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

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

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

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.

Okuda, J.

Beilstein J. Org. Chem. 2016, 12, 1314-1321, 10.3762/bjoc.12.124

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

DeGrado, W.F.; Lombardi, A.

Eur. J. Inorg. Chem. 2015, 2015, 3371-3390, 10.1002/ejic.201500470

A single polypeptide chain may provide an astronomical number of conformers. Nature selected only a trivial number of them through evolution, composing an alphabet of scaffolds, that can afford the complete set of chemical reactions needed to support life. These structural templates are so stable that they allow several mutations without disruption of the global folding, even having the ability to bind several exogenous cofactors. With this perspective, metal cofactors play a crucial role in the regulation and catalysis of several processes. Nature is able to modulate the chemistry of metals, adopting only a few ligands and slightly different geometries. Several scaffolds and metal‐binding motifs are representing the focus of intense interest in the literature. This review discusses the widespread four‐helix bundle fold, adopted as a scaffold for metal binding sites in the context of de novo protein design to obtain basic biochemical components for biosensing or catalysis. In particular, we describe the rational refinement of structure/function in diiron–oxo protein models from the due ferri (DF) family. The DF proteins were developed by us through an iterative process of design and rigorous characterization, which has allowed a shift from structural to functional models. The examples reported herein demonstrate the importance of the synergic application of de novo design methods as well as spectroscopic and structural characterization to optimize the catalytic performance of artificial enzymes.

Liu, J.

Chem. Soc. Rev. 2012, 41, 7890, 10.1039/c2cs35207a

Enzymes are nanometer-sized molecules with three-dimensional structures created by the folding and self-assembly of polymeric chain-like components through supramolecular interactions. They are capable of performing catalytic functions usually accompanied by a variety of conformational states. The conformational diversities and complexities of natural enzymes exerted in catalysis seriously restrict the detailed understanding of enzymatic mechanisms in molecular terms. A supramolecular viewpoint is undoubtedly helpful in understanding the principle of enzyme catalysis. The emergence of supramolecular artificial enzymes therefore provides an alternative way to approach the structural complexity and thus to unravel the mystery of enzyme catalysis. This critical review covers the recent development of artificial enzymes designed based on supramolecular scaffolds ranging from the synthetic macrocycles to self-assembled nanometer-sized objects. Such findings are anticipated to facilitate the design of supramolecular artificial enzymes as well as their potential uses in important fields, such as manufacturing and food industries, environmental biosensors, pharmaceutics and so on.

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.

Onoda, A.

Curr. Opin. Chem. Biol. 2015, 25, 133-140, 10.1016/j.cbpa.2014.12.041

Hydrogenase catalyses reversible transformation of H2 to H+ using an active site which includes an iron or nickel atom. Synthetic model complexes and molecular catalysts inspired by nature have unveiled the structural and functional basis of the active site with remarkable accuracy and this has led to the discovery of active synthetic catalysts. To further improve the activity of such molecular catalysts, both the first and outer coordination spheres should be well-organized and harmonized for an efficient shuttling of H+, electrons, and H2. This article reviews recent advances in the design and catalytic properties of artificial enzymes that mimic the hydrogenase active site and the outer coordination sphere in combination with a peptide or protein scaffold.

Artero, V.

ChemPlusChem 2016, 81, 1083-1089, 10.1002/cplu.201600218

The insertion of cobaloxime catalysts in the heme‐binding pocket of heme oxygenase (HO) yields artificial hydrogenases active for H2 evolution in neutral aqueous solutions. These novel biohybrids have been purified and characterized by using UV/visible and EPR spectroscopy. These analyses revealed the presence of two distinct binding conformations, thereby providing the cobaloxime with hydrophobic and hydrophilic environments, respectively. Quantum chemical/molecular mechanical docking calculations found open and closed conformations of the binding pocket owing to mobile amino acid residues. HO‐based biohybrids incorporating a {Co(dmgH)2} (dmgH2=dimethylglyoxime) catalytic center displayed up to threefold increased turnover numbers with respect to the cobaloxime alone or to analogous sperm whale myoglobin adducts. This study thus provides a strong basis for further improvement of such biohybrids, using well‐designed modifications of the second and outer coordination spheres, through site‐directed mutagenesis of the host protein.

