Biosynthesis of the O antigen of pathogenic Escherichia coli O157:H7. Characterization of α1,4-Fuc-transferase WbdO.

The O157:H7 strain of Escherichia coli is responsible for frequent outbreaks of hemorrhagic colitis worldwide. Its lipopolysaccharide is a virulence factor and contains an O antigen having repeating units with the tetrasaccharide structure [2-D-PerNAcα1-3-L-Fucα1-4-D-Glcβ1-3-D-GalNAcα1-]n. Genes encoding glycosyltransferases WbdN, WbdO, and WbdP are responsible for the biosynthesis of this repeating unit. We have previously characterized the second enzyme in the pathway, WbdN, which transfers Glc in β1-3 linkage to GalNAcα-O-PO3-PO3-(CH2)11-O-Ph (GalNAc-PP-PhU). In this work, Fuc-transferase WbdO from E. coli O157:H7 expressed in BL21 bacteria was characterized using the product of WbdN as the acceptor substrate. We showed that WbdO is specific for GDP-β-L-Fuc as the donor substrate. Compounds that contained terminal Glc or Glcβ1-3GalNAc structures but lacked the diphosphate group did not serve as acceptor substrates. The structure of the WbdO product was identified by mass spectrometry and Nuclear magnetic resonance (NMR) as L-Fucα1-4-D-Glcβ1-3-D-GalNAc PP-PhU. WbdO is an unusual bivalent metal ion-dependent Fuc-transferase classified as an inverting GT2 family enzyme that has 2 conserved sequences near the N-terminus. The Asp37 residue within the 36VDGGSTD42 sequence was found to be essential for catalysis. Mutation of Asp68 to Ala within the conserved 67YDAMNK72 sequence resulted in a 3-fold increase in activity. These studies show that WbdOO157 is a highly specific Fuc-transferase with little homology to other characterized Fuc-transferases.