The synthesis of glycogen in bacteria is regulated by ADP-glucose pyrophosphorylase (ADP-Glc PPase), which catalyzes the reaction between glucose 1-phosphate (Glc1P) and adenosine triphosphate (ATP) to form ADP-glucose (ADP-Glc) and pyrophosphate (PPi). In Agrobacterium tumefaciens, ADP-Glc PPase is activated by fructose 6-phosphate (Fru6P) and pyruvate and inhibited by AMP. In Escherichia coli, it is activated by fructose 1,6-bisphosphate (FBP) and inhibited by AMP. Based on previous studies a loop consisting of a span of residues in the ADP-Glc PPase from E. coli was identified as important to the allosteric activation pathway of the enzyme. Trp113 is part of this loop. When mutated to alanine, it showed no response to the allosteric activator FBP. It was found that Trp113 is highly conserved since it was present in all known ADP Glc-PPases. Trp106 was identified as the homologous residue in A. tumefaciens. When mutated to alanine it had no response for the activator Fru6P but in the presence of pyruvate, the enzyme behaved like the wild type (WT). Given that the ADP-Glc PPase from A. tumefaciens has two major different activators, E. coli has one and they both share the same inhibitor, the promiscuity of the allosteric site was investigated. The sugar phosphates studied had similar structures to the native activators of the enzyme. We found that these effectors fit into two categories based on their behavior, some as activators and others as inhibitors. Given the results of both studies, we analyzed how each of these molecules behave in the mutant W106A. The effectors exhibited similar results to the WT enzyme in the form of two distinct groups, activators, and inhibitors. None of them were able to activate the mutant, but they were able to bind indicating that the allosteric signal was disrupted. Here all the molecules exhibited an increase of activity at around 1.2- compared to the 9-fold displayed in the WT. The molecules that inhibited had a remaining activity of around 1.3% and in the WT, it ranged from 4-12% meaning that there was a larger difference between the initial activity (V0) and the final activity (V∞). The results from these studies demonstrate that the communication within the enzyme is different for both regulators since pyruvate was still able to activate W106A. It also reinforced the fact that Fru6P has a different allosteric site than pyruvate and that these molecules are all binding to the same site.

The role of W106A in the communication between the promiscuous allosteric site and the active site in the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase

Fall 2022 abstracts, posters, and presentations will launch in mid-September.

ACS Fall 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.