Artificial enzymes

Changing the catalytic metal centre of a non-haem iron dioxygenase to copper results in an enzyme capable of Lewis acid catalysis of new-to-nature enantioselective Conia-ene reactions.

Artificial metalloenzymes (ArM) hold great potential for the sustainable catalysis of complex new-to-nature reactions. To efficiently improve the catalytic efficacy of ArMs a rational approach is desirable, requiring detailed molecular insight into their conformational landscape. Lactococcal multidrug resistance regulator (LmrR) is a multi-purpose ArM scaffold protein that when bound to the Cu(II)-phenanthroline cofactor catalyzes the Friedel-Crafts Alkylation (FCA) of indoles. Previously, the M8D and A92E mutations were found to increase the efficiency of this reaction, but a molecular explanation has been lacking. We here determined the impact of these two activating mutations on the conformational landscape of LmrR in its apo, cofactor- and substrate-bound state. We found that the mutations cause a marked destabilization of the dimerization interface, resulting in a more opened central hydrophobic cavity and a dynamic equilibrium between dimer and monomer LmrR. While mutant and wild-type have similar pocket conformation in the cofactor-bound state, the mutant shows a distinct interaction with the substrate. Our results suggest that increased retention of the catalytic cofactor and widened plasticity improve activity of the mutant. Ultimately, these results help elucidating the intricate relationships between conformational dynamics of the protein scaffold, cofactor and substrates that define catalytic activity.

Genetic incorporation of noncanonical amino acids (ncAAs) harbouring catalytic side chains into proteins allows the creation of enzymes able to catalyse reactions that have no equivalent in nature. Here, we present for the first time the use of the ncAA 3-aminotyrosine (aY) as catalytic residue in a designer enzyme for iminium activation catalysis. Incorporation of aY into protein scaffold LmrR gave rise to an artificial Friedel-Crafts (FC) alkylase exhibiting complementary enantioselectivity to a previous FC-alkylase design using p-aminophenylalanine as catalytic residue in the same protein. The new FC-alkylase was optimized by directed evolution to afford a quadruple mutant that showed increased activity and excellent enantioselectivity (up to 95% ee). X-ray crystal structures of the parent and evolved designer enzymes suggest that the introduced mutations cause a narrowing of the active site and a reorientation of the catalytic -NH2 group. Furthermore, the evolved FC-alkylase was applied in whole-cell catalysis, facilitated by the straightforward incorporation of aY. Our work demonstrates that aY is a valuable addition to the biochemists toolbox for creating artificial enzymes. 

Artificial metalloenzymes (ArMs) are an attractive approach to achieving “new to nature” biocatalytic transformations. In this work, a novel copper-dependent artificial Michaelase (Cu_Michaelase) comprising a genetically encoded copper-binding ligand, i.e. (2,2-bipyridin-5-yl)alanine (BpyA), was developed. For the first time, such an ArM containing a non-canonical metal-binding amino acid was successfully optimized through directed evolution. The evolved Cu_Michaelase was applied in the copper-catalyzed asymmetric addition of 2-acetyl azaarenes to nitroalkenes, yielding various γ-nitro butyric acid derivatives, which are precursors for a range of high-value-added pharmaceutically relevant compounds, with good yields and high enantioselectivities (up to >99% yield and 99% ee). Additionally, the evolved variant could be further used in a preparative-scale synthesis, providing chiral products for diverse derivatizations. X-ray crystal structure analysis confirmed the binding of Cu(II) ions to the BpyA residues and showed that, in principle, there is sufficient space for the 2-acetyl azaarene substrate to coordinate. Kinetic studies showed that the increased catalytic efficiency of the evolved enzyme is due to improvements in apparent KM for both substrates and a notable threefold increase in apparent kcat for 2-acetyl pyridine. This work illustrates the potential of artificial metalloenzymes exploiting non-canonical metal-binding ligands for new-to-nature biocatalysis.

The evolution of a promiscuous enzyme for its various activities often results in catalytically specialized variants. This is an important natural mechanism to ensure the proper functioning of natural metabolic networks. It also acts as both a curse and blessing for enzyme engineers, where enzymes that have undergone directed evolution may exhibit exquisite selectivity at the expense of a diminished overall catalytic repertoire. We previously performed two independent directed evolution campaigns on a promiscuous designer enzyme that leverages the unique properties of a non-canonical amino acid (ncAA) para-aminophenylalanine (pAF) as catalytic residue, resulting in two evolved variants which are both catalytically specialized. Here, we combine mutagenesis, crystallography and computation to reveal the molecular basis of the specialization phenomenon. In one evolved variant, an unexpected change in quaternary structure biases substrate dynamics to promote enantioselective catalysis, whilst the other demonstrates synergistic cooperation between natural side chains and the pAF residue to form semi-synthetic catalytic machinery. 

