Molecular Cloning of Novel-Type Phospho<i>enol</i>pyruvate Carboxylase Isoforms in Pitaya (<i>Hylocereus undatus</i>)
Phospho<i>enol</i>pyruvate carboxylase (PEPC) is an important enzyme involved in the initial CO<sub>2</sub> fixation of crassulacean acid metabolism (CAM) photosynthesis. To understand the cultivation characteristics of a CAM plant pitaya, it is necessary to clarify the chara...
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MDPI AG
2020-09-01
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| Series: | Plants |
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| Online Access: | https://www.mdpi.com/2223-7747/9/9/1241 |
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| author | Keiichi Nomura Yuho Sakurai Mayu Dozono |
| author_facet | Keiichi Nomura Yuho Sakurai Mayu Dozono |
| author_sort | Keiichi Nomura |
| collection | DOAJ |
| description | Phospho<i>enol</i>pyruvate carboxylase (PEPC) is an important enzyme involved in the initial CO<sub>2</sub> 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, <i>HuPPC1</i>, <i>HuPPC2</i>, and <i>HuPPC3</i>, 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. |
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| spelling | doaj.art-f4dcfbebfa72441ab8c6176f0cfc12c32023-11-20T14:26:43ZengMDPI AGPlants2223-77472020-09-0199124110.3390/plants9091241Molecular Cloning of Novel-Type Phospho<i>enol</i>pyruvate Carboxylase Isoforms in Pitaya (<i>Hylocereus undatus</i>)Keiichi Nomura0Yuho Sakurai1Mayu Dozono2Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, JapanFaculty of Agricultural Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, JapanGraduate School of Agricultural Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, JapanPhospho<i>enol</i>pyruvate carboxylase (PEPC) is an important enzyme involved in the initial CO<sub>2</sub> 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, <i>HuPPC1</i>, <i>HuPPC2</i>, and <i>HuPPC3</i>, 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.https://www.mdpi.com/2223-7747/9/9/1241CAMisoformphospho<i>enol</i>pyruvate carboxylasepitaya (<i>Hylocereus undatus</i>) |
| spellingShingle | Keiichi Nomura Yuho Sakurai Mayu Dozono Molecular Cloning of Novel-Type Phospho<i>enol</i>pyruvate Carboxylase Isoforms in Pitaya (<i>Hylocereus undatus</i>) Plants CAM isoform phospho<i>enol</i>pyruvate carboxylase pitaya (<i>Hylocereus undatus</i>) |
| title | Molecular Cloning of Novel-Type Phospho<i>enol</i>pyruvate Carboxylase Isoforms in Pitaya (<i>Hylocereus undatus</i>) |
| title_full | Molecular Cloning of Novel-Type Phospho<i>enol</i>pyruvate Carboxylase Isoforms in Pitaya (<i>Hylocereus undatus</i>) |
| title_fullStr | Molecular Cloning of Novel-Type Phospho<i>enol</i>pyruvate Carboxylase Isoforms in Pitaya (<i>Hylocereus undatus</i>) |
| title_full_unstemmed | Molecular Cloning of Novel-Type Phospho<i>enol</i>pyruvate Carboxylase Isoforms in Pitaya (<i>Hylocereus undatus</i>) |
| title_short | Molecular Cloning of Novel-Type Phospho<i>enol</i>pyruvate Carboxylase Isoforms in Pitaya (<i>Hylocereus undatus</i>) |
| title_sort | molecular cloning of novel type phospho i enol i pyruvate carboxylase isoforms in pitaya i hylocereus undatus i |
| topic | CAM isoform phospho<i>enol</i>pyruvate carboxylase pitaya (<i>Hylocereus undatus</i>) |
| url | https://www.mdpi.com/2223-7747/9/9/1241 |
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