A novel bacterial class v dye-decolourizing peroxidase from the extremophile Deinococcus radiodurans
Autor(a) principal: | |
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Data de Publicação: | 2018 |
Outros Autores: | , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10362/85040 |
Resumo: | Deinococcus radiodurans is a bacterium with extreme resistance to desiccation and radiation. The resistance mechanism is unknown, but an efficient reactive oxygen species (ROS) scavenging system and DNA-repair and DNA-protection mechanisms are believed to play important roles. Here, the cloning and smalland medium-scale expression tests of a novel dye-decolourizing peroxidase from D. radiodurans (DrDyP) using three different Escherichia coli strains and three different temperatures in order to identify the optimum conditions for the expression of recombinant DrDyP are presented. The best expression conditions were used for large-scale expression and yielded ~10 mg recombinant DrDyP per litre of culture after purification. Initial characterization experiments demonstrated unusual features with regard to the haem spin state, which motivated the crystallization experiment. The obtained crystals were used for data collection and diffracted to 2.2 A - resolution. The crystals belonged to the trigonal space group P31 or P32, with unit-cell parameters a = b = 64.13, c = 111.32 A - , and are predicted to contain one DrDyP molecule per asymmetric unit. Structure determination by molecular replacement using previously determined structures of dye-decolourizing peroxidases with ~30% sequence identity at ~2 A- resolution as templates are ongoing. |
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A novel bacterial class v dye-decolourizing peroxidase from the extremophile Deinococcus radioduransCloning, expression optimization, purification, crystallization, initial characterization and X-ray diffraction analysisDeinococcus radioduransDye-decolourizing peroxidasesOxidative stressPeroxidasesRadiation resistanceBiophysicsStructural BiologyBiochemistryGeneticsCondensed Matter PhysicsDeinococcus radiodurans is a bacterium with extreme resistance to desiccation and radiation. The resistance mechanism is unknown, but an efficient reactive oxygen species (ROS) scavenging system and DNA-repair and DNA-protection mechanisms are believed to play important roles. Here, the cloning and smalland medium-scale expression tests of a novel dye-decolourizing peroxidase from D. radiodurans (DrDyP) using three different Escherichia coli strains and three different temperatures in order to identify the optimum conditions for the expression of recombinant DrDyP are presented. The best expression conditions were used for large-scale expression and yielded ~10 mg recombinant DrDyP per litre of culture after purification. Initial characterization experiments demonstrated unusual features with regard to the haem spin state, which motivated the crystallization experiment. The obtained crystals were used for data collection and diffracted to 2.2 A - resolution. The crystals belonged to the trigonal space group P31 or P32, with unit-cell parameters a = b = 64.13, c = 111.32 A - , and are predicted to contain one DrDyP molecule per asymmetric unit. Structure determination by molecular replacement using previously determined structures of dye-decolourizing peroxidases with ~30% sequence identity at ~2 A- resolution as templates are ongoing.Molecular, Structural and Cellular Microbiology (MOSTMICRO)Instituto de Tecnologia Química e Biológica António Xavier (ITQB)RUNFrade, Kelly Stefany TunaFernandes, Andreia Cecilia PimentaSilveira, Celia MarisaFraza-o, CarlosMoe, Elin2022-11-02T01:33:59Z2018-07-012018-07-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article6application/pdfhttp://hdl.handle.net/10362/85040eng2053-230XPURE: 12417605http://www.scopus.com/inward/record.url?scp=85049525371&partnerID=8YFLogxKhttps://doi.org/10.1107/S2053230X18008488info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-03-11T04:37:58Zoai:run.unl.pt:10362/85040Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:36:33.943585Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
A novel bacterial class v dye-decolourizing peroxidase from the extremophile Deinococcus radiodurans Cloning, expression optimization, purification, crystallization, initial characterization and X-ray diffraction analysis |
