The wbbD gene of E. coli strain VW187 (O7:K1) encodes a UDP-Gal: GlcNAc{alpha}-pyrophosphate-R {beta}1,3-galactosyltransferase involved in the biosynthesis of O7-specific lipopolysaccharide.
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 GlcNAcalpha-pyrophosphate covalently bound to a hydrophobic phenoxyundecyl moiety (GlcNAc alpha-O-PO(3)-PO(3)-(CH(2))(11)-O-phenyl). The WbbD galactosyltransferase had optimal activity at pH 7 in the presence of 2.5 mM MnCl(2). Detergents in the assay did not increase glycosyl transfer. Digestion of enzyme product by highly purified bovine testicular beta-galactosidase demonstrated a beta-linkage. Cleavage of product by pyrophosphatase and phosphatase, followed by HPLC and NMR analyses, revealed a disaccharide with the structure Gal beta1-3GlcNAc. Our results conclusively demonstrate that WbbD is a UDP-Gal: GlcNAcalpha-pyrophosphate-R beta1,3-galactosyltransferase and suggest that the novel synthetic glycolipid acceptor may be generally applicable to characterize other bacterial glycosyltransferases.
Riley
John G
JG
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.
Menggad
Mohammed
M
Montoya-Peleaz
Pedro J
PJ
Szarek
Walter A
WA
Marolda
Cristina L
CL
Valvano
Miguel A
MA
Schutzbach
John S
JS
Brockhausen
Inka
I
D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005700:Galactosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D019081:O Antigens / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D019081:O Antigens; D005700:Galactosyltransferases; C505073:WbbD protein, E coli