How allosteric control of Staphylococcus aureus penicillin binding protein 2a enables methicillin resistance and physiological function

6 pags, 4 figs, 1 tabThe expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the β-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The highmolecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to β-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the β-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to β-lactam acylation and successfully catalyzes the DD-transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with β-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain - a remarkable 60 Å distant from the DD-transpeptidase active site - discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA β-lactam antibiotic. The ability of an anti-MRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second β-lactam molecule, opens an unprecedented realm for β-lactam antibiotic structure-based design.Work in the United States was supported by National Institutes of Health Grants AI090818 and AI104987, and work in Spain was supported by Grants BFU2011-25326 (from the Spanish Ministry of Economy and Competitiveness) and S2010/BMD-2457 (from the Autonomous Government of Madrid)

A general reaction mechanism for carbapenem hydrolysis by mononuclear and binuclear metallo-β-lactamases

Carbapenem-resistant Enterobacteriaceae threaten human health, since carbapenems are last resort drugs for infections by such organisms. Metallo-β-lactamases (MβLs) 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 MβLs 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 MβLs. This common mechanism open avenues for rationally designed inhibitors of all MβLs, notwithstanding the profound differences between these enzymes' active site structure, β-lactam specificity and metal content.Fil: Lisa, María Natalia. 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. Instituto Pasteur de Montevideo; UruguayFil: 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: Aitha, Mahesh. Miami University; Estados UnidosFil: Gonzalez, Mariano Martin. 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: Moreno, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; ArgentinaFil: Crowder, Michael W.. Miami University; Estados UnidosFil: Bonomo, Robert A.. Case Western Reserve University; Estados Unidos. Louis Stokes Cleveland Department of Veterans Affairs Medical Center; Estados UnidosFil: Spencer, James. University Walk; Reino Unido. University of Bristol; Reino UnidoFil: Tierney, David L.. Miami University; Estados UnidosFil: 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. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Biológica. Área Biofísica; ArgentinaFil: 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. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Biológica. Área Biofísica; Argentina. Case Western Reserve University; Estados Unido

Dissection of Events in the Resistance to β-Lactam Antibiotics Mediated by the Protein BlaR1 from <i>Staphylococcus aureus</i>

A heterologous expression system was used to evaluate activation of BlaR1, a sensor/signal transducer protein of <i>Staphylococcus aureus</i> with a central role in resistance to β-lactam antibiotics. In the absence of other <i>S. aureus</i> proteins that might respond to antibiotics and participate in signal transduction events, we documented that BlaR1 fragmentation is autolytic, that it occurs in the absence of antibiotics, and that BlaR1 directly degrades BlaI, the gene repressor of the system. Furthermore, we disclosed that this proteolytic activity is metal ion-dependent and that it is not modulated directly by acylation of the sensor domain by β-lactam antibiotics

Water-Assisted Reaction Mechanism of Monozinc b-Lactamases

Hybrid Car-Parrinello QM/MM calcns. are used to investigate the reaction mechanism of hydrolysis of a common b-lactam substrate (cefotaxime) by the monozinc b-lactamase from Bacillus cereus (BcII). The calcns. suggest a fundamental role for an active site water in the catalytic mechanism. This water mol. binds the zinc ion in the first step of the reaction, expanding the zinc coordination no. and providing a proton donor adequately oriented for the second step. The free energy barriers of the two reaction steps are similar and consistent with the available exptl. data. The conserved hydrogen bond network in the active site, defined by Asp-120, Cys-221, and His-263, not only contributes to orient the nucleophile (as already proposed), but it also guides the second catalytic water mol. to the zinc ion after the substrate is bound. The hydrolysis reaction in water has a relatively high free energy barrier, which is consistent with the stability of cefotaxime in water soln. The modeled Michaelis complexes for other substrates are also characterized by the presence of an ordered water mol. in the same position, suggesting that this mechanism might be general for the hydrolysis of different b-lactam substrates. [on SciFinder (R)

Crystallization and preliminary X-ray diffraction analysis of the lytic transglycosylase MltE from Escherichia coli

3 pags, 2 figs, 1 tabMltE from Escherichia coli (193 amino acids, 21 380 Da) is a lytic trans-glycosylase that initiates the first step of cell-wall recycling. This enzyme is responsible for the cleavage of the cell-wall peptidoglycan at the Β-1,4-glycosidic bond between the N-acetylglucosamine and N-acetylmuramic acid units. At the end this reaction generates a disaccharide that is internalized and initiates the recycling process. To obtain insights into the biological functions of MltE, crystallization trials were performed and crystals of MltE protein that were suitable for X-ray diffraction analysis were obtained. The MltE protein of E. coli was crystallized using the hanging-drop vapour-diffusion method at 291 K. Crystals grew from a mixture consisting of 28% polyethylene glycol 4000, 0.1 M Tris pH 8.4 and 0.2 M magnesium chloride. Further optimization was performed using the microbatch technique. Single crystals were obtained that belonged to the orthorhombic space group C2221, with unit-cell parameters a = 123.32, b = 183.93, c = 35.29 Å, and diffracted to a resolution of 2.1 Å. © 2011 International Union of Crystallography. All rights reserved.This work was supported by grants from the Spanish Ministry of Science and Technology (BFU2008-01711 and the Factoría de Cristalización from CONSOLIDER-INGENIO 2010) and the COMBACT program (S-BIO-0260/2006). The work in the USA was supported by the National Institutes of Health. CA-R is a fellow of the Spanish Ministry of Education and Science (BFU2008-01711/BMC). LIL is a Pew Latin American Fellow in the Biomedical Sciences supported by The Pew Charitable Trusts. The opinions expressed are those of the authors and do not necessarily reflect the views of The Pew Charitable Trusts

High-resolution crystal structure of MltE, an outer membrane-anchored endolytic peptidoglycan lytic transglycosylase from Escherichia coli

El pdf del artículo es el manuscrito de autor (PMID:21341761).The crystal structure of the first endolytic peptidoglycan lytic transglycosylase MltE from Escherichia coli is reported here. The degradative activity of this enzyme initiates the process of cell wall recycling, which is an integral event in the existence of bacteria. The structure sheds light on how MltE recognizes its substrate, the cell wall peptidoglycan. It also explains the ability of this endolytic enzyme to cleave in the middle of the peptidoglycan chains. Furthermore, the structure reveals how the enzyme is sequestered on the inner leaflet of the outer membrane. © 2011 American Chemical Society.Este trabajo fue finanaciado por las concesiones bfu2008-01711 y eu-cp223111.Peer Reviewe

Regulation of the Expression of the β‑Lactam Antibiotic-Resistance Determinants in Methicillin-Resistant <i>Staphylococcus aureus</i> (MRSA)

β-Lactam antibiotics have faced obsolescence with the emergence of methicillin-resistant <i>Staphylococcus aureus</i> (MRSA). A complex set of events ensues upon exposure of MRSA to these antibiotics, which culminates in proteolysis of BlaI or MecI, two gene repressors, and results in the induction of resistance. We report studies on the mechanism of binding of these gene repressors to the operator regions by fluorescence anisotropy. Within the range of <i>in vivo</i> concentrations for BlaI and MecI, these proteins interact with their regulatory elements in a reversible manner, as both a monomer and a dimer