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: KEGG:D02011 (FAD)
5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Assimilatory nitrate reductase from Chlorella is a homotetramer which contains one of each of the prosthetic groups FAD, heme, and molybdenum per subunit. Besides the reduction of nitrate by NADH, nitrate reductase also catalyzes the partial activities NADH:cytochrome c reductase, NADH:ferricyanide reductase, and reduced methyl viologen:nitrate reductase. Incubation of native nitrate reductase with either trypsin, Staphylococcus aureus V8 protease, or a natural inactivator protease from corn results in a loss of NADH:nitrate reductase and NADH:cytochrome c reductase activities but no loss of reduced methyl viologen:nitrate reductase activity. Incubation of nitrate reductase with V8 protease or corn inactivator protease resulted in two different products, each of which retained a different partial activity. Reduced methyl viologen:nitrate reductase activity was associated with a homotetrameric fragment of about 260 kDa which contained heme and molybdenum but no FAD. The molecular mass of native nitrate reductase determined under the same conditions was 375 kDa. NADH:ferricyanide reductase activity was associated with a monomeric species of approximately 30 kDa which contained FAD and the NADH-binding site. These results are consistent with a structure-function model of nitrate reductase which has the following features: FAD/NADH-binding domains exposed on the surface of the molecule, a protease-sensitive hinge region which connects the nitrate-reducing and NADH dehydrogenase moieties, and the quaternary structure maintained via association sites on the heme/molybdenum domain.
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PMID:Functional domains of assimilatory NADH:nitrate reductase from Chlorella. 301 63

The sequence of nitrate reductase (EC 1.6.6.1) mRNA from the plant Arabidopsis thaliana has been determined. A 3.0-kilobase-long cDNA was isolated from a lambda gt10 cDNA library of Arabidopsis leaf poly(A)+ RNA. The cDNA hybridized to a 3.2-kilobase mRNA whose level increased 15-fold in response to treatment of the plant with nitrate. An open reading frame encoding a 917 amino acid protein was found in the sequence. This protein is very similar to tobacco nitrate reductase, being greater than 80% identical within a section of 450 amino acids. By comparing the Arabidopsis protein sequence with other protein sequences, three functional domains were deduced: (i) a molybdenum-pterin-binding domain that is similar to the molybdenum-pterin-binding domain of rat liver sulfite oxidase, (ii) a heme-binding domain that is similar to proteins in the cytochrome b5 superfamily, and (iii) an FAD-binding domain that is similar to NADH-cytochrome b5 reductase.
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PMID:Sequence and nitrate regulation of the Arabidopsis thaliana mRNA encoding nitrate reductase, a metalloflavoprotein with three functional domains. 339 28

Nitrate reductase of Mitsuokella multiacidus (formerly Bacteroides multiacidus) was solublized from the membrane fraction with 1% sodium deoxycholate and purified 40-fold by immunoaffinity chromatography on the antibody-Affi-Gel 10 column. The preparation showed a major band (86% of total protein) with enzyme activity and a minor band on polyacrylamide gel after disc electrophoresis in the presence of 0.1% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a major band, the relative mobility of which corresponded to a molecular weight of 160,000, and two minor bands. The molecular weight of the enzyme was determined to be 160,000 by gel filtration on Bio-Gel A-1.5 m in the presence of 0.1% deoxycholate. Molybdenum cofactor was detected in the enzyme by fluorescence spectroscopy and by complementation of nitrate reductase from the nit-1 mutant of Neurospora crassa. The M. multiacidus enzyme catalyzed reduction of nitrate, chlorate, and bromate using methyl viologen as an electron donor. The maximal activity was found at pH 6.2-7.5 for nitrate reduction. Either methyl or benzyl viologen served well as the electron donor, but FAD, FMN, and horse heart cytochrome c were not effective. Ferredoxin from Clostridium pasteurianum supplied electron to the nitrate reductase. The purified enzyme had Km values of 0.13 mM, 0.12 mM, and 0.22 mM for nitrate, methyl viologen, and ferredoxin, respectively. The enzyme activity was inhibited by cyanide (85% at 1 mM), azide (88% at 0.1 mM), and thiocyanate (75% at 10 mM).
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PMID:Purification and properties of nitrate reductase from Mitsuokella multiacidus. 371 Oct 52

