title: Control of prostaglandin stereochemistry at the 15-carbon by cyclooxygenases-1 and -2. A critical role for serine 530 and valine 349.
abstract: 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 (< or =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.
authors.affiliation: Department of Pharmacology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: Claus
authors.initials: C
authors.lastname: Schneider
authors.forename: William E
authors.initials: WE
authors.lastname: Boeglin
authors.forename: Jeffery J
authors.initials: JJ
authors.lastname: Prusakiewicz
authors.forename: Scott W
authors.initials: SW
authors.lastname: Rowlinson
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Marnett
authors.forename: Nigulas
authors.initials: N
authors.lastname: Samel
authors.forename: Alan R
authors.initials: AR
authors.lastname: Brash
mesh_terms: D000818:Animals; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000032:analysis; D007527:Isoenzymes / Q000502:physiology*; D008565:Membrane Proteins; D051379:Mice; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D011451:Prostaglandin-Endoperoxide Synthases / Q000502:physiology*; D011453:Prostaglandins / Q000737:chemistry*; D012694:Serine
chemical_list: D006893:Hydroxyeicosatetraenoic Acids; D007527:Isoenzymes; D008565:Membrane Proteins; D011453:Prostaglandins; D012694:Serine; C027356:11-hydroxy-5,8,12,14-eicosatetraenoic acid; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; C496512:Ptgs1 protein, mouse