A distant relative of catalase that is specialized for metabolism of a fatty acid hydroperoxide was identified. This heme peroxidase occurs in coral a s part of a fusion protein, the other component of which is a lipoxygenase that forms the hydroperoxide substrate. The end product is an unstable epoxide (an allene oxide) that is a potential precursor of prostaglandin-like molecules. These results extend the known chemistry of catalase-like proteins and reveal a distinct type of enzymatic construct involved in the metabolism of polyunsaturated fatty acids.

Prostaglandin synthesis by cyclooxygenases-1 and -2 (COX-1 and COX-2) involves an initial oxygenation of arachidonic acid at C-11, followed by endoperoxide and cyclopentane ring formation, and then a second reaction with molecular oxygen in the S configuration at C-15. The resulting 15S-hydroxyl group of prostaglandins is crucial for their bioactivity. Using human COX-1 and human and murine COX-2, we have identified two amino acids located in the oxygenase active site that control the stereochemistry at C-15. The most crucial determinant is Ser-530, the residue that is acetylated by aspirin. In COX-2, site-directed mutagenesis of Ser-530 to methionine, threonine, or valine produced highly active enzymes that formed 82-95% 15R-configuration prostaglandins; these have the opposite stereochemistry at C-15 to the natural products. In COX-1, the corresponding Ser-530 mutations inactivated the enzyme. The second residue, Val-349, exerts a more subtle influence. When Val-349 was replaced by isoleucine, the mutant COX-1 and COX-2 enzymes formed 41 and 65% 15R-prostaglandins, respectively. This change was highly specific for isoleucine, as mutations of Val-349 to alanine, leucine, asparagine, or threonine did not alter or only slightly altered (<13%) the S-configuration at C-15. These results establish a previously unrecognized role for Ser-530 and Val-349 in maintaining the correct S stereochemistry of the carbon-15 hydroxyl group during prostaglandin synthesis. The findings may also explain the absolute conservation of Ser-530, the target of aspirin, throughout the families of cyclooxygenase enzymes.The sequence of reactions that convert arachidonic acid to prostaglandin endoperoxide, the first stable product of the cyclooxygenase (COX) 1 enzymes, were elucidated over 30 years ago (1, 2). An initial enzyme-catalyzed removal of the pro-S hydrogen from the 13-carbon of arachidonic acid sparks a series of radical reactions that lead in turn to oxygenation at C-11, bridging of the oxygen between C-11 and C-9 to form an endoperoxide, covalent linkage of C-8 and C-12 to form the 5-membered carbon ring, and, finally, stereospecific reaction with a second molecule of oxygen in the S configuration at C-15. In the past 10 years, understanding the structural basis for control of these reactions by cyclooxygenase enzymes has been helped enormously by site-directed mutagenesis studies (3-5), and by the increasing availability of x-ray crystallographic data of COX enzymes with inhibitors, product, or substrate bound in the oxygenase active site (6 -11). Together, these data have helped elucidate the role of particular amino acids during the conversion of arachidonic acid to the prostaglandin product. Four highly conserved polar amino acid residues line the hydrophobic substrate binding channel in all cyclooxygenases (12, 13). Three of these residues are known to be involved in critical steps during the conversion of arachidonic acid to prostaglandin endoperoxide. Arg-120 and Tyr-355, located at the entrance of the COX active site, b...

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.

Background: Lipoxygenases vary in their catalytic specificity and regulation. Results: 11R-LOX, strictly Ca 2ϩ -dependent, displays novel structural features in the membrane-binding domain. Conclusion: A model for how access to an enclosed active site is linked to Ca 2ϩ -dependent membrane binding is proposed. Significance: The 11R-LOX model provides structural insights into the allosteric regulation of lipoxygenases.