© BioMed Central Ltd 2004
Published: 27 August 2004
Cyclooxygenases (COXs) catalyze the rate-limiting step in the production of prostaglandins, bioactive compounds involved in processes such as fever and sensitivity to pain, and are the target of aspirin-like drugs. COX genes have been cloned from coral, tunicates and vertebrates, and in all the phyla where they are found, there are two genes encoding two COX isoenzymes; it is unclear whether these genes arose from an early single duplication event or from multiple independent duplications in evolution. The intron-exon arrangement of COX genes is completely conserved in vertebrates and mostly conserved in all species. Exon boundaries largely define the four functional domains of the encoded protein: the amino-terminal hydrophobic signal peptide, the dimerization domain, the membrane-binding domain, and the catalytic domain. The catalytic domain of each enzyme contains distinct peroxidase and cyclooxygenase active sites; COXs are classified as members of the myeloperoxidase family. All COXs are homodimers and monotopic membrane proteins (inserted into only one leaflet of the membrane), and they appear to be targeted to the lumenal membrane of the endoplasmic reticulum, where they are N-glycosylated. In mammals, the two COX genes encode a constitutive isoenzyme (COX-1) and an inducible isoenzyme (COX-2); both are of significant pharmacological importance.
Gene organization and evolutionary history
Properties of human COX-1 and COX-2 and the genes encoding them
Chromosomal location of gene
Copy number of gene
About 22 kb
About 8 kb
Number of exons
Number of introns
Length of primary mRNA
Length of differentially polyadenylated variants
4.5 kb, 5.2 kb
4.0 kb, 2.8 kb
Lengths of splice variants
Length of coding region
Putative transcription regulatory elements found in:
5' upstream region*
AP-2, GATA-1, NF-IL6, NFκB, PEA-3, SP-1, SSRE
AP-2, C/EBP, CRE, GATA-1, GRE, NF-IL6, NFκB, PEA-3, SP-1
3' untranslated region
Inducible (by cytokines, growth factors and so on [10,35])
Length of protein (with signal peptide)
599 amino acids
604 amino acids
Length of mature protein (without signal peptide)
576 amino acids
581 amino acids
Number of glycosylation sites
Arachidonic acid and others
Endoplasmic reticulum and nuclear envelope
All vertebrates investigated, including cartilaginous fishes, bony fishes, birds, and mammals, have two COX genes: one encoding the constitutive COX-1 and another the inducible COX-2. COX-1 and COX-2 share approximately 60-65% amino-acid identity with each other; COX-1 orthologs (without the signal peptide) share approximately 70-95% amino-acid identity across vertebrate species and COX-2 orthologs share 70-90%. Additionally, coral (of the phylum Cnidaria) and sea squirt (ascidian) each have two COX genes, which may have arisen from gene-duplication events independent from those that produced vertebrate COX-1 and COX-2 . It is clear that the vertebrate, coral and ascidian COX genes all descend from a common ancestor.
Intron-exon junctions are highly conserved in all species, with a few notable exceptions: the vertebrate COX-1 genes contain an extra intron (intron 1), and the ascidian and coral genes have extra introns or lack some exons in the regions that encode exons 6, 7 or 11 in vertebrate COX-1 . The exon structures of COX genes largely reflect the domains encoded by the proteins (Figure 1b). The structure of human cyclooxygenase genes and their expression and regulation have been reviewed elsewhere .
COX genes have not been found in insects, unicellular organisms, or plants, although prostaglandins, their products, have been found in some of these organisms . Recently, an enzyme that catalyzes the synthesis of prostaglandin E2 from arachidonic acid (the substrate of COXs) was cloned from the protozoan Entamoeba histolytica. This enzyme shows no clear structural similarity to COXs, suggesting that alternative evolutionary paths to prostaglandin synthesis have evolved in some organisms .
