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 s equences 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