Physical association of the catalytic and helper modules of a family-9 glycoside hydrolase is essential for activity.
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.
Burstein
Tal
T
Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv 69978, Israel.
Shulman
Michal
M
Jindou
Sadanari
S
Petkun
Svetlana
S
Frolow
Felix
F
Shoham
Yuval
Y
Bayer
Edward A
EA
Lamed
Raphael
R
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
D002241:Carbohydrates; D006026:Glycoside Hydrolases