RESUMO
The treatment of hospital- and community-associated infections by methicillin-resistant Staphylococcus aureus (MRSA) is a perpetual challenge. This Gram-positive bacterium is resistant specifically to ß-lactam antibiotics, and generally to many other antibacterial agents. Its resistance mechanisms to ß-lactam antibiotics are activated only when the bacterium encounters a ß-lactam. This activation is regulated by the transmembrane sensor/signal transducer proteins BlaR1 and MecR1. Neither the transmembrane/metalloprotease domain, nor the complete MecR1 and BlaR1 proteins, are isolatable for mechanistic study. Here we propose a model for full-length MecR1 based on homology modeling, residue coevolution data, a new extensive experimental mapping of transmembrane topology, partial structures, molecular simulations, and available NMR data. Our model defines the metalloprotease domain as a hydrophilic transmembrane chamber effectively sealed by the apo-sensor domain. It proposes that the amphipathic helices inserted into the gluzincin domain constitute the route for transmission of the ß-lactam-binding event in the extracellular sensor domain, to the intracellular and membrane-embedded zinc-containing active site. From here, we discuss possible routes for subsequent activation of proteolytic action. This study provides the first coherent model of the structure of MecR1, opening routes for future functional investigations on how ß-lactam binding culminates in the proteolytic degradation of MecI.
Assuntos
Proteínas de Bactérias/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , beta-Lactamas/farmacologia , Proteínas de Bactérias/genética , Western Blotting , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Simulação de Acoplamento Molecular , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Espectrometria de Fluorescência , Esferoplastos/efeitos dos fármacos , Esferoplastos/genética , Resistência beta-Lactâmica/genéticaRESUMO
Carbapenems are "last resort" ß-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-ß-lactamases (MßLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all ß-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MßLs 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 MßLs 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 ß-lactams in all MßLs. 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 MßLs.
Assuntos
Antibacterianos/química , Ceftazidima/química , Imipenem/química , Meropeném/química , Zinco/química , beta-Lactamases/química , Sequência de Aminoácidos , Antibacterianos/metabolismo , Domínio Catalítico , Cefepima/química , Cefepima/metabolismo , Cefotaxima/química , Cefotaxima/metabolismo , Ceftazidima/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Imipenem/metabolismo , Cinética , Meropeném/metabolismo , Modelos Moleculares , Piperacilina/química , Piperacilina/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Zinco/metabolismo , Resistência beta-Lactâmica , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Carbapenem-resistant Enterobacteriaceae threaten human health, since carbapenems are last resort drugs for infections by such organisms. Metallo-ß-lactamases (MßLs) are the main mechanism of resistance against carbapenems. Clinically approved inhibitors of MBLs are currently unavailable as design has been limited by the incomplete knowledge of their mechanism. Here, we report a biochemical and biophysical study of carbapenem hydrolysis by the B1 enzymes NDM-1 and BcII in the bi-Zn(II) form, the mono-Zn(II) B2 Sfh-I and the mono-Zn(II) B3 GOB-18. These MßLs hydrolyse carbapenems via a similar mechanism, with accumulation of the same anionic intermediates. We characterize the Michaelis complex formed by mono-Zn(II) enzymes, and we identify all intermediate species, enabling us to propose a chemical mechanism for mono and binuclear MßLs. This common mechanism open avenues for rationally designed inhibitors of all MßLs, notwithstanding the profound differences between these enzymes' active site structure, ß-lactam specificity and metal content.Carbapenem-resistant bacteria pose a major health threat by expressing metallo-ß-lactamases (MßLs), enzymes able to hydrolyse these life-saving drugs. Here the authors use biophysical and computational methods and show that different MßLs share the same reaction mechanism, suggesting new strategies for drug design.
Assuntos
Carbapenêmicos/metabolismo , Zinco/metabolismo , beta-Lactamases/química , beta-Lactamases/metabolismo , Carbapenêmicos/química , Hidrólise , Imipenem/química , Imipenem/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Simulação de Dinâmica Molecular , Espectroscopia por Absorção de Raios XRESUMO
Metallo-ß-lactamases are the latest resistance mechanism of pathogenic and opportunistic bacteria against carbapenems, considered as last resort drugs. The worldwide spread of genes coding for these enzymes, together with the lack of a clinically useful inhibitor, have raised a sign of alarm. Inhibitor design has been mostly impeded by the structural diversity of these enzymes. Here we provide a critical review of mechanistic studies of the three known subclasses of metallo-ß-lactamases, analyzed at the light of structural and mutagenesis investigations. We propose that these enzymes present a modular structure in their active sites that can be dissected into two halves: one providing the attacking nucleophile, and the second one stabilizing a negatively charged reaction intermediate. These are common mechanistic elements in all metallo-ß-lactamases. Nucleophile activation does not necessarily requires a Zn(II) ion, but a Zn(II) center is essential for stabilization of the anionic intermediate. Design of a common inhibitor could be therefore approached based in these convergent mechanistic features despite the structural differences.
