57 publications

57 publications

A De Novo Designed Metalloenzyme for the Hydration of CO2

Pecoraro, V.L.

Angew. Chem. Int. Ed. 2014, 53, 7900-7903, 10.1002/anie.201404925

Protein design will ultimately allow for the creation of artificial enzymes with novel functions and unprecedented stability. To test our current mastery of nature’s approach to catalysis, a ZnII metalloenzyme was prepared using de novo design. α3DH3 folds into a stable single‐stranded three‐helix bundle and binds ZnII with high affinity using His3O coordination. The resulting metalloenzyme catalyzes the hydration of CO2 better than any small molecule model of carbonic anhydrase and with an efficiency within 1400‐fold of the fastest carbonic anhydrase isoform, CAII, and 11‐fold of CAIII.


Metal: Zn
Ligand type: Amino acid
Host protein: α3D peptide
Anchoring strategy: Dative
Optimization: Chemical & genetic
Max TON: ---
ee: ---
PDB: ---
Notes: kcat/KM ≈ 3.8*104 M-1*s-1

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.

A Designed Supramolecular Protein Assembly with In Vivo Enzymatic Activity

Tezcan, F.A.

Science 2014, 346, 1525-1528, 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, 135, 5384-5388, 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, 4, 900-906, 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 Ferri
Anchoring strategy: Dative
Optimization: Genetic
Reaction: N-Hydroxylation
Max TON: ---
ee: ---
PDB: 2LFD
Notes: ---

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

DeGrado, W.F.; Lombardi, A.

Nat. Chem. Biol. 2009, 5, 882-884, 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 Ferri
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 Ferri
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, 6, 736-740, 10.1002/cctc.201300995

Dative anchoring of a piano‐stool complex within ribonuclease S resulted in an artificial imine reductase. The catalytic performance was modulated upon variation of the coordinating amino acid residues in the S‐peptide. Binding of Cp*Ir (Cp*=C5Me5) to the native active site resulted in good conversions and moderate enantiomeric excess values for the synthesis of salsolidine.


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, 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: ---

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 Heme Enzymes for the Construction of Gold-Based Biomaterials

Lombardi, A.; Nastri, F.

Int. J. Mol. Sci. 2018, 19, 2896, 10.3390/ijms19102896

Many efforts are continuously devoted to the construction of hybrid biomaterials for specific applications, by immobilizing enzymes on different types of surfaces and/or nanomaterials. In addition, advances in computational, molecular and structural biology have led to a variety of strategies for designing and engineering artificial enzymes with defined catalytic properties. Here, we report the conjugation of an artificial heme enzyme (MIMO) with lipoic acid (LA) as a building block for the development of gold-based biomaterials. We show that the artificial MIMO@LA can be successfully conjugated to gold nanoparticles or immobilized onto gold electrode surfaces, displaying quasi-reversible redox properties and peroxidase activity. The results of this work open interesting perspectives toward the development of new totally-synthetic catalytic biomaterials for application in biotechnology and biomedicine, expanding the range of the biomolecular component aside from traditional native enzymes.


Metal: Fe
Ligand type: Amino acid; Porphyrin
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Reaction: Oxidation
Max TON: ---
ee: ---
PDB: ---
Notes: Immobilization of the ArM on gold surfaces via a lipoic acid anchor.

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

Fujieda, N.; Itoh, S.

