Editorial Cite This: J. Am. Chem. Soc. 2017, 139, 14331-14334
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Engineered Biomolecular Catalysts
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In recent years, a number of factors have converged to yield significant growth in the development of biomolecular catalysts for chemical applications. One such factor is the wider implementation of directed evolution. Furthermore, advances in computational chemistry made possible by methods development and increased computing power facilitate in silico directed evolution and rational design approaches to protein engineering, as well as screening and analysis of resultant variants. In bioinorganic chemistry, interest has grown in the importance of outer-sphere effects on metal site electronic structure and function. The introduction of these weak interactions in a systematic way to synthetic systems can be challenging, but placing an active site within a biomolecular scaffold affords a clear means to introduce and study these effects. In a related quest, chemists are pursuing how to efficiently deliver electrons and protons to and from catalyst active sites that perform multi-electron redox reactions. In this work, they turn to inspiration from electron- and protontransfer pathways present in redox enzymes, which are tuned to deliver electrons and protons at precise points along the reaction coordinate. The growth of biomaterials chemistry also has spurred important developments, including new approaches to engineering bioconjugates and new methods to arrange enzymes in arrays. Finally, the significant interest across chemistry in developing reactions and processes with environmental stewardship in mind has stimulated the use and engineering of biomolecular catalysts, which do not contain rare or expensive metals and do not require organic solvents. As a result of these developments, many examples of the engineering and implementation of biomolecule-based catalysts now exist across the broad field of chemistry. This ACS Select collection highlights recent publications from the Journal of the American Chemical Society, ACS Catalysis, and Inorganic Chemistry that report the development and application of biomolecules as catalysts. Engineering biocatalyst function and developing enabling technologies such as using biomolecules in heterogeneous reactions are described in this issue. Taken together, this collection illustrates the great promise of biomolecular catalysts and the diverse range of applications and technologies in this flourishing area of chemistry. In the realm of bioinorganic chemistry, performing metal substitutions to aid the study of protein structure and function is an established technique. However, there are relatively few examples of metal substitutions performed to yield a new biocatalyst. One case is nickel-substituted rubredoxin (NiRd), in which the native Fe is replaced with Ni to structurally and functionally mimic [NiFe]-hydrogenase. Shafaat et al. previously developed NiRd as an electrocatalyst for hydrogen production,7 and in the work featured here, Shafaat and coworkers probe its electronic structure. A combination of resonance Raman spectroscopy and density functional theory calculations reveals that the nickel site vibrational mode
hemists have long been inspired by the exquisite selectivity and efficiency of biomolecular catalysts. For example, cytochrome P450 enzymes are capable of chemoselective C−H bond activation in complex molecules under mild conditions, setting a high standard for researchers looking to replicate or model these synthetic transformations. Similarly, the efficiency of [FeFe]-hydrogenases has stimulated inorganic chemists to develop catalysts based on first-row transition metals to evolve hydrogen from water. An attractive approach to achieving enzyme-like activity in the lab is to make use of biomolecules themselves for the desired transformations. However, there are drawbacks to the use of biocatalysts in chemistry that have prevented their wide adoption. For most enzymes, three-dimensional structure and activity are highly sensitive to conditions such as solvent, temperature, pH, and pressure. The requirement of most enzymes for aqueous conditions and moderate temperatures has been a significant detriment to their wide implementation in the chemistry lab. Finally, the desired reactivity may not yet be known in nature. To take advantage of the enviable activities of enzymes for a range of applications, biochemists and engineers have found ways around these limitations. The discovery of extremophilic organisms and their associated biomolecules that resist denaturation provides access to enzymes that function at extremes of pH and temperature, and even in organic solvent. A major step toward engineering enzymes with desired properties is the development of directed evolution, in which an enzyme is subjected to an iterative process of random mutagenesis, screening, and selection. Early efforts have been toward enhancing an enzyme’s stability in organic solvent and/or at high temperature, but success has also been met in evolving enzyme activity.1−3 Enzymes may be evolved to accept new substrates, or even to show activities not known in nature. The evolution of enzymes has not taken place solely in the wet lab; computational chemists have also developed methods for in silico mutagenesis, screening, and selection.4 Taken together, these advances open a new space for the production and use of biomolecular catalysts for a range of applications across many areas of chemistry. As a complementary approach to directed evolution, rational design and redesign are attractive means for systematically building or altering biomolecule structure and activity.5,6 Articles in this ACS Select collection illustrate the redesign approach, in which targeted mutations and/or modifications are made to a biomolecule to alter its molecular and electronic structure, stability, and function. Enzyme (re)design elucidates structure−function relationships and fundamental chemical concepts. Because effects of mutations can be hard to predict, enzyme redesign may provide a circuitous path to the desired results. However, using this hypothesis-based method has yielded successes while providing fundamental insights into how enzymes work. As with directed evolution, computational methods have provided critical support for these efforts, with the introduction of mutations in silico along with screening for stability, substrate binding, and other properties.4 © 2017 American Chemical Society
