In eukaryotes, desaturation of fatty acyl chains is catalyzed by either soluble acyl-ACP desaturases or by integral membrane desaturases. These multi-protein complexes contain two structurally distinct classes of diiron centers in the catalytic active site. Together, they are responsible for the biosynthesis of all physiologically and nutritionally essential unsaturated fatty acids. However, despite the importance of these molecules, many details of the essential catalytic process leading to the formation of unsaturated fats are still poorly understood., including the reactivity of the diiron center, the contribution of protein-protein interactions, and the nature of oxidized substrate intermediates. The long-term objective of our research is to define the reaction mechanism of fatty acid desaturation at the molecular level. The proposed Aim 1 studies address the mechanism of 02 activation used by eukaryotic diiron enzymes. This reactivity is essential for many life processes, including biosynthesis, energy conservation, and protection from xenobiotics. Catalytic and spectroscopic approaches will be used to determine reactivity and to identify diiron intermediates formed during the reaction. The proposed Aim II studies address how protein-protein interactions control the efficiency and progress of the desaturase catalytic cycle. These studies are significant because protein-protein interactions are essential to many cellular processes. Biophysical and catalytic approaches will be used to identify and characterize the relevant complexes. The proposed Aim III studies provide information on oxidized states of the substrate, which thus provides unique insight into the chemical steps of desaturation catalysis. Chemical synthesis, enzyme reaction kinetics, and product determination studies will be undertaken. The proposed Aim IV studies dive detailed information on the reactivity of mammalian membrane desaturase isoforms. This work will impact our understanding of how cellular lipid composition is maintained. Since loss of this control is seen in atherosclerosis, cancer, heart disease, hypertension, obesity, and many other ailments, these studies have relevance to human health issues. The proposed studies will also reveal the molecular details of reaction with non-natural trans fatty acids, which are increasingly found in human diet from the consumption of partially hydrogenated plant oils.
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