论文总字数：29533字

摘 要

白藜芦醇，自然界中的植物在提升自身免疫性和抵御外来侵害时会产生一种抗毒素，经研究发现这类抗毒素是一种含有二苯乙烯结构的多酚化合物，被人们称为白藜芦醇。除此之外，它还能够调节血脂、抗氧化、抗癌、减肥降脂、降烧镇痛，具有多种有益于人类身体健康的功能。随着科学研究进展，发现糖基化可以改变受体分子的亲水性、药物代谢动力学特征、稳定性、活性及免疫原性，并有助于其在生物体内、细胞内的运输和贮藏。研究发现相对于白藜芦醇，白藜芦醇苷的稳定性、溶解性、药理活性均有明显提高。

糖基转移酶在获得糖基化活性化合物结构多样性的过程当中起着重要作用，定向改造能够提高酶对底物的催化活性、区域选择性和宽泛性。本论文通过对糖基转移酶ZS1进行蛋白质工程的改造，对底物结合域附近的氨基酸残基突变，获得相关突变体并进行测验糖基转移酶ZS1对白藜芦醇的区域选择性。其中I62G突变体催化白藜芦醇糖基化的单糖苷产物主要为3-O糖苷，即为虎杖苷。而野生型糖基转移酶催化白藜芦醇的产物为4’-O糖苷。

为了对野生型和突变型的催化机制进行解析，本论文对糖基转移酶ZS1进行同源建模，并利用计算机软件Autodock模拟预测糖基转移酶ZS1与底物的相互作用，分析造成对白藜芦醇的区域选择性糖基化差异的主要原因。Autodock进行分子对接的结果分析中，以自由结合能排序，以结合能最小的结果为结合效果最佳的。糖基转移酶中16位的组氨酸是关键催化残基，而我们发现16位组氨酸与白藜芦醇区域选择性有较大相关，结果中，白藜芦醇的羟基越靠近16位组氨酸，该羟基则越可能被糖基化。将来，如果能够获得糖基转移酶的晶体结构，会更有助于解析催化机制。

关键词：白藜芦醇 糖基转移酶 糖基化 区域选择性 Autodock 虎杖苷

Computer aided design for the engineering of glycosyltransferase ZS1 regioselectivity to resveratrol

Abstract

Resveratrol (C14H12O3) is a kind of polyphenol compounds with skeleton of toluylene. It has many functions, which are beneficial to human body, such as regulating blood lipid, antioxidant, prolonging life-span, reducing excess fat, fever and pain. With the progress of scientific research, it is found that glycosylation can change the hydrophily, pharmacokinetic characteristics, stability, activity and immunogenicity of the receptor molecules. And glycosylation is also helpful for the transportation and storage of the receptor molecules in the organism and intracellular. The researches show that, compared to reservatrol, reservatrol glycosides have higher stability, solubility and pharmacological activity.

Glycosyltransferases play an important role in obtaining diverse active compounds. Protein engineering can expand the range of substrates, improve or change the regioselectivity and promote the catalytic activity of transferases. This paper obtained mutants of glycosyltransferases ZS1 by protein engineering, and then tested the regioselectivity to resveratrol by these enzymes. Main monoglucoside product of mutant I62G is resveratrol 3-O-glucoside, defined as polydatin. And the primary product of wild type is resveratrol 4’-O-glucoside.

For analyzing the catalyzing mechanism of wild-type and mutant, this paper built a structure model of ZS1 by homology modeling. Predicting the interaction between ZS1 and the substrate by docking(autodock), and then find out the main factor for the differences in regioselectivity to resveratrol by glycosyltransferase. In the docking mechanism of autodock, the result would be ranked by free binding energy, and the one with lowest binding energy would be the best docking model. The 16His in glycosyltransferase is the key catalytic residues, and we find that 16H is very related to the regioselectivity. In the docking resulting models, the shorter distance between the hydroxy group of reservatrol and 16His of ZS1, the more likely this hydroxy group could be glycosylated. If the crystal structure of glycosyltransferase is available in the future, it would be of tremendous assistance to the analysis of catalytic mechanism.

Key Words: reservatrol; glycosyltransferase; glycosylation; regioselectivit; Autodock； polydatin

目 录

摘要 I

Abstract II

第一章 文 献 综 述 1

1.1选题意义 1

1.2白藜芦醇的研究进展 1

1.3白藜芦醇糖基化 1

1.4增加糖基化方法 2

1.5糖基转移酶的区域选择性改造 2

1.6计算机辅助设计 3

1.7 实验方法 3

第二章 糖基转移酶ZS1及突变体催化白藜芦醇糖基化 4

2.1 前言 4

2.2 实验材料 5

2.2.1 菌株及质粒 5

2.2.2 实验试剂 5

2.2.3 培养基配方 6

2.2.4 实验仪器 6

2.3 实验方法 7

2.3.1 糖基转移酶表达载体的构建及大肠杆菌的转化 7

2.3.2 全质粒PCR法点突变糖基转移酶ZS1 11

2.3.3 糖基转移酶ZS1的表达 12

2.3.4 利用His-tag标签纯化糖基转移酶ZS1 13

2.3.5 糖基转移酶ZS1体外催化白藜芦醇糖基化 13

2.4 结果与讨论 14

2.4.1 糖基转移酶ZS1 PCR产物琼脂糖凝胶电泳验证及表达载体的测序验证 14

2.4.2 糖基转移酶ZS1在大肠杆菌BL21中的表达验证 15

2.4.3 糖基转移酶ZS1的纯化 16

2.5 本章小结 19

第三章AUTODOCK的使用 20

3.1 Autodock简介 20

3.2 Autodock使用步骤 20

3.2.1 一配体和受体的PDB文件 20

3.2.2 野生型的糖基转移酶氨基酸序列ZS1 20

3.2.3 突变改造后的氨基酸序列ZS1-I62G 20

3.2.4 配体的化学式 21

3.2.5 步骤 21

3.3 通过ADT来使用autodock 22

3.3.1打开ADT 22

3.3.2编辑macromolecule文件 22

3.3.3 准备ligand文件 25

3.3.4 准备grid文件 26

3.3.5运行autogrid 28

3.3.6准备docking文件 29

3.3.7运行autodock 30

3.3.8 analyze分析对接结果 31

3.4 实验结果分析 32

第四章 总结与展望 37

参考文献 38

附录 40

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