Functionalized artificial bidomain proteins based on an α-solenoid protein repeat scaffold : a new class of artificial diels-alderases

This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.αRep is a family of entirely artificial repeat proteins. Within the previously described αRep library, some variants are homodimers displaying interdomain cavities. Taking advantage of these properties, one of these homodimers called αRep A3 was converted into entirely artificial single chain bidomain metalloenzymes. A nonmutated A3 domain was covalently linked with an A3' domain bearing a unique cysteine on a chosen mutated position (F119C or Y26C). This single mutation ensured the covalent coupling of a 1:1 copper(II)/phenanthroline or copper(II)/terpyridine complex as a catalytic center within the interdomain cavity which was maintained large enough to accommodate two substrates of the Diels-Alder (D-A) reaction. This allowed us to obtain four new artificial Diels-Alderases that were fully characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, UV-vis spectroscopy, and size exclusion chromatography analyses and were then further used for the catalysis of the D-A reaction. They were found to be able to catalyze the enantioselective D-A reaction of azachalcone with cyclopentadiene with up to 38% yield and 52% enantiomeric excess, which validates the proposed strategy. Moreover, the data were rationalized with a computational strategy suggesting the key factors of the selectivity. These results suggest that artificial metalloenzymes based on bidomain A3-A3 proteins modified with nitrogen donor ligands may be suitable for further catalyst optimization and may constitute valuable tools toward more efficient and selective artificial biocatalysts

New artificial metalloenzymes obtained by covalent coupling of metal complexes in a natural protein (Xylanase A) and in artificial proteins (αReps)

Dans un contexte de développement durable, les enzymes sont des outils biologiques puissants pour catalyser des réactions avec de très grandes efficacités et spécificités. Inspirée des enzymes et de la catalyse organométallique, l’élaboration de métalloenzymes artificielles émerge depuis plusieurs années comme une stratégie de choix pour fournir aux chimistes de nouveaux biocatalyseurs, en accord avec les principes de la chimie verte. Elles sont construites par l’insertion par interactions supramoléculaires ou couplage covalent, d’un ion ou d’un complexe métallique au sein d’une protéine, qui leur apporte un environnement hydrophobe protecteur et chiral. Lors de cette thèse, plusieurs métalloenzymes artificielles ont été construites par couplage covalent de complexes métalliques dans deux protéines hôtes, qui sont la Xylanase A (Xln) et les protéines artificielles de la famille des Reps. Dans un premier temps, une hydrogénase artificielle a été construite dans le mutant XlnS212C par ancrage covalent d’un complexe de fer appelé complexe de Knölker. L’hydrogénase artificielle obtenue, XlnS212CK, s’est avérée capable de catalyser l’hydrogénation par transfert d’hydrure de la trifluoroacétophénone, TFAC, sans excès énantiomérique. Dans un second temps, quatre Diels-Alderases artificielles ont été construites à partir de la protéine bidomaine (A3_A3’) de la famille des αReps. Les deux meilleures Diels-Alderases, qui ont conduit respectivement au meilleur rendement et la meilleure énantiosélectivité dans la réaction de l’azachalcone sur le cyclopentadiène, ont été élaborées respectivement par fixation covalente de complexe de cuivre de ligands phénanthroline et terpyridine dans un mutant F119C de A3_A3’ : (A3_A3’)F119Phen-Cu(II) et (A3_A3’)F119Terpy-Cu(II). Finalement, une nouvelle hémoprotéine artificielle a été construite par couplage covalent de la méso-tétraphénylporphyrine de manganèse Mn(III)TPP-NHMal dans le mutant (A3_A3’)Y26C. L’hémoprotéine artificielle formée BH MnTPP seule ne montre aucune activité catalytique pour l’oxydation de co-substrats par H2O2. Cependant, de manière inattendue, l’addition d’imidazole et d’une autre protéine αRep, bA3-2, qui se fixe de manière spécifique sur A3_A3 et provoque son ouverture, permet non seulement de déclencher l’activité peroxydase de BH MnTPP, mais également une activité monooxygénase qui catalyse la sulfoxydation du thioanisole par H2O2. Il s’agit du premier exemple décrit à ce jour de métalloenzyme artificielle dont l’activité peut être induite par la fixation d’une protéine partenaire.In a context of sustainable development, enzymes are powerful biological tools to catalyze reactions with very high efficiencies and specificities. Inspired by enzymes and organometallic catalysis, the development of artificial metalloenzymes has emerged for several years as a strategy of choice to provide the chemists with new biocatalysts, in accordance with the principles of green chemistry. They are constructed by the insertion by supramolecular interactions or covalent coupling of an ion or a metal complex within a protein, which provides them with a protective and chiral hydrophobic environment. In this thesis, several artificial metalloenzymes were constructed by covalent coupling of metal complexes into two host proteins, Xylanase A (Xln) and artificial proteins of the Reps family. Initially, an artificial hydrogenase was constructed in the XlnS212C mutant by covalent anchoring of an iron complex known as the Knölker complex. The artificial hydrogenase obtained, XlnS212CK, was found to be capable of catalyzing hydride hydrogenation of trifluoroacetophenone, TFAC, without enantiomeric excess. In a second time, four artificial Diel-Alderases were constructed from the bidomain protein (A3_A3') of the αReps family. The two best Diels-Alderases which led respectively to the best yield and the best enantioselectivity in the reaction of azachalcone on cyclopentadiene were developed respectively by covalent attachment of copper complex of phenanthroline and terpyridine ligands in a mutant F119C of A3_A3' (A3_A3')F119Phen-Cu (II) and (A3_A3')F119 Terpy-Cu (II). Finally, a new artificial hemoprotein was constructed by covalent coupling of the manganese meso-tetraphenylporphyrin Mn(III)TPP-NHMal in the (A3_A3')Y26C mutant. The artificial hemoprotein formed BH-MnTPP alone shows no catalytic activity for the oxidation of co-substrates by H2O2. However, unexpectedly, the addition of imidazole and another αRep protein, bA3-2, which binds specifically to A3_A3’ and causes it to be opened, not only triggers the BH-MnTPP peroxidase activity but also a monooxygenase activity which catalyzes the sulfoxidation of thioanisole by H2O2. This is the first example described to date of artificial metalloenzyme whose activity can be induced by the attachment of a partner protein

