Copper Ions and Coordination Complexes as Novel Carbapenem Adjuvants.

Carbapenem-resistant Enterobacteriaceae are urgent threats to global human health. These organisms produce ß-lactamases with carbapenemase activity, such as the metallo-ß-lactamase NDM-1, which is notable due to its association with mobile genetic elements and the lack of a clinically useful inhibitor. Here we examined the ability of copper to inhibit the activity of NDM-1 and explored the potential of a copper coordination complex as a mechanism to efficiently deliver copper as an adjuvant in clinical therapeutics. An NDM-positive Escherichia coli isolate, MS6192, was cultured from the urine of a patient with a urinary tract infection. MS6192 was resistant to antibiotics from multiple classes, including diverse ß-lactams (penicillins, cephalosporins, and carbapenems), aminoglycosides, and fluoroquinolones. In the presence of copper (range, 0 to 2 mM), however, the susceptibility of MS6192 to the carbapenems ertapenem and meropenem increased markedly. In standard checkerboard assays, copper decreased the MICs of ertapenem and meropenem against MS6192 in a dose-dependent manner, suggesting a synergistic mode of action. To examine the inhibitory effect of copper in the absence of other ß-lactamases, the blaNDM-1 gene from MS6192 was cloned and expressed in a recombinant E. coli K-12 strain. Analysis of cell extracts prepared from this strain revealed that copper directly inhibited NDM-1 activity, which was confirmed using purified recombinant NDM-1. Finally, delivery of copper at a low concentration of 10 µM by using the FDA-approved coordination complex copper-pyrithione sensitized MS6192 to ertapenem and meropenem in a synergistic manner. Overall, this work demonstrates the potential use of copper coordination complexes as novel carbapenemase adjuvants.

Intrinsic disorder and metal binding in UreG proteins from Archae hyperthermophiles: GTPase enzymes involved in the activation of Ni(II) dependent urease.

Urease is a Ni(II) enzyme present in every domain of life, in charge for nitrogen recycling through urea hydrolysis. Its activity requires the presence of two Ni(II) ions in the active site. These are delivered by the concerted action of four accessory proteins, named UreD, UreF, UreG and UreE. This process requires protein flexibility at different levels and some disorder-to-order transition events that coordinate the mechanism of protein-protein interaction. In particular, UreG, the GTPase in charge of nucleotide hydrolysis required for urease activation, presents a significant degree of intrinsic disorder, existing as a conformational ensemble featuring characteristics that recall a molten globule. Here, the folding properties of UreG were explored in Archaea hyperthermophiles, known to generally feature significantly low level of structural disorder in their proteome. UreG proteins from Methanocaldococcus jannaschii (Mj) and Metallosphaera sedula (Ms) were structurally and functionally analyzed by integrating circular dichroism, NMR, light scattering and enzymatic assays. Metal-binding properties were studied using isothermal titration calorimetry. The results indicate that, as the mesophilic counterparts, both proteins contain a significant amount of secondary structure but maintain a flexible fold and a low GTPase activity. As opposed to other UreGs, secondary structure is lost at high temperatures (68 and 75 °C, respectively) with an apparent two-state mechanism. Both proteins bind Zn(II) and Ni(II), with affinities two orders of magnitude higher for Zn(II) than for Ni(II). No major modifications of the average conformational ensemble are observed, but binding of Zn(II) yields a more compact dimeric form in MsUreG.

ß-Lactam antibiotic-degrading enzymes from non-pathogenic marine organisms: a potential threat to human health.

Metallo-ß-lactamases (MBLs) are a family of Zn(II)-dependent enzymes that inactivate most of the commonly used ß-lactam antibiotics. They have emerged as a major threat to global healthcare. Recently, we identified two novel MBL-like proteins, Maynooth IMipenemase-1 (MIM-1) and Maynooth IMipenemase-2 (MIM-2), in the marine organisms Novosphingobium pentaromativorans and Simiduia agarivorans, respectively. Here, we demonstrate that MIM-1 and MIM-2 have catalytic activities comparable to those of known MBLs, but from the pH dependence of their catalytic parameters it is evident that both enzymes differ with respect to their mechanisms, with MIM-1 preferring alkaline and MIM-2 acidic conditions. Both enzymes require Zn(II) but activity can also be reconstituted with other metal ions including Co(II), Mn(II), Cu(II) and Ca(II). Importantly, the substrate preference of MIM-1 and MIM-2 appears to be influenced by their metal ion composition. Since neither N. pentaromativorans nor S. agarivorans are human pathogens, the precise biological role(s) of MIM-1 and MIM-2 remains to be established. However, due to the similarity of at least some of their in vitro functional properties to those of known MBLs, MIM-1 and MIM-2 may provide essential structural insight that may guide the design of as of yet elusive clinically useful MBL inhibitors.

Identification and characterization of an unusual metallo-ß-lactamase from Serratia proteamaculans.

