Structural Analysis of ADP-Glucose Pyrophosphorylase from the Bacterium Agrobacterium tumefaciens,

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Abstract

ADP-glucose pyrophosphorylase (ADPGlc PPase) catalyzes the conversion of glucose 1-phosphate and ATP to ADP-glucose and pyrophosphate. As a key step in glucan synthesis, the ADPGlc PPases are highly regulated by allosteric activators and inhibitors in accord with the carbon metabolism pathways of the organism. Crystals of Agrobacterium tumefaciens ADPGlc PPase were obtained using lithium sulfate as a precipitant. A complete anomalous selenomethionyl derivative X-ray diffraction data set was collected with unit cell dimensions a = 85.38 A, b = 93.79 A, and c = 140.29 A (alpha = beta = gamma = 90 degrees ) and space group I 222. The A. tumefaciens ADPGlc PPase model was refined to 2.1 A with an R factor = 22% and R free = 26.6%. The model consists of two domains: an N-terminal alphabetaalpha sandwich and a C-terminal parallel beta-helix. ATP and glucose 1-phosphate were successfully modeled in the proposed active site, and site-directed mutagenesis of conserved glycines in this region (G20, G21, and G23) resulted in substantial loss of activity. The interface between the N- and the C-terminal domains harbors a strong sulfate-binding site, and kinetic studies revealed that sulfate is a competitive inhibitor for the allosteric activator fructose 6-phosphate. These results suggest that the interface between the N- and C-terminal domains binds the allosteric regulator, and fructose 6-phosphate was modeled into this region. The A. tumefaciens ADPGlc PPase/fructose 6-phosphate structural model along with sequence alignment analysis was used to design mutagenesis experiments to expand the activator specificity to include fructose 1,6-bisphosphate. The H379R and H379K enzymes were found to be activated by fructose 1,6-bisphosphate.

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