Deciphering the evolution of metallo-β-lactamases: A journey from the test tube to the bacterial periplasm
2022; Elsevier BV; Volume: 298; Issue: 3 Linguagem: Inglês
10.1016/j.jbc.2022.101665
ISSN1083-351X
AutoresCarolina López, juliana Delmonti, Robert A. Bonomo, Alejandro J. Vila,
Tópico(s)Vibrio bacteria research studies
ResumoUnderstanding the evolution of metallo-β-lactamases (MBLs) is fundamental to deciphering the mechanistic basis of resistance to carbapenems in pathogenic and opportunistic bacteria. Presently, these MBL-producing pathogens are linked to high rates of morbidity and mortality worldwide. However, the study of the biochemical and biophysical features of MBLs in vitro provides an incomplete picture of their evolutionary potential, since this limited and artificial environment disregards the physiological context where evolution and selection take place. Herein, we describe recent efforts aimed to address the evolutionary traits acquired by different clinical variants of MBLs in conditions mimicking their native environment (the bacterial periplasm) and considering whether they are soluble or membrane-bound proteins. This includes addressing the metal content of MBLs within the cell under zinc starvation conditions and the context provided by different bacterial hosts that result in particular resistance phenotypes. Our analysis highlights recent progress bridging the gap between in vitro and in-cell studies. Understanding the evolution of metallo-β-lactamases (MBLs) is fundamental to deciphering the mechanistic basis of resistance to carbapenems in pathogenic and opportunistic bacteria. Presently, these MBL-producing pathogens are linked to high rates of morbidity and mortality worldwide. However, the study of the biochemical and biophysical features of MBLs in vitro provides an incomplete picture of their evolutionary potential, since this limited and artificial environment disregards the physiological context where evolution and selection take place. Herein, we describe recent efforts aimed to address the evolutionary traits acquired by different clinical variants of MBLs in conditions mimicking their native environment (the bacterial periplasm) and considering whether they are soluble or membrane-bound proteins. This includes addressing the metal content of MBLs within the cell under zinc starvation conditions and the context provided by different bacterial hosts that result in particular resistance phenotypes. Our analysis highlights recent progress bridging the gap between in vitro and in-cell studies. The scourge of antimicrobial resistance represents a growing threat for public health. The indiscriminate use and abuse of antibiotics has fostered the selection of bacteria resistant even to our most potent antibiotics such as carbapenems (Fig. 1A) (1Walsh C. Molecular mechanisms that confer antibacterial drug resistance.Nature. 2000; 406: 775-781Google Scholar, 2Fisher J.F. Meroueh S.O. Mobashery S. 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Metallo-β-lactamases (MBLs) are Zn(II)-dependent enzymes, that represent one of the largest groups of carbapenemases for which clinical inhibitors are not yet commercially available (13Walsh T.R. Toleman M.A. Poirel L. Nordmann P. Metallo-β-lactamases: The quiet before the storm?.Clin. Microbiol. Rev. 2005; 18: 306-325Google Scholar, 14Nordmann P. Poirel L. The difficult-to-control spread of carbapenemase producers among Enterobacteriaceae worldwide.Clin. Microbiol. Infect. 2014; 20: 821-830Google Scholar, 15Bahr G. González L.J. Vila A.J. Metallo-β-lactamases in the age of multidrug resistance: From structure and mechanism to evolution, dissemination, and inhibitor design.Chem. Rev. 2021; 121: 7957-8094Google Scholar, 16Mojica M.F. Rossi M.