45 publications

45 publications

A Designed Metalloenzyme Achieving the Catalytic Rate of a Native Enzyme

Lu, Y.; Wang, J.

J. Am. Chem. Soc. 2015, 137, 11570-11573, 10.1021/jacs.5b07119

Terminal oxidases catalyze four-electron reduction of oxygen to water, and the energy harvested is utilized to drive the synthesis of adenosine triphosphate. While much effort has been made to design a catalyst mimicking the function of terminal oxidases, most biomimetic catalysts have much lower activity than native oxidases. Herein we report a designed oxidase in myoglobin with an O2 reduction rate (52 s–1) comparable to that of a native cytochrome (cyt) cbb3 oxidase (50 s–1) under identical conditions. We achieved this goal by engineering more favorable electrostatic interactions between a functional oxidase model designed in sperm whale myoglobin and its native redox partner, cyt b5, resulting in a 400-fold electron transfer (ET) rate enhancement. Achieving high activity equivalent to that of native enzymes in a designed metalloenzyme offers deeper insight into the roles of tunable processes such as ET in oxidase activity and enzymatic function and may extend into applications such as more efficient oxygen reduction reaction catalysts for biofuel cells.


Metal: Cu
Ligand type: Amino acid
Host protein: Myoglobin (Mb)
Anchoring strategy: Dative
Optimization: Genetic
Reaction: O2 reduction
Max TON: ---
ee: ---
PDB: ---
Notes: O2 reduction rates of 52 s-1 were achieved in combination with the native redox partner cyt b5.

A Highly Active Biohybrid Catalyst for Olefin Metathesis in Water: Impact of a Hydrophobic Cavity in a β-Barrel Protein

Okuda, J.

ACS Catal. 2015, 5, 7519-7522, 10.1021/acscatal.5b01792

A series of Grubbs–Hoveyda type catalyst precursors for olefin metathesis containing a maleimide moiety in the backbone of the NHC ligand was covalently incorporated in the cavity of the β-barrel protein nitrobindin. By using two protein mutants with different cavity sizes and choosing the suitable spacer length, an artificial metalloenzyme for olefin metathesis reactions in water in the absence of any organic cosolvents was obtained. High efficiencies reaching TON > 9000 in the ROMP of a water-soluble 7-oxanorbornene derivative and TON > 100 in ring-closing metathesis (RCM) of 4,4-bis(hydroxymethyl)-1,6-heptadiene in water under relatively mild conditions (pH 6, T = 25–40 °C) were observed.


Metal: Ru
Ligand type: Carbene
Host protein: Nitrobindin (Nb)
Anchoring strategy: Covalent
Optimization: Chemical
Reaction: Olefin metathesis
Max TON: 9900
ee: ---
PDB: ---
Notes: ROMP (cis/trans: 48/52)

Metal: Ru
Ligand type: Carbene
Host protein: Nitrobindin (Nb)
Anchoring strategy: Covalent
Optimization: Chemical
Reaction: Olefin metathesis
Max TON: 100
ee: ---
PDB: ---
Notes: RCM

Albumin as a Promiscuous Biocatalyst in Organic Synthesis

Review

Gaggero, N.

RSC Adv. 2015, 5, 10588-10598, 10.1039/C4RA11206G

Albumin emerged as a biocatalyst in 1980 and the continuing interest in this protein is proved by numerous papers.


Notes: ---

An Artificial Enzyme Made by Covalent Grafting of an FeII Complex into β-Lactoglobulin: Molecular Chemistry, Oxidation Catalysis, and Reaction-Intermediate Monitoring in a Protein

Banse, F.; Mahy, J.-P.

Chem. - Eur. J. 2015, 21, 12188-12193, 10.1002/chem.201501755

An artificial metalloenzyme based on the covalent grafting of a nonheme FeII polyazadentate complex into bovine β‐lactoglobulin has been prepared and characterized by using various spectroscopic techniques. Attachment of the FeII catalyst to the protein scaffold is shown to occur specifically at Cys121. In addition, spectrophotometric titration with cyanide ions based on the spin‐state conversion of the initial high spin (S=2) FeII complex into a low spin (S=0) one allows qualitative and quantitative characterization of the metal center’s first coordination sphere. This biohybrid catalyst activates hydrogen peroxide to oxidize thioanisole into phenylmethylsulfoxide as the sole product with an enantiomeric excess of up to 20 %. Investigation of the reaction between the biohybrid system and H2O2 reveals the generation of a high spin (S=5/2) FeIII(η2‐O2) intermediate, which is proposed to be responsible for the catalytic sulfoxidation of the substrate.


