37 publications

37 publications

A Hydroxyquinoline‐Based Unnatural Amino Acid for the Design of Novel Artificial Metalloenzymes

Roelfes, G.

ChemBioChem 2020, 21, 3077-3081, 10.1002/cbic.202000306

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


Metal: Cu
Ligand type: Hydroxyquinoline
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: 11
ee: 51
PDB: 3F8B
Notes: Also used Rh, but no reaction detected.

Metal: Cu
Ligand type: Hydroxyquinoline
Anchoring strategy: Supramolecular
Optimization: Genetic
Reaction: Water addition
Max TON: ---
ee: ---
PDB: 3F8B
Notes: ---

Metal: Zn
Ligand type: Hydroxyquinoline
Anchoring strategy: Supramolecular
Optimization: Genetic
Reaction: C-H activation
Max TON: ---
ee: ---
PDB: 3F8B
Notes: ---

Allosteric Cooperation in a De Novo-Designed Two-Domain Protein

DeGrado, W.F.; Lombardi, A.

Proc. Natl. Acad. Sci. U.S.A. 2020, 117, 33246-33253, 10.1073/pnas.2017062117

We describe the de novo design of an allosterically regulated protein, which comprises two tightly coupled domains. One domain is based on the DF (Due Ferri in Italian or two-iron in English) family of de novo proteins, which have a diiron cofactor that catalyzes a phenol oxidase reaction, while the second domain is based on PS1 (Porphyrin-binding Sequence), which binds a synthetic Zn-porphyrin (ZnP). The binding of ZnP to the original PS1 protein induces changes in structure and dynamics, which we expected to influence the catalytic rate of a fused DF domain when appropriately coupled. Both DF and PS1 are four-helix bundles, but they have distinct bundle architectures. To achieve tight coupling between the domains, they were connected by four helical linkers using a computational method to discover the most designable connections capable of spanning the two architectures. The resulting protein, DFP1 (Due Ferri Porphyrin), bound the two cofactors in the expected manner. The crystal structure of fully reconstituted DFP1 was also in excellent agreement with the design, and it showed the ZnP cofactor bound over 12 Å from the dimetal center. Next, a substrate-binding cleft leading to the diiron center was introduced into DFP1. The resulting protein acts as an allosterically modulated phenol oxidase. Its Michaelis–Menten parameters were strongly affected by the binding of ZnP, resulting in a fourfold tighter Km and a 7-fold decrease in kcat. These studies establish the feasibility of designing allosterically regulated catalytic proteins, entirely from scratch.


Metal: Fe; Zn
Ligand type: Amino acid
Host protein: Due Ferri
Anchoring strategy: Amino acid
Optimization: ---
Max TON: 10
ee: ---
PDB: 7JH6
Notes: diFe-DFP3: Km 2.9 mM, kcat 0.7 min-1, 10 turnovers for 1 mM substrate, 20 uM protein. On binding ZnP, Km decreased 4x, and kcat decreased 7x, resulting in a lower kcat/Km overall.

A Mechanistic Rationale Approach Revealed the Unexpected Chemoselectivity of an Artificial Ru-Dependent Oxidase: A Dual Experimental/Theoretical Approach

Marchi-Delapierre, C.

ACS Catal. 2020, 10, 5631-5645, 10.1021/acscatal.9b04904

Artificial enzymes represent an attractive alternative to design abiotic biocatalysis. EcNikA-Ru1, an artificial metalloenzyme developed by embedding a ruthenium-based catalyst into the cavity of the periplasmic nickel-binding protein NikA, was found to efficiently and selectively transform certain alkenes. The objective of this study was to provide a rationale on the enzymatic function and the unexpected substrate-dependent chemoselectivity of EcNikA-Ru1 thanks to a dual experimental/computational study. We observed that the de novo active site allows the formation of the terminal oxidant via the formation of a ruthenium aquo species that subsequently reacts with the hypervalent iodine of phenyl iodide diacetic acid. The oxidation process relies on a RuIV═O pathway via a two-step reaction with a radical intermediate, resulting in the formation of either a chlorohydrin or an epoxide. The results emphasize the impact of the protein scaffold on the kinetics of the reaction, through (i) the promotion of the starting oxidizing species via the exchange of a CO ligand with a water molecule; and (ii) the control of the substrate orientation on the intermediate structures, formed after the RuIV═O attack. When a Cα attack is preferred, chlorohydrins are formed while an attack on Cβ leads to an epoxide. This work provides evidence that artificial enzymes mimic the behavior of their natural counterparts.


Metal: Ru
Ligand type: Pyrazole
Host protein: NikA
Anchoring strategy: Hydrogen bond
Max TON: 175
ee: ---
PDB: 6R4Q
Notes: ---

An Artificial Hemoprotein with Inducible Peroxidase‐ and Monooxygenase‐Like Activities

Ricoux, R.

