Mechanisms of Leukotriene, Resolvin and Protectin Biosynthesis - The long-term objective of this project is to elucidate the biosynthesis of unstable fatty acid epoxides, leukotriene A4 (LTA4) and close relatives, involved in inflammation. Pro-inflammatory leukotrienes and eoxins formed from C20:4 arachidonic acid versus the pro-resolving resolvins, protectins, and maresins formed from C20:5 and C22:6 omega-3 fatty acids, their biosynthesis all involves a LTA-type intermediate. LTA epoxides are formed by a lipoxygenase (LOX) enzyme acting on a fatty acid hydroperoxide. While catalysis of LTA4 itself by leukocyte 5-LOX is well established, the biosynthesis and stereochemical structure of other LTAtype epoxides is not well defined. The extreme instability of these epoxides has impeded and probably also deterred a close examination of the mechanistic basis of the enzymatic reaction. In this project we will utilize methods developed for the handling of highly unstable fatty acid epoxides, including their enzymatic synthesis, extraction, purification and structural analysis by HPLC, UV, MS, and NMR. We will test a model implicating a dual role of the LOX iron in LTA epoxide synthesis, both in catalyzing the initial stereospecific hydrogen abstraction and in cleaving the hydroperoxide moiety, the two reactions occurring superficially. The product LTA-type epoxide may be of the cis or trans configuration, depending on the conformational relationship of the hydrogen abstraction to the hydroperoxide stereochemistry and to the head-to-tail orientation of the substrate in the LOX active site. This model and additional facets of the reaction will be investigated in Specific Aim 1.
In Specific Aim 2 the techniques for handling unstable epoxides and the principles established will be applied to enzymological and structural analysis of the synthesis of the pro-resolving metabolites of omega-3 fatty acids, the resolvins, protectins and maresins. The objectives are to bring the mechanistic concepts underlying LTA-type epoxide synthesis at least up to a par with the understanding of the primary LOX oxygenation reactions. The practical consequences are to further knowledge of hard-to-study biosynthetic pathways through an improved understanding of their mechanistic basis, and a foundation for rational development of therapeutics.
Inflammation is the redness, pain, and swelling associated with many diseases such as asthma, arthritis, and some forms of cancer. This study seeks to clarify how enzymes called lipoxygenases form so-called lipid mediators that are involved in either promoting or combating inflammation. Understanding this will help explain how these enzymes and their products participate in inflammation and allow for a more rational approach to its treatment.
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