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)

Human NADH-cytochrome b5 reductase (EC 1.6.2.2) contains 4 cyteine residues (Cys-203, -273, -283, and -297). Cys-283 was previously proposed to be involved in NADH binding by chemical modification (Hackett, C. S., Novoa, W. B., Ozols, J., and Strittmatter, P. (1986) J. Biol. Chem. 261, 9854-9857). In the present study the role of cysteines in the enzyme was probed by replacing these residues by Ser, Ala, or Gly employing site-directed mutagenesis and chemical modification. Four mutants, in which 1 of the 4 Cys residues was replaced by Ser, retained comparable kcat and Km values to those of the wild type. All of these mutants were as sensitive as the wild type to treatment with SH modifiers, while a double mutant, C273S/C283S was resistant. Since inhibition by SH modifiers was protected by NADH, Cys-273 and Cys-283 were implicated to be close to the NADH-binding site. C273A and C273A/C283A mutants showed approximately one-fifth of the enzyme-FAD reduction rate of the wild type as revealed by steady-state kinetics and by stopped-flow analysis. Anaerobic titration has shown that reduction and re-oxidation processes including formation of the red semiquinone of these mutants were not significantly altered from those of the wild type. From these results it was concluded that none of the Cys residues of the enzyme are essential in the catalytic reaction, but Cys-273 conserved among the enzymes homologous to NADH-cytochrome b5 reductase homologous to NADH-cytochrome b5 reductase plays role(s) in facilitating the reaction. A difference spectrum with a peak at 317 nm, which was formerly considered to be derived from the interaction between NAD+ and Cys-283 of the reduced enzyme, appeared upon binding of NAD+ not only to the reduced wild type enzyme but also to the C273A/C283A mutant in which both of the Cys residues close to the NADH-binding site were replaced.
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PMID:Role of cysteine residues in human NADH-cytochrome b5 reductase studied by site-directed mutagenesis. Cys-273 and Cys-283 are located close to the NADH-binding site but are not catalytically essential. 201 83

2,4-Pentadienoyl-CoA reductase from Clostridium aminovalericum was purified to homogeneity (170-182 kDa). PAGE in the presence of SDS revealed a single band (44 kDa) indicating a homotetrameric structure. The native enzyme had a green colour and contained 0.4 mol FAD/subunit. Its unusual ultraviolet/visible-spectrum showed absorption maxima at 270, 402 and 715 nm as well as shoulders at 278, 360, 450 and 500 nm. Removal of the prosthetic group at pH 2 in the presence of salt and charcoal yielded a colourless and completely inactive apoenzyme, which could be reconstituted with FAD (not with FMN) to an active holoenzyme showing a normal flavoprotein spectrum (peaks at 369 nm and 436 nm). Thereby the FAD content increased to 0.9 mol/subunit with a concomitant rise in activity to 200% of the original value. Anaerobic reduction of the green enzyme by dithionite and reoxidation by air afforded a green preparation with a spectrum similar to that of the native enzyme. Addition of excess FAD to the green reductase also increased the activity by a factor of two. The green enzyme catalysed the oxidation of (E)-3-pentenoyl-CoA or (E)-3-hexenoyl-CoA to 2,4-pentadienoyl-CoA or 2,4-hexenoyl-CoA, respectively. 2-Pentenoyl-CoA or 4-pentenoyl-CoA were not oxidised. Meldola blue (8-dimethylamino-2,3-benzophenoxazine) and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (V = 26 nkat/mg protein) or ferricenium hexafluorophosphate (V = 1900 nkat/mg), but not NAD(P), served as electron acceptors. Reduction of 2,4-pentadienoyl-CoA (V = 370 nkat/mg) was observed with reduced benzyl viologen, but not with NAD(P)H as an electron donor. Although the enzyme had some pentenoyl-CoA delta-isomerase activity (1.2 nkat/mg), the only product of the reduction was 3-pentenoyl-CoA rather than 2-pentenoyl-CoA.
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PMID:A green 2,4-pentadienoyl-CoA reductase from Clostridium aminovalericum. 204 Feb 89

In the three-dimensional structures of enzymes that bind NAD or FAD, there is an acidic residue that interacts with the 2'- and 3'-hydroxyl groups of the adenosine ribose of the coenzyme. The size and charge of the carboxylate might repel the binding of the 2'-phosphate group of NADP and explain the specificity for NAD. In the NAD-dependent alcohol dehydrogenases, Asp-223 (horse liver alcohol dehydrogenase sequence) appears to have this role. The homologous residue in yeast alcohol dehydrogenase I (residue 201 in the protein sequence) was substituted with Gly, and the D223G enzyme was expressed in yeast, purified, and characterized. The wild-type enzyme is specific for NAD. In contrast, the D223G enzyme bound and reduced NAD+ and NADP+ equally well, but, relative to wild-type enzyme, the dissociation constant for NAD+ was increased 17-fold, and the reactivity (V/K) on ethanol was decreased to 1%. Even though catalytic efficiency was reduced, yeast expressing the altered or wild-type enzyme grew at comparable rates, suggesting that equilibration of NAD and NADP pools is not lethal. Asp-223 participates in binding NAD and in excluding NADP, but it is not the only residue important for determining specificity for coenzyme.
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PMID:An aspartate residue in yeast alcohol dehydrogenase I determines the specificity for coenzyme. 205 45

