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

Abstract

Infections by carbapenem-resistant Enterobacteriaceae are difficult to manage owing to broad antibiotic resistance profiles and because of the inability of clinically used β-lactamase inhibitors to counter the activity of metallo-β-lactamases often harbored by these pathogens. Of particular importance is New Delhi metallo-β-lactamase (NDM), which requires a di-nuclear zinc ion cluster for catalytic activity. Here, we compare the structures and functions of clinical NDM variants 1-17. The impact of NDM variants on structure is probed by comparing melting temperature and refolding efficiency and also by spectroscopy (UV-visible, ¹H NMR, and EPR) of di-cobalt metalloforms. The impact of NDM variants on function is probed by determining the minimum inhibitory concentrations of various antibiotics, pre-steady-state and steady-state kinetics, inhibitor binding, and zinc dependence of resistance and activity. We observed only minor differences among the fully loaded di-zinc enzymes, but most NDM variants had more distinguishable selective advantages in experiments that mimicked zinc scarcity imposed by typical host defenses. Most NDM variants exhibited improved thermostability (up to ∼10 °C increased T_m ) and improved zinc affinity (up to ∼10-fold decreased K_{d, Zn2}). We also provide first evidence that some NDM variants have evolved the ability to function as mono-zinc enzymes with high catalytic efficiency (NDM-15, ampicillin: k_cat/K_m = 5 × 10⁶ m^-1 s^-1). These findings reveal the molecular mechanisms that NDM variants have evolved to overcome the combined selective pressures of β-lactam antibiotics and zinc deprivation.

Keywords: Co(II)-substituted enzyme; NDM-1; antibiotic resistance; antibiotics; enzyme kinetics; enzyme mutation; metallo-β-lactamase; metalloenzyme; protein evolution.

Figure 1.

Mutated residues in clinically derived NDM variants. Variants containing combinations of between 0 and 3 mutations are shown in relation to each other. NDM-10 (Table S1) is omitted. Solid lines indicate shared mutations. The dashed line indicates a shared site of mutation. NDM variants are colored to summarize their relative resistance in zinc-depleted conditions, as determined herein (Table 2): white for 0 to <2 dilution increases of MIC values in two or more antibiotics; yellow for ≥2 dilution increases in MIC values for two or more antibiotics; orange for ≥3 dilution increases in MIC values for two or more antibiotics; and red for ≥4 dilution increases in MIC values for two or more antibiotics.

Figure 2.

Position of residues mutated in clinically isolated variants of NDM. NDM is shown as a ribbon diagram with zinc atoms as spheres, coated by a transparent surface (all in gray). Hydrolyzed ampicillin is shown as sticks, with a transparent surface (blue). Positions mutated in clinically isolated variants are color-coded by the highest resistance variant in which each occurs (low in yellow, medium in orange, and high in red, see Fig. 1). Figure was prepared using Protein Data Bank code 4HL2 for di-zinc NDM-1 bound to hydrolyzed ampicillin.

Figure 3.

ITC determination of l-captopril/di-zinc NDM-1 dissociation constant. A representative experiment is shown for ITC of NDM-1 and l-captopril, with the thermograms and fits for the other NDM variants included in Fig. S4. Values for K_d of all variants are given in Table S5. Fits above give K_d and stoichiometry for di-zinc NDM1 and l-captopril binding as 4 ± 1 μm and 1.0 ± 0.1, respectively.

Figure 4.

Stopped-flow kinetics of chromacef turnover by di-zinc(II) NDM variants. Using conditions described under “Experimental procedures,” the amount of anionic intermediate that absorbs at 575 nm was monitored. Representative variants NDM-1 (black), NDM-4 (orange), and NDM-15 (red) are shown above, and kinetic traces for the remaining NDM variants are given in Fig. S3.

Figure 5.

Melting temperatures of NDM variants. SYPRO Orange fluorescent dye was mixed with each variant (5 μm) in HEPES buffer (20 mm) with NaCl (150 mm) at pH 7.5. Unfolding in response to heating was monitored by fluorescence at 570 nm.

Figure 6.

Relative refolding efficiencies of apo-NDM variants. Each purified metal-free variant at low (gray bars, 35 μm) or high (black bars, 350 μm) concentrations was refolded by dialysis and normalized by concentration of the variant before refolding. Each bar is the average of duplicate experiments.

Figure 7.

UV-visible spectra of cobalt(II)-loaded NDM variants. The spectrum of NDM-1 (red) is shown in bold for reference. All samples (300 μm) were in HEPES (50 mm), NaCl (150 mm) at pH 6.8.

Figure 8.

¹H NMR spectra (300 MHz) of cobalt(II)-substituted NDM variants.

Figure 9.

EPR spectra of representative di-cobalt(II) NDM variants. Spectra for all the NDM variants are given in supporting information. Perpendicular (A) and parallel (B) CW-EPR spectra of the metalloforms (0.5 mm), as labeled, are shown. Sharp spikes at 1600 G are due to a minor contamination from iron. The arrow in the right panel (800 G) indicates the feature associated with cobalt(II)–cobalt(II) coupling. NDM-1 is shown in black, NDM-4 in orange, and NDM-15 in red.

Figure 10.

Zinc(II) dependence of representative NDM variants for hydrolysis of ampicillin. Rates of ampicillin hydrolysis were determined with ampicillin (750 μm) as described under “Experimental procedures” and normalized to the largest rate for each variant set to 100%. Representative traces are shown for NDM-1 (black circle), NDM-4 (orange circle), and NDM-15 (red circle), but data for all variants tested are given in Table 4 and Fig. S9. As described under “Experimental procedures,” for each variant the transition is fit as a K_d value for Zn2 and a starting value that describes the relative activity under mono-zinc conditions.