Enzymatic Defluorination of Perfluorooctanoic Acid by an Evolutionarily Distinct Haloacid Dehalogenase.

Here, we demonstrate the successful purification of haloacid dehalogenase type II (HAD-II) enzyme, validating its catalytic capacity to directly mediate the cell-free defluorination of long-chain perfluorooctanoic acid (PFOA) by systematically cleaving the resilient C-F bond. While conventional remediation strategies rely on energy-intensive chemical methods, biological alternatives are limited to sluggish whole-microbiome consortia. We discovered a novel HAD-II enzyme from Achromobacter mucicolens harvested from PFAS-contaminated lacustrine sediment. Within 24 hours, the recombinant enzyme achieved cell-free PFOA defluorination, releasing 0.55 ppm of free fluoride (17% yield). Structural and phylogenetic analyses reveal that this HAD-II belongs to a deeply divergent lineage sharing only 25% sequence identity with the previously characterized Delftia homologue while preserving the core HAD-like catalytic fold. Comparative molecular docking elucidated that PFOA adopts a productive binding orientation near the conserved catalytic Asp15 within the A. mucicolens active-site pocket. Together, our work establishes a clean mechanistic paradigm for targeted environmental biotechnology.