Artificial iron hydrogenase made by covalent grafting of Knölker's complex into xylanase: Application in asymmetric hydrogenation of an aryl ketone in water

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An artificial hemoprotein with inducible peroxidase- and monooxygenase-like activities

A new inducible artificial metalloenzyme was obtained by covalent attachment of a Mn(III)-tetraphenylporphyrin to the artificial bidomain repeat protein (A3A3')Y26C. The biohybrid was characterized by MALDI-ToF MS, circular dichroism and UV-Vis spectroscopies. Its peroxidase activity was tested as well as its monooxygenase activity for the oxidation of thioanisole into sulfoxide by H 2 O 2 . Catalytic tests were performed on the original scaffold and with  several co-catalysts a) additional imidazole, b) a specific αRep bA3-2 that induces the opening of the (A3A3') interdomain region and c) a αRep bA3-2 bearing a His6-Tag (His6-bA3-2), and the best activity was obtained with His6-bA3-2. The mechanism was rationalized by a molecular modeling study including protein-ligand docking and large scale molecular dynamics. It appears that: 1) the hydrophobic Mn-porphyrin fits well into the (A3A3')Y26C cavity, becomes protected by the protein and fully soluble in water; 2) the biohybrid alone does not display any catalytic activity, even in the presence of imidazole because of a closed conformation of the system; 3) opening of the binding site by bA3-2 leads to a noticeable activity and, 4) the best activity is obtained in the presence of the His6-Tag with one or two of the histidines able to coordinate to the metal acting as co-catalyst in the reactions. To the best of our knowledge, this constitutes the first example of an entirely artificial metalloenzyme with inducible peroxidase and monooxygenase activities, reminiscent of allosteric regulation of natural enzymatic pathways

Encapsulation of Microperoxidase-8 in MIL-101(Cr)-X Nanoparticles: Influence of Metal-Organic Framework Functionalization on Enzymatic Immobilization and Catalytic Activity

International audienceMicroperoxidase 8 (MP8) was immobilized within MIL-101(Cr) bearing terephthalate linkers with functionalized groups (-NH2 and -SO3H). A synthesis protocol for MIL-101(Cr)-SO3H that avoids the use of toxic Cr(VI) and HF was developed. The electrostatic interactions between the MP8 molecules and the MOF matrices were found to be crucial for a successful immobilization. Raman spectroscopy revealed the dispersion of the immobilized MP8 molecules in MIL-101(Cr)-X matrices as monomers without aggregation. The presence of functional groups resulted in higher amounts of immobilized MP8 in comparison to the bare MIL-101(Cr). The catalytic activity of MP8@MIL-101(Cr)-NH2 per material mass was higher than that for MP8@MIL-101(Cr). The presence of free amino groups can thus improve the immobilization efficiency, leading to a higher amount of catalytically active species and improving the subsequent catalytic activity of the heterogeneous biocatalysts. MP8@MIL(Cr)-X also successfully catalyzed the selective oxidation of thioanisole derivatives into sulfoxides