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Structural basis for the hydrolytic dehalogenation of the fungicide chlorothalonil.

ABSTRACT: 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) from Pseudomonas sp. CTN-3, with 15 of its N-terminal residues truncated (Chd^T), using single-wavelength anomalous dispersion refined to 1.96 Å 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 α-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 Å) opening to bulk solvent and two hydrophilic branched channels. Each active-site Zn(II) ion resides in a distorted trigonal bipyramid geometry with His¹¹⁷, His²⁵⁷, Asp¹¹⁶, Asn²¹⁶, and a water/hydroxide as ligands. A conserved His residue, His¹¹⁴, 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 π-π interaction with Trp²²⁷ On the basis of these results, along with previously reported kinetics data, we propose a refined catalytic mechanism for Chd-mediated TPN dehalogenation.

SUBMITTER: Catlin DS

PROVIDER: S-EPMC7324493 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundContaminants have been implicated in declines of amphibians, a taxon with vital systems similar to those of humans. However, many chemicals have not been thoroughly tested on amphibians or do not directly kill them.ObjectiveOur goal in this study was to quantify amphibian responses to chlorothalonil, the most commonly used synthetic fungicide in the United States.MethodsWe reared Rana sphenocephala (southern leopard frog) and Osteopilus septentrionalis (Cuban treefrog) in outdoor mesocosms with or without 1 time (1×) and 2 times (2×) the expected environmental concentration (EEC) of chlorothalonil (~ 164 ?g/L). We also conducted two dose-response experiments on O. septentrionalis, Hyla squirella (squirrel treefrog), Hyla cinerea (green treefrog), and R. sphenocephala and evaluated the effects of chlorothalonil on the stress hormone corticosterone.ResultsFor both species in the mesocosm experiment, the 1× and 2× EEC treatments were associated with > 87% and 100% mortality, respectively. In the laboratory experiments, the approximate EEC caused 100% mortality of all species within 24 hr; 82 ?g/L killed 100% of R. sphenocephala, and 0.0164 ?g/L caused significant tadpole mortality of R. sphenocephala and H. cinerea. Three species showed a nonmonotonic dose response, with low and high concentrations causing significantly greater mortality than did intermediate concentrations or control treatments. For O. septentrionalis, corticosterone exhibited a similar nonmonotonic dose response and chlorothalonil concentration was inversely associated with liver tissue and immune cell densities (< 16.4 ?g/L).ConclusionsChlorothalonil killed nearly every amphibian at the approximate EEC; at concentrations to which humans are commonly exposed, it increased mortality and was associated with elevated corticosterone levels and changes in immune cells. Future studies should directly quantify the effects of chlorothalonil on amphibian populations and human health.

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Structural basis for the hydrolytic dehalogenation of the fungicide chlorothalonil.

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