-A. Vila A.J. Bonomo R.A. The urgent need for metallo-β-lactamase inhibitors: An unattended global threat.Lancet Infect. Dis. 2022; 22: e28-e34Google Scholar). Since MBLs are highly divergent in sequence, metal ligands at the active site, and Zn(II) stoichiometry, they have been divided into three subclasses: B1, B2, and B3 (Fig. 1B) (14Nordmann P. Poirel L. The difficult-to-control spread of carbapenemase producers among Enterobacteriaceae worldwide.Clin. Microbiol. Infect. 2014; 20: 821-830Google Scholar, 17Bebrone C. Metallo-β-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily.Biochem. Pharmacol. 2007; 74: 1686-1701Google Scholar, 18Garau G. García-Sáez I. Bebrone C. Anne C. Mercuri P. Galleni M. Frère J.M. Dideberg O. Update of the standard numbering scheme for class B β-lactamases.Antimicrob. Agents Chemother. 2004; 48: 2347-2349Google Scholar, 19Berglund F. Johnning A. Larsson D.G.J. Kristiansson E. An updated phylogeny of the metallo-β-lactamases.J. Antimicrob. Chemother. 2021; 76: 117-123Google Scholar). Most acquired, clinically relevant MBLs present in Gram-negative pathogens belong to subclass B1 (16Mojica M.F. Rossi M.-A. Vila A.J. Bonomo R.A. The urgent need for metallo-β-lactamase inhibitors: An unattended global threat.Lancet Infect. Dis. 2022; 22: e28-e34Google Scholar, 20Mojica M.F. Bonomo R.A. Fast W. B1-metallo-β-lactamases: Where do we stand?.Curr. Drug Targets. 2015; 17: 1029-1050Google Scholar, 21Palzkill T. Metallo-β-lactamase structure and function.Ann. N. Y. Acad. Sci. 2013; 1277: 91-104Google Scholar, 22Marshall S. Hujer A.M. Rojas L.J. Papp-Wallace K.M. Humphries R.M. Spellberg B. Hujer K.M. Marshall E.K. Rudin S.D. Perez F. Wilson B.M. Wasserman R.B. Chikowski L. Paterson D.L. Vila A.J. et al.Can ceftazidime-avibactam and aztreonam overcome β-lactam resistance conferred by metallo-β-lactamases in Enterobacteriaceae?.Antimicrob. Agents Chemother. 2017; 61e02243-16Google Scholar, 23Bush K. Bradford P.A. Epidemiology of β-lactamase-producing pathogens.Clin. Microbiol. Rev. 2020; 33e00047-19Google Scholar). B1 enzymes are active with two metal ions: Zn1 coordinated to three His ligands: His116, His118, and His196 (3H site) and Zn2 bound to Asp120, Cys221, and His263 (also called DCH site) (Fig. 1B) (15Bahr G. González L.J. Vila A.J. Metallo-β-lactamases in the age of multidrug resistance: From structure and mechanism to evolution, dissemination, and inhibitor design.Chem. Rev. 2021; 121: 7957-8094Google Scholar, 19Berglund F. Johnning A. Larsson D.G.J. Kristiansson E. An updated phylogeny of the metallo-β-lactamases.J. Antimicrob. Chemother. 2021; 76: 117-123Google Scholar, 24Rasmussen B.A. Bush K. Carbapenem-hydrolyzing β-lactamases.Antimicrob. Agents Chemother. 1997; 41: 223-232Google Scholar, 25Tooke C.L. Hinchliffe P. Bragginton E.C. Colenso C.K. Hirvonen V.H.A. Takebayashi Y. Spencer J. β-Lactamases and β-lactamase inhibitors in the 21st century.J. Mol. Biol. 2019; 431: 3472-3500Google Scholar). The coordination sphere is completed by strategically positioned water molecules (Fig. 1B). B3 enzymes are also binuclear, with a 3H ligand set in the Zn1 site, but with different ligands at the Zn2 site: Asp120, His121, and His263 (DHH site) (Fig. 1B). Finally, B2 MBLs are active in their mono-Zn(II) form, in which the metal ion is bound to the DCH site (Fig. 1B). In these enzymes, uptake of a second Zn(II) ion inhibits their activity (15Bahr G. González L.J. Vila A.J. Metallo-β-lactamases in the age of multidrug resistance: From structure and mechanism to evolution, dissemination, and inhibitor design.Chem. Rev. 2021; 121: 7957-8094Google Scholar). For a more comprehensive description of the structural and biochemical features of MBLs, the reader is referred to a recent review covering these aspects (15Bahr G. González L.J. Vila A.J. Metallo-β-lactamases in the age of multidrug resistance: From structure and mechanism to evolution, dissemination, and inhibitor design.Chem. Rev. 2021; 121: 7957-8094Google Scholar). β-Lactamases are a unique model to study protein evolution, since