39 publications

39 publications

A Chaperonin as Protein Nanoreactor for Atom-Transfer Radical Polymerization

Bruns, N.

Angew. Chem., Int. Ed., 2013, 10.1002/anie.201306798

The group II chaperonin thermosome (THS) from the archaea Thermoplasma acidophilum is reported as nanoreactor for atom‐transfer radical polymerization (ATRP). A copper catalyst was entrapped into the THS to confine the polymerization into this protein cage. THS possesses pores that are wide enough to release polymers into solution. The nanoreactor favorably influenced the polymerization of N‐isopropyl acrylamide and poly(ethylene glycol)methylether acrylate. Narrowly dispersed polymers with polydispersity indices (PDIs) down to 1.06 were obtained in the protein nanoreactor, while control reactions with a globular protein–catalyst conjugate only yielded polymers with PDIs above 1.84.


Metal: Cu
Host protein: Thermosome (THS)
Anchoring strategy: Covalent
Optimization: ---
Reaction: Polymerization
Max TON: ---
ee: ---
PDB: ---
Notes: Non-ROMP

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 Functional Metalloenzyme that Reduces O2 to H2O with Over One Thousand Turnovers

Lu, Y.

Angew. Chem., Int. Ed., 2012, 10.1002/anie.201201981

Rational design of functional enzymes with a high number of turnovers is a challenge, especially those with a complex active site, such as respiratory oxidases. Introducing two His and one Tyr residues into myoglobin resulted in enzymes that reduce O2 to H2O with more than 1000 turnovers (red line, see scheme) and minimal release of reactive oxygen species. The positioning of the Tyr residue is critical for activity.


Metal: Cu
Ligand type: Amino acid
Host protein: Myoglobin (Mb)
Anchoring strategy: Dative
Optimization: Chemical & genetic
Max TON: 1056
ee: ---
PDB: 4FWX
Notes: Sperm whale myoglobin

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.

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 Copper Enzymes for Asymmetric Diels–AlderReactions

Kamer, P. C. J.; Laan, W.

ChemCatChem, 2012, 10.1002/cctc.201200671


Metal: Cu
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Max TON: 9.6
ee: 25
PDB: 1IKT
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 Diels–Alderase based on the Transmembrane Protein FhuA

Okuda, J.

Beilstein J. Org. Chem., 2016, 10.3762/bjoc.12.124


Metal: Cu
Ligand type: Terpyridine
Host protein: FhuA
Anchoring strategy: Cystein-maleimide
Optimization: Chemical
Max TON: ---
ee: ---
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

Artificial Metalloenzymes with the Neocarzinostatin Scaffold: Toward a Biocatalyst for the Diels–Alder Reaction

Mahy, J.-P.; Ricoux, R.

ChemBioChem, 2016, 10.1002/cbic.201500445


Metal: Cu
Ligand type: Phenanthroline
Anchoring strategy: Supramolecular
Optimization: ---
Max TON: 33
ee: ---
PDB: ---
Notes: Up to endo/exo ratio 62:38

A Semisynthetic Metalloenzyme based on a Protein Cavity that Catalyzes the Enantioselective Hydrolysis of Ester and Amide Substrates

Distefano, M. D.

J. Am. Chem. Soc., 1997, 10.1021/JA970820K

In an effort to prepare selective and efficient catalysts for ester and amide hydrolysis, we are designing systems that position a coordinated metal ion within a defined protein cavity. Here, the preparation of a protein-1,10-phenanthroline conjugate and the hydrolytic chemistry catalyzed by this construct are described. Iodoacetamido-1,10-phenanthroline was used to modify a unique cysteine residue in ALBP (adipocyte lipid binding protein) to produce the conjugate ALBP-Phen. The resulting material was characterized by electrospray mass spectrometry, UV/vis and fluorescence spectroscopy, gel filtration chromatography, and thiol titration. The stability of ALBP-Phen was evaluated by guanidine hydrochloride denaturation experiments, and the ability of the conjugate to bind Cu(II) was demonstrated by fluorescence spectroscopy. ALBP-Phen-Cu(II) catalyzes the enantioselective hydrolysis of several unactivated amino acid esters under mild conditions (pH 6.1, 25 °C) at rates 32−280-fold above the background rate in buffered aqueous solution. In 24 h incubations 0.70 to 7.6 turnovers were observed with enantiomeric excesses ranging from 31% ee to 86% ee. ALBP-Phen-Cu(II) also promotes the hydrolysis of an aryl amide substrate under more vigorous conditions (pH 6.1, 37 °C) at a rate 1.6 × 104-fold above the background rate. The kinetics of this amide hydrolysis reaction fit the Michaelis−Menten relationship characteristic of enzymatic processes. The rate enhancements for ester and amide hydrolysis reported here are 102−103 lower than those observed for free Cu(II) but comparable to those previously reported for Cu(II) complexes.


Metal: Cu
Ligand type: Phenanthroline
Anchoring strategy: Covalent
Optimization: ---
Max TON: 1 to 8
ee: 39 to 86
PDB: ---
Notes: ---

Autoxidation of Ascorbic Acid Catalyzed by a Semisynthetic Enzyme

Kaiser, E. T.

Biopolymers, 1990, 10.1002/bip.360290107


Metal: Cu
Ligand type: Bipyridine
Host protein: Papain (PAP)
Anchoring strategy: Covalent
Optimization: ---
Reaction: Oxidation
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Bimetallic Copper-Heme-Protein-DNA Hybrid Catalyst for Diels Alder Reaction

Fruk, L.; Niemeyer, C. M.

