Cloning and Functional Characterization of Two Germacrene A Oxidases Isolated from <i>Xanthium sibiricum</i>
Sesquiterpene lactones (STLs) from the cocklebur <i>Xanthium sibiricum</i> exhibit significant anti-tumor activity. Although germacrene A oxidase (GAO), which catalyzes the production of Germacrene A acid (GAA) from germacrene A, an important precursor of germacrene-type STLs, has been r...
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MDPI AG
2022-05-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/27/10/3322 |
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| author | Dong-Mei Xie Qiang Zhang Ling-Kai Xin Guo-Kai Wang Cong-Bin Liu Min-Jian Qin |
| author_facet | Dong-Mei Xie Qiang Zhang Ling-Kai Xin Guo-Kai Wang Cong-Bin Liu Min-Jian Qin |
| author_sort | Dong-Mei Xie |
| collection | DOAJ |
| description | Sesquiterpene lactones (STLs) from the cocklebur <i>Xanthium sibiricum</i> exhibit significant anti-tumor activity. Although germacrene A oxidase (GAO), which catalyzes the production of Germacrene A acid (GAA) from germacrene A, an important precursor of germacrene-type STLs, has been reported, the remaining GAOs corresponding to various STLs’ biosynthesis pathways remain unidentified. In this study, 68,199 unigenes were studied in a de novo transcriptome assembly of <i>X. sibiricum</i> fruits. By comparison with previously published GAO sequences, two candidate <i>X. sibiricum</i> GAO gene sequences, <i>Xs</i>GAO1 (1467 bp) and <i>Xs</i>GAO2 (1527 bp), were identified, cloned, and predicted to encode 488 and 508 amino acids, respectively. Their protein structure, motifs, sequence similarity, and phylogenetic position were similar to those of other GAO proteins. They were most strongly expressed in fruits, according to a quantitative real-time polymerase chain reaction (qRT-PCR), and both <i>Xs</i>GAO proteins were localized in the mitochondria of tobacco leaf epidermal cells. The two <i>Xs</i>GAO genes were cloned into the expression vector for eukaryotic expression in <i>Saccharomyces cerevisiae</i>, and the enzyme reaction products were detected by gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS) methods. The results indicated that both <i>Xs</i>GAO1 and <i>Xs</i>GAO2 catalyzed the two-step conversion of germacrene A (GA) to GAA, meaning they are unlike classical GAO enzymes, which catalyze a three-step conversion of GA to GAA. This cloning and functional study of two GAO genes from <i>X. sibiricum</i> provides a useful basis for further elucidation of the STL biosynthesis pathway in <i>X. sibiricum</i>. |
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| last_indexed | 2024-03-10T03:18:37Z |
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| spelling | doaj.art-cef65b7a489b44e0be256a9115cada7d2023-11-23T12:24:37ZengMDPI AGMolecules1420-30492022-05-012710332210.3390/molecules27103322Cloning and Functional Characterization of Two Germacrene A Oxidases Isolated from <i>Xanthium sibiricum</i>Dong-Mei Xie0Qiang Zhang1Ling-Kai Xin2Guo-Kai Wang3Cong-Bin Liu4Min-Jian Qin5Department of Resources Science of Traditional Chinese Medicines, China Pharmaceutical University, Nanjing 210009, ChinaAnhui Province Key Laboratory of Research & Development of Chinese Medicine, Anhui University of Chinese Medicine, Hefei 230012, ChinaAnhui Province Key Laboratory of Research & Development of Chinese Medicine, Anhui University of Chinese Medicine, Hefei 230012, ChinaAnhui Province Key Laboratory of Research & Development of Chinese Medicine, Anhui University of Chinese Medicine, Hefei 230012, ChinaAnhui Province Key Laboratory of Research & Development of Chinese Medicine, Anhui University of Chinese Medicine, Hefei 230012, ChinaDepartment of Resources Science of Traditional Chinese Medicines, China Pharmaceutical University, Nanjing 210009, ChinaSesquiterpene lactones (STLs) from the cocklebur <i>Xanthium sibiricum</i> exhibit significant anti-tumor activity. Although germacrene A oxidase (GAO), which catalyzes the production of Germacrene A acid (GAA) from germacrene A, an important precursor of germacrene-type STLs, has been reported, the remaining GAOs corresponding to various STLs’ biosynthesis pathways remain unidentified. In this study, 68,199 unigenes were studied in a de novo transcriptome assembly of <i>X. sibiricum</i> fruits. By comparison with previously published GAO sequences, two candidate <i>X. sibiricum</i> GAO gene sequences, <i>Xs</i>GAO1 (1467 bp) and <i>Xs</i>GAO2 (1527 bp), were identified, cloned, and predicted to encode 488 and 508 amino acids, respectively. Their protein structure, motifs, sequence similarity, and phylogenetic position were similar to those of other GAO proteins. They were most strongly expressed in fruits, according to a quantitative real-time polymerase chain reaction (qRT-PCR), and both <i>Xs</i>GAO proteins were localized in the mitochondria of tobacco leaf epidermal cells. The two <i>Xs</i>GAO genes were cloned into the expression vector for eukaryotic expression in <i>Saccharomyces cerevisiae</i>, and the enzyme reaction products were detected by gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS) methods. The results indicated that both <i>Xs</i>GAO1 and <i>Xs</i>GAO2 catalyzed the two-step conversion of germacrene A (GA) to GAA, meaning they are unlike classical GAO enzymes, which catalyze a three-step conversion of GA to GAA. This cloning and functional study of two GAO genes from <i>X. sibiricum</i> provides a useful basis for further elucidation of the STL biosynthesis pathway in <i>X. sibiricum</i>.https://www.mdpi.com/1420-3049/27/10/3322<i>Xanthium sibiricum</i>sesquiterpene lactonesbiosynthesisfunctional analysisgermacrene A |
| spellingShingle | Dong-Mei Xie Qiang Zhang Ling-Kai Xin Guo-Kai Wang Cong-Bin Liu Min-Jian Qin Cloning and Functional Characterization of Two Germacrene A Oxidases Isolated from <i>Xanthium sibiricum</i> Molecules <i>Xanthium sibiricum</i> sesquiterpene lactones biosynthesis functional analysis germacrene A |
| title | Cloning and Functional Characterization of Two Germacrene A Oxidases Isolated from <i>Xanthium sibiricum</i> |
| title_full | Cloning and Functional Characterization of Two Germacrene A Oxidases Isolated from <i>Xanthium sibiricum</i> |
| title_fullStr | Cloning and Functional Characterization of Two Germacrene A Oxidases Isolated from <i>Xanthium sibiricum</i> |
| title_full_unstemmed | Cloning and Functional Characterization of Two Germacrene A Oxidases Isolated from <i>Xanthium sibiricum</i> |
| title_short | Cloning and Functional Characterization of Two Germacrene A Oxidases Isolated from <i>Xanthium sibiricum</i> |
| title_sort | cloning and functional characterization of two germacrene a oxidases isolated from i xanthium sibiricum i |
| topic | <i>Xanthium sibiricum</i> sesquiterpene lactones biosynthesis functional analysis germacrene A |
| url | https://www.mdpi.com/1420-3049/27/10/3322 |
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