In this work, a team led by Dr. Marc Garcia-Borràs report a computationally driven approach to access enantiodivergent enzymatic carbene N–H insertions catalyzed by P411 enzymes. Computational modeling, carried out by PhD student Carla Calvó-Tusell, 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, it was able to reverse the stereoselectivity of previously engineered S-selective P411 enzymes. The experimental work was carried out by the group of Prof. F. H. Arnold (Caltech) and Dr. Zhen Liu (National Institute of Biological Sciences). 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).
This collaborative work demonstrates that it is possible to geometrically control reactive carbene intermediates formed in enzyme active sites to modulate the selectivity of carbene transfer reactions. Beyond this example, there have been many more biocatalytic transformations, natural or non-natural, recruiting similar hydrogen bonds in enzyme active sites to drive stereoselectivity, but very few have demonstrated protein engineering to introduce a different hydrogen bond-anchoring point to reaction intermediates could alter the stereo- or site-selectivity. The team hope that this study will inspire more mechanism-driven protein engineering efforts, aiming to control key biocatalytic intermediates formed in enzyme active sites to enhance activity and control selectivity.
It has recently been published in the Angewandte Chemie International Edition:
C. Calvó-Tusell, Z. Liu*, K. Chen, F. H. Arnold, M. Garcia-Borràs*
“Reversing the Enantioselectivity of Enzymatic Carbene N–H Insertion Through Mechanism-Guided Protein Engineering”
Angew. Chem. Int. Ed., 2023, ASAP
DOI: 10.1002/anie.202303879