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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)

Nitrate reductase (EC 1.6.6.1) from Chlorella vulgaris, a flavin-cytochrome-molybdenum enzyme, catalyses two types of partial reactions: reduction of exogenous cytochrome c by NADH and reduction of nitrate to nitrite by reduced methyl viologen (reduced 1,1'-dimethyl-4,4'-dipyridine dichloride). Ferrate, an analogue of orthophosphate acting on the phosphate-binding region of the enzymes, abolishes the NADH-nitrate reductase as well as the NADH-cytochrome c activities. In addition, the ability of NADH to reduce the endogenous cytochrome b component of the enzyme is also impaired. The reduction of nitrate by reduced methyl viologen is only partially affected. The results indicate that the ferrate primarily disrupts the NADH site.
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PMID:Inactivation of the NADH-dependent activities of nitrate reductase by ferrate. 409 Aug 56

1. In Aspergillus nidulans nitrate and nitrite induce nitrate reductase, nitrite reductase and hydroxylamine reductase, and ammonium represses the three enzymes. 2. Nitrate reductase can donate electrons to a wide variety of acceptors in addition to nitrate. These artificial acceptors include benzyl viologen, 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride, cytochrome c and potassium ferricyanide. Similarly nitrite reductase and hydroxylamine reductase (which are possibly a single enzyme in A. nidulans) can donate electrons to these same artificial acceptors in addition to the substrates nitrite and hydroxylamine. 3. Nitrate reductase can accept electrons from reduced benzyl viologen in place of the natural donor NADPH. The NADPH-nitrate-reductase activity is about twice that of reduced benzyl viologen-nitrate reductase under comparable conditions. 4. Mutants at six gene loci are known that cannot utilize nitrate and lack nitrate-reductase activity. Most mutants in these loci are constitutive for nitrite reductase, hydroxylamine reductase and all the nitrate-induced NADPH-diaphorase activities. It is argued that mutants that lack nitrate-reductase activity are constitutive for the enzymes of the nitrate-reduction pathway because the functional nitrate-reductase molecule is a component of the regulatory system of the pathway. 5. Mutants are known at two gene loci, niiA and niiB, that cannot utilize nitrite and lack nitrite-reductase and hydroxylamine-reductase activities. 6. Mutants at the niiA locus possess inducible nitrate reductase and lack nitrite-reductase and hydroxylamine-reductase activities. It is suggested that a single enzyme protein is responsible for the reduction of nitrite to ammonium in A. nidulans and that the niiA locus is the structural gene for this enzyme. 7. Mutants at the niiB locus lack nitrate-reductase, nitrite-reductase and hydroxylamine-reductase activities. It is argued that the niiB gene is a regulator gene whose product is necessary for the induction of the nitrate-utilization pathway. The niiB mutants either lack or produce an incorrect product and consequently cannot be induced. 8. Mutants at the niiribo locus cannot utilize nitrate or nitrite unless provided with a flavine supplement. When grown in the absence of a flavine supplement the activities of some of the nitrate-induced enzymes are subnormal. 9. The growth and enzyme characteristics of a total of 123 mutants involving nine different genes indicate that nitrate is reduced to ammonium. Only two possible structural genes for enzymes concerned with nitrate utilization are known. This suggests that only two enzymes, one for the reduction of nitrate to nitrite, the other for the reduction of nitrite to ammonium, are involved in this pathway.
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PMID:Genetic and biochemical studies of nitrate reduction in Aspergillus nidulans. 438 27

The reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate oxidoreductase (EC 1.6.6.2) from Aspergillus nidulans was purified over 200-fold by use of salt fractionation, gel filtration, and ion-exchange chromatography. The purified enzyme was specific for NADPH and catalyzed reduction of nitrate, cytochrome c from isolated mitochondria of Aspergillus, and mammalian cytochrome c. An S(0.725) (20, w) of 7.8 was derived with sucrose density gradient centrifugation, and a Stokes radius of 6.4 nm was derived by gel filtration on Sephadex G-200. From these values, a molecular weight of 197,000 was computed, assuming v = 0.725 cm(3)/g. The spectral properties of the purified enzyme suggested a flavine component was present but revealed no pattern indicative of a hemoprotein. A cytochrome c, similar to the cytochrome c from isolated mitochondria, was found unassociated with the nitrate reductase after ion-exchange chromatography. No NADPH-nitrate reductase activity was detected in isolated mitochondria. Spectrally discernable reduction of the flavine component of the enzyme at 450 nm was noted after reaction with NADPH. This reduction was inhibited by p-chloromercuribenzoate but not by KCN. The addition of nitrate to NADPH reduced enzyme caused a reoxidation of the flavine component via a reaction which was inhibited by KCN but not by p-chloromercuribenzoate. The half-life of the purified enzyme at 37 C was 20 min for NADPH-nitrate reductase and 35 min for NADPH-cytochrome c reductase.
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PMID:Characterization of the reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase of Aspergillus nidulans. 439 43

The addition of nitrate to cultures of Spirillum itersonii incubated under low aeration produced a diauxic growth pattern in which the second exponential phase was preceded by the appearance of nitrite in the medium. The organism also grew anaerobically in the presence of nitrate. Nitrate reductase activity could be demonstrated in cell-free extracts by use of reduced methyl viologen as the electron donor. The enzyme was located in the supernatant fraction after centrifugation of extracts for 2 hr at 40,000 x g, and it sedimented as a single peak when centrifuged in a sucrose gradient. Nitrate reductase activity was found in cells grown with low aeration without nitrate, but was increased about twofold by addition of nitrate. Enzyme activity was negligible in cells grown with high aeration. The proportion of soluble cytochrome c was increased two- to threefold in cells grown with nitrate. The specific activities of nitrate reductase and soluble cytochrome c rose when nitrate or nitrite was added to cell suspensions incubated with low aeration; nitrite was more effective than nitrate during the early stages of incubation. A nitrate reductase-negative mutant synthesized increased amounts of soluble cytochrome c in response to nitrate or to nitrite in the cell suspension system. It is concluded that enhanced synthesis of soluble cytochrome c does not require the presence of a functional nitrate reductase.
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PMID:Nitrate reductase and soluble cytochrome c in Spirillum itersonii. 542 24

In order to identify the b-type cytochrome involved in the nitrate reduction in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans, the b-type cytochromes in the spheroplast membranes were characterized. Difference spectra at 77K of spheroplast membranes indicated the presence of two b-type cytochromes with a bands at 556.5 and 562 nm. Three components considered to be of the b-type cytochrome were resolved by anaerobic potentiometric titration at 560-572 nm. Their midpoint potentials at pH 7, Em,7, were - 135 mV, +40 mV and +175 nm and their approximate reduced minus oxidized maxima were determined to be at 565 nm (562 nm at 77K), 560 nm (556.5 nm) and 560 nm (556.5 nm), respectively. These values are almost the same as those reported for R. sphaeroides. The Em,7 value of the cytochrome c involved in the nitrate reductase of this denitrifier was determined to be 250 mV. A b-type cytochrome reduced with NADH and FMN was oxidized by nitrate in chromatophore membranes. The possibility that cytochrome b (Em,7 = 175 mV) is involved in the nitrate reduction is discussed.
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PMID:Redox properties of membrane-bound b-type cytochromes and a soluble c-type cytochrome of nitrate reductase in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans. 608 70