Metal: Fe
Ligand type: Poly-pyridine
Host protein: ß-lactoglobulin
Anchoring strategy: Covalent
Optimization: ---
Reaction: Sulfoxidation
Max TON: 5.6
ee: 20
PDB: ---
Notes: ---

Aqueous Light Driven Hydrogen Production by a Ru–Ferredoxin–Co Biohybrid

Utschig, L.M.

Chem. Commun. 2015, 51, 10628-10631, 10.1039/c5cc03006d

Long-lived charge separation facilitates photocatalytic H2 production in a mini reaction center/catalyst complex.


Metal: Co
Ligand type: Oxime
Host protein: Ferredoxin (Fd)
Anchoring strategy: Dative
Optimization: ---
Reaction: H2 evolution
Max TON: 210
ee: ---
PDB: ---
Notes: Recalculated TON

Artificial Diiron Enzymes with a De Novo Designed Four-Helix Bundle Structure

Review

DeGrado, W.F.; Lombardi, A.

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

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


Notes: ---

Artificial Hydrogenase: Biomimetic Approaches Controlling Active Molecular Catalysts

Review

Onoda, A.

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

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


Notes: ---

Artificial Hydrogenases: Biohybrid and Supramolecular Systems for Catalytic Hydrogen Production or Uptake

Review

Fontecave, M.

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

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


Notes: ---

Artificial Metalloenzymes Derived from Three-Helix Bundles

Review

Pecoraro, V.L.

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

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


Notes: ---

Artificial Metalloenzymes for Asymmetric Catalysis by Creation of Novel Active Sites in Protein and DNA Scaffolds

Review

Roelfes, G.

Isr. J. Chem. 2015, 55, 21-31, 10.1002/ijch.201400094

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.


Notes: ---

Artificial Metalloenzymes for the Diastereoselective Reduction of NAD+ to NAD2H

Ward, T.R.

Org. Biomol. Chem. 2015, 13, 357-360, 10.1039/c4ob02071e

Stereoselectively labelled isotopomers of NAD(P)H are highly relevant for mechanistic studies of enzymes which utilize them as redox equivalents.


Metal: Ir
Ligand type: Amino-sulfonamide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Artificial Metalloenzymes in Asymmetric Catalysis: Key Developments and Future Directions

Review

Bäckvall, J.E.; Diéguez, M.; Pàmies, O.

Adv. Synth. Catal. 2015, 357, 1567-1586, 10.1002/adsc.201500290

Artificial metalloenzymes combine the excellent selective recognition/binding properties of enzymes with transition metal catalysts, and therefore many asymmetric transformations can benefit from these entities. The search for new successful strategies in the construction of metal‐enzyme hybrid catalysts has therefore become a very active area of research. This review discusses all the developed strategies and the latest advances in the synthesis and application in asymmetric catalysis of artificial metalloenzymes with future directions for their design, synthesis and application (Sections 2–4). Finally, advice is presented (to the non‐specialist) on how to prepare and use artificial metalloenzymes (Section 5).


Notes: ---

Carbonic Anhydrase II as Host Protein for the Creation of a Biocompatible Artificial Metathesase

Ward, T.R.

Org. Biomol. Chem. 2015, 13, 5652-5655, 10.1039/c5ob00428d

We report an efficient artificial metathesase which combines an arylsulfonamide anchor within the protein scaffold human carbonic anhydrase II.


Metal: Ru
Ligand type: Carbene
Anchoring strategy: Dative
Optimization: Chemical & genetic
Reaction: Olefin metathesis
Max TON: 28
ee: ---
PDB: ---
Notes: Ring closing metathesis. 28 turnovers obtained under physiological conditions within 4 hours.