Assuntos
Biocatálise , beta-Lactamases/metabolismo , Bactérias/efeitos dos fármacos , Bactérias/enzimologia , Cristalografia por Raios X , Resistência Microbiana a Medicamentos , Humanos , Hidrólise , beta-Lactamases/químicaRESUMO
Pathogenic Gram-negative bacteria resistant to almost all ß-lactam antibiotics are a major public health threat. Zn(II)-dependent or metallo-ß-lactamases (MBLs) produced by these bacteria inactivate most ß-lactam antibiotics, including the carbapenems, which are "last line therapies" for life-threatening Gram-negative infections. NDM-1 is a carbapenemase belonging to the MBL family that is rapidly spreading worldwide. Regrettably, inhibitors of MBLs are not yet developed. Here we present the bisthiazolidine (BTZ) scaffold as a structure with some features of ß-lactam substrates, which can be modified with metal-binding groups to target the MBL active site. Inspired by known interactions of MBLs with ß-lactams, we designed four BTZs that behave as in vitro NDM-1 inhibitors with Ki values in the low micromolar range (from 7 ± 1 to 19 ± 3 µM). NMR spectroscopy demonstrated that they inhibit hydrolysis of imipenem in NDM-1-producing Escherichia coli. In vitro time kill cell-based assays against a variety of bacterial strains harboring blaNDM-1 including Acinetobacter baumannii show that the compounds restore the antibacterial activity of imipenem. A crystal structure of the most potent heterocycle (L-CS319) in complex with NDM-1 at 1.9 Å resolution identified both structural determinants for inhibitor binding and opportunities for further improvements in potency.
RESUMO
The production of ß-lactamase enzymes is one of the most distributed resistance mechanisms towards ß-lactam antibiotics. Metallo-ß-lactamases constitute a worrisome group of these kinds of enzymes, since they present a broad spectrum profile, being able to hydrolyze not only penicillins, but also the latest generation of cephalosporins and carbapenems, which constitute at present the last resource antibiotics. The VIM, IMP, and NDM enzymes comprise the main groups of clinically relevant metallo-ß-lactamases. Here we present an update of the features of the natural variants that have emerged and of the ones that have been engineered in the laboratory, in an effort to find sequence and structural determinants of substrate preferences. This knowledge is of upmost importance in novel drug design efforts. We also discuss the advances in knowledge achieved by means of in vitro directed evolution experiments, and the potential of this approach to predict natural evolution of metallo-ß-lactamases.
RESUMO
Metallo-beta-lactamases are enzymes capable of hydrolyzing all known classes of beta-lactam antibiotics, rendering them ineffective. The design of inhibitors active against all classes of metallo-beta-lactamases has been hampered by the heterogeneity in metal content in the active site and the existence of two different mononuclear forms. BcII is a B1 metallo-beta-lactamase which is found in both mononuclear and dinuclear forms. Despite very elegant studies, there is still controversy on the nature of the active BcII species. We carried out a non-steady-state study of the hydrolysis of penicillin G catalyzed by Co(II)-substituted BcII, and we followed the modifications occurring at the active site of the enzyme. Working at different metal/enzyme ratios we demonstrate that both mono-Co(II) and di-Co(II) BcII are active metallo-beta-lactamases. Besides, we here present evidence that during penicillin G hydrolysis catalyzed by mono-Co(II) BcII the metal is localized in the DCH site (the Zn2 site in B1 enzymes). These conclusions allow us to propose that both in mono-Co(II) and di-Co(II) BcII the substrate is bound to the enzyme through interactions with the Co(II) ion localized in the DCH site. The finding that the DCH site is able to give rise to an active lactamase suggests that the Zn2 site is a common feature to all subclasses of metallo-beta-lactamases and would play a similar role. This proposal provides a starting point for the design of inhibitors based on transition-state analogs, which might be effective against all MbetaLs.
Assuntos
Bacillus cereus/enzimologia , Cobalto/química , Cobalto/metabolismo , Zinco/química , Zinco/metabolismo , beta-Lactamases/química , beta-Lactamases/metabolismo , Bacillus cereus/genética , Cátions/química , Cinética , Modelos Moleculares , Processos Fotoquímicos , beta-Lactamases/genéticaRESUMO
Metallo-beta-lactamases hydrolyze most beta-lactam antibiotics. The lack of a successful inhibitor for them is related to the previous failure to characterize a reaction intermediate with a clinically useful substrate. Stopped-flow experiments together with rapid freeze-quench EPR and Raman spectroscopies were used to characterize the reaction of Co(II)-BcII with imipenem. These studies show that Co(II)-BcII is able to hydrolyze imipenem in both the mono- and dinuclear forms. In contrast to the situation met for penicillin, the species that accumulates during turnover is an enzyme-intermediate adduct in which the beta-lactam bond has already been cleaved. This intermediate is a metal-bound anionic species with a novel resonant structure that is stabilized by the metal ion at the DCH or Zn2 site. This species has been characterized based on its spectroscopic features. This represents a novel, previously unforeseen intermediate that is related to the chemical nature of carbapenems, as confirmed by the finding of a similar intermediate for meropenem. Since carbapenems are the only substrates cleaved by B1, B2, and B3 lactamases, identification of this intermediate could be exploited as a first step toward the design of transition-state-based inhibitors for all three classes of metallo-beta-lactamases.
