Department of Chemistry, Zhejiang University, Hangzhou 310027, China.
Chem Biol Drug Des. 2011 Aug;78(2):301-8. doi: 10.1111/j.1747-0285.2011.01097.x. Epub 2011 Jun 24.
The asymmetric catalysis, as the character of enzyme, attracts increasing attention from the scientific and industrial communities. In this study, the Bacillus subtilis lipase A, as a model enzyme, is studied systematically to dissect its stereoselectivity toward (rac)-ketoprofen vinyl ester using a combination scheme of molecular docking and quantum mechanical/molecular mechanical (QM/MM) analysis. In this procedure, the rational orientation of the two enantiomers of ketoprofen vinyl ester is obtained with the AutoDock performing, and then, the steric contacts between the enzyme and substrate in the docking outputs are examined visually at the atomic level with a small-probe technique. Subsequently, the binding energies of the enzyme-substrate complexes are calculated using an ONIOM (Our own N-layered Integrated Molecular Orbital + Molecular mechanics)-based QM/MM protocol. The results obtained from the theoretical studies show that the B. subtilis lipase A prefer to hydrolyze the (R )-ketoprofen vinyl ester when compared to its (S )-enantiomer, with a relatively high E (stereoselectivity) value of 31.28 charactering its enantioselectivity. Furthermore, to verify the conclusions from the computational analysis, the B. subtilis lipase A gene is cloned to overexpress the recombinant B. subtilis lipase A, and its stereoselectivity was determined. Satisfactorily, the experimental results are in well agreement with the theoretical predictions because the (R )-ketoprofen vinyl ester is found as the preferring enantiomer of the B. subtilis lipase A, with experimentally measured E value of 36.7. We therefore expect that this in silico-in vitro hybrid approach can provide a new and effective avenue to predict the catalytic activity of and to investigate the molecular mechanism of enzyme-mediated asymmetric catalysis and help in understanding the enzymatic process and in rational enzyme design.
不对称催化作为酶的特征，引起了科学界和工业界越来越多的关注。在这项研究中，枯草芽孢杆菌脂肪酶 A 作为一种模型酶，通过分子对接和量子力学/分子力学 (QM/MM) 分析相结合的方案，系统地研究了其对 (rac)-酮洛芬乙烯酯的立体选择性。在这个过程中，AutoDock 执行了酮洛芬乙烯酯两种对映体的合理取向，然后使用小探针技术在原子水平上直观地检查对接输出中酶和底物之间的立体接触。随后，使用基于 ONIOM（我们自己的 N 层集成分子轨道+分子力学）的 QM/MM 方案计算酶-底物复合物的结合能。理论研究结果表明，与 (S)-对映体相比，枯草芽孢杆菌脂肪酶 A 更喜欢水解 (R)-酮洛芬乙烯酯，其立体选择性 E（立体选择性）值相对较高，为 31.28。此外，为了验证计算分析的结论，克隆了枯草芽孢杆菌脂肪酶 A 基因以过表达重组枯草芽孢杆菌脂肪酶 A，并测定其立体选择性。令人满意的是，实验结果与理论预测非常吻合，因为 (R)-酮洛芬乙烯酯是枯草芽孢杆菌脂肪酶 A 的优先对映体，实验测量的 E 值为 36.7。因此，我们期望这种计算-体外混合方法可以为预测酶介导的不对称催化的催化活性和研究酶促反应的分子机制提供新的有效途径，并有助于理解酶的过程和合理的酶设计。
