Insights into the catalytic mechanism of a bacterial hydrolytic dehalogenase that degrades the fungicide chlorothalonil
Insights into the catalytic mechanism of a bacterial hydrolytic dehalogenase that degrades the fungicide chlorothalonil
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DOI：
10.1074/jbc.ra119.009094
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发表时间：
2019-07
期刊：
影响因子：
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通讯作者：
Xinhang Yang;B. Bennett;R. Holz
中科院分区：
文献类型：
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作者：
Xinhang Yang;B. Bennett;R. Holz
百菌清（2，4，5，6-四氯间苯二甲腈; TPN）是美国最常用的杀真菌剂之一。由于TPN的广泛应用、一般毒性和潜在致癌性，其生物降解性受到了广泛关注。在这里，我们开发了一种直接的分光光度法测定锌（II）依赖，百菌清水解脱卤酶假单胞菌CTN-3（Chd），使其金属结合性能的测定; pH依赖的动力学参数KCAT，Km，和KCAT/Km;和溶剂同位素效应。我们发现，一个单一的Zn（II）离子结合的Chd单体的Kd为0.17 μm，与电感耦合等离子体质谱数据的分离的Chd二聚体一致。我们观察到Chd对百菌清的最大活性在pH范围7.0-9.0，这些数据的拟合得到pKES 1为5.4 ± 0.2，pKES 2为9.9 ± 0.1（k′cat = 24 ± 2 s−1），pKE 1为5.4 ± 0.3，pKE 2为9.5 ± 0.1（k′cat/k′m = 220 ± 10 s−1 mm−1）。质子库存研究表明，一个质子转移的反应在pD 7.0的限速步骤。紫外可见停流数据的拟合表明了一个三步模型，并提供了中间体形成（即k '2为35.2 ± 0.1 s-1）和产物释放（即k' 3为1.1 ± 0.2 s-1）的表观速率常数，表明产物释放是催化中的缓慢步骤。在这些结果的基础上，沿着与以前报道的那些，我们提出了一个机制的Chd催化。
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.