Arnold William R, Baylon Javier L, Tajkhorshid Emad, Das Aditi
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.
Biochemistry. 2016 Dec 20;55(50):6969-6980. doi: 10.1021/acs.biochem.6b01037. Epub 2016 Dec 5.
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.
细胞色素P450（CYP）2J2是心脏中的主要环氧化酶，负责将ω-6多不饱和脂肪酸（PUFA）花生四烯酸（AA）环氧化为具有抗炎作用的环氧化物代谢产物。它还能将其他多不饱和脂肪酸如二十二碳六烯酸（DHA）、亚油酸（LA）和二十碳五烯酸（EPA）环氧化。在此，我们进行了详细的热力学和动力学分析，以确定DHA、LA和EPA如何调节CYP2J2对AA的代谢。我们使用纳米盘系统来稳定CYP2J2及其氧化还原伴侣CYP还原酶（CPR）。我们观察到，DHA强烈抑制CYP2J2介导的AA代谢，LA仅适度抑制AA代谢，而EPA的抑制作用不显著。我们还使用依巴斯汀竞争性结合试验对这些分子的结合进行了表征，结果表明DHA与CYP2J2的结合比AA、EPA或LA紧密得多。此外，我们利用分子动力学（MD）模拟和对接的组合方法来预测介导DHA紧密结合的关键残基。我们表明，尽管所有测试的脂肪酸与活性位点残基形成的接触相似，但由于DHA与残基Arg-321、Thr-318和Ser-493的相互作用，DHA对CYP2J2的亲和力更强。为了证明这些残基在结合中的重要性，我们对这些残基进行突变，制备了两个突变变体CYP2J2-T318A和CYP2J2-T318V/S493A。与野生型（WT）相比，这两个突变变体与DHA和AA的结合均较弱；与WT相比，突变体中DHA对AA的抑制作用也有所减轻。因此，通过结合实验和MD模拟方法，我们确定由于DHA与活性位点中特定残基的紧密结合，CYP2J2对DHA、EPA和LA对AA代谢的抑制作用是不对称的。
