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 enzyme nitrate reductase, which catalyzes the reduction of nitrate to nitrite, is a multi-redox center homodimeric protein. Each polypeptide subunit is approximately 100 kDa in size and contains three separate domains, one each for a flavin, a heme-iron, and a molybdopterin cofactor. The heme-iron domain of nitrate reductase has homology with the simple redox protein, cytochrome b5, whose crystal structure was used to predict a three-dimensional structure for the heme domain. Two histidine residues have been identified that appear to coordinate the iron of the heme moiety, while other residues may be important in the folding or the function of the heme pocket. Site-directed mutagenesis was employed to obtain mutants that encode nitrate reductase derivatives with eight different single amino acid substitutions within the heme domain, including the two central histidine residues. Replacement of one of these histidines by alanine resulted in a completely nonfunctional enzyme whereas replacement of the other histidine resulted in a stable and functional enzyme with a lower affinity for heme. Certain amino acid substitutions appeared to cause a rapid turnover of the heme domain, whereas other substitutions were tolerated and yielded a stable and fully active enzyme. Three different single amino acid replacements within the heme domain led to a dramatic change in regulation of nitrate reductase synthesis, with significant expression of the enzyme even in the absence of nitrate induction.
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PMID:Nitrate reductase of Neurospora crassa: the functional role of individual amino acids in the heme domain as examined by site-directed mutagenesis. 835 55

Alcaligenes eutrophus H16 shows three distinct nitrate reductase activities (U. Warnecke-Eberz and B. Friedrich, Arch. Microbiol. 159:405-409, 1993). The periplasmic enzyme, designated NAP (nitrate reductase, periplasmic), has been isolated. The 80-fold-purified heterodimeric enzyme catalyzed nitrate reduction with reduced viologen dyes as electron donors. The nap genes were identified in a library of A. eutrophus H16 megaplasmid DNA by using oligonucleotide probes based on the amino-terminal polypeptide sequences of the two NAP subunits. The two structural genes, designated napA and napB, code for polypeptides of 93 and 18.9 kDa, respectively. Sequence comparisons indicate that the putative gene products are translated with signal peptides of 28 and 35 amino acids, respectively. This is compatible with the fact that NAP activity was found in the soluble fraction of cell extracts and suggests that the mature enzyme is located in the periplasm. The deduced sequence of the large subunit, NAPA, contained two conserved amino-terminal stretches of amino acids found in molybdenum-dependent proteins such as nitrate reductases and formate dehydrogenases, suggesting that NAPA contains the catalytic site. The predicted sequence of the small subunit, NAPB, revealed two potential heme c-binding sites, indicating its involvement in the transfer of electrons. An insertion in the napA gene led to a complete loss of NAP activity but did not abolish the ability of A. eutrophus to use nitrate as a nitrogen source or as an electron acceptor in anaerobic respiration. Nevertheless, the NAP-deficient mutant showed delayed growth after transition from aerobic to anaerobic respiration, suggesting a role for NAP in the adaptation to anaerobic metabolism.
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PMID:Structure and function of a periplasmic nitrate reductase in Alcaligenes eutrophus H16. 837 34

Chimeric genes comprised of Rubisco small subunit transit peptide fused in frame with full-length and truncated sequences of a nitrate reductase (narB) structural gene of Synechococcus were constructed. Fusion proteins were synthesized in a rabbit reticulocyte system. In thylakoido integration of synthetic proteins resulted in the association of the full-length narB-coded protein to the Synechococcus photosynthetic membranes. The membrane-associated protein was sensitive to trypsin treatment but could not be removed by washing in the presence of NaBr. Trypsin pretreatment of thylakoids abolished the capability for association. The association of the narB-coded protein with thylakoids might require another membrane protein whose identity is not known. It is proposed that the Synechococcus narB polypeptide is a peripheral, membrane bound protein anchored to the thylakoids via a short hydrophobic domain while the major part of the protein resides on the outer side of the thylakoid membranes. The chimeric narB proteins were processed and imported by intact pea chloroplasts in vitro; however, the mature proteins were found localized in the stroma and not in the thylakoid membrane fraction. Similarly, the attempt to integrate the protein in vitro into isolated pea thylakoid membranes failed although these membranes incorporate early light-inducible proteins.
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PMID:Integration of a cyanobacterial protein involved in nitrate reduction (narB) into isolated Synechococcus but not into pea thylakoid membranes. 851 2