Fontecave, M.

Curr. Opin. Chem. Biol. 2015, 25, 36-47, 10.1016/j.cbpa.2014.12.018

There is an urgent need for cheap, abundant and efficient catalysts as an alternative to platinum for hydrogen production and oxidation in (photo)electrolyzers and fuel cells. Hydrogenases are attractive solutions. These enzymes use exclusively nickel and iron in their active sites and function with high catalytic rates at the thermodynamic equilibrium. As an alternative, a number of biomimetic and bioinspired catalysts for H2 production and/or uptake, based on Ni, Fe and Co, have been developed and shown to display encouraging performances. In this review we discuss specifically recent approaches aiming at incorporating these compounds within oligomeric and polymeric hosts. The latter are most often biological compounds (peptides, proteins, polysaccharides, etc.) but we also discuss non-biological scaffolds (synthetic polymers, Metal–organic-Frameworks, etc.) which can provide the appropriate environment to tune the activity and stability of the synthetic catalysts. These supramolecular catalytic systems thus define a class of original compounds so-called artificial hydrogenases.

Duhme-Klair, A.K.

RSC Chem. Biol. 2020, 1, 369-378, 10.1039/d0cb00113a

Biocatalytic imine reduction has been a topic of intense research by the artificial metalloenzyme community in recent years. Artificial constructs, together with natural enzymes, have been engineered to produce chiral amines with high enantioselectivity. This review examines the design of the main classes of artificial imine reductases reported thus far and summarises approaches to enhancing their catalytic performance using complementary methods. Examples of utilising these biocatalysts in vivo or in multi-enzyme cascades have demonstrated the potential that artIREDs can offer, however, at this time their use in biocatalysis remains limited. This review explores the current scope of artIREDs and the strategies used for catalyst improvement, and examines the potential for artIREDs in the future.

Mahy, J.-P.

Biotechnol. Appl. Biochem. 2020, 67, 563-573, 10.1002/bab.1906

We report a new artificial hydrogenase made by covalent anchoring of the iron Knölker's complex to a xylanase S212C variant. This artificial metalloenzyme was found to be able to catalyze efficiently the transfer hydrogenation of the benchmark substrate trifluoroacetophenone by sodium formate in water, yielding the corresponding secondary alcohol as a racemic. The reaction proceeded more than threefold faster with the XlnS212CK biohybrid than with the Knölker's complex alone. In addition, efficient conversion of trifluoroacetophenone to its corresponding alcohol was reached within 60 H with XlnS212CK, whereas a ≈2.5-fold lower conversion was observed with Knölker's complex alone as catalyst. Moreover, the data were rationalized with a computational strategy suggesting the key factors of the selectivity. These results suggested that the Knölker's complex was most likely flexible and could experience free rotational reorientation within the active-site pocket of Xln A, allowing it to access the subsite pocket populated by trifluoroacetophenone.

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.

Ward, T.R.

J. Am. Chem. Soc. 2008, 130, 8085-8088, 10.1021/ja8017219

Nature’s catalysts are specifically evolved to carry out efficient and selective reactions. Recent developments in biotechnology have allowed the rapid optimization of existing enzymes for enantioselective processes. However, the ex nihilo creation of catalytic activity from a noncatalytic protein scaffold remains very challenging. Herein, we describe the creation of an artificial enzyme upon incorporation of a vanadyl ion into the biotin-binding pocket of streptavidin, a protein devoid of catalytic activity. The resulting artificial metalloenzyme catalyzes the enantioselective oxidation of prochiral sulfides with good enantioselectivities both for dialkyl and alkyl-aryl substrates (up to 93% enantiomeric excess). Electron paragmagnetic resonance spectroscopy, chemical modification, and mutagenesis studies suggest that the vanadyl ion is located within the biotin-binding pocket and interacts only via second coordination sphere contacts with streptavidin.

Roelfes, G.

