Characterization of an Autonomously Activated Plant ADP-Glucose Pyrophosphorylase  

Abstract

ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step in starch biosynthesis in plants and changes in its catalytic and/or allosteric properties can lead to increased starch production. Recently, a maize (Zea mays)/potato (Solanum tuberosum) small subunit mosaic, MP ], containing the first 198 amino acids of the small subunit of the maize endosperm enzyme and the last 277 amino acids from the potato tuber enzyme, was expressed with the maize endosperm large subunit and was reported to have favorable kinetic and allosteric properties. Here, we show that this mosaic, in the absence of activator, performs like a wild-type AGPase that is partially activated with 3-phosphoglyceric acid (3-PGA). In the presence of 3-PGA, enzyme properties of Mos(1-198)/SH2 are quite similar to those of the wild-type maize enzyme. In the absence of 3-PGA, however, the mosaic enzyme exhibits greater activity, higher affinity for the substrates, and partial inactivation by inorganic phosphate. The Mos(1-198)/SH2 enzyme is also more stable to heat inactivation. The different properties of this protein were mapped using various mosaics containing smaller portions of the potato small subunit. Enhanced heat stability of Mos(1-198) was shown to originate from five potato-derived amino acids between 322 and 377. These amino acids were shown previously to be important in small subunit/large subunit interactions. These five potato-derived amino acids plus other potato-derived amino acids distributed throughout the carboxyl-terminal portion of the protein are required for the enhanced catalytic and allosteric properties exhibited by Mos(1-198)/SH2.

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

“…Since P i competitively reverses allosteric activation by ligands such as 3-PGA, the apparent inhibition constant for P i was measured in the presence of each of the three activators (Table III). In the absence of allosteric activators, very little inhibition by Pi is observed (Boehlein et al, 2009 (Fig. 4).…”

“…Except for the Sh2 R381A variant, site-directed mutagenesis involved PCR amplification of the entire plasmid as described previously (Boehlein et al, 2009). Two reverse-complementary primers containing the desired mutations were used to initiate amplification by vent DNA polymerase.…”

Genetic Engineering of Algae for Enhanced Biofuel Production

“…CELL on May 9, 2018 by guest http://ec.asm.org/ the introduction of an AGPase that does not require 3-PGA, such as the Mos(1-198)/SH2 AGPase, which still has activity even without the presence of an activator (14). Decreasing starch degradation in microalgae.…”

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

“…Phosphate exhibits a K i,app of only approximately 3.0 mM in the presence of 5.0 mM 3-PGA. We have also shown that concentrations of P i near the K i,app value inhibit activity only weakly in the absence of 3-PGA; in fact, P i actually activates the enzyme at low substrate concentrations (Boehlein et al, 2009). Fig.…”

“…In the presence of 5.0 mM P i or the absence of effector, 0.2 mg of enzyme was used and the ATP concentration was varied from 0.25 to 10 mM with G-1-P concentrations varied from 2.0 to 15 mM. Initial velocities were determined spectrophotometrically by following the production of PP i during ADP-Glc synthesis, as described previously (Boehlein et al, 2008(Boehlein et al, , 2009). Assays were duplicated and averages are presented.…”