concentrations, tungstate stimulates enzyme activation by molybdate; but at saturating molybdate concentrations, tungstate is inhibitory. These facts are regarded as an indication that molybdate and tungstate are both positive effectors of molybdenum incorporation, but that they are competitors for the active Mo center.
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PMID:Effect of reduced pyridine nucleotides and tungstate on the in vitro insertion of molybdenum into demolybdo-nitrate reductase of Chlorella vulgaris. 720 57

Eleven green individuals were isolated when 95000 M2 plants of barley (Hordeum vulgare L.), mutagenised with azide in the M1, were screened for nitrite accumulation in their leaves after nitrate treatment in the light. The selected plants were maintained in aerated liquid culture solution containing glutamine as sole nitrogen source. Not all plants survived to flowering and some others that did were not fertile. One of the selected plants, STA3999, from the cultivar Tweed could be crossed to the wild-type cultivar and analysis of the F2 progeny showed that leaf nitrite accumulation was due to a recessive mutation in a single nuclear gene, which has been designated Nir1. The homozygous nir1 mutant could be maintained to flowering in liquid culture with either glutamine or ammonium as sole nitrogen source, but died within 14 days after transfer to compost. The nitrite reductase cross-reacting material seen in nitrate-treated wild-type plants could not be detected in either the leaf or the root of the homozygous nir1 mutant. Nitrite reductase activity, measured with dithionite-reduced methyl viologen as electron donor, of the nitrate-treated homozygous nir1 mutant was much reduced but NADH-nitrate reductase activity was elevated compared to wild-type plants. We conclude that the Nir1 locus determines the formation of nitrite reductase apoprotein in both the leaf and root of barley and speculate that it represents either the nitrite reductase apoprotein gene locus or, less likely, a regulatory locus whose product is required for the synthesis of nitrite reductase, but not nitrate reductase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:nir1, a conditional-lethal mutation in barley causing a defect in nitrite reduction. 843 74

The C-terminal 268 residues of the spinach assimilatory NADH:nitrate reductase amino acid sequence that correspond to the flavin-containing domain of the enzyme have been selectively amplified and expressed as a recombinant protein in Escherichia coli. The recombinant protein, which was produced in both soluble and insoluble forms, was purified to homogeneity using a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP-agarose and FPLC gel filtration. The purified domain exhibited a molecular weight of approximately 30 kDa, estimated by polyacrylamide gel electrophoresis, and a molecular mass of 30,169 for the apoprotein determined by mass spectrometry, which also confirmed the presence of FAD. The UV/visible spectrum was typical of a flavoprotein, with maxima at 272, 386, and 461 nm in the oxidized form while CD spectroscopy yielded both positive and negative maxima at 313 and 382 nm and 461 and 484 nm, respectively. The purified domain showed immunological cross-reactivity with anti-spinach nitrate reductase polyclonal antibodies while both N-terminal and internal amino acid sequencing of isolated peptides confirmed the fidelity of the domain's primary sequence. The protein retained NADH-ferricyanide reductase activity (Vmax=84 micromol NADH consumer/min/nmol FAD) with Km's of 17 and 34 microM for NADH and ferricyanide, respectively, with a pH optimum of approximately 6.5 A variety of NADH-analogs could also function as electron donors, though with decreased efficiency, the most effective being reduced nicotinamide hypoxanthine dinucleotide (V(max) = 35 micromol NHDH consumer/min/nmol FAD) and Km = 22 microM). NAD+ was demonstrated to be a competitive inhibitor (Ki = 1.9 mM) while analysis of inhibition by a variety of NAD+-analogs indicated the most efficient inhibitor to be ADP (Ki = 0.2 mM), with analogs devoid of either the phosphate, ribose, or adenine moieties proving to be markedly less-efficient inhibitors. The isolated domain was also capable of reducing cytochrome b5 directly (V(max) = 1.2 micromol NADH consumed/min/nmol FAD, Km (cyt. b5) = 6 microM), supporting the FAD -> b557 -> Mo electron transfer sequence in spinach nitrate reductase.
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PMID:Spectroscopic and kinetic properties of a recombinant form of the flavin domain of spinach NADH: nitrate reductase. 861 85

The role of arbuscular mycorrhizal (AM) fungi in assisting their host plant in nitrate assimilation was studied. With polymerase chain reaction technology, part of the gene coding for the nitrate reductase (NR) apoprotein from either the AM fungus Glomus intraradices or from maize was specifically amplified and subsequently cloned and sequenced. Northern (RNA) blot analysis with these probes indicated that the mRNA level of the maize gene was lower in roots and shoots of mycorrhizal plants than in noncolonized controls, whereas the fungal gene was transcribed in roots of AM plants. The specific NR activity of leaves was significantly lower in AM-colonized maize than in the controls. Nitrite formation catalyzed by NR was mainly NADPH-dependent in roots of AM-colonized plants but not in those of the controls, which is consistent with the fact that NRs of fungi preferentially utilize NADPH as reductant. The fungal NR mRNA was detected in arbuscules but not in vesicles by in situ RNA hybridization experiments. This appears to be the first demonstration of differential formation of transcripts of a gene coding for the same function in both symbiotic partners.
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PMID:Expression of maize and fungal nitrate reductase genes in arbuscular mycorrhiza. 961 42

Two catalytically distinct molybdenum-free dissimilatory nitrate reductases, a soluble periplasmic and a membrane-bound one, were isolated from the vanadate-reducing facultatively anaerobic bacterium Pseudomonas isachenkovii and purified to electrophoretic homogeneity. The enzymes did not contain molybdenum, the periplasmic enzyme contained vanadium, whereas the membrane-bound enzyme was vanadium-free. Both nitrate reductases lacked molybdenum cofactor. This fact was proved by reconstitution of the apoprotein of the nitrate reductase of Neurospora crassa nit-1 mutant. This is the first demonstration of molybdenum-free and molybdenum cofactor-free nitrate reductases.
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PMID:Molybdenum-free nitrate reductases from vanadate-reducing bacteria. 988 95


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