Abstract
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.
Highlights
Chlorothalonil (2,4,5,6-tetrachloroisophtalonitrile; TPN) is one of the most commonly used fungicides in the United States
CTN-3 that encodes for Chd was synthesized with optimized Escherichia coli codon usage and includes a TEV protease cleavage site followed by a
CTN-3 atomic emission spectroscopy (ICP-AES) data obtained on purified Chd, which revealed that ϳ0.9 eq of zinc bind tightly to Chd per monomer
Summary
Chlorothalonil (2,4,5,6-tetrachloroisophtalonitrile; TPN) is one of the most commonly used fungicides in the United States. Chd contains a conserved Zn(II)-binding domain similar to enzymes in the metallo--lactamase superfamily and was proposed to be monomeric in solution [7] At least two His residues (His-128 and His-157) along with three Asp (Asp-45, Asp-130, and Asp-184), a Ser (Ser-126), and a Trp (Trp-241) were reported to be catalytically essential based on site-directed mutagenesis studies. We report a new continuous spectrophotometric assay for Chd that has allowed the detection of a Chd reaction intermediate using UV-visible stopped-flow spectroscopy with TPN as the substrate. From these stopped-flow data, along with metal-binding and kinetic studies including pH and solvent isotope effect studies, we propose the first catalytic mechanism for Chd
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have