J. Am. Chem. Soc. 2017, 139, 5149-5155, 10.1021/jacs.7b00675

Thermally stable TM1459 cupin superfamily protein from Thermotoga maritima was repurposed as an osmium (Os) peroxygenase by metal-substitution strategy employing the metal-binding promiscuity. This novel artificial metalloenzyme bears a datively bound Os ion supported by the 4-histidine motif. The well-defined Os center is responsible for not only the catalytic activity but also the thermodynamic stability of the protein folding, leading to the robust biocatalyst (Tm ≈ 120 °C). The spectroscopic analysis and atomic resolution X-ray crystal structures of Os-bound TM1459 revealed two types of donor sets to Os center with octahedral coordination geometry. One includes trans-dioxide, OH, and mer-three histidine imidazoles (O3N3 donor set), whereas another one has four histidine imidazoles plus OH and water molecule in a cis position (O2N4 donor set). The Os-bound TM1459 having the latter donor set (O2N4 donor set) was evaluated as a peroxygenase, which was able to catalyze cis-dihydroxylation of several alkenes efficiently. With the low catalyst loading (0.01% mol), up to 9100 turnover number was achieved for the dihydroxylation of 2-methoxy-6-vinyl-naphthalene (50 mM) using an equivalent of H2O2 as oxidant at 70 °C for 12 h. When octene isomers were dihydroxylated in a preparative scale for 5 h (2% mol cat.), the terminal alkene octene isomers was converted to the corresponding diols in a higher yield as compared with the internal alkenes. The result indicates that the protein scaffold can control the regioselectivity by the steric hindrance. This protein scaffold enhances the efficiency of the reaction by suppressing disproportionation of H2O2 on Os reaction center. Moreover, upon a simple site-directed mutagenesis, the catalytic activity was enhanced by about 3-fold, indicating that Os-TM1459 is evolvable nascent osmium peroxygenase.


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, 338, 500-503, 10.1126/science.1226132

Enzymes provide an exquisitely tailored chiral environment to foster high catalytic activities and selectivities, but their native structures are optimized for very specific biochemical transformations. Designing a protein to accommodate a non-native transition metal complex can broaden the scope of enzymatic transformations while raising the activity and selectivity of small-molecule catalysis. Here, we report the creation of a bifunctional artificial metalloenzyme in which a glutamic acid or aspartic acid residue engineered into streptavidin acts in concert with a docked biotinylated rhodium(III) complex to enable catalytic asymmetric carbon-hydrogen (C–H) activation. The coupling of benzamides and alkenes to access dihydroisoquinolones proceeds with up to nearly a 100-fold rate acceleration compared with the activity of the isolated rhodium complex and enantiomeric ratios as high as 93:7.


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: ---

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: ---

Catalysis by a De Novo Zinc-Mediated Protein Interface: Implications for Natural Enzyme Evolution and Rational Enzyme Engineering

Kuhlman, B.

Biochemistry 2012, 51, 3933-3940, 10.1021/bi201881p

Here we show that a recent computationally designed zinc-mediated protein interface is serendipitously capable of catalyzing carboxyester and phosphoester hydrolysis. Although the original motivation was to design a de novo zinc-mediated protein–protein interaction (called MID1-zinc), we observed in the homodimer crystal structure a small cleft and open zinc coordination site. We investigated if the cleft and zinc site at the designed interface were sufficient for formation of a primitive active site that can perform hydrolysis. MID1-zinc hydrolyzes 4-nitrophenyl acetate with a rate acceleration of 105 and a kcat/KM of 630 M–1 s–1 and 4-nitrophenyl phosphate with a rate acceleration of 104 and a kcat/KM of 14 M–1 s–1. These rate accelerations by an unoptimized active site highlight the catalytic power of zinc and suggest that the clefts formed by protein–protein interactions are well-suited for creating enzyme active sites. This discovery has implications for protein evolution and engineering: from an evolutionary perspective, three-coordinated zinc at a homodimer interface cleft represents a simple evolutionary path to nascent enzymatic activity; from a protein engineering perspective, future efforts in de novo design of enzyme active sites may benefit from exploring clefts at protein interfaces for active site placement.


Metal: Zn
Ligand type: Amino acid
Anchoring strategy: Dative
Optimization: Chemical & genetic
Max TON: >50
ee: ---
PDB: 3V1C
Notes: ---

Catalytic Cyclopropanation by Myoglobin Reconstituted with Iron Porphycene: Acceleration of Catalysis due to Rapid Formation of the Carbene Species

Hasegawa, J.-Y.; Lehnert, N.

