A Novel Hydrolytic Dehalogenase for the Chlorinated Aromatic Compound Chlorothalonil
A Novel Hydrolytic Dehalogenase for the Chlorinated Aromatic Compound Chlorothalonil
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一种新型氯化芳香族化合物百菌清水解脱卤酶
DOI：
10.1128/jb.01547-09
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发表时间：
2010-04
影响因子：
3.2
通讯作者：
李荣
中科院分区：
文献类型：
--
作者：
蒋建东;王光利;李顺鹏;李荣
脱卤酶在卤代芳烃的解毒过程中起着关键作用。有趣的是，只有一个水解脱卤酶卤代芳烃，4-氯苯甲酰辅酶A（CoA）脱卤酶，已被报道。在这里，我们表征了另一种新的水解脱卤酶的卤代芳香族化合物的2，4，5，6-四氯氰（百菌清）降解菌株假单胞菌属CTN-3，我们已命名为Chd。Chd催化百菌清的4-氯原子上的羟基取代。通过反相高效液相色谱（HPLC）、串联质谱（MS/MS）和核磁共振（NMR）鉴定了Chd脱卤的代谢产物4-羟基-三氯乙腈。Chd在厌氧和有氧条件下使百菌清脱卤，并且不需要辅酶A和ATP等辅助因子的存在。Chd含有金属-β-内酰胺酶超家族的一个推定的保守结构域，与几种金属水解酶具有最高的同源性（24 - 29%）。Chd是单体（36 kDa），其等电点（pI）估计为4.13。Chd对百菌清的解离常数（Km）为0.112 mM，总催化速率（kcat）为207 s−1。Chd被1，10-菲咯啉、焦碳酸二乙酯和N-溴代琥珀酸完全抑制。Chd的定点突变表明，组氨酸128和157，丝氨酸126，色氨酸45，130和184，色氨酸241是脱卤酶活性所必需的。基于氨基酸序列和催化机制的分析，Chd不同于其他已报道的水解脱卤酶。该研究为卤代芳香族化合物水解脱卤反应机理的研究提供了一个很好的脱卤酶候选者。
ABSTRACT 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 (Km ) 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.
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影响因子：
3.5
作者：
Christiansen, N;Ahring, BK;Diekert, G
通讯作者：
Diekert, G
影响因子：
4.4
作者：
T. Tsoi;E. G. Plotnikova;J. Cole;W. Guerin;M. Bagdasarian;J. Tiedje
通讯作者：
T. Tsoi;E. G. Plotnikova;J. Cole;W. Guerin;M. Bagdasarian;J. Tiedje
影响因子：
4.4
作者：
Thibodeau, J;Gauthier, A;Beaudet, R
通讯作者：
Beaudet, R
影响因子：
3.2
作者：
Shuisong Ni;James K. Fredrickson;L. Xun
通讯作者：
Shuisong Ni;James K. Fredrickson;L. Xun
影响因子：
15
作者：
Ya-Jun Zheng;R. Ornstein
通讯作者：
Ya-Jun Zheng;R. Ornstein