39 publications
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Abiotic reduction of ketones with silanes catalysed by carbonic anhydrase through an enzymatic zinc hydride
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Nat. Chem. 2021, 13, 312-318, 10.1038/s41557-020-00633-7
Enzymatic reactions through mononuclear metal hydrides are unknown in nature, despite the prevalence of such intermediates in the reactions of synthetic transition-metal catalysts. If metalloenzymes could react through abiotic intermediates like these, then the scope of enzyme-catalysed reactions would expand. Here we show that zinc-containing carbonic anhydrase enzymes catalyse hydride transfers from silanes to ketones with high enantioselectivity. We report mechanistic data providing strong evidence that the process involves a mononuclear zinc hydride. This work shows that abiotic silanes can act as reducing equivalents in an enzyme-catalysed process and that monomeric hydrides of electropositive metals, which are typically unstable in protic environments, can be catalytic intermediates in enzymatic processes. Overall, this work bridges a gap between the types of transformation in molecular catalysis and biocatalysis.
Metal: ZnLigand type: Histidine residuesHost protein: Human carbonic anhydrase II (hCAII)Anchoring strategy: NativeOptimization: ChemicalNotes: ---
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A De Novo‐Designed Artificial Metallopeptide Hydrogenase: Insights into Photochemical Processes and the Role of Protonated Cys
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ChemSusChem 2021, 14, 2237-2246, 10.1002/cssc.202100122
Hydrogenase enzymes produce H2 gas, which can be a potential source of alternative energy. Inspired by the [NiFe] hydrogenases, we report the construction of a de novo-designed artificial hydrogenase (ArH). The ArH is a dimeric coiled coil where two cysteine (Cys) residues are introduced at tandem a/d positions of a heptad to create a tetrathiolato Ni binding site. Spectroscopic studies show that Ni binding significantly stabilizes the peptide producing electronic transitions characteristic of Ni-thiolate proteins. The ArH produces H2 photocatalytically, demonstrating a bell-shaped pH-dependence on activity. Fluorescence lifetimes and transient absorption spectroscopic studies are undertaken to elucidate the nature of pH-dependence, and to monitor the reaction kinetics of the photochemical processes. pH titrations are employed to determine the role of protonated Cys on reactivity. Through combining these results, a fine balance is found between solution acidity and the electron transfer steps. This balance is critical to maximize the production of NiI-peptide and protonation of the NiII−H− intermediate (Ni−R) by a Cys (pKa≈6.4) to produce H2.
Metal: NiLigand type: Amino acidHost protein: Synthetic peptideAnchoring strategy: DativeOptimization: ChemicalNotes: ---
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Alternative Strategy to Obtain Artificial Imine Reductase by Exploiting Vancomycin/D-Ala-D-Ala Interactions with an Iridium Metal Complex
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Inorg. Chem. 2021, 60, 2976-2982, 10.1021/acs.inorgchem.0c02969
Based on the supramolecular interaction between vancomycin (Van), an antibiotic glycopeptide, and D-Ala-D-Ala (DADA) dipeptides, a novel class of artificial metalloenzymes was synthesized and characterized. The presence of an iridium(III) ligand at the N-terminus of DADA allowed the use of the metalloenzyme as a catalyst in the asymmetric transfer hydrogenation of cyclic imines. In particular, the type of link between DADA and the metal-chelating moiety was found to be fundamental for inducing asymmetry in the reaction outcome, as highlighted by both computational studies and catalytic results. Using the [IrCp*(m-I)Cl]Cl ⊂ Van complex in 0.1 M CH3COONa buffer at pH 5, a significant 70% (S) e.e. was obtained in the reduction of quinaldine B.
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An Artificial Cofactor Catalyzing the Baylis‐Hillman Reaction with Designed Streptavidin as Protein Host
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ChemBioChem 2021, 22, 1573-1577, 10.1002/cbic.202000880
An artificial cofactor based on an organocatalyst embedded in a protein has been used to conduct the Baylis-Hillman reaction in a buffered system. As protein host, we chose streptavidin, as it can be easily crystallized and thereby supports the design process. The protein host around the cofactor was rationally designed on the basis of high-resolution crystal structures obtained after each variation of the amino acid sequence. Additionally, DFT-calculated intermediates and transition states were used to rationalize the observed activity. Finally, repeated cycles of structure determination and redesign led to a system with an up to one order of magnitude increase in activity over the bare cofactor and to the most active proteinogenic catalyst for the Baylis-Hillman reaction known today.
