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)

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To understand the evolution of Verona integron-encoded metallo-β-lactamase (VIM) genes (blaVIM) and their clinical impact, microbiological, biochemical, and structural studies were conducted. Forty-five clinically derived VIM variants engineered in a uniform background and expressed in Escherichia coli afforded increased resistance toward all tested antibiotics; the variants belonging to the VIM-1-like and VIM-4-like families exhibited higher MICs toward five out of six antibiotics than did variants belonging to the widely distributed and clinically important VIM-2-like family. Generally, maximal MIC increases were observed when cephalothin and imipenem were tested. Additionally, MIC determinations under conditions with low zinc availability suggested that some VIM variants are also evolving to overcome zinc deprivation. The most profound increase in resistance was observed in VIM-2-like variants (e.g., VIM-20 H229R) at low zinc availability. Biochemical analyses reveal that VIM-2 and VIM-20 exhibited similar metal binding properties and steady-state kinetic parameters under the conditions tested. Crystal structures of VIM-20 in the reduced and oxidized forms at 1.25 Å and 1.37 Å resolution, respectively, show that Arg229 forms an additional salt bridge with Glu171. Differential scanning fluorimetry of purified proteins and immunoblots of periplasmic extracts revealed that this difference increases thermostability and resistance to proteolytic degradation when zinc availability is low. Therefore, zinc scarcity appears to be a selective pressure driving the evolution of multiple metallo-β-lactamase families, although compensating mutations use different mechanisms to enhance resistance. IMPORTANCE Antibiotic resistance is a growing clinical threat. One of the most serious areas of concern is the ability of some bacteria to degrade carbapenems, drugs that are often reserved as last-resort antibiotics. Resistance to carbapenems can be conferred by a large group of related enzymes called metallo-β-lactamases that rely on zinc ions for function and for overall stability. Here, we studied an extensive panel of 45 different metallo-β-lactamases from a subfamily called VIM to discover what changes are emerging as resistance evolves in clinical settings. Enhanced resistance to some antibiotics was observed. We also found that at least one VIM variant developed a new ability to remain more stable under conditions where zinc availability is limited, and we determined the origin of this stability in atomic detail. These results suggest that zinc scarcity helps drive the evolution of this resistance determinant.

Proteotoxicity from insufficient clearance of misfolded/damaged proteins underlies many diseases. Carboxyl terminus of Hsc70-interacting protein (CHIP) is an important regulator of proteostasis in many cells, having E3-ligase and chaperone functions and often directing damaged proteins towards proteasome recycling. While enhancing CHIP functionality has broad therapeutic potential, prior efforts have all relied on genetic upregulation. Here we report that CHIP-mediated protein turnover is markedly post-translationally enhanced by direct protein kinase G (PKG) phosphorylation at S20 (mouse, S19 human). This increases CHIP binding affinity to Hsc70, CHIP protein half-life, and consequent clearance of stress-induced ubiquitinated-insoluble proteins. PKG-mediated CHIP-pS20 or expressing CHIP-S20E (phosphomimetic) reduces ischemic proteo- and cytotoxicity, whereas a phospho-silenced CHIP-S20A amplifies both. In vivo, depressing PKG activity lowers CHIP-S20 phosphorylation and protein, exacerbating proteotoxicity and heart dysfunction after ischemic injury. CHIP-S20E knock-in mice better clear ubiquitinated proteins and are cardio-protected. PKG activation provides post-translational enhancement of protein quality control via CHIP.

This review article highlights recent developments in the field of photochemistry and photochemical reversible deactivation radical polymerization applied to aqueous polymerizations. Photochemistry is a topic of significant interest in the fields of organic, polymer, and materials chemistry because it allows challenging reactions to be performed under mild conditions. Aqueous polymerization is of significant interest because water is an environmentally benign solvent, and the use of water enables complex polymer self-assembly and bioconjugation processes to occur. This review focuses on powerful new developments in photochemical aqueous polymerization reactions and their applications to the synthesis of well-defined polymer nano-objects and bioconjugates. It is anticipated that these aqueous photopolymerizations will enable the next generation of self-assembled structures and biohybrid materials to be developed under mild and environmentally friendly conditions.