Incorporating noble metals in artificial metalloenzymes (ArMs) is challenging due to the lack of suitable soft coordinating ligands among natural amino acids. We present a new class of ArMs featuring a genetically encoded noble-metal-binding site based on a non-canonical thiophenol-based amino acid, 4-mercaptophenylalanine (pSHF), incorporated in the transcriptional regulator LmrR via stop codon suppression. We demonstrate that pSHF is an excellent ligand for noble metals in their low oxidation states. The corresponding gold(I) enzyme was characterised by mass spectrometry, UV-vis spectroscopy and X-ray crystallography and successfully catalysed hydroamination reactions of 2-ethynyl anilines with over 50 turnover numbers. Interestingly, two equivalents of gold(I) per protein dimer proved to be required for activity. Up to 98% regioselectivity in the hydroamination of an ethynylphenylurea substrate was obtained, yielding the corresponding phenyl-dihydroquinazolinone product, consistent with a π-activation mechanism by single gold centres. The ArM was optimized by site saturation mutagenesis, using an on-bead screening protocol. This resulted in a single mutant that showed higher activity on one class of substrates. This work brings the power of noble-metal catalysis into the realm of enzyme engineering and establishes thiophenols as alternative ligands for noble metals, providing new opportunities in coordination chemistry and catalysis.

Genetically encoded noncanonical amino acids can introduce new-to-nature activation modes into enzymes. While these amino acids can act as catalysts on their own due to their inherent chemical properties, interactions with adjacent residues in an enzyme, such as those present in natural catalytic dyads or triads, unlock a higher potential for designer enzymes. We incorporated a boron-containing amino acid into the protein scaffold RamR to create an active enzyme for the kinetic resolution of α-hydroxythioesters. We found that a closely positioned lysine residue is crucial for the catalytic activity of the designer enzyme by forming a hybrid catalytic dyad with the boronic acid residue. The enzyme is capable of resolving differently substituted α-hydroxythioesters with good selectivities. High-resolution mass spectrometry, 11B NMR spectroscopy, and crystal structure analysis of the designer enzyme gave insight into the three steps of the mechanism (substrate binding, hydroxide transfer, product release). Mutations of a residue around the catalytic dyad led to a variant of the enzyme with 2-fold improvement of catalytic activity and selectivity.

The development of artificial metalloenzymes (ArMs) aims to expand the capabilities of enzymatic catalysis, most notably towards new reaction mechanisms. Frequently, ArMs harness metal cofactors that are not naturally found in enzymes and embed these in specifically selected or designed protein scaffolds. ArMs have been developed for a wide range of natural and non-natural reactions, underscoring their potential to revolutionize fields such as biocatalysis or metabolic engineering. At the same time, replicating the catalytic prowess of natural enzymes is a highly challenging task, and several limitations need to be overcome to make ArM catalysis widely applicable. In this Primer, we introduce the state of the art in designing and engineering ArMs, describing best practices and important examples and achievements. Moreover, we consider potential applications of ArMs, as well as outstanding challenges, and discuss how these may be addressed in the coming years.

Biocatalysis has become an important component of modern organic chemistry, presenting an efficient and environmentally friendly approach to synthetic transformations. Advances in molecular biology, computational modeling, and protein engineering have unlocked the full potential of enzymes in various industrial applications. However, the inherent limitations of the natural building blocks have sparked a revolutionary shift. In vivo genetic incorporation of noncanonical amino acids exceeds the conventional 20 amino acids, opening new avenues for innovation. This review provides a comprehensive overview of applications of noncanonical amino acids in biocatalysis. We aim to examine the field from multiple perspectives, ranging from their impact on enzymatic reactions to the creation of novel active sites, and subsequent catalysis of new-to-nature reactions. Finally, we discuss the challenges, limitations, and promising opportunities within this dynamic research domain. 

Combining a natural decarboxylase and an artificial metathase, a microbial cell factory is created that enables the synthesis of cycloalkenes from fatty diacids in a whole-cell hybrid biocatalytic cascade process.

