Addressable DNA–Myoglobin Photocatalysis
Chem. - Asian J. 2009, 4, 1064-1069, 10.1002/asia.200900082
A hybrid myoglobin, containing a single‐stranded DNA anchor and a redox‐active ruthenium moiety tethered to the heme center can be used as a photocatalyst. The catalyst can be selectively immobilized on a surface‐bound complementary DNA molecule and thus readily recycled from complex reaction mixtures. This principle may be applied to a range of heme‐dependent enzymes allowing the generation of novel light‐triggered photocatalysts. Photoactivatable myoglobin containing a DNA oligonucleotide as a structural anchor was designed by using the reconstitution of artificial heme moieties containing Ru3+ ions. This semisynthetic DNA–enzyme conjugate was successfully used for the oxidation of peroxidase substrates by using visible light instead of H2O2 for the activation. The DNA anchor was utilized for the immobilization of the enzyme on the surface of magnetic microbeads. Enzyme activity measurements not only indicated undisturbed biofunctionality of the tethered DNA but also enabled magnetic separation‐based enrichment and recycling of the photoactivatable biocatalyst.
Host protein: Myoglobin (Mb)Optimization: ---Reaction: PhotooxidationMax TON: ---ee: ---PDB: ---Notes: Horse heart myoglobin
Aqueous Oxidation of Alcohols Catalyzed by Artificial Metalloenzymes Based on the Biotin–Avidin Technology
J. Organomet. Chem. 2005, 690, 4488-4491, 10.1016/j.jorganchem.2005.02.001
Based on the incorporation of biotinylated organometallic catalyst precursors within (strept)avidin, we have developed artificial metalloenzymes for the oxidation of secondary alcohols using tert-butylhydroperoxide as oxidizing agent. In the presence of avidin as host protein, the biotinylated aminosulfonamide ruthenium piano stool complex 1 (0.4 mol%) catalyzes the oxidation of sec-phenethyl alcohol at room temperature within 90 h in over 90% yield. Gel electrophoretic analysis of the reaction mixture suggests that the host protein is not oxidatively degraded during catalysis.
Ligand type: Amino-sulfonamide; BenzeneMax TON: 200ee: ---PDB: ---Notes: ---
Ligand type: Amino-sulfonamide; BenzeneHost protein: Avidin (Av)Max TON: 230ee: ---PDB: ---Notes: ---
Ligand type: Bipyridine; C6Me6Max TON: 173ee: ---PDB: ---Notes: ---
Max TON: 7.5ee: ---PDB: ---Notes: ---
Max TON: 30ee: ---PDB: ---Notes: ---
Atroposelective Antibodies as a Designed Protein Scaffold for Artificial Metalloenzymes
Sci. Rep. 2019, 9, 10.1038/s41598-019-49844-0
Design and engineering of protein scaffolds are crucial to create artificial metalloenzymes. Herein we report the first example of C-C bond formation catalyzed by artificial metalloenzymes, which consist of monoclonal antibodies (mAbs) and C2 symmetric metal catalysts. Prepared as a tailored protein scaffold for a binaphthyl derivative (BN), mAbs bind metal catalysts bearing a 1,1?-bi-isoquinoline (BIQ) ligand to yield artificial metalloenzymes. These artificial metalloenzymes catalyze the Friedel-Crafts alkylation reaction. In the presence of mAb R44E1, the reaction proceeds with 88% ee. The reaction catalyzed by Cu-catalyst incorporated into the binding site of mAb R44E1 is found to show excellent enantioselectivity with 99% ee. The protein environment also enables the use of BIQ-based catalysts as asymmetric catalysts for the first time.
Host protein: AntibodyAnchoring strategy: AntigenReaction: Friedel-Crafts alkylationMax TON: 2ee: 88PDB: ---Notes: ---
Autoxidation of Ascorbic Acid Catalyzed by a Semisynthetic Enzyme
Biopolymers 1990, 29, 39-43, 10.1002/bip.360290107
The semisyntehtic enzyme 6 was prepared by alkylation of the cysteine‐25 sulfhydryl group of papain with the bipyridine 5 and was shown to stoichiometrically bind copper ion; 7 catalyzed the autoxidation of ascorbic acid derivatives with saturation kinetics approximately 20‐fold faster than a model system using 3‐Cu(II).
