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 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 variants—CYP2J2-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.

Arachidonic Acid Metabolism by Human Cardiovascular CYP2J2 Is Modulated by Doxorubicin

“…EBS is a substrate of CYP2J2 with a similar K D as DOX (8.16 μM) and demonstrates one-site binding to the active site. 25, 59 EBS competitively inhibited DOX binding to CYP2J2, with a K i of 13.8 ± 1.6 μM that agrees well with the K D and K m values for EBS. 25 These data conclude that DOX and EBS are competing for binding to the active site of CYP2J2 and that AA weakly inhibits DOX binding (Figure 4A).…”

“…We used AA at a concentration near the previously determined K m . 25 The concentration ranges for DOX covers plasma levels upon immediate exposure (~1–10 μM) to the toxic cumulative dose (~50 μM). 56, 57 We see that DOX inhibits the total AA metabolism with an IC 50 of 5.48 ± 0.04 μM and a K i of 3.11 ± 0.02 μM.…”

“…We determined the K m of EBS metabolism by CYP2J2 to be 19.4 μM. 25 Therefore, we looked at the effect that DOX has on the CYP2J2-mediated metabolism with 20 μM of EBS. DOX inhibited EBS metabolism with an IC 50 of 17.9 ± 0.0 μM and a K i of 8.97 ± 0.01 μM (Figure 4C).…”

“…The simulations suggest that the regioselectivity shift is in part due to how 7-de-aDOX sits in the active site of CYP2J2 (Figure 6A). A specific orientation of 7-de-aDOX located near the active site interacts with key residues that have been shown to be important for AA binding to CYP2J2 (i.e., R321 and S493) 25 . In this configuration, 7-de-aDOX prevents AA from fully extending to place its main epoxidation sites (i.e., carbons C11,C12 and C14,C15) close to the heme, and instead favors positioning of other sites (carbons C8,C9 and to a lesser extent C5,C6) at potentially productive distances from the heme (< 5 °A for C8,C9) during the majority of the simulation (Figure 6B).…”

“…Reactions were started with 6 mM NADPH and terminated after 5 min with 100 μL acetic acid. Metabolites were extracted and quantified as previously described 25 . AA epoxidation by CYP2J2 was determined to be linear up to 8 min under these conditions.…”