Insight into the Functional Role of the ADP-glucose Pyrophosphorylase Subunits

MATÍAS D. ASENCION DIEZ; MISTY L. KHUN; ALBERTO A. IGLESIAS; DANISA M.L. FERRERO; MIGUEL A. BALLICORA

Honolulu, Hawaii

Congreso; ASPB Meeting; 2018

American Society of Plant Biologists

Starch is one of the most important sources of nutritional energy for numerous animals, including humans. ADP-glucose pyrophosphorylase (ADPGlcPPase, EC 2.7.7.27) is a key enzyme in plant starch biosynthesis, catalyzing the conversion of ATP and glucose-1-phosphate into pyrophosphate and ADP-glucose, the latter being the glucosyl-donor to elongate α-1,4-glucosidic chains in starch. Plant ADPGlcPPases are heterotetramers comprising small (S) and large (L) subunits with unequal catalytic and regulatory roles. These enzymes are allosterically regulated by 3-phosphoglycerate (3PGA, activator) and inorganic orthophosphate (Pi, inhibitor). However, the ADPGlcPPase from wheat (Triticum aestivum) endosperm (TaeE) has distinctive regulatory properties within the enzyme from plants. To better understand quaternary structure-function relationships in ADPGlcPPases, we synthesized de novo genes encoding the wheat endosperm small (TaeES) and large (TaeEL) subunits and developed a heterologous expression system in Escherichia coli to produce each subunit separately or co-expressed (TaeES/TaeEL). Proteins were purified to homogeneity and then kinetically characterized. As previously reported for Tae E purified form the source, the recombinant Tae ES/Tae EL was not significantly activated by 3PGA. To further explore the regulatory properties of ADPGlcPPase we utilized the potato (Solanum tuberosum) tuber enzyme (StuT), which is highly sensitive to 3PGA activation, constructing hybrid proteins by co-expressing the StuTS and StuTL subunits and the subunits of TaeES. The resulting proteins having TaeEL (TaeES/TaeEL and Stu TS/Tae EL) were not activated by 3PGA whereas, conversely, those with Stu TL (Tae ES/Stu TL and Stu TS/Stu TL) were sensitive to the activator. The regulatory analysis of these hybrids suggests that sensitivity/insensitivity to activation of plant ADPGlcPPases would be determined by the L subunit. We propose that L subunit plays a critical role for regulation of the enzyme, possibly through inter-subunits interaction. This work contributes to the understanding of mechanisms modulating activity of plant ADPGlcPPases, which is not only important as a case study of enzymatic regulation, but also for possible biotechnological developments