Metal: ---Ligand type: ---Host protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & computational designNotes: Organocatalyst
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An Artificial Ruthenium-Containing β-Barrel Protein for Alkene–Alkyne Coupling Reaction
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Org. Biomol. Chem. 2021, 19, 2912-2916, 10.1039/d1ob00279a
A modified Cp*Ru complex, equipped with a maleimide group, was covalently attached to a cysteine of an engineered variant of Ferric hydroxamate uptake protein component: A (FhuA). This synthetic metalloprotein catalyzed the intermolecular alkene–alkyne coupling of 3-butenol with 5-hexynenitrile. When compared with the protein-free Cp*Ru catalyst, the biohybrid catalyst produced the linear product with higher regioselectivity.
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Artificial Metalloproteins with Dinuclear Iron–Hydroxido Centers
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J. Am. Chem. Soc. 2021, 143, 2384-2393, 10.1021/jacs.0c12564
Dinuclear iron centers with a bridging hydroxido or oxido ligand form active sites within a variety of metalloproteins. A key feature of these sites is the ability of the protein to control the structures around the Fe centers, which leads to entatic states that are essential for function. To simulate this controlled environment, artificial proteins have been engineered using biotin–streptavidin (Sav) technology in which Fe complexes from adjacent subunits can assemble to form [FeIII–(μ-OH)–FeIII] cores. The assembly process is promoted by the site-specific localization of the Fe complexes within a subunit through the designed mutation of a tyrosinate side chain to coordinate the Fe centers. An important outcome is that the Sav host can regulate the Fe···Fe separation, which is known to be important for function in natural metalloproteins. Spectroscopic and structural studies from X-ray diffraction methods revealed uncommonly long Fe···Fe separations that change by less than 0.3 Å upon the binding of additional bridging ligands. The structural constraints imposed by the protein host on the di-Fe cores are unique and create examples of active sites having entatic states within engineered artificial metalloproteins.
Reaction: ---Max TON: ---ee: ---PDB: ---Notes: PDB: 6VOZ, 6VO9
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Binding of Vanadium Ions and Complexes to Proteins and Enzymes in Aqueous Solution
Review -
Coord. Chem. Rev. 2021, 449, 214192, 10.1016/j.ccr.2021.214192
The understanding of the role of vanadium enzymes and of vanadium compounds (VCs) in biology, as well as the design of new vanadium-based species for catalysis, materials science and medicinal chemistry has exponentially increased during the last decades. In biological systems, VCs may rapidly interconvert under physiological conditions and several V-containing moieties may be formed and bind to proteins. These interactions play key roles in the form transported in blood, in the uptake by cells, in inhibition properties and mechanism of action of essential and pharmacologically active V species. In this review, we focus on the recent advances made, namely in the application of the theoretical methodologies that allowed the description of the coordinative and non-covalent VC–protein interactions. The text is organized in six main topics: a general overview of the most important experimental and computational techniques useful to study these systems, a discussion on the nature of binding process, the recent advances on the comprehension of the V-containing natural and artificial enzymes, the interaction of mononuclear VCs with blood and other physiologically relevant proteins, the binding of polyoxidovanadates(V) to proteins and, finally, the biological and therapeutic implications of the interaction of pharmacologically relevant VCs with proteins and enzymes. Recent developments on vanadium-containing nitrogenases, haloperoxidases and nitrate reductases, and binding of VCs to transferrin, albumins, immunoglobulins, hemoglobin, lysozyme, myoglobin, ubiquitin and cytochrome c are discussed. Challenges and ideas about desirable features and potential drawbacks of VCs in biology and medicine and future directions to explore this chemistry area are also presented. The deeper understanding of the interactions of V-species with proteins, and the discussed data may provide the basis to undertake the investigation, design and development of new potentially active VCs with a more solid knowledge to predict their binding to biological receptors at a molecular point of view.
