蛋白质3D建模,酶与底物分子模拟对接 autodock

摘要

多环芳烃（polycylic aromatic hydrocarbons，PAHs）是一类典型的芳香烃类有机污染物，其种类繁多，常见的共有16种。近年来多环芳烃的污染已经引起人们的高度重视，随着对PAHs 微生物降解研究的深入，已经发现大量在耗氧条件下对四环以下PAHs有降解能力的细菌，但微生物对五环及五环以上PAHs的降解能力较低，为了提高菌群的PAHs底物范围，对其降解途径中的关键酶进行分子改造具有非常重要的意义。萘双加氧酶（Naphthalene dioxygenase，NDO）是多环芳烃降解途径中的关键酶，。本论文通过计算机模拟的方式研究不同来源的萘双加氧酶与多环芳烃的相互作用规律，考察影响其活性中心口袋大小的关键氨基酸，为使用定点突变等基因工程技术提高萘双加氧酶的降解效率提供参考。本实验从数据库下载了9种来源不同的萘双加氧酶的α 亚基氨基酸序列，采用3种方式进行同源建模，经过3种方法对模型进行评价，选取质量最好的一组模型与16个PAHs分子进行对接。通过比较这些不同菌种来源的NDO与PAHs的对接结果，寻找影响其相互作用的关键氨基酸。实验结论如下：通过同源模建及模型评价，发现工具Phyre2获得的模型质量相对较好；使用Autodock Tools（ADT）将模型与PAHs进行对接后获得了不同来源NDO与PAHs相互作用的特征曲线，PAHs环数的多少会显著影响NDO与PAHs的结合能力；通过对对接结果的统计，发现来自Rhodococcus sp.的萘双加氧酶（Q9X3R9）和PAHs的结合能最低，结合能力最强。通过统计9种不同来源的NDO活性中心18个氨基酸的突变情况和偏移量发现，相对于实验室的JM-2序列，比较保守的氨基酸包括N205、F206、D209、H212、H217、G255、V264、D368、G208。而这些不同来源的BDO活性中心氨基酸组成差异主要发生于V213、L257、H301、N303、T316、L364、A412七个位置，其变异性较强，结构位置不稳定，对七个氨基酸进行改造，增大NDO的活性口袋，能增强酶对高环PAHs的结合能力，为NDO的分子改造提供参考。。

关键词：萘双加氧酶；同源建模；分子对接；活性中心；蛋白设计

Molecular simulation of the interaction of naphthalene dioxygenase and

polycylic aromatic hydrocarbons

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are a class of typical aromatic hydrocarbons organic pollutants which include 16 common congeners. In recent years，pollution due to PAHs has aroused great attention, as the research of biological degradation of PAHs presently, lots of microbe strains have been found with different abilities of degrading PAHs. Naphthalene dioxygenase (NDO) is a key enzyme in biologically degrading of PAHs. It can oxygenate a benzene ring of polycyclic aromatic hydrocarbons, and then metabolizes PAHs with the synergistic effect of other enzyme. In this paper, we mainly research molecular simulation of the interaction of naphthalene dioxygenase and polycylic aromatic hydrocarbons. We expect to understand the key animo acids in the active pocket. which can serve as a reference to improve the degradation efficiency of NDO using Site-direct mutagenesis or other gene engineering technology in the future. Results: in this experiment, we first download many amino acids sequences of α-subunit of Naphthalene dioxygenase derived from different strains and obtained 3D models by homology modeling, then chose the best model through different model assessing methods and ran molecular docking with 16 PAHs congeners. Conclusion:Through homology modeling and model assessing, the quality of model created by Phyre2 are found better; According to the docking results of model with PAHs using Autodock Tools(ADT), we draw the characteristic curve of interaction between PAHs and NDO which derived from different strains, and it reveals the numbers of the benzene ring observably effect on the combination; By the statistics of the docking results we find that the NDO(Q9X3R9) which from Rhodococcus sp. has the minimum bind energy and strongest affinity. make observably effect on the combination; We align the 9 models of NDO derived from different strains to the template 2BMQ_A and measure the shift distance of the 18 residues in the active site, the conservative amino acid N205, F206, D209, H212, H217, G255, V264, D368, G208, their conformations are conserved. However, compared to the NDO which from our lab JM-2, the main difference of NDO derived from different strains focus on seven sites V213、L257、H301、N303、T316、L364、A412 in the active site. If the active pocket became bigger after the mutagenesis of the seven sites, the substrate-binding ability of the enzyme would trend to improve. Finally, some mutagenesis according to the active center amino acids arranged of mod6(Q9X3R9) are made in order to enhance the catalytic ability of the Lab naphthalene dioxygenase(JM-2).

Keyword: Naphthalene dioxygenase; homology modeling; dock; active sites; mutation

目录

摘要 ..................................................................................................................................... I Abstract ............................................................................................................................. II 1.文献综述 ......................................................................................................................... 2 1.1微生物降解多环芳烃的研究现状 .............................................................................. 2

1.1.1 多环芳烃的理化性质 ...................................................................................... 2 1.1.2 PAHs降解菌株的来源 ..................................................................................... 3 1.1.3主要的萘双加氧酶的种类 ............................................................................... 3 1.1.4 萘双加氧酶对PAHs降解情况 ....................................................................... 6 1.2同源建模发展情况 ...................................................................................................... 7

1.2.1同源建模的意义 ............................................................................................... 7 1.2.2同源建模的概念 ............................................................................................... 8 1.2.3 同源建模的一般流程 ...................................................................................... 8 1.3 蛋白质和蛋白质结构数据库 ..................................................................................... 9

