Lewis, J.C.

ChemBioChem 2014, 15, 223-227, 10.1002/cbic.201300661

Strain‐promoted azide–alkyne cycloaddition (SPAAC) can be used to generate artificial metalloenzymes (ArMs) from scaffold proteins containing a p‐azido‐L‐phenylalanine (Az) residue and catalytically active bicyclononyne‐substituted metal complexes. The high efficiency of this reaction allows rapid ArM formation when using Az residues within the scaffold protein in the presence of cysteine residues or various reactive components of cellular lysate. In general, cofactor‐based ArM formation allows the use of any desired metal complex to build unique inorganic protein materials. SPAAC covalent linkage further decouples the native function of the scaffold from the installation process because it is not affected by native amino acid residues; as long as an Az residue can be incorporated, an ArM can be generated. We have demonstrated the scope of this method with respect to both the scaffold and cofactor components and established that the dirhodium ArMs generated can catalyze the decomposition of diazo compounds and both SiH and olefin insertion reactions involving these carbene precursors.

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.

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

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

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

Lewis, J.C.

Acc. Chem. Res. 2019, 52, 576-584, 10.1021/acs.accounts.8b00625

Transition metal catalysis is a powerful tool for chemical synthesis, a standard by which understanding of elementary chemical processes can be measured, and a source of awe for those who simply appreciate the difficulty of cleaving and forming chemical bonds. Each of these statements is amplified in cases where the transition metal catalyst controls the selectivity of a chemical reaction. Enantioselective catalysis is a challenging but well-established phenomenon, and regio- or site-selective catalysis is increasingly common. On the other hand, transition-metal-catalyzed reactions are typically conducted under highly optimized conditions. Rigorous exclusion of air and water is common, and it is taken for granted that only a single substrate (of a particular class) will be present in a reaction, a desired site selectivity can be achieved by installing a directing group, and undesired reactivity can be blocked with protecting groups. These are all reasonable synthetic strategies, but they also highlight limits to catalyst control. The utility of transition metal catalysis could be greatly expanded if catalysts possessed the ability to regulate which molecules they encounter and the relative orientation of those molecules. The rapid and widespread adoption of stoichiometric bioorthogonal reactions illustrates the utility of robust reactions that proceed with high selectivity and specificity under mild reaction conditions. Expanding this capability beyond preprogrammed substrate pairs via catalyst control could therefore have an enormous impact on molecular science. Many metalloenzymes exhibit this level of catalyst control, and directed evolution can be used to rapidly improve the catalytic properties of these systems. On the other hand, the range of reactions catalyzed by enzymes is limited relative to that developed by chemists. The possibility of imparting enzyme-like activity, selectivity, and evolvability to reactions catalyzed by synthetic transition metal complexes has inspired the creation of artificial metalloenzymes (ArMs). The increasing levels of catalyst control exhibited by ArMs developed to date suggest that these systems could constitute a powerful platform for bioorthogonal transition metal catalysis and for selective catalysis in general. This Account outlines the development of a new class of ArMs based on a prolyl oligopeptidase (POP) scaffold. Studies conducted on POP ArMs containing a covalently linked dirhodium cofactor have shown that POP can impart enantioselectivity to a range of dirhodium-catalyzed reactions, increase reaction rates, and improve the specificity for reaction of dirhodium carbene intermediates with targeted organic substrates over components of cell lysate, including bulk water. Several design features of these ArMs enabled their evolution via random mutagenesis, which revealed that mutations throughout the POP scaffold, beyond the second sphere of the dirhodium cofactor, were important for ArM activity and selectivity. While it was anticipated that the POP scaffold would be capable of encapsulating and thus controlling the selectivity of bulky cofactors, molecular dynamics studies also suggest that POP conformational dynamics plays a role in its unique efficacy. These advances in scaffold selection, bioconjugation, and evolution form the basis of our ongoing efforts to control transition metal reactivity using protein scaffolds with the goal of enabling unique synthetic capabilities, including bioorthogonal catalysis.

