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Cloning and Functional Characterization of Two Germacrene A Oxidases Isolated from Xanthium sibiricum
Sesquiterpene lactones (STLs) from the cocklebur Xanthium sibiricum 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 remain...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9145264/ https://www.ncbi.nlm.nih.gov/pubmed/35630799 http://dx.doi.org/10.3390/molecules27103322 |
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author | Xie, Dong-Mei Zhang, Qiang Xin, Ling-Kai Wang, Guo-Kai Liu, Cong-Bin Qin, Min-Jian |
author_facet | Xie, Dong-Mei Zhang, Qiang Xin, Ling-Kai Wang, Guo-Kai Liu, Cong-Bin Qin, Min-Jian |
author_sort | Xie, Dong-Mei |
collection | PubMed |
description | Sesquiterpene lactones (STLs) from the cocklebur Xanthium sibiricum 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 X. sibiricum fruits. By comparison with previously published GAO sequences, two candidate X. sibiricum GAO gene sequences, XsGAO1 (1467 bp) and XsGAO2 (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 XsGAO proteins were localized in the mitochondria of tobacco leaf epidermal cells. The two XsGAO genes were cloned into the expression vector for eukaryotic expression in Saccharomyces cerevisiae, 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 XsGAO1 and XsGAO2 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 X. sibiricum provides a useful basis for further elucidation of the STL biosynthesis pathway in X. sibiricum. |
format | Online Article Text |
id | pubmed-9145264 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-91452642022-05-29 Cloning and Functional Characterization of Two Germacrene A Oxidases Isolated from Xanthium sibiricum Xie, Dong-Mei Zhang, Qiang Xin, Ling-Kai Wang, Guo-Kai Liu, Cong-Bin Qin, Min-Jian Molecules Article Sesquiterpene lactones (STLs) from the cocklebur Xanthium sibiricum 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 X. sibiricum fruits. By comparison with previously published GAO sequences, two candidate X. sibiricum GAO gene sequences, XsGAO1 (1467 bp) and XsGAO2 (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 XsGAO proteins were localized in the mitochondria of tobacco leaf epidermal cells. The two XsGAO genes were cloned into the expression vector for eukaryotic expression in Saccharomyces cerevisiae, 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 XsGAO1 and XsGAO2 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 X. sibiricum provides a useful basis for further elucidation of the STL biosynthesis pathway in X. sibiricum. MDPI 2022-05-22 /pmc/articles/PMC9145264/ /pubmed/35630799 http://dx.doi.org/10.3390/molecules27103322 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Xie, Dong-Mei Zhang, Qiang Xin, Ling-Kai Wang, Guo-Kai Liu, Cong-Bin Qin, Min-Jian Cloning and Functional Characterization of Two Germacrene A Oxidases Isolated from Xanthium sibiricum |
title | Cloning and Functional Characterization of Two Germacrene A Oxidases Isolated from Xanthium sibiricum |
title_full | Cloning and Functional Characterization of Two Germacrene A Oxidases Isolated from Xanthium sibiricum |
title_fullStr | Cloning and Functional Characterization of Two Germacrene A Oxidases Isolated from Xanthium sibiricum |
title_full_unstemmed | Cloning and Functional Characterization of Two Germacrene A Oxidases Isolated from Xanthium sibiricum |
title_short | Cloning and Functional Characterization of Two Germacrene A Oxidases Isolated from Xanthium sibiricum |
title_sort | cloning and functional characterization of two germacrene a oxidases isolated from xanthium sibiricum |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9145264/ https://www.ncbi.nlm.nih.gov/pubmed/35630799 http://dx.doi.org/10.3390/molecules27103322 |
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