RESUMEN
Layered two-dimensional transition metal dichalcogenides and their heterostructures are of current interest, owing to the diversity of their applications in many areas of materials nanoscience and technologies. With this in mind, we have examined the three molecular dimers of the tungsten dichalcogenide series, (WCh2)2 (Ch = S, Se, Te), using density functional theory to provide insight into which interactions, and their specific characteristics, are responsible for the interfacial/interlayer region in the room temperature 2H phase of WCh2 crystals. Our calculations at various levels of theory suggested that the Te···Te chalcogen bonding in (WTe2)2 is weak, whereas the Se···Se and S···S bonding interactions in (WSe2)2 and (WS2)2, respectively, are of the van der Waals type. The presence and character of Ch···Ch chalcogen bonding interactions in the dimers of (WCh2)2 are examined with a number of theoretical approaches and discussed, including charge-density-based approaches, such as the quantum theory of atoms in molecules, interaction region indicator, independent gradient model, and reduced density gradient non-covalent index approaches. The charge-density-based topological features are shown to be concordant with the results that originate from the extrema of potential on the electrostatic surfaces of WCh2 monomers. A natural bond orbital analysis has enabled us to suggest a number of weak hyperconjugative charge transfer interactions between the interacting monomers that are responsible for the geometry of the (WCh2)2 dimers at equilibrium. In addition to other features, we demonstrate that there is no so-called van der Waals gap between the monolayers in two-dimensional layered transition metal tungsten dichalcogenides, which are gapless, and that the (WCh2)2 dimers may be prototypes for a basic understanding of the physical chemistry of the chemical bonding environments associated with the local interfacial/interlayer regions in layered 2H-WCh2 nanoscale systems.
Asunto(s)
Calcógenos/química , Compuestos de Tungsteno/química , Tungsteno/química , Calcógenos/metabolismo , Enlace de Hidrógeno , Modelos Moleculares , Teoría Cuántica , Electricidad Estática , Elementos de Transición , Compuestos de Tungsteno/metabolismoRESUMEN
We report on the synthesis of the tetrasubstituted sandwich-type Keggin silicotungstates as the pure Na salts Na14[(A-α-SiW10O37)2{Co4(OH)2(H2O)2}]·37H2O (Na{SiW10Co2}2) and Na14[(A-α-SiW10O37)2{Ni4(OH)2(H2O)2}]·77.5H2O (Na{SiW10Ni2}2), which were prepared by applying a new synthesis protocol and characterized thoroughly in the solid state by single-crystal and powder X-ray diffraction, IR spectroscopy, thermogravimetric analysis, and elemental analysis. Proteinase K was applied as a model protein and the polyoxotungstate (POT)-protein interactions of Na{SiW10Co2}2 and Na{SiW10Ni2}2 were studied side by side with the literature-known K5Na3[A-α-SiW9O34(OH)3{Co4(OAc)3}]·28.5H2O ({SiW9Co4}) featuring the same number of transition metals. Testing the solution behavior of applied POTs under the crystallization conditions (sodium acetate buffer, pH 5.5) by time-dependent UV/vis spectroscopy and electrospray ionization mass spectrometry speciation studies revealed an initial dissociation of the sandwich POTs to the disubstituted Keggin anions HxNa5-x[SiW10Co2O38]3- and HxNa5-x[SiW10Ni2O38]3- ({SiW10M2}, M = CoII and NiII) followed by partial rearrangement to the monosubstituted compounds (α-{SiW11Co} and α-{SiW11Ni}) after 1 week of aging. The protein crystal structure analysis revealed monosubstituted α-Keggin POTs in two conserved binding positions for all three investigated compounds, with one of these positions featuring a covalent attachment of the POT anion to an aspartate carboxylate. Despite the presence of both mono- and disubstituted anions in a crystallization mixture, proteinase K selectively binds to monosubstituted anions because of their preferred charge density for POT-protein interaction.
