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Poly-β(1,6)- N -acetyl-D-glucosamine (PNAG) is a major biofilm component of many pathogenic bacteria. The production, modification, and export of PNAG in  Escherichia coli  and  Bordetella  species require the protein products encoded by the  pgaABCD  operon. PgaB is a two-domain periplasmic protein that contains an N-terminal deacetylase domain and a C-terminal PNAG binding domain that is critical for export. However, the exact function of the PgaB C-terminal domain remains unclear. Herein, we show that the C-terminal domains of  Bordetella bronchiseptica  PgaB (PgaB  Bb  ) and  E .  coli  PgaB (PgaB  Ec  ) function as glycoside hydrolases. These enzymes hydrolyze purified deacetylated PNAG (dPNAG) from  Staphylococcus aureus , disrupt PNAG-dependent biofilms formed by  Bordetella pertussis ,  Staphylococcus carnosus ,  Staphylococcus epidermidis , and  E .  coli , and potentiate bacterial killing by gentamicin. Furthermore, we found that PgaB  Bb   was only able to hydrolyze PNAG produced  in situ  by the  E .  coli  PgaCD synthase complex when an active deacetylase domain was present. Mass spectrometry analysis of the PgaB-hydrolyzed dPNAG substrate showed a GlcN-GlcNAc-GlcNAc motif at the new reducing end of detected fragments. Our 1.76 Å structure of the C-terminal domain of PgaB  Bb   reveals a central cavity within an elongated surface groove that appears ideally suited to recognize the GlcN-GlcNAc-GlcNAc motif. The structure, in conjunction with molecular modeling and site directed mutagenesis led to the identification of the dPNAG binding subsites and D474 as the probable catalytic acid. This work expands the role of PgaB within the PNAG biosynthesis machinery, defines a new glycoside hydrolase family GH153, and identifies PgaB as a possible therapeutic agent for treating PNAG-dependent biofilm infections.

PgaB orthologues contain a glycoside hydrolase domain that cleaves deacetylated poly-β(1,6)-N-acetylglucosamine and can disrupt bacterial biofilms