肝素骨架合酶pmHS2单糖供体选择机制的研究

Glycosyltransferase (GT) is widely used in the enzymatic synthesis of oligosaccharides in vitro. However, strict substrate specificity limits the number and type of glycan structures synthesized by GTs. Heparosan synthase pmHS2, important enzyme used in enzymatic synthesis approach of heparin oligosaccharides, shows narrow donor substrate promiscuity, which is the bottleneck for effective synthesis. This project aims at mechanism of pmHS2 donor substrate promiscuity. Firstly, uncover the reason of desired monosaccharide transferase activity deficiency of pmHS2, followed by obtain mutations with desired activity using GT substrate engineering directed evolution platform, achieve the mechanism of pmHS2 donor substrate promiscuity; Secondly, to uncover the causes of that substrate specificities of pmHS2 in the cell system was more selective than that of recombinant protein, by rapidly assessing a large number of insertional transposon mutagenesis, the factors influencing the donor substrate promiscuity were determined by high-throughput screening of "click-reaction" signal of target glycosyltransferase reaction. Finally, new tool enzymes with desired activity were obtained based on the evolution platform established by the project. The collusions should have important theoretical value; besides, the pmHS2 mutations of this project can be directly applied to the synthesis of HP oligosaccharides, and make the enzymatic synthesis approach of heparin one step closer toward “application”.

糖基转移酶（GT）广泛的用于糖链的体外酶法制备，但其严格的底物特异性限制了酶法的合成能力。如肝素骨架合酶pmHS2单糖供体选择性是制约肝素酶法合成效率的瓶颈所在。项目以该酶为研究对象。一方面，通过对特定单糖供体转移活性缺失原因的初步分析和底物工程定向进化平台的建立，有针对性的筛选并分析供体选择性变化的突变体，揭示GT选择单糖供体的分子机理；另一方面，针对pmHS2在细胞体系内供体选择性较体外重组蛋白更加严格的现象，设计基于转座子标签技术和“Click”反应的菌体单糖供体适应性快速分析方法并分选基因组突变菌体，确定细胞体系内影响糖基转移活性供体选择的因素并分析其作用机理。最后，在理论研究基础上，借助项目建立的定向进化平台，获得具有特定转糖基活性的工具酶分子，应用于肝素寡糖的酶法合成。项目结论对Leloir型GT研究具有理论价值，且成果可以直接有助于肝素寡糖合成体系瓶颈问题的突破。

糖基转移酶（Glycosyltransferases ，GTs）广泛的用于糖链的体外酶法制备，但其严格的底物特异性限制了酶法的合成能力。项目以微生物来源的肝素骨架合成糖基转移酶为模式分子，通过新酶分子的发掘和催化活性分析，有效拓展了pmHS2底物选择机制的“氨基酸位点-催化特点”的信息素材。基于底物亲和分析等有效数据，对潜在关键氨基酸位点开展定点突变及体外活性变化的分析，确定了第三“柔性环”对双功能酶肝素骨架合酶底物亲和的显著影响；同时确定了一系列影响各类型肝素骨架合成糖基转移酶活性、稳定性的关键氨基酸位点。此外，项目建立的基于YND1的糖基转移酶活性快速分析与定向进化体系，非常适合以UDP-sugar为单糖供体的Leloir家族GTs的反应筛选；项目建立的基于生物传感器和生物正交反应的细胞体系内的单糖供体适应性快速分析方法，可以有效的应用于细胞体系内GT底物特异性分析。在理论研究基础上，借助项目建立的高通量筛选体系，通过定向进化获得具特定单糖引入效率显著提升的工具酶分子，并建立、优化了由两种细菌来源糖基转移酶催化的肝素寡糖可控合成体系，有效避免了原有合成体系中糖链降解的可逆反应。项目相关结论不但对Leloir家族GTs糖基转移催化研究具有重要理论价值，成果可直接有助于肝素寡糖化学酶法合成体系的规模化应用，使肝素寡糖化学酶法合成技术体系向“实际应用”再走近一步。