Host protein: Papain (PAP)Optimization: ---Reaction: OxidationMax TON: ---ee: ---PDB: ---Notes: ---
Bimetallic Copper-Heme-Protein-DNA Hybrid Catalyst for Diels Alder Reaction
Croat. Chem. Acta 2011, 84, 269-275, 10.5562/cca1828
A bimetallic heme-DNA cofactor, containing an iron and a copper center, was synthesized for the design of novel hybrid catalysts for stereoselective synthesis. The cofactor was used for the reconstitution of apo-myoglobin. Both the cofactor alone and its myoglobin adduct were used to catalyze a model Diels Alder reaction. Stereoselectivity of this conversion was analyzed by chiral HPLC. Reactions carried out in the presence of myoglobin-heme-Cu-DNA catalyst showed greater product conversion and stereoselectivity than those carried out with the heme-Cu-DNA cofactor. This observation suggested that the protein shell plays a significant role in the catalytic conversion.
Host protein: Myoglobin (Mb)Optimization: ---Reaction: Diels-Alder reactionMax TON: 7.1ee: 18PDB: ---Notes: Horse heart myoglobin
Biocatalytic Cross-Coupling of Aryl Halides with a Genetically Engineered Photosensitizer Artificial Dehalogenase
J. Am. Chem. Soc. 2021, 143, 617-622, 10.1021/jacs.0c10882
Devising artificial photoenzymes for abiological bond-forming reactions is of high synthetic value but also a tremendous challenge. Disclosed herein is the first photobiocatalytic cross-coupling of aryl halides enabled by a designer artificial dehalogenase, which features a genetically encoded benzophenone chromophore and site-specifically modified synthetic NiII(bpy) cofactor with tunable proximity to streamline the dual catalysis. Transient absorption studies suggest the likelihood of energy transfer activation in the elementary organometallic event. This design strategy is viable to significantly expand the catalytic repertoire of artificial photoenzymes for useful organic transformations.
Metal: NiHost protein: CO2-reducing photosensitizer protein (PSP)Reaction: Cross-couplingMax TON: 223ee: ---PDB: ---Notes: ---
Biosynthesis of a Site-Specific DNA Cleaving Protein
J. Am. Chem. Soc. 2008, 130, 13194-13195, 10.1021/ja804653f
An E. coli catabolite activator protein (CAP) has been converted into a sequence-specific DNA cleaving protein by genetically introducing (2,2′-bipyridin-5-yl)alanine (Bpy-Ala) into the protein. The mutant CAP (CAP-K26Bpy-Ala) showed comparable binding affinity to CAP-WT for the consensus operator sequence. In the presence of Cu(II) and 3-mercaptopropionic acid, CAP-K26Bpy-Ala cleaves double-stranded DNA with high sequence specificity. This method should provide a useful tool for mapping the molecular details of protein−nucleic acid interactions.
Host protein: Catabolite activator protein (CAP)Anchoring strategy: ---Max TON: ---ee: ---PDB: ---Notes: Catabolite activator protein from E. coli
Metal: FeHost protein: Catabolite activator protein (CAP)Anchoring strategy: ---Max TON: ---ee: ---PDB: ---Notes: Catabolite activator protein from E. coli
Controlled Ligand Exchange Between Ruthenium Organometallic Cofactor Precursors and a Naïve Protein Scaffold Generates Artificial Metalloenzymes Catalysing Transfer Hydrogenation
Angew. Chem. Int. Ed. 2021, 60, 10919-10927, 10.1002/anie.202015834
Many natural metalloenzymes assemble from proteins and biosynthesised complexes, generating potent catalysts by changing metal coordination. Here we adopt the same strategy to generate artificial metalloenzymes (ArMs) using ligand exchange to unmask catalytic activity. By systematically testing RuII(η6-arene)(bipyridine) complexes designed to facilitate the displacement of functionalised bipyridines, we develop a fast and robust procedure for generating new enzymes via ligand exchange in a protein that has not evolved to bind such a complex. The resulting metal cofactors form peptidic coordination bonds but also retain a non-biological ligand. Tandem mass spectrometry and 19F NMR spectroscopy were used to characterise the organometallic cofactors and identify the protein-derived ligands. By introduction of ruthenium cofactors into a 4-helical bundle, transfer hydrogenation catalysts were generated that displayed a 35-fold rate increase when compared to the respective small molecule reaction in solution.
