Identification of the metal center of chlorothalonil hydrolytic dehalogenase and enhancement of catalytic efficiency by directed evolution

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Honghong Chen, Hui Wang, Tongtong Wang, Songren Huang, Xiaoxia Zang, Shunpeng Li, Jiandong Jiang


Chlorothalonil hydrolytic dehalogenase (Chd) is one of two reported hydrolytic dehalogenases for halogenated aromatics, and its catalysis is independent of coenzyme A and ATP. Earlier studies have established that the catalytic activity of Chd requires zinc ions. In this study, the metal center of Chd was systematically investigated. The metal content of Chd was determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES), and there were 2.14 equivalents of zinc/mol of protein, indicating that Chd contains a binuclear (Zn2+-Zn2+) center. It was found that other divalent cations, such as cobalt (Co2+) and cadmium (Cd2+), could substitute zinc (Zn2+) leading to relative activities of 91.6% and 120.0%, whereas manganese (Mn2+) and calcium (Ca2+) could substitute Zn2+ leading to relative activities of 29.1% and 57.0%, respectively. The enzymatic properties of these different metal ion-substituted Chd variants were also compared. Error-prone PCR and DNA shuffling methods were applied to directly evolve Chd to generate variants with higher catalytic efficiencies of chlorothalonil. Enhanced Chd variants were selected based on the formation of clear haloes on Luria-Bertani plates supplemented with chlorothalonil. One variant, Q146R/N168Y/S303G, exhibited a 4.43-fold increase in catalytic efficiency, showing the potential for application in the dehalogenation and detoxification of chlorothalonil contaminated-sites.


chlorothalonil hydrolytic dehalogenase;metallo-β-lactamase; binuclear center; metal ion substitution; directed evolution

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