糖调节β-葡萄糖苷酶酶活力及稳定性的机制

β-Glucosidases are a heterogeneous group of enzymes that hydrolyze the β-1,4-glycosidic bond presented in disaccharides, oligosaccharides, aryl-, and alkyl β-glucosides, and release the nonreducing terminal glucosyl residues. These enzymes have attracted considerable interests because of their potential applications in a variety of biological processes. However, most β-glucosidases are feed-back inhibited by the product glucose, which restricts their application in industries. Recently, the activities of a few microbial β-glucosidases have been reported to be upregulated by sugars, such as glucose, xylose, and sucrose. Furthermore the thermodynamic stability of some β-glucosidases has been reported to be improved in the presence of sugar. Elucidation of the mechanisms how sugars influence enzyme activity and improve the protein thermodynamic stability will be essential to enhance the sugar tolerance of β-glucosidases by protein engineering, and thus contribute to their improvment in biotechnology. Previous reports indicated that allosteric effect plays important roles in the modulation of β-glucosidases’ properties by sugars. In this project, we will apply statistical coupling analysis (SCA) to define the coevolving networks i.e. “sectors” of β-glucosidases. We will identify the key amino acids in the sectors associated with allosteric regulation, and finally to reveal the potential roles and functioning mechanism of allosteric regulation in modulation of the enzyme activity. Furthermore, we will clarify the interaction pattern between the sugars and the sectors, and reveal how sugars improve the protein thermodynamic stability. The findings will contribute to improving the performance of β-glucosidases in industral applications.

β-葡萄糖苷酶水解糖苷或寡糖，释放葡萄糖及糖苷配体，具有重要工业应用价值。多数β-葡萄糖苷酶受产物葡萄糖的反馈抑制，而另有一些酶的酶活力及热力学稳定性在糖分子存在条件下得到提升。除底物通道特征及转糖苷作用，变构调节在β-葡萄糖苷酶的糖耐受方面可能发挥了重要作用。氨基酸残基共进化网络中位于蛋白质表面的相关位点是变构调节的热点，且网络中氨基酸残基间的精细平衡对蛋白质的热力学稳定性有重要作用。本课题拟以序列一致性较高而糖耐受差异显著的β-葡萄糖苷酶Bgl1A和Bgl1B为对象，研究酶蛋白氨基酸残基共进化网络，确定其中可能参与变构调节的位点，揭示变构调节在糖影响β-葡萄糖苷酶酶活力方面扮演的角色和作用机制；分析糖分子与氨基酸残基共进化网络之间的作用模式，明晰糖提升酶分子热力学稳定性的机制，为改善β-葡萄糖苷酶的工业应用性能奠定理论和技术基础。

收集GH1家族β-葡萄糖苷酶的序列信息和结构信息，以多序列比对结果为基础，采用统计耦联分析法，研究β-葡萄糖苷酶Bgl1A的氨基酸残基共进化网络，共获得10条独立分量（Independent component, IC），明晰与酶的活性及糖耐受相关的独立分量IC1、IC6和IC7。选择β-葡萄糖苷酶Bgl1A的氨基酸残基共进化网络中位于蛋白质表面的氨基酸残基位点为突变位点，明晰了独立分量中与糖耐受关系密切的多个氨基酸残基位点，如独立分量IC1中的W325，独立分量IC7中的M323、K412以及独立分量IC6中的F172、C173、N226等。另一方面，基于序列和结构比对，以及氨基酸保守性分析，明晰葡萄糖耐受关键位点为位于底物通道外部的D322位点。以糖耐受性能发生显著改变的W325和D322位突变体为例，进行同源建模、分子对接等研究，明晰糖与氨基酸残基共进化网络之间的作用模式。依据氨基酸残基共进化网络与糖耐受性能的关系，选取关键的突变位点，以糖敏感的β-葡萄糖苷酶Bgl3A为例进行理性改造，获得多个糖耐受性提升的突变酶，突变酶的葡萄糖耐受性得以提升，显示出良好的应用潜力。