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

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)-alpha-L-Rhap-(1 -> 3)-beta-L-Rhap-(1 -> 2)-alpha-L-Rhap-(1 ->](n) (=) (13-18), a 2-O-methyl group capping the terminal repeating unit at the nonreducing end and a -> 2)-alpha-L- Rhap-[(1 -> 3)-alpha-L-Rhap](n) (=) (1-2)(1 -> 3)- adaptor are linked via a beta-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-beta-L-Rha precursor to the nascent S-layer glycan, catalyzing the formation of the alpha 1,3-(WsaC and WsaD) and beta 1,2-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 Sadenosylmethionine-dependent methylation reaction of the terminal alpha 1,3- linked L-rhamnose residue, and the central and C-terminal portions are involved in the transfer of L-rhamnose from dTDP-beta-L-rhamnose to the adaptor saccharide to form the alpha 1,2- and alpha 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.