Overcoming differences: The catalytic mechanism of metallo‐β‐lactamases

Abstract

Metallo-β-lactamases are the latest resistance mechanism of pathogenic and opportunistic bacteria against carbapenems, considered as last resort drugs. The worldwide spread of genes coding for these enzymes, together with the lack of a clinically useful inhibitor, have raised a sign of alarm. Inhibitor design has been mostly impeded by the structural diversity of these enzymes. Here we provide a critical review of mechanistic studies of the three known subclasses of metallo- β-lactamases, analyzed at the light of structural and mutagenesis investigations. We propose that these enzymes present a modular structure in their active sites that can be dissected into two halves: one providing the attacking nucleophile, and the second one stabilizing a negatively charged reaction intermediate. These are common mechanistic elements in all metallo-β-lactamases. Nucleophile activation does not necessarily requires a Zn(II) ion, but a Zn(II) center is essential for stabilization of the anionic intermediate. Design of a common inhibitor could be therefore approached based in these convergent mechanistic features despite the structural differences.

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

“…A conservative mutation of this Cys residue to a Ser (29) preserves the enzyme activity, supporting this hypothesis. The role of the Zn1 site in MBLs is to facilitate deprotonation of the bound water to provide an active nucleophile (1). This function cannot be properly fulfilled by the Zn1 ion in B2 enzymes, resulting in enzyme inhibition (16), but instead is fully preserved in GOB enzymes.…”

“…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.…”

“…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).…”

“…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).…”

“…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).…”