title: The structural basis of endocannabinoid oxygenation by cyclooxygenase-2.
abstract: The cyclooxygenases (COX-1 and COX-2) oxygenate arachidonic acid (AA) in the committed step of prostaglandin biogenesis. Substitutions of I434V, H513R, and I523V constitute the only differences in residues lining the cyclooxygenase channel between COX-1 and COX-2. These changes create a hydrophobic pocket in COX-2, with Arg-513 located at the base of the pocket, which has been exploited in the design of COX-2-selective inhibitors. Previous studies have shown that COX-2, but not COX-1, can oxygenate endocannabinoid substrates, including 2-arachidonoyl glycerol (2-AG). To investigate the isoform-specific structural basis of endocannabinoid binding to COX-2, we determined the crystal structure of the 2-AG isomer 1-arachidonoyl glycerol (1-AG) in complex with wild type and R513H murine (mu) COX-2 to 2.2 and 2.35 Å, respectively, and R513H muCOX-2 in complex with AA to 2.45 Å resolution. The 2,3-dihydroxypropyl moiety of 1-AG binds near the opening of the cyclooxygenase channel in the space vacated by the movement of the Leu-531 side chain, validating our previous hypothesis implicating the flexibility of the Leu-531 side chain as a determinant for the ability of COX-2 to oxygenate endocannabinoid substrates. Functional analyses carried out to compliment our structural findings indicated that Y355F and R513H muCOX-2 constructs had no effect on the oxygenation of 1-AG and 2-AG, whereas substitutions that resulted in a shortened side chain for Leu-531 had only modest effects. Both AA and 1-AG bind to R513H muCOX-2 in conformations similar to those observed in the co-crystal structures of these substrates with wild type enzyme.
authors.affiliation: Department of Structural Biology, Hauptman-Woodward Medical Research Institute,The State University of New York, Buffalo, New York 14203, USA.
authors.forename: Alex J
authors.initials: AJ
authors.lastname: Vecchio
authors.forename: Michael G
authors.initials: MG
authors.lastname: Malkowski
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001095:Arachidonic Acids / Q000737:chemistry* / Q000378:metabolism; D063385:Cannabinoid Receptor Modulators / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D051546:Cyclooxygenase 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D063388:Endocannabinoids; D005989:Glycerides / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D001095:Arachidonic Acids; D063385:Cannabinoid Receptor Modulators; D063388:Endocannabinoids; D005989:Glycerides; C094503:glyceryl 2-arachidonate; C077623:Ptgs2 protein, mouse; D051546:Cyclooxygenase 2