Exploring the role of residue 228 in substrate and inhibitor recognition by VIM Metallo-β-lactamases

Autores
Mojica, Maria F.; Mahler, S. Graciela; Bethel, Christopher R.; Taracila, Magdalena A.; Kosmopoulou, Magda; Papp Wallace, Krisztina M.; Llarrull, Leticia Irene; Wilson, Brigid M.; Marshall, Steven H.; Wallace, Christopher J.; Villegas, Maria V.; Harris, Michael E.; Vila, Alejandro Jose; Spencer, James; Bonomo, Robert A.
Año de publicación
2015
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
β-Lactamase inhibitors (BLIs) restore the efficacy of otherwise obsolete β-lactams. However, commercially available BLIs are not effective against metallo-β-lactamases (MBLs), which continue to be disseminated globally. One group of the most clinically important MBLs is the VIM family. The discovery of VIM-24, a natural variant of VIM-2, possessing an R228L substitution and a novel phenotype, compelled us to explore the role of this position and its effects on substrate specificity. We employed mutagenesis, biochemical and biophysical assays, and crystallography. VIM-24 (R228L) confers enhanced resistance to cephems and increases the rate of turnover compared to that of VIM-2 (kcat/KM increased by 6- and 10-fold for ceftazidime and cefepime, respectively). Likely the R → L substitution relieves steric clashes and accommodates the C3N-methyl pyrrolidine group of cephems. Four novel bisthiazolidine (BTZ) inhibitors were next synthesized and tested against these MBLs. These inhibitors inactivated VIM-2 and VIM-24 equally well (Ki∗ values of 40-640 nM) through a two-step process in which an initial enzyme (E)-inhibitor (I) complex (EI) undergoes a conformational transition to a more stable species, E∗I. As both VIM-2 and VIM-24 were inhibited in a similar manner, the crystal structure of a VIM-2-BTZ complex was determined at 1.25 Å and revealed interactions of the inhibitor thiol with the VIM Zn center. Most importantly, BTZs also restored the activity of imipenem against Klebsiella pneumoniae and Pseudomonas aeruginosa in whole cell assays producing VIM-24 and VIM-2, respectively. Our results suggest a role for position 228 in defining the substrate specificity of VIM MBLs and show that BTZ inhibitors are not affected by the R228L substitution.
Fil: Mojica, Maria F.. Case Western Reserve University; Estados Unidos. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos
Fil: Mahler, S. Graciela. Universidad de la República; Uruguay
Fil: Bethel, Christopher R.. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos
Fil: Taracila, Magdalena A.. Case Western Reserve University; Estados Unidos. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos
Fil: Kosmopoulou, Magda. University of Bristol; Reino Unido
Fil: Papp Wallace, Krisztina M.. Case Western Reserve University; Estados Unidos. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos
Fil: Llarrull, Leticia Irene. 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: Wilson, Brigid M.. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos
Fil: Marshall, Steven H.. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos
Fil: Wallace, Christopher J.. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos
Fil: Villegas, Maria V.. Centro Internacional de Entrenamiento e Investigaciones Medicas; Colombia
Fil: Harris, Michael E.. Case Western Reserve University; Estados Unidos
Fil: Vila, Alejandro Jose. 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: Spencer, James. University of Bristol; Reino Unido
Fil: Bonomo, Robert A.. Case Western Reserve University; Estados Unidos. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos
Materia
Vim
Metallo-Beta-Lactamase
Inhibition
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:11336/51578
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/51578