Enzymes play an increasingly important role in improving the benignity and efficiency of chemical production, yet the diversity of their applications lags heavily behind chemical catalysts as a result of the relatively narrow range of reaction mechanisms of enzymes. The creation of enzymes containing non-biological functionalities facilitates reaction mechanisms outside nature’s canon and paves the way towards fully programmable biocatalysis. Here we present a completely genetically encoded boronic-acid-containing designer enzyme with organocatalytic reactivity not achievable with natural or engineered biocatalysts. This boron enzyme catalyses the kinetic resolution of hydroxyketones by oxime formation, in which crucial interactions with the protein scaffold assist in the catalysis. A directed evolution campaign led to a variant with natural-enzyme-like enantioselectivities for several different substrates. The unique activation mode of the boron enzyme was confirmed using X-ray crystallography, high-resolution mass spectrometry (HRMS) and 11B NMR spectroscopy. Our study demonstrates that genetic-code expansion can be used to create evolvable enantioselective enzymes that rely on xenobiotic catalytic moieties such as boronic acids and access reaction mechanisms not reachable through catalytic promiscuity of natural or engineered enzymes.

Artificial enzymes are valuable biocatalysts able to perform new-to-nature transformations with the precision and (enantio-)selectivity of natural enzymes. Although they are highly engineered biocatalysts, they often cannot reach catalytic rates akin those of their natural counterparts, slowing down their application in real-world industrial processes. Typically, their designs only optimise the chemistry inside the active site, while overlooking the role of protein dynamics on catalysis. In this work, we show how the catalytic performance of an already engineered artificial enzyme can be further improved by distal mutations that affect the conformational equilibrium of the protein. To this end, we subjected a specialised artificial enzyme based on the lactococcal multidrug resistance regulator (LmrR) to an innovative algorithm that quickly inspects the whole protein sequence space for hotpots which affect the protein dynamics. From an initial predicted selection of 73 variants, two variants with mutations distant by more than 11 Å from the catalytic pAF residue showed increased catalytic activity towards the new-to-nature hydrazone formation reaction. Their recombination displayed a 66% higher turnover number and 14 °C higher thermostability. Microsecond time scale molecular dynamics simulations evidenced a shift in the distribution of productive enzyme conformations, which are the result of a cascade of interactions initiated by the introduced mutations.

Friedel-Crafts alkylation and acylation reactions are important methodologies in synthetic and industrial chemistry for the construction of aryl-alkyl and aryl-acyl linkages that are ubiquitous in bioactive molecules. Nature also exploits these reactions in many biosynthetic processes. Much work has been done to expand the synthetic application of these enzymes to unnatural substrates through directed evolution. The promise of such biocatalysts is their potential to supersede inefficient and toxic chemical approaches to these reactions, with mild operating conditions - the hallmark of enzymes. Complementary work has created many bio-hybrid Friedel-Crafts catalysts consisting of chemical catalysts anchored into biomolecular scaffolds, which display many of the same desirable characteristics. In this Review, we summarise these efforts, focussing on both mechanistic aspects and synthetic considerations, concluding with an overview of the frontiers of this field and routes towards more efficient and benign Friedel-Crafts reactions for the future of humankind.

The incorporation of organocatalysts into protein scaffolds holds the promise of overcoming some of the limitations of this powerful catalytic approach. Previously, we showed that incorporation of the non-canonical amino acid para-aminophenylalanine into the non-enzymatic protein scaffold LmrR forms a proficient and enantioselective artificial enzyme (LmrR_pAF) for the Friedel-Crafts alkylation of indoles with enals. The unnatural aniline side-chain is directly involved in catalysis, operating via a well-known organocatalytic iminium-based mechanism. In this study, we show that LmrR_pAF can enantioselectively form tertiary carbon centres not only during C-C bond formation, but also by enantioselective protonation, delivering a proton to one face of a prochiral enamine intermediate. The importance of various side-chains in the pocket of LmrR is distinct from the Friedel-Crafts reaction without enantioselective protonation, and two particularly important residues were probed by exhaustive mutagenesis.