Metallo-ß-lactamases (MBLs) are a family of metalloenzymes that are capable of hydrolyzing ß-lactam antibiotics and are an important means by which bacterial pathogens use to inactivate antibiotics. A database search of the available amino acid sequences from Serratia proteamaculans indicates the presence of an unusual MBL. A full length amino acid sequence alignment indicates overall homology to B3-type MBLs, but also suggests considerable variations in the active site, notably among residues that are relevant to metal ion binding. Steady-state kinetic measurements further indicate functional differences and identify two relevant pK a values for catalysis (3.8 for the enzyme-substrate complex and 7.8 for the free enzyme) and a preference for penams with modest reactivity towards some cephalosporins. An analysis of the metal ion content indicates the presence of only one zinc ion per active site in the resting enzyme. In contrast, kinetic data suggest that the enzyme may operate as a binuclear enzyme, and it is thus proposed that a catalytically active di-Zn(2+) center is formed only once the substrate is present.

Structure and mechanism of potent bifunctional ß-lactam- and homoserine lactone-degrading enzymes from marine microorganisms.

Genes that confer antibiotic resistance can rapidly be disseminated from one microorganism to another by mobile genetic elements, thus transferring resistance to previously susceptible bacterial strains. The misuse of antibiotics in health care and agriculture has provided a powerful evolutionary pressure to accelerate the spread of resistance genes, including those encoding ß-lactamases. These are enzymes that are highly efficient in inactivating most of the commonly used ß-lactam antibiotics. However, genes that confer antibiotic resistance are not only associated with pathogenic microorganisms, but are also found in non-pathogenic (i.e. environmental) microorganisms. Two recent examples are metal-dependent ß-lactamases (MBLs) from the marine organisms Novosphingobium pentaromativorans and Simiduia agarivorans. Previous studies have demonstrated that their ß-lactamase activity is comparable to those of well-known MBLs from pathogenic sources (e.g. NDM-1, AIM-1) but that they also possess efficient lactonase activity, an activity associated with quorum sensing. Here, we probed the structure and mechanism of these two enzymes using crystallographic, spectroscopic and fast kinetics techniques. Despite highly conserved active sites both enzymes demonstrate significant variations in their reaction mechanisms, highlighting both the extraordinary ability of MBLs to adapt to changing environmental conditions and the rather promiscuous acceptance of diverse substrates by these enzymes.

Promiscuous metallo-ß-lactamases: MIM-1 and MIM-2 may play an essential role in quorum sensing networks.

MIM-1 and MIM-2 are two recently identified metallo-ß-lactamases (MBLs) from Novosphingobium pentaromativorans and Simiduia agarivorans, respectively. Since these organisms are non-pathogenic we speculated that the biological role(s) of MIM-1 and MIM-2 may not be related to their MBL activity. Although both sequence comparison and homology modeling indicate that these proteins are homologous to well-known MBLs such as AIM-1, the sequence analysis also indicated that MIM-1 and MIM-2 share similarities with N-acyl homoserine lactonases (AHLases) and glyoxalase II (GLX-II). Steady-state kinetic assays using a series of lactone substrates confirm that MIM-1 and MIM-2 are efficient lactonases, with catalytic efficiencies resembling those of well-known AHLases. Interestingly, unlike their MBL activity the AHLase activity of MIM-1 and MIM-2 is not dependent on the metal ion composition with Zn(II), Co(II), Cu(II), Mn(II) and Ca(II) all being able to reconstitute catalytic activity (with Co(II) being the most efficient). However, these enzymes do not turn over S-lactoylglutathione, a substrate characteristic for GLX-II activity. Since lactonase activity is linked to the process of quorum sensing the bifunctional activity of "non-pathogenic" MBLs such as MIM-1 and MIM-2 may provide insight into one possible evolutionary pathway for the emergence of antibiotic resistance.

Catalytic mechanisms of metallohydrolases containing two metal ions.

At least one-third of enzymes contain metal ions as cofactors necessary for a diverse range of catalytic activities. In the case of polymetallic enzymes (i.e., two or more metal ions involved in catalysis), the presence of two (or more) closely spaced metal ions gives an additional advantage in terms of (i) charge delocalisation, (ii) smaller activation barriers, (iii) the ability to bind larger substrates, (iv) enhanced electrostatic activation of substrates, and (v) decreased transition-state energies. Among this group of proteins, enzymes that catalyze the hydrolysis of ester and amide bonds form a very prominent family, the metallohydrolases. These enzymes are involved in a multitude of biological functions, and an increasing number of them gain attention for translational research in medicine and biotechnology. Their functional versatility and catalytic proficiency are largely due to the presence of metal ions in their active sites. In this chapter, we thus discuss and compare the reaction mechanisms of several closely related enzymes with a view to highlighting the functional diversity bestowed upon them by their metal ion cofactors.