the survival of the bacterial cell challenged with antibiotics can be correlated with the biochemical and biophysical traits of a single protein (26Andersson D.I. Balaban N.Q. Baquero F. Courvalin P. Glaser P. Gophna U. Kishony R. Molin S. Tønjum T. Antibiotic resistance: Turning evolutionary principles into clinical reality.FEMS Microbiol. Rev. 2021; 44: 171-188Google Scholar). The serine-β-lactamase (SBL) TEM (named for patient Temoneira) has been an excellent model for the study of protein evolution (27Orencia M.C. Yoon J.S. Ness J.E. Stemmer W.P.C. Stevens R.C. Predicting the emergence of antibiotic resistance by directed evolution and structural analysis.Nat. Struct. Biol. 2001; 8: 238-242Google Scholar, 28Sideraki V. Huang W. Palzkill T. Gilbert H.F. A secondary drug resistance mutation of TEM-1 β-lactamase that suppresses misfolding and aggregation.Proc. Natl. Acad. Sci. 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The essential Zn(II) ions in MBLs represent an additional constraint for protein fitness that does not allow to extrapolate evolutionary studies on SBLs to MBLs (15Bahr G. González L.J. Vila A.J. Metallo-β-lactamases in the age of multidrug resistance: From structure and mechanism to evolution, dissemination, and inhibitor design.Chem. Rev. 2021; 121: 7957-8094Google Scholar, 34Ambler R.P. Daniel M. Fleming J. Hermoso J.M. Pang C. Waley S.G. The amino acid sequence of the zinc-requiring β-lactamase II from the bacterium Bacillus cereus 569.FEBS Lett. 1985; 189: 207-211Google Scholar, 35Hussain M. Carlino A. Madonna M.J. Lampen J.O. Cloning and sequencing of the metallothioprotein β-lactamase II gene of Bacillus cereus 569/H in Escherichia coli.J. Bacteriol. 1985; 164: 223-229Google Scholar, 36Baier F. Tokuriki N. Connectivity between catalytic landscapes of the metallo-β-lactamase superfamily.J. Mol. Biol. 2014; 426: 2442-2456Google Scholar, 37Meini M.R. Llarrull L.I. Vila A.J. Overcoming differences: The catalytic mechanism of metallo-β-lactamases.FEBS Lett. 2015; 589: 3419-3432Google Scholar). The study of the biochemical and biophysical features of purified proteins has provided clues for understanding the impact of substitutions in the evolution of β-lactamases (27Orencia M.C. Yoon J.S. Ness J.E. Stemmer W.P.C. Stevens R.C. Predicting the emergence of antibiotic resistance by directed evolution and structural analysis.Nat. Struct. Biol. 2001; 8: 238-242Google Scholar, 28Sideraki V. Huang W. Palzkill T. Gilbert H.F. A secondary drug resistance mutation of TEM-1 β-lactamase that suppresses misfolding and aggregation.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 283Google Scholar, 29Wang X. Minasov G. Shoichet B.K. Evolution of an antibiotic resistance enzyme constrained by stability and activity trade-offs.J. Mol. Biol. 2002; 320: 85-95Google Scholar, 30Bershtein S. Segal M. Bekerman R. Tokuriki N. Tawfik D.S. Robustness-epistasis link shapes the fitness landscape of a randomly drifting protein.Nature. 2006; 444: 929-932Google Scholar, 31Weinreich D.M. Delaney N.F. DePristo M.A. Hartl D.L. Darwinian evolution can follow only very few mutational paths to fitter proteins.Science. 2006; 312: 111-114Google Scholar, 32Salverda M.L.M. Dellus E. Gorter F.A. Debets A.J.M. van der Oost J. Hoekstra R.F. Tawfik D.S. de Visser J.A.G.M. Initial mutations direct alternative pathways of protein evolution.PLoS Genet. 2011; 7e1001321Google Scholar, 33Gong L.I. Bloom J.D. Epistatically interacting substitutions are enriched during adaptive protein evolution.PLoS Genet. 