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Westman, Erin L., McNally, David J., Charchoglyan, Armen, Brewer, Dyanne, Field, Robert A. 
ORCID: https://orcid.org/0000-0001-8574-0275 and Lam, Joseph S.
(2009)
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.
Journal of Biological Chemistry, 284 (18).
pp. 11854-11862.
ISSN 0021-9258
Abstract
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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | biochemistry,molecular biology,cell biology ,/dk/atira/pure/subjectarea/asjc/1300/1303 |
| Faculty \ School: | Faculty of Science > School of Biological Sciences Faculty of Science > School of Chemistry, Pharmacy and Pharmacology |
| Related URLs: | |
| Depositing User: | LivePure Connector |
| Date Deposited: | 06 Sep 2024 13:35 |
| Last Modified: | 25 Sep 2024 18:07 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/96597 |
| DOI: | 10.1074/jbc.M808583200 |
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