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)

Six mutant strains (301, 102, 203, 104, 305, and 307) affected in their nitrate assimilation capability and their corresponding parental wild-type strains (6145c and 21gr) from Chlamydomonas reinhardii have been studied on different nitrogen sources with respect to NAD(P)H-nitrate reductase and its associated activities (NAD(P)H-cytochrome c reductase and reduced benzyl viologen-nitrate reductase) and to nitrite reductase activity. The mutant strains lack NAD(P)H-nitrate reductase activity in all the nitrogen sources. Mutants 301, 102, 104, and 307 have only NAD(P)H-cytochrome c reductase activity whereas mutant 305 solely has reduced benzyl viologen-nitrate reductase activity. Both activities are repressible by ammonia but, in contrast to the nitrate reductase complex of wild-type strains, require neither nitrate nor nitrite for their induction. Moreover, the enzyme from mutant 305 is always obtained in active form whereas nitrate reductase from wild-types needs to be reactivated previously with ferricyanide to be fully detected. Wild-type strains and mutants 301, 102, 104, and 307, when properly induced, exhibit an NAD(P)H-cytochrome c reductase distinguishable electrophoretically from constitutive diaphorases as a rapidly migrating band. Nitrite reductase from wild-type and mutant strains is also repressible by ammonia and does not require nitrate or nitrite for its synthesis. These facts are explained in terms of a regulation of nitrate reductase synthesis by the enzyme itself.
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PMID:Regulation of the nitrate-reducing system enzymes in wild-type and mutant strains of Chlamydomonas reinhardii. 681 63

Nitrate reductase (NAD(P)H:nitrate oxidoreductase, EC 1.6.6.2) of the unicellular alga Cyanidium caldarium can exist in two interconvertible forms; one catalytically active and one inactive. The inactive nitrate reductase can be activated by mild treatment with denaturing agents of protein. By treatment with urea or mersalyl, activation of both the NADPH and benzyl viologen activities can be realized under mild conditions, whereas by treatment with heat, the activation of benzyl viologen activity is concomitant with loss of the NADPH activity. On the other hand, both activities are activated and destroyed concomitantly by ethylene glycol. In the present of FAD, either activation of benzyl viologen activity or loss of NADPH activity upon heating occur only at higher temperatures. The existence of a controlling region in the nitrate reductase molecule is postulated.
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PMID:Active and inactive nitrate reductase. Effects of mild treatment with denaturing agents of protein. 718 70

Molybdenum is required for induction of nitrate reductase and of NAD-linked formate dehydrogenase activities in suspensions of wild type Paracoccus denitrificans; tungsten prevents the development of these enzyme activities. The wild type forms a membrane protein Mr150,000 when incubated with tungsten and inducers of nitrate reductase and this is presumed to represent an inactive form of the enzyme. Suspensions of mutuant M-1 did not develop nitrate reductase or formate dehydrogenase activities but the membrane protein Mr150,000 was formed under all conditions tested, including without inducers and without molybdenum. Analysis of membranes, solubilized with deoxycholate, by polyacrylamide gel electrophoresis under nondenaturing conditions showed that the mutant protein had similar electrophoretic mobility to the active nitrate reductase formed by the wild type. Autoradiography of preparations from cells incubated with 55Fe showed that the mutant and wild type proteins contained iron. However, in similar experiments with 99Mo, incorporation of molybdenum into the mutant protein was not detectable. We conclude that mutant M-1 is defective in one or more steps required to process molybdenum for incorporation into molybdoenzymes. This failure affects the normal regulation of nitrate reductase protein with respect to the role of inducers.
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PMID:Effects of molybdenum and tungsten on induction of nitrate reductase and formate dehydrogenase in wild type and mutant Paracoccus denitrificans. 719 82

Assimilatory nitrate reductase [NAD(P)H] (EC 1.6.6.2) from Ankistrodesmus braunii has been purified to homogeneity by a simple procedure that utilizes as the main step affinity chromatography on Blue-Sepharose. The best enzyme preparation has a specific activity of 61.25 units/mg protein. The enzyme has a sedimentation coefficient of 10.9 S by sucrose-density-gradient centrifugation, and a Stokes radius of 9.8 nm was estimated by gel filtration techniques. Its molecular weight is 460000, but only one single band of 58000 was detected after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The native enzyme seems thus to be composed of eight subunits. The nitrate reductase absorption spectrum shows wavelengths maxima at 280 and 416 nm and a broad shoulder at 450 nm. Reduced enzyme shows maxima at 424 (Soret), 527 (beta) and 557 (alpha) nm, and a bleaching at 450 nm. The reduced extracted heme chromophore, in pyridine and KOH, shows absorption bands at 414, 522 and 552 nm. These properties indicate the presence of a b-type cytochrome and flavin as prosthetic groups of A. braunii nitrate reductase. A minimum of four molecules of heme has been calculated per molecule of the enzyme complex. Redox titration of the enzyme shows a midpoint potential for the heme of -73 mV at pH 7.0. In the presence of p-hydroxymercuribenzoate, which inhibits the NAD(P)H-dependent activities of the complex, the enzyme-bound heme can be reduced with dithionite, but not with NAD(P)H.
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PMID:Purification and properties of assimilatory nitrate reductase [NAD(P)H] from Ankistrodesmus braunii. 720 Apr 26