1. The assimilatory nitrite reductase of the N(2)-fixing bacterium Azotobacter chroococcum was prepared in a soluble form from cells grown aerobically with nitrate as the nitrogen source, and some of its properties have been studied. 2. The enzyme is a FAD-dependent metalloprotein (mol.wt. about 67000), which stoicheiometrically catalyses the direct reduction of nitrite to NH(3) with NADH as the electron donor. 3. NADH-nitrite reductase can exist in two either active or inactive interconvertible forms. Inactivation in vitro can be achieved by preincubation with NADH. Nitrite can specifically protect the enzyme against this inactivation and reverse the process once it has occurred. 4. A. chroococcum nitrite reductase is an adaptive enzyme whose formation depends on the presence of either nitrate or nitrite in the nutrient solution. 5. Tungstate inhibits growth of the microorganism very efficiently, by competition with molybdate, when nitrate is the nitrogen source, but does not interfere when nitrite or NH(3) is substituted for nitrate. The addition of tungstate to the culture media results in the loss of nitrate reductase activity but does not affect nitrite reductase.
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PMID:Reduced nicotinamide-adenine dinucleotide-nitrite reductase from Azotobacter chroococcum. 414 87

The reductase enzymes in Nitrosomonas and Nitrobacter were studied under anaerobic conditions when the oxidase enzymes were inactive. The most effective electron-donor systems for nitrate reductase in Nitrobacter were reduced benzyl viologen alone, phenazine methosulphate with either NADH or NADPH, and FMN or FAD with NADH. Nitrite and hydroxylamine reductases were found in both nitrifying bacteria, and optimum activity for each enzyme was obtained with NADH or NADPH with either FMN or FAD. The product of both these enzymes was identified as ammonia. In extracts of Nitrosomonas the ammonia was further utilized by an NADPH-specific glutamate dehydrogenase. (15)N-labelled nitrite, hydroxylamine and ammonia were rapidly incorporated into cell protein by Nitrosomonas, and Nitrobacter in addition incorporated [(15)N]nitrate. Relatively gentle methods of cell disruption were compared with ultrasonic treatment, to enable a more exact study to be undertaken of the intracellular distribution of the oxidase and reductase enzymes. The functional relationship of these opposing enzyme systems in the nitrifying bacteria is considered.
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PMID:Properties of some reductase enzymes in the nitrifying bacteria and their relationship to the oxidase systems. 438 32

The molecular basis for the action of two natural inactivator proteins, isolated from rice and corn, on a purified assimilatory nitrate reductase has been examined by several physical techniques. Incubation of purified Chlorella nitrate reductase with either rice inactivator protein or corn inactivator protein results in a loss of NADH:nitrate reductase and the associated partial activity, NADH:cytochrome c reductase, but no loss in nitrate-reducing activity with reduced methyl viologen as the electron donor. The molecular weight of the reduced methyl viologen:nitrate reductase species, determined by sedimentation equilibrium in the Beckman airfuge after complete inactivation with rice inactivator protein or with corn inactivator protein, was 595,000 and 283,000, respectively, compared to a molecular weight of 376,000 for the untreated control determined under the same conditions. Two protein peaks were observed after molecular-sieve chromatography on Sephacryl S-300 of nitrate reductase inactivated by corn inactivator protein. The Stokes radii of these fragments were 68 and 24 A, compared to a value of 81 A for untreated nitrate reductase. The large fragment contained molybdenum and heme but no flavin, and had nitrate-reducing activity with reduced methyl viologen as electron donor. The small fragment contained FAD but had no NADH:cytochrome c reductase or nitrate-reducing activities. Molecular weights determined by sodium dodecyl sulfate-gel electrophoresis were 67,000 and 28,000 for the large and small fragments, respectively, compared to a subunit molecular weight of 99,000 determined for the untreated control. No change in subunit molecular weight of nitrate reductase after inactivation by rice inactivator protein was observed. These results indicate that rice inactivator protein acts by binding to nitrate reductase. The stoichiometry of binding is 1-2 molecules of rice inactivator protein to one tetrameric molecule of nitrate reductase. Corn inactivator protein, in contrast, acts by cleavage of a Mr 30,000 fragment from nitrate reductase which is associated with FAD. The remaining fragment is a tetramer of Mr 70,000 subunits which retains nitrate-reducing activity and contains molybdenum and heme but has no NADH:dehydrogenase activity. The action of rice inactivator protein was partially prevented by NADH and completely prevented by a combination of NADH and cyanide, while the action of corn inactivator protein was not significantly affected by these effectors.
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PMID:Mode of action of natural inactivator proteins from corn and rice on a purified assimilatory nitrate reductase. 654 59

