47 publications

47 publications

8-Amino-5,6,7,8-tetrahydroquinoline in Iridium(III) Biotinylated Cp* Complex as Artificial Imine Reductase

Rimoldi, I.

New J. Chem. 2018, 42, 18773-18776, 10.1039/C8NJ04558E

The imine reductase formed by the (R)-CAMPY ligand bound to the S112M Sav mutant showed an 83% ee in the asymmetric transfer hydrogenation of 6,7-dimethoxy-1-methyl-3,4-dihydroisoquinoline.


Metal: Ir
Ligand type: Cp*; Diamine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 32
ee: 83
PDB: ---
Notes: ---

Metal: Ir
Ligand type: Cp*; Diamine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 99
ee: 13
PDB: ---
Notes: ---

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

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

Nat. Commun. 2018, 9, 10.1038/s41467-018-04440-0

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


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

A Designed Heme-[4Fe-4S] Metalloenzyme Catalyzes Sulfite Reduction like the Native Enzyme

Lu, Y.

Science 2018, 361, 1098-1101, 10.1126/science.aat8474

Multielectron redox reactions often require multicofactor metalloenzymes to facilitate coupled electron and proton movement, but it is challenging to design artificial enzymes to catalyze these important reactions, owing to their structural and functional complexity. We report a designed heteronuclear heme-[4Fe-4S] cofactor in cytochrome c peroxidase as a structural and functional model of the enzyme sulfite reductase. The initial model exhibits spectroscopic and ligand-binding properties of the native enzyme, and sulfite reduction activity was improved—through rational tuning of the secondary sphere interactions around the [4Fe-4S] and the substrate-binding sites—to be close to that of the native enzyme. By offering insight into the requirements for a demanding six-electron, seven-proton reaction that has so far eluded synthetic catalysts, this study provides strategies for designing highly functional multicofactor artificial enzymes.


Metal: Fe
Host protein: Cytochrome c peroxidase
Anchoring strategy: Dative
Optimization: Chemical & genetic
Reaction: Sulfite reduction
Max TON: ---
ee: ---
PDB: ---
Notes: Designed heteronuclear heme-[4Fe-4S] cofactor in cytochrome c peroxidase

An Artificial Heme Enzyme for Cyclopropanation Reactions

Roelfes, G.

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

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


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

An Artificial Metalloenzyme for Carbene Transfer Based on a Biotinylated Dirhodium Anchored Within Streptavidin

Ward, T.R.

Cat. Sci. Technol. 2018, 8, 2294-2298, 10.1039/C8CY00646F

We report an artificial carbenoid transferase which combines a biotinylated dirhodium moiety within streptavidin scaffold.


Metal: Rh
Ligand type: Carboxylate
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Cyclopropanation
Max TON: ~60
ee: ---
PDB: ---
Notes: Cyclopropanation reaction was also performed in the E. coli periplasm.

Metal: Rh
Ligand type: Carboxylate
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: C-H insertion
Max TON: ~60
ee: ---
PDB: ---
Notes: ---

A Noncanonical Proximal Heme Ligand Affords an Efficient Peroxidase in a Globin Fold

Green, A.P.; Hilvert, D.

J. Am. Chem. Soc. 2018, 140, 1535-1543, 10.1021/jacs.7b12621

Expanding the range of genetically encoded metal coordination environments accessible within tunable protein scaffolds presents excellent opportunities for the creation of metalloenzymes with augmented properties and novel activities. Here, we demonstrate that installation of a noncanonical Nδ-methyl histidine (NMH) as the proximal heme ligand in the oxygen binding protein myoglobin (Mb) leads to substantial increases in heme redox potential and promiscuous peroxidase activity. Structural characterization of this catalytically modified myoglobin variant (Mb NMH) revealed significant changes in the proximal pocket, including alterations to hydrogen-bonding interactions involving the prosthetic porphyrin cofactor. Further optimization of Mb NMH via a combination of rational modification and several rounds of laboratory evolution afforded efficient peroxidase biocatalysts within a globin fold, with activities comparable to those displayed by nature’s peroxidases.


