10 publications

10 publications

An Enantioselective Artificial Metallo-Hydratase

Roelfes, G.

Chem. Sci. 2013, 4, 3578, 10.1039/c3sc51449h

Direct addition of water to alkenes to generate important chiral alcohols as key motif in a variety of natural products still remains a challenge in organic chemistry. Here, we report the first enantioselective artificial metallo-hydratase, based on the transcription factor LmrR, which catalyses the conjugate addition of water to generate chiral β-hydroxy ketones with enantioselectivities up to 84% ee. A mutagenesis study revealed that an aspartic acid and a phenylalanine located in the active site play a key role in achieving efficient catalysis and high enantioselectivities.


Metal: Cu
Ligand type: Phenanthroline
Host protein: LmrR
Anchoring strategy: Covalent
Optimization: Genetic
Max TON: 30
ee: 84
PDB: 3F8B
Notes: ---

An Enantioselective Artificial Suzukiase Based on the Biotin–Streptavidin Technology

Ward, T.R.

Chem. Sci. 2016, 7, 673-677, 10.1039/c5sc03116h

Introduction of a biotinylated monophosphine palladium complex within streptavidin affords an enantioselective artificial Suzukiase. Site-directed mutagenesis allowed the optimization of the activity and the enantioselectivity of this artificial metalloenzyme. A variety of atropisomeric biaryls were produced in good yields and up to 90% ee.


Metal: Pd
Ligand type: Allyl; Phosphine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 88
ee: 80
PDB: ---
Notes: ---

Metal: Pd
Ligand type: Allyl; Carbene
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 5
ee: ---
PDB: ---
Notes: ---

A Structural View of Synthetic Cofactor Integration into [FeFe]-Hydrogenases

Apfel, U.-P.; Happe, T.; Kurisu, G.

Chem. Sci. 2016, 7, 959-968, 10.1039/C5SC03397G

Crystal structures of semisynthetic [FeFe]-hydrogenases with variations in the [2Fe] cluster show little structural differences despite strong effects on activity.


Metal: Fe
Ligand type: CN; CO; Dithiolate
Anchoring strategy: Dative
Optimization: Chemical
Reaction: H2 evolution
Max TON: ---
ee: ---
PDB: 4XDC
Notes: H2 evolution activity of the ArM: 2874 (mmol H2)*min-1*(mg protein)-1.

Design of an Enantioselective Artificial Metallo-Hydratase Enzyme Containing an Unnatural Metal-Binding Amino Acid

Maréchal, J.-D.; Roelfes, G.

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.


Metal: Cu
Ligand type: Bipyridine
Host protein: LmrR
Anchoring strategy: ---
Optimization: Genetic
Reaction: Hydration
Max TON: 9
ee: 64
PDB: ---
Notes: ---

E. coli Surface Display of Streptavidin for Directed Evolution of an Allylic Deallylase

Ward, T.R.

Chem. Sci. 2018, 9, 5383-5388, 10.1039/c8sc00484f

Artificial metalloenzymes (ArMs hereafter) combine attractive features of both homogeneous catalysts and enzymes and offer the potential to implement new-to-nature reactions in living organisms. Herein we present an E. coli surface display platform for streptavidin (Sav hereafter) relying on an Lpp-OmpA anchor. The system was used for the high throughput screening of a bioorthogonal CpRu-based artificial deallylase (ADAse) that uncages an allylcarbamate-protected aminocoumarin 1. Two rounds of directed evolution afforded the double mutant S112M–K121A that displayed a 36-fold increase in surface activity vs. cellular background and a 5.7-fold increased in vitro activity compared to the wild type enzyme. The crystal structure of the best ADAse reveals the importance of mutation S112M to stabilize the cofactor conformation inside the protein.


Metal: Ru
Ligand type: Cp; Quinoline
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Reaction: Deallylation
Max TON: 148
ee: ---
PDB: 6FH8
Notes: ---

Enzyme Repurposing of a Hydrolase as an Emergent Peroxidase Upon Metal Binding

Fujieda, N.; Ward, T.R.

Chem. Sci. 2015, 6, 4060-4065, 10.1039/c5sc01065a

Adding a metal cofactor to a protein bearing a latent metal binding site endows the macromolecule with nascent catalytic activity.


