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The cyclooxygenase isoforms COX-1 and COX-2 catalyse the rate-limiting step of prostaglandin biosynthesis. While COX-1 has mainly "housekeeping" function, COX-2 is found to be upregulated in rheumatoid arthritis, colorectal cancer and Alzheimer's disease. Development of COX-2 selective inhibitors to control inflammatory and neoplastic activities of the COX-2 isozyme is continuously of great interest. This has further spurred research into the mechanisms of cyclooxygenase catalysis and into the structural and mechanistic basis of the subtle differences between COX-1 and COX-2. This proposal is aimed at further understanding of crucial aspects of prostaglandin synthesis by comparison of mammalian cyclooxygenases with the unique properties of the coral evolutionary ancestors. The coral cyclooxygenases are the first non-vertebrate cyclooxygenases discovered, and our initial characterisation showed that they have significant catalytic differences together with high structural similarity to the mammalian COX isozymes. Therefore they constitute an important model for helping to unravel mechanistic issues of mammalian COX catalysis that are still poorly understood. The following aspects of COX catalysis will be investigated in the current study: 1) control of the oxygenation stereospecificity at C-15 of arachidonic acid in cyclooxygenase catalysis; 2) basis of unusually low peroxidase activity of coral COX and its impact in "sparking" and maintenance of oxygenase reaction; 3) effect of N-glycosylation pattern and each of the potent glycosylation sites on the catalytic activity of cyclooxygenases; and 4) gene structures of coral COX enzymes and insights into the origin of COX-1 and COX-2 genes. |