Metal: Fe
Host protein: Myoglobin (Mb)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Oxidation
Max TON: ~1650
ee: ---
PDB: 5OJ9
Notes: Oxidation of amplex red

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.

Artificial Metalloenzyme Design with Unnatural Amino Acids and Non-Native Cofactors

Review

Wang, J.

ACS Catal. 2018, 8, 1851-1863, 10.1021/acscatal.7b03754

There are 20 proteinogenic amino acids and a limited number of cofactors naturally available to build enzymes. Genetic codon expansion enables us to incorporate more than 200 unnatural amino acids into proteins using cell translation machinery, greatly expanding structures available to protein chemists. Such tools enable scientists to mimic the active site of an enzyme to tune enzymatic activity, anchor cofactors, and immobilize enzymes on electrode surfaces. Non-native cofactors can be incorporated into the protein through covalent or noncovalent interactions, expanding the reaction scope of existing enzymes. The review discusses strategies to incorporate unnatural amino acids and non-native cofactors and their applications in tuning and expanding enzymatic activities of artificial metalloenzymes.


Notes: ---

Artificial Metalloenzymes as Catalysts for Oxidative Lignin Degradation

Jarvis, A.G.

ACS Sustainable Chem. Eng. 2018, 6, 15100-15107, 10.1021/acssuschemeng.8b03568

We report novel artificial metalloenzymes (ArMs), containing tris(pyridylmethyl)amine (TPA), for the atom economic oxidation of lignin β-O-4 model compounds, using hydrogen peroxide. The protein scaffold alters the selectivity of the reaction from a low yielding cleavage reaction when using the parent Fe-tpa complex to a high yielding benzylic alcohol oxidation when using the complex incorporated into a protein scaffold, SCP-2L A100C. Engineering the protein scaffold to incorporate glutamic acid was found to improve the ArM activity, showing that rational design of the protein environment using metal binding amino acids can be a first step toward improving the overall activity of an artificial metalloenzyme.


Metal: Fe
Anchoring strategy: Cystein-maleimide
Optimization: Chemical & genetic
Reaction: Lignin oxidation
Max TON: 20
ee: ---
PDB: ---
Notes: Reaction performed with a lignin model compound and hydrogen peroxide as oxidizing agent

Artificial Metalloenzymes for Hydrogenation and Transfer Hydrogenation Reactions

Review

Klein Gebbink, R.J.M.

Artificial Metalloenzymes and MetalloDNAzymes in Catalysis: From Design to Applications 2018, 171-197, 10.1002/9783527804085.ch6

The development of artificial hydrogenases (AHases) and transfer hydrogenases (ATHases) has played a leading and guiding role for the field of artificial metalloenzymes. Starting from the early studies by Whitesides and coworkers, this chapter showcases the conceptual development of AHases and ATHases, highlighting the different conjugation strategies used for their construction and exemplifying the stereoselective control in product formation that can be reached.


Notes: Book chapter

Artificial Metalloenzymes on the Verge of New-to-Nature Metabolism

Review

Jeschek, M.

Trends Biotechnol. 2018, 36, 60-72, 10.1016/j.tibtech.2017.10.003

Residing at the interface of chemistry and biotechnology, artificial metalloenzymes (ArMs) offer an attractive technology to combine the versatile reaction repertoire of transition metal catalysts with the exquisite catalytic features of enzymes. While earlier efforts in this field predominantly comprised studies in well-defined test-tube environments, a trend towards exploiting ArMs in more complex environments has recently emerged. Integration of these artificial biocatalysts in enzymatic cascades and using them in whole-cell biotransformations and in vivo opens up entirely novel prospects for both preparative chemistry and synthetic biology. We highlight selected recent developments with a particular focus on challenges and opportunities in the in vivo application of ArMs.


Notes: ---

Artificial Metalloenzymes: Reaction Scope and Optimization Strategies

Review

Lewis, J.C.; Ward, T.R.

