IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Catalytic Mechanism and Evolutionary Traits of zinc β-lactamases: Does it take two to tango?
Autor/es:
VILA, A J
Lugar:
Frankfurt
Reunión:
Conferencia; 3rd European Conference on Chemistry for Life Sciences; 2009
Institución organizadora:
EuCheMS
Resumen:
b-lactamases represent the prevalent
resistance mechanism to b-lactam antibiotics. 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. The exponential growth of MBL sequences being
characterized has revealed an initially unforeseen structural diversity, that
gives rise to the presence of mono- and dinuclear metal sites. MBL´s have been
recently subdivided into classes B1, B2 and B3, each of them displaying
different zinc ligands and coordination geometries (1).
We have studied the structural features of MBL´s from
different subclasses with the aim of finding common structural and catalytic
features. By means of mutagenesis, functional and structural studies, we
conclude that a Zn site, previously regarded as non essential for catalysis,
plays a major role in substrate binding and catalysis (2-5).
Non-steady state kinetic studies, aided by time-resolved
electronic, EPR and Resonance Raman spectroscopy have allowed us to trap a key
intermediate in β-lactam hydrolysis, and to assess the role of each metal
binding site in the mechanism and stabilization of this intermediate (6-8).
Finally, directed evolution was used as an evolutionary
engineering tool to explore the effect of challenging MBLs towards different
antibiotics. BcII (the MBL from B.cereus)
has been considered as a precursor of more efficient MBLs present in
pathogenic bacteria. In vitro
evolution experiments on BcII by DNA shuffling with a cephalosporin substrate
resulted in a expanded substrate spectrum of this enzyme, without sacrificing
its stability nor the hydrolytic efficiency towards classical substrates of
BcII (9,10). 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 zinc ions, expected to play a supramolecular
control of reactivity. These results suggest that evolution of the chemical
landscape can be predicted by means of this approach.