Publications

• Elucidation of the CMP-pseudaminic acid pathway in Helicobacter pylori: synthesis from UDP-N-acetylglucosamine by a single enzymatic reaction.

  Schoenhofen I.C., McNally D.J., Brisson J.R., Logan S.M.

  Flagellin glycosylation is a necessary modification allowing flagellar assembly, bacterial motility, colonization, and hence virulence for the gastrointestinal pathogen Helicobacter pylori [Josenhans, C., Vossebein, L., Friedrich, S., and Suerbaum, S. (2002) FEMS Microbiol. Lett., 210, 165-172; Sc ... >> More

  Flagellin glycosylation is a necessary modification allowing flagellar assembly, bacterial motility, colonization, and hence virulence for the gastrointestinal pathogen Helicobacter pylori [Josenhans, C., Vossebein, L., Friedrich, S., and Suerbaum, S. (2002) FEMS Microbiol. Lett., 210, 165-172; Schirm, M., Schoenhofen, I.C., Logan, S.M., Waldron, K.C., and Thibault, P. (2005) Anal. Chem., 77, 7774-7782]. A causative agent of gastric and duodenal ulcers, H. pylori, heavily modifies its flagellin with the sialic acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-alpha-l-manno-nonulosonic acid (pseudaminic acid). Because this sugar is unique to bacteria, its biosynthetic pathway offers potential as a novel therapeutic target. We have identified six H. pylori enzymes, which reconstitute the complete biosynthesis of pseudaminic acid, and its nucleotide-activated form CMP-pseudaminic acid, from UDP-N-acetylglucosamine (UDP-GlcNAc). The pathway intermediates and final product were identified from monitoring sequential reactions with nuclear magnetic resonance (NMR) spectroscopy, thereby confirming the function of each biosynthetic enzyme. Remarkably, the conversion of UDP-GlcNAc to CMP-pseudaminic acid was achieved in a single reaction combining six enzymes. This represents the first complete in vitro enzymatic synthesis of a sialic acid-like sugar and sets the groundwork for future small molecule inhibitor screening and design. Moreover, this study provides a strategy for efficient large-scale synthesis of novel medically relevant bacterial sugars that has not been attainable by chemical methods alone. << Less

  Glycobiology 16:8C-14C(2006) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• PseG of pseudaminic acid biosynthesis: a UDP-sugar hydrolase as a masked glycosyltransferase.

  Liu F., Tanner M.E.

  The flagellin proteins in pathogenic bacteria such as Campylobacter jejuni and Helicobacter pylori are heavily glycosylated with the nine-carbon alpha-keto acid, pseudaminic acid. The presence of this posttranslational modification is absolutely required for assembly of functional flagella. Since ... >> More

  The flagellin proteins in pathogenic bacteria such as Campylobacter jejuni and Helicobacter pylori are heavily glycosylated with the nine-carbon alpha-keto acid, pseudaminic acid. The presence of this posttranslational modification is absolutely required for assembly of functional flagella. Since motility is required for colonization, pseudaminic acid biosynthesis represents a virulence factor in these bacteria. Pseudaminic acid is generated from UDP-N-acetylglucosamine in five biosynthetic steps. The final step has been shown to involve the condensation of 2,4-diacetamido-2,4,6-trideoxy-L-altrose (6-deoxy-Altdi-NAc) with phosphoenolpyruvate as catalyzed by the enzyme pseudaminic acid synthase, NeuB3. The 6-deoxy-AltdiNAc used in this process is generated from its nucleotide-linked form, UDP-6-deoxy-AltdiNAc, by the action of a hydrolase that cleaves the glycosidic bond and releases UDP. This manuscript describes the first characterization of a UDP-6-deoxy-AltdiNAc hydrolase, namely PseG (Cj1312) from C. jejuni. The activity of this enzyme is independent of the presence of divalent metal ions, and the values of the catalytic constants were found to be k(cat) = 27 s(-1) and K(m) = 174 microm. The enzyme was shown to hydrolyze the substrate with an overall inversion of stereochemistry at C-1 and to utilize a C-O bond cleavage mechanism during catalysis. These results, coupled with homology comparisons, suggest that the closest ancestors to the hydrolase are members of the metal-independent GT-B family of glycosyltransferases that include the enzyme MurG. << Less

  J. Biol. Chem. 281:20902-20909(2006) [PubMed] [EuropePMC]