Differences in the amino acid sequence between the bispecific NAD(P)H-nitrate reductase of birch (Betula pendula Roth) and the monospecific NADH-nitrate reductases of a variety of other higher plants have been found at the dinucleotide-binding site in the FAD domain. To pinpoint amino acid residues that determine the choice of reducing substrate, we introduced mutations into the cDNA coding for birch nitrate reductase. These mutations were aimed at replacing certain amino acids of the NAD(P)H-binding site by conserved amino acids located at identical positions in NADH-monospecific enzymes. The mutated cDNAs were integrated into the genome of tobacco by Agrobacterium-mediated transformation. Transgenic tobacco (Nicotiana tabacum) plants were grown on a medium containing ammonium as the sole nitrogen source to keep endogenous tobacco nitrate reductase activity low. Whereas some of the mutated enzymes showed a slight preference for NADPH, as does the nonmutated birch enzyme, the activity of some others greatly depended on the availability of NADH and was low with NADPH alone. Comparison of the mutations reveals that replacement of a single amino acid in the birch sequence (alanine871 by proline) is critical for the use of reducing substrate.
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PMID:The choice of reducing substrate is altered by replacement of an alanine by a proline in the FAD domain of a bispecific NAD(P)H-nitrate reductase from birch. 778 4

In order to study the variation of nitrate reductase (NR) genes among grass species, gene number, intron size and number, and the heme-hinge fragment sequence of 25 grass species were compared. Genomic DNA cut with six restriction enzymes and hybridized with the barley NAD(P)H and NADH NR gene probes revealed a single NAD(P)H NR gene copy and two or more NADH NR gene copies per haploid genome in most of the species examined. Major exceptions were Hordeum vulgare, H. vulgare ssp. spontaneum, and Avena strigosa, which appeared to have a single NADH NR gene copy. The NADH NR gene intron number and lengths were examined by polymerase chain reaction amplification. Introns I and III appeared to be absent in at least one of the NADH NR genes in the grass species, while intron II varied from 0.8 to 2.4 kilobases in length. The NADH NR gene heme-hinge regions were amplified and sequenced. The estimated average overall nucleotide substitution rate in the sequenced region was 7.8 x 10(-10) substitutions/site per year. The synonymous substitution rate was 2.11 x 10(-9) substitutions/synonymous site per year and the nonsynonymous substitution rate was 4.10 x 10(-10) substitutions/nonsynonymous site per year. Phylogenetic analyses showed that all of the wild Hordeum species examined clustered in a group separate from H. vulgare and H. vulgare ssp. spontaneum.
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PMID:Variation of nitrate reductase genes in selected grass species. 853 1

Nitrate is a significant nitrogen source for plants and microorganisms. Recent molecular genetic analyses of representative bacterial species have revealed structural and regulatory genes responsible for the nitrate-assimilation phenotype. Together with results from physiological and biochemical studies, this information has unveiled fundamental aspects of bacterial nitrate assimilation and provides the foundation for further investigations. Well-studied genera are: the cyanobacteria, including the unicellular Synechococcus and the filamentous Anabaena; the gamma-proteobacteria Klebsiella and Azotobacter; and a Gram-positive bacterium, Bacillus. Nitrate uptake in most of these groups seems to involve a periplasmic binding protein-dependent system that presumably is energized by ATP hydrolysis (ATP-binding cassette transporters). However, Bacillus may, like fungi and plants, utilize electrogenic uptake through a representative of the major facilitator superfamily of transport proteins. Nitrate reductase contains both molybdenum cofactor and an iron-sulfur cluster. Electron donors for the enzymes from cyanobacteria and Azotobacter are ferredoxin and flavodoxin, respectively, whereas the Klebsiella and Bacillus enzymes apparently accept electrons from a specific NAD(P)H-reducing subunit. These subunits share sequence similarity with the reductase components of bacterial aromatic ring-hydroxylating dehydrogenases such as toluene dioxygenase. Nitrite reductase contains sirohaem and an iron-sulfur cluster. The enzymes from cyanobacteria and plants use ferredoxin as the electron donor, whereas the larger enzymes from other bacteria and fungi contain FAD and NAD(P)H binding sites. Nevertheless, the two forms of nitrite reductase share recognizable sequence and structural similarity. Synthesis of nitrate assimilation enzymes and uptake systems is controlled by nitrogen limitation in all bacteria examined, but the relevant regulatory proteins exhibit considerable structural and mechanistic diversity in different bacterial groups. A second level of control, pathway-specific induction by nitrate and nitrite in Klebsiella, involves transcription antitermination. Several issues await further experimentation, including the mechanism and energetics of nitrate uptake, the pathway(s) for nitrite uptake, the nature of electron flow during nitrate reduction, and the action of transcriptional regulatory circuits. Fundamental knowledge of nitrate assimilation physiology should also enhance the study of nitrate metabolism in soil, water and other natural environments, a challenging topic of considerable interest and importance.
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PMID:Nitrate assimilation by bacteria. 932 45

