The highest concentrations of prostaglandins in nature are found in the Caribbean gorgonian Plexaura homomalla. Depending on its geographical location, this coral contains prostaglandins with typical mammalian stereochemistry (15S-hydroxy) or the unusual 15R-prostaglandins. Their metabolic origin has remained the subject of mechanistic speculations for three decades. Here, we report the structure of a type of cyclooxygenase (COX) that catalyzes transformation of arachidonic acid into 15R-prostaglandins. Using a homology-based reverse transcriptase-PCR strategy, we cloned a cDNA corresponding to a COX protein from the R variety of P. homomalla. The deduced peptide sequence shows 80% identity with the 15S-specific coral COX from the Arctic soft coral Gersemia fruticosa and Ϸ50% identity to mammalian COX-1 and COX-2. The predicted tertiary structure shows high homology with mammalian COX isozymes having all of the characteristic structural units and the amino acid residues important in catalysis. Some structural differences are apparent around the peroxidase active site, in the membrane-binding domain, and in the pattern of glycosylation. When expressed in Sf9 cells, the P. homomalla enzyme forms a 15R-prostaglandin endoperoxide together with 11R-hydroxyeicosatetraenoic acid and 15R-hydroxyeicosatetraenoic acid as byproducts. The endoperoxide gives rise to 15R-prostaglandins and 12R-hydroxyheptadecatrienoic acid, identified by comparison to authentic standards. Evaluation of the structural differences of this 15R-COX isozyme should provide new insights into the substrate binding and stereospecificity of the dioxygenation reaction of arachidonic acid in the cyclooxygenase active site.

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In vertebrates, the synthesis of prostaglandin hormones is catalyzed by cyclooxygenase (COX)-1, a constitutively expressed enzyme with physiological functions, and COX-2, induced in inflammation and cancer. Prostaglandins have been detected in high concentrations in certain corals, and previous evidence suggested their biosynthesis through a lipoxygenase-allene oxide pathway. Here we describe the discovery of an ancestor of cyclooxygenases that is responsible for prostaglandin biosynthesis in coral. Using a homology-based polymerase chain reaction cloning strategy, the cDNA encoding a polypeptide with ϳ50% amino acid identity to both mammalian COX-1 and COX-2 was cloned and sequenced from the Arctic soft coral Gersemia fruticosa. , implicated in the binding of certain COX inhibitors, is represented as alanine. Expression of the G. fruticosa cDNA afforded a functional cyclooxygenase that converted exogenous arachidonic acid to prostaglandins. The biosynthesis was inhibited by indomethacin, whereas the selective COX-2 inhibitor nimesulide was ineffective. We conclude that the cyclooxygenase occurs widely in the animal kingdom and that vertebrate COX-1 and COX-2 are evolutionary derivatives of the invertebrate precursor.Prostaglandins have been found in a diverse range of vertebrates and invertebrates (1, 2). In vertebrates, they are synthesized by prostaglandin-H 2 synthase, known also as cyclooxygenase (COX) 1 (3-5). COX is a hemoprotein with two distinct catalytic activities: the cyclooxygenase activity involved in the formation of PGG 2 from arachidonic acid and the peroxidase activity that catalyzes the reduction of PGG 2 to PGH 2 (3). There are two COX isozymes called COX-1 and COX-2 (6, 7). COX-1 is expressed constitutively in nearly all mammalian tissues and forms prostaglandins with housekeeping functions. COX-2, although absent from most cells, can be rapidly induced in many cell types upon treatment with inflammatory cytokines, growth factors, and tumor promoters (8). These two isoforms share ϳ60% amino acid sequence identity. They have similar structural topology and an identical catalytic mechanism (4, 9). The three-dimensional x-ray crystal structures of COX-1 and COX-2 are virtually superimposable. The residues that form the substrate-binding channel, catalytic sites, and residues immediately adjacent are all identical except for some small variations (9 -12). These small differences in sequence lead to clear biochemical differences in substrate selectivity and sensitivity to various nonsteroidal anti-inflammatory drugs (4, 6). The mechanism of prostaglandin biosynthesis in invertebrates, particularly in the prostaglandin-containing corals (13,14), has been the object of intense studies and speculations over the years. A proposal that coral uses a fundamentally different mechanism from the mammalian pathway, i.e. a lipoxygenaseallene oxide synthase route similar to the jasmonic acid pathway in plants (15-17), has not found experimental support more recently. The highly active peroxidase-lipoxygenase ...