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

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 interce...

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Bibliographic Details
Published in:The Journal of biological chemistry 2012-09, Vol.287 (37), p.31126-31137
Main Authors: Little, Dustin J., Poloczek, Joanna, Whitney, John C., Robinson, Howard, Nitz, Mark, Howell, P.Lynne
Format: Article
Language:English
Subjects:
Acetylation
Amidohydrolases - chemistry
Amidohydrolases - genetics
Amidohydrolases - metabolism
beta-Glucans - chemistry
beta-Glucans - metabolism
Biofilm
Biofilms
Carbohydrate Esterase
Carbohydrate Processing
Crystallography, X-Ray
De-N-acetyalase
Enzyme Catalysis
Enzyme Structure
Escherichia coli - enzymology
Escherichia coli - genetics
Escherichia coli Proteins - chemistry
Escherichia coli Proteins - genetics
Escherichia coli Proteins - metabolism
Exopolysaccharide Biosynthesis
Iron - chemistry
Iron - metabolism
Microbiology
Nickel - chemistry
Nickel - metabolism
PNAG
Protein Structure, Tertiary
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Summary: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 Ni2+ and Fe3+ 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 Co2+ and Ni2+ under aerobic conditions, and Co2+, Ni2+ and Fe2+ under anaerobic conditions, but decreased activity with Zn2+. 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. Background: Polysaccharide intercellular adhesin-dependent biofilm formation in E. coli requires the de-N-acetylation of poly-β-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 Co2+, Ni2+, and Fe2+ ions. Significance: The structure of PgaB will guide inhibitor design to combat biofilm formation.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M112.390005