Structural Insights for Core Scaffold and Substrate Specificity of B1, B2, and B3 Metallo-β-Lactamases

Metallo-β-lactamases (MBLs) hydrolyze almost all β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems; however, no effective inhibitors are currently clinically available. MBLs are classified into three subclasses: B1, B2, and B3. Although the amino acid sequences of MBLs are varied, their overall scaffold is well conserved. In this study, we systematically studied the primary sequences and crystal structures of all subclasses of MBLs, especially the core scaffold, the zinc-coordinating residues in the active site, and the substrate-binding pocket. We presented the conserved structural features of MBLs in the same subclass and the characteristics of MBLs of each subclass. The catalytic zinc ions are bound with four loops from the two central β-sheets in the conserved αβ/βα sandwich fold of MBLs. The three external loops cover the zinc site(s) from the outside and simultaneously form a substrate-binding pocket. In the overall structure, B1 and B2 MBLs are more closely related to each other than they are to B3 MBLs. However, B1 and B3 MBLs have two zinc ions in the active site, while B2 MBLs have one. The substrate-binding pocket is different among all three subclasses, which is especially important for substrate specificity and drug resistance. Thus far, various classes of β-lactam antibiotics have been developed to have modified ring structures and substituted R groups. Currently available structures of β-lactam-bound MBLs show that the binding of β-lactams is well conserved according to the overall chemical structure in the substrate-binding pocket. Besides β-lactam substrates, B1 and cross-class MBL inhibitors also have distinguished differences in the chemical structure, which fit well to the substrate-binding pocket of MBLs within their inhibitory spectrum. The systematic structural comparison among B1, B2, and B3 MBLs provides in-depth insight into their substrate specificity, which will be useful for developing a clinical inhibitor targeting MBLs.

Inhibition of the urea-urease reaction by the components of the zeolite imidazole frameworks-8 and the formation of urease-zinc-imidazole hybrid compound

In the past decade, much effort has been devoted to using chemical clock-type reactions in material design and driving the self-assembly of various building blocks. Urea-urease enzymatic reaction has chemical pH clock behavior in an unbuffered medium, in which the induction time and the final pH can be programmed by the concentrations of the reagents. The urea-urease reaction can offer a new alternative in material synthesis, where the pH and its course in time are crucial factors in the synthesis. However, before using it in any synthesis method, it is important to investigate the possible effects of the reagents on the enzymatic reaction. Here we investigate the effect of the reagents of the zeolite imidazole framework-8 (zinc ions and 2-methylimidazole) on the urea-urease reaction. We have chosen the zeolite imidazole framework-8 because its formation serves as a model reaction for the formation of other metal–organic frameworks. We found that, besides the inhibition effect of the zinc ions which is well-known in the literature, 2-methylimidazole inhibits the enzymatic reaction as well. In addition to the observed inhibition effect, we report the formation of a hybrid urease-zinc-2-methylimidazole hybrid material. To support the inhibition effect, we developed a kinetic model which reproduced qualitatively the experimentally observed kinetic curves.

Influence of aeration rate and method of process activation on the degree of purification of zinc-containing waste water by ferritization

The aeration rate for the degree of purification of highly concentrated galvanic wastewater from zinc and ferrum ions was investigated using various activation methods. It is shown that the intensity of aeration has a significant effect on the quality of wastewater treatment and the characteristics of water treatment sludge. The efficiency of the use of an energy-saving method for activating the ferritization process with the use of electromagnetic pulses for the extraction of zinc ions from wastewater has been confirmed. It was determined that with an increase in the aeration rate to 3.5 dm3/min per 1 dm3 of the reaction mixture and the use of thermal activation of the process, the residual concentration of zinc ions remains within the range of 0.12÷0.2 mg/dm3. In this case, the concentration of ferrum ions decreases to values of 0.08÷0.14 mg/dm3. It was found that at an aeration rate of 2.5 dm3/min and the use of pulsed electromagnetic (EMP) activation, the residual concentrations of heavy metal ions decrease to values of 0.08÷0.16 mg/dm3. Comparison of the results indicates the advisability of using low rates of aeration of the reaction mixture. This, together with the use of resource-saving EMR process activation, allows to achieve a significant reduction in energy costs. The quantitative phase composition of ferritization precipitates was determined, in which the crystalline phases of zinc ferrite Zn2Fe2O4 and magnetite Fe3O4, as well as ferrum oxyhydroxide FeO (OH) and sodium sulfate Na2SO4, prevail. It is found that with an increase in the volumetric aeration rate, the proportion of the ferrite phase increases. At an aeration rate of 2.0 dm3/min, more than 85 % of the zinc ferrite phase was found in the sediments. Taking into account the qualitative and quantitative composition of precipitates, it is recommended to use them in the production of building materials. The experimental results obtained make it possible to provide a comprehensive processing of liquid galvanic waste.