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Exploring the role of residue 228 in substrate and inhibitor recognition by VIM Metallo-β-lactamasesMojica, Maria F.Mahler, S. GracielaBethel, Christopher R.Taracila, Magdalena A.Kosmopoulou, MagdaPapp Wallace, Krisztina M.Llarrull, Leticia IreneWilson, Brigid M.Marshall, Steven H.Wallace, Christopher J.Villegas, Maria V.Harris, Michael E.Vila, Alejandro JoseSpencer, JamesBonomo, Robert A.VimMetallo-Beta-LactamaseInhibitionhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1β-Lactamase inhibitors (BLIs) restore the efficacy of otherwise obsolete β-lactams. However, commercially available BLIs are not effective against metallo-β-lactamases (MBLs), which continue to be disseminated globally. One group of the most clinically important MBLs is the VIM family. The discovery of VIM-24, a natural variant of VIM-2, possessing an R228L substitution and a novel phenotype, compelled us to explore the role of this position and its effects on substrate specificity. We employed mutagenesis, biochemical and biophysical assays, and crystallography. VIM-24 (R228L) confers enhanced resistance to cephems and increases the rate of turnover compared to that of VIM-2 (kcat/KM increased by 6- and 10-fold for ceftazidime and cefepime, respectively). Likely the R → L substitution relieves steric clashes and accommodates the C3N-methyl pyrrolidine group of cephems. Four novel bisthiazolidine (BTZ) inhibitors were next synthesized and tested against these MBLs. These inhibitors inactivated VIM-2 and VIM-24 equally well (Ki∗ values of 40-640 nM) through a two-step process in which an initial enzyme (E)-inhibitor (I) complex (EI) undergoes a conformational transition to a more stable species, E∗I. As both VIM-2 and VIM-24 were inhibited in a similar manner, the crystal structure of a VIM-2-BTZ complex was determined at 1.25 Å and revealed interactions of the inhibitor thiol with the VIM Zn center. Most importantly, BTZs also restored the activity of imipenem against Klebsiella pneumoniae and Pseudomonas aeruginosa in whole cell assays producing VIM-24 and VIM-2, respectively. Our results suggest a role for position 228 in defining the substrate specificity of VIM MBLs and show that BTZ inhibitors are not affected by the R228L substitution.Fil: Mojica, Maria F.. Case Western Reserve University; Estados Unidos. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados UnidosFil: Mahler, S. Graciela. Universidad de la República; UruguayFil: Bethel, Christopher R.. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados UnidosFil: Taracila, Magdalena A.. Case Western Reserve University; Estados Unidos. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados UnidosFil: Kosmopoulou, Magda. University of Bristol; Reino UnidoFil: Papp Wallace, Krisztina M.. Case Western Reserve University; Estados Unidos. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados UnidosFil: Llarrull, Leticia Irene. 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: Wilson, Brigid M.. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados UnidosFil: Marshall, Steven H.. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados UnidosFil: Wallace, Christopher J.. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados UnidosFil: Villegas, Maria V.. Centro Internacional de Entrenamiento e Investigaciones Medicas; ColombiaFil: Harris, Michael E.. Case Western Reserve University; Estados UnidosFil: Vila, Alejandro Jose. 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: Spencer, James. University of Bristol; Reino UnidoFil: Bonomo, Robert A.. Case Western Reserve University; Estados Unidos. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados UnidosAmerican Chemical Society2015-05info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/51578Mojica, Maria F.; Mahler, S. Graciela; Bethel, Christopher R.; Taracila, Magdalena A.; Kosmopoulou, Magda; et al.; Exploring the role of residue 228 in substrate and inhibitor recognition by VIM Metallo-β-lactamases; American Chemical Society; Biochemistry; 54; 20; 5-2015; 3183-31960006-2960CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1021/acs.biochem.5b00106info:eu-repo/semantics/altIdentifier/url/https://pubs.acs.org/doi/10.1021/acs.biochem.5b00106info:eu-repo/semantics/altIdentifier/url/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4700511/info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:45:29Zoai:ri.conicet.gov.ar:11336/51578instacron: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 10:45:29.588CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Exploring the role of residue 228 in substrate and inhibitor recognition by VIM Metallo-β-lactamases
title Exploring the role of residue 228 in substrate and inhibitor recognition by VIM Metallo-β-lactamases
spellingShingle Exploring the role of residue 228 in substrate and inhibitor recognition by VIM Metallo-β-lactamases
Mojica, Maria F.
