Metallo‐β‐lactamases and their Synthetic Mimics: Structure, Function, and Catalytic Mechanism

Umayal, Muthaiah; Tamilselvi, A.; Mugesh, Govindasamy

doi:10.1002/9781118148235.ch7

Cited by 6 publications

(11 citation statements)

References 165 publications

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“…The existence of a water molecule bound to Zn2 in the apoenzyme is also debated. 33 Based on the existing studies on NDM-1 and other di-Zn Class B1 MBLs, [33][34][35] the hydrolysis is initialized by the nucleophilic attack (ES → EI) where the bridging hydroxide attacks the β-lactam ring of the drug molecules (see Figure 1). This leads to the intermediate structure EI where the β-lactam N of the ring-opened drug molecule is bound to Zn2.…”

Section: Introductionmentioning

confidence: 99%

Hydrolysis of cephalexin and meropenem by New Delhi metallo-β-lactamase: the substrate protonation mechanism is drug dependent

Das

Nair

2017

Phys. Chem. Chem. Phys.

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Emergence of antibiotic resistance due to New Delhi metallo-β-lactamase (NDM-1) bacterial enzymes is of great concern due to their ability to hydrolyze a wide range of antibiotics. There are ongoing efforts to obtain the atomistic details of the hydrolysis mechanism in order to develop inhibitors for NDM-1. In particular, it remains elusive how drug molecules of different families of antibiotics are hydrolyzed by NDM-1 in an efficient manner. Here we report the detailed molecular mechanism of NDM-1 catalyzed hydrolysis of cephalexin, a cephalosporin family drug, and meropenem, a carbapenem family drug. This study employs molecular dynamics (MD) simulations using hybrid quantum mechanical/molecular mechanical (QM/MM) methods at the density functional theory (DFT) level, based on which reaction pathways and the associated free energies are obtained. We find that the mechanism and the free energy barrier for the ring-opening step are the same for both the drug molecules, while the subsequent protonation step differs. In particular, we observe that the mechanism of the protonation step depends on the R2 group of the drug molecule. Our simulations show that allylic carbon protonation occurs in the case of the cephalexin drug molecule where Lys211 is the proton donor, and the proton transfer occurs via a water chain formed (only) at the ring-opened intermediate structure. Based on the free energy profiles, the overall kinetics of drug hydrolysis is discussed. Finally, we show that the proposed mechanisms and free energy profiles could explain various experimental observations.

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Section: Introductionmentioning

confidence: 99%

Hydrolysis of cephalexin and meropenem by New Delhi metallo-β-lactamase: the substrate protonation mechanism is drug dependent

Das

Nair

2017

Phys. Chem. Chem. Phys.

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“…The structurally diverse dinuclear metallohydrolases include ureases, the purple acid phosphatases (PAPs), phosphotriesterases, exonucleases and metallo-β-lactamases (MβLs), among others. − These metalloenzymes employ a dinuclear metal ion center to catalyze the hydrolysis of amides and esters of carboxylic and phosphoric acids . Interest in these systems arises from their potential as targets for drug design and application in bioremediation. ,,, The metallohydrolases display similarities in the first coordination sphere across the entire family but exhibit variations in metal ion specificity and basic mechanism .…”

Section: Introductionmentioning

confidence: 99%

Cadmium(II) Complexes: Mimics of Organophosphate Pesticide Degrading Enzymes and Metallo-β-lactamases

Daumann

Gahan

Comba³

et al. 2012

Inorg. Chem.

