漆酶—介质体系的分子机制研究及新介质的发现

发布时间：2018-08-25 16:39

【摘要】：第一部分变色栓菌漆酶1KYA活性位点结构特点及分子对接模型的建立目的:以变色栓菌漆酶-底物复合物的晶体结构(PDB注册号:1KYA)为模板,探究1KYA活性位点结构特点与生物功能,并基于分子对接技术,建立变色栓菌漆酶-底物对接模型,揭示分子间相互作用关系,为后期有关LMS的研究及数据库筛选提供依据。方法:以1KYA为模板,利用分子模拟和分子设计软件包MOE(Molecular Operation Environment)的图形显示功能,分别从二维(2D)和三维(3D)水平分析漆酶催化氧化活性位点结构特点及其与底物的结合模式;通过分子对接得到的漆酶-底物对接模型,以确定最佳的分子对接参数。结果:1漆酶活性位点的结构特点是,其中的Phe162、Leu164、Asn264、Phe265、Pro391、Gly392、Ala393等氨基酸为中性氨基酸,可与底物产生较强的疏水作用,对底物的结合起到重要作用,酸性氨基酸Asp206可接受来自底物的质子,碱性氨基酸His458为电子受体。2利用分子对接在不同条件将1KYA中的原配体2,5-二甲苯胺对接回活性位点,当重现出与晶体结构最相近的结合模式时,确定分子对接最佳参数设置为:温度300K,p H 5.0,分子对接力场设置为MMFF94x,放置函数(Placement)设置为Triangle Mather,打分函数(Rescoring)设置为London d G,优化函数设置(Refinement)设置为Forcefield。第二部分基于分子对接技术探究变色栓菌漆酶与氢转移机制介质间的结合模式和分子机制目的:为深入探讨漆酶与氢转移机制介质间的相互作用,基于分子对接模拟了漆酶与氢转移机制介质间的结合模式,并在分子水平阐明了漆酶与介质的相互作用机制。方法:基于分子对接技术,构建漆酶-介质复合物模型,揭示氢转移机制介质的结构特点及与漆酶作用的分子机制,为介质的结构改造提供方向和思路。结果:分子对接研究发现氢转移机制介质与漆酶作用过程中,中性氨基酸与介质发生疏水作用,Asp206作为质子受体,通过与介质结构中的-OH发生氢键作用,介质生成氮氧自由基或苯氧自由基中间体。第三部分基于分子对接技术探究变色栓菌漆酶与电子转移机制介质间的结合模式和分子机制目的:采用分子对接方法探究电子转移机制介质与漆酶的结合模式和作用机制。方法:基于分子对接技术,构建漆酶-介质复合物模型,揭示电子转移机制介质与漆酶作用的分子机制,为介质的结构改造提供方向和思路。结果:电子转移机制介质与漆酶作用过程中,中性氨基酸与介质发生疏水作用,His458作为电子受体,介质失去电子转化为阳离子中间体。第四部分漆酶-介质-有机磷毒物三元复合物体系的分子机制研究目的:本研究通过分子模拟技术探究LMO作用模式,为后期漆酶与介质的理性设计,进一步提高LMS的作用范围和催化效率奠定理论基础。方法:基于分子对接技术,构建LMO模型,揭示三元体系分子间的结合模式和作用机制;基于分子动力学方法模拟LMO模型相互作用过程中受体Cα原子的变化,考察三元体系的稳定性和动态研究LMS对有机磷毒物的降解机制。结果:在LMO中,介质结合于漆酶活性位点内部,与Asp206产生氢键作用,与内部中性氨基酸产生疏水作用和π-π堆积作用;介质在以上作用力的共同作用下生成介质中间体,进而与结合于漆酶活性位点外缘的有机磷毒物发生相互作用;分子动力学模拟发现,当三元复合物达到稳定时漆酶构象发生了突变,其中电子受体His458靠近了介质远离了Ⅰ型铜离子(T1Cu),有利于介质与漆酶之间的电子传递,且造成T1Cu的“缺电子特性”,从而氧化电势升高,电子传递速率加快。第五部分构建具有木质素降解产物基本结构单元的小分子数据库目的:通过文献检索搜集更多具有木质素降解产物基本结构单元的小分子化合物,构建具有木质素降解产物基本结构单元的小分子数据库(Molecular with Basic unit of Lignin degradation products Database,MBLD),从中挖掘新介质。方法:对文献报道的具有木质素降解产物结构单元的小分子化合物进行总结,利用MOE数据库模块建立用于挖掘新介质的MBLD。结果:通过文献检索和整理,收集到27个具有木质素降解产物结构单元的化合物,构建了MBLD,该数据库包含了化合物的2D和3D结构信息,为后续的数据库筛选提供了化学信息学基础。第六部分基于分子对接和分子相似性搜索数据库筛选漆酶新介质目的:通过对MBLD和中药化学数据库(Traditional Chinese Medicine Database,TCMD)进行筛选,从中发现漆酶新介质。方法:综合运用分子对接和分子相似性搜索对MBLD和购买的中药化学数据库(Traditional Chinese Medicine Database,TCMD)进行筛选并对筛选所得化合物进行结构优化和理论验证,以发现漆酶新介质。结果:基于分子对接和分子相似性搜索分别对MBLD和TCMD中近一万个化合物进行筛选,根据结合模式分析优先筛选出26个潜在的漆酶新介质,这些化合物与常见的天然介质在结构上十分相似,差异主要体现在取代基的种类和位置上。第七部分漆酶介质分子的结构改造与理论验证目的:本研究以紫丁香醇和丁香酸甲酯为模板,通过改变邻位取代基尝试不同的结构改造方案并确定邻位取代基的最佳改造方案,并进行理论验证。方法:基于分子对接方法,将改造后的分子与漆酶对接,进行理论验证。结果:利用分子模型和分子对接技术,探明了介质与漆酶活性位点结合与反应的结构特点。结果表明,介质酚羟基邻位取代基可保持与漆酶作用过程中介质构象的稳定,邻位取代基给电子能力可增强介质中间体的稳定性;介质酚羟基对位取代基的吸电子性有利于加强与Phe265间的π-π堆积作用,但对位取代基吸电性过强反而不利于提高介质反应活性及苯氧自由基中间体的稳定性。第八部分新介质用于漆酶-介质体系降解有机磷毒物的理论研究目的:将丁香亭和去氢二异丁香酚及其结构改造后的新化合物用于LMS降解有机磷毒物,进一步在理论层面说明丁香亭和去氢二异丁香酚及其结构改造后的化合物成为新介质的可能性。方法:基于分子动力学方法,验证新介质降解有机磷毒物的效果。结果:从虚拟筛选所得化合物中,选择丁香亭和去氢二异丁香酚作为研究对象,根据机制研究结果和化学结构理论对两者进行结构改造,原分子结构中酚羟基邻位取代基改造为两个丙烯酸基,将改造后的新化合物放于LMO体系进行验证,新化合物均能稳定存在于LMO体系,且与漆酶之间相互作用力种类和数目均有增多,结合自由能更低,与漆酶结合更加稳定。结论:1漆酶与介质相互作用过程中,漆酶活性位点的中性氨基酸主要与介质产生疏水作用,稳定介质的结合构象,Asp206可作为质子受体,His458为电子受体。2介质分子结构中,酚羟基邻位取代基可保持与漆酶作用过程中介质构象的稳定,且其给电子效应可增强介质中间体的稳定性;酚羟基对位取代基的吸电子性可加强与Phe265间的π-π堆积作用,但吸电性过强反而不利于提高介质反应活性及介质中间体稳定性。3在LMO中,介质结合于漆酶活性位点内部,有机磷毒物结合于漆酶活性位点外缘,漆酶通过介质中间体降解有机磷毒物。在LMO达到稳定时漆酶构象发生突变,该构象变化提高了漆酶氧化电势,促进了漆酶与介质间的电子传递,更有利于有机磷毒物的降解。4从MBLD和TCMD中优先选出26个先导物,综合结合模式分析、分子结构特点、结合自由能和氢传递距离等因素,发现丁香亭和去氢二异丁香酚最具成为漆酶新介质的潜力,作为后期研究重点。5对常见天然介质酚羟基邻位取代基进行结构改造发现,将其改造为两个丙烯酸基后与漆酶结合效果最佳。邻位取代基为丙烯酸基的新化合物与漆酶结合构象更稳定,且避免了不利因素分子内氢键的产生。6对丁香亭和去氢二异丁香酚进行结构改造,新化合物能稳定存在于三元复合物中,且结合模式较改造前更好,初步验证了改造的合理性,预测新化合物作为漆酶介质催化效率会有明显提高。
