title: Asymmetric Binding and Metabolism of Polyunsaturated Fatty Acids (PUFAs) by CYP2J2 Epoxygenase.
abstract: Cytochrome P450 (CYP) 2J2 is the primary epoxygenase in the heart and is responsible for the epoxidation of arachidonic acid (AA), an ω-6 polyunsaturated fatty acid (PUFA), into anti-inflammatory epoxide metabolites. It also epoxidizes other PUFAs such as docosahexaenoic acid (DHA), linoleic acid (LA), and eicosapentaenoic acid (EPA). Herein, we have performed detailed thermodynamic and kinetic analyses to determine how DHA, LA, and EPA modulate the metabolism of AA by CYP2J2. We use the Nanodisc system to stabilize CYP2J2 and its redox partner, CYP reductase (CPR). We observe that DHA strongly inhibits CYP2J2-mediated AA metabolism, LA only moderately inhibits AA metabolism, and EPA exhibits insignificant inhibition. We also characterized the binding of these molecules using ebastine competitive binding assays and show that DHA binds significantly tighter to CYP2J2 than AA, EPA, or LA. Furthermore, we utilize a combined approach of molecular dynamics (MD) simulations and docking to predict key residues mediating the tight binding of DHA. We show that although all the tested fatty acids form similar contacts to the active site residues, the affinity of DHA for CYP2J2 is tighter because of the interaction of DHA with residues Arg-321, Thr-318, and Ser-493. To demonstrate the importance of these residues in binding, we mutated these residues to make two mutant variants, CYP2J2-T318A and CYP2J2-T318V/S493A. Both mutant variants showed weaker binding than the wild type (WT) to DHA and AA; DHA inhibition of AA was also mitigated in the mutants compared to the WT. Therefore, using a combined experimental and MD simulation approach, we establish that CYP2J2 inhibition of AA metabolism by DHA, EPA, and LA is asymmetric because of tighter binding of DHA to select residues in the active site.
authors.affiliation: Department of Comparative Biosciences, ‡Department of Biochemistry, §Center for Biophysics and Quantitative Biology, ∥Beckman Institute for Advanced Science and Technology, and ⊥Neuroscience Program, Department of Bioengineering, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: William R
authors.initials: WR
authors.lastname: Arnold
authors.affiliation: Department of Comparative Biosciences, ‡Department of Biochemistry, §Center for Biophysics and Quantitative Biology, ∥Beckman Institute for Advanced Science and Technology, and ⊥Neuroscience Program, Department of Bioengineering, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Javier L
authors.initials: JL
authors.lastname: Baylon
authors.affiliation: Department of Comparative Biosciences, ‡Department of Biochemistry, §Center for Biophysics and Quantitative Biology, ∥Beckman Institute for Advanced Science and Technology, and ⊥Neuroscience Program, Department of Bioengineering, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Emad
authors.initials: E
authors.lastname: Tajkhorshid
authors.affiliation: Department of Comparative Biosciences, ‡Department of Biochemistry, §Center for Biophysics and Quantitative Biology, ∥Beckman Institute for Advanced Science and Technology, and ⊥Neuroscience Program, Department of Bioengineering, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Aditi
authors.identifier: http://orcid.org/0000-0002-6731-6726
authors.initials: A
authors.lastname: Das
mesh_terms: D020134:Catalytic Domain; D002853:Chromatography, Liquid; D000090762:Cytochrome P-450 CYP2J2; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004281:Docosahexaenoic Acids / Q000737:chemistry / Q000378:metabolism*; D015118:Eicosapentaenoic Acid / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D019787:Linoleic Acid / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D053719:Tandem Mass Spectrometry
chemical_list: C000717350:CYP2J2 protein, human; D004281:Docosahexaenoic Acids; D003577:Cytochrome P-450 Enzyme System; D019787:Linoleic Acid; D015118:Eicosapentaenoic Acid; D000090762:Cytochrome P-450 CYP2J2