کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1999610 1065865 2007 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Expression and characterization of mutations in human very long-chain acyl-CoA dehydrogenase using a prokaryotic system
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Expression and characterization of mutations in human very long-chain acyl-CoA dehydrogenase using a prokaryotic system
چکیده انگلیسی

Very long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the first enzymatic step in the mitochondrial β-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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Molecular Genetics and Metabolism - Volume 91, Issue 2, June 2007, Pages 138–147
نویسندگان
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