title: Identification and biochemical characterization of two novel UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronic acid 2-epimerases from respiratory pathogens.
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, beta-D-ManNAc3NAcA (2,3-diacetamido-2,3-dideoxy-beta-D-mannuronic acid). The biosynthesis of the precursor for this sugar is proposed to require five steps, through which UDP-alpha-D-GlcNAc (UDP-N-acetyl-alpha-D-glucosamine) is converted via four steps into UDP-alpha-D-GlcNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronic acid), and this intermediate compound is then epimerized by WbpI (P. aeruginosa), or by its orthologue, WlbD (B. pertussis), to form UDP-alpha-D-ManNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-mannuronic acid). UDP-alpha-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-alpha-D-GlcNAc3NAcA as a substrate, and reacted optimally in sodium phosphate buffer (pH 6.0). Neither enzyme utilized UDP-alpha-D-GlcNAc, UDP-alpha-D-GlcNAcA (UDP-2-acetamido-2,3-dideoxy-alpha-D-glucuronic acid) or UDP-alpha-D-GlcNAc3NAc (UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucose) as substrates. His6-WbpI or His6-WlbD reactions with UDP-alpha-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-alpha-D-GlcNAc3NAcA, converting it into UDP-alpha-D-ManNAc3NAcA. Collectively, these results provide evidence that WbpI and WlbD are UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronic acid 2-epimerases.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1.
authors.forename: Erin L
authors.initials: EL
authors.lastname: Westman
authors.forename: David J
authors.initials: DJ
authors.lastname: McNally
authors.forename: Martin
authors.initials: M
authors.lastname: Rejzek
authors.forename: Wayne L
authors.initials: WL
authors.lastname: Miller
authors.forename: Vellupillai Sri
authors.initials: VS
authors.lastname: Kannathasan
authors.forename: Andrew
authors.initials: A
authors.lastname: Preston
authors.forename: Duncan J
authors.initials: DJ
authors.lastname: Maskell
authors.forename: Robert A
authors.initials: RA
authors.lastname: Field
authors.forename: Jean-Robert
authors.initials: JR
authors.lastname: Brisson
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Lam
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001886:Bordetella pertussis / Q000201:enzymology / Q000472:pathogenicity; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D006801:Humans; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000472:pathogenicity; D013379:Substrate Specificity; D014535:Uridine Diphosphate Glucuronic Acid / Q000737:chemistry / Q000378:metabolism*; D014574:Uronic Acids / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008070:Lipopolysaccharides; D014574:Uronic Acids; D014535:Uridine Diphosphate Glucuronic Acid; D006639:Histidine; C035278:2,3-diacetamido-2,3-dideoxymannuronic acid; D002238:Carbohydrate Epimerases; C521149:WbpI protein, Pseudomonas aeruginosa; C521150:WlbD protein, Bordetella pertussis