Dependence of the bi-functional nature of a sialyltransferase from Neisseria meningitidis on a single amino acid substitution.
The L1 immunotype strain 126E of Neisseria meningitidis has been shown to have an N-acetyl-neuraminic acid-containing lipooligosaccharide in which an alpha-linked galactose from a P(k) epitope is substituted at the O6 position (Wakarchuk, W. W., Gilbert, M., Martin, A., Wu, Y., Brisson, J. R., Thibault, P., and Richards, J. C. (1998) Eur. J. Biochem. 254, 626-633). Using a synthetic P(k)-epitope containing acceptor in glycosyltransferase reactions, we were able to show by NMR analysis of the reaction product that the 126E(L1)-derived sialyltransferase can make both alpha-2,3 and alpha-2,6 linkages to the terminal galactose. Gene disruption experiments showed that the lst gene in 126E(L1) was responsible for the in vivo addition of the alpha-2,6-linked N-acetyl-neuraminic acid residue. By site-directed mutagenesis it was possible to change the MC58(L3)-derived enzyme into a bifunctional enzyme with a single amino acid change at position 168, where a glycine was changed to an isoleucine. We performed a gene replacement experiment where the 126E(L1) alpha-2,3/6-sialyltransferase was replaced by allelic exchange with the monofunctional MC58(L3) alpha-2,3-sialyltransferase and with the mutant MC58(L3) allele G168I. We observed that the level of LOS sialylation with the G168I allele was very similar to that of the wild type 126E(L1), indicating that residue 168 is the critical residue for the alpha-2,6-sialyltransferase activity in vitro as well as in vivo.
Wakarchuk
W W
WW
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada, K1A 0R6. warren.wakarchuk@nrc.ca
Watson
D
D
St  Michael
F
F
Li
J
J
Wu
Y
Y
Brisson
J R
JR
Young
N M
NM
Gilbert
M
M
D019943:Amino Acid Substitution; D002240:Carbohydrate Sequence; D006027:Glycosides / Q000096:biosynthesis / Q000737:chemistry; D016695:Glycosyltransferases / Q000378:metabolism; D008070:Lipopolysaccharides / Q000096:biosynthesis* / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009345:Neisseria meningitidis / Q000145:classification / Q000201:enzymology* / Q000276:immunology; D019906:Nuclear Magnetic Resonance, Biomolecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012799:Sialyltransferases / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D006027:Glycosides; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; C023023:lipid-linked oligosaccharides; D016695:Glycosyltransferases; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase