Asymmetric Binding and Metabolism of Polyunsaturated Fatty Acids (PUFAs) by CYP2J2 Epoxygenase.
Asymmetric Binding and Metabolism of Polyunsaturated Fatty Acids (PUFAs) by CYP2J2 Epoxygenase.
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DOI：
10.1021/acs.biochem.6b01037
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发表时间：
2016-12-20
期刊：
影响因子：
2.9
通讯作者：
Das A
中科院分区：
文献类型：
--
作者：
Arnold WR;Baylon JL;Tajkhorshid E;Das A
细胞色素P450（CYP）2 J2是心脏中的主要环氧酶，负责花生四烯酸（AA）（ω-6多不饱和脂肪酸（PUFA））环氧化成抗炎环氧化物代谢物。它还环氧化其他PUFA，如二十二碳六烯酸（DHA），亚油酸（LA）和二十碳五烯酸（EPA）。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如何通过CYP 2 J2调节AA代谢。我们使用Nanodisc（ND）系统来稳定CYP 2 J2及其氧化还原伴侣β-还原酶（CPR）。我们观察到，DHA强烈抑制CYP 2 J2介导的AA代谢，而LA仅中度抑制，EPA表现出不显著的抑制作用。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，与AA、EPA或LA相比，DHA与CYP 2 J2的结合显著更紧密。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，虽然所有测试的脂肪酸形成类似的接触的活性位点残基，DHA结合CYP 2 J2的亲和力更紧密，由于DHA与残基Arg-321，Thr-318和Ser-493的相互作用。为了证明这些残基在结合中的重要性，我们突变这些残基以产生两个突变变体-CYP 2 J2-T318 A和CYP 2 J2-T318 V/S493 A。与WT相比，这两种变体均显示出对DHA和AA较弱的结合亲和力，并且在这些突变体中减轻了WT中DHA对AA的较强抑制。因此，使用实验和MD模拟相结合的方法，我们确定CYP 2 J2对DHA、EPA和LA对AA代谢的抑制是不对称的，因为DHA与活性位点中的选定残基结合更紧密。
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.
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影响因子：
3.3
作者：
Klauda, Jeffery B.;Venable, Richard M.;Freites, J. Alfredo;O'Connor, Joseph W.;Tobias, Douglas J.;Mondragon-Ramirez, Carlos;Vorobyov, Igor;MacKerell, Alexander D., Jr.;Pastor, Richard W.
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影响因子：
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