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

J Biol Chem. 2009 May 1;284(18):11854-62. doi: 10.1074/jbc.M808583200. Epub 2009 Mar 12.


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 His(6)-WbpB and His(6)-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 (UDP-d-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-d-GlcNAc3NAcA) was identified. Preparative quantities of UDP-d-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyltransferases / chemistry
  • Acyltransferases / genetics
  • Acyltransferases / metabolism*
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Lipopolysaccharides / biosynthesis*
  • Lipopolysaccharides / chemistry
  • Pseudomonas aeruginosa / enzymology*
  • Pseudomonas aeruginosa / genetics
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods
  • Transaminases / chemistry
  • Transaminases / genetics
  • Transaminases / metabolism*
  • Uronic Acids / metabolism*


  • Bacterial Proteins
  • Lipopolysaccharides
  • Recombinant Fusion Proteins
  • UDP-2,3-diacetamido-2,3-dideoxy-D-mannuronic acid
  • Uronic Acids
  • Acyltransferases
  • Transaminases