title: Functional assignment of motifs conserved in beta 1,3-glycosyltransferases.
abstract: The beta 1,3-glycosyltransferase enzymes identified to date share several conserved regions and conserved cysteine residues, all being located in the putative catalytic domain. To investigate the importance of these motifs and cysteines for the enzymatic activity, 14 mutants of the murine beta 1,3-galactosyltransferase-I gene were constructed and expressed in Sf9 insect cells. Seven mutations abolished the galactosyltransferase activity. Kinetic analysis of the other seven active mutants revealed that three of them showed a threefold to 21-fold higher apparent K(m) with regard to the donor substrate UDP-galactose relative to the wild-type enzyme, while two mutants had a sixfold to 7.5-fold increase of the apparent K(m) value for the acceptor substrate N-acetylglucosamine-beta-p-nitrophenol. Taken together, our results indicate that the conserved residues W101 and W162 are involved in the binding of the UDP-galactose donor, the residue W315 in the binding of the N-acetylglucosamine-beta-p-nitrophenol acceptor, and the domain including E264 appears to participate in the binding of both substrates.
authors.affiliation: Institute of Physiology, University of Zürich, Switzerland.
authors.forename: Martine
authors.initials: M
authors.lastname: Malissard
authors.forename: André
authors.initials: A
authors.lastname: Dinter
authors.forename: Eric G
authors.initials: EG
authors.lastname: Berger
authors.forename: Thierry
authors.initials: T
authors.lastname: Hennet
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence; D005700:Galactosyltransferases / Q000737:chemistry* / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018411:Spodoptera; D014531:Uridine Diphosphate Galactose / Q000378:metabolism*
chemical_list: D014531:Uridine Diphosphate Galactose; D005700:Galactosyltransferases; D000117:Acetylglucosamine