DISSECTING THE EVOLUTIONARY TRAITS IN METALLO-B-LACTAMASE MEDIATED CATALYSIS

TOMATIS, P. E.; SIMONA, F.; FABIANE, S. M.; CARLONI, P; VILA, A. J.

Montevideo, Uruguay

Congreso; 6th International Conference of Biological Physics; 2007

Sociedad Argentina de Biofisica

Metallo-beta-lactamases (MBLs) represent the latest generation of β-lactamases. The structural diversity and broad substrate profile of MBLs allows them to confer resistance to most β-lactam antibiotics. In order to explore the evolutionary potential of these rather incipient enzymes, we have already reported a directed molecular evolution study with the Bacillus cereus MBL (BcII). We selected mutants displaying increased resistance towards a cephalosporin (cephalexin). The clone named m5 (containing only 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. The crystal structure of mutant m5 (solved and refined to 2.8 Å) 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. Molecular dynamic simulations confirmed the stability of this hydrogen-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. The G262S mutation significantly increases the enzymatic activities towars nitrocefin and cephalexin. Instead, the catalytic efficiency of mutant N70S is severely compromised for all the analyzed antibiotics. Interestingly, the catalytic efficiency of the double mutant N70S/G262S was enhanced for all the substrates respect to BcII WT. Pre steady state kinetic studies on G262S have shown that, unlike the observed for BcII WT, the hydrolysis of chromophoric substrate nitrocefin proceeds through an anionic intermediate similar that the one observed on other MBLs. This results indicated that evolution has taken place by stabilization of an intermediate, thus acting on the rate limiting step of the catalytic mechanism.