Carbapenem-resistant Enterobacteriaceae threaten human health, since carbapenems are last resort drugs for infections by such organisms. Metallo-β-lactamases (MβLs) are the main mechanism of resistance against carbapenems. Clinically approved inhibitors of MBLs are currently unavailable as design has been limited by the incomplete knowledge of their mechanism. Here, we report a biochemical and biophysical study of carbapenem hydrolysis by the B1 enzymes NDM-1 and BcII in the bi-Zn(II) form, the mono-Zn(II) B2 Sfh-I and the mono-Zn(II) B3 GOB-18. These MβLs hydrolyse carbapenems via a similar mechanism, with accumulation of the same anionic intermediates. We characterize the Michaelis complex formed by mono-Zn(II) enzymes, and we identify all intermediate species, enabling us to propose a chemical mechanism for mono and binuclear MβLs. This common mechanism open avenues for rationally designed inhibitors of all MβLs, notwithstanding the profound differences between these enzymes’ active site structure, β-lactam specificity and metal content.

Metallo-β-lactamases (MBLs) are major culprits of resistance to carbapenems in bacteria. A series of thiazolidines are potent MBL inhibitors, restoring the activity of carbapenems. Metal binding and sulphur–π interactions are key to inhibition.

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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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Pseudomonas aeruginosa is one of the most virulent and resistant non-fermenting Gram-negative pathogens in the clinic. Unfortunately, P. aeruginosa has acquired genes encoding metallo-β-lactamases (MβLs), enzymes able to hydrolyze most β-lactam antibiotics. SPM-1 is an MβL produced only by P. aeruginosa, while other MβLs are found in different bacteria. Despite similar active sites, the resistance profile of MβLs towards β-lactams changes from one enzyme to the other. SPM-1 is unique among pathogen-associated MβLs in that it contains “atypical” second sphere residues (S84, G121). Codon randomization on these positions and further selection of resistance-conferring mutants was performed. MICs, periplasmic enzymatic activity, Zn(II) requirements, and protein stability was assessed. Our results indicated that identity of second sphere residues modulates the substrate preferences and the resistance profile of SPM-1 expressed in P. aeruginosa. The second sphere residues found in wild type SPM-1 give rise to a substrate selectivity that is observed only in the periplasmic environment. These residues also allow SPM-1 to confer resistance in P. aeruginosa under Zn(II)-limiting conditions, such as those expected under infection. By optimizing the catalytic efficiency towards β-lactam antibiotics, the enzyme stability and the Zn(II) binding features, molecular evolution meets the specific needs of a pathogenic bacterial host by means of substitutions outside the active site.

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