A Novel Hydrolytic Dehalogenase for the Chlorinated Aromatic Compound Chlorothalonil

Dehalogenases play key roles in the detoxification of halogenated aromatics. Interestingly, only one hydrolytic dehalogenase for halogenated aromatics, 4-chlorobenzoyl-coenzyme A (CoA) dehalogenase, has been reported. Here, we characterize another novel hydrolytic dehalogenase for a halogenated aromatic compound from the 2,4,5,6-tetrachloroisophthalonitrile (chlorothalonil)-degrading strain of Pseudomonas sp. CTN-3, which we have named Chd. Chd catalyzes a hydroxyl substitution at the 4-chlorine atom of chlorothalonil. The metabolite of the Chd dehalogenation, 4-hydroxy-trichloroisophthalonitrile, was identified by reverse-phase high-performance liquid chromatography (HPLC), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR). Chd dehalogenates chlorothalonil under anaerobic and aerobic conditions and does not require the presence of cofactors such as CoA and ATP. Chd contains a putative conserved domain of the metallo-β-lactamase superfamily and shows the highest identity with several metallohydrolases (24 to 29%). Chd is a monomer (36 kDa), and the isoelectric point (pI) of Chd is estimated to be 4.13. Chd has a dissociation constant (K_m) of 0.112 mM and an overall catalytic rate (k_cat) of 207 s⁻¹ for chlorothalonil. Chd is completely inhibited by 1,10-phenanthroline, diethyl pyrocarbonate, and N-bromosuccinic acid. Site-directed mutagenesis of Chd revealed that histidines 128 and 157, serine 126, aspartates 45, 130 and 184, and tryptophan 241 were essential for the dehalogenase activity. Chd differs from other reported hydrolytic dehalogenases based on the analysis of amino acid sequences and catalytic mechanisms. This study provides an excellent dehalogenase candidate for mechanistic study of hydrolytic dehalogenation of halogenated aromatic compound.Halogenated aromatic compounds are widely used in agriculture and industry as solvents, defatting agents, herbicides, and fungicides. A variety of these compounds have been identified as priority organic pollutants by the United Nations and the U.S. Environmental Protection Agency. Therefore, the remediation of these pollutants is desirable. Microorganisms play key roles in the detoxification of halogenated aromatics. The use of microbial enzymes for bioremediation has received increasing attention as an efficient and cost-effective biotechnological approach. Halogen removal from halogenated aromatics reduces both the recalcitrance to biodegradation and the risk of forming toxic intermediates during subsequent metabolic steps. As a result, the key reaction for microbial detoxification of halogenated aromatics is the actual dehalogenation (35).Investigation of the microbial degradation of different halogenated aromatics has led to the detection and elucidation of various dehalogenases that catalyze the removal of the halogen atom under aerobic and anaerobic conditions (8, 11, 17). Four dehalogenation mechanisms of halogenated aromatics are known, including reductive, thiolytic, oxidative, and hydrolytic mechanisms (41). Reductive dehalogenation plays important roles in the degradation of chlorinated aromatics under anaerobic conditions (36, 44). Several anaerobic bacteria are capable of using chlorinated benzenes (2) or polychlorinated dibenzodioxins (5) as the terminal electron acceptors in their energy metabolism. These bacteria couple reductive dehalogenation to electron transport phosphorylation (15). Several enzymes catalyzing the respiratory reductive dechlorination of halogenated aromatics have also been characterized (1, 4, 7, 20, 28, 38, 40). Under aerobic conditions, some chlorinated aromatics can also be reductively dehalogenated by thiolytic substitution in the presence of glutathione (12, 19, 25, 46, 48). In this dehalogenation system, chlorine atoms are displaced by the nucleophilic attack of the thiolate anion of glutathione. The nucleophilic attack of the thiolate anion is catalyzed by glutathione S-transferases (43). Besides the two well-characterized mechanisms for aryl halide reductive dehalogenation, two other mechanisms have been reported, including reduced NADPH-dependent reductive dechlorination of 2,4-dichlorobenzoyl-coenzyme A (CoA) to 4-chlorobenzoyl-CoA in Corynebacterium sepedonicum KZ-4 and coryneform bacterium strain NTB-1 (31), as well as a CoA-mediated reductive dehalogenation of 3-chlorobenzoate in Rhodopseudomonas palustris RCB100 using 3-chlorobenzoate as the carbon source rather than as a terminal electron acceptor (13). Oxidative dehalogenation of halogenated aromatics is catalyzed by monooxygenase (29), dioxygenases (34, 37, 39, 45), and peroxidase (30).Even though several hydrolytic dehalogenases involved in dehalogenation of halogenated aliphatic hydrocarbons and halogenated carboxylic acids have been characterized, only one kind of hydrolytic dehalogenase for halogenated aromatics has been reported. The only hydrolytic dehalogenase identified to date is 4-chlorobenzoyl-CoA dehalogenase in the 4-chlorobenzoate degradation system (32, 33). For the hydrolytic substitution of the chlorine atom of 4-chlorobenzoate with a hydroxyl group, activation by the CoA thioester formation is required. Initially, 4-chlorobenzoate-CoA ligase adenylates the carboxyl group in a reaction requiring ATP, followed by the replacement of AMP with CoA and the formation of a thioester. This intermediate is sufficiently energized to facilitate the replacement of the hydroxyl group with a 4-chlorine atom; this is catalyzed by 4-chlorobenzoyl-CoA dehalogenase. Finally, 4-hydroxybenzoyl-CoA thioesterase removes the CoA (Fig. ​(Fig.1a).1a). Three separate enzymes are involved in this system, and cofactors including CoA and ATP are needed (32, 33).Open in a separate windowFIG. 1.Dechlorination mechanism of 4-chlorobenzoate in Pseudomonas sp. CBS3 and Acinetobacter sp. strain 4-CB1 (a) and the first-step dechlorination mechanism of 2,4,5,6-tetrachloroisophthalonitrile (chlorothalonil) in Pseudomonas sp. CTN-3 (b).Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile), a broad-spectrum chlorinated aromatic fungicide, is the second most widely used agricultural fungicide in the United States, with 5 million kilograms applied annually (9). Chlorothalonil is highly toxic to fish, birds, and aquatic invertebrates (6) and is commonly detected in ecosystems (18). The bacterial strain Pseudomonas sp. CTN-3, capable of efficiently transforming chlorothalonil, was isolated in our laboratory from long-term chlorothalonil-contaminated soil in the Jiangsu Province in China. In Ochrobactrum anthropi SH35B, a glutathione-dependent glutathione S-transferase was reported to be able to catalyze the nucleophilic substitution of chlorine atoms of chlorothalonil (19). The glutathione S-transferase in our CTN-3 bacterial strain showed 84% identity with that of O. anthropi SH35B. However, the glutathione S-transferase from CTN-3 was not functionally expressed. Here, we report for the first time the characterization of a novel chlorothalonil hydrolytic dehalogenase (Chd) that contains a conserved domain of the metallo-β-lactamase superfamily. Chd is the second hydrolytic dehalogenase for chlorinated aromatic compounds to be identified. The hydrolytic dehalogenation of chlorothalonil catalyzed by Chd is independent of CoA and ATP. Based on the analysis of amino acid sequences and catalytic mechanisms, Chd is unique from the other reported hydrolytic dehalogenases.