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.

Keywords: Antibiotic resistance; Drug design; Mechanism; Metallo-β-lactamase; Zinc enzyme.

Figure 1

A) Reaction mechanism for penicillin hydrolysis by class A serine-β-lactamases. This reaction scheme is based in the one reported in reference [14], which reviews original data. B) Reaction mechanism for penicillin hydrolysis by di-Zn(II) B1 enzymes. The reaction scheme is based on the results on penicillin G hydrolysis by di-Co(II)-BcII from reference [22]. The representation of E is taken from the crystallographic structures of di-Zn(II) BcII (PDB 1BC2) and di- Co(II) BcII (PDB 3I11). The representation of EP is based on the crystallographic structures of NDM- 1 complexed with hydrolyzed penicillins (see section 3.1). W1 stands for Wat1 and W2 stands for Wat2.

Figure 2

A) Reaction mechanism for nitrocefin hydrolysis by di-Zn(II) B1 and B3 enzymes. EI is the experimentally characterized anionic intermediate interacting with Zn2 via the N atom and the carboxylate [66,91]. The same scheme would be valid for other chromogenic cephalosporins such as cromaceph and CENTA [92]. B) Reaction mechanism for imipenem hydrolysis by di-Zn(II) B1 and B3 enzymes. EI¹ and EI² are the experimentally characterized anionic intermediates [23], stabilized by interaction with Zn2. The representation of EI² is based on the crystallographic structures of NDM-1 in complex with hydrolyzed meropenem (PDB 4EYL, see Figure 4) [44]. W1 stands for Wat1 and W2 stands for Wat2.

Figure 3. Minimum reaction mechanism for carbapenem hydrolysis by mono-Zn(II)-B2 enzymes

The arrangement of water molecules is based on the crystallographic structures of CphA, free and in complex with hydrolyzed biapenem [47,61], and of the free form of Sfh-I [62]. W1 stands for Wat1 and W2 stands for Wat2.

Figure 4. Active sites of free, EI and EP adducts solved by crystallography

A) Free form of NDM-1 (PDB 3SPU). B) Free form of L1 (PDB 2FM6). C) Free form of CphA (PDB 1X8G). D) NDM-1 in complex with hydrolyzed ampicillin (PDB 3Q6X) E) L1 in complex with hydrolyzed moxolactam (PDB 2AIO). F) CphA in complex with hydrolyzed biapenem (PDB 1X8I). G) NDM-1 in complex with hydrolyzed meropenem (4EYL). H) Reaction mechanism for hydrolysis of cephalosporins with good R2 leaving groups (i.e., cefuroxime) by NDM-1 [79]. I) NDM-1 in complex with a cefuroxime hydrolysis intermediate (4RLO) corresponding to species EI² in panel H. W1 stands for Wat1 and W2 stands for Wat2.