Asunto(s)
Cobalto/química , Complejos de Coordinación/química , Endopeptidasa K/química , Silicio/química , Elementos de Transición/química , Compuestos de Tungsteno/química , Cobalto/metabolismo , Complejos de Coordinación/metabolismo , Cristalografía por Rayos X , Endopeptidasa K/metabolismo , Hypocreales/enzimología , Modelos Moleculares , Estructura Molecular , Silicio/metabolismo , Elementos de Transición/metabolismo , Compuestos de Tungsteno/metabolismoRESUMEN
The biological enzyme-mimetic activity of cerium oxide nanoparticles (CeNPs) is well known to scavenge the reactive oxygen and nitrogen species in cell culture and animal models, imparting protection from the deleterious effects of oxidative and nitrosative stress. The superoxide dismutase (SOD)- and catalase-mimicking activity of CeNPs is reported to be controlled by the oxidation state of the surface "Ce" ions, where a high ratio of Ce3+/4+ or Ce4+/3+ has been considered for the displayed SOD and catalase-like activity, respectively. However, the redox behavior of CeNPs can be controlled by certain ligands that could offer changes in their enzyme-mimetic properties. Therefore, in this work, we have studied the enzyme-mimetic activities of CeNPs under the influence of polyoxometalates [phosphomolybdic acid (PMA) and phosphotungstic acid (PTA)], which are electron-dense molecules displaying quick and reversible multielectron redox reactions. Results revealed that the interaction of PMA with CeNPs results in the inhibition of the SOD-like activity; however, it has no impact on the catalase-like activity. Contrary to this, the interaction of PTA with CeNPs improved the SOD as well as catalase-like activities of CeNPs (3+), which generally do not exhibit catalase activity in the bare form. Although CeNPs (3+) did not show any peroxidase-like activity, CeNPs (4+) showed excellent activity, which was enhanced after the interaction with polyoxometalates. Further, the autoregeneration ability of CeNPs was found to be intact even after PTA or PMA interaction; however, the full catalytic activity was observed in the case of PTA but partially with PMA.
Asunto(s)
Cerio/metabolismo , Nanopartículas/metabolismo , Superóxido Dismutasa/metabolismo , Compuestos de Tungsteno/metabolismo , Células Cultivadas , Cerio/química , Humanos , Nanopartículas/química , Tamaño de la Partícula , Especies Reactivas de Oxígeno/análisis , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/química , Propiedades de Superficie , Compuestos de Tungsteno/químicaRESUMEN
This Viewpoint brings awareness of the challenges and subsequent breakthroughs at the intersection of different disciplines, illustrated by the example of the influence biological entities exerted on a huge class of inorganic coordination compounds, called polyoxometalates (POMs). We highlight the possible effects of biological systems on POMs that need to be considered, thereby emphasizing the depth and complexity of interdisciplinary work. We map POMs' structural, electrochemical, and stability properties in the presence of biomolecules and stress the potential challenges related to inorganic coordination chemistry carried out in biological systems. This Viewpoint shows that new chemistry is available at the intersections between disciplines and aims to guide the community toward a discussion about current as well as future trends in truly interdisciplinary work.
