The reaction mechanism of metallo-beta-lactamases is tuned by the conformation of an active site mobile loop

Autores
Palacios, Antonela Rocio; Mojica, María F.; Giannini, Estefanía; Taracila, Magdalena A.; Bethel, Christopher R.; Alzari, Pedro M.; Otero, Lisandro Horacio; Klinke, Sebastian; Llarrull, Leticia Irene; Bonomo, Robert A.; Vila, Alejandro Jose
Año de publicación
2018
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Carbapenems are "last resort" β-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-β-lactamases (MβLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all β-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MβLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MβLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different β-lactams in all MβLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MβLs.
Fil: Palacios, Antonela Rocio. 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: Mojica, María F.. Case Western Reserve University; Estados Unidos
Fil: Giannini, Estefanía. 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: Taracila, Magdalena A.. Case Western Reserve University; Estados Unidos. Louis Stokes Veterans Affairs Medical Center; Estados Unidos
Fil: Bethel, Christopher R.. Louis Stokes Veterans Affairs Medical Center; Estados Unidos
Fil: Alzari, Pedro M.. Institut Pasteur de Paris; Francia
Fil: Otero, Lisandro Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
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: Bonomo, Robert A.. 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
Materia
NEW DELHI METALLO-BETA-LACTAMASE
ANTIBIOTIC RESISTANCE
ENZYME MECHANISM
ENZYME STRUCTURE
METALLO-BETA-LACTAMASE
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/105019
Acceder
id CONICETDig_5ac9d6f57ba7071cdc082e4e4708665f
oai_identifier_str oai:ri.conicet.gov.ar:11336/105019
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling The reaction mechanism of metallo-beta-lactamases is tuned by the conformation of an active site mobile loopPalacios, Antonela RocioMojica, María F.Giannini, EstefaníaTaracila, Magdalena A.Bethel, Christopher R.Alzari, Pedro M.Otero, Lisandro HoracioKlinke, SebastianLlarrull, Leticia IreneBonomo, Robert A.Vila, Alejandro JoseNEW DELHI METALLO-BETA-LACTAMASEANTIBIOTIC RESISTANCEENZYME MECHANISMENZYME STRUCTUREMETALLO-BETA-LACTAMASEhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Carbapenems are "last resort" β-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-β-lactamases (MβLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all β-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MβLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MβLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different β-lactams in all MβLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MβLs.Fil: Palacios, Antonela Rocio. 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: Mojica, María F.. Case Western Reserve University; Estados UnidosFil: Giannini, Estefanía. 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: Taracila, Magdalena A.. Case Western Reserve University; Estados Unidos. Louis Stokes Veterans Affairs Medical Center; Estados UnidosFil: Bethel, Christopher R.. Louis Stokes Veterans Affairs Medical Center; Estados UnidosFil: Alzari, Pedro M.. Institut Pasteur de Paris; FranciaFil: Otero, Lisandro Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: 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: Bonomo, Robert A.. 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; ArgentinaAmerican Society for Microbiology2018-12info: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/11336/105019Palacios, Antonela Rocio; Mojica, María F.; Giannini, Estefanía; Taracila, Magdalena A.; Bethel, Christopher R.; et al.; The reaction mechanism of metallo-beta-lactamases is tuned by the conformation of an active site mobile loop; American Society for Microbiology; Antimicrobial Agents and Chemotherapy; 63; 1; 12-2018; e01754-180066-48041098-6596CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://aac.asm.org/content/63/1/e01754-18.longinfo:eu-repo/semantics/altIdentifier/doi/10.1128/AAC.01754-18info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T10:11:43Zoai:ri.conicet.gov.ar:11336/105019instacron: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:34982025-09-03 10:11:44.287CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv The reaction mechanism of metallo-beta-lactamases is tuned by the conformation of an active site mobile loop
title The reaction mechanism of metallo-beta-lactamases is tuned by the conformation of an active site mobile loop
spellingShingle The reaction mechanism of metallo-beta-lactamases is tuned by the conformation of an active site mobile loop
Palacios, Antonela Rocio
NEW DELHI METALLO-BETA-LACTAMASE
ANTIBIOTIC RESISTANCE
ENZYME MECHANISM
ENZYME STRUCTURE
METALLO-BETA-LACTAMASE
title_short The reaction mechanism of metallo-beta-lactamases is tuned by the conformation of an active site mobile loop
title_full The reaction mechanism of metallo-beta-lactamases is tuned by the conformation of an active site mobile loop
title_fullStr The reaction mechanism of metallo-beta-lactamases is tuned by the conformation of an active site mobile loop
title_full_unstemmed The reaction mechanism of metallo-beta-lactamases is tuned by the conformation of an active site mobile loop
title_sort The reaction mechanism of metallo-beta-lactamases is tuned by the conformation of an active site mobile loop
dc.creator.none.fl_str_mv Palacios, Antonela Rocio
Mojica, María F.
