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Molecular Characterization and Functional Analysis of Cytochrome b5 Reductase (CBR) Encoding Genes from the Carotenogenic Yeast Xanthophyllomyces dendrorhous
The eukaryotic microsomal cytochrome P450 systems consist of a cytochrome P450 enzyme (P450) and a cytochrome P450 redox partner, which generally is a cytochrome P450 reductase (CPR) that supplies electrons from NADPH. However, alternative electron donors may exist such as cytochrome b5 reductase an...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Public Library of Science
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4605618/ https://www.ncbi.nlm.nih.gov/pubmed/26466337 http://dx.doi.org/10.1371/journal.pone.0140424 |
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author | Gutiérrez, María Soledad Rojas, María Cecilia Sepúlveda, Dionisia Baeza, Marcelo Cifuentes, Víctor Alcaíno, Jennifer |
author_facet | Gutiérrez, María Soledad Rojas, María Cecilia Sepúlveda, Dionisia Baeza, Marcelo Cifuentes, Víctor Alcaíno, Jennifer |
author_sort | Gutiérrez, María Soledad |
collection | PubMed |
description | The eukaryotic microsomal cytochrome P450 systems consist of a cytochrome P450 enzyme (P450) and a cytochrome P450 redox partner, which generally is a cytochrome P450 reductase (CPR) that supplies electrons from NADPH. However, alternative electron donors may exist such as cytochrome b5 reductase and cytochrome b5 (CBR and CYB5, respectively) via, which is NADH-dependent and are also anchored to the endoplasmic reticulum. In the carotenogenic yeast Xanthophyllomyces dendrorhous, three P450-encoding genes have been described: crtS is involved in carotenogenesis and the CYP51 and CYP61 genes are both implicated in ergosterol biosynthesis. This yeast has a single CPR (encoded by the crtR gene), and a crtR (-) mutant does not produce astaxanthin. Considering that this mutant is viable, the existence of alternative cytochrome P450 electron donors like CBR and CYB5 could operate in this yeast. The aim of this work was to characterize the X. dendrorhous CBR encoding gene and to study its involvement in P450 reactions in ergosterol and carotenoid biosynthesis. Two CBRs genes were identified (CBR.1 and CBR.2), and deletion mutants were constructed. The two mutants and the wild-type strain showed similar sterol production, with ergosterol being the main sterol produced. The crtR (-) mutant strain produced a lower proportion of ergosterol than did the parental strain. These results indicate that even though one of the two CBR genes could be involved in ergosterol biosynthesis, crtR complements their absence in the cbr (-) mutant strains, at least for ergosterol production. The higher NADH-dependent cytochrome c reductase activity together with the higher transcript levels of CBR.1 and CYB5 in the crtR (-) mutant as well as the lower NADH-dependent activity in CBS-cbr.1(-) strongly suggest that CBR.1-CYB5 via participates as an alternative electron donor pathway for P450 enzymes involved in ergosterol biosynthesis in X. dendrorhous. |
format | Online Article Text |
id | pubmed-4605618 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-46056182015-10-29 Molecular Characterization and Functional Analysis of Cytochrome b5 Reductase (CBR) Encoding Genes from the Carotenogenic Yeast Xanthophyllomyces dendrorhous Gutiérrez, María Soledad Rojas, María Cecilia Sepúlveda, Dionisia Baeza, Marcelo Cifuentes, Víctor Alcaíno, Jennifer PLoS One Research Article The eukaryotic microsomal cytochrome P450 systems consist of a cytochrome P450 enzyme (P450) and a cytochrome P450 redox partner, which generally is a cytochrome P450 reductase (CPR) that supplies electrons from NADPH. However, alternative electron donors may exist such as cytochrome b5 reductase and cytochrome b5 (CBR and CYB5, respectively) via, which is NADH-dependent and are also anchored to the endoplasmic reticulum. In the carotenogenic yeast Xanthophyllomyces dendrorhous, three P450-encoding genes have been described: crtS is involved in carotenogenesis and the CYP51 and CYP61 genes are both implicated in ergosterol biosynthesis. This yeast has a single CPR (encoded by the crtR gene), and a crtR (-) mutant does not produce astaxanthin. Considering that this mutant is viable, the existence of alternative cytochrome P450 electron donors like CBR and CYB5 could operate in this yeast. The aim of this work was to characterize the X. dendrorhous CBR encoding gene and to study its involvement in P450 reactions in ergosterol and carotenoid biosynthesis. Two CBRs genes were identified (CBR.1 and CBR.2), and deletion mutants were constructed. The two mutants and the wild-type strain showed similar sterol production, with ergosterol being the main sterol produced. The crtR (-) mutant strain produced a lower proportion of ergosterol than did the parental strain. These results indicate that even though one of the two CBR genes could be involved in ergosterol biosynthesis, crtR complements their absence in the cbr (-) mutant strains, at least for ergosterol production. The higher NADH-dependent cytochrome c reductase activity together with the higher transcript levels of CBR.1 and CYB5 in the crtR (-) mutant as well as the lower NADH-dependent activity in CBS-cbr.1(-) strongly suggest that CBR.1-CYB5 via participates as an alternative electron donor pathway for P450 enzymes involved in ergosterol biosynthesis in X. dendrorhous. Public Library of Science 2015-10-14 /pmc/articles/PMC4605618/ /pubmed/26466337 http://dx.doi.org/10.1371/journal.pone.0140424 Text en © 2015 Gutiérrez et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Gutiérrez, María Soledad Rojas, María Cecilia Sepúlveda, Dionisia Baeza, Marcelo Cifuentes, Víctor Alcaíno, Jennifer Molecular Characterization and Functional Analysis of Cytochrome b5 Reductase (CBR) Encoding Genes from the Carotenogenic Yeast Xanthophyllomyces dendrorhous |
title | Molecular Characterization and Functional Analysis of Cytochrome b5 Reductase (CBR) Encoding Genes from the Carotenogenic Yeast Xanthophyllomyces dendrorhous
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title_full | Molecular Characterization and Functional Analysis of Cytochrome b5 Reductase (CBR) Encoding Genes from the Carotenogenic Yeast Xanthophyllomyces dendrorhous
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title_fullStr | Molecular Characterization and Functional Analysis of Cytochrome b5 Reductase (CBR) Encoding Genes from the Carotenogenic Yeast Xanthophyllomyces dendrorhous
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title_full_unstemmed | Molecular Characterization and Functional Analysis of Cytochrome b5 Reductase (CBR) Encoding Genes from the Carotenogenic Yeast Xanthophyllomyces dendrorhous
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title_short | Molecular Characterization and Functional Analysis of Cytochrome b5 Reductase (CBR) Encoding Genes from the Carotenogenic Yeast Xanthophyllomyces dendrorhous
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title_sort | molecular characterization and functional analysis of cytochrome b5 reductase (cbr) encoding genes from the carotenogenic yeast xanthophyllomyces dendrorhous |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4605618/ https://www.ncbi.nlm.nih.gov/pubmed/26466337 http://dx.doi.org/10.1371/journal.pone.0140424 |
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