Fragment-based screening of the donor substrate specificity of human blood group B galactosyltransferase using saturation transfer difference NMR

Astrid Blume, Jesus Angulo, Thorsten Biet, Hannelore Peters, Andrew J. Benie, Monica Palcic, Thomas Peters

Research output: Contribution to journalArticlepeer-review

27 Citations (Scopus)


Saturation transfer difference NMR experiments on human blood group B a-(1,3)-galactosyltransferase (GTB) for the first time provide a comprehensive set of binding epitopes of donor substrate analogs in relation to the natural donor UDP-Gal. This study revealed that the enzyme binds several UDP-activated sugars, including UDP-Glc, UDP-GlcNAc, and UDP-GalNAc. In all cases, UDP is the dominant binding epitope. To identify the minimum requirements for specific binding, a detailed analysis utilizing a fragment-based approach was employed. The binding of donor substrate to GTB is essentially controlled by the base as a "molecular anchor." Uracil represents the smallest fragment that is recognized, whereas CDP, AMP, and GDP do not exhibit any significant binding affinity for the enzyme. The ribose and ß-phosphate moieties increase the affinity of the ligands, whereas the pyranose sugar apparently weakens the binding, although this part of the molecule controls the specificity of the enzyme. Accordingly, UDP represents the best binder. The binding affinities of UDP-Gal, UDP-Glc, and UMP are about the same, but lower than that of UDP. Furthermore, we observed that ß-D-galactose and a-D-galactose bind weakly to GTB. Whereas ß-D-galactose binds to the acceptor and donor sites, it is suggested that a-D-galactose occupies a third hitherto unknown binding pocket. Finally, our experiments revealed that modulation of enzymatic activity by metal ions critically depends on the total enzyme concentration, raising the question as to which of the bivalent metal cations Mg and Mn is more relevant under physiological conditions.
Original languageEnglish
Pages (from-to)32728-32740
Number of pages13
JournalJournal of Biological Chemistry
Issue number43
Publication statusPublished - 27 Oct 2006

Cite this