Site-directed mutagenesis identified the key active site residues of 2,3-oxidosqualene cyclase HcOSC6 responsible for cucurbitacins biosynthesis in Hemsleya chinensis
Hemsleya chinensis is a Chinese traditional medicinal plant, containing cucurbitacin IIa (CuIIa) and cucurbitacin IIb (CuIIb), both of which have a wide range of pharmacological effects, including antiallergic, anti-inflammatory, and anticancer properties. However, few studies have been explored on...
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| Format: | Article |
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Frontiers Media S.A.
2023-03-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fpls.2023.1138893/full |
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| author | Xia Li Xia Li Geng Chen Geng Chen Qing-Qing Gao Qing-Qing Gao Chun-Fan Xiang Chun-Fan Xiang Cheng-Xiao Yuan Cheng-Xiao Yuan Xiao-Ning Li Yan-Yu Shu Yan-Yu Shu Guang-Hui Zhang Guang-Hui Zhang Yan-Li Liang Yan-Li Liang Sheng-Chao Yang Chen-Xi Zhai Yan Zhao Yan Zhao |
| author_facet | Xia Li Xia Li Geng Chen Geng Chen Qing-Qing Gao Qing-Qing Gao Chun-Fan Xiang Chun-Fan Xiang Cheng-Xiao Yuan Cheng-Xiao Yuan Xiao-Ning Li Yan-Yu Shu Yan-Yu Shu Guang-Hui Zhang Guang-Hui Zhang Yan-Li Liang Yan-Li Liang Sheng-Chao Yang Chen-Xi Zhai Yan Zhao Yan Zhao |
| author_sort | Xia Li |
| collection | DOAJ |
| description | Hemsleya chinensis is a Chinese traditional medicinal plant, containing cucurbitacin IIa (CuIIa) and cucurbitacin IIb (CuIIb), both of which have a wide range of pharmacological effects, including antiallergic, anti-inflammatory, and anticancer properties. However, few studies have been explored on the key enzymes that are involved in cucurbitacins biosynthesis in H. chinensis. Oxidosqualene cyclase (OSC) is a vital enzyme for cyclizing 2,3-oxidosqualene and its analogues. Here, a gene encoding the oxidosqualene cyclase of H. chinensis (HcOSC6), catalyzing to produce cucurbitadienol, was used as a template of mutagenesis. With the assistance of AlphaFold2 and molecular docking, we have proposed for the first time to our knowledge the 3D structure of HcOSC6 and its binding features to 2,3-oxidosqualene. Mutagenesis experiments on HcOSC6 generated seventeen different single-point mutants, showing that single-residue changes could affect its activity. Three key amino acid residues of HcOSC6, E246, M261 and D490, were identified as a prominent role in controlling cyclization ability. Our findings not only comprehensively characterize three key residues that are potentially useful for producing cucurbitacins, but also provide insights into the significant role they could play in metabolic engineering. |
| first_indexed | 2024-04-09T21:22:30Z |
| format | Article |
| id | doaj.art-3044bc9d04fa4ef0be30e1476ed089d1 |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-04-09T21:22:30Z |
| publishDate | 2023-03-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-3044bc9d04fa4ef0be30e1476ed089d12023-03-28T04:32:06ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2023-03-011410.3389/fpls.2023.11388931138893Site-directed mutagenesis identified the key active site residues of 2,3-oxidosqualene cyclase HcOSC6 responsible for cucurbitacins biosynthesis in Hemsleya chinensisXia Li0Xia Li1Geng Chen2Geng Chen3Qing-Qing Gao4Qing-Qing Gao5Chun-Fan Xiang6Chun-Fan Xiang7Cheng-Xiao Yuan8Cheng-Xiao Yuan9Xiao-Ning Li10Yan-Yu Shu11Yan-Yu Shu12Guang-Hui Zhang13Guang-Hui Zhang14Yan-Li Liang15Yan-Li Liang16Sheng-Chao Yang17Chen-Xi Zhai18Yan Zhao19Yan Zhao20Key Laboratory of Medicinal Plant Biology of Yunnan Province, National and Local Joint Engineering Research Center on Germplasms Innovation and Utilization of Chinese Medicinal Materials in Southwest China, Yunnan Agricultural University, Kunming, ChinaCollege of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming, ChinaKey Laboratory of Medicinal Plant Biology of Yunnan Province, National and Local Joint Engineering Research Center on Germplasms Innovation and Utilization of Chinese Medicinal Materials in Southwest China, Yunnan Agricultural University, Kunming, ChinaCollege of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming, ChinaKey Laboratory of Medicinal Plant Biology of Yunnan Province, National and Local Joint Engineering Research Center on Germplasms Innovation and Utilization of Chinese Medicinal Materials in Southwest China, Yunnan Agricultural University, Kunming, ChinaCollege of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming, ChinaKey Laboratory of Medicinal Plant Biology of Yunnan Province, National and Local Joint Engineering Research Center on Germplasms Innovation and Utilization of Chinese Medicinal Materials in Southwest China, Yunnan Agricultural University, Kunming, ChinaCollege of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming, ChinaKey Laboratory of Medicinal Plant Biology of Yunnan Province, National and Local Joint Engineering Research Center on Germplasms Innovation and Utilization of Chinese Medicinal Materials in Southwest China, Yunnan Agricultural University, Kunming, ChinaCollege of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming, ChinaKey Laboratory of Medicinal Plant