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.