Chem. Rev. 2018, 118, 142-231, 10.1021/acs.chemrev.7b00014

The incorporation of a synthetic, catalytically competent metallocofactor into a protein scaffold to generate an artificial metalloenzyme (ArM) has been explored since the late 1970’s. Progress in the ensuing years was limited by the tools available for both organometallic synthesis and protein engineering. Advances in both of these areas, combined with increased appreciation of the potential benefits of combining attractive features of both homogeneous catalysis and enzymatic catalysis, led to a resurgence of interest in ArMs starting in the early 2000’s. Perhaps the most intriguing of potential ArM properties is their ability to endow homogeneous catalysts with a genetic memory. Indeed, incorporating a homogeneous catalyst into a genetically encoded scaffold offers the opportunity to improve ArM performance by directed evolution. This capability could, in turn, lead to improvements in ArM efficiency similar to those obtained for natural enzymes, providing systems suitable for practical applications and greater insight into the role of second coordination sphere interactions in organometallic catalysis. Since its renaissance in the early 2000’s, different aspects of artificial metalloenzymes have been extensively reviewed and highlighted. Our intent is to provide a comprehensive overview of all work in the field up to December 2016, organized according to reaction class. Because of the wide range of non-natural reactions catalyzed by ArMs, this was done using a functional-group transformation classification. The review begins with a summary of the proteins and the anchoring strategies used to date for the creation of ArMs, followed by a historical perspective. Then follows a summary of the reactions catalyzed by ArMs and a concluding critical outlook. This analysis allows for comparison of similar reactions catalyzed by ArMs constructed using different metallocofactor anchoring strategies, cofactors, protein scaffolds, and mutagenesis strategies. These data will be used to construct a searchable Web site on ArMs that will be updated regularly by the authors.


Notes: ---

Artificial Metalloproteins Containing Co4O4 Cubane Active Sites

Borovik, A.S.; Don Tilley, T.

J. Am. Chem. Soc. 2018, 140, 2739-2742, 10.1021/jacs.7b13052

Artificial metalloproteins (ArMs) containing Co4O4 cubane active sites were constructed via biotin–streptavidin technology. Stabilized by hydrogen bonds (H-bonds), terminal and cofacial CoIII–OH2 moieties are observed crystallographically in a series of immobilized cubane sites. Solution electrochemistry provided correlations of oxidation potential and pH. For variants containing Ser and Phe adjacent to the metallocofactor, 1e–/1H+ chemistry predominates until pH 8, above which the oxidation becomes pH-independent. Installation of Tyr proximal to the Co4O4 active site provided a single H-bond to one of a set of cofacial CoIII–OH2 groups. With this variant, multi-e–/multi-H+ chemistry is observed, along with a change in mechanism at pH 9.5 that is consistent with Tyr deprotonation. With structural similarities to both the oxygen-evolving complex of photosystem II (H-bonded Tyr) and to thin film water oxidation catalysts (Co4O4 core), these findings bridge synthetic and biological systems for water oxidation, highlighting the importance of secondary sphere interactions in mediating multi-e–/multi-H+ reactivity.


Metal: Co
Ligand type: OAc; Pyridine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: ---
ee: ---
PDB: 6AUC
Notes: Co-complex in Sav WT

Metal: Co
Ligand type: OAc; Pyridine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: ---
ee: ---
PDB: 6AUE
Notes: Co-complex in Sav S112Y

A Whole Cell E. coli Display Platform for Artificial Metalloenzymes: Poly(phenylacetylene) Production with a Rhodium–Nitrobindin Metalloprotein

Schwaneberg, U.

ACS Catal. 2018, 8, 2611-2614, 10.1021/acscatal.7b04369

Whole cell catalysis is, in many cases, a prerequisite for the cost-effective production of chemicals by biotechnological means. Synthetic metal catalysts for bioorthogonal reactions can be inactivated within cells due to abundant thiol derivatives. Here, a cell surface display-based whole cell biohybrid catalyst system (termed ArMt bugs) is reported as a generally applicable platform to unify cost-effective whole cell catalysis with biohybrid catalysis. An inactivated esterase autotransporter is employed to display the nitrobindin protein scaffold with a Rh catalyst on the E. coli surface. Stereoselective polymerization of phenylacetylene yielded a high turnover number (TON) (39 × 106 cell–1) for the ArMt bugs conversion platform.


