Kumar Rajalakshmi, Jayaraman Manikandan, Ramadas Krishna, Chandrasekaran Adithan
Central Inter-Disciplinary Research Facility, Sri Balaji Vidyapeeth (Deemed to be University), Pillayarkuppam, Puducherry, 607 402, India.
Centre for Bioinformatics, School of Life Sciences, Pondicherry University, Puducherry, 605 014, India.
J Mol Graph Model. 2020 Nov;100:107708. doi: 10.1016/j.jmgm.2020.107708. Epub 2020 Aug 5.
Cytochrome P450 oxidoreductase (POR) is a steroidogenic and drug-metabolizing enzyme. It helps in the NADPH dependent transfer of electrons to cytochrome P450 (CYP) enzymes for their biological activity. In this study, we employed integrative computational approaches to decipher the impact of proline to leucine missense mutation at position 384 (P384L) in the connecting/hinge domain region which is essential for the catalytic activity of POR. Analysis of protein stability using DUET, MUpro, CUPSAT, I-Mutant2.0, iStable and SAAFEC servers predicted that mutation might alter the structural stability of POR. The significant conformational changes induced by the mutation to the POR structure were analyzed by long-range molecular dynamics simulation. The results revealed that missense mutation decreased the conformational stability of POR as compared to wild type (WT). The PCA based FEL analysis described the mutant-specific conformational alterations and dominant motions essential for the biological activity of POR. The LIGPLOT interaction analysis showed the different binding architecture of FMN, FAD, and NADPH as a result of mutation. The increased number of hydrogen bonds in the FEL conformation of WT proved the strong binding of cofactors in the binding pocket as compared to the mutant. The porcupine plot analysis associated with cross-correlation analysis depicted the high-intensity flexible motion exhibited by functionally important FAD and NADPH binding domain regions. The computational findings unravel the impact of mutation at the structural level, which could be helpful in understanding the molecular mechanism of drug metabolism.
细胞色素P450氧化还原酶（POR）是一种类固醇生成和药物代谢酶。它有助于将电子以NADPH依赖性方式转移至细胞色素P450（CYP）酶，以实现其生物活性。在本研究中，我们采用综合计算方法来解读连接/铰链结构域区域第384位（P384L）脯氨酸到亮氨酸错义突变的影响，该区域对POR的催化活性至关重要。使用DUET、MUpro、CUPSAT、I-Mutant2.0、iStable和SAAFEC服务器对蛋白质稳定性进行分析，预测该突变可能会改变POR的结构稳定性。通过长程分子动力学模拟分析了该突变对POR结构诱导的显著构象变化。结果显示，与野生型（WT）相比，错义突变降低了POR的构象稳定性。基于主成分分析（PCA）的自由能景观（FEL）分析描述了突变体特异性的构象改变以及对POR生物活性至关重要的主导运动。LIGPLOT相互作用分析表明，由于突变，FMN、FAD和NADPH的结合结构不同。与突变体相比，WT的FEL构象中氢键数量增加证明了辅因子在结合口袋中的强结合。与互相关分析相关的豪猪图分析描绘了功能重要的FAD和NADPH结合结构域区域表现出的高强度灵活运动。这些计算结果揭示了突变在结构水平上的影响，这有助于理解药物代谢的分子机制。
