Functional Characterization of the Initiation Enzyme of S-Layer Glycoprotein Glycan Biosynthesis in Geobacillus stearothermophilus NRS 2004/3a

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Abstract
The glycan chain of the S-layer glycoprotein of Geobacillus stearothermophilus NRS 2004/3a is composed of repeating units [→2)-α-l-Rhap-(1→3)-β-l-Rhap-(1→2)-α-l-Rhap-(1→], with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain, a core saccharide composed of two or three α-l-rhamnose residues, and a β-d-galactose residue as a linker to the S-layer protein. In this study, we report the biochemical characterization of WsaP of the S-layer glycosylation gene cluster as a UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase that primes the S-layer glycoprotein glycan biosynthesis of Geobacillus stearothermophilus NRS 2004/3a. Our results demonstrate that the enzyme transfers in vitro a galactose-1-phosphate from UDP-galactose to endogenous phosphoryl-polyprenol and that the C-terminal half of WsaP carries the galactosyltransferase function, as already observed for the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WbaP from Salmonella enterica. To confirm the function of the enzyme, we show that WsaP is capable of reconstituting polysaccharide biosynthesis in WbaP-deficient strains of Escherichia coli and Salmonella enterica serovar Typhimurium.

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