18 publications

18 publications

A "Broad Spectrum" Carbene Transferase for Synthesis of Chiral α-Trifluoromethylated Organoborons

Roelfes, G.

ACS Cent. Sci. 2019, 5, 206-208, 10.1021/acscentsci.9b00015

Directed evolution generated an enzyme for the enantioselective synthesis of α-trifluoromethylated organoborons—potentially attractive synthons for fluorinated compounds.


Metal: Fe
Ligand type: Porphyrin
Host protein: Cytochrome c
Anchoring strategy: Native
Optimization: Genetic
Reaction: B-H insertion
Max TON: 2900
ee: 95
PDB: ---
Notes: ---

A Hydroxyquinoline‐Based Unnatural Amino Acid for the Design of Novel Artificial Metalloenzymes

Roelfes, G.

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: Cu
Ligand type: Hydroxyquinoline
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: 11
ee: 51
PDB: 3F8B
Notes: Also used Rh, but no reaction detected.

Metal: Cu
Ligand type: Hydroxyquinoline
Anchoring strategy: Supramolecular
Optimization: Genetic
Reaction: Water addition
Max TON: ---
ee: ---
PDB: 3F8B
Notes: ---

Metal: Zn
Ligand type: Hydroxyquinoline
Anchoring strategy: Supramolecular
Optimization: Genetic
Reaction: C-H activation
Max TON: ---
ee: ---
PDB: 3F8B
Notes: ---

A Metal Ion Regulated Artificial Metalloenzyme

Roelfes, G.

Dalton Trans. 2017, 46, 4325-4330, 10.1039/C7DT00533D

An artificial metalloenzyme containing both a regulatory and a catalytic domain is selectively activated in presence of Fe2+ ions.


Metal: Fe
Ligand type: Bypyridine
Anchoring strategy: Covalent
Optimization: Genetic
Max TON: 14
ee: 75
PDB: ---
Notes: ---

Metal: Zn
Ligand type: Bypyridine
Anchoring strategy: Covalent
Optimization: Genetic
Max TON: 6
ee: 80
PDB: ---
Notes: ---

An Artificial Heme Enzyme for Cyclopropanation Reactions

Roelfes, G.

Angew. Chem. Int. Ed. 2018, 57, 7785-7789, 10.1002/anie.201802946

An artificial heme enzyme was created through self‐assembly from hemin and the lactococcal multidrug resistance regulator (LmrR). The crystal structure shows the heme bound inside the hydrophobic pore of the protein, where it appears inaccessible for substrates. However, good catalytic activity and moderate enantioselectivity was observed in an abiological cyclopropanation reaction. We propose that the dynamic nature of the structure of the LmrR protein is key to the observed activity. This was supported by molecular dynamics simulations, which showed transient formation of opened conformations that allow the binding of substrates and the formation of pre‐catalytic structures.


Metal: Fe
Ligand type: Protoporphyrin IX
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Cyclopropanation
Max TON: 449
ee: 51
PDB: 6FUU
Notes: ---

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
Anchoring strategy: Covalent
Optimization: Genetic
Max TON: 30
ee: 84
PDB: 3F8B
Notes: ---

Artificial Metalloenzymes

Review

Roelfes, G.

ChemCatChem 2010, 2, 916-927, 10.1002/cctc.201000011

Artificial metalloenzymes have emerged as a promising approach to merge the attractive properties of homogeneous catalysis and biocatalysis. The activity and selectivity, including enantioselectivity, of natural metalloenzymes are due to the second coordination sphere interactions provided by the protein. Artificial metalloenzymes aim at harnessing second coordination sphere interactions to create transition metal complexes that display enzyme‐like activities and selectivities. In this Review, the various approaches that can be followed for the design and optimization of an artificial metalloenzyme are discussed. An overview of the synthetic transformations that have been achieved using artificial metalloenzymes is provided, with a particular focus on recent developments. Finally, the role that the second coordination sphere plays in artificial metalloenzymes and their potential for synthetic applications are evaluated.


