Glycobiology Advance Access published online on December 29, 2004
Glycobiology, doi:10.1093/glycob/cwi038
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1 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;
In this work, we demonstrate that the wbbD gene of the O7 lipopolysaccharide (LPS) biosynthesis cluster in E. coli strain VW187 (O7:K1) encodes a galactosyltransferase involved in the synthesis of the O7-polysaccharide repeating unit. The galactosyltransferase catalyzed the transfer of galactose (Gal) from UDP-Gal to the Nacetylglucosamine (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
Received September 16, 2004
Revised November 18, 2004
Accepted December 23, 2004
Article
The wbbD gene of Escherichia coli strain VW187 (O7:K1) encodes a UDP-Gal: GlcNAc
-pyrophosphate-R 
1,3-galactosyltransferase involved in the biosynthesis of O7-specific lipopolysaccharide.*
2 Department of Chemistry, Queen’s University, Kingston, Ontario K7L 3N6, Canada;
3 Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, Canada.
Abstract 
-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 
-galactosidase demonstrated a â-linkage. Cleavage of product by pyrophosphatase and phosphatase, followed by HPLC and NMR analyses revealed a disaccharide with the structure Gal 1-3GlcNAc. Our results conclusively demonstrate that WbbD is a UDP-Gal: GlcNAc-pyrophosphate-R 1,3-galactosyltransferase and suggest that the novel synthetic glycolipid acceptor may be generally applicable to characterize other bacterial glycosyltransferases.
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