Enantioselective protonation is conceptually one of the most attractive methods to generate an α-chiral center. However, enantioselective protonation presents major challenges, especially in water. Herein, we report a tandem Michael addition/enantioselective protonation reaction catalyzed by an artificial enzyme employing two abiological catalytic sites in a synergistic fashion: a genetically encoded noncanonical p-aminophenylalanine residue and a Lewis acid Cu(II) complex. The exquisite stereocontrol achieved in the protonation of the transient enamine intermediate is illustrated by up to >20:1 dr and >99% ee of the product. These results illustrate the potential of exploiting synergistic catalysis in artificial enzymes for challenging reactions

Heme enzymes are some of the most versatile catalysts in nature. In recent years it has been found that they can also catalyze reactions for which there are no equivalents in nature. This development has been driven by the abiological catalytic reactivity reported for bio-inspired and biomimetic iron porphyrin complexes. This review focuss es on heme enzymes for catalysis of cyclopropanation reactions. The two most important approaches used to create enzymes for cyclopropanation are repurposing of heme enzymes and the various strategies used to improve these enzymes such as mutagenesis and heme replacement, and artificial heme enzymes. These strategies are introduced and compared. Moreover, lessons learned with regard to mechanism and design principles are discussed.

Artificial metalloenzymes (ArMs), which are hybrids of catalytically active transition metal complexes and proteins, have emerged as promising approach to the creation of biocatalysts for reactions that have no equivalent in nature. Here we report the assembly and application in catalysis of ArMs in the cytoplasm of E. coli cells based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneously added copper(II)‐phenanthroline (Cu(II)‐phen) complex. The ArMs are spontaneously assembled by addition of Cu(II)‐phen to E. coli cells that express LmrR and it is shown that the ArM containing whole cells are active in the catalysis of the enantioselective vinylogous Friedel‐Crafts alkylation of indoles. The ArM assembly in E. coli is further supported by a combination of cell‐ fractionation and inhibitor experiments and confirmed by in‐cell solid‐state NMR. A mutagenesis study showed that the same trends in catalytic activity and enantioselectivity in response to mutations of LmrR were observed for the ArM containing whole cells and the isolated ArMs. This made it possible to perform a directed evolution study using ArMs in whole cells, which gave rise to a mutant, LmrR_A92E_M8D that showed increased activity and enantioselectivity in the catalyzed vinylogous Friedel‐Crafts alkylation of a variety of indoles. The unique aspect of this whole‐cell ArM system is that no engineering of the microbial host, the protein scaffold or the cofactor is required to achieve ArM assembly and catalysis. This makes this system attractive for applications in whole cell biocatalysis and directed evolution, as demonstrated here. Moreover, our findings represent important step forward towards achieving the challenging goal of a hybrid metabolism by integrating artificial metalloenzymes in biosynthetic pathways.

The construction and engineering of artificial enzymes consisting of abiological catalytic moieties incorporated into protein scaffolds is a promising strategy to realise new mechanisms in biocatalysis. Here, we show that incorporation of the non-canonical amino acid para-aminophenylalanine (pAF) into the non-enzymatic protein scaffold LmrR creates a proficient and stereoselective artificial enzyme (LmrR_pAF) for the vinylogous Friedel-Crafts alkylation between alpha,beta-unsaturated aldehydes and indoles. pAF acts as a catalytic residue, activating enal substrates towards conjugate addition via the formation of intermediate iminium ion species, whilst the protein scaffold provides rate acceleration and enantio-induction. Improved LmrR_pAF variants were identified by low-throughput directed evolution advised by alanine-scanning to obtain a triple mutant that provided higher yields and enantioselectivities for a range of aliphatic enals and substituted indoles. Analysis of Michaelis-Menten kinetics of LmrR_pAF and evolved mutants reveals that new activities emerge via evolutionary pathways that diverge from one another and specialise catalytic reactivity. Translating this iminium-based catalytic mechanism into an enzymatic context will enable many new biocatalytic transformations inspired by organocatalysis.

We present an artificial metalloenzymes based on the transcriptional regulator LmrR that exhibits dynamics involving the positioning of its abiological metal cofactor. The position of the cofactor, in turn, was found to be related to the preferred catalytic reactivity, which is either the enantioselective Friedel-Crafts alkylation of indoles with beta-substituted enones or the tandem Friedel-Crafts alkylation / enantioselective protonation of indoles with alpha-substituted enones. The artificial metalloenzyme could be specialized for one of these catalytic reactions introducing a single mutation in the protein. The relation between cofactor dynamics and activity and selectivity in catalysis has not been described for natural enzymes and, to date, appears to be particular for artificial metalloenzymes.

