Glycobiology Advance Access originally published online on December 29, 2004
Glycobiology 2005 15(6):605-613; doi:10.1093/glycob/cwi038
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Glycobiology vol. 15 no. 6 © Oxford University Press 2004; all rights reserved.
The wbbD gene of E. coli strain VW187 (O7:K1) encodes a UDP-Gal: GlcNAc
-pyrophosphate-R ß1,3-galactosyltransferase involved in the biosynthesis of O7-specific lipopolysaccharide
2 Department of Medicine, Department of Biochemistry, The Arthritis Centre and Human Mobility Research Centre, Queen’s University, Kingston General Hospital, Kingston, Ontario K7L 2V7, Canada; 3 Department of Chemistry, Queen’s University, Kingston, Ontario K7L 3N6, Canada; 4 Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, Canada
1 To whom correspondence should be addressed; email: brockhau{at}post.queensu.com
Received on September 16, 2004; revised on November 18, 2004; accepted on December 23, 2004
In this work, we demonstrate that the wbbD gene of the O7 lipopolysaccharide (LPS) biosynthesis cluster in Escherichia coli strain VW187 (O7:K1) encodes a galactosyltransferase involved in the synthesis of the O7-polysaccharide repeating unit. The galactosyltransferase catalyzed the transfer of Gal from UDP-Gal to the GlcNAc residue of a GlcNAc-pyrophosphate-lipid acceptor. A mutant strain with a defective wbbD gene was unable to form O7 LPS and lacked this specific galactosyltransferase activity. The normal phenotype was restored by complementing the mutant with the cloned wbbD gene. To characterize the WbbD galactosyltransferase, we used a novel acceptor substrate containing GlcNAc
-pyrophosphate covalently bound to a hydrophobic phenoxyundecyl moiety (GlcNAc -O-PO3-PO3-(CH2)11-O-phenyl). The WbbD galactosyltransferase had optimal activity at pH 7 in the presence of 2.5 mM MnCl2. Detergents in the assay did not increase glycosyl transfer. Digestion of enzyme product by highly purified bovine testicular ß-galactosidase demonstrated a ß-linkage. Cleavage of product by pyrophosphatase and phosphatase, followed by HPLC and NMR analyses, revealed a disaccharide with the structure Gal ß1-3GlcNAc. Our results conclusively demonstrate that WbbD is a UDP-Gal: GlcNAc-pyrophosphate-R ß1,3-galactosyltransferase and suggest that the novel synthetic glycolipid acceptor may be generally applicable to characterize other bacterial glycosyltransferases.
Key words: enzyme assay / galactosyltransferase / O antigen synthesis / undecaprenol-P
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