ChemCatChem 2010, 2, 916-927, 10.1002/cctc.201000011

Artificial metalloenzymes have emerged as a promising approach to merge the attractive properties of homogeneous catalysis and biocatalysis. The activity and selectivity, including enantioselectivity, of natural metalloenzymes are due to the second coordination sphere interactions provided by the protein. Artificial metalloenzymes aim at harnessing second coordination sphere interactions to create transition metal complexes that display enzyme‐like activities and selectivities. In this Review, the various approaches that can be followed for the design and optimization of an artificial metalloenzyme are discussed. An overview of the synthetic transformations that have been achieved using artificial metalloenzymes is provided, with a particular focus on recent developments. Finally, the role that the second coordination sphere plays in artificial metalloenzymes and their potential for synthetic applications are evaluated.

Ward, T.R.

Effects of Nanoconfinement on Catalysis 2017, 49-82, 10.1007/978-3-319-50207-6_3

While chemists are developing confined environments for catalysis, nature has evolved highly elaborate compartments to carry out reactions. Proteins offer such catalytic nano-environments that accept specific substrates to yield highly enantioenriched products. Metalloenzymes form a subclass that combines the functional diversity of proteins with the promiscuous activities of metals. In recent years, a variety of artificial metalloenzymes (ArMs) has been created upon incorporation of metal complexes into a protein scaffold. The following chapter discusses some of the protein scaffolds exploited for the creation of artificial metalloenzymes. Focus is laid on artificial metalloenzymes that catalyze abiotic and asymmetric reactions. Each subchapter presents the unique characteristics of a scaffold followed by a description of the reactions that were performed with it.

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.

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.

Reetz, M.T.

Chem. Rec. 2012, 12, 391-406, 10.1002/tcr.201100043

Numerous enzymes are useful catalysts in synthetic organic chemistry, but they cannot catalyze the myriad transition‐metal‐mediated transformations customary in daily chemical work. For this reason the concept of directed evolution of hybrid catalysts was proposed some time ago. A synthetic ligand/transition‐metal moiety is anchored covalently or non‐covalently to a host protein, thereby generating a single artificial metalloenzyme which can then be optimized by molecular biological methods. In the quest to construct an appropriate experimental platform for asymmetric Diels–Alder reactions amenable to this Darwinian approach to catalysis, specifically those not currently possible using traditional chiral transition‐metal catalysts, two strategies have been developed which are reviewed here. One concerns the supramolecular anchoring of a Cu(II)‐phthalocyanine complex to serum albumins; the other is based on the design of a Cu(II)‐specific binding site in a thermostable protein host (tHisF), leading to 46–98% ee in a model Diels–Alder reaction. This sets the stage for genetic fine‐tuning using the methods of directed evolution.

Ward, T.R.

Coord. Chem. Rev. 2008, 252, 751-766, 10.1016/j.ccr.2007.09.016

The fusion of homogeneous and enzymatic catalysis has recently drawn attention due to reported novel activities and high selectivities. The incorporation of metal-catalysts into proteins combines the advantages of both catalytic strategies. Herein we summarize recent approaches of artificial metalloenzymes applied to catalysis. The discussion includes different strategies of anchoring and screening for improved selectivity.

Ward, T.R.

Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 4683-4687, 10.1073/pnas.0409684102

Most physiological and biotechnological processes rely on molecular recognition between chiral (handed) molecules. Manmade homogeneous catalysts and enzymes offer complementary means for producing enantiopure (single-handed) compounds. As the subtle details that govern chiral discrimination are difficult to predict, improving the performance of such catalysts often relies on trial-and-error procedures. Homogeneous catalysts are optimized by chemical modification of the chiral environment around the metal center. Enzymes can be improved by modification of gene encoding the protein. Incorporation of a biotinylated organometallic catalyst into a host protein (avidin or streptavidin) affords versatile artificial metalloenzymes for the reduction of ketones by transfer hydrogenation. The boric acid·formate mixture was identified as a hydrogen source compatible with these artificial metalloenzymes. A combined chemo-genetic procedure allows us to optimize the activity and selectivity of these hybrid catalysts: up to 94% (R) enantiomeric excess for the reduction of p-methylacetophenone. These artificial metalloenzymes display features reminiscent of both homogeneous catalysts and enzymes.

Distefano, M.D.

Bioorg. Med. Chem. Lett. 1999, 9, 79-84, 10.1016/S0960-894X(98)00684-2

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

Roelfes, G.