Hepatocyte-Specific Co-Delivery of Zinc Ions and Plasmid DNA by Lactosylated Poly(1-vinylimidazole) for Suppression of Insulin Receptor Internalization

The lactosylated poly(1-vinylimidazole) (PVIm-Lac) with various lactosylated degrees has been synthesized for the co-delivery of zinc ions (Zn) and plasmid DNA (pDNA). The Zn/DNA/PVIm-Lac complex formation has achieved the specific delivery of zinc ions to HepG2 cells. Especially, the resulting hepatocyte-specific delivery of zinc ions has increased the number of insulin receptors on the cell surface. Consequently, the Zn/DNA/PVIm-Lac complexes have suppressed insulin receptor internalization on the surface of the HepG2 cells, expecting to offer unique therapy to inhibit hepatic insulin clearance.

Mechanism of Zn2+ and Ca2+ Binding to Human S100A1

S100A1 is a member of the S100 family of small ubiquitous Ca2+-binding proteins, which participates in the regulation of cell differentiation, motility, and survival. It exists as homo- or heterodimers. S100A1 has also been shown to bind Zn2+, but the molecular mechanisms of this binding are not yet known. In this work, using ESI-MS and ITC, we demonstrate that S100A1 can coordinate 4 zinc ions per monomer, with two high affinity (KD~4 and 770 nm) and two low affinity sites. Using competitive binding experiments between Ca2+ and Zn2+ and QM/MM molecular modeling we conclude that Zn2+ high affinity sites are located in the EF-hand motifs of S100A1. In addition, two lower affinity sites can bind Zn2+ even when the EF-hands are saturated by Ca2+, resulting in a 2Ca2+:S100A1:2Zn2+ conformer. Finally, we show that, in contrast to calcium, an excess of Zn2+ produces a destabilizing effect on S100A1 structure and leads to its aggregation. We also determined a higher affinity to Ca2+ (KD~0.16 and 24 μm) than was previously reported for S100A1, which would allow this protein to function as a Ca2+/Zn2+-sensor both inside and outside cells, participating in diverse signaling pathways under normal and pathological conditions.

Silymarin Dehydroflavonolignans Chelate Zinc and Partially Inhibit Alcohol Dehydrogenase

Silymarin is known for its hepatoprotective effects. Although there is solid evidence for its protective effects against Amanita phalloides intoxication, only inconclusive data are available for alcoholic liver damage. Since silymarin flavonolignans have metal-chelating activity, we hypothesized that silymarin may influence alcoholic liver damage by inhibiting zinc-containing alcohol dehydrogenase (ADH). Therefore, we tested the zinc-chelating activity of pure silymarin flavonolignans and their effect on yeast and equine ADH. The most active compounds were also tested on bovine glutamate dehydrogenase, an enzyme blocked by zinc ions. Of the six flavonolignans tested, only 2,3-dehydroderivatives (2,3-dehydrosilybin and 2,3-dehydrosilychristin) significantly chelated zinc ions. Their effect on yeast ADH was modest but stronger than that of the clinically used ADH inhibitor fomepizole. In contrast, fomepizole strongly blocked mammalian (equine) ADH. 2,3-Dehydrosilybin at low micromolar concentrations also partially inhibited this enzyme. These results were confirmed by in silico docking of active dehydroflavonolignans with equine ADH. Glutamate dehydrogenase activity was decreased by zinc ions in a concentration-dependent manner, and this inhibition was abolished by a standard zinc chelating agent. In contrast, 2,3-dehydroflavonolignans blocked the enzyme both in the absence and presence of zinc ions. Therefore, 2,3-dehydrosilybin might have a biologically relevant inhibitory effect on ADH and glutamate dehydrogenase.