Ostreococcus tauri ADP-glucose Pyrophosphorylase Reveals Alternative Paths for the Evolution of Subunit Roles

Abstract

ADP-glucose pyrophosphorylase controls starch synthesis in plants and is an interesting case to study the evolution and differentiation of roles in heteromeric enzymes. It includes two homologous subunits, small (S) and large (L), that originated from a common photosynthetic eukaryotic ancestor. In present day organisms, these subunits became complementary after loss of certain roles in a process described as subfunctionalization. For instance, the potato tuber enzyme has a noncatalytic L subunit that complements an S subunit with suboptimal allosteric properties. To understand the evolution of catalysis and regulation in this family, we artificially synthesized both subunit genes from the unicellular alga Ostreococcus tauri. This is among the most ancient species in the green lineage that diverged from the ancestor of all green plants and algae. After heterologous gene expression, we purified and characterized the proteins. The O. tauri enzyme was not redox-regulated, suggesting that redox regulation of ADP-glucose pyrophosphorylases appeared later in evolution. The S subunit had a typical low apparent affinity for the activator 3-phosphoglycerate, but it was atypically defective in the catalytic efficiency (V max /K m ) for the substrate Glc-1-P. The L subunit needed the S subunit for soluble expression. In the presence of a mutated S subunit (to avoid interference), the L subunit had a high apparent affinity for 3-phosphoglycerate and substrates suggesting a leading role in catalysis. Therefore, the subfunctionalization of the O. tauri enzyme was different from previously described cases. To the best of our knowledge, this is the first biochemical description of a system with alternative subfunctionalization paths.Starch synthesis in photosynthetic eukaryotes such as higher plants and unicellular algae is controlled by a heterotetrameric ADP-glucose pyrophosphorylase (ADP-Glc PPase, 4 EC 2.7.7.27). This enzyme catalyzes the reaction of ATP and Glc-1-P to form ADP-glucose (ADP-Glc) and PP i , and it is primarily activated by 3-phosphoglycerate (3-PGA) and inhibited by P i . In photosynthetic eukaryotes the enzyme includes two distinct S and L homologous subunits (S 2 L 2 or ␣ 2 ␤ 2 ), but in photosynthetic bacteria the enzyme is a homotetramer (␣ 4 ). There has been debate regarding the role of the different subunits of ADP-Glc PPase. The S subunit homotetramer from potato (Solanum tuberosum) tuber (StuS), Arabidopsis thaliana (APS1), and barley (Hordeum vulgare) endosperm have a catalytic function with defective regulatory properties (1-3). It is not clear if there is a set of universal roles for the L subunit in plants. The L subunit from potato tuber (StuL) is catalytically deficient and plays more of a regulatory role by modifying the apparent affinity of the S subunit toward allosteric regulators (1, 4). On the other hand, the Arabidopsis APL1 and APL2 isoforms have both catalytic and regulatory functions, whereas the APL3 and APL4 isoforms behave like StuL (2, 5). The maize (Zea mays) endosperm L subunit...

Microarray data can predict diurnal changes of starch content in the picoalga Ostreococcus

“…To explore this hypothesis more narrowly, based on literature and sequence databases (Uniprot, JGI), we checked whether enzymes of the starch metabolic pathway in O. tauri contain the target sequences reported as responsible for the redox-related modifications for plant species, namely, for AGPase, GWD and β-amylase enzymes [16-18]. Our sequence analysis revealed that in addition to already published results for O. tauri [25] and C. reinhardtii [24], neither AGPases of O. lucimarinus nor both Micromonas species contain the redox-target sequences (Figure 6). The same is true for another known redox regulated enzyme, GWD, which is believed to initiate the starch mobilization process.…”

The Crystal Structure of Nitrosomonas europaea Sucrose Synthase Reveals Critical Conformational Changes and Insights into Sucrose Metabolism in Prokaryotes

“…Site-directed mutagenesis was performed by PCR overlap extension as previously described using Phusion DNA polymerase (30,31). The plasmid encoding the N. europaea sucrose synthase (pNESS2) was used as a template for mutagenesis.…”

The ancestral activation promiscuity of ADP-glucose pyrophosphorylases from oxygenic photosynthetic organisms

“…It is believed that the ancestral enzyme may have been a bacterial subunit with both catalytic and regulatory functions [2]. Based on the roles of different S and L subunits from plants, it seems that after gene duplication ADP-Glc PPase has undergone a subfunctionalization process in which each copy adopted complementary roles [4]. The interaction between the product of these gene copies is now necessary to maintain the same overall set of functions of the common ancestor [4].…”

“…Based on the roles of different S and L subunits from plants, it seems that after gene duplication ADP-Glc PPase has undergone a subfunctionalization process in which each copy adopted complementary roles [4]. The interaction between the product of these gene copies is now necessary to maintain the same overall set of functions of the common ancestor [4]. This duplication and divergence increased the sequence landscape and enabled ADP-Glc PPase to evolve towards more complex regulation.…”

“…each subunit retained and/or lost particular properties that were already present in the common ancestor [4]. The S subunit from the potato tuber ADP-Glc PPase has catalytic activity and a low affinity (mM range) for the allosteric activator (3-phosphoglycerate, 3-PGA), whereas the L subunit is non-catalytic but its co-expression with the S subunit increases the affinity (μM range) for the activator of the resulting heterotetramer [5].…”