Ligand type: Arene; BipyridineHost protein: Cytochrome b562Optimization: ---Max TON: ---ee: ---PDB: ---Notes: 35 fold rate increase
Ligand type: Arene; BipyridineHost protein: UbiquitinOptimization: ---Max TON: ---ee: ---PDB: ---Notes: 35 fold rate increase
Design of an Enantioselective Artificial Metallo-Hydratase Enzyme Containing an Unnatural Metal-Binding Amino Acid
Chem. Sci. 2017, 8, 7228-7235, 10.1039/C7SC03477F
The design of artificial metalloenzymes is a challenging, yet ultimately highly rewarding objective because of the potential for accessing new-to-nature reactions. One of the main challenges is identifying catalytically active substrate–metal cofactor–host geometries. The advent of expanded genetic code methods for the in vivo incorporation of non-canonical metal-binding amino acids into proteins allow to address an important aspect of this challenge: the creation of a stable, well-defined metal-binding site. Here, we report a designed artificial metallohydratase, based on the transcriptional repressor lactococcal multidrug resistance regulator (LmrR), in which the non-canonical amino acid (2,2′-bipyridin-5yl)alanine is used to bind the catalytic Cu(II) ion. Starting from a set of empirical pre-conditions, a combination of cluster model calculations (QM), protein–ligand docking and molecular dynamics simulations was used to propose metallohydratase variants, that were experimentally verified. The agreement observed between the computationally predicted and experimentally observed catalysis results demonstrates the power of the artificial metalloenzyme design approach presented here.
Anchoring strategy: ---Reaction: HydrationMax TON: 9ee: 64PDB: ---Notes: ---
Design of Artificial Metalloenzymes for the Reduction of Nicotinamide Cofactors
J. Inorg. Biochem. 2021, 220, 111446, 10.1016/j.jinorgbio.2021.111446
Artificial metalloenzymes result from the insertion of a catalytically active metal complex into a biological scaffold, generally a protein devoid of other catalytic functionalities. As such, their design requires efforts to engineer substrate binding, in addition to accommodating the artificial catalyst. Here we constructed and characterised artificial metalloenzymes using alcohol dehydrogenase as starting point, an enzyme which has both a cofactor and a substrate binding pocket. A docking approach was used to determine suitable positions for catalyst anchoring to single cysteine mutants, leading to an artificial metalloenzyme capable to reduce both natural cofactors and the hydrophobic 1-benzylnicotinamide mimic. Kinetic studies revealed that the new construct displayed a Michaelis-Menten behaviour with the native nicotinamide cofactors, which were suggested by docking to bind at a surface exposed site, different compared to their native binding position. On the other hand, the kinetic and docking data suggested that a typical enzyme behaviour was not observed with the hydrophobic 1-benzylnicotinamide mimic, with which binding events were plausible both inside and outside the protein. This work demonstrates an extended substrate scope of the artificial metalloenzymes and provides information about the binding sites of the nicotinamide substrates, which can be exploited to further engineer artificial metalloenzymes for cofactor regeneration.
Ligand type: Amino-sulfonamide; Bipyridine; Cp*Host protein: Alcohol dehydrogenaseReaction: Nicotinamide reductionMax TON: ---ee: ---PDB: 1YKFNotes: ---
Dual Modification of a Triple-Stranded β-Helix Nanotube with Ru and Re Metal Complexes to Promote Photocatalytic Reduction of CO2
Chem. Commun. 2011, 47, 2074, 10.1039/C0CC03015E
We have constructed a robust β-helical nanotube from the component proteins of bacteriophage T4 and modified this nanotube with RuII(bpy)3 and ReI(bpy)(CO)3Cl complexes. The photocatalytic system arranged on the tube catalyzes the reduction of CO2 with higher reactivity than that of the mixture of the monomeric forms.
