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)

The regulatory protein Fnr is required for anaerobic expression of several anaerobic respiratory enzymes in Escherichia coli. To gain insight into how Fnr activity is regulated by oxygen, we have isolated Fnr mutants that increase expression of the nitrate reductase operon in the presence of oxygen (Fnr* mutants). Seven single-amino-acid substitutions that mapped within two regions of Fnr have been characterized. Two mutants mapped adjacent to two Cys residues in the N-terminal Cys cluster. Five Fnr* substitutions mapped to a region of Fnr that is similar to the cyclic AMP-binding domain of the catabolite activator protein (CAP). Within this group, four mutants were clustered in a region analogous to the CAP C helix, which is important in CAP dimer subunit interactions. Taken together, these data implicate regions in Fnr that may be important either in sensing oxygen deprivation or in the conformational change proposed to be necessary for Fnr activation under anaerobic conditions.
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PMID:Fnr mutants that activate gene expression in the presence of oxygen. 189 18

The three-dimensional structure of spinach ferredoxin-NADP+ reductase (NADP+, nicotinamide adenine dinucleotide phosphate) has been determined by x-ray diffraction at 2.6 angstroms (A) resolution and initially refined to an R factor of 0.226 at 2.2 A resolution. The model includes the flavin-adenine dinucleotide (FAD) prosthetic group and the protein chain from residue 19 through the carboxyl terminus at residue 314 and is composed of two domains. The FAD binding domain (residues 19 to 161) has an antiparallel beta barrel core and a single alpha helix for binding the pyrophosphate of FAD. The NADP binding domain (residues 162 to 314) has a central five-strand parallel beta sheet and six surrounding helices. Binding of the competitive inhibitor 2'-phospho-AMP (AMP, adenosine monophosphate) places the NADP binding site at the carboxyl-terminal edge of the sheet in a manner similar to the nucleotide binding of the dehydrogenase family. The structures reveal the key residues that function in cofactor binding and the catalytic center. With these key residues as a guide, conclusive evidence is presented that the ferredoxin reductase structure is a prototype for the nicotinamide dinucleotide and FAD binding domains of the enzymes NADPH-cytochrome P450 reductase, NADPH-sulfite reductase, NADH-cytochrome b5 reductase, and NADH-nitrate reductase. Thus this structure provides a structural framework for the NADH- or NADPH-dependent flavoenzyme parts of five distinct enzymes involved in photosynthesis, in the assimilation of inorganic nitrogen and sulfur, in fatty-acid oxidation, in the reduction of methemoglobin, and in the metabolism of many pesticides, drugs, and carcinogens.
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PMID:Atomic structure of ferredoxin-NADP+ reductase: prototype for a structurally novel flavoenzyme family. 198 12

Nitrate reductase was purified from leaves of Nicotiana plumbaginifolia using either 5'AMP-Sepharose chromatography or two steps of immunoaffinity chromatography involving monoclonal antibodies directed against nitrate reductase from maize and against ribulose-1,5-bisphosphate carboxylase from N. plumbaginifolia. Nitrate reductase obtained by the first method was purified 1000-fold to a specific activity of 9 units/mg protein. The second method produced an homogenous enzyme, purified 21,000-fold to a specific activity of 80 units/mg protein. SDS/PAGE of nitrate reductase always resulted in two bands of 107 and 99.5 kDa. The 107-kDa band was the nitrate reductase subunit of N. plumbaginifolia; the smaller one of 99.5 kDa is thought, as commonly reported, to result from proteolysis of the larger protein. The molecular mass of 107 kDa is close to the values calculated from the coding sequences of the two nitrate reductase genes recently cloned from tobacco (Nicotiana tabacum cv Xanthi).
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PMID:Purification of nitrate reductase from Nicotiana plumbaginifolia by affinity chromatography using 5'AMP-sepharose and monoclonal antibodies. 292 Jul 29

