Evolution of Metallo-β-lactamases: Trends Revealed by Natural Diversity and in vitro Evolution

Meini, María Rocío; Llarrull, Leticia I.; Vila, Alejandro J.

doi:10.3390/antibiotics3030285

Cited by 74 publications

(57 citation statements)

References 111 publications

(214 reference statements)

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“…An earlier study by Fast, Wang, and Benkovic (16) showed that a single mutation, C109R (C121R, according to the standard MBL numbering scheme [17]) in the MBL CcrA has a significant effect on the accumulation of nitrocefin intermediate and alters the rate-limiting step from the protonation to formation of I by ␤-lactam amide bond cleavage, which also results in altered k cat and K m under steady-state conditions. Thus, mutations of second-shell residues, such as 70, 121, 235, and 262, as they occur in the course of MBL evolution (18,19) certainly have a role in fine-tuning both the overall activity and catalytic pathway of these enzymes. As new ␤-lactams and MBL inhibitors, including mechanism-based inhibitors, are being developed, such subtle changes in the catalytic mechanism deserve our attention.…”

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confidence: 99%

Meropenem and Chromacef Intermediates Observed in IMP-25 Metallo-β-Lactamase-Catalyzed Hydrolysis

Oelschlaeger

Aitha

Yang

et al. 2015

Antimicrob Agents Chemother

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Metallo-␤-lactamases (MBLs) efficiently inactivate most ␤-lactam antibacterials and have recently raised concerns due to this broad substrate spectrum, their global spread in various Gram-negative bacteria, and the absence of inhibitors for clinical use (1-3). Zn(II)-bound anionic intermediates of chromogenic ␤-lactams, such as nitrocefin, have been observed during their hydrolysis catalyzed by the MBLs CcrA (4), L1 (5), NDM-1 (6), and VIM-2 (7). For imipenemase (IMP-1), there might be a nitrocefin intermediate (8), although other studies negate this (9, 10). Tioni and coworkers also observed imipenem and meropenem intermediates in BcII (11). Recently, we found that the variant IMP-25 hydrolyzes meropenem more efficiently than IMP-1 and IMP-6 (12). IMP-6 was previously reported to confer resistance to carbapenems, especially meropenem (13). The increasing k cat for meropenem hydrolysis in the order IMP-1 ¡ IMP-6 ¡ IMP-25 (22 Ϯ 1 s Ϫ1 ¡ 60 Ϯ 10 s Ϫ1 ¡ 100 Ϯ 10 s Ϫ1 , respectively) also translates into increasing MICs of meropenem (16 g/ml ¡ 64 g/ml ¡ 128 g/ml, respectively) (12).To study in more detail the basis of the increased k cat in IMP-25 versus IMP-1, we carried out pre-steady-state kinetic experiments (6) for the hydrolysis of meropenem [6°C, 25 M enzyme (E) and substrate (S) ([E]-to-[S] ratio of 1:1) in 50 mM MOPS (morpholinepropanesulfonic acid) (pH 7.0) supplemented with 100 M Zn(II) ions]. The meropenem substrate (S) was tracked at 310 nm (due to noise at 300 nm), product (P) was tracked at 342 nm, and intermediate (I) was tracked at 390 nm. Details on the determination of extinction coefficients and conversion of the spectral data into concentrations are provided in the supplemental material. With IMP-1, a very small amount of intermediate was observed (Fig. 1E, black line), and the data could not be fitted to a two-phase association/decay function. Substrate disappeared at approximately the same exponential rate (25 Ϯ 3 s Ϫ1 ) at which product appeared (38 Ϯ 12 s Ϫ1 ) (Table 1). In contrast, with IMP-25 (Fig. 1A and B and Table 1), an initial appearance of I at 300 Ϯ 100 s Ϫ1 was followed by its disappearance at 24 Ϯ 8 s Ϫ1 . As with IMP-1, the exponential disappearance rate of S (27 Ϯ 1 s Ϫ1 ) was similar to the appearance rate of P (30 Ϯ 20 s Ϫ1 ). Thus, the observed rates of S disappearance and P appearance are indistinguishable between IMP-1 and IMP-25 under these conditions, and the uncertainty of the product formation rates was rather high. However, a clear formation and decay of intermediate was observed in IMP-25. Intermediate accumulated to no more than 6% of the initial [S], which equals [E], meaning that only up to 6% of the E molecules were bound to I.In order to explore if a clearer picture could be obtained at conditions that are more similar to steady-state conditions, we increased [S] to 100 M, 200 M, and 500 M. Several complications became apparent for the accurate quantification of S and P and, consequently, their disappearance and appearance rates, respectively, at higher concentr...

