Identification of isobutyryl-CoA dehydrogenase and its deficiency in humans. J. Vockley¹, T.V. Nguyen¹, S. Ghisla², A.-W. Mohsen¹, D. Roe³, C.R. Roe³. 1) Dept Medical Genetics, Mayo Clinic, Rochester, MN; 2) Faculty of Biology, Univ. Konstanz, Konstanz, Germany; 3) Institute of Metabolic Disease, Baylor Univ., Dallas, TX.
   The acyl-CoA dehydrogenases (ACDs) are a family of related enzymes which catalyze the a,b-dehydrogenation of acyl-CoA esters, transferring electrons to electron transferring flavoprotein. Two homologues active in branched chain amino acid metabolism have previously been identified. One of these, short-branched chain acyl-CoA dehydrogenase, has been postulated to catalyze reactions in both the valine and isoleucine catabolic pathways, but the human enzyme utilizes isobutyryl-CoA as substrate poorly. Recently, a new gene of unproven function has been identified and postulated to be an ACD on the basis of sequence homology (ACD8). We have used expression in E. coli to produce and characterize the substrate specificity of this enzyme. Testing with different acyl-CoA substrates shows that the expressed enzyme has maximal activity with isobutyryl-CoA (23% activity with S-2 methylbutyryl-CoA as substrate as compared to isobutyryl-CoA) and no activity with butyryl-, valeryl-, or isovaleryl-CoA. A single patient has previously been described in whom fibroblast metabolic loading studies revealed an increase in accumulation of isobutyrylcarnitine, and a decreased oxidation of labeled valine. Metabolism of labeled isoleucine was normal. Amplified ACD8 cDNA made from fibroblast mRNA from this patient is homozygous for a single nucleotide change (G905A) compared to mRNA from control cells. This predicts an R302N alteration in the precursor protein. Molecular modeling studies indicate that this mutation is located at the monomer interface of the holoenzyme tetramer, and predicts that the aberrant protein will be unstable. Expression of this mutant enzyme in our E. coli system leads to the production of an aggregated, inactive protein. Our findings unequivocally demonstrate the presence of a new ACD in humans specific to valine catabolism (isobutyryl-CoA dehydrogenase), along with the first definitive identification of a patient deficient in this enzyme.