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 reductase from the yeast Candida nitratophila was found to contain one molecule of cytochrome b557 and one atom of molybdenum per subunit. FAD/haem-dependent diaphorase activity (haem domain) was associated with a 40 kDa tryptic fragment of the subunit. The 50 amino-terminal residues of this fragment were determined, and the sequence did not show significant similarity to deduced sequences of other nitrate reductases previously published. Increasing ionic strength in vitro had a stimulatory effect on enzymic activity via stimulation of the molybdenum-dependent terminal nitrate-reducing activity. Stimulation of activity by exogenous protein (bovine serum albumin or casein) also appeared to be an ionic effect. Stimulation of catalytic activity by phosphate was a separate effect.
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PMID:Further characterization of the assimilatory nitrate reductase from the yeast Candida nitratophila. 847 56

The C-terminal 268 residues of the spinach assimilatory NADH:nitrate reductase amino acid sequence that correspond to the flavin-containing domain of the enzyme have been selectively amplified and expressed as a recombinant protein in Escherichia coli. The recombinant protein, which was produced in both soluble and insoluble forms, was purified to homogeneity using a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP-agarose and FPLC gel filtration. The purified domain exhibited a molecular weight of approximately 30 kDa, estimated by polyacrylamide gel electrophoresis, and a molecular mass of 30,169 for the apoprotein determined by mass spectrometry, which also confirmed the presence of FAD. The UV/visible spectrum was typical of a flavoprotein, with maxima at 272, 386, and 461 nm in the oxidized form while CD spectroscopy yielded both positive and negative maxima at 313 and 382 nm and 461 and 484 nm, respectively. The purified domain showed immunological cross-reactivity with anti-spinach nitrate reductase polyclonal antibodies while both N-terminal and internal amino acid sequencing of isolated peptides confirmed the fidelity of the domain's primary sequence. The protein retained NADH-ferricyanide reductase activity (Vmax=84 micromol NADH consumer/min/nmol FAD) with Km's of 17 and 34 microM for NADH and ferricyanide, respectively, with a pH optimum of approximately 6.5 A variety of NADH-analogs could also function as electron donors, though with decreased efficiency, the most effective being reduced nicotinamide hypoxanthine dinucleotide (V(max) = 35 micromol NHDH consumer/min/nmol FAD) and Km = 22 microM). NAD+ was demonstrated to be a competitive inhibitor (Ki = 1.9 mM) while analysis of inhibition by a variety of NAD+-analogs indicated the most efficient inhibitor to be ADP (Ki = 0.2 mM), with analogs devoid of either the phosphate, ribose, or adenine moieties proving to be markedly less-efficient inhibitors. The isolated domain was also capable of reducing cytochrome b5 directly (V(max) = 1.2 micromol NADH consumed/min/nmol FAD, Km (cyt. b5) = 6 microM), supporting the FAD -> b557 -> Mo electron transfer sequence in spinach nitrate reductase.
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PMID:Spectroscopic and kinetic properties of a recombinant form of the flavin domain of spinach NADH: nitrate reductase. 861 85

Incubation of either Chlorella nitrate reductase or the recombinant flavin domain of spinach nitrate reductase with reagents specific for modification of cysteine residues, such as N-ethylmaleimide, resulted in a time-dependent inactivation of NADH:ferricyanide reductase activity which could be prevented by incubation in the presence of NADH. At 25 degrees C and employing a fixed enzyme:modifier ratio, the rate of inactivation for both the Chlorella and spinach enzymes followed the order p-chloromercuribenzoate > methyl methanethiosulfonate > 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid > N-ethylmaleimide. For the spinach flavin domain, inactivation by methyl methanethiosulfonate or p-chloromercuribenzoate was found to be concentration independent suggesting the absence of nonspecific modifications. Initial rate studies of the methyl methanethiosulfonate-modified flavin domain indicated a reduction in NADH:ferricyanide activity (Vmax) from 85 to 44 micromol NADH consumed/min/nmol FAD and an increase in the Km for NADH from 12 to 35 microM when compared to the native enzyme, confirming a role for cysteine residue(s) in maintaining diaphorase activity. Site-directed mutagenesis of the four individual cysteines (residues 17, 54, 62, and 240) in the recombinant spinach flavin domain resulted in mutant proteins with visible and CD spectra very similar to those of the wild-type domain. Initial rate studies indicated that only substitutions of serine for cysteine 240 decreased diaphorase activity with maximal NADH:ferricyanide activity for the C240S mutant corresponding to 51 micromol NADH consumed/min/nmol FAD with a Km for NADH of 14 microM. Mutation of C240 to Ala or Gly resulted in greater loss of activity. The thermal stability of the four serine mutants was slightly decreased compared to the wild-type domain with the C62S mutant exhibiting the greatest instability. In contrast to the effects on diaphorase activity, square wave voltammetric studies indicated changes in the oxidation-reduction midpoint potential for the FAD/FADH2 couple in the C54S (E0'= -197 mV), C62S (E0' = -226 mV), and C240S (E0' = -219 mV) mutants compared to the wild-type domain (E0' = -268 mV). These results indicate that of the four cysteine residues in the spinach nitrate reductase flavin domain, only C240 plays a role in maintaining diaphorase activity, while C54 has the greatest influence on flavin redox potential and that no correlation between changes in catalytic activity and flavin redox potential was observed.
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PMID:Thiol modification and site directed mutagenesis of the flavin domain of spinach NADH:nitrate reductase. 866 Jun 90

