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Loss of Function in Zeaxanthin Epoxidase of Dunaliella tertiolecta Caused by a Single Amino Acid Mutation within the Substrate-Binding Site
The zea1 mutant of marine microalga Dunaliella tertiolecta accumulates zeaxanthin under normal growth conditions, and its phenotype has been speculated to be related to zeaxanthin epoxidase (ZEP). In this study, we isolated the ZEP gene from both wild-type D. tertiolecta and the mutant. We found tha...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6266236/ https://www.ncbi.nlm.nih.gov/pubmed/30388729 http://dx.doi.org/10.3390/md16110418 |
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author | Kim, Minjae Kang, Jisu Kang, Yongsoo Kang, Beom Sik Jin, EonSeon |
author_facet | Kim, Minjae Kang, Jisu Kang, Yongsoo Kang, Beom Sik Jin, EonSeon |
author_sort | Kim, Minjae |
collection | PubMed |
description | The zea1 mutant of marine microalga Dunaliella tertiolecta accumulates zeaxanthin under normal growth conditions, and its phenotype has been speculated to be related to zeaxanthin epoxidase (ZEP). In this study, we isolated the ZEP gene from both wild-type D. tertiolecta and the mutant. We found that the zea1 mutant has a point mutation of the 1337th nucleotide of the ZEP sequence (a change from guanine to adenine), resulting in a change of glycine to aspartate in a highly conserved region in the catalytic domain. Similar expression levels of ZEP mRNA and protein in both wild-type and zea1 were confirmed by using qRT-PCR and western blot analysis, respectively. Additionally, the enzyme activity analysis of ZEPs in the presence of cofactors showed that the inactivation of ZEP in zea1 was not caused by deficiency in the levels of cofactors. From the predicted three-dimensional ZEP structure of zea1, we observed a conformational change on the substrate-binding site in the ZEP. A comparative analysis of the ZEP structures suggested that the conformational change induced by a single amino acid mutation might impact the interaction between the substrate and substrate-binding site, resulting in loss of zeaxanthin epoxidase function. |
format | Online Article Text |
id | pubmed-6266236 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-62662362018-12-06 Loss of Function in Zeaxanthin Epoxidase of Dunaliella tertiolecta Caused by a Single Amino Acid Mutation within the Substrate-Binding Site Kim, Minjae Kang, Jisu Kang, Yongsoo Kang, Beom Sik Jin, EonSeon Mar Drugs Article The zea1 mutant of marine microalga Dunaliella tertiolecta accumulates zeaxanthin under normal growth conditions, and its phenotype has been speculated to be related to zeaxanthin epoxidase (ZEP). In this study, we isolated the ZEP gene from both wild-type D. tertiolecta and the mutant. We found that the zea1 mutant has a point mutation of the 1337th nucleotide of the ZEP sequence (a change from guanine to adenine), resulting in a change of glycine to aspartate in a highly conserved region in the catalytic domain. Similar expression levels of ZEP mRNA and protein in both wild-type and zea1 were confirmed by using qRT-PCR and western blot analysis, respectively. Additionally, the enzyme activity analysis of ZEPs in the presence of cofactors showed that the inactivation of ZEP in zea1 was not caused by deficiency in the levels of cofactors. From the predicted three-dimensional ZEP structure of zea1, we observed a conformational change on the substrate-binding site in the ZEP. A comparative analysis of the ZEP structures suggested that the conformational change induced by a single amino acid mutation might impact the interaction between the substrate and substrate-binding site, resulting in loss of zeaxanthin epoxidase function. MDPI 2018-11-01 /pmc/articles/PMC6266236/ /pubmed/30388729 http://dx.doi.org/10.3390/md16110418 Text en © 2018 by the authors. 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 (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Kim, Minjae Kang, Jisu Kang, Yongsoo Kang, Beom Sik Jin, EonSeon Loss of Function in Zeaxanthin Epoxidase of Dunaliella tertiolecta Caused by a Single Amino Acid Mutation within the Substrate-Binding Site |
title | Loss of Function in Zeaxanthin Epoxidase of Dunaliella tertiolecta Caused by a Single Amino Acid Mutation within the Substrate-Binding Site |
title_full | Loss of Function in Zeaxanthin Epoxidase of Dunaliella tertiolecta Caused by a Single Amino Acid Mutation within the Substrate-Binding Site |
title_fullStr | Loss of Function in Zeaxanthin Epoxidase of Dunaliella tertiolecta Caused by a Single Amino Acid Mutation within the Substrate-Binding Site |
title_full_unstemmed | Loss of Function in Zeaxanthin Epoxidase of Dunaliella tertiolecta Caused by a Single Amino Acid Mutation within the Substrate-Binding Site |
title_short | Loss of Function in Zeaxanthin Epoxidase of Dunaliella tertiolecta Caused by a Single Amino Acid Mutation within the Substrate-Binding Site |
title_sort | loss of function in zeaxanthin epoxidase of dunaliella tertiolecta caused by a single amino acid mutation within the substrate-binding site |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6266236/ https://www.ncbi.nlm.nih.gov/pubmed/30388729 http://dx.doi.org/10.3390/md16110418 |
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