KEGG:D02011 - FACTA Search

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

Adrenodoxin reductase is an NADP dependent flavoenzyme which functions as the reductase of mitochondrial P 450 systems. We sequenced two adrenodoxin reductase cDNAs isolated from a bovine adrenal cortex cDNA library. The deduced amino acid sequence shows no similarity to the sequence of the microsomal P 450 systems or other known protein sequences. Nonetheless, by sequence analysis and c comparisons with known sequences of dinucleotide-binding folds of two NADP-binding flavoenzymes, two regions of adrenodoxin reductase sequence were identified as the FAD- and NADP-binding sites. These analyses revealed a consensus sequence for the NADP-binding dinucleotide fold (GXGXXAXXXAXXXXXXG, in one-letter amino acid code) that differs from FAD and NAD-binding dinucleotide-fold sequences. In the data base of protein sequences, the NADP-binding-site sequence appears solely in NADP-dependent enzymes, the binding sites of which were not known to date. Thus, this sequence may be used for identification of a certain type of NADP-binding site of enzymes that show no significant sequence similarity.
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PMID:cDNA sequence of adrenodoxin reductase. Identification of NADP-binding sites in oxidoreductases. 292 77

The 31P NMR spectra of NADPH-adrenodoxin reductase and its complex with NADP+ are reported. The spectrum of adrenodoxin reductase showed two doublets arising from the phosphorus nuclei in the pyrophosphate group of FAD. Both doublets were shifted upfield to different extents in comparison with those of free FAD. Further, one of the doublets of phosphorus nuclei of the pyrophosphate group of bound NADP+ in the complex of adrenodoxin reductase and NADP+ was considerably shifted upfield in comparison with that of free NADP+. The spectrum of the complex of the reductase and NADP+ showed that the resonance of the 2'-phosphate group of NADP+ bound to the reductase was shifted downfield by 1.37 ppm compared with that of free NADP+ in the dianionic state. The 2'-phosphate resonance of bound NADP+ was independent of pH within the physiological range, whereas that of free NADP+ changed according to its ionization. The resonance of the 2'-phosphate group of NADP+ bound to the reductase also revealed that the ratio for the complex of NADP+ and the reductase was 1:1, and that this complex formation was inhibited by a high KCl concentration. These results were confirmed by electronic spectroscopic studies.
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PMID:Phosphorus-31 nuclear magnetic resonance and electronic spectroscopic studies of adrenodoxin reductase and its binary complex with NADP+. 299 64

Chemical modification of rat hepatic NADPH-cytochrome P-450 reductase by sodium 2,4,6-trinitrobenzenesulfonate (TNBS) resulted in a time-dependent loss of the reducing activity for cytochrome c. The inactivation exhibited pseudo-first-order kinetics with a reaction order approximately one, and a second-order constant of 4.8 min-1 X M-1. The reducing activities for 2,6-dichloroindophenol and K3Fe(CN)6 were also decreased by TNBS. Almost complete protection of the NADPH-cytochrome P-450 reductase from inactivation by TNBS was achieved by NADP(H), while partial protection was obtained with a high concentration of NADH. NAD, FAD and FMN showed no effect against the inactivation. 3-Acetylpyridine-adenine dinucleotide phosphate, adenosine 2',5'-bisphosphate and 2'AMP protected the enzyme against the chemical modification. Stoichiometric studies showed that the complete inactivation was caused by modification of three lysine residues per molecule of the enzyme. But, under the conditions where the inactivation was almost protected by NADPH, two lysine residues were modified. From those results, we propose that one residue of lysine is located at the binding site of the 2'-phosphate group on the adenosine ribose of NADP(H), and plays an essential role in the catalytic function of the NADPH-cytochrome P-450 reductase.
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PMID:Chemical modification of NADPH-cytochrome P-450 reductase. Presence of a lysine residue in the rat hepatic enzyme as the recognition site of 2'-phosphate moiety of the cofactor. 300 31

