Directed Evolution of a Fluorinase for Improved Fluorination Efficiency with a Non-native Substrate

Fluorinases offer an environmentally friendly alternative for selective fluorination under mild conditions. However, their diversity is limited in nature and they have yet to be engineered through directed evolution. Herein, we report the directed evolution of the fluorinase FlA1 for improved conversion of the non-native substrate 5′-chloro-5′-deoxyadenosine (5′-ClDA) into 5′-fluoro-5′-deoxyadenosine (5′-FDA). The evolved variants, fah2081 (A279Y) and fah2114 (F213Y, A279L), were successfully applied in the radiosynthesis of 5′-[¹⁸F]FDA, with overall radiochemical conversion (RCC) more than 3-fold higher than wild-type FlA1. Kinetic studies of the two-step reaction revealed that the variants show a significantly improved k_catvalue in the conversion of 5′-ClDA into S-adenosyl-l-methionine (SAM) but a reduced k_catvalue in the conversion of SAM into 5′-FDA.