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 α-2 and α-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-[3H]Rha. Using 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 ∆wbpZ 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. IMPORTANCE: 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.
O-antigen, Pseudomonas aeruginosa, glycosyltransferase, common polysaccharide antigen, D-Rha-transferase, WbpZ
NCBI PubMed ID: 25845842Publication DOI: 10.1128/JB.02590-14Journal NLM ID: 2985120RPublisher: American Society for Microbiology
Correspondence: brockhau@queensu.ca (Inka Brockhausen)
Institutions: N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia, Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1, Department of Medicine, Department of Biomedical and Molecular Sciences, Queens University, Kingston, Ontario, Canada K7L 3N6, Department of Chemistry, Queens University, Kingston, Ontario, Canada K7L 3N6
Methods: 13C NMR, 1H NMR, NMR-2D, PCR, SDS-PAGE, DNA techniques, glycosyltransferase assays, kinetics assays, ESI-MS, Western blotting, NMR-1D, genetic methods, biochemical methods, radioactivity measurement, HPLC, mutation analysis, RP-HPLC, enzymatic synthesis
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