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 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.
Westman
Erin L
EL
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
McNally
David J
DJ
Charchoglyan
Armen
A
Brewer
Dyanne
D
Field
Robert A
RA
Lam
Joseph S
JS
D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000096:biosynthesis* / Q000737:chemistry; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D000637:Transaminases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014574:Uronic Acids / Q000378:metabolism*
D001426:Bacterial Proteins; D008070:Lipopolysaccharides; D011993:Recombinant Fusion Proteins; C506191:UDP-2,3-diacetamido-2,3-dideoxy-D-mannuronic acid; D014574:Uronic Acids; D000217:Acyltransferases; D000637:Transaminases