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

• Published in: The Journal of biological chemistry Vol. 294; no. 36; pp. 13411 - 13420
• Main Authors: Yang, Xinhang, Bennett, Brian, Holz, Richard C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 06-09-2019; American Society for Biochemistry and Molecular Biology
• Subjects: Biocatalysis; bioremediation; Chd; chlorothalonil; dehalogenase; enzyme catalysis; enzyme kinetics; enzyme mechanism; Enzymology; Fungicides, Industrial - chemistry; Fungicides, Industrial - metabolism; hydrolase; Hydrolases - metabolism; Hydrolysis; Molecular Structure; Nitriles - chemistry; Nitriles - metabolism; Pseudomonas - enzymology; stopped-flow; zinc; stopped-flow; chlorothalonil; bioremediation; hydrolase; enzyme catalysis; dehalogenase; enzyme mechanism; zinc; enzyme kinetics; Chd
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA119.009094

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 Pseudomonas sp. CTN-3 (Chd), enabling determination of its metal-binding properties; pH dependence of the kinetic parameters kcat, Km, and kcat/Km; and solvent isotope effects. We found that a single Zn(II) ion binds a Chd monomer with a Kd 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 pKES1 of 5.4 ± 0.2, a pKES2 of 9.9 ± 0.1 (k′cat = 24 ± 2 s−1), a pKE1 of 5.4 ± 0.3, and a pKE2 of 9.5 ± 0.1 (k′cat/k′m = 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.e. a k′2 of 35.2 ± 0.1 s−1) and product release (i.e. a k′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.