Assimilatory nitrate reductase (NAD(P)H-nitrate oxidoreductase, EC 1.6.6.2) from the green alga Ankistrodesmus braunii can be purified to homogeneity by dye-ligand chromatography on blue-Sepharose. The purified enzyme, whose turnover number is 623 s-1, presents an optimum pH of 7.5 and Km values of 13 microM, 23 microM and 0.15 mM for NADH, NADPH and nitrate, respectively. The NADH-nitrate reductase activity exhibits an iso ping pong bi bi kinetic mechanism. The molecular weight of the native nitrate reductase is 467 400, while that of its subunits is 58 750. These values suggest an octameric structure for the enzyme, which has been confirmed by electron microscopy. As deduced from spectrophotometric and fluorimetric studies, the enzyme contains FAD and cytochrome b-557 as prosthetic groups. FAD is not covalently bound to the protein and is easily dissociated in diluted solutions from the enzyme. Its apparent Km value is 4 nM, indicative of a high affinity of the enzyme for FAD. The results of the quantitative analyses of prosthetic groups indicate that nitrate reductase contains four molecules of flavin, four heme irons, and two atoms of molybdenum. The three components act sequentially transferring electrons from reduced pyridine nucleotides to nitrate, thus forming a short electron transport chain along the protein. A mechanism is proposed for the redox interconversion of the nitrate reductase activity. Inactivation seems to occur by formation of a stable complex of reduced enzyme with cyanide or superoxide, while reactivation is a consequence of reoxidation of the inactive enzyme. Both reactions imply the transfer of only one electron.
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PMID:Assimilatory nitrate reductase from the green alga Ankistrodesmus braunii. 668 79

