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)

Glutaryl-CoA dehydrogenase, a flavoprotein, catalyzes the reaction -OOCCH3CH2--CH2COSR (FAD leads to FADH2) leads to CH3CH = CHCOSR + CO2 (SR = CoA or pantetheine). With the isolated enzyme, a dye serves as the final electron acceptor. The enzyme from Pseudomonas fluorescens (ATCC 11250) has been purified to homogeneity. It was established with appropriate isotopic substitutions that the proton which is added to the gamma position of the product, subsequent to decarboxylation, is not derived from the solvent but is derived from the alpha position of the substrate. Under conditions where no net conversion of substrate occurs, i.e., in the absence of electron acceptor, the enzyme catalyzes the exchange of the beta hydrogen of the substrate with solvent protons. Butyryl-CoA dehydrogenase (M. elsedenii), which catalyzes an analogous reaction, catalyzes the exchange of both the alpha and beta hydrogens with solvent protons in the absence of electron acceptor. Glutaryl-CoA dehydrogenase and butyryl-CoA dehydrogenase are irreversibly inactivated by the substrate analogues 3-butynoylpantetheine and 3-pentynoylpantetheine. These inactivators do not form an adduct with the flavin and probably react with a nucleophile at the active site. Upon inactivation, the spectrum of the enzyme-bound flavin is essentially unchanged, and the flavin can be reduced by Na2S2O4. We suggest that inactivation involves intermediate allene formation. We proposed that these results support an oxidation mechanism for glutaryl-CoA dehydrogenase and butyryl-CoA dehydrogenase which is initiated by proton abstraction. With glutaryl-CoA dehydrogenase, the base, which abstracts the substrate alpha proton, is shielded from the solvent and is then used to protonate the carbanion (CH2--CH--CHCOSCoA) formed after oxidation and decarboxylation.
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PMID:Mechanism of action of glutaryl-CoA and butyryl-CoA dehydrogenases. Purification of glutaryl-CoA dehydrogenase. 626 96

Butyryl-CoA dehydrogenase from Megasphera elsdenii catalyzes the exchange of the alpha- and beta-hydrogens of substrate with solvent [Gomes, B., Fendrich, G., & Abeles, R. H. (1981) Biochemistry 20, 1481-1490]. The stoichiometry of this exchange was determined by using 3H2O label as 1.94 +/- 0.1 per substrate molecule. The rate of 3H label incorporation into substrate under anaerobic conditions is monophasic, indicating that both the alpha- and beta-hydrogens exchange at the same rate. The exchange in 2H2O leads to incorporation of one 2H each into the alpha- and the beta-positions of butyryl-CoA, as determined by companion 1H NMR experiments and confirmed by mass spectroscopic analysis. In contrast, with general acyl-CoA dehydrogenase from pig kidney, only exchange of the alpha-hydrogen was found. The beta-hydrogen is the one that is transferred (reversibly) to the flavin 5-position during substrate dehydrogenation. This was demonstrated by reacting 5-3H- and 5-2H-reduced 5-deaza-FAD-general acyl-CoA dehydrogenase with crotonyl-CoA. Only one face of the reduced flavin analogue is capable of transferring hydrogen to substrate. The rate of this reaction is 11.1 s-1 for 5-deaza-FAD-enzyme and 2.2 s-1 for [5-2H]deaza-FAD-enzyme, yielding an isotope effect of 5. These values compare with a rate of 2.6 s-1 for the reaction of native reduced enzyme with crotonyl-CoA. The two reduced enzymes (normal vs. 5-deaza-FAD-enzyme) thus react at similar rates, indicating a similar mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mechanistic studies with general acyl-CoA dehydrogenase and butyryl-CoA dehydrogenase: evidence for the transfer of the beta-hydrogen to the flavin N(5)-position as a hydride. 646 35

