Publications

• Purification of human very-long-chain acyl-coenzyme A dehydrogenase and characterization of its deficiency in seven patients.

  Aoyama T., Souri M., Ushikubo S., Kamijo T., Yamaguchi S., Kelley R.I., Rhead W.J., Uetake K., Tanaka K., Hashimoto T.

  Mitochondrial very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) was purified from human liver. The molecular masses of the native enzyme and the subunit were estimated to be 154 and 70 kD, respectively. The enzyme was found to catalyze the major part of mitochondrial palmitoylcoenzyme A dehydr ... >> More

  Mitochondrial very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) was purified from human liver. The molecular masses of the native enzyme and the subunit were estimated to be 154 and 70 kD, respectively. The enzyme was found to catalyze the major part of mitochondrial palmitoylcoenzyme A dehydrogenation in liver, heart, skeletal muscle, and skin fibroblasts (89-97, 86-99, 96-99, and 78-87%, respectively). Skin fibroblasts from 26 patients suspected of having a disorder of mitochondrial beta-oxidation were analyzed for VLCAD protein using immunoblotting, and 7 of them contained undetectable or trace levels of the enzyme. The seven deficient fibroblast lines were characterized by measuring acyl-coenzyme A dehydrogenation activities, overall palmitic acid oxidation, and VLCAD protein synthesis using pulse-chase, further confirming the diagnosis of VLCAD deficiency. These results suggested the heterogenous nature of the mutations causing the deficiency in the seven patients. Clinically, all patients with VLCAD deficiency exhibited cardiac disease. At least four of them presented with hypertrophic cardiomyopathy. This frequency (> 57%) was much higher than that observed in patients with other disorders of mitochondrial long-chain fatty acid oxidation that may be accompanied by cardiac disease in infants. << Less

  J. Clin. Invest. 95:2465-2473(1995) [PubMed] [EuropePMC]

• Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. I. Purification and properties of very-long-chain acyl-coenzyme A dehydrogenase.

  Izai K., Uchida Y., Orii T., Yamamoto S., Hashimoto T.

  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 su ... >> More

  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. << Less

  J. Biol. Chem. 267:1027-1033(1992) [PubMed] [EuropePMC]

• Structural basis for substrate fatty acyl chain specificity: crystal structure of human very-long-chain acyl-CoA dehydrogenase.

  McAndrew R.P., Wang Y., Mohsen A.W., He M., Vockley J., Kim J.J.

  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 t ... >> More

  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. << Less

  J. Biol. Chem. 283:9435-9443(2008) [PubMed] [EuropePMC]