双功能纤维二糖磷酸化酶TaCBP宽泛的底物特异性的结构基础研究

自然界微生物利用纤维素的部分降解模式——纤维素经部分降解生成的纤维二糖和纤维寡糖被直接吸收到胞内利用——已成为纤维素高效降解和利用的新的发展方向。相比于β-葡萄糖苷酶，纤维二糖磷酸化酶（CBP）和纤维寡糖磷酸化酶（CDP）催化的胞内纤维二糖和寡糖的利用途径具有显著的优势，因此发掘新型的CBP和CDP具有重要的理论意义和应用价值。项目组鉴定的新型耐热CBP（TaCBP）具有独特的兼具CBP和CDP活性的双功能催化活性，本项目拟利用X射线晶体学技术解析TaCBP及其与糖类底物复合物的晶体结构，确定TaCBP的催化活性中心，揭示其宽泛的底物特异性的结构基础，并结合酶与底物的相互作用等研究，确定TaCBP双功能催化的关键氨基酸残基，阐明TaCBP同时磷解纤维二糖和纤维寡糖的双功能催化机制，为CBP和CDP底物特异性的改造提供理论依据，为其在纤维素高效降解和利用中的应用奠定理论基础。

The partial hydrolysis paradigm requiring partial hydrolysis of cellulose yielding cellobiose and cellodextrin which in turn are directly transported and assimilated in the cell, inspired by naturally cellulolytic microorganism, has been considered as a promising strategy for high-efficient cellulose degradation and utilization. Compared to hydrolysis catalyzed by β-glucosidase, hydrolysis of cellobiose and cellodextrin in the cell catalyzed by cellobiose phosphorylase (CBP) and cellodextrin phosphorylase (CDP) has compelling advantages. So it is significant to discovery novel CBP and CDP. The thermophilic CBP from Thermosipho africanus (TaCBP) is characterized as a bifunctional enzyme with phosphorolytic activity on both cellobiose and cellodextrin. Here we project to solve the crystal structures of TaCBP and complexes with cellobiose and cellodextrin, in order to determine the substrate binding sites and catalytic residues and to reveal the structural basis for its broad substrate specificity. Combining the structural and biochemical information, the key bifunctional catalytic residues and the mechanism of bifunctional TaCBP catalysis on both cellobiose and cellodextrin will be proposed, thus to lay the foundation for engineering of substrate specificity of other CBPs and CDPs and for their industrial application of high-efficient cellulose utilization.