Reduced pyridine nucleotide dependent glutamate synthase [L-glutamate: NADP+ oxidoreductase (transaminating); EC 1.4.1.13] was purified to homogeneity from Bacillus subtilis PCI 219. The molecular weight of the enzyme was 210,000, and the enzyme was composed of two nonidentical subunits with molecular weights of 160,000 and 56,000. The absorption and CD spectra of the enzyme indicated that the enzyme is an iron-sulfur flavoprotein. The enzyme was found to contain 1:1:7.4:8.7 mol of FMN, FAD, iron atoms, and acid-labile sulfur atoms per mol (MW 210,000). EPR measurements of the NADPH-reduced enzyme at 77K revealed the formation of a stable flavin semiquinone intermediate; however, none of the signals originating from the iron-sulfur cluster was observed. Still at 4.2K the EPR signals in the region of g = 2, which may originate from the paramagnetic iron-sulfur cluster, were clearly observed for both the isolated and dithionite-reduced states of the enzyme. The enzyme exhibited a wide coenzyme specificity, and either NADPH or NADH could be used as electron donor, although the latter was less effective. The enzyme activity was also expressed when ammonium chloride was substituted for L-glutamine. The optimum pHs for NADPH-Gln-, NADH-Gln-, and NADPH-NH3-dependent reactions were 7.8, 6.9, and 9.4, respectively. The apoenzyme exhibited substantial inactivation of the Gln-dependent activities but still retained the NH3-dependent activities. Enzyme reduction-oxidation experiments, initial velocity experiments, and product inhibition patterns revealed that both the NADPH-Gln- and NADH-Gln-dependent reactions coincided with the two-site ping-pong uni-uni bi-bi kinetic mechanism, while the NADPH-NH3-dependent reaction deviated from Michaelis-Menten kinetics. The Gln-dependent activities were inhibited by several TCA cycle members, especially L-malate and fumarate, as well as L-methionine-SR-sulfoximine, pyridoxal-5'-phosphate, and pCMB. The regulation of the glutamate synthase, glutamine synthetase [EC 6.3.1.2], and glutamate dehydrogenase [EC 1.4.1.3] activities was examined with cultures of cells grown with various nitrogen and carbon sources.
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PMID:Glutamate synthase from Bacillus subtilis PCI 219. 301 66

The activities of the mitochondrial enzymes citrate synthase (citrate oxaloacetatelyase, EC 4.1.3.7), NADP-linked isocitrate dehydrogenase (threo-Ds-isocitrate:NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42), and succinate dehydrogenase (succinate: FAD oxidoreductase, EC 1.3.99.1) as well as their kinetic behavior in the two developmental forms of Trypanosoma cruzi at insect vector stage, epimastigotes and infective metacyclic trypomastigotes, were studied. The results presented in this work clearly demonstrate a higher mitochondrial metabolism in the metacyclic forms as is shown by the extraordinary enhanced activities of metacyclic citrate synthase, isocitrate dehydrogenase, and succinate dehydrogenase. In epimastigotes, the specific activities of citrate synthase at variable concentrations of oxalacetate and acetyl-CoA were 24.6 and 26.6 mU/mg of protein, respectively, and the Michaelis constants were 7.88 and 6.84 microM for both substrates. The metacyclic enzyme exhibited the following kinetic parameters: a specific activity of 228.4 mU/mg and Km of 3.18 microM for oxalacetate and 248.5 mU/mg and 2.75 microM, respectively, for acetyl-CoA. NADP-linked isocitrate dehydrogenase specific activities for epimastigotes and metacyclics were 110.2 and 210.3 mU/mg, whereas the apparent Km's were 47.9 and 12.5 microM, respectively. No activity for the NAD-dependent isozyme was found in any form of T. cruzi differentiation. The particulated succinate dehydrogenase showed specific activities of 8.2 and 39.1 mU/mg for epimastigotes and metacyclic trypomastigotes, respectively, although no significant changes in the Km (0.46 and 0.48 mM) were found. The cellular role and the molecular mechanism that probably take place during this significant shift in the mitochondrial metabolism during the T. cruzi differentiation have been discussed.
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PMID:Differential energetic metabolism during Trypanosoma cruzi differentiation. I. Citrate synthase, NADP-isocitrate dehydrogenase, and succinate dehydrogenase. 305 38

