Development of Mono-de-N-acetylated β-(1→6)-N-acetyl-D-glucosamine Substrates for Characterization of PgaB Hydrolase Activity

Many bacteria form biofilms, surface-attached communities embedded in an extracellular matrix, as a means for survival. The production of the partially de-N-acetylated poly-β-(1→6)-N-acetyl-D-glucosamine (dPNAG) exopolysaccharide is required for biofilm formation in many clinically relevant bacteria. The PgaABCD biosynthetic machinery is responsible for the synthesis and export of dPNAG in Gram-negative bacteria. PgaB is a two-domain periplasmic protein that processes PNAG prior to its export to the extracellular space. ΔpgaB strains have an impaired biofilm-forming phenotype, making PgaB a potential therapeutic target. The N-terminal domain of PgaB is a family 4 carbohydrate esterase that partially de-N-acetylates PNAG. The C-terminal domain is a family 153 glycoside hydrolase whose role in vivo is not fully understood. This domain can degrade dPNAG-based biofilms, demonstrating the potential for the enzyme to be used as a therapeutic treatment for biofilm-mediated infections. It is known that the PgaB C-terminal domain is active on dPNAG, but further characterization of the enzyme’s activity, substrate specificity, and mechanism of hydrolysis has been hampered by a lack of pure mono-de-N-acetylated β-(1→6)-N-acetyl-D-glucosamine (GlcNAc) oligosaccharides to be used as substrates. This thesis describes the synthesis of defined mono-de-N-acetylated β-(1→6)-GlcNAc penta- and hepta-saccharides by using a convergent synthetic method and a multistep selective deprotection. These synthetic oligosaccharides were validated as Bordetella bronchiseptica PgaB C-terminal domain substrates through the development of a fluorescence-based thin-layer chromatography (TLC) assay. Mass spectrometry experiments and nuclear magnetic resonance (NMR) spectroscopy analysis provided further insights into the requirements for substrate recognition and mechanism of hydrolysis. These results are consistent with the previously reported observations that the site of de-N-acetylation drives the selectivity of hydrolysis by PgaB.

Department dc:contributor.department

Chemistry

Year dc:date.issued

2020

Author dc:creator

• Forman, Adam Steven

Advisor dc:contributor.advisor

• Nitz, Mark

• Biofilms
• Biological chemistry
• Carbohydrate chemistry
• Exopolysaccharides
• Glycobiology
• Oligosaccharide synthesis

Statement dc:rights

• Attribution-NonCommercial 4.0 International

Licence dc:rights.uri

• http://creativecommons.org/licenses/by-nc/4.0/

Handle dc:identifier.uri

http://hdl.handle.net/1807/101001

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/101001