岩藻糖基转移酶对非天然供体底物选择性的分子改造

项目名称： 岩藻糖基转移酶对非天然供体底物选择性的分子改造

项目编号： No.31470788

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 生物科学

项目作者： 杨广宇

作者单位： 上海交通大学

项目金额： 85万元

中文摘要： 岩藻糖基转移酶(FucT)催化反应将供体底物岩藻糖鸟苷二磷酸(GDP-岩藻糖)上的岩藻糖转移至受体底物，在药物相关寡糖合成中有重要价值，但其大规模应用受到昂贵的天然供体底物(GDP-岩藻糖)限制。我们前期研究发现，来源于幽门螺杆菌的FucT对廉价的、非天然糖基供体(α-氟代岩藻糖)也具有一定活性，进一步提升后有望替代GDP-岩藻糖为寡糖合成提供一条新途径。对FucT的结构分析表明，其活性中心附近的5段短LOOP区可能是控制酶供体底物选择性的关键。为探讨酶对非天然底物的识别机制、构建高效进化酶，本项目拟对酶活性中心进行杂化，结合酶家族序列分析构建小型、智能半理性设计突变库；应用我们前期发展的FACS筛选策略，建立针对FucT活性的超高通量筛选方法，筛选获得对非天然底物具有高活性的进化酶；对突变体结构-功能关系分析，阐明FucT供体底物选择性机制，为糖基转移酶的分子改造提供理论指导。

中文关键词： 岩藻糖转移酶；底物选择性；半理性设计；高通量筛选；荧光激活细胞分选

英文摘要： Fucosyltransferases are important biocatalysts and have shown enormous potential in pharmaceutical industries for the synthesis of oligosaccharides and glycoconjugates. However, the broad applications of fucosyltransferases have been hampered by the high cost of its sugar nucleotide donor substrate (GDP-fucose). Recently, we have found that the fucosyltransferase from Helicobacter pylori (FucT) showed promiscuous activity on cheap artificial glycoside substrate (fucosyl fluoride), thus may provide an inexpensive alternative for the enzymatic synthesis of fucose-containing compounds. However, the activity of FucT against fucosyl fluoride is quite low, which needs further improvement before substituting the sugar nucleotide. By analyzing the crystal structure, we found five short loop-regions in the active-site of FucT may play crucial roles in substrate recognition. In order to better understand the mechanism of the substrate specificity of FucT, and to improve its non-natural catalytic activity, this project aims to construct focused mutagenesis libraries in these substrate-binding loops using a semi-rational design strategy. A FACS-based screening method will be developed for the unnatural activity of FucT. Then consecutive rounds of screening will be carried out to identify the mutant enzymes with increased activity. Structural and functional analysis of the mutants may provide insights into the mechanism of substrate recognition of FucT, and help for the further engineering of other glycosyltransferases.

英文关键词： fucosyltransferase;specificity;semi-rational design;high-throughput screening;FACS