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)

This study reports the molecular characterization of the nitrate-assimilation gene cluster from the opportunistic fungal pathogen Aspergillus fumigatus. A genomic fragment was isolated which contained the entire structural gene encoding nitrite reductase (niiA), plus segments of the nitrate reductase (niaD) and the nitrate transporter (crnA) genes. Nitrate-assimilation genes in A. fumigatus are physically linked and transcribed in the same direction as in A. nidulans. The nitrate-assimilation gene cluster is on the largest chromosome (5.3 Mb). The nitrite reductase (niiA) gene encodes a protein of 1110 amino acids that contains regions corresponding to FAD, NADPH, FeS and siroheme binding sites. Eight small introns interrupt the niiA open reading frame. The niaD-niiA intergenic regulatory region contains promoter consensus sequences including TATA, CAAT, and binding sites for the areA and nirA gene products. Northern analysis indicated that the expression of niaD, niiA and crnA are induced by nitrate and repressed by ammonium at the transcriptional level.
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PMID:Mapping of the nitrate-assimilation gene cluster (crnA-niiA-niaD) and characterization of the nitrite reductase gene (niiA) in the opportunistic fungal pathogen Aspergillus fumigatus. 950 95

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

Nitric oxide synthase (NOS) is distributed within the brain, and nitric oxide (NO) is felt to be involved in the pathophysiology of deterioration after head injury and cerebral ischemia. This study determined the levels of the stable end products of NOS (NOx=nitrite+nitrate) after traumatic brain injury (TBI) and transient cerebral ischemia. A fluorometric assay using nitrate reductase and the NADPH regenerating system was used to quantitate NOx in ultrafiltered (10-kDa cutoff) cortical and hippocampal extracts after reduction of nitrate. In TBI rats, both the plasma and tissue showed a sharp increase in NOx levels 5 min after injury. Plasma NOx returned to control levels by 2 h after injury. Ipsilateral-cortex NOx levels returned to control levels approximately 6 h after injury and remained constant from 6-24 h. Contralateral-cortex returned near to control levels after 1 h. Hippocampus also followed a similar trend. In gerbils, there was a significant elevation in tissue NOx levels immediately after 10 min transient cerebral ischemia, which gradually returned to control levels over 24 h reperfusion. This striking burst of NO synthesis immediately after injury is clearly evident whether the injury is head trauma or ischemia, or whether the measurements were performed on tissue or plasma. It is unknown whether endothelial NOS, neuronal NOS, or both caused the elevation of the NO end products seen after the CNS insults.
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PMID:Fluorometric assay of nitrite and nitrate in brain tissue after traumatic brain injury and cerebral ischemia. 963 Jun 67

Hansenula polymorpha (syn. Pichia angusta) is able to grow on nitrate as sole nitrogen source. Nitrate reductase (NR) assays, optimized in crude extracts from nitrate-grown cells, revealed that NR preferentially used NADPH, but also used NADH, as electron donor and required FAD for maximum activity. NR activity was present in nitrate-grown and nitrite-grown cells, and was absent in cells grown in ammonium, glutamate and methylamine. Addition of reduced nitrogen compounds to nitrate-grown cells led to loss of NR activity, even if added with nitrate. Under nitrogen starvation, NR activity was not observed; however, following growth on nitrate, NR activity is maintained in the absence of nitrate. Increases but not decreases in NR activity were dependent on protein synthesis. Conditions for chlorate selection were optimized, and Nit- (nitrate-) mutants were isolated. Some of these mutants showed reduced or absent NR activity. Sixty-one NR- mutants revealed the monogenic recessive nature of their lesions and were grouped in 10 complementation classes. These mutants will be used in gene cloning experiments aimed at identifying structural and regulatory elements involved in the first step of nitrate reduction.
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PMID:Nitrate reduction and the isolation of Nit- mutants in Hansenula polymorpha. 972 55