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Biocatalytic Cross-Coupling of Aryl Halides with a Genetically Engineered Photosensitizer Artificial Dehalogenase
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J. Am. Chem. Soc. 2021, 143, 617-622, 10.1021/jacs.0c10882
Devising artificial photoenzymes for abiological bond-forming reactions is of high synthetic value but also a tremendous challenge. Disclosed herein is the first photobiocatalytic cross-coupling of aryl halides enabled by a designer artificial dehalogenase, which features a genetically encoded benzophenone chromophore and site-specifically modified synthetic NiII(bpy) cofactor with tunable proximity to streamline the dual catalysis. Transient absorption studies suggest the likelihood of energy transfer activation in the elementary organometallic event. This design strategy is viable to significantly expand the catalytic repertoire of artificial photoenzymes for useful organic transformations.
Metal: NiLigand type: BipyridineHost protein: CO2-reducing photosensitizer protein (PSP)Anchoring strategy: CovalentOptimization: Chemical & geneticNotes: ---
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Catalytic Principles from Natural Enzymes and Translational Design Strategies for Synthetic Catalysts
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ACS Cent. Sci. 2021, 7, 72-80, 10.1021/acscentsci.0c01556
As biocatalysts, enzymes are characterized by their high catalytic efficiency and strong specificity but are relatively fragile by requiring narrow and specific reactive conditions for activity. Synthetic catalysts offer an opportunity for more chemical versatility operating over a wider range of conditions but currently do not reach the remarkable performance of natural enzymes. Here we consider some new design strategies based on the contributions of nonlocal electric fields and thermodynamic fluctuations to both improve the catalytic step and turnover for rate acceleration in arbitrary synthetic catalysts through bioinspired studies of natural enzymes. With a focus on the enzyme as a whole catalytic construct, we illustrate the translational impact of natural enzyme principles to synthetic enzymes, supramolecular capsules, and electrocatalytic surfaces.
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Chemical Modifications of Proteins and Their Applications in Metalloenzyme Studies
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Synth. Syst. Biotechnol. 2021, 6, 32-49, 10.1016/j.synbio.2021.01.001
Protein chemical modifications are important tools for elucidating chemical and biological functions of proteins. Several strategies have been developed to implement these modifications, including enzymatic tailoring reactions, unnatural amino acid incorporation using the expanded genetic codes, and recognition-driven transformations. These technologies have been applied in metalloenzyme studies, specifically in dissecting their mechanisms, improving their enzymatic activities, and creating artificial enzymes with non-natural activities. Herein, we summarize some of the recent efforts in these areas with an emphasis on a few metalloenzyme case studies.
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Chemogenetic Evolution of a Peroxidase-like Artificial Metalloenzyme
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ACS Catal. 2021, 11, 5079-5087, 10.1021/acscatal.1c00134
Directed evolution has helped enzyme engineering to remarkable successes in the past. A main challenge in directed evolution is to find the most suitable starting point, that is, an enzyme that allows maximum “evolvability”. Consisting of a synthetic cofactor embedded in a protein scaffold, artificial metalloenzymes (ArMs) are reminiscent of rough-hewn ancestral metalloproteins and thus could provide an evolutionarily clean slate. Here, we report the design and directed evolution of an ArM with peroxidase-like properties based on the nitrobindin variant, NB4. After identifying a suitable artificial metal cofactor, two rounds of directed evolution were sufficient to elevate the ArM’s activity to levels akin to those of some natural peroxidases (up to kcat = 14.1 s–1 and kcat/Km = 52,800 M–1 s–1). A substitution to arginine in the distal cofactor environment (position 76) was the key to boost the peroxidase activity. Molecular dynamics simulations reveal a remarkable flexibility in the distal site of the NB4 scaffold that is absent in the nitrobindin wildtype and which allows the unrestricted movement of the catalytically important Arg76. In addition to the oxidation of the common redox mediators (ABTS, syringaldehyde, and 2,6-dimethoxyphenol), the ArM proved efficient in the decolorization of three recalcitrant dyes (indigo carmine, reactive blue 19, and reactive black 5) and was amenable to several rounds of ArM recycling.
