ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step of bacterial glycogen and plant starch biosynthesis, the most common carbon storage polysaccharides in nature. A major challenge is to understand how AGPase activity is regulated by metabolites in the energetic flux within the cell.
Here we report the first crystal structures of the paradigmatic homotetrameric AGPase from Escherichia coli in complex with its physiological positive and negative allosteric regulators, fructose-1,6-bisphosphate (FBP) and AMP, and sucrose in the active site.
FBP and AMP bind to partially overlapping sites located in a deep cleft between glycosyltransferase A-like and left-handed beta-helix domains of neighboring protomers, accounting for the fact that sensitivity to inhibition by AMP is modulated by the concentration of the activator FBP. Single point mutations of key residues in the AMP-binding site decrease its inhibitory effect but also clearly abolish the overall AMP-mediated stabilization effect in wild-type EcAGPase. Single point mutations of key residues for FBP binding did not revert the AMP-mediated stabilization. Strikingly, an EcAGPase-R130A mutant displayed a dramatic increase in activity when compared with wild-type EcAGPase, and this increase correlated with a significant increment of glycogen content in vivo.
Altogether, we propose a model in which the energy reporters regulate EcAGPase catalytic activity by intra-protomer interactions and inter-protomer crosstalk, with a sensory motif and two regulatory loops playing a prominent role.