Asunto(s)
Citocromos c/química , NAD/química , Compuestos de Tungsteno/química , Ubiquinona/química , Citocromos c/metabolismo , NAD/metabolismo , Compuestos de Tungsteno/metabolismo , Ubiquinona/metabolismoRESUMEN
The development of artificial proteases is challenging, but important for many applications in modern proteomics and biotechnology. The hydrolysis of hydrophobic or unstructured proteins is particularly difficult due to their poor solubility, which often requires the presence of surfactants. Herein, it is shown that a zirconium(IV)-substituted Keggin polyoxometalate (POM), (Et2 NH2 )10 [Zr(α-PW11 O39 )2 ] (1), is able to selectively hydrolyze ß-casein, which is an intrinsically unstructured protein at pHâ 7.4 and 60 °C. Four surfactants (sodium dodecyl sulfate (SDS), N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (ZW3-12), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and polyethylene glycol tert-octylphenyl ether (TX-100)), which differ in the nature of their polar groups, were investigated for their role in influencing the selectivity and efficiency of protein hydrolysis. Under experimental conditions, ß-casein forms micellar structures in which the hydrophilic part of the protein is water accessible and able to interact with 1. Identical fragmentation patterns of ß-casein in the presence of 1 were observed through SDS poly(acrylamide) gel electrophoresis both in the presence and absence of surfactants, but the rate of hydrolysis varied, depending on the nature of surfactant. Whereas TX-100 surfactant, which has a neutral polar head, caused only a slight decrease in the hydrolysis rate, stronger inhibition was observed in the presence surfactants with charges in their polar heads (CHAPS, ZW3-12, SDS). These results were consistent with those of tryptophan fluorescencequenching studies, which showed that the binding between ß-casein and 1 decreased with increasing repulsion between the POM and the polar heads of the surfactants. In all cases, the micellar structure of ß-casein was not significantly affected by the presence of POM or surfactants, as indicated by circular dichroism spectroscopy.
Asunto(s)
Micelas , Péptido Hidrolasas/metabolismo , Péptidos/química , Compuestos de Tungsteno/química , Compuestos de Tungsteno/metabolismo , Circonio/química , Hidrólisis/efectos de los fármacos , Péptido Hidrolasas/química , Péptidos/metabolismo , Tensoactivos/farmacologíaRESUMEN
Two-dimentional layered WS2 nanosheets with rich active edge exhibit intrinsic peroxidase-mimic activity, which make them an ideal material for sensor design. However, there is still lack of research on the catalysis and regulation mechanisms of the layered WS2 nanosheets as well as their application in the detection of hazardous substances. Herein, the regulatory effect of Pb(II) on the peroxidase-mimic activity of the layered WS2 nanosheets was firstly investigated, which enable us to construct a novel and facile colorimetric sensor for ultrasensitive and selective detection of Pb(II). To improve the performance of colorimetric sensor, some important parameters like buffer conditions, substrates and temperature have been investigated. Under the optimal conditions, the catalytic kinetics of layered WS2 nanosheets were extensively investigated. The peroxidase-mimic catalytic reaction was proved to be the "ping pong" mechanism, and the regulatory effect of Pb(II) on layered WS2 nanosheets was agreed with noncompetitive inhibition. The absorbance variation of colorimetric sensor is proportionally related to the concentration of heavy metals, which enable us to easily distinguish whether Pb(II) exceeds the permissible level in less than 20 min even by the naked eyes. The limit of detection (LOD) and the limit of quantification (LOQ) of the proposed colorimetric sensor for Pb(II) were determined as low as 4 µg L-1 and 13.3 µg L-1, and displays excellent selectivity against other competitive metal ions. Moreover, the further studies also validate the applicability of colorimetric sensor in several actual samples, indicating that our strategy may has prospective applications for Pb(II) detection in environment and biological samples.
