Substituting a conserved histidine alleviates difluoroacetate-dependent isoaspartate formation in a fluoroacetate dehalogenase

Organofluorides are ubiquitous in modern society, but their persistence poses significant environmental and health risks. Fluoroacetate dehalogenases (FAcDs) are promising candidates for the bioremediation of polyfluorinated substances, as they can hydrolyze C–F bonds efficiently under mild conditions. However, their activity toward gem-difluorinated carboxylic acids, such as 2,2-difluoroacetate (F ₂ A), is typically poor, with rates orders of magnitude lower than for monofluorinated substrates (e.g. fluoroacetate). Here, we combine kinetic studies, mass spectrometric and biochemical analyses, and computational modeling to pinpoint the F ₂ A-induced isomerization of the catalytic aspartate (Asp105) as a major inactivation pathway that severely limits sustained F ₂ A turnover. To overcome this inhibition mechanism, we discovered that a highly conserved histidine (His104) acts as a gatekeeper for sustained F ₂ A activity. Substituting His104 with the noncanonical N ³ -methylhistidine increased activity 14-fold, while replacement with asparagine boosted activity almost 50-fold, yielding a potent F ₂ A defluorinase with an initial degradation rate of 0.3 s ⁻¹ . Computational studies elucidated that these substitutions reshape the conformational landscape of Asp105, suppressing isoAsp-promoting conformations while preserving catalytically productive poses for F ₂ A conversion. Notably, transplanting the His-to-Asn substitution into two homologous FAcDs drastically reduced their activity, highlighting H1 as a uniquely suitable scaffold for engineering promiscuous F ₂ A conversion. Overall, this study demonstrates that targeting mechanistic intricacies can significantly enhance FAcD performance on F ₂ A, providing a foundation for further engineering of H1 to degrade polyfluorinated pollutants.