The NarX, NarQ, and NarL proteins make up a nitrate-responsive regulatory system responsible for control of the anaerobic respiratory pathway genes in Escherichia coli, including nitrate reductase (narGHJI), dimethyl sulfoxide/trimethylamine-N-oxide reductase (dmsABC), and fumarate reductase (frdABCD) operons among others. The two membrane-bound proteins NarX and NarQ can independently sense the presence of nitrate and transfer this signal to the DNA-binding regulatory protein NarL, which controls gene expression by transcriptional activation or repression. To establish the role of protein phosphorylation in this process and to determine whether the NarX and NarQ proteins differ in their interaction with NarL, the cytoplasmic domains of NarX and NarQ were overproduced and purified. Both proteins were autophosphorylated in the presence of [gamma-32P]ATP and MgCl2 but not with [alpha-32P]ATP. Whereas these autophosphorylation reactions were unaffected by the presence of nitrate, molybdate, GTP, or AMP, ADP was an inhibitor. The phosphorylated forms of 'NarX and 'NarQ were stable for hours at room temperature. Each protein transferred its phosphoryl group to purified NarL protein, although 'NarQ-phosphate catalyzed the transfer reaction at an apparently much faster rate than did 'NarX-phosphate. In addition, NarL was autophosphorylated with acetyl phosphate but not with ATP as a substrate. NarL-phosphate remained phosphorylated for at least 3 h. However, addition of 'NarX resulted in rapid dephosphorylation of NarL-phosphate. In contrast, 'NarQ exhibited a much slower phosphatase activity with NarL-phosphate. These studies establish that the cytoplasmic domains of the two nitrate sensors 'NarX and 'NarQ differ in their ability to interact with NarL.
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PMID:Phosphorylation and dephosphorylation of the NarQ, NarX, and NarL proteins of the nitrate-dependent two-component regulatory system of Escherichia coli. 805 Oct 11

The synthesis of proteins necessary for the respiratory reduction of nitrate to dinitrogen is induced in most denitrifying bacteria by a shift to anaerobiosis. A homolog of the fur gene, which encodes a redox-active transcriptional activator in Escherichia coli, was isolated from Pseudomonas stutzeri by using the anr gene of Pseudomonas aeruginosa as the hybridization probe (R. G. Sawers, Mol. Microbiol. 5:1469-1481, 1991). The coding region was located on a 3-kb SmaI fragment. An open reading frame of 735 nucleotides, designated fnrA, had the coding potential for a protein of 244 amino acids (M(r) = 27,089) with 51.2% positional identity to the Fnr protein of E. coli and 86.1% to the Anr protein of P. aeruginosa. The fnrA gene gave a single transcript of 0.85 kb and complemented nitrate-dependent anaerobic growth of an fnr deletion mutant of E. coli. An open reading frame immediately downstream of fnrA encoded adenine phosphoribosyltransferase (EC 2.4.2.7). Mutations in fnrA were generated in vitro by insertional mutagenesis followed by gene replacement. Gene inactivation was shown by loss of the fnrA transcript and detection of an arginine deiminase (EC 3.5.3.6)-negative phenotype in the mutants. However, neither the enzymatic activities nor the levels of anaerobic expression of the respiratory enzymes nitrate reductase (EC 1.7.99.4), nitrate reductase (EC 1.9.3.2), NO reductase (EC 1.7.99.7), and N2O reductase (EC 1.7.99.6) were changed in fnrA mutants versus the P. stutzeri wild type. A promoter-probe vector for Fnr-dependent transcription was activated anaerobically in the fnrA mutants, suggesting the existence of a second Fnr homolog in the same bacterium. The Fnr-binding motifs, apparent in the promoter region of genes encoding denitrification components of P. stutzeri, are likely to be recognized by this second Fnr homolog. Preliminary evidence indicates also the presence of the catabolite activator protein, Crp, in P. stutzeri.
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PMID:Anaerobic control of denitrification in Pseudomonas stutzeri escapes mutagenesis of an fnr-like gene. 822 70