Metal: MnLigand type: PorphyrinHost protein: Nitrobindin (Nb)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: kcat = 14.1 s−1 and kcat/Km = 52,800 M−1 s −1
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Controlled Ligand Exchange Between Ruthenium Organometallic Cofactor Precursors and a Naïve Protein Scaffold Generates Artificial Metalloenzymes Catalysing Transfer Hydrogenation
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Angew. Chem. Int. Ed. 2021, 60, 10919-10927, 10.1002/anie.202015834
Many natural metalloenzymes assemble from proteins and biosynthesised complexes, generating potent catalysts by changing metal coordination. Here we adopt the same strategy to generate artificial metalloenzymes (ArMs) using ligand exchange to unmask catalytic activity. By systematically testing RuII(η6-arene)(bipyridine) complexes designed to facilitate the displacement of functionalised bipyridines, we develop a fast and robust procedure for generating new enzymes via ligand exchange in a protein that has not evolved to bind such a complex. The resulting metal cofactors form peptidic coordination bonds but also retain a non-biological ligand. Tandem mass spectrometry and 19F NMR spectroscopy were used to characterise the organometallic cofactors and identify the protein-derived ligands. By introduction of ruthenium cofactors into a 4-helical bundle, transfer hydrogenation catalysts were generated that displayed a 35-fold rate increase when compared to the respective small molecule reaction in solution.
Notes: 35 fold rate increase
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De Novo Designed Coiled Coils as Scaffolds for Lanthanides, Including Novel Imaging Agents with a Twist
Review -
Chem. Commun. 2021, 57, 6851-6862, 10.1039/d1cc02013g
For much of their history, lanthanides were thought to be biologically inert. However, the last decade has seen the discovery and development of the field of native lanthanide biochemistry. Lanthanides exhibit a variety of interesting photophysical properties from which many useful applications derive. The development of effective functional lanthanide complexes requires control of their coordination sphere; something proteins manage very effectively through their 3D metal-binding sites. α-Helical coiled coil peptides are miniature scaffolds which can be designed de novo and can retain the favourable properties of larger proteins within a much simplified system. Metal binding sites, including those which bind lanthanides can be engineered into the coiled coil sequence. This review will highlight the opportunities presented by the use of coiled coil peptides as scaffolds for lanthanide binding and the potential to control the coordination environment by simple modifications to peptide sequence. Designed lanthanide coiled coils offer opportunities to gain greater insight into native lanthanide biochemistry as well as to develop new functional complexes, including imaging agents.
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De Novo Design, Solution Characterization, and Crystallographic Structure of an Abiological Mn–Porphyrin-Binding Protein Capable of Stabilizing a Mn(V) Species
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J. Am. Chem. Soc. 2021, 143, 252-259, 10.1021/jacs.0c10136
De novo protein design offers the opportunity to test our understanding of how metalloproteins perform difficult transformations. Attaining high-resolution structural information is critical to understanding how such designs function. There have been many successes in the design of porphyrin-binding proteins; however, crystallographic characterization has been elusive, limiting what can be learned from such studies as well as the extension to new functions. Moreover, formation of highly oxidizing high-valent intermediates poses design challenges that have not been previously implemented: (1) purposeful design of substrate/oxidant access to the binding site and (2) limiting deleterious oxidation of the protein scaffold. Here we report the first crystallographically characterized porphyrin-binding protein that was programmed to not only bind a synthetic Mn–porphyrin but also maintain binding site access to form high-valent oxidation states. We explicitly designed a binding site with accessibility to dioxygen units in the open coordination site of the Mn center. In solution, the protein is capable of accessing a high-valent Mn(V)–oxo species which can transfer an O atom to a thioether substrate. The crystallographic structure is within 0.6 Å of the design and indeed contained an aquo ligand with a second water molecule stabilized by hydrogen bonding to a Gln side chain in the active site, offering a structural explanation for the observed reactivity.
