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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...

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Autores principales: Xie, Dong-Mei, Zhang, Qiang, Xin, Ling-Kai, Wang, Guo-Kai, Liu, Cong-Bin, Qin, Min-Jian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
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.
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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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