It was found that when Escherichia coli is grown in the presence of 0.2-0.3 mM menadione (2-methyl-1,4-naphthoquinone), an FMN-dependent NADH-quinone reductase increases more than 20-fold in the cytoplasmic fraction. The menadione-induced quinone reductase was isolated from the cytoplasmic fraction of induced cells. The purified enzyme had an Mr of 24 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme required flavin as a cofactor and a half-maximum activity was obtained with 0.54 microM FMN or 16.5 microM FAD. The enzyme had a broad pH optimum at pH 7.0-8.0 and reacted with NADH, but not with NADPH. The reaction followed a ping-pong mechanism and the intrinsic Km values for NADH and menadione were estimated to be 132 microM and 2.0 microM, respectively. Dicoumarol was a simple competitive inhibitor with respect to NADH with a Ki value of 0.22 microM. The electron acceptor specificity of this enzyme was very similar to that of NAD(P)H: (quinone acceptor) oxidoreductase (EC 1.6.99.2, DT-diaphorase) from rat liver. Since menadione is reduced by the two-electron reduction pathway to menadiol, the induction of this enzyme is likely to be an adaptive response of E. coli to partially alleviate the toxicity of menadione.
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PMID:Characterization of FMN-dependent NADH-quinone reductase induced by menadione in Escherichia coli. 211 86

Hypusine formation on an 18,000-dalton cellular protein is a unique spermidine-dependent, post-translational modification that appears to be ubiquitous in mammalian cells. To determine whether this modification also exists in lower eukaryotes, we examined possible labeling in vitro and in vivo of cellular protein(s) by [3H]spermidine in a mutant strain of Neurospora crassa (arge-12 ota aga) in which ornithine and polyamine synthesis could be nutritionally manipulated. Because of poor uptake of polyamines in this organism, [3H]ornithine, the immediate precursor of polyamines, was used for the in vivo labeling experiment. Both in vitro and in vivo labeling resulted in a specific labeling of a 21,000-dalton protein. Radioactive hypusine was recovered from radiolabeled 21,000-dalton protein following acid hydrolysis. The in vitro labeling of the 21,000-dalton protein was dramatically stimulated by NAD+ and NADP+, but not by FMN or FAD, suggesting that an NAD+/NADP(+)-dependent oxidative cleavage of spermidine is involved in deoxyhypusine formation. Isoelectric focusing/sodium dodecyl sulfate two-dimensional gel analysis revealed three isoforms of the in vitro labeled 21,000-dalton protein, with pI values ranging from 5.2 to 6.5. In contrast, the 21,000-dalton protein metabolically labeled in vivo gave only one spot with a pI value of approx. 3.5.
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PMID:Deoxyhypusine/hypusine formation on a 21,000-dalton cellular protein in a Neurospora crassa mutant in vivo and in vitro. 213 13

The effects of butylated hydroxyanisole (BHA), a commonly used food antioxidant, on oxygen consumption, ATPase activity, and the redox state of some electron carriers of rat liver mitochondria have been studied. It was observed that BHA slightly stimulated state 4 respiration but strongly inhibited ADP- and uncoupler-stimulated respiration on NAD(+)- and FAD-linked substrates. ATPase activity and vectorial H+ ejection were affected only slightly by BHA, suggesting that BHA predominantly inhibits mitochondrial electron flow. Experiments to determine its site of action showed that BHA did not noticeably affect electron flow through cytochrome oxidase; in contrast, NADH:duroquinone reductase activity and electron flow through ubiquinone-cytochrome b-cytochrome c complex were inhibited strongly because the oxidation of duroquinol was affected markedly. The BHA block of electron transport was bypassed by both N,N,N',N'-tetramethyl-p-phenylenediamine and 2,6-dichlorophenolindophenol. Also, the presence of BHA changed the redox state of cytochrome b and c1 to a more oxidized level. These observations suggest that electron transport is inhibited by BHA at the NADH-ubiquinone and at the ubiquinone-cytochrome b levels. From Hill plots, it is clear that more than one binding site is involved in complete inhibition; in addition, available evidence suggests that there may be two sites at the substrate side of ubiquinone and another two sites at the oxygen side of ubiquinone. Consequently, mitochondrial ATP synthesis would be interrupted. This event could be related to the toxicity of BHA.
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PMID:Effect of butylated hydroxyanisole on electron transport in rat liver mitochondria. 214 54