Biology of Yunnan Province, National and Local Joint Engineering Research Center on Germplasms Innovation and Utilization of Chinese Medicinal Materials in Southwest China, Yunnan Agricultural University, Kunming, ChinaKey Laboratory of Medicinal Plant Biology of Yunnan Province, National and Local Joint Engineering Research Center on Germplasms Innovation and Utilization of Chinese Medicinal Materials in Southwest China, Yunnan Agricultural University, Kunming, ChinaCollege of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming, ChinaKey Laboratory of Medicinal Plant Biology of Yunnan Province, National and Local Joint Engineering Research Center on Germplasms Innovation and Utilization of Chinese Medicinal Materials in Southwest China, Yunnan Agricultural University, Kunming, ChinaCollege of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming, ChinaKey Laboratory of Medicinal Plant Biology of Yunnan Province, National and Local Joint Engineering Research Center on Germplasms Innovation and Utilization of Chinese Medicinal Materials in Southwest China, Yunnan Agricultural University, Kunming, ChinaCollege of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming, ChinaKey Laboratory of Medicinal Plant Biology of Yunnan Province, National and Local Joint Engineering Research Center on Germplasms Innovation and Utilization of Chinese Medicinal Materials in Southwest China, Yunnan Agricultural University, Kunming, ChinaSibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, NY, United StatesKey Laboratory of Medicinal Plant Biology of Yunnan Province, National and Local Joint Engineering Research Center on Germplasms Innovation and Utilization of Chinese Medicinal Materials in Southwest China, Yunnan Agricultural University, Kunming, ChinaCollege of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming, ChinaHemsleya chinensis is a Chinese traditional medicinal plant, containing cucurbitacin IIa (CuIIa) and cucurbitacin IIb (CuIIb), both of which have a wide range of pharmacological effects, including antiallergic, anti-inflammatory, and anticancer properties. However, few studies have been explored on the key enzymes that are involved in cucurbitacins biosynthesis in H. chinensis. Oxidosqualene cyclase (OSC) is a vital enzyme for cyclizing 2,3-oxidosqualene and its analogues. Here, a gene encoding the oxidosqualene cyclase of H. chinensis (HcOSC6), catalyzing to produce cucurbitadienol, was used as a template of mutagenesis. With the assistance of AlphaFold2 and molecular docking, we have proposed for the first time to our knowledge the 3D structure of HcOSC6 and its binding features to 2,3-oxidosqualene. Mutagenesis experiments on HcOSC6 generated seventeen different single-point mutants, showing that single-residue changes could affect its activity. Three key amino acid residues of HcOSC6, E246, M261 and D490, were identified as a prominent role in controlling cyclization ability. Our findings not only comprehensively characterize three key residues that are potentially useful for producing cucurbitacins, but also provide insights into the significant role they could play in metabolic engineering.https://www.frontiersin.org/articles/10.3389/fpls.2023.1138893/fullHcOSC6cucurbitacinsmolecular dockingsite-directed mutagenesisHemsleya chinensis |
| spellingShingle | Xia Li Xia Li Geng Chen Geng Chen Qing-Qing Gao Qing-Qing Gao Chun-Fan Xiang Chun-Fan Xiang Cheng-Xiao Yuan Cheng-Xiao Yuan Xiao-Ning Li Yan-Yu Shu Yan-Yu Shu Guang-Hui Zhang Guang-Hui Zhang Yan-Li Liang Yan-Li Liang Sheng-Chao Yang Chen-Xi Zhai Yan Zhao Yan Zhao Site-directed mutagenesis identified the key active site residues of 2,3-oxidosqualene cyclase HcOSC6 responsible for cucurbitacins biosynthesis in Hemsleya chinensis Frontiers in Plant Science HcOSC6 cucurbitacins molecular docking site-directed mutagenesis Hemsleya chinensis |
| title | Site-directed mutagenesis identified the key active site residues of 2,3-oxidosqualene cyclase HcOSC6 responsible for cucurbitacins biosynthesis in Hemsleya chinensis |
| title_full | Site-directed mutagenesis identified the key active site residues of 2,3-oxidosqualene cyclase HcOSC6 responsible for cucurbitacins biosynthesis in Hemsleya chinensis |
| title_fullStr | Site-directed mutagenesis identified the key active site residues of 2,3-oxidosqualene cyclase HcOSC6 responsible for cucurbitacins biosynthesis in Hemsleya chinensis |
| title_full_unstemmed | Site-directed mutagenesis identified the key active site residues of 2,3-oxidosqualene cyclase HcOSC6 responsible for cucurbitacins biosynthesis in Hemsleya chinensis |
| title_short | Site-directed mutagenesis identified the key active site residues of 2,3-oxidosqualene cyclase HcOSC6 responsible for cucurbitacins biosynthesis in Hemsleya chinensis |
| title_sort | site directed mutagenesis identified the key active site residues of 2 3 oxidosqualene cyclase hcosc6 responsible for cucurbitacins biosynthesis in hemsleya chinensis |
| topic | HcOSC6 cucurbitacins molecular docking site-directed mutagenesis Hemsleya chinensis |
| url | https://www.frontiersin.org/articles/10.3389/fpls.2023.1138893/full |
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