A very simple synthesis of GlcNAc-α-pyrophosphoryl-decanol: A substrate for the assay of a bacterial galactosyltransferase

Brockhausen, Inka; Larsson, Andreas; Hindsgaul, Ole

doi:10.1016/j.bmcl.2007.11.031

Cited by 18 publications

(5 citation statements)

References 36 publications

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“…It is not known, however, if the ␣ linkage of GalNAc is required and if both phosphate groups are necessary for high activity. 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).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2014

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c Escherichia coli displays O antigens on the outer membrane that play an important role in bacterial interactions with the environment. The O antigens of enterohemorrhagic E. coli O104 and O5 contain a Gal␤1-3GalNAc disaccharide at the reducing end of the repeating unit. Several other O antigens contain this disaccharide, which is identical to the mammalian O-glycan core 1 or the cancer-associated Thomsen-Friedenreich (TF) antigen. We identified the wbwC genes responsible for the synthesis of the disaccharide in E. coli serotypes O104 and O5. To functionally characterize WbwC, an acceptor substrate analog, GalNAc␣-diphosphate-phenylundecyl, was synthesized. WbwC reaction products were isolated by high-pressure liquid chromatography and analyzed by mass spectrometry, nuclear magnetic resonance, galactosidase and O-glycanase digestion, and anti-TF antibody. The results clearly showed that the Gal␤1-3GalNAc␣ linkage was synthesized, confirming WbwC ECO104 and WbwC ECO5 as UDP-Gal:GalNAc␣-diphosphate-lipid ␤1,3-Gal-transferases. Sequence analysis revealed a conserved DxDD motif, and mutagenesis showed the importance of these Asp residues in catalysis. The purified enzymes require divalent cations (Mn 2؉ ) for activity and are specific for UDP-Gal and GalNAc-diphosphate lipid substrates. WbwC was inhibited by bis-imidazolium salts having aliphatic chains of 18 to 22 carbons. This work will help to elucidate mechanisms of polysaccharide synthesis in pathogenic bacteria and provide technology for vaccine synthesis.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2014

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“…Substrates and substrate analogs, as well as GlcNActerminating oligosaccharide derivatives, were synthesized as reported previously (6,7,22). The synthesis of GlcNAc-pyrophosphate-lipid derivatives will be described elsewhere.…”

Section: Methodsmentioning

confidence: 99%

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

Brockhausen

Liu

et al. 2008

J Bacteriol

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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.

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“…Similarly, for WbbD, a specific length or structure of the lipid chain of the substrate does not appear to be required. The enzyme also does not require the O-phenyl group in the acceptor since GlcNAcα-PP-(CH 2 ) 9 -CH 3 is an active substrate for WbbD [16]. Although the lipid chain has 55 carbons in the natural substrate, synthetic compounds with lipid chains containing 12 to 22 carbons are also compatible with high activity.…”

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confidence: 94%

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

Brockhausen¹,

Riley²,

Joynt³

et al. 2008

Glycoconj J

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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.

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A very simple synthesis of GlcNAc-α-pyrophosphoryl-decanol: A substrate for the assay of a bacterial galactosyltransferase

Cited by 18 publications

References 36 publications

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

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

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

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

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