64 publications

64 publications

A Cell-Penetrating Artificial Metalloenzyme Regulates a Gene Switch in a Designer Mammalian Cell

Fussenegger, M.; Matile, S.; Ward, T. R.

Nat. Commun., 2018, 10.1038/s41467-018-04440-0

Complementing enzymes in their native environment with either homogeneous or heterogeneous catalysts is challenging due to the sea of functionalities present within a cell. To supplement these efforts, artificial metalloenzymes are drawing attention as they combine attractive features of both homogeneous catalysts and enzymes. Herein we show that such hybrid catalysts consisting of a metal cofactor, a cell-penetrating module, and a protein scaffold are taken up into HEK-293T cells where they catalyze the uncaging of a hormone. This bioorthogonal reaction causes the upregulation of a gene circuit, which in turn leads to the expression of a nanoluc-luciferase. Relying on the biotin–streptavidin technology, variation of the biotinylated ruthenium complex: the biotinylated cell-penetrating poly(disulfide) ratio can be combined with point mutations on streptavidin to optimize the catalytic uncaging of an allyl-carbamate-protected thyroid hormone triiodothyronine. These results demonstrate that artificial metalloenzymes offer highly modular tools to perform bioorthogonal catalysis in live HEK cells.


Metal: Ru
Ligand type: Cp; Quinoline
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Reaction: Deallylation
Max TON: 33
ee: ---
PDB: ---
Notes: ---

A Cofactor Approach to Copper-Dependent Catalytic Antibodies

Janda, K. D.; Nicholas, K. M.

Proc. Natl. Acad. Sci. U. S. A., 2002, 10.1073/pnas.052001099

A strategy for the preparation of semisynthetic copper(II)-based catalytic metalloproteins is described in which a metal-binding bis-imidazole cofactor is incorporated into the combining site of the aldolase antibody 38C2. Antibody 38C2 features a large hydrophobic-combining site pocket with a highly nucleophilic lysine residue, LysH93, that can be covalently modified. A comparison of several lactone and anhydride reagents shows that the latter are the most effective and general derivatizing agents for the 38C2 Lys residue. A bis-imidazole anhydride (5) was efficiently prepared from N-methyl imidazole. The 38C2–5-Cu conjugate was prepared by either (i) initial derivatization of 38C2 with 5 followed by metallation with CuCl2, or (ii) precoordination of 5 with CuCl2 followed by conjugation with 38C2. The resulting 38C2–5-Cu conjugate was an active catalyst for the hydrolysis of the coordinating picolinate ester 11, following Michaelis–Menten kinetics [kcat(11) = 2.3 min−1 and Km(11) 2.2 mM] with a rate enhancement [kcat(11)kuncat(11)] of 2.1 × 105. Comparison of the second-order rate constants of the modified 38C2 and the Cu(II)-bis-imidazolyl complex k(6-CuCl2) gives a rate enhancement of 3.5 × 104 in favor of the antibody complex with an effective molarity of 76.7 M, revealing a significant catalytic benefit to the binding of the bis-imidazolyl ligand into 38C2.


Metal: Cu
Ligand type: Bisimidazol
Host protein: Antibody 38C2
Anchoring strategy: Covalent
Optimization: Genetic
Max TON: ---
ee: ---
PDB: ---
Notes: ---

A Designed Metalloenzyme Achieving the Catalytic Rate of a Native Enzyme

Lu, Y.; Wang, J.

J. Am. Chem. Soc., 2015, 10.1021/jacs.5b07119

Terminal oxidases catalyze four-electron reduction of oxygen to water, and the energy harvested is utilized to drive the synthesis of adenosine triphosphate. While much effort has been made to design a catalyst mimicking the function of terminal oxidases, most biomimetic catalysts have much lower activity than native oxidases. Herein we report a designed oxidase in myoglobin with an O2 reduction rate (52 s–1) comparable to that of a native cytochrome (cyt) cbb3 oxidase (50 s–1) under identical conditions. We achieved this goal by engineering more favorable electrostatic interactions between a functional oxidase model designed in sperm whale myoglobin and its native redox partner, cyt b5, resulting in a 400-fold electron transfer (ET) rate enhancement. Achieving high activity equivalent to that of native enzymes in a designed metalloenzyme offers deeper insight into the roles of tunable processes such as ET in oxidase activity and enzymatic function and may extend into applications such as more efficient oxygen reduction reaction catalysts for biofuel cells.


