title: Mechanistic insights into the allosteric regulation of bacterial ADP-glucose pyrophosphorylases.
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 fromEscherichia 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 modulatesEcAGPase 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-typeEcAGPase. Single point mutations of key residues for FBP binding did not revert the AMP-mediated stabilization. Strikingly, anEcAGPase-R130A mutant displayed a dramatic increase in activity when compared with wild-typeEcAGPase, and this increase correlated with a significant increment of glycogen contentin vivoThe crystal structure ofEcAGPase-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.
authors.affiliation: From the Structural Biology Unit, CIC bioGUNE, Bizkaia Technology Park, 48160 Derio, Spain.
authors.forename: Natalia
authors.initials: N
authors.lastname: Comino
authors.affiliation: From the Structural Biology Unit, CIC bioGUNE, Bizkaia Technology Park, 48160 Derio, Spain.
authors.forename: Javier O
authors.initials: JO
authors.lastname: Cifuente
authors.affiliation: From the Structural Biology Unit, CIC bioGUNE, Bizkaia Technology Park, 48160 Derio, Spain.
authors.forename: Alberto
authors.initials: A
authors.lastname: Marina
authors.affiliation: From the Structural Biology Unit, CIC bioGUNE, Bizkaia Technology Park, 48160 Derio, Spain.
authors.forename: Ane
authors.initials: A
authors.lastname: Orrantia
authors.affiliation: From the Structural Biology Unit, CIC bioGUNE, Bizkaia Technology Park, 48160 Derio, Spain.
authors.forename: Ander
authors.initials: A
authors.lastname: Eguskiza
authors.affiliation: From the Structural Biology Unit, CIC bioGUNE, Bizkaia Technology Park, 48160 Derio, Spain, mrcguerin@cicbiogune.es.|Unidad de Biofísica, Centro Mixto Consejo Superior de Investigaciones Científicas-Universidad del País Vasco/Euskal Herriko Unibertsitatea (CSIC,UPV/EHU), Barrio Sarriena s/n, Leioa, 48940 Bizkaia, Spain.|Departamento de Bioquímica, Universidad del País Vasco, Leioa, 48940 Bizkaia, Spain, and.|IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain.
authors.forename: Marcelo E
authors.initials: ME
authors.lastname: Guerin
mesh_terms: D000249:Adenosine Monophosphate / Q000737:chemistry* / Q000378:metabolism; D000494:Allosteric Regulation; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005635:Fructosediphosphates / Q000737:chemistry* / Q000378:metabolism; D051599:Glucose-1-Phosphate Adenylyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation
chemical_list: D029968:Escherichia coli Proteins; D005635:Fructosediphosphates; D000249:Adenosine Monophosphate; D051599:Glucose-1-Phosphate Adenylyltransferase; C029063:fructose-1,6-diphosphate
keywords: allosteric regulation; carbohydrate biosynthesis; crystal structure; enzyme mechanism; glycobiology; glycogen; starch