RESUMEN
The biocatalyzed oxidative detoxification of the V-series simulant PhX, by mean of the microperoxidase AcMP11, affords the corresponding phosphonothioate as the prominent product instead of the classical P-S and P-O bond cleavage. While PhX is structurally very close to the live agent VX (the methyl group is replaced by a phenyl), assessment with other surrogates missing the nucleophilic amino function displayed more resistance under the same conditions with no phosphonothioate observed. These encouraging results highlight 1) the efficacy of AcMP11 microperoxidase to efficiently detoxify V-series organophosphorus nerve agents (OPNA), and 2) the necessity to use representative alkyl or aryl phosphonothioates simulants such as PhX bearing the appropriate side chain as well as the P-O and P-S cleavable bond to mimic accurately the V-series OPNA to prevent false positive or false negative results.
Asunto(s)
Agentes Nerviosos , Compuestos Organotiofosforados , Peroxidasas , Agentes Nerviosos/química , Agentes Nerviosos/metabolismo , Compuestos Organotiofosforados/química , Compuestos Organotiofosforados/metabolismo , Peroxidasas/metabolismo , Peroxidasas/química , Estructura Molecular , Biocatálisis , Oxidación-ReducciónRESUMEN
A binuclear Cu(II) cofactor was covalently bound to a lauric acid anchor. The resulting conjugate was characterized then combined with beta-lactoglobulin (ßLG) to generate a new biohybrid following the so-called "Trojan horse" strategy. This biohybrid was examined for its effectiveness in the oxidation of a catechol derivative to the corresponding quinone. The resulting biohybrid did not exhibit the sought after catecholase activity, likely due to its ability to bind and stabilize the semiquinone radical intermediate DTB-SQ. This semi-quinone radical was stabilized only in the presence of the protein and was characterized using optical and magnetic spectroscopic techniques, demonstrating stability for over 16â hours. Molecular docking studies revealed that this stabilization could occur owing to interactions of the semi-quinone with hydrophobic amino acid residues of ßLG.
RESUMEN
The covalent insertion of a cobalt heme into the cavity of an artificial protein named alpha Rep (αRep) leads to an artificial cobalt hemoprotein that is active as a catalyst not only for the photo-induced production of H2, but also for the reduction of CO2 in a neutral aqueous solution. This new artificial metalloenzyme has been purified and characterized by Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS), circular dichroism, and UltraViolet-Visible spectroscopy. Using theoretical experiments, the structure of this biohybrid and the positioning of the residues near the metal complex were examined, which made it possible to complete the coordination of the cobalt ion by an axial glutamine Gln283 ligand. While the Co(III)-porphyrin catalyst alone showed weak catalytic activity for both reactions, 10 times more H2 and four times more CO2 were produced when the Co(III)-porphyrin complex was buried in the hydrophobic cavity of the protein. This study thus provides a solid basis for further improvement of these biohybrids using well-designed modifications of the second and outer coordination sphere by site-directed mutagenesis of the host protein.
Asunto(s)
Complejos de Coordinación , Hemoproteínas , Porfirinas , Cobalto/química , Dióxido de Carbono/química , Complejos de Coordinación/química , Catálisis , Hidrógeno/químicaRESUMEN
The Diels-Alder (DA) reaction is a cycloaddition of a conjugated diene and an alkene (dienophile) leading to the formation of a cyclohexene derivative through a concerted mechanism. As DA reactions generally proceed with a high degree of regio- and stereoselectivity, they are widely used in synthetic organic chemistry. Considering eco-conscious public and governmental movements, efforts are now directed towards the development of synthetic processes that meet environmental concerns. Artificial enzymes, which can be developed to catalyze abiotic reactions, appear to be important synthetic tools in the synthetic biology field. This review describes the different strategies used to develop protein-based artificial enzymes for DA reactions, including for in cellulo approaches.
Asunto(s)
Ciclohexenos/síntesis química , Albúmina Sérica/química , Animales , Reacción de Cicloadición , Ciclohexenos/química , Humanos , Modelos Moleculares , Estructura Molecular , PorcinosRESUMEN
The selective targeting of protein-protein interactions remains a significant determinant for the proper modulation and regulation of cell apoptosis. Prototypic galectins such as human galectin-7 (GAL-7) are characterized by their ability to form homodimers that control the molecular fate of a cell by mediating subtle yet critical glycan-dependent interactions between pro- and anti-apoptotic molecular partners. Altering the structural architecture of GAL-7 can therefore result in resistance to apoptosis in various human cancer cells, further illustrating its importance in cell survival. In this study, we used a combination of biophysical and cellular assays to illustrate that binding of a water-soluble meso-tetraarylporphyrin molecule to GAL-7 induces protein oligomerization and modulation of GAL-7-induced apoptosis in human Jurkat T cells. Our results suggest that the integrity of the GAL-7 homodimer architecture is essential for its molecular function, in addition to providing an interesting porphyrin binding modulator for controlling apoptosis in mammalian cells.