Metal: Cu
Ligand type: Amino acid
Host protein: Myoglobin (Mb)
Anchoring strategy: Dative
Optimization: Genetic
Reaction: O2 reduction
Max TON: ---
ee: ---
PDB: ---
Notes: O2 reduction rates of 52 s-1 were achieved in combination with the native redox partner cyt b5.

A Designed Supramolecular Protein Assembly with In Vivo Enzymatic Activity

Tezcan, F. A.

Science, 2014, 10.1126/science.1259680

The generation of new enzymatic activities has mainly relied on repurposing the interiors of preexisting protein folds because of the challenge in designing functional, three-dimensional protein structures from first principles. Here we report an artificial metallo-β-lactamase, constructed via the self-assembly of a structurally and functionally unrelated, monomeric redox protein into a tetrameric assembly that possesses catalytic zinc sites in its interfaces. The designed metallo-β-lactamase is functional in the Escherichia coli periplasm and enables the bacteria to survive treatment with ampicillin. In vivo screening of libraries has yielded a variant that displays a catalytic proficiency [(kcat/Km)/kuncat] for ampicillin hydrolysis of 2.3 × 106 and features the emergence of a highly mobile loop near the active site, a key component of natural β-lactamases to enable substrate interactions.


Metal: Zn
Ligand type: Amino acid
Host protein: Cytochrome cb562
Anchoring strategy: Dative
Optimization: Genetic
Max TON: ---
ee: ---
PDB: 4U9E
Notes: ---

A Dual Anchoring Strategy for the Localization and Activation of Artificial Metalloenzymes Based on the Biotin−Streptavidin Technology

Ward, T. R.

J. Am. Chem. Soc., 2013, 10.1021/ja309974s

Artificial metalloenzymes result from anchoring an active catalyst within a protein environment. Toward this goal, various localization strategies have been pursued: covalent, supramolecular, or dative anchoring. Herein we show that introduction of a suitably positioned histidine residue contributes to firmly anchor, via a dative bond, a biotinylated rhodium piano stool complex within streptavidin. The in silico design of the artificial metalloenzyme was confirmed by X-ray crystallography. The resulting artificial metalloenzyme displays significantly improved catalytic performance, both in terms of activity and selectivity in the transfer hydrogenation of imines. Depending on the position of the histidine residue, both enantiomers of the salsolidine product can be obtained.


Metal: Ir
Ligand type: Amino acid; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: 14
ee: 11
PDB: ---
Notes: ---

Metal: Rh
Ligand type: Amino acid; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: 100
ee: 79
PDB: ---
Notes: ---

Alteration of the Oxygen-Dependent Reactivity of De Novo Due Ferri Proteins

DeGrado, W. F.

Nat. Chem., 2012, 10.1038/NCHEM.1454

De novo proteins provide a unique opportunity to investigate the structure–function relationships of metalloproteins in a minimal, well-defined and controlled scaffold. Here, we describe the rational programming of function in a de novo designed di-iron carboxylate protein from the Due Ferri family. Originally created to catalyse the O2-dependent, two-electron oxidation of hydroquinones, the protein was reprogrammed to catalyse the selective N-hydroxylation of arylamines by remodelling the substrate access cavity and introducing a critical third His ligand to the metal-binding cavity. Additional second- and third-shell modifications were required to stabilize the His ligand in the core of the protein. These structural changes resulted in at least a 106-fold increase in the relative rate between the arylamine N-hydroxylation and hydroquinone oxidation reactions. This result highlights the potential for using de novo proteins as scaffolds for future investigations of the geometric and electronic factors that influence the catalytic tuning of di-iron active sites.


Metal: Fe
Ligand type: Amino acid
Host protein: Due Ferro 1
Anchoring strategy: Dative
Optimization: Genetic
Reaction: N-Hydroxylation
Max TON: ---
ee: ---
PDB: 2LFD
Notes: ---

A Metal Ion Regulated Artificial Metalloenzyme

Roelfes, G.

