TAILIEUCHUNG - Báo cáo Y học: Functional assignment of motifs conserved in b1,3-glycosyltransferases A mutagenesis study of murine UDP-galactose:b-N-acetylglucosamine b1,3-galactosyltransferase-I

Theb1,3-glycosyltransferase enzymes identi®ed to date share several conserved regions and conserved cysteine res-idues, 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 b1,3-galactosyltransferase-I gene were constructed and expressed in Sf9 insect cells. Seven mutations abolished the galacto-syltransferase activity. | Eur. J. Biochem. 269 233-239 2002 FEBS 2002 Functional assignment of motifs conserved in pi 3-glycosyltransferases A mutagenesis study of murine UDP-galactose p-Macetylglucosamine p1 3-galactosyltransferase-I Martine Malissard Andre Dinter Eric G. Berger and Thierry Hennet Institute of Physiology University of Zurich Switzerland The pi 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 pi 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 Km with regard to the donor substrate UDP-galactose relative to the wild-type enzyme while two mutants had a sixfold to increase of the apparent Km value for the acceptor substrate N-acetylglu-cosamine-p-p-nitrophenol. Taken together our results indicate that the conserved residues Wi0i and Wi62 are involved in the binding of the UDP-galactose donor the residue W3i5 in the binding of the N-acetylglucosamine- p-p-nitrophenol acceptor and the domain including E264 appears to participate in the binding of both substrates. Keywords Gal transferase GlcNAc transferase mutagenesis gene family. Glycosyltransferase enzymes account for the structural diversity of glycoconjugates found in all organisms. Based on amino-acid sequence similarity glycosyltransferases can be classified into at least 27 different families 1 . In contrast to the common classification based on the reaction catalyzed and the substrate specificity the association by structural similarity allows one to putatively assign a glycosyltransferase function to proteins not previously suspected to catalyze such a reaction 2 3 . .

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