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.

Structured summary:

MINT-6946626:

Cel9I (uniprotkb:Q02934) and Cel9I (uniprotkb:Q02934) bind (MI:0407) by comigration in non-denaturing gel electrophoresis (MI:0404)

MINT-6946649:

Cel9I (uniprotkb:Q02934) and Cel9I (uniprotkb:Q02934) bind (MI:0407) by molecular sieving (MI:0071)

MINT-6946687:

Cel9I (uniprotkb:Q02934) and Cel9I (uniprotkb:Q02934) bind (MI:0407) by isothermal titration calorimetry (MI:0065)

MINT-6946706:

Cel9I (uniprotkb:Q02934) binds (MI:0407) to Cel9I (uniprotkb:Q02934) by pull down (MI:0096)

Abbreviations: Cel9I, full-length cellulase 9I from C. thermocellum, GH9, catalytic module (family-9 GH) of Cel9I, CBM3c, family-3c carbohydrate-binding module with intact N-terminal linker, CBM3b, family-3b carbohydrate-binding module with intact N-terminal linker, CBM3c–CBM3b, tandem dyad of the two CBM3’s, CMC, carboxymethyl cellulose

Keywords: Family-3 carbohydrate-binding module (CBM3), Family-9 glycoside hydrolases (GH9), Cellulose, Cellulase, Endoglucanase