Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.7.1.2 (nitrate reductase)
 3,861 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The complete primary structure of rubredoxin (Rd) isolated from Clostridium perfringens was sequenced to be: MKKFICDVCGYIYDPAVGDPDNGVEPGTEFKDIPDDWVCPLCGVDKSQFSETEE. The sequence was highly homologous to that of C. pasteurianum Rd but was different at 13 sites out of the total 54 amino acid residues (76% homology). It contained 1 Fe atom, 4 cysteine residues, and no labile sulfur, had a molecular weight of 6,056, and shared the general properties of classical anaerobic Rds. The pI was 4.4. The Rd was reduced with NADH in the presence of a specific NAD(P)H oxidoreductase preparation from the bacterium. The Km value of nitrate reductase for Rd as an electron-donor was 12 microM, a value comparable to that of the 13 microM for ferredoxin (Fd). These results taken together provide additional support for its role as the electron carrier in the nitrate reductase system [Seki, S., Ikeda, A., and Ishimoto, M. (1988) J. Biochem. 103, 583-584].
 ...
PMID:Rubredoxin from Clostridium perfringens: complete amino acid sequence and participation in nitrate reduction. 255 84

The NAD(P)H-dependent nitrate reductase system in Clostridium perfringens was reconstituted with rubredoxin (Rd), nitrate reductase (NaR), and an unadsorbed fraction, on a DEAE-cellulose column, of the extract (designated as fraction A), under nitrogen gas. Ferredoxin in place of Rd was not effective as an electron carrier in this reconstituted system. NAD(P)H-dependent nitrate reducing activity was also obtained by replacing fraction A with ferredoxin-NADP+ reductase from spinach. We propose the following scheme for the electron transfer in this NAD(P)H dependent nitrate reduction system. NAD(P)H----NAD(P)H-Rd reductase----Rd----NaR----NO3-.
 ...
PMID:Rubredoxin as an intermediary electron carrier for nitrate reduction by NAD(P)H in Clostridium perfringens. 290 73

Nitrate reductase activity is usually measured by colorimetric determination of the nitrite formed. Since reduced pyridine nucleotides interfere with color formation, the use of NADPH or NADH in the assay requires a specific postassay treatment to remove excess substrate. A "stop mix" containing 1.5 mM phenazine methosulfate and 4.0 mM ferricyanide (final concentrations 0.136 and 0.36 mM, respectively) can remove excess NAD(P)H and terminate the enzymatic reaction quickly in a single, time-saving step. For activity tests containing dithionite we recommend the use of a 1:1 mixture of the two color reagents to avoid incomplete color formation. This may occur during longer time intervals between addition of the color reagents due to destruction of the diazonium salt formed with the first reagent by oxidation product(s) of dithionite.
 ...
PMID:Nitrate reductase activity test: phenazine methosulfate-ferricyanide stop reagent replaces postassay treatment. 293 67

Incubation of the complex metalloflavoprotein, assimilatory nitrate reductase with N-ethylmaleimide, or a spin-labeled analog, 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl, resulted in a time-dependent inactivation of NADH:nitrate reductase and NADH: cytochrome-c reductase activity with no effect on reduced methyl viologen:nitrate reductase activity. Inactivation of the enzyme, which could be prevented by incubation in the presence of NADH, was achieved following modification of a single sulfhydryl group determined from [3H]N-ethylmaleimide incorporation and quantitation of the EPR spectrum of the spin-labeled enzyme. Sulfhydryl group modification precluded reduction of the enzyme by NADH and NAD+ binding. The EPR spectrum of the spin-labeled enzyme revealed the presence of a single species with the nitroxide retaining substantial motional freedom. Cleavage of the spin-labeled enzyme using corn-inactivating protease and separation into its flavin and molybdenum/heme domains followed by EPR spectroscopy revealed the modified sulfhydryl group to be associated with the latter fragment suggesting a close interaction of these domains in the region of the nucleotide-binding site.
 ...
PMID:The role of the essential sulfhydryl group in assimilatory NADH: nitrate reductase of Chlorella. 300 65

