Institute of Complex Systems: Structural Biochemistry , Forschungszentrum Jülich , 52425 Jülich , Germany.
Institute of Biochemistry , Heinrich Heine University Düsseldorf , Universitätsstraße 1 , 40225 Düsseldorf , Germany.
J Chem Inf Model. 2018 Apr 23;58(4):848-858. doi: 10.1021/acs.jcim.8b00043. Epub 2018 Mar 23.
Engineering high chemo-, regio-, and stereoselectivity is a prerequisite for enzyme usage in organic synthesis. Cytochromes P450 can oxidize a broad range of substrates, including macrocycles, which are becoming popular scaffolds for therapeutic agents. However, a large conformational space explored by macrocycles not only reduces the selectivity of oxidation but also impairs computational enzyme design strategies based on docking and molecular dynamics (MD) simulations. We present a novel design workflow that uses enhanced-sampling Hamiltonian replica exchange (HREX) MD and focuses on quantifying the substrate binding for suggesting the mutations to be made. This computational approach is applied to P450 BM3 with the aim to shift regioselectively toward one of the numerous possible positions during β-cembrenediol oxidation. The predictions are experimentally tested and the resulting product distributions validate our design strategy, as single mutations led up to 5-fold regioselectivity increases. We thus conclude that the HREX-MD-based workflow is a promising tool for the identification of positions for mutagenesis aiming at P450 enzymes with improved regioselectivity.
在有机合成中使用酶的前提条件是实现高化学选择性、区域选择性和立体选择性。细胞色素 P450 可以氧化广泛的底物，包括大环化合物，这些化合物正成为治疗药物的热门支架。然而，大环化合物探索的大构象空间不仅降低了氧化的选择性，而且还损害了基于对接和分子动力学 (MD) 模拟的计算酶设计策略。我们提出了一种新的设计工作流程，该流程使用增强采样哈密顿复制交换 (HREX) MD，并侧重于量化底物结合以提出要进行的突变。该计算方法应用于 P450 BM3，目的是在β-cembrenediol 氧化过程中选择性地转移到众多可能位置之一。预测结果经过实验验证，所得产物分布验证了我们的设计策略，因为单个突变可使区域选择性提高 5 倍。因此，我们得出结论，基于 HREX-MD 的工作流程是确定 P450 酶提高区域选择性的突变位置的有前途的工具。