Asunto(s)
Colorimetría , Contaminantes Ambientales/análisis , Plomo/análisis , Nanoestructuras/química , Peroxidasa/metabolismo , Sulfuros/metabolismo , Compuestos de Tungsteno/metabolismo , Animales , Biocatálisis , Monitoreo del Ambiente , Contaminantes Ambientales/metabolismo , Peces , Radical Hidroxilo/análisis , Plomo/metabolismo , Tamaño de la Partícula , Peroxidasa/química , Sulfuros/química , Propiedades de Superficie , Compuestos de Tungsteno/químicaRESUMEN
Small- and wide-angle X-ray scattering (SAXS/WAXS/SWAXS) have evolved to be accurate tools used to gain structural information of biomolecules in solution. However, the interpretation of SWAXS data remains challenging owing to the low information content of the data and scattering contributions from the solvent. In recent years, methods for the interpretation of SWAXS data based on explicit-solvent molecular dynamics (MD) simulations have become increasingly popular. The physicochemical information in the MD force fields complements the low-information SWAXS data, thereby greatly reducing the risk of overfitting, and the explicit-solvent models may accurately account for scattering contributions from the solvent. In this chapter, we provide a practical introduction to MD-based methods for the interpretation of SWAXS data. First, we present the back-calculation of a SWAXS curve from an MD trajectory as required to validate an MD simulation against experimental SWAXS data. Second, we present the structure refinement of an atomic model against SWAXS data using SAXS-driven MD simulations. Common technical problems together with appropriate solutions are discussed.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Simulación de Dinámica Molecular , Dispersión del Ángulo Pequeño , Solventes/química , Difracción de Rayos X/métodos , Transportadoras de Casetes de Unión a ATP/metabolismo , Humanos , Compuestos de Tungsteno/metabolismoRESUMEN
Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single nanometer-scale pore. The signal is characteristic of the molecule's physicochemical properties and its interactions with the pore. We demonstrate that the nanopore formed by the bacterial protein exotoxin Staphylococcus aureus alpha hemolysin (αHL) can detect polyoxometalates (POMs, anionic metal oxygen clusters), at the single molecule limit. Moreover, multiple degradation products of 12-phosphotungstic acid POM (PTA, H3PW12O40) in solution are simultaneously measured. The single molecule sensitivity of the nanopore method allows for POMs to be characterized at significantly lower concentrations than required for nuclear magnetic resonance (NMR) spectroscopy. This technique could serve as a new tool for chemists to study the molecular properties of polyoxometalates or other metallic clusters, to better understand POM synthetic processes, and possibly improve their yield. Hypothetically, the location of a given atom, or the rotation of a fragment in the molecule, and the metal oxidation state could be investigated with this method. In addition, this new technique has the advantage of allowing the real-time monitoring of molecules in solution.
Asunto(s)
Nanopartículas del Metal/química , Toxinas Bacterianas/metabolismo , Proteínas Hemolisinas/metabolismo , Nanoporos , Nanotecnología , Ácidos Fosfóricos/metabolismo , Staphylococcus aureus , Compuestos de Tungsteno/análisis , Compuestos de Tungsteno/metabolismoRESUMEN
Tungsten (W) is a valuable element with considerable industrial and economic importance that belongs to the European Union list of critical metals with a high supply risk. Therefore, the development of effective and new methods for W recovery is essential to ensure a sustainable supply. In the present study, the Sulfitobacter dubius W transport system TupABC was explored in order to demonstrate both its functionality in Escherichia coli cells and to construct a bioaccumulator (EcotupW). The complete gene cluster tupBCA or partial gene cluster tupBC were cloned in an expression vector and transformed into E. coli. Metal accumulation was evaluated when each construct strain was grown with three separate metal oxyanions (tungstate, molybdate or chromate). The specificity of the bioaccumulator was determined by competition assays using cells grown with mixed solutions of metal oxyanions (W/Mo and W/Cr). The results showed the relevance of the TupA protein in the TupABC transporter system to W-uptake and also allowed Mo and Cr accumulations, although with amounts 1.7 and 2.9-fold lower than W, respectively. To identify the importance of the valine residue in the accumulation efficiency of the VTTS motif, site-directed mutagenesis of tupA was performed. A mutant with a threonine residue, instead of the respective valine, confirmed that W was internalized by nearly double the amount compared to the native form. The findings indicated that cells carrying the native S. dubius TupABC system were great W-bioaccumulators and could be promising tools for W recovery.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Proteínas Bacterianas/genética , Escherichia coli/genética , Rhodobacteraceae/genética , Tungsteno/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Cromatos/metabolismo , Conservación de los Recursos Naturales , Expresión Génica , Molibdeno/metabolismo , Familia de Multigenes , Mutación , Unión Proteica , Compuestos de Tungsteno/metabolismoRESUMEN
5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), two of the best-studied DNA modifications, play crucial roles in normal development and disease in mammals. Although 5-methylcytidine (m5C) and 5-hydroxymethylcytidine (hm5C) have also been identified in RNA, their distribution and biological function in RNA remain largely unexplored, due to the lack of suitable sequencing methods. Here, we report a base-resolution sequencing method for hm5C in RNA. We applied the selective oxidation of hm5C to trihydroxylated-thymine (thT) mediated by peroxotungstate. thT was subsequently converted to T during cDNA synthesis using a thermostable group II intron reverse transcriptase (TGIRT). Base-resolution analysis of the hm5C sites in RNA was performed using Sanger sequencing. Furthermore, in combination with the TET enzyme oxidation of m5C to hm5C in RNA, we expand the use of peroxotungstate oxidation to detect m5C in RNA at base-resolution. By using this method, we confirmed three known m5C sites in human tRNA, demonstrating the applicability of our method in analyzing real RNA samples.
