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

Authors:
ORCiD logo; ; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL), Upton, NY (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1409517
Report Number(s):
BNL-114569-2017-JA¿¿¿
Journal ID: ISSN 2041-1723
DOE Contract Number:
SC0012704
Resource Type:
Journal Article
Resource Relation:
Journal Name: Nature Communications; Journal Volume: 8; Journal Issue: 1
Country of Publication:
United States
Language:
English

Citation Formats

Lisa, María-Natalia, Palacios, Antonela R., Aitha, Mahesh, González, Mariano M., Moreno, Diego M., Crowder, Michael W., Bonomo, Robert A., Spencer, James, Tierney, David L., Llarrull, Leticia I., and Vila, Alejandro J. A general reaction mechanism for carbapenem hydrolysis by mononuclear and binuclear metallo-β-lactamases. United States: N. p., 2017. Web. doi:10.1038/s41467-017-00601-9.
Lisa, María-Natalia, Palacios, Antonela R., Aitha, Mahesh, González, Mariano M., Moreno, Diego M., Crowder, Michael W., Bonomo, Robert A., Spencer, James, Tierney, David L., Llarrull, Leticia I., & Vila, Alejandro J. A general reaction mechanism for carbapenem hydrolysis by mononuclear and binuclear metallo-β-lactamases. United States. doi:10.1038/s41467-017-00601-9.
Lisa, María-Natalia, Palacios, Antonela R., Aitha, Mahesh, González, Mariano M., Moreno, Diego M., Crowder, Michael W., Bonomo, Robert A., Spencer, James, Tierney, David L., Llarrull, Leticia I., and Vila, Alejandro J. 2017. "A general reaction mechanism for carbapenem hydrolysis by mononuclear and binuclear metallo-β-lactamases". United States. doi:10.1038/s41467-017-00601-9.
@article{osti_1409517,
title = {A general reaction mechanism for carbapenem hydrolysis by mononuclear and binuclear metallo-β-lactamases},
author = {Lisa, María-Natalia and Palacios, Antonela R. and Aitha, Mahesh and González, Mariano M. and Moreno, Diego M. and Crowder, Michael W. and Bonomo, Robert A. and Spencer, James and Tierney, David L. and Llarrull, Leticia I. and Vila, Alejandro J.},
abstractNote = {},
doi = {10.1038/s41467-017-00601-9},
journal = {Nature Communications},
number = 1,
volume = 8,
place = {United States},
year = 2017,
month = 9
}
  • The carbapenem-hydrolyzing class D β-lactamases OXA-23 and OXA-24/40 have emerged world-wide as causative agents for β-lactam antibiotic resistance in Acinetobacter species. Many variants of these enzymes have appeared clinically, including OXA-160 and OXA-225, which both contain a P→S substitution at homologous positions in the OXA-24/40 and OXA-23 backgrounds respectively. We purified OXA-160 and OXA-225 and used steady-state kinetic analysis to compare the substrate profiles of these variants to their parental enzymes, OXA-24/40 and OXA-23. OXA-160 and OXA-225 possess greatly enhanced hydrolytic activities against aztreonam, ceftazidime, cefotaxime and ceftriaxone when compared to OXA-24/40 and OXA-23. These enhanced activities are the resultmore » of much lower Km values, suggesting that the P→S substitution enhances the binding affinity of these drugs. We have determined the structures of the acylated forms of OXA-160 (with ceftazidime and aztreonam) and OXA-225 (ceftazidime). These structures show that the R1 oxyimino side-chain of these drugs occupies a space near the β5-β6 loop and the omega loop of the enzymes. The P→S substitution found in OXA-160 and OXA-225 results in a deviation of the β5-β6 loop, relieving the steric clash with the R1 side-chain carboxypropyl group of aztreonam and ceftazidime. We found that these results reveal worrying trends in the enhancement of substrate spectrum of class D β-lactamases, but may also provide a map for β-lactam improvement.« less
  • Metallo-?-lactamases (M?Ls) are bacterial Zn(II)-dependent hydrolases that confer broad-spectrum resistance to ?-lactam antibiotics. These enzymes can be subdivided into three subclasses (B1, B2 and B3) that differ in their metal binding sites and their characteristic tertiary structure. To date there are no clinically useful pan-M?L inhibitors available, mainly due to the unawareness of key catalytic features common to all M?L brands. Here we have designed, expressed and characterized two double mutants of BcII, a di-Zn(II) B1-M?L from Bacillus cereus, namely BcII-R121H/C221D (BcII-HD) and BcII-R121H/C221S (BcII-HS). These mutants display modified environments at the so-called Zn2 site or DCH site, reproducing themore » metal coordination environments of structurally related metallohydrolases. Through a combination of structural and functional studies, we found that BcII-HD is an impaired ?-lactamase even as a di-Zn(II) enzyme, whereas BcII-HS exhibits the ability to exist as mono or di-Zn(II) species in solution, with different catalytic performances. We show that these effects result from an altered position of Zn2, which is incapable of providing a productive interaction with the substrate ?-lactam ring. These results indicate that the position of Zn2 is essential for a productive substrate binding and hydrolysis.« less
  • Rates and kinetic solvent isotope effects for the title substrates have been studied at 15 deg . Isotope effects k H2O/k D2O on the order of 2.5, essentially independent of substituent, were found for the uncatslyzed'' hydrolysis of both substrates, as well as benzoylimidazolium ions, and for the methylimidazole-catalyzed hydrolysis latter -k HO-of 1.2 to 1.4, larger for more electron-supplying substituents, as expected for simple ratedetermining nucleophilic attack on the carbonyl carbon atom. The lyoxide term for the benzoylimidazoles has isootope effects near 1.0. It is concluded that general base catalyzed hydrolysis, with water, methylimidazole, or lyoxide as general basemore » as the case may require, is the problem mechanism for all terms studied (except the one case of simple nucleophilic attack), and that this mechanism consists of rate-determining nucleophilic attack by water with one proton partly transferred to the general base but not being transferred as part of the reaction coordinate motion.« less
  • Metallo-{beta}-lactamases (M{beta}Ls) 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 M{beta}Ls 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 metalmore » 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 M{beta}L 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.« less
  • The cleavage of organotransition metal-carbon bonds by H{sub 2} (hydrogenolysis) is a ubiquitous step in many catalytic processes, but its mechanism is not well understood. In an effort to study this process directly, the mechanism of hydrogenolysis of the title compound (1) has been investigated. Hydrogenolysis of 1 takes place under mild conditions, and leads to two moles of methane and ( {eta}{sup 5}-cyclopentadienylbis- (triphenylphosphine)cobalt(I) (2). The reaction is autocatalytic; the rate is inhibited by added phosphine and accelerated by the addition of product 2 to the initial reaction mixture. A mechanism is proposed which postulates that 2 reacts withmore » dihydrogen to give ({eta}{sup 5}-cyclopentadienyl)- (triphenylphosphine)dihydridocobalt(III) (5) as a transient species, and methane is formed by reaction of 5 with 1. Dihydride 5 is unknown and could not be prepared to test this hypothesis directly; however, it was found possible to generate solutions of the corresponding pentamethylcyclopentadienyl dihydride 7 by hydrogenolysis of 6, the pentamethylcyclopentadienyl analog of 1. As predicted, 7 and 6 react readily to give methane, and the kinetics of this reaction are consistent with the mechanism postulated for the hydrogenolysis of 1.« less