The FAD-containing short-chain acyl-CoA dehydrogenase was purified from ox liver mitochondria by using (NH4)2SO4 fractionation, DEAE-Sephadex A-50 and chromatofocusing on PBE 94 resin. The enzyme is a tetramer, with a native Mr of approx. 162 000 and a subunit Mr of 41 000. Short-chain acyl-CoA dehydrogenases are usually isolated in a green form. The chromatofocusing step in the purification presented here partially resolved the enzyme into a green form and a yellow form. In the dye-mediated assay system, the enzyme exhibited optimal activity towards 50 microM-butyryl-CoA at pH 7.1. Kinetic parameters were also determined for a number of other straight-chain acyl-CoA substrates. The u.v.- and visible-absorption characteristics of the native forms of the enzyme are described, together with complexes formed by addition of butyryl-CoA, acetoacetyl-CoA and CoA persulphide.
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PMID:The purification and properties of ox liver short-chain acyl-CoA dehydrogenase. 671 27

Removal of the green colour of butyryl-CoA dehydrogenase by reagents specific for nucleophilic sulphur is shown to involve chemical modification of the tightly bound CoA persulphide. 5,5'-Dithiobis-(2-nitrobenzoic acid) (Ellman's reagent) de-greens the enzyme essentially irreversibly, with a stoicheiometry of approx. 1 mol/mol of FAD. A compound separated by subsequent gel filtration is eluted at the same position as a CoA-thionitrobenzoate standard. The 35S-labelled distal sulphur atom of CoA persulphide is separated from this material. The enzyme remains fully active. Phenylmercuric acetate also de-greens the enzyme. The extent to which thiols restore the green colour declines with time. Gel filtration of mercurial-treated enzyme separates low-Mr material containing a CoA moiety, an extra S atom and a phenylmercury moiety. This material, added to yellow butyryl-CoA dehydrogenase with an excess of thiol, re-forms green enzyme, but it loses this ability on storage. The results are explicable if it is assumed that the thionitrobenzoate derivative of CoA persulphide loses the extra sulphur atom much more readily than does the phenylmercury derivative.
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PMID:The effect on butyryl-CoA dehydrogenase of reagents specific for nucleophilic sulphur. 688 59

Yellow butyryl-CoA dehydrogenase and general acyl-CoA dehydrogenase are "greened" by a mixture of coenzyme A plus elemental sulfur. The resultant stable complex contains an identical ligand with that present in native green butyryl-CoA dehydrogenase and has the same broad absorption band centered at 710 nm. Evidence is presented that the greening ligand is a CoA persulfide, possibly a mimic of the substrate carbanion thought to be generated early in the normal catalytic cycle. Variation in the position of the long wavelength band on replacement of FAD by a series of analogs of differing oxidation-reduction potential is consistent with a charge-transfer complex between a persulfide as the donor and oxidized flavin as the acceptor. The possible physiological and metabolic significance is discussed.
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PMID:Evidence that the greening ligand in native butyryl-CoA dehydrogenase is a CoA persulfide. 706 37

