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Legionaminic acid is a nine-carbon α-keto acid that is similar in structure to other members of the sialic acid family that includes neuraminic acid and pseudaminic acid. It is found as a component of the lipopolysaccharide in several bacterial species and is perhaps best known for its presence in the O-antigen of the causative agent of Legionnaires' disease, Legionella pneumophila. In this work, the enzymes responsible for the biosynthesis and activation of N, N'-diacetyllegionaminic acid are identified for the first time. A cluster of three L. pneumophila genes bearing homology to known sialic acid biosynthetic genes (neuA,B,C) were cloned and overexpressed in Escherichia coli. The NeuC homologue was found to be a hydrolyzing UDP- N, N'-diacetylbacillosamine 2-epimerase that converts UDP- N, N'-diacetylbacillosamine into 2,4-diacetamido-2,4,6-trideoxymannose and UDP. Stereochemical and isotopic labeling studies showed that the enzyme utilizes a mechanism involving an initial anti elimination of UDP to form a glycal intermediate and a subsequent syn addition of water to generate product. This is similar to the hydrolyzing UDP- N-acetylglucosamine 2-epimerase (NeuC) of sialic acid biosynthesis, but the L. pneumophila enzyme would not accept UDP-GlcNAc as an alternate substrate. The NeuB homologue was found to be a N, N'-diacetyllegionaminic acid synthase that condenses 2,4-diacetamido-2,4,6-trideoxymannose with phosphoenolpyruvate (PEP), although the in vitro activity of the recombinant enzyme (isolated as a MalE fusion protein) was very low. The synthase activity was dependent on the presence of a divalent metal ion, and the reaction proceeded via a C-O bond cleavage process, similar to the reactions catalyzed by the sialic acid and pseudaminic acid synthases. Finally, the NeuA homologue was shown to possess the CMP- N, N'-diacetyllegionaminic acid synthetase activity that generates the activated form of legionaminic acid used in lipopolysaccharide biosynthesis. Together, the three enzymes constitute a pathway that converts a UDP-linked bacillosamine derivative into a CMP-linked legionaminic acid derivative

O-antigen, lipopolysaccharide biosynthesis, pseudaminic acid, neuraminic acid, Legionella pneumophila, legionaminic acid, Legionella, bacillosamine

NCBI PubMed ID: 18275154
Journal NLM ID: 0370623
Publisher: American Chemical Society
Correspondence: mtanner@chem.ubc.ca
Institutions: Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1
Methods: 13C NMR, 1H NMR, NMR-2D, SDS-PAGE, 31P NMR, genetic methods, biochemical methods

The sialic acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-nonulosonic acid, or legionaminic acid, is found as a virulence-associated cell-surface glycoconjugate in the Gram-negative bacteria Legionella pneumophila and Campylobacter coli. L. pneumophila serogroup 1 strains, causative agents of Legionnaire's disease, contain an α2,4-linked homopolymer of legionaminic acid within their lipopolysaccharide O-chains, whereas the gastrointestinal pathogen C. coli modifies its flagellin with this monosaccharide via O-linkage. In this work, we have purified and biochemically characterized eleven candidate biosynthetic enzymes from C. jejuni, thereby fully reconstituting the biosynthesis of legionaminic acid and its CMP-activated form, starting from fructose-6-P. This pathway involves unique GDP-linked intermediates, likely providing a cellular mechanism for differentiating between this and similar UDP-linked pathways, such as UDP-2,4-diacetamido-bacillosamine biosynthesis involved in N-linked protein glycosylation. Importantly, these findings provide a facile method for efficient large-scale synthesis of legionaminic acid, and since legionaminic acid and sialic acid share the same D-glycero-D-galacto absolute configuration, this sugar may now be evaluated for its potential as a sialic acid mimic.

Campylobacter jejuni, sialic acid, legionaminic acid, flagellin glycosylation, neuraminic aid

NCBI PubMed ID: 19282391
Publication DOI: 10.1093/glycob/cwp039
Journal NLM ID: 9104124
Publisher: IRL Press at Oxford University Press
Correspondence: ian.schoenhofen@nrc-cnrc-gc.ca
Institutions: Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada K1A 0R6
Methods: 13C NMR, 1H NMR, SDS-PAGE, genetic methods, biochemical methods, CE, CE-MS

Tannerella forsythia is an anaerobic, Gram-negative periodontal pathogen. A unique O-linked oligosaccharide decorates the bacterium's cell surface proteins and was shown to modulate the host immune response. In our study, we investigated the biosynthesis of the nonulosonic acid (NulO) present at the terminal position of this glycan. A bioinformatic analysis of T. forsythia genomes revealed a gene locus for the synthesis of pseudaminic acid (Pse) in the type strain ATCC 43037 while strains FDC 92A2 and UB4 possess a locus for the synthesis of legionaminic acid (Leg) instead. In contrast to the NulO in ATCC 43037, which has been previously identified as a Pse derivative (5-N-acetimidoyl-7-N-glyceroyl-3,5,7,9-tetradeoxy-l-glycero-l-manno-NulO), glycan analysis of strain UB4 performed in this study indicated a 350-Da, possibly N-glycolyl Leg (3,5,7,9-tetradeoxy-d-glycero-d-galacto-NulO) derivative with unknown C5,7 N-acyl moieties. We have expressed, purified and characterized enzymes of both NulO pathways to confirm these genes' functions. Using capillary electrophoresis (CE), CE-mass spectrometry and NMR spectroscopy, our studies revealed that Pse biosynthesis in ATCC 43037 essentially follows the UDP-sugar route described in Helicobacter pylori, while the pathway in strain FDC 92A2 corresponds to Leg biosynthesis in Campylobacter jejuni involving GDP-sugar intermediates. To demonstrate that the NulO biosynthesis enzymes are functional in vivo, we created knockout mutants resulting in glycans lacking the respective NulO. Compared to the wild-type strains, the mutants exhibited significantly reduced biofilm formation on mucin-coated surfaces, suggestive of their involvement in host-pathogen interactions or host survival. This study contributes to understanding possible biological roles of bacterial NulOs.

Campylobacter jejuni, Helicobacter, Biofilm, biosynthesis pathway, pseudaminic and legionaminic acid, bacterium

NCBI PubMed ID: 27986835
Publication DOI: 10.1093/glycob/cww129
Journal NLM ID: 9104124
Publisher: IRL Press at Oxford University Press
Correspondence: christina.schaeffer@boku.ac.at; Ian.Schoenhofen@nrc-cnrc.gc.ca
Institutions: Department of NanoBiotechnology, NanoGlycobiology Unit, Universität für Bodenkultur Wien, Muthgasse 11, A-1190 Vienna, Austria, Department of Chemistry, Universität für Bodenkultur Wien, Muthgasse 18, A-1190 Vienna, Austria, National Research Council, Human Health Therapeutics Portfolio, 100 Sussex Drive, Ottawa, ON, Canada K1A 0R6, Department of Oral Biology, School of Dental Medicine, University at Buffalo, 311 Foster Hall, 3435 Main St. Buffalo, New York 14214, USA
Methods: 13C NMR, 1H NMR, PCR, SDS-PAGE, DNA techniques, b-elimination, genetic methods, biochemical methods, CE, CE-MS, bioinformatic analysis, LC-ESI-MS/MS, biofilm assays