Defining the Role of Tyrosine and Rational Tuning of Oxidase Activity by Genetic Incorporation of Unnatural Tyrosine Analogs

Lu, Y.; Wang, J.

J. Am. Chem. Soc. 2015, 137, 4594-4597, 10.1021/ja5109936

While a conserved tyrosine (Tyr) is found in oxidases, the roles of phenol ring pKa and reduction potential in O2 reduction have not been defined despite many years of research on numerous oxidases and their models. These issues represent major challenges in our understanding of O2 reduction mechanism in bioenergetics. Through genetic incorporation of unnatural amino acid analogs of Tyr, with progressively decreasing pKa of the phenol ring and increasing reduction potential, in the active site of a functional model of oxidase in myoglobin, a linear dependence of both the O2 reduction activity and the fraction of H2O formation with the pKa of the phenol ring has been established. By using these unnatural amino acids as spectroscopic probe, we have provided conclusive evidence for the location of a Tyr radical generated during reaction with H2O2, by the distinctive hyperfine splitting patterns of the halogenated tyrosines and one of its deuterated derivatives incorporated at the 33 position of the protein. These results demonstrate for the first time that enhancing the proton donation ability of the Tyr enhances the oxidase activity, allowing the Tyr analogs to augment enzymatic activity beyond that of natural Tyr.


Metal: Cu
Ligand type: Porphyrin
Host protein: Myoglobin (Mb)
Anchoring strategy: Dative
Optimization: Chemical & genetic
Max TON: 1200
ee: ---
PDB: 4FWX
Notes: Sperm whale myoglobin

De Novo Protein Design as a Methodology for Synthetic Bioinorganic Chemistry

Review

Pecoraro, V.L.

Acc. Chem. Res. 2015, 48, 2388-2396, 10.1021/acs.accounts.5b00175

The major advances in molecular and structural biology and automated peptide and DNA synthesis of the 1970s and 1980s generated fertile conditions in the 1990s for the exploration of designed proteins as a new approach for inorganic chemists to generate biomolecular mimics of metalloproteins. This Account follows the development of the TRI peptide family of three-stranded coiled coils (3SCC) and α3D family of three-helix bundles (3HB) as scaffolds for the preparation of metal binding sites within de novo designed constructs. The 3SCC were developed using the concept of a heptad repeat (abcdefg) putting hydrophobes in the a and d positions. The TRI peptides contain four heptads with capping glycines. Via substitution of leucine hydrophobes, metal ligands can be introduced into the a or d sites in order to bind metals. First, the ability to use cysteine-substituted 3SCC aggregates to impose higher or lower coordination numbers on Hg(II) and Cd(II) or matching the coordination preferences of As(III) and Pb(II) is discussed. Then, methods to develop dual site peptides capable of discriminating metals based on their type (e.g., Cd(II) vs Pb(II)), their preference for a vs d sites, and then their coordination number is described. Once these principles of metal site differentiation are described, we shift to building dual site peptides using both cysteine and histidine metal binding sites. This approach provides a construct with both a Hg(II) structural and a Zn(II) hydrolytic center, the latter of which is capable of hydrating CO2. With these Zn(II) proteins, we consider the relative importance of the location of the catalytic center along the primary sequence of the peptide and show that only minor perturbations in catalytic efficiencies are observed based on metal location. We then assess the feasibility of preparing enzymes competent to reduce nitrite with copper centers in a histidine-rich environment. As part of this discussion, we examine the influence of surface residues on catalyst reduction potentials and catalytic efficiencies. We end describing approaches to prepare asymmetric proteins that can incorporate acid–base catalysts or water channels. In this respect, we highlight modifications of a helix–turn–helix–turn–helix motif called α3D and show how this 3HB can be modified to bind heavy metals or to make Zn(II) centers, which are active hydrolytic catalysts. A comparison is made to the comparable parallel 3SCC.


Notes: ---

Directed Evolution of Artificial Metalloenzymes

Review

Reetz, M.T.