The cytochrome b reductase fragment of Neurospora crassa NADPH:nitrate reductase (EC 1.6.6.3) was overexpressed in Escherichia coli with a His-tag for purification after mutation of the NADPH binding site. The recombinant enzyme fragment was altered by site-directed mutagenesis guided by the three-dimensional structure of cytochrome b reductase fragment of corn NADH:nitrate reductase (EC 1.6.6.1). Substitution of Asp for Ser920 (using residue numbering for holo-NADPH:nitrate reductase of N. crassa) greatly increased preference for NADH. This mutant had nearly the same NADH:ferricyanide reductase kcat as wild-type with NADPH. Substitutions for Arg921 had little influence on coenzyme specificity, while substitution of Ser or Gln for Arg932 did. The cytochrome b reductase mutant with greatest preference for NADH over NADPH was the doubly substituted form, Asp for Ser920/Ser for Arg932, but it had low activity and low affinity for coenzymes, which indicated a general loss of specificity in the binding site. Steady-state kinetic constants were determined for wild type and mutants with NADPH and NADH. Wild type had a specificity ratio of 1100, which was defined as the catalytic efficiency (kcat/Km) for NADPH divided by catalytic efficiency for NADH, while Asp for Ser920 mutant had a ratio of 0.17. Thus, the specificity ratio was reversed by over 6000-fold by a single mutation. Preference for NADPH versus NADH is strongly influenced by presence/absence of a negatively charged amino acid side chain in the binding site for the 2' phosphate of NADPH in nitrate reductase, which may partially account for existence of bispecific NAD(P)H:nitrate reductases (EC 1.6.6.2).
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PMID:Engineering of pyridine nucleotide specificity of nitrate reductase: mutagenesis of recombinant cytochrome b reductase fragment of Neurospora crassa NADPH:Nitrate reductase. 975 Jan 71

2-Nitropropane (2-NP), a rat hepatocarcinogen, is denitrified to nitrite and acetone by rat liver microsomes; the denitrification rate is increased using microsomes from phenobarbital (PB)-pretreated rats. To obtain evidence that denitrification of 2-NP also occurs in vivo, we attempted to determine nitrite and nitrate levels in blood sera and urines of 2-NP-treated (1.5 mmol/kg, ip, once) rats with and without PB pretreatment (80 mg/kg, ip, once daily, 3 days), using enzymatic reduction followed by the standard Griess reaction. However, due to various interfering factors, including pigment from methemoglobinemia, we found the assay had to be modified as follows: (a) reduction of nitrate to nitrite was accomplished using NADPH and nitrate reductase, (b) excess NADPH, proteins, and interfering pigments were precipitated using zinc acetate and Na(2)CO(3), and (c) the Griess reagents were prepared in 3 N HCl rather than 5% H(3)PO(4). With these modifications it became possible to show that 2-NP is indeed metabolized to nitrite in vivo and that the metabolism is increased by PB pretreatment. Two hours after 2-NP administration, rat blood serum nitrate plus nitrite levels were approximately 1600 microM (PB-pretreated) and 940 microM (vehicle-pretreated controls). The PB-pretreated and control rats, respectively, excreted 250 and 120 micromol nitrate/nitrite in the 24-h urine post 2-NP treatment. The modifications described make the method more specific, reproducible, and more widely applicable.
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PMID:Analysis of nitrite/nitrate in biological fluids: denitrification of 2-nitropropane in F344 rats. 1070 89

