The active site of the Escherichia coli glycogen synthase is similar to the active site of retaining GT-B glycosyltransferases.

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.