13 publications
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A General Method for Artificial Metalloenzyme Formationthrough Strain-Promoted Azide–Alkyne Cycloaddition
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ChemBioChem 2014, 15, 223-227, 10.1002/cbic.201300661
Strain‐promoted azide–alkyne cycloaddition (SPAAC) can be used to generate artificial metalloenzymes (ArMs) from scaffold proteins containing a p‐azido‐L‐phenylalanine (Az) residue and catalytically active bicyclononyne‐substituted metal complexes. The high efficiency of this reaction allows rapid ArM formation when using Az residues within the scaffold protein in the presence of cysteine residues or various reactive components of cellular lysate. In general, cofactor‐based ArM formation allows the use of any desired metal complex to build unique inorganic protein materials. SPAAC covalent linkage further decouples the native function of the scaffold from the installation process because it is not affected by native amino acid residues; as long as an Az residue can be incorporated, an ArM can be generated. We have demonstrated the scope of this method with respect to both the scaffold and cofactor components and established that the dirhodium ArMs generated can catalyze the decomposition of diazo compounds and both SiH and olefin insertion reactions involving these carbene precursors.
Metal: RhLigand type: Poly-carboxylic acidHost protein: tHisFAnchoring strategy: CovalentOptimization: ---Notes: ---
Metal: RhLigand type: Poly-carboxylic acidHost protein: tHisFAnchoring strategy: CovalentOptimization: ---Notes: ---
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A Hydroxyquinoline‐Based Unnatural Amino Acid for the Design of Novel Artificial Metalloenzymes
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ChemBioChem 2020, 21, 3077-3081, 10.1002/cbic.202000306
We have examined the potential of the noncanonical amino acid (8-hydroxyquinolin-3-yl)alanine (HQAla) for the design of artificial metalloenzymes. HQAla, a versatile chelator of late transition metals, was introduced into the lactococcal multidrug-resistance regulator (LmrR) by stop codon suppression methodology. LmrR_HQAla was shown to complex efficiently with three different metal ions, CuII, ZnII and RhIII to form unique artificial metalloenzymes. The catalytic potential of the CuII-bound LmrR_HQAla enzyme was shown through its ability to catalyse asymmetric Friedel-Craft alkylation and water addition, whereas the ZnII-coupled enzyme was shown to mimic natural Zn hydrolase activity.
Metal: CuLigand type: HydroxyquinolineHost protein: Lactoccal multidrug resistant regulator (LmrR)Anchoring strategy: SupramolecularOptimization: GeneticNotes: Also used Rh, but no reaction detected.
Metal: CuLigand type: HydroxyquinolineHost protein: Lactoccal multidrug resistant regulator (LmrR)Anchoring strategy: SupramolecularOptimization: GeneticNotes: ---
Metal: ZnLigand type: HydroxyquinolineHost protein: Lactoccal multidrug resistant regulator (LmrR)Anchoring strategy: SupramolecularOptimization: GeneticNotes: ---
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An Artificial Cofactor Catalyzing the Baylis‐Hillman Reaction with Designed Streptavidin as Protein Host
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ChemBioChem 2021, 22, 1573-1577, 10.1002/cbic.202000880
An artificial cofactor based on an organocatalyst embedded in a protein has been used to conduct the Baylis-Hillman reaction in a buffered system. As protein host, we chose streptavidin, as it can be easily crystallized and thereby supports the design process. The protein host around the cofactor was rationally designed on the basis of high-resolution crystal structures obtained after each variation of the amino acid sequence. Additionally, DFT-calculated intermediates and transition states were used to rationalize the observed activity. Finally, repeated cycles of structure determination and redesign led to a system with an up to one order of magnitude increase in activity over the bare cofactor and to the most active proteinogenic catalyst for the Baylis-Hillman reaction known today.
Metal: ---Ligand type: ---Host protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & computational designNotes: Organocatalyst
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Artificial Metalloenzymes for Enantioselective Catalysis: Recent Advances
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ChemBioChem 2006, 7, 1845-1852, 10.1002/cbic.200600264
Creating new catalytic function in proteins. Anchoring an organometallic moiety within a protein affords artificial metalloenzymes for enantioselective catalysis. Both chemical and genetic tools can be applied in the optimization of such systems, which lie at the interface between homogeneous and enzymatic catalysis. This minireview presents the latest developments in the field of artificial metalloenzymes.
