CONICET | Buscador de Institutos y Recursos Humanos

Phosphoenolpyruvate (PEP) carboxylase from orange fruit juice sacs (CsiPEPCase) 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, as it has been reported for other plant PEPCs. Regarding the structural properties, the enzyme presents an unusual high molecular mass in the absence of PEP, while in its presence it displays a more common tetrameric arrangement. Although different conformational states of the CsiPEPCase could be postulated, it is unclear at present whether the reversible association-dissociation processes could be of importance for in vivo enzyme regulation and the role that PEP would play in it. The overall properties of the enzyme suggest that its role in the fruit could be the production of organic acids in the early stages and the reoxidation of NADH in the environment of reduced respiration of the mature fruit. The present study provides the first analysis of a recombinant fruit PEPCase.