Metal: MnLigand type: PorphyrinHost protein: Manganese Porphyrin-binding Protein 1Anchoring strategy: CovalentOptimization: ---Notes: ---
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Design and Engineering of Artificial Metalloproteins: From De Novo Metal Coordination to Catalysis
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Protein Eng. Des. Sel. 2021, 34, 10.1093/protein/gzab003
Metalloproteins are essential to sustain life. Natural evolution optimized them for intricate structural, regulatory and catalytic functions that cannot be fulfilled by either a protein or a metal ion alone. In order to understand this synergy and the complex design principles behind the natural systems, simpler mimics were engineered from the bottom up by installing de novo metal sites in either natural or fully designed, artificial protein scaffolds. This review focuses on key challenges associated with this approach. We discuss how proteins can be equipped with binding sites that provide an optimal coordination environment for a metal cofactor of choice, which can be a single metal ion or a complex multinuclear cluster. Furthermore, we highlight recent studies in which artificial metalloproteins were engineered towards new functions, including electron transfer and catalysis. In this context, the powerful combination of de novo protein design and directed evolution is emphasized for metalloenzyme development.
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Design of Artificial Metalloenzymes for the Reduction of Nicotinamide Cofactors
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J. Inorg. Biochem. 2021, 220, 111446, 10.1016/j.jinorgbio.2021.111446
Artificial metalloenzymes result from the insertion of a catalytically active metal complex into a biological scaffold, generally a protein devoid of other catalytic functionalities. As such, their design requires efforts to engineer substrate binding, in addition to accommodating the artificial catalyst. Here we constructed and characterised artificial metalloenzymes using alcohol dehydrogenase as starting point, an enzyme which has both a cofactor and a substrate binding pocket. A docking approach was used to determine suitable positions for catalyst anchoring to single cysteine mutants, leading to an artificial metalloenzyme capable to reduce both natural cofactors and the hydrophobic 1-benzylnicotinamide mimic. Kinetic studies revealed that the new construct displayed a Michaelis-Menten behaviour with the native nicotinamide cofactors, which were suggested by docking to bind at a surface exposed site, different compared to their native binding position. On the other hand, the kinetic and docking data suggested that a typical enzyme behaviour was not observed with the hydrophobic 1-benzylnicotinamide mimic, with which binding events were plausible both inside and outside the protein. This work demonstrates an extended substrate scope of the artificial metalloenzymes and provides information about the binding sites of the nicotinamide substrates, which can be exploited to further engineer artificial metalloenzymes for cofactor regeneration.
Metal: RhHost protein: Alcohol dehydrogenaseAnchoring strategy: CovalentOptimization: Chemical & geneticNotes: ---
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Design of Artificial Metalloenzymes with Multiple Inorganic Elements: The More the Merrier
Review -
J. Inorg. Biochem. 2021, 223, 111552, 10.1016/j.jinorgbio.2021.111552
A large fraction of metalloenzymes harbors multiple metal-centers that are electronically and/or functionally arranged within their proteinaceous environments. To explore the orchestration of inorganic and biochemical components and to develop bioinorganic catalysts and materials, we have described selected examples of artificial metalloproteins having multiple metallocofactors that were grouped depending on their initial protein scaffolds, the nature of introduced inorganic moieties, and the method used to propagate the number of metal ions within a protein. They demonstrated that diverse inorganic moieties can be selectively grafted and modulated in protein environments, providing a retrosynthetic bottom-up approach in the design of versatile and proficient biocatalysts and biomimetic model systems to explore fundamental questions in bioinorganic chemistry.
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Directed Evolution of a Cp*RhIII‐Linked Biohybrid Catalyst Based on a Screening Platform with Affinity Purification
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ChemBioChem 2021, 22, 679-685, 10.1002/cbic.202000681
Directed evolution of Cp*RhIII-linked nitrobindin (NB), a biohybrid catalyst, was performed based on an in vitro screening approach. A key aspect of this effort was the establishment of a high-throughput screening (HTS) platform that involves an affinity purification step employing a starch-agarose resin for a maltose binding protein (MBP) tag. The HTS platform enables efficient preparation of the purified MBP-tagged biohybrid catalysts in a 96-well format and eliminates background influence of the host E. coli cells. Three rounds of directed evolution and screening of more than 4000 clones yielded a Cp*RhIII-linked NB(T98H/L100K/K127E) variant with a 4.9-fold enhanced activity for the cycloaddition of acetophenone oximes with alkynes. It is confirmed that this HTS platform for directed evolution provides an efficient strategy for generating highly active biohybrid catalysts incorporating a synthetic metal cofactor.
