Biochemical characterization of Neurospora crassa glycogenin (GNN), the self-glucosylating initiator of glycogen synthesis.
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.
de Paula
Renato M
RM
Instituto de Química, UNESP, Departamento de Bioquímica e Tecnologia Química, R. Professor Francisco Degni, s/n, 14800-900 Araraquara, SP, Brazil.
Wilson
Wayne A
WA
Roach
Peter J
PJ
Terenzi
Héctor F
HF
Bertolini
Maria Célia
MC
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*
D017931:DNA Primers; D006023:Glycoproteins; C051887:glycogenin; D006003:Glycogen; D005964:Glucosyltransferases; D006006:Glycogen Synthase