[Abstract]:The first part is about the structural characteristics of 1KYA active site and the establishment of molecular docking model of the laccase-substrate complex of Thrombococcus discolor. Methods: Using 1KYA as a template and using the graphical display function of Molecular Operation Environment (MOE), the structure characteristics of laccase catalytic oxidation sites were analyzed at two-dimensional (2D) and three-dimensional (3D) levels, respectively. Results: 1. The structure of laccase sites was characterized by the neutral amino acids such as Phe162, Leu164, Asn264, Phe265, Pro391, Gly392 and Ala393, which could produce strong hydrophobic interaction with the substrate. Acidic amino acid Asp206 accepts protons from substrates, and basic amino acid His458 is an electron acceptor. 2 Docking of proligand 2,5-dimethylaniline in 1KYA to the active site under different conditions is used to determine the optimal molecular docking when the most similar binding mode is reproduced. The parameters are as follows: temperature 300K, P H 5.0, molecular butt relay field MMFF94x, placement Triangle Mather, Rescoring London DG and Refinement Forcefield. The second part explores the mechanism between chameleon laccase and hydrogen transfer media based on molecular butt technology. The binding modes and molecular mechanisms of laccase and hydrogen transfer mediators were investigated. The binding modes between laccase and hydrogen transfer mediators were simulated based on molecular docking, and the interaction mechanism between laccase and mediator was clarified at the molecular level. Results: Molecular docking studies showed that neutral amino acids had hydrophobic interaction with laccase, Asp206 acted as a proton acceptor through the interaction with laccase. In the third part, based on the molecular docking technology, the binding mode and molecular mechanism between the laccase and the electron transfer mechanism medium of T. chromotropica were explored. The binding mode and interaction between the electron transfer mechanism medium and the laccase were explored by the molecular docking method. Mechanisms. Methods: Based on the molecular docking technique, a laccase-mediator complex model was constructed to reveal the molecular mechanism of the interaction between the electron transfer mechanism medium and laccase, and to provide directions and ideas for the structural modification of the medium. Part IV. Molecular mechanism of laccase-mediator-organophosphorus toxicant ternary complex system. Objective: This study explored the mode of action of LMO by molecular simulation technology, laying a theoretical foundation for the rational design of laccase and mediator, and further improving the range of action and catalytic efficiency of LMS. METHODS: Based on the molecular docking technique, a LMO model was constructed to reveal the binding mode and interaction mechanism of the ternary system. The changes of receptor C alpha atoms during the interaction of the LMO model were simulated by