Single polysaccharide assembly protein that integrates polymerization, termination, and chain-length quality control

Area of Science:

• Bacterial glycobiology
• Molecular microbiology
• Biochemistry

Background:

• Lipopolysaccharides (LPS) are crucial outer membrane components of Gram-negative bacteria.
• O-antigenic polysaccharide (OPS) biosynthesis utilizes diverse strategies, including ATP-binding cassette (ABC) transporter-dependent pathways.
• Understanding these pathways is key to deciphering bacterial surface structure and function.

Purpose of the Study:

• To characterize the Raoultella terrigena WbbB protein and its role in OPS biosynthesis.
• To elucidate the function of novel glycosyltransferase (GT) modules within WbbB.
• To investigate the mechanism of polysaccharide chain assembly and termination.

Main Methods:

• Protein purification and characterization of Raoultella terrigena WbbB.
• In vitro enzymatic assays to determine glycosyltransferase activity.
• Structural analysis of protein domains and polysaccharide products.

Main Results:

• WbbB integrates three distinct glycosyltransferase (GT) modules into a single polypeptide.
• Two novel GT families (GT102 and GT103) were identified, forming a polymerase for [4)-α-Rhap-(1→3)-β-GlcNAc-(1→] repeat units.
• A GT99 module terminates the chain with a Kdo residue, and a coiled-coil structure acts as a molecular ruler for chain length.

Conclusions:

• WbbB represents a prototype for a family of multifunctional proteins in bacterial polysaccharide synthesis.
• The modular organization allows for the generation of diverse OPS structures, contributing to antigenic variation.
• These findings offer potential for glycoengineering applications and a deeper understanding of bacterial immunity.