Asunto(s)
Citidina/análogos & derivados , ARN/genética , ARN/metabolismo , Análisis de Secuencia de ARN/métodos , Compuestos de Tungsteno/química , Compuestos de Tungsteno/metabolismo , Secuencia de Bases , Citidina/metabolismoRESUMEN
Acid-redox bifunctional Dawson-type polyoxometalates K6P2W18O62 (P2W18) and K10P2W17O61 (P2W17) were introduced as the new-type catalysts in oxidative decomposition of lignocellulose. The lignin and hemicellulose ingredients of lignocellulose could be decomposed by P2W17 to produce diisobutyl phthalate with the selectivity of 75.67% and other aromatic and aliphatic compounds under mild conditions, evidently differed from other POMs-catalyzed lignocellulose depolymerization in which aromatic ketones and phenols were the main compounds. Diisobutyl phthalate was obtained from the oxidation of Cα-OR and α-OH of the phenyl structure. The catalyst could be recycled for three times without obvious deactivation. This is the first report of lignocellulose decomposition catalyzed by Dawson-type polyoxometalates to selectively produce phthalates.
Asunto(s)
Biocatálisis , Lignina/metabolismo , Ácidos Ftálicos/metabolismo , Polisacáridos/metabolismo , Compuestos de Tungsteno/metabolismo , Oxidación-Reducción , Estrés OxidativoRESUMEN
Bacterial genes for molybdenum-containing and tungsten-containing enzymes are often differentially regulated depending on the metal availability in the environment. Here, we describe a new family of transcription factors with an unusual DNA-binding domain related to excisionases of bacteriophages. These transcription factors are associated with genes for various molybdate and tungstate-specific transporting systems as well as molybdo/tungsto-enzymes in a wide range of bacterial genomes. We used a combination of computational and experimental techniques to study a member of the TF family, named TaoR (for tungsten-containing aldehyde oxidoreductase regulator). In Desulfovibrio vulgaris Hildenborough, a model bacterium for sulfate reduction studies, TaoR activates expression of aldehyde oxidoreductase aor and represses tungsten-specific ABC-type transporter tupABC genes under tungsten-replete conditions. TaoR binding sites at aor promoter were identified by electrophoretic mobility shift assay and DNase I footprinting. We also reconstructed TaoR regulons in 45 Deltaproteobacteria by comparative genomics approach and predicted target genes for TaoR family members in other Proteobacteria and Firmicutes.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Proteínas Bacterianas/metabolismo , Desulfovibrio vulgaris/genética , Desulfovibrio vulgaris/metabolismo , Molibdeno/metabolismo , Factores de Transcripción/metabolismo , Compuestos de Tungsteno/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/genética , Sitios de Unión , Transporte Biológico , Desulfovibrio vulgaris/aislamiento & purificación , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Familia de Multigenes , Regiones Promotoras Genéticas , Regulón , Factores de Transcripción/genéticaRESUMEN
