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 Tm ) and improved zinc affinity (up to ∼10-fold decreased Kd, 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: kcat/Km = 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.
Highlights
Infections by carbapenem-resistant Enterobacteriaceae are difficult to manage owing to broad antibiotic resistance profiles and because of the inability of clinically used -lactamase inhibitors to counter the activity of metallo--lactamases often harbored by these pathogens
We provide first evidence that some New Delhi metallo--lactamase (NDM) variants have evolved the ability to function as mono-zinc enzymes with high catalytic efficiency (NDM-15, ampicillin: kcat/Km ؍5 ؋ 106 M؊1 s؊1)
We subsequently found that the selective advantages conferred by the M154L mutation, found in more than half of NDM variants, were only revealed under experimental conditions where zinc availability was limited, mimicking the paucity of free zinc ions at common infection sites [4]
Summary
Biochemical, and biophysical characterization of the clinically-derived NDM variants NDM-1 through NDM-17 to determine whether there were any shared characteristics that might reveal which selective pressures are driving the evolution of the corresponding genes. With the exception of NDM-10, these variants contain 0 –3 mutated residues relative to NDM-1 (Fig. 1), often occurring in combinations, notably with the most resistant variants containing the M154L substitution Each of these substitutions occurs at a site that does not directly interact with zinc or -lactam substrates (Fig. 2). The structure of each enzyme was probed by using multiple spectroscopic techniques, denaturation and refolding experiments, and metal content analysis Taken together, these findings prompted additional studies on zinc affinity and the zinc dependence of antibiotic hydrolysis. Characteristics found to be common among the most resistant NDM variants include an increase in thermostability and refolding efficiency, a structurally-perturbed Zn2 site, and increased Zn2 affinity, indicating that protein instability and zinc scarcity may be two selective pressures driving the evolution of blaNDM in the clinic. NDM-1 is only active as a di-zinc protein, but some clinically-derived variants have evolved high catalytic efficiency as mono-zinc enzymes
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
