Membrane-associated maturation of the heterotetrameric nitrate reductase of Thermus thermophilus

The nar operon, coding for the respiratory nitrate reductase of Thermus thermophilus (NRT), encodes a di-heme b-type (NarJ) and a di-heme c-type (NarC) cytochrome. The role of both cytochromes and that of a putative chaperone (NarJ) in the synthesis and maturation of NRT was studied. Mutants of T. t...

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Detalles Bibliográficos
Autores: Zafra, Olga, Cava, Felipe, Blasco, Francis, Magalon, Axel, Berenguer, José
Tipo de recurso: artículo
Fecha de publicación:2005
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/8188
Acceso en línea:http://hdl.handle.net/10261/8188
Access Level:acceso abierto
Palabra clave:Thermus thermophilus
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spelling Membrane-associated maturation of the heterotetrameric nitrate reductase of Thermus thermophilusZafra, OlgaCava, FelipeBlasco, FrancisMagalon, AxelBerenguer, JoséThermus thermophilusThe nar operon, coding for the respiratory nitrate reductase of Thermus thermophilus (NRT), encodes a di-heme b-type (NarJ) and a di-heme c-type (NarC) cytochrome. The role of both cytochromes and that of a putative chaperone (NarJ) in the synthesis and maturation of NRT was studied. Mutants of T. thermophilus lacking either NarI or NarC synthesized a soluble form of NarG, suggesting that a putative NarCI complex constitutes the attachment site for the enzyme. Interestingly, the NarG protein synthesized by both mutants was inactive in nitrate reduction and misfolded, showing that membrane attachment was required for enzyme maturation. Consistent with its putative role as a specific chaperone, inactive and misfolded NarG was synthesized by narJ mutants, but in contrast to its Escherichia coli homologue, NarJ was also required for the attachment of the thermophilic enzyme to the membrane. A bacterial two-hybrid system was used to demonstrate the putative interactions between the NRT proteins suggested by the analysis of the mutants. Strong interactions were detected between NarC and NarI and between NarG and NarJ. Weaker interaction signals were detected between NarI, but not NarC, and both NarG and NarH. These results lead us to conclude that the NRT is a heterotetrameric (NarC/NarI/NarG/NarH) enzyme, and we propose a model for its synthesis and maturation that is distinct from that of E. coli. In the synthesis of NRT, a NarCI membrane complex and a soluble NarGJH complex are synthesized in a first step. In a second step, both complexes interact at the cytoplasmic face of the membrane, where the enzyme is subsequently activated with the concomitant conformational change and release of the NarJ chaperone from the mature enzyme.This work has been supported by project BIO2004-02671 from the "Ministerio de Educación y Ciencia". An institutional grant from the Fundación Ramón Areces to CBMSO is acknowledged. O. Zafra and F. Cava held fellowships from the Comunidad de Madrid and the "Ministerio de Educación y Ciencia," respectivelyPeer reviewedAmerican Society for MicrobiologyMinisterio de Educación y Ciencia (España)Fundación Ramón ArecesComunidad de Madrid200820082005info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501211591 bytesapplication/pdfhttp://hdl.handle.net/10261/8188reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Ingléshttp://dx.doi.org/10.1128/JB.187.12.3990-3996.2005info:eu-repo/semantics/openAccessoai:digital.csic.es:10261/81882026-05-22T06:33:51Z
dc.title.none.fl_str_mv Membrane-associated maturation of the heterotetrameric nitrate reductase of Thermus thermophilus
title Membrane-associated maturation of the heterotetrameric nitrate reductase of Thermus thermophilus
spellingShingle Membrane-associated maturation of the heterotetrameric nitrate reductase of Thermus thermophilus
Zafra, Olga
Thermus thermophilus
title_short Membrane-associated maturation of the heterotetrameric nitrate reductase of Thermus thermophilus
title_full Membrane-associated maturation of the heterotetrameric nitrate reductase of Thermus thermophilus
title_fullStr Membrane-associated maturation of the heterotetrameric nitrate reductase of Thermus thermophilus
title_full_unstemmed Membrane-associated maturation of the heterotetrameric nitrate reductase of Thermus thermophilus
title_sort Membrane-associated maturation of the heterotetrameric nitrate reductase of Thermus thermophilus
dc.creator.none.fl_str_mv Zafra, Olga
Cava, Felipe
Blasco, Francis
Magalon, Axel
Berenguer, José
author Zafra, Olga
author_facet Zafra, Olga
Cava, Felipe
Blasco, Francis
Magalon, Axel
Berenguer, José
author_role author
author2 Cava, Felipe
Blasco, Francis
Magalon, Axel
Berenguer, José
author2_role author
author
author
author
dc.contributor.none.fl_str_mv Ministerio de Educación y Ciencia (España)
Fundación Ramón Areces
Comunidad de Madrid
dc.subject.none.fl_str_mv Thermus thermophilus
topic Thermus thermophilus
description The nar operon, coding for the respiratory nitrate reductase of Thermus thermophilus (NRT), encodes a di-heme b-type (NarJ) and a di-heme c-type (NarC) cytochrome. The role of both cytochromes and that of a putative chaperone (NarJ) in the synthesis and maturation of NRT was studied. Mutants of T. thermophilus lacking either NarI or NarC synthesized a soluble form of NarG, suggesting that a putative NarCI complex constitutes the attachment site for the enzyme. Interestingly, the NarG protein synthesized by both mutants was inactive in nitrate reduction and misfolded, showing that membrane attachment was required for enzyme maturation. Consistent with its putative role as a specific chaperone, inactive and misfolded NarG was synthesized by narJ mutants, but in contrast to its Escherichia coli homologue, NarJ was also required for the attachment of the thermophilic enzyme to the membrane. A bacterial two-hybrid system was used to demonstrate the putative interactions between the NRT proteins suggested by the analysis of the mutants. Strong interactions were detected between NarC and NarI and between NarG and NarJ. Weaker interaction signals were detected between NarI, but not NarC, and both NarG and NarH. These results lead us to conclude that the NRT is a heterotetrameric (NarC/NarI/NarG/NarH) enzyme, and we propose a model for its synthesis and maturation that is distinct from that of E. coli. In the synthesis of NRT, a NarCI membrane complex and a soluble NarGJH complex are synthesized in a first step. In a second step, both complexes interact at the cytoplasmic face of the membrane, where the enzyme is subsequently activated with the concomitant conformational change and release of the NarJ chaperone from the mature enzyme.
publishDate 2005
dc.date.none.fl_str_mv 2005
2008
2008
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
format article
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/8188
url http://hdl.handle.net/10261/8188
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv http://dx.doi.org/10.1128/JB.187.12.3990-3996.2005
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv 211591 bytes
application/pdf
dc.publisher.none.fl_str_mv American Society for Microbiology
publisher.none.fl_str_mv American Society for Microbiology
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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