42 publications

42 publications

A Chaperonin as Protein Nanoreactor for Atom-Transfer Radical Polymerization

Bruns, N.

Angew. Chem. Int. Ed. 2014, 53, 1443-1447, 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, 99, 2648-2653, 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, 51, 5589-5592, 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, 137, 11570-11573, 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, 49, 5151-5155, 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, 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: ---

Artificial Copper Enzymes for Asymmetric Diels–AlderReactions

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

ChemCatChem 2013, 5, 1184-1191, 10.1002/cctc.201200671

The development of artificial copper enzymes from sterol carrier protein type 2 like domain (SCP‐2L) for the use in asymmetric catalysis was explored. For this purpose, proteins were modified with various nitrogen donor ligands. Maleimide‐containing ligands were found most suitable for selective cysteine bio‐conjugation. Fluorescence spectroscopy was used to confirm copper binding to an introduced phenanthroline ligand, which was introduced in two unique cysteine containing SCP‐2L mutants. Copper adducts of several modified SCP‐2L templates were applied in asymmetric Diels–Alder reactions. A clear influence of both the protein environment and the introduced ligand was found in the asymmetric Diels–Alder reaction between azachalcone and cyclopentadiene. A promising enantioselectivity of 25 % ee was obtained by using SCP‐2L V83C modified with phenanthroline–maleimide ligand. Good endo selectivity was observed for SCP‐2L modified with the dipicolylamine‐based nitrogen donor ligand. These artificial metalloenzymes provide a suitable starting point for the implementation of various available techniques to optimise the performance of this system.


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, 7, 1203-1207, 10.1002/asia.201101014

Teaching metalloenzymes new tricks: An artificial type III dicopper oxidase has been developed using a hydrolytic enzyme, metallo‐β‐lactamase, as a metal‐binding platform. The triple mutant D88G/S185H/P224G redesigned by computer‐assisted structural analysis showed spectroscopic features similar to those of type III copper proteins and exhibited a high catalytic activity in the oxidation of catechols under aerobic conditions.


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, 12, 1314-1321, 10.3762/bjoc.12.124

Copper(I) and copper(II) complexes were covalently linked to an engineered variant of the transmembrane protein Ferric hydroxamate uptake protein component A (FhuA ΔCVFtev). Copper(I) was incorporated using an N-heterocyclic carbene (NHC) ligand equipped with a maleimide group on the side arm at the imidazole nitrogen. Copper(II) was attached by coordination to a terpyridyl ligand. The spacer length was varied in the back of the ligand framework. These biohybrid catalysts were shown to be active in the Diels–Alder reaction of a chalcone derivative with cyclopentadiene to preferentially give the endo product.


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, 9, 79-84, 10.1016/S0960-894X(98)00684-2

In an effort to construct catalysts with enzyme-like properties, we are employing a small, cavity-containing protein as a scaffold for the attachment of catalytic groups. In earlier work we demonstrated that a phenanthroline ligand could be introduced into the cavity of the protein ALBP and used to catalyze ester hydrolysis. To examine the effect of positioning the phenanthroline catalyst at different locations wthin the protein cavity, three new constucts — Phen60, Phen72 and Phen104 — were prepared. Each new conjugate was characterized by UV/vis spectroscopy, fluorescence spectroscopy, guanidine hydrochloride denaturation, gel filtration chromatography, and CD spectroscopy to confirm the preparation of the desired contruct. Analysis of reactions containing Ala-OiPr showed that Phen60 catalyzed ester hydrolysis with less selectivity than ALBP-Phen while Phen72 promoted this same reaction with higher selectivity. Reactions with Tyr-OMe were catalyzed with higher selectivity by Phen60 and more rapidly by Phen104. These results demonstrate that both the rates and selectivities of hydrolysis reactions catalyzed by these constructs are dependent on the precise site of attachment of the metal ligand within the protein cavity.


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, 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: 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, 119, 11643-11652, 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: ---

Atroposelective Antibodies as a Designed Protein Scaffold for Artificial Metalloenzymes

Harada, A.; Yamaguchi, H.

Sci. Rep. 2019, 9, 10.1038/s41598-019-49844-0

Design and engineering of protein scaffolds are crucial to create artificial metalloenzymes. Herein we report the first example of C-C bond formation catalyzed by artificial metalloenzymes, which consist of monoclonal antibodies (mAbs) and C2 symmetric metal catalysts. Prepared as a tailored protein scaffold for a binaphthyl derivative (BN), mAbs bind metal catalysts bearing a 1,1?-bi-isoquinoline (BIQ) ligand to yield artificial metalloenzymes. These artificial metalloenzymes catalyze the Friedel-Crafts alkylation reaction. In the presence of mAb R44E1, the reaction proceeds with 88% ee. The reaction catalyzed by Cu-catalyst incorporated into the binding site of mAb R44E1 is found to show excellent enantioselectivity with 99% ee. The protein environment also enables the use of BIQ-based catalysts as asymmetric catalysts for the first time.