Asunto(s)
Galectinas/química , Galectinas/metabolismo , Mesoporfirinas/química , Mesoporfirinas/metabolismo , Apoptosis/efectos de los fármacos , Sitios de Unión/efectos de los fármacos , Galectinas/farmacología , Humanos , Técnicas In Vitro , Células Jurkat , Simulación del Acoplamiento Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Estructura Cuaternaria de Proteína/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Dispersión del Ángulo Pequeño , Solubilidad , Difracción de Rayos XRESUMEN
A novel inducible artificial metalloenzyme obtained by covalent attachment of a manganese(III)-tetraphenylporphyrin (MnTPP) to the artificial bidomain repeat protein, (A3A3')Y26C, is reported. The protein is part of the αRep family. The biohybrid was fully characterized by MALDI-ToF mass spectrometry, circular dichroism and UV/Vis spectroscopies. The peroxidase and monooxygenase activities were evaluated on the original and modified scaffolds including those that have a)â an additional imidazole, b)â a specific αRep bA3-2 that is known to induce the opening of the (A3A3') interdomain region and c)â a derivative of the αRep bA3-2 inducer extended with a His6 -Tag (His6 -bA3-2). Catalytic profiles are highly dependent on the presence of co-catalysts with the best activity obtained with His6 -bA3-2. The entire mechanism was rationalized by an integrative molecular modeling study that includes protein-ligand docking and large-scale molecular dynamics. This constitutes the first example of an entirely artificial metalloenzyme with inducible peroxidase and monooxygenase activities, reminiscent of allosteric regulation of natural enzymatic pathways.
Asunto(s)
Oxigenasas de Función Mixta/metabolismo , Catálisis , Metaloproteínas , PeroxidasasRESUMEN
The elusive flavin semiquinone intermediate found in flavoproteins such as cryptochromes has been obtained in aqueous solution by single electron reduction of the natural FMN cofactor using sodium ascorbate. This species was formed in the local hydrophobic microenvironment of a modified polyethyleneimine and characterized by UV-Visible, fluorescence and EPR spectroscopies.
RESUMEN
We report a new artificial hydrogenase made by covalent anchoring of the iron Knölker's complex to a xylanase S212C variant. This artificial metalloenzyme was found to be able to catalyze efficiently the transfer hydrogenation of the benchmark substrate trifluoroacetophenone by sodium formate in water, yielding the corresponding secondary alcohol as a racemic. The reaction proceeded more than threefold faster with the XlnS212CK biohybrid than with the Knölker's complex alone. In addition, efficient conversion of trifluoroacetophenone to its corresponding alcohol was reached within 60 H with XlnS212CK, whereas a ≈2.5-fold lower conversion was observed with Knölker's complex alone as catalyst. Moreover, the data were rationalized with a computational strategy suggesting the key factors of the selectivity. These results suggested that the Knölker's complex was most likely flexible and could experience free rotational reorientation within the active-site pocket of Xln A, allowing it to access the subsite pocket populated by trifluoroacetophenone.
Asunto(s)
Proteínas Bacterianas/química , Endo-1,4-beta Xilanasas/química , Hidrogenasas/química , Proteínas Hierro-Azufre/química , Streptomyces lividans/enzimología , Proteínas Bacterianas/genética , Catálisis , Endo-1,4-beta Xilanasas/genética , Hidrogenasas/genética , Hidrogenación , Proteínas Hierro-Azufre/genética , Streptomyces lividans/genética , AguaRESUMEN
In the context of developing ecofriendly chemistry, artificial enzymes are now considered as promising tools for synthesis. They are prepared in particular with the aim to catalyze reactions that are rarely, if ever, catalyzed by natural enzymes. We discovered that 1-aminocyclopropane carboxylic acid oxidase reconstituted with CuII served as an efficient artificial Diels-Alderase. The kinetic parameters of the catalysis of the cycloaddition of cyclopentadiene and 2-azachalcone were determined (KM =230â µm, kapp =3â h-1 ), which gave access to reaction conditions that provided quantitative yield and >99 % ee of the (1S,2R,3R,4R) product isomer. This unprecedented performance was rationalized by molecular modeling as only one docking pose of 2-azachalcone was possible in the active site of the enzyme and this was the one that leads to the (1S,2R,3R,4R) product isomer.
