Structure‐Guided Surface Engineering of an Artificial Metallolyase for Highly Enantioselective Michael Addition

A nonheme copper protein based on a TM1459 cupin fold was engineered to catalyze the stereoselective Michael addition of 2‐nitropropane to 2‐azachalcone. Metal‐center screening combined with secondary coordination‐sphere engineering enabled the initial control of enantioselectivity. X‐ray crystallographic analysis further identified nonspecific metal‐binding sites on the protein surface, and targeted surface‐residue mutations effectively suppressed undesired background reactions. This structure‐guided approach significantly enhanced catalytic performance, affording up to 98% enantiomeric excess ( ee ) ( S ) and 99% ee ( R ). These results demonstrate that the visualization‐driven selection of mutation sites provides a powerful and generalizable strategy for optimizing artificial metalloenzymes.