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

IGLESIAS AA; KUHN ML; ASENCION DIEZ MD; BALLICORA MA; FERRERO DML

Honolulu

Congreso; The Annual Plant Biologist Meeting 2017, American Society for Plant Biologists (ASPB); 2017

American Society for Plant Biologists

Starchis one of the most important sources of nutritional energy for numerousanimals, including humans. ADP-glucosepyrophosphorylase (ADPGlcPPase, EC 2.7.7.27) is a key enzyme in plant starchbiosynthesis, catalyzing the conversion of ATP and glucose-1-phosphate intopyrophosphate and ADP-glucose, the latter being the glucosyl-donor to elongate α-1,4-glucosidic chains in starch. Plant ADP-GlcPPase isa heterotetramer of a catalytic small (S) and a regulatory large (L) subunit,which is allosterically regulated by 3-phosphoglycerate (3PGA, activator) andinorganic orthophosphate (Pi, inhibitor). The ADPGlcPPase from wheat endosperm(TaeE) has distinctive regulatory properties within the enzyme fromplants. To better understand quaternary structure-function relationships inADPGlcPPases, we synthesized de novo genes encoding the wheat small (TaeES)and large (TaeEL) subunits and developed a heterologous expressionsystem in Escherichia coli to produce each subunit separately orco-expressed (TaeES/TaeEL). Proteins were purified to homogeneityand then kinetically characterized. As previously reported for TaeE purifiedform the source, the recombinant TaeES/TaeEL was notsignificantly activated by 3PGA. To further explore the regulatory propertiesof ADPGlcPPase we utilized the potato tuber enzyme (StuE), which ishighly sensitive to 3PGA activation, constructing hybrid proteins byco-expressing the StuES and StuEL subunits and the subunits of TaeEs.The resulting proteins having TaeEL (TaeES/TaeEL and StuES/TaeEL)were not activated by 3PGA whereas, conversely, those with StuEL (TaeES/StuELand StuES/StuEL) exhibited sensitivity to the activator. Theregulatory analysis of these hybrids suggests that sensitivity/insensitivity toactivation of plant ADPGlcPPases would be determined by the L subunit. Wepropose that L subunit plays a critical role for regulation of the enzyme,possibly through inter-subunits interaction. This work contributes to the understanding of mechanismsmodulating activity of plant ADPGlcPPases, which is not only important as acase study of enzymatic regulation, but also for possible biotechnologicaldevelopments