Isr. J. Chem. 2015, 55, 51-60, 10.1002/ijch.201400087

Transition metal catalysis in asymmetric transformations plays a pivotal role in modern synthetic organic chemistry, with these catalysts being tuned by systematic variation of the chiral ligand. More than three decades ago it was recognized that an alternative approach is possible, namely the anchoring of an achiral ligand/metal entity in an appropriate protein host, with formation of an artificial metalloenzyme (hybrid catalyst). However, this procedure delivers a single transition metal catalyst, with high enantioselectivity being a matter of chance. In view of this restriction, we proposed in 2001/2002 the concept of directed evolution of such hybrid catalysts. The most intensively studied system involves biotinylated phosphine/metal entities which are non‐covalently anchored to streptavidin. The present review summarizes progress in this intriguing area of research. It includes the assessment of the requirements of a given Darwinian system to be successful, and offers hints on how to achieve success in future studies.


Notes: ---

Direct Hydrogenation of Carbon Dioxide by an Artificial Reductase Obtained by Substituting Rhodium for Zinc in the Carbonic Anhydrase Catalytic Center. A Mechanistic Study

Marino, T.

ACS Catal. 2015, 5, 5397-5409, 10.1021/acscatal.5b00185

Recently, a new artificial carbonic anhydrase enzyme in which the native zinc cation has been replaced with a Rh(I) has been proposed as a new reductase that is able to efficiently catalyze the hydrogenation of olefins. In this paper, we propose the possible use of this modified enzyme in the direct hydrogenation of carbon dioxide. In our theoretical investigation, we have considered different reaction mechanisms such as reductive elimination and σ-bond metathesis. In addition, the release of the formic acid and the restoring of the catalytic cycle have also been studied. Results show that the σ-bond metathesis potential energy surface lies below the reactant species. The rate-determining step is the release of the product with an energy barrier of 12.8 kcal mol–1. On the basis of our results, we conclude that this artificial enzyme can efficiently catalyze the conversion of CO2 to HCOOH by a direct hydrogenation reaction.


Metal: Rh
Ligand type: Amino acid
Anchoring strategy: Metal substitution
Optimization: ---
Reaction: Hydrogenation
Max TON: ---
ee: ---
PDB: ---
Notes: Computational study of the reaction mechanism of the formation of HCOOH from CO2

Engineering a Dirhodium Artificial Metalloenzyme for Selective Olefin Cyclopropanation

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.


Metal: Rh
Ligand type: Poly-carboxylic acid
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Reaction: Cyclopropanation
Max TON: 74
ee: 92
PDB: ---
Notes: ---

Enzyme Repurposing of a Hydrolase as an Emergent Peroxidase Upon Metal Binding

Fujieda, N.; Ward, T.R.

Chem. Sci. 2015, 6, 4060-4065, 10.1039/c5sc01065a

Adding a metal cofactor to a protein bearing a latent metal binding site endows the macromolecule with nascent catalytic activity.


Metal: Cu
Ligand type: Amino acid
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 35
ee: ---
PDB: ---
Notes: ---

From Enzyme Maturation to Synthetic Chemistry: The Case of Hydrogenases

Review

Fontecave, M.

