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)

A simple and rapid procedure to make yeast cells permeable by agitating with toluene-ethanol, (TE) 1:4, v/v was developed. The permeated cells retained their ability to catalyze certain enzyme reactions. Temperature and duration of agitation during TE treatment played an important role in retention of the catalytic potential of permeated cells. The in situ assay using permeated cell preparations was more sensitive even in the absence of added cofactors than in the vitro assay in detecting assimilatory nitrate reductase (NAD(P)H:nitrate oxidoreductase, EC 1.6.6.2) (NAR) activity in Candida utilis. Using in situ assay technique, different mechanisms regulating the biosynthesis of NAR in C. utilis were investigated. Nitrogen starvation did not lead to derepression of NAR. NO3-ions were absolutely essential for induction and maintenance of high levels of NAR activity. Cells grown on ammonium nitrate possessed relatively lower levels of NAR. Kinetics of NAR induction were followed as a function of time and inducer concentration. The influence of various cations on the induction of NAR by nitrate was investigated. A wide range of D-amino acids induced NAR synthesis. Of 22 L-amino acids tested only phenylalanine induced significant levels of NAR. Various intermediates of the pathway of nitrate reduction influenced the rate of NAR induction. There was a rapid disappearance of in vivo activity of the enzyme of induced yeast cells on nitrogen starvation, and the rate of loss was accelerated by the presence of NH4+.
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PMID:Regulatory properties of yeast nitrate reductase in situ. 94 16

The described bacterium was isolated by enrichment culture in peptone broth inoculated with garden soil, pasteurized and then put to incubate under N2O at 32 degrees. It is a Gram-negative rod, motile with peritrichous flagella, and producing oval spores without exosporium in swollen sporangia. However, cells have the thick walls, mesosomes and persistant septa characteristic of Gram-positive bacteria. It lacks fermentative activity, does not attack carbohydrates, has complex growth requirements, and will grow anaerobically only if one of the following electron acceptors is present: NO3, NO2, N2O, S4O6, and fumarate. Nitrate, nitrite, and nitrous oxide are denitrified with production of N2. The microorganism is mesophilic, gives a positive oxidase reaction, synthesizes a type of c cytochrome, and does not hydrolyse gelatin, starch nor "Tween 80". The following enzymes are present: nitrate reductase A, respiratory nitrite reductase, tetrathionate and fumarate reductases, L-glutamate dehydrogenase, and superoxide dismutase. The following enzymes are absent: thiosulfate reductase, urease, lecithinase, arginine dihydrolase, L-alanine dehydrogenase, phenylalanine desaminase, and catalase. The GC% of its DNA is 39. The bacterium described can be considered to be a new species. We propose the name Bacillus azotoformans n. sp.
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PMID:[A new, sporulating, denitrifying, mesophilic bacterium: Bacillus azotoformans N. SP. (author's transl)]. 102 Aug 72

We have used site-directed mutagenesis to alter the [Fe-S] cluster composition of Escherichia coli dimethyl sulfoxide (DMSO) reductase (DmsABC). The electron-transfer subunit (DmsB) of this enzyme contains 16 Cys residues arranged in 4 groups (I-IV) which provide ligands to 4 [4Fe-4S] clusters [Cammack, R., & Weiner, J. H. (1990) Biochemistry 29, 8410-8416]. Strong homologies exist between these Cys groups and the four Cys groups of the electron-transfer subunit (NarH) of E. coli nitrate reductase (NarGHJI), which contains a [3Fe-4S] cluster in addition to multiple [4Fe-4S] clusters. The Cys group primarily involved in providing ligands to the [3Fe-4S] cluster of NarH has a Trp residue at a position equivalent to Cys102 of DmsB. We have mutated Cys102 to Trp, Ser, Tyr, and Phe and have investigated the altered enzymes in terms of their enzymatic activities and EPR properties. The mutant enzymes do not support electron transfer from menaquinol to DMSO, although they retain high rates of electron transport from reduced benzyl viologen to DMSO. The mutations cause major changes in the EPR properties of the enzyme in the fully reduced and oxidized states. In the oxidized state, new species are observed in all the mutants; these have spectral features comprising a peak at g = 2.03 (gz) and a peak-trough at g = 2.00 (gxy). The temperature dependencies, microwave power dependencies, and spin quantitations of these species are consistent with the Trp102, Ser102, Phe102, and Tyr102 mutations causing conversion of one of the [4Fe-4S] clusters present in the wild-type enzyme into [3Fe-4S] clusters in the mutant enzymes.
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PMID:Alteration of the iron-sulfur cluster composition of Escherichia coli dimethyl sulfoxide reductase by site-directed mutagenesis. 165 10

