title: Biosynthesis of the Common Polysaccharide Antigen of Pseudomonas aeruginosa PAO1: Characterization and Role of GDP-D-Rhamnose:GlcNAc/GalNAc-Diphosphate-Lipid α1,3-D-Rhamnosyltransferase WbpZ.
abstract: The opportunistic pathogen Pseudomonas aeruginosa produces two major cell surface lipopolysaccharides, characterized by distinct O antigens, called common polysaccharide antigen (CPA) and O-specific antigen (OSA). CPA contains a polymer of D-rhamnose (D-Rha) in α1-2 and α1-3 linkages. Three putative glycosyltransferase genes, wbpX, wbpY, and wbpZ, are part of the CPA biosynthesis cluster. To characterize the enzymatic function of the wbpZ gene product, we chemically synthesized the donor substrate GDP-D-Rha and enzymatically synthesized GDP-D-[(3)H]Rha. Using nuclear magnetic resonance (NMR) spectroscopy, we showed that WbpZ transferred one D-Rha residue from GDP-D-Rha in α1-3 linkage to both GlcNAc- and GalNAc-diphosphate-lipid acceptor substrates. WbpZ is also capable of transferring D-mannose (D-Man) to these acceptors. Therefore, WbpZ has a relaxed specificity with respect to both acceptor and donor substrates. The diphosphate group of the acceptor, however, is required for activity. WbpZ does not require divalent metal ion for activity and exhibits an unusually high pH optimum of 9. WbpZ from PAO1 is therefore a GDP-D-Rha:GlcNAc/GalNAc-diphosphate-lipid α1,3-D-rhamnosyltransferase that has significant activity of GDP-D-Man:GlcNAc/GalNAc-diphosphate-lipid α1,3-D-mannosyltransferase. We used site-directed mutagenesis to replace the Asp residues of the two DXD motifs with Ala. Neither of the mutant constructs of wbpZ (D172A or D254A) could be used to rescue CPA biosynthesis in the ΔwbpZ knockout mutant in a complementation assay. This suggested that D172 and D254 are essential for WbpZ function. This work is the first detailed characterization study of a D-Rha-transferase and a critical step in the development of CPA synthesis inhibitors.
  
  
  OBJECTIVE
  This is the first characterization of a D-rhamnosyltransferase and shows that it is essential in Pseudomonas aeruginosa for the synthesis of the common polysaccharide antigen.
authors.affiliation: Department of Medicine, Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada.
authors.forename: Shuo
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Youai
authors.initials: Y
authors.lastname: Hao
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Lam
authors.affiliation: Department of Chemistry, Queen's University, Kingston, Ontario, Canada.
authors.forename: Jason Z
authors.initials: JZ
authors.lastname: Vlahakis
authors.affiliation: Department of Chemistry, Queen's University, Kingston, Ontario, Canada.
authors.forename: Walter A
authors.initials: WA
authors.lastname: Szarek
authors.affiliation: Department of Medicine, Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia.
authors.forename: Anna
authors.initials: A
authors.lastname: Vinnikova
authors.affiliation: N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia.
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Veselovsky
authors.affiliation: Department of Medicine, Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada brockhau@queensu.ca.
authors.forename: Inka
authors.initials: I
authors.lastname: Brockhausen
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011135:Polysaccharides, Bacterial / Q000276:immunology / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D013379:Substrate Specificity
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D011135:Polysaccharides, Bacterial; D016695:Glycosyltransferases
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