title: Physical association of the catalytic and helper modules of a family-9 glycoside hydrolase is essential for activity.
abstract: Clostridium thermocellum cellulase 9I (Cel9I) is a non-cellulosomal tri-modular enzyme, consisting of a family-9 glycoside hydrolase (GH9) catalytic module and two family-3 carbohydrate-binding modules (CBM3c and CBM3b). The presence of CBM3c was previously shown to be essential for activity, however the mechanism by which it functions is unclear. We expressed the three recombinant modules independently in Escherichia coli and examined their interactions. Non-denaturing gel electrophoresis, isothermal titration calorimetry, and affinity purification of the GH9-CBM3c complex revealed a specific non-covalent binding interaction between the GH9 module and CBM3c. Their physical association was shown to recover 60-70% of the intact Cel9I endoglucanase activity.
authors.affiliation: Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv 69978, Israel.
authors.forename: Tal
authors.initials: T
authors.lastname: Burstein
authors.forename: Michal
authors.initials: M
authors.lastname: Shulman
authors.forename: Sadanari
authors.initials: S
authors.lastname: Jindou
authors.forename: Svetlana
authors.initials: S
authors.lastname: Petkun
authors.forename: Felix
authors.initials: F
authors.lastname: Frolow
authors.forename: Yuval
authors.initials: Y
authors.lastname: Shoham
authors.forename: Edward A
authors.initials: EA
authors.lastname: Bayer
authors.forename: Raphael
authors.initials: R
authors.lastname: Lamed
mesh_terms: D055162:Biocatalysis; D002151:Calorimetry; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry; D003001:Cloning, Molecular; D048013:Acetivibrio thermocellus / Q000201:enzymology / Q000235:genetics; D004789:Enzyme Activation; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization
chemical_list: D002241:Carbohydrates; D006026:Glycoside Hydrolases