Lewis, J.C.

Acc. Chem. Res. 2019, 52, 576-584, 10.1021/acs.accounts.8b00625

Transition metal catalysis is a powerful tool for chemical synthesis, a standard by which understanding of elementary chemical processes can be measured, and a source of awe for those who simply appreciate the difficulty of cleaving and forming chemical bonds. Each of these statements is amplified in cases where the transition metal catalyst controls the selectivity of a chemical reaction. Enantioselective catalysis is a challenging but well-established phenomenon, and regio- or siteselective catalysis is increasingly common. On the other hand, transition-metal-catalyzed reactions are typically conducted under highly optimized conditions. Rigorous exclusion of air and water is common, and it is taken for granted that only a single substrate (of a particular class) will be present in a reaction, a desired site selectivity can be achieved by installing a directing group, and undesired reactivity can be blocked with protecting groups. These are all reasonable synthetic strategies, but they also highlight limits to catalyst control. The utility of transition metal catalysis could be greatly expanded if catalysts possessed the ability to regulate which molecules they encounter and the relative orientation of those molecules. The rapid and widespread adoption of stoichiometric bioorthogonal reactions illustrates the utility of robust reactions that proceed with high selectivity and specificity under mild reaction conditions. Expanding this capability beyond preprogrammed substrate pairs via catalyst control could therefore have an enormous impact on molecular science. Many metalloenzymes exhibit this level of catalyst control, and directed evolution can be used to rapidly improve the catalytic properties of these systems. On the other hand, the range of reactions catalyzed by enzymes is limited relative to that developed by chemists. The possibility of imparting enzyme-like activity, selectivity, and evolvability to reactions catalyzed by synthetic transition metal complexes has inspired the creation of artificial metalloenzymes (ArMs). The increasing levels of catalyst control exhibited by ArMs developed to date suggest that these systems could constitute a powerful platform for bioorthogonal transition metal catalysis and for selective catalysis in general. This Account outlines the development of a new class of ArMs based on a prolyl oligopeptidase (POP) scaffold. Studies conducted on POP ArMs containing a covalently linked dirhodium cofactor have shown that POP can impart enantioselectivity to a range of dirhodium-catalyzed reactions, increase reaction rates, and improve the specificity for reaction of dirhodium carbene intermediates with targeted organic substrates over components of cell lysate, including bulk water. Several design features of these ArMs enabled their evolution via random mutagenesis, which revealed that mutations throughout the POP scaffold, beyond the second sphere of the dirhodium cofactor, were important for ArM activity and selectivity. While it was anticipated that the POP scaffold would be capable of encapsulating and thus controlling the selectivity of bulky cofactors, molecular dynamics studies also suggest that POP conformational dynamics plays a role in its unique efficacy. These advances in scaffold selection, bioconjugation, and evolution form the basis of our ongoing efforts to control transition metal reactivity using protein scaffolds with the goal of enabling unique synthetic capabilities, including bioorthogonal catalysis.

Seelig, B.

Trends Biotechnol. 2010, 28, 340-345, 10.1016/j.tibtech.2010.04.003

Enzymes offer cheap, environmentally responsible and highly efficient alternatives to chemical catalysts. The past two decades have seen a significant rise in the use of enzymes in industrial settings. Although many natural enzymes have been modified through protein engineering to better suit practical applications, these approaches are often insufficient. A key goal of enzyme engineers is to build enzymes de novo – or, ‘from scratch’. To date, several technologies have been developed to achieve this goal: namely, computational design, catalytic antibodies and mRNA display. These methods rely on different principles, trading off rational protein design against an entirely combinatorial approach of directed evolution of vast protein libraries. The aim of this article is to review and compare these methods and their potential for generating truly de novo biocatalysts.

Lewis, J.C.