Natural proteins often present binding or functional promiscuity. In  biocatalysis, this promiscuity has been exploited for accessing new‐ to‐nature  reactions.  Here,  we    report  an  unexpected  catalytic  activity  for  the  regulatory  protein  QacR  from  the  TetR  family  of  Multidrug  Resistance  Regulators.  QacR_C72A_C141S  is  able  to  catalyze the tandem Friedel‐Crafts/enantioselective protonation of  indoles with α‐substituted conjugated enones with 38% yield and up to 83% ee. Mutagenesis and computational studies support the hypothesis  that  an  acidic  residue  in  the  binding  pocket  of  the  protein is responsible for protonating the reaction intermediate.

Here we examine the potential of the non-canonical amino acid (8-hydroxyquinolin-3-yl)alanine (HQAla) for the design of artificial metalloenzymes. HQAla, a versatile chelator of late transition metals, was introduced into the lactococcal multidrug resistance regulator (LmrR) via stop codon suppression methodology. LmrR_HQAla was shown to efficiently complex with three different metal ions, CuII, ZnII and RhIII to form unique artificial metalloenzymes. The catalytic potential of the CuII bound LmrR_HQAla enzyme was shown through its ability to catalyze asymmetric Friedel-Craft alkylation and water addition reactions, whereas the ZnII coupled enzyme was shown to mimic natural Zn-hydrolase activity.

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.

Artificial enzymes, which are hybrids of proteins with abiological catalytic groups, have emerged as a powerful approach towards the creation of enzymes for new-to-nature reactions. Typically, only a single abiological catalytic moiety is incorporated. Here we introduce a design of an artificial enzyme that comprises two different abiological catalytic moieties and show that these can act synergistically to achieve high activity and enantioselectivity (up to >99% e.e.) in the catalysed Michael addition reaction. The design is based on the lactococcal multidrug resistance regulator as the protein scaffold and combines a genetically encoded unnatural p-aminophenylalanine residue (which activates an enal through iminium ion formation) and a supramolecularly bound Lewis acidic Cu(ii) complex (which activates the Michael donor by enolization and delivers it to one preferred prochiral face of the activated enal). This study demonstrates that synergistic combination of abiological catalytic groups is a robust way to achieve catalysis that is normally outside of the realm of artificial enzymes.

The emergence of robust methods to expand the genetic code allows incorporation of non-canonical amino acids into the polypeptide chain of proteins, thus making it possible to introduce unnatural chemical functionalities in enzymes. In this Perspective, we show how this powerful methodology is used to create enzymes with improved and novel, even new-to-nature, catalytic activities. We provide an overview of the current state of the art, and discuss the potential benefits of developing and using enzymes with genetically encoded non-canonical amino acids compared with enzymes containing only canonical amino acids.

The ability of one enzyme to catalyse multiple, mechanistically distinct transformations likely played a crucial role in organisms' abilities to adapt to changing external stimuli in the past and can still be observed in extant enzymes. Given the importance of catalytic promiscuity in nature, enzyme designers have recently begun to create catalytically promiscuous enzymes in order to expand the canon of transformations catalysed by proteins. This article aims to both critically review different strategies for the design of enzymes that display catalytic promiscuity for new- to-nature reactions and highlight the successes of subsequent directed- evolution efforts to fine- tune these novel reactivities. For the former, we put a particular emphasis on the creation, stabilization and repurposing of reaction intermediates, which are key for unlocking new activities in an existing or designed active site. For the directed evolution of the resulting catalysts, we contrast approaches for enzyme design that make use of components found in nature and those that achieve new reactivities by incorporating synthetic components. Following the critical analysis of selected examples that are now available, we close this Review by providing a set of considerations and design principles for enzyme engineers, which will guide the future generation of efficient artificial enzymes for synthetically useful, abiotic transformations.

We report the late stage chemical modification of ribosomally synthesized and posttranslationally modified peptides (RIPPs) by Diels‐Alder cycloadditions to naturally occurring dehydroalanines. The tail region of the thiopeptide thiostrepton could be modified selectively and efficiently under microwave heating and transition metal free conditions. The Diels‐Alder adducts were isolated and the different site‐ and endo/exo isomers were identified by 1D/2D 1H NMR. Via efficient modification of the thiopeptide nosiheptide and the lanthipeptide nisin Z the generality of the method was established. MIC assays of the purified thiostrepton Diels‐Alder products against thiostrepton‐susceptible strains displayed high activities comparable to that of native thiostrepton. These Diels‐Alder products were also subjected successfully to Inverse‐electron‐demand Diels‐Alder reactions with a variety of functionalized tetrazines, demonstrating the utility of this method for labeling of RiPPs.

