Metallo-β-lactamases (MβLs) are the main mechanism of bacterial resistance against last generation β-lactam antibiotics such as carbapenems. Most MβLs display unusual structural features in their active sites, such as binuclear zinc centers without carboxylate bridging ligands, and/or a Cys ligand in a catalytic zinc site. Cys221 is an essential residue for catalysis conserved in B1 and B2 lactamases, while most B3 enzymes present a Ser in this position. GOB lactamases stand as an exception within this picture, with a Met residue in position 221. Then, we obtained a series of GOB-18 point mutants in order to analyze the role of this unusual Met221 residue. We found that Met221 is essential for the protein stability, most likely due to its involvement in a hydrophobic core. In contrast to other known MβLs, residue 221 is not involved in metal binding or in catalysis in GOB enzymes, according to spectroscopic and kinetic studies. Our findings show that the essential catalytic features are maintained despite the structural heterogeneity among MβLs, and suggest that a strategy to design general inhibitors should be undertaken based on mechanistic rather than on structural information.