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 (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...

NDM Metallo-β-Lactamases and Their Bacterial Producers in Health Care Settings

“…unclear. Variants containing V88L also have other substitutions, and a recent study, using the natural promoter of bla NDM-1 for cloning, failed to report any difference in carbapenem MICs between strains producing NDM-5 or NDM-17 (V88L-containing variants) and a strain producing NDM-1 (30). A variant exists which contains solely the V88L substitution, NDM-24, and the true ability of this substitution to enhance activity will be known when the phenotypic characteristics of this variant are reported.…”

Investigation of Dipicolinic Acid Isosteres for the Inhibition of Metallo‐β‐Lactamases

“…[28] The spectrum of CoCo-NDM-1 shows an intense peak between 320-350 nm, which has been assigned to ac ysteine-to-Co II ligand-to-metal charget ransfer (LMCT) band,a nd multiple peaks between 500-650nm, which have been assignedt oC o II ligand field bands (Figure4). [22,47] The inclusion of captopril to the sample resultsi nl arge changes in the ligand field transitions, and an increase in the LMCT band.…”

“…[4,14,21] Some NDM variants exhibit an increasei nt hermal stability, Zn II affinity,a nd mononuclear Zn II activity that may provide an evolutionary advantage when Zn II availability is low. [22][23][24] Unfortunately,e ven with the rapid spread of the bla NDM gene and the evolution of NDM variants,l ittle advancement has been made in inhibitor development. There are currently % 525 inhibitors reported in literature( representative structures shown in Figure 1), [25] but many inhibitors share similars tructuralf eatures and none have progressed to clinicalt rials.…”

Capillary electrophoresis‐based enzyme assays for β‐lactamase enzymes

“…For example, a K m value of 65 ± 5 µM has been reported for penicillinase using PG as substrate in a photometric assay . In case of NDM‐1, K m = 110 ± 23 µM or 102 ± 9 µM were determined using ampicillin as substrate, while values of 49 µM or 70 µM were reported for VIM‐2 and PG as substrate.…”