Ⅲ型聚酮合酶CsyB的底物特异性关键氨基酸残基的研究

abstract：Type Ⅲ polyketide synthases (PKS) is widely distributed in plants, bacteria, fungi, which is responsible for adding two-carbon unit as an pivotal biosynthetic enzyme. It exhibits remarkable diversity substrate specificity for substrates. The structure and catalytic mechanism of Type Ⅲ PKS CsyB is novel, but relatively few researches have done about the mechanism how key amino acid residues influence the substrate specificity of PKS enzyme. In this project, the substrate specificity of type Ⅲ PKS CsyB will perform a comprehensive and detailed study combined with two kinds of tools, the enzymatic crystal structure and the " non-natural " substrates. In addition, the key amino acid residues will be validated by site-directed mutagenesis. Key amino acid mutant enzymes crystallization experiment will be done. From the crystal structure point of view, it is clarified that how these amino acid residues affect the amino acid residues substrate specificity of this enzyme; at the same time, from the perspective of enzyme kinetics, key amino acid mutant enzymes’ kinetic parameters of Km, Kcat, Km / Kcat will be measured and quantitative analysis will also be done for explaining the specific role of these amino acid residues in the enzyme-catalyzed process. Based on these above results, it is theoretical interpretation that how CsyB key amino acid residues effect on substrate specificity. This project aims to reveal the relationship between important amino acid residues and its substrate specificity in the CsyB, to lay the theoretical and technical basis for elucidation the reaction mechanism of type Ⅲ polyketide synthase enzyme and effective enzymatic engineering.

摘要：Ⅲ型聚酮合酶是在植物、细菌、真菌中，大量存在的负责添加碳二单元的关键生物合成酶。这类酶具有丰富的底物多样性。真菌来源的Ⅲ型聚酮合酶CsyB具有新的结构和催化机制，然而，其影响底物特异性的关键氨基酸残基尚不完全明确。本项目以该酶为研究对象，结合酶晶体结构和“非天然”底物两种手段，对酶的底物特异性做全面的考察，并对影响其特性的关键氨基酸残基进行点突变。进而，对这些关键氨基酸突变型酶进行结晶试验，从晶体结构学角度，阐明这些氨基酸残基如何影响酶的底物特异性；同时，对关键氨基酸突变型酶的酶动力学参数Km、Kcat、Km/ Kcat进行测定，从酶促动力学角度，定量分析这些氨基酸残基在酶催化过程中所起得具体作用；综合以上结果，解释CsyB关键氨基酸残基对底物特异性影响的理论原因。本项目旨在揭示CsyB重要氨基酸残基与其底物特异性的规律，为深入地理解Ⅲ型聚酮合酶的反应机制及有效地酶分子改造奠定理论和技术基础。

本项目以真菌来源的III型聚酮合酶CsyB具为研究对象，该酶具有新的结构和催化机制，其影响底物特异性的关键氨基酸残基尚不完全明确。采用结合酶晶体结构和“非天然”底物两种手段，对酶的底物特异性做全面的考察，确定了负责催化克莱森缩合反应的关键氨基酸C155/H310/N343、负责激活底物的关键氨基酸H377以及负责底物多样性的关键氨基酸I375，并对这些影响其特性的关键氨基酸残基进行定点突变。进而，对这些关键氨基酸突变型酶进行结晶试验，从晶体结构学角度，阐明这些氨基酸残基如何影响酶的底物特异性，负责激活底物的关键氨基酸H377与一分子水分子形成氢键网络，完成底物分子的活化，负责底物多样性的关键氨基酸I375则是通过改变了底物的活性口袋来实现对底物多样性的控制;同时，对关键氨基酸突变型酶的酶动力学参数Km、Kcat、Km/ Kcat进行测定，从酶促动力学角度，定量分析这些氨基酸残基在酶催化过程中所起得具体作用;综合以上结果，解释CsyB关键氨基酸残基对底物特异性影响的理论原因。本项目旨在揭示CsyB重要氨基酸残基与其底物特异性的规律，为深入地理解III型聚酮合酶的反应机制及有效地酶分子改造奠定理论和技术基础。