The NADH-dependent nitrate reductase (NR, EC 1.6.6.1) in roots of hydroponically grown barley seedlings was extracted, desalted and the activity measured in buffer containing either Mg2+ (10 mM) or EDTA (5 mM). The former gives the actual NR activity (NRact) equivalent to dephospho-NR, whereas the latter gives the maximum NR capacity of the dephospho-form (NRmax). Both values together permit an estimation of the NR-phosphorylation state. Changes in NRact and NRmax were followed in response to root aeration or to shoot illumination or shoot removal, and were correlated with sugar contents and adenylate levels. Ethanol formation was also measured in roots differing in NR activity in order to obtain information on the relation between anaerobic alcoholic fermentation and nitrate reduction. In aerated roots, NR was highly phosphorylated (about 80%) and largely inactive. It was partly dephosphorylated (activated) by anoxia or by cellular acidification (pH 4.8 plus propionic acid). Anaerobic activation (dephosphorylation) of NR was stronger at acidic external pH (5) than at slightly alkaline pH (8), although ATP levels decreased and AMP levels increased at pH 5 and at pH 8 to the same extent. Thus, rapid changes in the NR-phosphorylation state in response to anaerobiosis were not directly triggered by the adenylate pool, but rather by cytosolic pH. Under prolonged darkness (24 h) or after shoot removal. NRmax decreased slowly without a large change in the phosphorylation state. This decrease of NRmax was correlated with a large decrease in the sugar content, and was prevented by glucose feeding, which had only minor effects on the phosphorylation state. Cycloheximide also prevented the decrease in NRmax without affecting the phosphorylation state. In contrast, anaerobiosis or cellular acidification prevented the decrease of NRmax and at the same time decreased the NR-phosphorylation state. It is suggested that NR turnover in roots is controlled by several factors: NR synthesis appears to depend on sugar availability, which has little effect on the phosphorylation state; in addition, NR degradation appears to be strongly affected by the phosphorylation state in such a way that the inactive phospho-NR is a better substrate for NR degradation than the dephospho-form. The rate of anaerobic ethanol formation was not affected by NR activity, indicating that the purpose of NR activation under hypoxia or anoxia is not to decrease or prevent alcoholic fermentation.
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PMID:Nitrate reductase activation state in barley roots in relation to the energy and carbohydrate status. 915 51

Direct electrochemical studies, utilizing two voltammetric methods-square-wave voltammetry (SWV) and cyclic voltammetry (CV)-have been performed on recombinant forms of the flavin domain of spinach assimilatory nitrate reductase in the presence of NAD+ analogs. The reduction potentials (E degrees ') of the flavin domains have been determined at an edge pyrolytic graphite electrode utilizing MgCl2 as a redox-inactive promoter. Under identical experimental conditions (pH 7.0, 25 degrees C), the two-electron reduction potential for the FAD/FADH2 couple has been determined to be -274 and -257 mV by SWV and CV, respectively. In contrast, the reduction potentials of free FAD have been determined to be -234 and -227 mV by SWV and CV, respectively. The reduction potentials of the complex formed between the FAD prosthetic group in the recombinant flavin domain and various NAD+ analogs have been determined to be as follows: NAD+ (E degrees ' = -192 mV), 5'-ADP ribose (E degrees ' = -199 mV), ADP (E degrees ' = -154 mV), AMP (E degrees ' = -196 mV), adenosine (E degrees ' = -192 mV), adenine (E degrees ' = -220 mV), and NMN (E degrees ' = -208 mV). In contrast to these positive shifts in reduction potential, nicotinamide (E degrees ' = -268 mV) had very little effect on the reduction potential of this flavin complex. Moreover, addition of NAD+ to the FAD prosthetic group in a variety of mutant forms of the recombinant flavin domain resulted in positive shifts in the reduction potential of the complex, although the magnitude of the shifts varied from a minimum of 6 mV obtained for the C240A mutant to a maximum of 79 mV obtained for the C62S mutant. These results represent the first extensive application of direct electrochemistry to examine the redox properties of assimilatory nitrate reductase and indicate that complex formation with NAD+, or various NAD+ analogs, results in a positive shift in the flavin reduction potential, with the magnitude of the shift correlating well with the efficiency of the inhibitor.
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PMID:Direct electrochemistry of the flavin domain of assimilatory nitrate reductase: effects of NAD+ and NAD+ analogs. 928 15

