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

Abstract

BackgroundADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in the synthesis of glycogen in bacteria and starch in algae and plants. In oxygenic photosynthetic organisms, ADP-Glc PPase is mainly activated by 3-phosphoglycerate (3-PGA) and to a lesser extent by other metabolites. In this work, we analyzed the activation promiscuity of ADP-Glc PPase subunits from the cyanobacterium Anabaena PCC 7120, the green alga Ostreococcus tauri, and potato (Solanum tuberosum) tuber by comparing a specificity constant for 3-PGA, fructose-1,6-bisphosphate (FBP), fructose-6-phosphate, and glucose-6-phosphate.ResultsThe 3-PGA specificity constant for the enzymes from Anabaena (homotetramer), O. tauri, and potato tuber was considerably higher than for other activators. O. tauri and potato tuber enzymes were heterotetramers comprising homologous small and large subunits. Conversely, the O. tauri small subunit (OtaS) homotetramer was more promiscuous because its FBP specificity constant was similar to that for 3-PGA. To explore the role of both OtaS and OtaL (O. tauri large subunit) in determining the specificity of the heterotetramer, we knocked out the catalytic activity of each subunit individually by site-directed mutagenesis. Interestingly, the mutants OtaSD148A/OtaL and OtaS/OtaLD171A had higher specificity constants for 3-PGA than for FBP.ConclusionsAfter gene duplication, OtaS seemed to have lost specificity for 3-PGA compared to FBP. This was physiologically and evolutionarily feasible because co-expression of both subunits restored the specificity for 3-PGA of the resulting heterotetrameric wild type enzyme. This widespread promiscuity seems to be ancestral and intrinsic to the enzyme family. Its presence could constitute an efficient evolutionary mechanism to accommodate the ADP-Glc PPase regulation to different metabolic needs.

Resurrecting the Regulatory Properties of the Ostreococcus tauri ADP-Glucose Pyrophosphorylase Large Subunit

“…Recombinant proteins were expressed in Escherichia coli AC70R1-504, which is deficient in ADP-Glc PPase activity ( Iglesias et al, 1993 ). Enzyme purification was performed as described by Kuhn et al (2013) . Briefly, crude extracts were loaded on a 10 ml DEAE-Sepharose column (GE Healthcare, Piscataway, NJ, United States) and proteins were eluted with a linear NaCl gradient.…”

“…Kinetic experiments were performed at least twice with similar results. The net activation fold (i.e., the activation that is over one) was calculated as ( V max − v 0 )/ v 0 ( Kuhn et al, 2013 ).…”

“…In this particular case, the S subunit ( Ota S) displays the typical low apparent affinity for the activator (3PGA) and is defective for catalysis, whereas the L subunit ( Ota L) shows higher affinity for substrates, suggesting a leading role in catalysis ( Kuhn et al, 2009 ). Moreover, Ota L could be considered an activator “specifier,” as it specifically increases the apparent affinity for 3PGA of Ota S ( Kuhn et al, 2013 ).…”

Glucosamine-6P and glucosamine-1P, respectively an activator and a substrate of rhodococcal ADP-glucose pyrophosphorylases, show a hint to ascertain (actino)bacterial glucosamine metabolism

“…These results reinforce the view that GlcN-6P constitutes an efficient activator of Rfa ADP-GlcPPase, and it would signal a high carbon availability to be led to glycogen synthesis, as we above described for the Rjo ADP-GlcPPase. From data in Table 1, it is worthy to calculate the specificity constant for each activator, defined as the ratio between net activation and the respective A 0.5 [15], which evaluates the capacity of the respective allosteric effector with the different substrates. Figure 3 illustrates that for Rjo ADP-GlcPPase, the Glc-6P specificity constant is higher (∼9-fold) compared to GlcN-6P with either substrate.…”

“…Hill plots were used to calculate the Hill coefficient ( n H ), the maximal velocity ( V max ), and the kinetic constants that correspond to the activator or substrate, concentrations giving 50% of the maximal activation ( A 0.5 ) or velocity ( S 0.5 ), respectively. The specificity constant for activators were obtained as reported in [15], defined as the ratio between net activation the respective A 0.5 . The net activation was calculated as ( V max - v 0 )/ v 0 , where V max is the activity obtained in presence of the respective effector while v 0 is the activity without effector.…”