β-葡萄糖醛酸苷酶水解与合成特性的转换机制

Glycosides have been widely used in pharmacy, food and chemical engineering due to their high biological activity and medicinal value. Chemical synthesis of glycosides has drawbacks including poor stereoselectivity and complicated technology, in contrast, enzymatic synthesis of glycosides has good stereoselectivity thus making it a promising industrial technique. Currently, most glycosynthases are derived from glycosidases through molecular modification, however, the conformation switch mechanism between hydrolysis and synthesis of glycosidases is still unclear resulting in the low efficiency of the molecular modification. Therefore, in this project, the catalytic model of the hydrolysis and synthesis property of glycosidase is established through database analysis and mining; Then, based on the crystal structure of β-glucuronidase from Penicillium purpurogenum Li-3 (PGUS) resolved in our lab, PGUS mutants with glycosynthesis capability were obtained by molecular dynamics simulation and molecular modification, and a new protocol is developed for mono-glucuronide glycyrrhizin synthesis; Finally, the functional switch mechanism of glycosidase from hydrolysis to synthesis is explored by molecular docking of enzyme and substrate, and structure analysis. The above work will provide a new insight into the rational design of other glycosidases for glycosides synthesis, further boosting the high-efficiency synthesis and industrial application of glycosides.

糖苷化合物因其具有重要的生物活性和药用价值被广泛地应用于医药、食品和化工等领域。相比化学合成糖苷存在立体选择性差、工艺复杂等问题，酶在催化合成糖苷过程中具有良好的立体选择性，而现有糖苷合成酶大多数是从其水解特性改造而来，由于糖苷酶水解与合成特性的结构转换机制不清楚，仍存在着改造难度大及合成效率低的问题。为此，本项目拟通过对糖苷酶数据库的分析与挖掘，建立糖苷水解和合成的催化模型及结构特征文库；以课题组前期解析的产紫青霉β-葡萄糖醛酸苷酶的晶体结构为基础，通过动力学模拟和分子改造使之具有合成糖苷的特性，并以甘草次酸为苷元构建酶法合成单葡萄糖醛酸基甘草次酸的新方法；通过对β-葡萄糖醛酸苷酶与底物分子甘草次酸和葡萄糖醛酸的分子对接和结构分析，探索糖苷酶的水解与合成特性之间的结构转换及其分子调控机制。研究也为理性改造其它糖苷酶的合成特性提供新思路，对糖苷化合物的高效合成与工业化应用起到推动作用。

糖苷化合物因其具有重要的生物活性和药用价值被广泛地应用于医药、食品和化工等领域。相比化学合成糖苷存在立体选择性差、工艺复杂等问题，酶在催化合成糖苷过程中具有良好的立体选择性.针对糖苷酶水解与合成特性的结构转换机制不清楚，存在着改造难度大及合成效率低的问题。本项目通过对糖苷酶、糖基转移酶数据库的分析与挖掘，建立糖苷水解和合成的催化模型及结构特征文库；以解析的β-葡萄糖醛酸苷酶的晶体结构为基础，通过动力学模拟和分子改造使之具有合成糖苷的特性，并以甘草次酸为苷元构建酶法合成单葡萄糖醛酸基甘草次酸的新方法。项目完成了来源于A. oryzae的PGUS及其底物复合物GL、GAMG的晶体结构解析，阐明其识别底物的分子机制；挖掘并表征了能特异性糖基化五环三萜苷元底物的欧洲山芥（Barbarea vulgaris）和刺茄(Solanum aculeatissimum)的糖基转移酶BvEc和SaEc；通过分子改造、筛选，获得定向水解GL生成GAMG的突变体，底物特异性大于95%，实现GAMG的可控水解；通过分子动力学模拟和分子对接，筛选一系列具有转糖基的突变体，实现了糖基水解与合成的功能转化。