Biosynthetic Mechanisms of LTA-type Epoxides and Novel Bioactive Lipid Mediators

Abstract

Biosynthesis of lipid mediators including the leukotrienes, lipoxins, eoxins, resolvins, and protectins entails the lipoxygenase (LOX) catalyzed synthesis of an allylic epoxide intermediate, designated as leukotriene A4 (LTA4) and LTA analogues, and heretofore considered too unstable for direct structural characterization. In this dissertation I developed methods involving biphasic reaction conditions for the LOX-catalyzed synthesis of LTA epoxides and their structural analysis by NMR. As proof of concept, human 15-LOX-1 was shown to convert 15S-hydroperoxyeicosatetraenoic acid (15S-HPETE) to the LTA analogue, 14,15-LTA4 (thus identifying eoxin A4). Using this methodology I then showed that recombinant Arabidopsis AtLOX1, an arachidonate 5-LOX, converts 5S-HPETE to the trans epoxide LTA4, and 5R-HPETE to the cis epoxide 5-epi-LTA4. The results are reconciled with a mechanism based on a dual role of the LOX non-heme iron in LTA epoxide biosynthesis. The same methodology was used to structurally characterize the LTA-related epoxides from omega-3 fatty acids, including identification of the proposed epoxide intermediate in protectin biosynthesis.