Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Respiratory nitrate reductase purified from the cell membrane of Escherichia coli is composed of three subunits, alpha, beta, and gamma, which are encoded, respectively, by the narG, narH, and narI genes of the narGHJI operon. The product of the narJ gene was deduced previously to be a highly charged, acidic protein which was not found to be associated with any of the purified preparations of the enzyme and which, in studies with putative narJ mutants, did not appear to be absolutely required for formation of the membrane-bound enzyme. To test this latter hypothesis, the narJ gene was disrupted in a plasmid which contained the complete narGHJI operon, and the operon was expressed in a narG::Tn10 insertion mutant. The chromosomal copy of the narJ gene of a wild-type strain was also replaced by the disrupted narJ gene. In both cases, when nar operon expression was induced, the alpha and beta subunits accumulated in a form which expressed only very low activity with either reduced methyl viologen (MVH) or formate as electron donors, although an alpha-beta complex separated from the gamma subunit is known to catalyze full MVH-linked activity but not the formate-linked activity associated with the membrane-bound complex. The low-activity forms of the alpha and beta subunits also accumulated in the absence of the NarJ protein when the gamma subunit (NarI) was provided from a multicopy plasmid, indicating that NarJ is essential for the formation of the active, membrane-bound complex. When both NarJ and NarI were provided from a plasmid in the narJ mutant, fully active, membrane-bound activity was formed. When NarJ only was provided from a plasmid in the narJ mutant, a cytosolic form of the alpha and beta subunits, which expressed significantly increased levels of the MVH-dependent activity, accumulated, and the alpha subunit appeared to be protected from the proteolytic clipping which occurred in the absence of NarJ. We conclude that NarJ is indispensible for the biogenesis of membrane-bound nitrate reductase and is involved either in the maturation of a soluble, active alpha-beta complex or in facilitating the interaction of the complex with the membrane-bound gamma subunit.
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PMID:The narJ gene product is required for biogenesis of respiratory nitrate reductase in Escherichia coli. 173 20

When grown anaerobically on nitrate-containing medium, Bacillus halodenitrificans exhibited a membrane-bound nitrate reductase (NR) that was solubilized by 2% Triton X-100 but not by 1% cholate or deoxycholate. Purification on columns of DE-52, hydroxylapatite, and Sephacryl S-300 yielded reduced methyl viologen NR (MVH-NR) with specific activities of 20 to 35 U/mg of protein that was stable when stored in 40% sucrose at -20 degrees C for 6 weeks. 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxypropone-1-sulfonat e (CHAPSO) and dodecyl-beta-D-maltoside stimulated enzyme activity three- to fourfold. Membrane extractions yielded purified NR that separated after electrophoresis into a 145-kDa alpha subunit, a 58-kDa beta subunit, and a 23-kDa gamma subunit. The electronic spectrum of dithionite-reduced, purified NR displayed peaks at 424.6, 527, and 557 nm, indicative of the presence of a cytochrome b, an interpretation consistent with the pyridine hemochrome spectrum formed. Analyses revealed a molybdenum-heme-non-heme iron ratio of 1:1:8 for the NR and the presence of molybdopterin. Electron paramagnetic resonance (EPR) signals characteristic of iron-sulfur centers were detected at low temperature. EPR also revealed a minor signal centered in the g = 2 region of the spectra. Upon reduction with dithionite, the enzyme displayed signals at g = 2.064, 2.026, 1.906, and 1.888, indicative of the presence of low-potential iron-sulfur centers, which resolve most probably as two [4Fe-4S]+1 clusters. With menadiol as the substrate for nitrate reduction, the Km for nitrate was 50-fold less than that seen when MVH was the electron donor. The cytochrome b557-containing enzyme from B. halodenitrificans is characterized as a menaquinol-nitrate:oxidoreductase.
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PMID:Menaquinol-nitrate oxidoreductase of Bacillus halodenitrificans. 201 72

Under anaerobic circumstances in the presence of nitrate Paracoccus denitrificans is able to denitrify. The properties of the reductases involved in nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase are described. For that purpose not only the properties of the enzymes of P. denitrificans are considered but also those from Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas stutzeri. Nitrate reductase consists of three subunits: the alpha subunit contains the molybdenum cofactor, the beta subunit contains the iron sulfur clusters, and the gamma subunit is a special cytochrome b. Nitrate is reduced at the cytoplasmic side of the membrane and evidence for the presence of a nitrate-nitrite antiporter is presented. Electron flow is from ubiquinol via the specific cytochrome b to the nitrate reductase. Nitrite reductase (which is identical to cytochrome cd1) and nitrous oxide reductase are periplasmic proteins. Nitric oxide reductase is a membrane-bound enzyme. The bc1 complex is involved in electron flow to these reductases and the whole reaction takes place at the periplasmic side of the membrane. It is now firmly established that NO is an obligatory intermediate between nitrite and nitrous oxide. Nitrous oxide reductase is a multi-copper protein. A large number of genes is involved in the acquisition of molybdenum and copper, the formation of the molybdenum cofactor, and the insertion of the metals. It is estimated that at least 40 genes are involved in the process of denitrification. The control of the expression of these genes in P. denitrificans is totally unknown. As an example of such complex regulatory systems the function of the fnr, narX, and narL gene products in the expression of nitrate reductase in E. coli is described. The control of the effects of oxygen on the reduction of nitrate, nitrite, and nitrous oxide are discussed. Oxygen inhibits reduction of nitrate by prevention of nitrate uptake in the cell. In the case of nitrite and nitrous oxide a competition between reductases and oxidases for a limited supply of electrons from primary dehydrogenases seems to play an important role. Under some circumstances NO formed from nitrite may inhibit oxidases, resulting in a redistribution of electron flow from oxygen to nitrite. P. denitrificans contains three main oxidases: cytochrome aa3, cytochrome o, and cytochrome co. Cytochrome o is proton translocating and receives its electrons from ubiquinol. Some properties of cytochrome co, which receives its electrons from cytochrome c, are reported.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Metabolic regulation including anaerobic metabolism in Paracoccus denitrificans. 205 Jun 53