Metal: Rh
Ligand type: COD; Cp
Host protein: Nitrobindin variant NB4
Anchoring strategy: Cystein-maleimide
Optimization: ---
Max TON: 3046
ee: ---
PDB: ---
Notes: Calculated in vivo TON assuming 12800 metalloenzymes per E. coli cell

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

Hilvert, D.

Nat. Catal. 2018, 1, 578-584, 10.1038/s41929-018-0105-6

Non-canonical amino acid ligands are useful for fine-tuning the catalytic properties of metalloenzymes. Here, we show that recombinant replacement of the histidine ligand proximal to haem in myoglobin with Nδ-methylhistidine enhances the protein’s promiscuous carbene-transfer chemistry, enabling efficient styrene cyclopropanation in the absence of reductant, even under aerobic conditions. The increased electrophilicity of the modified Fe(iii) centre, combined with subtle structural adjustments at the active site, allows direct attack of ethyl diazoacetate to produce a reactive carbenoid adduct, which has an unusual bridging Fe(iii)–C–N(pyrrole) configuration as shown by X-ray crystallography. Quantum chemical calculations suggest that the bridged complex equilibrates with the more reactive end-on isomer, ensuring efficient cyclopropanation. These findings underscore the potential of non-canonical ligands for extending the capabilities of metalloenzymes by opening up new reaction pathways and facilitating the characterization of reactive species that would not otherwise accumulate.


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

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

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 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.

Chimeric Streptavidins as Host Proteins for Artificial Metalloenzymes

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

ACS Catal. 2018, 8, 1476-1484, 10.1021/acscatal.7b03773

The streptavidin scaffold was expanded with well-structured naturally occurring motifs. These chimeric scaffolds were tested as hosts for biotinylated catalysts as artificial metalloenzymes (ArM) for asymmetric transfer hydrogenation, ring-closing metathesis and anion−π catalysis. The additional second coordination sphere elements significantly influence both the activity and the selectivity of the resulting hybrid catalysts. These findings lead to the identification of propitious chimeric streptavidins for future directed evolution efforts of artificial metalloenzymes.


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

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

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

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

Design of Artificial Enzymes by Supramolecular Strategies

Review

Liu, J.

Curr. Opin. Struct. Biol. 2018, 51, 19-27, 10.1016/j.sbi.2018.02.003

Enzymes are biomacromolecules with three-dimensional structures composed of peptide polymers via supramolecular interactions. Owing to the incredible catalytic efficiency and unique substrate selectivity, enzymes arouse considerable attention. To rival natural enzymes, various artificial enzymes have been developed over the last decades. Since supramolecular interactions play important roles in both substrate recognition and the process of enzymatic catalysis, designing artificial enzymes using supramolecular strategies is undoubtedly significant. Here we discuss the recent advances in constructing artificial enzymes using supramolecular platforms.


Notes: ---

Design of Artificial Metalloproteins/Metalloenzymes by Tuning Noncovalent Interactions

Review

Hirota, S.; Lin, Y.-W.

J. Biol. Inorg. Chem. 2018, 23, 7-25, 10.1007/s00775-017-1506-8

Noncovalent weak interactions [hydrophobic interaction and hydrogen (H)-bond] play crucial roles in controlling the functions of biomolecules, and thus have been used to design artificial metalloproteins/metalloenzymes during the past few decades. In this review, we focus on the recent progresses in protein design by tuning the noncovalent interactions, including hydrophobic and H-bonding interactions. The topics include redesign and reuse of the heme pocket and other protein scaffolds, design of the heme protein interface, and de novo design of metalloproteins. The informations not only give insights into the metalloenzyme reaction mechanisms but also provide new reactions for future applications.


Notes: ---

Development of De Novo Copper Nitrite Reductases: Where we are and where we need to go

Review

Pecoraro, V.L.