J. Am. Chem. Soc. 2017, 139, 17265-17268, 10.1021/jacs.7b10154

Myoglobin reconstituted with iron porphycene catalyzes the cyclopropanation of styrene with ethyl diazoacetate. Compared to native myoglobin, the reconstituted protein significantly accelerates the catalytic reaction and the kcat/Km value is 26-fold enhanced. Mechanistic studies indicate that the reaction of the reconstituted protein with ethyl diazoacetate is 615-fold faster than that of native myoglobin. The metallocarbene species reacts with styrene with the apparent second-order kinetic constant of 28 mM–1 s–1 at 25 °C. Complementary theoretical studies support efficient carbene formation by the reconstituted protein that results from the strong ligand field of the porphycene and fewer intersystem crossing steps relative to the native protein. From these findings, the substitution of the cofactor with an appropriate metal complex serves as an effective way to generate a new biocatalyst.


Metal: Fe
Ligand type: Amino acid; Porphycene
Host protein: Myoglobin (Mb)
Anchoring strategy: Reconstitution
Optimization: ---
Reaction: Cyclopropanation
Max TON: ---
ee: ---
PDB: ---
Notes: Cyclopropanation of styrene with ethyl diazoacetate: kcat/KM = 1.3 mM-1 * s-1, trans/cis = 99:1

Catalytic Properties and Specificity of the Extracellular Nuclease of Staphylococcus Aureus

Cuatrecasas, P.

J. Biol. Chem. 1967, n/a

A spectrophotometric assay is described for staphylococcal nuclease, based on the increase in absorbance at 260 mp which accompanies deoxyribonucleic acid and RNA hy- drolysis. Initial velocities are proportional to enzyme con- centration over a 70-fold range. The enzyme has greater aflinity for DNA than for RNA, and activity is greater with heat-denatured DNA than with native DNA. No inhibitory products accumulate during the reaction. The enzyme is stable at pH values as low as 0.1, and in a concentration of 0.15 mg per ml there is no loss of activity after boiling (20 min). Dilute solutions are protected from heat inactivation by a mixture of albumin and Ca++ as well as by denatured DNA. The optimum pH for RNase and DNase activities is be- tween 9 and 10, depending on the Ca++ concentration. At higher pH values, less Ca+f is required. The inhibitory effect of high Ca+f concentrations is more pronounced at higher pH values. Considerable DNase but no RNase activity results if Ca++ is replaced by Sr+f, while Fe++ and C&f cause minimal activation. A number of heavy metal cations inhibit DNase and RNase activities competitively with Ca++; Hg++, Zn++, and Cd++ are the most potent of these. Activities resulting from combinations of DNA and RNA with Ca+f or Sr+f suggest that these substrates are hy- drolyzed by the same or closely related regions on the en- zyme. Enzyme activity toward DNA and RNA is strongly in- hibited by 5’-phosphoryl (not by 2’- or 3’-phosphoryl) deriva- tives of deoxyadenylic, adenylic, and deozythymidylic acids, and deozythymidine 3’,5’-diphosphate is the most po- tent inhibitor. High activity is obtained with polyadenylic acid compared to polyuridylic acid, polycytidylic acid, and RNA. These tidings are consistent with the known action of the enzyme (cleavage of the 5’-phosphoryl ester bond), and suggest that the differential activity toward DNA and RNA results at least in part from differences in the afhnity toward the constituent bases of these nucleic acids.


Metal: Sr
Ligand type: Amino acid
Host protein: Nuclease from S. aureus
Anchoring strategy: Metal substitution
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: PMID 4290246; DNA cleavage

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

Catalytic Water Oxidation by Iridium-Modified Carbonic Anhydrase

Lee, S.-Y.

