论文总字数：21687字

摘 要

周环反应是用于形成碳-碳键和碳-杂原子键的最有力的合成反应中的一种，其应用了区域和立体选择性的方式，虽然周环反应在有机合成中比较普遍，但只有少数天然存在的酶可以催化它。甲基转移酶是生命活动反应中最重要的一种酶，而S-腺苷甲硫氨酸(SAM)是生化反应中极为重要的甲基供体。最近科学家发现并证实了一种SAM依赖的多功能酶LepI，生化实验显示LepI可以催化立体选择性脱水，产生(E)-醌甲基化物，该化合物可以发生分子内Diels-Alder反应(IMDA)和杂原子参与的Diels-Alder反应(HAD)生成leporin C及其异构体。在LepI的催化下，IMDA的产物还可以发生逆Claisen重排反应，最终生成2-吡啶酮天然产物leporin C。目前已知SAM结合LepI的X射线晶体结构和与底物类似物的复合物，结合生化实验Lep I的突变体H133A、H133F、H133N、H133Q、R295A、R295E、R295H、R295N和R295Q等的酶活数据，及为了更深入地研究催化机制，开展了突变体H133N与底物Leporin C的复合物的研究，对其进行表达、纯化和结晶。

关键词：周环反应 酶促作用 晶体结构 催化机制

Structural Basis for Catalysis by the SAM-Dependent

Pericyclase LepI Mutant H133N

Abstract

The pericyclic reaction is one of the most potent synthetic reactions for the formation of carbon-carbon bonds and carbon-heteroatom bonds, which employs a regional and stereoselective approach, although pericyclic reactions are more prevalent in organic synthesis. But only a few naturally occurring enzymes can catalyze it. Methyltransferase is the most important enzyme in life-active reactions, and S-adenosylmethionine (SAM) is a very important methyl donor in biochemical reactions. Recently, scientists have discovered and confirmed a SAM-dependent multifunctional enzyme LepI. Biochemical experiments show that LepI can catalyze stereoselective dehydration to produce (E)-quinone methide, which can undergo intramolecular Diels-Alder reaction (IMDA)and the Diels-Alder reaction (HDA) in which heteroatoms participate in the production of leporin C and its isomers. Under the catalysis of LepI, the product of IMDA can also undergo reverse Claisen rearrangement reaction to finally form the 2-pyridone natural product leporin C. We have known that the X-ray crystal structure of SAM-bound LepI and its complex with substrate analogs, combined with biochemical experiments LepI mutants H133A, H133F, H133N, H133Q, R295A, R295E, R295H, R295N and R295Q, etc. In order to study the catalytic mechanism in more depth, a complex of mutant H133N and

substrate Leporin C was expressed, purified and crystallized.

Key words: Pericyclic reactions; enzymatic catalysis; crystal structure; molecular mechanism

目 录

摘要···························I

ABSTRACT···························II

第一章 引言···························1

1.1 X射线衍射·························1

1.1.1 X射线衍射的发展···················1

1.1.2 X射线衍射在结构生物学中的应用简述··········1

1.2课题背景介绍························2

1.3本课题的研究意义和初步方案·················8

1.3.1研究意义·······················8

1.3.2初步方案·······················8

第二章 实验材料和方法····················9

2.1实验材料··························9

2.1.1实验仪器·······················9

2.1.2实验试剂·······················10

2.1.3菌株和试剂盒·····················11

2.1.4常用培养基及溶液···················11

2.2实验原理与方法·······················14

2.2.1.普通感受态细胞的制备和转化···············14

2.2.2蛋白质的表达·····························14

2.2.3.蛋白质的纯化·······························14

2.2.4蛋白质的结晶·······························15

第三章 LepI突变体H133N的表达、纯化、结晶·········16

3.1突变体H133N的构建·······························16

3.2突变体H133N的表达纯化··················16

3.2.1细菌破壁·······················17

3.2.2 镍亲和柱层析纯化·························17

3.2.3凝胶排阻色谱分离····························17

3.2.4蛋白浓度确定·····················19

3.3突变体H133N与底物的复合物晶体筛选与数据收集和处理····19

3.3.1复合物晶体的常见制备方法···············19

3.3.2突变体H133N与Leporin C的复合物晶体筛选与数据采集··19

3.3.3蛋白晶体的冷冻保护剂选择···············20

3.4结果与讨论·······························21

第四章 结论与展望·····································23

4.1结论···········································23

4.2展望···········································23

参考文献···············································24

主要符号对照表·························27

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