The cyclooxygenases (COX-1 and COX-2) are membrane-associated heme-containing homodimers that generate prostaglandin H 2 from arachidonic acid (AA). Although AA is the preferred substrate, other fatty acids are oxygenated by these enzymes with varying efficiencies. We determined the crystal structures of AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) bound to Co 3؉ -protoporphyrin IX-reconstituted murine COX-2 to 2.1, 2.4, and 2.65 Å , respectively. AA, EPA, and docosahexaenoic acid bind in different conformations in each monomer constituting the homodimer in their respective structures such that one monomer exhibits nonproductive binding and the other productive binding of the substrate in the cyclooxygenase channel. The interactions identified between protein and substrate when bound to COX-1 are conserved in our COX-2 structures, with the only notable difference being the lack of interaction of the carboxylate of AA and EPA with the side chain of Arg-120. Leu-531 exhibits a different side chain conformation when the nonproductive and productive binding modes of AA are compared. Unlike COX-1, mutating this residue to Ala, Phe, Pro, or Thr did not result in a significant loss of activity or substrate binding affinity. Determination of the L531F:AA crystal structure resulted in AA binding in the same global conformation in each monomer. We speculate that the mobility of the Leu-531 side chain increases the volume available at the opening of the cyclooxygenase channel and contributes to the observed ability of COX-2 to oxygenate a broad spectrum of fatty acid and fatty ester substrates.The cyclooxygenase enzymes (COX-1 and COX-2) are membrane-associated heme-containing bifunctional enzymes that catalyze the first committed step in prostaglandin (PG) 2 biosynthesis (reviewed in Refs. 1, 2). The product, PGH 2 , is produced as a result of two sequential reactions that are performed in separate but functionally linked active sites. In the first reaction, arachidonic acid (AA; 20:4 -6) bound in the cyclooxygenase channel undergoes a bis-oxygenation to form the intermediate PGG 2 . The released intermediate is then bound in the peroxidase active site, where the 15-hydroperoxide group of PGG 2 is reduced to form PGH 2 in the second reaction. Both COX-1 and COX-2 require a preliminary catalytic turnover at the peroxidase active site to generate an oxy-ferryl porphyrin radical that is subsequently transferred to Tyr-385 for the initiation of cyclooxygenase catalysis.Understanding the similarities and differences associated with the structure and function of COX-1 and COX-2 and the rationale for the existence of two isoforms has been the focus of much recent research (3-5). COX-1 and COX-2, which are encoded by separate genes (6), display ϳ60% sequence identity within the same species and greater than 85% sequence identity among orthologs from different species (7). Accordingly, the crystal structures of COX-1 and COX-2 are virtually superimposable, and the catalytic mechanism is conserved between isoform...

Pathogen-inducible oxygenase (PIOX) oxygenates fatty acids into 2R-hydroperoxides. PIOX belongs to the fatty acid ␣-dioxygenase family, which exhibits homology to cyclooxygenase enzymes (COX-1 and COX-2). Although these enzymes share common catalytic features, including the use of a tyrosine radical during catalysis, little is known about other residues involved in the dioxygenase reaction of PIOX. We generated a model of linoleic acid (LA) bound to PIOX based on computational sequence alignment and secondary structure predictions with COX-1 and experimental observations that governed the placement of carbon-2 of LA below the catalytic Tyr-379. Examination of the model identified His-311, Arg-558, and Arg-559 as potential molecular determinants of the dioxygenase reaction. Substitutions at His-311 and Arg-559 resulted in mutant constructs that retained virtually no oxygenase activity, whereas substitutions of Arg-558 caused only moderate decreases in activity. Arg-559 mutant constructs exhibited increases of greater than 140-fold in K m , whereas no substantial