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

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

doi:10.3389/fpls.2018.01564

Abstract

ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step for the synthesis of glycogen in cyanobacteria and starch in green algae and plants. The enzyme from cyanobacteria is homotetrameric (α4), while that from green algae and plants is heterotetrameric (α2β2). These ADP-Glc PPases are allosterically regulated by 3-phosphoglycerate (3PGA, activator) and inorganic orthophosphate (Pi, inhibitor). Previous studies on the cyanobacterial and plant enzymes showed that 3PGA binds to two highly conserved Lys residues located in the C-terminal domain. We observed that both Lys residues are present in the small (α) subunit of the Ostreococcus tauri enzyme; however, one of these Lys residues is replaced by Arg in the large (β) subunit. In this work, we obtained the K443R and R466K mutants of the O. tauri small and large subunits, respectively, and co-expressed them together or with their corresponding wild type counterparts. Our results show that restoring the Lys residue in the large subunit enhanced 3PGA affinity, whereas introduction of an Arg residue in the small subunit reduced 3PGA affinity of the heterotetramers. Inhibition kinetics also showed that heterotetramers containing the K443R small subunit mutant were less sensitive to Pi inhibition, but only minor changes were observed for those containing the R466K large subunit mutant, suggesting a leading role of the small subunit for Pi inhibition of the heterotetramer. We conclude that, during evolution, the ADP-Glc PPase large subunit from green algae and plants acquired mutations in its regulatory site. The rationale for this could have been to accommodate sensitivity to particular metabolic needs of the cell or tissue.

Highlights

ADP-glucose pyrophosphorylase (ADP-Glc PPase; EC 2.7.7.27) catalyzes the conversion of glucose 1-phosphate (Glc1P) and ATP into ADP-glucose and inorganic pyrophosphate (PPi), in the presence of a divalent cation (Mg2+)
In the homotetrameric enzyme from cyanobacteria the Lys residue is usually followed by Asn (Figure 3F), while in S subunits from algae and plants the Lys residue is always followed by Asp (Figures 3G,H)
Chemical modification studies performed with pyridoxalphosphate on the ADP-Glc PPase from spinach leaf demonstrated that 3PGA binds to Lys residues within the C-terminal domain of both S and L subunits (Morell et al, 1988; Ball and Preiss, 1994)

Summary

Introduction

ADP-glucose pyrophosphorylase (ADP-Glc PPase; EC 2.7.7.27) catalyzes the conversion of Glc1P and ATP into ADP-glucose and inorganic pyrophosphate (PPi), in the presence of a divalent cation (Mg2+) This is the first committed step for glycogen biosynthesis in bacteria and starch biosynthesis in green algae and plants. The sequence identity between α subunits ( called small, S) from different species is higher than that of β subunits ( called large, L), and it has been proposed that L subunits differentially emerged through a process of gene duplication followed by subfunctionalization (Ballicora et al, 2005; Georgelis et al, 2008; Kuhn et al, 2009). The L subunit can be catalytically active in some organisms or in different isoforms within the same organism (Ventriglia et al, 2008; Kuhn et al, 2009)

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Conclusion