IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
69. Mechanistic clues for the inhibition of zinc beta-lactamases,
Autor/es:
ALEJANDRO J VILA
Lugar:
San Francisco
Reunión:
Conferencia; 52 ICAAC; 2012
Institución organizadora:
American Society For Microbiology
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.2-5,10,11
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.5,6
Finally, directed evolution was used
as an evolutionary engineering tool to explore the effect of challenging MBLs
towards different antibiotics. 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.7,8 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. Moreover, we found that zinc binding is limiting within
the bacterial periplasm to elicit resistance and can be tuned during evolution.9