Publications

• Insights into the catalytic mechanism of a bacterial hydrolytic dehalogenase that degrades the fungicide chlorothalonil.

  Yang X., Bennett B., Holz R.C.

  Chlorothalonil (2,4,5,6-tetrachloroisophtalonitrile; TPN) is one of the most commonly used fungicides in the United States. Given TPN's widespread use, general toxicity, and potential carcinogenicity, its biodegradation has garnered significant attention. Here, we developed a direct spectrophotome ... >> More

  Chlorothalonil (2,4,5,6-tetrachloroisophtalonitrile; TPN) is one of the most commonly used fungicides in the United States. Given TPN's widespread use, general toxicity, and potential carcinogenicity, its biodegradation has garnered significant attention. Here, we developed a direct spectrophotometric assay for the Zn(II)-dependent, chlorothalonil-hydrolyzing dehalogenase from <i>Pseudomonas</i> sp. <i>CTN-3</i> (Chd), enabling determination of its metal-binding properties; pH dependence of the kinetic parameters <i>k</i>_cat, <i>K_m</i> , and <i>k</i>_cat/<i>K_m</i> ; and solvent isotope effects. We found that a single Zn(II) ion binds a Chd monomer with a <i>K_d</i> of 0.17 μm, consistent with inductively coupled plasma MS data for the as-isolated Chd dimer. We observed that Chd was maximally active toward chlorothalonil in the pH range 7.0-9.0, and fits of these data yielded a p<i>K</i>_ES1 of 5.4 ± 0.2, a p<i>K</i>_ES2 of 9.9 ± 0.1 (<i>k</i>'_cat = 24 ± 2 s^-1), a p<i>K</i>_E1 of 5.4 ± 0.3, and a p<i>K</i>_E2 of 9.5 ± 0.1 (<i>k</i>'_cat/<i>k</i>' <i>_m</i> = 220 ± 10 s^-1 mm^-1). Proton inventory studies indicated that one proton is transferred in the rate-limiting step of the reaction at pD 7.0. Fits of UV-visible stopped-flow data suggested a three-step model and provided apparent rate constants for intermediate formation (<i>i.e.</i> a <i>k</i>'₂ of 35.2 ± 0.1 s^-1) and product release (<i>i.e.</i> a <i>k</i>'₃ of 1.1 ± 0.2 s^-1), indicating that product release is the slow step in catalysis. On the basis of these results, along with those previously reported, we propose a mechanism for Chd catalysis. << Less

  J Biol Chem 294:13411-13420(2019) [PubMed] [EuropePMC]

• Structural basis for the hydrolytic dehalogenation of the fungicide chlorothalonil.

  Catlin D.S., Yang X., Bennett B., Holz R.C., Liu D.

  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 <i>Pseudomonas</i> sp. CTN-3, with 15 of its N-terminal residues truncated (Chd^T), using single ... >> More

  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 <i>Pseudomonas</i> 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. << Less

  J Biol Chem 295:8668-8677(2020) [PubMed] [EuropePMC]