Combined experimental and molecular dynamics approach towards a rational design of the YfeX biocatalyst for enhanced carbene transferase reactivity†

Pharmaceutical synthesis has been a driving force behind advancements in the field of biocatalysis. In this paper, we report the further optimization of the heme protein YfeX for regio- and stereoselective carbene transfer reactions using mutagenesis to explore the role of important amino acids in the active site for catalysis. In this way, we identified YfeX variants that are efficient and selective carbene transferases towards primary and secondary amines, olefins, and indoles. Molecular dynamic simulations reveal that mutations within the second coordination sphere induce distinct alterations in the conformation and electrostatic properties within the active site. These changes, in turn, affect substrate positioning both within the active site and at its entrance, which explains the distinct and sometimes surprising variations in selectivity observed experimentally between select YfeX variants. Our results show that the I230A single variant identified here is one of the most active N–H insertion catalysts known, producing >90% yields in only 1 hour (typical reaction times in the literature are 8–24 hours). On the other hand, the R232A variant catalyzes the C–H insertion of unprotected indole in up to 21% yield. The capacity to selectively act on unprotected indole offers a cost-effective, environmentally-friendly approach for late-stage functionalization of indoles and similar precursors in pharmaceuticals. In addition, YfeX is an efficient and fast biocatalyst that shows no structural degradation or heme loss during turnover, underscoring YfeX's robustness as a viable biocatalyst for both industrial and academic applications.