Published: October 4, 2017 14331
DOI: 10.1021/jacs.7b09896 J. Am. Chem. Soc. 2017, 139, 14331−14334
Journal of the American Chemical Society
Editorial
linked matrix of lipase B. In this case, the catalytic activity of the synthetic catalyst and of the biomolecular scaffold is called for duty. Palladium catalyzes cycloisomerization of 4-pentynoic acid to a lactone, which serves as an acyl donor to the lipase for the kinetic resolution of sec-alcohols. Remarkably, the enzyme remains active in an aggregated state, which also enhances its robustness and allows it to be recycled. Introduction of non-native metals and metal complexes into biomolecules is one approach to engineering biocatalysts by rational design, as established knowledge of the metal site’s reactivity provides a preview of the expected engineered enzyme’s activity. An alternative rational design strategy is to judiciously select mutations to confer a new function or refine an existing function of an enzyme. Huang and co-workers use iterative rational design to prepare an amine transaminase variant that yields an enantiomerically pure product. Their approach combines docking, molecular dynamics simulations, quantum mechanics calculations, in silico analysis of stability, and in vitro screening of selected variants. The result of this process is a rationally designed enzyme with a >1700-fold enhanced activity toward a target amine substrate to produce an enantiomerially pure amine product. Because transaminases exhibit significant promiscuity in their activity, they provide an attractive route to a wide range of chiral amines after refinement. A rational design approach is also employed by Lescar, Wu, and co-workers to engineer a ligase to join the Nand C-termini of peptidyl substrates, a valuable reaction for protein labeling, peptide modification, and semisynthesis. Starting with the X-ray crystal structure and knowledge of the ligase mechanism, the authors prepare mutants proposed to accommodate the intended amine substrate and also to exclude water, thus inhibiting the competing hydrolysis reaction. Directed evolution is an increasingly popular alternative to rational design, and this trend is reflected in this special issue. This approach may be applied to alter an enzyme’s function, to introduce a new catalytic function, and/or to impact protein stability and conditions for optimal activity. The work described above by Hartwig and colleagues utilizes metal substitution to confer an abiological activity on cytochrome P450, followed by directed evolution to refine that activity by enhancing chemoselectivity. Another example of activity refinement by directed evolution is the work by Hilvert and co-workers. This study uses an artificial retro-aldolase previously engineered using computational design. Here, this enzyme is shown to promote Knoevenagel condensations of electron-rich aldehydes and activated methylene donors to form C−C bonds, making use of an iminium intermediate formed with an active-site lysine. Refinement of activity through directed evolution identifies an efficient enzyme variant with a catalytic proficiency of >108-fold enhancement over simple amines. Directed evolution of rebeccamycin halogenase (RebH) to expand substrate scope by Lewis et al. previously yielded enzyme variants for the selective halogenation of aromatic compounds.10 Here, Lewis and co-workers demonstrate the application of these RebH variants in a two-step chemoenzymatic process for selective C−H functionalization. Directed evolution may also be applied to confer a novel function on a protein. Arnold has been a pioneer in this area, and in this issue Arnold, Buller, and co-workers demonstrate the synthesis of β-branched tryptophan amino acid derivatives through evolution of tryptophan synthase, providing a route to these derivatives that circumvents a three-enzyme pathway used in nature. Heme proteins have been rich targets in this work,
intensities are influenced by coupling to the peptide backbone, reflecting contributions of outer-sphere interactions to the metal electronic structure. Tuning the electronic structure and activity of NiRd and, indeed, of any engineered metalloenzyme ideally will consider the importance of these outer-sphere effects. The metal substitution approach to protein engineering also may yield a catalyst with abiological activity. To prepare a new enzyme for carbene insertion reactions, Lehnert and coworkers substitute the Fe-protoporphyrin IX (heme) of myoglobin with Ru-mesoporphyrin IX. The myoglobin variants chosen are inspired by results of Fasan and co-workers demonstrating carbene-transfer activity of myoglobin.8 The researchers here select ruthenium for its low oxygen sensitivity and its high activity toward carbene-transfer reactions. The result is that carbene insertion into N−H bonds and cyclopropanation of olefins occur with higher activity than for myoglobin with its native iron. However, this higher activity has the side effect of accelerating catalyst degradation. Finally, by