molecular dynamics method, and the stability and dynamics of the ternary system were investigated. Medium bound to the site of laccase activity, hydrogen bonding with Asp206, hydrophobic interaction with internal neutral amino acids and pion-pion stacking; Medium formed medium intermediates under the combined action of the above forces, and then interacted with organic phosphorus poisons bound to the outer edge of laccase activity site; Molecular dynamics simulation hair Now, when the ternary complex stabilized, the conformation of laccase changed. The electron acceptor His458 was close to the medium and away from type I copper ion (T1Cu), which was beneficial to the electron transfer between the medium and laccase, and resulted in the "electron deficiency" of T1Cu, thus increasing the oxidation potential and accelerating the electron transfer rate. OBJECTIVE: To collect more small molecular compounds with basic structural units of lignin degradation products and construct a small molecular database (MB) with basic structural units of lignin degradation products. METHODS: Small molecular compounds with structural units of lignin degradation products reported in the literature were summarized, and MBLD for mining new media was established by using MOE database module. The database contains 2D and 3D structural information of compounds, which provides a chemical informatics basis for subsequent database screening. Part VI Screening of new laccase media based on molecular docking and molecular similarity search database Objective: To screen MBLD and Traditional Chinese Medicine Database (TCMD). Methods: MBLD and TCMD were screened by molecular docking and molecular similarity search, and the structures of the compounds were optimized and verified theoretically to find new laccase media. Similarity search screened nearly 10,000 compounds in MBLD and TCMD respectively. According to the binding pattern analysis, 26 potential laccase mediators were screened out. These compounds were very similar to the common natural media in structure, and the differences were mainly reflected in the types and positions of substituents. Part VII: Structural modification of laccase mediators In this study, syringol and methyl eugenolate were used as templates to try different structural modification schemes by changing the ortho-substituents and to determine the best modification scheme of the ortho-substituents. Methods: Based on the molecular docking method, the modified molecules were docked with laccase to verify the theory. The results showed that the phenolic hydroxyl ortho-substituents could maintain the stability of the medium conformation during the interaction with laccase, and the electron donor ability of the ortho-substituents could enhance the stability of the medium. The electron absorption of the group is beneficial to the enhancement of the pi-pi stacking with Phe 265, but the strong electro-absorption of the p-substituent is not conducive to the enhancement of the reactivity of the medium and the stability of the phenoxy free radical intermediates. Part VIII Theoretical study on the degradation of organophosphorus poisons by laccase-medium system The degradability