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

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 a-proton in the alpha beta-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.