Acc. Chem. Res. 2015, 48, 2380-2387, 10.1021/acs.accounts.5b00157

Water splitting into oxygen and hydrogen is one of the most attractive strategies for storing solar energy and electricity. Because the processes at work are multielectronic, there is a crucial need for efficient and stable catalysts, which in addition have to be cheap for future industrial developments (electrolyzers, photoelectrochemicals, and fuel cells). Specifically for the water/hydrogen interconversion, Nature is an exquisite source of inspiration since this chemistry contributes to the bioenergetic metabolism of a number of living organisms via the activity of fascinating metalloenzymes, the hydrogenases. In this Account, we first briefly describe the structure of the unique dinuclear organometallic active sites of the two classes of hydrogenases as well as the complex protein machineries involved in their biosynthesis, their so-called maturation processes. This knowledge allows for the development of a fruitful bioinspired chemistry approach, which has already led to a number of interesting and original catalysts mimicking the natural active sites. More specifically, we describe our own attempts to prepare artificial hydrogenases. This can be achieved via the standard bioinspired approach using the combination of a synthetic bioinspired catalyst and a polypeptide scaffold. Such hybrid complexes provide the opportunity to optimize the system by manipulating both the catalyst through chemical synthesis and the protein component through mutagenesis. We also raise the possibility to reach such artificial systems via an original strategy based on mimicking the enzyme maturation pathways. This is illustrated in this Account by two examples developed in our laboratory. First, we show how the preparation of a lysozyme–{MnI(CO)3} hybrid and its clean reaction with a nickel complex led us to generate a new class of binuclear Ni-Mn H2-evolving catalysts mimicking the active site of [NiFe]-hydrogenases. Then we describe how we were able to rationally design and prepare a hybrid system, displaying remarkable structural similarities to an [FeFe]-hydrogenase, and we show here for the first time that it is catalytically active for proton reduction. This system is based on the combination of HydF, a protein involved in the maturation of [FeFe]-hydrogenase (HydA), and a close mimic of the active site of this class of enzymes. Moreover, the synthetic [Fe2(adt)(CO)4(CN)2]2– (adt2–= aza-propanedithiol) mimic, alone or within a HydF hybrid system, was shown to be able to maturate and activate a form of HydA itself lacking its diiron active site. We discuss the exciting perspectives this “synthetic maturation” opens regarding the “invention” of novel hydrogenases by the chemists.


Notes: ---

From "Hemoabzymes" to "Hemozymes": Towards new Biocatalysts for Selective Oxidations

Review

Mahy, J.-P.

Chem. Commun. 2015, 51, 2476-2494, 10.1039/c4cc08169b

The design of artificial hemoproteins that could catalyze selective oxidations using clean oxidants such as O2 or H2O2 under ecocompatible conditions constitutes a real challenge for a wide range of industrial applications. In vivo, such reactions are performed by heme-thiolate proteins, cytochromes P450, which catalyze the oxidation of substrates by dioxygen in the presence of electrons delivered from NADPH by cytochrome P450 reductase. Several strategies were used to design new artificial hemoproteins that mimic these enzymes. The first one involved the non-covalent association of synthetic hemes with monoclonal antibodies raised against these cofactors. This led to the first generation of artificial hemoproteins or “hemoabzymes” that displayed a peroxidase activity, and in some cases catalyzed the regioselective nitration of phenols by H2O2/NO2 and the stereoselective oxidation of sulfides by H2O2. The second one involved the non-covalent association of easily affordable non-relevant proteins with metalloporphyrin derivatives, using either the “Trojan Horse strategy” or the “host–guest” strategy. This led to a second generation of artificial hemoproteins or “hemozymes”, some of which were found able to catalyze the stereoselective oxidation of organic compounds such as sulfides and alkenes by H2O2 and KHSO5.


Notes: ---

Generation of New Artificial Metalloproteins by Cofactor Modification of Native Hemoproteins

Review

Hayashi, T

Isr. J. Chem. 2015, 55, 76-84, 10.1002/ijch.201400123

Heme can be removed from a number of native hemoproteins, thus forming corresponding apoproteins, each of which provides a site for binding of a metal complex. In one example, myoglobin, an O2 storage protein, can be reconstituted with iron porphycene to dramatically enhance the O2 affinity. Although it is known that myoglobin has poor enzymatic activity, the insertion of iron corrole or iron porphycene into apomyoglobin increases its H2O2‐dependent peroxidase/peroxygenase activities. Furthermore, reconstitution with manganese porphycene promotes hydroxylation of an inert CH bond. It is also of interest to insert a non‐porphyrinoid complex into an apoprotein. A cavity of apocytochrome c has been found to bind a diiron carbonyl complex, serving as a functional model of diiron hydrogenase. Aponitrobindin has a rigid β‐barrel structure that provides an excellent cavity for covalently anchoring a metal complex. A rhodium complex embedded in the cavity of genetically modified nitrobindin has been found to promote stereoselective polymerization of phenylacetylene.


Notes: ---

Hybrid [FeFe]-Hydrogenases with Modified Active Sites Show Remarkable Residual Enzymatic Activity

Lubitz, W.; Reijerse, E.