engineering the heme pocket, substantial impacts on reaction stereoselectivity are observed, pointing to opportunities to further refine this catalyst. Combining metal substitution and directed evolution, Hartwig et al. previously developed the use of Ir(Me) porphyrin-substituted heme proteins to produce catalysts for carbene insertion.9 More recently, Hartwig and colleagues describe Ir(Me)-substituted cytochrome P450 variants that perform chemoselective nitrene insertion into C−H bonds. The reactions proceed with high yields and high enantiomeric ratios. While Lehnert and Hartwig incorporate heavy-metalsubstituted cofactors into proteins, Fujieda, Itoh, and coworkers substitute the native manganese with osmium in a thermally stable cuprin-like protein to prepare a peroxidase for the cis-hydroxylation of olefins. Incorporation of heavy metals into proteins in a site-specific manner is challenging because of these metals’ inert nature, and indeed the researchers isolate two osmium-protein forms, only one of which is catalytically active. Notably, the Os active site has a coordination structure reminiscent of the synthetic catalyst osmium tris(2pyridylmethyl)amine developed for the same chemistry. Stereoselectivity is modest for both the protein and the related synthetic catalysts, but the Os-protein derivative offers the possibility of designing or evolving an active-site structure to improve this result. The work of Fujieda and Itoh described above demonstrates a “chemomimetic” strategy in which an active site is built within a protein to resemble a synthetic catalyst. A complementary approach is to introduce a known inorganic catalyst complex into a biomolecular scaffold. Goals of these efforts include conferring water solubility on a catalyst, creating a substratebinding pocket that enhances reactivity and/or stereoselectivity, and making use of the catalyst in the context of a medical application. Alternatively, as illustrated by Ward and colleagues, this strategy can be used to develop a cascade reaction involving enzymes and a protein-bound synthetic catalyst. Here, the authors incorporate a biotinylated synthetic iridium hydridetransfer catalyst into the protein streptavidin. The resulting artificial transfer hydrogenase (ATHase) uses NAD(P)H as a hydride source for reduction of a range of substrates and racemization of alcohols and amines. Pairing the ATHase with glucose dehydrogenase regenerates the NAD(P)H, making glucose the terminal reductant. Johnston, Bäckvall, and coworkers describe a related semisynthetic approach in which the researchers embed palladium nanoparticle catalysts in a cross14332
DOI: 10.1021/jacs.7b09896 J. Am. Chem. Soc. 2017, 139, 14331−14334
Journal of the American Chemical Society
Editorial
(sorbitol methacrylate) matrix, which is proposed to act as a surrogate for water. Remarkably, this “dry” enzyme retains 25% of its activity seen in aqueous buffer. A motivation for this approach is that desired substrates of this enzyme are highly volatile, and a solvent-free system facilitates its action on gaseous substrates. Furthermore, enzyme immobilization provides a device for use in the field, for example in environmental remediation. To engineer an amidase that functions in anhydrous organic solvent, Wu, Janssen, and colleagues employ computational protein engineering along with energy calculations and molecular dynamics simulations. The engineered amidase catalyzes a range of C-terminal peptide modifications in high yield (>85%) in anhydrous solvents. Achieving anhydrous conditions is essential for this goal because the reverse hydrolysis reaction is favored in water. Attaching enzymes to surfaces or other substrates is employed for a range of applications and can enhance enzyme stability and recyclability. The surface attachment may also provide a means to alter or control enzyme activity. It was noted above that Jeuken, Butt, Reisner, and co-workers observe efficient peroxide reduction catalyzed by an electron-transfer cytochrome adsorbed on indium tin oxide. Electrochemistry is also employed by De Lacey, Pita, and co-workers to alter the function of laccase, a copper enzyme that catalyzes the reduction of dioxygen to water. To force laccase to perform the water oxidation reaction, they attach it to a photoanode consisting of a fluorinated tin oxide electrode modified with the visible-light-absorbing n-type semiconductor In2S3. Irradiation with visible light and application of a modest electrochemical overpotential drive laccase to oxidize water with turnover frequencies rivaling those of Photosystem II. Another motivation for immobilizing an enzyme on a surface is to achieve a desired spatial arrangement to impact activity. Morii and co-workers employ this approach to prepare an artificial enzyme cascade. Using the conversion of xylose to xylulose via the intermediate xylitol to illustrate this concept, the authors assemble xylose reductase and xylitol dehydrogenase onto a DNA origami scaffold using DNA-binding protein adaptors to place the enzymes close to each other in space. Simulations show that when bimolecular transport (in this case, xylitol and NAD+) is required for a reaction, interenzyme distance is a key determinant of reaction rate. Enzyme engineering has come a long way during the four decades since site-directed mutagenesis was first developed. The wide use of directed evolution and computational approaches along with improved methods for characterizing resultant enzyme variants has opened a new frontier in biocatalyst development. At the same time, the field seems to be at a point where the possibilities offered by these approaches are just being grasped. I hope that this collection of publications illustrates that engineered biocatalysts have wide applications and great potential.
