Biochemical characterization of Neurospora crassa glycogenin (GNN), the self-glucosylating initiator of glycogen synthesis |
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Authors: | de Paula Renato M Wilson Wayne A Roach Peter J Terenzi Héctor F Bertolini Maria Célia |
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Institution: | Instituto de Química, UNESP, Departamento de Bioquímica e Tecnologia Química, R. Professor Francisco Degni, s/n, 14800-900 Araraquara, SP, Brazil. |
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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. |
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Keywords: | DTT dithiothreitol GAPD glyceraldehyde-3-phosphate dehydrogenase GNIP glycogenin-interacting protein GS glycogen synthase GST glutathione-S-transferase HEPES N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) MW molecular weight PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PCR polymerase chain reaction PMSF phenylmethylsulfonyl fluoride SDS sodium dodecyl sulfate TLCK l-lysine choromethyl ketone" target="_blank">N-p-tosyl-l-lysine choromethyl ketone UDPG uridine diphosphoglucose VM Vogel minimum WT wild type |
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