Department of Chemistry, University of Southern California, Los Angeles, CA 90089-1062, USA.
Chembiochem. 2012 Jan 23;13(2):215-23. doi: 10.1002/cbic.201100600. Epub 2011 Dec 21.
The prospect for consistent computer-aided refinement of stereoselective enzymes is explored by simulating the hydrolysis of enantiomers of an α-substituted ester by wild-type and mutant Candida antarctica lipase A, using several strategies. In particular, we focused on the use of the empirical valence bond (EVB) method in a quantitative screening for enantioselectivity, and evaluate both k(cat) and k(cat)/K(M) of the R and S stereoisomers. We found that an extensive sampling is essential for obtaining converging results. This requirement points towards possible problems with approaches that use a limited conformational sampling. However, performing the proper sampling appears to give encouraging results and to offer a powerful tool for the computer-aided design of enantioselective enzymes. We also explore faster strategies for identifying mutations that will help in augmenting directed-evolution experiments, but these approaches require further refinement.
通过使用多种策略，模拟野生型和突变型南极假丝酵母脂肪酶 A 对 α-取代酯的对映异构体的水解，探索了一致的计算机辅助立体选择性酶改进的前景。特别是，我们专注于使用经验价键（EVB）方法进行定量筛选对映选择性，并评估 R 和 S 立体异构体的 k(cat) 和 k(cat)/K(M)。我们发现，为了获得收敛的结果，广泛的采样是必不可少的。这一要求指出了使用有限构象采样的方法可能存在的问题。然而，进行适当的采样似乎会产生令人鼓舞的结果，并为计算机辅助设计对映选择性酶提供强大的工具。我们还探索了更快的策略来识别有助于增强定向进化实验的突变，但这些方法需要进一步改进。
