Title | Synthesis of defined mono-de-N-acetylated β-(1→6)-N-acetyl-d-glucosamine oligosaccharides to characterize PgaB hydrolase activity. |
Publication Type | Journal Article |
Year of Publication | 2019 |
Authors | Forman, A, Pfoh, R, Eddenden, A, P Howell, L, Nitz, M |
Journal | Org Biomol Chem |
Volume | 17 |
Issue | 43 |
Pagination | 9456-9466 |
Date Published | 2019 11 06 |
ISSN | 1477-0539 |
Keywords | Acetylation, Acetylglucosamine, Amidohydrolases, Biofilms, Escherichia coli Proteins, Hydrolysis, Molecular Conformation, Oligosaccharides |
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. |
DOI | 10.1039/c9ob02079a |
Alternate Journal | Org Biomol Chem |
PubMed ID | 31642455 |