2014; 10e1004328Google Scholar). However, it provides an incomplete and sometimes biased picture, since the impact of the cellular environment is disregarded. In the case of Gram-negative organisms, survival of the bacterial cell in the presence of β-lactam antibiotics depends on the successful expression, translocation, processing, and the appropriate localization of these enzymes. Moreover, MBLs can be present in different bacterial hosts with different resistance phenotypes. The in vitro study of the purified mature protein also overlooks the impact of mutations in the signal peptide (SP) that may affect the translocation and levels of functional protein in the periplasm (38Socha R.D. Chen J. Tokuriki N. The molecular mechanisms underlying hidden phenotypic variation among metallo-β-lactamases.J. Mol. Biol. 2019; 431: 1172-1185Google Scholar, 39López C. Ayala J.A. Bonomo R.A. González L.J. Vila A.J. Protein determinants of dissemination and host specificity of metallo-β-lactamases.Nat. Commun. 2019; 10: 3617Google Scholar), their cellular localization, and even the correct folding or stability. The description of protein evolution based solely on in vitro studies in purified proteins is an example of the "streetlight effect," in which a person who is intoxicated is looking under a lamppost for the keys lost somewhere else because the light is there. The understanding of the differences between the features displayed by a protein in vitro and within the cell is crucial for disentangling the driving forces in evolution. Evolution results from the accumulation of mutations and the selection of particular traits under defined conditions. However, bacteria are continuously exposed to changes in their environment. Two significant factors are usually disregarded when describing MBL evolution: the exposure of bacteria to antibiotics is not permanent, and bacteria cycle between restrictive and permissive conditions (with and without antibiotics, respectively) (2Fisher J.F. Meroueh S.O. Mobashery S. Bacterial resistance to β-lactam antibiotics: Compelling opportunism, compelling opportunity.Chem. Rev. 2005; 105: 395-424Google Scholar, 39López C. Ayala J.A. Bonomo R.A. González L.J. Vila A.J. Protein determinants of dissemination and host specificity of metallo-β-lactamases.Nat. Commun. 2019; 10: 3617Google Scholar, 40Hughes D. Andersson D.I. Environmental and genetic modulation of the phenotypic expression of antibiotic resistance.FEMS Microbiol. Rev. 2017; 41: 374-391Google Scholar, 41Pereira C. Larsson J. Hjort K. Elf J. Andersson D.I. The highly dynamic nature of bacterial heteroresistance impairs its clinical detection.Commun. Biol. 2021; 4: 1-12Google Scholar) at the sites of infection, the immune system response elicits a massive metal starvation, limiting the amount of available Zn(II), which impacts on MBL-mediated resistance (42Corbin B.D. Seeley E.H. Raab A. Feldmann J. Miller M.R. Torres V.J. Anderson K.L. Dattilo B.M. Dunman P.M. Gerads R. Caprioli R.M. Nacken W. Chazin W.J. Skaar E.P. Metal chelation and inhibition of bacterial growth in tissue abscesses.Science. 2008; 319: 962-965Google Scholar, 43Kehl-Fie T.E. Skaar E.P. Nutritional immunity beyond iron: A role for manganese and zinc.Curr. Opin. Chem. Biol. 2010; 14: 218-224Google Scholar, 44González L.J. Bahr G. Nakashige T.G. Nolan E.M. Bonomo R.A. Vila A.J. Membrane anchoring stabilizes and favors secretion of New Delhi metallo-β-lactamase.Nat. Chem. Biol. 2016; 12: 516-522Google Scholar, 45Antelo G.T. Vila A.J. Giedroc D.P. Capdevila D.A. Molecular evolution of transition metal bioavailability at the host–pathogen interface.Trends Microbiol. 2021; 29: 441-457Google Scholar). These conditions define the driving forces in the evolutionary landscape of MBLs. Here, we present and discuss recent advances and new challenges in the study of the evolution of MBLs, encompassing methodological approaches that enable a quantitative measurement of biochemical and biophysical parameters in cell-like environments. We also review the "state of the art" regarding the physiology (and life cycle) of MBLs in bacterial cells, which is intimately related to their evolutionary landscape. In the last section, we also discuss the biochemical and biophysical traits that have been