Host-specific enzyme-substrate interactions in SPM-1 metallo-β-lactamase are modulated by second sphere residues

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dc.citation.titlePLoS Pathogens
dc.citation.volume10
dc.creatorGonzález, Lisandro Javier
dc.creatorMoreno, Diego M.
dc.creatorBonomo, Robert A.
dc.creatorVila, Alejandro J.
dc.date.accessioned2024-08-28T12:00:25Z
dc.date.available2024-08-28T12:00:25Z
dc.date.issued2014-01-02
dc.description.abstractPseudomonas aeruginosa is one of the most virulent and resistant non-fermenting Gram-negative pathogens in the clinic. Unfortunately, P. aeruginosa has acquired genes encoding metallo-β-lactamases (MβLs), enzymes able to hydrolyze most β-lactam antibiotics. SPM-1 is an MβL produced only by P. aeruginosa, while other MβLs are found in different bacteria. Despite similar active sites, the resistance profile of MβLs towards β-lactams changes from one enzyme to the other. SPM-1 is unique among pathogen-associated MβLs in that it contains “atypical” second sphere residues (S84, G121). Codon randomization on these positions and further selection of resistance-conferring mutants was performed. MICs, periplasmic enzymatic activity, Zn(II) requirements, and protein stability was assessed. Our results indicated that identity of second sphere residues modulates the substrate preferences and the resistance profile of SPM-1 expressed in P. aeruginosa. The second sphere residues found in wild type SPM-1 give rise to a substrate selectivity that is observed only in the periplasmic environment. These residues also allow SPM-1 to confer resistance in P. aeruginosa under Zn(II)-limiting conditions, such as those expected under infection. By optimizing the catalytic efficiency towards β-lactam antibiotics, the enzyme stability and the Zn(II) binding features, molecular evolution meets the specific needs of a pathogenic bacterial host by means of substitutions outside the active site.
dc.description.abstractThe presence of Zn(II)-containing metallo-β-lactamases (MβLs) that confer resistance to all penicillins, cephalosporins and carbapenems in Pseudomonas aeruginosa adds significantly to the threat of this pathogen in our health care system. SPM-1 is an MβLs widely distributed in South America and only found in P. aeruginosa. In common with all MβLs, the active site residues are highly conserved. In this work we asked the following question: how would substrate specificity evolve in SPM-1 if the active site residues are highly uniform and do not permit substitutions. To this end, we explored the role of two amino acids (S84 and G121) that are outside the active site (second sphere) and are unique in the SPM-1 β-lactamase. We discovered that replacing these amino acids impacts resistance to cephalosporins and carbapenems and that this resistance profile depends on the enzymatic behavior and the availability of Zn(II) in the environment. This work demonstrates how protein evolution by means of subtle substitutions outside the active site meets the specific needs of a pathogenic bacterial host.
dc.description.filFil: González, Lisandro Javier. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET). Área Biofísica; Argentina.
dc.description.filFil: Moreno, Diego M. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química de Rosario (IQUIR-CONICET); Argentina.
dc.description.filFil: Bonomo, Robert A. Louis Stokes Cleveland Department of Veterans Affairs Medical Center. Research Service; United States.
dc.description.filFil: Bonomo, Robert A. Case Western Reserve University. School of Medicine. Departments of Medicine, Pharmacology, Molecular Biology and Microbiology, Biochemistry, Proteomics and Bioinformatics; United States.
dc.description.filFil: Vila, Alejandro J. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET). Área Biofísica; Argentina.
dc.description.sponsorshipConsejo Nacional de Investigaciones Científicas y Técnicas (CONICET)
dc.description.sponsorshipAgencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación (Agencia I+D+i)
dc.description.sponsorshipNational Institutes of Health (NIH): 1R01AI100560, R01AI072219, R01AI063517, R01AI100560
dc.description.sponsorshipLouis Stokes Cleveland Department of Veterans Affairs Medical Center
dc.description.sponsorshipThe Geriatric Research, Education, and Clinical Centers (GRECCs)
dc.description.versionpeerreviewed
dc.format.extent1-12
dc.identifier.e-issn1553-7374
dc.identifier.issn1553-7366
dc.identifier.urihttps://hdl.handle.net/2133/27660
dc.language.isoen
dc.publisherPublic Library of Science (PLOS)
dc.relation.publisherversionhttps://doi.org/10.1371/journal.ppat.1003817
dc.relation.publisherversionhttps://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1003817
dc.rightsopenAccess
dc.rights.holderGonzález, Lisandro Javier
dc.rights.holderMoreno, Diego M.
dc.rights.holderBonomo, Robert A.
dc.rights.holderVila, Alejandro J.
dc.rights.holderUniversidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas
dc.rights.textCC0 1.0 Universalen
dc.rights.urihttps://creativecommons.org/publicdomain/zero/1.0/
dc.subjectPseudomonas aeruginosa
dc.subjectMetallo-β-lactamases
dc.subjectBeta-lactam resistance
dc.subjectDrug resistance, bacterial
dc.subjectCodon
dc.subjectAmino Acids
dc.titleHost-specific enzyme-substrate interactions in SPM-1 metallo-β-lactamase are modulated by second sphere residues
dc.typearticulo
dc.type.versionpublishedVersion

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