Infections by carbapenem-resistant Enterobacteriaceae (CRE) 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 dinuclear 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 comparing melting temperature and refolding efficiency and also by spectroscopy (UV-Vis, 1 H-NMR, and EPR) of di-cobalt metalloforms. The impact of NDM variants on function is probed by determining of minimum inhibitory concentrations of various antibiotics, pre-steady state and steadystate kinetics, inhibitor binding, and zincdependence of resistance and activity. We observed only minor differences among the fullloaded dizinc enzymes, but most NDM variants had more distinguishable selective advantages in experiments that mimicked zinc scarcity imposed by typical host defenses. Most NDM variants http://www.jbc.org/ Downloaded from 2 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 monozinc enzymes with high catalytic efficiency (NDM-15, ampicillin: kcat/KM = 5 × 10 6 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.Carbapenem-resistant Enterobacteriaceae (CRE) continue to be classified as an "urgent threat," the highest hazard level assigned by the Centers for Disease Control and Prevention(1). The five carbapenemases currently of primary public concern include Klebsiella pneumonia carbapanemase (KPC), New Delhi metallo-β-lactamase (NDM), Verona integrin encoded metallo-β-lactamase (VIM), imipenemase (IMP), and oxacillinase-48-like carbapenemase (OXA-48)(2). Three of these carbapenemases (NDM, VIM, and IMP) are metal-dependent β-lactamases that are not susceptible to any of the β-lactamase inhibitors incorporated into combination drugs used in the clinic. Of these three β-lactamases, NDM is the most widespread in U.S. patients, with infections bearing a blaNDM gene reported in 34 / 50 states (as of December 2017)(3).The genes encoding NDM continue to evolve, with discovery of more than 20 variants (NDM-1 through NDM-21 at the time of writing, 16 at the start of this project). Most of these mutations occur at sites distant from the active site, and the functions they confer are not immediately obvious. A comparison of NDM-1 through NDM-8 showed only minor differences in kcat/KM values (≤ 5-fold) for a panel of diverse β-lactam drugs(4). However, a considerable increase in thermostability was noted for many of the variants, suggesting the functional impact of NDM...

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.

Peak assignments of the Fourier-transform infrared spectra and solution NMR spectra of the synthesized SMADs and representative TEM images of SMADLPs made from POPC liposomes (PDF)

No abstract

No abstract