The Agrobacterium tumefaciens ADP-glucose Pyrophosphorylase G329D Mutant Exists in a Pre-Activated State

B.L. HILL; R. MASCAREHAS; A.M. KOENIG; F. HUERTA; S. KHAN; A.A. IGLESIAS; D. LIU; M.A. BALLICORA

San Diego

Congreso; ASBMB Annual Meeting; 2016

ASBMB

Allosteric regulation of ADP-glucose (ADP-Glc PPase, EC 2.7.7.27) is critical for determining the amount of glycogen and starch that gets synthesized in bacteria and plants, respectively [1]. This enzyme combines ATP and glucose-1-phosphate to form ADP-glucose, which serves as the glucose moiety donor for polyglucan synthesis. ADP-Glc PPases are regulated by intermediates of the major carbon assimilatory pathway that exists within a given species. To date, all identified regulators have a negative charge. The ADP-Glc PPase from Agrobacterium tumefaciens is strongly activated by pyruvate and fructose-6-phosphate (Fru-6P). Since ADP-Glc PPase serves as a major control point for the production of glycogen and starch, which are renewable and biodegradable carbon sources, these enzymes have been attractive targets for protein engineering [1,2,3]. A growing body of evidence suggests that the interface between the enzyme?s distinct C-ter and N-ter domains is important for allosteric regulation [2,4,5]. Here, we report on the aspartate mutant of Gly329 in the A. tumefaciens enzyme, which, based on the crystal structure [2], is a residue that residues near this cleft. When this enzyme was characterized, it was found to be in a pre-activated state, meaning that it did not need allosteric activators present in order to achieve levels of activity similar to the activated wild-type enzyme. We suspect that the positioning of an aspartate group in this region mimics the action of negatively charged activators like pyruvate. Furthermore, these findings could help pinpoint the exact location of the allosteric binding site. This work could provide important information into the allosteric regulation of ADP-Glc PPases.