Metal: Cu
Ligand type: Amino acid
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 35
ee: ---
PDB: ---
Notes: ---

Human Carbonic Anhydrase II as Host Protein for the Creation of Artificial Metalloenzymes: The Asymmetric Transfer Hydrogenation of Imines

Ward, T.R.

Chem. Sci. 2013, 4, 3269, 10.1039/c3sc51065d

In the presence of human carbonic anhydrase II, aryl-sulfonamide-bearing IrCp* pianostool complexes catalyze the asymmetric transfer hydrogenation of imines. Critical cofactor–protein interactions revealed by the X-ray structure of [(η5-Cp*)Ir(pico 4)Cl] 9 ⊂ WT hCA II were genetically optimized to improve the catalytic performance of the artificial metalloenzyme (68% ee, kcat/KM 6.11 × 10−3 min−1 mM−1).


Metal: Ir
Ligand type: Amino-sulfonamide; Cp*
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 47
ee: 70
PDB: ---
Notes: ---

Novel Artificial Metalloenzymes by In Vivo Incorporation of Metal-Binding Unnatural Amino Acids

Roelfes, G.

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.


Metal: Cu
Ligand type: Bipyridine
Host protein: LmrR
Anchoring strategy: ---
Optimization: Genetic
Max TON: 10.4
ee: 83
PDB: 3F8B
Notes: ---

Ru–protein–Co Biohybrids Designed for Solar Hydrogen Production: Understanding Electron Transfer Pathways Related to Photocatalytic Function

Utschig, L.M.

Chem. Sci. 2016, 7, 7068-7078, 10.1039/c6sc03121h

A series of Ru–protein–Co biohybrids have been prepared using the electron transfer proteins ferredoxin (Fd) and flavodoxin (Fld) as scaffolds for photocatalytic hydrogen production. The light-generated charge separation within these hybrids has been monitored by transient optical and electron paramagnetic resonance spectroscopies. Two distinct electron transfer pathways are observed. The Ru–Fd–Co biohybrid produces up to 650 turnovers of H2 utilizing an oxidative quenching mechanism for Ru(II)* and a sequential electron transfer pathway via the native [2Fe–2S] cluster to generate a Ru(III)–Fd–Co(I) charge separated state that lasts for ∼6 ms. In contrast, a direct electron transfer pathway occurs for the Ru–ApoFld–Co biohybrid, which lacks an internal electron relay, generating Ru(I)–ApoFld–Co(I) charge separated state that persists for ∼800 μs and produces 85 turnovers of H2 by a reductive quenching mechanism for Ru(II)*. This work demonstrates the utility of protein architectures for linking donor and catalytic function via direct or sequential electron transfer pathways to enable stabilized charge separation which facilitates photocatalysis for solar fuel production.


Metal: Co
Ligand type: Oxime
Host protein: Ferredoxin (Fd)
Anchoring strategy: Dative
Optimization: Chemical
Reaction: H2 evolution
Max TON: 650
ee: ---
PDB: ---
Notes: Recalculated TON

Significant Improvement of Oxidase Activity Through the Genetic Incorporation of a Redox-Active Unnatural Amino Acid

Lu, Y.; Wang, J.

Chem. Sci. 2015, 6, 3881-3885, 10.1039/C5SC01126D

While nature employs various covalent and non-covalent strategies to modulate tyrosine (Y) redox potential and pKa in order to optimize enzyme activities, such approaches have not been systematically applied for the design of functional metalloproteins. Through the genetic incorporation of 3-methoxytyrosine (OMeY) into myoglobin, we replicated important features of cytochrome c oxidase (CcO) in this small soluble protein, which exhibits selective O2 reduction activity while generating a small amount of reactive oxygen species (ROS). These results demonstrate that the electron donating ability of a tyrosine residue in the active site is important for CcO function. Moreover, we elucidated the structural basis for the genetic incorporation of OMeY into proteins by solving the X-ray structure of OMeY specific aminoacyl-tRNA synthetase complexed with OMeY.


Metal: Cu
Ligand type: Amino acid
Host protein: Myoglobin (Mb)
Anchoring strategy: Dative
Optimization: Genetic
Reaction: O2 reduction
Max TON: >1100
ee: ---
PDB: ---
Notes: Reduction potential was lowered by incorporation of the unnatural amino acid 3-methoxy tyrosine.