Metallo-?-lactamases: two binding sites for one catalytic metal ion?

Heinz, Uwe; Adolph, Hans-Werner

doi:10.1007/s00018-004-4214-9

Cited by 82 publications

(105 citation statements)

References 121 publications

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“…2, 26,42,43 Similarly, it appears that some MBLs can operate as mononuclear enzymes, while others require two metal ions. 5, 44,45 In contrast, the present study supports a mechanistic model for GpdQ whereby the mononuclear form is inactive and a substrate-and metal-ioninduced fit leads to the formation of a catalytically active binuclear center, providing an intricate regulatory mechanism for enzyme activity ("on-off switch").…”

Section: Resultssupporting

confidence: 72%

“…Furthermore, the catalytic mechanism of GpdQ may also be related to other binuclear metallohydrolases, such as methionine aminopeptidase (MetAP) 2,26,42,43 and metallo--lactamases (MBLs). 5, 44,45 However, MetAP is flexible in that it can operate as either a mononuclear or binuclear hydrolase, with each form having a distinct reaction mechanism. 2, 26,42,43 Similarly, it appears that some MBLs can operate as mononuclear enzymes, while others require two metal ions.…”

Section: Resultsmentioning

confidence: 99%

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Structural Flexibility Enhances the Reactivity of the Bioremediator Glycerophosphodiesterase by Fine-Tuning Its Mechanism of Hydrolysis

et al. 2009

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Abstract:The glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) belongs to the family of binuclear metallohydrolases and has attracted recent attention due to its potential in bioremediation. Formation of a catalytically competent binuclear center is promoted by the substrate (Hadler et al. J. Am. Chem. Soc. 2008, 130, 14129). Using the paramagnetic properties of Mn(II), we estimated the K d values for the metal ions in the R and sites to be 29 and 344 µM, respectively, in the absence of a substrate analogue. In its presence, the affinity of the site increases substantially (K d ) 56 µM), while that of the R site is not greatly affected (K d ) 17 µM). Stopped-flow fluorescence measurements identified three distinct phases in the catalytic turnover, associated with the initial binding of substrate to the active site (k obs1 ), the assembly of a catalytically active binuclear center (k obs2 ), and subsequent slower structural rearrangements to optimize catalysis (k obs3 ). These three phases depend on the concentration of substrate ([S]), with k obs1 and k obs2 reaching maximum values at high [S] (354 and 38 s -1 , respectively), whereas k obs3 is reduced as [S] is increased. The k cat for the hydrolysis of the substrate bis(para-nitrophenyl) phosphate (∼1 s -1 ) gradually increases from the moment of initiating the reaction, reaching a maximum when the structural change associated with k obs3 is complete. This structural change is mediated via an extensive hydrogen-bond network that connects the coordination sphere with the substrate binding pocket, as demonstrated by mutation of two residues in this network (His81 and His217). The identities of both the substrate and the metal ion also affect interactions within this H-bond network, thus leading to some mechanistic variations. Overall, the mechanism employed by GpdQ is a paradigm of a substrate-and metal-ion-induced fit to optimize catalysis.

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Section: Resultssupporting

confidence: 72%

Section: Resultsmentioning

confidence: 99%

Structural Flexibility Enhances the Reactivity of the Bioremediator Glycerophosphodiesterase by Fine-Tuning Its Mechanism of Hydrolysis

et al. 2009

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“…Based on the very tight binding of only one of the metal ions in the active site it was suggested that MBLs may only require one Zn(II) for catalysis under physiological conditions, especially since it is estimated that the concentration of free Zn(II) in cells may be in the picomolar range or lower [12]. However, this inter- pretation raises a serious issue in terms of why all known MBLs have two closely spaced metal ion binding sites in their catalytic centres-as already mentioned in a previous report it appears unlikely that such a structural arrangement would be conserved throughout evolution if there was no functional benefit for the enzyme [110].…”

Section: Interactions Between Mbls and Metal Ionsmentioning

confidence: 99%

Metallo-β-Lactamases: A Major Threat to Human Health

Phelan¹,

Miraula²,

Selleck³

et al. 2014

AJMB

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Antibiotic resistance is one of the most significant challenges facing global healthcare. Since the 1940s, antibiotics have been used to fight infections, initially with penicillin and subsequently with various derivatives including cephalosporins, carbapenams and monobactams. A common characteristic of these antibiotics is the four-membered β-lactam ring. Alarmingly, in recent years an increasing number of bacteria have become resistant to these antibiotics. A major strategy employed by these pathogens is to use Zn(II)-dependent enzymes, the metallo-β-lactamases (MBLs), which hydrolyse the β-lactam ring. Clinically useful MBL inhibitors are not yet available. Consequently, MBLs remain a major threat to human health. In this review biochemical properties of MBLs are discussed, focusing in particular on the interactions between the enzymes and the functionally essential metal ions. The precise role(s) of these metal ions is still debated and may differ between different MBLs. However, since they are required for catalysis, their binding site may present an alternative target for inhibitor design.

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“…Zinc-bound ␤-lactamases appear to be more stable than their corresponding metal-depleted forms (9), and removal of the metal is known to induce conformational changes (40,46,50). No detailed information on the conformational stability of MBLs has, however, been reported to date (for a brief review of the literature, see under "Discussion").…”

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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Metallo-?-lactamases: two binding sites for one catalytic metal ion?

Cited by 82 publications

References 121 publications

Structural Flexibility Enhances the Reactivity of the Bioremediator Glycerophosphodiesterase by Fine-Tuning Its Mechanism of Hydrolysis

Structural Flexibility Enhances the Reactivity of the Bioremediator Glycerophosphodiesterase by Fine-Tuning Its Mechanism of Hydrolysis

Metallo-β-Lactamases: A Major Threat to Human Health

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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