Electron spin resonance (ESR) measurements on solutions and isolated powders provide direct evidence for the involvement of Cr(V) species in the reduction of Cr(VI) by NAD(P)H. ESR analysis of an isolated Cr(V)-NAD(P)H solid yields g parallel = 1.9831 and g perpendicular = 1.9772, indicating that the unpaired electron occupies the dz2 orbital of the Cr(V) ion, with square-pyramidal geometry. Addition of hydrogen peroxide (H2O2) to the NAD(P)H-Cr(VI) reaction mixtures suppresses the Cr(V) species and generates hydroxyl (.OH) radicals. The .OH radicals were detected via ESR spin trapping, employing 5,5-dimethyl-1-pyrroline-N-oxide and alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone as spin traps. The dependence of Cr(V) and .OH radical formation on the H2O2 and Cr(VI) concentrations indicates that the Cr(V) species react with H2O2 to generate the .OH radicals. Similar results were obtained by using various diols (arabinose, cellobiose, FAD, fructose, glyceraldehyde, ribose, and tartaric acid), alpha-hydroxycarboxylic acids, and glutathione. Investigations with superoxide dismutase showed no significant participation of O2- in the generation of .OH radicals. These results thus indicate that the Cr(V) complexes, produced in the reduction of Cr(VI) by cellular reductants, react with H2O2 to generate .OH radicals, which might be initiators of the primary events in the Cr(VI) cytotoxicity.
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PMID:On the hydroxyl radical formation in the reaction between hydrogen peroxide and biologically generated chromium(V) species. 215 79

The effect of halothane, a potent and popular volatile anesthetic, on isolated rat liver mitochondria was examined. Halothane inhibited state 3 and dinitrophenol-induced uncoupled respiration with NAD(+)-linked substrates, but not with FAD-linked substrates, and did not affect the oxidation-reduction state of mitochondrial cytochromes. Moreover, halothane increased state 4 respiration and ATPase activity and decreased the extra-mitochrondrial pH change coupled to ATP synthesis. These results indicate that halothane impairs mitochondrial ATP production by interfering with both the electron transport from NAD+ to FAD and the coupling of oxidative phosphorylation. Halothane only slightly affected the membrane potential, which is commonly dissipated by typical classical uncouplers. Moreover, halothane inhibited both ATP-driven and respiration-driven Ca2+ accumulation in mitochondria and stimulated Ca2+ release from mitochondrial stores at concentrations higher than those at which it inhibited ATP production. These findings indicate that the uncoupling action of halothane is not classical. During halothane anesthesia, these mitochondrial abnormalities may contribute to hepatocyte dysfunctions.
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PMID:Halothane impairs the bioenergetic functions of isolated rat liver mitochondria. 216 74

Highly-purified bidirectional hydrogenase (hydrogenase 1) of Clostridium pasteurianum could rapidly reduce several 2-, 4- and 5-nitroimidazole compounds via an electron carrier-coupled mechanism. Hydrogenase 1 was also shown to reduce a 2-nitroimidazole (misonidazole) and a 4-nitroimidazole in the presence of its required electron carriers including ferredoxin, the flavin coenzymes FAD and FMN, and the low potential electron carrier dyes methyl- and benzyl-viologen. No drug reduction by hydrogenase 1 occurred when any one of these electron carriers was replaced by nicotinamide electron carriers (NAD and NADP), or was omitted from the reaction mixture. The rates of reduction of the nitroimidazole compounds correlated with their one electron reduction potentials at pH 7(E7(1)); the higher the drug's E7(1), the faster its rate of reduction by the enzyme. Reduction rates for the drugs did not correlate with the antibacterial activity of these compounds against C. pasteurianum, suggesting that other factors are also important in determining the antimicrobial potencies of nitroimidazoles.
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PMID:Reduction of 2-, 4- and 5-nitroimidazole drugs by hydrogenase 1 in Clostridium pasteurianum. 218 Aug 90

Several NAD(P)H-dependent ferri-reductase activities were detected in sub-cellular extracts of the yeast Saccharomyces cerevisiae. Some were induced in cells grown under iron-deficient conditions. At least two cytosolic iron-reducing enzymes having different substrate specificities could contribute to iron assimilation in vivo. One enzyme was purified to homogeneity: it is a flavoprotein (FAD) of 40 kDa that uses NADPH as electron donor and Fe(III)-EDTA as artificial electron acceptor. Isolated mitochondria reduced a variety of ferric chelates, probably via an 'external' NADH dehydrogenase, but not the siderophore ferrioxamine B. A plasma membrane-bound ferri-reductase system functioning with NADPH as electron donor and FMN as prosthetic group was purified 100-fold from isolated plasma membranes. This system may be involved in the reductive uptake of iron in vivo.
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PMID:Iron-reductases in the yeast Saccharomyces cerevisiae. 218 97


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