Biochemistry 2015, 54, 1474-1483, 10.1021/bi501391d

[FeFe]-hydrogenases are to date the only enzymes for which it has been demonstrated that the native inorganic binuclear cofactor of the active site Fe2(adt)(CO)3(CN)2 (adt = azadithiolate = [S-CH2-NH-CH2-S]2–) can be synthesized on the laboratory bench and subsequently inserted into the unmaturated enzyme to yield fully functional holo-enzyme (Berggren, G. et al. (2013) Nature 499, 66–70; Esselborn, J. et al. (2013) Nat. Chem. Biol. 9, 607–610). In the current study, we exploit this procedure to introduce non-native cofactors into the enzyme. Mimics of the binuclear subcluster with a modified bridging dithiolate ligand (thiodithiolate, N-methylazadithiolate, dimethyl-azadithiolate) and three variants containing only one CN– ligand were inserted into the active site of the enzyme. We investigated the activity of these variants for hydrogen oxidation as well as proton reduction and their structural accommodation within the active site was analyzed using Fourier transform infrared spectroscopy. Interestingly, the monocyanide variant with the azadithiolate bridge showed ∼50% of the native enzyme activity. This would suggest that the CN– ligands are not essential for catalytic activity, but rather serve to anchor the binuclear subsite inside the protein pocket through hydrogen bonding. The inserted artificial cofactors with a propanedithiolate and an N-methylazadithiolate bridge as well as their monocyanide variants also showed residual activity. However, these activities were less than 1% of the native enzyme. Our findings indicate that even small changes in the dithiolate bridge of the binuclear subsite lead to a rather strong decrease of the catalytic activity. We conclude that both the Brønsted base function and the conformational flexibility of the native azadithiolate amine moiety are essential for the high catalytic activity of the native enzyme.


Metal: Fe
Ligand type: CN; CO; Dithiolate
Anchoring strategy: Dative
Optimization: Chemical
Max TON: ---
ee: ---
PDB: ---
Notes: H2 evolution: TOF = 450 s-1. H2 oxidation: TOF = 150 s-1.

Hybrid Ruthenium ROMP Catalysts Based on an Engineered Variant of β-Barrel Protein FhuA ΔCVFtev: Effect of Spacer Length

Okuda, J.

Chem. - Asian J. 2015, 10, 177-182, 10.1002/asia.201403005

A biohybrid ring‐opening olefin metathesis polymerization catalyst based on the reengineered β‐barrel protein FhuA ΔCVFtev was chemically modified with respect to the covalently anchored Grubbs–Hoveyda type catalyst. Shortening of the spacer (1,3‐propanediyl to methylene) between the N‐heterocyclic carbene ligand and the cysteine site 545 increased the ROMP activity toward a water‐soluble 7‐oxanorbornene derivative. The cis/trans ratio of the double bond in the polymer was influenced by the hybrid catalyst.


Metal: Ru
Ligand type: Carbene
Host protein: FhuA ΔCVFtev
Anchoring strategy: Covalent
Optimization: Chemical
Reaction: Olefin metathesis
Max TON: 555
ee: ---
PDB: ---
Notes: ROMP; cis/trans = 58/42

Improving the Catalytic Performance of an Artificial Metalloenzyme by Computational Design

Baker, D.; Ward, T.R.

J. Am. Chem. Soc. 2015, 137, 10414-10419, 10.1021/jacs.5b06622

Artifical metalloenzymes combine the reactivity of small molecule catalysts with the selectivity of enzymes, and new methods are required to tune the catalytic properties of these systems for an application of interest. Structure-based computational design could help to identify amino acid mutations leading to improved catalytic activity and enantioselectivity. Here we describe the application of Rosetta Design for the genetic optimization of an artificial transfer hydrogenase (ATHase hereafter), [(η5-Cp*)Ir(pico)Cl] ⊂ WT hCA II (Cp* = Me5C5–), for the asymmetric reduction of a cyclic imine, the precursor of salsolsidine. Based on a crystal structure of the ATHase, computational design afforded four hCAII variants with protein backbone-stabilizing and hydrophobic cofactor-embedding mutations. In dansylamide-competition assays, these designs showed 46–64-fold improved affinity for the iridium pianostool complex [(η5-Cp*)Ir(pico)Cl]. Gratifyingly, the new designs yielded a significant improvement in both activity and enantioselectivity (from 70% ee (WT hCA II) to up to 92% ee and a 4-fold increase in total turnover number) for the production of (S)-salsolidine. Introducing additional hydrophobicity in the Cp*-moiety of the Ir-catalyst provided by adding a propyl substituent on the Cp* moiety yields the most (S)-selective (96% ee) ATHase reported to date. X-ray structural data indicate that the high enantioselectivity results from embedding the piano stool moiety within the protein, consistent with the computational model.


