CPS (capsular polysaccharide) is a major virulence factor in Streptococcus pneumoniae. Biosynthesis of CPS RU (repeat unit) proceeds by sequential transfer of sugar residues from the appropriate sugar donor to an activated lipid carrier by committed GTs (glycosyltransferases). While the nucleotide sequence of many cps loci is already known, the real substrate specificity of the hypothetical GTs, as well as the sequence of sugar addition is unclear. In the present paper, we report the biochemical characterization of one α-galactosyltransferase, WciS (Cap8H), a member of GT family 4. This enzyme is implicated in the tetrasaccharide RU biosynthetic pathway of Strep. pneumoniae CPS 8 ([→4)-α-D-Glcp-(1→4)-α-D-Galp-(1→4)-β-D-GlcAp-(1→4)-β-D-Glcp-(1→]n). Expression of WciS–His6 in Escherichia coli BL21 (DE3) strains or BL21 (DE3)/ΔgalU strain resulted in synthesis of a 39 kDa membrane-associated protein identified by N-terminal sequencing and recognized by anti-His6-tag antibody. This protein was capable of adding a galactose residue cellobiuronic acid [β-D-GlcAp-(1→4)-D-Glcp]-pyrophosphate-polyprenol from UDP-Gal. The newly added galactose residue is removed by α-galactosidase, indicating that WciS is a retaining GT. Our results suggest that WciS catalyses the addition of the third sugar residue of the CPS 8 RU. The recombinant WciS–His6 was solubilized and purified as a soluble multimer, opening the way for structural studies.
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Research Article|
June 21 2005
The capsular polysaccharide biosynthesis of Streptococcus pneumoniae serotype 8: functional identification of the glycosyltransferase WciS (Cap8H)
Nehmé SAKSOUK;
Nehmé SAKSOUK
*Laboratoire Adaptation et Pathogénie des Micro-organismes, CNRS UMR 5163, Bâtiment Jean Roget, Faculté de Médecine Pharmacie, La Tronche, France
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Ludovic PELOSI;
Ludovic PELOSI
*Laboratoire Adaptation et Pathogénie des Micro-organismes, CNRS UMR 5163, Bâtiment Jean Roget, Faculté de Médecine Pharmacie, La Tronche, France
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Pierre COLIN-MOREL;
Pierre COLIN-MOREL
†Laboratoire d'Ecologie Alpine, CNRS UMR 5553, Université Joseph Fourier, 38041 Grenoble cedex 9, France
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Manel BOUMEDIENNE;
Manel BOUMEDIENNE
*Laboratoire Adaptation et Pathogénie des Micro-organismes, CNRS UMR 5163, Bâtiment Jean Roget, Faculté de Médecine Pharmacie, La Tronche, France
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Patricia L. ABDIAN;
Patricia L. ABDIAN
‡Instituto de Investigaciones Bioquímicas, Fundación Leloir, Buenos Aires, Argentina
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Roberto A. GEREMIA
Roberto A. GEREMIA
1
*Laboratoire Adaptation et Pathogénie des Micro-organismes, CNRS UMR 5163, Bâtiment Jean Roget, Faculté de Médecine Pharmacie, La Tronche, France
†Laboratoire d'Ecologie Alpine, CNRS UMR 5553, Université Joseph Fourier, 38041 Grenoble cedex 9, France
1To whom correspondence should be addressed (email roberto.geremia@ujf-grenoble.fr).
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Publisher: Portland Press Ltd
Received:
February 04 2005
Accepted:
March 15 2005
Accepted Manuscript online:
March 15 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 389 (1): 63–72.
Article history
Received:
February 04 2005
Accepted:
March 15 2005
Accepted Manuscript online:
March 15 2005
Citation
Nehmé SAKSOUK, Ludovic PELOSI, Pierre COLIN-MOREL, Manel BOUMEDIENNE, Patricia L. ABDIAN, Roberto A. GEREMIA; The capsular polysaccharide biosynthesis of Streptococcus pneumoniae serotype 8: functional identification of the glycosyltransferase WciS (Cap8H). Biochem J 1 July 2005; 389 (1): 63–72. doi: https://doi.org/10.1042/BJ20050217
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