Swapping FAD Binding Motifs between Plastidic and Bacterial Ferredoxin-NADP(H) Reductases

Autores
Musumeci, Matias Alejandro; Botti, Horacio; Buschiazzo, Alejandro; Ceccarelli, Eduardo Augusto
Año de publicación
2011
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Plant-type ferredoxin-NADP(H) reductases (FNRs) are grouped in two classes, plastidic with an extended FAD conformation and high catalytic rates and bacterial with a folded flavin nucleotide and low turnover rates. The 112-123 β-hairpin from a plastidic FNR and the carboxy-terminaltryptophan of a bacterial FNR, suggested to be responsible for the FAD differential conformation, were mutually exchanged. The plastidic FNR lacking the β-hairpin was unable to fold properly. An extra tryptophan at the carboxy terminus, emulating the bacterial FNR, resulted in an enzyme withdecreased affinity for FAD and reduced diaphorase and ferredoxin-dependent cytochrome c reductase activities. The insertion of the β-hairpin into the corresponding position of the bacterialFNR increased FAD affinity but did not affect its catalytic properties. The same insertion with simultaneous deletion of the carboxyterminal tryptophan produced a bacterial chimera emulating the plastidic architecture with an increased kcat and an increased catalytic efficiency for the diaphorase activity and a decrease in the enzyme?s ability to react with its substrates ferredoxin and flavodoxin. Crystallographic structures of the chimeras showed no significant changes in their overall structure, although alterations in the FAD conformations were observed. Plastidic and bacterial FNRs thus reveal differential effects of key structural elements. While the 112-123 β-hairpin modulates the catalytic efficiency of plastidic FNR, it seems not to affect the bacterial FNR behavior, which instead can be improved by the loss of the C-terminal tryptophan. This report highlights the role of the FAD moiety conformation and the structural determinants involved in stabilizing it, ultimately modulating the functional output of FNRs.
Fil: Musumeci, Matias Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Botti, Horacio. Instituto Pasteur de Montevideo; Uruguay
Fil: Buschiazzo, Alejandro. Instituto Pasteur de Montevideo; Uruguay
Fil: Ceccarelli, Eduardo Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina
Materia
Amino Acid Motifs
Enzyme Stability
Ferredoxin-NADP Reductase
Protein Unfolding
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/268125

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network_name_str CONICET Digital (CONICET)
spelling Swapping FAD Binding Motifs between Plastidic and Bacterial Ferredoxin-NADP(H) ReductasesMusumeci, Matias AlejandroBotti, HoracioBuschiazzo, AlejandroCeccarelli, Eduardo AugustoAmino Acid MotifsEnzyme StabilityFerredoxin-NADP ReductaseProtein Unfoldinghttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Plant-type ferredoxin-NADP(H) reductases (FNRs) are grouped in two classes, plastidic with an extended FAD conformation and high catalytic rates and bacterial with a folded flavin nucleotide and low turnover rates. The 112-123 β-hairpin from a plastidic FNR and the carboxy-terminaltryptophan of a bacterial FNR, suggested to be responsible for the FAD differential conformation, were mutually exchanged. The plastidic FNR lacking the β-hairpin was unable to fold properly. An extra tryptophan at the carboxy terminus, emulating the bacterial FNR, resulted in an enzyme withdecreased affinity for FAD and reduced diaphorase and ferredoxin-dependent cytochrome c reductase activities. The insertion of the β-hairpin into the corresponding position of the bacterialFNR increased FAD affinity but did not affect its catalytic properties. The same insertion with simultaneous deletion of the carboxyterminal tryptophan produced a bacterial chimera emulating the plastidic architecture with an increased kcat and an increased catalytic efficiency for the diaphorase activity and a decrease in the enzyme?s ability to react with its substrates ferredoxin and flavodoxin. Crystallographic structures of the chimeras showed no significant changes in their overall structure, although alterations in the FAD conformations were observed. Plastidic and bacterial FNRs thus reveal differential effects of key structural elements. While the 112-123 β-hairpin modulates the catalytic efficiency of plastidic FNR, it seems not to affect the bacterial FNR behavior, which instead can be improved by the loss of the C-terminal tryptophan. This