Biochemical characterization and structural analysis of a bifunctional cellulase/xylanase from Clostridium thermocellum

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

doi:10.1074/jbc.M114.604454

Biochemical characterization and structural analysis of a bifunctional cellulase/xylanase from Clostridium thermocellum

J Biol Chem. 2015 Feb 27;290(9):5739-48. doi: 10.1074/jbc.M114.604454. Epub 2015 Jan 9.

Authors

Shuo-Fu Yuan¹, Tzu-Hui Wu², Hsiao-Lin Lee³, Han-Yu Hsieh¹, Wen-Ling Lin³, Barbara Yang¹, Chih-Kang Chang³, Qian Li⁴, Jian Gao⁴, Chun-Hsiang Huang⁴, Meng-Chiao Ho⁵, Rey-Ting Guo⁶, Po-Huang Liang⁷

Affiliations

¹ the Institute of Biochemical Sciences, and.
² the Institute of Biotechnology, National Taiwan University, Taipei 10617, Taiwan and.
³ From the Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
⁴ the Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
⁵ the Institute of Biochemical Sciences, and From the Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan, [email protected].
⁶ the Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China [email protected].
⁷ the Institute of Biochemical Sciences, and From the Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan, [email protected].

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.

Keywords: Biodegradation; Biofuel; Site-directed Mutagenesis; Substrate Specificity; X-ray Crystallography.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Amino Acids / chemistry
Amino Acids / genetics
Amino Acids / metabolism
Bacterial Proteins / chemistry*
Bacterial Proteins / genetics
Bacterial Proteins / metabolism
Binding Sites / genetics
Catalytic Domain
Cellobiose / chemistry
Cellobiose / metabolism
Cellulase / chemistry*
Cellulase / genetics
Cellulase / metabolism
Clostridium thermocellum / enzymology*
Clostridium thermocellum / genetics
Crystallography, X-Ray
Disaccharides / chemistry
Disaccharides / metabolism
Endo-1,4-beta Xylanases / chemistry*
Endo-1,4-beta Xylanases / genetics
Endo-1,4-beta Xylanases / metabolism
Enzyme Assays
Kinetics
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Protein Binding
Protein Structure, Tertiary*
Recombinant Proteins / chemistry
Recombinant Proteins / metabolism
Sequence Homology, Amino Acid
Substrate Specificity
Thermotoga maritima / enzymology
Thermotoga maritima / genetics

Substances

Amino Acids
Bacterial Proteins
Disaccharides
Recombinant Proteins
Cellobiose
Cellulase
Endo-1,4-beta Xylanases
xylobiose