TCB Publications - Abstract

William R. Arnold, Javier L. Baylon, Emad Tajkhorshid, and Aditi Das. Asymmetric binding and metabolism of polyunsaturated fatty acids (PUFAs) by CYP2J2 epoxygenase. Biochemistry, 55:6969-6980, 2016. (PMC: PMC5558443)

ARNO2016-ET Cytochrome P450 (CYP) 2J2 is the primary epoxygenase in the heart and is responsible for the epoxidation of arachidonic acid (AA), an $\omega$-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 AA metabolism by CYP2J2. We use the Nanodisc (ND) system to stabilize CYP2J2 and its redox partner CYP reductase (CPR). We observe that DHA strongly inhibits CYP2J2-mediated AA metabolism, while LA only moderately inhibits 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 as compared to 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 binding to CYP2J2 is tighter due to 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 variantsóCYP2J2-T318A and CYP2J2-T318V/S493A. Both of these variants showed weaker binding affinity to DHA and AA compared to the WT and the stronger inhibition of AA by DHA in the WT is mitigated in these mutants. Therefore, using a combined experimental and MD simulations approach, we establish that CYP2J2 inhibition of AA metabolism by DHA, EPA and LA is asymmetric due to tighter binding of DHA to select residues in the active site.

Download Full Text

We're sorry, but this article is not available for download.