P174E substitution in GES-1 and GES-5 {beta}-lactamases improves catalytic efficiency towards carbapenems [PublishAheadOfPrint]

GES-type β-lactamases are a group of enzymes that have evolved their hydrolytic activity towards carbapenems. In this study the role of residue 174 inside the -loop of GES-1 and GES-5 was investigated. GES-1^P174E and GES-5^P174Emutants, selected by site-saturation mutagenesis, were purified and kinetically characterized. Comparing with GES-1 and GES-5 wild-type enzymes, GES-1^P174E and GES-5^P174E mutants exhibited lower k_cat and k_cat/K_m values for cephalosporins and penicillins. Concerning carbapenems, the two mutants shared higher k_cat values but lower K_m values respect to those calculated for GES-1 and GES-5. The GES-1^P174E and GES-5^P174E showed high hydrolytic efficiency for imipenem with k_cat/K_m values 100- and 660-fold higher than GES-1. Clavulanic acid and tazobactam are good inhibitors for both GES-1^P174E and GES-5^P174E. Molecular dynamic simulations (MDs) carried out for GES-1, GES-5, GES-1^P174E and GES-5^P174E complexed with imipenem and meropenem have shown that mutation at position 174 induces a drastic increase of enzymes flexibility, in particular in the -loop. The CD spectroscopy spectra of the four enzymes indicates that substitution P174E in GES-1 and GES-5 does not affect the secondary structural content of the enzymes.

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