单序列结构域双功能乙酰酯-木糖苷水解酶CLH10的底物识别及催化机制

Enzymolysis of xylan is one of the main bioway to produce xylo-oligosaccharide which is a kind of prebiotics. However, effective enzymolysis of xylan has to be carried out in the multi-enzymatic system, because the conformation of xylan is complex due to the interaction between the main chain and side chain of xylan. Hence, bifunctional enzymes capable of simultaneous hydrolysis of main and side chains are effective for enzymolysis of xylan. In the previous work, the thermophilic hydrolase CLH10 was obtained by genetic mining. Sequence alignment indicated that the single sequence of CLH10 contains both the conserved catalytic residues of glycosidase and the conserved catalytic residues of esterase. CLH10 could hydrolyze both the main chain (β-xyloside bond) and the side chain (acetyl ester bond) of xylan in activity assay, indicating that CLH10 was valuable for constructing the enzymolysis system of xylan for xylo-oligosaccharide production. However, the unknown substrate recognitive and catalytic mechanism of CLH10 restricts the application of CLH10. In the present work, x-ray diffraction was employed to resolve the crystal structure of CLH10 (Apo-) and CLH10-substrate complex (Holo-); molecular dynamic simulation was employed to reveal the substrate recognitive and catalytic mechanism of CLH10 at the molecule level; reaction kinetics of enzyme was employed to demonstrate the substrate recognitive and catalytic mechanism of CLH10 at the reaction system level. This work aims to solve the substrate recognitive and catalytic mechanism of CLH10, and pave the way for the application of CLH10.

酶解木聚糖是生物制备木寡糖（益生元）的主要方法之一。然而，由于主链与侧链交联形成的网络化结构，致使木聚糖需要复杂的多酶协同催化才可实现有效降解。因此，挖掘兼备降解木聚糖主链和侧链的双功能酶具有重要意义。本研究前期利用基因挖掘策略获得了嗜热水解酶CLH10，序列比对表明该酶的单序列结构域兼备糖苷酶和酯酶的保守催化残基。酶活测定首次发现CLH10兼备水解木聚糖主链（β-木糖苷键）和侧链（乙酰酯键）的活力，具备较大的酶解木聚糖制备木寡糖（益生元）的应用开发潜力。然而，CLH10实现双功能的底物识别及催化机制尚不清楚。本研究拟从酶的“序列-结构-功能”关系入手，首先通过X射线衍射手段，解析该酶及其与底物复合体的晶体结构，进而联合分子动力学模拟手段阐释该酶底物识别和催化的分子机制，最后在反应体系层面通过酶促反应动力学研究进一步阐明该酶的底物识别及催化机制，为该酶的后续应用开发奠定理论基础。

酶解木聚糖是生物制备木寡糖（益生元）的主要方法之一。然而，由于主链与侧链交联形成的网络化结构，致使木聚糖需要复杂的多酶协同催化才可实现有效降解。因此，挖掘兼备降解木聚糖主链和侧链的双功能酶具有重要意义。本研究利用基因挖掘策略获得了双功能乙酰酯-木糖苷水解酶（CLH10）。CLH10的氨基酸序列包含离散的保守酯酶序列片段和保守糖苷酶序列片段，系单序列结构域双功能酶。晶体结构揭示CLH10的氨基酸序列折叠形成了一个球形蛋白分子，该分子的两个独立结构区域分别承担了其对于乙酰酯和木糖苷类底物的识别催化能力。因此，CLH10具备“一酶多用”作用于木聚糖主链和侧链高效酶解乙酰化木聚糖分子，进而制备木寡糖的应用潜力。值得注意的是，CLH10是一个保守的酯酶分子，其β-1,4-木糖苷键水解活力的获得机制为，基于酯酶结构骨架的Loop结构区域偶然获得了糖苷键水解关键残基对（Asp10\Glu139），且灵活的Loop结构实现了对于木糖苷底物的有效识别。上述发现提示，以酶分子的Loop区域作为定向进化或理性设计靶向区域可成为拓展酶催化功能的潜在研究焦点。