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), a2-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 alpha1,3- (WsaC and WsaD) and beta1,2-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 alpha1,3-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 alpha1,2- and alpha1,3-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.
Steiner
Kerstin
K
Center for NanoBiotechnology, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
Novotny
René
R
Werz
Daniel B
DB
Zarschler
Kristof
K
Seeberger
Peter H
PH
Hofinger
Andreas
A
Kosma
Paul
P
Schäffer
Christina
C
Messner
Paul
P
D002855:Chromatography, Thin Layer / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D001411:Geobacillus stearothermophilus / Q000378:metabolism*; D006031:Glycosylation; D009682:Magnetic Resonance Spectroscopy; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008954:Models, Biological; D008956:Models, Chemical; D005810:Multigene Family; D010957:Plasmids / Q000378:metabolism; D011134:Polysaccharides / Q000096:biosynthesis / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization
D008562:Membrane Glycoproteins; D011134:Polysaccharides; D011994:Recombinant Proteins; C055764:S-layer proteins