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Cadmium(II) complexes of ethyl 4-hydroxy-3,5-bis(((2-hydroxyethyl)(pyridin-2-ylmethyl)amino)methyl)benzoate (CO(2)EtH(3)L1) and ethyl 4-hydroxy-3,5-bis(((2-methoxyethyl)(pyridin-2-ylmethyl)amino)methyl)benzoate (CO(2)EtHL2) are described. The two ligands possess an ethyl ester (CO(2)Et-) at the position para to the phenolic -OH; CO(2)EtHL2, with methyl ether donors in contrast to potentially nucleophilic alkoxide donors in CO(2)EtH(3)L1, offers a direct comparison of potential ligand-centered nucleophiles. The complex with CO(2)EtH(3)L1 was characterized using (1)H and (13)C NMR spectroscopy, mass spectrometry and microanalysis; X-ray crystallography defined a tetranuclear structure [Cd(4)(CO(2)EtH(2)L1)(2)(CH(3)COO)(3.75)Cl(0.25)(H(2)O)(2)](PF(6))(2). Functional studies of the cadmium(II) complexes were undertaken with the substrates bis(2,4-dinitrophenyl)phosphate (BDNPP), and nitrocefin to assess their phosphatase and β-lactamase activities, respectively. The complexes with CO(2)EtH(3)L1 and CO(2)EtHL2 are competent phosphoesterase mimics with K(M) = 9.4 ± 2.1 mM and 10.1 ± 3.4 mM, k(cat) = 9.4 ± 0.2 × 10(-3) s(-1) and 9.7 ± 2.7 × 10(-3) s(-1), respectively. Use of a solvent mixture containing H(2)(18)O/H(2)(16)O in the reaction with BDNPP showed that for the complex with CO(2)EtH(3)L1 the (18)O label was incorporated in the reaction product suggesting that the nucleophile involved is a Cd-OH moiety and not a metal bound alkoxide; for CO(2)EtHL2 the presence of the methyl-ether dictates that the active nucleophile must also be a hydroxide. The cadmium(II) complex with CO(2)EtH(3)L1 was furthermore found to be a competent β-lactamase mimic with k(cat) = 1.39 × 10(-2) ± 3 × 10(-3) s(-1), K(M) = 0.11 ± 0.03 mM, and pK(a) = 7.9 ± 0.1. Mass spectral evidence suggested that the active nucleophile in this reaction is the alkoxide; lack of β-lactamase activity of the complex with CO(2)EtHL2 supports this assignment. Similar to enzyme-catalyzed reactions, a blue reaction intermediate in the β-lactamase reaction of the CO(2)EtH(3)L1 complex was also identified. It is proposed that the Cd(II) complexes of CO(2)EtH(3)L1 and CO(2)EtHL2 react identically as phosphatases, with a terminal hydroxide as the nucleophile; the former exhibits β-lactamase activity with the alkoxide as a nucleophile, while the latter, without a potentially nucleophilic alkoxide, is inactive.

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“…In recent years preorganized dizinc complexes have been developed as model systems for mβl active sites. Some of them showed catalytic activity in the hydrolysis of β-lactam substrates, which may help to understand the mechanistic pathways of the enzymes. − In addition, some insight into the binding modes of β-lactam substrates at mono- and dizinc sites could be obtained. − ,, β-Lactam antibiotics like benzylpenicillin (pen, a in Chart ) bear several potential donor groups that are able to coordinate to zinc atoms: (i) the carboxylate group, (ii) the lactam amide moiety, (iii) the thioether group, and (iv) the side-chain amide group.…”

Section: Introductionmentioning

confidence: 99%

Binding of β-Lactam Antibiotics to a Bioinspired Dizinc Complex Reminiscent of the Active Site of Metallo-β-lactamases

et al. 2012

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Metallo-β-lactamases (mβls) cause bacterial resistance toward a broad spectrum of β-lactam antibiotics by catalyzing the hydrolytic cleavage of the four-membered β-lactam ring, thus inactivating the drug. Minutiae of the mechanism of these enzymes are still not well understood, and reports about binding studies of the substrates to the enzymes as well as to synthetic model systems are rare. Here we report a new pyrazolate-based bioinspired dizinc complex (1) reminiscent of the active site of binuclear mβls. Since 1 does not mediate hydrolytic degradation of β-lactams, the binding of a series of common β-lactam antibiotics (benzylpenicillin, cephalotin, 6-aminopenicillanic acid, ampicillin) as well as the inhibitor sulbactam and the simplest β-lactam, 2-azetidinone, to the dizinc core of 1 could now be studied in detail by NMR and IR spectroscopy as well as mass spectrometry. X-ray crystallographic information was obtained for 1 and its complexes with 2-azetidinone (2) and sulbactam (3); the latter represents the first structurally characterized dizinc complex with a bound β-lactam drug. While 2-azetidinone was found deprotonated and bridging in the clamp of the two zinc ions in 2, in 3 and all other cases the substrates preferentially bind via their carboxylate group within the bimetallic pocket. The relevance of this binding mode for mβls and consequences for the design of functional model systems are discussed.

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Metallo‐β‐lactamases and their Synthetic Mimics: Structure, Function, and Catalytic Mechanism

Cited by 6 publications

References 165 publications

Hydrolysis of cephalexin and meropenem by New Delhi metallo-β-lactamase: the substrate protonation mechanism is drug dependent

Hydrolysis of cephalexin and meropenem by New Delhi metallo-β-lactamase: the substrate protonation mechanism is drug dependent

Cadmium(II) Complexes: Mimics of Organophosphate Pesticide Degrading Enzymes and Metallo-β-lactamases

Binding of β-Lactam Antibiotics to a Bioinspired Dizinc Complex Reminiscent of the Active Site of Metallo-β-lactamases

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