Asunto(s)
Aminoácido Oxidorreductasas/química , Aminoácido Oxidorreductasas/metabolismo , Cobre/química , Dominio Catalítico , Tecnología Química Verde , Modelos Moleculares , Conformación ProteicaRESUMEN
We demonstrate that the devised incorporation of an alkylamine group into the second coordination sphere of an FeII complex allows to switch its reactivity with H2 O2 from the usual formation of FeIII species towards the selective generation of an FeIV -oxo intermediate. The FeIV -oxo species was characterized by UV/Vis absorption and Mössbauer spectroscopy. Variable-temperature kinetic analyses point towards a mechanism in which the heterolytic cleavage of the O-O bond is triggered by a proton transfer from the proximal to the distal oxygen atom in the FeII -H2 O2 complex with the assistance of the pendant amine. DFT studies reveal that this heterolytic cleavage is actually initiated by an homolytic O-O cleavage immediately followed by a proton-coupled electron transfer (PCET) that leads to the formation of the FeIV -oxo and release of water through a concerted mechanism.
RESUMEN
Artificial metalloenzymes are known to be promising tools for biocatalysis, but their recent compartmentalization has led to compatibly with cell components thus shedding light on possible therapeutic applications. We prepared and characterized artificial metalloenzymes based on the A2A adenosine receptor embedded in the cytoplasmic membranes of living human cells. The wild type receptor was chemically engineered into metalloenzymes by its association with strong antagonists that were covalently bound to copper(II) catalysts. The resulting cells enantioselectively catalyzed the abiotic Diels-Alder cycloaddition reaction of cyclopentadiene and azachalcone. The prospects of this strategy lie in the organ-confined in vivo preparation of receptor-based artificial metalloenzymes for the catalysis of reactions exogenous to the human metabolism. These could be used for the targeted synthesis of either drugs or deficient metabolites and for the activation of prodrugs, leading to therapeutic tools with unforeseen applications.
Asunto(s)
Metaloproteínas/química , Receptor de Adenosina A2A/química , Receptores Artificiales/química , Biocatálisis , Catálisis , Línea Celular , Chalcona/análogos & derivados , Cobre , Reacción de Cicloadición , Ciclopentanos/química , Humanos , Modelos Moleculares , EstereoisomerismoRESUMEN
Direct incorporation of molecular oxygen into small organic molecules has attracted much attention for the development of new environmentally friendly oxidation processes. In line with this approach, bioinspired systems mimicking enzyme activities are of particular interest since they may perform catalysis in aqueous media. Demonstrated herein is the incorporation of a natural flavin cofactor (FMN) into the specific microenvironment of a water-soluble polymer which allows the efficient reduction of the FMN by NADH in aqueous solution. Once reduced, this artificial flavoenzyme can then activate molecular dioxygen under aerobic conditions and result in the Baeyer-Villiger reaction at room temperature in water.
Asunto(s)
Materiales Biomiméticos/química , Mononucleótido de Flavina/química , Oxígeno/química , Polietileneimina/química , Catálisis , Flavoproteínas/química , Modelos Moleculares , NADP/química , Oxidación-Reducción , Solubilidad , Agua/químicaRESUMEN
Microperoxidase-8, a small, peroxidase-type enzyme was immobilized into nanoparticles of the mesoporous and ultra-stable metal-organic framework (MOF) MIL-101(Cr). The immobilized enzyme fully retained its catalytic activity and exhibited enhanced resistance to acidic conditions. The biocatalyst was reusable and showed a long-term stability. By exploiting the properties of the MOF's framework, we demonstrated, for the first time, that the MOF matrix could act in synergy with the enzyme (Microperoxidase-8) and enhance selectivity the oxidation reaction of dyes. The oxidation rate of the harmful negatively charged dye (methyl orange) was significantly increased after enzyme immobilization, probably as a result of the pre-concentration of the methyl orange reactant owing to a charge matching between this dye and the MOF.
