Fatty acid ?-oxidation is the major energy-producing process in the liver, heart, and type 1 muscle fiber. It is carried out by a series of four reactions that successively cleave acetyl-CoA from fatty acyl-CoA. The rate of this process can be altered by diet (fed/fasting), physiological status (pregnancy), or disease (diabetes). The first of the four reactions in this process is initiated by a family of flavoproteins, acyl-CoA dehydrogenases (ADs). Electron transfer from ADs to the main mitochondrial respiratory chain is catalyzed by electron transfer flavoprotein (ETF) and the membrane-bound ETF-ubiquinone oxidoreductase (ETF-QO). There are at least seven soluble ADs for catalyzing short chain acyl-CoAs (three for fatty acid metabolism and four for amino acid catabolism) and two membrane-bound ADs specific for long chain fatty acyl-CoAs, very long chain acyl-CoA dehydrogenase (VLCAD) and ACAD9. The three remaining reactions of ?-oxidation for long chain fatty acids are carried out by the trifunctional protein (TFP), a membrane-bound multienzyme complex. The critical metabolic roles of the enzymes involved in this process (the nine identified ADs, TFP, ETF, and ETF-QO) are illustrated by the severity of human diseases resulting from inherited deficiencies of each of these enzymes. Recessively inherited fatty acid oxidation disorders together occur in 1:4000 newborns, present as sudden infant death syndrome and cardiomyopathy, and are the most common cause of lipid myopathy in older children and young adults. We have determined the crystal structures of all but one of the soluble ADs, as well as one membrane-bound AD (VLCAD), ETF, and ETF-QO. The proposed investigations are focused on two membrane-bound enzymes, VLCAD and TFP, and interactions of ETF with its electron transfer partners, including ADs, sarcosine dehydrogenase (SD), and ETF-QO. SD functions in choline metabolism and is not a member of the AD family, but donates electrons to ETF. To further our understanding of interactions among these different electron transfer partners, we propose: 1a) to complete structural studies of long chain acyl- CoA dehydrogenase and 1b) to determine the orientation of VLCAD in the membrane and to assess the subunit arrangement of VLCAD upon binding to TFP by EPR spectroscopy. 2) to initiate structural studies of TFP in order to understand how its three distinct active sites communicate with each other;3) to investigate the domain movement of ETF and its interactions with three representative electron donors (medium chain acyl-CoA dehydrogenase, VLCAD, and SD) and with its electron acceptor, ETF-QO;and 4) to complete the structural studies of pig SD in order to determine the mechanism of oxidative demethylation and 1-carbon transfer reactions.
All cells in the human body need energy to maintain their structure and to carry out their vital functions. A great portion of this energy comes from the oxidation of fatty acids in heart, liver, and muscle cells;an imbalance of this process results in disease states, such as obesity and diabetes. A better understanding of how these enzymes work may help in the design of inhibitors or agonists that could be used in clinical settings.
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