Abstract
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.
Highlights
Phosphoenolpyruvate (PEP) carboxylase (PEPC, EC 4.1.1.31) is an enzyme that catalyzes the carboxylation of PEP, followed by the release of oxaloacetate (OAA) and inorganic phosphate (Pi).PEPC is widely distributed in plant kingdom and some microorganisms
HuPPC3 corresponds to a general crassulacean acid metabolism (CAM)-related PEPC, whereas HuPPC1 and HuPPC2 are related to carbon and/or nitrogen metabolism, with a characteristic regulation mechanism similar to those of Keywords: CAM; isoform; phosphoenolpyruvate carboxylase; pitaya (Hylocereus undatus)
Encoding 942 amino acid residues, HuPPC2 consisted of 3318 bp with a 2802 bp open reading frame (ORF) encoding 934 amino acids, and HuPPC3 was 3337 bp in length with a 2898 bp ORF encoding 966 amino acid residues
Summary
Phosphoenolpyruvate (PEP) carboxylase (PEPC, EC 4.1.1.31) is an enzyme that catalyzes the carboxylation of PEP, followed by the release of oxaloacetate (OAA) and inorganic phosphate (Pi).PEPC is widely distributed in plant kingdom and some microorganisms. There are two types of PEPC: plant-type PEPC (PTPC) and bacterial-type PEPC (BTPC) [1]. The former, usually called Class-1, PEPC consists of a homotetramer of a ~110 kDa subunit. The latter, which has a larger molecular mass (~117 kDa) and forms a hetero-octamer, is called Class-2 PEPC and consists of Class-1 PEPC and four BTPC subunits. PTPCs can be broadly categorized further into two groups according to their functions: photosynthetic and nonphotosynthetic isozymes. The main function of nonphotosynthetic PEPC was thought to be its contribution to an anaplerotic reaction [1,2]
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