NADPH-cytochrome P-450 reductase releases FAD upon dilution into slightly acidic potassium bromide. Chromatography on high performance hydroxylapatite resolved the FAD-dependent reductase from holoreductase. The FAD dependence was matched by a low FAD content, with the ratio of FAD to FMN as low as 0.015. The aporeductase had negligible activity toward cytochrome c, ferricyanide, menadione, dichlorophenolindophenol, nitro blue tetrazolium, and an analogue of NADP, acetylpyridine adenine dinucleotide phosphate. A 4-min incubation in FAD reconstituted from one-half to all of the enzyme activity, as compared to the untreated reductase, depending upon the substrate. After a 2-h reconstitution, the reductase eluted from hydroxylapatite at the same location in the elution profile as did the untreated holoreductase. The reconstituted reductase had little flavin dependence, was nearly equimolar in FMN and FAD, and had close to the specific activity, per mol of flavin, of untreated reductase. The dependence upon FAD implies that FMN is not a competent electron acceptor from NADPH. Thus, the FAD site must be the only point of electron uptake from NADPH.
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PMID:Preparation and characterization of FAD-dependent NADPH-cytochrome P-450 reductase. 308 19

The effect of nicotinamide and flavin coenzymes on the 5-lipoxygenase activity has been determined in cell-free extracts from rat polymorphonuclear leukocytes. 5-lipoxygenase was assayed in the presence of 5-hydroperoxyeicosatetraenoic acid (5-HPETE), which caused a 3 to 4-fold stimulation in the maximal conversion of radiolabeled arachidonic acid to 5-hydroxyeicosatetraenoic acid (5-HETE) and 5,12-dihydroxyeicosatetraenoic acid (5,12-di-HETE). Addition of FMN or FAD to the assay mixture had little effect on the 5-lipoxygenase activity and caused inhibition only at high concentrations (IC50 greater than 100 microM). NADH markedly potentiated the inhibition of lipoxygenase by flavins with a 100-fold decrease in the FMN concentration required to inhibit the enzyme (IC50 approximately equal to 2 microM). Similar effects were observed for FAD although this flavin derivative was slightly less potent than FMN (IC50 congruent to 10 microM). NADH could be substituted by NADPH but not by NAD or NADP, indicating that the inhibition was not due to the production of the oxidized forms of these co-factors. These results show that the 5-lipoxygenase activity is stimulated by 5-HPETE and inhibited by flavin-dependent redox transformations.
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PMID:Modulation of rat polymorphonuclear leukocyte 5-lipoxygenase activity by 5-HPETE and NADH-dependent flavin inhibition. 309 90

Microsomal NADPH-cytochrome P-450 reductase is the only mammalian flavoprotein known to contain both FAD and FMN as prosthetic groups. The discovery of the air-stable semiquinone [Masters, B. S. S., Kamin, H., Gibson, Q. H., & Williams, C. H., Jr. (1965) J. Biol. Chem. 240, 921-931] and its identification as a one-electron-reduced state [Iyanagi, T., & Mason, H. S. (1973) Biochemistry 12, 2297-2308] have engendered a number of studies to elucidate its unique catalytic mechanism. In this paper, 31P NMR spectroscopy is utilized to probe the localization of the free radical in this air-stable semiquinone form and to ascertain the environments of the FAD and FMN prosthetic groups as affected by the paramagnetic ion Mn(II). Consistent with conclusions drawn from studies utilizing FMN-free reductase [Vermilion, J. L., & Coon, M. J. (1978) J. Biol. Chem. 253, 8812-8819], the free radical was shown to reside on the FMN moiety by the broadening of its characteristic resonance in the 31P NMR spectrum. In addition, the effect of the paramagnetic ion Mn(II) was determined on the four resonances attributable to FAD and FMN and the additional ones contributed by NADP+ resulting from the oxidation of the physiological reductant NADPH. The addition of Mn(II) had little effect on the line widths of the FMN and FAD signals but resulted in an increase in their intensities due to a decrease in T1 relaxation times.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Localization of the free radical on the flavin mononucleotide of the air-stable semiquinone state of NADPH-cytochrome P-450 reductase using 31P NMR spectroscopy. 309 32