ACS Catal. 2018, 8, 8046-8057, 10.1021/acscatal.8b02153

The development of redox-active metalloprotein catalysts is a challenging objective of de novo protein design. Within this Perspective we detail our efforts to create a redox-active Cu nitrite reductase (NiR) by incorporating Cu into the hydrophobic interior of well-defined three-stranded coiled coils (3SCCs). The scaffold contains three histidine residues that provide a layer of three nitrogen donors that mimic the type 2 catalytic site of NiR. We have found that this strategy successfully produces an active and stable CuNiR model that functions for over 1000 turnovers. Spectroscopic evidence indicates that the Cu(I) site has a lower coordination number in comparison to the enzyme, whereas the Cu(II) geometry may more faithfully reproduce the NiR type 2 center. Mutations at the helical interface successfully produce a hydrogen bond between an interfacial Glu residue and the Cu-ligating His residue, which allows for the tuning of the redox potential over a 100 mV range. We successfully created constructs with as much as a 120-fold improvement from the original design by modifying the steric bulk above or below the Cu binding site. These systems are now the most active water-soluble and stable artificial NiR catalysts yet produced. Several avenues for improving the catalytic efficiency of later designs are detailed within this Perspective, including adjustment of their resting oxidation state, the use of asymmetric scaffolds to allow for single amino acid mutation within the second coordination sphere, and the design of hydrogen-bonding networks to tune residue orientation and electronics. Through these studies the TRI-H system has given insight into the difficulties that arise in creating a de novo redox active enzyme. Work to improve upon this model will provide strategies by which redox-active de novo enzymes may be tuned and detail how native enzymes accomplish catalytic efficiencies through proton gated redox catalysis.


Notes: ---

Directed Evolution of an Artificial Imine Reductase

Maréchal, J.-D.; Ward, T.R.

Angew. Chem. Int. Ed. 2018, 57, 1863-1868, 10.1002/anie.201711016

Artificial metalloenzymes, resulting from incorporation of a metal cofactor within a host protein, have received increasing attention in the last decade. The directed evolution is presented of an artificial transfer hydrogenase (ATHase) based on the biotin‐streptavidin technology using a straightforward procedure allowing screening in cell‐free extracts. Two streptavidin isoforms were yielded with improved catalytic activity and selectivity for the reduction of cyclic imines. The evolved ATHases were stable under biphasic catalytic conditions. The X‐ray structure analysis reveals that introducing bulky residues within the active site results in flexibility changes of the cofactor, thus increasing exposure of the metal to the protein surface and leading to a reversal of enantioselectivity. This hypothesis was confirmed by a multiscale approach based mostly on molecular dynamics and protein–ligand dockings.


Metal: Ir
Ligand type: Amino-sulfonamide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 380
ee: 95
PDB: 6ESS
Notes: Salsolidine formation; Sav mutant S112A-N118P-K121A-S122M: (R)-selective

Metal: Ir
Ligand type: Amino-sulfonamide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 220
ee: 85
PDB: 6ESU
Notes: Salsolidine formation; Sav mutant S112R-N118P-K121A-S122M-L124Y: (S)-selective

Directed Evolution of Artificial Metalloenzymes: Bridging Synthetic Chemistry and Biology

Review

Arnold, F.H.

Artificial Metalloenzymes and MetalloDNAzymes in Catalysis: From Design to Applications 2018, 137-170, 10.1002/9783527804085.ch5

Directed evolution is a powerful algorithm for engineering proteins to have novel and useful properties. However, we do not yet fully understand the characteristics of an evolvable system. In this chapter, we present examples where directed evolution has been used to enhance the performance of metalloenzymes, focusing first on “classical” cases such as improving enzyme stability or expanding the scope of natural reactivity. We then discuss how directed evolution has been extended to artificial systems, in which a metalloprotein catalyzes reactions using abiological reagents or in which the protein utilizes a nonnatural cofactor for catalysis. These examples demonstrate that directed evolution can also be applied to artificial systems to improve catalytic properties, such as activity and enantioselectivity, and to favor a different product than that favored by small‐molecule catalysts. Future work will help define the extent to which artificial metalloenzymes can be altered and optimized by directed evolution and the best approaches for doing so.