optimized in the clinical evolution of different MBLs. The first challenge in extrapolating in vitro to in cell data is the attempt to correlate the catalytic activities of the β-lactamases with the resistance phenotype in different bacteria. The resistance phenotype is quantitated based on the minimum inhibitory concentration (MIC) of an antibiotic that inhibits bacterial growth. Most B1 MBLs exhibit catalytic efficiencies (kcat/KM) within a range of 105–106 M−1s−1 (15Bahr G. González L.J. Vila A.J. Metallo-β-lactamases in the age of multidrug resistance: From structure and mechanism to evolution, dissemination, and inhibitor design.Chem. Rev. 2021; 121: 7957-8094Google Scholar, 17Bebrone C. Metallo-β-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily.Biochem. Pharmacol. 2007; 74: 1686-1701Google Scholar), differing at the most by one order of magnitude. In contrast, MIC values are quite variable, particularly among different hosts (38Socha R.D. Chen J. Tokuriki N. The molecular mechanisms underlying hidden phenotypic variation among metallo-β-lactamases.J. Mol. Biol. 2019; 431: 1172-1185Google Scholar, 39López C. Ayala J.A. Bonomo R.A. González L.J. Vila A.J. Protein determinants of dissemination and host specificity of metallo-β-lactamases.Nat. Commun. 2019; 10: 3617Google Scholar). These different resistance phenotypes are due in most cases to changes in the cell permeability and the presence of other, complementary, and resistance mechanisms (15Bahr G. González L.J. Vila A.J. Metallo-β-lactamases in the age of multidrug resistance: From structure and mechanism to evolution, dissemination, and inhibitor design.Chem. Rev. 2021; 121: 7957-8094Google Scholar). In this review, we ask the reader to also appreciate the different environments present in each host that impact on the expression levels, processing, and activity of the MBLs. This notion regards the so-called "quinary structure of proteins," a term coined by McConkey (46McConkey E.H. Molecular evolution, intracellular organization, and the quinary structure of proteins.Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 3236-3240Google Scholar), who considers macromolecular interactions within the cell, and it has been addressed by Pielak, Gruebele, Shekhtman, and others (47Monteith W.B. Cohen R.D. Smith A.E. Guzman-Cisneros E. Pielak G.J. Quinary structure modulates protein stability in cells.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 1739-1742Google Scholar, 48Cohen R.D. Pielak G.J. A cell is more than the sum of its (dilute) parts: A brief history of quinary structure.Protein Sci. 2017; 26: 403-413Google Scholar, 49Guin D. Gruebele M. Weak chemical interactions that drive protein evolution: Crowding, sticking, and quinary structure in folding and function.Chem. Rev. 2019; 119: 10691-10717Google Scholar) by measuring the biochemical and biophysical properties of proteins under conditions mimicking the physiological environment. In Gram-negative bacteria, MBLs are synthesized as cytoplasmic precursors with an N-terminal signal sequence (the SP), which directs them to the secretion machinery for their translocation into the cell envelope, where they perform their hydrolytic activity (Fig. 2). So far, all known MBLs are exported by the SecA–SecYEG system as unfolded polypeptides, recognized and processed by type I signal peptidase (SPase I) or by type II lipoprotein signal peptidase (SPase II), depending on whether their final localization is the periplasm or the outer membrane, respectively (Figs. 2 and 3) (50Pradel N. Delmas J. Wu L.F. Santini C.L. Bonnet R. Sec- and Tat-dependent translocation of β-lactamases across the Escherichia coli inner membrane.Antimicrob. Agents Chemother. 2009; 53: 242-248Google Scholar, 51Du Plessis D.J.F. Nouwen N. Driessen A.J.M. The Sec translocase.Biochim. Biophys. Acta Biomembr. 