Metal: RhLigand type: CpHost protein: Nitrobindin (Nb)Anchoring strategy: CovalentOptimization: GeneticNotes: ---
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Diversifying Metal–Ligand Cooperative Catalysis in Semi‐Synthetic [Mn]‐Hydrogenases
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Angew. Chem. Int. Ed. 2021, 60, 13350-13357, 10.1002/anie.202100443
The reconstitution of [Mn]-hydrogenases using a series of MnI complexes is described. These complexes are designed to have an internal base or pro-base that may participate in metal–ligand cooperative catalysis or have no internal base or pro-base. Only MnI complexes with an internal base or pro-base are active for H2 activation; only [Mn]-hydrogenases incorporating such complexes are active for hydrogenase reactions. These results confirm the essential role of metal–ligand cooperation for H2 activation by the MnI complexes alone and by [Mn]-hydrogenases. Owing to the nature and position of the internal base or pro-base, the mode of metal–ligand cooperation in two active [Mn]-hydrogenases is different from that of the native [Fe]-hydrogenase. One [Mn]-hydrogenase has the highest specific activity of semi-synthetic [Mn]- and [Fe]-hydrogenases. This work demonstrates reconstitution of active artificial hydrogenases using synthetic complexes differing greatly from the native active site.
Metal: MnHost protein: Apo-[Fe]-hydrogenase from M. jannaschiiAnchoring strategy: ReconstitutionOptimization: ChemicalNotes: ---
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Efficient Lewis Acid Catalysis of an Abiological Reaction in a De Novo Protein Scaffold
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Nat. Chem. 2021, 13, 231-235, 10.1038/s41557-020-00628-4
New enzyme catalysts are usually engineered by repurposing the active sites of natural proteins. Here we show that design and directed evolution can be used to transform a non-natural, functionally naive zinc-binding protein into a highly active catalyst for an abiological hetero-Diels–Alder reaction. The artificial metalloenzyme achieves >104 turnovers per active site, exerts absolute control over reaction pathway and product stereochemistry, and displays a catalytic proficiency (1/KTS = 2.9 × 1010 M−1) that exceeds all previously characterized Diels–Alderases. These properties capitalize on effective Lewis acid catalysis, a chemical strategy for accelerating Diels–Alder reactions common in the laboratory but so far unknown in nature. Extension of this approach to other metal ions and other de novo scaffolds may propel the design field in exciting new directions.
Metal: ZnLigand type: Amino acidHost protein: De novo-designed proteinAnchoring strategy: DativeOptimization: GeneticNotes: PDB: 3V1C, 7BWW
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Engineered and Artificial Metalloenzymes for Selective C–H Functionalization
Review -
Curr. Opin. Green Sustain. Chem. 2021, 31, 100494, 10.1016/j.cogsc.2021.100494
The direct functionalization of C–H bonds constitutes a powerful strategy to construct and diversify organic molecules. However, controlling the chemo- and site-selectivity of this transformation, particularly in complex molecular settings, represents a significant challenge. Metalloenzymes are ideal platforms for achieving catalyst-controlled selective C–H bond functionalization as their reactivities can be tuned by protein engineering and/or redesign of their cofactor environment. In this review, we highlight recent progress in the development of engineered and artificial metalloenzymes for C–H functionalization, with a focus on biocatalytic strategies for selective C–H oxyfunctionalization and halogenation as well as C–H amination and C–H carbene insertion via abiological nitrene and carbene transfer chemistries. Engineered heme and nonheme iron dependent enzymes have emerged as promising scaffolds for executing these transformations with high chemo-, regio-, and stereocontrol as well as tunable selectivity. These emerging systems and methodologies have expanded the toolbox of sustainable strategies for organic synthesis and created new opportunities for the generation of chiral building blocks, the late-stage C–H functionalization of complex molecules, and the total synthesis of natural products.
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Engineering and Emerging Applications of Artificial Metalloenzymes with Whole Cells
Review -
Nat. Catal. 2021, 4, 814-827, 10.1038/s41929-021-00673-3
The field of artificial metalloenzymes (ArMs) is rapidly growing and ArMs are attracting increasing attention, for example, in the fields of biosensing and drug therapy. Protein-engineering methods that are commonly used to tailor the properties of natural enzymes are more frequently included in the design of ArMs. In particular, directed evolution allows the fine-tuning of ArMs, ultimately assisting in the development of their enormous potential. The integration of ArMs in whole cells enables their in vivo application and facilitates high-throughput directed-evolution methodologies. In this Review, we highlight the recent progress of whole-cell conversions and applications of ArMs and critically discuss their limitations and prospects. To focus on ArMs and their specific properties, advantages and challenges, the evolution of natural enzymes for non-natural reactions will not be covered.
