Leiden-Amsterdam Center for Drug Research, Division of Molecular and Computational Toxicology, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
J Chem Inf Model. 2012 Aug 27;52(8):2139-48. doi: 10.1021/ci300243n. Epub 2012 Jul 19.
Previously, stereoselective hydroxylation of α-ionone by Cytochrome P450 BM3 mutants M01 A82W and M11 L437N was observed. While both mutants hydroxylate α-ionone in a regioselective manner at the C3 position, M01 A82W catalyzes formation of trans-3-OH-α-ionone products whereas M11 L437N exhibits opposite stereoselectivity, producing trans-(3S,6S)-OH-α-ionone and cis-(3S,6R)-OH-α-ionone. Here, we explore the stereoselective C3 hydroxylation of α-ionone by Cytochrome P450 BM3 mutants M01 A82W and M11 L437N using molecular dynamics-based free energy calculations to study the interaction between the enzyme and both the substrates and the products. The one-step perturbation approach is applied using an optimized reference state for substrates and products. While the free energy differences between the substrates free in solution amount to ~0 kJ mol(-1), the differences in mutant M01 A82W agree with the experimentally obtained dissociation constants K(d). Moreover, a correlation with experimentally observed trends in product formation is found in both mutants. The trans isomers show the most favorable relative binding free energy in the range of all four possible hydroxylated diastereomers for mutant M01 A82W, while the trans product from (6S)-α-ionone and the cis product from (6R)-α-ionone show highest affinity for mutant M11 L437N. Marcus theory is subsequently used to relate the thermodynamic stability to transition state energies and rates of formation.
先前，细胞色素 P450 BM3 突变体 M01 A82W 和 M11 L437N 对α-紫罗兰酮进行了立体选择性羟化。虽然这两种突变体都以区域选择性方式在 C3 位置将α-紫罗兰酮羟化，但 M01 A82W 催化反式-3-OH-α-紫罗兰酮产物的形成，而 M11 L437N 表现出相反的立体选择性，生成反式-(3S,6S)-OH-α-紫罗兰酮和顺式-(3S,6R)-OH-α-紫罗兰酮。在这里，我们使用基于分子动力学的自由能计算来探索细胞色素 P450 BM3 突变体 M01 A82W 和 M11 L437N 对α-紫罗兰酮的立体选择性 C3 羟化，以研究酶与底物和产物之间的相互作用。应用一步扰动方法，使用优化的参考状态对底物和产物进行处理。虽然游离在溶液中的底物的自由能差异约为~0 kJ mol(-1)，但突变体 M01 A82W 的差异与实验获得的离解常数 K(d)一致。此外，在两种突变体中都发现了与实验观察到的产物形成趋势的相关性。在突变体 M01 A82W 中，所有四个可能的羟化非对映异构体中，反式异构体的相对结合自由能最有利，而来自(6S)-α-紫罗兰酮的反式产物和来自(6R)-α-紫罗兰酮的顺式产物对突变体 M11 L437N 具有最高亲和力。随后，Marcus 理论被用于将热力学稳定性与过渡态能量和形成速率联系起来。
