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:1.7.1.1 (nitrate reductase)
3,728 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

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

The distribution of the Mo-enzymes aldehyde oxidase (AO; EC 1.2.3.1) xanthine dehydrogenase (XDH; EC 1.2.1.37) and nitrate reductase (NAD(P)H NR; EC 1.6.6.1-2) was studied along the longitudinal and transversal axes of maize (Zea mays L. cv. Jubily) nodal roots as affected by nitrogen sources and salinity. Activities of the Mo-enzymes were considerably enhanced under mild saline conditions. The activities of AO and XDH increased following addition of ammonium to the nutrient solution. Immunoblot analysis with antibodies raised against maize AO protein revealed increased levels of AO proteins in root tips of ammonium fed plants. Application of salinity to nitrate fed plants did not affect the enzyme protein level, although it enhanced the activity of the Mo-hydroxylases. The specific activities of the Mo-enzymes were the highest in root tips (0-1 cm segments) while on the transversal axis maximal activity was observed in the stele or vascular cylinder. Activity staining of AO after native PAGE of root extracts revealed four bands of AO proteins (AO1-4) capable of oxidizing a number of aliphatic and aromatic aldehydes. Increased AO activity in maize nodal roots grown with ammonium, and salinity were observed mainly at the AO3 and AO4 bands. Tips and stele contained primarily AO3 and AO4, and only traces of AO1 and AO2. SDS-PAGE of root extracts followed by Western blots revealed, besides the major 150 kD subunit of AO, two polypeptides with molecular masses of 72 and 85 kD located specifically in the cortex. Part of the polymorphism of AO in plant roots may be related to the allocation of distinct isoforms to different regions of the root, although the specific metabolic roles of the different bands have not been established.
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PMID:Distribution of the Mo-enzymes aldehyde oxidase, xanthine dehydrogenase and nitrate reductase in maize (Zea mays L.) nodal roots as affected by nitrogen and salinity. 1077 39

In Chlamydomonas reinhardtii, the expression of the Nia1 gene encoding NAD(P)H nitrate reductase is controlled at the transcriptional level, positively by light and negatively by ammonium. Previous work has shown that the region -279 to +269 with respect to the start site of transcription was sufficient to confer regulated expression of a promoterless arylsulfatase (Ars) reporter gene. To understand the mechanisms underlying this regulation, the -279 to +2 sequence was analysed for the presence of ammonium-responsive elements using either pJD54 (promoterless Ars gene) or pJD100 (minimal beta-tubulin promoter-driven Ars gene). The region lying between -195 and -120 was shown to be dispensable. Essential responsive elements were found in four distinct regions between -231 and -219, -120 and -100, -76 and -65 and -33 and -8. Each of these sequences is required for maximal expression in the absence of ammonium and a conserved GGA/TAGGGT motif is present in two of these regions. Several deletions within the region -33 to -77 were shown to partially relieve the transformants from the negative effect of ammonium. These experiments demonstrate that Nia1 expression is promoted by at least four elements between -231 and -8 and suggest that part of the repression by ammonium takes place through a proximal element located in the -51 to -33 sequence.
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PMID:Identification of short promoter regions involved in the transcriptional expression of the nitrate reductase gene in Chlamydomonas reinhardtii. 1128 12

Barley (Hordeum vulgare L.) has NADH-specific and NAD(P)H-bispecific nitrate reductase isozymes. Four isogenic lines with different nitrate reductase isozyme combinations were used to determine the role of NADH and NAD(P)H nitrate reductases on nitrate transport and assimilation in barley seedlings. Both nitrate reductase isozymes were induced by nitrate and were required for maximum nitrate assimilation in barley seedlings. Genotypes lacking the NADH isozyme (Az12) or the NAD(P)H isozyme (Az70) assimilated 65 or 85%, respectively, as much nitrate as the wild type. Nitrate assimilation by genotype (Az12;Az70) which is deficient in both nitrate reductases, was only 13% of the wild type indicating that the NADH and NAD(P)H nitrate reductase isozymes are responsible for most of the nitrate reduction in barley seedlings. For all genotypes, nitrate assimilation rates in the dark were about 55% of the rates in light. Hypotheses that nitrate reductase has direct or indirect roles in nitrate uptake were not supported by this study. Induction of nitrate transporters and the kinetics of net nitrate uptake were the same for all four genotypes indicating that neither nitrate reductase isozyme has a direct role in nitrate uptake in barley seedlings.
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PMID:Nitrate transport is independent of NADH and NAD(P)H nitrate reductases in barley seedlings. 1153 65


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