Reversing the Enantioselectivity of Enzymatic Carbene N-H Insertion Through Mechanism-Guided Protein Engineering

We report a computationally driven approach to access enantiodivergent enzymatic carbene N-H insertions catalyzed by P411 enzymes. Computational modeling was employed to rationally 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. This H-bonding interaction controls the relative orientation of the reactive carbene intermediate, orienting it for an enantioselective N-nucleophilic attack by the amine substrate. By combining MD simulations and site-saturation mutagenesis and screening targeted to only two key residues, we were able to reverse the stereoselectivity of previously engineered S-selective P411 enzymes. The resulting variant, L5_FL-B3, accepts a broad scope of amine substrates for N-H insertion with excellent yields (up to >99 %), high efficiency (up to 12 300 TTN), and good enantiocontrol (up to 7 : 93 er).

Automated summary

We successfully engineered P411 enzymes for enantiodivergent enzymatic carbene N-H insertions through computational modeling and molecular simulations. By introducing a new H-bond anchoring point, we could control the accessible conformations of the lactone-carbene intermediate and direct the reactive carbene for an enantioselective N-nucleophilic attack. With site-saturation mutagenesis and screening, we reversed the stereoselectivity of previously engineered S-selective P411 enzymes. The resulting variant, L5_FL-B3, displayed broad substrate scope, high yields (>99%), high efficiency (up to 12,300 TTN), and good enantiocontrol (up to 7:93 er).