Cloning, expression, purification and physical and kinetic characterization of the phosphoenolpyruvate carboxylase from orange (Citrus sinensis osbeck var. Valencia) fruit juice sacs

Phosphoenolpyruvate (PEP) carboxylase (PEPCase) from orange fruit juice sacs has been cloned and heterogously expressed in high yield. The purified recombinant enzyme displays properties typical of plant PEPCase, including activation by sugar phosphates and inhibition by malate and citrate. Malate inhibition is weak in the physiological pH range, and the enzyme is also poorly affected by Glu and Asp, known inhibitors of C3 plants PEPCases. However, it is strongly inhibited by citrate. Orange fruit PEPCase phosphorylation by mammalian protein kinase A decreased inhibition by malate. The enzyme presents an unusual high molecular mass in the absence of PEP, while in its presence it displays a more common tetrameric arrangement. The overall properties of the enzyme suggest that it is suited for organic acid synthesis and NADH reoxidation in the mature fruit. The present study provides the first analysis of a recombinant fruit PEPCase.

Research highlights▶ A C3-type PEP carboxylase from orange fruit juice sacs has been cloned and heterogously expressed for the first time. ▶ The citrus fruit enzyme is less responsive to the common inhibitors (malate, Asp, Glu) than other C3 PEP carboxylases. ▶ The enzyme presents an unusual high molecular mass quaternary structure, that is dependent on the presence of PEP and malate.