Giannini, Estefanía
Taracila, Magdalena A.
Bethel, Christopher R.
Alzari, Pedro M.
Otero, Lisandro Horacio
Klinke, Sebastian
Llarrull, Leticia Irene
Bonomo, Robert A.
Vila, Alejandro Jose
author Palacios, Antonela Rocio
author_facet Palacios, Antonela Rocio
Mojica, María F.
Giannini, Estefanía
Taracila, Magdalena A.
Bethel, Christopher R.
Alzari, Pedro M.
Otero, Lisandro Horacio
Klinke, Sebastian
Llarrull, Leticia Irene
Bonomo, Robert A.
Vila, Alejandro Jose
author_role author
author2 Mojica, María F.
Giannini, Estefanía
Taracila, Magdalena A.
Bethel, Christopher R.
Alzari, Pedro M.
Otero, Lisandro Horacio
Klinke, Sebastian
Llarrull, Leticia Irene
Bonomo, Robert A.
Vila, Alejandro Jose
author2_role author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv NEW DELHI METALLO-BETA-LACTAMASE
ANTIBIOTIC RESISTANCE
ENZYME MECHANISM
ENZYME STRUCTURE
METALLO-BETA-LACTAMASE
topic NEW DELHI METALLO-BETA-LACTAMASE
ANTIBIOTIC RESISTANCE
ENZYME MECHANISM
ENZYME STRUCTURE
METALLO-BETA-LACTAMASE
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Carbapenems are "last resort" β-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-β-lactamases (MβLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all β-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MβLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MβLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different β-lactams in all MβLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MβLs.
Fil: Palacios, Antonela Rocio. 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: Mojica, María F.. Case Western Reserve University; Estados Unidos
Fil: Giannini, Estefanía. 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: Taracila, Magdalena A.. Case Western Reserve University; Estados Unidos. Louis Stokes Veterans Affairs Medical Center; Estados Unidos
Fil: Bethel, Christopher R.. Louis Stokes Veterans Affairs Medical Center; Estados Unidos
Fil: Alzari, Pedro M.. Institut Pasteur de Paris; Francia
Fil: Otero, Lisandro Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
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: Bonomo, Robert A.. 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
description Carbapenems are "last resort" β-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-β-lactamases (MβLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all β-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MβLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MβLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different β-lactams in all MβLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MβLs.
publishDate 2018
dc.date.none.fl_str_mv 2018-12
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/11336/105019
Palacios, Antonela Rocio; Mojica, María F.; Giannini, Estefanía; Taracila, Magdalena A.; Bethel, Christopher R.; et al.; The reaction mechanism of metallo-beta-lactamases is tuned by the conformation of an active site mobile loop; American Society for Microbiology; Antimicrobial Agents and Chemotherapy; 63; 1; 12-2018; e01754-18
0066-4804
1098-6596
CONICET Digital
CONICET
url http://hdl.handle.net/11336/105019
identifier_str_mv Palacios, Antonela Rocio; Mojica, María F.; Giannini, Estefanía; Taracila, Magdalena A.; Bethel, Christopher R.; et al.; The reaction mechanism of metallo-beta-lactamases is tuned by the conformation of an active site mobile loop; American Society for Microbiology; Antimicrobial Agents and Chemotherapy; 63; 1; 12-2018; e01754-18
0066-4804
1098-6596
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://aac.asm.org/content/63/1/e01754-18.long
info:eu-repo/semantics/altIdentifier/doi/10.1128/AAC.01754-18
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/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 Society for Microbiology
publisher.none.fl_str_mv American Society for Microbiology
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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