鞘糖脂糖苷合成酶的全局构效关系解析及其分子改造

Endoglycoceramide glycosynthases is an artificial enzyme derived from the catalytic nucleophilic residue mutant of endoglycoceramide glycosidase (EGC) in GH5 family. It shows enormous potential in the enzymatic synthesis of glycosphingolipid medicines. However, the application of EGC glycosynthases is subject to several limitations, such as low catalytic activity and stringent substrate specificity. The design and optimization of more efficient EGC glycosynthase is hampered by the limited understanding of the catalytic mechanism of this class of enzymes. Recently, we cloned a new EGC endoglycosidase with high activity and broad substrates specificity, solved its crystal structure, and converted it into an EGC glycosynthases. Also we established an ultra-high throughput screening technology (>10^7 clones per day) for both the endoglycosidase and glycosynthase activities. In this project, we aim to reveal the “sequence-structure-function” relationships of EGC glycosynthases by combineing the ultra-high throughput screening technology and next generation sequencing technology through the analysis of overall single site-saturated libraries of the enzyme. Optimize mutants with high activity and broad substrate spectrum will be rapidly obtained by recombination of positive mutations guided by the structure-function relationship information. This project will be helpful to reveal the catalytic mechanism of glycosynthases, and can also lay the foundation for the establishment of novel, high-efficiency enzyme engineering systems.

鞘糖脂糖苷合成酶可以催化氟化寡糖与鞘氨醇合成溶血鞘糖脂，在鞘糖脂类药物的酶法合成中具有重要应用潜力。但由于目前对于糖苷合成酶的催化和活性调节机制了解不充分，构建新的高效糖苷合成酶或对现有酶进行分子改造难度较大。在前期研究中，我们克隆了具有高活性和宽泛底物选择性的鞘糖脂内切糖苷酶、解析了其晶体结构、将其突变为鞘糖脂糖苷合成酶，并建立了基于单细胞微反应器的酶活性超高通量筛选方法。在此基础上，本项目拟构建该酶所有氨基酸位点的全局饱和突变库，结合高通量筛选和二代测序技术对酶进行“序列-结构-功能”关系的全面分析；进而依据结构信息提取对酶功能有正向作用的突变位点进行合理重组，快速构建具有高活性、宽底物谱的新型糖苷合成酶。本项目将有助于深入理解该酶的催化机制，并为建立新的高效酶分子改造技术体系奠定基础。

鞘糖脂内切糖苷酶（EGCase）能够特异性水解鞘糖脂中的糖苷键，从而将其裂解为寡糖链和神经酰胺两个部分。将EGCase活性中心的亲核残基进行突变，则可以将其转化为糖苷键合成酶。糖苷合成酶水解鞘糖脂的活性完全消失，但可以利用氟代寡糖和鞘氨醇合成糖基鞘氨醇，产率高达近100%。EGCase水解和合成的双功能特性使其在鞘糖脂组学分析及鞘糖脂药物合成中有着重要应用，然而天然EGCase的催化活性低、合成活性底物谱窄，亟待进行分子改造提升其性能。本项目通过进化分析，从马红球菌中克隆了一种新鞘糖脂内切酶（103S_EGCase I），对其进行了系统的结构-酶学功能研究，我们发现该酶构成了GH5家族一个新的亚家族，而且表现出极高的催化活性和宽泛的底物谱，体现了良好的应用潜力。我们首次解析了103S_EGCase I与GM1的复合体结构，阐明了该酶具有高催化活性和宽底物特异性的结构基础；我们将EGC I与EGCase II进行了结构比较分析，鉴定了影响EGCase 活力的关键氨基酸，并基于理性设计构建并获得了具有更高活力及底物谱的EGCase II突变酶，并对水解活性提高的机制进行了探索；我们针对突变酶进行了鞘糖脂合成的催化体系优化，显著提高了乳糖鞘氨醇的产量及转化率，为实现神经节苷脂类药物的高效制备提供了技术基础，为今后进一步进行疾病标志物检测以及新药研发开拓新的思路。

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