Structural basis for binding uronic acids by family 32 carbohydrate-binding modules

Area of Science:

• Biochemistry
• Enzymology
• Carbohydrate Chemistry

Background:

• Alginate lyases are enzymes that degrade alginate, a polysaccharide found in brown algae.
• Carbohydrate-binding modules (CBMs) are protein domains that bind carbohydrates and are often found associated with enzymes.
• The CBM32 family is known to bind various carbohydrate structures, but their specificities are not fully understood.

Purpose of the Study:

• To elucidate the binding mechanism of the CBM32 domain (AlyQ_B) from alginate lyase AlyQ.
• To determine the structural basis for AlyQ_B's specificity towards cleaved alginate.
• To compare the binding properties of AlyQ_B with another CBM32 domain (YeCBM32).

Main Methods:

• Co-crystallisation of AlyQ_B with cleaved alginate.
• X-ray crystallography to determine the structure of the AlyQ_B-alginate complex.
• Molecular docking analysis of YeCBM32 with oligogalacturonic acid.

Main Results:

• AlyQ_B binds to the 4,5-unsaturated mannuronic acid at the non-reducing end of cleaved alginate.
• Key residues R248 and W303 in AlyQ_B are crucial for binding unsaturated mannuronic acid.
• AlyQ_B cannot bind saturated mannuronic or guluronic acids due to steric clashes.
• Docking analysis suggests YeCBM32 has a broader binding capability, accommodating both saturated and unsaturated galacturonic acid.

Conclusions:

• AlyQ_B exhibits high specificity for the non-reducing end of cleaved alginate, targeting unsaturated mannuronic acid.
• The structural insights explain the selective binding of AlyQ_B and its inability to bind saturated sugars.
• Comparison with YeCBM32 highlights variations in binding mechanisms within the CBM32 family.