Notes: ---

Artificial Metalloenzymes based on TetR Proteins and Cu(II) for Enantioselective Friedel‐Crafts Alkylation Reactions

Roelfes, G.

ChemCatChem 2020, 12, 3190-3194, 10.1002/cctc.202000245

The supramolecular approach is among the most convenient methodologies for creating artificial metalloenzymes (ArMs). Usually this approach involves the binding of a transition metal ion complex to a biomolecular scaffold via its ligand, which also modulates the catalytic properties of the metal ion. Herein, we report ArMs based on the proteins CgmR, RamR and QacR from the TetR family of multidrug resistance regulators (MDRs) and Cu2+ ions, assembled without the need of a ligand. These ArMs catalyze the enantioselective vinylogous Friedel-Crafts alkylation reaction with up to 75 % ee. Competition experiments with ethidium and rhodamine 6G confirm that the reactions occur in the chiral environment of the hydrophobic pocket. It is proposed that the Cu2+-substrate complex is bound via a combination of electrostatic and π-stacking interactions provided by the second coordination sphere. This approach constitutes a fast and straightforward way to assemble metalloenzymes and may facilitate future optimization of the protein scaffolds via mutagenesis or directed evolution approaches.


Metal: Cu
Ligand type: Amino acid
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Max TON: 78
ee: 75
PDB: 1JTY
Notes: ---

Artificial Metalloenzymes for Asymmetric Catalysis by Creation of Novel Active Sites in Protein and DNA Scaffolds

Review

Roelfes, G.

Isr. J. Chem. 2015, 55, 21-31, 10.1002/ijch.201400094

Artificial metalloenzymes have emerged as a promising new approach to asymmetric catalysis. In our group, we are exploring novel artificial metalloenzyme designs involving creation of a new active site in a protein or DNA scaffold that does not have an existing binding pocket. In this review, we give an overview of the developments in the two approaches to artificial metalloenzymes for asymmetric catalysis investigated in our group: creation of a novel active site on a peptide or protein dimer interface and using DNA as a scaffold for artificial metalloenzymes.


Notes: ---

Artificial Metalloenzymes for Enantioselective Catalysis

Review

Roelfes, G.

Curr. Opin. Chem. Biol. 2014, 19, 135-143, 10.1016/j.cbpa.2014.02.002

Artificial metalloenzymes have emerged over the last decades as an attractive approach towards combining homogeneous catalysis and biocatalysis. A wide variety of catalytic transformations have been established by artificial metalloenzymes, thus establishing proof of concept. The field is now slowly transforming to take on new challenges. These include novel designs, novel catalytic reactions, some of which have no equivalent in both homogenous catalysis and biocatalysis and the incorporation of artificial metalloenzymes in chemoenzymatic cascades. Some of these developments represent promising steps towards integrating artificial metalloenzymes in biological systems. This review will focus on advances in this field and perspectives discussed.


Notes: ---

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
Anchoring strategy: ---
Optimization: Genetic
Reaction: Hydration
Max TON: 9
ee: 64
PDB: ---
Notes: ---

Enantioselective Artificial Metalloenzymes by Creation of a Novel Active Site at the Protein Dimer Interface

Roelfes, G.

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).


Metal: Cu
Ligand type: Bipyridine; Phenanthroline
Anchoring strategy: Covalent
Optimization: Genetic
Max TON: 32.7
ee: 97
PDB: 3F8B
Notes: ---

Hybrid Catalysts as Lewis Acid

Review

Roelfes, G.

Artificial Metalloenzymes and MetalloDNAzymes in Catalysis: From Design to Applications 2018, 225-251, 10.1002/9783527804085.ch8

Lewis acid catalysis is undisputedly of great significance for synthetic chemistry. Hence, many hybrid catalysts have been designed that can function as Lewis acid. These hybrid catalysts are based on DNA, protein, or peptide scaffolds. In this chapter an overview of the hybrid catalysts reported for three important classes of Lewis acid‐catalyzed reactions is given: C–C bond‐forming reactions, C–X bond‐forming reactions, and hydrolysis reactions.


