Characterization of Two β-1,3-Glucosyltransferases fromEscherichia coliSerotypes O56 and O152

Abstract

The O antigens of outer membrane-bound lipopolysaccharides (LPS) in gram-negative bacteria are oligosaccharides consisting of repeating units with various structures and antigenicities. The O56 and O152 antigens of Escherichia coli both contain a Glc-␤1-3-GlcNAc linkage within the repeating unit. We have cloned and identified the genes (wfaP in O56 and wfgD in O152) within the two O-antigen gene clusters that encode glucosyltransferases involved in the synthesis of this linkage. A synthetic substrate analog of the natural acceptor substrate undecaprenol-pyrophosphate-lipid [GlcNAc-␣-PO 3 -PO 3 -(CH 2 ) 11 -O-phenyl] was used as an acceptor and UDP-Glc as a donor substrate to demonstrate that both wfgD and wfaP encode glucosyltransferases. Enzyme products from both glucosyltransferases were isolated by high-pressure liquid chromatography and analyzed by nuclear magnetic resonance. The spectra showed the expected Glc-␤1-3-GlcNAc linkage in the products, confirming that both WfaP and WfgD are forms of UDP-Glc: GlcNAc-pyrophosphate-lipid ␤-1,3-glucosyltransferases. Both WfaP and WfgD have a DxD sequence, which is proposed to interact with phosphate groups of the nucleotide donor through the coordination of a metal cation, and a short hydrophobic sequence at the C terminus that may help to associate the enzymes with the inner membrane. We showed that the enzymes have similar properties and substrate recognition. They both require a divalent cation (Mn 2؉ or Mg 2؉ ) for activity, are deactivated by detergents, have a broad pH optimum, and require the pyrophosphate-sugar linkage in the acceptor substrate for full activity. Substrates lacking phosphate or pyrophosphate linked to GlcNAc were inactive. The length of the aliphatic chain of acceptor substrates also contributes to the activity.

Characterization of Two UDP-Gal:GalNAc-Diphosphate-Lipid β1,3-Galactosyltransferases WbwC from Escherichia coli Serotypes O104 and O5

“…Human core 1 Gal-transferase (C1GalT1), which also forms a Gal␤1-3GalNAc linkage, shows very low sequence identity to WbwC ECO104 (12%) and to WbwC ECO5 (11%). WbwC is predicted to form a GT-A glycosyltransferase fold and has been classified in the CAZy GT2 family, which includes many other bacterial and mammalian inverting ␤-glycosyltransferases (22)(23)(24)(37)(38)(39). All of the WbwC homologs have a DxD sequence that might be involved in catalysis (40).…”

“…Mixtures were incubated for 10 min at 37°C, and reactions were quenched by the addition of 700 l of ice-cold water and freezing. Enzyme reaction product was isolated using Sep-Pak C 18 columns, eluted first in water and then in MeOH, and quantified by scintillation counting as described previously (22,25). High-pressure liquid chromatography (HPLC) separations were carried out as described previously (24), using a C 18 column and acetonitrile-water as the mobile phase.…”

“…Dried product was dissolved in D 2 O and analyzed by 600-MHz NMR. Spectra were collected in one-dimensional (1D) and 2D experiments using a Bruker spectrometer as described previously (22).…”

“…We have characterized several Gal and Glc transferases that catalyze the second step in O antigen repeating-unit synthesis (22)(23)(24)(25). The synthesis of a natural substrate analog, GlcNAc␣-diphosphate phenylundecyl [GlcNAc␣-O-PO 3 -PO 3 -(CH 2 ) 11 -O-Ph, GlcNAc-PP-PhU], numbered 14 in Fig.…”

A Membrane-located Glycosyltransferase Complex Required for Biosynthesis of the d-Galactan I Lipopolysaccharide O Antigen in Klebsiella pneumoniae

“…Although this is not sufficient to span the membrane, this region could potentially intercalate into the membrane (31). WbbO does contain a number of additional helices (particularly the segment between residues 326 and 342), which contain hydrophobic residues and could also participate in its interaction with the membrane; there is precedent for this in other glycosyltransferases (32,33).…”