Metal: ReLigand type: Bipyridine; COAnchoring strategy: Cystein-maleimideOptimization: ---Reaction: CO2 reductionMax TON: ---ee: ---PDB: ---Notes: ---
Anchoring strategy: Lysine-succinimideReaction: CO2 reductionMax TON: ---ee: ---PDB: ---Notes: ---
Enantioselective Artificial Metalloenzymes by Creation of a Novel Active Site at the Protein Dimer Interface
Angew. Chem. Int. Ed. 2012, 51, 7472-7475, 10.1002/anie.201202070
A game of two halves: Artificial metalloenzymes are generated by forming a novel active site on the dimer interface of the transcription factor LmrR. Two copper centers are incorporated by binding to ligands in each half of the dimer. With this system up to 97 % ee was obtained in the benchmark CuII catalyzed Diels–Alder reaction (see scheme).
Ligand type: Bipyridine; PhenanthrolineReaction: Diels-Alder reactionMax TON: 32.7ee: 97PDB: 3F8BNotes: ---
Expanding the Chemical Diversity in Artificial Imine Reductases Based on the Biotin–Streptavidin Technology
ChemCatChem 2014, 6, 1010-1014, 10.1002/cctc.201300825
We report on the optimization of an artificial imine reductase based on the biotin‐streptavidin technology. With the aim of rapidly generating chemical diversity, a novel strategy for the formation and evaluation of biotinylated complexes is disclosed. Tethering the biotin‐anchor to the Cp* moiety leaves three free coordination sites on a d6 metal for the introduction of chemical diversity by coordination of a variety of ligands. To test the concept, 34 bidentate ligands were screened and a selection of the 6 best was tested in the presence of 21 streptavidin (Sav) isoforms for the asymmetric imine reduction by the resulting three legged piano stool complexes. Enantiopure α‐amino amides were identified as promising bidentate ligands: up to 63 % ee and 190 turnovers were obtained in the formation of 1‐phenyl‐1,2,3,4‐tetrahydroisoquinoline with [IrCp*biotin(L‐ThrNH2)Cl]⊂SavWT as a catalyst.
Ligand type: Amino acid; Cp*Max TON: 188ee: 43PDB: ---Notes: ---
Ligand type: Amino carboxylic acid; Cp*Max TON: 4ee: 21PDB: ---Notes: ---
Ligand type: Cp*Notes: ---
Ligand type: Cp*; Pyrazine amideMax TON: 26ee: 16PDB: ---Notes: ---
Max TON: 12ee: 13PDB: ---Notes: ---
Max TON: 102ee: 14PDB: ---Notes: ---
Ligand type: Amino acid; Cp*Max TON: 94ee: 67PDB: ---Notes: ---
Ligand type: Amino amide; Cp*Max TON: 10ee: 7PDB: ---Notes: ---
Ligand type: Amino carboxylic acid; Cp*Max TON: 8ee: 1PDB: ---Notes: ---
Ligand type: Cp*Notes: ---
Ligand type: Cp*; Pyrazine amideNotes: ---
Max TON: 4ee: 6PDB: ---Notes: ---
Max TON: 8ee: 0PDB: ---Notes: ---
Metal-Mediated Protein Assembly Using a Genetically Incorporated Metal-Chelating Amino Acid
Biomacromolecules 2020, 21, 5021-5028, 10.1021/acs.biomac.0c01194
Many natural proteins function in oligomeric forms, which are critical for their sophisticated functions. The construction of protein assemblies has great potential for biosensors, enzyme catalysis, and biomedical applications. In designing protein assemblies, a critical process is to create protein–protein interaction (PPI) networks at defined sites of a target protein. Although a few methods are available for this purpose, most of them are dependent on existing PPIs of natural proteins to some extent. In this report, a metal-chelating amino acid, 2,2′-bipyridylalanine (BPA), was genetically introduced into defined sites of a monomeric protein and used to form protein oligomers. Depending on the number of BPAs introduced into the protein and the species of metal ions (Ni2+ and Cu2+), dimers or oligomers with different oligomerization patterns were formed by complexation with a metal ion. Oligomer sizes could also be controlled by incorporating two BPAs at different locations with varied angles to the center of the protein. When three BPAs were introduced, the monomeric protein formed a large complex with Ni2+. In addition, when Cu2+ was used for complex formation with the protein containing two BPAs, a linear complex was formed. The method proposed in this report is technically simple and generally applicable to various proteins with interesting functions. Therefore, this method would be useful for the design and construction of functional protein assemblies.
