title: Biochemical characterization of Neurospora crassa glycogenin (GNN), the self-glucosylating initiator of glycogen synthesis.
abstract: Glycogenin acts in the initiation step of glycogen biosynthesis by catalyzing a self-glucosylation reaction. In a previous work [de Paula et al., Arch. Biochem. Biophys. 435 (2005) 112-124], we described the isolation of the cDNA gnn, which encodes the protein glycogenin (GNN) in Neurospora crassa. This work presents a set of biochemical and functional studies confirming the GNN role in glycogen biosynthesis. Kinetic experiments showed a very low GNN K(m) (4.41 microM) for the substrate UDP-glucose. Recombinant GNN was produced in Escherichia coli and analysis by mass spectroscopy identified a peptide containing an oligosaccharide chain attached to Tyr196 residue. Site-directed mutagenesis and functional complementation of a Saccharomyces cerevisiae mutant strain confirmed the participation of this residue in the GNN self-glucosylation and indicated the Tyr198 residue as an additional, although less active, glucosylation site. The physical interaction between GNN and glycogen synthase (GSN) was analyzed by the two-hybrid assay. While the entire GSN was required for full interaction, the C-terminus in GNN was more important. Furthermore, mutation in the GNN glucosylation sites did not impair the interaction with GSN.
authors.affiliation: Instituto de Química, UNESP, Departamento de Bioquímica e Tecnologia Química, R. Professor Francisco Degni, s/n, 14800-900 Araraquara, SP, Brazil.
authors.forename: Renato M
authors.initials: RM
authors.lastname: de Paula
authors.forename: Wayne A
authors.initials: WA
authors.lastname: Wilson
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Roach
authors.forename: Héctor F
authors.initials: HF
authors.lastname: Terenzi
authors.forename: Maria Célia
authors.initials: MC
authors.lastname: Bertolini
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D005964:Glucosyltransferases; D006003:Glycogen / Q000096:biosynthesis*; D006006:Glycogen Synthase / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D009492:Neurospora crassa / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D006023:Glycoproteins; C051887:glycogenin; D006003:Glycogen; D005964:Glucosyltransferases; D006006:Glycogen Synthase