Reversing the enantioselectivity of enzymatic carbene N–H insertion through mechanism-guided protein engineering

18 August 2022, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


In this work, we report a computationally driven approach to access enantiodivergent enzymatic carbene N–H bond insertions catalyzed by P411 enzyme variants. Computational modeling was employed to guide engineering efforts to control the accessible conformations of a key lactone-carbene (LAC) intermediate in the enzyme active site by installing a new H-bond anchoring point. By combining MD simulations and protein engineering, a reversed (R-selective) P411 enzyme variant, L5_FL-B3, was obtained in a single round of semi-rational directed evolution. L5_FL-B3 accepts a broad scope of amine substrates with excellent yields (up to >99%), high efficiency (up to 12,300 TTN) and good enantiocontrol (up to 7:93 er), which complements the previously engineered S-selective P411-L7_LF variant.


Asymmetric Catalysis
Carbene Transfer
Computational Modeling

Supplementary materials

Supporting Information
Experimental and computational methods, supplementary figures, and graphs.


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