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dc.contributor.author
Blancato, Victor Sebastian
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dc.contributor.author
Pagliai, Fernando A.
dc.contributor.author
Magni, Christian
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dc.contributor.author
Gonzalez, Claudio Fabricio
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dc.contributor.author
Lorca, Graciela L.
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dc.date.available
2018-10-17T17:47:12Z
dc.date.issued
2016-02
dc.identifier.citation
Blancato, Victor Sebastian; Pagliai, Fernando A.; Magni, Christian; Gonzalez, Claudio Fabricio; Lorca, Graciela L.; Functional analysis of the citrate activator CitO from Enterococcus faecalis implicates a divalent metal in ligand binding; Frontiers Research Foundation; Frontiers in Microbiology; 7; FEB; 2-2016; 1-12
dc.identifier.issn
1664-302X
dc.identifier.uri
http://hdl.handle.net/11336/62579
dc.description.abstract
The regulator of citrate metabolism, CitO, from Enterococcus faecalis belongs to the FCD family within the GntR superfamily. In the presence of citrate, CitO binds to cis-acting sequences located upstream of the cit promoters inducing the expression of genes involved in citrate utilization. The quantification of the molecular binding affinities, performed by isothermal titration calorimetry (ITC), indicated that CitO has a high affinity for citrate (KD = 1.2 ± 0.2 μM), while it did not recognize other metabolic intermediates. Based on a structural model of CitO where a putative small molecule and a metal binding site were identified, it was hypothesized that the metal ion is required for citrate binding. In agreement with this model, citrate binding to CitO sharply decreased when the protein was incubated with EDTA. This effect was reverted by the addition of Ni2+, and Zn2+ to a lesser extent. Structure-based site-directed mutagenesis was conducted and it was found that changes to alanine in residues Arg97 and His191 resulted in decreased binding affinities for citrate, as determined by EMSA and ITC. Further assays using lacZ fusions confirmed that these residues in CitO are involved in sensing citrate in vivo. These results indicate that the molecular modifications induced by a ligand and a metal binding in the C-terminal domain of CitO are required for optimal DNA binding activity, and consequently, transcriptional activation.
dc.format
application/pdf
dc.language.iso
eng
dc.publisher
Frontiers Research Foundation
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dc.rights
info:eu-repo/semantics/openAccess
dc.rights.uri
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.subject
Citrate
dc.subject
Enterococcus
dc.subject
Fadr Family
dc.subject
Fcd Domain
dc.subject
Metalloprotein
dc.subject.classification
Otras Ciencias Biológicas
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dc.subject.classification
Ciencias Biológicas
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dc.subject.classification
CIENCIAS NATURALES Y EXACTAS
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dc.title
Functional analysis of the citrate activator CitO from Enterococcus faecalis implicates a divalent metal in ligand binding
dc.type
info:eu-repo/semantics/article
dc.type
info:ar-repo/semantics/artículo
dc.type
info:eu-repo/semantics/publishedVersion
dc.date.updated
2018-10-12T13:53:21Z
dc.journal.volume
7
dc.journal.number
FEB
dc.journal.pagination
1-12
dc.journal.pais
Suiza
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dc.description.fil
Fil: Blancato, Victor Sebastian. 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
dc.description.fil
Fil: Pagliai, Fernando A.. University of Florida; Estados Unidos
dc.description.fil
Fil: Magni, Christian. 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
dc.description.fil
Fil: Gonzalez, Claudio Fabricio. University of Florida; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
dc.description.fil
Fil: Lorca, Graciela L.. University of Florida; Estados Unidos
dc.journal.title
Frontiers in Microbiology
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/doi/http://dx.doi.org/10.3389/fmicb.2016.00101
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/url/https://www.frontiersin.org/articles/10.3389/fmicb.2016.00101/full
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