title: The family 1 glycoside hydrolase from Clostridium cellulolyticum H10 is a cellodextrin glucohydrolase.
abstract: The only family 1 glycoside hydrolase in Clostridium cellulolyticum H10 (CcGH1) is annotated as a beta-galactosidase but has high sequence homology with many beta-glucosidases. Given the possible importance of beta-glucosidase in cellulose utilization by C. cellulolyticum, the encoding open reading frame Ccel_0374 was cloned and expressed in E. coli as a soluble fusion protein with thioredoxin. After tag cleavage, the purified enzyme had a molecular mass of 52 kDa and was active in dimeric form on a broad range of substrates, including cellobiose, cellotriose, cellotetraose, p-nitrophenyl-beta-glucopyranoside, lactose, and o-nitrophenyl-beta-galactopyranoside. The enzyme showed lower K(m) and higher catalytic efficiency (k (cat)/K(m)) on cellodextrins with degree of polymerization from 2 to 4 than on lactose, and the k (cat)/K (m) values on cellodextrins increased in the order of cellobiose < cellotriose < cellotetraose, suggesting that CcGH1 was a cellodextrin glucohydrolase (EC 3.2.1.74). The high K(m) (69 mM) on cellobiose implies that CcGH1 likely has a minimal role in the intracellular hydrolysis of cellobiose in C. cellulolyticum. The three-dimensional structure model of CcGH1 generated by homology modeling showed a typical (alpha/beta)(8) barrel topology characteristic of family 1 glycoside hydrolases.
authors.affiliation: Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, 210-A Seitz Hall, Blacksburg, VA 24061, USA.
authors.forename: Wenjin
authors.initials: W
authors.lastname: Liu
authors.forename: David R
authors.initials: DR
authors.lastname: Bevan
authors.forename: Y-H Percival
authors.initials: YH
authors.lastname: Zhang
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000031:analogs & derivatives* / Q000378:metabolism; D046973:Ruminiclostridium cellulolyticum / Q000201:enzymology*; D003912:Dextrins / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D003912:Dextrins; D011993:Recombinant Fusion Proteins; D002482:Cellulose; C068994:cellodextrin; D006026:Glycoside Hydrolases