2. D-Glucuronic Acid 2-Epimerases from Respiratory Pathogens" style="margin:15px;">
    Author's Information
    Erin L. Westman
    Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1

    David J. Mcnally
    Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada, K1A 0R6

    Martin Rejzek
    Centre for Carbohydrate Chemistry, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, NR4 7TJ, U.K.

    Wayne L. Miller
    Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1

    Vellupillai Sri Kannathasan
    Centre for Carbohydrate Chemistry, School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, U.K.

    Andrew Preston
    Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1

    Duncan J. Maskell
    Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, U.K.

    Robert A. Field
    Centre for Carbohydrate Chemistry, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, NR4 7TJ, U.K.

    Jean-Robert Brisson
    Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada, K1A 0R6

    Joseph S. Lam
    Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1
  3. D-Glucuronic Acid 2-Epimerases from Respiratory Pathogens" style="margin:15px;">
    Abstract
    The heteropolymeric O-antigen of the lipopolysaccharide from Pseudomonas aeruginosa serogroup O5 as well as the band-A trisaccharide from Bordetella pertussis contain the di-N-acetylated mannosaminuronic acid derivative, β-D-ManNAc3NAcA (2,3-diacetamido-2,3-dideoxy-β-D-mannuronic acid). The biosynthesis of the precursor for this sugar is proposed to require five steps, through which UDP-α-D-GlcNAc (UDP-N-acetyl-α-D-glucosamine) is converted via four steps into UDP-α-D-GlcNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucuronic acid), and this intermediate compound is then epimerized by WbpI (P. aeruginosa), or by its orthologue, WlbD (B. pertussis), to form UDP-α-D-ManNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-α-D-mannuronic acid). UDP-α-D-GlcNAc3NAcA, the proposed substrate for WbpI and WlbD, was obtained through chemical synthesis. His6–WbpI and His6–WlbD were overexpressed and then purified by affinity chromatography using FPLC. Capillary electrophoresis was used to analyse reactions with each enzyme, and revealed that both enzymes used UDP-α-D-GlcNAc3NAcA as a substrate, and reacted optimally in sodium phosphate buffer (pH 6.0). Neither enzyme utilized UDP-α-D-GlcNAc, UDP-α-D-GlcNAcA (UDP-2-acetamido-2,3-dideoxy-α-D-glucuronic acid) or UDP-α-D-GlcNAc3NAc (UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucose) as substrates. His6–WbpI or His6–WlbD reactions with UDP-α-D-GlcNAc3NAcA produce a novel peak with an identical retention time, as shown by capillary electrophoresis. To unambiguously characterize the reaction product, enzyme–substrate reactions were allowed to proceed directly in the NMR tube and conversion of substrate into product was monitored over time through the acquisition of a proton spectrum at regular intervals. Data collected from one- and two-dimensional NMR experiments showed that His6–WbpI catalysed the 2-epimerization of UDP-α-D-GlcNAc3NAcA, converting it into UDP-α-D-ManNAc3NAcA. Collectively, these results provide evidence that WbpI and WlbD are UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucuronic acid 2-epimerases.
    Keywords
    2-epimerase, lipopolysaccharide, mannosaminuronic acid biosynthesis, O antigen, sugar–nucleotide metabolism, UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucuronic acid
    DOI: 10.1042/BJ20070017
    ADLID: 32215-v4
  4. D-Glucuronic Acid 2-Epimerases from Respiratory Pathogens" style="margin:15px;">
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  1. Keywords
    2-epimerase lipopolysaccharide mannosaminuronic acid biosynthesis O antigen sugar–nucleotide metabolism UDP-2 3-diacetamido-2 3-dideoxy-α-D-glucuronic acid
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