This proposal is to extend the structural studies of acyl-C0A dehydrogenases, a family of enzymes that are involved in both the first oxidative step in the catabolism of fatty acids and in the catabolism of some amino acids. It is also proposed to determine the structure of electron transfer flavoprotein (ETF), the physiological oxidant of the acyl-CioA dehydrogenases, that funnels the reducing equivalents to the main mitochondrial respiratory chain via ETF-ubiquinone oxidoreductase. Fatty acid oxidation is the principal energy yielding process in liver, heart, kidney, and skeletal muscle. The rate of this process can be altered by diet, physiological status, and disease, such as starvation, pregnancy, and diabetes, respectively. The critical roles of these dehydrogenases and ETF in metabolism is illustrated by the severity of human diseases attributive to inherited deficiencies of each of these dehydrogenases and ETF. The crystal structures of medium chain acyl CoA dehydrogenase (MCAD) from pig liver, with and without substrates, and the structures of two short chain-specific enzymes have been determined. It is proposed to extend these studies to site-directed mutants of these three enzymes and to other members of the dehydrogenase family, including long chain acyl-CoA dehydrogenase (LCAD) and isovaleryl-CoA dehydrogenase (IVD). LCAD and IVD are unique in that they a glycine residue in lieu of the glutamate found in all other dehydrogenases for which sequences are known. A high resolution data set of IVD and crystals of lCAD have been obtained. The structures of lCAD and iVD will confirm/determine their catalytic residues identified by molecular modeling and site-directed mutagenesis and also will reveal the structural basis for this unique substitution. Comparison of the structures of different members of this family of dehydrogenases will enable us to study the detailed interactions between these enzymes and their acyl-CoA substrates and the molecular basis of their substrate specificities. It is also proposed to probe the mechanism of the MCAD action using several inhibitors, including methylenecyclopropyl-acetyl-CoA, a metabolite of hypoglycine that causes Jamaican vomiting sickness. Human ETF has been cloned, expressed, and crystallized in a form suitable for high resolution X-ray analysis. An electron density map at 3.5 A resolution has been obtained and further phase refinements are in progress. The high resolution structure of ETF together with those of various acyl C0A, a metabolite of hypoglycine that causes Jamaican vomiting sickness. Human ETF has been cloned, expressed, and crystallized in a form suitable for high resolution X-ray analysis. An electron density map at 3.5 A resolution has been obtained and further phase refinements are in progress. The high resolution structure of ETF together with those of various acyl CoA dehydrogenases will enable us to study the molecular basis of electron transfer between the dehydrogenases and ETF and, perhaps, flavoprotein- flavoprotein interactions in general.
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