Premium
Probing the Role of E304, K310, and P288 in the Allosteric Regulation of Agrobacterium tumefaciens ADPGlucose Pyrophosphorylase
Author(s) -
Sayed Hoomai,
Duong Vanessa,
Meyer Christopher
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.891.12
Subject(s) - agrobacterium tumefaciens , allosteric regulation , chemistry , microbiology and biotechnology , biology , biochemistry , enzyme , gene , transformation (genetics)
ADP‐Glucose Pyrophosphorylase (ADPG PPase) catalyzes the rate limiting step in glucan biosynthesis. As glucans serve as renewable and carbon sources, ADPG PPase an attractive target for engineering. The x ray structure of the Agrobacterium tumefaciens (Ag. t.) ADPG PPase (pdb: 3BRK) is a useful tool for probing the role of a loop structure (containing P288) that joins the N terminus to the C‐terminus as well as specific amino acids in the allosteric site such as E304. The Ag. t. ADPG PPase is activated by F6P and pyruvate. The altered proteins E304A and E304D were generated, purified and characterized. Substitution of the E304 residue resulted in changes in allosteric behavior with the largest effect being desensitization to allosteric effectors. The E304D mutant displayed a higher Vmax (~2.5‐fold) in the absence of activators; the E304N enzyme has been generated for analyses to further probe this site. The P288D enzyme displayed higher activity in the absence of activators and complete desensitization to effector molecules. Molecular modeling suggests that the K310 residue could form a salt bridge with the aspartate residue at 288 resulting in an activated form. To test this hypothesis, the K310A and P288D/K310A altered proteins were generated. The K310A enzyme displayed a drastic decrease in activity, but still displayed activation kinetics similar to wild‐type. The addition of the P288D mutation resulted in increased activity, decreased apparent affinity for the substrate ATP, and the same desensitization to F6P and pyruvate seen with the P288D mutation alone. Generation and characterization of the K310R and P288D/K310R enzymes are underway to further probe this region. Supported in part by NSF (BIO MCB 0448676).