title: P. aeruginosa SGNH hydrolase-like proteins AlgJ and AlgX have similar topology but separate and distinct roles in alginate acetylation.
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.
authors.affiliation: Program in Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada.
authors.forename: Perrin
authors.initials: P
authors.lastname: Baker
authors.affiliation: Program in Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada; Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Tyler
authors.initials: T
authors.lastname: Ricer
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Moynihan
authors.affiliation: Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Elena N
authors.initials: EN
authors.lastname: Kitova
authors.affiliation: Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.
authors.forename: Marthe T C
authors.initials: MT
authors.lastname: Walvoort
authors.affiliation: Program in Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada; Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Dustin J
authors.initials: DJ
authors.lastname: Little
authors.affiliation: Program in Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada; Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.
authors.forename: John C
authors.initials: JC
authors.lastname: Whitney
authors.affiliation: Program in Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada; Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Karen
authors.initials: K
authors.lastname: Dawson
authors.affiliation: Program in Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada.
authors.forename: Joel T
authors.initials: JT
authors.lastname: Weadge
authors.affiliation: Photon Sciences Division, Brookhaven National Laboratory, Upton, New York, United States of America.
authors.forename: Howard
authors.initials: H
authors.lastname: Robinson
authors.affiliation: Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center and McGuire Veterans Affairs Medical Center, Richmond, Virginia, United States of America.
authors.forename: Dennis E
authors.initials: DE
authors.lastname: Ohman
authors.affiliation: Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.
authors.forename: Jeroen D C
authors.initials: JD
authors.lastname: Codée
authors.affiliation: Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: John S
authors.initials: JS
authors.lastname: Klassen
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Clarke
authors.affiliation: Program in Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada; Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.
authors.forename: P Lynne
authors.initials: PL
authors.lastname: Howell
mesh_terms: D000107:Acetylation; D000464:Alginates / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D020723:Glucuronic Acid / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
chemical_list: C098844:AlgJ protein, Pseudomonas aeruginosa; D000464:Alginates; D001426:Bacterial Proteins; D006603:Hexuronic Acids; D020723:Glucuronic Acid