Structural Superimposition

Abstact

Enzymes that catalyze non-natural C-C bond-forming reactions are powerful tools in asymmetric synthesis, yet reprogramming their active sites to invert stereochemical outcome remains challenging. Building on our recently engineered S(N)Arase, S(N)Ar1.3, which performs enantioselective nucleophilic aromatic substitutions with carbon nucleophiles, we now report the evolution of an enantiocomplementary biocatalyst (eS(N)Ar1.3) that displays enhanced activity and expanded substrate scope. Structural and computational analyses uncover both conserved and divergent features between S(N)Ar1.3 and eS(N)Ar1.3. Despite retaining similar electrophile binding poses and a conserved catalytic arginine, the halide-binding pocket of S(N)Ar1.3 has been abandoned in eS(N)Ar1.3. Instead, His23 has emerged as a key motif that works with Arg124 to accurately position the nucleophilic substrate. Calculations reveal that Arg124 also plays a crucial role in facilitating halide release during catalysis. Our study demonstrates how evolution can reshape enzyme mechanisms in unforeseen ways, highlighting the importance of exploring diverse trajectories to access new functions.