Dalton Trans., 2017, 10.1039/C7DT00533D

Regulation of enzyme activity is essential in living cells. The rapidly increasing number of designer enzymes with new-to-nature activities makes it necessary to develop novel strategies for controlling their catalytic activity. Here we present the development of a metal ion regulated artificial metalloenzyme created by combining two anchoring strategies, covalent and supramolecular, for introducing a regulatory and a catalytic site, respectively. This artificial metalloenzyme is activated in the presence of Fe2+ ions, but only marginally in the presence of Zn2+.


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

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

An Artificial Di-Iron Oxo-Orotein with Phenol Oxidase Activity

DeGrado, W. F.; Lombardi, A.

Nat. Chem. Biol., 2009, 10.1038/nchembio.257

Here we report the de novo design and NMR structure of a four-helical bundle di-iron protein with phenol oxidase activity. The introduction of the cofactor-binding and phenol-binding sites required the incorporation of residues that were detrimental to the free energy of folding of the protein. Sufficient stability was, however, obtained by optimizing the sequence of a loop distant from the active site.


Metal: Fe
Ligand type: Amino acid
Host protein: Due Ferro 1
Anchoring strategy: Dative
Optimization: Genetic
Reaction: Alcohol oxidation
Max TON: >50
ee: ---
PDB: 2KIK
Notes: kcat/KM ≈ 1380 M-1*min-1

Metal: Fe
Ligand type: Amino acid
Host protein: Due Ferro 1
Anchoring strategy: Dative
Optimization: Genetic
Reaction: Amine oxidation
Max TON: ---
ee: ---
PDB: 2KIK
Notes: kcat/KM ≈ 83 M-1*min-1

An Artificial Imine Reductase Based on the Ribonuclease S Scaffold

Ward, T. R.

ChemCatChem, 2014, 10.1002/cctc.201300995


Metal: Ir
Ligand type: Amino acid; Cp*
Host protein: Ribonuclease S
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: 4
ee: 18
PDB: ---
Notes: ---

An Artificial Metalloenzyme: Creation of a Designed Copper Binding Site in a Thermostable Protein

Reetz, M. T.

Angew. Chem., Int. Ed., 2010, 10.1002/anie.201002106

Guided by nature: A designed binding site comprising the His/His/Asp motif for CuII complexation has been constructed in a robust protein by site‐specific mutagenesis (see picture). The artificial metalloenzyme catalyzes an enantioselective Diels–Alder reaction.


Metal: Cu
Ligand type: Amino acid
Host protein: tHisF
Anchoring strategy: Dative
Optimization: Genetic
Max TON: 6.7
ee: 46
PDB: ---
Notes: ---

An Enantioselective Artificial Metallo-Hydratase

Roelfes, G.

Chem. Sci., 2013, 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: ---

Artificial Dicopper Oxidase: Rational Reprogramming of Bacterial Metallo- b-lactamase into a Catechol Oxidase

Fujieda, N.; Itoh, S.

Chem. - Asian J., 2012, 10.1002/asia.201101014


Metal: Cu
Ligand type: Amino acid
Host protein: β-lactamase
Anchoring strategy: Dative
Optimization: Genetic
Reaction: Catechol oxidation
Max TON: ---
ee: ---
PDB: 2FU7
Notes: ---

Artificial Metalloenzyme for Enantioselective Sulfoxidation Based on Vanadyl-Loaded Streptavidin

Ward, T. R.

J. Am. Chem. Soc., 2008, 10.1021/ja8017219


Metal: V
Ligand type: Water
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Reaction: Sulfoxidation
Max TON: 27
ee: 93
PDB: ---
Notes: ---

Artificial Metalloenzymes based on Protein Cavities: Exploring the Effect of Altering the Metal Ligand Attachment Position by Site Directed Mutagenesis

Distefano, M. D.

Bioorg. Med. Chem. Lett., 1999, 10.1016/S0960-894X(98)00684-2


Metal: Cu
Ligand type: Phenanthroline
Anchoring strategy: Covalent
Optimization: Genetic
Max TON: 1 to 4
ee: 61 to 94
PDB: ---
Notes: Varied attachment position

A Site-Selective Dual Anchoring Strategy for Artificial Metalloprotein Design

Lu, Y.

