Structural and mutagenetic analyses of a 1,3–1,4-β-glucanase from Paecilomyces thermophila |
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Authors: | 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 |
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Institution: | 1. Genozyme Biotechnology Inc., Taipei 106, Taiwan;2. AsiaPac Biotechnology Co., Ltd., Dongguan 523808, China;3. Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China;4. Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan;5. Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan;6. Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan;g Department of Animal Science and Technology, National Taiwan University, Taipei 106, Taiwan |
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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 1.80 Å to 2.25 Å 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. |
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Keywords: | CBI cellobiose CTT cellotetraose DEAE diethylaminoethyl GLC glucose GTI 1 3&ndash 1 4-β-glucotriose MR molecular replacement PEG polyethyleneglycol RMSD root mean square deviation Tris tris (hydroxymethyl) aminomethane |
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