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Biochemical characterization of Neurospora crassa glycogenin (GNN), the self‐glucosylating initiator of glycogen synthesis
Author(s) -
de Paula Renato M.,
Wilson Wayne A.,
Roach Peter J.,
Terenzi Héctor F.,
Bertolini Maria Célia
Publication year - 2005
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2005.02.075
Subject(s) - neurospora crassa , biochemistry , chemistry , glycogen , glycogen synthase , gene , mutant
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 μM) 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.