Biochemical and microbiological studies were conducted to characterize the mechanism of bacterial formation of N-nitrosomorpholine from morpholine and nitrite at neutral pH. Nitrosating activity was markedly induced when bacteria were cultured anaerobically in minimal culture medium containing nitrate, while the presence of cysteine or tungsten in the medium inhibited induction. Of various metals, coenzymes and inhibitors tested for their effects on in vitro nitrosation of morpholine, potassium cyanide, sodium azide, NAD(P)H and nitrate strongly inhibited nitrosation. Several mutants of Escherichia coli A10 strain were prepared in order to examine whether nitrosation activity is linked to specific loci. Niridazole-resistant mutants, which lack nitroreductase, had as much nitrosating activity as the original E. coli A10, but chlorate-resistant mutants had completely lost this activity. A good correlation was observed between nitrate reductase activity and nitrosating activity in these mutants. These results indicate that bacterial nitrosation is an enzyme-mediated reaction closely associated with molybdenoenzymes such as the nitrate reductase/formate hydrogenlyase system.
 ...
PMID:Biochemical studies on the catalysis of nitrosation by bacteria. 330 Oct 45

Expression of the tripeptide permease gene tppB is anaerobically induced. This induction is independent of the fnr (oxrA) gene product, which is known to be required for the anaerobic induction of several respiratory enzymes. We isolated, characterized, and mapped mutations in two genes, oxrC and tppR, which prevent the anaerobic induction of tppB expression. Mutations in oxrC were highly pleiotropic, preventing the anaerobic expression of the formate dehydrogenase component of formate hydrogen lyase (fhl), a tripeptidase (pepT), and two of the three known hydrogenase isoenzymes (hydrogenases 1 and 3). On the other hand, expression of nitrate reductase, fumarate reductase, and a number of other fnr (oxrA)-dependent enzymes was not affected by mutations in oxrC. Thus, there appeared to be at least two distinct classes of anaerobically induced genes, those which required fnr for their expression and those which required oxrC. It seems that fnr-dependent enzymes perform primarily respiratory functions, whereas oxrC-dependent enzymes served fermentative or biosynthetic roles. We found the primary defect of oxrC mutants to be a deficiency in phosphoglucose isomerase activity, implying that a product of glycolysis functions as an anaerobic regulatory signal. Mutations in tppR were specific for tppB and did not affect expression of other oxrC-dependent genes. However, tppR did exhibit phenotypes other than the regulation of tppB. Both oxrC and tppR mutants were hypersensitive to the toxic NAD analog 6-aminonicotinic acid. This suggests that oxrC and tppR may play a role in the regulation of NAD biosynthesis or, alternatively, that NAD or a related nucleotide serves as the anaerobic signal for oxrC-dependent enzymes.
 ...
PMID:Two genetically distinct pathways for transcriptional regulation of anaerobic gene expression in Salmonella typhimurium. 353 Nov 76

The oxidation-reduction midpoint potential for the heme prosthetic group present in assimilatory nitrate reductase from Chlorella vulgaris has been determined by optical potentiometric titrations in the presence of dye mediators. At pH 7, the midpoint potential was determined to be -160 mV and corresponds to a reversible n = 1 redox process. The midpoint potential was unaltered by the use of NADH as reductant, unaffected by the presence of NAD+, cytochrome c, phosphate, cyanide, or alkaline pH. In addition, the redox potential of the heme was independent of modifications to the enzyme such as substitution of the molybdenum center with tungsten, or cleavage and separation of the enzyme into its flavin and heme/molybdenum domains. In contrast, the midpoint potential increased on decreasing the pH yielding a pH dependence of approximately 20 mV/pH unit within the range 5.5 to 7, suggesting the presence of a single, redox-associated, ionizable functional group on the protein with pKox = 5.8 and pKred = 6.1. At pH 7 and within the range 12 to 38 degrees C, the midpoint potential of the heme decreased by approximately 1 mV/degree. Values for delta S0 and delta H0 were calculated to be -25.6 e.u. and -4.0 kcal/mol.
 ...
PMID:Thermodynamic properties of the heme prosthetic group in assimilatory nitrate reductase. 370 Mar 73

