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)

Nitrate uptake and its regulation were investigated using an ion-specific nitrate electrode for denitrifying Flexibacter canadensis under anaerobic conditions. Glucose supported a greater rate of nitrate uptake than did glycerol, glutamate, lactose, cellobiose, or ethanol. Nitrate uptake closely approximated Michaelis--Menten kinetics; the estimated Ks(glucose) and apparent Km(nitrate) for nitrate uptake were 21 and 44 microM, respectively. Nitrate disappearance was correlated with nitrite accumulation, and nitrate had an inhibitory effect on nitrite reduction. Oxygen inhibition of nitrate uptake increased as the percent air saturation increased, and reversed readily as the percent air saturation decreased. The minimal air saturation showing inhibition of nitrate uptake was about 2-4%. Azide and cyanide completely inhibited nitrate uptake. No nitrate uptake was observed in cells grown in the presence of 1 or 5 mM tungstate (no added molybdate). When molybdate (100-200 microM) was present in the medium, nitrate uptake was exhibited by organisms grown with 1 mM, but not with 5 mM, tungstate, indicating that nitrate uptake was dependent on the presence of an active nitrate reductase, and that competition between tungsten and molybdenum occurred during the formation of nitrate reductase. Nitrite production from nitrate by whole cells but not cell-free extracts was inhibited by 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone, indicating that nitrate and (or) nitrite transport depended upon the electrochemical proton gradient.
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PMID:Cellular regulation of nitrate uptake in denitrifying Flexibacter canadensis. 807 52

Tungsten resistant (Wr) mutants of Het+Nif+Nia+, Het+Nif-Nia+ and Het+Nif+Nia- strains of Nostoc muscorum were isolated with severely defective molybdate transport activity. All such mutants showed vanadium (V)-dependent nitrogenase activity and/or nitrate reductase activity and V-dependent growth on N2-nitrogen and/or NO3(-)-nitrogen and V-dependent NO3(-)-repression of heterocyst formation and nitrogenase activity. None of them grew with molybdenum (Mo) under parallel growth condition. Results strongly suggest the ability of V to replace Mo in N2-assimilation or NO3(-)-assimilation under Mo-deficiency.
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PMID:Mutational replacement of molybdenum by vanadium in assimilation of N2 or NO3- as nitrogen source in the cyanobacterium Nostoc muscorum. 833 16

Nuclear transformation of the unicellular green alga Chlamydomonas reinhardtii has thus far been characterized by integration of the introduced DNA into nonhomologous sites. In this study, the occurrence of homologous recombination events during transformation was investigated with the intent of developing strategies for gene targeting and gene disruption. Homologous recombination was monitored by using nonfunctional 5' and 3' deletion derivatives of the wild-type C. reinhardtii nit1 gene (encodes nitrate reductase) as selectable markers (p5' delta and p3' delta respectively) and the low reverting nit1-305 strain as the transformation recipient. After introduction of the DNA into the cell, intermolecular recombination between p5' delta and p3' delta occurs at a high frequency to restore a functional nit1 gene, indicating the presence of homologous recombination machinery in mitotic cells. Gene-targeting events at the nit1 locus were selected by restoring nit1-305 cells to prototrophy after transformation with only p5' delta and were confirmed by analysis of genomic DNA. By comparing the number of transformants obtained after transformation with p5' delta to the number obtained after transformation with a functional nit1 gene, the frequency of homologous-to-random integration events ranged between 1:1000 after glass bead-mediated transformation and 1:24 after bombardment with DNA-coated tungsten microprojectiles.
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PMID:Homologous recombination in the nuclear genome of Chlamydomonas reinhardtii. 841 77

Among higher plants, soybean is unique in that biochemically it has been characterized as having two constitutive nitrate reductase (cNR) isoforms and one substrate-inducible nitrate reductase (iNR) isoform in leaves. All three NR isoforms are expressed in cv. Williams 82 while the nr1 mutant expresses only the iNR isoform. The genetic and molecular mechanisms for regulation of these isoforms have not been elucidated. We describe here the isolation, by reverse transcription-polymerase chain reaction (RT-PCR), of two cDNA clones encoding soybean NR. They were designated as iNR1 and iNR2, respectively, since both were inducible by nitrate. The iNR1 and iNR2 cDNAs cover total encoding regions of 2661 and 2673 nucleotides, respectively. The iNR1 clone shows a 12 bp deletion at the 5' end, relative to iNR2. They show overall similarity of 89% at the nucleotide level, and 87% at the amino acid level. Like all plant NRs cloned so far, deduced amino acid sequences between iNR1 and iNR2 show greatest variation at the N-terminal region while no difference was observed at the C-terminus. Soybean iNR mRNAs were found to be different from those of maize and tobacco in response to tungsten inhibitor treatment, since the inhibitor decreased the steady-state levels of mRNA for soybean iNR and for NiR. Using the same 5' regions of both cDNAs as the probes, Southern blot analysis of genomic DNA revealed differences in organization between iNR1 and iNR2. The genomic DNA from wild-type Williams 82 soybean was shown to have three Eco RI fragments while the nr1 mutant lacked an 8 kb fragment when probed with iNR1 cDNA. Likewise, the nr1 mutant lacked three Hae III restriction fragments when probed with iNR1 cDNA. When probed with iNR2, both wildtype and nr1 mutant showed one identical Eco RI band and two identical Hae III bands. In northern blots, the steady-state level of iNR1 mRNA was similar for the nr1 mutant and the wild-type parent after 20 to 48 h induction by nitrate. Based on the Eco RI and Hae III restriction enzyme digestion patterns observed in Southern blot analysis of soybean DNA, it is concluded that in soybean iNR1 is encoded by a small multiple gene family and iNR2 is a single gene.
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PMID:Identification of cDNA clones corresponding to two inducible nitrate reductase genes in soybean: analysis in wild-type and nr1 mutant. 853 48

