title: Biochemical characterization and structural analysis of a bifunctional cellulase/xylanase from Clostridium thermocellum.
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.
authors.affiliation: the Institute of Biochemical Sciences, and.
authors.forename: Shuo-Fu
authors.initials: SF
authors.lastname: Yuan
authors.affiliation: the Institute of Biotechnology, National Taiwan University, Taipei 10617, Taiwan and.
authors.forename: Tzu-Hui
authors.initials: TH
authors.lastname: Wu
authors.affiliation: From the Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
authors.forename: Hsiao-Lin
authors.initials: HL
authors.lastname: Lee
authors.affiliation: the Institute of Biochemical Sciences, and.
authors.forename: Han-Yu
authors.initials: HY
authors.lastname: Hsieh
authors.affiliation: From the Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
authors.forename: Wen-Ling
authors.initials: WL
authors.lastname: Lin
authors.affiliation: the Institute of Biochemical Sciences, and.
authors.forename: Barbara
authors.initials: B
authors.lastname: Yang
authors.affiliation: From the Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
authors.forename: Chih-Kang
authors.initials: CK
authors.lastname: Chang
authors.affiliation: the 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: the 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: the Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Chun-Hsiang
authors.initials: CH
authors.lastname: Huang
authors.affiliation: the Institute of Biochemical Sciences, and From the Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan, joeho@gate.sinica.edu.tw.
authors.forename: Meng-Chiao
authors.initials: MC
authors.lastname: Ho
authors.affiliation: the Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China guo_rt@tib.cas.cn.
authors.forename: Rey-Ting
authors.initials: RT
authors.lastname: Guo
authors.affiliation: the Institute of Biochemical Sciences, and From the Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan, phliang@gate.sinica.edu.tw.
authors.forename: Po-Huang
authors.initials: PH
authors.lastname: Liang
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain; D002475:Cellobiose / Q000737:chemistry / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D048013:Acetivibrio thermocellus / Q000201:enzymology* / Q000235:genetics; D018360:Crystallography, X-Ray; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057075:Enzyme Assays; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology / Q000235:genetics
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D004187:Disaccharides; D011994:Recombinant Proteins; D002475:Cellobiose; D002480:Cellulase; D043364:Endo-1,4-beta Xylanases; C004173:xylobiose
keywords: Biodegradation; Biofuel; Site-directed Mutagenesis; Substrate Specificity; X-ray Crystallography