Structure‐Function Studies of the ADPGlucose Pyrophosphorylase from Agrobacterium tumefaciens: Defining the Roles of the K310 and P288 Residues in Allosteric Regulation

Abstract

ADP‐glucose pyrophosphorylase (ADPG PPase, EC 2.7.7.27) is the enzyme that catalyzes the rate limiting step in the glucan biosynthesis pathway. By identifying important residues of the protein a more active enzyme may be engineered thus yielding more renewable carbon. Agrobacterium tumefaciens (Ag. t) ADPG PPase is activated by fructose‐6‐phosphate (F6P) and pyruvate and is inhibited by inorganic phosphate and sulfate. It is believed these effectors bind at a common allosteric site. Based on previous experiments it is believed the P288 residue, which is part of the loop region connecting the N‐terminus domain to the C‐terminus domain, is involved in allosteric regulation. The P288D mutant displayed higher activity than the wild‐type (WT) in the absence of activators and it displayed desensitization to effectors. Molecular modeling suggests reveals the K310 residue could potentially be forming a salt bridge with the aspartate residue at the substituted P288 site. It had been previously shown that the K310A enzyme displayed very low activity that was similar to WT in its response to activators; the K301A enzyme activity was increased by the presence of the P288D mutation which also desensitized the enzyme to effectors. To further probe the roles of these two residues with respect to charge and size, the K310Q, K310R, P288D/K310Q and P288D/K310R were successfully generated. The altered proteins were generated by site‐directed mutagenesis (QuikChange Kit, Stratagene) and expressed in E.coli glgC− cells. The K310Q and K310R mutants were successfully generated and purified using Phenyl Sepharose, Diethylaminoethanol, and Cibracon Blue chromatography. Substitution of the P288 residue resulted in changes in allosteric behavior with the largest effects being desensitization to activators. Preliminary studies on the crude extract of the K310Q and K310R mutants show a 2.85 and 17.46 fold increase activity compared to the WT, respectively, indicating the importance of shape and charge for optimal activity. For the each double mutation the activity was diminished, for P288D/K310Q and P288D/K310R, a 6.5 and 1.6 fold decrease in activity compared to WT, respectively. Complete kinetic and structural characterization of the single and double mutant enzymes will allow for a comprehensive comparison to the WT enzyme and a more detailed understanding of structure‐function relationships.Support or Funding InformationSupported in part by NSF and NSF BIO MCB grant #0448676.