Characterization of ligand binding to the bifunctional key enzyme in the sialic acid biosynthesis by NMR: I. Investigation of the UDP-GlcNAc 2-epimerase functionality

Astrid Blume, Andrew J. Benie, Florian Stolz, Richard R. Schmidt, Werner Reutter, Stephan Hinderlich*, Thomas Peters

*Korrespondierende/r Autor/-in für diese Arbeit
20 Zitate (Scopus)

Abstract

The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N- acetylmannosamine kinase is the key enzyme for the biosynthesis of sialic acids. As terminal components of glycoconjugates, sialic acids are associated with a variety of pathological processes such as inflammation and cancer. For the first time, this study reveals characteristics of the interaction of the epimerase site of the enzyme with its natural substrate, UDP-N-acetylglucosamine (UDP-GlcNAc) and derivatives thereof at atomic resolution. Saturation transfer difference NMR experiments were crucial in obtaining ligand binding epitopes and to rank ligands according to their binding affinities. Employing a fragment based approach, it was possible to assign the major component of substrate recognition to the UDP moiety. In particular, the binding epitopes of the uridine moieties of UMP, UDP, UDP-GalNAc, and UDP-GlcNAc are rather similar, suggesting that the binding mode of the UDP moiety is the same in all cases. In contrast, the hexopyranose units of UDP-GlcNAc and UDP-GalNAc display small differences reflecting the inability of the enzyme to process UDP-GalNAc. Surprisingly, saturation transfer difference NMR titrations show that UDP has the largest binding affinity to the epimerase site and that at least one phosphate group is required for binding. Consequently, this study provides important new data for rational drug design.

OriginalspracheEnglisch
ZeitschriftJournal of Biological Chemistry
Jahrgang279
Ausgabenummer53
Seiten (von - bis)55715-55721
Seitenumfang7
ISSN0021-9258
DOIs
PublikationsstatusVeröffentlicht - 31.12.2004

Strategische Forschungsbereiche und Zentren

  • Forschungsschwerpunkt: Infektion und Entzündung - Zentrum für Infektions- und Entzündungsforschung Lübeck (ZIEL)

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