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

FIGUEROA, CARLOS M.; KUHN, MISTY L.; HILL, BENJAMIN L.; IGLESIAS*, ALBERTO A.; BALLICORA*, MIGUEL A.

Frontiers in Plant Science

Frontiers

Lugar: Lussane; Año: 2018 vol. 9

ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed stepfor the synthesis of glycogen in cyanobacteria and starch in green algae and plants.The enzyme from cyanobacteria is homotetrameric (a4), while that from green algaeand plants is heterotetrameric (a2b2). These ADP-Glc PPases are allosterically regulatedby 3-phosphoglycerate (3PGA, activator) and inorganic orthophosphate (Pi, inhibitor).Previous studies on the cyanobacterial and plant enzymes showed that 3PGA binds totwo highly conserved Lys residues located in the C-terminal domain. We observed thatboth Lys residues are present in the small (a) subunit of the Ostreococcus tauri enzyme;however, one of these Lys residues is replaced by Arg in the large (b) subunit. In thiswork, we obtained the K443R and R466K mutants of the O. tauri small and largesubunits, respectively, and co-expressed them together or with their correspondingwild type counterparts. Our results show that restoring the Lys residue in the largesubunit enhanced 3PGA affinity, whereas introduction of an Arg residue in the smallsubunit reduced 3PGA affinity of the heterotetramers. Inhibition kinetics also showedthat heterotetramers containing the K443R small subunit mutant were less sensitiveto Pi inhibition, but only minor changes were observed for those containing the R466Klarge subunit mutant, suggesting a leading role of the small subunit for Pi inhibition of theheterotetramer. We conclude that, during evolution, the ADP-Glc PPase large subunitfrom green algae and plants acquired mutations in its regulatory site. The rationale forthis could have been to accommodate sensitivity to particular metabolic needs of thecell or tissue.