Premium
Probing the Allosteric Regulation of the Agrobacterium tumefaciens ADPGlucose Pyrophosphorylase
Author(s) -
Bains Gagandeep,
Bohmer Nadine,
Sanders Jamie,
Guzman David,
Meyer Christopher R
Publication year - 2011
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.25.1_supplement.765.3
Subject(s) - agrobacterium tumefaciens , allosteric regulation , enzyme , biochemistry , allosteric enzyme , chemistry , octopine , activator (genetics) , biology , gene , transformation (genetics)
ADP‐glucose pyrophosphorylase (ADPG PPase) is the rate limiting enzyme in the glucan synthesis pathways of bacteria and plants and is an attractive target for protein engineering to increase the production of renewable carbon. The Agrobacterium tumefaciens ( Ag.t. ) ADPGlc PPase is activated by F6P and pyruvate; these effectors are believed to bind at a common allosteric site. Our recent crystal structure of this enzyme (pdb: 3BRK ) indicates that Y39 is part of the allosteric site. We have probed the role of this residue as well as the role of a proline residue in a loop between the N and C‐terminus domains shown to be important in regulation of the E. coli ADPG PPase. The Y39A, Y39F, P288G, and P288D enzymes were successfully expressed and purified. The Y39A, Y39F, and P288D enzymes displayed higher activity than the wild‐type enzyme in the absence of activator. While the P288G enzyme displayed significantly less activity than the wild‐type enzyme, all of the enzymes were significantly desensitized to activators. Complete kinetic and structural characterization of these enzymes will allow for a more detailed picture of structure‐function relationships to emerge. Supported by NSF Award 0448676.