[Source: Antimicrobial Agents and Chemotherapy, full page: (LINK). Abstract, edited.]
Y229W substitution in NDM-1/L209F variant restores the hydrolytic activity of the enzyme towards penicillins, cephalosporins and carbapenems: kinetic profile and molecular dynamic studies
Alessandra Piccirilli, Fabrizia Brisdelli, Massimiliano Aschi, Giuseppe Celenza, Gianfranco Amicosante, Mariagrazia Perilli
NDM-1 enzyme is the most common metallo-β-lactamase identified in many Gram-negative bacteria causing severe nosocomial infections. The aim of this study was to focus the attention on non-active site residues, L209 and Y229, of NDM-1 and to investigate their role in the catalytic mechanism. Specifically, the effect of Y229W substitution in L209F variant was evaluated by antimicrobial susceptibility testing, kinetic and molecular dynamic (MD) studies. The Y229W single mutant and L209F/Y229W double mutant were generated by site-directed mutagenesis. The Km, kcat and kcat/Km kinetic constants, calculated for the two mutants, were compared with those of NDM-1 and L209F variants. Compared to L209F single mutant, L209F/Y229W mutant showed a remarkable increase of kcat values of 100-, 240-, 250- and 420-fold for imipenem, meropenem, benzylpenicillin and cefepime, respectively. In L209F/Y229W enzyme we observed a remarkable increase of kcat/Km of 370-, 140- and 80-fold for cefepime, meropenem and cefazolin, respectively. The same behavior was stated by antimicrobial susceptibility test. MD simulations were carried out on both L209F and L209F/Y229W enzymes complexed with benzylpenicillin focusing the attention on the overall mechanical features and on the differences between the two systems. With respect to L209F variant, the L209F/Y229W double mutant showed a mechanical stabilization of Loop 10 and N-terminal region but a destabilization of C-terminal and 149-154 regions. The epistatic effect of Y229W mutation jointly with stabilization of Loop 10 lead to a better catalytic efficiency of β-lactams. NDM numbering is used in order to facilitate the comparison with other NDM-1 studies.
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Keywords: Antibiotics; Drugs Resistance; Carbapenem; Beta-lactams; NDM1; Imipenem; Meropenem; Penicillins.