In this study, a combined molecular docking (rigid and flexible) and all-atom molecular dynamics simulations technique have been employed to investigate interactions of 1:1 Zr-containing Keggin polyoxometalate (ZrK) with four chemically distinct cleavage sites [Arg114-Leu115 (site 1), Ala257-Asp258 (site 2), Lys313-Asp314 (site 3), and Cys392-Glu393 (site 4)] of human serum albumin (HSA). The ZrK-HSA complexations were analyzed using electrostatic potentials, the chemical nature of amino acid residues, binding free energies, and secondary structures as parameters. They suggested that ZrK binds in a rather distinct manner to different cleavage sites, and its association was dominated by hydrogen bonding, both direct and solvent mediated, and electrostatic interactions, as suggested experimentally. The computed binding free interaction energies (-57.5, -24.2, -50.8, and -91.2 kJ/mol for sites 1, 2, 3, and 4, respectively) predicted the existence of one major binding site (site 4) and three minor binding sites (site 1, site 2, and site 3). The strong exothermicity of the binding was also supported by isothermal calorimetry experiments. Additionally, the binding of ZrK did not alter the overall α-helical secondary structure of HSA, which was in line with experimental observation. Furthermore, hydrolysis of the peptide bonds of the substrate was found to retain its overall structure. These results have provided a deeper understanding of the complex ZrK interactions with proteins, and they will lead to the design of the next generation of catalytically active polyoxometalates with improved hydrolytic activities.
Asunto(s)
Albúmina Sérica Humana/química , Compuestos de Tungsteno/química , Sitios de Unión , Calorimetría , Humanos , Enlace de Hidrógeno , Simulación del Acoplamiento Molecular , Unión Proteica , Estructura Secundaria de Proteína , Teoría Cuántica , Albúmina Sérica Humana/metabolismo , Electricidad Estática , Termodinámica , Compuestos de Tungsteno/metabolismoRESUMEN
Heteropolyacids (HPAs) are a class of polyoxometallates (POMs) with oxygen-rich surfaces. Herein, we have developed an Fe-containing heteropolyacid by cation-exchange and employed KFePW12O40 nanostructures for Fenton, photo-Fenton and enzyme-mimetic reactions. The as-prepared KFePW12O40 catalyst exhibits efficient degradation of Rhodamine B (RhB) via the photo-Fenton reaction. As an enzyme-mimetic, this material can effectively oxidize TMB and dopamine. The obtained nanomaterials were characterized via SEM, TEM, XPS, BET surface area, TGA, UV-Vis spectroscopy, FT-IR, and XRD techniques. The photocatalyst has a relatively large surface area of 38 m2 g-1, and the Keggin structure of phosphotungstic ions is kept intact during the preparation. The RhB dye pollutants can be efficiently bleached and degraded up to about 80% within a one hour photo-Fenton reaction under visible light irradiation. Our results indicate that the KFePW12O40 nanomaterial can effectively mimic the enzyme cascade reaction of horseradish peroxidase (HRP). It also has a high affinity toward 3,3',5,5'-tetramethylbenzidine (TMB) for oxidation and henceforth, it has been used for the colorimetric assay of dopamine and H2O2. Overall, our study suggests that KFePW12O40 can be used for the efficient degradation of environmental pollutants. The KFePW12O40 catalyst is stable and can be easily separated from the reaction system for reuse without an obvious loss of activity.
