P. aeruginosa SGNH Hydrolase-Like Proteins AlgJ and AlgX Have Similar Topology but Separate and Distinct Roles in Alginate Acetylation

Authors

Perrin Baker, The Hospital for Sick Children
Tyler Ricer, University of Toronto
Patrick J. Moynihan, University of Guelph
Elena N. Kitova, University of Alberta
Marthe T. C. Walvoort, Leiden University
Dustin J. Little, The Hospital for Sick Children, University of Toronto
John C. Whitney, The Hospital for Sick Children, University of Toronto
Karen Dawson, The Hospital for Sick Children, University of Toronto
Joel T. Weadge, The Hospital for Sick Children
Howard Robinson, Brookhaven National Laboratory
Dennis E. Ohman, Virginia Commonwealth UniversityFollow
Jeroen D. C. Codee, Leiden University
John S. Klassen, University of Alberta
Anthony J. Clarke, University of Guelph
P. Lynne Howell, The Hospital for Sick Children, University of Toronto

Document Type

Article

Original Publication Date

2014

Journal/Book/Conference Title

PLOS Pathogens

Volume

10

DOI

10.1371/journal.ppat.1004334

Comments

Originally Published at http://dx.doi.org/10.1371/journal.ppat.1004334

Date of Submission

November 2014

Abstract

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.

Rights

Copyright: © 2014 Baker et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Is Part Of

VCU Microbiology and Immunology Publications