Vim
Metallo-Beta-Lactamase
Inhibition
title_short Exploring the role of residue 228 in substrate and inhibitor recognition by VIM Metallo-β-lactamases
title_full Exploring the role of residue 228 in substrate and inhibitor recognition by VIM Metallo-β-lactamases
title_fullStr Exploring the role of residue 228 in substrate and inhibitor recognition by VIM Metallo-β-lactamases
title_full_unstemmed Exploring the role of residue 228 in substrate and inhibitor recognition by VIM Metallo-β-lactamases
title_sort Exploring the role of residue 228 in substrate and inhibitor recognition by VIM Metallo-β-lactamases
dc.creator.none.fl_str_mv Mojica, Maria F.
Mahler, S. Graciela
Bethel, Christopher R.
Taracila, Magdalena A.
Kosmopoulou, Magda
Papp Wallace, Krisztina M.
Llarrull, Leticia Irene
Wilson, Brigid M.
Marshall, Steven H.
Wallace, Christopher J.
Villegas, Maria V.
Harris, Michael E.
Vila, Alejandro Jose
Spencer, James
Bonomo, Robert A.
author Mojica, Maria F.
author_facet Mojica, Maria F.
Mahler, S. Graciela
Bethel, Christopher R.
Taracila, Magdalena A.
Kosmopoulou, Magda
Papp Wallace, Krisztina M.
Llarrull, Leticia Irene
Wilson, Brigid M.
Marshall, Steven H.
Wallace, Christopher J.
Villegas, Maria V.
Harris, Michael E.
Vila, Alejandro Jose
Spencer, James
Bonomo, Robert A.
author_role author
author2 Mahler, S. Graciela
Bethel, Christopher R.
Taracila, Magdalena A.
Kosmopoulou, Magda
Papp Wallace, Krisztina M.
Llarrull, Leticia Irene
Wilson, Brigid M.
Marshall, Steven H.
Wallace, Christopher J.
Villegas, Maria V.
Harris, Michael E.
Vila, Alejandro Jose
Spencer, James
Bonomo, Robert A.
author2_role author
author
author
author
author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Vim
Metallo-Beta-Lactamase
Inhibition
topic Vim
Metallo-Beta-Lactamase
Inhibition
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv β-Lactamase inhibitors (BLIs) restore the efficacy of otherwise obsolete β-lactams. However, commercially available BLIs are not effective against metallo-β-lactamases (MBLs), which continue to be disseminated globally. One group of the most clinically important MBLs is the VIM family. The discovery of VIM-24, a natural variant of VIM-2, possessing an R228L substitution and a novel phenotype, compelled us to explore the role of this position and its effects on substrate specificity. We employed mutagenesis, biochemical and biophysical assays, and crystallography. VIM-24 (R228L) confers enhanced resistance to cephems and increases the rate of turnover compared to that of VIM-2 (kcat/KM increased by 6- and 10-fold for ceftazidime and cefepime, respectively). Likely the R → L substitution relieves steric clashes and accommodates the C3N-methyl pyrrolidine group of cephems. Four novel bisthiazolidine (BTZ) inhibitors were next synthesized and tested against these MBLs. These inhibitors inactivated VIM-2 and VIM-24 equally well (Ki∗ values of 40-640 nM) through a two-step process in which an initial enzyme (E)-inhibitor (I) complex (EI) undergoes a conformational transition to a more stable species, E∗I. As both VIM-2 and VIM-24 were inhibited in a similar manner, the crystal structure of a VIM-2-BTZ complex was determined at 1.25 Å and revealed interactions of the inhibitor thiol with the VIM Zn center. Most importantly, BTZs also restored the activity of imipenem against Klebsiella pneumoniae and Pseudomonas aeruginosa in whole cell assays producing VIM-24 and VIM-2, respectively. Our results suggest a role for position 228 in defining the substrate specificity of VIM MBLs and show that BTZ inhibitors are not affected by the R228L substitution.
