Clinical Variants of New Delhi Metallo-β-Lactamase Are Evolving To Overcome Zinc Scarcity

Abstract

Use and misuse of antibiotics has driven the evolution of serine β-lactamases to better recognize new generations of β-lactam drugs, but the selective pressures driving evolution of metallo-β-lactamases are less clear. Here, we present evidence that New Delhi Metallo-β-lactamase (NDM) is evolving to overcome the selective pressure of zinc(II) scarcity. Studies of NDM-1, NDM-4 (M154L), and NDM-12 (M154L, G222D) demonstrate that the point mutant M154L, contained in 50% of clinical NDM variants, selectively enhances resistance to the penam ampicillin at low zinc(II) concentrations relevant to infection sites. Each of the clinical variants is shown to be progressively more thermostable and to bind zinc(II) more tightly than NDM-1, but a selective enhancement of penam turnover at low zinc(II) concentrations indicates that most of the improvement derives from catalysis rather than stability. X-ray crystallography of NDM-4 and NDM-12, as well as bioinorganic spectroscopy of dizinc(II), zinc(II)/cobalt(II), and dicobalt (II) metalloforms probe the mechanism of enhanced resistance and reveal perturbations of the dinuclear metal cluster that underlie improved catalysis. These studies support the proposal that zinc(II) scarcity, rather than changes in antibiotic structure, is driving the evolution of new NDM variants in clinical settings.

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

“…Resonances at 64, 72, and 79 ppm were previously assigned to solvent-exchangeable protons on His120, His122, and His189 in the Zn1 site, while resonances at 48, 107, and 165 ppm have been assigned, respectively, to protons on Asp124, His250 and Cys208 at the Zn2 site (14). The spectrum of dicobalt (II) NDM-1 shown here ( Figure 8) reproduces what we have reported earlier (5,15). In comparison, all of the variants differ from NDM-1, yet they are similar to one another.…”

“…The change in resistance to ampicillin (a penam) was most impacted by mutations found in the clinic, and resistance to cephems (especially cephalothin) was the least impacted. This rank order is similar to what we reported earlier for the zinc dependence on in vitro activity of the M154L substitution, a mutation that is shared by the majority of the NDM variants (5). So, we next prepared purified NDM proteins and characterized their structure and function in vitro.…”

“…We note that increased zinc affinity and stabilization of folded NDM are likely not unrelated. Zinc binding stabilizes the folded structure of metallo-β-lactamases in general, and NDM in particular (4,5,23). The increased ability of NDM variants to refold in the absence of zinc ions and to maintain their folded structures will increase the concentration of fully-formed zinc binding sites available to better scavenge zinc ions.…”

“…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.…”

“…However, a considerable increase in thermostability was noted for many of the variants, suggesting the functional impact of NDM mutations may be to confer stability. We subsequently found that the selective advantages conferred by the M154L mutation, found in more than half of NDM variants, was only revealed under experimental conditions where zinc availability was limited, mimicking the paucity of free zinc ions at common infection sites (5). Improvements in resistance conferred by enzyme activity correlate with an improved affinity for the more weakly bound zinc ion (Zn2, ligated by D124, C208, H250) in the dinuclear zinc ion cluster required for NDM catalysis.…”