Probing the role of the hinge segment of cytochrome P450 oxidoreductase in the interaction with cytochrome P450
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: | https://doi.org/10.3390/ijms19123914 |
Resumo: | NADPH-cytochrome P450 reductase (CPR) is the unique redox partner of microsomal cytochrome P450s (CYPs). CPR exists in a conformational equilibrium between open and closed conformations throughout its electron transfer (ET) function. Previously, we have shown that electrostatic and flexibility properties of the hinge segment of CPR are critical for ET. Three mutants of human CPR were studied (S243P, I245P and R246A) and combined with representative human drug-metabolizing CYPs (isoforms 1A2, 2A6 and 3A4). To probe the effect of these hinge mutations different experimental approaches were employed: CYP bioactivation capacity of pre-carcinogens, enzyme kinetic analysis, and effect of the ionic strength and cytochrome b5 (CYB5) on CYP activity. The hinge mutations influenced the bioactivation of pre-carcinogens, which seemed CYP isoform and substrate dependent. The deviations of Michaelis-Menten kinetic parameters uncovered tend to confirm this discrepancy, which was confirmed by CYP and hinge mutant specific salt/activity profiles. CPR/CYB5 competition experiments indicated a less important role of affinity in CPR/CYP interaction. Overall, our data suggest that the highly flexible hinge of CPR is responsible for the existence of a conformational aggregate of different open CPR conformers enabling ET-interaction with structural varied redox partners. |
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Probing the role of the hinge segment of cytochrome P450 oxidoreductase in the interaction with cytochrome P450Cytochrome b5 (CYB5)Electron-transfer (ET)Microsomal cytochrome p450 (CYP)NADPH-cytochrome P450 reductase (CPR)Protein dynamicsProtein-protein interactionCatalysisMolecular BiologySpectroscopyComputer Science ApplicationsPhysical and Theoretical ChemistryOrganic ChemistryInorganic ChemistryNADPH-cytochrome P450 reductase (CPR) is the unique redox partner of microsomal cytochrome P450s (CYPs). CPR exists in a conformational equilibrium between open and closed conformations throughout its electron transfer (ET) function. Previously, we have shown that electrostatic and flexibility properties of the hinge segment of CPR are critical for ET. Three mutants of human CPR were studied (S243P, I245P and R246A) and combined with representative human drug-metabolizing CYPs (isoforms 1A2, 2A6 and 3A4). To probe the effect of these hinge mutations different experimental approaches were employed: CYP bioactivation capacity of pre-carcinogens, enzyme kinetic analysis, and effect of the ionic strength and cytochrome b5 (CYB5) on CYP activity. The hinge mutations influenced the bioactivation of pre-carcinogens, which seemed CYP isoform and substrate dependent. The deviations of Michaelis-Menten kinetic parameters uncovered tend to confirm this discrepancy, which was confirmed by CYP and hinge mutant specific salt/activity profiles. CPR/CYB5 competition experiments indicated a less important role of affinity in CPR/CYP interaction. Overall, our data suggest that the highly flexible hinge of CPR is responsible for the existence of a conformational aggregate of different open CPR conformers enabling ET-interaction with structural varied redox partners.Centre for Toxicogenomics and Human Health (ToxOmics)NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM)RUNCampelo, DianaEsteves, FranciscoPalma, Bernardo BritoGomes, Bruno CostaRueff, JoseLautier, ThomasUrban, PhilippeTruan, GillesKranendonk, Michel2018-12-27T23:28:31Z2018-12-012018-12-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttps://doi.org/10.3390/ijms19123914eng1661-6596PURE: 10978041http://www.scopus.com/inward/record.url?scp=85058293841&partnerID=8YFLogxKhttps://doi.org/10.3390/ijms19123914info: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:RCAAP2024-09-23T01:37:50Zoai:run.unl.pt:10362/55830Portal AgregadorONGhttps://www.rcaap.pt/oai/openairemluisa.alvim@gmail.comopendoar:71602024-09-23T01:37:50Repositório 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 |
Probing the role of the hinge segment of cytochrome P450 oxidoreductase in the interaction with cytochrome P450 |
title |
Probing the role of the hinge segment of cytochrome P450 oxidoreductase in the interaction with cytochrome P450 |
spellingShingle |