given the large range of functions that heme proteins carry out. The heme protein myoglobin is converted into a catalyst for olefin cyclopropanation using directed evolution performed by Fasan and colleagues. The researchers express myoglobin variants in E. coli and, remarkably, demonstrate that the catalyst is active in whole cells. In fact, the catalyst is more stable within cells and displays the same high stereoselectivity that is found for purified protein. This approach circumvents the need for time-consuming protein purification steps, facilitating screening and implementation. Furthermore, analysis of the active variants indicates that the heme pocket shape plays a key role in determining stereoselectivity. Martins and colleagues also target a heme protein in their work, evolving a dye-decolorizing peroxidase into an enzyme that degrades lignin phenolic substrates. The evolved enzyme displays a 100-fold increase in catalytic efficiency for the lignin model substrate 2,6dimethoxyphenol over the parent peroxidase. Furthermore, this evolved enzyme is active at alkaline pH values, conditions preferred for use in industry, while lignin peroxidase requires acidic pH. Enzyme engineering that considers primarily the activity conferred on the enzyme active site may be missing key opportunities. While stability has received significant attention, other properties and functions of enzymes have not. Nature’s enzymes control not only the chemistry at the active site but also substrate delivery and product release, as well as protein− protein interactions critical for delivering electrons and/or regulating activity. These aspects of enzyme activity involve large groups of amino acids and are particularly challenging to understand and engineer. In this collection, there are some notable successes in engineering “supporting functions” into enzymes. Damborsky and co-workers successfully take on the challenge of introducing a new substrate tunnel into a haloalkane dehalogenase. The authors analyze the enzyme’s X-ray crystal structure to identify potential new substrate pathways and bottleneck residues that block those pathways. Introducing a disulfide bond blocks the native substrate channel, and this variant is subjected to directed evolution to introduce a new pathway for substrate delivery to the active site. In reactions that require protons, enzymes have in place pathways of hydrogen-bonding amino acids and water molecules to transport protons between the protein surface and the active site. In this collection, Hoffman, Lu, and colleagues investigate Lu’s rationally designed myoglobin-based model of a heme copper oxidase that catalyzes the fourelectron, four-proton reduction of dioxygen to water. Through a combination of X-ray crystallography and pulsed electron paramagnetic resonance on cryoreduced forms of the oxyprotein, the authors identify a network for proton transport necessary for the high dioxygen reduction activity. The delivery of electrons to the active site is also a critical component of the work of Jeuken, Butt, Reisner, and co-workers, who report high activity for the electrocatalytic reduction of hydrogen peroxide to water by the decaheme electron-transfer protein MtrC immobilized on an indium tin oxide electrode. The data support the proposal that the multiheme protein “wire” is critical for efficient electron delivery to the hemes that perform the catalysis. The use of a biomolecular catalyst in the lab, in industry, or in the field may require it to be active under abiological conditions, including in the absence of water. To render an enzyme active in a solvent-free environment, Marsh and coworkers immobilize a haloalkane dehalogenase in a poly-
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Kara L. Bren, Associate Editor FEATURED ARTICLES
Slater, J. W.; Marguet, S. C.; Cirino, S. L.; Maugeri, P. T.; Shafaat, H. S. Inorg. Chem. 2017, 56, 3926. Wolf, M. W.; Vargas, D. A.; Lehnert, N. Inorg. Chem. 2017, 56, 5623. Dydio, P.; Key, H. M.; Hayashi, H.; Clark, D. S.; Hartwig, J. F. J. Am. Chem. Soc. 2017, 139, 1750. DOI: 10.1021/jacs.7b09896 J. Am. Chem. Soc. 2017, 139, 14331−14334
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(7) Slater, J. W.; Shafaat, H. S. J. Phys. Chem. Lett. 2015, 6, 3731. (8) Bordeaux, M.; Tyagi, V.; Fasan, R. Angew. Chem., Int. Ed. 2015, 54, 1744. (9) Key, H. M.; Dydio, P.; Clark, D. S.; Hartwig, J. F. Nature 2016, 534, 534. (10) Payne, J. T.; Poor, C. B.; Lewis, J. C. Angew. Chem., Int. Ed. 2015, 54, 4226.