ChemCatChem 2020, 12, 3190-3194, 10.1002/cctc.202000245

The supramolecular approach is among the most convenient methodologies for creating artificial metalloenzymes (ArMs). Usually this approach involves the binding of a transition metal ion complex to a biomolecular scaffold via its ligand, which also modulates the catalytic properties of the metal ion. Herein, we report ArMs based on the proteins CgmR, RamR and QacR from the TetR family of multidrug resistance regulators (MDRs) and Cu2+ ions, assembled without the need of a ligand. These ArMs catalyze the enantioselective vinylogous Friedel-Crafts alkylation reaction with up to 75 % ee. Competition experiments with ethidium and rhodamine 6G confirm that the reactions occur in the chiral environment of the hydrophobic pocket. It is proposed that the Cu2+-substrate complex is bound via a combination of electrostatic and π-stacking interactions provided by the second coordination sphere. This approach constitutes a fast and straightforward way to assemble metalloenzymes and may facilitate future optimization of the protein scaffolds via mutagenesis or directed evolution approaches.

Ward, T.R.

Acc. Chem. Res. 2011, 44, 47-57, 10.1021/ar100099u

Artificial metalloenzymes are created by incorporating an organometallic catalyst within a host protein. The resulting hybrid can thus provide access to the best features of two distinct, and often complementary, systems: homogeneous and enzymatic catalysts. The coenzyme may be positioned with covalent, dative, or supramolecular anchoring strategies. Although initial reports date to the late 1970s, artificial metalloenzymes for enantioselective catalysis have gained significant momentum only in the past decade, with the aim of complementing homogeneous, enzymatic, heterogeneous, and organic catalysts. Inspired by a visionary report by Wilson and Whitesides in 1978, we have exploited the potential of biotin−avidin technology in creating artificial metalloenzymes. Owing to the remarkable affinity of biotin for either avidin or streptavidin, covalent linking of a biotin anchor to a catalyst precursor ensures that, upon stoichiometric addition of (strept)avidin, the metal moiety is quantitatively incorporated within the host protein. In this Account, we review our progress in preparing and optimizing these artificial metalloenzymes, beginning with catalytic hydrogenation as a model and expanding from there. These artificial metalloenzymes can be optimized by both chemical (variation of the biotin−spacer−ligand moiety) and genetic (mutation of avidin or streptavidin) means. Such chemogenetic optimization schemes were applied to various enantioselective transformations. The reactions implemented thus far include the following: (i) The rhodium-diphosphine catalyzed hydrogenation of N-protected dehydroaminoacids (ee up to 95%); (ii) the palladium-diphosphine catalyzed allylic alkylation of 1,3-diphenylallylacetate (ee up to 95%); (iii) the ruthenium pianostool-catalyzed transfer hydrogenation of prochiral ketones (ee up to 97% for aryl-alkyl ketones and ee up to 90% for dialkyl ketones); (iv) the vanadyl-catalyzed oxidation of prochiral sulfides (ee up to 93%). A number of noteworthy features are reminiscent of homogeneous catalysis, including straightforward access to both enantiomers of the product, the broad substrate scope, organic solvent tolerance, and an accessible range of reactions that are typical of homogeneous catalysts. Enzyme-like features include access to genetic optimization, an aqueous medium as the preferred solvent, Michaelis−Menten behavior, and single-substrate derivatization. The X-ray characterization of artificial metalloenzymes provides fascinating insight into possible enantioselection mechanisms involving a well-defined second coordination sphere environment. Thus, such artificial metalloenzymes combine attractive features of both homogeneous and enzymatic kingdoms. In the spirit of surface borrowing, that is, modulating ligand affinity by harnessing existing protein surfaces, this strategy can be extended to selectively binding streptavidin-incorporated biotinylated ruthenium pianostool complexes to telomeric DNA. This application paves the way for chemical biology applications of artificial metalloenzymes.

Ward, T.R.

