Biochemical Characterization and Structural Analysis of a Bifunctional Cellulase/Xylanase from Clostridium thermocellum |
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| Authors: | Shuo-Fu Yuan Tzu-Hui Wu Hsiao-Lin Lee Han-Yu Hsieh Wen-Ling Lin Barbara Yang Chih-Kang Chang Qian Li Jian Gao Chun-Hsiang Huang Meng-Chiao Ho Rey-Ting Guo Po-Huang Liang |
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| Affiliation: | From the ¶Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.;the ‡Institute of Biochemical Sciences, and ;the §Institute of Biotechnology, National Taiwan University, Taipei 10617, Taiwan and ;the ‖Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China |
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| Abstract: | We expressed an active form of CtCel5E (a bifunctional cellulase/xylanase from Clostridium thermocellum), performed biochemical characterization, and determined its apo- and ligand-bound crystal structures. From the structures, Asn-93, His-168, His-169, Asn-208, Trp-347, and Asn-349 were shown to provide hydrogen-bonding/hydrophobic interactions with both ligands. Compared with the structures of TmCel5A, a bifunctional cellulase/mannanase homolog from Thermotoga maritima, a flexible loop region in CtCel5E is the key for discriminating substrates. Moreover, site-directed mutagenesis data confirmed that His-168 is essential for xylanase activity, and His-169 is more important for xylanase activity, whereas Asn-93, Asn-208, Tyr-270, Trp-347, and Asn-349 are critical for both activities. In contrast, F267A improves enzyme activities. |
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| Keywords: | Biodegradation Biofuel Site-directed Mutagenesis Substrate Specificity X-ray Crystallography |
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