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

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.

Probing Allosteric Binding Sites of the Maize Endosperm ADP-Glucose Pyrophosphorylase

“…Both available AGPase crystal structures show two domains in each subunit: an N-terminal catalytic domain, which resembles previously reported pyrophosphorylase structures (Jin et al, 2005;Cupp-Vickery et al, 2008) and a C-terminal domain that makes strong hydrophobic interactions with the catalytic domain. In the potato small subunit homotetramer, two of the three bound sulfate ions (per monomer) are located in a crevice between the N-and C-terminal domains, separated by 7.24 Å .…”

“…The three-dimensional structure of a bacterial homotetrameric enzyme from A. tumefaciens has recently been solved (Cupp-Vickery et al, 2008). Only one crystal structure is available for a higher plant AGPase: a nonnative, low-activity form of the enzyme from potato tuber (small subunit homotetramer; Jin et al, 2005).…”

“…On the other hand, one of the sulfate ions originally found in site 3 is lost when ATP is bound, despite the large distance between their respective binding sites. The A. tumefaciens AGPase homotetramer binds a single sulfate ion (per monomer) with 100% occupancy (Cupp-Vickery et al, 2008).…”

Structural Insights into the Catalytic Mechanism of Bacterial Guanosine-diphospho-d-mannose Pyrophosphorylase and Its Regulation by Divalent Ions

“…1). The dimer interface is formed by a parallel tail-to-tail arrangement of the left-handed ␤-helices from each monomer, as previously observed for the ADP-Glc PPase from potato tuber (7) and Agrobacterium tumefaciens (26). This tight arrangement results in an elongated and continuous ␤-helix structure central to the dimeric assembly.…”

Studies of the Kinetic Mechanism of Maize Endosperm ADP-Glucose Pyrophosphorylase Uncovered Complex Regulatory Properties

“…Crystal structures of a bacterial AGPase and a nonnative, small subunit homotetramer derived from the potato tuber enzyme have been described recently (Jin et al, 2005;Cupp-Vickery et al, 2008). Unfortunately, since both structures were determined in the presence of high sulfate concentrations, both enzymes are in inactive forms.…”