Issue 43, 2019

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

Abstract

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.

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

Supplementary files

Article information

Article type
Paper
Submitted
24 Sep 2019
Accepted
10 Oct 2019
First published
23 Oct 2019

Org. Biomol. Chem., 2019,17, 9456-9466

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

A. Forman, R. Pfoh, A. Eddenden, P. L. Howell and M. Nitz, Org. Biomol. Chem., 2019, 17, 9456 DOI: 10.1039/C9OB02079A

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