Nitrate reductase (NaR) catalyses the reduction of nitrate to nitrite via a two-electron transfer. In fungi, the electron donor for NaR is NADPH whereas plants can have two enzymes, NADH:NaR and a bispecific NAD(P)H:NaR. PCR mutagenesis was employed to introduce mutations into the niaD gene of Aspergillus nidulans in order to identify residues involved in co-enzyme specificity. The niaD3000 mutation (NiaD T813D, K814Q) altered co-enzyme specificity: the new enzyme had high levels of NADH:NaR activity in vitro, whilst all NADPH-associated activity was lost. However, strains carrying this mutation did not grow on nitrate. Enzyme assays suggested that this was not due to inhibition of the mutant enzyme by NADPH. All revertants of the niaD3000 mutants had restored NADPH activity and lost NADH activity. Sequence analysis of these revertants showed that they all contained a single amino acid change at Asp-813, suggesting that this position is crucial to co-enzyme specificity. Further studies have shown that the mutant enzyme was not protected from deactivation by either co-factor in cell-free extracts (unlike the wild-type), and that induction of the glucose-6-phosphate dehydrogenase occurred independently of NADPH levels. These data highlight the importance of functional tests in vivo under physiological conditions.
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PMID:Structure-function analysis of NADPH:nitrate reductase from Aspergillus nidulans: analysis of altered pyridine nucleotide specificity in vivo. 1084 18

Recombinant Arabidopsis NADH:nitrate reductase was expressed in Pichia pastoris using fermentation. Large enzyme quantities were purified for pre-steady-state kinetic analysis, which had not been done before with any eukaryotic nitrate reductase. Basic biochemical properties of recombinant nitrate reductase were similar to natural enzyme forms. Molybdenum content was lower than expected, which was compensated for by activity calculation on molybdenum basis. Stopped-flow rapid-scan spectrophotometry showed that the enzyme FAD and heme were rapidly reduced by NADH with and without nitrate present. NADPH reduced FAD at less than one-tenth of NADH rate. Reaction of NADH-reduced enzyme with nitrate yielded rapid initial oxidation of heme with slower oxidation of flavin. Rapid-reaction freeze-quench EPR spectra revealed molybdenum was maintained in a partially reduced state during turnover. Rapid-reaction chemical quench for quantifying nitrite production showed that the rate of nitrate reduction was initially greater than the steady-state rate, but rapidly decreased to near steady-state turnover rate. However, rates of internal electron transfer and nitrate reduction were similar in magnitude with no one step in the catalytic process appearing to be much slower than the others. This leads to the conclusion that the catalytic rate is determined by a combination of rates with no overall rate-limiting individual process.
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PMID:Pre-steady-state kinetic analysis of recombinant Arabidopsis NADH:nitrate reductase: rate-limiting processes in catalysis. 1135 30

Haloferax mediterranei can use nitrate as sole nitrogen source during aerobic growth. We report here the purification and biochemical characterisation of the assimilatory nitrate reductase (EC 1.6.6.2) from H. mediterranei. The enzyme, as isolated, was composed of two subunits (105+/-1.3 kDa and 50+/-1.3 kDa) and behaved as a dimer during gel filtration (132+/-6 kDa). A pH of 9 and elevated temperatures up to 80 degrees C (at 3.1 M NaCl) are necessary for optimum activity. The enzyme stability and activity of the enzyme depend upon the salt concentration. Reduced methyl viologen was as effective as the natural electron donor ferredoxin in the catalytic process. In contrast, NADPH and NADH, which are electron donors in nitrate reductases from different non-photosynthetic bacteria, were ineffective.
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PMID:Assimilatory nitrate reductase from the haloarchaeon Haloferax mediterranei: purification and characterisation. 1173 Nov 52

The incubation of peroxynitrite (PN)-pretreated histone III-S (NH) with Escherichia coli nitrate reductase (cytochrome, NADPH/GSH-independent) and that of NADPH-treated NH (NHNADPH) with liver cytochrome P-450 reductase (NADPH-dependent) resulted in decreased 3-nitrotyrosine immunoreactivity found in Western blot analysis. Additionally, increased nitrate was noted as an end product of these reactions. These findings imply that varied enzymatic denitration/modification of NO/PN-reacted protein, either with or without a reductant, may be important in regulating related signal transduction cascade(s) and relieving oxidative stress.
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PMID:Protein denitration/modification by Escherichia coli nitrate reductase and mammalian cytochrome P-450 reductase. 1181 90


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