P174E Substitution in GES-1 and GES-5 beta-Lactamases Improves Catalytic Efficiency toward Carbapenems.

[en] 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-1(P174E) and GES-5(P174E) mutants exhibited lower kcat and kcat/Km values for cephalosporins and penicillins. Concerning carbapenems, GES-1(P174E) shared higher kcat values but lower Km values than those calculated for GES-1. The GES-1(P174E) and GES-5(P174E) mutants showed high hydrolytic efficiency for imipenem, with kcat/Km 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(P174E) 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.

Disciplines :

Biochemistry, biophysics & molecular biology

Author, co-author :

Piccirilli, Alessandra

Mercuri, Paola ; Université de Liège - ULiège > Département des sciences de la vie > Macromolécules biologiques

Galleni, Moreno ; Université de Liège - ULiège > Département des sciences de la vie > Macromolécules biologiques

Aschi, Massimiliano

Matagne, André ; Université de Liège - ULiège > Département des sciences de la vie > Enzymologie et repliement des protéines

Amicosante, Gianfranco

Perilli, Mariagrazia

Language :

English

Title :

P174E Substitution in GES-1 and GES-5 beta-Lactamases Improves Catalytic Efficiency toward Carbapenems.

Publication date :

2018

Journal title :

Antimicrobial Agents and Chemotherapy

ISSN :

0066-4804

eISSN :

1098-6596

Publisher :

American Society for Microbiology, Washington, United States - District of Columbia

Volume :

Issue :

Peer reviewed :

Peer Reviewed verified by ORBi

Commentary :

Available on ORBi :

since 08 July 2018

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