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

We have analyzed a temperature-sensitive mutant, ts4, of Nicotiana tabacum, together with its newly isolated second revertant, Rt1, both in cell culture and as a regenerated plant. Cells of the temperature-sensitive mutant will grow only below 30 degrees C, whereas the revertant can grow at temperatures above that but not at those as high as will wild-type cells. Enzyme assays of phenylalanine ammonia lyase and nitrate reductase showed that ts4 had substantial increases and decreases, respectively. The revertant resembled ts4 for these activities. The decrease in nitrate reductase is associated with resistance of ts4 to a pulse of chlorate that kills wild-type. Amino acid and polyamine analyses showed that ornithine levels rise in ts4 during 24 hr at the high temperature, while spermine and spermidine levels fall. The ts4 mutant has low levels of ornithine decarboxylase activity and s-adnosyl-methionine decarboxylase activity, whereas the revertant has restored levels of only ornithine decarboxylase. This is the only enzyme activity in which Rt1 resembles wild-type more than it resembles ts4, suggesting that it is the site of metabolic alteration of the mutation. In regenerated plants, ts4 has a low chlorophyll content, while the revertant has a fully restored chlorophyll content. Flowers on the revertant plant are both male and female sterile, with the male sterility associated with anthers converted into petals. The correlations among these ts4 and Rt1 alterations demonstrate novel regulatory interactions between nitrate reductase and the two decarboxylase enzymes, and also suggest that we have partially unravelled the molecular basis for a particular developmental endpoint.
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PMID:Biochemical, cellular and developmental characterization of a temperature-sensitive mutant of Nicotiana tabacum and its second site revertant. 719 6

We have used site-directed mutagenesis to alter the ligands to the iron-sulfur centers of Escherichia coli nitrate reductase A. The beta subunit of this enzyme contains four Cys groups which are thought to accommodate the single [3Fe-4S] center and the three [4Fe-4S] centers involved in the electron-transfer process from quinol to nitrate. The third Cys group (group III) contains a Trp at a site occupied by a Cys residue in typical ferredoxin arrangements or in the DmsB subunit of dimethyl sulfoxide (DMSO) reductase. In an attempt to determine the coordination site of the different iron-sulfur centers in the amino acid sequence, we have changed the Trp of group III to Cys, Ala, Phe, and Tyr and the first Cys residue of groups II-IV to Ala and Ser. Physiological, biochemical, and EPR studies were performed on the mutated enzymes. Substitution of Ala for either Cys184, Cys217, or Cys244 results in the full loss of all four iron-sulfur centers present in the wild-type enzyme. These inactive enzymes still possess the alpha,beta, and gamma polypeptides associated in a membrane-bound complex. These Cys have important structural roles and are very likely involved in the coordination of the iron-sulfur centers. Substitution of Cys184 with a Ser residue produces an enzyme containing the four iron-sulfur centers, but displaying reduced activity. EPR studies suggest that Cys184 is a ligand of the [4Fe-4S] center whose midpoint potential is -200 mV in the native enzyme. All substitutions performed in this study on Trp220 lead to mutant enzymes harboring the four iron-sulfur centers and a nitrate reductase activity close to that of the wild-type. In spite of the high similarity between the NarH and DmsB subunits, the Trp220-->Cys substitution does not allow the conversion of the [3Fe-4S] center of the nitrate reductase into a [4Fe-4S] center. Therefore, Trp220 does not seem to play any major role in the beta subunit.
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PMID:Site-directed mutagenesis of conserved cysteine residues within the beta subunit of Escherichia coli nitrate reductase. Physiological, biochemical, and EPR characterization of the mutated enzymes. 838 31

14-3-3 proteins bind to the hinge 1 region of nitrate reductase (NR) and inhibit its activity. To determine which residues of NR are required for 14-3-3-inhibitory interactions, wild-type and mutant forms of Arabidopsis NR were examined in the yeast two-hybrid system and in vitro inhibition assays. NR fragments with or without hinge 1 were introduced into yeast with one of seven Arabidopsis 14-3-3 isoforms (called GF14s). NR fragments (residues 1-562 or 487-562) containing hinge 1 interacted with all GF-14s tested; an NR fragment (residues 1-487) lacking hinge 1 did not. GF14 binding to NR fragments was dependent on Ser-534, since Asp or Ala substitutions at this site blocked the interaction. Revertants with second site substitutions restoring interaction between GF14omega and the Ala- or Asp-substituted NR fragments were identified. One isolate had a Lys to Glu substitution at position 531, which is in hinge 1, and six isolates had Ile to Leu or Phe substitutions at 561 in the heme binding region. Double mutant forms of holo-NR (S534D plus K531E, I561F, or I561L) were constructed and found to be partially inhibited by protein extracts from Arabidopsis containing 14-3-3 proteins. Wild-type NR is phosphorylated and inhibited by these extracts, but S534D single mutant forms are not. These results show that inhibitory NR/14-3-3 interactions are dependent on Ser-534 but only in the context of the wild-type sequence, since substitutions at second sites render 14-3-3 binding and in vitro NR inhibition independent of Ser-534.
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PMID:Ser-534 in the hinge 1 region of Arabidopsis nitrate reductase is conditionally required for binding of 14-3-3 proteins and in vitro inhibition. 993 11

