Mechanism-Guided Engineering of Fluorinase Unlocks Efficient Nucleophilic Biofluorination

The fluorinase enzyme, the only known biocatalyst forming stable carbon–fluorine bonds, operates with extremely low efficiency— catalyzing one reaction every 2–12 minutes. This severely limits its utility for sustainable biofluorination, and its sluggish activity remains poorly understood. We suppressed its aggregation through directed mutagenesis and elucidated the kinetic mechanism using a novel mathematical framework that fit complex kinetic and oligomerization data. This analysis revealed that >80% of enzyme molecules are inactive under standard conditions due to two dead-end pathways. A designed W50F+A279R mutant showed a 2-fold improvement in efficiency and expanded operational tolerance. Combined with mechanism-based medium optimization, the catalytic rate reached 10.1 ± 3.1 min ^-1 . Our work provides a mechanistic blueprint for fluorinase enhancement and a generalizable mathematical framework for analyzing kinetics of multimeric enzymes.