Structural characterization of enzymatic products in the dTDP-d-Qui4NFo biosynthetic pathway using electrospray ionization tandem mass spectrometry

Authors

  • Dian-Ge Li,

    1. TEDA School of Biological Sciences and Biotechnology, Tianjin Key Laboratory of Microbial Functional Genomics, Tianjin Research Center for Functional Genomics and Biochip, the Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, Tianjin, China
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  • Bin Liu,

    1. TEDA School of Biological Sciences and Biotechnology, Tianjin Key Laboratory of Microbial Functional Genomics, Tianjin Research Center for Functional Genomics and Biochip, the Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, Tianjin, China
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  • Da-Wei Zhou

    Corresponding author
    • TEDA School of Biological Sciences and Biotechnology, Tianjin Key Laboratory of Microbial Functional Genomics, Tianjin Research Center for Functional Genomics and Biochip, the Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, Tianjin, China
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Correspondence to: D.-W. Zhou, TEDA School of Biological Sciences and Biotechnology, Tianjin Key Laboratory of Microbial Functional Genomics, Tianjin Research Center for Functional Genomics and Biochip, the Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, 23 Hongda Street, TEDA, Tianjin 300457, China.

E-mail: daweizhou@nankai.edu.cn

Abstract

RATIONALE

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.

METHODS

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.

RESULTS

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.

CONCLUSIONS

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.

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