Role of mutations in a chemoenzymatic enantiodivergent C(sp(3))–H insertion: exploring the mechanism and origin of stereoselectivity

New-to-nature enzymes have emerged as powerful catalysts in recent years for streamlining various stereoselective organic transformations. While synthetic strategies employing engineered enzymes have witnessed proliferating success, there is limited clarity on the mechanistic front and more so when considering molecular-level insights into the role of selected mutations, dramatically escalating catalytic competency and selectivity. We have investigated the mechanism and correlation between mutations and exquisite stereoselectivity of a lactone carbene insertion into the C(sp(3))–H bond of substituted aniline, catalyzed by two mutants of a cytochrome P450 variant, “P411” (engineered through directed evolution) in which the axial cysteine has been mutated to serine, utilizing various computational tools. The pivotal role of S264 and L/R328 mutations in the active site has been delineated computationally using two cluster models, thus rationalizing the enantiodivergence. This report provides much-needed insights into the origin of enantiodivergence, furnishing a mechanistic framework for understanding the anchoring effects of H-bond donor residues with the lactone ring. This study is expected to have important implications in the rational design of stereodivergent enzymes and toward successful in silico enzyme designing.