title: Expression and characterization of mutations in human very long-chain acyl-CoA dehydrogenase using a prokaryotic system.
abstract: Very long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the first enzymatic step in the mitochondrial beta-oxidation of fatty acids 14-20 carbons in length. More than 100 cases of VLCAD deficiency have been reported with the disease varying from a severe, often fatal neonatal form to a mild adult-onset form. VLCAD is distinguished from matrix-soluble acyl-CoA dehydrogenases by its unique C-terminal domain, homodimeric structure, and localization to the inner mitochondrial membrane. We have for the first time expressed and purified VLCAD using a bacterial system. Recombinant VLCAD had similar biochemical properties to those reported for native VLCAD and the bacterial system was used to study six previously described disease-causing missense mutations including the two most common mild mutations (T220M, V243A), a mutation leading to the severe disease phenotype (R429W), and three mutations in the C-terminal domain (A450P, L462P, and R573W). Of particular interest was the finding that the A450P and L462P bacterial extracts had normal or increased amounts of VLCAD antigen and activity. In the pure form L462P had roughly 30% of wild-type activity while A450P was normal. Using computer modeling both mutations were mapped to a predicted charged surface of VLCAD that we postulate interacts with the mitochondrial membrane. In a membrane pull down assay both mutants showed greatly reduced mitochondrial membrane association, suggesting a mechanism for the disease in these patients. In summary, the bacterial expression system developed here will significantly advance our understanding of both the clinical aspects of VLCAD deficiency and the basic biochemistry of the enzyme.
authors: Department of Pediatrics, School of Medicine, University of Pittsburgh, Children's Hospital of Pittsburgh, PA 15213, USA.
authors: Eric S
authors: ES
authors: Goetzman
authors: Yudong
authors: Y
authors: Wang
authors: Miao
authors: M
authors: He
authors: Al-Walid
authors: AW
authors: Mohsen
authors: Brittani K
authors: BK
authors: Ninness
authors: Jerry
authors: J
authors: Vockley
mesh_terms: D044942:Acyl-CoA Dehydrogenase, Long-Chain / Q000096:biosynthesis* / Q000172:deficiency / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology; D051336:Mitochondrial Membranes / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D044942:Acyl-CoA Dehydrogenase, Long-Chain