In Aspergillus nidulans the positive-acting, wide domain regulatory gene areA mediates nitrogen metabolite repression. Previous analysis demonstrated that the C-terminal 153 residues of the areA product (AREA) are inessential for at least partial expression of most genes subject to regulation by areA. Paradoxically, areAr2, a -1 frameshift replacing the wild-type 122 C-terminal residues with a mutant peptide of 117 amino acids, leads to general loss of function. To determine the basis for the areAr2 mutant phenotype, and as a means of delineating functional domains within the C-terminal region of AREA, we have selected and characterised areAr2 revertants. Deletion analysis, utilising direct gene replacement, extended this analysis. A mutant areA product truncated immediately after the last residue of the highly conserved GATA (DNA-binding) domain retains partial function. The areAr2 product retains some function with respect to the expression of uaZ (encoding urate oxidase) and the mutant allele is partially dominant with respect to nitrate reductase levels. Consistent with the areAr2 product having a debilitating biological activity, we have demonstrated that a polypeptide containing both the wild-type DNA-binding domain and the mutant C-terminus of AREA2 is able to bind DNA in vitro but no longer shows specificity for GATA sequences.
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PMID:Mutational analysis of the C-terminal region of AREA, the transcription factor mediating nitrogen metabolite repression in Aspergillus nidulans. 856 80

The narB gene from the cyanobacterium Synechococcus sp. PCC 7942 was cloned downstream from the LacI-regulated promoter Ptrc in the Escherichia coli vector pTrc99A, rendering plasmid pCSLM1. Addition of isopropyl-beta-D-thiogalactoside to E. coli (pCSLM1) resulted in the parallel expression of a 76 kDa polypeptide and a nitrate reductase activity with properties identical to those known for nitrate reductase isolated from Synechococcus cells. As is the case for nitrate reductase from Synechococcus cells, either reduced methyl viologen or reduced ferredoxin could be used as an electron donor for the reduction of nitrate catalyzed by E. coli (pCSLM1) extracts. This data shows that narB is a cyanobacterial structural gene for nitrate reductase.
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PMID:A cyanobacterial narB gene encodes a ferredoxin-dependent nitrate reductase. 862 15

Mutants of Anabaena sp. strain PCC 7120 that form heterocysts when grown on nitrate-containing media were isolated following nitrosoguanidine mutagenesis. Six independent mutants were isolated, and the characterization of one mutant, strain AMC260, which forms 6 to 8% heterocysts in the presence of nitrate, is presented. A 1.8-kb chromosomal fragment that complemented the AMC260 mutant was sequenced, and a 1.2-kb open reading frame, named moeA, was identified. The deduced amino acid sequence of the predicted Anabaena sp. strain PCC 7120 MoeA polypeptide shows 37% identity to MoeA from Escherichia coli, which is required for the synthesis of molybdopterin cofactor. Molybdopterin is required by various molybdoenzymes, such as nitrate reductase. Interruption of the moeA gene in Anabaena sp. strain PCC 7120 resulted in a strain, AMC364, that showed a phenotype similar to that of AMC260. We show that AMC260 and AMC364 lack methyl viologen-supported nitrate reductase activity. We conclude that the inability of the moeA mutants to metabolize nitrate results in heterocyst formation on nitrate-containing media. Northern (RNA) analysis detected a 1.5-kb moeA transcript in wild-type cells grown in the presence or absence of a combined nitrogen source.
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PMID:Nitrate reductase activity and heterocyst suppression on nitrate in Anabaena sp. strain PCC 7120 require moeA. 868 95

An operon including two new genes (nasS and nasT) has been defined, cloned and sequenced. The deduced NASS protein is homologous to NRTA from Synechococcus sp. and to NASF from Klebsiella pneumoniae, two proteins involved in nitrate uptake. The predicted NAST polypeptide is homologous to the regulator proteins of the two-component regulatory systems. NASS plays a negative regulatory role in the synthesis of the nitrate and nitrite reductase. NAST is required for the expression of the nitrite-nitrate reductase operon (nasAB). Expression of the nasST operon is not under the control of the NTR system and is not regulated by the nitrogen source. A Phi(nasA-lacZ) fusion has been used to analyse expression of the nasAB operon in three different genetic backgrounds with altered nitrate reductase activity. Beta-galactosidase activity in two of them was independent of nitrate but in a mutant unable to reduce nitrate, nas-4, it was normally induced by nitrate.
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PMID:nasST, two genes involved in the induction of the assimilatory nitrite-nitrate reductase operon (nasAB) of Azotobacter vinelandii. 874 40