Asunto(s)
Peróxido de Hidrógeno/metabolismo , Hierro/metabolismo , Nanoestructuras/química , Peroxidasa/metabolismo , Compuestos de Tungsteno/metabolismo , Peróxido de Hidrógeno/química , Hierro/química , Peroxidasa/química , Procesos Fotoquímicos , Compuestos de Tungsteno/químicaRESUMEN
AP-2 gamma (AP-2γ) is a transcription factor that plays pivotal roles in breast cancer biology. To search for small molecule inhibitors of AP-2γ, we performed a high-throughput fluorescence anisotropy screen and identified a polyoxometalate compound with Wells-Dawson structure K6[P2Mo18O62] (Dawson-POM) that blocks the DNA-binding activity of AP-2γ. We showed that this blocking activity is due to the direct binding of Dawson-POM to AP-2γ. We also provided evidence to show that Dawson-POM decreases AP-2γ-dependent transcription similar to silencing the gene. Finally, we demonstrated that Dawson-POM contains anti-proliferative and pro-apoptotic effects in breast cancer cells. In summary, we identified the first small molecule inhibitor of AP-2γ and showed Dawson-POM-mediated inhibition of AP-2γ as a potential avenue for cancer therapy.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Factor de Transcripción AP-2/antagonistas & inhibidores , Compuestos de Tungsteno/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/fisiopatología , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Cinética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Factor de Transcripción AP-2/genética , Factor de Transcripción AP-2/metabolismo , Compuestos de Tungsteno/química , Compuestos de Tungsteno/metabolismoRESUMEN
Despite belonging to the phosphoserine- and phosphothreonine-specific phosphoprotein phosphatase (PPP) family, Arabidopsis thaliana Rhizobiales-like phosphatase 2 (RLPH2) strongly prefers substrates bearing phosphorylated tyrosine residues. We solved the structures of RLPH2 crystallized in the presence or absence of sodium tungstate. These structures revealed the presence of a central domain that forms a binding site for two divalent metal ions that closely resembles that of other PPP-family enzymes. Unique structural elements from two flanking domains suggest a mechanism for the selective dephosphorylation of phosphotyrosine residues. Cocrystallization with the phosphate mimetic tungstate also suggests how positively charged residues that are highly conserved in the RLPH2 class form an additional pocket that is specific for a phosphothreonine residue located near the phosphotyrosine residue that is bound to the active site. Site-directed mutagenesis confirmed that this auxiliary recognition element facilitates the recruitment of dual-phosphorylated substrates containing a pTxpY motif.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Fosfotirosina/metabolismo , Tirosina/metabolismo , Secuencia de Aminoácidos , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sitios de Unión/genética , Dominio Catalítico/genética , Cristalografía por Rayos X , Modelos Moleculares , Mutación , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Fosforilación , Fosfotirosina/química , Fosfotirosina/genética , Unión Proteica , Conformación Proteica , Especificidad por Sustrato , Compuestos de Tungsteno/química , Compuestos de Tungsteno/metabolismo , Tirosina/química , Tirosina/genéticaRESUMEN
We present a method, the bacterial mineral excretion (BME), for synthesizing two kinds of microcapsules, sodium tungstate and sodium molybdate, and the two metal oxides' corresponding nanoparticles-the former being as small as 22 nm and the latter 15 nm. We fed two strains of bacteria, Shewanella algae and Pandoraea sp., with various concentrations of tungstate or molybdate ions. The concentrations of tungstate and molybdate were adjusted to make microcapsules of different length-to-diameter ratios. We found that the higher the concentration the smaller the nanoparticles were. The nanoparticles came in with three length-to-diameter ratios: 10:1, 3:1 and 1:1, which were achieved by feeding the bacteria respectively with a low concentration, a medium concentration, and a high concentration. The images of the hollow microcapsules were taken via the scanning electron microsphere (SEM). Their crystal structures were verified by X-ray diffraction (XRD)-the crystal structure of molybdate microcapsules is Na2MoO4 and that of tungstate microcapsules is Na2WO4 with Na2W2O7. These syntheses all were accomplished under a near ambient condition.