The unusual ability of Thiosphaera pantotropha to catalyze respiratory nitrate reduction under aerobic conditions is shown to correlate with the activity of a periplasmic nitrate reductase that is expressed under both aerobic and anaerobic growth conditions. The organism also synthesizes, but only under anaerobic conditions, a membrane-bound nitrate reductase which resembles the corresponding enzyme in Paracoccus denitrificans in respect of both catalytic properties and inhibition of activity in intact cells in the presence of oxygen.
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PMID:Periplasmic and membrane-bound respiratory nitrate reductases in Thiosphaera pantotropha. The periplasmic enzyme catalyzes the first step in aerobic denitrification. 236 57

The membrane-bound respiratory particle complex of Pseudomonas aeruginosa, which reduces nitrate to nitrite using formate as the electron donor, was prepared and characterized by e.p.r. and low-temperature magnetic c.d. (m.c.d.) spectroscopy. The particle complex has two enzymic components, namely nitrate reductase (NiR) and formate dehydrogenase (FDH), which are multi-centred proteins containing molybdenum, iron-sulphur clusters and cytochrome. By using results from work on the purified extracted enzymes NiR and FDH to aid in the assignment, it has been possible to observe spectroscopically all the components of the electron-transfer chain in the intact particle. This led to a proposal for the organization of the metal components of the FDH-NiR chain. Molybdenum ions are at opposite ends of the chain and interact with, respectively, the formate-CO2 couple and the nitrate-nitrite couple. The molybdenum ion at the low-potential end of the chain passes electrons to cytochrome b of FDH, a bishistidine-co-ordinated haem with unusual steric restraint at the iron. The next component is a [4Fe-4S] cluster. This comprises all the components of FDH. Electrons are passed to the molybdenum of NiR via a number, probably two, of [4Fe-4S] clusters. No evidence has been found in this work for the presence of a quinone to mediate electron transfer between FDH and NiR. Cytochrome c appears to be able to feed electrons into the chain at the level of one of the [4Fe-4S] centres of NiR.
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PMID:Electron-paramagnetic-resonance and magnetic-circular-dichroism studies on the formate dehydrogenase-nitrate reductase particle from Pseudomonas aeruginosa. 303 83

Nitrate reductase, released and purified from membrane fractions of Escherichia coli, is composed of three subunits. Formation of the enzyme depends on induction of the nar operon, narGHJI, which is composed of four open reading frames (ORF). Previous studies established that the first two genes in the operon narG and narH encode the alpha and beta subunits, respectively, while formation of the gamma subunit, cytochrome bNR, depends on expression of the promoter distal genes. The narJ and narI genes were subcloned separately into plasmids where each was under the control of the nar promoter. Expression of these plasmids in a mutant which forms only alpha and beta subunits revealed that expression of the narI gene is sufficient to restore normal levels of cytochrome bNR, but expression of both genes is required for assembly of fully active, membrane-bound nitrate reductase. The amino acid composition, the N-terminal sequence, and the sequence of cyanogen bromide fragments derived from the isolated gamma subunit corresponds to that expected for a protein produced by the narI ORF. A protein corresponding to the narJ ORF did not appear to be associated with the purified nitrate reductase complex or with the complex immunoprecipitated from Triton X-100-solubilized membrane preparations. We conclude that narI encodes the gamma subunit (cytochrome bNR) and that while the product of the narJ gene is required for assembly of fully active membrane-bound enzyme it is not tightly associated with the active enzyme.
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PMID:Roles of the narJ and narI gene products in the expression of nitrate reductase in Escherichia coli. 305 88

