Structural characterization of enzymatic products in the dTDP-d-Qui4NFo biosynthetic pathway using electrospray ionization tandem mass spectrometry
Article first published online: 17 FEB 2013
Copyright © 2013 John Wiley & Sons, Ltd.
Rapid Communications in Mass Spectrometry
Volume 27, Issue 6, pages 681–690, 30 March 2013
How to Cite
Li, D.-G., Liu, B. and Zhou, D.-W. (2013), Structural characterization of enzymatic products in the dTDP-d-Qui4NFo biosynthetic pathway using electrospray ionization tandem mass spectrometry. Rapid Commun. Mass Spectrom., 27: 681–690. doi: 10.1002/rcm.6501
- Issue published online: 17 FEB 2013
- Article first published online: 17 FEB 2013
- Manuscript Accepted: 3 JAN 2013
- Manuscript Revised: 4 DEC 2012
- Manuscript Received: 15 APR 2012
- Program of China. Grant Number: grant 2012CB721101, 2012CB721001, 2009CB522603, 2011CB504900
- National Natural Science Foundation of China (NSFC) Key Program. Grant Number: Grant 31030002
- NSFC General Program. Grant Number: Grant 30900041 and 81171524
- Tianjin Research Program of Application Foundation and Advanced Technology. Grant Number: 10JCYBJC10000
- Research Fund for the Doctoral Program of Higher Education of China. Grant Number: 20090031120023
- Fundamental Research Funds for the Central Universities. Grant Number: 65020121 and 65020061
Structural characterization of biosynthetic precursors is very important in assigning enzymatic function to proteins that have been identified as functional homologs on the basis of sequence homology alone. The objective of this study is to demonstrate the use of electrospray ionization tandem mass spectrometry (ESI-MS/MS) as a powerful technique for the characterization of enzymatic products in the biosynthetic pathway of deoxythymidine 5'-diphosphate-4-formamido-4,6-dideoxy-D-glucose (dTDP-D-Qui4NFo) in Providencia alcalifaciens O30.
The glucose-1-phosphate thymidyltransferase (RmlA), dTDP-d-glucose 4,6-dehydratase (RmlB), dTDP-4-keto-6-deoxy-d-glucose aminotransferase (VioA), and formyltransferase (VioF) catalyzed reactions were directly monitored by ESI-MS, followed by a detailed structural characterization of the final enzymatic products using ESI-MS/MS in the negative-ion mode after minimal cleanup.
The biosynthetic pathway of dTDP-D-Qui4NFo, beginning from α-D-glucose-1-phosphate in four reaction steps catalyzed by RmlA, RmlB, VioA and VioF, was characterized solely by ESI-MS/MS. The results obtained were in good agreement with that of traditional high-performance liquid chromatography (HPLC) monitoring and preparation, as well as nuclear magnetic resonance (NMR) and ESI-MS structural characterization.
MS provides efficient and simple characterization of important unusual dTDP-sugar biosynthetic pathways in the O-chains of bacterial lipopolysaccharides. Copyright © 2013 John Wiley & Sons, Ltd.