A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency

Autores
Partipilo, Michele; Yang, Guang; Mascotti, María Laura; Wijma, Hein J.; Slotboom, Dirk Jan; Fraaije, Marco Wilhelmus
Año de publicación
2022
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Soluble pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equivalents between the two nicotinamide cofactors. The soluble transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equivalents toward NADPH-requiring reactions. However, mechanistic insight into SthA function is still lacking. In this work, we present a biochemical characterization of SthA, focusing for the first time on the reactivity of the flavoenzyme with molecular oxygen. We report on oxidase activity of SthA that takes place both during transhydrogenation and in the absence of an oxidized nicotinamide cofactor as an electron acceptor. We find that this reaction produces the reactive oxygen species hydrogen peroxide and superoxide anion. Furthermore, we explore the evolutionary significance of the well-conserved CXXXXT motif that distinguishes STHs from the related family of flavoprotein disulfide reductases in which a CXXXXC motif is conserved. Our mutational analysis revealed the cysteine and threonine combination in SthA leads to better coupling efficiency of transhydrogenation and reduced reactive oxygen species release compared to enzyme variants with mutated motifs. These results expand our mechanistic understanding of SthA by highlighting reactivity with molecular oxygen and the importance of the evolutionarily conserved sequence motif.
Fil: Partipilo, Michele. University of Groningen; Países Bajos
Fil: Yang, Guang. University of Groningen; Países Bajos
Fil: Mascotti, María Laura. University of Groningen; Países Bajos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina
Fil: Wijma, Hein J.. University of Groningen; Países Bajos
Fil: Slotboom, Dirk Jan. University of Groningen; Países Bajos
Fil: Fraaije, Marco Wilhelmus. University of Groningen; Países Bajos
Materia
FLAVOPROTEIN
NICOTINAMIDE COFACTORS
PROTEIN ENGINEERING
REACTIVE OXYGEN SPECIES
SOLUBLE TRANSHYDROGENASE
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:11336/211142
Acceder
id CONICETDig_90ca36304331c7c4e9672baf5dc6f42d
oai_identifier_str oai:ri.conicet.gov.ar:11336/211142
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiencyPartipilo, MicheleYang, GuangMascotti, María LauraWijma, Hein J.Slotboom, Dirk JanFraaije, Marco WilhelmusFLAVOPROTEINNICOTINAMIDE COFACTORSPROTEIN ENGINEERINGREACTIVE OXYGEN SPECIESSOLUBLE TRANSHYDROGENASEhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Soluble pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equivalents between the two nicotinamide cofactors. The soluble transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equivalents toward NADPH-requiring reactions. However, mechanistic insight into SthA function is still lacking. In this work, we present a biochemical characterization of SthA, focusing for the first time on the reactivity of the flavoenzyme with molecular oxygen. We report on oxidase activity of SthA that takes place both during transhydrogenation and in the absence of an oxidized nicotinamide cofactor as an electron acceptor. We find that this reaction produces the reactive oxygen species hydrogen peroxide and superoxide anion. Furthermore, we explore the evolutionary significance of the well-conserved CXXXXT motif that distinguishes STHs from the related family of flavoprotein disulfide reductases in which a CXXXXC motif is conserved. Our mutational analysis revealed the cysteine and threonine combination in SthA leads to better coupling efficiency of transhydrogenation and reduced reactive oxygen species release compared to enzyme variants with mutated motifs. These results expand our mechanistic understanding of SthA by highlighting reactivity with molecular oxygen and the importance of the evolutionarily conserved sequence motif.Fil: Partipilo, Michele. University of Groningen; Países BajosFil: Yang, Guang. University of Groningen; Países BajosFil: Mascotti, María Laura. University of Groningen; Países Bajos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Wijma, Hein J.. University of Groningen; Países BajosFil: Slotboom, Dirk Jan. University of Groningen; Países BajosFil: Fraaije, Marco Wilhelmus. University of Groningen; Países BajosAmerican Society for Biochemistry and Molecular Biology2022-09info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/211142Partipilo, Michele; Yang, Guang; Mascotti, María Laura; Wijma, Hein J.; Slotboom, Dirk Jan; et al.; A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency; American Society for Biochemistry and Molecular Biology; Journal of Biological Chemistry (online); 298; 9; 9-2022; 1-130021-9258CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.jbc.2022.102304info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0021925822007463info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T11:58:41Zoai:ri.conicet.gov.ar:11336/211142instacron: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:34982025-10-22 11:58:42.255CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency
title A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency
spellingShingle A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency
Partipilo, Michele
FLAVOPROTEIN
NICOTINAMIDE COFACTORS
PROTEIN ENGINEERING
REACTIVE OXYGEN SPECIES
SOLUBLE TRANSHYDROGENASE
title_short A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency
title_full A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency
title_fullStr A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency
title_full_unstemmed A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency
title_sort A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency
dc.creator.none.fl_str_mv Partipilo, Michele
Yang, Guang
Mascotti, María Laura
Wijma, Hein J.
