Background: Polysaccharide intercellular adhesin-dependent biofilm formation in E. coli requires the de-N-acetylation of poly-␤-1,6-N-acetyl-D-glucosamine by PgaB. Results: Nickel-and iron-bound structures of PgaB have been determined, and the metal-dependent de-N-acetylase activity of the enzyme has been characterized. Conclusion: PgaB has low catalytic efficiency and shows preference for Co 2ϩ , Ni 2ϩ , and Fe 2ϩ ions. Significance: The structure of PgaB will guide inhibitor design to combat biofilm formation.

Background:The pathways governing biosynthesis of the Aspergillus fumigatus exopolysaccharide galactosaminogalactan are poorly understood. Results: The structure of Sph3 revealed a (␤/␣) 8 barrel fold. The enzyme hydrolyzes galactosaminogalactan and is required for the synthesis of this exopolysaccharide. Conclusion: Sph3 is a glycoside hydrolase (GH) whose activity is essential for galactosaminogalactan biosynthesis. Significance: Sph3 defines a new glycoside hydrolase superfamily, GH family 135.

Background:Bacillus subtilis is a model organism for analyzing bacterial biofilms, but the carbohydrate components are undescribed. Results: Genes in the epsHIJK locus needed for biofilm formation encode proteins synthesizing the conserved bacterial polysaccharide poly-N-acetylglucosamine (PNAG). Conclusion: PNAG is a major carbohydrate component of B. subtilis biofilms. Significance: PNAG production is essential for formation of B. subtilis biofilms.

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The O-acetylation of the essential cell wall polymer peptidoglycan occurs in most Gram-positive bacterial pathogens, including species of Staphylococcus, Streptococcus and Enterococcus. This modification to peptidoglycan protects these pathogens from the lytic action of the lysozymes of innate immunity systems and, as such, is recognized as a virulence factor. The key enzyme involved, peptidoglycan O-acetyltransferase A (OatA) represents a particular challenge to biochemical study since it is a membrane associated protein whose substrate is the insoluble peptidoglycan cell wall polymer. OatA is predicted to be bimodular, being comprised of an N-terminal integral membrane domain linked to a C-terminal extracytoplasmic domain. We present herein the first biochemical and kinetic characterization of the C-terminal catalytic domain of OatA from two important human pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Using both pseudosubstrates and novel biosynthetically-prepared peptidoglycan polymers, we characterized distinct substrate specificities for the two enzymes. In addition, the high resolution crystal structure of the C-terminal domain reveals an SGNH/GDSL-like hydrolase fold with a catalytic triad of amino acids but with a non-canonical oxyanion hole structure. Site-specific replacements confirmed the identity of the catalytic and oxyanion hole residues. A model is presented for the O-acetylation of peptidoglycan whereby the translocation of acetyl groups from a cytoplasmic source across the cytoplasmic membrane is catalyzed by the N-terminal domain of OatA for their transfer to peptidoglycan by its C-terminal domain. This study on the structure-function relationship of OatA provides a molecular and mechanistic understanding of this bacterial resistance mechanism opening the prospect for novel chemotherapeutic exploration to enhance innate immunity protection against Gram-positive pathogens.

The O-acetylation of polysaccharides is a common modification used by pathogenic organisms to protect against external forces. Pseudomonas aeruginosa secretes the anionic, O-acetylated exopolysaccharide alginate during chronic infection in the lungs of cystic fibrosis patients to form the major constituent of a protective biofilm matrix. Four proteins have been implicated in the O-acetylation of alginate, AlgIJF and AlgX. To probe the biological function of AlgJ, we determined its structure to 1.83 Å resolution. AlgJ is a SGNH hydrolase-like protein, which while structurally similar to the N-terminal domain of AlgX exhibits a distinctly different electrostatic surface potential. Consistent with other SGNH hydrolases, we identified a conserved catalytic triad composed of D190, H192 and S288 and demonstrated that AlgJ exhibits acetylesterase activity in vitro. Residues in the AlgJ signature motifs were found to form an extensive network of interactions that are critical for O-acetylation of alginate in vivo. Using two different electrospray ionization mass spectrometry (ESI-MS) assays we compared the abilities of AlgJ and AlgX to bind and acetylate alginate. Binding studies using defined length polymannuronic acid revealed that AlgJ exhibits either weak or no detectable polymer binding while AlgX binds polymannuronic acid specifically in a length-dependent manner. Additionally, AlgX was capable of utilizing the surrogate acetyl-donor 4-nitrophenyl acetate to catalyze the O-acetylation of polymannuronic acid. Our results, combined with previously published in vivo data, suggest that the annotated O-acetyltransferases AlgJ and AlgX have separate and distinct roles in O-acetylation. Our refined model for alginate acetylation places AlgX as the terminal acetlytransferase and provides a rationale for the variability in the number of proteins required for polysaccharide O-acetylation.

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 (PgaB 310-672 ) directly binds PNAG oligomers with dissociation constants of ∼1-3 mM, and the structures of PgaB 310-672 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, PgaB 310-672 contains a β-hairpin loop (βHL) important for binding PNAG that was disordered in previous PgaB 42-655 structures and is highly dynamic in the MD simulations. We propose that conformational changes in PgaB 310-672 mediated by the βHL on binding of PNAG/dPNAG play an important role in the targeting of the polymer for export and its release.exopolysaccharide biosynthesis | glycobiology | carbohydrate binding | deacetylase

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that expresses type IVa pili. The pilus assembly system, which promotes surface-associated twitching motility and virulence, is composed of inner and outer membrane subcomplexes, connected by an alignment subcomplex composed of PilMNOP. PilM binds to the N terminus of PilN, and we hypothesize that this interaction causes functionally significant structural changes in PilM. To characterize this interaction, we determined the crystal structures of PilM and a PilM chimera where PilM was fused to the first 12 residues of PilN (PilM⅐PilN(1-12)).