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Abstract：
The thermostable 1,3-1,4-β-glucanase PtLic16A from the fungus Paecilomyces thermophila catalyzes stringent hydrolysis of barley β-glucan and lichenan with an outstanding efficiency and has great potential for broad industrial applications. Here, we report the crystal structures of PtLic16A and an inactive mutant E113A in ligand-free form and in complex with the ligands cellobiose, cellotetraose and glucotriose at 1.80Å to 2.25Å resolution. PtLic16A adopts a typical β-jellyroll fold with a curved surface and the concave face forms an extended ligand binding cleft. These structures suggest that PtLic16A might carry out the hydrolysis via retaining mechanism with E113 and E118 serving as the nucleophile and general acid/base, respectively. Interestingly, in the structure of E113A/1,3-1,4-β-glucotriose complex, the sugar bound to the -1 subsite adopts an intermediate-like (α-anomeric) configuration. By combining all crystal structures solved here, a comprehensive binding mode for a substrate is proposed. These findings not only help understand the 1,3-1,4-β-glucanase catalytic mechanism but also provide a basis for further enzymatic engineering.
摘要：
来自真菌嗜热拟青霉的热稳定的1,3-1,4-β-葡聚糖酶PtLic16A催化大麦β-葡聚糖和地衣聚糖的严格水解，具有出色的效率，具有广泛的工业应用潜力。这里,我们报告了PtLic16A和无配体形式的无活性突变体E113A的晶体结构，并与配体纤维二糖复合，细胞四糖和葡萄糖三糖在1.80埃至2.25埃分辨率。PtLic16A采用具有弯曲表面的典型β-果冻折叠,并且凹面形成延伸的配体结合裂隙。这些结构表明，PtLic16A可能通过保留机制进行水解，E113和E118作为亲核试剂和一般的酸/碱，分别。有趣的是,在E113A/1,3-1,4-β-葡萄糖三糖复合物的结构中，与-1亚位点结合的糖采用中间样（α-异头）构型。通过结合这里解决的所有晶体结构，提出了一种底物的综合结合模式。这些发现不仅有助于理解1,3-1,4-β-葡聚糖酶的催化机理，而且为进一步的酶工程提供了基础。