change in K m was observed for His-311 or Arg-558 mutant constructs. Thermal shift assays used to measure ligand binding affinity show that the binding of LA is significantly reduced in a Y379F/ R559A mutant construct compared with that observed for Y379F/R558A construct. Although Oryza sativa PIOX exhibited oxygenase activity against a variety of 14 -20-carbon fatty acids, the enzyme did not oxygenate substrates containing modifications at the carboxylate, carbon-1, or carbon-2. Taken together, these data suggest that Arg-559 is required for high affinity binding of substrates to PIOX, whereas His-311 is involved in optimally aligning carbon-2 below Tyr-379 for catalysis.Pathogen attack on plants brings about the activation of multiple enzyme systems that results in the production of oxylipins from 18 -22 carbon fatty acid precursors. The generation of these bioactive lipid mediators initiates and sustains the defense reaction of the plant against insects, bacteria, fungi, and other pathogens (1, 2). One of the enzymes up-regulated during the host defense response is pathogen-inducible oxygenase (PIOX), 2 which catalyzes a non-lipoxygenase type of fatty acid oxygenation (3). PIOX belongs to a larger family of heme-containing proteins that oxygenate fatty acids (4), which include the mammalian cyclooxygenases (COX-1 and COX-2; (5)), linoleate diol synthase (LDS) from the fungus Gaeumannomyces graminis (6, 7), and a Pseudomonas alcalignes protein of unknown function encoded by OrfX (8). PIOX has also been identified in many plant species, including Nicotiana attenuata (9), Nicotiana tabacum (3), Arabidopsis thaliana (3, 10), O. sativa (11), Capsicum annuum (12), and Lycopersicon esculentum (13).PIOX utilizes stereoselective oxygenation to convert linoleic acid (LA) (18:2, n-6) and other fatty acid substrates to their corresponding 2R-hydroperoxides, generating a novel class of oxylipins (3,11,14,15). The resulting 2R-hydroperoxides undergo spontaneous deca...

DP-155 is a lipid prodrug of indomethacin that comprises the latter conjugated to lecithin at position sn-2 via a 5-carbon length linker. It is cleaved by phospholipase A 2 (PLA) 2 to a greater extent than similar compounds with linkers of 2, 3, and 4 carbons. Indomethacin is the principal metabolite of DP-155 in rat serum and, after DP-155 oral administration, the half-life of the metabolite was 22 and 93 h in serum and brain, respectively, compared to 10 and 24 h following indomethacin administration. The brain to serum ratio was 3.5 times higher for DP-155 than for indomethacin. In vitro studies demonstrated that DP-155 is a selective cyclooxygenase (COX)-2 inhibitor. After it is cleaved, its indomethacin derivative nonselectively inhibits both COX-1 and -2. DP-155 showed a better toxicity profile probably due to the sustained, low serum levels and reduced maximal concentration of its indomethacin metabolite. DP-155 did not produce gastric toxicity at the highest acute dose tested (0.28 mmol/kg), while indomethacin caused gastric ulcers at a dose 33-fold lower. Furthermore, after repeated oral dosing, gastrointestinal and renal toxicity was lower (10-and 5-fold, respectively) and delayed with DP-155 compared to indomethacin. In addition to reduced toxicity, DP-155 had similar ameliorative effects to * Address correspondence and reprint requests to: Eran Dvir, DVM, MMedVet (Med), Dipl. ECVIM-CA (Internal DP-155 a Lecithin Derivative of Indomethacin 261indomethacin in antipyretic and analgesia models. Moreover, DP-155 and indomethacin were equally efficacious in reducing levels of amyloid ß (Aß)42 in transgenic Alzheimer's disease mouse (Tg2576) brains as well as reducing Aß42 intracellular uptake, neurodegeneration, and inflammation in an in vitro AD model. The relatively high brain levels of indomethacin after DP-155 administration explain the equal efficacy of DP-155 despite its low systemic blood concentrations. Compared to indomethacin, the favored safety profile and equal efficacy of DP-155 establish the compound as a potential candidate for chronic use to treat AD-related pathology and for analgesia.