Asunto(s)
Colorantes/química , Estructuras Metalorgánicas/química , Peroxidasas/química , Colorantes/metabolismo , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Concentración de Iones de Hidrógeno , Estructuras Metalorgánicas/metabolismo , Modelos Moleculares , Estructura Molecular , Nanopartículas/química , Nanopartículas/metabolismo , Tamaño de la Partícula , Peroxidasas/metabolismo , Porosidad , Propiedades de SuperficieRESUMEN
αRep refers to a new family of artificial proteins based on a thermostable α-helical repeated motif. One of its members, αRep A3, forms a stable homo-dimer with a wide cleft that is able to accommodate metal complexes and thus appears to be suitable for generating new artificial biocatalysts. Based on the crystal structure of αRep A3, two positions (F119 and Y26) were chosen, and independently changed into cysteine residues. A phenanthroline ligand was covalently attached to the unique cysteine residue of each protein variant, and the corresponding biohybrids were purified and characterized. Once mutated and coupled to phenanthroline, the protein remained folded and dimeric. Copper(II) was specifically bound by the two biohybrids with two different binding modes. Furthermore, the holo-biohybrid A3F119NPH was found to be capable of enantioselectively catalyzing Diels-Alder (D-A) cycloadditions with up to 62 % ee. This study validates the choice of the αRep A3 dimer as a protein scaffold and provides a promising new route for the design and production of new enantioselective biohybrids based on entirely artificial proteins obtained from a highly diverse library.
Asunto(s)
Metaloproteínas/metabolismo , Secuencia de Aminoácidos , Catálisis , Dicroismo Circular , Cobre/química , Reacción de Cicloadición , Dimerización , Espectroscopía de Resonancia por Spin del Electrón , Espectrometría de Masas , Metaloproteínas/química , Fenantrolinas/química , Pliegue de Proteína , Alineación de Secuencia , EstereoisomerismoRESUMEN
A copper(II) cofactor coupled to a testosterone anchor, copper(II)-(5-(Piperazin-1-yl)-1,10-phenanthroline)testosterone-17-hemisuccinamide (10) was synthesized and associated with a neocarzinostatin variant, NCS-3.24 (KD =3â µm), thus generating a new artificial metalloenzyme by following a "Trojan horse" strategy. Interestingly, the artificial enzyme was able to efficiently catalyze the Diels-Alder cyclization reaction of cyclopentadiene (1) with 2-azachalcone (2). In comparison with what was observed with cofactor 10 alone, the artificial enzymes favored formation of the exo products (endo/exo ratios of 84:16 and 62:38, respectively, after 12â h). Molecular modeling studies assigned the synergy between the copper complex and the testosterone (KD =13â µm) moieties in the binding of 10 to good van der Waals complementarity. Moreover, by pushing the modeling exercise to its limits, we hypothesize on the molecular grounds that are responsible for the observed selectivity.
Asunto(s)
Enzimas/metabolismo , Metaloproteínas/metabolismo , Cinostatina/metabolismo , Biocatálisis , Espectroscopía de Resonancia Magnética con Carbono-13 , Reacción de Cicloadición , Simulación del Acoplamiento Molecular , Espectroscopía de Protones por Resonancia Magnética , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
An artificial metalloenzyme based on the covalent grafting of a nonheme Fe(II) polyazadentate complex into bovine ß-lactoglobulin has been prepared and characterized by using various spectroscopic techniques. Attachment of the Fe(II) catalyst to the protein scaffold is shown to occur specifically at Cys121. In addition, spectrophotometric titration with cyanide ions based on the spin-state conversion of the initial high spin (S=2) Fe(II) complex into a low spin (S=0) one allows qualitative and quantitative characterization of the metal center's first coordination sphere. This biohybrid catalyst activates hydrogen peroxide to oxidize thioanisole into phenylmethylsulfoxide as the sole product with an enantiomeric excess of up to 20 %. Investigation of the reaction between the biohybrid system and H2 O2 reveals the generation of a high spin (S=5/2) Fe(III) (η(2) -O2 ) intermediate, which is proposed to be responsible for the catalytic sulfoxidation of the substrate.
