Today, enzymes can be reprogrammed beyond biology's natural reactions. This is achieved through noncovalent interactions decorating and templating enzyme active sites to stabilize a single transition state amid competing alternatives. We engineered a flavin-dependent oxidoreductase to generate tertiary radicals within its active site and channel them into stereoselective C–N coupling with unsubstituted anilines, delivering chiral α-tertiary amines with good yields and high chemo- and enantioselectivity, under visible light irradiation—without metal cofactors.Six rounds of directed evolution install a π-stacking/hydrogen-bond network that templates lone pair–radical hyperconjugation, overriding arene addition to enforce C(sp³)–N formation. DFT and multivariate statistical modeling reveal how this microenvironment flips innate radical reactivity toward N-alkylation. This establishes biocatalytic spatial programming as a general platform for non-natural radical transformations beyond both evolution and small-molecule catalysis.
