Mechanistic insights into the allosteric regulation of bacterial ADP-glucose pyrophosphorylases

Natalia Comino,Javier O Cifuente,Alberto Marina,Ane Orrantia,Ander Eguskiza,Marcelo E Guerin

doi:10.1074/jbc.m116.773408

Abstract

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, understanding 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-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 The crystal structure of EcAGPase-R130A 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 interprotomer cross-talk, 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.

Highlights

ADP-glucose pyrophosphorylase (AGPase) controls bacterial glycogen and plant starch biosynthetic pathways, the most common carbon storage polysaccharides in nature
The crystal structures of the EcAGPase-adenosine 5؅-monophosphate (AMP)-SUC (PDB code 5L6V) and EcAGPase-FBP (PDB code 5L6S) complexes revealed that the allosteric regulators AMP and FBP bind to a common regulatory cleft defined by the GT-A-like and left-handed ␤-helix domain (L␤H) domains of neighboring protomers [26]
The addition of FBP to the EcAGPase-AMP complex triggered a clear reduction in the apparent melting temperatures (Tm) values, indicating that FBP is able to compete with AMP and to modify the structural arrangement of the EcAGPase-AMP complex, leading to a less stable structure [26, 45]

Summary

Introduction

ADP-glucose pyrophosphorylase (AGPase) controls bacterial glycogen and plant starch biosynthetic pathways, the most common carbon storage polysaccharides in nature. 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. We propose a model in which the positive and negative energy reporters regulate AGPase catalytic activity via intra- and interprotomer cross-talk, with a “sensory motif” and two loops, RL1 and RL2, flanking the ATP-binding site playing a significant role. Eukaryotes utilize UDP-glucose as the activated nucleotide donor for glycogen biosynthesis, whereas archaebacteria and bacteria have selected ADP-Glc, defining two different pathways with distinct regulatory mechanisms and rate-controlling steps (2, 4 –7). Glycogen degradation is carried out by glycogen phosphorylase (GP; Ref. 22), which functions as a depolymerizing enzyme, and the debranching enzyme that catalyzes the removal of ␣-(136)-linked ramifications [23]

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Results

Conclusion