Chem. - Asian J. 2018, 13, 334-341, 10.1002/asia.201701543

Carbonic anhydrase (CA) is a ubiquitous metalloenzyme with a Zn cofactor coordinated to trigonal histidine imidazole moieties in a tetrahedral geometry. Removal of the Zn cofactor in CA and subsequent binding of Ir afforded CA[Ir]. Under mild and neutral conditions (30 °C, pH 7), CA[Ir] exhibited water‐oxidizing activity with a turnover frequency (TOF) of 39.8 min−1, which is comparable to those of other Ir‐based molecular catalysts. Coordination of Ir to the apoprotein of CA is thermodynamically preferred and is associated with an exothermic energy change (ΔH) of −10.8 kcal mol−1, which implies that the CA apoprotein is stabilized by Ir binding. The catalytic oxygen‐evolving activity of CA[Ir] is displayed only if Ir is bound to CA, which functions as an effective biological scaffold that activates the Ir center for catalysis. The results of this study indicate that the histidine imidazoles at the CA active site could be exploited as beneficial biological ligands to provide unforeseen biochemical activity by coordination to a variety of transition‐metal ions.


Metal: Ir
Ligand type: Amino acid
Anchoring strategy: Metal substitution
Optimization: Chemical
Reaction: Water oxidation
Max TON: ---
ee: ---
PDB: ---
Notes: Sodium periodate as sacrificial oxidant. TOF at pH 7 and 30°C is 39.8 min-1.

Computational Redesign of a Mononuclear Zinc Metalloenzyme for Organophosphate Hydrolysis

Baker, D.

Nat. Chem. Biol. 2012, 8, 294-300, 10.1038/NChemBio.777

The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency (kcat/Km) of ∼104 M−1 s−1 after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzes the hydrolysis of the RP isomer of a coumarinyl analog of the nerve agent cyclosarin, and it shows marked substrate selectivity for coumarinyl leaving groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities.


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

De Novo Design of Catalytic Proteins

DeGrado, W.F.

Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 11566-11570, 10.1073/pnas.0404387101

The de novo design of catalytic proteins provides a stringent test of our understanding of enzyme function, while simultaneously laying the groundwork for the design of novel catalysts. Here we describe the design of an O2-dependent phenol oxidase whose structure, sequence, and activity are designed from first principles. The protein catalyzes the two-electron oxidation of 4-aminophenol (k cat/K M = 1,500 M·1·min·1) to the corresponding quinone monoimine by using a diiron cofactor. The catalytic efficiency is sensitive to changes of the size of a methyl group in the protein, illustrating the specificity of the design.


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

De Novo Design of Four-Helix Bundle Metalloproteins: One Scaffold, Diverse Reactivities

DeGrado, W.F.

Acc. Chem. Res. 2019, 10.1021/acs.accounts.8b00674

De novo protein design represents anattractive approach for testing and extending our under-standing of metalloprotein structure and function. Here, we describe our work on the design of DF (Due Ferri or two-ironin Italian), a minimalist model for the active sites of muchlarger and more complex natural diiron and dimanganeseproteins. In nature, diiron and dimanganese proteins protypi-cally bind their ions in 4-Glu, 2-His environments, and theycatalyze diverse reactions, ranging from hydrolysis, to O2-dependent chemistry, to decarbonylation of aldehydes. In the design of DF, the position of each atom including the backbone, the first-shell ligands, the second-shell hydrogen-bonded groups, and the well-packed hydrophobic core was bespoke using precise mathematical equations and chemical principles. The first member of the DF family was designed to be of minimal size and complexity and yet to display the quintessential elements required for binding the dimetal cofactor. After thoroughly characterizing its structural, dynamic, spectroscopic, and functional properties, we added additional complexity in a rational stepwise manner to achieve increasingly sophisticated catalytic functions, ultimately demonstrating substrate-gated four-electron reduction of O2to water. We also briefly describe the extension of these studies to the design of proteins that bind non biological metal cofactors (a synthetic porphyrin and a tetranuclear cluster), and a Zn2+/proton antiporting membrane protein. Together these studies demonstrate a successful and generally applicable strategy for de novo metalloprotein design, which might indeed mimic the process by which primordial metalloproteins evolved. We began the design process with a highly symmetrical backbone and binding site, by using point-group symmetry to assemble the secondary structures that position the amino acid side chains required for binding. The resulting models provided a rough starting point and initial parameters for the subsequent precise design of thefinal protein using modern methods of computational protein design. Unless the desired site is itself symmetrical, this process requires reduction of the symmetry or lifting it altogether. Nevertheless, the initial symmetrical structure can be helpful to restrain the search space during assembly of the backbone. Finally, the methods described here should be generally applicable to the design of highly stable and robust catalysts and sensors. There is considerable potential in combining the efficiency and knowledge base associated with homogeneous metal catalysis with the programmability, biocompatibility, and versatility of proteins. While the work reported here focuses on testing and learning the principles of natural metalloproteins by designing and studying proteins one at a time, there is also considerable potential for using designed proteins that incorporate both biological and non biological metal ion cofactors for the evolution of novel catalysts.