report highlights the role of the FAD moiety conformation and the structural determinants involved in stabilizing it, ultimately modulating the functional output of FNRs.Fil: Musumeci, Matias Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Botti, Horacio. Instituto Pasteur de Montevideo; UruguayFil: Buschiazzo, Alejandro. Instituto Pasteur de Montevideo; UruguayFil: Ceccarelli, Eduardo Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaAmerican Chemical Society2011-03info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/268125Musumeci, Matias Alejandro; Botti, Horacio; Buschiazzo, Alejandro; Ceccarelli, Eduardo Augusto; Swapping FAD Binding Motifs between Plastidic and Bacterial Ferredoxin-NADP(H) Reductases; American Chemical Society; Biochemistry; 50; 12; 3-2011; 2111-21220006-2960CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/http://pubs.acs.org/doi/abs/10.1021/bi101772ainfo:eu-repo/semantics/altIdentifier/doi/10.1021/bi101772ainfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T09:46:53Zoai:ri.conicet.gov.ar:11336/268125instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 09:46:53.797CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Swapping FAD Binding Motifs between Plastidic and Bacterial Ferredoxin-NADP(H) Reductases
title Swapping FAD Binding Motifs between Plastidic and Bacterial Ferredoxin-NADP(H) Reductases
spellingShingle Swapping FAD Binding Motifs between Plastidic and Bacterial Ferredoxin-NADP(H) Reductases
Musumeci, Matias Alejandro
Amino Acid Motifs
Enzyme Stability
Ferredoxin-NADP Reductase
Protein Unfolding
title_short Swapping FAD Binding Motifs between Plastidic and Bacterial Ferredoxin-NADP(H) Reductases
title_full Swapping FAD Binding Motifs between Plastidic and Bacterial Ferredoxin-NADP(H) Reductases
title_fullStr Swapping FAD Binding Motifs between Plastidic and Bacterial Ferredoxin-NADP(H) Reductases
title_full_unstemmed Swapping FAD Binding Motifs between Plastidic and Bacterial Ferredoxin-NADP(H) Reductases
title_sort Swapping FAD Binding Motifs between Plastidic and Bacterial Ferredoxin-NADP(H) Reductases
dc.creator.none.fl_str_mv Musumeci, Matias Alejandro
Botti, Horacio
Buschiazzo, Alejandro
Ceccarelli, Eduardo Augusto
author Musumeci, Matias Alejandro
author_facet Musumeci, Matias Alejandro
Botti, Horacio
Buschiazzo, Alejandro
Ceccarelli, Eduardo Augusto
author_role author
author2 Botti, Horacio
Buschiazzo, Alejandro
Ceccarelli, Eduardo Augusto
author2_role author
author
author
dc.subject.none.fl_str_mv Amino Acid Motifs
Enzyme Stability
Ferredoxin-NADP Reductase
Protein Unfolding
topic Amino Acid Motifs
Enzyme Stability
Ferredoxin-NADP Reductase
Protein Unfolding
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Plant-type ferredoxin-NADP(H) reductases (FNRs) are grouped in two classes, plastidic with an extended FAD conformation and high catalytic rates and bacterial with a folded flavin nucleotide and low turnover rates. The 112-123 β-hairpin from a plastidic FNR and the carboxy-terminaltryptophan of a bacterial FNR, suggested to be responsible for the FAD differential conformation, were mutually exchanged. The plastidic FNR lacking the β-hairpin was unable to fold properly. An extra tryptophan at the carboxy terminus, emulating the bacterial FNR, resulted in an enzyme withdecreased affinity for FAD and reduced diaphorase and ferredoxin-dependent cytochrome c reductase activities. The insertion of the β-hairpin into the corresponding position of the bacterialFNR increased FAD affinity but did not affect its catalytic properties. The same insertion with simultaneous deletion of the carboxyterminal tryptophan produced a bacterial chimera emulating the plastidic architecture with an increased kcat and an increased catalytic efficiency for the diaphorase activity and a decrease in the enzyme?s ability to react with its substrates ferredoxin and flavodoxin. Crystallographic structures of the chimeras showed no significant changes in their overall structure, although alterations in the FAD conformations were observed. Plastidic and bacterial FNRs thus reveal differential effects of key structural elements. While the 112-123 β-hairpin modulates the catalytic efficiency of plastidic FNR, it seems not to affect the bacterial FNR behavior, which instead can be improved by the loss of the C-terminal tryptophan. This report highlights the role of the FAD moiety conformation and the structural determinants involved in stabilizing it, ultimately modulating the functional output of FNRs.
