title: Octamerization is essential for enzymatic function of human UDP-glucose pyrophosphorylase.
abstract: Uridine diphosphate-glucose pyrophosphorylase (UGP) occupies a central position in carbohydrate metabolism in all kingdoms of life, since its product uridine diphosphate-glucose (UDP-glucose) is essential in a number of anabolic and catabolic pathways and is a precursor for other sugar nucleotides. Its significance as a virulence factor in protists and bacteria has given momentum to the search for species-specific inhibitors. These attempts are, however, hampered by high structural conservation of the active site architecture. A feature that discriminates UGPs of different species is the quaternary organization. While UGPs in protists are monomers, di- and tetrameric forms exist in bacteria, and crystal structures obtained for the enzyme from yeast and human identified octameric UGPs. These octamers are formed by contacts between highly conserved amino acids in the C-terminal β-helix. Still under debate is the question whether octamerization is required for the functionality of the human enzyme. Here, we used single amino acid replacements in the C-terminal β-helix to interrogate the impact of highly conserved residues on octamer formation and functional activity of human UGP (hUGP). Replacements were guided by the sequence of Arabidopsis thaliana UGP, known to be active as a monomer. Correlating the data obtained in blue native PAGE, size exclusion chromatography and enzymatic activity testing, we prove that the octamer is the active enzyme form. This new insight into structure-function relationships in hUGP does not only improve the understanding of the catalysis of this important enzyme, but in addition broadens the basis for studies aimed at designing drugs that selectively inhibit UGPs from pathogens.
authors.affiliation: Institute for Cellular Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Führing
authors.forename: Sebastian
authors.initials: S
authors.lastname: Damerow
authors.forename: Roman
authors.initials: R
authors.lastname: Fedorov
authors.forename: Julia
authors.initials: J
authors.lastname: Schneider
authors.forename: Anja-Katharina
authors.initials: AK
authors.lastname: Münster-Kühnel
authors.forename: Rita
authors.initials: R
authors.lastname: Gerardy-Schahn
mesh_terms: D017360:Arabidopsis / Q000201:enzymology; D020134:Catalytic Domain; D017124:Conserved Sequence; D006801:Humans; D009154:Mutation; D055503:Protein Multimerization; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000737:chemistry* / Q000235:genetics
chemical_list: D005957:UTP-Glucose-1-Phosphate Uridylyltransferase