Characterization of WbpB, WbpE, and WbpD and reconstitution of a pathway for the biosynthesis of UDP-2,3-diacetamido-2,3-dideoxy-D-mannuronic acid in Pseudomonas aeruginosa

The lipopolysaccharide of Pseudomonas aeruginosa PAO1 contains an unusual sugar, 2, 3-diacetamido-2, 3-dideoxy-D-mannuronic acid (D-ManNAc3NAcA). wbpB, wbpE, and wbpD are thought to encode oxidase, transaminase, and N-acetyltransferase enzymes. To characterize their functions, recombinant proteins were overexpressed and purified from heterologous hosts. Activities of His6-WbpB and His6-WbpE were detected only when both proteins were combined in the same reaction. Using a direct MALDI-TOF mass spectrometry approach, we identified ions that corresponded to the predicted products of WbpB (UDP-3-keto-D-GlcNAcA) and WbpE (UDPD-GlcNAc3NA) in the coupled enzyme-substrate reaction. Additionally, in reactions involving WbpB, WbpE, and WbpD, an ion consistent with the expected product of WbpD (UDP-DGlcNAc3NAcA) was identified. Preparative quantities of UDPD-GlcNAc3NA and UDP-D-GlcNAc3NAcA were enzymatically synthesized. These compounds were purified by high-performance liquid chromatography, and their structures were elucidated by NMR spectroscopy. This is the first report of the functional characterization of these proteins, and the enzymatic synthesis of UDP-D-GlcNAc3NA and UDP-D-GlcNAc3NAcA. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.