Biochemical characterization of New Delhi metallo-β-lactamase variants reveals differences in protein stability

Objectives: Metallo-b-lactamase (MBL)-based resistance is a threat to the use of most b-lactam antibiotics. Multiple variants of the New Delhi MBL (NDM) have recently been reported. Previous reports indicate that the substitutions affect NDM activity despite being located outside the active site. This study compares the biochemical properties of seven clinically reported NDM variants. Methods:NDMvariantsweregeneratedbysite-directedmutagenesis;recombinantproteinswerepurifiedtonear homogeneity. Thermal stability and secondary structures of the variants were investigated using differential scanning fluorimetry and circular dichroism; kinetic parameters and MIC values were investigated for representative carbapenem, cephalosporin and penicillin substrates. Results: The substitutions did not affect the overall folds of the NDM variants, within limits of detection; however, differences in thermal stabilities were observed. NDM-8 was the most stable variant with a melting temperature of 728C compared with 608C for NDM-1. In contrast to some previous studies, kcat/KM values were similar for carbapenem and penicillin substrates for NDM variants, but differences in kinetics were observed for cephalosporin substrates. Apparent substrate inhibition was observed with nitrocefin for variants containing the M154L substitution. In all cases, cefoxitin and ceftazidime were poorly hydrolysed with kcat/KM values , 1s 21 mM 21 . Conclusions:TheseresultsdonotdefinemajordifferencesinthecatalyticefficienciesofthestudiedNDMvariants and carbapenem or penicillin substrates. Differences in the kinetics of cephalosporin hydrolysis were observed. The results do reveal that the clinically observed substitutions can make substantial differences in thermodynamic stability, suggesting that this may be a factor in MBL evolution.