Slotboom, Dirk Jan
Fraaije, Marco Wilhelmus
author Partipilo, Michele
author_facet Partipilo, Michele
Yang, Guang
Mascotti, María Laura
Wijma, Hein J.
Slotboom, Dirk Jan
Fraaije, Marco Wilhelmus
author_role author
author2 Yang, Guang
Mascotti, María Laura
Wijma, Hein J.
Slotboom, Dirk Jan
Fraaije, Marco Wilhelmus
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv FLAVOPROTEIN
NICOTINAMIDE COFACTORS
PROTEIN ENGINEERING
REACTIVE OXYGEN SPECIES
SOLUBLE TRANSHYDROGENASE
topic FLAVOPROTEIN
NICOTINAMIDE COFACTORS
PROTEIN ENGINEERING
REACTIVE OXYGEN SPECIES
SOLUBLE TRANSHYDROGENASE
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Soluble pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equivalents between the two nicotinamide cofactors. The soluble transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equivalents toward NADPH-requiring reactions. However, mechanistic insight into SthA function is still lacking. In this work, we present a biochemical characterization of SthA, focusing for the first time on the reactivity of the flavoenzyme with molecular oxygen. We report on oxidase activity of SthA that takes place both during transhydrogenation and in the absence of an oxidized nicotinamide cofactor as an electron acceptor. We find that this reaction produces the reactive oxygen species hydrogen peroxide and superoxide anion. Furthermore, we explore the evolutionary significance of the well-conserved CXXXXT motif that distinguishes STHs from the related family of flavoprotein disulfide reductases in which a CXXXXC motif is conserved. Our mutational analysis revealed the cysteine and threonine combination in SthA leads to better coupling efficiency of transhydrogenation and reduced reactive oxygen species release compared to enzyme variants with mutated motifs. These results expand our mechanistic understanding of SthA by highlighting reactivity with molecular oxygen and the importance of the evolutionarily conserved sequence motif.
Fil: Partipilo, Michele. University of Groningen; Países Bajos
Fil: Yang, Guang. University of Groningen; Países Bajos
Fil: Mascotti, María Laura. University of Groningen; Países Bajos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina
Fil: Wijma, Hein J.. University of Groningen; Países Bajos
Fil: Slotboom, Dirk Jan. University of Groningen; Países Bajos
Fil: Fraaije, Marco Wilhelmus. University of Groningen; Países Bajos
description Soluble pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equivalents between the two nicotinamide cofactors. The soluble transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equivalents toward NADPH-requiring reactions. However, mechanistic insight into SthA function is still lacking. In this work, we present a biochemical characterization of SthA, focusing for the first time on the reactivity of the flavoenzyme with molecular oxygen. We report on oxidase activity of SthA that takes place both during transhydrogenation and in the absence of an oxidized nicotinamide cofactor as an electron acceptor. We find that this reaction produces the reactive oxygen species hydrogen peroxide and superoxide anion. Furthermore, we explore the evolutionary significance of the well-conserved CXXXXT motif that distinguishes STHs from the related family of flavoprotein disulfide reductases in which a CXXXXC motif is conserved. Our mutational analysis revealed the cysteine and threonine combination in SthA leads to better coupling efficiency of transhydrogenation and reduced reactive oxygen species release compared to enzyme variants with mutated motifs. These results expand our mechanistic understanding of SthA by highlighting reactivity with molecular oxygen and the importance of the evolutionarily conserved sequence motif.
publishDate 2022
dc.date.none.fl_str_mv 2022-09
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/11336/211142
Partipilo, Michele; Yang, Guang; Mascotti, María Laura; Wijma, Hein J.; Slotboom, Dirk Jan; et al.; A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency; American Society for Biochemistry and Molecular Biology; Journal of Biological Chemistry (online); 298; 9; 9-2022; 1-13
0021-9258
CONICET Digital
CONICET
url http://hdl.handle.net/11336/211142
identifier_str_mv Partipilo, Michele; Yang, Guang; Mascotti, María Laura; Wijma, Hein J.; Slotboom, Dirk Jan; et al.; A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency; American Society for Biochemistry and Molecular Biology; Journal of Biological Chemistry (online); 298; 9; 9-2022; 1-13
0021-9258
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1016/j.jbc.2022.102304
info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0021925822007463
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
dc.publisher.none.fl_str_mv American Society for Biochemistry and Molecular Biology
publisher.none.fl_str_mv American Society for Biochemistry and Molecular Biology
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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score 12.982451

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