Burkholderia xenovorans LB400联苯双加氧酶及突变体对DDT的降解机制研究
孙成成
学位类型硕士
导师胡晓珂
2021-05
培养单位中国科学院烟台海岸带研究所
学位授予单位中国科学院大学
学位授予地点中国科学院烟台海岸带研究所
学位名称工程硕士
学位专业生物工程
关键词DDT联苯双加氧酶Burkholderia xenovorans LB400立体特异性酶催化DDTPolychlorinated biphenylsBurkholderia xenovorans LB400StereospecificityEnzyme catalysis
摘要DDT（Dichlorodiphenyltrichloroethane，1，1，1-三氯-二氯二苯基乙烷）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DDT，但目前对环境中DDT的检测发现它仍然广泛存在且具有新的输入源。DDT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。DDT及其脱氯产物在自然条件下极难分解原因之一在于缺乏有效的酶降解系统。目前很少发现可以将DDT完全矿化的降解酶系。由于DDT与多氯联苯等芳香化合物结构类似，随着可以降解DDT的多氯联苯降解菌被发现，联苯双加氧酶（Biphenyl dioxygenase，BPDO）也成为了DDT降解酶系的重要补充。但是，目前有关联苯双加氧酶降解DDT的研究较少。因此，本研究选取了多氯联苯（Polychlorinated biphenyls，PCBs）降解模式菌株Burkholderia xenovorans LB400的联苯双加氧酶作为研究对象，试图探讨联苯双加氧酶对DDT的降解特性及机制。之前的许多研究表明，底物范围和区域特异性主要由联苯双加氧酶末端氧化酶α亚基C末端的残基决定，总共分为4个区域。有****对Ⅲ、Ⅳ区域氨基酸的功能进行了研究，但目前对Ⅰ、Ⅱ区域氨基酸残基功能研究较少。本研究以BphAELB400（Biphenyl dioxygenase terminal oxidase of Burkholderia xenovorans LB400，Burkholderia xenovorans LB400联苯双加氧酶末端氧化酶）为亲本，通过晶体结构解析确定位于酶催化口袋入口处Ⅱ区域的283位氨基酸为影响BphAELB400与DDT结合的关键氨基酸残基，将BphAELB400 283位的Ser突变为Met，获得了酶突变体BphAES283M。通过比较BphAELB400和BphAES283M分别以p,p’-DDT和o,p’-DDT为底物时的代谢产物、酶动力学参数、结构分析以及分子对接分析，试图探讨联苯双加氧酶对DDT的催化机理，并以此提高BphAELB400的底物范围和对DDT的催化代谢能力。同时为如何扩大其他BPDO的底物范围和提高降解率的机制研究奠定理论基础。主要研究内容和结论如下：（1）通过对联苯双加氧酶的序列和晶体分析计算发现，280-287片段B值很高，说明该片段具有较高的柔性即在底物结合过程中可以发生构象的改变，同时283位氨基酸残基的突变可以改变283位氨基酸本身以及催化口袋内其他氨基酸与DDT间的作用力。因此，结合其他联苯双加氧酶的比对，对该位点进行突变，构建了pET14b[LB400-S283M-bphAE]，并转化至Escherichia coli C41中，诱导表达，成功获得了目标蛋白BphAES283M。（2）生理生化数据表明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（BphAELB400未观察到相应代谢产物）。稳态动力学数据表明，经Ser283Met替换的突变体Km值约为4.6 μmol/L，kcat值为0.1/s，整体kcat/Km为24.3 L/（μmol?s），说明突变体与DDT间的结合能力增强，并实现了催化效率的突破。同时，也对实验室保有的其他Ⅲ、Ⅳ区域氨基酸突变的突变体进行了DDT的降解特征实验，未观察到代谢产物和稳态动力学数据。这意味着处于柔性片段上的Ser283Met突变是催化腔匹配DDT分子构象的关键，对于联苯双加氧酶催化其他空间构象较大化合物2，3位的双氧化具有重要意义。（3）结构分析发现Ser283Met可以扩大催化腔体积，并使催化腔内DDT的空间朝向发生改变。BphAE催化中心包括Met231、Tyr232、Gln226、Phe227、Gln322、His323等氨基酸残基，同时蛋白表面有多处负电势位点。BphAES283M的催化活性空腔体积增大，这很可能有助于BphAES283M与DDT的有效结合。BphAELB400和BphAES283M都无法降解p,p’-DDT的原因可能与其对称结构和对位氯原子的空间构象有关，在分子对接实验中p,p’-DDT反应环始终无法对接到有效结合的位置。苯环上的氯原子影响了2，3位的羟基化反应，这使得BphAELB400仍然无法降解o,p’-DDT；Ser283Met突变使得反应环的两个邻近碳原子与单核铁原子催化中心处于反应距离当中，从而实现2，3位的氧化。最后，残基267和残基247-260组成的片段对于确定对DDT的立体特异性至关重要，但这些残基的作用机制有待进一步探索。对于与BphAELB400结构特征相似的DDT降解酶以及联苯双加氧酶，结合晶体结构解析将其Ⅱ区域关键氨基酸残基如283位点进行定点突变或将是进一步增强其对DDT催化活性的有效策略。
其他摘要Dicholodiphenyltrichloroethanes (DDT) 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 have new input sources although their usage in agriculture has been banned in China and other countries. Numerous concerns have arisen over the past decades about the adverse environmental impacts (including harm to offshore ecosystem and human health) of DDT. DDT and its reductive dechlorination products are degraded difficultly under natural conditions, one of the reasons is the lack of effective enzyme degradation system. At present, there is no degradation enzyme system that can completely mineralize DDT. Because of the similar aromatic compounds between DDT and PCBs, biphenyl dioxygenase (BPDO) has become an important supplement to DDT degrading enzymes with the discovery of PCBs degrading bacteria. A comparison of α subunit protein sequences of LB400 and other strains identified four regions (designated I, II, III, and IV) in which specific sequences were consistently associated with either a broad or narrow substrate specificity. Some scholars have studied the function of amino acids in regions III and IV, but there are few studies on the function of amino acid residues in regions I and II. In order to explore the degradation