Nat. Commun. 2015, 6, 10.1038/ncomms8789

Artificial metalloenzymes (ArMs) formed by incorporating synthetic metal catalysts into protein scaffolds have the potential to impart to chemical reactions selectivity that would be difficult to achieve using metal catalysts alone. In this work, we covalently link an alkyne-substituted dirhodium catalyst to a prolyl oligopeptidase containing a genetically encoded L-4-azidophenylalanine residue to create an ArM that catalyses olefin cyclopropanation. Scaffold mutagenesis is then used to improve the enantioselectivity of this reaction, and cyclopropanation of a range of styrenes and donor–acceptor carbene precursors were accepted. The ArM reduces the formation of byproducts, including those resulting from the reaction of dirhodium–carbene intermediates with water. This shows that an ArM can improve the substrate specificity of a catalyst and, for the first time, the water tolerance of a metal-catalysed reaction. Given the diversity of reactions catalysed by dirhodium complexes, we anticipate that dirhodium ArMs will provide many unique opportunities for selective catalysis.

Lewis, J.C.; Roux, B.

Angew. Chem. Int. Ed. 2021, 60, 23672-23677, 10.1002/anie.202107982

Artificial metalloenzymes (ArMs) are commonly used to control the stereoselectivity of catalytic reactions, but controlling chemoselectivity remains challenging. In this study, we engineer a dirhodium ArM to catalyze diazo cross-coupling to form an alkene that, in a one-pot cascade reaction, is reduced to an alkane with high enantioselectivity (typically >99 % ee) by an alkene reductase. The numerous protein and small molecule components required for the cascade reaction had minimal effect on ArM catalysis. Directed evolution of the ArM led to improved yields and E/Z selectivities for a variety of substrates, which translated to cascade reaction yields. MD simulations of ArM variants were used to understand the structural role of the cofactor on ArM conformational dynamics. These results highlight the ability of ArMs to control both catalyst stereoselectivity and chemoselectivity to enable reactions in complex media that would otherwise lead to undesired side reactions.

Lewis, J.C.

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

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

Lewis, J.C.

Tetrahedron 2014, 70, 4245-4249, 10.1016/j.tet.2014.03.008

New catalysts for non-directed hydrocarbon functionalization have great potential in organic synthesis. We hypothesized that incorporating a Mn-terpyridine cofactor into a protein scaffold would lead to artificial metalloenzymes (ArMs) in which the selectivity of the Mn cofactor could be controlled by the protein scaffold. We designed and synthesized a maleimide-substituted Mn-terpyridine cofactor and demonstrated that this cofactor could be incorporated into two different scaffold proteins to generate the desired ArMs. The structure and reactivity of one of these ArMs was explored, and the broad oxygenation capability of the Mn-terpyridine catalyst was maintained, providing a robust platform for optimization of ArMs for selective hydrocarbon functionalization.

Lewis, J.C.

Curr. Opin. Chem. Biol. 2015, 25, 27-35, 10.1016/j.cbpa.2014.12.016

Metallopeptide catalysts and artificial metalloenzymes built from peptide scaffolds and catalytically active metal centers possess a number of exciting properties that could be exploited for selective catalysis. Control over metal catalyst secondary coordination spheres, compatibility with library based methods for optimization and evolution, and biocompatibility stand out in this regard. A wide range of unnatural amino acids (UAAs) have been incorporated into peptide and protein scaffolds using several distinct methods, and the resulting UAAs containing scaffolds can be used to create novel hybrid metal–peptide catalysts. Promising levels of selectivity have been demonstrated for several hybrid catalysts, and these provide a strong impetus and important lessons for the design of and optimization of hybrid catalysts.

Lewis, J.C.

Artificial Metalloenzymes and MetalloDNAzymes in Catalysis: From Design to Applications 2018, 1-40, 10.1002/9783527804085.ch1

Transition metal catalysts and enzymes are ubiquitous tools for chemical synthesis. Potential benefits of combining complementary properties of these catalysts have driven efforts to create artificial metalloenzymes (ArMs), hybrid constructs comprised of synthetic metal centers embedded within protein scaffolds. This unique composition necessitates the use of synthetic chemistry, bioconjugation methodology, and protein engineering for ArM formation. Despite this challenge, a range of approaches for ArM formation has been developed. This chapter provides an overview of these different approaches and discussion of potential advantages and disadvantages of each.

