Substrate recognition by metallo-beta-lactamases: Selectivity, promiscuity and evolution

ALEJANDRO J, VILA

Sevilla-España

Simposio; International Symposium on Molecular Recognition Phenomena in Biopolymers; 2007

Real Academia Sevillana de Ciencias

    Beta-lactam-based drugs represent more than 50% of the antibiotics in the market nowadays. The increasing use of these drugs in the clinical setting places high selective pressure that favors selection of pathogenic microorganisms through the development of different resistance mechanisms. Among them, the expression of b-lactamases is largely prevalent. In the last decade, the dissemination of genes coding for metallo-b-lactamases (MBL´s) has become an emergent clinical problem. MBL´s are zinc-dependent enzymes, that hydrolyze almost all b-lactam based drugs. There are no clinically useful inhibitors for MBL´s yet. In addition, their catalytic mechanism has not been fully dissected.      Sequence analysis of MBLs reveals a large structural diversity, that gives rise to the presence of mono- and dinuclear metal sites. MBLs have been recently subdivided into classes B1, B2 and B3, each of them displaying different zinc ligands and coordination geometries. The broad substrate spectrum displayed by these enzymes is in line with the lack of a defined recognition patch in the enzyme active ite.        We have studied the B1 MBL from B.cereus (BcII) and analyzed the minimal structural requirements for activity and substrate recognition in these enzymes. By using fluorescence spectroscopy with rapid mixing techniques, we have been able to determine that the two Zn(II) ions provide main anchoring points for a minimal ligand recognition, giving rise to a large substrate promiscuity. We have engineered the metal site of this lactamase in order to alter this behavior.        Directed evolution on BcII by DNA shuffling resulted in a expanded substrate spectrum of this enzyme, without sacrificing its stability nor the hydrolytic efficiency towards classical substrates of BcII. The mutations that give rise to these effects parallel others naturally found in MBL´s from pathogenic bacteria, and are related to the second-shell ligands of the second zinc ion, expected to play a supramolecular control of reactivity. Interestingly, we have shown that in this case, evolution does not operate on substrate recognition, but on the rate-determining step of the catalytic mechanism.