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

Abstract

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.

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