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Functional Characterization of Staphylococcus epidermidis IcaB, a De-N-acetylase Important for Biofilm Formation

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† Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada
‡ Program in Molecular Structure & Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada
§ Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada
*E-mail [email protected], phone 416-946-0640 (M.N.).
Cite this: Biochemistry 2013, 52, 32, 5463–5471
Publication Date (Web):July 18, 2013
https://doi.org/10.1021/bi400836g
Copyright © 2013 American Chemical Society
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    Abstract

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    A polymer of partially de-N-acetylated β-1,6-linked N-acetylglucosamine (dPNAG), also known as the polysaccharide intercellular adhesin (PIA), is an important component of many bacterial biofilm matrices. In Staphyloccocus epidermidis, the poly-N-acetylglucosamine polymer is partially de-N-acetylated by the extracellular protein IcaB. To understand the mechanism of action of IcaB, the enzyme was overexpressed and purified. IcaB demonstrates metal-dependent de-N-acetylase activity on β-1,6-linked N-acetylglucosamine oligomers with a broad preference for divalent metals. Steady-state kinetic analysis reveals the low catalytic efficiency (pentasaccharide kcat/KM 0.03 M–1 s–1) of the enzyme toward the oligomeric substrates. While IcaB displays similar rates of de-N-acetylation with tri- through hexasaccharide PNAG oligomers, position specific de-N-acetylation was only observed with penta- and hexasaccharides. The enzyme preferentially de-N-acetylates the second residue from the reducing terminus in the pentasaccharide and second and third residues from the reducing terminus in the hexasaccharide. The data described here represent an important step toward a detailed understanding of dPNAG biosynthesis in S. epidermidis, an important nosocomial pathogen, as well as in other Gram-positive bacteria. The low catalytic activity of IcaB is consistent with reports of other enzymes which act on biofilm-related polysaccharides, and this emerging trend may indicate a common feature among this group of polysaccharide processing enzymes.

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    IcaBHis10 expression and purification SDS-PAGE gel, gel filtration chromatography of IcaBHis10 and IcaB, ESI-MS/MS analysis of the [M + H]+ precursor ion, structures and calculated molecular weights of each fragment of monode-N-acetylated GlcNAc trisaccharides, tetrasaccharides, and hexasaccharides. This material is available free of charge via the Internet at http://pubs.acs.org.

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