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Artificial Metalloenzymes Through Cysteine-Selective Conjugation of Phosphines to Photoactive Yellow Protein
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ChemBioChem 2010, 11, 1236-1239, 10.1002/cbic.201000159
Pinning phosphines on proteins: A method for the cysteine‐selective bioconjugation of phosphines has been developed. The photoactive yellow protein has been site‐selectively functionalized with phosphine ligands and phosphine transition metal complexes to afford artificial metalloenzymes that are active in palladium‐catalysed allylic nucleophilic substitution reactions.
Metal: PdHost protein: Photoactive Yellow Protein (PYP)Anchoring strategy: CovalentOptimization: Chemical & geneticNotes: ---
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Artificial Metalloenzymes with the Neocarzinostatin Scaffold: Toward a Biocatalyst for the Diels–Alder Reaction
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ChemBioChem 2016, 17, 433-440, 10.1002/cbic.201500445
A new artificial enzyme formed by associating NCS‐3.24 with a copper complex catalyzed the Diels–Alder cyclization of cyclopentadiene with 2‐azachalcone and led to an increase in the formation of the exo‐products. Molecular modeling proposed the preferred relative positioning of both the Trojan horse complex and the two substrates.
Metal: CuLigand type: PhenanthrolineHost protein: Neocarzinostatin (variant 3.24)Anchoring strategy: SupramolecularOptimization: ---Notes: Up to endo/exo ratio 62:38
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Design and Evaluation of Artificial Hybrid Photoredox Biocatalysts
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ChemBioChem 2020, 21, 3146-3150, 10.1002/cbic.202000362
A pair of 9-mesityl-10-phenyl acridinium (Mes−Acr+) photoredox catalysts were synthesized with an iodoacetamide handle for cysteine bioconjugation. Covalently tethering of the synthetic Mes−Acr+ cofactors with a small panel of thermostable protein scaffolds resulted in 12 new artificial enzymes. The unique chemical and structural environment of the protein hosts had a measurable effect on the photophysical properties and photocatalytic activity of the cofactors. The constructed Mes−Acr+ hybrid enzymes were found to be active photoinduced electron-transfer catalysts, controllably oxidizing a variety of aryl sulfides when irradiated with visible light, and possessed activities that correlated with the photophysical characterization data. Their catalytic performance was found to depend on multiple factors including the Mes−Acr+ cofactor, the protein scaffold, the location of cofactor immobilization, and the substrate. This work provides a framework toward adapting synthetic photoredox catalysts into artificial cofactors and includes important considerations for future bioengineering efforts.
Metal: ---Ligand type: 9-mesityl-10-phenyl acridiniumHost protein: Aspertate dehydrogenaseAnchoring strategy: CovalentOptimization: Chemical & geneticNotes: Maximum conversion is 95%; In most cases, a comparable yield or modest increase in yield was observed for the protein-bound catalyst compared to the unbound cofactor.
Metal: ---Ligand type: 9-mesityl-10-phenyl acridiniumHost protein: Phosphoribosylamine - glycine ligaseAnchoring strategy: CovalentOptimization: Chemical & geneticNotes: Maximum conversion is 95%; In most cases, a comparable yield or modest increase in yield was observed for the protein-bound catalyst compared to the unbound cofactor.
Metal: ---Ligand type: 9-mesityl-10-phenyl acridiniumHost protein: Folypolyglutamate synthaseAnchoring strategy: CovalentOptimization: Chemical & geneticNotes: Maximum conversion is 95%; In most cases, a comparable yield or modest increase in yield was observed for the protein-bound catalyst compared to the unbound cofactor.
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Directed Evolution of a Cp*RhIII‐Linked Biohybrid Catalyst Based on a Screening Platform with Affinity Purification
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ChemBioChem 2021, 22, 679-685, 10.1002/cbic.202000681
Directed evolution of Cp*RhIII-linked nitrobindin (NB), a biohybrid catalyst, was performed based on an in vitro screening approach. A key aspect of this effort was the establishment of a high-throughput screening (HTS) platform that involves an affinity purification step employing a starch-agarose resin for a maltose binding protein (MBP) tag. The HTS platform enables efficient preparation of the purified MBP-tagged biohybrid catalysts in a 96-well format and eliminates background influence of the host E. coli cells. Three rounds of directed evolution and screening of more than 4000 clones yielded a Cp*RhIII-linked NB(T98H/L100K/K127E) variant with a 4.9-fold enhanced activity for the cycloaddition of acetophenone oximes with alkynes. It is confirmed that this HTS platform for directed evolution provides an efficient strategy for generating highly active biohybrid catalysts incorporating a synthetic metal cofactor.
