Molecular Cloning of Novel-Type Phospho enol pyruvate Carboxylase Isoforms in Pitaya (Hylocereus undatus)

Phosphoenolpyruvate carboxylase (PEPC) is an important enzyme involved in the initial CO₂ fixation of crassulacean acid metabolism (CAM) photosynthesis. To understand the cultivation characteristics of a CAM plant pitaya, it is necessary to clarify the characteristics of PEPC in this species. Here, we cloned three PEPC cDNAs in pitaya, HuPPC1, HuPPC2, and HuPPC3, which encode 942, 934, and 966 amino acid residues, respectively. Phylogenetic analysis indicated that these PEPC belonged to plant-type PEPC (PTPC), although HuPPC1 and HuPPC2 have no Ser-phosphorylation motif in N-terminal region, which is a crucial regulation site in PTPC and contributes to CAM periodicity. HuPPC1 and HuPPC2 phylogenetically unique to the Cactaceae family, whereas HuPPC3 was included in a CAM clade. Two isoforms were partially purified at the protein level and were assigned as HuPPC2 and HuPPC3 using MASCOT analysis. The most distinct difference in enzymatic properties between the two was sensitivity to malate and aspartate, both of which are allosteric inhibitors of PEPC. With 2 mM malate, HuPPC3 was inhibited to 10% of the initial activity, whereas HuPPC2 activity was maintained at 70%. Aspartate inhibited HuPPC3 activity by approximately 50% at 5 mM; however, such inhibition was not observed for HuPPC2 at 10 mM. These results suggest that HuPPC3 corresponds to a general CAM-related PEPC, whereas HuPPC1 and HuPPC2 are related to carbon and/or nitrogen metabolism, with a characteristic regulation mechanism similar to those of Cactaceae plants.