Characterization and crystal structure of a thermostable glycoside hydrolase family 45 1,4- β-endoglucanase from Thielavia terrestris

Publication date: Available online 17 January 2017 Source:Enzyme and Microbial Technology Author(s): Jian Gao, Jian-Wen Huang, Qian Li, Weidong Liu, Tzu-Ping Ko, Yingying Zheng, Xiansha Xiao, Chih-Jung Kuo, Chun-Chi Chen, Rey-Ting Guo 1,4-β-Endoglucanase is one of the most important biocatalysts in modern industries. Here, a glycoside hydrolase (GH) family 45 endoglucanase from thermophilic fungus Theilavia terrestris (TtCel45A) was expressed in Pichia pastoris. The recombinant protein shows optimal activity at 60°C, pH 4-5. The enzyme exhibits extraordinary thermostability that more than 80% activity was detected after heating at 80°C for 2.5hours. The high resolution crystal structures of apo-form enzyme and that in complex with cellobiose and cellotetraose were solved to 1.36-1.58Å. The protein folds into two overall regions: one is a six-stranded β-barrel, and the other one consists of several extended loops. Between the two regions lies the substrate-binding channel, which is an open cleft spanning across the protein surface. A continuous substrate-binding cleft from subsite −4 to +3 were clearly identified in the complex structures. Notably, the flexible V–VI loop (113Gly-114Gly-115Asp-116Leu-117Gly-118Ser) is found to open in the presence of −1 sugar, with D115 and L116 swung away to yield a space to accommodate the catalytic acid D122 and the 2,5 B boat conformation of −1 sugar during transition state. Collectively, we characterized the enzyme...
Source: Enzyme and Microbial Technology - Category: Biotechnology Source Type: research
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