Structural basis for the hydrolytic dehalogenation of the fungicide chlorothalonil

Cleavage of aromatic carbon?chlorine bonds is critical for the degradation of toxic industrial compounds. Here, we solved the X-ray crystal structure of chlorothalonil dehalogenase (Chd) fromPseudomonassp. CTN-3, with 15 of its N-terminal residues truncated (Chd(T)), using single-wavelength anomalous dispersion refined to 1.96 ? resolution. Chd has low sequence identity (<15%) compared with all other proteins whose structures are currently available, and to the best of our knowledge, we present the first structure of a Zn(II)-dependent aromatic dehalogenase that does not require a coenzyme. Chd(T)forms a ?head-to-tail? homodimer, formed between two ?-helices from each monomer, with three Zn(II)-binding sites, two of which occupy the active sites, whereas the third anchors a structural site at the homodimer interface. The catalytic Zn(II) ions are solvent-accessible via a large hydrophobic (8.5 ? 17.8 ?) opening to bulk solvent and two hydrophilic branched channels. Each active-site Zn(II) ion resides in a distorted trigonal bipyramid geometry with His(117), His(257), Asp(116), Asn(216), and a water/hydroxide as ligands. A conserved His residue, His(114), is hydrogen-bonded to the Zn(II)-bound water/hydroxide and likely functions as the general acid-base. We examined substrate binding by docking chlorothalonil (2,4,5,6-tetrachloroisophtalonitrile, TPN) into the hydrophobic channel and observed that the most energetically favorable pose includes a TPN orientation that coordinates to the active-site Zn(II) ions via a CN and that maximizes a ??? interaction with Trp(227). On the basis of these results, along with previously reported kinetics data, we propose a refined catalytic mechanism for Chd-mediated TPN dehalogenation.