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Journal of Bacteriology, December 2007, p. 8626-8635, Vol. 189, No. 23
0021-9193/07/$08.00+0     doi:10.1128/JB.00777-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Biochemical Characterization of dTDP-D-Qui4N and dTDP-D-Qui4NAc Biosynthetic Pathways in Shigella dysenteriae Type 7 and Escherichia coli O7{triangledown} ,{dagger}

Ying Wang,1,2,3 Yanli Xu,1,2,3 Andrei V. Perepelov,4 Yuanyuan Qi,1,2 Yuriy A. Knirel,4 Lei Wang,1,2,3 and Lu Feng1,2,3*

TEDA School of Biological Sciences and Biotechnology, Nankai University,1 Tianjin Key Laboratory of Microbial Functional Genomics,2 Tianjin Research Center for Functional Genomics and Biochip, 23 Hongda Street, TEDA, Tianjin 300457, People's Republic of China,3 N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russian Federation4

Received 18 May 2007/ Accepted 19 September 2007

O-antigen variation due to the presence of different types of sugars and sugar linkages is important for the survival of bacteria threatened by host immune systems. The O antigens of Shigella dysenteriae type 7 and Escherichia coli O7 contain 4-(N-acetylglycyl)amino-4,6-dideoxy-D-glucose (D-Qui4NGlyAc) and 4-acetamido-4,6-dideoxy-D-glucose (D-Qui4NAc), respectively, which are sugars not often found in studied polysaccharides. In this study, we characterized the biosynthetic pathways for dTDP-D-Qui4N and dTDP-D-Qui4NAc (the nucleotide-activated precursors of D-Qui4NGlyAc and D-Qui4NAc in O antigens). Predicted genes involved in the synthesis of the two sugars were cloned, and the gene products were overexpressed and purified as His-tagged fusion proteins. In vitro enzymatic reactions were carried out using the purified proteins, and the reaction products were analyzed by capillary electrophoresis, electrospray ionization-mass spectrometry, and nuclear magnetic resonance spectroscopy. It is shown that in S. dysenteriae type 7 and E. coli O7, dTDP-D-Qui4N is synthesized from {alpha}-D-glucose-1-phosphate in three reaction steps catalyzed by glucose-1-phosphate thymidyltransferase (RmlA), dTDP-D-glucose 4,6-dehydratase (RmlB), and dTDP-4-keto-6-deoxy-D-glucose aminotransferase (VioA). An additional acetyltransferase (VioB) catalyzes the conversion of dTDP-D-Qui4N into dTDP-D-Qui4NAc in E. coli O7. Kinetic parameters and some other properties of VioA and VioB are described and differences between VioA proteins from S. dysenteriae type 7 (VioAD7) and E. coli O7 (VioAO7) discussed. To our knowledge, this is the first time that functions of VioA and VioB have been biochemically characterized. This study provides valuable enzyme sources for the production of dTDP-D-Qui4N and dTDP-D-Qui4NAc, which are potentially useful in the pharmaceutical industry for drug development.

* Corresponding author. Mailing address: TEDA School of Biological Sciences and Biotechnology, Nankai University, 23 Hongda Street, TEDA, Tianjin 300457, People's Republic of China. Phone: 86-22-66229592. Fax: 86-22-66229596. E-mail: fenglu63{at}nankai.edu.cn

{triangledown} Published ahead of print on 28 September 2007.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, December 2007, p. 8626-8635, Vol. 189, No. 23
0021-9193/07/$08.00+0     doi:10.1128/JB.00777-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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