Synthesis of defined mono-de--acetylated β-(1→6)--acetyl--glucosamine oligosaccharides to characterize PgaB hydrolase activity

Many clinically-relevant biofilm-forming bacterial strains produce partially de- N -acetylated poly-β-(1→6)- N -acetyl- d -glucosamine (dPNAG) as an exopolysaccharide. In Gram-negative bacteria, the periplasmic protein PgaB is responsible for partial de- N -acetylation of PNAG prior to its export to the extracellular space. In addition to de- N -acetylase activity found in the N-terminal domain, PgaB contains a C-terminal hydrolase domain that can disrupt dPNAG-dependent biofilms and hydrolyzes dPNAG but not fully acetylated PNAG. The role of this C-terminal domain in biofilm formation has yet to be determined in vivo . Further characterization of the enzyme's hydrolase activity has been hampered by a lack of specific dPNAG oligosaccharides. Here, we report the synthesis of a defined mono de- N -acetylated dPNAG penta- and hepta-saccharide. Using mass spectrometry analysis and a fluorescence-based thin-layer chromatography (TLC) assay, we found that our defined dPNAG oligosaccharides are hydrolase substrates. In addition to the expected cleavage site, two residues to the reducing side of the de- N -acetylated residue, minor cleavage products on the non-reducing side of the de- N -acetylation site were observed. These findings provide quantitative data to support how PNAG is processed in Gram-negative bacteria. Mono-de- N -acetylated β-(1→6)- N -acetyl- d -glucosamine penta- and hepta-saccharides were obtained using a convergent synthesis. The site of de- N -acetylation drives the selectivity of hydrolysis by PgaB.