Acc. Chem. Res. 2016, 49, 1711-1721, 10.1021/acs.accounts.6b00235

The biotin–streptavidin technology offers an attractive means to engineer artificial metalloenzymes (ArMs). Initiated over 50 years ago by Bayer and Wilchek, the biotin–(strept)avidin techonology relies on the exquisite supramolecular affinity of either avidin or streptavidin for biotin. This versatile tool, commonly referred to as “molecular velcro”, allows nearly irreversible anchoring of biotinylated probes within a (strept)avidin host protein. Building upon a visionary publication by Whitesides from 1978, several groups have been exploiting this technology to create artificial metalloenzymes. For this purpose, a biotinylated organometallic catalyst is introduced within (strept)avidin to afford a hybrid catalyst that combines features reminiscent of both enzymes and organometallic catalysts. Importantly, ArMs can be optimized by chemogenetic means. Combining a small collection of biotinylated organometallic catalysts with streptavidin mutants allows generation of significant diversity, thus allowing optimization of the catalytic performance of ArMs. Pursuing this strategy, the following reactions have been implemented: hydrogenation, alcohol oxidation, sulfoxidation, dihydroxylation, allylic alkylation, transfer hydrogenation, Suzuki cross-coupling, C–H activation, and metathesis. In this Account, we summarize our efforts in the latter four reactions. X-ray analysis of various ArMs based on the biotin–streptavidin technology reveals the versatility and commensurability of the biotin-binding vestibule to accommodate and interact with transition states of the scrutinized organometallic transformations. In particular, streptavidin residues at positions 112 and 121 recurrently lie in close proximity to the biotinylated metal cofactor. This observation led us to develop a streamlined 24-well plate streptavidin production and screening platform to optimize the performance of ArMs. To date, most of the efforts in the field of ArMs have focused on the use of purified protein samples. This seriously limits the throughput of the optimization process. With the ultimate goal of complementing natural enzymes in the context of synthetic and chemical biology, we outline the milestones required to ultimately implement ArMs within a cellular environment. Indeed, we believe that ArMs may allow signficant expansion of the natural enzymes’ toolbox to access new-to-nature reactivities in vivo. With this ambitious goal in mind, we report on our efforts to (i) activate the biotinylated catalyst precursor upon incorporation within streptavidin, (ii) minimize the effect of the cellular environment on the ArM’s performance, and (iii) demonstrate the compatibility of ArMs with natural enzymes in cascade reactions.

Ward, T.R.

Acc. Chem. Res. 2019, 52, 585-595, 10.1021/acs.accounts.8b00618

Artificial metalloenzymes (ArMs) result from anchoring a metal-containing moiety within a macromolecular scaffold (protein or oligonucleotide). The resulting hybrid catalyst combines attractive features of both homogeneous catalysts and enzymes. This strategy includes the possibility of optimizing the reaction by both chemical (catalyst design) and genetic means leading to achievement of a novel degree of (enantio)selectivity, broadening of the substrate scope, or increased activity, among others. In the past 20 years, the Ward group has exploited, among others, the biotin–(strept)avidin technology to localize a catalytic moiety within a well-defined protein environment. Streptavidin has proven versatile for the implementation of ArMs as it offers the following features: (i) it is an extremely robust protein scaffold, amenable to extensive genetic manipulation and mishandling, (ii) it can be expressed in E. coli to very high titers (up to >8 g·L–1 in fed-batch cultures), and (iii) the cavity surrounding the biotinylated cofactor is commensurate with the size of a typical metal-catalyzed transition state. Relying on a chemogenetic optimization strategy, varying the orientation and the nature of the biotinylated cofactor within genetically engineered streptavidin, 12 reactions have been reported by the Ward group thus far. Recent efforts within our group have focused on extending the ArM technology to create complex systems for integration into biological cascade reactions and in vivo. With the long-term goal of complementing in vivo natural enzymes with ArMs, we summarize herein three complementary research lines: (i) With the aim of mimicking complex cross-regulation mechanisms prevalent in metabolism, we have engineered enzyme cascades, including cross-regulated reactions, that rely on ArMs. These efforts highlight the remarkable (bio)compatibility and complementarity of ArMs with natural enzymes. (ii) Additionally, multiple-turnover catalysis in the cytoplasm of aerobic organisms was achieved with ArMs that are compatible with a glutathione-rich environment. This feat is demonstrated in HEK-293T cells that are engineered with a gene switch that is upregulated by an ArM equipped with a cell-penetrating module. (iii) Finally, ArMs offer the fascinating prospect of “endowing organometallic chemistry with a genetic memory.” With this goal in mind, we have identified E. coli’s periplasmic space and surface display to compartmentalize an ArM, while maintaining the critical phenotype–genotype linkage. This strategy offers a straightforward means to optimize by directed evolution the catalytic performance of ArMs. Five reactions have been optimized following these compartmentalization strategies: ruthenium-catalyzed olefin metathesis, ruthenium-catalyzed deallylation, iridium-catalyzed transfer hydrogenation, dirhodium-catalyzed cyclopropanation and carbene insertion in C–H bonds. Importantly, >100 turnovers were achieved with ArMs in E. coli whole cells, highlighting the multiple turnover catalytic nature of these systems.