Preincubation of the oxidized form of the flavoenzyme mercuric reductase with the reducing substrate, NADPH, or with a high concentration of cysteine (30 mM) results in a substantial increase of the catalytic activity as measured in a standard spectrophotometric assay. Also NADH has some activating effect but NADP+ or EDTA have no effect. In the presence of 1 mM cysteine only one equivalent of NADPH per FAD seems to be required for full activation which occurs after an incubation time of about 10 min. Activated mercuric reductase appears to be stable under anaerobic conditions but eventually returns to the original level of activity in the presence of oxygen. The activated state seems to be stabilized by 1 mM cysteine. Activation of mercuric reductase does not seem to be correlated with a change in the number of reactive thiol groups. The chemical nature of the activation process is not yet understood. Stopped-flow studies have shown that the nonactivated enzyme is practically inactive prior to contact with the substrates. The enzyme is gradually activated during the assay. The kinetics of activation of the 'native' enzyme is biphasic but 'clipped' enzyme, lacking an 85-residue N-terminal domain, is activated in a single first-order process. The progress curves obtained with preactivated enzyme are approximately exponential even at saturating concentrations of NADPH (Km = 0.4 microM at 25 degrees C, pH 7.3) and Hg2+ (Km = 3.2 microM in the presence of 1 mM cysteine). The initial rates yield kcat values of about 13 s-1 per FAD molecule (25 degrees C, pH 7.3). We find no evidence for a thiol-dependent change from a rapid to a slow kinetic phase. The shape of the progress curves presumably depends on product inhibition, but NADP+ is not a sufficiently effective inhibitor to explain the effect fully.
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PMID:Activation of mercuric reductase by the substrate NADPH. 310 42

Pyruvate:NADP+ oxidoreductase was homogeneously purified from crude extract of Euglena gracilis. The Mr of the enzyme was estimated to be 309,000 by gel filtration. The enzyme migrated as a single protein band with Mr of 166,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the enzyme consists of two identical polypeptides. The absorption spectrum of the native enzyme exhibited maxima at 278, 380, and 430 nm, and a broad shoulder was observed around 480 nm; the maximum at 430 nm was eliminated by reduction of the enzyme with dithionite. Reduction of the enzyme with pyruvate and CoA and reoxidation with NADP+ were proved from changes of absorption spectra. The enzyme contained 2 molecules of FAD and 8 molecules of iron. It was also indicated that the enzyme was thiamine pyrophosphate-dependent. The enzyme was oxygen-sensitive, and the reaction was affected by the presence of oxygen. Pyruvate was the most active substrate, but the enzyme was slightly active for 2-oxobutyrate, 3-hydroxypyruvate, and oxalacetate, but not for glyoxylate and 2-oxoglutarate. The native electron acceptor was NADP+, whereas NAD+ was completely inactive. Methyl viologen, benzyl viologen, FAD, and FMN were utilized as artificial electron acceptors, whereas spinach and Clostridium ferredoxins were inactive. Pyruvate synthesis by reductive carboxylation of acetyl-CoA with NADPH as the electron donor occurred by the reverse reaction of the enzyme. The enzyme also catalyzed a pyruvate-CO2 exchange reaction and electron-transfer reaction from NADPH to other electron acceptors like methyl viologen. These results indicate that pyruvate:NADP+ oxidoreductase in E. gracilis is clearly distinct from either the pyruvate dehydrogenase multienzyme complex or pyruvate:ferredoxin oxidoreductase.
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PMID:Purification and characterization of pyruvate:NADP+ oxidoreductase in Euglena gracilis. 311 Jan 54

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