Characteristic structural features
COXs are all close to 600 amino acids in size and have a similar primary structure [13, 14] (Figure 1b). The crystal structure of sheep COX-1 (minus the post-translationally cleaved signal peptide), was obtained in 1994 ; human and mouse COXs have since been crystallized and show strikingly similar features [16, 17]. After the signal peptide, the amino terminus of the protein contains a single epidermal growth factor (EGF) module with conserved disulfide bonds that functions as a dimerization domain. This is followed by a series of four amphipathic helices that anchors the protein to one leaflet of the membrane. This 'monotopic' type of insertion into a membrane has been found only in this enzyme and a few other proteins such as squalene cyclase and S-mandelate dehydrogenase [18, 19]. The remainder of the protein consists of the catalytic domain, which has two distinct cyclooxygenase and peroxidase active sites.
COXs are highly conserved, and few significant differences are seen in the dimerization, membrane-binding and catalytic domains between COXs from different species. The amino-terminal hydrophobic signal peptides differ significantly in length between species. In the case of two splice variants of canine COX-1, the signal peptide has been found not to be cleaved from the enzyme when expressed in insect cells .
Localization and function
COX-1 is ubiquitously and constitutively expressed in mammalian tissues and cells, whereas COX-2 is highly inducible and is generally present in mammalian tissues at very low levels, unless increased by one of many types of stimuli such as cytokines and growth factors.
Both classes of COX are bifunctional enzymes with two distinct catalytic activities: cyclooxygenase (or bis-dioxygenase) activity and peroxidase activity (Figure 3a). The primary products of COXs were first detected in human seminal fluid by clinicians studying uterine contraction . Thought to be the product of the prostate gland, these highly potent bioactive compounds were given the name prostaglandins. They are synthesized in virtually all tissues in vertebrates, however, and some organisms that lack prostate glands, such as corals, also synthesize prostaglandins. Thus, in many respects the term prostaglandin is a misnomer. Initially, the enzyme activity that synthesized prostaglandins was frequently called prostaglandin synthetase, but because it does not require ATP it is now called prostaglandin G/H synthase to fit the nomenclature convention. It is more popularly known as cyclooxygenase, a name that only partially describes the enzyme since it refers to only one of its two enzymatic activities.
Prostaglandin isomers - including thromboxane and prostaglandins D2, E2, F2α, and I2 (prostacyclin; Figure 3b) - function in numerous physiological and pathophysiological processes, such as pyresis (fever), algesia (sensitivity to pain), inflammation, thrombosis, parturition, mitogenesis, vasodilation and vasoconstriction, ovulation, and renal function. Prostaglandin isomers act upon G-protein-coupled receptors , and there are multiple receptors for some isoforms (such as prostaglandin E2). Prostaglandins are short-lived in vivo (with half-lives of seconds to minutes), and act in an autocrine or a paracrine rather than an endocrine fashion. COX-1 was first studied in tissue and cell homogenates, and in this context was shown by Vane  to be the inhibitory target of NSAIDs.
The cyclooxygenase activity of COXs oxygenates arachidonic acid to produce prostaglandin G2, a cyclopentane hydroperoxy endoperoxide; the peroxidase activity of COXs then reduces this to prostaglandin H2 (Figure 3a). The two reactions are functionally interconnected (see below and Figure 3c). A branch-chain reaction mechanism for COX, indicating that the two reaction cycles are coupled, was first proposed by Ruf and colleagues . The mechanism by which arachidonic acid is converted to prostaglandin H2 has been the subject of excellent reviews [31, 32]. The newly synthesized COX enzyme needs to be activated at Tyr384 (in human COX-1; Tyr371 in human COX-2) to produce a tyrosyl radical; this activation involves the heme in the peroxidase site (see Figure 3b). The tyrosyl radical converts arachidonic acid to an arachidonyl radical, which reacts with two molecules of oxygen to yield prostaglandin G2 This then diffuses to the peroxidase site and is reduced to prostaglandin H2 by the peroxidase activity. The cyclooxygenase activity is dependent on heme oxidation - that is, on the peroxidase activity - but continuous peroxidase activity is not necessary for cyclooxygenase activity, as the tyrosyl radical is regenerated in each catalytic cycle (Figure 3c). Prostaglandin H2 is the root prostaglandin from which prostaglandin isomers such as thromboxane and prostacyclin are made by downstream synthases, via isomerization and oxidation or reduction reactions (Figure 3b). Cyclooxygenases have short catalytic life spans (frequently 1-2 minutes at Vmax in vitro) because the enzyme is autoinactivated. The mechanism of autoinactivation is unknown, but reactive tyrosyl radicals may cause internal protein modification.
The exact distinct functions of COX-1 and COX-2 are still being unraveled . There is increasing evidence for the involvement of COXs in the development and progression of cancer, Alzheimer's disease and other pathophysiological states. Development of therapeutic and diagnostic tools to treat these diseases is being actively investigated. Moreover, variants of cyclooxygenase derived from alternative splicing have been reported (reviewed in [13, 34]). Elucidation of the roles played by these variants could provide greater insight into the roles of COXs in physiology and disease.
This work was supported by National Institute of Health grant AR 46688 and Merck, USA. We wish to thank K.L.T. Roos, D. Melville and C. Gurney for helping us in numerous ways.
- Daiyasu H, Toh H: Molecular evolution of the myeloperoxidase family. J Mol Evol. 2000, 51: 433-445. An evolutionary analysis of the myeloperoxidasesPubMedGoogle Scholar
- Hemler M, Lands WEM, Smith WL: Purification of the cyclooxygenase that forms prostaglandins: demonstration of two forms of iron in the holoenzyme. J Biol Chem. 1976, 251: 5575-5579. The first isolation and purification of cyclooxygenase from sheep seminal vesiclesPubMedGoogle Scholar
- Miyamoto T, Ogino N, Yamamoto S, Hayaishi O: Purification of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J Biol Chem. 1976, 251: 2629-2636. The first isolation and purification of cyclooxygenase enzyme from bovine seminal vesiclesPubMedGoogle Scholar
- Yokoyama C, Tanabe T: Cloning of human gene encoding prostaglandin endoperoxide synthase and primary structure of the enzyme. Biochem Biophys Res Commun. 1989, 165: 888-894. The cloning of human cyclooxygenase-1 (COX-1)PubMedView ArticleGoogle Scholar
- Merlie JP, Fagan D, Mudd J, Needleman P: Isolation and characterization of the complementary DNA for sheep seminal vesicle prostaglandin endoperoxide synthase (cyclooxygenase). J Biol Chem. 1988, 263: 3550-3553. The cloning of the first cyclooxygenase gene from sheepPubMedGoogle Scholar
- DeWitt DL, Smith WL: Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence. Proc Natl Acad Sci USA. 1988, 85: 1412-1416. The cloning of the first cyclooxygenase gene from sheepPubMedPubMed CentralView ArticleGoogle Scholar
- Xie W, Chipman JG, Robertson DL, Erikson RL, Simmons DL: Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing. Proc Natl Acad Sci USA. 1991, 88: 2692-2696. The first report on the cloning and characterization of a second isozyme of cyclooxygenase (COX-2)PubMedPubMed CentralView ArticleGoogle Scholar
- Kujubu DA, Fletcher BS, Varnum BC, Lim RW, Herschman HR: TIS10, a phorbol ester tumor promoter-inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/cyclooxygenase homologue. J Biol Chem. 1991, 266: 12866-12872. Cloning and characterization of a second isozyme of cyclooxygenase (COX-2)PubMedGoogle Scholar
- Jarving R, Jarving I, Kurg R, Brash AR, Samel N: On the evolutionary origin of cyclooxygenase (COX) isozymes: characterization of marine invertebrate COX genes points to independent duplication events in vertebrate and invertebrate lineages. J Biol Chem. 2004, 279: 13624-13633. 10.1074/jbc.M313258200. An evolutionary analysis of invertebrate cyclooxygenasesPubMedView ArticleGoogle Scholar
- Tanabe T, Tohnai N: Cyclooxygenase isozymes and their gene structures and expression. Prostaglandins Other Lipid Mediat. 2002, 68-69: 95-114. 10.1016/S0090-6980(02)00024-2. A review on the gene structures and expression of COX-1 and COX-2PubMedView ArticleGoogle Scholar