Despite the increasing efforts in the last few years, the identification of efficient catalysts able to perform the enantioselective addition of water to double bonds has not been achieved yet. Natural hydratases represent an interesting pool of biocatalysts to generate chiral alcohols, but modifying their substrate scope remains an issue. The use of artificial metalloenzymes (ArMs) appears as a promising solution in this field. In the last few years, Roelfes and co-workers have been designing a variety of DNA- and protein-based ArMs able to carry out the copper-mediated addition of water to conjugated alkenes with promising enantioselective levels. Still, from a mechanistic point of view, the copper-mediated hydration reaction remains unclear and a matter of debate. This lack of information greatly hampers further designs and optimizations of the LmrR-based copper hydratases in terms of substrates and/or enantioselective profiles. In this study, we aim to provide a better understanding of the copper-catalyzed hydration of alkenes occurring both in water solvent and into the context of the LmrR protein as designed by Roelfes and co-workers. For that purpose, we make use of an integrated computational protocol that combines quantum mechanics (QM) (including small and large cluster models as well as ab initio molecular dynamics (AIMD)) and force-field approaches (including protein-ligand docking and classical molecular dynamics (MD) simulation). This integrative study sheds light on the general doubts around the copper-catalyzed hydration mechanism and also paves the way toward more conscious designs of ArMs able to efficiently catalyze the enantioselective addition of water to double bonds.

The biotechnological revolution has made it possible to create enzymes for many reactions by directed evolution. However, because of the immense number of possibilities, the availability of enzymes that possess a basal level of the desired catalytic activity is a prerequisite for success. For new-to-nature reactions, artificial metalloenzymes (ARMs), which are rationally designed hybrids of proteins and catalytically active transition-metal complexes, can be such a starting point. This Account details our efforts toward the creation of ARMs for the catalysis of new-to-nature reactions. Key to our approach is the notion that the binding of substrates, that is, effective molarity, is a key component to achieving large accelerations in catalysis. For this reason, our designs are based on the multidrug resistance regulator LmrR, a dimeric transcription factor with a large, hydrophobic binding pocket at its dimer interface. In this pocket, there are two tryptophan moieties, which are important for promiscuous binding of planar hydrophobic conjugated compounds by π-stacking. The catalytic machinery is introduced either by the covalent linkage of a catalytically active metal complex or via the ligand or supramolecular assembly, taking advantage of the two central tryptophan moieties for noncovalent binding of transition-metal complexes. Designs based on the chemical modification of LmrR were successful in catalysis, but this approach proved too laborious to be practical. Therefore, expanded genetic code methodologies were used to introduce metal binding unnatural amino acids during LmrR biosynthesis in vivo. These ARMs have been successfully applied in Cu(II) catalyzed Friedel-Crafts alkylation of indoles. The extension to MDRs from the TetR family resulted in ARMs capable of providing the opposite enantiomer of the Friedel-Crafts product. We have employed a computationally assisted redesign of these ARMs to create a more active and selective artificial hydratase, introducing a glutamate as a general base at a judicious position so it can activate and direct the incoming water nucleophile. A supramolecularly assembled ARM from LmrR and copper(II)-phenanthroline was successful in Friedel-Crafts alkylation reactions, giving rise to up to 94% ee. Also, hemin was bound, resulting in an artificial heme enzyme for enantioselective cyclopropanation reactions. The importance of structural dynamics of LmrR was suggested by computational studies, which showed that the pore can open up to allow access of substrates to the catalytic iron center, which, according to the crystal structure, is deeply buried inside the protein. Finally, the assembly approaches were combined to introduce both a catalytic and a regulatory domain, resulting in an ARM that was specifically activated in the presence of Fe(II) salts but not Zn(II) salts. Our work demonstrates that LmrR is a privileged scaffold for ARM design: It allows for multiple assembly methods and even combinations of these, it can be applied in a variety of different catalytic reactions, and it shows significant structural dynamics that contribute to achieving the desired catalytic activity. Moreover, both the creation via expanded genetic code methods as well as the supramolecular assembly make LmrR-based ARMs highly suitable for achieving the ultimate goal of the integration of ARMs in biosynthetic pathways in vivo to create a hybrid metabolism.