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Engineering Dirhodium Artificial Metalloenzymes for Diazo Coupling Cascade Reactions
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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.
Metal: RhLigand type: DirhodiumHost protein: Prolyl oligopeptidase (POP)Anchoring strategy: CovalentOptimization: ---Notes: 61% max combined yield for cascade reactions
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Engineering Thermostability in Artificial Metalloenzymes to Increase Catalytic Activity
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ACS Catal. 2021, 11, 3620-3627, 10.1021/acscatal.0c05413
Protein engineering has shown widespread use in improving the industrial application of enzymes and broadening the conditions they are able to operate under by increasing their thermostability and solvent tolerance. Here, we show that protein engineering can be used to increase the thermostability of an artificial metalloenzyme. Thermostable variants of the human steroid carrier protein 2L, modified to bind a metal catalyst, were created by rational design using structural data and a 3DM database. These variants were tested to identify mutations that enhanced the stability of the protein scaffold, and a significant increase in melting temperature was observed with a number of modified metalloenzymes. The ability to withstand higher reaction temperatures resulted in an increased activity in the hydroformylation of 1-octene, with more than fivefold improvement in turnover number, whereas the selectivity for linear aldehyde remained high up to 80%.
Metal: RhLigand type: PhosphineHost protein: Steroid Carrier Protein 2L (SCP-2L)Anchoring strategy: CovalentOptimization: GeneticNotes: ---
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Exploring and Adapting the Molecular Selectivity of Artificial Metalloenzymes
Review -
BCSJ 2021, 94, 382-396, 10.1246/bcsj.20200316
In recent years, artificial metalloenzymes (ArMs) have become a major research interest in the field of biocatalysis. With the ability to facilitate new-to-nature reactions, researchers have generally prepared them either through intensive protein engineering studies or through the introduction of abiotic transition metals. The aim of this review will be to summarize the major types of ArMs that have been recently developed, as well as to highlight their general reaction scope. A point of emphasis will also be made to discuss the promising ways that the molecular selectivity of ArMs can be applied to in areas of pharmaceutical synthesis, diagnostics, and drug therapy.
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Histidine orientation in artificial peroxidase regioisomers as determined by paramagnetic NMR shifts
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Chem. Commun. 2021, 57, 990-993, 10.1039/d0cc06676a
Fe-Mimochrome VI*a is a synthetic peroxidase and peroxygenase, featuring two different peptides that are covalently-linked to deuteroheme. To perform a systematic structure/function correlation, we purposely shortened the distance between the distal peptide and the heme, allowing for the separation and characterization of two regioisomers. They differ in both His axial-ligand orientation, as determined by paramagnetic NMR shifts, and activity. These findings highlight that synthetic metalloenzymes may provide an efficient tool for disentangling the role of axial ligand orientation over peroxidase activity.
Metal: FeLigand type: Deuteroporphyrin IXHost protein: Synthetic peptideAnchoring strategy: CovalentOptimization: ---Notes: NMR studies of the complexes, no catalysis
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In Vivo Assembly of Artificial Metalloenzymes and Application in Whole‐Cell Biocatalysis
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Angew. Chem. Int. Ed. 2021, 60, 5913-5920, 10.1002/anie.202014771
We report the supramolecular assembly of artificial metalloenzymes (ArMs), based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneous copper(II)–phenanthroline complex, in the cytoplasm of E. coli cells. A combination of catalysis, cell-fractionation, and inhibitor experiments, supplemented with in-cell solid-state NMR spectroscopy, confirmed the in-cell assembly. The ArM-containing whole cells were active in the catalysis of the enantioselective Friedel–Crafts alkylation of indoles and the Diels–Alder reaction of azachalcone with cyclopentadiene. Directed evolution resulted in two different improved mutants for both reactions, LmrR_A92E_M8D and LmrR_A92E_V15A, respectively. The whole-cell ArM system required no engineering of the microbial host, the protein scaffold, or the cofactor to achieve ArM assembly and catalysis. We consider this a key step towards integrating abiological catalysis with biosynthesis to generate a hybrid metabolism.
