Probing the Role of Proline‐288 in the Regulation of Agrobacterium tumefaciens ADPGlucose Pyrophosphorylase

Abstract

ADP‐glucose pyrophosphorylase (ADPG PPase) catalyzes the rate limiting step in glucan synthesis pathways of bacteria and plants and is an attractive target for protein engineering to increase the production of renewable carbon. Agrobacterium tumefaciens (Ag.t.) ADPG PPase is activated by F6P and pyruvate and inhibited by Pi; these effectors are believed to bind at allosteric site(s) located between the catalytic N‐terminal and C‐terminal domains. We probed the role of highly conserved P288 in the loop region between the two domains that has shown to be vital in regulation of the E. coli ADPG PPase. The P288A and P288D mutants were successfully expressed and purified while P288G was found to be unstable. P288A displayed a decrease in activity and fold activation by activators versus wild‐type while the P288D enzyme showed ~5‐fold higher activity than wild‐type and insensitivity to activation and inhibition by effectors. The mutations may alter the loop conformation that positions the domains, resulting in enzyme activation, particularly when negative charge is at this site. Molecular modeling indicates the loop region may interact with another subunit in the tetrameric structure of the enzyme. Efforts are underway to test this hypothesis by additional mutagenesis and structural studies. Supported by NSF Award 0448676.