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 fits complex kinetic and oligomerization data. This analysis revealed that >80% of enzyme molecules are inactive under standard conditions due to two dead-end pathways. The designed W50F+A279R mutant preferentially formed hexamers and displayed enhanced catalytic efficiency in this oligomeric state. When coupled with mechanism-based optimization of the reaction medium, including enzymatic removal of the inhibitory product, the catalytic turnover rate reached 12.5 ± 2.1 min⁻¹, representing ∼60-fold increase compared with previously reported turnover rates of the wild-type enzyme. Our work provides a mechanistic blueprint for fluorinase enhancement and a generalizable mathematical framework for analyzing kinetics of multimeric enzymes.