Asunto(s)
Burkholderiaceae/metabolismo , Molibdeno/metabolismo , Shewanella/metabolismo , Compuestos de Tungsteno/metabolismo , Cápsulas , Molibdeno/química , Nanopartículas/química , Nanopartículas/metabolismo , Compuestos de Tungsteno/químicaRESUMEN
Considering the increasing incorporation of manufactured nano-material into consumer products, there is a concern about its potential impacts in biological wastewater treatment. In this study, the response of anaerobic sludge to the presence of Bi2WO6 nano-particles (NPs) was investigated in the anaerobic membrane bioreactor (AnMBR). As the concentration of Bi2WO6 in the reactor was controlled around 1 mg/L, there was no significant difference in effluent water quality or bacterial activities before and after NP exposure, partially due to the microbial-induced NP aggregation and stable complex formation. However, with the increasing dosage of Bi2WO6 from 5 to 40 mg/L, great influences on the AnMBR performance were observed, including the reduction of COD removal efficiency, inhibition of the mechanization step, increased production of soluble microbial products, and enhanced secretion of extracellular polymer substrates. Additional investigation with high-throughput sequencing was conducted, clearly demonstrating that Bi2WO6 NPs induced changes in the bacterial community.
Asunto(s)
Bacterias/metabolismo , Bismuto/metabolismo , Nanopartículas del Metal , Aguas del Alcantarillado/microbiología , Compuestos de Tungsteno/metabolismo , Eliminación de Residuos Líquidos , Aguas Residuales/microbiología , Contaminantes Químicos del Agua/metabolismo , Anaerobiosis , Análisis de la Demanda Biológica de Oxígeno , Reactores Biológicos/microbiologíaRESUMEN
Lignin valorization is a pending issue for the integrated conversion of lignocellulose in consumer goods. Lignosulfonates (LS) are the main technical lignins commercialized today. However, their molecular weight should be enlarged to meet application requirements as additives or dispersing agents. Oxidation of lignosulfonates with fungal oxidoreductases, such as laccases, can increase the molecular weight of lignosulfonates by the cross-linking of lignin phenols. To advance in this direction, we describe here the development of a high-throughput screening (HTS) assay for the directed evolution of laccases, with lignosulfonate as substrate and the Folin-Ciocalteau reagent (FCR), to detect the decrease in phenolic content produced upon polymerization of lignosulfonate by the enzyme. Once the reaction conditions were adjusted to the 96-well-plate format, the enzyme for validating the assay was selected from a battery of high-redox-potential laccase variants functionally expressed in S. cerevisiae (the preferred host for the directed evolution of fungal oxidoreductases). The colorimetric response (absorbance at 760 nm) correlated with laccase activity secreted by the yeast. The HTS assay was reproducible (coefficient of variation (CV) = 15%) and sensitive enough to detect subtle differences in activity among yeast clones expressing a laccase mutant library obtained by error-prone PCR (epPCR). The method is therefore feasible for screening thousands of clones during the precise engineering of laccases toward valorization of lignosulfonates.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Lacasa/metabolismo , Lignina/análogos & derivados , Lignina/metabolismo , Fenoles/metabolismo , Ingeniería Genética/métodos , Lacasa/genética , Molibdeno/metabolismo , Oxidación-Reducción , Polimerizacion , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Compuestos de Tungsteno/metabolismoRESUMEN
Molybdenum and tungsten are taken up by bacteria and archaea as their soluble oxyanions through high affinity transport systems belonging to the ATP-binding cassette (ABC) transporters. The component A (ModA/TupA) of these transporters is the first selection gate from which the cell differentiates between MoO42-, WO42- and other similar oxyanions. We report the biochemical characterization and the crystal structure of the apo-TupA from Desulfovibrio desulfuricans G20, at 1.4 Å resolution. Small Angle X-ray Scattering data suggests that the protein adopts a closed and more stable conformation upon ion binding. The role of the arginine 118 in the selectivity of the oxyanion was also investigated and three mutants were constructed: R118K, R118E and R118Q. Isothermal titration calorimetry clearly shows the relevance of this residue for metal discrimination and oxyanion binding. In this sense, the three variants lost the ability to coordinate molybdate and the R118K mutant keeps an extremely high affinity for tungstate. These results contribute to an understanding of the metal-protein interaction, making it a suitable candidate for a recognition element of a biosensor for tungsten detection.