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

KUHN ML; FIGUEROA CM; IGLESIAS AA; BALLICORA MA

Minneapolis

Congreso; Plant Biology 2011; 2011

American Society of Plant Biologists

ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in the synthesis of bacterial glycogen and starch in plants. In oxygenic photosynthetic organisms, it is mainly activated by 3-phosphoglycerate (3-PGA). Here, we analyzed the activation promiscuity of ADP-Glc PPase from Anabaena (cyanobacteria), and the subunits from the unicellular algae Ostreococcus tauri (OtaS/OtaL), and potato tuber (StuS/StuL) by comparing the Activation/A0.5 specificity constant for 3-PGA, fructose-1,6-bisphosphate (FBP), fructose-6-phosphate, and glucose-6-phosphate. The 3-PGA specificity constants for the Anabaena, OtaS/OtaL and StuS/StuL enzymes (73.3, 60.7, and 1259 mM-1) were higher than for other activators (18-, 9.4-, and 34-fold higher than the second activator, respectively). This apparent omnipresent promiscuity in divergent organisms suggests a very distant common ancestry. Interestingly, the OtaS homotetramer was even more promiscuous with an FBP specificity constant similar to the one for 3-PGA. To explore the role of OtaS and OtaL in determining the specificity of the heterotetramer, we knocked the catalytic activity of each subunit by mutagenesis. Both constructs OtaSD149A/OtaL and OtaS/OtaLD171A had higher specificity constants for 3-PGA than for FBP (2527 and 153 mM-1 compared with 0.61 and 3.9 mM-1, respectively). After gene duplication, OtaS seemed to have lost specificity for 3-PGA compared to FBP. This was physiologically possible because co-expression of both subunits restored the specificity for 3-PGA of the resulting heterotetrameric wild type enzyme. This ancestral promiscuity could constitute an efficient evolutionary mechanism to accommodate different metabolic needs and ADP-Glc PPase regulation. Funded by NSF MCB-1024945.