Tungsten in the presence of molybdenum stimulates nitrate reductase activity and growth of the salt-tolerant yeast Rhodotorula glutinis on medium with nitrates. Tungsten is not incorporated in proteins possessing nitrate reductase activity. A significant increase in molybdenum cofactor in cells grown on medium with equimolar amounts of molybdenum and tungsten may relate to the stimulatory action of tungsten.
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PMID:Stimulation of nitrate reductase activity of the salt-tolerant yeast Rhodotorula glutinis by tungsten in the presence of molybdenum. 1071 48

The X-ray absorption spectra at the molybdenum and selenium K-edges and the tungsten L2,3-edges are acquired for a set of 14 Mo(IV) and W(IV,VI) bis(dithiolene) complexes related to the active sites of molybdo- and tungstoenzymes. The set includes square pyramidal [MoIVL(S2C2Me2)2]- (L = O2-, R3SiO-, RO-, RS-, RSe-) and [WIV(OR)(S2C2Me2)2]-, distorted trigonal prismatic [MoIV(CO)(SeR)(S2C2Me2)2]- and [WIV(CO)L(S2C2Me2)2]- (L = RS-, RSe-), and distorted octahedral [WVIO(OR)(S2C2Me2)2]-. The dithiolene simulates the pterin-dithiolene cofactor ligand, and L represents a protein ligand. Bond lengths are determined by EXAFS analysis using the GNXAS protocol. Normalized edge spectra, non-phase-shift-corrected Fourier transforms, and EXAFS data and fits are presented. Bond lengths determined by EXAFS and X-ray crystallography agree to < or = 0.02 A as do the M-Se distances determined by both metal and selenium EXAFS. The complexes [MoIV(QR)(S2C2Me2)2]- simulate protein ligation by the DMSO reductase family of enzymes, including DMSO reductase itself (Q = O), dissimilatory nitrate reductase (Q = S), and formate dehydrogenase (Q = Se). Edge shifts of these complexes correlate with the ligand electronegativities. Terminal ligand binding is clearly distinguished in the presence of four Mo-S(dithiolene) interactions. Similarly, five-coordinate [ML(S2C2Me2)2]- and six-coordinate [M(CO)L(S2C2Me2)2]- are distinguishable by edge and EXAFS spectra. This study expands a previous XAS investigation of bis(dithiolene)metal(IV,V,VI) complexes (Musgrave, K. B.; Donahue, J. P.; Lorber, C.; Holm, R. H.; Hedman, B.; Hodgson, K. O. J. Am. Chem. Soc. 1999, 121, 10297) by including a larger inventory of molecules with variant physiologically relevant terminal ligation. The previous and present XAS results should prove useful in characterizing and refining metric features and structures of enzyme sites.
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PMID:X-ray spectroscopy of enzyme active site analogues and related molecules: bis(dithiolene)molybdenum(IV) and -tungsten(IV,VI) complexes with variant terminal ligands. 1115 82

The tungsten-containing formate dehydrogenase (W-FDH) isolated from Desulfovibrio gigas has been crystallized in space group P2(1), with cell parameters a = 73.8 A, b = 111.3 A, c = 156.6 A and beta = 93.7 degrees. These crystals diffract to beyond 2.0 A on a synchrotron radiation source. W-FDH is a heterodimer (92 kDa and 29 kDa subunits) and two W-FDH molecules are present in the asymmetric unit. Although a molecular replacement solution was found using the periplasmic nitrate reductase as a search model, additional phasing information was needed. A multiple-wavelength anomalous dispersion (MAD) dataset was collected at the W- and Fe-edges, at four different wavelengths. Anomalous and dispersive difference data allowed us to unambiguously identify the metal atoms bound to W-FDH as one W atom with a Se-cysteine ligand as well as one [4Fe-4S] cluster in the 92 kDa subunit, and three additional [4Fe-4S] centers in the smaller 29 kDa subunit. The D. gigas W-FDH was previously characterized based on metal analysis and spectroscopic data. One W atom was predicted to be bound to two molybdopterin guanine dinucleotide (MGD) pterin cofactors and two [4Fe-4S] centers were proposed to be present. The crystallographic data now reported reveal a selenium atom (as a Se-cysteine) coordinating to the W site, as well as two extra [4Fe-4S] clusters not anticipated before. The EPR data were re-evaluated in the light of these new results.
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PMID:Tungsten-containing formate dehydrogenase from Desulfovibrio gigas: metal identification and preliminary structural data by multi-wavelength crystallography. 1137 98

