Spectroscopic and Mechanistic Studies of Heterodimetallic Forms of Metallo-β-lactamase NDM-1

In an effort to characterize the roles of each metal ion in metallo-β-lactamase NDM-1, heterodimetallic analogues (CoCo-, ZnCo-, and CoCd-) of the enzyme were generated and characterized. UV–vis, 1H NMR, EPR, and EXAFS spectroscopies were used to confirm the fidelity of the metal substitutions, including the presence of a homogeneous, heterodimetallic cluster, with a single-atom bridge. This marks the first preparation of a metallo-β-lactamase selectively substituted with a paramagnetic metal ion, Co(II), either in the Zn1 (CoCd-NDM-1) or in the Zn2 site (ZnCo-NDM-1), as well as both (CoCo-NDM-1). We then used these metal-substituted forms of the enzyme to probe the reaction mechanism, using steady-state and stopped-flow kinetics, stopped-flow fluorescence, and rapid-freeze-quench EPR. Both metal sites show significant effects on the kinetic constants, and both paramagnetic variants (CoCd- and ZnCo-NDM-1) showed significant structural changes on reaction with substrate. These changes are discussed in terms of a minimal kinetic mechanism that incorporates all of the data

Engineered mononuclear variants in Bacillus cereus metallo-beta-lactamase BcII are inactive.

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

Real‐Time Monitoring of New Delhi Metallo‐β‐Lactamase Activity in Living Bacterial Cells by ¹H NMR Spectroscopy

Disconnections between invitro responses and those observed in whole cells confound many attempts to design drugs in areas of serious medical need. A method based on 1D 1H NMR spectroscopy is reported that affords the ability to monitor the hydrolytic decomposition of the carbapenem antibiotic meropenem inside Escherichia coli cells expressing New Delhi metallo-β-lactamase subclass 1 (NDM-1), an emerging antibiotic-resistance threat. Cell-based NMR studies demonstrated that two known NDM-1 inhibitors, L-captopril and ethylenediaminetetraacetic acid (EDTA), inhibit the hydrolysis of meropenem invivo. NDM-1 activity in cells was also shown to be inhibited by spermine, a porin inhibitor, although in an invitro assay, the influence of spermine on the activity of isolated NDM-1 protein is minimal. This new approach may have generic utility for monitoring reactions involving diffusible metabolites in other complex biological matrices and whole-cell settings, including mammalian cells

Beta-Lactamase Repressor BlaI Modulates Staphylococcus aureus Cathelicidin Antimicrobial Peptide Resistance and Virulence

BlaI is a repressor of BlaZ, the beta-lactamase responsible for penicillin resistance in Staphylococcus aureus. Through screening a transposon library in S. aureus Newman for susceptibility to cathelicidin antimicrobial peptide, we discovered BlaI as a novel cathelicidin resistance factor. Additionally, through integrational mutagenesis in S. aureus Newman and MRSA Sanger 252 strains, we confirmed the role of BlaI in resistance to human and murine cathelidicin and showed that it contributes to virulence in human whole blood and murine infection models. We further demonstrated that BlaI could be a target for innate immune-based antimicrobial therapies; by removing BlaI through subinhibitory concentrations of 6-aminopenicillanic acid, we were able to sensitize S. aureus to LL-37 killing

Dinuclear cobalt(II) omplexes as metallo--lactamase mimics

The -lactamase activity of two previously reported dinuclear cobalt(II) complexes is described. The two complexes, [Co2(CO2EtH2L1)(CH3COO)2](PF6) (CO2EtH3L1 = ethyl 4-hydroxy-3,5-bis{[(2-hydroxyethyl)(pyridin-2-ylmethyl)amino]methyl}benzoate) and [Co2(CO2EtL2)(CH3COO)2](PF6) (CO2EtHL2 = ethyl 4-hydroxy-3,5-bis{[(2-methoxyethyl)(pyridin-2-ylmethyl)amino]methyl}benzoate), differ in that the latter has methyl ether donors in contrast to potentially nucleophilic alkoxide donors in the former. They thus offer a direct comparison of potential ligand-centered nucleophiles. The complexes were treated with the antibiotic penicillin G and the commonly used lactamase substrate nitrocefin. On the basis of mass spectrometry, UV/Vis, and infrared spectroscopy measurements in solution, it was shown that only [Co2(CO2EtH2L1)(CH3COO)2](PF6) was capable of hydrolyzing both penicillin and nitrocefin, and that the hydrolysis-initiating nucleophile was an alkoxide donor. Analysis of kinetic data showed that nitrocefin binding occurs more rapidly {k1 = [(2.5x103)+/-(1.9x101)] M-1min-1} than its subsequent hydrolysis {k2 = [(1.6x10-1)+/-(8.1x10-4)] min-1}. The pH dependence of nitrocefin hydrolysis by [Co2(CO2EtH2L1)(CH3COO)2]+ displays two pKa values (6.88 +/- 0.74; 8.45 +/- 0.68), the first of which is attributed to the deprotonation of a CoII alcohol, and the second of which is proposed to arise from CoII-OH2. For [Co2(CO2EtL2)(CH3COO)2]+, only one relevant pKa1 (8.47 +/- 0.14) is evident, assigned to a terminal water molecule. By using variable-temperature/variable-field magnetic circular dichroism (VTVH MCD), it was demonstrated that the sign of the magnetic exchange coupling parameter (J) for the parent dinuclear cobalt(II) complexes changes upon binding of the substrate. This work presents one of the few cobalt(II) -lactamase model complexes that is capable of facile hydrolysis of -lactam substrates, an outcome that provides a good benchmark to investigate the reaction mechanism(s) applicable to the enzyme systems