Metal: Ir
Ligand type: Cp*; Pyridine sulfonamide
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: 100
ee: 96
PDB: ---
Notes: ---

Latest Developments in Metalloenzyme Design and Repurposing

Review

Ward, T.R.

Eur. J. Inorg. Chem. 2015, 2015, 3406-3418, 10.1002/ejic.201500408

In the past decade, artificial metalloenzymes (AMEs) have emerged as attractive alternatives to more traditional homogeneous catalysts and enzymes. This microreview presents a selection of recent achievements in the design of such hybrid catalysts. These include artificial zinc hydrolases and metathesases, the heme‐protein repurposing for C–H, N–H, and S–H insertion reactions, novel light‐driven redox hybrid catalysts, novel scaffold proteins, and metallocofactor anchoring techniques and metalloenzyme models.


Notes: ---

Lipase Active Site Covalent Anchoring of Rh(NHC) Catalysts: Towards Chemoselective Artificial Metalloenzymes

Klein Gebbink, R.J.M.

Chem. Commun. 2015, 51, 6792-6795, 10.1039/c4cc09700a

A Rh(NHC) phosphonate complex reacts with the lipases cutinase and Candida antarctica lipase B resulting in the first (soluble) artificial metalloenzymes formed by covalent active site-directed hybridization. When compared to unsupported complexes, these new robust hybrids show enhanced chemoselectivity in the (competitive) hydrogenation of olefins over ketones.


Metal: Rh
Ligand type: Carbene
Host protein: Cutinase
Anchoring strategy: Covalent
Optimization: ---
Reaction: Hydrogenation
Max TON: 20
ee: rac.
PDB: 1CEX
Notes: ---

Metal: Rh
Ligand type: Carbene
Anchoring strategy: Covalent
Optimization: ---
Reaction: Hydrogenation
Max TON: 20
ee: rac.
PDB: 4K6G
Notes: ---

Metal-Binding Promiscuity in Artificial Metalloenzyme Design

Review

Pordea, A.

Curr. Opin. Chem. Biol. 2015, 25, 124-132, 10.1016/j.cbpa.2014.12.035

This review presents recent examples of metal-binding promiscuity in protein scaffolds and highlights the effect of metal variation on catalytic functionality. Naturally evolved binding sites, as well as unnatural amino acids and cofactors can bind a diverse range of metals, including non-biological transition elements. Computational screening and rational design have been successfully used to create promiscuous binding-sites. Incorporation of non-native metals into proteins expands the catalytic range of transformations catalysed by enzymes and enhances their potential for application in chemicals synthesis.


Notes: ---

Metallopeptide Catalysts and Artificial Metalloenzymes Containing Unnatural Amino Acids

Review

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.


Notes: ---

Molecular Recognition in Protein Modification with Rhodium Metallopeptides

Review

Ball, Z.T.

Curr. Opin. Chem. Biol. 2015, 25, 98-102, 10.1016/j.cbpa.2014.12.017

Chemical manipulation of natural, unengineered proteins is a daunting challenge which tests the limits of reaction design. By combining transition-metal or other catalysts with molecular recognition ideas, it is possible to achieve site-selective protein reactivity without the need for engineered recognition sequences or reactive sites. Some recent examples in this area have used ruthenium photocatalysis, pyridine organocatalysis, and rhodium(II) metallocarbene catalysis, indicating that the fundamental ideas provide opportunities for using diverse reactivity on complex protein substrates and in complex cell-like environments.


Notes: ---