Thermodynamic insights into the structural basis governing the donor substrate recognition by human β1,4-galactosyltransferase 7

Abstract

Human β1,4-GalT (galactosyltransferase)7 is involved in the biosynthesis of the tetrasaccharide linker protein region (GlcAβ1→3Galβ1→3Galβ1→4Xylβ1) (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 β1,4-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 β-phosphate, and to a lesser extent the α-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 β1,4-GalT7. Surprisingly, in contrast with other GalTs, soluble β1,4-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.