Thermodynamic insights into the structural basis governing the donor substrate recognition by the human β1,4-galactosyltransferase 7 - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Biochemical Journal Année : 2009

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

Résumé

Human β1,4-galactosyltransferase7 (β1,4-GalT7) is involved in the biosynthesis of the tetrasaccharide linker protein region (GlcAβ1-3Galβ1-3Galβ1-4Xylβ1) of proteoglycans, by catalysing the transfer of galactose from the uridine 5'-diphosphogalactose, to xylose residue. This reaction is rate-limiting in the glycosaminoglycan biosynthesis. In the present study, we established a large scale production system of the 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 breaking down of donor substrate into smaller fragments and the characterisation of their respective binding to the enzyme by isothermal titration calorimetry. The β-phosphate, and more moderately the α-phosphate, largely contributed to the binding energy. However the uridine moiety was found to be essential for an optimal positioning of the donor substrate within the binding site. Unexpectedly, the contribution of the galactose moiety in substrate recognition was found negligible. Indeed, UDP-Gal, but also various UDP-sugars could bind to β1,4-GalT7. Surprisingly, in contrast to other galactosyltransferases, soluble β1,4-GalT7 was able to transfer Glc, Xyl, and more moderately GlcA and GlcNAc to acceptor sugars while UDP-Man and UDP-GalNAc were not substrates.

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hal-00479032 , version 1 (30-04-2010)

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Franck Daligault, Sophie Rahuel-Clermont, Sandrine Gulberti, Manh-Thong Cung, Guy Branlant, et al.. Thermodynamic insights into the structural basis governing the donor substrate recognition by the human β1,4-galactosyltransferase 7. Biochemical Journal, 2009, 418 (3), pp.605-614. ⟨10.1042/BJ20081093⟩. ⟨hal-00479032⟩
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