Metal: RhLigand type: CpHost protein: Nitrobindin (Nb)Anchoring strategy: CovalentOptimization: GeneticNotes: ---
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Incorporation of Manganese Complexes into Xylanase: New Artificial Metalloenzymes for Enantioselective Epoxidation
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ChemBioChem 2012, 13, 240-251, 10.1002/cbic.201100659
Enantioselective epoxidation: An artificial metalloenzyme obtained by noncovalent insertion of MnIII‐meso‐tetrakis(para‐carboxyphenyl)porphyrin Mn(TpCPP) into xylanase 10A from Streptomyces lividans as a host protein was able to catalyse the oxidation of para‐methoxystyrene by KHSO5 with a 16 % yield and the best enantioselectivity (80 % in favour of the R isomer) ever reported for an artificial metalloenzyme.
Metal: MnLigand type: PorphyrinHost protein: Xylanase A (XynA)Anchoring strategy: SupramolecularOptimization: ---Notes: ---
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Orthogonal Expression of an Artificial Metalloenzyme for Abiotic Catalysis
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ChemBioChem 2017, 18, 2380-2384, 10.1002/cbic.201700397
Engineering an (Ir)regular cytochrome P450: Mutations within the heme‐binding pocket of a cytochrome P450 enabled the selective incorporation of an artificial Ir‐porphyrin cofactor into the protein, in cells. This orthogonal metalloprotein showed enhanced behavior in unnatural carbene‐mediated cyclopropanation of aliphatic and electron‐deficient olefins.
Metal: IrHost protein: Cytochrome BM3hAnchoring strategy: ReconstitutionOptimization: Chemical & geneticNotes: Reaction of styrene with ethyl diazoacetate, cis:trans = 29:71
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Polymer Enzyme Conjugates as Chiral Ligands for Sharpless Dihydroxylation of Alkenes in Organic Solvents
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ChemBioChem 2015, 16, 83-90, 10.1002/cbic.201402339
Count Os in: We report organosoluble artificial metalloenzymes, generated from poly(2‐methyl‐oxazoline) enzyme conjugates and osmate as a promising new catalytic system for the dihydroxylation of alkenes in organic media.
Metal: OsLigand type: Amino acidHost protein: LaccaseAnchoring strategy: Metal substitutionOptimization: ChemicalNotes: ---
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The Protein Environment Drives Selectivity for Sulfide Oxidation by an Artificial Metalloenzyme
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ChemBioChem 2009, 10, 545-552, 10.1002/cbic.200800595
Magic Mn–salen metallozyme: The design of an original, artificial, inorganic, complex‐protein adduct, has led to a better understanding of the synergistic effects of both partners. The exclusive formation of sulfoxides by the hybrid biocatalyst, as opposed to sulfone in the case of the free inorganic complex, highlights the modulating role of the inorganic‐complex‐binding site in the protein.
Metal: MnLigand type: SalenHost protein: Human serum albumin (HSA)Anchoring strategy: SupramolecularOptimization: ChemicalNotes: ---
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Transforming Carbonic Anhydrase into Epoxide Synthase by Metal Exchange
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ChemBioChem 2006, 7, 1013-1016, 10.1002/cbic.200600127
Enantioselective epoxidation of styrene was observed in the presence of manganese‐containing carbonic anhydrase as catalyst. The probable oxygen‐transfer reagent is peroxymonocarbonate, which has a structural similarity with the hydrogenocarbonate substrate of the natural reaction. Styrene was chosen as the enzyme possesses a small hydrophobic cavity close to the active site.
Metal: MnLigand type: Amino acidHost protein: Bovine carbonic anhydrase II (CA)Anchoring strategy: Metal substitutionOptimization: Chemical & geneticNotes: ---
Metal: MnLigand type: Amino acidHost protein: Human carbonic anhydrase II (hCAII)Anchoring strategy: Metal substitutionOptimization: Chemical & geneticNotes: ---