Functional characterization of the initiation enzyme of S-layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus NRS 2004/3a.
The glycan chain of the S-layer glycoprotein of Geobacillus stearothermophilus NRS 2004/3a is composed of repeating units [-->2)-alpha-l-Rhap-(1-->3)-beta-l-Rhap-(1-->2)-alpha-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 alpha-l-rhamnose residues, and a beta-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.
Steiner
Kerstin
K
Zentrum für NanoBiotechnologie, Universität für Bodenkultur Wien, A-1180 Wien, Austria.
Novotny
René
R
Patel
Kinnari
K
Vinogradov
Evgenij
E
Whitfield
Chris
C
Valvano
Miguel A
MA
Messner
Paul
P
Schäffer
Christina
C
D000595:Amino Acid Sequence; D001406:Bacillaceae / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002240:Carbohydrate Sequence; D017124:Conserved Sequence; D005700:Galactosyltransferases / Q000235:genetics; D008070:Lipopolysaccharides / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D011134:Polysaccharides / Q000096:biosynthesis*; D011487:Protein Conformation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D011134:Polysaccharides; C055764:S-layer proteins; D005700:Galactosyltransferases