Okuda, J.

ACS Catal. 2015, 5, 7519-7522, 10.1021/acscatal.5b01792

A series of Grubbs–Hoveyda type catalyst precursors for olefin metathesis containing a maleimide moiety in the backbone of the NHC ligand was covalently incorporated in the cavity of the β-barrel protein nitrobindin. By using two protein mutants with different cavity sizes and choosing the suitable spacer length, an artificial metalloenzyme for olefin metathesis reactions in water in the absence of any organic cosolvents was obtained. High efficiencies reaching TON > 9000 in the ROMP of a water-soluble 7-oxanorbornene derivative and TON > 100 in ring-closing metathesis (RCM) of 4,4-bis(hydroxymethyl)-1,6-heptadiene in water under relatively mild conditions (pH 6, T = 25–40 °C) were observed.

Marchi-Delapierre, C.

ACS Catal. 2020, 10, 5631-5645, 10.1021/acscatal.9b04904

Artificial enzymes represent an attractive alternative to design abiotic biocatalysis. EcNikA-Ru1, an artificial metalloenzyme developed by embedding a ruthenium-based catalyst into the cavity of the periplasmic nickel-binding protein NikA, was found to efficiently and selectively transform certain alkenes. The objective of this study was to provide a rationale on the enzymatic function and the unexpected substrate-dependent chemoselectivity of EcNikA-Ru1 thanks to a dual experimental/computational study. We observed that the de novo active site allows the formation of the terminal oxidant via the formation of a ruthenium aquo species that subsequently reacts with the hypervalent iodine of phenyl iodide diacetic acid. The oxidation process relies on a RuIV═O pathway via a two-step reaction with a radical intermediate, resulting in the formation of either a chlorohydrin or an epoxide. The results emphasize the impact of the protein scaffold on the kinetics of the reaction, through (i) the promotion of the starting oxidizing species via the exchange of a CO ligand with a water molecule; and (ii) the control of the substrate orientation on the intermediate structures, formed after the RuIV═O attack. When a Cα attack is preferred, chlorohydrins are formed while an attack on Cβ leads to an epoxide. This work provides evidence that artificial enzymes mimic the behavior of their natural counterparts.

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.

Lewis, J.C.

ACS Catal. 2013, 3, 2954-2975, 10.1021/cs400806a

Transition metal catalysts and enzymes possess unique and often complementary properties that have made them important tools for chemical synthesis. The potential practical benefits of catalysts that combine these properties and a desire to understand how the structure and reactivity of metal and peptide components affect each other have driven researchers to create hybrid metal–peptide catalysts since the 1970s. The hybrid catalysts developed to date possess unique compositions of matter at the inorganic/biological interface that often pose significant challenges from design, synthesis, and characterization perspectives. Despite these obstacles, researchers have developed systems in which secondary coordination sphere effects impart selectivity to metal catalysts, accelerate chemical reactions, and are systematically optimized via directed evolution. This perspective outlines fundamental principles, key developments, and future prospects for the design, preparation, and application of peptide- and protein-based hybrid catalysts for organic transformations.

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.

Okuda, J.; Schwaneberg, U.

ACS Catal. 2021, 11, 5079-5087, 10.1021/acscatal.1c00134

Directed evolution has helped enzyme engineering to remarkable successes in the past. A main challenge in directed evolution is to find the most suitable starting point, that is, an enzyme that allows maximum “evolvability”. Consisting of a synthetic cofactor embedded in a protein scaffold, artificial metalloenzymes (ArMs) are reminiscent of rough-hewn ancestral metalloproteins and thus could provide an evolutionarily clean slate. Here, we report the design and directed evolution of an ArM with peroxidase-like properties based on the nitrobindin variant, NB4. After identifying a suitable artificial metal cofactor, two rounds of directed evolution were sufficient to elevate the ArM’s activity to levels akin to those of some natural peroxidases (up to kcat = 14.1 s–1 and kcat/Km = 52,800 M–1 s–1). A substitution to arginine in the distal cofactor environment (position 76) was the key to boost the peroxidase activity. Molecular dynamics simulations reveal a remarkable flexibility in the distal site of the NB4 scaffold that is absent in the nitrobindin wildtype and which allows the unrestricted movement of the catalytically important Arg76. In addition to the oxidation of the common redox mediators (ABTS, syringaldehyde, and 2,6-dimethoxyphenol), the ArM proved efficient in the decolorization of three recalcitrant dyes (indigo carmine, reactive blue 19, and reactive black 5) and was amenable to several rounds of ArM recycling.

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.

