The structure- and metal-dependent activity of Escherichia coli PgaB provides insight into the partial de-N-acetylation of poly-β-1,6-N-acetyl-D-glucosamine

Abstract

Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In Escherichia coli, partial de-N-acetylation of the exopolysaccharide poly-β-1,6-N-acetyl-D-glucosamine (PNAG) by the periplasmic protein PgaB is required for polysaccharide intercellular adhesin-dependent biofilm formation. To understand the molecular basis for PNAG de-N-acetylation, the structure of PgaB in complex with Ni(2+) and Fe(3+) have been determined to 1.9 and 2.1 Å resolution, respectively, and its activity on β-1,6-GlcNAc oligomers has been characterized. The structure of PgaB reveals two (β/α)(x) barrel domains: a metal-binding de-N-acetylase that is a member of the family 4 carbohydrate esterases (CE4s) and a domain structurally similar to glycoside hydrolases. PgaB displays de-N-acetylase activity on β-1,6-GlcNAc oligomers but not on the β-1,4-(GlcNAc)(4) oligomer chitotetraose and is the first CE4 member to exhibit this substrate specificity. De-N-acetylation occurs in a length-dependent manor, and specificity is observed for the position of de-N-acetylation. A key aspartic acid involved in de-N-acetylation, normally seen in other CE4s, is missing in PgaB, suggesting that the activity of PgaB is attenuated to maintain the low levels of de-N-acetylation of PNAG observed in vivo. The metal dependence of PgaB is different from most CE4s, because PgaB shows increased rates of de-N-acetylation with Co(2+) and Ni(2+) under aerobic conditions, and Co(2+), Ni(2+) and Fe(2+) under anaerobic conditions, but decreased activity with Zn(2+). The work presented herein will guide inhibitor design to combat biofilm formation by E. coli and potentially a wide range of medically relevant bacteria producing polysaccharide intercellular adhesin-dependent biofilms.

FIGURE 1.

PgaB structure. A and B, overall structure of PgaB shown in cartoon (A) and topology (B) representations with the secondary structural elements labeled. The canonical (β/α)_x folds are colored blue (β-strands) and red (α-helices), respectively. Additional secondary structure elements are colored green, except the CE4 capping helix, which is shown in purple. Residues not modeled in the topology diagram are colored cyan. C, electrostatic surface representation of PgaB (generated in PyMOL using APBS) shown in the same orientation as in A. The face of the C-terminal domain contains a pronounced electronegative groove that extends toward the N-terminal domain. Quantitative electrostatics are colored from red (−20 kT) to blue (+20 kT).

FIGURE 2.

Conservation of the CE4 fold. A, superposition of the PgaB N-terminal domain (green) with SpPgdA C-terminal domain (magenta) in cartoon representation shows the structural similarities between the de-N-acetylase domains. B, active site residues of PgaB and SpPgdA shown in stick representation and colored as in A show differences in the canonical CE4 motifs. C, superposition of the PgaB N-terminal domain with other CE4 structures shown in cartoon representation reveals four β-hairpins, β2–3, β6–7, β9–10, and β11–12, that are unique to PgaB. The structures and corresponding RMSDs to the PgaB N-terminal domain (green) are: SpPgdA C-terminal domain (magenta), 2.6 Å over 141 equivalent Cα atoms; CE4 from Streptomyces lividans (blue), 2.8 Å over 141 equivalent Cα atoms; CDA from C. lindemuthianum (orange), 2.9 Å over 146 equivalent Cα atoms; and PgdA from S. mutans (yellow), 3.3 Å over 141 equivalent Cα atoms.

FIGURE 3.

The C-terminal domain of PgaB has structural similarity to glycoside hydrolases. A, superposition of the C-terminal domain of PgaB (green) and β-amylase from B. cereus (blue) shown in cartoon representation. B, residues lining the electronegative groove with a bound MES molecule (orange) shown in stick representation, and calcium ion (gray) with the |F_o − F_c| electron density map contoured at 3.0 σ. The residues indicated with an asterisk are ≥90% conserved among PgaB homologues for bacteria that have been shown to produce dPNAG in their biofilms.

FIGURE 4.

Substrate dependence of PgaB. Fluorescamine assay for Ni-loaded MBP-PgaB incubated with varying length β-1,6-GlcNAc oligomers showing increased rates of de-N-acetylation with increasing oligomer lengths (A) and β-1,6-(GlcNAc)₄ oligomer or β-1,4-(GlcNAc)₄ chitotetraose showing de-N-acetylation specificity for the β-1,6-(GlcNAc)₄ oligomer (B). The bars represent duplicate experiments with standard deviations.

FIGURE 5.

Metal dependence of PgaB. A, fluorescamine activity assay of MBP-PgaB (as isolated) in the presence of various metals. Increased rates were observed with the addition of Co²⁺ and Ni²⁺. The bars represent duplicate experiments with standard deviations. B, superposition of the nickel- and iron-complexed PgaB de-N-acetylase active sites shown in stick representation and wall-eyed stereo view. Nickel and coordinating residues are shown in cyan, whereas iron and coordinating residues are shown in orange. Additional active site residues conserved among CE4s are colored blue (nickel complex) and yellow (iron complex). The |F_o − F_c| electron density map for the nickel-complexed structure is shown in gray and contoured at 3.0 σ. C, fluorescamine activity assay comparison of MBP-PgaB (as isolated), nickel-loaded MBP-PgaB, and iron-loaded MBP-PgaB under aerobic and anaerobic conditions. The bars represent duplicate experiments with standard deviations.

FIGURE 6.

HPLC analysis of the de-N-acetylation position. A, analysis of β-1,6-GlcNAc standards with arrows indicating elution time of GlcNAc through (GlcNAc)₅. AU, absorbance units. B and C, analysis of re-N-acetylated SpHex degradation products of MBP-PgaB treated (B) β-1,6-(GlcNAc)₄ and (C) β-1,6-(GlcNAc)₅. The peaks were identified by comparing elution time to the β-1,6-(GlcNAc)_n standards and confirmed by MALDI-MS. D, representation of productive binding mode of β-1,6-(GlcNAc)₅ to PgaB, showing de-N-acetylation preference for the central or O-subsite GlcNAc.