Nitrogen-fixing Azotobacter chroococcum cells, but not ammonium- or nitrate-grown cells, exhibited two polypeptide components of 22 and 35 kDa, respectively, that we termed P22 and P35. Bidimensional polyacrylamide gel electrophoresis analysis of preparations from N2-fixing cells that had been transferred to nitrate medium and then incubated for 2 h revealed that P22 had shifted to a more acidic part of the gel while P35 did not change its electrophoretic pattern. Using [32P]orthophosphoric acid it could be demonstrated that the shift in mobility of P22 was due to the phosphorylation of the polypeptide dependent on nitrate (nitrite). The A. chroococcum TR1 strain, which is unable to use nitrate as a nitrogen source and displays activities of nitrogenase, nitrate reductase and nitrite reductase, exhibited both polypeptides. In contrast, P22 and P35 were absent from A. chroococcum MCD1, a mutant strain that cannot assimilate nitrate and lacks the nitrate-reducing enzymatic system. The results suggest that P22 could act as a sensor protein for nitrate in A. chroococcum.
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PMID:A sensor protein involved in induction of nitrate assimilation in Azotobacter chroococcum. 880 13

The bacterioferritin-associated ferredoxin (Bfd) of Escherichia coli is a 64-residue polypeptide encoded by the bfd gene located upstream of the gene (bfr) encoding the iron-storage haemoprotein, bacterioferritin. The Bfd sequence resembles those of the approximately 60-residue domains found in NifU proteins (required for metallocluster assembly), nitrite reductases, and Klebsiella pneumoniae nitrate reductase. These related-domains contain four well-conserved cysteine residues, which are thought to function as ligands to a [2Fe-2S] cluster. The Bfd protein was over-produced, purified, and characterised. Bfd was found to be a positively-charged monomer containing two iron atoms and two labile sulphides. Ultraviolet-visible, EPR, variable-temperature magnetic-circular dichroism and resonance Raman spectroscopies, together with cyclic voltogram measurements, revealed the presence of a [2Fe-2S]2+,+ centre (E1/2 = -254 mV) having remarkably similar properties to the Fe-S cluster of NifU. Bfd may thus be a 2Fe ferredoxin participating either in release/delivery of iron from/to bacterioferritin (or other iron complexes), or in iron-dependent regulation of bfr expression.
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PMID:Spectroscopic and voltammetric characterisation of the bacterioferritin-associated ferredoxin of Escherichia coli. 895 50

A 13-kb genomic region of Paracoccus dentrificans GB17 is involved in lithotrophic thiosulfate oxidation. Adjacent to the previously reported soxB gene (C. Wodara, S. Kostka, M. Egert, D. P. Kelly, and C. G. Friedrich, J. Bacteriol. 176:6188-6191, 1994), 3.7 kb were sequenced. Sequence analysis revealed four additional open reading frames, soxCDEF. soxC coded for a 430-amino-acid polypeptide with an Mr of 47,339 that included a putative signal peptide of 40 amino acids (Mr of 3,599) with a RR motif present in periplasmic proteins with complex redox centers. The mature soxC gene product exhibited high amino acid sequence similarity to the eukaryotic molybdoenzyme sulfite oxidase and to nitrate reductase. We constructed a mutant, GBsoxC delta, carrying an in-frame deletion in soxC which covered a region possibly coding for the molybdenum cofactor binding domain. GBsoxC delta was unable to grow lithoautotrophically with thiosulfate but grew well with nitrate as a nitrogen source or as an electron acceptor. Whole cells and cell extracts of mutant GBsoxC delta contained 10% of the thiosulfate-oxidizing activity of the wild type. Only a marginal rate of sulfite-dependent cytochrome c reduction was observed from cell extracts of mutant GBsoxC delta. These results demonstrated that sulfite dehydrogenase was essential for growth with thiosulfate of P. dentrificans GB17. soxD coded for a periplasmic diheme c-type cytochrome of 384 amino acids (Mr of 39,983) containing a putative signal peptide with an Mr of 2,363. soxE coded for a periplasmic monoheme c-type cytochrome of 236 amino acids (Mr of 25,926) containing a putative signal peptide with an Mr of 1,833. SoxD and SoxE were highly identical to c-type cytochromes of P. denitrificans and other organisms. soxF revealed an incomplete open reading frame coding for a peptide of 247 amino acids with a putative signal peptide (Mr of 2,629). The deduced amino acid sequence of soxF was 47% identical and 70% similar to the sequence of the flavoprotein of flavocytochrome c of Chromatium vinosum, suggesting the involvement of the flavoprotein in thiosulfate oxidation of P. denitrificans GB17.
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PMID:Cloning and characterization of sulfite dehydrogenase, two c-type cytochromes, and a flavoprotein of Paracoccus denitrificans GB17: essential role of sulfite dehydrogenase in lithotrophic sulfur oxidation. 926 Sep 41


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