Characterization of Purified New Delhi Metallo-β-lactamase-1

Abstract

New Delhi metallo-β-lactmase-1 (NDM-1) has recently emerged as a global threat because of its ability to confer resistance to almost all clinically used β-lactam antibiotics, its presence within an easily transmissible plasmid bearing a number of other antibiotic resistance determinants, its carriage in a variety of enterobacteria, and its presence in both nosocomial and community-acquired infections. To improve our understanding of the molecular basis of this threat, NDM-1 was purified and characterized. Recombinant NDM-1 bearing its native leader sequence was expressed in Escherichia coli BL21 cells. The major processed form found to be released into culture media contains a 35-residue truncation at the N-terminus. This form of NDM-1 is monomeric and can be purified with 1.8 or 1.0 equiv of zinc ion, depending on the experimental conditions. Treatment of dizinc NDM-1 with EDTA results in complete removal of both zinc ions, but the relatively weaker chelator PAR chelates only 1 equiv of zinc ion from folded protein but 1.9 equiv of zinc ion from denatured protein, indicating different affinities for each metal binding site. UV-vis spectroscopy of the dicobalt metalloform along with molecular dynamics simulations of the dizinc metallo form indicates that the dinuclear metal cluster at the active site of NDM-1 is similar in structure to other class B1 metallo-β-lactamases. Supplementation of excess zinc ions to monozinc NDM-1 has differential effects on enzyme activity with respect to three different classes of β-lactam substrates tested, penems, cephems, and carbapenems, and likely reflects dissimilar contributions of the second equivalent of metal ion to the catalysis of the hydrolysis of these substrates. Fits to these concentration dependencies are used to approximate the K(d) value of the more weakly bound zinc ion (2 μM). NDM-1 achieved maximal activity with all substrates tested when supplemented with approximately 10 μM ZnSO(4), displaying k(cat)/K(M) values ranging from 1.4 × 10(6) to 2.0 × 10(7) M(-1) s(-1), and a slight preference for cephem substrates. This work provides a foundation for an improved understanding of the molecular basis of NDM-1-mediated antibiotic resistance and should allow more quantitative studies to develop targeted therapeutics.

Molecular Mechanisms of Substrate Recognition and Specificity of New Delhi Metallo-β-Lactamase

“…Furthermore, the kinetic assay indicated that NDM-1 showed approximately 2-fold increase of hydrolytic activity on ampicillin and meropenem in the presence of 50 M ZnSO 4 compared to the condition in the absence of zinc in the assay buffer (data not shown). Our kinetic constants determined under our assay condition supplemented with 50 M ZnSO 4 were similar to those of other reports (25), suggesting that under our assay condition, the NDM-1 protein was likely in its dizinc form.…”

“…S2C in the supplemental material). It is widely accepted that a hydroxide/water between Zn1 and Zn2 is ready for the nucleophilic attack in a binding geometry where the carbonyl oxygen of the ␤-lactam ring associates with Zn1, while one of the carboxylate oxygens of the fused ring coordinates with Zn2 (14,17,25,26). The approach of molecular docking following the suggested binding mode of ␤-lactam moieties to two Zn ions enabled us to model the promising structures of NDM-1 with different cephalosporins.…”

Evolution of New Delhi metallo-β-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity

“…The final assay solution used to monitor ampicillin hydrolysis (as described above) contained 0.3 mL of 50 mM Hepes, pH 7, 0.6 mM ampicillin, 20 nM NDM variant, with addition of ZnSO4 to the final concentrations of 0.0001, 0.01, 0.1, 0.5, 1, 5, 10, 50, and 100 µM. Observed rates were converted into percentages, using the highest value for each variant as 100 %, as described earlier (5,16). Since most NDM variants showed increasing activity followed by a loss of activity as zinc concentrations were increased, data were fitted using the equation below, with [Zinc] being the concentration of ZnSO4, x being the activity (%) at no added zinc, y being the Kd,Zn2, and z being Kd for binding an inhibitory zinc at high concentrations.…”

Kinetic Study of Laboratory Mutants of NDM-1 Metallo-β-Lactamase and the Importance of an Isoleucine at Position 35

“…It is commonly suggested that the zinc ion acts as a Lewis acid to stabilize the transient tetrahedral intermediate formed by the nucleophilic attachment of a hydroxide ion to the carboxyl group of the ␤-lactam ring (10). NDM-1 enzyme shows a great ability to hydrolyze all ␤-lactam antibiotics (11). Several crystal structures of NDM-1 have been solved, and the enzyme displays the typical ␣␤/␤␣ fold of MBLs (12,13,14).…”