title: Characterization and crystal structure of a thermostable glycoside hydrolase family 45 1,4-β-endoglucanase from Thielavia terrestris.
abstract: 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 2.5h. 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 the2,5B boat conformation of -1 sugar during transition state. Collectively, we characterized the enzyme properties of P. pastoris-expressed TtCel45A and solved high-resolution crystal structures of the enzyme. These results are of great interests in industrial applications and provide new insights into the fundamental understanding of enzyme catalytic mechanism of GH45 endoglucanases.
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Gao
authors.affiliation: AsiaPac Biotechnology Co., Ltd, Dongguan, 523808, China.
authors.forename: Jian-Wen
authors.initials: JW
authors.lastname: Huang
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Li
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
authors.forename: Weidong
authors.initials: W
authors.lastname: Liu
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei, 11529, Taiwan.
authors.forename: Tzu-Ping
authors.initials: TP
authors.lastname: Ko
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
authors.forename: Yingying
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
authors.forename: Xiansha
authors.initials: X
authors.lastname: Xiao
authors.affiliation: Department of Veterinary Medicine, National Chung Hsing University, Taichung, City 402, Taiwan.
authors.forename: Chih-Jung
authors.initials: CJ
authors.lastname: Kuo
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China. Electronic address: ccckate0722@gmail.com.
authors.forename: Chun-Chi
authors.initials: CC
authors.lastname: Chen
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China. Electronic address: guo_rt@tib.cas.cn.
authors.forename: Rey-Ting
authors.initials: RT
authors.lastname: Guo
mesh_terms: D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D007218:Industrial Microbiology; D008958:Models, Molecular; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020037:Sordariales / Q000201:enzymology* / Q000235:genetics; D055672:Static Electricity
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D002480:Cellulase
keywords: Crystal structure; Endoglucanase; Pichia pastoris; Thermostable; Thielavia terrestris