2011; 1808: 851-865Google Scholar, 52Denks K. Vogt A. Sachelaru I. Petriman N.A. Kudva R. Koch H.G. The Sec translocon mediated protein transport in prokaryotes and eukaryotes.Mol. Membr. Biol. 2014; 31: 58-84Google Scholar, 53Paetzel M. Karla A. Strynadka N.C.J. Dalbey R.E. Signal peptidases.Chem. Rev. 2002; 102: 4549-4580Google Scholar, 54Natale P. Brüser T. Driessen A.J.M. Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane-distinct translocases and mechanisms.Biochim. Biophys. Acta Biomembr. 2008; 1778: 1735-1756Google Scholar). Finally, the enzymes fold and bind Zn(II) in the periplasmic space (55Morán-Barrio J. Limansky A.S. Viale A.M. Secretion of GOB metallo-β-lactamase in Escherichia coli depends strictly on the cooperation between the cytoplasmic DnaK chaperone system and the Sec machinery: Completion of folding and Zn(II) ion acquisition occur in the bacterial periplasm.Antimicrob. Agents Chemother. 2009; 53: 2908-2917Google Scholar). Most MBLs are soluble periplasmic enzymes, except for New Delhi metallo-β-lactamase (NDM) enzymes, which are lipoproteins anchored to the inner leaflet of the outer membrane (Fig. 2) (44González L.J. Bahr G. Nakashige T.G. Nolan E.M. Bonomo R.A. Vila A.J. Membrane anchoring stabilizes and favors secretion of New Delhi metallo-β-lactamase.Nat. Chem. Biol. 2016; 12: 516-522Google Scholar, 56King D. Strynadka N. Crystal structure of New Delhi metallo-β-lactamase reveals molecular basis for antibiotic resistance.Protein Sci. 2011; 20: 1484-1491Google Scholar). The functional levels of MBLs in the periplasmic space vary considerably among different bacterial hosts, finally impacting on the resistance phenotype (38Socha R.D. Chen J. Tokuriki N. The molecular mechanisms underlying hidden phenotypic variation among metallo-β-lactamases.J. Mol. Biol. 2019; 431: 1172-1185Google Scholar, 44González L.J. Bahr G. Nakashige T.G. Nolan E.M. Bonomo R.A. Vila A.J. Membrane anchoring stabilizes and favors secretion of New Delhi metallo-β-lactamase.Nat. Chem. Biol. 2016; 12: 516-522Google Scholar).Figure 3The adaptability of MBLs to different bacterial hosts depends on the impact of protein expression on biological fitness. This model describes the adaptability of MBLs to different bacterial hosts based on the processing of their SPs (39López C. Ayala J.A. Bonomo R.A. González L.J. Vila A.J. Protein determinants of dissemination and host specificity of metallo-β-lactamases.Nat. Commun. 2019; 10: 3617Google Scholar). An MBL is confined to a narrow range of bacteria (left panel) when its expression in nonfrequent hosts causes the accumulation of toxic precursor forms (shown as green misfolded protein) in the periplasmic face of the inner membrane because of an inefficient translocation and processing that compromises the bacterial growth (bottom left panel). This toxicity correlates with an increase in the production of outer membrane vesicles (OMVs) that incorporate both the mature (folded protein) and the precursor protein (misfolded) to alleviate the envelope stress; thus acting as a vesicle-mediated detoxification mechanism. By contrast, a broad host MBL (right panel) is produced by different bacteria without fitness cost (bottom right panel). These MBLs are efficiently translocated and processed. In these cases, the level of selective packaging into OMVs depends on the interaction of the MBL with the outer membrane, either by membrane anchoring (violet folded protein) or by specific electrostatic interactions (cyan folded protein) through a positively charged patch. The double-headed dashed arrow represents the binding equilibrium of soluble periplasmic MBL with the outer membrane, which favors protein packaging into OMVs. EV, cells carrying the empty vector; MBL, metallo-β-lactamase; SP, signal peptide.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Based on this knowledge, one simple strategy to fill this gap is to measure the catalytic efficiency of the enzymes in periplasmic extracts, using immunoblotting quantitation to retrieve apparent kcat values. kcat/KM values measured in the periplasm show a significant correlation with MIC values (57Meini M.