Structure‐function studies of the ADPglucose pyrophosphorylase from Thermotoga maritima: probing the role of a conserved aspartate residue in the glgD and glgC subunits (769.20)

Abstract

ADPGlucose pyrophosphorylase (ADPG PPase, glgC gene) catalyzes the rate‐limiting step of glucan biosynthesis in plants and bacteria. Structure‐function studies of the diverse forms of the enzyme will allow for the engineering of a more active enzyme to produce more renewable carbon. In Thermotoga maritima (T.ma) there are two genes, glgC and glgD, which code for two subunits of this ADPG PPase. The recombinant His‐tagged subunits were successfully purified using a heat step and nickel chromatography; the wild type (WT) glgD alone had little to no activity, while the glgC protein displayed a specific activity of ~3 Units/mg with S0.5 values for ATP and magnesium (Mg) of 5.4 mM and 18.7 mM, respectively. However, the WT glgC/D complex was stimulated ~5‐fold compared to glgC alone and also activated ~2‐fold by FBP. Previous alignment studies and molecular modeling indicated that glgC D126 plays a role in catalysis so the D126A enzyme was generated to test this hypothesis. The glgC D126A protein displayed a modest increase in apparent affinity for substrates compared to WT but a 25‐fold decrease in Vmax indicating a role in optimal catalysis. Interestingly, the inactive glgD protein also harbors an aspartate (D125) at this position and the D125A glgD enzyme was generated for characterization. Surprisingly, the altered protein was found to have activity with S0.5 values for ATP, Mg, and G1P of 4.90 mM, 14.8 mM, and 39.2 µM, respectively, with a Vmax of 0.26 U/mg. In preliminary assays, we found that the glgD D125A/glgC complex displayed only a small stimulation compared to glgC alone while the glgD/glgC D126A complex displayed stimulation comparable to WT; a very large stimulation of activity was found when the glgD D125A/glgC D126A was combined, rising to the level of the glgD D125A/glgC2 complex. These aspartate residues may affect activity and stimulation of the subunits in a complex in a synergistic manner. Complete kinetic and physical characterization of the altered enzyme complexes is underway.Grant Funding Source: Supported by NSF award 0448676.