榴菌素生物合成中糖基与苷元母核分子内环化机制研究

糖基化修饰在药物开发中具有较大的应用价值，但碳氧糖苷键易于水解，常成为限制化合物成药的关键因素。糖基-苷元分子内环化修饰可显著提高糖苷的水解抗性，但化学合成难以实现。寻找能催化这类分子内环化的酶，利用酶催化修饰可以较好实现这一策略。在榴菌素的结构中，糖基与母核通过两个碳碳键连接，形成非常独特的糖基附着方式。前期研究表明，糖基C1'与母核C9在糖基转移酶的作用下形成第一个碳碳键，随后C4'与C10可能通过类似羟醛反应的机制形成第二个碳碳键，使糖基与苷元母核发生分子内环化。独特的糖基附着方式蕴含着独特的酶学催化机制。本项目在前期研究的基础上，利用基因编辑、异源表达等方法来定位参与分子内环化的相关基因，并通过体外酶促反应，对基因编码产物的功能进行验证，进一步通过蛋白结构解析、分子对接、点突变等手段，在分子水平上阐明其催化机制，为糖基-苷元分子内环化修饰在药物开发中的潜在应用提供理论基础和酶资源。

Glycosylation has great application value in drug development. However, the glycosidic C-O bond is easy to be hydrolyzed, which often becomes a key limiting factor for druggability. The hydrolysis resistance of glycosides can be significantly improved by the intramolecular cyclization between the sugar and the aglycone, but it is difficult to achieve chemical synthesis. This strategy can be realized by enzymatic modifications with the efforts of successful screening and characterization of enzymes that catalyze such kind of cyclization. In the structure of granaticin, the sugar and the chromophore are linked by two carbon-carbon bonds, forming a unique pattern of sugar attachment. Previous studies have shown that the first carbon-carbon bond between C1' and C9 was catalyzed by a C-glycosyltransferase, while the second carbon-carbon bond between C4' and C10 might be subsequently catalyzed by an unknown enzyme through an aldol like reaction, thus making the intramolecular cyclization between the sugar moeity and the chromophore. Unique sugar attachments indicate unique enzymatic mechanisms. On the basis of previous studies, this project aims to: locate the related gene(s) involved in the intramolecular cyclization between the sugar and the aglycone by gene editing and heterologous expression; verify the function of the encoded products through in vitro enzymatic reactions; and unveil its catalytic mechanism at molecular level by means of protein structure determination, molecular docking and rational mutagenesis. This would provide basic knowledge and enzyme resources for the potential application of the sugar-aglycone intramolecular cyclization strategy in drug development.