Metal: Fe
Ligand type: Amino acid
Host protein: Due Ferri
Anchoring strategy: Dative
Optimization: Computational design
Reaction: Oxidation
Max TON: ---
ee: ---
PDB: 1EC5
Notes: Additional PDB: 1LT1

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

Kim, H.S.

Science 2006, 311, 535-538, 10.1126/science.1118953

The design of enzymes with new functions and properties has long been a goal in protein engineering. Here, we report a strategy to change the catalytic activity of an existing protein scaffold. This was achieved by simultaneous incorporation and adjustment of functional elements through insertion, deletion, and substitution of several active site loops, followed by point mutations to fine-tune the activity. Using this approach, we were able to introduce β-lactamase activity into the αβ/βα metallohydrolase scaffold of glyoxalase II. The resulting enzyme, evMBL8 (evolved metallo β-lactamase 8), completely lost its original activity and, instead, catalyzed the hydrolysis of cefotaxime with a (kcat /Km)app of 1.8 × 102 (mole/liter)–1 second–1, thus increasing resistance to Escherichia coli growth on cefotaxime by a factor of about 100.


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

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 a Switchable Eliminase

DeGrado, W.F.

Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 6823-6827, 10.1073/pnas.1018191108

The active sites of enzymes are lined with side chains whose dynamic, geometric, and chemical properties have been finely tuned relative to the corresponding residues in water. For example, the carboxylates of glutamate and aspartate are weakly basic in water but become strongly basic when dehydrated in enzymatic sites. The dehydration of the carboxylate, although intrinsically thermodynamically unfavorable, is achieved by harnessing the free energy of folding and substrate binding to reach the required basicity. Allosterically regulated enzymes additionally rely on the free energy of ligand binding to stabilize the protein in a catalytically competent state. We demonstrate the interplay of protein folding energetics and functional group tuning to convert calmodulin (CaM), a regulatory binding protein, into AlleyCat, an allosterically controlled eliminase. Upon binding Ca(II), native CaM opens a hydrophobic pocket on each of its domains. We computationally identified a mutant that (i) accommodates carboxylate as a general base within these pockets, (ii) interacts productively in the Michaelis complex with the substrate, and (iii) stabilizes the transition state for the reaction. Remarkably, a single mutation of an apolar residue at the bottom of an otherwise hydrophobic cavity confers catalytic activity on calmodulin. AlleyCat showed the expected pH-rate profile, and it was inactivated by mutation of its active site Glu to Gln. A variety of control mutants demonstrated the specificity of the design. The activity of this minimal 75-residue allosterically regulated catalyst is similar to that obtained using more elaborate computational approaches to redesign complex enzymes to catalyze the Kemp elimination reaction.


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

Direct Hydrogenation of Carbon Dioxide by an Artificial Reductase Obtained by Substituting Rhodium for Zinc in the Carbonic Anhydrase Catalytic Center. A Mechanistic Study

Marino, T.

