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Physical association of the catalytic and helper modules of a family-9 glycoside hydrolase is essential for activity |
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| Authors: | Tal Burstein Sadanari Jindou Felix Frolow Edward A Bayer Raphael Lamed |
| Institution: | a Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv 69978, Israel b Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel c Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel |
| 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) |
| Keywords: | 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&rsquo s CMC carboxymethyl cellulose |
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