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confidence: 99%

Meropenem and Chromacef Intermediates Observed in IMP-25 Metallo-β-Lactamase-Catalyzed Hydrolysis

Oelschlaeger

Aitha

Yang

et al. 2015

Antimicrob Agents Chemother

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“…These enzymes catalyze the hydrolysis of the amide bond in the ␤-lactam ring characteristic of this family of drugs (1)(2)(3)(4)(5). MBLs are metal-dependent hydrolases which generally use Zn(II) as a Lewis acid to activate a water molecule for the nucleophilic attack.…”

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confidence: 99%

“…Crystal structures of MBLs from the three subclasses have revealed that these enzymes present a common ␣␤/␤␣ sandwich fold, with the active site located within a groove at the interface between these two halves (1-6). The Zn(II)-binding residues vary among different subclasses, giving rise to diverse metal site architectures and metal contents required for activity (1)(2)(3)(4)(5)(6). B1 and B3 MBLs are broad-spectrum enzymes that hydrolyze penicillins, cephalosporins, and carbapenems with a wide variety of in vitro catalytic efficiencies, displaying a broad range of resistance profiles in vivo (1)(2)(3)(4)(5)8).…”

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confidence: 99%

“…The Zn(II)-binding residues vary among different subclasses, giving rise to diverse metal site architectures and metal contents required for activity (1)(2)(3)(4)(5)(6). B1 and B3 MBLs are broad-spectrum enzymes that hydrolyze penicillins, cephalosporins, and carbapenems with a wide variety of in vitro catalytic efficiencies, displaying a broad range of resistance profiles in vivo (1)(2)(3)(4)(5)8). The di-Zn(II) form of B1 MBLs has been shown to be the active form in the bacterial periplasm, despite contradictory data obtained from in vitro studies (8)(9)(10).…”

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confidence: 99%

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Crystal Structure of the Metallo-β-Lactamase GOB in the Periplasmic Dizinc Form Reveals an Unusual Metal Site

Morán-Barrio

Lisa

Larrieux

et al. 2016

Antimicrob Agents Chemother

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f Metallo-beta-lactamases (MBLs) are broad-spectrum, Zn(II)-dependent lactamases able to confer resistance to virtually every ␤-lactam antibiotic currently available. The large diversity of active-site structures and metal content among MBLs from different sources has limited the design of a pan-MBL inhibitor. GOB-18 is a divergent MBL from subclass B3 that is expressed by the opportunistic Gram-negative pathogen Elizabethkingia meningoseptica. This MBL is atypical, since several residues conserved in B3 enzymes (such as a metal ligand His) are substituted in GOB enzymes. Here, we report the crystal structure of the periplasmic di-Zn(II) form of GOB-18. This enzyme displays a unique active-site structure, with residue Gln116 coordinating the Zn1 ion through its terminal amide moiety, replacing a ubiquitous His residue. This situation contrasts with that of B2 MBLs, where an equivalent His116Asn substitution leads to a di-Zn(II) inactive species. Instead, both the mono-and di-Zn(II) forms of GOB-18 are active against penicillins, cephalosporins, and carbapenems. In silico docking and molecular dynamics simulations indicate that residue Met221 is not involved in substrate binding, in contrast to Ser221, which otherwise is conserved in most B3 enzymes. These distinctive features are conserved in recently reported GOB orthologues in environmental bacteria. These findings provide valuable information for inhibitor design and also posit that GOB enzymes have alternative functions.T he expression of ␤-lactamases is the main mechanism of bacterial resistance against ␤-lactam antibiotics. These enzymes catalyze the hydrolysis of the amide bond in the ␤-lactam ring characteristic of this family of drugs (1-5). MBLs are metal-dependent hydrolases which generally use Zn(II) as a Lewis acid to activate a water molecule for the nucleophilic attack. These enzymes are refractive to clinically employed lactamase inhibitors (1) and have a particular relevance in the clinical setting as they can hydrolyze a broad spectrum of ␤-lactam substrates, being able to inactivate carbapenems, the "last-resort" antibiotics in antibacterial therapy (6).MBLs have been classified into subclasses B1, B2, and B3 based on sequence identity (7). Crystal structures of MBLs from the three subclasses have revealed that these enzymes present a common ␣␤/␤␣ sandwich fold, with the active site located within a groove at the interface between these two halves (1-6). The Zn(II)-binding residues vary among different subclasses, giving rise to diverse metal site architectures and metal contents required for activity (1-6). B1 and B3 MBLs are broad-spectrum enzymes that hydrolyze penicillins, cephalosporins, and carbapenems with a wide variety of in vitro catalytic efficiencies, displaying a broad range of resistance profiles in vivo (1)(2)(3)(4)(5)8). The di-Zn(II) form of B1 MBLs has been shown to be the active form in the bacterial periplasm, despite contradictory data obtained from in vitro studies (8-10). These enzymes display a conserved metal binding m...

