Structurally Characterized Haloacid Dehalogenase 4A Catalyzes Fluoroacetate Defluorination and Provides a Scaffold for Exploring Organofluorine C─F Bond Cleavage

Haloacid dehalogenases (HADs) catalyze hydrolytic carbon–halogen bond cleavage and are a tractable enzyme family for studying C─F bond cleavage. In this study, we characterized HAD 4A from Delftia acidovorans strain D4B. Recombinant 4A was expressed, purified, and crystallized, yielding a 1.59 Å apo structure (PDB 9C9E) with an accessible aspartate (Asp22)‐centered active site. Under the reported assay condition, purified 4A catalyzed hydrolytic defluorination of fluoroacetate, as supported by enzyme‐dependent fluoride release and liquid chromatography–mass spectrometry detection of glycolate. The docking model placed the fluoroacetate α ‐carbon near Asp22, providing computational structural context consistent with Asp22 acting as the putative catalytic nucleophile. Compared with related HAD‐like dehalogenases, 4A showed a more open active‐site entrance, suggesting a possible scaffold for organofluorine accommodation and structure‐guided engineering. Exploratory reactions with PFOA (0.5 mM) and high 4A loading (500 µM, 120 h) produced only weak apparent fluoride release (~6 µM). These data do not demonstrate PFOA defluorination but identify 4A as a validated fluoroacetate‐active HAD scaffold for future organofluorine biocatalysis.