Structural Biology of Eicosanoid Biosynthesis

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The dietary or essential fatty acids linoleate and alpha-linolenate play extremely important roles in mammalian physiology. As they cannot be synthesized by mammals, the biosynthesis of all C-20, C-22 and C-24 polyunsaturated fatty acids depends on the availability of essential fatty acids in the diet, primarily from plant sources. A large group of bioactive, oxygenated C-18, C-20 and C-22 fatty acids, collectively known as eicosanoids, are derived from n-3 and n-6 PUFAs. The oxygenated products of polyunsaturated fatty acids, from endogenous and exogenous sources, have potent and wide-ranging effects on many physiological processes in humans including cell proliferation, immune responses, inflammation, renal function, reproduction, and cardiovascular homeostasis. Subtle changes in the regio- and stereochemistry of these compounds can also substantially alter their biological properties. A clear molecular understanding of the chemistry leading to stereochemically unique oxygenated products and how these products are converted into the bioactive target molecules is lacking. The primary focus of this renewal application is on elucidating, by X-ray crystallography and mutagenesis, (a) the structural mechanisms for the recognition and binding of polyunsaturated fatty acids, (b) how the regio- and stereochemistry of bis- or monooxygenation are controlled, and (c) how regio- and stereochemistry affect the further metabolism of the oxygenated polyunsaturated fatty acids. Three classes of enzymes in the metabolism of polyunsaturated fatty acids and their oxygenated products are being studied: sheep and human prostaglandin synthase, rat prostacyclin synthase, and 3 related enzymes of the oxylipin metabolic pathways in plants. These enzymes utilize heme to activate the substrates and are integral membrane proteins. In the previous grant period, the crystal structures of native prostaglandin synthase-1 with bound arachidonic acid and other fatty acids were determined, overexpression systems for recombinant prostaglandin synthases-1 and -2 were developed for crystallographic studies, and over 50 mutants of prostaglandin synthases-1 and -2 were engineered for structural analysis. Thus, the structural details of bis- or monooxygenation reactions can be studied. Overexpression of rat prostacyclin synthase and divinylether synthase also make crystallographic studies on these two membrane-bound P450 enzymes tractable.