Croat. Chem. Acta, 2011, 10.5562/cca1828


Metal: Cu
Ligand type: Bipyridine
Host protein: Myoglobin (Mb)
Anchoring strategy: Supramolecular
Optimization: ---
Max TON: 7.1
ee: 18
PDB: ---
Notes: Horse heart myoglobin

Biosynthesis of a Site-Specific DNA Cleaving Protein

Schultz, P. G.

J. Am. Chem. Soc., 2008, 10.1021/ja804653f


Metal: Cu
Ligand type: Bipyridine
Anchoring strategy: ---
Optimization: Chemical & genetic
Max TON: ---
ee: ---
PDB: ---
Notes: Catabolite activator protein from E. coli

Metal: Fe
Ligand type: Bipyridine
Anchoring strategy: ---
Optimization: Chemical & genetic
Max TON: ---
ee: ---
PDB: ---
Notes: Catabolite activator protein from E. coli

Building Reactive Copper Centers in Human Carbonic Anhydrase II

Emerson, J. P.

J. Biol. Inorg. Chem., 2013, 10.1007/s00775-013-1009-1


Metal: Cu
Ligand type: Amino acid
Anchoring strategy: Dative
Optimization: ---
Reaction: Oxidation
Max TON: ---
ee: ---
PDB: 1RZC
Notes: Oxidation of 2-aminophenol with subsequent formation of 2-aminophenoxazinone. Reaction rate = 0.09 s-1

Carbene in Cupredoxin Protein Scaffolds: Replacement of a Histidine Ligand in the Active Site Substantially Alters Copper Redox Properties

Albrecht, M.; Paradisi, F.

Angew. Chem., Int. Ed., 2018, 10.1002/ange.201807168


Metal: Cu
Host protein: Azurin
Anchoring strategy: Dative
Optimization: Chemical & genetic
Reaction: Electron transfer
Max TON: ---
ee: ---
PDB: ---
Notes: ---

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

Chemical Conversion of a DNA-Binding Protein into a Site-Specific Nuclease

Sigman, D. S.

Science, 1987, 10.1126/science.2820056


Metal: Cu
Ligand type: Phenanthroline
Anchoring strategy: Covalent
Optimization: ---
Reaction: Oxidative cleavage
Max TON: <1
ee: ---
PDB: ---
Notes: Engineered sequence specificity

Construction of a Hybrid Biocatalyst Containing a Covalently-Linked Terpyridine Metal Complex within a Cavity of Aponitrobindin

Onoda, A.

J. Inorg. Biochem., 2016, 10.1016/j.jinorgbio.2015.12.026


Metal: Cu
Ligand type: Terpyridine
Host protein: Nitrobindin (Nb)
Anchoring strategy: Cystein-maleimide
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Construction of Robust Bio-Nanotubes using the Controlled Self-Assembly of Component Proteins of Bacteriophage T4

Ueno, T.

Small, 2010, 10.1002/smll.201000772


Metal: Cu
Ligand type: Flavin
Host protein: [(gp5βf)3]2
Anchoring strategy: Lysine-succinimide
Optimization: ---
Reaction: Cycloaddition
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Conversion of a Helix-Turn-Helix Motif Sequence-Specific DNA Binding Protein into a Site-Specific DNA Cleavage Agent

Ebright, R. H.; Gunasekeram, A.

Proc. Natl. Acad. Sci. U. S. A., 1990, 10.1073/pnas.87.8.2882


Metal: Cu
Ligand type: Phenanthroline
Anchoring strategy: Covalent
Optimization: ---
Reaction: Oxidative cleavage
Max TON: <1
ee: ---
PDB: ---
Notes: Engineered sequence specificity

Copper–Phthalocyanine Conjugates of Serum Albumins as Enantioselective Catalysts in Diels–Alder Reactions

Reetz, M. T.

Angew. Chem., Int. Ed., 2005, 10.1002/anie.200504561


Metal: Cu
Ligand type: Phthalocyanine
Anchoring strategy: Supramolecular
Optimization: Chemical
Max TON: 45.5
ee: 98
PDB: ---
Notes: ---

Defining the Role of Tyrosine and Rational Tuning of Oxidase Activity by Genetic Incorporation of Unnatural Tyrosine Analogs

Lu, Y.; Wang, J.

J. Am. Chem. Soc., 2015, 10.1021/ja5109936


Metal: Cu
Ligand type: Porphyrin
Host protein: Myoglobin (Mb)
Anchoring strategy: Dative
Optimization: Chemical & genetic
Max TON: 1200
ee: ---
PDB: 4FWX
Notes: Sperm whale myoglobin

Designing a Functional Type 2 Copper Center that has Nitrite Reductase Activity Within α-Helical Coiled Coils

Pecoraro, V. L.

Proc. Natl. Acad. Sci. U. S. A., 2012, 10.1073/pnas.1212893110


Metal: Cu
Ligand type: Amino acid
Host protein: TRI peptide
Anchoring strategy: Dative
Optimization: Chemical & genetic
Max TON: >5
ee: ---
PDB: ---
Notes: Nitrite reduction

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: ---

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

Roelfes, G.

Angew. Chem., Int. Ed., 2012, 10.1002/anie.201202070


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

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

Fujieda, N.; Ward, T. R.

Chem. Sci., 2015, 10.1039/c5sc01065a


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

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

Roelfes, G.

Chem. Sci., 2015, 10.1039/c4sc01525h


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

Peroxide Activation Regulated by Hydrogen Bonds within Artificial Cu Proteins

Borovik, A. S.

J. Am. Chem. Soc., 2017, 10.1021/jacs.7b10452


Metal: Cu
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Oxidation
Max TON: ---
ee: ---
PDB: 6ANX
Notes: ---