ADP-glucose pyrophosphorylase (AGPase) controls bacterial glycogen and plant starch biosynthetic pathways, the most common carbon storage polysaccharides in nature. AGPase activity is allosterically regulated by a series of metabolites in the energetic flux within the cell. Very recently, we reported the first crystal structures of the paradigmatic AGPase from Escherichia coli (EcAGPase) in complex with its preferred physiological negative and positive allosteric regulators, adenosine-5′-monophosphate (AMP) and fructose-1,6-bisphosphate (FBP), respectively. However, the understanding of the molecular mechanism by which AMP and FBP allosterically modulates EcAGPase enzymatic activity still remains enigmatic. Here we found that 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·Arg130Ala mutant displayed a dramatic increase in the activity when compared with wild-type EcAGPase, and this increase correlated with a significant increment of glycogen content in vivo. The crystal structure of EcAGPase·Arg130Ala revealed unprecedented conformational changes in structural elements involved in the allosteric signal transmission. Altogether, we propose a model in which the positive and negative energy reporters regulate AGPase catalytic activity via intra- and inter-protomer crosstalk, with a ′sensory motif′ and two loops RL1 and RL2 flanking the ATP binding site playing a significant role. The information reported herein provides exciting possibilities for industrial/biotechnological applications.
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