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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 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 π–π 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.
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Degradation of Chlorothalonil by Catalytic Biomaterials
Chlorothalonil (2,4,5,6-tetrachloro-1,3-benzenedicarbonitrile, TPN, CAS: 1897-45-6) is a halogenated fungicide currently widely applied to a large variety of crops. Its carcinogenicity, embryo lethality, and high chronic oral toxicity in mammals, among other effects on a variety of organisms, has made its biodegradation of great interest. Chlorothalonil dehalogenase (Chd) from the bacterium Pseudomonas sp. CTN-3 offers a potential solution by catalyzing the first step in the degradation of chlorothalonil. Reported herein are active biomaterials of Chd when encapsulated in tetramethylorthosilicate (TMOS) gels using the sol–gel method (Chd/sol), alginate beads (Chd/alginate), and chitosan-coated alginate beads (Chd/chitosan). Both Chd/sol and Chd/chitosan increased protection from the endopeptidase trypsin as well as imparted stability over a pH range from 5 to 9. Chd/sol outperformed Chd/alginate and Chd/chitosan in long-term storage and reuse experiments, retaining similar activity to soluble Chd stored under similar conditions. All three materials showed a level of increased thermostability, with Chd/sol retaining >60% activity up to 70 °C. All materials showed activity in 40% methanol, suggesting the possibility for organic solvents to improve TPN solubility. Overall, Chd/sol offers the best potential for bioremediation of TPN using Chd.
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- Award ID(s):
- 2003861
- PAR ID:
- 10560312
- Publisher / Repository:
- MDPI
- Date Published:
- Journal Name:
- Catalysts
- Volume:
- 14
- Issue:
- 11
- ISSN:
- 2073-4344
- Page Range / eLocation ID:
- 805
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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