Structural basis for the de-N-acetylation of poly-{beta}-1,6-N-acetyl-D-glucosamine in Gram-positive bacteria [Microbiology]

October 30th, 2014 by Little, D. J., Bamford, N. C., Podrovskaya, V., Robinson, H., Nitz, M., Howell, P. L.

Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In staphylococci, partial de-N-acetylation of the exopolysaccharide poly-β-1,6-N-acetyl-D-glucosamine (PNAG) by the extracellular protein IcaB is required for biofilm formation. To understand the molecular basis for PNAG de-N-acetylation, the structure of IcaB from Ammonifex degensii (AmIcaB) has been determined to 1.7 Å resolution. The structure of AmIcaB reveals a (β/α)7 barrel common to the family four carbohydrate esterases (CE4s), with the canonical motifs circularly permuted. The metal dependence of AmIcaB is similar to most CE4s showing the maximum rates of de-N-acetylation with Ni2+, Co2+ and Zn2+. Docking studies with β-1,6-GlcNAc oligomers and structural comparison to PgaB from Escherichia coli, the Gram-negative homologue of IcaB, we identify R45, Y67, and W180 as key residues for PNAG binding during catalysis. The absence of these residues in PgaB provides a rationale for the requirement of a C-terminal domain for efficient deacetylation of PNAG in Gram-negative species. Mutational analysis of conserved active site residues suggests that IcaB uses an altered catalytic mechanism in comparison to other characterized CE4 members. Furthermore, we identified a conserved surface-exposed hydrophobic loop found only in Gram-positive homologues of IcaB. This loop is required for membrane association, and likely anchors IcaB to the membrane during polysaccharide biosynthesis. The work presented herein will help guide the design of IcaB inhibitors to combat biofilm formation by staphylococci.