Catalytic and FAD-binding residues of mitochondrial very long chain acyl-coenzyme a dehydrogenase

Abstract

Very long-chain acyl-CoA dehydrogenase (VLCAD) is one of four flavoproteins which catalyze the initial step of the mitochondrial β- oxidation spiral. By sequence comparison with other acyl-CoA dehydrogenases, Glu422 of VLCAD has been presumed to be the catalytic residue that abstracts the α-proton in the αβ-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 V(max) 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.