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)

Freeze-thawed rat liver mitochondria were extensively washed with potassium phosphate, pH 7.5, and the residue was extracted with 10 mM potassium phosphate, pH 7.5, 1% (w/v) sodium cholate, 0.5 M KCl. The four beta-oxidation enzyme activities of the washes and the last extract were assayed with substrates of various carbon chain lengths. Our data suggest that the last extract contains a novel acyl-CoA dehydrogenase and long-chain 3-hydroxyacyl-CoA dehydrogenase. A novel acyl-CoA dehydrogenase was purified. The molecular masses of the native enzyme and the subunit were estimated to be 150 and 71 kDa, respectively. One mole of enzyme contained 2 mole of FAD. These properties and immunochemical properties of the enzyme differed from those of three other acyl-CoA dehydrogenases: short-, medium-, and long-chain acyl-CoA dehydrogenases. Carbon chain length specificity of the enzyme differed from that of other acyl-CoA dehydrogenases. The enzyme was active toward CoA esters of long- and very-long-chain fatty acids, but not toward those of medium- and short-chain fatty acids. The specific enzyme activity was greater than 10 times that of long-chain acyl-CoA dehydrogenase when palmitoyl-CoA was used as substrate. We propose the name "very-long-chain acyl-CoA dehydrogenase" for this enzyme.
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PMID:Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. I. Purification and properties of very-long-chain acyl-coenzyme A dehydrogenase. 173 Jun 32

Short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases were purified to homogeneity from rat liver mitochondria by sequential chromatography on DEAE-Sephadex A-50, hydroxyapatite, Matrex Gel Blue A, agarose-hexane-CoA, and Bio-Gel A-0.5m. Molecular, immunological, and catalytic properties of the pure acyl-CoA dehydrogenases were investigated. The native molecular weights of these three enzymes were 160,000, 180,000, and 180,000, respectively. The subunit molecular weights of the three enzymes were estimated to be 41,000, 45,000, and 45,000, respectively, indicating that these enzymes are each composed of four subunits of equal size. The FAD content was calculated to be 1 mol/mol of subunit. While FAD binding by short-chain acyl-CoA dehydrogenase was very tight, that by medium-chain acyl-CoA and long-chain acyl-CoA dehydrogenases was less tight. The medium- and long-chain acyl-CoA dehydrogenases were also purified to homogeneity as FAD-free apoenzymes. The apoenzymes were converted to the fully active holoenzymes by incubation with FAD. The three acyl-CoA dehydrogenases were immunologically distinct from each other, i.e. the antibodies raised against the individual enzymes were monospecific and did not cross-react with any other acyl-CoA dehydrogenases. Our preparations of the three enzymes exhibited substrate specificities (as defined in Vappmax and Kappmax) significantly more specific than those of the previous preparations isolated from other sources. The substrate specificities were assessed also by measuring the activities in mitochondrial sonicates after selectively precipitating each enzyme with their individual monospecific antibodies. Butyryl-CoA was almost exclusively dehydrogenated by short-chain acyl-CoA dehydrogenase while C6-C10 acyl-CoAs were mainly dehydrogenated by medium-chain acyl-CoA dehydrogenase. C14-C22 acyl-CoAs were exclusively dehydrogenated by long-chain acyl-CoA dehydrogenase. C24 acyl-CoAs were not dehydrogenated by this enzyme. Lauroyl-CoA appeared to be jointly dehydrogenated by the latter two enzymes. Branched-chain acyl-CoAs were not dehydrogenated by short-chain acyl-CoA dehydrogenase. In the presence of electron-transfer flavoprotein or phenazine methosulfate, 2-enoyl-CoAs were identified as products from the corresponding enzyme/acyl-CoA reactions.
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PMID:Purification and characterization of short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases from rat liver mitochondria. Isolation of the holo- and apoenzymes and conversion of the apoenzyme to the holoenzyme. 396 63

Very long-chain acyl-CoA dehydrogenase (VLCAD) is one of four flavoproteins which catalyze the initial step of the mitochondrial beta-oxidation spiral. By sequence comparison with other acyl-CoA dehydrogenases, Glu-422 of VLCAD has been presumed to be the catalytic residue that abstracts the alpha-proton in the alphabeta-dehydrogenation reaction. Replacing Glu-422 with glutamine (E422Q) caused a loss of enzyme activity by preventing the formation of a charge transfer complex between VLCAD and palmitoyl-CoA. This result provides further evidence for Glu-422 being part of the active site of VLCAD. F418L is a disease-causing mutation in human VLCAD deficiency. Unlike wild-type VLCAD, F418L and F418V contained no bound FAD when expressed at extremely high levels in the baculovirus expression system. Although F418T and F418Y bound FAD at a level similar to that of wild-type VLCAD, both showed reduced Vmax values toward palmitoyl-CoA, most likely due to a decrease in the rate of enzyme-bound FAD reduction. These data suggest that Phe-418 is involved in the binding and subsequent reduction of FAD. FAD-deficient VLCADs (F418L, F418V, and apo-VLCAD) showed increased sensitivity to trypsinization. Loss of FAD may change the folding of VLCAD subunit.
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PMID:Catalytic and FAD-binding residues of mitochondrial very long chain acyl-coenzyme A dehydrogenase. 946 20

