IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Metallo-beta-lactamases: antibiotic resistance and the role of periplasmic Zn(II)
Autor/es:
MARÍA-ROCÍO MEINI; GONZALEZ MARIANO; GONZALEZ, L; PALACIOS, ANTONELA; BARH, GUILLERMO; LLARRULL, L; A. J. VILA
Lugar:
Lanzarote
Reunión:
Conferencia; Zing Conference; Bioinorganic Chemistry Conference; 2013
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