Directed evolution generated an enzyme for the enantioselective synthesis of α-trifluoromethylated organoborons, potentially attractive synthons for fluorinated compounds.

The impressive rate accelerations that enzymes display in nature often result from boosting the inherent catalytic activities of side chains by their precise positioning inside a protein binding pocket. Here we show that such fine‐tuning is also possible for catalytic unnatural amino acids. Specifically, we report the directed evolution of a recently described designer enzyme, which utilizes an aniline side chain to promote a model hydrazone formation reaction. Consecutive rounds of directed evolution identified a number of mutations in the promiscuous binding pocket, in which the unnatural amino acid is embedded in the starting catalyst. When combined, these mutations boost the turnover frequency (kcat) of the designer enzyme by almost 100‐fold. Crucially, these gains result from strengthening the catalytic contribution of the unnatural amino acid, as the engineered designer enzymes outperform variants, in which the aniline side chain is replaced with a catalytically inactive tyrosine residue, by >200‐fold.

Creating designer enzymes with the ability to catalyse abiological transformations is a formidable challenge. Efforts toward this goal typically consider only canonical amino acids in the initial design process. However, incorporating unnatural amino acids that feature uniquely reactive side chains could significantly expand the catalytic repertoire of designer enzymes. To explore the potential of such artificial building blocks for enzyme design, here we selected p-aminophenylalanine as a potentially novel catalytic residue. We demonstrate that the catalytic activity of the aniline side chain for hydrazone and oxime formation reactions is increased by embedding p-aminophenylalanine into the hydrophobic pore of the multidrug transcriptional regulator from Lactococcus lactis. Both the recruitment of reactants by the promiscuous binding pocket and a judiciously placed aniline that functions as a catalytic residue contribute to the success of the identified artificial enzyme. We anticipate that our design strategy will prove rewarding to significantly expand the catalytic repertoire of designer enzymes in the future.

Lewis acid catalysis is undisputedly of great significance for synthetic chemistry. Hence, many hybrid catalysts have been designed that can function as Lewis acid. These hybrid catalysts are based on DNA, protein, or peptide scaffolds. In this chapter an overview of the hybrid catalysts reported for three important classes of Lewis acid-catalyzed reactions is given: CC bond-forming reactions, CX bond-forming reactions, and hydrolysis reactions.

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.

The interaction of a number of first-row transition-metal ions with a 2,2'-bipyridyl alanine (bpyA) unit incorporated into the lactococcal multidrug resistance regulator (LmrR) scaffold is reported. The composition of the active site is shown to influence binding affinities. In the case of Fe(II), we demonstrate the need of additional ligating residues, in particular those containing carboxylate groups, in the vicinity of the binding site. Moreover, stabilization of di-tert-butylsemiquinone radical (DTB-SQ) in water was achieved by binding to the designed metalloproteins, which resulted in the radical being shielded from the aqueous environment. This allowed the first characterization of the radical semiquinone in water by resonance Raman spectroscopy.

The design of artificial metalloenzymes is a challenging, yet ultimately highly rewarding objective because of the potential for accessing new-to-nature reactions. One of the main challenges is identifying catalytically active substrate-metal cofactor-host geometries. The advent of expanded genetic code methods for the in vivo incorporation of non-canonical metal-binding amino acids into proteins allow to address an important aspect of this challenge: the creation of a stable, well-defined metal-binding site. Here, we report a designed artificial metallohydratase, based on the transcriptional repressor lactococcal multidrug resistance regulator (LmrR), in which the non-canonical amino acid (2,2′-bipyridin-5yl)alanine is used to bind the catalytic Cu(II) ion. Starting from a set of empirical pre-conditions, a combination of cluster model calculations (QM), protein-ligand docking and molecular dynamics simulations was used to propose metallohydratase variants, that were experimentally verified. The agreement observed between the computationally predicted and experimentally observed catalysis results demonstrates the power of the artificial metalloenzyme design approach presented here.