-R. Tomatis P.E. Weinreich D.M. Vila A.J. Quantitative description of a protein fitness landscape based on molecular features.Mol. Biol. Evol. 2015; 32: 1774-1787Google Scholar, 58González J.M. Meini M.-R. Tomatis P.E. Medrano Martín F.J. Cricco J.A. Vila A.J. Metallo-β-lactamases withstand low Zn(II) conditions by tuning metal-ligand interactions.Nat. Chem. Biol. 2012; 8: 698-700Google Scholar, 59González L.J. Moreno D.M. Bonomo R.A. Vila A.J. Host-specific enzyme-substrate interactions in SPM-1 metallo-β-lactamase are modulated by second sphere residues.PLoS Pathog. 2014; 10e1003817Google Scholar). This approach has been useful to describe the stepwise evolution of an enzyme evolved in the laboratory (57Meini M.-R. Tomatis P.E. Weinreich D.M. Vila A.J. Quantitative description of a protein fitness landscape based on molecular features.Mol. Biol. Evol. 2015; 32: 1774-1787Google Scholar), to account for differences in the activities in vitro and MICs in MBL mutants (57Meini M.-R. Tomatis P.E. Weinreich D.M. Vila A.J. Quantitative description of a protein fitness landscape based on molecular features.Mol. Biol. Evol. 2015; 32: 1774-1787Google Scholar, 58González J.M. Meini M.-R. Tomatis P.E. Medrano Martín F.J. Cricco J.A. Vila A.J. Metallo-β-lactamases withstand low Zn(II) conditions by tuning metal-ligand interactions.Nat. Chem. Biol. 2012; 8: 698-700Google Scholar, 59González L.J. Moreno D.M. Bonomo R.A. Vila A.J. Host-specific enzyme-substrate interactions in SPM-1 metallo-β-lactamase are modulated by second sphere residues.PLoS Pathog. 2014; 10e1003817Google Scholar), and to study the resistance phenotype of a series of MBLs (either chromosomal or acquired) in different bacterial hosts (38Socha R.D. Chen J. Tokuriki N. The molecular mechanisms underlying hidden phenotypic variation among metallo-β-lactamases.J. Mol. Biol. 2019; 431: 1172-1185Google Scholar). In all these cases, the activity measured in these extracts showed a tight correlation with MIC data. In the case of membrane-bound lactamases, such as NDM-1, similar experiments have been optimized for the measurement of kinetic parameters in spheroplasts (60Giannini E. González L.J. Vila A.J. A simple protocol to characterize bacterial cell-envelope lipoproteins in a native-like environment.Protein Sci. 2019; 28: 2004-2010Google Scholar). This approach can also be applied to quantitate the thermodynamic stability by measuring the enzymatic activity after incubating the periplasmic extracts at different temperatures (57Meini M.-R. Tomatis P.E. Weinreich D.M. Vila A.J. Quantitative description of a protein fitness landscape based on molecular features.Mol. Biol. Evol. 2015; 32: 1774-1787Google Scholar, 59González L.J. Moreno D.M. Bonomo R.A. Vila A.J. Host-specific enzyme-substrate interactions in SPM-1 metallo-β-lactamase are modulated by second sphere residues.PLoS Pathog. 2014; 10e1003817Google Scholar), providing an apparent Tm value that reflects the thermal stability of the MBLs in conditions mimicking the physiological ones (57Meini M.-R. Tomatis P.E. Weinreich D.M. Vila A.J. Quantitative description of a protein fitness landscape based on molecular features.Mol. Biol. Evol. 2015; 32: 1774-1787Google Scholar, 59González L.J. Moreno D.M. Bonomo R.A. Vila A.J. Host-specific enzyme-substrate interactions in SPM-1 metallo-β-lactamase are modulated by second sphere residues.PLoS Pathog. 2014; 10e1003817Google Scholar). This parameter describes how the equilibrium between the folded and unfolded forms of the protein is affected by the temperature. However, this information does not provide clues regarding the kinetic stability of the protein. The kinetic stability, instead, is related
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