Notes: Book chapter

In Vivo Assembly of Artificial Metalloenzymes and Application in Whole‐Cell Biocatalysis

Roelfes, G.

Angew. Chem. Int. Ed. 2021, 60, 5913-5920, 10.1002/anie.202014771

We report the supramolecular assembly of artificial metalloenzymes (ArMs), based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneous copper(II)–phenanthroline complex, in the cytoplasm of E. coli cells. A combination of catalysis, cell-fractionation, and inhibitor experiments, supplemented with in-cell solid-state NMR spectroscopy, confirmed the in-cell assembly. The ArM-containing whole cells were active in the catalysis of the enantioselective Friedel–Crafts alkylation of indoles and the Diels–Alder reaction of azachalcone with cyclopentadiene. Directed evolution resulted in two different improved mutants for both reactions, LmrR_A92E_M8D and LmrR_A92E_V15A, respectively. The whole-cell ArM system required no engineering of the microbial host, the protein scaffold, or the cofactor to achieve ArM assembly and catalysis. We consider this a key step towards integrating abiological catalysis with biosynthesis to generate a hybrid metabolism.


Metal: Cu
Ligand type: Phenanthroline
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: ---
ee: 98
PDB: 3F8F
Notes: ---

Metal: Cu
Ligand type: Phenanthroline
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: ---
ee: 84
PDB: 3F8F
Notes: ---

LmrR: A Privileged Scaffold for Artificial Metalloenzymes

Review

Roelfes, G.

Acc. Chem. Res. 2019, 52, 545-556, 10.1021/acs.accounts.9b00004

The biotechnological revolution has made it possible to create enzymes for many reactions by directed evolution. However, because of the immense number of possibilities, the availability of enzymes that possess a basal level of the desired catalytic activity is a prerequisite for success. For new-to-nature reactions, artificial metalloenzymes (ARMs), which are rationally designed hybrids of proteins and catalytically active transition-metal complexes, can be such a starting point. This Account details our efforts toward the creation of ARMs for the catalysis of new-to-nature reactions. Key to our approach is the notion that the binding of substrates, that is, effective molarity, is a key component to achieving large accelerations in catalysis. For this reason, our designs are based on the multidrug resistance regulator LmrR, a dimeric transcription factor with a large, hydrophobic binding pocket at its dimer interface. In this pocket, there are two tryptophan moieties, which are important for promiscuous binding of planar hydrophobic conjugated compounds by π-stacking. The catalytic machinery is introduced either by the covalent linkage of a catalytically active metal complex or via the ligand or supramolecular assembly, taking advantage of the two central tryptophan moieties for noncovalent binding of transition-metal complexes. Designs based on the chemical modification of LmrR were successful in catalysis, but this approach proved too laborious to be practical. Therefore, expanded genetic code methodologies were used to introduce metal binding unnatural amino acids during LmrR biosynthesis in vivo. These ARMs have been successfully applied in Cu(II) catalyzed Friedel–Crafts alkylation of indoles. The extension to MDRs from the TetR family resulted in ARMs capable of providing the opposite enantiomer of the Friedel–Crafts product. We have employed a computationally assisted redesign of these ARMs to create a more active and selective artificial hydratase, introducing a glutamate as a general base at a judicious position so it can activate and direct the incoming water nucleophile. A supramolecularly assembled ARM from LmrR and copper(II)–phenanthroline was successful in Friedel–Crafts alkylation reactions, giving rise to up to 94% ee. Also, hemin was bound, resulting in an artificial heme enzyme for enantioselective cyclopropanation reactions. The importance of structural dynamics of LmrR was suggested by computational studies, which showed that the pore can open up to allow access of substrates to the catalytic iron center, which, according to the crystal structure, is deeply buried inside the protein. Finally, the assembly approaches were combined to introduce both a catalytic and a regulatory domain, resulting in an ARM that was specifically activated in the presence of Fe(II) salts but not Zn(II) salts. Our work demonstrates that LmrR is a privileged scaffold for ARM design: It allows for multiple assembly methods and even combinations of these, it can be applied in a variety of different catalytic reactions, and it shows significant structural dynamics that contribute to achieving the desired catalytic activity. Moreover, both the creation via expanded genetic code methods as well as the supramolecular assembly make LmrR-based ARMs highly suitable for achieving the ultimate goal of the integration of ARMs in biosynthetic pathways in vivo to create a hybrid metabolism.


