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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-beta-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.
...plasmid containing the chd gene was amplified by using Primestart DNA polymerase and phosphorylated primers (synthesized in GenScript) that introduced the desired changes. The amplified linear PCR product containing the desired mutation was circularized with...
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