IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Dissecting The Evolutionary Traits In Metallo-b-Lactamase Mediated Catalysis
Autor/es:
PABLO E. TOMATIS, FABIO SIMONA, STELLA M. FABIANE, PAOLO CARLONI AND ALEJANDRO J. VILA
Lugar:
Rosario, Argentina
Reunión:
Congreso; SAB XXXV Annual Meeting; 2006
Resumen:
Metallo-beta-lactamases (MBLs) represent the latest generation of lactamases. The structural diversity and broad substrate profile of MBLs allows them to confer resistance to most lactam antibiotics. In order to explore the evolutionary potential of these rather incipient enzymes, we recently reported a directed molecular evolution study with the Bacillus cereus MBL (BcII) [1]. Using this method, we selected several clones displaying increased hydrolytic efficiency towards cephalexin. One clone (named m5 hereafter) containing the four most frequent mutation (N70S, V112A, L250S and G262S) was chosen for a more detailed analysis. A systematic study of the hydrolytic profile, substrate binding and active site features of the evolved lactamase revealed that directed evolution has shaped the active site by means of remote mutations to better hydrolyze cephalosporins with small C-3 substituents, without loosing the broad subtrate profile. Two single mutants (N70S and G262S) and the double mutant N70S/G262S were studied. The G262S mutation significantly increases the enzymatic activities towars nitrocefin and cephalexin. In contrast, the rates of  benzylpenicillin, imipenem and cefotaxime hydrolysis did not show substantial changes compared with the ones of the WT enzyme. Instead, the catalytic efficiency of the single mutant N70S is compromised for all the substrates respect to the WT enzyme. Interestingly, the catalytic efficiency of the double mutant N70S/G262S was enhanced for all the substrates respect to BcII WT. The crystal structure of mutant m5 revealed a hydrogen-bond between OH@Ser262 and S@Cys221, a metal ligand. This new interaction was also revealed by electronic spectra of Co(II)-substituted enzymes harboring the G262S mutation. MD simulations with mutants confirmed the stability of this H-bond. MD on the free enzymes and adducts with cephalexin showed that the conformational changes induced by the G262S mutation are compensated with a higher degree of mobility on the substrate binding pocket when  Ser70 is present. Pre steady state kinetic studies on G262S have shown that, unlike the observed for BcII WT, the hydrolysis of cromophoric susbtrate nitrocefin proceeds through an anionic intermediate similar that the one observed on CcrA and L1 MBLs [2]. This results indicated that evolution has taken place by stabilization of an intermediate, thus acting  on the rate limiting step.References: [1] Pablo E. Tomatis, Rodolfo M. Rasia, Lorenzo Segovia and Alejandro J. Vila, PNAS, 2005, 102, 13761 - 13766. [2] Zhigang Wang, Walter Fast and Stephen Benkovic, JACS, 1998, 120, 10788-10789.