Catalytic Properties of Caseinolytic Protease Subunit of Plasmodium knowlesi and Its Inhibition by a Member of δ-Lactone, Hyptolide
The caseinolytic protease (Clp) system plays an essential role in the protein homeostasis of the malaria parasite, particularly at the stage of apicoplast development. The inhibition of this protein is known to have a lethal effect on the parasite and is therefore considered an interesting avenue fo...
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| Format: | Article |
| Language: | English English |
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Multidisciplinary Digital Publishing Institute (MDPI)
2022
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| Online Access: | https://eprints.ums.edu.my/id/eprint/33185/5/Catalytic%20Properties%20of%20Caseinolytic%20Protease%20Subunit%20of%20Plasmodium%20knowlesi%20and%20Its%20Inhibition%20by%20a%20Member%20of%20%CE%B4-Lactone%2C%20Hyptolide.pdf https://eprints.ums.edu.my/id/eprint/33185/3/Catalytic%20Properties%20of%20Caseinolytic%20Protease%20Subunit%20of%20Plasmodium%20knowlesi%20and%20Its%20Inhibition%20by%20a%20Member%20of%20%CE%B4-Lactone%2C%20Hyptolide%20_ABSTRACT.pdf |
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| author | Cahyo Budiman Raimalynah Abd Razak Angelesa Runin Unggit Rafida Razali Meiny Suzery Ruzaidi Azli Mohd Mokhtar Lee, Ping Chin Didik Huswo Utomo |
| author_facet | Cahyo Budiman Raimalynah Abd Razak Angelesa Runin Unggit Rafida Razali Meiny Suzery Ruzaidi Azli Mohd Mokhtar Lee, Ping Chin Didik Huswo Utomo |
| author_sort | Cahyo Budiman |
| collection | UMS |
| description | The caseinolytic protease (Clp) system plays an essential role in the protein homeostasis of the malaria parasite, particularly at the stage of apicoplast development. The inhibition of this protein is known to have a lethal effect on the parasite and is therefore considered an interesting avenue for antimalaria drugs discovery. The catalytic activity of the Clp system is modulated by its proteolytic subunit (ClpP), which belongs to the serine protease family member and is therefore extensively studied for further inhibitors development. Among many inhibitors, the group of β-lactone is known to be a specific inhibitor for ClpP. Nevertheless, other groups of lactones have never been studied. This study aims to characterize the catalytic properties of ClpP of Plasmodium knowlesi (Pk-ClpP) and the inhibition properties of a δ-lactone hyptolide against this protein. Accordingly, a codon-optimized synthetic gene encoding Pk-ClpP was expressed in Escherichia coli BL21(DE3) and purified under a single step of Ni2+-affinity chromatography, yielding a 2.20 mg from 1 L culture. Meanwhile, size-exclusion chromatography indicated that Pk-ClpP migrated primarily as homoheptameric with a size of 205 kDa. The specific activity of pure Pk-ClpP was 0.73 U µg−1, with a catalytic efficiency kcat/KM of 0.05 µM−1 s−1, with optimum temperature and pH of 50 °C and 7.0–7.5, respectively. Interestingly, hyptolide, a member of δ-lactone, was shown to inhibit Pk-ClpP with an IC50 value of 17.36 ± 1.44 nM. Structural homology modelling, secondary structure prediction, and far-UV CD spectra revealed that helical structures dominate this protein. In addition, the structural homology modeling showed that this protein forms a barrel-shaped homoheptamer. Docking simulation revealed that the inhibition was found to be a competitive inhibition in which hyptolide was able to dock into the catalytic site and block the substrate. The competitiveness of hyptolide is due to the higher binding affinity of this molecule than the substrate. |
| first_indexed | 2024-03-06T03:17:40Z |
| format | Article |
| id | ums.eprints-33185 |
| institution | Universiti Malaysia Sabah |
| language | English English |
| last_indexed | 2024-03-06T03:17:40Z |
| publishDate | 2022 |
| publisher | Multidisciplinary Digital Publishing Institute (MDPI) |
| record_format | dspace |