The transcription factor NNR from Paracoccus denitrificans was expressed in a strain of Escherichia coli carrying a plasmid-borne fusion of the melR promoter to lacZ, with a consensus FNR-binding site 41.5 bp upstream of the transcription start site. This promoter was activated by NNR under anaerobic growth conditions in media containing nitrate, nitrite, or the NO(+) donor sodium nitroprusside. Activation by nitrate was abolished by a mutation in the molybdenum cofactor biosynthesis pathway, indicating a requirement for nitrate reductase activity. Activation by nitrate was modulated by the inclusion of reduced hemoglobin in culture media, because of the ability of hemoglobin to sequester nitric oxide and nitrite. The ability of nitrate and nitrite to activate NNR is likely due to the formation of NO (or related species) during nitrate and nitrite respiration. Amino acids potentially involved in NNR activity were replaced by site-directed mutagenesis, and the activities of NNR derivatives were tested in the E. coli reporter system. Substitutions at Cys-103 and Tyr-35 significantly reduced NNR activity but did not abolish the response to reactive nitrogen species. Substitutions at Phe-82 and Tyr-93 severely impaired NNR activity, but the altered proteins retained the ability to repress an FNR-repressible promoter, so these mutations have a "positive control" phenotype. It is suggested that Phe-82 and Tyr-93 identify an activating region of NNR that is involved in an interaction with RNA polymerase. Replacement of Ser-96 with alanine abolished NNR activity, and the protein was undetectable in cell extracts. In contrast, NNR in which Ser-96 was replaced with threonine retained full activity.
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PMID:Heterologous NNR-mediated nitric oxide signaling in Escherichia coli. 1105 88

A pot experiment was conducted to evaluate the effects of applying L-methionine (L-MET), L-phenylalanine (LPHE) and L-tryptophan (L-TRP) on the growth of Zea mays and its nutrient uptake, and to determine the optimal application rate of them. The results showed that L-MET, L-PHE and L-TRP could improve the shoot height, shoot and root dry weights, root activity, nitrate reductase and hydrogen peroxidase activities, and N, P, K and Zn uptake of corn. The optimal application rate of L-MET, L- PHE and L-TRP was 0.0185 - 0.185 mg x kg)(-1) soil, 0.2 mg x kg(-1) soil, and 0.03 - 0.3 mg x kg(-1) soil, respectively, and L-PHE and L-TRP were superior to L-MET.
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PMID:[Effects of applying L-methionine, L-phenylalanine and L-tryptophan on Zea mays growth and its nutrient uptake]. 1618 Jul 48

The size of tissue amino acid pools in plants may indicate nitrogen status and provide a signal that can regulate nitrate uptake and assimilation. The effects of treating barley roots with glutamine have been examined, first to identify the transport system for the uptake of the amino acid and then to measure root NR activity and cellular pools of nitrate. Treating N replete roots with glutamine elicited a change in the cell membrane potential and the size of this response was concentration dependent. In addition, the size of the electrical change depended on the previous exposures of the root to glutamine and was lost after a few cycles of treatment. Whole root tissue pools of glutamine and phenylalanine increased when roots were incubated in a nutrient solution containing 10 mM nitrate and 1 mM glutamine. Treating roots with 1 mM glutamine increased cytosolic nitrate activity from 3 mM to 7 mM and this change peaked after 2 h of treatment. Parallel measurements of root nitrate reductase activity during treatment with 1 mM glutamine showed a decrease. These measurements provide evidence for feedback regulation on NR activity that result in changes in cytosolic nitrate activity. After 6 h in glutamine both root NR activity and cytosolic nitrate activity returned to pretreatment values, while tissue concentrations of glutamine and phenylalanine remained elevated. The data are discussed in terms of the mechanisms that are most likely to be responsible for the changes in cytosolic nitrate.
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PMID:Glutamine transport and feedback regulation of nitrate reductase activity in barley roots leads to changes in cytosolic nitrate pools. 1654 28


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