Notes: Book chapter

E. coli Surface Display of Streptavidin for Directed Evolution of an Allylic Deallylase

Ward, T.R.

Chem. Sci. 2018, 9, 5383-5388, 10.1039/c8sc00484f

Artificial metalloenzymes (ArMs hereafter) combine attractive features of both homogeneous catalysts and enzymes and offer the potential to implement new-to-nature reactions in living organisms. Herein we present an E. coli surface display platform for streptavidin (Sav hereafter) relying on an Lpp-OmpA anchor. The system was used for the high throughput screening of a bioorthogonal CpRu-based artificial deallylase (ADAse) that uncages an allylcarbamate-protected aminocoumarin 1. Two rounds of directed evolution afforded the double mutant S112M–K121A that displayed a 36-fold increase in surface activity vs. cellular background and a 5.7-fold increased in vitro activity compared to the wild type enzyme. The crystal structure of the best ADAse reveals the importance of mutation S112M to stabilize the cofactor conformation inside the protein.


Metal: Ru
Ligand type: Cp; Quinoline
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Reaction: Deallylation
Max TON: 148
ee: ---
PDB: 6FH8
Notes: ---

Engineered Metalloenzymes with Non-Canonical Coordination Environments

Review

Green, A.P.; Hilvert, D.

Chem. - Eur. J. 2018, 24, 11821-11830, 10.1002/chem.201800975

Nature employs a limited number of genetically encoded, metal‐coordinating residues to create metalloenzymes with diverse structures and functions. Engineered components of the cellular translation machinery can now be exploited to encode non‐canonical ligands with user‐defined electronic and structural properties. This ability to install “chemically programmed” ligands into proteins can provide powerful chemical probes of metalloenzyme mechanism and presents excellent opportunities to create metalloprotein catalysts with augmented properties and novel activities. In this Concept article, we provide an overview of several recent studies describing the creation of engineered metalloenzymes with interesting catalytic properties, and reveal how characterization of these systems has advanced our understanding of nature's bioinorganic mechanisms. We also highlight how powerful laboratory evolution protocols can be readily adapted to allow optimization of metalloenzymes with non‐canonical ligands. This approach combines beneficial features of small molecule and protein catalysis by allowing the installation of a greater variety of local metal coordination environments into evolvable protein scaffolds, and holds great promise for the future creation of powerful metalloprotein catalysts for a host of synthetically valuable transformations.


Notes: ---

Evolving Artificial Metalloenzymes via Random Mutagenesis

Lewis, J.C.

Nat. Chem. 2018, 10, 318-324, 10.1038/nchem.2927

Random mutagenesis has the potential to optimize the efficiency and selectivity of protein catalysts without requiring detailed knowledge of protein structure; however, introducing synthetic metal cofactors complicates the expression and screening of enzyme libraries, and activity arising from free cofactor must be eliminated. Here we report an efficient platform to create and screen libraries of artificial metalloenzymes (ArMs) via random mutagenesis, which we use to evolve highly selective dirhodium cyclopropanases. Error-prone PCR and combinatorial codon mutagenesis enabled multiplexed analysis of random mutations, including at sites distal to the putative ArM active site that are difficult to identify using targeted mutagenesis approaches. Variants that exhibited significantly improved selectivity for each of the cyclopropane product enantiomers were identified, and higher activity than previously reported ArM cyclopropanases obtained via targeted mutagenesis was also observed. This improved selectivity carried over to other dirhodium-catalysed transformations, including N–H, S–H and Si–H insertion, demonstrating that ArMs evolved for one reaction can serve as starting points to evolve catalysts for others.


Metal: Rh
Ligand type: OAc
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Reaction: Cyclopropanation
Max TON: 66
ee: 94
PDB: 5T88
Notes: Mutagenesis of the ArM by error-prone PCR

Metal: Rh
Ligand type: OAc
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Reaction: N-H Insertion
Max TON: 73
ee: 40
PDB: 5T88
Notes: Mutagenesis of the ArM by error-prone PCR

Metal: Rh
Ligand type: OAc
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Reaction: S-H insertion
Max TON: 64
ee: 32
PDB: 5T88
Notes: Mutagenesis of the ArM by error-prone PCR

Metal: Rh
Ligand type: OAc
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Reaction: Si-H insertion
Max TON: 35
ee: 64
PDB: 5T88
Notes: Mutagenesis of the ArM by error-prone PCR

Ferritin Encapsulation of Artificial Metalloenzymes: Engineering a Tertiary Coordination Sphere for an Artificial Transfer Hydrogenase

Ward, T.R.