The nitrate reductase gene (niaD) and nitrite reductase gene (niiA) of Aspergillus parasiticus are clustered and are divergently transcribed from a 1.6-kb intergenic region (niaD-niiA). The deduced aminoacid sequence of the A. parasiticus nitrate reductase demonstrated a high degree of homology to those of other Aspergillus species, as well as to Leptosphaeria maculans, Fusarium oxysporum, Gibberella fujikuroi and Neurospora crassa, particularly in the cofactor-binding domains for molybdenum, heme and FAD. A portion of the deduced nitrite reductase sequence was homologous to those of A. nidulans and N. crassa. The nucleotide sequences in niaD-niiA of A. parasiticus and of A. oryzae were 95% identical, indicating that these two species are closely related. Several GATA motifs, the recognition sites for the N. crassa positive-acting global regulatory protein NIT2 in nitrogen metabolism, were found in A. parasiticus niaD-niiA. Two copies of the palindrome TCCGCGGA and other partial palindromic sequences similar to the target sites for the pathway specific regulatory proteins, N. crassa NIT4 and A. nidulans NirA, in nitrate assimilation, were also identified. A recombinant protein containing the A. nidulans AreA (the NIT2 equivalent) zinc finger and an adjacent basic region was able to bind to segments of niaD-niiA encompassing the GATA motifs. These results suggest that the catalytic and regulatory mechanisms of nitrate assimilation are well conserved in Aspergillus.
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PMID:Characterization of the Aspergillus parasiticus niaD and niiA gene cluster. 866 12

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 assimilatory nitrate reductase from the phototrophic bacterium Rhodobacter capsulatus has been purified to electrophoretic homogeneity and its molecular and kinetic parameters determined. The native nitrate reductase is a dimer of 144 kDa composed of two subunits of 46 and 95 kDa. The purified enzyme catalyzes the electron transfer from NADH, reduced bromophenol blue or reduced viologens to nitrate. The nitrate reductase contains 1 mol FAD per mole of enzyme and also reduces cytochrome c or dichlorophenol indophenol with NADH as the electron donor. The diaphorase activity is located in the small subunit.
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PMID:The assimilatory nitrate reductase from the phototrophic bacterium, Rhodobacter capsulatus E1F1, is a flavoprotein. 930 29

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

This study reports the molecular characterization of the nitrate-assimilation gene cluster from the opportunistic fungal pathogen Aspergillus fumigatus. A genomic fragment was isolated which contained the entire structural gene encoding nitrite reductase (niiA), plus segments of the nitrate reductase (niaD) and the nitrate transporter (crnA) genes. Nitrate-assimilation genes in A. fumigatus are physically linked and transcribed in the same direction as in A. nidulans. The nitrate-assimilation gene cluster is on the largest chromosome (5.3 Mb). The nitrite reductase (niiA) gene encodes a protein of 1110 amino acids that contains regions corresponding to FAD, NADPH, FeS and siroheme binding sites. Eight small introns interrupt the niiA open reading frame. The niaD-niiA intergenic regulatory region contains promoter consensus sequences including TATA, CAAT, and binding sites for the areA and nirA gene products. Northern analysis indicated that the expression of niaD, niiA and crnA are induced by nitrate and repressed by ammonium at the transcriptional level.
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PMID:Mapping of the nitrate-assimilation gene cluster (crnA-niiA-niaD) and characterization of the nitrite reductase gene (niiA) in the opportunistic fungal pathogen Aspergillus fumigatus. 950 95

Hansenula polymorpha (syn. Pichia angusta) is able to grow on nitrate as sole nitrogen source. Nitrate reductase (NR) assays, optimized in crude extracts from nitrate-grown cells, revealed that NR preferentially used NADPH, but also used NADH, as electron donor and required FAD for maximum activity. NR activity was present in nitrate-grown and nitrite-grown cells, and was absent in cells grown in ammonium, glutamate and methylamine. Addition of reduced nitrogen compounds to nitrate-grown cells led to loss of NR activity, even if added with nitrate. Under nitrogen starvation, NR activity was not observed; however, following growth on nitrate, NR activity is maintained in the absence of nitrate. Increases but not decreases in NR activity were dependent on protein synthesis. Conditions for chlorate selection were optimized, and Nit- (nitrate-) mutants were isolated. Some of these mutants showed reduced or absent NR activity. Sixty-one NR- mutants revealed the monogenic recessive nature of their lesions and were grouped in 10 complementation classes. These mutants will be used in gene cloning experiments aimed at identifying structural and regulatory elements involved in the first step of nitrate reduction.
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PMID:Nitrate reduction and the isolation of Nit- mutants in Hansenula polymorpha. 972 55

Chemical modification of purified nitrate reductase (NR) from sunflower leaves by white light-irradiated rose bengal was studied. NADH:NR activity was inhibited by light-activated rose bengal in both a concentration- and time-dependent manner. MV:NR activity was less sensitive to inhibition than NADH:NR activity, especially when the enzyme was preincubated with NADH. Preincubation of the enzyme with FAD protected inhibition of NADH:NR activity but not the MV:NR activity. These results suggest that sunflower NR contains sensitive histidine residue which interacts with reduced FAD during catalytic electron transfer. Most importantly, NADH-reduced NR was more sensitive to the irradiated dye, indicating that conformation of the oxidized and reduced enzyme forms were different.
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PMID:Inactivation of sunflower NADH:nitrate reductase by white light-activated rose bengal. 1042 32


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