The choice of protein scaffolds is an important element in the design of artificial metalloenzymes. Herein, we introduce Multidrug Resistance Regulators (MDRs) from the TetR family as a viable class of protein scaffolds for artificial metalloenzyme design. In vivo incorporation of the metal binding amino acid (2,2-bipyridin-5yl)alanine (BpyA) by stop codon suppression methods was used to create artificial metalloenzymes from three members of the TetR family of MDRs: QacR, CgmR and RamR. Excellent results were achieved with QacR Y123BpyA in the Cu(2+) catalyzed enantioselective vinylogous Friedel-Crafts alkylation reaction with ee's up to 94% of the opposite enantiomer that was achieved with other mutants and the previously reported LmrR-based artificial metalloenzymes.

Supramolecular anchoring of transition metal complexes to a protein scaffold is an attractive approach to the construction of artificial metalloenzymes since this is conveniently achieved by self-assembly. Here, we report a novel design for supramolecular artificial metalloenzymes that exploits the promiscuity of the central hydrophobic cavity of the transcription factor Lactococcal multidrug resistance Regulator (LmrR) as a generic binding site for planar coordination complexes that do not provide specific protein binding interactions. The success of this approach is manifested in the excellent enantioselectivities that are achieved in the Cu(II) catalyzed enantioselective Friedel–Crafts alkylation of indoles.

Supramolecular assembly has been used to design and create new proteins capable of performing biomimetic functions in complex environments such as membranes and inside living cells.

Artificial metalloenzymes have emerged as an attractive new approach to enantioselective catalysis. Herein, we introduce a novel strategy for preparation of artificial metalloenzymes utilizing amber stop codon suppression methodology for the in vivo incorporation of metal-binding unnatural amino acids. The resulting artificial metalloenzymes were applied in catalytic asymmetric Friedel-Crafts alkylation reactions and up to 83% ee for the product was achieved.

Artificial metalloenzymes have emerged as a promising new approach to asymmetric catalysis. In our group, we are exploring novel artificial metalloenzyme designs involving creation of a new active site in a protein or DNA scaffold that does not have an existing binding pocket. In this review, we give an overview of the developments in the two approaches to artificial metalloenzymes for asymmetric catalysis investigated in our group: creation of a novel active site on a peptide or protein dimer interface and using DNA as a scaffold for artificial metalloenzymes.

Artificial metalloenzymes have emerged over the last decades as an attractive approach towards combining homogeneous catalysis and biocatalysis. A wide variety of catalytic transformations have been established by artificial metalloenzymes, thus establishing proof of concept. The field is now slowly transforming to take on new challenges. These include novel designs, novel catalytic reactions, some of which have no equivalent in both homogenous catalysis and biocatalysis and the incorporation of artificial metalloenzymes in chemoenzymatic cascades. Some of these developments represent promising steps towards integrating artificial metalloenzymes in biological systems. This review will focus on advances in this field and perspectives discussed.

Direct addition of water to alkenes to generate important chiral alcohols as key motif in a variety of natural products still remains a challenge in organic chemistry. Here, we report the first enantioselective artificial metallo-hydratase, based on the transcription factor LmrR, which catalyses the conjugate addition of water to generate chiral β-hydroxy ketones with enantioselectivities up to 84% ee. A mutagenesis study revealed that an aspartic acid and a phenylalanine located in the active site play a key role in achieving efficient catalysis and high enantioselectivities.

A game of two halves: Artificial metalloenzymes are generated by forming a novel active site on the dimer interface of the transcription factor LmrR. Two copper centers are incorporated by binding to ligands in each half of the dimer. With this system up to 97 % ee was obtained in the benchmark CuII catalyzed Diels-Alder reaction (see scheme).

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.

The recently developed concept of DNA-based asymmetric catalysis involves the transfer of chirality from the DNA double helix in reactions using a noncovalently bound catalyst. To date, two generations of DNA-based catalysts have been reported that differ in the design of the ligand for the metal. Herein we present a study of the first generation of DNA-based catalysts, which contain ligands comprising a metal-binding domain linked through a spacer to a 9-aminoacridine moiety. Particular emphasis has been placed on determining the effect of DNA on the structure of the CuII complex and the catalyzed Diels-Alder reaction. The most important findings are that the role of DNA is limited to being a chiral scaffold; no rate acceleration was observed in the presence of DNA. Furthermore, the optimal DNA sequence for obtaining high enantioselectivities proved to contain alternating GC nucleotides. Finally, DNA has been shown to interact with the CuII complex to give a chiral structure. Comparison with the second generation of DNA-based catalysts, which bear bipyridine-type ligands, revealed marked differences, which are believed to be related to the DNA microenvironment in which the catalyst resides and where the reaction takes place.