title: The active site of the Escherichia coli glycogen synthase is similar to the active site of retaining GT-B glycosyltransferases.
abstract: Bacterial glycogen synthases transfer a glucosyl unit, retaining the anomeric configuration, from ADP-glucose to the non-reducing end of glycogen. We modeled the Escherichia coli glycogen synthase based on three glycosyltransferases with a GT-B fold. Comparison between the model and the structure of the active site of crystallized retaining GT-B glycosyltransferases identified conserved residues with the same topology. To confirm the importance of these residues predicted by the model, we studied them in E. coli glycogen synthase by site-directed mutagenesis. Mutations D137A, R300A, K305A, and H161A decreased the specific activity 8100-, 2600-, 1200-, and 710-fold, respectively. None of these mutations increased the Km for glycogen and only H161A and R300A had a higher Km for ADP-Glc of 11- and 8-fold, respectively. These residues were essential, validating the model that shows a strong similarity between the active site of E. coli glycogen synthase and the other retaining GT-B glycosyltransferases known to date.
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Yep
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Ballicora
authors.forename: Jack
authors.initials: J
authors.lastname: Preiss
mesh_terms: D000409:Alanine / Q000737:chemistry; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology*; D006006:Glycogen Synthase / Q000737:chemistry*; D016695:Glycosyltransferases / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D016695:Glycosyltransferases; D006006:Glycogen Synthase; D000409:Alanine