Probing the Acceptor Active Site Organization of the Human Recombinant beta 1,4-Galactosyltransferase 7 and Design of Xyloside-based Inhibitors - Université de Lorraine Accéder directement au contenu
Article Dans Une Revue Journal of Biological Chemistry Année : 2015

Probing the Acceptor Active Site Organization of the Human Recombinant beta 1,4-Galactosyltransferase 7 and Design of Xyloside-based Inhibitors

Résumé

Background: Glycosyltransferase inhibitors have important applications in therapeutics and as chemical biology tools. Results: The human 1,4-galactosyltransferase 7 enzyme active site was mapped by modeling, mutagenesis, and in vitro/in cellulo assays, and novel inhibitors were synthesized. Conclusion: An efficient inhibitor of 1,4-galactosyltransferase 7 and glycosaminoglycan synthesis was obtained. Significance: This inhibitory molecule can be exploited to investigate glycosaminoglycan biology and modulate glycosaminoglycan synthesis in therapeutics. Among glycosaminoglycan (GAG) biosynthetic enzymes, the human 1,4-galactosyltransferase 7 (h4GalT7) is characterized by its unique capacity to take over xyloside derivatives linked to a hydrophobic aglycone as substrates and/or inhibitors. This glycosyltransferase is thus a prime target for the development of regulators of GAG synthesis in therapeutics. Here, we report the structure-guided design of h4GalT7 inhibitors. By combining molecular modeling, in vitro mutagenesis, and kinetic measurements, and in cellulo analysis of GAG anabolism and decorin glycosylation, we mapped the organization of the acceptor binding pocket, in complex with 4-methylumbelliferone-xylopyranoside as prototype substrate. We show that its organization is governed, on one side, by three tyrosine residues, Tyr(194), Tyr(196), and Tyr(199), which create a hydrophobic environment and provide stacking interactions with both xylopyranoside and aglycone rings. On the opposite side, a hydrogen-bond network is established between the charged amino acids Asp(228), Asp(229), and Arg(226), and the hydroxyl groups of xylose. We identified two key structural features, i.e. the strategic position of Tyr(194) forming stacking interactions with the aglycone, and the hydrogen bond between the His(195) nitrogen backbone and the carbonyl group of the coumarinyl molecule to develop a tight binder of h4GalT7. This led to the synthesis of 4-deoxy-4-fluoroxylose linked to 4-methylumbelliferone that inhibited h4GalT7 activity in vitro with a K-i 10 times lower than the K-m value and efficiently impaired GAG synthesis in a cell assay. This study provides a valuable probe for the investigation of GAG biology and opens avenues toward the development of bioactive compounds to correct GAG synthesis disorders implicated in different types of malignancies.

Dates et versions

hal-01451902 , version 1 (01-02-2017)

Identifiants

Citer

Mineem Saliba, Nick Ramalanjaona, Sandrine Gulberti, Isabelle Bertin-Jung, Aline Thomas, et al.. Probing the Acceptor Active Site Organization of the Human Recombinant beta 1,4-Galactosyltransferase 7 and Design of Xyloside-based Inhibitors. Journal of Biological Chemistry, 2015, 290 (12), pp.7658-7670. ⟨10.1074/jbc.M114.628123⟩. ⟨hal-01451902⟩
229 Consultations
0 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More