Fil: Mojica, Maria F.. Case Western Reserve University; Estados Unidos. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos
Fil: Mahler, S. Graciela. Universidad de la República; Uruguay
Fil: Bethel, Christopher R.. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos
Fil: Taracila, Magdalena A.. Case Western Reserve University; Estados Unidos. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos
Fil: Kosmopoulou, Magda. University of Bristol; Reino Unido
Fil: Papp Wallace, Krisztina M.. Case Western Reserve University; Estados Unidos. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos
Fil: Llarrull, Leticia Irene. 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: Wilson, Brigid M.. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos
Fil: Marshall, Steven H.. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos
Fil: Wallace, Christopher J.. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos
Fil: Villegas, Maria V.. Centro Internacional de Entrenamiento e Investigaciones Medicas; Colombia
Fil: Harris, Michael E.. Case Western Reserve University; Estados Unidos
Fil: Vila, Alejandro Jose. 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: Spencer, James. University of Bristol; Reino Unido
Fil: Bonomo, Robert A.. Case Western Reserve University; Estados Unidos. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos
description β-Lactamase inhibitors (BLIs) restore the efficacy of otherwise obsolete β-lactams. However, commercially available BLIs are not effective against metallo-β-lactamases (MBLs), which continue to be disseminated globally. One group of the most clinically important MBLs is the VIM family. The discovery of VIM-24, a natural variant of VIM-2, possessing an R228L substitution and a novel phenotype, compelled us to explore the role of this position and its effects on substrate specificity. We employed mutagenesis, biochemical and biophysical assays, and crystallography. VIM-24 (R228L) confers enhanced resistance to cephems and increases the rate of turnover compared to that of VIM-2 (kcat/KM increased by 6- and 10-fold for ceftazidime and cefepime, respectively). Likely the R → L substitution relieves steric clashes and accommodates the C3N-methyl pyrrolidine group of cephems. Four novel bisthiazolidine (BTZ) inhibitors were next synthesized and tested against these MBLs. These inhibitors inactivated VIM-2 and VIM-24 equally well (Ki∗ values of 40-640 nM) through a two-step process in which an initial enzyme (E)-inhibitor (I) complex (EI) undergoes a conformational transition to a more stable species, E∗I. As both VIM-2 and VIM-24 were inhibited in a similar manner, the crystal structure of a VIM-2-BTZ complex was determined at 1.25 Å and revealed interactions of the inhibitor thiol with the VIM Zn center. Most importantly, BTZs also restored the activity of imipenem against Klebsiella pneumoniae and Pseudomonas aeruginosa in whole cell assays producing VIM-24 and VIM-2, respectively. Our results suggest a role for position 228 in defining the substrate specificity of VIM MBLs and show that BTZ inhibitors are not affected by the R228L substitution.
publishDate 2015
dc.date.none.fl_str_mv 2015-05
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/11336/51578
Mojica, Maria F.; Mahler, S. Graciela; Bethel, Christopher R.; Taracila, Magdalena A.; Kosmopoulou, Magda; et al.; Exploring the role of residue 228 in substrate and inhibitor recognition by VIM Metallo-β-lactamases; American Chemical Society; Biochemistry; 54; 20; 5-2015; 3183-3196
0006-2960
CONICET Digital
CONICET
url http://hdl.handle.net/11336/51578
identifier_str_mv Mojica, Maria F.; Mahler, S. Graciela; Bethel, Christopher R.; Taracila, Magdalena A.; Kosmopoulou, Magda; et al.; Exploring the role of residue 228 in substrate and inhibitor recognition by VIM Metallo-β-lactamases; American Chemical Society; Biochemistry; 54; 20; 5-2015; 3183-3196
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/doi/10.1021/acs.biochem.5b00106
info:eu-repo/semantics/altIdentifier/url/https://pubs.acs.org/doi/10.1021/acs.biochem.5b00106
info:eu-repo/semantics/altIdentifier/url/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4700511/
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
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)
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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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