Nitrate reductase (NADPH:nitrate oxidoreductase; EC 1.6.6.1-3) was purified to apparent homogeneity from mycelium of Penicillium chrysogenum. The final preparation catalyzed the NADPH-dependent, FAD-mediated reduction of nitrate with a specific activity of 170-225 units X mg of protein-1. Gel filtration and glycerol density centrifugation yielded, respectively, a Stokes radius of 6.3 nm and an s20,w of 7.4. The molecular weight was calculated to be 199,000. On sodium dodecyl sulfate gels, the enzyme displayed two almost contiguous dye-staining bands corresponding to molecular weights of about 97,000 and 98,000. The enzyme prefers NADPH to NADH (kspec ratio = 2813), FAD to FMN (kspec ratio = 141), FAD (+ NADPH) to FADH2 (kspec ratio = 12,000), and nitrate to chlorate (kspec ratio = 4.33), where the kspec (the specificity constant for a given substrate) represents Vmax/Km. The Penicillium enzyme will also catalyze te NADPH-dependent, FAD-mediated reduction of cytochrome c with a specific activity of 647 units X mg of protein-1 (Kmcyt = 1.25 X 10(-5) M), and the reduced methyl viologen (MVH2, i.e. methyl viologen + dithionite)-dependent, NADPH and FAD-independent reduction of nitrate with a specific activity of 250 units X mg of protein-1 kmMVH2 = 3.5 X 10(-6) M). Initial velocity studies showed intersecting NADPH-FAD and nitrate-FAD reciprocal plot patterns. The NADPH-nitrate pattern was a series of parallel lines at saturating and unsaturating FAD levels. NADP+ was competitive with NADPH, uncompetitive with nitrate (at saturating and unsaturating FAD levels), and a mixed-type inhibitor with respect to FAD. Nitrite was competitive with nitrate, uncompetitive with NADPH (at saturating and unsaturating FAD levels), and a mixed-type inhibitor with respect to FAD. At unsaturating nitrate and FAD, NADPH exhibited substrate inhibition, perhaps as a result of binding to the FAD site(s). At very low FAD concentrations, low concentrations of NADP+ activated the reaction slightly. The initial velocity and product inhibition patterns are consistent with either of the two kinetic mechanisms. One (rather unlikely) mechanism involves the rapid equilibrium random binding of all ligands with (a) NADP+ and NADPH mutually exclusive, (b) nitrate and nitrite mutually exclusive, (c) the binding of NADPH strongly inhibiting the binding of nitrate and vice versa, (d) the binding of NADPH strongly promoting the binding of nitrite and vice versa, and (e) the binding of nitrate strongly promoting the binding of NADP+ and vice versa...
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PMID:Nitrate reductase from Penicillium chrysogenum. Purification and kinetic mechanism. 679 May 45

NADPH-nitrate reductase [NADPH : nitrate oxidoreductase, EC 1.6.6.3] was purified 500-fold from Aspergillus nidulans with an overall yield of about 20%. The purified enzyme catalyzed NADPH-nitrate, NADPH-cytochrome c, FADH2-nitrate and reduced methyl viologen-nitrate reductase activities. Its molecular weight was estimated to be 180,000 from the Stokes radius and sedimentation coefficient. The oxidized enzyme exhibited an absorption spectrum having a peak at 412 nm and a broad shoulder at about 450 nm. When reduced with NADPH, absorption peaks appeared at 423 (Soret), 527 (beta) and 557 (alpha) nm, and absorption in the 450 nm region decreased. Upon treatment of the reduced enzyme with KNO3, the spectrum returned to that of the oxidized enzyme. The presence of protoheme in the enzyme was confirmed by the absorption spectrum of reduced pyridine hemochromogen. It was concluded that a b-type cytochrome ("cytochrome b-557") is present in the enzyme and is involved in the intramolecular electron transport from NADPH to nitrate. The NADPH-nitrate and NADPH-cytochrome c reductase activities, but not the other two activities, were significantly decreased by incubation of the enzyme at 37 degrees C in the absence of FAD. Analysis by SDS slab gel electrophoresis suggested that the nitrate reductase consists of two each of two subunits of 59,000 and 38,000 daltons and that a dissociation of 38,000 subunits from the native enzyme occurs during heat treatment, resulting in alteration of the catalytic activity.
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PMID:Purification and characterization of the assimilatory NADPH-nitrate reductase of Aspergillus nidulans. 704 1

Nitrate reductase (NAD(P)H:nitrate oxidoreductase, EC 1.6.6.2) of the unicellular alga Cyanidium caldarium can exist in two interconvertible forms; one catalytically active and one inactive. The inactive nitrate reductase can be activated by mild treatment with denaturing agents of protein. By treatment with urea or mersalyl, activation of both the NADPH and benzyl viologen activities can be realized under mild conditions, whereas by treatment with heat, the activation of benzyl viologen activity is concomitant with loss of the NADPH activity. On the other hand, both activities are activated and destroyed concomitantly by ethylene glycol. In the present of FAD, either activation of benzyl viologen activity or loss of NADPH activity upon heating occur only at higher temperatures. The existence of a controlling region in the nitrate reductase molecule is postulated.
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PMID:Active and inactive nitrate reductase. Effects of mild treatment with denaturing agents of protein. 718 70


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