Chem. Eur. J. 2020, 26, 14929-14937, 10.1002/chem.202002434

A novel inducible artificial metalloenzyme obtained by covalent attachment of a manganese(III)-tetraphenylporphyrin (MnTPP) to the artificial bidomain repeat protein, (A3A3′)Y26C, is reported. The protein is part of the αRep family. The biohybrid was fully characterized by MALDI-ToF mass spectrometry, circular dichroism and UV/Vis spectroscopies. The peroxidase and monooxygenase activities were evaluated on the original and modified scaffolds including those that have a) an additional imidazole, b) a specific αRep bA3-2 that is known to induce the opening of the (A3A3′) interdomain region and c) a derivative of the αRep bA3-2 inducer extended with a His6-Tag (His6-bA3-2). Catalytic profiles are highly dependent on the presence of co-catalysts with the best activity obtained with His6-bA3-2. The entire mechanism was rationalized by an integrative molecular modeling study that includes protein–ligand docking and large-scale molecular dynamics. This constitutes the first example of an entirely artificial metalloenzyme with inducible peroxidase and monooxygenase activities, reminiscent of allosteric regulation of natural enzymatic pathways.


Metal: Mn
Ligand type: Porphyrin
Anchoring strategy: Covalent
Optimization: ---
Reaction: Peroxidation
Max TON: ---
ee: ---
PDB: ---
Notes: ---

An Artificial Metalloenzyme for Catalytic Cancer-Specific DNA Cleavage and Operando Imaging

Gao, X.; Zhao, L.

Sci. Adv. 2020, 6, 10.1126/sciadv.abb1421

Metalloenzymes are promising anticancer candidates to overcome chemoresistance by involving unique mechanisms. To date, it is still a great challenge to obtain synthetic metalloenzymes with persistent catalytic performance for cancer-specific DNA cleavage and operando imaging. Here, an artificial metalloenzyme, copper cluster firmly anchored in bovine serum albumin conjugated with tumor-targeting peptide, is exquisitely constructed. It is capable of persistently transforming hydrogen peroxide in tumor microenvironment to hydroxyl radical and oxygen in a catalytic manner. The stable catalysis recycling stems from the electron transfer between copper cluster and substrate with well-matched energy levels. Notably, their high biocompatibility, tumor-specific recognition, and persistent catalytic performance ensure the substantial anticancer efficacy by triggering DNA damage. Meanwhile, by coupling with enzyme-like reactions, the operando therapy effect is expediently traced by chemiluminescence signal with high sensitivity and sustainability. It provides new insights into synthesizing biocompatible metalloenzymes on demand to visually monitor and efficiently combat specific cancers.


Metal: Cu
Ligand type: Copper cluster
Anchoring strategy: Dative
Optimization: Chemical
Reaction: DNA cleavage
Max TON: ---
ee: ---
PDB: ---
Notes: ---

An Evolutionary Path to Altered Cofactor Specificity in a Metalloenzyme

Kehl-Fie, T.E.; Waldron, K.J.

Nat. Commun. 2020, 11, 10.1038/s41467-020-16478-0

AbstractAlmost half of all enzymes utilize a metal cofactor. However, the features that dictate the metal utilized by metalloenzymes are poorly understood, limiting our ability to manipulate these enzymes for industrial and health-associated applications. The ubiquitous iron/manganese superoxide dismutase (SOD) family exemplifies this deficit, as the specific metal used by any family member cannot be predicted. Biochemical, structural and paramagnetic analysis of two evolutionarily related SODs with different metal specificity produced by the pathogenic bacterium Staphylococcus aureus identifies two positions that control metal specificity. These residues make no direct contacts with the metal-coordinating ligands but control the metal’s redox properties, demonstrating that subtle architectural changes can dramatically alter metal utilization. Introducing these mutations into S. aureus alters the ability of the bacterium to resist superoxide stress when metal starved by the host, revealing that small changes in metal-dependent activity can drive the evolution of metalloenzymes with new cofactor specificity.


Metal: Fe; Mn
Ligand type: Amino acid
Anchoring strategy: Dative
Optimization: Genetic
Reaction: Metal substitution
Max TON: ---
ee: ---
PDB: ---
Notes: PDB: 6EX3, 6EX4, 6EX5, 6QV8, 6QV9

A Palladium-Catalyst Stabilized in the Chiral Environment of a Monoclonal Antibody in Water

Arada, H.; Yamaguchi, H.

Chem. Commun. 2020, 56, 1605-1607, 10.1039/c9cc08756g

We report the first preparation of a monoclonal antibody (mAb) that can immobilize a palladium (Pd)-complex. The allylic amination reaction using a supramolecular catalyst of the Pd-complex with mAb selectively gives the (R)-enantiomer product.


Metal: Pd
Ligand type: Allyl; Phosphine
Host protein: Antibody
Anchoring strategy: Supramolecular
Optimization: ---
Reaction: Allylic amination
Max TON: 600
ee: 98
PDB: ---
Notes: Recalculated TON

A Positive Charge in the Outer Coordination Sphere of an Artificial Enzyme Increases CO2 Hydrogenation

Shaw, W.J.