of organophosphorus poisons in LMS by carvol and its new compounds after structural modification was further explained theoretically. METHODS: Based on molecular dynamics method, the degradability of organophosphorus poisons in the new media was verified. Among the selected compounds, eugenol and dehydrodiisoeugenol were selected as the research objects. According to the results of mechanism research and chemical structure theory, the structures of the two compounds were reconstructed. The ortho-substituents of phenolic hydroxyl groups in the original molecular structure were reconstructed into two acrylic groups, and the new compounds were put into LMO system to verify the stability of the new compounds. The type and number of interaction forces between laccase and laccase increased, the binding free energy was lower, and the binding with laccase was more stable. CONCLUSION: 1 In the process of interaction between laccase and medium, the neutral amino acids of laccase activity site mainly produced hydrophobic interaction with medium, and the binding conformation of stable medium was stable, Asp206 could be used as proton. Receptor, His458 is the electron acceptor. In the molecular structure of the medium, the phenolic hydroxyl ortho-substituent can maintain the stability of the medium conformation during the interaction with laccase, and its electron-donating effect can enhance the stability of the medium. The electron-absorbing property of the phenolic hydroxy ortho-substituent can enhance the pion-pion stacking interaction with Phe 265, but the strong electro-absorbing property does not. In LMO, the medium is bound to the interior of the laccase activity site, the organophosphorus poison is bound to the outer edge of the laccase activity site, and the laccase degrades the organophosphorus poison through the medium. When the LMO is stable, the laccase conformation mutates, which improves the laccase oxidation potential and promotes the laccase oxidation. Electron transfer between laccase and medium is more conducive to the degradation of organophosphorus poisons. 4 Twenty-six precursors were selected from MBLD and TCMD. Combined with mode analysis, molecular structure characteristics, free energy and hydrogen transfer distance, it was found that eugenol and dehydrodiisoeugenol had the most potential to be new laccase media, and could be used as a new laccase in the later stage. 5. The structure modification of phenolic hydroxyl ortho-substituents in common natural media showed that the ortho-substituents had the best binding effect with laccase. New compounds with ortho-substituents of acrylic group had more stable binding conformation with laccase and avoided the formation of intramolecular hydrogen bonds. The structure of isoeugenol was reconstructed, and the new compound could exist stably in the ternary complex, and the binding mode was better than before. The rationality of the modification was preliminarily verified. It was predicted that the catalytic efficiency of the new compound as laccase medium would be significantly improved.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q55

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