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“…The first class B enzyme was isolated from an innocuous strain of Bacillus cereus (37). This protein, known as BcII, is the archetype, the most extensively studied model of enzymes of the largest ubiquitous and clinically relevant B1 subclass, such as VIM-, IMP-, and NDM-type MBLs (all transferable broad spectrum ␤-lactamases) (38). BcII consists of 227 residues in the mature form (M r ϭ 24,960/25,088 in the absence/presence of zinc ions), and its three-dimensional structure ( Fig.…”

Section: ؊1mentioning

confidence: 99%

The Role of Active Site Flexible Loops in Catalysis and of Zinc in Conformational Stability of Bacillus cereus 569/H/9 β-Lactamase

Montagner¹,

Nigen²,

Jacquin³

et al. 2016

Journal of Biological Chemistry

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Metallo-␤-lactamases catalyze the hydrolysis of most ␤-lactam antibiotics and hence represent a major clinical concern. The development of inhibitors for these enzymes is complicated by the diversity and flexibility of their substrate-binding sites, motivating research into their structure and function. In this study, we examined the conformational properties of the Bacillus cereus ␤-lactamase II in the presence of chemical denaturants using a variety of biochemical and biophysical techniques. The apoenzyme was found to unfold cooperatively, with a Gibbs free energy of stabilization (⌬G 0 ) of 32 ؎ 2 kJ⅐mol ؊1. For holoBcII, a first non-cooperative transition leads to multiple interconverting native-like states, in which both zinc atoms remain bound in an apparently unaltered active site, and the protein displays a well organized compact hydrophobic core with structural changes confined to the enzyme surface, but with no catalytic activity. Two-dimensional NMR data revealed that the loss of activity occurs concomitantly with perturbations in two loops that border the enzyme active site. A second cooperative transition, corresponding to global unfolding, is observed at higher denaturant concentrations, with ⌬G 0 value of 65 ؎ 1.4 kJ⅐mol ؊1 .These combined data highlight the importance of the two zinc ions in maintaining structure as well as a relatively well defined conformation for both active site loops to maintain enzymatic activity.␤-Lactamases catalyze hydrolysis of the ␤-lactam ring of antibiotics belonging to the penicillin family (1-3). Synthesis of these enzymes represents the major cause of bacterial resistance to ␤-lactam antibiotics (4 -7). They are classified into two structural superfamilies (8), namely the active site serine enzymes (both ␤-lactamases and penicillin-binding proteins) and the metallo-␤-lactamases (MBLs). 4 The latter, also referred to as class B ␤-lactamases, require one or two zinc ions for activity (9 -11). They show no structural similarity with the active site serine ␤-lactamases, and they are part of a remarkable set of enzymes (i.e. the zinc metallo-hydrolase family of the ␤-lactamase fold or, more simply, the MBL superfamily (10 -16)) that exhibit a wide variety of functions related to hydrolysis and redox reactions and DNA and RNA metabolism. Seventeen groups of enzymes have been identified on the basis of their biological functions (13), which all share a novel ␣␤/␤␣ fold. Most of the three-dimensional structures reveal a binuclear center with metal ligands located on loops connecting secondary structure elements (15, 17).Zinc ␤-lactamases have been found in many bacterial species, including pathogenic strains (18, 19). Most of them are able to hydrolyze almost all ␤-lactam antibiotics (20, 21), including carbapenems (i.e. a family of last resort ␤-lactams that generally escape the activity of the most widespread serine ␤-lactamases), and they are not sensitive to the classical inactivators of serine ␤-lactamases, such as clavulanate, sulbactam, and tazobactam (22,23). Furt...

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Evolution of Metallo-β-lactamases: Trends Revealed by Natural Diversity and in vitro Evolution

Cited by 74 publications

References 111 publications

Meropenem and Chromacef Intermediates Observed in IMP-25 Metallo-β-Lactamase-Catalyzed Hydrolysis

Meropenem and Chromacef Intermediates Observed in IMP-25 Metallo-β-Lactamase-Catalyzed Hydrolysis

Crystal Structure of the Metallo-β-Lactamase GOB in the Periplasmic Dizinc Form Reveals an Unusual Metal Site

The Role of Active Site Flexible Loops in Catalysis and of Zinc in Conformational Stability of Bacillus cereus 569/H/9 β-Lactamase

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