Maréchal, J.-D.

ACS Catal. 2014, 4, 833-842, 10.1021/cs400921n

We present a computational study that combines protein–ligand docking, quantum mechanical, and quantum mechanical/molecular mechanical calculations to scrutinize the mechanistic behavior of the first artificial enzyme able to enantioselectively reduce cyclic imines. We applied a novel strategy that allows the characterization of transition state structures in the protein host and their associated reaction paths. Of the most striking results of our investigation is the identification of major conformational differences between the transition state geometries of the lowest energy paths leading to (R)- and (S)-reduction products. The molecular features of (R)- and (S)-transition states highlight distinctive patterns of hydrophobic and polar complementarities between the substrate and the binding site. These differences lead to an activation energy gap that stands in very good agreement with the experimentally determined enantioselectivity. This study sheds light on the mechanism by which transfer hydrogenases operate and illustrates how the change of environment (from homogeneous solution conditions to the asymmetric protein frame) affect the reactivity of the organometallic cofactor. It provides novel insights on the complexity in integrating unnatural organometallic compounds into biological scaffolds. The modeling strategy that we pursued, based on the generation of “pseudo transition state” structures, is computationally efficient and suitable for the discovery and optimization of artificial enzymes. Alternatively, this approach can be applied on systems for which a large conformational sampling is needed to identify relevant transition states.

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.

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.

Ward, T.R.

ACS Catal. 2016, 6, 3553-3557, 10.1021/acscatal.6b00258

NADH mimics (mNADHs) have been shown to accelerate and orthogonally activate ene reductase-catalyzed reactions. However, existing regeneration methods of NAD(P)H fail for mNADHs. Catalysis with artificial metalloenzymes based on streptavidin (Sav) variants and a biotinylated iridium cofactor enable mNADH regeneration with formate. This regeneration can be coupled with ene reductase-catalyzed asymmetric reduction of α,β-unsaturated compounds, because of the protective compartmentalization of the organometallic cofactor. With 10 mol % mNAD+, a preparative scale reaction (>100 mg) gave full conversion with 98% ee, where TTNs reached 2000, with respect to the Ir cofactor under ambient atmosphere in aqueous medium.

Li, C.

ACS Catal. 2020, 10, 6561-6567, 10.1021/acscatal.0c01203

Developing high-performance DNA-based biocatalysts for desired stereoselective syntheses remains a formidable challenge. Here, we report promising DNA-based catalysts comprised of G-quadruplex (G4) and Fe porphyrin for asymmetric olefin cyclopropanation. After the G4-based catalysts are optimized by several rounds of site mutation, their catalytic enantioselectivities achieve +81% and −86% enantiomeric excess (eetrans) at a turnover number (TON) as high as 500. The Fe porphyrin, binding upon the 5′,3′-end G-quartet, constitutes the active center for olefin cyclopropanation via an iron porphyrin carbene intermediate. The findings provide an opportunity for generating high-value chiral cyclopropane blocks via G4 biocatalysts and shed light on the potential of DNA as protein enzymes for catalysis.

Jarvis, A.G.

ACS Catal. 2021, 11, 3620-3627, 10.1021/acscatal.0c05413

Protein engineering has shown widespread use in improving the industrial application of enzymes and broadening the conditions they are able to operate under by increasing their thermostability and solvent tolerance. Here, we show that protein engineering can be used to increase the thermostability of an artificial metalloenzyme. Thermostable variants of the human steroid carrier protein 2L, modified to bind a metal catalyst, were created by rational design using structural data and a 3DM database. These variants were tested to identify mutations that enhanced the stability of the protein scaffold, and a significant increase in melting temperature was observed with a number of modified metalloenzymes. The ability to withstand higher reaction temperatures resulted in an increased activity in the hydroformylation of 1-octene, with more than fivefold improvement in turnover number, whereas the selectivity for linear aldehyde remained high up to 80%.

Ward, T.R.

ACS Catal. 2013, 3, 1752-1755, 10.1021/cs400428r

Artificial metalloenzymes enable the engineering of the reaction microenvironment of the active metal catalyst by modification of the surrounding host protein. We report herein the optimization of an artificial imine reductase (ATHase) based on biotin–streptavidin technology. By introduction of lipophilic amino acid residues around the active site, an 8-fold increase in catalytic efficiency compared with the wild type imine reductase was achieved. Whereas substrate inhibition was encountered for the free cofactor and wild type ATHase, two engineered systems exhibited classical Michaelis–Menten kinetics, even at substrate concentrations of 150 mM with measured rates up to 20 min–1.

