Thermodynamic insights into the structural basis governing the donor substrate recognition by human beta1,4-galactosyltransferase 7.
Human beta1,4-GalT (galactosyltransferase)7 is involved in the biosynthesis of the tetrasaccharide linker protein region (GlcAbeta1-->3Galbeta1-->3Galbeta1-->4Xylbeta1) (where GlcA is glucuronic acid and Xyl is xylose) of proteoglycans, by catalysing the transfer of Gal (galactose) from the uridine 5'-diphosphogalactose to a Xyl residue. This reaction is rate-limiting in glycosaminoglycan biosynthesis. In the present study, we established a large-scale production system of beta1,4-GalT7 fused with the maltose-binding protein to study substrate recognition. Calorimetric binding studies showed that the binding of the donor substrate UDP-Gal largely promoted binding of the acceptor substrate. To identify the structural basis governing substrate recognition, we used a fragment-based approach involving the artificial breakdown of the donor substrate into smaller fragments and characterization of their respective binding to the enzyme by isothermal titration calorimetry. The beta-phosphate, and to a lesser extent the alpha-phosphate, largely contributed to the binding energy. However, the uridine moiety was found to be essential for the optimal positioning of the donor substrate within the binding site. Unexpectedly, the contribution of the Gal moiety in substrate recognition was found to be negligible. Indeed, UDP-Gal, but also various UDP-sugars, could bind to beta1,4-GalT7. Surprisingly, in contrast with other GalTs, soluble beta1,4-GalT7 was able to transfer Glc (glucose), Xyl and, to a lesser extent GlcA and GlcNAc (N-acetyl glucosamine), to acceptor sugars, whereas UDP-Man (mannose) and UDP-GalNAc (N-acetyl galactosamine) were not substrates.
Daligault
Franck
F
Physiopathologie et Pharmacologie Articulaires, Unité Mixte de Recherche CNRS-UHP 7561, Nancy Université, Faculté de Médecine, BP 184 Vandoeuvre-Les-Nancy, France.
Rahuel-Clermont
Sophie
S
Gulberti
Sandrine
S
Cung
Manh-Thong
MT
Branlant
Guy
G
Netter
Patrick
P
Magdalou
Jacques
J
Lattard
Virginie
V
D002352:Carrier Proteins / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D005700:Galactosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D058619:Maltose-Binding Proteins; D019906:Nuclear Magnetic Resonance, Biomolecular; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D013379:Substrate Specificity; D013816:Thermodynamics; D014539:Uridine Diphosphate Sugars / Q000378:metabolism
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D014539:Uridine Diphosphate Sugars; D005700:Galactosyltransferases; C063053:xylosylprotein 4-beta-galactosyltransferase