Asunto(s)
Compuestos Aza/química , Complejos de Coordinación/química , Compuestos Ferrosos/química , Peróxido de Hidrógeno/química , Lactoglobulinas/química , Proteínas/síntesis química , Animales , Biocatálisis , Catálisis , Bovinos , Oxidación-Reducción , Proteínas/químicaRESUMEN
A new zinc(II)-cofactor coupled to a testosterone anchor, zinc(II)-N,N-bis(2-pyridylmethyl)-1,3-diamino-propa-2-ol-N'(17'-succinimidyltestosterone) (Zn-Testo-BisPyPol) 1-Zn has been synthesized and fully characterized. It has been further associated with a neocarzinostatin variant, NCS-3.24, to generate a new artificial metalloenzyme following the so-called 'Trojan horse' strategy. This new 1-Zn-NCS-3.24 biocatalyst showed an interesting catalytic activity as it was found able to catalyze the hydrolysis of the RNA model HPNP with a good catalytic efficiency (kcat/KM=13.6M(-1)s(-1) at pH 7) that places it among the best artificial catalysts for this reaction. Molecular modeling studies showed that a synergy between the binding of the steroid moiety and that of the BisPyPol into the protein binding site can explain the experimental results, indicating a better affinity of 1-Zn for the NCS-3.24 variant than testosterone and testosterone-hemisuccinate themselves. They also show that the artificial cofactor entirely fills the cavity, the testosterone part of 1-Zn being bound to one the two subdomains of the protein providing with good complementarities whereas its metal ion remains widely exposed to the solvent which made it a valuable tool for the catalysis of hydrolysis reactions, such as that of HPNP. Some possible improvements in the 'Trojan horse' strategy for obtaining better catalysts of selective reactions will be further studied.
Asunto(s)
Biocatálisis , Compuestos Organometálicos/metabolismo , Ribonucleasas/metabolismo , Zinc/metabolismo , Cinostatina/metabolismo , Modelos Moleculares , Estructura Molecular , Compuestos Organometálicos/química , Zinc/química , Cinostatina/químicaRESUMEN
Here we report the best artificial metalloenzyme to date for the selective oxidation of aromatic alkenes; it was obtained by noncovalent insertion of Mn(III)-meso-tetrakis(p-carboxyphenyl)porphyrin [Mn(TpCPP), 1-Mn] into a host protein, xylanase 10A from Streptomyces lividans (Xln10A). Two metallic complexes-N,N'-ethylene bis(2-hydroxybenzylimine)-5,5'-dicarboxylic acid Mn(III) [(Mn-salen), 2-Mn] and 1-Mn-were associated with Xln10A, and the two hybrid biocatalysts were characterised by UV-visible spectroscopy, circular dichroism and molecular modelling. Only the artificial metalloenzyme based on 1-Mn and Xln10A was studied for its catalytic properties in the oxidation of various substituted styrene derivatives by KHSO(5): after optimisation, the 1-Mn-Xln10A artificial metalloenzyme was able to catalyse the oxidation of para-methoxystyrene by KHSO(5) with a 16 % yield and the best enantioselectivity (80 % in favour of the R isomer) ever reported for an artificial metalloenzyme.
Asunto(s)
Endo-1,4-beta Xilanasas/química , Compuestos Epoxi/química , Manganeso/química , Metaloproteínas/química , Catálisis , Dicroismo Circular , Endo-1,4-beta Xilanasas/metabolismo , Manganeso/metabolismo , Metaloproteínas/metabolismo , Modelos Moleculares , Estructura Molecular , Oxidación-Reducción , Espectrofotometría Ultravioleta , Estereoisomerismo , Estireno/químicaRESUMEN
Direct amination of C-H or CîC bonds using unprotected amino groups is very challenging, especially with earth abundant metal ions. Here we show that a bioinspired iron(II) complex catalyses the double amination of its dangling benzyl branch in the presence of hydroxylamine derivatives as the unprotected amine donor and that the replacement of the benzyl branch by a methyl group also allows the aziridination of styrene.
Asunto(s)
Aminas , Hierro , Aminas/química , Hierro/química , Metales , Aminación , Compuestos FerrososRESUMEN
Engineering non-native metal active sites into proteins using canonical amino acids offers many advantages but is hampered by significant challenges. The TIM barrel protein, imidazole glycerol phosphate synthase from the hyperthermophilic organism Thermotoga maritima (tHisF), is well-suited for the construction of artificial metalloenzymes by this approach. To this end, we have generated a tHisF variant (tHisFEHH) with a Glu/His/His motif for metal ion coordination. Crystal structures of ZnII:tHisFEHH and NiII:tHisFEHH reveal that both metal ions bind to the engineered histidines. However, the two metals bind at distinct sites with different geometries, demonstrating the adaptability of tHisF. Only ZnII additionally ligates the Glu residue and adopts a tetrahedral geometry. The pseudo-octahedral NiII site comprises the two His and a native Ser residue. NiII:tHisFEHH catalyzes the oxidative cleavage of the flavanols quercetin and myricetin, providing an unprecedented example of an artificial metalloprotein with quercetinase activity.