Dalton Trans. 2018, 47, 10837-10841, 10.1039/C8DT02224K

Ferritin, a naturally occuring iron-storage protein, plays an important role in nanoengineering and biomedical applications. Upon iron removal, apoferritin was shown to allow the encapsulation of an artificial transfer hydrogenase (ATHase) based on the streptavidin-biotin technology. The third coordination sphere, provided by ferritin, significantly influences the catalytic activity of an ATHase for the reduction of cyclic imines.


Metal: Ir
Ligand type: Amino-sulfonamide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 3874
ee: 75
PDB: ---
Notes: ---

Functionalization of Protein Crystals with Metal Ions, Complexes and Nanoparticles

Review

Ueno, T.

Curr. Opin. Chem. Biol. 2018, 43, 68-76, 10.1016/j.cbpa.2017.11.015

Self-assembled proteins have specific functions in biology. With inspiration provided by natural protein systems, several artificial protein assemblies have been constructed via site-specific mutations or metal coordination, which have important applications in catalysis, material and bio-supramolecular chemistry. Similar to natural protein assemblies, protein crystals have been recognized as protein assemblies formed of densely-packed monomeric proteins. Protein crystals can be functionalized with metal ions, metal complexes or nanoparticles via soaking, co-crystallization, creating new metal binding sites by site-specific mutations. The field of protein crystal engineering with metal coordination is relatively new and has gained considerable attention for developing solid biomaterials as well as structural investigations of enzymatic reactions, growth of nanoparticles and catalysis. This review highlights recent and significant research on functionalization of protein crystals with metal coordination and future prospects.


Notes: ---

Generation of a Functional, Semisynthetic [FeFe]-Hydrogenase in a Photosynthetic Microorganism

Berggren, G.; Lindblad, P.

Energy Environ. Sci. 2018, 11, 3163-3167, 10.1039/C8EE01975D

[FeFe]-Hydrogenases are hydrogen producing metalloenzymes with excellent catalytic capacities, highly relevant in the context of a future hydrogen economy. Here we demonstrate the synthetic activation of a heterologously expressed [FeFe]-hydrogenase in living cells of Synechocystis PCC 6803, a photoautotrophic microbial chassis with high potential for biotechnological energy applications. H2-Evolution assays clearly show that the non-native, semi-synthetic enzyme links to the native metabolism in living cells.


Metal: Fe
Ligand type: CN; CO
Anchoring strategy: Reconstitution
Optimization: Chemical & genetic
Reaction: H2 evolution
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Genetic Engineering of an Artificial Metalloenzyme for Transfer Hydrogenation of a Self-Immolative Substrate in Escherichia coli’s Periplasm

Ward, T.R.

J. Am. Chem. Soc. 2018, 140, 13171-13175, 10.1021/jacs.8b07189

Artificial metalloenzymes (ArMs), which combine an abiotic metal cofactor with a protein scaffold, catalyze various synthetically useful transformations. To complement the natural enzymes’ repertoire, effective optimization protocols to improve ArM’s performance are required. Here we report on our efforts to optimize the activity of an artificial transfer hydrogenase (ATHase) using Escherichia coli whole cells. For this purpose, we rely on a self-immolative quinolinium substrate which, upon reduction, releases fluorescent umbelliferone, thus allowing efficient screening. Introduction of a loop in the immediate proximity of the Ir-cofactor afforded an ArM with up to 5-fold increase in transfer hydrogenation activity compared to the wild-type ATHase using purified mutants.


Metal: Ir
Ligand type: Amino-sulfonamide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 1000
ee: 76
PDB: 6GMI
Notes: ---