Some Surprising Implications of NMR-directed Simulations of Substrate Recognition and Binding by Cytochrome P450(cam) (CYP101A1).
Some Surprising Implications of NMR-directed Simulations of Substrate Recognition and Binding by Cytochrome P450(cam) (CYP101A1).
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DOI：
10.1016/j.jmb.2018.03.014
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发表时间：
2018-04-27
影响因子：
5.6
通讯作者：
Pochapsky TC
中科院分区：
文献类型：
--
作者：
Asciutto EK;Pochapsky TC
细胞色素P450 cam（CYP 101 A1）催化D-樟脑通过分子氧的立体特异性5-外羟基化。以前，残留偶极耦合（RDC）测量的骨干酰胺1H-15 N相关性在无底物和结合形式的CYP 101 A1被用作软退火分子动力学（MD）模拟的限制，以确定平均构象的酶与和无底物结合。确定了远离酶活性位点的多个底物依赖性构象变化，并且定点诱变和活性测定证实了这些变化在底物识别中的重要性。目前的工作利用扰动响应扫描（PRS）和伞形采样MD的RDC衍生的CYP 101 A1结构，以探测远程结构特征的作用，在强制执行的区域和立体特异性的CYP 101 A1催化的羟基化反应。定义了一个不适当的二面角θ，并用于维持底物在CYP 101 A1活性位点的方向，并观察到不同的θ值导致不同的PRS响应图。伞形采样法表明，体系的自由能对酶的构象变化敏感，结合底物是酶结构传递机械耦合的重要机械环节。最后，提出了一种定性的方法来解释PRS图的二级结构特征的作用。
Cytochrome P450cam (CYP101A1) catalyzes the stereospecific 5-exo hydroxylation of d-camphor by molecular oxygen. Previously, residual dipolar couplings (RDCs) measured for backbone amide 1H-15N correlations in both substrate-free and bound forms of CYP101A1 were used as restraints in soft annealing molecular dynamics (MD) simulations in order to identify average conformations of the enzyme with and without substrate bound. Multiple substrate-dependent conformational changes remote from the enzyme active site were identified, and site-directed mutagenesis and activity assays confirmed the importance of these changes in substrate recognition. The current work makes use of perturbation response scanning (PRS) and umbrella sampling MD of the RDC-derived CYP101A1 structures to probe the roles of remote structural features in enforcing the regio- and stereospecific nature of the hydroxylation reaction catalyzed by CYP101A1. An improper dihedral angle Ψ was defined and used to maintain substrate orientation in the CYP101A1 active site, and it was observed that different values of Ψ result in different PRS response maps. Umbrella sampling methods show that the free energy of the system is sensitive to Ψ, and bound substrate forms an important mechanical link in the transmission of mechanical coupling through the enzyme structure. Finally, a qualitative approach to interpreting PRS maps in terms of the roles of secondary structural features is proposed.
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