Dependence of the bi-functional nature of a sialyltransferase from Neisseria meningitidis on a single amino acid substitution

Téléchargement	Voir la version finale : Dependence of the bi-functional nature of a sialyltransferase from Neisseria meningitidis on a single amino acid substitution (PDF, 337 Kio)
DOI	Trouver le DOI : https://doi.org/10.1074/jbc.M011293200
Auteur	Rechercher : Wakarchuk, Warren W.¹; Rechercher : Watson, David¹; Rechercher : St. Michael, Frank¹; Rechercher : Li, Jianjun¹; Rechercher : Wu, Yuyang¹; Rechercher : Brisson, Jean-Robert¹; Rechercher : Young, N. Martin¹; Rechercher : Gilbert, Michel¹
Affiliation	Conseil national de recherches Canada. Institut des sciences biologiques du CNRC
Format	Texte, Article
Sujet	acids; alpha; amino acid; amino acid substitution; biosynthesis; Canada; carbohydrate sequence; enzymes; enzymology; epitope; exchange; galactose; gene; glycine; glycosides; glycosyltransferases; immunology; In vitro; isoleucine; linkages; lipooligosaccharide; metabolism; molecular sequence data; mutagenesis; mutagenesis, site-directed; mutant; Neisseria meningitidis; NMR; NMR analysis; nuclear magnetic resonance, biomolecular; oligosaccharides; protein; recombinant proteins; sialyltransferases; strains; substrate specificity; synthetic
Résumé	The L1 immunotype strain 126E of Neisseria meningitidis has been shown to have an N-acetyl-neuraminic acid-containing lipooligosaccharide in which an α-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 α-2,3 and α-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 α-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) α-2,3/6-sialyltransferase was replaced by allelic exchange with the monofunctional MC58(L3) α-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 α-2,6-sialyltransferase activity in vitro as well as in vivo
Date de publication	2001-04-20
Maison d’édition	American Society for Biochemistry and Molecular Biology
Dans	The Journal of Biological Chemistry 276, nº 16 : 12785–12790.
Langue	anglais
Publications évaluées par des pairs	Oui
Identificateur	NRC-IBS-WAKARCHUK2001
Numéro NPARC	9379056
Exporter la notice	Exporter en format RIS
Signaler une correction	Signaler une correction (s'ouvre dans un nouvel onglet)
Identificateur de l’enregistrement	d1b3c8c3-dbd7-4644-a1fe-c9d1cf7b92af
Enregistrement créé	2009-07-10
Enregistrement modifié	2020-05-27

Détails de la page

Par :

Conseil national de recherches Canada

Date de modification :: 2026-04-20