title: Endocannabinoids anandamide and 2-arachidonoylglycerol are substrates for human CYP2J2 epoxygenase.
abstract: The endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are arachidonic acid (AA) derivatives that are known to regulate human cardiovascular functions. CYP2J2 is the primary cytochrome P450 in the human heart and is most well known for the metabolism of AA to the biologically active epoxyeicosatrienoic acids. In this study, we demonstrate that both 2-AG and AEA are substrates for metabolism by CYP2J2 epoxygenase in the model membrane bilayers of nanodiscs. Reactions of CYP2J2 with AEA formed four AEA-epoxyeicosatrienoic acids, whereas incubations with 2-AG yielded detectable levels of only two 2-AG epoxides. Notably, 2-AG was shown to undergo enzymatic oxidative cleavage to form AA through a NADPH-dependent reaction with CYP2J2 and cytochrome P450 reductase. The formation of the predominant AEA and 2-AG epoxides was confirmed using microsomes prepared from the left myocardium of porcine and bovine heart tissues. The nuances of the ligand-protein interactions were further characterized using spectral titrations, stopped-flow small-molecule ligand egress, and molecular modeling. The experimental and theoretical data were in agreement, which showed that substitution of the AA carboxylic acid with the 2-AG ester-glycerol changes the binding interaction of these lipids within the CYP2J2 active site, leading to different product distributions. In summary, we present data for the functional metabolomics of AEA and 2-AG by a membrane-bound cardiovascular epoxygenase.
authors.affiliation: Department of Comparative Biosciences (D.R.M., A.D.), Department of Biochemistry (A.K., D.D.M., A.D.), and Medical Scholars Program (D.R.M.), Beckman Institute for Advanced Science and Technology, and Department of Bioengineering (A.D.), University of Illinois Urbana-Champaign, Urbana, Illinois.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: McDougle
authors.affiliation: Department of Comparative Biosciences (D.R.M., A.D.), Department of Biochemistry (A.K., D.D.M., A.D.), and Medical Scholars Program (D.R.M.), Beckman Institute for Advanced Science and Technology, and Department of Bioengineering (A.D.), University of Illinois Urbana-Champaign, Urbana, Illinois.
authors.forename: Amogh
authors.initials: A
authors.lastname: Kambalyal
authors.affiliation: Department of Comparative Biosciences (D.R.M., A.D.), Department of Biochemistry (A.K., D.D.M., A.D.), and Medical Scholars Program (D.R.M.), Beckman Institute for Advanced Science and Technology, and Department of Bioengineering (A.D.), University of Illinois Urbana-Champaign, Urbana, Illinois.
authors.forename: Daryl D
authors.initials: DD
authors.lastname: Meling
authors.affiliation: Department of Comparative Biosciences (D.R.M., A.D.), Department of Biochemistry (A.K., D.D.M., A.D.), and Medical Scholars Program (D.R.M.), Beckman Institute for Advanced Science and Technology, and Department of Bioengineering (A.D.), University of Illinois Urbana-Champaign, Urbana, Illinois aditidas@illinois.edu.
authors.forename: Aditi
authors.initials: A
authors.lastname: Das
mesh_terms: D000818:Animals; D001095:Arachidonic Acids / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D000090762:Cytochrome P-450 CYP2J2; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D063388:Endocannabinoids / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D005989:Glycerides / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008297:Male; D053284:Polyunsaturated Alkamides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D013379:Substrate Specificity / Q000502:physiology; D013552:Swine
chemical_list: D001095:Arachidonic Acids; C000717350:CYP2J2 protein, human; D063388:Endocannabinoids; D005989:Glycerides; D053284:Polyunsaturated Alkamides; C094503:glyceryl 2-arachidonate; D003577:Cytochrome P-450 Enzyme System; D000090762:Cytochrome P-450 CYP2J2; C078814:anandamide