Fil: Musumeci, Matias Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Botti, Horacio. Instituto Pasteur de Montevideo; Uruguay
Fil: Buschiazzo, Alejandro. Instituto Pasteur de Montevideo; Uruguay
Fil: Ceccarelli, Eduardo Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina
description Plant-type ferredoxin-NADP(H) reductases (FNRs) are grouped in two classes, plastidic with an extended FAD conformation and high catalytic rates and bacterial with a folded flavin nucleotide and low turnover rates. The 112-123 β-hairpin from a plastidic FNR and the carboxy-terminaltryptophan of a bacterial FNR, suggested to be responsible for the FAD differential conformation, were mutually exchanged. The plastidic FNR lacking the β-hairpin was unable to fold properly. An extra tryptophan at the carboxy terminus, emulating the bacterial FNR, resulted in an enzyme withdecreased affinity for FAD and reduced diaphorase and ferredoxin-dependent cytochrome c reductase activities. The insertion of the β-hairpin into the corresponding position of the bacterialFNR increased FAD affinity but did not affect its catalytic properties. The same insertion with simultaneous deletion of the carboxyterminal tryptophan produced a bacterial chimera emulating the plastidic architecture with an increased kcat and an increased catalytic efficiency for the diaphorase activity and a decrease in the enzyme?s ability to react with its substrates ferredoxin and flavodoxin. Crystallographic structures of the chimeras showed no significant changes in their overall structure, although alterations in the FAD conformations were observed. Plastidic and bacterial FNRs thus reveal differential effects of key structural elements. While the 112-123 β-hairpin modulates the catalytic efficiency of plastidic FNR, it seems not to affect the bacterial FNR behavior, which instead can be improved by the loss of the C-terminal tryptophan. This report highlights the role of the FAD moiety conformation and the structural determinants involved in stabilizing it, ultimately modulating the functional output of FNRs.
publishDate 2011
dc.date.none.fl_str_mv 2011-03
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/268125
Musumeci, Matias Alejandro; Botti, Horacio; Buschiazzo, Alejandro; Ceccarelli, Eduardo Augusto; Swapping FAD Binding Motifs between Plastidic and Bacterial Ferredoxin-NADP(H) Reductases; American Chemical Society; Biochemistry; 50; 12; 3-2011; 2111-2122
0006-2960
CONICET Digital
CONICET
url http://hdl.handle.net/11336/268125
identifier_str_mv Musumeci, Matias Alejandro; Botti, Horacio; Buschiazzo, Alejandro; Ceccarelli, Eduardo Augusto; Swapping FAD Binding Motifs between Plastidic and Bacterial Ferredoxin-NADP(H) Reductases; American Chemical Society; Biochemistry; 50; 12; 3-2011; 2111-2122
0006-2960
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://pubs.acs.org/doi/abs/10.1021/bi101772a
info:eu-repo/semantics/altIdentifier/doi/10.1021/bi101772a
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv American Chemical Society
publisher.none.fl_str_mv American Chemical Society
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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