ACS Catal. 2015, 5, 5397-5409, 10.1021/acscatal.5b00185

Recently, a new artificial carbonic anhydrase enzyme in which the native zinc cation has been replaced with a Rh(I) has been proposed as a new reductase that is able to efficiently catalyze the hydrogenation of olefins. In this paper, we propose the possible use of this modified enzyme in the direct hydrogenation of carbon dioxide. In our theoretical investigation, we have considered different reaction mechanisms such as reductive elimination and σ-bond metathesis. In addition, the release of the formic acid and the restoring of the catalytic cycle have also been studied. Results show that the σ-bond metathesis potential energy surface lies below the reactant species. The rate-determining step is the release of the product with an energy barrier of 12.8 kcal mol–1. On the basis of our results, we conclude that this artificial enzyme can efficiently catalyze the conversion of CO2 to HCOOH by a direct hydrogenation reaction.


Metal: Rh
Ligand type: Amino acid
Anchoring strategy: Metal substitution
Optimization: ---
Reaction: Hydrogenation
Max TON: ---
ee: ---
PDB: ---
Notes: Computational study of the reaction mechanism of the formation of HCOOH from CO2

Diruthenium Diacetate-Catalyzed Aerobic Oxidation of Hydroxylamines and Improved Chemoselectivity by Immobilization to Lysozyme

Cardona, F.; Goti, A.; Messori, L.

ChemCatChem 2017, 9, 4225-4230, 10.1002/cctc.201701083

A new green method for the preparation of nitrones through the aerobic oxidation of the corresponding N,N‐disubstituted hydroxylamines has been developed upon exploring the catalytic activity of a diruthenium catalyst, that is, [Ru2(OAc)4Cl]), in aqueous or alcoholic solution under mild reaction conditions (0.1 to 1 mol % catalyst, air, 50 °C) and reasonable reaction times. Notably, the catalytic activity of the dimetallic centre is retained after its binding to the small protein lysozyme. Interestingly, this new artificial metalloenzyme conferred complete chemoselectivity to the oxidation of cyclic hydroxylamines, in contrast to the diruthenium catalyst.


Metal: Ru
Ligand type: Amino acid; OAc
Host protein: Lysozyme
Anchoring strategy: Dative
Optimization: Chemical
Max TON: 1000
ee: ---
PDB: ---
Notes: ---

Engineered Metal Regulation of Trypsin Specificity

Craik, C.S.

Biochemistry 1995, 34, 2172-2180, 10.1021/bi00007a010

Histidine substrate specificity has been engineered into trypsin by creating metal binding sites for Ni2+ and Zn2+ ions. The sites bridge the substrate and enzyme on the leaving-group side of the scissile bond. Application of simple steric and geometric criteria to a crystallographically derived enzyme- substrate model suggested that histidine specificity at the P2' position might be acheived by a tridentate site involving amino acid residues 143 and 151 of trypsin. Trypsin N143H/E151H hydrolyzes a P2'- His-containing peptide (AGPYAHSS) exclusively in the presence of nickel or zinc with a high level of catalytic efficiency. Since cleavage following the tyrosine residue is normally highly disfavored by trypsin, this result demonstrates that a metal cofactor can be used to modulate specificity in a designed fashion. The same geometric criteria applied in the primary SI binding pocket suggested that the single-site mutation D189H might effect metal-dependent His specificity in trypsin. However, kinetic and crystallographic analysis of this variant showed that the design was unsuccessful because His 189 rotates away from substrate causing a large perturbation in adjacent surface loops. This observation suggests that the reason specificity modification at the trypsin S1 site requires extensive mutagenesis is because the pocket cannot deform locally to accommodate alternate PI side chains. By taking advantage of the extended subsites, an alternate substrate specificity has been engineered into trypsin.


Metal: Zn
Ligand type: Amino acid
Host protein: Trypsin
Anchoring strategy: Dative
Optimization: Genetic
Max TON: ---
ee: ---
PDB: ---
Notes: Substrate specificty

Metal: Ni
Ligand type: Amino acid
Host protein: Trypsin
Anchoring strategy: Dative
Optimization: Genetic
Max TON: ---
ee: ---
PDB: ---
Notes: Substrate specificty

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: ---