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Both Subunits of Thermotoga maritima ADPGlucose Pyrophosphorylase are Required for Optimal Acitvity and Allosteric Regulation
Author(s) -
Matsui Mikiko,
Lim Shan,
Harake Tala,
Guandique Erick,
Tran Margaret,
Reyes David,
Orry Andrew,
Meyer Christopher R
Publication year - 2009
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.23.1_supplement.503.3
Subject(s) - allosteric regulation , thermotoga maritima , effector , chemistry , biology , biochemistry , microbiology and biotechnology , enzyme , gene , escherichia coli
The T. maritima genome harbors two ADP‐glucose pyrophosphorylases (ADPG PPases), glgC and glgD, which share only 36.9% identity. Activity assays revealed negligible glgD activity alone in accord with molecular modeling predictions. However, glgD showed 20‐fold stimulation when combined with glgC. The kinetic parameters for glgC alone and the glgD/C complex were determined. The glgC protein was insensitive to a screen of 15 known ADPG PPase effector molecules, as predicted from modeling, but glgD/C exhibited 2.2‐fold activation by FBP. The following mutations were introduced to restore glgD activity: K12R, S13G, D14S, S25K, A26P, F189S, N240D. This altered protein was inactive alone but in complex with glgC displayed a 2.7‐fold increase over wild‐type glgD/C, indicating that the pyramided mutations effectively resurrected glgD activity in the presence of glgC. The glgD (R23A,D344N) and glgC (I24R,A347D,E348R,N349G) proteins were generated based on the molecular modeling of a hypothetical salt bridge between the N and C‐terminal domain involved in allostery. The glgD AN protein was not as responsive to FBP and the glgC RDRG enzyme exhibited partial FBP activation. Supported in part by NSF Grant 0448676.