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.
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Research Article|
June 13 2007
Identification and biochemical characterization of two novel UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucuronic acid 2-epimerases from respiratory pathogens
Erin L. Westman;
Erin L. Westman
*Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1
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David J. Mcnally;
David J. Mcnally
†Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada, K1A 0R6
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Martin Rejzek;
Martin Rejzek
1
‡Centre for Carbohydrate Chemistry, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, NR4 7TJ, U.K.
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Wayne L. Miller;
Wayne L. Miller
*Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1
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Vellupillai Sri Kannathasan;
Vellupillai Sri Kannathasan
§Centre for Carbohydrate Chemistry, School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, U.K.
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Andrew Preston;
Andrew Preston
*Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1
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Duncan J. Maskell;
Duncan J. Maskell
∥Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, U.K.
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Robert A. Field;
Robert A. Field
1
‡Centre for Carbohydrate Chemistry, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, NR4 7TJ, U.K.
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Jean-Robert Brisson;
Jean-Robert Brisson
†Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada, K1A 0R6
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Joseph S. Lam
Joseph S. Lam
2
*Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1
2To whom correspondence should be addressed (email jlam@uoguelph.ca).
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Publisher: Portland Press Ltd
Received:
January 02 2007
Revision Received:
February 15 2007
Accepted:
February 19 2007
Accepted Manuscript online:
February 19 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2007 Biochemical Society
2007
Biochem J (2007) 405 (1): 123–130.
Article history
Received:
January 02 2007
Revision Received:
February 15 2007
Accepted:
February 19 2007
Accepted Manuscript online:
February 19 2007
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Citation
Erin L. Westman, David J. Mcnally, Martin Rejzek, Wayne L. Miller, Vellupillai Sri Kannathasan, Andrew Preston, Duncan J. Maskell, Robert A. Field, Jean-Robert Brisson, Joseph S. Lam; Identification and biochemical characterization of two novel UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucuronic acid 2-epimerases from respiratory pathogens. Biochem J 1 July 2007; 405 (1): 123–130. doi: https://doi.org/10.1042/BJ20070017
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