Probing the role of the E304 and P288 residues in the allosteric site of Agrobacterium tumefaciens ADPglucose pyrophosphorylase (769.19)

Abstract

ADP‐glucose pyrophosphorylase (ADPG PPase) is the rate limiting enzyme in the glucan biosynthesis pathway, a vital source for renewable and biodegradable carbon. Thus ADPG PPase is an attractive target for protein engineering to produce a more active enzyme to increase the biomass. Agrobacterium tumefaciens (Ag. t) ADPG PPase is activated by F6P and pyruvate and inhibited by phosphate and sulfate via common allosteric site(s). The Ag.t ADPG PPase structure (pdb: 3BRK) indicates that the E304 residue is part of the allosteric site. The P288 residue, which is part of the loop region connecting the N‐terminus domain to the C‐terminus domain, is also involved in allosteric regulation. To probe the roles of these two residues the E304A, E304D, and P288E enzymes were generated, purified and kinetically characterized. The purification scheme included Phenyl Sepharose, anion exchange (DEAE or UNO Q), and Blue A chromatography. Substitution of the P288 residue resulted in changes in allosteric behavior with the largest effects being desensitization to activators as well as higher activity in the absence of activators. A decrease in Vmax as well as stability was observed in the P288E mutant in both the absence and presence of activators. Substitution of the E304 residue also resulted in changes in allosteric behavior with the largest effect being desensitization to both activators and inhibitors. The E304D mutant displayed a higher Vmax (~2.5‐fold) in the absence of activators and it was activated by FBP, a gain of function compared to wild type (WT). Future studies include the characterization of E304N and the generation and characterization of K310A (in the presence and absence of P288D) as this residue may be forming a salt bridge with the introduced aspartate residue. Efforts to crystallize the P288D enzyme are underway to compare to the WT structure.Grant Funding Source: Supported by NSF award 0448676