Novel clostridial cell-surface hemicellulose-binding CBM3 proteins

Area of Science:

• Structural biology
• Biochemistry
• Microbiology

Background:

• Family 3 carbohydrate-binding modules (CBM3s) are typically known for binding cellulose.
• A highly expressed CBM3 in Clostridium thermocellum (CtCBM3-0271) was investigated for its substrate specificity.

Purpose of the Study:

• To determine the substrate binding preference and structural basis of CtCBM3-0271 and its orthologue CcCBM3-1192.
• To understand the role of calcium ions in CBM3 structure and function.

Main Methods:

• Gene expression analysis in Clostridium thermocellum.
• Crystallization and X-ray crystallography to determine the 3D structures of CtCBM3-0271 and CcCBM3-1192.
• Comparative structural analysis and functional binding assays.

Main Results:

• Both CtCBM3-0271 and CcCBM3-1192 exhibit binding to xylan, not cellulose, due to a lack of key binding residues.
• CcCBM3-1192 lacks a structural calcium ion, leading to conformational changes in loop regions compared to calcium-bound CBM3s.
• The calcium-binding site residues are conserved in CcCBM3-1192 despite the absence of the ion.

Conclusions:

• The studied CBM3s possess an unexpected substrate specificity for xylan, challenging the general understanding of CBM3 function.
• Structural differences, including the absence of calcium in CcCBM3-1192, influence CBM3 conformation and potentially substrate interaction.
• Further research is needed to reconcile the high expression of CtCBM3-0271 on cellulose with its observed xylan-binding activity.