Engineered P450 Atom-Transfer Radical Cyclases are Bifunctional Biocatalysts: Reaction Mechanism and Origin of Enantioselectivity

作者：Yue Fu, Heyu Chen, Wen‐Zhen Fu, Marc Garcia‐Borràs, Yang Yang, Peng Liu · 发表于：Journal of the American Chemical Society · 年份：2022 · DOI：10.1021/jacs.2c04937 · 被引用次数：48 · 研究领域：Metal-Catalyzed Oxygenation Mechanisms、CO2 Reduction Techniques and Catalysts、Catalytic C–H Functionalization Methods

New-to-nature radical biocatalysis has recently emerged as a powerful strategy to tame fleeting open-shell intermediates for stereoselective transformations. In 2021, we introduced a novel metalloredox biocatalysis strategy that leverages the innate redox properties of the heme cofactor of P450 enzymes, furnishing new-to-nature atom-transfer radical cyclases (ATRCases) with excellent activity and stereoselectivity. Herein, we report a combined computational and experimental study to shed light on the mechanism and origins of enantioselectivity for this system. Molecular dynamics and quantum mechanics/molecular mechanics (QM/MM) calculations revealed an unexpected role of the key beneficial mutation I263Q. The glutamine residue serves as an essential hydrogen bond donor that engages with the carbonyl moiety of the substrate to promote bromine atom abstraction and enhance the enantioselectivity of radical cyclization. Therefore, the evolved ATRCase is a bifunctional biocatalyst, wherein the heme cofactor enables atom-transfer radical biocatalysis, while the hydrogen bond donor residue further enhances the activity and enantioselectivity. Unlike many enzymatic stereocontrol rationales based on a rigid substrate binding model, our computations demonstrate a high degree of rotational flexibility of the allyl moiety in an enzyme-substrate complex and succeeding intermediates. Therefore, the enantioselectivity is controlled by the radical cyclization transition states rather than th...