Metal: CuLigand type: PhenanthrolineHost protein: Lactoccal multidrug resistant regulator (LmrR)Anchoring strategy: SupramolecularOptimization: GeneticNotes: ---
Metal: CuLigand type: PhenanthrolineHost protein: Lactoccal multidrug resistant regulator (LmrR)Anchoring strategy: SupramolecularOptimization: GeneticNotes: ---
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Modular Design of G-Quadruplex MetalloDNAzymes for Catalytic C–C Bond Formations with Switchable Enantioselectivity
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J. Am. Chem. Soc. 2021, 143, 3555-3561, 10.1021/jacs.0c13251
Metal-binding DNA structures with catalytic function are receiving increasing interest. Although a number of metalloDNAzymes have been reported to be highly efficient, the exact coordination/position of their catalytic metal center is often unknown. Here, we present a new approach to rationally develop metalloDNAzymes for Lewis acid-catalyzed reactions such as enantioselective Michael additions. Our strategy relies on the predictable folding patterns of unimolecular DNA G-quadruplexes, combined with the concept of metal-mediated base-pairing. Transition-metal coordination environments were created in G-quadruplex loop regions, accessible by substrates. Therefore, protein-inspired imidazole ligandoside L was covalently incorporated into a series of G-rich DNA strands by solid-phase synthesis. Iterative rounds of DNA sequence design and catalytic assays allowed us to select tailored metalloDNAzymes giving high conversions and excellent enantioselectivities (≥99%). Based on their primary sequence, folding pattern, and metal coordination mode, valuable information on structure–activity relationships could be extracted. Variation of the number and position of ligand L within the sequence allowed us to control the formation of (S) and (R) enantiomeric reaction products, respectively.
Metal: CuLigand type: DNA (G quadruplex)Host protein: metalloDNAzymeAnchoring strategy: Imidazole ligandosideOptimization: GeneticNotes: Km 35.2 uM, vmax-8.2 nM min-1
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Molecular Understanding of Heteronuclear Active Sites in Heme–Copper Oxidases, Nitric Oxide Reductases, and Sulfite Reductases Through Biomimetic Modelling
Review -
Chem. Soc. Rev. 2021, 50, 2486-2539, 10.1039/d0cs01297a
Heme–copper oxidases (HCO), nitric oxide reductases (NOR), and sulfite reductases (SiR) catalyze the multi-electron and multi-proton reductions of O2, NO, and SO32−, respectively. Each of these reactions is important to drive cellular energy production through respiratory metabolism and HCO, NOR, and SiR evolved to contain heteronuclear active sites containing heme/copper, heme/nonheme iron, and heme–[4Fe–4S] centers, respectively. The complexity of the structures and reactions of these native enzymes, along with their large sizes and/or membrane associations, make it challenging to fully understand the crucial structural features responsible for the catalytic properties of these active sites. In this review, we summarize progress that has been made to better understand these heteronuclear metalloenzymes at the molecular level though study of the native enzymes along with insights gained from biomimetic models comprising either small molecules or proteins. Further understanding the reaction selectivity of these enzymes is discussed through comparisons of their similar heteronuclear active sites, and we offer outlook for further investigations.
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Nitrene Transfers Mediated by Natural and Artificial Iron Enzymes
Review -
J. Inorg. Biochem. 2021, 225, 111613, 10.1016/j.jinorgbio.2021.111613
Amines are ubiquitous in biology and pharmacy. As a consequence, introducing N functionalities in organic molecules is attracting strong continuous interest. The past decade has witnessed the emergence of very efficient and selective catalytic systems achieving this goal thanks to engineered hemoproteins. In this review, we examine how these enzymes have been engineered focusing rather on the rationale behind it than the methodology employed. These studies are put in perspective with respect to in vitro and in vivo nitrene transfer processes performed by cytochromes P450. An emphasis is put on mechanistic aspects which are confronted to current molecular knowledge of these reactions. Forthcoming developments are delineated.
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