Fujieda, N.; Nakano, T.; Taniguchi, Y.; Ichihashi, H.; Sugimoto, H.; Morimoto, Y.; Nishikawa, Y.; Kurisu, G.; Itoh, S. J. Am. Chem. Soc. 2017, 139, 5149. Okamoto, Y.; Kohler, V.; Ward, T. R. J. Am. Chem. Soc. 2016, 138, 5781. Görbe, T.; Gustafson, K. P. J.; Verho, O.; Kervefors, G.; Zheng, H. Q.; Zou, X. D.; Johnston, E. V.; Bäckvall, J. E. ACS Catal. 2017, 7, 1601. Dourado, D.; Pohle, S.; Carvalho, A. T. P.; Dheeman, D. S.; Caswell, J. M.; Skvortsov, T.; Miskelly, I.; Brown, R. T.; Quinn, D. J.; Allen, C. C. R.; Kulakov, L.; Huang, M. L.; Moody, T. S. ACS Catal. 2016, 6, 7749. Yang, R. L.; Wong, Y. H.; Nguyen, G. K. T.; Tam, J. P.; Lescar, J.; Wu, B. J. Am. Chem. Soc. 2017, 139, 5351. Garrabou, X.; Wicky, B. I. M.; Hilvert, D. J. Am. Chem. Soc. 2016, 138, 6972. Durak, L. J.; Payne, J. T.; Lewis, J. C. ACS Catal. 2016, 6, 1451. Herger, M.; van Roye, P.; Romney, D. K.; BrinkmannChen, S.; Buller, A. R.; Arnold, F. H. J. Am. Chem. Soc. 2016, 138, 8388. Tinoco, A.; Steck, V.; Tyagi, V.; Fasan, R. J. Am. Chem. Soc. 2017, 139, 5293. Brissos, V.; Tavares, D.; Sousa, A. C.; Robalo, M. P.; Martins, L. O. ACS Catal. 2017, 7, 3454. Brezovsky, J.; Babkova, P.; Degtjarik, O.; Fortova, A.; Gora, A.; Iermak, I.; Rezacova, P.; Dvorak, P.; Smatanova, I. K.; Prokop, Z.; Chaloupkova, R.; Damborsky, J. ACS Catal. 2016, 6, 7597. Petrik, I. D.; Davydov, R.; Ross, M.; Zhao, X.; Hoffman, B.; Lu, Y. J. Am. Chem. Soc. 2016, 138, 1134. Reuillard, B.; Ly, K. H.; Hildebrandt, P.; Jeuken, L. J. C.; Butt, J. N.; Reisner, E. J. Am. Chem. Soc. 2017, 139, 3324. Badieyan, S.; Wang, Q. M.; Zou, X. Q.; Li, Y. X.; Herron, M.; Abbott, N. L.; Chen, Z.; Marsh, E. N. G. J. Am. Chem. Soc. 2017, 139, 2872. Wu, B.; Wijma, H. J.; Song, L.; Rozeboom, H. J.; Poloni, C.; Tian, Y.; Arif, M. I.; Nuijens, T.; Quaedflieg, P.; Szymanski, W.; Feringa, B. L.; Janssen, D. B. ACS Catal. 2016, 6, 5405. Tapia, C.; Shleev, S.; Conesa, J. C.; De Lacey, A. L.; Pita, M. ACS Catal. 2017, 7, 4881. Ngo, T. A.; Nakata, E.; Saimura, M.; Morii, T. J. Am. Chem. Soc. 2016, 138, 3012.
AUTHOR INFORMATION
ORCID
Kara L. Bren: 0000-0002-8082-3634 Notes
Views expressed in this editorial are those of the author and not necessarily the views of the ACS.
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REFERENCES
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DOI: 10.1021/jacs.7b09896 J. Am. Chem. Soc. 2017, 139, 14331−14334
