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Burkholderia xenovorans LB400联苯双加氧酶及突变体对DDTs的降解
引用本文:孙成成,王媛,李俊德,陈卫卫,闵军,胡晓珂. Burkholderia xenovorans LB400联苯双加氧酶及突变体对DDTs的降解[J]. 微生物学报, 2021, 61(11): 3471-3482
作者姓名:孙成成  王媛  李俊德  陈卫卫  闵军  胡晓珂
作者单位:中国科学院烟台海岸带研究所, 海岸带生物学与生物资源利用重点实验室, 山东 烟台 264003;青岛海洋科学与技术试点国家实验室, 海洋生物学与生物技术功能实验室, 山东 青岛 266237;中国科学院大学, 北京 100049;中国科学院烟台海岸带研究所, 海岸带生物学与生物资源利用重点实验室, 山东 烟台 264003;烟台大学海洋学院, 山东 烟台 264003
基金项目:国家自然科学基金(32070112,32070102,92051119,42077305);国家重点研发计划(2020YFD0901003);河北省重点研发计划(19273802D)
摘    要:DDTs(dichlorodiphenyltrichloroethane,1,1,1-三氯-2,2-双氯苯基乙烷)是一种典型的持久性有机污染物,曾在疟疾防治和农业除虫方面被广泛应用。虽然包括我国在内的很多国家已经禁止使用DDTs,但目前对环境中DDTs的检测发现它仍然广泛存在且具有新的输入源。DDTs的持续存在对近海生态系统和人类健康具有一定危害,因此它所造成的环境污染问题仍然值得关注。由于Rieske型芳香羟化双加氧酶能够起始多种持久性污染物的降解,过去的几十年里一直是芳香化合物降解领域的焦点。[目的] 为探讨联苯双加氧酶对DDTs的降解特性及机制,本研究选取了食异生素伯克霍尔德氏菌LB400(Burkholderia xenovorans)联苯双加氧酶及突变体对p,p''-DDT和o,p''-DDT的降解过程进行研究。[方法] 以BphAELB400为亲本,通过两步定点突变将283位的丝氨酸突变为蛋氨酸,获得突变体BphAES283M。通过比较亲本酶与突变体对DDTs的催化性能,模拟突变蛋白结构和分子对接等方法,探究其降解特性及机制。[结果] BphAELB400和突变体BphAES283M都无法降解对位的p,p''-DDT,但突变体BphAES283M可以代谢o,p''-DDT并产生2个立体异构体。对接p,p''-DDT的BphAELB400和BphAES283M的结构分析表明,BphAELB400和BphAES283Mp,p''-DDT的反应环均不与原晶体结构中的联苯反应环重合。而对接o,p''-DDT的BphAES283M的结构分析表明o,p''-DDT的反应环与晶体结构中的联苯反应环距离很近,且2、3位的碳原子与单核铁原子催化中心的距离在0.5 nm以内,此外,BphAES283M的催化腔表面积和体积比BphAELB400更大,这很可能有助于BphAES283Mo,p''-DDT的结合。[结论] 283位氨基酸是影响BphAELB400对DDTs的催化代谢能力的关键氨基酸残基,它可以通过调节反应碳原子与催化中心的距离以及催化腔的大小来影响底物特异性。本次研究进一步阐明了283位氨基酸残基的影响机理,为更有效修复DDTs污染提供理论依据和技术支持。

关 键 词:DDTs  联苯双加氧酶  Burkholderia xenovorans LB400  底物特异性
收稿时间:2021-01-23
修稿时间:2021-03-18

The degradation of DDTs by Burkholderia xenovorans LB400 biphenyl dioxygenase and mutants
Chengcheng Sun,Yuan Wang,Junde Li,Weiwei Chen,Jun Min,Xiaoke Hu. The degradation of DDTs by Burkholderia xenovorans LB400 biphenyl dioxygenase and mutants[J]. Acta microbiologica Sinica, 2021, 61(11): 3471-3482
Authors:Chengcheng Sun  Yuan Wang  Junde Li  Weiwei Chen  Jun Min  Xiaoke Hu
Affiliation:Key Laboratory of Coastal Biology and Bio-resource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, Shandong Province, China;Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, Shandong Province, China;University of Chinese Academy of Sciences, Beijing 100049, China;Key Laboratory of Coastal Biology and Bio-resource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, Shandong Province, China;Ocean School, Yantai University, Yantai 264003, Shandong Province, China
Abstract:Dicholodiphenyltrichloroethanes (DDTs) is probably the best known and typical persistent organic pollutant in the world, which has been widely used in malaria control and agricultural deworming. They are still detected in various environmental matrices and has new input sources although their usage in agriculture has been banned in China and others. Numerous concerns have arisen over the past decades about the adverse environmental impacts(including harm to offshore ecosystem and human health) of DDTs. There has been a considerable interest over the last decades for the Rieske-type arylhydroxylating dioxygenases (RHDs) as they are seen as potentially capable of initiating their degradation. [Objective] In order to explore the degradation characteristics and mechanism of biphenyl dioxygenase(BPDO) on DDTs, we selected Burkholderia xenovorans LB400 biphenyl dioxygenase and its mutants to explore the degradation process of p,p''-DDT and o,p''-DDT. [Methods] Using BphAELB400 as parent, the mutant BphAES283M was obtained by two-step site-directed mutagenesis from Ser to Met. The degradation characteristics and mechanism of wild type and mutant were explored by comparing the catalytic performance of wild type and mutant to DDTs, simulating the structure of mutant protein and molecular docking. [Results] The data showed that BphAELB400 and BphAES283Mcould not be degraded p,p''-DDT, but BphAES283M metabolized o,p''-DDT and produced two stereoisomers. The structural analysis of BphAELB400 and BphAES283M showed that the reaction ring of p,p''-DDT did not coincide with the biphenyl reaction ring in the original crystal structure. In the o,p''-DDT-BphAES283M conformation, the proximal ring did not fit the biphenyl reactive ring as well, but its orientation toward the catalytic Fe2+ places two vicinal atoms at a distance(within 0.5 nm) that would allow a catalytic reaction. In addition, the surface area and volume of the catalytic cavity of BphAES283M is larger than that of BphAELB400, which is likely to contribute to the combination of BphAES283M and o,p''-DDT. [Conclusion] 283 is the key amino acid residue that affects the catalytic metabolism of BPDO to DDTs. It can affect the substrate specificity by adjusting the distance between the reaction carbon atom and the catalytic center and the size of the catalytic cavity. This will provide better insights about the bases for BPDO broad substrate range and about the mechanisms by which the enzyme evolves to change or expand its substrate range and its stereo- and regiospecificity.
Keywords:DDTs  BPDO  Burkholderia xenovorans LB400  substrate specificity
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