1.3.1蛋白质结构数据库 ........................................................................................... 9 1.3.2蛋白质数据库 ................................................................................................. 10 1.4序列比对 .................................................................................................................... 11

1.4.1 序列对比的意义 ............................................................................................ 11 1.4.2原理和方法 ..................................................................................................... 11 1.4.3 算法和工具 .................................................................................................... 12 1.5 分子对接 ................................................................................................................... 13

1.5.1历史背景 ......................................................................................................... 13 1.5.2原理和方法 ..................................................................................................... 13 1.5.3 对接工具 ........................................................................................................ 14 2.萘双加氧酶的序列比对 ............................................................................................... 15 2.1实验材料和方法 ........................................................................................................ 15 2.2 实验结果与讨论 ....................................................................................................... 15 2.3 小结 ........................................................................................................................... 16 3. 萘双加氧酶的同源建模 ............................................................................................. 17 3.1建模工具 .................................................................................................................... 17 3.2 实验结果与讨论 ....................................................................................................... 17 3.2.1萘双加氧酶模板筛选 ............................................................................................. 17 3.2.2萘双加氧酶的同源建模 ......................................................................................... 19 3.2.3模型评价 ................................................................................................................. 19

3.3 小结 ........................................................................................................................... 20 4. 萘双加氧酶与PAHs的分子对接 .............................................................................. 21 4.1实验准备 .................................................................................................................... 21

4.1.1分子对接工具准备 ......................................................................................... 21 4.1.2 PAHs分子构建与优化 ................................................................................... 21 4.1.3初始模型的修饰 ............................................................................................. 21 4.2预对接 ........................................................................................................................ 22 4.3优化后对接 ................................................................................................................ 22 4.4数据整理与分析 ........................................................................................................ 23

4.4.1 预对接结果 .................................................................................................... 24 4.4.2 优化后对接结果 ............................................................................................ 26 4.4.3 优化前后的实验结果对比 ............................................................................ 28 4.5 小结 ........................................................................................................................... 29 5.蛋白设计 ....................................................................................................................... 31 5.1质心和C到Fe距离分析 ......................................................................................... 31 5.2氨基酸突变 ................................................................................................................ 34

5.2.1突变过程 ......................................................................................................... 34 5.2.2结果验证 ......................................................................................................... 41 5.3作出假设 .................................................................................................................... 45 5.4小结 ............................................................................................................................ 46 6. 结论与展望 ................................................................................................................. 48 6.1 实验结果讨论 ........................................................................................................... 48 6.2 展望 ........................................................................................................................... 48 参考文献 .......................................................................................................................... 49

前言

多环芳烃（Polycyclic Aromatic Hydrocarbons PAHs）是一类含有两个或两个以上苯环或者杂环的有机化合物。是煤、石油、烟草、木材等有机物在不完全燃烧产生的状态下都能够产生多环芳烃。产生的多环芳烃对土壤、空气和水体造成污染，由于这类物质具有脂溶性的特点，水溶性很差，几乎不能自然降解，即使极低浓度的污染物经年累积，也会达到有害浓度。同时，由于多环芳烃能够通过食物链或者直接被人体摄入，尤其是大环多环芳烃，如苯并芘【α】和苯并【α】蒽，具有极强的致癌性。

近年来，有关对PAHs污染土壤的修复问题一直是研究热点。修复PAHs污染一般有物理、化学、生物的方法，其中生物降解法具有环保、花费较低和不会造成二次污染的优势，被认为是最具有前景的PAHs污染方法。在多环芳烃的降解过程中有一种至关重要的酶---萘双加氧酶。萘双加氧酶是一个多组分酶系统，包括由phnAc和phnAd组成的铁硫蛋白酶，由phnAa构成的还原酶（reductase）和phnAb构成的铁氧化还原酶（ferredoxin）。还原酶组分从NAD（P）H中释放电子，然后把电子转移到铁氧化还原酶。铁氧化还原酶然后再将电子转移到加氧酶。最终，加氧酶组分负责催化PAHs的双羟基化反应。其中，加氧酶组分phnAc（α）和phnAd（β），是α3β3的四级结构，六聚体，蘑菇状。3个大亚基构成蘑菇的伞盖，3个小亚基构成蘑菇的伞柄。phnAc作为α亚基包含两个区域：Rieske区域和催化区域。Rieske区域中心是由2个Fe和2个S构成，其中一个Fe和His82、His103配位，另一个和Cys80、Cys100配位。催化区域活性中心由1个Fe构成，这个Fe与三个保守残基His207、His212和Asp360配位相连。而phnAd（β）作为小亚基，它的主要作用是稳定结构。

Rieske 加氧酶系统是催化PAHs降解的关键步骤---苯环的加氧，所以微生物降解PAHs的能力很大程度上取决于萘双加氧酶的催化活性。并且萘双加氧酶的催化产物顺式二醇也是很重要的工业原料。本文通过序列对比和同源模建，构建出萘双加氧酶的三维结构，研究不同菌种来源的萘双加氧酶与PAHs的相互作用，寻找其活性中心起关键作用的氨基酸，为萘双加氧酶的分子改造提供依据和参考。实验方案为：从数据库下载了9种不同菌种来源的萘双加氧酶的α亚基氨基酸序列，进行同源建模，经过不同方法评价，选取结果最好的一组模型和16个PAHs同类物进行分子对接，分析对接结果，寻找影响其相互作用的关键氨基酸。并以提高酶对PAHs的底物范围为目的，依据酶与底物的作用规律对实验室的酶进行分子改造，为NDO的定向改造提供参考。

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