Assuntos
Bacillus cereus/enzimologia , Carbapenêmicos/química , Carbapenêmicos/metabolismo , Cobalto/química , Cobalto/metabolismo , beta-Lactamases/química , beta-Lactamases/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Hidrólise , Cinética , Modelos Biológicos , Estrutura Terciária de Proteína , Análise Espectral Raman , EstereoisomerismoRESUMO
Metallo-beta-lactamases (MbetaLs) are zinc enzymes able to hydrolyze almost all beta-lactam antibiotics, rendering them inactive, at the same time endowing bacteria high levels of resistance. The design of inhibitors active against all classes of MbetaLs has been hampered by their structural diversity and by the heterogeneity in metal content in enzymes from different sources. BcII is the metallo-beta-lactamase from Bacillus cereus, which is found in both the mononuclear and dinuclear forms. Despite extensive studies, there is still controversy about the nature of the active BcII species. Here we have designed two mutant enzymes in which each one of the metal binding sites was selectively removed. Both mutants were almost inactive, despite preserving most of the structural features of each metal site. These results reveal that neither site isolated in the MbetaL scaffold is sufficient to render a fully active enzyme. This suggests that only the dinuclear species is active or that the mononuclear variants can be active only if aided by other residues that would be metal ligands in the dinuclear species.
Assuntos
Bacillus cereus/enzimologia , Engenharia de Proteínas , beta-Lactamases/genética , beta-Lactamases/metabolismo , Sítios de Ligação , Modelos Moleculares , Mutação , Conformação Proteica , beta-Lactamases/químicaRESUMO
Metallo-beta-lactamases are zinc-dependent hydrolases that inactivate beta-lactam antibiotics, rendering bacteria resistant to them. Asp-120 is fully conserved in all metallo-beta-lactamases and is central to catalysis. Several roles have been proposed for Asp-120, but so far there is no agreed consensus. We generated four site-specifically substituted variants of the enzyme BcII from Bacillus cereus as follows: D120N, D120E, D120Q, and D120S. Replacement of Asp-120 by other residues with very different metal ligating capabilities severely impairs the lactamase activity without abolishing metal binding to the mutated site. A kinetic study of these mutants indicates that Asp-120 is not the proton donor, nor does it play an essential role in nucleophilic activation. Spectroscopic and crystallographic analysis of D120S BcII, the least active mutant bearing the weakest metal ligand in the series, reveals that this enzyme is able to accommodate a dinuclear center and that perturbations in the active site are limited to the Zn2 site. It is proposed that the role of Asp-120 is to act as a strong Zn2 ligand, locating this ion optimally for substrate binding, stabilization of the development of a partial negative charge in the beta-lactam nitrogen, and protonation of this atom by a zinc-bound water molecule.
Assuntos
Ácido Aspártico/química , Bacillus cereus/enzimologia , Metais/química , Zinco/química , beta-Lactamases/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/metabolismo , Cinética , Ligantes , Modelos Químicos , Mutagênese Sítio-Dirigida , Ligação Proteica , EspectrofotometriaRESUMO
Malonyl-CoA is an essential intermediate in fatty acid synthesis in all living cells. Here we demonstrate a new role for this molecule as a global regulator of lipid homeostasis in Gram-positive bacteria. Using in vitro transcription and binding studies, we demonstrate that malonyl-CoA is a direct and specific inducer of Bacillus subtilis FapR, a conserved transcriptional repressor that regulates the expression of several genes involved in bacterial fatty acid and phospholipid synthesis. The crystal structure of the effector-binding domain of FapR reveals a homodimeric protein with a thioesterase-like 'hot-dog' fold. Binding of malonyl-CoA promotes a disorder-to-order transition, which transforms an open ligand-binding groove into a long tunnel occupied by the effector molecule in the complex. This ligand-induced modification propagates to the helix-turn-helix motifs, impairing their productive association for DNA binding. Structure-based mutations that disrupt the FapR-malonyl-CoA interaction prevent DNA-binding regulation and result in a lethal phenotype in B. subtilis, suggesting this homeostatic signaling pathway as a promising target for novel chemotherapeutic agents against Gram-positive pathogens.