Probing the role of the hinge segment of cytochrome P450 oxidoreductase in the interaction with cytochrome P450 Campelo, Diana Cytochrome b5 (CYB5) Electron-transfer (ET) Microsomal cytochrome p450 (CYP) NADPH-cytochrome P450 reductase (CPR) Protein dynamics Protein-protein interaction Catalysis Molecular Biology Spectroscopy Computer Science Applications Physical and Theoretical Chemistry Organic Chemistry Inorganic Chemistry |
title_short |
Probing the role of the hinge segment of cytochrome P450 oxidoreductase in the interaction with cytochrome P450 |
title_full |
Probing the role of the hinge segment of cytochrome P450 oxidoreductase in the interaction with cytochrome P450 |
title_fullStr |
Probing the role of the hinge segment of cytochrome P450 oxidoreductase in the interaction with cytochrome P450 |
title_full_unstemmed |
Probing the role of the hinge segment of cytochrome P450 oxidoreductase in the interaction with cytochrome P450 |
title_sort |
Probing the role of the hinge segment of cytochrome P450 oxidoreductase in the interaction with cytochrome P450 |
author |
Campelo, Diana |
author_facet |
Campelo, Diana Esteves, Francisco Palma, Bernardo Brito Gomes, Bruno Costa Rueff, Jose Lautier, Thomas Urban, Philippe Truan, Gilles Kranendonk, Michel |
author_role |
author |
author2 |
Esteves, Francisco Palma, Bernardo Brito Gomes, Bruno Costa Rueff, Jose Lautier, Thomas Urban, Philippe Truan, Gilles Kranendonk, Michel |
author2_role |
author author author author author author author author |
dc.contributor.none.fl_str_mv |
Centre for Toxicogenomics and Human Health (ToxOmics) NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM) RUN |
dc.contributor.author.fl_str_mv |
Campelo, Diana Esteves, Francisco Palma, Bernardo Brito Gomes, Bruno Costa Rueff, Jose Lautier, Thomas Urban, Philippe Truan, Gilles Kranendonk, Michel |
dc.subject.por.fl_str_mv |
Cytochrome b5 (CYB5) Electron-transfer (ET) Microsomal cytochrome p450 (CYP) NADPH-cytochrome P450 reductase (CPR) Protein dynamics Protein-protein interaction Catalysis Molecular Biology Spectroscopy Computer Science Applications Physical and Theoretical Chemistry Organic Chemistry Inorganic Chemistry |
topic |
Cytochrome b5 (CYB5) Electron-transfer (ET) Microsomal cytochrome p450 (CYP) NADPH-cytochrome P450 reductase (CPR) Protein dynamics Protein-protein interaction Catalysis Molecular Biology Spectroscopy Computer Science Applications Physical and Theoretical Chemistry Organic Chemistry Inorganic Chemistry |
description |
NADPH-cytochrome P450 reductase (CPR) is the unique redox partner of microsomal cytochrome P450s (CYPs). CPR exists in a conformational equilibrium between open and closed conformations throughout its electron transfer (ET) function. Previously, we have shown that electrostatic and flexibility properties of the hinge segment of CPR are critical for ET. Three mutants of human CPR were studied (S243P, I245P and R246A) and combined with representative human drug-metabolizing CYPs (isoforms 1A2, 2A6 and 3A4). To probe the effect of these hinge mutations different experimental approaches were employed: CYP bioactivation capacity of pre-carcinogens, enzyme kinetic analysis, and effect of the ionic strength and cytochrome b5 (CYB5) on CYP activity. The hinge mutations influenced the bioactivation of pre-carcinogens, which seemed CYP isoform and substrate dependent. The deviations of Michaelis-Menten kinetic parameters uncovered tend to confirm this discrepancy, which was confirmed by CYP and hinge mutant specific salt/activity profiles. CPR/CYB5 competition experiments indicated a less important role of affinity in CPR/CYP interaction. Overall, our data suggest that the highly flexible hinge of CPR is responsible for the existence of a conformational aggregate of different open CPR conformers enabling ET-interaction with structural varied redox partners. |
publishDate |
2018 |
dc.date.none.fl_str_mv |
2018-12-27T23:28:31Z 2018-12-01 2018-12-01T00:00:00Z |
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 |
https://doi.org/10.3390/ijms19123914 |
url |
https://doi.org/10.3390/ijms19123914 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
1661-6596 PURE: 10978041 http://www.scopus.com/inward/record.url?scp=85058293841&partnerID=8YFLogxK https://doi.org/10.3390/ijms19123914 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
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) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
repository.mail.fl_str_mv |
mluisa.alvim@gmail.com |
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1817545666207940608 |