Alternate Heme Ligation Steers Activity and Selectivity in Engineered Cytochrome P450-Catalyzed Carbene-Transfer Reactions

We report a biocatalytic platform of engineered cytochrome P450 enzymes to carry out carbene-transfer reactions using a lactone-based carbene precursor. By simply altering the heme-ligating residue, we obtained two enzymes that catalyze olefin cyclopropanation (Ser) or S–H bond insertion (Cys). Both enzymes exhibit high catalytic efficiency and stereo­selectivity, thus enabling facile access to structurally diverse spiro[2.4]­lactones and α-thio-γ-lactones. Computational studies revealed the mechanism of carbene S–H insertion and explain how the axial ligand controls reactivity and selectivity. This work expands the catalytic repertoire of heme­proteins and offers insights into how these enzymes can be tuned for new chemistry.

本研究报道了一种工程化细胞色素P450酶（cytochrome P450 enzymes）的生物催化平台，可利用内酯类卡宾前体（lactone-based carbene precursor）开展卡宾转移反应（carbene-transfer reactions）。仅需调整血红素配位残基（heme-ligating residue），我们即可获得两种酶：其一可催化烯烃环丙烷化反应（olefin cyclopropanation，对应丝氨酸残基型酶），其二可催化S-H键插入反应（S–H bond insertion，对应半胱氨酸残基型酶）。两种酶均具备优异的催化效率与立体选择性（stereoselectivity），能够便捷地合成结构多样的螺[2.4]内酯（spiro[2.4]lactones）与α-硫代-γ-内酯（α-thio-γ-lactones）。计算研究揭示了卡宾S-H键插入的反应机制，并阐明了轴向配体（axial ligand）如何调控反应活性与选择性。本研究拓展了血红素蛋白（hemeproteins）的催化谱库，同时为通过改造这类酶以适配全新化学反应提供了理论见解。