- Bundy GL: Nonmammalian sources of eicosanoids. Adv Prostaglandin Thromboxane Leukot Res. 1985, 14: 229-262. A review on prostaglandins from nonmammalian sourcesPubMedGoogle Scholar
- Dey I, Keller K, Belley A, Chadee K: Identification and characterization of a cyclooxygenase-like enzyme from Entamoeba histolytica. Proc Natl Acad Sci USA. 2003, 100: 13561-13566. 10.1073/pnas.1835863100. The cloning of a prostaglandin-producing enzyme unrelated to the cyclooxygenasesPubMedPubMed CentralView ArticleGoogle Scholar
- Simmons DL, Botting RM, Hla T: Cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition. Pharmacol Rev. A review on the biology of prostaglandin synthesis and inhibition by non-steroidal anti-inflammatory drugs (NSAIDs)Google Scholar
- Garavito RM, Malkowski MG, DeWitt DL: The structures of prostaglandin endoperoxide H synthases-1 and -2. Prostaglandins Other Lipid Mediat. 2002, 68-69: 129-152. 10.1016/S0090-6980(02)00026-6. A review on the structure of cyclooxygenasesPubMedView ArticleGoogle Scholar
- Picot D, Loll P, Garavito M: The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1. Nature. 1994, 367: 243-249. 10.1038/367243a0. A landmark study elucidating the three dimensional structure of sheep COX-1PubMedView ArticleGoogle Scholar
- Luong C, Miller A, Barnett J, Chow J, Ramesha C, Browner MF: Flexibility of the NSAID binding site in the structure of human cyclooxygenase-2. Nat Struct Biol. 1996, 3: 927-933. 10.1038/nsb1196-927. The first report on the crystal structure of human COX-2PubMedView ArticleGoogle Scholar
- Kurumbail RG, Stevens AM, Gierse JK, McDonald JJ, Stegeman RA, Pak JY, Gildehaus D, Miyashiro JM, Penning TD, Seibert K, et al: Structural basis for selective inhibition of cyclooxygenase-2 by anti-inflammatory agents. Nature. 1996, 384: 644-648. 10.1038/384644a0. The crystal structure of murine COX-2PubMedView ArticleGoogle Scholar
- Wendt KU, Poralla K, Schulz GE: Structure and function of a squalene cyclase. Science. 1997, 277: 1811-1815. 10.1126/science.277.5333.1811. Describes the crystal structure of squalene cyclase, which has membrane-binding properties similar to those of cyclooxygenasePubMedView ArticleGoogle Scholar
- Sukumar N, Xu Y, Gatti DL, Mitra B, Mathews FS: Structure of an active soluble mutant of the membrane-associated (S)-mandelate dehydrogenase. Biochemistry. 2001, 40: 9870-9878. 10.1021/bi010938k. Describes the crystal structure of S-mandelate dehydrogenase, which has membrane-binding properties similar to those of cyclooxygenasePubMedView ArticleGoogle Scholar
- Chandrasekharan NV, Dai H, Roos KLT, Evanson NK, Tomsik J, Elton TS, Simmons DL: COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression. Proc Natl Acad Sci USA. 2002, 99: 13926-13931. 10.1073/pnas.162468699. The cloning and characterization of canine COX-1 variantsPubMedPubMed CentralView ArticleGoogle Scholar
- Otto JC, DeWitt DL, Smith WL: N-glycosylation of prostaglandin endoperoxide synthase-1 and -2 and their orientations in the endoplasmic reticulum. J Biol Chem. 1993, 268: 18234-18242. Describes the role of glycosylation in cyclooxygenasesPubMedGoogle Scholar
- Thuresson ED, Lakkides KM, Rieke CJ, Sun Y, Wingerd BA, Micielli R, Mulichack AM, Malkowski MG, Garavito RM, Smith WL: Prostaglandin endoperoxide H synthase-1: the function of cyclooxygenase active site residues in the binding, positioning, and oxygenation of arachidonic acid. J Biol Chem. 2001, 276: 10347-10358. 10.1074/jbc.M009377200. A mutational analysis of the cyclooxygenase active sitePubMedView ArticleGoogle Scholar