characteristics and mechanism of biphenyl dioxygenase on DDT, we selected Burkholderia xenovorans LB400 biphenyl dioxygenase as parent, and 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 DDT, simulating the structure of mutant protein and molecular docking. The purpose of this study is to explore the catalytic mechanism of biphenyl dioxygenase on DDT, and to improve the substrate range of BphAELB400 and its catalytic capacity for DDT. At the same time, it lays a theoretical foundation for how to expand the range of other BPDO substrates and improve the degradation rate. The main contents and results are summarized as follow:(1) Through the sequence analysis and crystal calculation of biphenyl dioxygenase, it was found that the B-factor of 280-287 fragment was higher, which indicated that the fragment had higher flexibility. Meanwhile, the conformation could be changed in the process of substrate binding, and the mutation of 283 amino acid residue could change the force between 283 amino acid itself and other amino acids in the catalytic pocket. Therefore, pET14b[LB400-S283M-bphAE] was constructed and transformed into Escherichia coli C41. The target protein BphAES283M was successfully obtained. (2) Physiology and biochemical data showed that neither BphAELB400 nor the mutant BphAES283M could degrade p,p’-DDT, but the mutant BphAES283M could metabolize o,p’-DDT and produced two stereoisomers. Steady-state kinetics data showed that the Km value and kcat value of the mutants were about 4.6 mol/L and 0.1/s, and the overall kcat/ Km value was 24.3 L/(μmol?s), indicating that the binding ability between the mutants and DDT was enhanced. This implies that the Ser283Met mutation, which is on the flexible fragment, is the key to catalytic lumen-matching substrate conformation and is important for biphenyl dioxygenase to catalyze the double oxidation of other compounds with larger spatial conformation at the 2,3 position.(3) Structural analysis revealed that the Ser283Met mutation expanded the catalytic cavity volume and altered the spatial orientation of DDT. The catalytic center of BphAE includes amino acid residues Met231, Tyr232, Gln226, Phe227, Gln322 and His323, along with multiple negative potential sites on the protein surface. The volume of the catalytic activity of BphAES283M increased, which may be conducive to the effective binding of BphAES283M to DDT. The p,p’-DDT could notbe degraded by BphAELB400 or BphAES283M, which may be related to its symmetrical structure and spatial conformation to chlorine atoms. The chlorine atoms in the benzene ring affect the hydroxylation reaction at positions 2 and 3, which makes BphAELB400 still unable to degrade o,p’-DDT. The mutation of Ser283Met makes the two adjacent carbon atoms in the reaction ring and the catalytic center of mononuclear iron atom in the middle of the reaction distance, thus resulting in the oxidation of 2,3 position. Finally, the residue 267 and the fragment 247-260 are very important for determining the stereospecificity of DDT, but the mechanism of action of these residues needs to be further explored. For DDT degrading enzyme and others, site directed mutagenesis of key amino acid residues such as 283 site in region II combined with crystal structure analysis may be an effective strategy to further enhance its DDT catalytic activity.
语种中文
文献类型学位论文
条目标识符http://ir.yic.ac.cnhttp://ir.yic.ac.cn/handle/133337/29344
专题中科院烟台海岸带研究所知识产出

推荐引用方式
GB/T 7714孙成成. Burkholderia xenovorans LB400联苯双加氧酶及突变体对DDT的降解机制研究[D]. 中国科学院烟台海岸带研究所. 中国科学院大学,2021.


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