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is a member of the family of acyl-CoA dehydrogenases (ACADs). Unlike the other ACADs, which are soluble homotetramers, VLCAD is a homodimer associated with the mitochondrial membrane. VLCAD also possesses an additional 180 residues in the C terminus that are not present in the other ACADs. We have determined the crystal structure of VLCAD complexed with myristoyl-CoA, obtained by co-crystallization, to 1.91-A resolution. The overall fold of the N-terminal approximately 400 residues of VLCAD is similar to that of the soluble ACADs including medium-chain acyl-CoA dehydrogenase (MCAD). The novel C-terminal domain forms an alpha-helical bundle that is positioned perpendicular to the two N-terminal helical domains. The fatty acyl moiety of the bound substrate/product is deeply imbedded inside the protein; however, the adenosine pyrophosphate portion of the C14-CoA ligand is disordered because of partial hydrolysis of the thioester bond and high mobility of the CoA moiety. The location of Glu-422 with respect to the C2-C3 of the bound ligand and FAD confirms Glu-422 to be the catalytic base. In MCAD, Gln-95 and Glu-99 form the base of the substrate binding cavity. In VLCAD, these residues are glycines (Gly-175 and Gly-178), allowing the binding channel to extend for an additional 12A and permitting substrate acyl chain lengths as long as 24 carbons to bind. VLCAD deficiency is among the more common defects of mitochondrial beta-oxidation and, if left undiagnosed, can be fatal. This structure allows us to gain insight into how a variant VLCAD genotype results in a clinical phenotype.
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PMID:Structural basis for substrate fatty acyl chain specificity: crystal structure of human very-long-chain acyl-CoA dehydrogenase. 1822 65

SIRT3 and SIRT5 have been shown to regulate mitochondrial fatty acid oxidation but the molecular mechanisms behind the regulation are lacking. Here, we demonstrate that SIRT3 and SIRT5 both target human very long-chain acyl-CoA dehydrogenase (VLCAD), a key fatty acid oxidation enzyme. SIRT3 deacetylates and SIRT5 desuccinylates K299 which serves to stabilize the essential FAD cofactor in the active site. Further, we show that VLCAD binds strongly to cardiolipin and isolated mitochondrial membranes via a domain near the C-terminus containing lysines K482, K492, and K507. Acetylation or succinylation of these residues eliminates binding of VLCAD to cardiolipin. SIRT3 deacetylates K507 while SIRT5 desuccinylates K482, K492, and K507. Sirtuin deacylation of recombinant VLCAD rescues membrane binding. Endogenous VLCAD from SIRT3 and SIRT5 knockout mouse liver shows reduced binding to cardiolipin. Thus, SIRT3 and SIRT5 promote fatty acid oxidation by converging upon VLCAD to promote its activity and membrane localization. Regulation of cardiolipin binding by reversible lysine acylation is a novel mechanism that is predicted to extrapolate to other metabolic proteins that localize to the inner mitochondrial membrane.
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PMID:SIRT3 and SIRT5 regulate the enzyme activity and cardiolipin binding of very long-chain acyl-CoA dehydrogenase. 2581 81

The fatty acid oxidation enzyme long-chain acyl-CoA dehydrogenase (LCAD) is expressed at high levels in human alveolar type II (ATII) cells in the lung. A common polymorphism causing an amino acid substitution (K333Q) was previously linked to a loss of LCAD antigen in the lung tissue in sudden infant death syndrome. However, the effects of the polymorphism on LCAD function has not been tested. The present work evaluated recombinant LCAD K333Q. Compared to wild-type LCAD protein, LCAD K333Q exhibited significantly reduced enzymatic activity. Molecular modeling suggested that K333 is within interacting distance of the essential FAD cofactor, and the K333Q protein showed a propensity to lose FAD. Exogenous FAD only partially rescued the activity of LCAD K333Q. LCAD K333Q protein was less stable than wild-type when incubated at physiological temperatures, likely explaining the observation of dramatically reduced LCAD antigen in primary ATII cells isolated from individuals homozygous for K333Q. Despite the effect of K333Q on activity, stability, and antigen levels, the frequency of the polymorphism was not increased among infants and children with lung disease.
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PMID:The common K333Q polymorphism in long-chain acyl-CoA dehydrogenase (LCAD) reduces enzyme stability and function. 3238 75