Identification of key functional residues in the active site of human {beta}1,4-galactosyltransferase 7: a major enzyme in the glycosaminoglycan synthesis pathway.
Fiche publication
Date publication
novembre 2010
Auteurs
Membres identifiés du Cancéropôle Est :
Dr FOURNEL-GIGLEUX Sylvie, Dr OUZZINE Mohamed
Tous les auteurs :
Talhaoui I, Bui C, Oriol R, Mulliert G, Gulberti S, Netter P, Coughtrie MW, Ouzzine M, Fournel-Gigleux S
Lien Pubmed
Résumé
Glycosaminoglycans (GAGs) play a central role in many pathophysiological events, and exogenous xyloside substrates of beta1,4-galactosyltransferase 7 (beta4GalT7), a major enzyme of GAG biosynthesis, have interesting biomedical applications. To predict functional peptide regions important for substrate binding and activity of human beta4GalT7, we conducted a phylogenetic analysis of the beta1,4-galactosyltransferase family and generated a molecular model using the x-ray structure of Drosophila beta4GalT7-UDP as template. Two evolutionary conserved motifs, (163)DVD(165) and (221)FWGWGREDDE(230), are central in the organization of the enzyme active site. This model was challenged by systematic engineering of point mutations, combined with in vitro and ex vivo functional assays. Investigation of the kinetic properties of purified recombinant wild-type beta4GalT7 and selected mutants identified Trp(224) as a key residue governing both donor and acceptor substrate binding. Our results also suggested the involvement of the canonical carboxylate residue Asp(228) acting as general base in the reaction catalyzed by human beta4GalT7. Importantly, ex vivo functional tests demonstrated that regulation of GAG synthesis is highly responsive to modification of these key active site amino acids. Interestingly, engineering mutants at position 224 allowed us to modify the affinity and to modulate the specificity of human beta4GalT7 toward UDP-sugars and xyloside acceptors. Furthermore, the W224H mutant was able to sustain decorin GAG chain substitution but not GAG synthesis from exogenously added xyloside. Altogether, this study provides novel insight into human beta4GalT7 active site functional domains, allowing manipulation of this enzyme critical for the regulation of GAG synthesis. A better understanding of the mechanism underlying GAG assembly paves the way toward GAG-based therapeutics.
Référence
J Biol Chem. 2010 Nov 26;285(48):37342-58