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

Autor:	Estefanía Giannini, Lisandro H. Otero, Antonela Rocio Palacios, Magdalena A. Taracila, Pedro M. Alzari, Sebastián Klinke, Christopher R. Bethel, Maria F. Mojica, Robert A. Bonomo, Leticia I. Llarrull, Alejandro J. Vila
Přispěvatelé:	Mojica, María Fernanda [0000-0002-1380-9824], Instituto de Biología Molecular y Celular de Rosario [Rosario] (IBR), Consejo Nacional de Investigaciones Científicas y Técnicas [Buenos Aires] (CONICET)-Universidad Nacional de Rosario [Santa Fe], Department of Biochemistry, School of Medicine, Case Western Reserve University, Research Institute of University Hospitals of Cleveland-University Hospitals of Cleveland-Case Western Reserve University [Cleveland], Louis Stokes Cleveland Veterans Affairs Medical Center, Case Western Reserve University School of Medicine, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Fundación Instituto Leloir [Buenos Aires], Plataforma de Biología Estructural y Metabolómica [Rosario] (PLABEM), Facultad de Ciencias Bioquımicas y Farmaceuticas [Rosario] (FBIOyF), Universidad Nacional de Rosario [Santa Fe], CWRU-Cleveland VAMC Center for Antimicrobial Resistance and Epidemiology (CARES), A.R.P. and E.G. received doctoral fellowships from CONICET. M.F.M. received a doctoral scholarship from COLCIENCIAS. L.H.O., S.K., L.I.L., and A.J.V. are CONICET staff members. This study was supported by a grant from ANPCyT (A.J.V.), National Institutes of Health (NIH) grant R01 AI100560 (A.J.V. and R.A.B.), NIH grant R01 AI063517, NIH grant R01 AI072219 (R.A.B.), the ECOS-MinCyT collaborative project (A.J.V. and P.M.A.), the Cleveland Department of Veterans Affairs and Development (1I01BX001974 [R.A.B.]), and the Geriatric Research Education and Clinical Center (R.A.B.)., We thank Ahmed Haouz and Patrick Weber (Institut Pasteur) for help with the robot-driven crystallization screenings. We acknowledge the synchrotron sources Soleil (Saint-Aubin, France) and ESRF (Grenoble, France) for granting access to their facilities, and we thank their staff members for helpful assistance., Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)
Jazyk:	angličtina
Rok vydání:	2019
Předmět:	antibiotic resistance MESH: Ceftazidime Enzyme mechanism MESH: Sequence Homology Amino Acid Antibiotic resistance MESH: beta-Lactamases MESH: Amino Acid Sequence Protein Engineering Substrate Specificity MESH: Recombinant Proteins Pharmacology (medical) Catálisis chemistry.chemical_classification 0303 health sciences [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry Molecular Biology/Structural Biology [q-bio.BM] MESH: Escherichia coli 3. Good health enzyme structure MESH: Protein Engineering Zinc METALLO-BETA-LACTAMASE MESH: Models Molecular MESH: Piperacillin MESH: Gene Expression Stereochemistry metallo-β-lactamase MESH: Sequence Alignment Reaction intermediate beta-Lactam Resistance Ciencias Biológicas 03 medical and health sciences MESH: Anti-Bacterial Agents MESH: Protein Binding Protein Interaction Domains and Motifs MESH: Cloning Molecular Amino Acid Sequence Pharmacology MESH: Protein Conformation alpha-Helical MESH: Protein Interaction Domains and Motifs 030306 microbiology Active site Substrate (chemistry) Meropenem chemistry Protein Conformation beta-Strand [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM] Function (biology) Models Molecular Protein Conformation alpha-Helical Enzyme structure Gene Expression MESH: Catalytic Domain Cefotaxime Crystallography X-Ray Ceftazidime MESH: Zinc MESH: Meropenem purl.org/becyt/ford/1 [https] MESH: Genetic Vectors Catalytic Domain Cloning Molecular Cefepime MESH: Imipenem biology MESH: Kinetics MESH: Cefepime MESH: Cefotaxime NEW DELHI METALLO-BETA-LACTAMASE Recombinant Proteins Anti-Bacterial Agents Infectious Diseases MESH: Protein Conformation beta-Strand CIENCIAS NATURALES Y EXACTAS Protein Binding Cristalografía por rayos X [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph] Genetic Vectors Protonation [SDV.BC]Life Sciences [q-bio]/Cellular Biology beta-Lactamases Mechanisms of Resistance Hydrolase [CHIM.CRIS]Chemical Sciences/Cristallography Escherichia coli enzyme mechanism [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology purl.org/becyt/ford/1.6 [https] 030304 developmental biology Piperacillin Sequence Homology Amino Acid MESH: Crystallography X-Ray Biofísica MESH: beta-Lactam Resistance Imipenem Kinetics Enzyme New Delhi metallo-β-lactamase biology.protein MESH: Substrate Specificity Sequence Alignment
Zdroj:	Repositorio U. El Bosque Universidad El Bosque instacron:Universidad El Bosque Antimicrobial Agents and Chemotherapy Antimicrobial Agents and Chemotherapy, American Society for Microbiology, 2019, 63 (1), ⟨10.1128/AAC.01754-18⟩ CONICET Digital (CONICET) Consejo Nacional de Investigaciones Científicas y Técnicas instacron:CONICET Antimicrobial Agents and Chemotherapy, 2019, 63 (1), ⟨10.1128/AAC.01754-18⟩
ISSN:	0066-4804 1098-6596
DOI:	10.1128/AAC.01754-18⟩
Popis:	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
Databáze:	OpenAIRE
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