Redox potentials of short-chain acyl-CoA dehydrogenase from the anaerobe, Megasphaera elsdenii, have been determined by means of uv-visible spectroelectrochemistry in the presence of substrate analogs. During redox titrations in the presence of 2-azabutyryl-CoA, up to 85% anionic FAD semiquinone was stabilized with a molar absorbance at 387 nm of 19 mM-1 cm-1. Despite a slow reduction of short-chain acyl-CoA dehydrogenase by 2-azabutyryl-CoA (< 2% reduction/h), a dissociation constant of 0.7 microM was measured and redox potentials, E1(0') and E2(0'), of -0.07 and -0.17 V, respectively, were determined at pH 7.0 for the first and second electrons in reduction of the FAD of short-chain acyl-CoA dehydrogenase. The analog, 2-azoctanoyl-CoA, did not reduce short-chain acyl-CoA dehydrogenase, bound with a dissociation constant of 2 microM, stabilized up to 47% anionic FAD semiquinone, and gave values of -0.08 and -0.11 V for E1(0') and E2(0') at pH 6.9. In contrast to 2-aza-acyl-CoA, the thioethers, butyl-CoA, octyl-CoA, and allyl-CoA, and the thioester, acetyl-CoA, did not bind strongly (Kd > or = 50 microM) and caused no significant change in the redox properties of short-chain acyl-CoA dehydrogenase. The two-electron redox potential, Em, remained at -0.08 V at pH 7.0 and there was no stabilization of FAD semiquinone in the presence of these analogs. These results show that no single feature of substrate structure, the thioester carbonyl, the presence of 2,3-unsaturation, or a fatty alkyl chain of appropriate length, can account for the 0.06-V positive change in redox potential which is observed in the presence of the substrate couple, crotonyl-CoA/butyryl-CoA (M. T. Stankovich and S. Soltysik (1987) Biochemistry 26 2627-2632). As outlined above, 2-azabutyryl-CoA or 2-azaoctanoyl-CoA did cause marked changes in the redox properties of short-chain acyl-CoA dehydrogenase, but the preferential stabilization of FAD semiquinone and negative change in Em distinguish the effects of 2-azaacyl-CoA from those of the substrate couple.
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PMID:Oxidation-reduction properties of short-chain acyl-CoA dehydrogenase: effects of substrate analogs. 808 Feb 71

1. Isolated colonic epithelial cells of the rat were incubated for 40 min with [6-14C]glucose and n-[1-14C]butyrate in the presence of 0.1-2.0 mmol/l NaHS, a concentration range found in the human colon. Metabolic products, 14CO2, acetoacetate, beta-hydroxybutyrate and lactate, were measured and injury to cells was judged by diminished production of metabolites. 2. Oxidation of n-butyrate to CO2 and acetoacetate was reduced at 0.1 and 0.5 mmol/l NaHS, whereas glucose oxidation remained unimpaired. At 1.0-2.0 mmol/l NaHS, n-butyrate and glucose oxidation were dose-dependently reduced at the same rate. 3. To bypass short-chain acyl-CoA dehydrogenase activity necessary for butyrate oxidation, ketogenesis from crotonate was measured in the presence of 1.0 mmol/l NaHS. Suppression by sulphide of ketogenesis from crotonate (-10.5 +/- 6.1%) compared with control conditions was not significant, whereas suppression of ketogenesis from n-butyrate (-36.00 +/- 5.14%) was significant (P = < 0.01). Inhibition of FAD-linked oxidation was more affected by NaHS than was NAD-linked oxidation. 4. L-Methionine (5.0 mmol/l) significantly redressed the impaired beta-oxidation induced by NaHS. Methionine equally improved CO2 and ketone body production, suggesting a global reversal of the action of sulphide. 5. Sulphide-induced oxidative changes closely mirror the impairment of beta-oxidation observed in colonocytes of patients with ulcerative colitis. A hypothesis for the disease process of ulcerative colitis is that sulphides may form persulphides with butyryl-CoA, which would inhibit cellular short-chain acyl-CoA deHydrogenase and beta-oxidation to induce an energy-deficiency state in colonocytes and mucosal inflammation.
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PMID:Sulphide impairment of substrate oxidation in rat colonocytes: a biochemical basis for ulcerative colitis? 828 51