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex molybdenum-, cytochrome b(557)- and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. To facilitate structure/function studies of the individual molybdenum center, we have developed bacterial expression systems for the heterologous production of the 541 residue amino-terminal, molybdenum center-containing domain of spinach nitrate reductase either as a six-histidine-tagged variant or as a glutathione-S-transferase-tagged fusion protein. Expression of the his-tagged molybdenum domain in Escherichia coli BL21(DE3) cells under anaerobic conditions yielded a 55-kDa domain with a specific activity of 1.5 micromol NO(3)(-) consumed/min/nmol enzyme and with a K(mapp)(NO(3)(-)) of 8 mciroM. In contrast, expression of the molybdenum domain as a GST-tagged fusion protein in E. coli TP1000(MobA(-) strain) cells under aerobic conditions yielded an 85-kDa fusion protein with a specific activity of 10.8 micromol NO(3)(-) consumed/min/nmol enzyme and with a K(mapp)(NO(3)(-)) of 12 microM. Fluorescence analysis indicated that both forms of the molybdenum domain contained the cofactor, MPT, although the MPT content was higher in the GST-fusion domain. Inductively coupled plasma mass spectrometric analysis of both the his-tagged and GST-fusion protein domain samples indicated Mo/protein ratios of 0.44 and 0.93, respectively, confirming a very high level of Mo incorporation in the GST-fusion protein. Expression of the GST-fusion protein in TP1000 cells in the presence of elevated tungsten concentrations resulted in an 85-kDa fusion protein that contained MPT but which was devoid of nitrate-reducing activity. Partial reduction of the molybdenum domain resulted in the generation of an axial Mo(V) EPR species with g values of 1.9952, 1.9693, and 1.9665, respectively, and exhibiting superhyperfine coupling to a single exchangeable proton, analogous to that previously observed for the native enzyme. In contrast, the tungsten-substituted MPT-containing domain yielded a W(V) EPR species with g values of 1.9560, 1.9474, and 1.9271, respectively, with unresolved superhyperfine interaction. NADH:nitrate reductase activity could be reconstituted using the GST-molybdenum domain fusion protein in the presence of the recombinant forms of the spinach nitrate reductase' flavin- and heme-containing domains.
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PMID:Bacterial expression of the molybdenum domain of assimilatory nitrate reductase: production of both the functional molybdenum-containing domain and the nonfunctional tungsten analog. 1213 73

Desulfovibrio gigas formate dehydrogenase is the first representative of a tungsten-containing enzyme from a mesophile that has been structurally characterized. It is a heterodimer of 110 and 24 kDa subunits. The large subunit, homologous to E. coli FDH-H and to D. desulfuricans nitrate reductase, harbors the W site and one [4Fe-4S] center. No small subunit ortholog containing three [4Fe-4S] clusters has been reported. The structural homology with E. coli FDH-H shows that the essential residues (SeCys158, His159, and Arg407) at the active site are conserved. The active site is accessible via a positively charged tunnel, while product release may be facilitated, for H(+) by buried waters and protonable amino acids and for CO(2) through a hydrophobic channel.
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PMID:Gene sequence and the 1.8 A crystal structure of the tungsten-containing formate dehydrogenase from Desulfovibrio gigas. 1222 Apr 97

Heterocyst-forming filamentous cyanobacteria, such as Anabaena variabilis ATCC 29413, require molybdenum as a component of two essential cofactors for the enzymes nitrate reductase and nitrogenase. A. variabilis efficiently transported (99)Mo (molybdate) at concentrations less than 10(-9) M. Competition experiments with other oxyanions suggested that the molybdate-transport system of A. variabilis also transported tungstate but not vanadate or sulfate. Although tungstate was probably transported, tungsten did not function in place of molybdenum in the Mo-nitrogenase. Transport of (99)Mo required prior starvation of the cells for molybdate, suggesting that the Mo-transport system was repressed by molybdate. Starvation, which required several generations of growth for depletion of molybdate, was enhanced by growth under conditions that required synthesis of nitrate reductase or nitrogenase. These data provide evidence for a molybdate storage system in A. variabilis. NtcA, a regulatory protein that is essential for synthesis of nitrate reductase and nitrogenase, was not required for transport of molybdate. The closely related strain Anabaena sp. PCC 7120 transported (99)Mo in a very similar way to A. variabilis.
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PMID:Transport of molybdate in the cyanobacterium Anabaena variabilis ATCC 29413. 1247 4


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