Seelig, B.; Szostak, J.W.

Nature 2007, 448, 828-831, 10.1038/nature06032

Enzymes are exceptional catalysts that facilitate a wide variety of reactions under mild conditions, achieving high rate-enhancements with excellent chemo-, regio- and stereoselectivities. There is considerable interest in developing new enzymes for the synthesis of chemicals and pharmaceuticals1,2,3 and as tools for molecular biology. Methods have been developed for modifying and improving existing enzymes through screening, selection and directed evolution4,5. However, the design and evolution of truly novel enzymes has relied on extensive knowledge of the mechanism of the reaction6,7,8,9,10. Here we show that genuinely new enzymatic activities can be created de novo without the need for prior mechanistic information by selection from a naive protein library of very high diversity, with product formation as the sole selection criterion. We used messenger RNA display, in which proteins are covalently linked to their encoding mRNA11, to select for functional proteins from an in vitro translated protein library of >1012independent sequences without the constraints imposed by any in vivo step. This technique has been used to evolve new peptides and proteins that can bind a specific ligand12,13,14,15,16,17,18, from both random-sequence libraries12,14,15,16 and libraries based on a known protein fold17,18. We now describe the isolation of novel RNA ligases from a library that is based on a zinc finger scaffold18,19, followed by in vitro directed evolution to further optimize these enzymes. The resulting ligases exhibit multiple turnover with rate enhancements of more than two-million-fold.

Lewis, J.C.

Curr. Opin. Chem. Biol. 2017, 37, 48-55, 10.1016/j.cbpa.2016.12.027

Catalytic CH bond functionalization has become an important tool for organic synthesis. Metalloenzymes offer a solution to one of the foremost challenges in this field, site-selective CH functionalization, but they are only capable of catalyzing a subset of the CH functionalization reactions known to small molecule catalysts. To overcome this limitation, metalloenzymes have been repurposed by exploiting the reactivity of their native cofactors toward substrates not found in nature. Additionally, new reactivity has been accessed by incorporating synthetic metal cofactors into protein scaffolds to form artificial metalloenzymes. The selectivity and activity of these catalysts has been tuned using directed evolution. This review covers the recent progress in developing and optimizing both repurposed and artificial metalloenzymes as catalysts for selective CH bond functionalization.

Seelig, B.

Nat. Chem. Biol. 2013, 9, 81-83, 10.1038/nchembio.1138

Engineering functional protein scaffolds capable of carrying out chemical catalysis is a major challenge in enzyme design. Starting from a noncatalytic protein scaffold, we recently generated a new RNA ligase by in vitro directed evolution. This artificial enzyme lost its original fold and adopted an entirely new structure with substantially enhanced conformational dynamics, demonstrating that a primordial fold with suitable flexibility is sufficient to carry out enzymatic function.

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.

Happe, T.; Hemschemeier, A.

Nat. Rev. Chem. 2018, 2, 231-243, 10.1038/s41570-018-0029-3

Metal cofactors considerably widen the catalytic space of naturally occurring enzymes whose specific and enantioselective catalytic activity constitutes a blueprint for economically relevant chemical syntheses. To optimize natural enzymes and uncover novel reactivity, we need a detailed understanding of cofactor–protein interactions, which can be challenging to obtain in the case of enzymes with sophisticated cofactors. As a case study, we summarize recent research on the [FeFe]-hydrogenases, which interconvert protons, electrons and dihydrogen at a unique iron-based active site. We can now chemically synthesize the complex cofactor and incorporate it into an apo-protein to afford functional enzymes. By varying both the cofactor and the polypeptide components, we have obtained detailed knowledge on what is required for a metal cluster to process H2. In parallel, the design of artificial proteins and catalytically active nucleic acids are advancing rapidly. In this Perspective, we introduce these fields and outline how chemists and biologists can use this knowledge to develop novel tailored semisynthetic catalysts.