J. Am. Chem. Soc., 2004, 10.1021/ja046908x

Introducing nonnative metal ions or metal-containing prosthetic groups into a protein can dramatically expand the repertoire of its functionalities and thus its range of applications. Particularly challenging is the control of substrate-binding and thus reaction selectivity such as enantioselectivity. To meet this challenge, both non-covalent and single-point attachments of metal complexes have been demonstrated previously. Since the protein template did not evolve to bind artificial metal complexes tightly in a single conformation, efforts to restrict conformational freedom by modifying the metal complexes and/or the protein are required to achieve high enantioselectivity using the above two strategies. Here we report a novel site-selective dual anchoring (two-point covalent attachment) strategy to introduce an achiral manganese salen complex (Mn(salen)), into apo sperm whale myoglobin (Mb) with bioconjugation yield close to 100%. The enantioselective excess increases from 0.3% for non-covalent, to 12.3% for single point, and to 51.3% for dual anchoring attachments. The dual anchoring method has the advantage of restricting the conformational freedom of the metal complex in the protein and can be generally applied to protein incorporation of other metal complexes with minimal structural modification to either the metal complex or the protein.


Metal: Mn
Ligand type: Salen
Host protein: Myoglobin (Mb)
Anchoring strategy: Covalent
Optimization: Genetic
Reaction: Sulfoxidation
Max TON: 3.9
ee: 51
PDB: 1MBO
Notes: Sperm whale myoglobin

A Well-Defined Osmium–Cupin Complex: Hyperstable Artificial Osmium Peroxygenase

Fujieda, N.; Itoh, S.

J. Am. Chem. Soc., 2017, 10.1021/jacs.7b00675


Metal: Os
Ligand type: Amino acid
Host protein: TM1459 cupin
Anchoring strategy: Metal substitution
Optimization: Genetic
Reaction: Dihydroxylation
Max TON: 45
ee: ---
PDB: 5WSE
Notes: Exclusively cis dihydroxylation product obtained

Metal: Os
Ligand type: Amino acid
Host protein: TM1459 cupin
Anchoring strategy: Metal substitution
Optimization: Genetic
Reaction: Dihydroxylation
Max TON: 45
ee: ---
PDB: 5WSF
Notes: Exclusively cis dihydroxylation product obtained

Biotinylated Rh(III) Complexes in Engineered Streptavidin for Accelerated Asymmetric C–H Activation

Rovis, T.; Ward, T. R.

Science, 2012, 10.1126/science.1226132


Metal: Rh
Ligand type: Amino acid; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Reaction: C-H activation
Max TON: 95
ee: 82
PDB: ---
Notes: ---

Capture and Characterization of a Reactive Haem– Carbenoid Complex in an Artificial Metalloenzyme

Hilvert, D.

Nat. Catal., 2018, 10.1038/s41929-018-0105-6


Metal: Fe
Host protein: Myoglobin (Mb)
Anchoring strategy: ---
Optimization: Genetic
Reaction: Cyclopropanation
Max TON: 1000
ee: 99
PDB: 6F17
Notes: Structure of the Mb*(NMH) haem-iron complex

Metal: Fe
Host protein: Myoglobin (Mb)
Anchoring strategy: ---
Optimization: Genetic
Reaction: Cyclopropanation
Max TON: 1000
ee: 99
PDB: 6G5B
Notes: Structure of the Mb*(NMH) haem-iron–carbenoid complex

Catalytic Reduction of NO to N2O by a Designed Heme Copper Center in Myoglobin: Implications for the Role of Metal Ions

Lu, Y.

J. Am. Chem. Soc., 2006, 10.1021/ja058822p


Metal: Cu
Ligand type: Amino acid; Porphyrin
Host protein: Myoglobin (Mb)
Anchoring strategy: Dative
Optimization: Genetic
Max TON: 2400
ee: ---
PDB: ---
Notes: Sperm whale myoglobin

Chimeric Streptavidins as Host Proteins for Artificial Metalloenzymes

Ward, T. R.; Woolfson, D. N.

