Synthesis of the Ostreococcus tauri ADP‐glucose pyrophosphorylase genes and characterization of their expressed subunits

Abstract

The ADP‐glucose pyrophosphorylase small (S) and large (L) subunit genes from the unicellular algae Ostreococcus tauri were synthesized by PCR, subcloned, and the recombinant expression products characterized. This enzyme regulates the biosynthesis of starch in photosynthetic eukaryotes and has a reversible reaction in vitro. In higher plants, the L subunit makes the S subunit more sensitive to the activator. To understand the evolution of the subunit roles, we partially purified the S+L heterotetramer and S homotetramer from O. tauri, the simplest and smallest photosynthetic eukaryote. On the other hand, the L subunit could not be expressed alone with activity. We found that S+L is more sensitive to 3‐phosphoglycerate (activator) and phosphate (inhibitor) (A0.5 1.35 mM, I0.5 0.21 mM, forward direction, A0.5 0.80 mM, I0.5 0.51 mM reverse) than the homotetramer S (A0.5 2.20 mM, I0.5 1.40 mM forward, A0.5 2.80 mM, I0.5 1.20 mM reverse) with an activation of 33.5‐ and 7.6‐fold in the forward and 22.0‐ and 9.9‐fold in the reverse direction. The difference in regulation between the homotetramer (S) and heterotetramer (S+L) is not as pronounced as found in other plant ADP‐glucose pyrophosphorylases. These subunits in unicellular algae may not have evolved to play distinct roles as in higher plants. This research was supported by NSF grant no. MCB 0615982.