Molecular basis of S-layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus

Abstract

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)-α-L-Rhap- (1→3)-β-L-Rhap-(1→2)-α-L-Rhap-(1→] n = 13-18, a 2-O-methyl group capping the terminal repeating unit at the nonreducing end and a →2)-α-L-Rhap- [(1→3)-α-L-Rhap] n = 1-2(1→3)- 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 α1,3- (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 α1,3-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 α1,2- and α1,3-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. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.