A gentamicin-resistant mutant of Pseudomonas aeruginosa PAO503 was selected after ethyl methane sulfonate mutagenesis. The strain, P. aeruginosa PAO2401 had increased resistance to all aminoglycosides tested but exhibited no change for other antibiotics. The mutation designated aglA (aminoglycoside resistance) was 50% cotransducible with the 8-min ilvB,C marker on the P. aeruginosa chromosome. It showed a marked reduction in cytochrome c(552) and nitrate reductase (Nar) and a change in terminal oxidase activity. Cytochrome c(552) is a component of the P. aeruginosa Nar. No changes in succinate and reduced nicotinamide adenine dinucleotide dehydrogenases, ubiquinone content, Mg(2+)/Ca(2+) membrane adenosine triphosphatase, and energy coupling of electron transport to adenosine 5'-triphosphate synthesis were detected. Transport of gentamicin and dihydrostreptomycin was impaired in PAO2401, but transport of proline, arginine, glutamine, glucose or the polyamine spermidine was not reduced. Ribosomes of PAO2401, and PAO503 bound dihydrostreptomycin equally well, and cell extracts did not inactivate gentamicin or dihydrostreptomycin. Strain PAO2401 is resistant to gentamicin and dihydrostreptomycin because of impaired transport of these compounds. The transport studies indicate a selective coupling of dihydrostreptomycin and gentamicin transport with terminal electron transport. This conclusion was supported by results from another mutant (PAO417-T2) with increased Nar activity, enhanced dihydrostreptomycin and gentamicin transport and a reduction in resistance to these drugs. These results are discussed in relation to a refined model for aminoglycoside transport and briefly relative to plasmid-mediated aminoglycoside resistance.
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PMID:Aminoglycoside-resistant mutation of Pseudomonas aeruginosa defective in cytochrome c552 and nitrate reductase. 624 53

Nitrate reductase (NADPH:nitrate oxidoreductase; EC 1.6.6.1-3) was purified to apparent homogeneity from mycelium of Penicillium chrysogenum. The final preparation catalyzed the NADPH-dependent, FAD-mediated reduction of nitrate with a specific activity of 170-225 units X mg of protein-1. Gel filtration and glycerol density centrifugation yielded, respectively, a Stokes radius of 6.3 nm and an s20,w of 7.4. The molecular weight was calculated to be 199,000. On sodium dodecyl sulfate gels, the enzyme displayed two almost contiguous dye-staining bands corresponding to molecular weights of about 97,000 and 98,000. The enzyme prefers NADPH to NADH (kspec ratio = 2813), FAD to FMN (kspec ratio = 141), FAD (+ NADPH) to FADH2 (kspec ratio = 12,000), and nitrate to chlorate (kspec ratio = 4.33), where the kspec (the specificity constant for a given substrate) represents Vmax/Km. The Penicillium enzyme will also catalyze te NADPH-dependent, FAD-mediated reduction of cytochrome c with a specific activity of 647 units X mg of protein-1 (Kmcyt = 1.25 X 10(-5) M), and the reduced methyl viologen (MVH2, i.e. methyl viologen + dithionite)-dependent, NADPH and FAD-independent reduction of nitrate with a specific activity of 250 units X mg of protein-1 kmMVH2 = 3.5 X 10(-6) M). Initial velocity studies showed intersecting NADPH-FAD and nitrate-FAD reciprocal plot patterns. The NADPH-nitrate pattern was a series of parallel lines at saturating and unsaturating FAD levels. NADP+ was competitive with NADPH, uncompetitive with nitrate (at saturating and unsaturating FAD levels), and a mixed-type inhibitor with respect to FAD. Nitrite was competitive with nitrate, uncompetitive with NADPH (at saturating and unsaturating FAD levels), and a mixed-type inhibitor with respect to FAD. At unsaturating nitrate and FAD, NADPH exhibited substrate inhibition, perhaps as a result of binding to the FAD site(s). At very low FAD concentrations, low concentrations of NADP+ activated the reaction slightly. The initial velocity and product inhibition patterns are consistent with either of the two kinetic mechanisms. One (rather unlikely) mechanism involves the rapid equilibrium random binding of all ligands with (a) NADP+ and NADPH mutually exclusive, (b) nitrate and nitrite mutually exclusive, (c) the binding of NADPH strongly inhibiting the binding of nitrate and vice versa, (d) the binding of NADPH strongly promoting the binding of nitrite and vice versa, and (e) the binding of nitrate strongly promoting the binding of NADP+ and vice versa...
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PMID:Nitrate reductase from Penicillium chrysogenum. Purification and kinetic mechanism. 679 May 45

