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J. Biol. Chem., Vol. 283, Issue 30, 21120-21133, July 25, 2008

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Molecular Basis of S-layer Glycoprotein Glycan Biosynthesis in Geobacillus stearothermophilus^*

Kerstin Steiner¹, René Novotny², Daniel B. Werz³, Kristof Zarschler, Peter H. Seeberger, Andreas Hofinger^¶, Paul Kosma^¶, Christina Schäffer⁴, and Paul Messner⁵

From the Center for NanoBiotechnology and the ^¶Department of Chemistry, University of Natural Resources and Applied Life Sciences, Wien, Austria, and the Laboratory for Organic Chemistry, Swiss Federal Institute of Technology, Zürich, Switzerland

The Gram-positive bacterium Geobacillus stearothermophilus NRS 2004/3a possesses a cell wall containing an oblique surface layer (S-layer) composed of glycoprotein subunits. O-Glycans with the structure [2)--L-Rhap-(13)-β-L-Rhap-(12)--L-Rhap-(1]_{n = 13-18}, a2-O-methyl group capping the terminal repeating unit at the nonreducing end and a 2)--L-Rhap-[(13)--L-Rhap]_{n = 1-2}(13)- adaptor are linked via a β-D-Galp residue to distinct sites of the S-layer protein SgsE. S-layer glycan biosynthesis is encoded by a polycistronic slg (surface layer glycosylation) gene cluster. Four assigned glycosyltransferases named WsaC-WsaF, were investigated by a combined biochemical and NMR approach, starting from synthetic octyl-linked saccharide precursors. We demonstrate that three of the enzymes are rhamnosyltransferases that are responsible for the transfer of L-rhamnose from a dTDP-β-L-Rha precursor to the nascent S-layer glycan, catalyzing the formation of the 1,3- (WsaC and WsaD) and β1,2-linkages (WsaF) present in the adaptor saccharide and in the repeating units of the mature S-layer glycan, respectively. These enzymes work in concert with a multifunctional methylrhamnosyltransferase (WsaE). The N-terminal portion of WsaE is responsible for the S-adenosylmethionine-dependent methylation reaction of the terminal 1,3-linked L-rhamnose residue, and the central and C-terminal portions are involved in the transfer of L-rhamnose from dTDP-β-L-rhamnose to the adaptor saccharide to form the 1,2- and 1,3-linkages during S-layer glycan chain elongation, with the methylation and the glycosylation reactions occurring independently. Characterization of these enzymes thus reveals the complete molecular basis for S-layer glycan biosynthesis.

Received for publication, March 6, 2008 , and in revised form, May 23, 2008.

^* This work was supported by Austrian Science Fund Projects P19047 [GenBank] -B12 (to C. S.) and P18013 [GenBank] -B10 (to P. M.), Hochschuljubilaümsstiftung der Stadt Wien Project H-1809/2006 (to C. S.), ETH Zürich, and the Swiss National Science Foundation (to P. H. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1-S3 and Figs. S1-S7.

¹ Present address: Centre of Biomolecular Sciences, University of St. Andrews, St. Andrews, UK.

² Present address: Institute for Applied Genetics and Cell Biology, University of Natural Resources and Applied Life Sciences (Wien, Austria).

³ Recipient of a Deutsche Forschungsgemeinschaft Emmy Noether Fellowship. Present address: Institute for Organic and Biomolecular Chemistry, Georg-August-Universität (Göttingen, Germany).

⁴ To whom correspondence may be addressed. Tel.: 43-1-47654-2203; Fax: 43-1-4789112; E-mail: christina.schaeffer{at}boku.ac.at.

⁵ To whom correspondence may be addressed. Tel.: 43-1-47654-2202; Fax: 43-1-4789112; E-mail: paul.messner{at}boku.ac.at.

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