Korendovych, I.V.

ACS Catal. 2019, 9, 9265-9275, 10.1021/acscatal.9b02509

The field of protein design has grown enormously in the past few decades. In this review, we discuss the minimalist approach to the design of artificial enzymes, in which protein sequences are created with the minimum number of elements for folding and function. This method relies on identifying starting points in catalytically inert scaffolds for active site installation. The progress of the field from the original helical assemblies of the 1980s to the more complex structures of the present day is discussed, highlighting the variety of catalytic reactions which have been achieved using these methods. We outline the strengths and weaknesses of the minimalist approaches, describe representative design cases, and put it in the general context of the de novo design of proteins.

Onoda, A.

ACS Catal. 2014, 4, 2645-2648, 10.1021/cs500392e

The hydrogen-evolving diiron complex, (μ-S)2Fe2(CO)6 with a tethered maleimide moiety was synthesized and covalently embedded within the cavity of a rigid β-barrel protein matrix by coupling a maleimide moiety to a cysteine residue within the β-barrel. The (μ-S)2Fe2(CO)6 core within the cavity was characterized by UV–vis absorption and a characteristic CO vibration determined by IR measurements. The diiron complex embedded within the cavity retains the necessary catalytic activity (TON up to 130 for 6 h) to evolve H2 via a photocatalytic cycle with a Ru photosensitizer in a solution of 100 mM ascorbate and 50 mM Tris/HCl at pH 4.0 and 25 °C.

Liu, X.; Tian, C.; Wang, J.

ACS Catal. 2021, 11, 5628-5635, 10.1021/acscatal.1c00287

Photosystem I (PSI) is a very large membrane protein complex (∼1000 kDa) harboring P700*, the strongest reductant known in biological systems, which is responsible for driving NAD(P)+ and ultimately for CO2 reduction. Although PSI is one of the most important components in the photosynthesis machinery, it has remained difficult to enhance PSI functions through genetic engineering due to its enormous complexity. Inspired by PSI’s ability to undergo multiple-step photo-induced electron hopping from P700* to iron–sulfur [Fe4S4] clusters, we designed a 33 kDa miniature photocatalytic CO2-reducing enzyme (mPCE) harboring a chromophore (BpC) and two [Fe4S4] clusters (FeA/FeB). Through reduction potential fine-tuning, we optimized the multiple-step electron hopping from BpC to FeA/FeB, culminating in a CO2/HCOOH conversion quantum efficiency of 1.43%. As mPCE can be overexpressed with a high yield in Escherichia coli cells without requiring synthetic cofactors, further development along this route may result in rapid photo-enzyme quantum yield improvement and functional expansion through an efficient directed evolution process.

Lin, Y.-W.

ACS Catal. 2020, 10, 14359-14365, 10.1021/acscatal.0c04572

Design of artificial nucleases is essential in biotechnology and biomedicine, whereas few artificial nucleases can both cleave and degrade DNA molecules. Heme proteins are potential enzymes for DNA cleavage. Using a small heme protein, myoglobin (Mb), as a model protein, we engineered a metal-binding motif of [1-His-1-Glu] (native His64 and mutated Glu29) in the heme distal site. The single mutant of L29E Mb was capable of not only efficient DNA cleavage but also DNA degradation upon Mg2+ binding to the heme distal site, as shown by an X-ray crystal structure of the Mg2+-L29E Mb complex. Molecular docking of the protein–DNA complex revealed multiple hydrogen-bonding interactions at their interfaces, involving both minor and major grooves of DNA. Moreover, both the distal Arg45 and the ligand Glu29 were identified as critical residues for the nuclease activity. This study reports the structure of a water-bridged heterodinuclear center of Mg-heme (Mg2+-H2O-Fe3+), showing a similar function as the homodinuclear center (MgA2+-H2O–MgB2+) in natural nuclease, which indicates that the Mg2+-L29E Mb complex is an effective artificial nuclease.

Arold, S.T.; Eppinger, J.; Groll, M.

ACS Catal. 2019, 9, 11371-11380, 10.1021/acscatal.9b02896

Artificial metalloenzymes (ArMs) have high potential in biotechnological applications as they combine the versatility of transition-metal catalysis with the substrate selectivity of enzymes. An ideal host protein should allow high-yield recombinant expression, display thermal and solvent stability to withstand harsh reaction conditions, lack nonspecific metal-binding residues, and contain a suitable cavity to accommodate the artificial metal site. Moreover, to allow its rational functionalization, the host should provide an intrinsic reporter for metal binding and structural changes, which should be readily amendable to high-resolution structural characterization. Herein, we present the design, characterization, and de novo functionalization of a fluorescent ArM scaffold, named mTFP*, that achieves these characteristics. Fluorescence measurements allowed direct assessment of the scaffold’s structural integrity. Protein X-ray structures and transition metal Förster resonance energy transfer (tmFRET) studies validated the engineered metal coordination sites and provided insights into metal binding dynamics at the atomic level. The implemented active metal centers resulted in ArMs with efficient Diels–Alderase and Friedel–Crafts alkylase activities.

