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

Misty L Kuhn,Carlos M Figueroa,Alberto A Iglesias,Miguel A Ballicora

doi:10.1186/1471-2148-13-51

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.

Highlights

ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in the synthesis of glycogen in bacteria and starch in algae and plants
The biosynthesis of these polyglucans occurs by the usage of “activated” glucose molecules: UDP-glucose serves as glycosyl donor for the synthesis of glycogen in fungi and mammals, while ADP-glucose (ADP-Glc) is the initial substrate for the
When we analyzed the homotetrameric O. tauri ADP-Glc PPase (i.e. O. tauri small subunit (OtaS) without OtaL) we found that the specificity constant for 3-PGA and FBP were similar (Table 1 and Figure 2D)

Summary

Introduction

ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in the synthesis of glycogen in bacteria and starch in algae and plants. Green algae and plants accumulate starch, which is composed of two different molecules: amylose and amylopectin [1,2]. The biosynthesis of these polyglucans occurs by the usage of “activated” glucose molecules: UDP-glucose serves as glycosyl donor for the synthesis of glycogen in fungi and mammals, while ADP-glucose (ADP-Glc) is the initial substrate for the ADP-Glc pyrophosphorylase (ADP-Glc PPase, EC 2.7.7.27) catalyzes the synthesis of ADP-Glc from ATP and glucose-1-phosphate (Glc1P), in presence of a divalent cation (Mg2+) [1,2]. Even though both subunits seem to have evolved from the same ancestor, the higher homology among the S subunits suggests stronger evolutionary constraints for them than for the L subunits [2,3]

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