ACS Catal., 2018, 10.1021/acscatal.7b03773


Metal: Ir
Ligand type: Cp*; Diamine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: 970
ee: 13
PDB: ---
Notes: ---

Metal: Ir
Ligand type: Cp*; Diamine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: 158
ee: 82
PDB: ---
Notes: ---

Metal: Ru
Ligand type: Carbene
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Reaction: Olefin metathesis
Max TON: 105
ee: ---
PDB: ---
Notes: RCM, biotinylated Hoveyda-Grubbs second generation catalyst

Metal: ---
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Reaction: Anion-π catalysis
Max TON: 6
ee: 41
PDB: ---
Notes: No metal

Computational Insights on an Artificial Imine Reductase Based on the Biotin-Streptavidin Technology

Maréchal, J.-D.

ACS Catal., 2014, 10.1021/cs400921n


Metal: Ir
Ligand type: Cp*; Diamine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: ---
ee: 96
PDB: 3PK2
Notes: Prediction of the enantioselectivity by computational methods.

Computational Redesign of a Mononuclear Zinc Metalloenzyme for Organophosphate Hydrolysis

Baker, D.

Nat. Chem. Biol., 2012, 10.1038/NChemBio.777


Metal: Zn
Ligand type: Amino acid
Anchoring strategy: Dative
Optimization: Genetic
Max TON: >140
ee: ---
PDB: 3T1G
Notes: kcat/KM ≈ 104 M-1*s-1

Construction and In Vivo Assembly of a Catalytically Proficient and Hyperthermostable De Novo Enzyme

Anderson, J. L. R.

Nat. Commun., 2017, 10.1038/s41467-017-00541-4


Metal: Fe
Ligand type: Porphyrin
Anchoring strategy: Supramolecular
Optimization: Genetic
Reaction: Oxidation
Max TON: ---
ee: ---
PDB: ---
Notes: Oxidation of 2,2′-azino-bis(3-ethylbenzothiazo-line-6-sulfonic acid (ABTS)

Definite Coordination Arrangement of Organometallic Palladium Complexes Accumulated on the Designed Interior Surface of Apo-Ferritin

Ueno, T.

Chem. Commun., 2011, 10.1039/C0CC02221G


Metal: Pd
Ligand type: Allyl
Host protein: Ferritin
Anchoring strategy: Dative
Optimization: Genetic
Reaction: Suzuki coupling
Max TON: ---
ee: ---
PDB: ---
Notes: ---

De Novo Design of Catalytic Proteins

DeGrado, W. F.

Proc. Natl. Acad. Sci. U. S. A., 2004, 10.1073/pnas.0404387101


Metal: Fe
Ligand type: Amino acid
Host protein: Due Ferro 1
Anchoring strategy: Dative
Optimization: Genetic
Reaction: Alcohol oxidation
Max TON: >100
ee: ---
PDB: ---
Notes: kcat/KM ≈ 1540 M-1*min-1

Design and Evolution of New Catalytic Activity with an Existing Protein Scaffold

Kim, H. S.

Science, 2006, 10.1126/science.1118953


Metal: Zn
Ligand type: Amino acid
Host protein: Glyoxalase II (Human)
Anchoring strategy: Dative
Optimization: Genetic
Max TON: ---
ee: ---
PDB: 2F50
Notes: kcat/KM ≈ 184 M-1*s-1

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

Maréchal, J.-D.; Roelfes, G.

Chem. Sci., 2017, 10.1039/C7SC03477F


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

Design of a Switchable Eliminase

DeGrado, W. F.

Proc. Natl. Acad. Sci. U. S. A., 2011, 10.1073/pnas.1018191108


Metal: Ca
Ligand type: Amino acid
Anchoring strategy: Dative
Optimization: Genetic
Reaction: Kemp elimination
Max TON: >40
ee: ---
PDB: 2KZ2
Notes: Ca acts as allosteric regulator, catalytically active site contains no metal

Directed Evolution of Artificial Metalloenzymes for In Vivo Metathesis

Panke, S.; Ward, T. R.

Nature, 2016, 10.1038/nature19114


Metal: Ru
Ligand type: Carbene
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Reaction: Olefin metathesis
Max TON: 610
ee: ---
PDB: ---
Notes: Reaction in the periplasm

Directed Evolution of Hybrid Enzymes: Evolving Enantioselectivity of an Achiral Rh-Complex Anchored to a Protein

Reetz, M. T.

Chem. Commun., 2006, 10.1039/b610461d


Metal: Rh
Ligand type: COD; Phosphine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Reaction: Hydrogenation
Max TON: 4500
ee: 65
PDB: ---
Notes: ---