Structural and mutagenetic analyses of a 1,3-1,4-尾-glucanase from Paecilomyces thermophila

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• 作者：Ya-Shan Cheng ; Chun-Hsiang Huang ; Chun-Chi Chen ; Ting-Yung Huang ; Tzu-Ping Ko ; Jian-Wen Huang ; Tzu-Hui Wu ; Je-Ruei Liu ; Rey-Ting Guo
• 关键词：CBI ; cellobiose ; CTT ; cellotetraose ; DEAE ; diethylaminoethyl ; GLC ; glucose ; GTI ; 1 ; 3&ndash ; 1 ; 4-尾-glucotriose ; MR ; molecular replacement ; PEG ; polyethyleneglycol ; RMSD ; root mean square deviation ; Tris ; tris (hydroxymethyl) aminomethane
• 刊名：Biochimica et Biophysica Acta - Proteins and Proteomics
• 出版年：February, 2014
• 年：2014
• 卷：1844
• 期：2
• 页码：366-373
• 全文大小：1796 K

文摘

The thermostable 1,3-1,4-尾-glucanase PtLic16A from the fungus Paecilomyces thermophila catalyzes stringent hydrolysis of barley 尾-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 1.80 脜 to 2.25 脜 resolution. PtLic16A adopts a typical 尾-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-尾-glucotriose complex, the sugar bound to the 鈭?#xA0;1 subsite adopts an intermediate-like (伪-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-尾-glucanase catalytic mechanism but also provide a basis for further enzymatic engineering.