The acyl-CoA dehydrogenases are a family of related enzymes that share high structural homology and a common catalytic mechanism which involves abstraction of an alpha-proton from the substrate by an active site glutamate residue. Several lines of investigation have shown that the position of the catalytic glutamate is conserved in most of these dehydrogenases (the E2 site), but is in a different location in two other family members (the E1 site). Using site specific in vitro mutagenesis, a double mutant rat short chain acyl-CoA dehydrogenase (rSCAD) has been constructed in which the catalytic glutamate is moved from the E2 to the E1 site (Glu368Gly/Gly247Glu). This mutant enzyme is catalytically active, but utilizes substrate less efficiently than the native enzyme (K(m) = 0.6 and 2.0 microM, and Vmax = 2.8 and 0.3 s-1 for native and mutant enzyme respectively). In this study we show that both the wild-type and mutant rSCADs display identical stereochemical preference for catalysis--abstraction of the alpha-HR from the substrate followed by transfer of the beta-HR to the FAD coenzyme. These results, in conjunction with molecular modeling of the native and double mutant SCAD indicate that the catalytic base in the E1 and E2 sites are topologically similar and catalytically competent. However, analysis of the 1H NMR spectra of the incubation products of these two enzymes revealed that, in contrast to the wild-type rSCAD, the Gly368Glu/Gly247Glu rSCAD could not perform gamma-proton exchange of the product with the solvent, a property inherent to most acyl-CoA dehydrogenases. It is evident that the base in the mutant enzyme has access to the alpha-HR but is far removed from the gamma-Hs. These findings provide further support for a one base mechanism of alpha- and gamma-reprotonation/deprotonation catalysis by acyl-CoA dehydrogenases.
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PMID:Redesigning the active-site of an acyl-CoA dehydrogenase: new evidence supporting a one-base mechanism. 945 13

The genes that encode the two different subunits of the novel electron-transferring flavoprotein (ETF) from Megasphaera elsdenii were identified by screening a partial genomic DNA library with a probe that was generated by amplification of genomic sequences using the polymerase chain reaction. The cloned genes are arranged in tandem with the coding sequence for the beta-subunit in the position 5' to the alpha-subunit coding sequence. Amino acid sequence analysis of the two subunits revealed that there are two possible dinucleotide-binding sites on the alpha-subunit and one on the beta-subunit. Comparison of M. elsdenii ETF amino acid sequence to other ETFs and ETF-like proteins indicates that while homology occurs with the mitochondrial ETF and bacterial ETFs, the greatest similarity is with the putative ETFs from clostridia and with fixAB gene products from nitrogen-fixing bacteria. The recombinant ETF was isolated from extracts of Escherichia coli. It is a heterodimer with subunits identical in size to the native protein. The isolated enzyme contains approximately 1 mol of FAD, but like the native protein it binds additional flavin to give a total of about 2 mol of FAD/dimer. It serves as an electron donor to butyryl-CoA dehydrogenase, and it also has NADH dehydrogenase activity.
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PMID:Cloning and analysis of the genes for a novel electron-transferring flavoprotein from Megasphaera elsdenii. Expression and characterization of the recombinant protein. 969 53

The flavoprotein nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the respective aldehydes with production of nitrite and hydrogen peroxide. The sequences of several peptides from the fungal enzyme were used to design oligonucleotides for the isolation of a portion of the NAO gene from an F. oxysporum genomic DNA preparation. This sequence was used to clone the cDNA for NAO from an F. oxysporum cDNA library. The sequence of the cloned cDNA showed that NOA is a member of the acyl-CoA dehydrogenase (ACAD) superfamily. The members of this family share with NAO a mechanism that is initiated by proton removal from carbon, suggesting a common chemical reaction for this superfamily. NAO was expressed in Escherichia coli and the recombinant enzyme was characterized. Recombinant NAO has identical kinetic parameters to enzyme isolated from F. oxysporum but is isolated with oxidized FAD rather than the nitrobutyl-FAD found in the fungal enzyme. NAO purified from E. coli or from F. oxysporum has no detectable ACAD activity on short- or medium-chain acyl CoAs, and medium-chain acyl-CoA dehydrogenase and short-chain acyl-CoA dehydrogenase are unable to catalyze oxidation of nitroalkanes.
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PMID:Cloning of nitroalkane oxidase from Fusarium oxysporum identifies a new member of the acyl-CoA dehydrogenase superfamily. 1186 31


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