The addition of nitrate, EDTA and dithiothreitol to the enzyme extraction buffer resulted in improved stability of the assimilatory nitrate reductase activity from the food yeast Candida utilis at both 4 degrees C and -10 degrees C. By incorporating this critical step in the following sequence the yeast NAD(P)H: nitrate oxidoreductase (EC 1.6.6.2) was purified approximately 68-fold by protamine sulphate precipitation, calcium gel adsorption, ion exchange chromatography and gel filtration. Both NADPH-nitrate reductase and NADH-nitrate reductase activities remained in constant association and ratio (2:3) during the entire course of purification. The enzyme showed an absolute requirement of NADPH or NADH for its activity. Maximal enzyme activity was obtained with 10-120 micrograms protein in a 10 min assay at 30 degrees C at pH 6.5, with an apparent Michaelis constant of 0.69 mM for nitrate as substrate. The enzyme is a molybdoflavo-protein involving sulphydryl groups, and is highly sensitive to free reducing agents, heavy metal ions and electron-transfer inhibitors. The results also suggested possible involvement of a second metal ion, perhaps iron, which was hypothesized to participate in the electron transfer scheme catalysed by this enzyme.
 ...
PMID:Partial purification and properties of the assimilatory nitrate reductase of the food yeast Candida utilis. 378 22

Chlorella nitrate reductase catalyzes the reduction of nitrate to nitrite by NADH. Initial velocity studies showed that the kinetic mechanism is sequential, indicating that both substrates must bind to the enzyme before any products are released. Product inhibition with NAD and nitrite showed that competitive inhibition was observed when the inhibitor was similar to the varied substrate, while noncompetitive inhibition was observed when the inhibitor was dissimilar to the varied substrate. Likewise, dead-end inhibition with adenosine 5'-diphosphoribose and thiocyanate showed competitive inhibition when the inhibitor was similar to the varied substrate and noncompetitive inhibition when the inhibitor was dissimilar to the varied substrate. These results indicate that Chlorella nitrate reductase follows a random bi bi kinetic mechanism. Phosphate was found to stimulate NADH:nitrate reductase activity and 2-fold. The NADH:cytochrome c reductase activity associated with nitrate reductase was not affected by phosphate suggesting the effect of phosphate is on the nitrate-reducing moiety of the enzyme. Phosphate increases Vmax but has no effect on the apparent Km for nitrate.
 ...
PMID:Kinetic mechanism of assimilatory NADH:nitrate reductase from Chlorella. 627 5

When strains and mutants of the strictly aerobic hydrogen-oxidizing bacterium Alcaligenes eutrophus are grown heterotrophically on gluconate or fructose and are subsequently exposed to anaerobic conditions in the presence of the organic substrates, molecular hydrogen is evolved. Hydrogen evolution started immediately after the suspension was flushed with nitrogen, reached maximum rates of 70 to 100 mumol of H2 per h per g of protein, and continued with slowly decreasing rates for at least 18 h. The addition of oxygen to an H2-evolving culture, as well as the addition of nitrate to cells (which had formed the dissimilatory nitrate reductase system during the preceding growth), caused immediate cessation of hydrogen evolution. Formate is not the source of H2 evolution. The rates of H2 evolution with formate as the substrate were lower than those with gluconate. The formate hydrogenlyase system was not detectable in intact cells or crude cell extracts. Rather the cytoplasmic, NAD-reducing hydrogenase is involved by catalyzing the release of excessive reducing equivalents under anaerobic conditions in the absence of suitable electron acceptors. This conclusion is based on the following experimental results. H2 is formed only by cells which had synthesized the hydrogenases during growth. Mutants lacking the membrane-bound hydrogenase were still able to evolve H2. Mutants lacking the NAD-reducing or both hydrogenases were unable to evolve H2.
 ...
PMID:Hydrogen evolution by strictly aerobic hydrogen bacteria under anaerobic conditions. 637 84


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>