Genes different from those of the narGHI operon and encoding a nitrate reductase activity have been cloned by Bonnefoy et al. (unpublished results). We have shown by the use of well-known assay methods that the encoded enzyme activity is catalyzed by a true nitrate reductase and not by trimethylamine-N-oxide reductase or formate dehydrogenase. The biochemical and immunological study, employing anti-(nitrate reductase) serum raised against the known enzyme, revealed that Escherichia coli contains a second nitrate reductase (nitrate reductase Z) which shares some similarities as well as differences with the known enzyme. By using a strain with a deletion of the narGHI operon and carrying a multicopy plasmid having the nitrate reductase Z genes, we have shown that nitrate reductase Z is a membrane-bound molybdoenzyme able to couple formate oxidation with nitrate reduction. Like the known nitrate reductase, this enzyme has chlorate reductase activity. The molecular mass and pH and temperature dependence of enzyme Z are similar to these of the known enzyme. On the other hand the two enzymes have significant difference in their electrophoretic mobility on polyacrylamide gels. Unlike the known enzyme, enzyme Z is synthesized in small amounts; the expression of its structural genes does not seem to be induced by nitrate, repressed by oxygen or activated by the product of the fnr gene. The immunological comparison of the two enzymes was performed by rocket immunoelectrophoresis, double diffusion on agar plates and immunoblots. These techniques disclosed a difference between the two enzymes in their recognition by the antiserum and showed that E. coli has two types of nitrate reductase.
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PMID:Biochemical and immunological evidence for a second nitrate reductase in Escherichia coli K12. 331 49

Lysozyme digestion and sonication of sodium dodecyl sulfate (SDS)-purified Klebsiella aerogenes murein sacculi resulted in the quantitative release of both subunits of nitrate reductase, as well as a number of other cytoplasmic membrane polypeptides (5.2%, by weight, of the total membrane proteins). Similar results were obtained after lysozyme digestion of SDS-prepared peptidoglycan fragments, which excluded the phenomenon of simple trapping of the polypeptides by the surrounding peptidoglycan matrix. About 28% of membrane-bound nitrate reductase appears to be tightly associated with the peptidoglycan. Additional evidence for this association was demonstrated by positive immunogold labeling of SDS-murein sacculi and thin sections of plasmolyzed bacteria. Qualitative amino acid analysis of trypsin-treated sacculi, a tryptic product of holo-nitrate reductase, and amino- and carboxypeptidase digests of both nitrate reductase subunits indicated the possible existence of a terminal anchoring peptide containing the following amino acids: (Gly)n, Trp, Ser, Pro, Ile, Leu, Phe, Cys, Tyr, Asp, and Lys.
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PMID:Part of respiratory nitrate reductase of Klebsiella aerogenes is intimately associated with the peptidoglycan. 354 73

An enzymatic assay system for nitrate employing the membrane-bound nitrate reductase (EC 1.7.99.4) of E. coli is described. Contrary to previous enzymatic assay systems, the present method is a kinetic one, i.e. the substrate, nitrate, is assayed by measuring the reaction rate of the nitrate reductase-catalyzed reaction. Based on the observation that the nitrate reductase-catalyzed reaction obeys pseudo-first order kinetics, a test system is described allowing the assay of nitrate at a concentration as low as 1 ppm. The relatively high Michaelis-Menten constant for nitrate (0.3 mM) of the E. coli nitrate reductase favours nitrate assay by the kinetic method.
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PMID:Enzymatic nitrate assay by a kinetic method employing Escherichia coli nitrate reductase. 388 90

White bands resulting from precipitation of dodecan-1-ol liberated by hydrolysis of sodium dodecyl sulfate and decan-5-ol released by hydrolysis of decan-5-yl sulfate produced zymograms of the primary and secondary alkylsulfatases from Pseudomonas C(12)B. Gas-liquid chromatographic analyses of ether extracts of the precipitate-containing segments of the zymograms confirmed the identity of the alcohols which were not discerned in extracts of segments of the gels other than those containing precipitates. beta-Galactosidase from Escherichia coli was marked on zymograms by the liberation of o-nitrophenol from o-nitrophenyl-beta-D-galactoside, and arylsulfatase from Pseudomonas C(12)B was marked in gels by liberation of p-nitrophenol from p-nitrophenyl sulfate. Membrane-associated dissimilatory nitrate reductases from a nitrate respirer (Enterobacter aerogenes) and a denitrifier (Pseudomonas perfectomarinus) did not penetrate either 6.8 or 3% polyacrylamide gel but were demonstrable at the top of the gels. In the membrane-bound state, formate served as electron donor for nitrate reductase from E. aerogenes, and reduced nicotinamide adenine dinucleotide (NADH) served as donor for nitrate reductase from P. perfectomarinus. Both enzymes reduced nitrate at the expense of reduced benzyl viologen as well. Assimilatory nitrate reductase from E. aerogenes moved easily into the 6.8% gels (R(f) = 0.43 under the conditions of these experiments). The reduced dye served as electron donor for the assimilatory reductase, but formate and NADH did not. Incubation of the membrane-associated nitrate reductases with 2% Triton X-100 solubilized the enzymes and removed the capacity of formate and NADH to serve as electron donors. Both retained the ability to reduce nitrate at the expense of reduced benzyl viologen. The solubilized dissimilatory reductase from E. aerogenes moved further in the gels (R(f) = 0.49) than the soluble assimilatory reductase; the solubilized dissimilatory reductase from the denitrifier, P. perfectomarinus, moved further in the gels (R(f) = 0.64) than either of the enzymes from E. aerogenes.
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PMID:Methods for visualization of enzymes in polyacrylamide gels. 435 59


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