| spelling | ums.eprints-331852022-07-12T20:48:06Z https://eprints.ums.edu.my/id/eprint/33185/ Catalytic Properties of Caseinolytic Protease Subunit of Plasmodium knowlesi and Its Inhibition by a Member of δ-Lactone, Hyptolide Cahyo Budiman Raimalynah Abd Razak Angelesa Runin Unggit Rafida Razali Meiny Suzery Ruzaidi Azli Mohd Mokhtar Lee, Ping Chin Didik Huswo Utomo QR1-502 Microbiology The caseinolytic protease (Clp) system plays an essential role in the protein homeostasis of the malaria parasite, particularly at the stage of apicoplast development. The inhibition of this protein is known to have a lethal effect on the parasite and is therefore considered an interesting avenue for antimalaria drugs discovery. The catalytic activity of the Clp system is modulated by its proteolytic subunit (ClpP), which belongs to the serine protease family member and is therefore extensively studied for further inhibitors development. Among many inhibitors, the group of β-lactone is known to be a specific inhibitor for ClpP. Nevertheless, other groups of lactones have never been studied. This study aims to characterize the catalytic properties of ClpP of Plasmodium knowlesi (Pk-ClpP) and the inhibition properties of a δ-lactone hyptolide against this protein. Accordingly, a codon-optimized synthetic gene encoding Pk-ClpP was expressed in Escherichia coli BL21(DE3) and purified under a single step of Ni2+-affinity chromatography, yielding a 2.20 mg from 1 L culture. Meanwhile, size-exclusion chromatography indicated that Pk-ClpP migrated primarily as homoheptameric with a size of 205 kDa. The specific activity of pure Pk-ClpP was 0.73 U µg−1, with a catalytic efficiency kcat/KM of 0.05 µM−1 s−1, with optimum temperature and pH of 50 °C and 7.0–7.5, respectively. Interestingly, hyptolide, a member of δ-lactone, was shown to inhibit Pk-ClpP with an IC50 value of 17.36 ± 1.44 nM. Structural homology modelling, secondary structure prediction, and far-UV CD spectra revealed that helical structures dominate this protein. In addition, the structural homology modeling showed that this protein forms a barrel-shaped homoheptamer. Docking simulation revealed that the inhibition was found to be a competitive inhibition in which hyptolide was able to dock into the catalytic site and block the substrate. The competitiveness of hyptolide is due to the higher binding affinity of this molecule than the substrate. Multidisciplinary Digital Publishing Institute (MDPI) 2022 Article PeerReviewed text en https://eprints.ums.edu.my/id/eprint/33185/5/Catalytic%20Properties%20of%20Caseinolytic%20Protease%20Subunit%20of%20Plasmodium%20knowlesi%20and%20Its%20Inhibition%20by%20a%20Member%20of%20%CE%B4-Lactone%2C%20Hyptolide.pdf text en https://eprints.ums.edu.my/id/eprint/33185/3/Catalytic%20Properties%20of%20Caseinolytic%20Protease%20Subunit%20of%20Plasmodium%20knowlesi%20and%20Its%20Inhibition%20by%20a%20Member%20of%20%CE%B4-Lactone%2C%20Hyptolide%20_ABSTRACT.pdf Cahyo Budiman and Raimalynah Abd Razak and Angelesa Runin Unggit and Rafida Razali and Meiny Suzery and Ruzaidi Azli Mohd Mokhtar and Lee, Ping Chin and Didik Huswo Utomo (2022) Catalytic Properties of Caseinolytic Protease Subunit of Plasmodium knowlesi and Its Inhibition by a Member of δ-Lactone, Hyptolide. Molecules, 27 (3787). pp. 1-21. ISSN 1420-3049 https://www.mdpi.com/1420-3049/27/12/3787/htm https://doi.org/10.3390/molecules27123787 https://doi.org/10.3390/molecules27123787 |
| spellingShingle | QR1-502 Microbiology Cahyo Budiman Raimalynah Abd Razak Angelesa Runin Unggit Rafida Razali Meiny Suzery Ruzaidi Azli Mohd Mokhtar Lee, Ping Chin Didik Huswo Utomo Catalytic Properties of Caseinolytic Protease Subunit of Plasmodium knowlesi and Its Inhibition by a Member of δ-Lactone, Hyptolide |
| title | Catalytic Properties of Caseinolytic Protease Subunit of Plasmodium knowlesi and Its Inhibition by a Member of δ-Lactone, Hyptolide |
| title_full | Catalytic Properties of Caseinolytic Protease Subunit of Plasmodium knowlesi and Its Inhibition by a Member of δ-Lactone, Hyptolide |
| title_fullStr | Catalytic Properties of Caseinolytic Protease Subunit of Plasmodium knowlesi and Its Inhibition by a Member of δ-Lactone, Hyptolide |
| title_full_unstemmed | Catalytic Properties of Caseinolytic Protease Subunit of Plasmodium knowlesi and Its Inhibition by a Member of δ-Lactone, Hyptolide |
| title_short | Catalytic Properties of Caseinolytic Protease Subunit of Plasmodium knowlesi and Its Inhibition by a Member of δ-Lactone, Hyptolide |
| title_sort | catalytic properties of caseinolytic protease subunit of plasmodium knowlesi and its inhibition by a member of δ lactone hyptolide |
| topic | QR1-502 Microbiology |
| url | https://eprints.ums.edu.my/id/eprint/33185/5/Catalytic%20Properties%20of%20Caseinolytic%20Protease%20Subunit%20of%20Plasmodium%20knowlesi%20and%20Its%20Inhibition%20by%20a%20Member%20of%20%CE%B4-Lactone%2C%20Hyptolide.pdf https://eprints.ums.edu.my/id/eprint/33185/3/Catalytic%20Properties%20of%20Caseinolytic%20Protease%20Subunit%20of%20Plasmodium%20knowlesi%20and%20Its%20Inhibition%20by%20a%20Member%20of%20%CE%B4-Lactone%2C%20Hyptolide%20_ABSTRACT.pdf |
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