Host protein: Maltose-binding protein (MBP)Optimization: ---Reaction: ---Max TON: ---ee: ---PDB: ---Notes: ---
Novel Artificial Metalloenzymes by In Vivo Incorporation of Metal-Binding Unnatural Amino Acids
Chem. Sci. 2015, 6, 770-776, 10.1039/c4sc01525h
Artificial metalloenzymes have emerged as an attractive new approach to enantioselective catalysis. Herein, we introduce a novel strategy for preparation of artificial metalloenzymes utilizing amber stop codon suppression methodology for the in vivo incorporation of metal-binding unnatural amino acids. The resulting artificial metalloenzymes were applied in catalytic asymmetric Friedel–Crafts alkylation reactions and up to 83% ee for the product was achieved.
Anchoring strategy: ---Reaction: Fridel-Crafts reactionMax TON: 10.4ee: 83PDB: 3F8BNotes: ---
Photo-Driven Hydrogen Evolution by an Artificial Hydrogenase Utilizing the Biotin-Streptavidin Technology
Helv. Chim. Acta 2018, 101, e1800036, 10.1002/hlca.201800036
Photocatalytic hydrogen evolution by an artificial hydrogenase based on the biotin‐streptavidin technology is reported. A biotinylated cobalt pentapyridyl‐based hydrogen evolution catalyst (HEC) was incorporated into different mutants of streptavidin. Catalysis with [Ru(bpy)3]Cl2 as a photosensitizer (PS) and ascorbate as sacrificial electron donor (SED) at different pH values highlighted the impact of close lying amino acids that may act as a proton relay under the reaction conditions (Asp, Arg, Lys). In the presence of a close‐lying lysine residue, both, the rates were improved, and the reaction was initiated much faster. The X‐ray crystal structure of the artificial hydrogenase reveals a distance of 8.8 Å between the closest lying Co‐moieties. We thus suggest that the hydrogen evolution mechanism proceeds via a single Co centre. Our findings highlight that streptavidin is a versatile host protein for the assembly of artificial hydrogenases and their activity can be fine‐tuned via mutagenesis.
Metal: CoLigand type: Bipyridine; PyridineReaction: H2 evolutionMax TON: >1800ee: ---PDB: 6FRYNotes: ---
Semi-Synthesis of an Artificial Scandium(III) Enzyme with a β-Helical Bio-Nanotube
Dalton Trans. 2012, 41, 11424, 10.1039/C2DT31030A
We have succeeded in preparing semi-synthesized proteins bound to Sc3+ ion which can promote an epoxide ring-opening reaction. The Sc3+ binding site was created on the surface of [(gp5βf)3]2 (N. Yokoi et al., Small, 2010, 6, 1873) by introducing a cysteine residue for conjugation of a bpy moiety using a thiol–maleimide coupling reaction. Three cysteine mutants [(gp5βf_X)3]2 (X = G18C, L47C, N51C) were prepared to introduce a bpy in different positions because it had been reported that Sc3+ ion can serve as a Lewis-acid catalyst for an epoxide ring-opening reaction upon binding of epoxide to bpy and two –ROH groups. G18C_bpy with Sc3+ can accelerate the rate of catalysis of the epoxide ring-opening reaction and has the highest rate of conversion among the three mutants. The value is more than 20 times higher than that of the mixtures of [(gp5βf)3]2/2,2′-bipyridine and L-threonine/2,2′-bipyridine. The elevated activity was obtained by the cooperative effect of stabilizing the Sc3+ coordination and accumulation of substrates on the protein surface. Thus, we expect that the semi-synthetic approach can provide insights into new rational design of artificial metalloenzymes.
Metal: ScAnchoring strategy: Cystein-maleimideReaction: Epoxide ring opening reactionMax TON: ---ee: ---PDB: ---Notes: ---
Semisynthesis of Bipyridyl-Alanine Cytochrome c Mutants: Novel Proteins with Enhanced Electron-Transfer Properties
J. Am. Chem. Soc. 1993, 115, 8455-8456, 10.1021/ja00071a068
Ligand type: Bipyridine; PorphyrinHost protein: Horse heart cytochrome cOptimization: ---Reaction: Electron transferMax TON: ---ee: ---PDB: ---Notes: No catalysis