Unveiling the molecular basis of selective fluorination of SAM-dependent fluorinases.

S-Adenosylmethionine (SAM)-dependent fluorinases have emerged as environmentally friendly enzymatic alternatives for organofluorine chemical synthesis. However, their use remains limited by their rarity; only 16 fluorinases have been found in nature so far. Here we report two new fluorinases, FLA(Sbac) from Streptosporangiales bacterium and a modified FLA(Adig_Nter) from Actinoplanes digitatis. Through molecular dynamics (MD) simulations, we have identified the crucial roles of the SAM-binding site and an ion-egress site (IES) for fluorination reaction, particularly regarding its preference for fluoride ions. We have validated these findings by testing mutants of the two new fluorinases and the known fluorinase from Streptomyces sp. MA37 (FLA(MA37)). Through these targeted mutations, we identified, for the first time, specific sites in certain variants that significantly enhance the enzyme's specificity for fluorination over chlorination while maintaining its fluorination activity. In these particular variants, this refinement led to a remarkable increase in fluorine preference, improving from approximately 10-fold to over 200-fold. Overall, this research advances our fundamental understanding of enzymatic fluorination, providing a basis for further exploration of fluorinase optimization. In turn, these advancements could open new opportunities for the pharmaceutical industry in the development of organofluorine drugs and other fluorine-reliant biotechnologies.