Ward, T.R.

ACS Cent. Sci. 2019, 5, 1120-1136, 10.1021/acscentsci.9b00397

Artificial metalloenzymes (ArMs) result from the incorporation of an abiotic metal cofactor within a protein scaffold. From the earliest techniques of transition metals adsorbed on silk fibers, the field of ArMs has expanded dramatically over the past 60 years to encompass a range of reaction classes and inspired approaches: Assembly of the ArMs has taken multiple forms with both covalent and supramolecular anchoring strategies, while the scaffolds have been intuitively selected and evolved, repurposed, or designed in silico. Herein, we discuss some of the most prominent recent examples of ArMs to highlight the challenges and opportunities presented by the field.

Ward, T.R.

Synlett 2009, 2009, 3225-3236, 10.1055/s-0029-1218305

By combining homogeneous with enzymatic catalysis, artificial metalloenzymes offer new perspectives for conferring unnatural activities to biomolecules. The article reassembles the important advances in the field of these hybrid catalysts and summarizes the contributions of our group to this continuously growing field of research.

Onoda, A.; Salmain, M.

Bioorganometallic Chemistry: Applications in Drug Discovery, Biocatalysis, and Imaging 2014, 305-338, 10.1002/9783527673438.ch10

Enzymes are the catalysts of the living world. Nature has tailored proteins to catalyze an incredibly wide range of reactions with exquisite selectivity and efficiency under very mild conditions of temperature, pH, pressure, and so on. Protein engineering combined with molecular modeling techniques affords tailor‐made biocatalysts for the industrial production of chiral synthons. Nonetheless, endowing a given protein scaffold with a totally new activity remains a challenging task for the biochemist. Among the current strategies to impart proteins with unnatural activity, those dealing with the construction of artificial metalloenzymes are particularly promising. By definition, artificial metalloenzymes are hybrid catalysts resulting from the incorporation of a transition metal species within a biomacromolecular scaffold. The rationale behind this concept is to combine the wide catalytic scope of transition metal complexes with the high activity and selectivity of biocatalysts. In most of the hybrid catalysts reported so far, the roles devoted to both partners are clearly separated: the metal complex being responsible for reactivity, while the protein environment is used to induce selectivity in the chemical process. In that, artificial metalloenzymes truly resemble enzymes whose efficiency relies on both the active site and the second sphere of coordination (also called the outer coordination sphere). In this chapter, we intend to give an overview of the various anchoring strategies reported over the last decade for the controlled, site‐selective attachment of nonnative metal cofactors within protein matrices together with the activity/selectivity displayed by these hybrid enzymes.

Salmain, M.

Dalton Trans. 2014, 43, 5482-5489, 10.1039/c3dt53253d

Protein hybrids resulting from the supramolecular anchoring to bovine β-lactoglobulin of fatty acid-derived Rh(iii) diimine complexes catalysed the asymmetric transfer hydrogenation of trifluoroacetophenone with up to 32% ee.

Pecoraro, V.L.

Curr. Opin. Chem. Biol. 2015, 25, 65-70, 10.1016/j.cbpa.2014.12.034

Three-helix bundles and coiled-coil motifs are well-established de novo designed scaffolds that have been investigated for their metal-binding and catalytic properties. Satisfaction of the primary coordination sphere for a given metal is sufficient to introduce catalytic activity and a given structure may catalyze different reactions dependent on the identity of the incorporated metal. Here we describe recent contributions in the de novo design of metalloenzymes based on three-helix bundles and coiled-coil motifs, focusing on non-heme systems for hydrolytic and redox chemistry.