title |
A novel bacterial class v dye-decolourizing peroxidase from the extremophile Deinococcus radiodurans |
spellingShingle |
A novel bacterial class v dye-decolourizing peroxidase from the extremophile Deinococcus radiodurans Frade, Kelly Stefany Tuna Deinococcus radiodurans Dye-decolourizing peroxidases Oxidative stress Peroxidases Radiation resistance Biophysics Structural Biology Biochemistry Genetics Condensed Matter Physics |
title_short |
A novel bacterial class v dye-decolourizing peroxidase from the extremophile Deinococcus radiodurans |
title_full |
A novel bacterial class v dye-decolourizing peroxidase from the extremophile Deinococcus radiodurans |
title_fullStr |
A novel bacterial class v dye-decolourizing peroxidase from the extremophile Deinococcus radiodurans |
title_full_unstemmed |
A novel bacterial class v dye-decolourizing peroxidase from the extremophile Deinococcus radiodurans |
title_sort |
A novel bacterial class v dye-decolourizing peroxidase from the extremophile Deinococcus radiodurans |
author |
Frade, Kelly Stefany Tuna |
author_facet |
Frade, Kelly Stefany Tuna Fernandes, Andreia Cecilia Pimenta Silveira, Celia Marisa Fraza-o, Carlos Moe, Elin |
author_role |
author |
author2 |
Fernandes, Andreia Cecilia Pimenta Silveira, Celia Marisa Fraza-o, Carlos Moe, Elin |
author2_role |
author author author author |
dc.contributor.none.fl_str_mv |
Molecular, Structural and Cellular Microbiology (MOSTMICRO) Instituto de Tecnologia Química e Biológica António Xavier (ITQB) RUN |
dc.contributor.author.fl_str_mv |
Frade, Kelly Stefany Tuna Fernandes, Andreia Cecilia Pimenta Silveira, Celia Marisa Fraza-o, Carlos Moe, Elin |
dc.subject.por.fl_str_mv |
Deinococcus radiodurans Dye-decolourizing peroxidases Oxidative stress Peroxidases Radiation resistance Biophysics Structural Biology Biochemistry Genetics Condensed Matter Physics |
topic |
Deinococcus radiodurans Dye-decolourizing peroxidases Oxidative stress Peroxidases Radiation resistance Biophysics Structural Biology Biochemistry Genetics Condensed Matter Physics |
description |
Deinococcus radiodurans is a bacterium with extreme resistance to desiccation and radiation. The resistance mechanism is unknown, but an efficient reactive oxygen species (ROS) scavenging system and DNA-repair and DNA-protection mechanisms are believed to play important roles. Here, the cloning and smalland medium-scale expression tests of a novel dye-decolourizing peroxidase from D. radiodurans (DrDyP) using three different Escherichia coli strains and three different temperatures in order to identify the optimum conditions for the expression of recombinant DrDyP are presented. The best expression conditions were used for large-scale expression and yielded ~10 mg recombinant DrDyP per litre of culture after purification. Initial characterization experiments demonstrated unusual features with regard to the haem spin state, which motivated the crystallization experiment. The obtained crystals were used for data collection and diffracted to 2.2 A - resolution. The crystals belonged to the trigonal space group P31 or P32, with unit-cell parameters a = b = 64.13, c = 111.32 A - , and are predicted to contain one DrDyP molecule per asymmetric unit. Structure determination by molecular replacement using previously determined structures of dye-decolourizing peroxidases with ~30% sequence identity at ~2 A- resolution as templates are ongoing. |
publishDate |
2018 |
dc.date.none.fl_str_mv |
2018-07-01 2018-07-01T00:00:00Z 2022-11-02T01:33:59Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10362/85040 |
url |
http://hdl.handle.net/10362/85040 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
2053-230X PURE: 12417605 http://www.scopus.com/inward/record.url?scp=85049525371&partnerID=8YFLogxK https://doi.org/10.1107/S2053230X18008488 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
6 application/pdf |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
instname_str |
Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
instacron_str |
RCAAP |
institution |
RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
collection |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository.name.fl_str_mv |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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1799137983889145856 |