Metal: Cu; Pd; Pt
Ligand type: Bipyridine
Host protein: Antibody
Anchoring strategy: Antigen
Optimization: Genetic
Max TON: 2
ee: 88
PDB: ---
Notes: ---

Autoxidation of Ascorbic Acid Catalyzed by a Semisynthetic Enzyme

Kaiser, E.T.

Biopolymers 1990, 29, 39-43, 10.1002/bip.360290107

The semisyntehtic enzyme 6 was prepared by alkylation of the cysteine‐25 sulfhydryl group of papain with the bipyridine 5 and was shown to stoichiometrically bind copper ion; 7 catalyzed the autoxidation of ascorbic acid derivatives with saturation kinetics approximately 20‐fold faster than a model system using 3‐Cu(II).


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, 84, 269-275, 10.5562/cca1828

A bimetallic heme-DNA cofactor, containing an iron and a copper center, was synthesized for the design of novel hybrid catalysts for stereoselective synthesis. The cofactor was used for the reconstitution of apo-myoglobin. Both the cofactor alone and its myoglobin adduct were used to catalyze a model Diels Alder reaction. Stereoselectivity of this conversion was analyzed by chiral HPLC. Reactions carried out in the presence of myoglobin-heme-Cu-DNA catalyst showed greater product conversion and stereoselectivity than those carried out with the heme-Cu-DNA cofactor. This observation suggested that the protein shell plays a significant role in the catalytic conversion.


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, 130, 13194-13195, 10.1021/ja804653f

An E. coli catabolite activator protein (CAP) has been converted into a sequence-specific DNA cleaving protein by genetically introducing (2,2′-bipyridin-5-yl)alanine (Bpy-Ala) into the protein. The mutant CAP (CAP-K26Bpy-Ala) showed comparable binding affinity to CAP-WT for the consensus operator sequence. In the presence of Cu(II) and 3-mercaptopropionic acid, CAP-K26Bpy-Ala cleaves double-stranded DNA with high sequence specificity. This method should provide a useful tool for mapping the molecular details of protein−nucleic acid interactions.


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, 18, 595-598, 10.1007/s00775-013-1009-1

Reengineering metalloproteins to generate new biologically relevant metal centers is an effective a way to test our understanding of the structural and mechanistic features that steer chemical transformations in biological systems. Here, we report thermodynamic data characterizing the formation of two type-2 copper sites in carbonic anhydrase and experimental evidence showing one of these new, copper centers has characteristics similar to a variety of well-characterized copper centers in synthetic models and enzymatic systems. Human carbonic anhydrase II is known to bind two Cu2+ ions; these binding events were explored using modern isothermal titration calorimetry techniques that have become a proven method to accurately measure metal-binding thermodynamic parameters. The two Cu2+-binding events have different affinities (K a approximately 5 × 1012 and 1 × 1010), and both are enthalpically driven processes. Reconstituting these Cu2+ sites under a range of conditions has allowed us to assign the Cu2+-binding event to the three-histidine, native, metal-binding site. Our initial efforts to characterize these Cu2+ sites have yielded data that show distinctive (and noncoupled) EPR signals associated with each copper-binding site and that this reconstituted enzyme can activate hydrogen peroxide to catalyze the oxidation of 2-aminophenol.


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, 130, 10837-10842, 10.1002/ange.201807168

Im Tausch gegen NHC: Die Einfügung eines N‐heterocyclischen Carbenliganden (grün/blau) als Ersatz für His in das aktive Zentrum des Redoxenzyms Azurin rekonstituiert das T1‐Kupferzentrum. Der resultierende Komplex ist spektroskopisch kaum unterscheidbar von der N‐Bindung von His oder N‐Methylimidazol, senkt aber signifikant das Reduktionspotential des Kupferzentrums und erleichtert dadurch Elektronentransferprozesse.


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, 128, 6766-6767, 10.1021/ja058822p

