GES-type beta-lactamases are a group of enzymes that have evolved their hydrolytic activity against carbapenems. In this study, the role of residue 174 inside the Omega-loop of GES-1 and GES-5 was investigated. GES-1(P174E) and GES-5(P174E) mutants, selected by site saturation mutagenesis, were purified and kinetically characterized. In comparison with GES-1 and GES-5 wild-type enzymes, GES-1P174E and GES-5P174E mutants exhibited lower k(cat) and k(cat)/K-m values for cephalosporins and penicillins. Concerning carbapenems, GES-1(P174E) shared higher k(cat) values but lower K-m values than those calculated for GES-1. The GES-1(P174E) and GES-5(P174E) mutants showed high hydrolytic efficiency for imipenem, with k(cat)/K-m values 100- and 660-fold higher, respectively, than those of GES-1. Clavulanic acid and tazobactam are good inhibitors for both GES-1(P174E) and GES-5(P174E). Molecular dynamic (MD) simulations carried out for GES-1, GES-5, GES-1(P174E), and GES-5(P174)E complexed with imipenem and meropenem have shown that mutation at position 174 induces a drastic increase of enzyme flexibility, in particular in the Omega-loop. The circular dichroism (CD) spectroscopy spectra of the four enzymes indicate that the P174E substitution in GES-1 and GES-5 does not affect the secondary structural content of the enzymes.
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