The inactivation of phosphorylated nitrate reductase (NR) by the binding of 14-3-3 proteins is one of a very few unambiguous biological functions for 14-3-3 proteins. We report here that serine and threonine residues at the +6 to +8 positions, relative to the known regulatory binding site involving serine-543, are important in the interaction with GF14omega, a recombinant plant 14-3-3. Also shown is that an increase in ionic strength with KCl or inorganic phosphate, known physical effectors of NR activity, directly disrupts the binding of protein and peptide ligands to 14-3-3 proteins. Increased ionic strength attributable to KCl caused a change in conformation of GF14omega, resulting in reduced surface hydrophobicity, as visualized with a fluorescent probe. Similarly, it is shown that the 5' isomer of AMP was specifically able to disrupt the inactive phosphorylated NR:14-3-3 complex. Using the 5'-AMP fluorescent analog trinitrophenyl-AMP, we show that there is a probable AMP-binding site on GF14omega.
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PMID:Phosphorylated nitrate reductase and 14-3-3 proteins. Site of interaction, effects of ions, and evidence for an amp-binding site on 14-3-3 proteins. 980 49

The proteins commonly referred to as 14-3-3s have recently come to prominence in the study of protein:protein interactions, having been shown to act as allosteric or steric regulators and possibly scaffolds. The binding of 14-3-3 proteins to the regulatory phosphorylation site of nitrate reductase (NR) was studied in real-time by surface plasmon resonance, using primarily an immobilized synthetic phosphopeptide based on spinach NR-Ser543. Both plant and yeast 14-3-3 proteins were shown to bind the immobilized peptide ligand in a Mg2+-stimulated manner. Stimulation resulted from a reduction in KD and an increase in steady-state binding level (Req). As shown previously for plant 14-3-3s, fluorescent probes also indicated that yeast BMH2 interacted directly with cations, which bind and affect surface hydrophobicity. Binding of 14-3-3s to the phosphopeptide ligand occurred in the absence of divalent cations when the pH was reduced below neutral, and the basis for enhanced binding was a reduction in K(D). At pH 7.5 (+Mg2+), AMP inhibited binding of plant 14-3-3s to the NR based peptide ligand. The binding of AMP to 14-3-3s was directly demonstrated by equilibrium dialysis (plant), and from the observation that recombinant plant 14-3-3s have a low, but detectable, AMP phosphatase activity.
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PMID:Modulation of 14-3-3 protein interactions with target polypeptides by physical and metabolic effectors. 1084 67

Despite 14-3-3 proteins being implicated in the control of the eukaryotic cell cycle, metabolism, cell signalling and survival, little is known about the global regulation or functions of the phosphorylation-dependent binding of 14-3-3s to diverse target proteins. We identified Arabidopsis cytosolic proteins that bound 14-3-3s in competition with a 14-3-3-binding phosphopeptide, including nitrate reductase, glyceraldehyde- 3-phosphate dehydrogenase, a calcium-dependent protein kinase, sucrose-phosphate synthase (SPS) and glutamyl-tRNA synthetase. Remarkably, in cells starved of sugars or fed with non-metabolizable glucose analogues, all 14-3-3 binding was lost and the target proteins were selectively cleaved into proteolytic fragments. 14-3-3 binding reappeared after several hours of re-feeding with sugars. Starvation-induced degradation was blocked by 5-amino imidazole-4-carboxamide riboside (which is converted to an AMP-mimetic) or the protease inhibitor MG132 (Cbz-leu-leu-leucinal). Extracts of sugar-starved (but not sugar-fed) Arabidopsis cells contained an ATP-independent, MG132-sensitive, neutral protease that cleaved Arabidopsis SPS, and the mammalian 14-3-3-regulated transcription factor, FKHR. Cleavage of SPS and phosphorylated FKHR in vitro was blocked by binding to 14-3-3s. The finding that 14-3-3s participate in a nutrient-sensing pathway controlling cleavage of many targets may underlie the effects of these proteins on plant development.
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PMID:14-3-3s regulate global cleavage of their diverse binding partners in sugar-starved Arabidopsis cells. 1085 32


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