The effects of metal ions on the reduction of nitric oxide (NO) with a designed heme copper center in myoglobin (F43H/L29H sperm whale Mb, CuBMb) were investigated under reducing anaerobic conditions using UV−vis and EPR spectroscopic techniques as well as GC/MS. In the presence of Cu(I), catalytic reduction of NO to N2O by CuBMb was observed with turnover number of 2 mol NO·mol CuBMb-1·min-1, close to 3 mol NO·mol enzyme-1·min-1 reported for the ba3 oxidases from T. thermophilus. Formation of a His-heme-NO species was detected by UV−vis and EPR spectroscopy. In comparison to the EPR spectra of ferrous-CuBMb-NO in the absence of metal ions, the EPR spectra of ferrous-CuBMb-NO in the presence of Cu(I) showed less-resolved hyperfine splitting from the proximal histidine, probably due to weakening of the proximal His-heme bond. In the presence of Zn(II), formation of a five-coordinate ferrous-CuBMb-NO species, resulting from cleavage of the proximal heme Fe-His bond, was shown by UV−vis and EPR spectroscopic studies. The reduction of NO to N2O was not observed in the presence of Zn(II). Control experiments using wild-type myoglobin indicated no reduction of NO in the presence of either Cu(I) or Zn(II). These results suggest that both the identity and the oxidation state of the metal ion in the CuB center are important for NO reduction. A redox-active metal ion is required to deliver electrons, and a higher oxidation state is preferred to weaken the heme iron−proximal histidine toward a five-coordinate key intermediate in NO reduction.


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, 237, 1197-1201, 10.1126/science.2820056

The tryptophan gene (trp) repressor of Escherichia coli has been converted into a site-specific nuclease by covalently attaching it to the 1,10-phenanthroline-copper complex. In its cuprous form, the coordination complex with hydrogen peroxide as a coreactant cleaves DNA by oxidatively attacking the deoxyribose moiety. The chemistry for the attachment of 1,10-phenanthroline to the trp repressor involves modification of lysyl residues with iminothiolane followed by alkylation of the resulting sulfhydryl groups with 5-iodoacetamido-1,10-phenanthroline. The modified trp repressor cleaves the operators of aroH and trpEDCBA upon the addition of cupric ion and thiol in a reaction dependent on the corepressor L-tryptophan. Scission was restricted to the binding site for the repressor, defined by deoxyribonuclease I footprinting. Since DNA-binding proteins have recognition sequences approximately 20 base pairs long, the nucleolytic activities derived from them could be used to isolate long DNA fragments for sequencing or chromosomal mapping.


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, 158, 55-61, 10.1016/j.jinorgbio.2015.12.026

A hybrid biocatalyst containing a metal terpyridine (tpy) complex within a rigid β-barrel protein nitrobindin (NB) is constructed. A tpy ligand with a maleimide group, N-[2-([2,2′:6′,2′′-terpyridin]-4′-yloxy)ethyl]maleimide (1), was covalently linked to Cys96 inside the cavity of NB to prepare a conjugate NB–1. Binding of Cu2 +, Zn2 +, or Co2 + ion to the tpy ligand in NB–1 was confirmed by UV–vis spectroscopy and ESI–TOF MS measurements. Cu2 +-bound NB–1 is found to catalyze a Diels–Alder reaction between azachalcone and cyclopentadiene in 22% yield, which is higher than that of the Cu2 +–tpy complex without the NB matrix. The results suggest that the hydrophobic cavity close to the copper active site within the NB scaffold supports the binding of the two substrates, dienophile and diene, to promote the reaction.


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, 6, 1873-1879, 10.1002/smll.201000772

The synthesis of a robust bio‐nanotube consisting of the β‐helical tubular component proteins of bacteriophage T4 is described. The crystal structure indicates that it has a well‐defined nanoscale length of 10 nm as a result of the head‐to‐head dimerization of β‐helices. Surprisingly, the tube assembly has high thermal stability, high tolerance to organic solvents, and a wide pH‐stability range.


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, 87, 2882-2886, 10.1073/pnas.87.8.2882

Escherichia coli catabolite gene activator protein (CAP) is a helix-turn-helix motif sequence-specific DNA binding protein [de Crombrugghe, B., Busby, S. & Buc, H. (1984) Science 224, 831-838; and Pabo, C. & Sauer, R. (1984) Annu. Rev. Biochem. 53, 293-321]. In this work, CAP has been converted into a site-specific DNA cleavage agent by incorporation of the chelator 1,10-phenanthroline at amino acid 10 of the helix-turn-helix motif. [(N-Acetyl-5-amino-1,10-phenanthroline)-Cys178]CAP binds to a 22-base-pair DNA recognition site with Kobs = 1 x 10(8) M-1. In the presence of Cu(II) and reducing agent, [(N-acetyl-5-amino-1,10-phenanthroline)-Cys178]CAP cleaves DNA at four adjacent nucleotides on each DNA strand within the DNA recognition site. The DNA cleavage reaction has been demonstrated using 40-base-pair and 7164-base-pair DNA substrates. The DNA cleavage reaction is not inhibited by dam methylation of the DNA substrate. Such semisynthetic site-specific DNA cleavage agents have potential applications in chromosome mapping, cloning, and sequencing.


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. 2006, 45, 2416-2419, 10.1002/anie.200504561

Chirality from blood: Serum albumins form strong complexes with CuII–phthalocyanines, leading to protein conjugates. These hybrid catalysts promote enantioselective Diels–Alder reactions, such as that of azachalcones 1 with cyclopentadiene (2) to give products 3 with 85–98 % ee.


