Emergence of a Distinctive Nucleophile Activation Mechanism in an Enantiocomplementary SNArase

Nucleophilic aromatic substitutions (SNAr) are among the most widely used transformations in the pharmaceutical and agrochemical industries, however examples of selective catalytic processes are scarce. To address this limitation, our lab recently reported the development of an engineered enzyme for nucleophilic aromatic substitutions, SNAr1.3, that allows construction of all-carbon quaternary stereocentres with high levels of stereocontrol. Here, starting from a common an-cestor we report the evolution of an enantiocomplementary SNArase, eSNAr1.3, that displays enhanced activities compared with our previously reported enzyme and can mediate a wide range of selective C–C bond forming processes. A series of biochemical, structural and computational studies reveal some features that are common to both SNAr1.3 and eSNAr1.3, including similar electrophile binding poses and the presence of an essential catalytic arginine responsible for nucleo-phile activation. However, divergent evolution has led to key mechanistic differences; the well-defined halide binding site of SNAr1.3 has been abandoned in eSNAr1.3, and instead His23 has emerged as a key catalytic motif that works in synergy with Arg124 to activate the nucleophilic coupling partner. Interestingly, these differences manifest in SNAr1.3 and eSNAr1.3 displaying opposite preferences for the halide leaving group of the electrophilic coupling partner. This study highlights how laboratory evolution can result in unanticipated mechanistic outcomes, underscoring the importance of sampling multiple trajectories when developing enzymes with unprecedented functions.