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
Anchoring strategy: ---
Optimization: Genetic
Max TON: 10.4
ee: 83
PDB: 3F8B
Notes: ---

Supramolecular Assembly of Artificial Metalloenzymes Based on the Dimeric Protein LmrR as Promiscuous Scaffold

Roelfes, G.

J. Am. Chem. Soc. 2015, 137, 9796-9799, 10.1021/jacs.5b05790

Supramolecular anchoring of transition metal complexes to a protein scaffold is an attractive approach to the construction of artificial metalloenzymes since this is conveniently achieved by self-assembly. Here, we report a novel design for supramolecular artificial metalloenzymes that exploits the promiscuity of the central hydrophobic cavity of the transcription factor Lactococcal multidrug resistance Regulator (LmrR) as a generic binding site for planar coordination complexes that do not provide specific protein binding interactions. The success of this approach is manifested in the excellent enantioselectivities that are achieved in the Cu(II) catalyzed enantioselective Friedel–Crafts alkylation of indoles.


Metal: Cu
Ligand type: Phenanthroline
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: 11.1
ee: 94
PDB: 3F8B
Notes: ---

Synergistic Catalysis in an Artificial Enzyme by Simultaneous Action of Two Abiological Catalytic Sites

Roelfes, G.

Nat. Catal. 2020, 3, 289-294, 10.1038/s41929-019-0420-6

Artificial enzymes, which are hybrids of proteins with abiological catalytic groups, have emerged as a powerful approach towards the creation of enzymes for new-to-nature reactions. Typically, only a single abiological catalytic moiety is incorporated. Here we introduce a design of an artificial enzyme that comprises two different abiological catalytic moieties and show that these can act synergistically to achieve high activity and enantioselectivity (up to >99% e.e.) in the catalysed Michael addition reaction. The design is based on the lactococcal multidrug resistance regulator as the protein scaffold and combines a genetically encoded unnatural p-aminophenylalanine residue (which activates an enal through iminium ion formation) and a supramolecularly bound Lewis acidic Cu(ii) complex (which activates the Michael donor by enolization and delivers it to one preferred prochiral face of the activated enal). This study demonstrates that synergistic combination of abiological catalytic groups is a robust way to achieve catalysis that is normally outside of the realm of artificial enzymes.


Metal: Cu
Ligand type: Amino acid
Anchoring strategy: Covalent; Supramolecular
Optimization: Genetic
Reaction: Michael addition
Max TON: ---
ee: >99
PDB: ---
Notes: 6:1 d.r

The Importance of Catalytic Promiscuity for Enzyme Design and Evolution

Review

Mayer, C.; Roelfes, G.

Nat. Rev. Chem. 2019, 3, 687-705, 10.1038/s41570-019-0143-x

The ability of one enzyme to catalyse multiple, mechanistically distinct transformations likely played a crucial role in organisms’ abilities to adapt to changing external stimuli in the past and can still be observed in extant enzymes. Given the importance of catalytic promiscuity in nature, enzyme designers have recently begun to create catalytically promiscuous enzymes in order to expand the canon of transformations catalysed by proteins. This article aims to both critically review different strategies for the design of enzymes that display catalytic promiscuity for new-to-nature reactions and highlight the successes of subsequent directed-evolution efforts to fine-tune these novel reactivities. For the former, we put a particular emphasis on the creation, stabilization and repurposing of reaction intermediates, which are key for unlocking new activities in an existing or designed active site. For the directed evolution of the resulting catalysts, we contrast approaches for enzyme design that make use of components found in nature and those that achieve new reactivities by incorporating synthetic components. Following the critical analysis of selected examples that are now available, we close this Review by providing a set of considerations and design principles for enzyme engineers, which will guide the future generation of efficient artificial enzymes for synthetically useful, abiotic transformations.


Notes: ---