Metal: Cu
Ligand type: Phthalocyanine
Anchoring strategy: Supramolecular
Optimization: Chemical
Max TON: 45.5
ee: 98
PDB: ---
Notes: Chirality from blood: Serum albumins form strong complexes with CuII–phthalocyanines, leading to protein conjugates. These hybrid catalysts promote enantioselective Diels–Alder reactions, such as that of azachalcones 1 with cyclopentadiene (2) to give products 3 with 85–98 % ee.

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, 137, 4594-4597, 10.1021/ja5109936

While a conserved tyrosine (Tyr) is found in oxidases, the roles of phenol ring pKa and reduction potential in O2 reduction have not been defined despite many years of research on numerous oxidases and their models. These issues represent major challenges in our understanding of O2 reduction mechanism in bioenergetics. Through genetic incorporation of unnatural amino acid analogs of Tyr, with progressively decreasing pKa of the phenol ring and increasing reduction potential, in the active site of a functional model of oxidase in myoglobin, a linear dependence of both the O2 reduction activity and the fraction of H2O formation with the pKa of the phenol ring has been established. By using these unnatural amino acids as spectroscopic probe, we have provided conclusive evidence for the location of a Tyr radical generated during reaction with H2O2, by the distinctive hyperfine splitting patterns of the halogenated tyrosines and one of its deuterated derivatives incorporated at the 33 position of the protein. These results demonstrate for the first time that enhancing the proton donation ability of the Tyr enhances the oxidase activity, allowing the Tyr analogs to augment enzymatic activity beyond that of natural Tyr.


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, 109, 21234-21239, 10.1073/pnas.1212893110

One of the ultimate objectives of de novo protein design is to realize systems capable of catalyzing redox reactions on substrates. This goal is challenging as redox-active proteins require design considerations for both the reduced and oxidized states of the protein. In this paper, we describe the spectroscopic characterization and catalytic activity of a de novo designed metallopeptide Cu(I/II)(TRIL23H)3+/2+, where Cu(I/II) is embeded in α-helical coiled coils, as a model for the CuT2 center of copper nitrite reductase. In Cu(I/II)(TRIL23H)3+/2+, Cu(I) is coordinated to three histidines, as indicated by X-ray absorption data, and Cu(II) to three histidines and one or two water molecules. Both ions are bound in the interior of the three-stranded coiled coils with affinities that range from nano- to micromolar [Cu(II)], and picomolar [Cu(I)]. The Cu(His)3 active site is characterized in both oxidation states, revealing similarities to the CuT2 site in the natural enzyme. The species Cu(II)(TRIL23H)32+ in aqueous solution can be reduced to Cu(I)(TRIL23H)3+ using ascorbate, and reoxidized by nitrite with production of nitric oxide. At pH 5.8, with an excess of both the reductant (ascorbate) and the substrate (nitrite), the copper peptide Cu(II)(TRIL23H)32+ acts as a catalyst for the reduction of nitrite with at least five turnovers and no loss of catalytic efficiency after 3.7 h. The catalytic activity, which is first order in the concentration of the peptide, also shows a pH dependence that is described and discussed.


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, 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: ---

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
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, 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: ---

Highly Efficient Cyclic Dinucleotide Based Artificial Metalloribozymes for Enantioselective Friedel–Crafts Reactions in Water

Chen, Y.; Wang, C.

Angew. Chem. Int. Ed. 2020, 59, 3444-3449, 10.1002/anie.201912962

The diverse secondary structures of nucleic acids are emerging as attractive chiral scaffolds to construct artificial metalloenzymes (ArMs) for enantioselective catalysis. DNA‐based ArMs containing duplex and G‐quadruplex scaffolds have been widely investigated, yet RNA‐based ArMs are scarce. Here we report that a cyclic dinucleotide of c‐di‐AMP and Cu2+ ions assemble into an artificial metalloribozyme (c‐di‐AMP⋅Cu2+) that enables catalysis of enantioselective Friedel–Crafts reactions in aqueous media with high reactivity and excellent enantioselectivity of up to 97 % ee. The assembly of c‐di‐AMP⋅Cu2+ gives rise to a 20‐fold rate acceleration compared to Cu2+ ions. Based on various biophysical techniques and density function theory (DFT) calculations, a fine coordination structure of c‐di‐AMP⋅Cu2+ metalloribozyme is suggested in which two c‐di‐AMP form a dimer scaffold and the Cu2+ ion is located in the center of an adenine‐adenine plane through binding to two N7 nitrogen atoms and one phosphate oxygen atom.


Metal: Cu
Ligand type: RNA
Host protein: RNA
Anchoring strategy: Dative
Optimization: Chemical
Max TON: 20
ee: 97
PDB: ---
Notes: ---