Reversing the Enantioselectivity of Enzymatic Carbene N−H Insertion Through Mechanism‐Guided Protein Engineering**

Author:

Calvó‐Tusell Carla1ORCID,Liu Zhen23ORCID,Chen Kai24ORCID,Arnold Frances H.2ORCID,Garcia‐Borràs Marc1ORCID

Affiliation:

1. Institut de Química Computacional i Catàlisi and Departament de Química Universitat de Girona C/M. Aurèlia Capmany, 69 17003 Girona Spain

2. Division of Chemistry and Chemical Engineering California Institute of Technology 1200 E California Blvd. Pasadena CA 91125 USA

3. National Institute of Biological Sciences Beijing 102206 China

4. Innovative Genomics Institute University of California Berkeley CA USA

Abstract

AbstractWe 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).

Funder

Generalitat de Catalunya

Ministerio de Ciencia e Innovación

Division of Molecular and Cellular Biosciences

Publisher

Wiley

Subject

General Chemistry,Catalysis

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