Acceptor substrate specificity of UDP-Gal: GlcNAc-R β1,3-galactosyltransferase (WbbD) from Escherichia coli O7:K1

Abstract

Most of the glycosyltransferases involved in O antigen biosynthesis have not yet been characterized. We recently demonstrated that the wbbD gene of the O7 lipopolysaccharide biosynthesis cluster in E. coli strain VW187 (O7:K1) encodes WbbD, a UDP-Gal: GlcNAcalpha-pyrophosphate-lipid beta1,3-Gal-transferase (EC 2.4.1., accession number AAC27537) that transfers the second sugar moiety in the assembly of the O7 repeating unit. The enzyme utilizes undecaprenol-pyrophosphate-GlcNAc as a natural acceptor substrate, but can also transfer Gal to GlcNAcalpha-PO(3)-PO(3)-(CH(2))(11)-O-phenyl (GlcNAc-PP-PhU). A number of acceptor substrate analogs have now been tested to further characterize the acceptor specificity of WbbD and to determine the roles of the pyrophosphate bond and the lipid moiety in the acceptor substrate. The enzyme was found to have a low activity with a substrate containing only one phosphate group directly alpha-linked to GlcNAc, and the enzyme was inactive when the phosphate was absent or further removed from the anomeric carbon of GlcNAc. Modifications of the lipid chain yielded substrates with variable activities. GlcNAc derivatives that were inactive as substrates did not inhibit WbbD suggesting that these compounds did not bind to the active site of the enzyme. The specificity of mammalian beta4-galactosyltransferase I has been compared to that of WbbD. The results indicate that the bacterial WbbD enzyme has a distinct specificity for GlcNAc-PP-lipid, and that WbbD recognition of its acceptor substrate is very different from that of the ubiquitous mammalian beta4-galactosyltransferase I. These studies help to understand mechanisms of O antigen synthesis, to develop methods to synthesize defined oligosaccharide structures and to develop specific O antigen inhibitors.

Biochemical Characterization of UDP-Gal:GlcNAc-Pyrophosphate-Lipid β-1,4-Galactosyltransferase WfeD, a New Enzyme fromShigella boydiiType 14 That Catalyzes the Second Step in O-Antigen Repeating-Unit Synthesis

“…Modifications of the pyrophosphate group eliminated the activity and binding to the enzyme, although the compound having a single phosphate did have 11% activity. None of the GlcNAc analogs tested, which were inactive as substrates, bound to and inhibited WfeD, and we previously reported similar findings for WbbD (8) and for WfgD and WfaP (6). Thus, the pyrophosphate group may be an essential group responsible for the binding of the acceptor to the enzymes that recognize GlcNAc-PP-lipid.…”

“…The advantage of GlcNAc␣-PP-(CH 2 ) 11 -OPh is that the substrate and the enzymatic reaction product can be readily detected and separated by HPLC. We have previously shown that this compound was also an excellent substrate for the ␤1,3-Gal-transferase WbbD from E. coli strain VW187 (8,17) and the ␤1,3-Glc-transferases WfgD and WfaP from E. coli serogroups O152 and O56, respectively (6). A similar compound was also shown to be an acceptor for a GalNAc-transferase from E. coli O86:H2 (28).…”

“…In order to define further the structural requirements of WfeD for the acceptor substrate, a number of neutral and negatively charged phosphate-containing analogs of GlcNAc (5,7,8,13,18) were tested as acceptor substrates ( Table 2). The neutral compounds included both ␣-and ␤-anomers of GlcNAc-benzyl; ␤-anomers of GlcNBu-benzyl, GlcNAc-naphthyl, GlcNAc-isoquinolinyl (5), as well as GlcNAc␣-OCOCH 2 -COO(CH 2 ) 11 -OPh; …”

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

“…Based on our experience, we expect that at least one phosphate residue directly linked to GalNAc␣ is an absolute requirement for WbwC, but this remains to be shown upon synthesis of the appropriate acceptor analogs (26,50,51). Previous studies showed that different lipid chains in the acceptor were compatible with high activities of Gal-and Glc-transferases (22,24,48). In the current study, we used two acceptors that differed in the terminal ring structure in the aglycone, suggesting that the structure of the lipid moiety may be of minor importance for in vitro activity.…”

Defining Function of Lipopolysaccharide O-antigen Ligase WaaL Using Chemoenzymatically Synthesized Substrates

“…Some evidence has demonstrated that bacterial glycosyltransferases acting on Und-linked acceptors are relaxed in their recognition of lipid moiety (21,27,28). Such flexibility allows for the in vitro construction of an oligosaccharide on an unnatural lipid mimic rather than Und.…”