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

Integrated bioelectrocatalytically active electrodes are assembled by the deposition of enzymes onto respective electrically contacted affinity matrices and further cross-linking of the enzyme monolayers. A catalyst-NAD(+)-dyad for the binding of the NAD(+)-dependent enzymes and cytochrome-like molecules for the binding of the heme-protein-dependent enzymes are used to construct integrated electrically contacted biocatalytic systems. NAD(+)-dependent lactate dehydrogenase (LDH) is assembled onto a pyrroloquinoline quinone-NAD+ monolayer. The redox-active monolayer is organized via covalent attachment of pyrroloquinoline quinone (PQQ) to a cystamine monolayer associated with a Au-electrode, followed by covalent linkage of N6-(2-aminoethyl)-NAD+ to the monolayer. The interface modified with the PQQ-NAD(+)-dyad provides temporary affinity binding for LDH and allows cross-linking of the enzyme monolayer. The cross-linked LDH is bioelectrocatalytically active towards oxidation of lactate. The bioelectrocatalyzed process involves the PQQ-mediated oxidation of the immobilized NADH. Integrated, electrically contacted bioelectrodes are produced by the affinity binding and further cross-linking of nitrate reductase (NR) (cytochrome-dependent, E.C. 1.9.6.1 from E. coli) or CoII-protoporphyrin IX reconstituted myoglobin (CoII-Mb) atop the microperoxidase-11 (MP-11) monolayer associated with a Au-electrode. The MP-11 monolayer provides an affinity interface for the temporary binding of the enzymes, that allows the cross-linkage of the enzyme molecules. The MP-11 assembly acts as electron transfer mediator for the reduction of the secondary enzyme layer. The integrated bioelectrodes consisting of NR and CoII-Mb show catalytic activities for NO3- reduction and acetylene-dicarboxylic acid hydrogenation, respectively. Two FeIII-protoporphyrin IX units are reconstituted into a four alpha-helix bundle de novo protein assembled as a monolayer on a Au-electrode. Vectorial electron transfer proceeds in the synthetic heme-protein monolayer. Cross-linking of an affinity complex generated between the FeIII-protoporphyrin IX reconstituted de novo protein monolayer and NR yields an integrated, electrically contacted enzyme electrode that stimulates the bioelectrocatalyzed reduction of nitrate.
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PMID:Fully integrated biocatalytic electrodes based on bioaffinity interactions. 982 68

The NAD(P)H nitrate reductase (NR) from Chlamydomonas reinhardtii is encoded by the structural gene Nia1. Numerous data from the literature indicate that this enzyme is submitted to complex regulation mechanisms involving multiple controls at transcriptional and post-transcriptional levels. To specifically investigate the regulation of the Nia1 gene at the transcriptional level, NR+ and NR- transformed cells harbouring the Nia1:Ars construct (Nia1 promoter fused to the arylsulfatase (ARS)-encoding Ars reporter gene) were cultivated under various experimental conditions and the ARS activities were recorded. ARS levels were very low in cells grown in the presence of NH4Cl and dramatically increased on agar medium deprived of any nitrogen source or containing nitrate, nitrite, urea, arginine or glutamine. Compared to nitrogen-free medium, a slight positive effect of nitrate in the NR+ strain and a significant negative effect of nitrite in both NR+ and NR- strains were observed. The ARS activities were high in the light and very low in the dark or in the light in the presence of DCMU, indicating that Nia1 transcription is strikingly dependent on photosynthetic activity. Acetate used as a carbon source in the dark did not substitute for light in stimulating Nia1:Ars expression. Inactivation of NR by tungstate treatment of the NR+ strain resulted in a dramatic increase of ARS level suggesting that in Chlamydomonas, like in higher plants, active NR negatively regulates the transcription of the NR structural gene. Deleting the major part of the Nia1 leader sequence still present in the chimeric gene resulted in a decrease of ARS level but did not modify the regulation pattern.
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PMID:Transcriptional regulation of the Nia1 gene encoding nitrate reductase in Chlamydomonas reinhardtii: effects of various environmental factors on the expression of a reporter gene under the control of the Nia1 promoter. 1064 29


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