Insights into the catalytic mechanism of a bacterial hydrolytic dehalogenase that degrades the fungicide chlorothalonil. J Biol Chem 2019 Sep 06;294(36):13411-13420
Date
07/25/2019Pubmed ID
31331935Pubmed Central ID
PMC6737215DOI
10.1074/jbc.RA119.009094Scopus ID
2-s2.0-85071831587 (requires institutional sign-in at Scopus site) 8 CitationsAbstract
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.
Author List
Yang X, Bennett B, Holz RCAuthor
Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette UniversityMESH terms used to index this publication - Major topics in bold
BiocatalysisFungicides, Industrial
Hydrolases
Hydrolysis
Molecular Structure
Nitriles
Pseudomonas