NADPH-nitrate reductase [NADPH : nitrate oxidoreductase, EC 1.6.6.3] was purified 500-fold from Aspergillus nidulans with an overall yield of about 20%. The purified enzyme catalyzed NADPH-nitrate, NADPH-cytochrome c, FADH2-nitrate and reduced methyl viologen-nitrate reductase activities. Its molecular weight was estimated to be 180,000 from the Stokes radius and sedimentation coefficient. The oxidized enzyme exhibited an absorption spectrum having a peak at 412 nm and a broad shoulder at about 450 nm. When reduced with NADPH, absorption peaks appeared at 423 (Soret), 527 (beta) and 557 (alpha) nm, and absorption in the 450 nm region decreased. Upon treatment of the reduced enzyme with KNO3, the spectrum returned to that of the oxidized enzyme. The presence of protoheme in the enzyme was confirmed by the absorption spectrum of reduced pyridine hemochromogen. It was concluded that a b-type cytochrome ("cytochrome b-557") is present in the enzyme and is involved in the intramolecular electron transport from NADPH to nitrate. The NADPH-nitrate and NADPH-cytochrome c reductase activities, but not the other two activities, were significantly decreased by incubation of the enzyme at 37 degrees C in the absence of FAD. Analysis by SDS slab gel electrophoresis suggested that the nitrate reductase consists of two each of two subunits of 59,000 and 38,000 daltons and that a dissociation of 38,000 subunits from the native enzyme occurs during heat treatment, resulting in alteration of the catalytic activity.
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PMID:Purification and characterization of the assimilatory NADPH-nitrate reductase of Aspergillus nidulans. 704 1

Chlorella vulgaris was cultured on an ammonia-mineral salts medium until the nitrate reductase content reached a minimal level. These ammonia-grown cells were then induced by nitrate in the absence of molybdenum and of tungsten. A demolybdo nitrate reductase developed and reached high levels. This protein contained very little nitrate-reducing capacity, but had the full cytochrome c-reducing capacity of normal nitrate reductase. It was purified to homogeneity by the same procedures previously developed for the purification of nitrate reductase. The purified enzyme contained 1 molecule of heme and 1 molecule of FAD/subunit, but no detectable molybdenum or tungsten. This cytochrome c reductase was completely inhibited by antibodies raised against purified nitrate reductase of Chlorella. Mixtures prepared from normal nitrate reductase and the demolybdoenzyme could not be resolved by disc gel electrophoresis or by centrifugation in a density gradient. By a two-step enzyme induction (1, incubation with nitrate in absence of Mo; 2, incubation with Mo in absence of nitrate) the process of nitrate reductase synthesis could be cleanly separated from growth into two steps: Step 1, induction of cytochrome c reductase, was completely inhibited by cycloheximide. Step 2 was unaffected by cycloheximide, and most of the nitrate reductase synthesized accumulated in the form of the reversibly inactivated HCN complex of the enzyme.
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PMID:Purification and characterization of demolybdo nitrate reductase (NADH-cytochrome c oxidoreductase) of Chlorella vulgaris. 719 74

The napEDABC locus coding for the periplasmic nitrate reductase of Thiosphaera pantotropha has been cloned and sequenced. The large and small subunits of the enzyme are coded by napA and napB. The sequence of NapA indicates that this protein binds the GMP-conjugated form of the molybdopterin cofactor. Cysteine-181 is proposed to ligate the molybdenum atom. It is inferred that the active site of the periplasmic nitrate reductase is structurally related to those of the molybdenum-dependent formate dehydrogenases and bacterial assimilatory nitrate reductases, but is distinct from that of the membrane-bound respiratory nitrate reductases. A four-cysteine motif at the N-terminus of NapA binds a [4Fe-4S] cluster. The DNA- and protein-derived primary sequence of NapB confirm that this protein is a dihaem c-type cytochrome and, together with spectroscopic data, indicate that both NapB haems have bis-histidine ligation. napC is predicted to code for a membrane-anchored tetrahaem c-type cytochrome that shows sequence similarity to the NirT cytochrome c family. NapC may be the direct electron donor to the NapAB complex. napD is predicted to encode a soluble cytoplasmic protein and napE a monotopic integral membrane protein, napDABC genes can be discerned at the aeg-46.5 locus of Escherichia coli K-12, suggesting that this operon encodes a periplasmic nitrate reductase system, while napD and napC are identified adjacent to the napAB genes of Alcaligenes eutrophus H16.
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PMID:The napEDABC gene cluster encoding the periplasmic nitrate reductase system of Thiosphaera pantotropha. 763 19


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