Further studies on the role of plant ADP-glucose pyrophosphorylase subunits

ASENCION DIEZ, MATIAS D.; IGLESIAS, ALBERTO A.; FERRERO, DANISA M. L.; BALLICORA, MIGUEL A.; PIATTONI, CLAUDIA V.

Paraná

Congreso; SAIB - LIV Reunión anual; 2018

Sociedad Argentina de Investigación Bioquímica y Biología Moecular

Starch is a highly important source of nutritional energy for humans and numerous animals. ADP-glucose pyrophosphorylase (ADPGlcPPase, EC 2.7.7.27) is the key enzyme in starch biosynthesis, producing the glucosyl-donor (ADPGlc) to elongate the glucan α-1,4-glucosidic chains. Plant ADPGlcPPases are heterotetramers comprising small (S) and large (L) subunits with unequal catalytic and regulatory roles. These are allosteric enzymes regulated by 3- phosphoglycerate (3PGA, activator) and inorganic orthophosphate (Pi, inhibitor). Amongst them, the ADPGlcPPase from wheat endosperm has unique regulatory properties compared to others, since it is modulated by the ratio activator/inhibitor: 3PGA activation occurs after partial inhibition by Pi. We expressed the wheat endosperm ADPGlcPPase S and L subunit genes in Escherichiacoli and also, we co-expressed them with well characterized subunits from the potato tuber ADPGlcPPase to obtain S/L hybrids. To further understand their distinctive roles, we characterized the recombinant proteins and analyzed its sensitiveness towards classic effectors (3PGA and Pi) as well as fructose-6P, phosphoenolpyruvate and fructose 1,6-bisphosphate. After kinetic analysis with one or a combination of two effectors, results show that ADPGlcPPases with the wheat endosperm L subunit (independently of the partner S subunit) are insensitive to assayed metabolites; whereas those versions harboring potato L subunits were highly activated. This approach offers new molecular tools to understand the regulatory mechanisms governing plant ADPGlcPPases activity and starch synthesis.