- Bozza PT, Yu W, Penrose JF, Morgan ES, Dvorak AM, Weller PF: Eosinophil lipid bodies: specific, inducible intracellular sites for enhanced eicosanoid formation. J Exp Med. 1997, 186: 909-920. 10.1084/jem.186.6.909. Cyclooxygenases in lipid bodiesPubMedPubMed CentralView ArticleGoogle Scholar
- Liou JY, Deng WG, Gilroy DW, Shyue SK, Wu KK: Colocalization and interaction of cyclooxygenase-1 with caveolin-1 in human fibroblasts. J Biol Chem. 2001, 276: 34975-34982. 10.1074/jbc.M105946200. Cyclooxygenases in vesiclesPubMedView ArticleGoogle Scholar
- Coffey RJ, Hawkey CJ, Damstrup L, Graves-Deal R, Daniel VC, Dempsey PJ, Chimery R, Kirkland SC, DuBois RN, Jetton TL, Morrow JD: Epidermal growth factor receptor activation induces nuclear targeting of cyclooxygenase-2, basolateral release of prostaglandins, and mitogenesis in polarizing colon cancer cells. Proc Natl Acad Sci USA. 1997, 94: 657-662. 10.1073/pnas.94.2.657. The nuclear localization of COX-2PubMedPubMed CentralView ArticleGoogle Scholar
- Liou JY, Shyue SK, Tsai MJ, Chung CL, Chu KY, Wu KK: Colocalization of prostacyclin synthase with prostaglandin H synthase-1 (PGHS-1) but not phorbol ester-induced PGHS-2 in cultured endothelial cells. J Biol Chem. 2000, 275: 15314-15320. 10.1074/jbc.275.20.15314. Demonstration of the colocalization of cyclooxygenase with filamentous structuresPubMedView ArticleGoogle Scholar
- Goldblatt MW: A depressor substance in seminal fluid. J Soc Chem Ind. 1933, 52: 1056-1057. The first detection of the products of COXs in human seminal fluidGoogle Scholar
- Narumiya S, Sugimoto Y, Ushikubi F: Prostanoid receptors: structures properties and functions. Physiol Rev. 1999, 79: 1193-1226. A review on prostanoid receptorsPubMedGoogle Scholar
- Vane JR: Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature. 1971, 231: 232-235. The first report indicating that the target for aspirin-like drugs is cyclooxygenaseGoogle Scholar
- Dietz R, Nastainczyk W, Ruf HH: Higher oxidation states of prostaglandin H synthase. Rapid electronic spectroscopy detected two spectral intermediates during the peroxidase reaction with prostaglandin G2. Eur J Biochem. 1988, 171: 321-328. The first paper to propose a branch chain mechanism for cyclooxygenase catalysisPubMedView ArticleGoogle Scholar
- Rouzer CA, Marnett LJ: Mechanism of free radical oxygenation of polyunsaturated fatty acids by cyclooxygenases. Chem Rev. 2003, 103: 2239-2304. 10.1021/cr000068x. A review on the reaction mechanism of cyclooxygenasePubMedView ArticleGoogle Scholar
- van der Donk WA, Tsai A-L, Kulmacz RJ: The cyclooxygenase reaction mechanism. Biochemistry. 2002, 41: 15451-15457. 10.1021/bi026938h. A review on the reaction mechanism of cyclooxygenasePubMedView ArticleGoogle Scholar
- Loftin CD, Tiano HF, Langenbach R: Phenotypes of the COX-deficient mice indicate physiological and pathophysiological roles for COX-1 and COX-2. Prostaglandins Other Lipid Mediat. 2002, 68-69: 177-185. 10.1016/S0090-6980(02)00028-X. Determination of the roles of COX-1 and COX-2 using COX-1-/-, COX-2-/-, and COX-1-/- COX-2-/- micePubMedView ArticleGoogle Scholar
- Simmons DL: Variants of cyclooxygenase-1 and their roles in medicine. Thrombosis Res. 2003, 110: 265-268. 10.1016/S0049-3848(03)00380-3. A review on variants of COX-1View ArticleGoogle Scholar
- Smith WL, De Witt DL, Garavito RM: Cyclooxygenases: structural, cellular and molecular biology. Annu Rev Biochem. 2000, 69: 145-182. 10.1146/annurev.biochem.69.1.145. A review on cyclooxygenasesPubMedView ArticleGoogle Scholar