Lu, Y.; Zhang, J.-L.

ACS Catal. 2011, 1, 1083-1089, 10.1021/cs200258e

Two questions important to the success in metalloenzyme design are how to attach or anchor metal cofactors inside protein scaffolds and in what way such positioning affects enzymatic properties. We have previously reported a dual anchoring method to position a nonnative cofactor, MnSalen (1), inside the heme cavity of apo sperm whale myoglobin (Mb) and showed that the dual anchoring can increase both the activity and enantioselectivity over single anchoring methods, making this artificial enzyme an ideal system to address the above questions. Here, we report systematic investigations of the effect of different covalent attachment or anchoring positions on reactivity and selectivity of sulfoxidation by the MnSalen-containing Mb enzymes. We have found that changing the left anchor from Y103C to T39C has an almost identical effect of increasing rate by 1.8-fold and increasing selectivity by +15% for S, whether the right anchor is L72C or S108C. At the same time, regardless of the identity of the left anchor, changing the right anchor from S108C to L72C increases the rate by 4-fold and selectivity by +66%. The right anchor site was observed to have a greater influence than the left anchor site on the reactivity and selectivity in sulfoxidation of a wide scope of other ortho-, meta- and para-substituted substrates. The 1·Mb(T39C/L72C) showed the highest reactivity (TON up to 2.32 min–1) and selectivity (ee % up to 83%) among the different anchoring positions examined. Molecular dynamic simulations indicate that these changes in reactivity and selectivity may be due to the steric effects of the linker arms inside the protein cavity. These results indicate that small differences in the anchor positions can result in significant changes in reactivity and enantioselectivity, probably through steric interactions with substrates when they enter the substrate-binding pocket, and that the effects of right and left anchor positions are independent and additive in nature. The finding that the anchoring arms can influence both the positioning of the cofactor and steric control of substrate entrance will help design better functional metalloenzymes with predicted catalytic activity and selectivity.

Lin, Y.-W.

ACS Catal. 2019, 9, 7888-7893, 10.1021/acscatal.9b02226

Approaches to degradation of industrial dyes are desirable, of which bioremediation is more favorable. In addition to the use of native enzymes, rational design of artificial enzymes provides an alternative approach. Meanwhile, few designs can achieve a catalytic activity comparable to that of native enzymes. We have previously designed two generations of artificial dye-decolorizing peroxidases (DyPs) in myoglobin (Mb) by introduction of Tyr43 and Trp138 in the heme pocket; however, the activity is moderate. To improve the activity of the artificial DyP, we herein designed a third generation by introduction of an additional Trp (P88W) to the protein surface, named F43Y/F138W/P88W Mb. The third generation of artificial DyP was shown to exhibit a catalytic efficiency exceeding that of various native DyPs and comparable to that of the most efficient native DyPs. Titration of reactive blue 19 (RB19) and molecular docking studies revealed crucial roles of Trp88 in substrate binding and oxidation, which acts as a catalytic site. This study not only provides clues for heme protein design but also suggests that the artificial DyP has potential applications for bioremediation in the future.

Maréchal, J.-D.

ACS Catal. 2015, 5, 2469-2480, 10.1021/acscatal.5b00010

The development of artificial enzymes aims at expanding the scope of biocatalysis. Over recent years, artificial metalloenzymes based on the insertion of homogeneous catalysts in biomolecules have received an increasing amount of attention. Rational or pseudorational design of these composites is a challenging task because of the complexity of the identification of efficient complementarities among the cofactor, the substrate, and the biological partner. Molecular modeling represents an interesting alternative to help in this task. However, little attention has been paid to this field so far. In this manuscript, we aim at reviewing our efforts in developing strategies efficient to computationally drive the design of artificial metalloenzymes. From protein–ligand dockings to multiscale approaches, we intend to demonstrate that modeling could be useful at the different steps of the design. This Perspective ultimately aims at providing computational chemists with illustration of the applications of their tools for artificial metalloenzymes and convincing enzyme designers of the capabilities, qualitative and quantitative, of computational methodologies.