2XFG

Reassembly and co-crystallization of a family 9 processive endoglucanase from separately expressed GH9 and CBM3c modules

Summary for 2XFG

• Entry DOI: 10.2210/pdb2xfg/pdb
• Descriptor: ENDOGLUCANASE 1, CALCIUM ION, CHLORIDE ION, ... (5 entities in total)
• Functional Keywords: hydrolase-sugar binding protein complex, family-9 glycoside hydrolase, hydrolase, sugar binding protein, hydrolase/sugar binding protein
• Biological source: CLOSTRIDIUM THERMOCELLUM; More
• Total number of polymer chains: 2
• Total formula weight: 72586.18

Authors

Petkun, S.,Lamed, R.,Jindou, S.,Burstein, T.,Yaniv, O.,Shoham, Y.,Shimon, J.W.L.,Bayer, E.A.,Frolow, F. (deposition date: 2010-05-24, release date: 2011-06-22, Last modification date: 2023-12-20)

Primary citation

Petkun, S.,Grinberg, I.R.,Lamed, R.,Jindou, S.,Burstein, T.,Yaniv, O.,Shoham, Y.,Shimon, J.W.L.,Bayer, E.A.,Frolow, F.
Reassembly and Co-Crystallization of a Family 9 Processive Endoglucanase from its Component Parts: Structural and Functional Significance of Intermodular Linker
Peerj, 3:E1126-, 2015

PubMed Abstract: Non-cellulosomal processive endoglucanase 9I (Cel9I) from Clostridium thermocellum is a modular protein, consisting of a family-9 glycoside hydrolase (GH9) catalytic module and two family-3 carbohydrate-binding modules (CBM3c and CBM3b), separated by linker regions. GH9 does not show cellulase activity when expressed without CBM3c and CBM3b and the presence of the CBM3c was previously shown to be essential for endoglucanase activity. Physical reassociation of independently expressed GH9 and CBM3c modules (containing linker sequences) restored 60-70% of the intact Cel9I endocellulase activity. However, the mechanism responsible for recovery of activity remained unclear. In this work we independently expressed recombinant GH9 and CBM3c with and without their interconnecting linker in Escherichia coli. We crystallized and determined the molecular structure of the GH9/linker-CBM3c heterodimer at a resolution of 1.68 Å to understand the functional and structural importance of the mutual spatial orientation of the modules and the role of the interconnecting linker during their re-association. Enzyme activity assays and isothermal titration calorimetry were performed to study and compare the effect of the linker on the re-association. The results indicated that reassembly of the modules could also occur without the linker, albeit with only very low recovery of endoglucanase activity. We propose that the linker regions in the GH9/CBM3c endoglucanases are important for spatial organization and fixation of the modules into functional enzymes.

PubMed: 26401442
DOI: 10.7717/PEERJ.1126

Experimental method

X-RAY DIFFRACTION (1.679 Å)