This project is aimed at gaining insights into the structure-function relationships of the beta-oxidation multienzyme complexes from prokaryotic and eukaryotic cells and ultimately at identifying genetic defect in the genes coding for the subunits of the human mitochondrial membrane-bound beta-oxidation complex. The discovery of a long-chain fatty acid oxidation enzyme system in mitochondria has prompted us to suggest that the beta-oxidation spiral of long-chain fatty acyl-CoA proceeds mainly on the mitochondrial inner membrane. The identification of a new disease, human long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency, indicated that the catalytic function of the mitochondrial membrane-bound beta-oxidation complex is of great importance to health. In order to test the hypothesis and to identify the molecular defects of CHAD deficiency, the primary structures of the subunits of the pig and human mitochondrial membrane-bound beta-oxidation complexes and the structural organizations of the genes coding for the subunits of the human mitochondrial membrane-bound beta-oxidation complex will be determined. Since a comparison of the structure of the mitochondrial membrane-bound beta-oxidation complex with the E. coli fatty acid oxidation complex should lead to a better understanding of the structure-function relationships of such multienzyme complexes, the E. coli fatty acid oxidation protein by site-directed mutagenesis of the fadB gene. The structural factors that stabilize the quaternary structure of the E. coli fatty acid oxidation complex will also be explored. Finally, the genetic defects of human CHAD deficiency will be characterized by cloning and sequencing the patients' cDNAs and genes coding for the mitochondrial membrane-bound beta-oxidation complex. Based on such information a simple method of detecting the mutant alleles will be developed to facilitate the diagnosis and the prevention of this disease. This project will only fill in the gaps of our knowledge of the b-oxidation enzymes, but also shed light on inborn errors of fatty acid oxidation.
