Key Lab of Colloid and Interface Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering , Shandong University , Jinan , Shandong 250100 , China.
College of Geography and Environmental Sciences , Zhejiang Normal University , Jinhua , Zhejiang 321004 , China.
Inorg Chem. 2018 Sep 17;57(18):11738-11745. doi: 10.1021/acs.inorgchem.8b01875. Epub 2018 Aug 29.
Engineered P450s can catalyze some non-natural reactions with high efficiency and excellent selectivity, such as the carbine transfer, nitrene transfer, C-H insertion, and C-H amination, opening alternative routes for sustainable production of chemicals. Recent experiments revealed that two engineered cytochrome P450 enzymes (P450-CIS and P411-CIS) show different efficiencies and stereoselectivities in the olefin cyclopropanation, but key factors that affect the activity remain unclear. In this work, both quantum mechanics (QM) and QM/molecular mechanics (MM) methods were employed to explore the catalytic reactions and selectivity of these two engineered cytochrome P450 enzymes. On the basis of our results, the cyclopropanation of styrene is suggested to mainly occur on the open-shell singlet (OSS) and triplet state surfaces, which contain two elementary steps. The reactive iron(III)-porphyrin carbene (IPC) radical first attacks the terminal alkenyl group of styrene to form a C-radical intermediate, which then undergoes a cyclization reaction affording the cyclopropanation products. Importantly, it is found that the stereoselectivity of cyclopropanations is elucidated only if considering the real protein environment, and the stereoselectivity is determined by multiple factors, such as the relative orientation of IPC to styrene, the binding affinity of the substrate, and the reaction barriers of rate-limiting steps. It is the enzymatic environment that makes the reaction highly stereoselective, which provides useful clues for designing whole-cell catalysts for non-natural chemical reactions.
工程化 P450 可以高效且高选择性地催化一些非天然反应，例如卡宾转移、氮宾转移、C-H 插入和 C-H 胺化，为可持续生产化学品开辟了替代途径。最近的实验表明，两种工程化细胞色素 P450 酶（P450-CIS 和 P411-CIS）在烯烃环丙烷化反应中表现出不同的效率和立体选择性，但影响活性的关键因素仍不清楚。在这项工作中，我们同时采用量子力学（QM）和 QM/分子力学（MM）方法来探索这两种工程化细胞色素 P450 酶的催化反应和选择性。基于我们的结果，推测苯乙烯的环丙烷化主要发生在开壳单重态（OSS）和三重态表面上，其中包含两个基本步骤。反应性铁（III）-卟啉碳烯（IPC）自由基首先攻击苯乙烯的末端烯基基团，形成 C-自由基中间体，然后进行环化反应，生成环丙烷化产物。重要的是，只有考虑到真实的蛋白质环境，才能阐明环丙烷化的立体选择性，而立体选择性取决于多个因素，例如 IPC 与苯乙烯的相对取向、底物的结合亲和力以及限速步骤的反应势垒。正是酶的环境使反应具有高度的立体选择性，为设计用于非天然化学反应的全细胞催化剂提供了有用的线索。
