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The Carbohydrate Esterase family 4 contains virulence factors which modify peptidoglycan and biofilm-related exopolysaccharides. Despite the importance of this family of enzymes, a potent mechanism-based inhibition strategy has yet to emerge. Based on the postulated tridentate binding mode of the tetrahedral de-N-acetylation intermediate, GlcNAc derivatives bearing metal chelating groups at the 2 and 3 positions were synthesized. These scaffolds include 2-C phosphonate, 2-C sulfonamide, 2-thionoacetamide warheads as well as derivatives bearing thiol, amine and azide substitutions at the 3-position. The inhibitors were assayed against a representative peptidoglycan deacetylase, Pgda from Streptococcus pneumonia, and a representative biofilm-related exopolysaccharide deacetylase, PgaB from Escherichia coli. Of the inhibitors evaluated, the 3-thio derivatives showed weak to moderate inhibition of Pgda. The strongest inhibitor was benzyl 2,3-dideoxy-2-thionoacetamide-3-thio-β-d-glucoside, whose inhibitory potency showed an unexpected dependence on metal concentration and was found to have a partial mixed inhibition mode (K i  = 2.9 ± 0.6 μM)., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bordetella pertussis and Bordetella bronchiseptica are the causative agents of whooping cough in humans and a variety of respiratory diseases in animals, respectively. Bordetella species produce an exopolysaccharide, known as the Bordetella polysaccharide (Bps), which is encoded by the bpsABCD operon. Bps is required for Bordetella biofilm formation, colonization of the respiratory tract, and confers protection from complement-mediated killing. In this report, we have investigated the role of BpsB in the biosynthesis of Bps and biofilm formation by B. bronchiseptica. BpsB is a two-domain protein that localizes to the periplasm and outer membrane. BpsB displays metal- and length-dependent deacetylation on poly-β-1,6-N-acetyl-d-glucosamine (PNAG) oligomers, supporting previous immunogenic data that suggests Bps is a PNAG polymer. BpsB can use a variety of divalent metal cations for deacetylase activity and showed highest activity in the presence of Ni(2+) and Co(2+). The structure of the BpsB deacetylase domain is similar to the PNAG deacetylases PgaB and IcaB and contains the same circularly permuted family four carbohydrate esterase motifs. Unlike PgaB from Escherichia coli, BpsB is not required for polymer export and has unique structural differences that allow the N-terminal deacetylase domain to be active when purified in isolation from the C-terminal domain. Our enzymatic characterizations highlight the importance of conserved active site residues in PNAG deacetylation and demonstrate that the C-terminal domain is required for maximal deacetylation of longer PNAG oligomers. Furthermore, we show that BpsB is critical for the formation and complex architecture of B. bronchiseptica biofilms., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Poly-β-1,6-N-acetyl-D-glucosamine (PNAG) is an exopolysaccharide produced by a wide variety of medically important bacteria. Polyglucosamine subunit B (PgaB) is responsible for the de-N-acetylation of PNAG, a process required for polymer export and biofilm formation. PgaB is located in the periplasm and likely bridges the inner membrane synthesis and outer membrane export machinery. Here, we present structural, functional, and molecular simulation data that suggest PgaB associates with PNAG continuously during periplasmic transport. We show that the association of PgaB's N- and C-terminal domains forms a cleft required for the binding and de-N-acetylation of PNAG. Molecular dynamics (MD) simulations of PgaB show a binding preference for N-acetylglucosamine (GlcNAc) to the N-terminal domain and glucosammonium to the C-terminal domain. Continuous ligand binding density is observed that extends around PgaB from the N-terminal domain active site to an electronegative groove on the C-terminal domain that would allow for a processive mechanism. PgaB's C-terminal domain (PgaB310-672) directly binds PNAG oligomers with dissociation constants of ∼1-3 mM, and the structures of PgaB310-672 in complex with β-1,6-(GlcNAc)6, GlcNAc, and glucosamine reveal a unique binding mode suitable for interaction with de-N-acetylated PNAG (dPNAG). Furthermore, PgaB310-672 contains a β-hairpin loop (βHL) important for binding PNAG that was disordered in previous PgaB42-655 structures and is highly dynamic in the MD simulations. We propose that conformational changes in PgaB310-672 mediated by the βHL on binding of PNAG/dPNAG play an important role in the targeting of the polymer for export and its release.