Identification of Two Glutathione-dependent 3,6-Dichlorogentisate Dehalogenases and Their Roles in the Catabolism of the Herbicide Dicamba in Rhizorhabdus dicambivorans Ndbn-20
Identification of Two Glutathione-dependent 3,6-Dichlorogentisate Dehalogenases and Their Roles in the Catabolism of the Herbicide Dicamba in Rhizorhabdus dicambivorans Ndbn-20
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两种谷胱甘肽依赖性 3,6-二氯龙胆酸脱卤酶的鉴定及其在除草剂麦草畏分解代谢中的作用 Rhizorhabdus dicambivorans Ndbn-20
DOI：
10.1128/aem.00623-18
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发表时间：
2018
影响因子：
4.4
通讯作者：
Jian-dong Jiang
中科院分区：
文献类型：
--
作者：
Na Li;Ren-Lei Tong;Li Yao;Qing Chen;Xin Yan;De-Rong Ding;Ji-Guo Qiu;Jian He;Jian-dong Jiang
除草剂敌草畏在双目根霉NDBN-20中首先被脱甲基化为3，6-二氯水杨酸酯(3，6-DCSA)，然后被5-羟基化为3，6-二氯己酸酯(3，6-DCGA)。在本研究中，在菌株NDBN-20中鉴定了两个依赖谷胱甘肽的3，6-DCGA脱卤酶DsmH1和DsmH2。DsmH2与Sphingobium氯苯二酚ATCC 39723脱卤酶的同源性较低(仅为31%)，而DsmH1与四氯对苯二酚脱卤酶的同源性较高(79%)。在相关谷胱甘肽转移酶(GSTs)的系统发育树中，DsmH1和DsmH2与PCPC和2，5-二氯对苯二酚脱卤酶Lind形成一个独立的分支。在大肠杆菌BL21中合成了DsmH1和DsmH2，并纯化为His标记的酶。这两种酶都需要谷胱甘肽(GSH)作为辅因子，在体外都能将3，6-DCGA脱氯为3-氯代葡萄糖酸。DsmH2对3，6-DCGA的催化效率明显高于DsmH1。转录和中断分析表明，在NDBN-20菌株体内，3，6-DCGA的6-脱氯是由DsmH2而不是DsmH1负责的。此外，我们提出了一种新的ETA类GST来适应四种细菌脱卤酶PCPC、LIND、DsmH1和DsmH2。进口敌草畏是一种重要的除草剂，自2015年大规模种植抗转基因麦草畏作物以来，其使用量和向环境中的渗漏大幅增加。然而，麦草畏的完全分解代谢途径尚不清楚，这限制了对该除草剂的生态毒理学研究。我们先前的研究表明，3，6-DCGA是菌株NDBN-20降解麦草畏的中间产物。在这项研究中，我们鉴定了两个依赖谷胱甘肽的3，6-DCGA脱卤酶DsmH1和DsmH2，并证明DsmH2在NDBN-20菌株中对3，6-DCGA的6-脱氯起生理作用。GSTS在多种内源和外源有毒物质的解毒和降解过程中发挥着重要作用。根据它们的序列同源性、系统发育地位和功能，四种细菌依赖GSH的脱卤酶(PCPC、Lind、DsmH1和DsmH2)被重新归类为一个新的ETA类GSTs。这项研究有助于阐明麦草畏的微生物分解代谢，并加深我们对GSTs多样性和功能的理解。
The herbicide dicamba is initially demethylated to 3,6-dichlorosalicylate (3,6-DCSA) in Rhizorhabdus dicambivorans Ndbn-20 and is subsequently 5-hydroxylated to 3,6-dichlorogentisate (3,6-DCGA). In the present study, two glutathione-dependent 3,6-DCGA dehalogenases, DsmH1 and DsmH2, were identified in strain Ndbn-20. DsmH2 shared a low identity (only 31%) with the tetrachlorohydroquinone (TCHQ) dehalogenase PcpC from Sphingobium chlorophenolicum ATCC 39723, while DsmH1 shared a high identity (79%) with PcpC. In the phylogenetic tree of related glutathioneS-transferases (GSTs), DsmH1 and DsmH2, together with PcpC and the 2,5-dichlorohydroquinone dehalogenase LinD, formed a separate clade. DsmH1 and DsmH2 were synthesized in Escherichia coli BL21 and purified as His-tagged enzymes. Both enzymes required glutathione (GSH) as a cofactor and could 6-dechlorinate 3,6-DCGA to 3-chlorogentisatein vitro. DsmH2 had a significantly higher catalytic efficiency toward 3,6-DCGA than DsmH1. Transcription and disruption analysis revealed that DsmH2 but not DsmH1 was responsible for the 6-dechlorination of 3,6-DCGA in strain Ndbn-20in vivo. Furthermore, we propose a novel eta class of GSTs to accommodate the four bacterial dehalogenases PcpC, LinD, DsmH1, and DsmH2.IMPORTANCEDicamba is an important herbicide, and its use and leakage into the environment have dramatically increased since the large-scale planting of genetically modified (GM) dicamba-resistant crops in 2015. However, the complete catabolic pathway of dicamba has remained unknown, which limits ecotoxicological studies of this herbicide. Our previous study revealed that 3,6-DCGA was an intermediate of dicamba degradation in strain Ndbn-20. In this study, we identified two glutathione-dependent 3,6-DCGA dehalogenases, DsmH1 and DsmH2, and demonstrated that DsmH2 is physiologically responsible for the 6-dechlorination of 3,6-DCGA in strain Ndbn-20. GSTs play an important role in the detoxification and degradation of a variety of endogenous and exogenous toxic compounds. On the basis of their sequence identities, phylogenetic status, and functions, the four bacterial GSH-dependent dehalogenases (PcpC, LinD, DsmH1, and DsmH2) were reclassified as a new eta class of GSTs. This study helps us to elucidate the microbial catabolism of dicamba and enhances our understanding of the diversity and functions of GSTs.