Organometallics 2020, 39, 1532-1544, 10.1021/acs.organomet.9b00843

The protein scaffold around the active site of enzymes is known to influence catalytic activity, but specific scaffold features responsible for favorable influences are often not known. This study focuses on using an artificial metalloenzyme to probe one specific feature of the scaffold, the position of a positive charge in the outer coordination sphere around the active site. Previous work showed that a small molecular complex, [Rh(PEt2NglycinePEt2)2]−, immobilized covalently within a protein scaffold was activated for CO2 hydrogenation. Here, using an iterative design where the effect of arginine, histidine, or lysine residues placed in the outer coordination sphere of the catalytic active site were evaluated, we tested the hypothesis that positively charged groups facilitate CO2 hydrogenation with seven unique constructs. Single-, double-, and triple-point mutations were introduced to directly compare catalytic activity, as monitored by turnover frequencies (TOFs) measured in real time with 1H NMR spectroscopy, and evaluate related structural and electronic properties. Two of the seven constructs showed a 2- and 3-fold increase relative to the wild type, with overall rates ranging from 0.2 to 0.7 h–1, and a crystal structure of the fastest of these shows the positive charge positioned next to the active site. A crystal structure of the arginine-containing complex shows that the arginines are positioned near the metal. Molecular dynamics (MD) studies also suggest that the positive charge is oriented next to the active site in the two constructs with faster rates but not in the others and that the positive charge near the active site holds the CO2 near the metal, all consistent with a positive charge appropriately positioned in the scaffold benefiting catalysis. The MD studies also suggest that changes in the water distribution around the active site may contribute to catalytic activity, while modest structural changes and movement of the complex within the scaffold do not.


Metal: Rh
Ligand type: Bisdiphosphine
Anchoring strategy: Covalent
Reaction: Hydrogenation
Max TON: 33
ee: ---
PDB: 6VWE
Notes: ---

Artificial Imine Reductases: Developments and Future Directions

Review

Duhme-Klair, A.K.

RSC Chem. Biol. 2020, 1, 369-378, 10.1039/d0cb00113a

Biocatalytic imine reduction has been a topic of intense research by the artificial metalloenzyme community in recent years. Artificial constructs, together with natural enzymes, have been engineered to produce chiral amines with high enantioselectivity. This review examines the design of the main classes of artificial imine reductases reported thus far and summarises approaches to enhancing their catalytic performance using complementary methods. Examples of utilising these biocatalysts in vivo or in multi-enzyme cascades have demonstrated the potential that artIREDs can offer, however, at this time their use in biocatalysis remains limited. This review explores the current scope of artIREDs and the strategies used for catalyst improvement, and examines the potential for artIREDs in the future.


Notes: ---

Artificial Iron Hydrogenase Made by Covalent Grafting of Knölker's Complex into Xylanase: Application in Asymmetric Hydrogenation of an Aryl Ketone in Water

Mahy, J.-P.

Biotechnol. Appl. Biochem. 2020, 67, 563-573, 10.1002/bab.1906

We report a new artificial hydrogenase made by covalent anchoring of the iron Knölker's complex to a xylanase S212C variant. This artificial metalloenzyme was found to be able to catalyze efficiently the transfer hydrogenation of the benchmark substrate trifluoroacetophenone by sodium formate in water, yielding the corresponding secondary alcohol as a racemic. The reaction proceeded more than threefold faster with the XlnS212CK biohybrid than with the Knölker's complex alone. In addition, efficient conversion of trifluoroacetophenone to its corresponding alcohol was reached within 60 H with XlnS212CK, whereas a ≈2.5-fold lower conversion was observed with Knölker's complex alone as catalyst. Moreover, the data were rationalized with a computational strategy suggesting the key factors of the selectivity. These results suggested that the Knölker's complex was most likely flexible and could experience free rotational reorientation within the active-site pocket of Xln A, allowing it to access the subsite pocket populated by trifluoroacetophenone.


Metal: Fe
Ligand type: Cyclopentadienyl
Host protein: Xylanase A (XynA)
Anchoring strategy: Covalent
Optimization: ---
Max TON: 9
ee: ---
PDB: ---
Notes: ---

Artificial Metalloenzymes based on TetR Proteins and Cu(II) for Enantioselective Friedel‐Crafts Alkylation Reactions

Roelfes, G.

ChemCatChem 2020, 12, 3190-3194, 10.1002/cctc.202000245

The supramolecular approach is among the most convenient methodologies for creating artificial metalloenzymes (ArMs). Usually this approach involves the binding of a transition metal ion complex to a biomolecular scaffold via its ligand, which also modulates the catalytic properties of the metal ion. Herein, we report ArMs based on the proteins CgmR, RamR and QacR from the TetR family of multidrug resistance regulators (MDRs) and Cu2+ ions, assembled without the need of a ligand. These ArMs catalyze the enantioselective vinylogous Friedel-Crafts alkylation reaction with up to 75 % ee. Competition experiments with ethidium and rhodamine 6G confirm that the reactions occur in the chiral environment of the hydrophobic pocket. It is proposed that the Cu2+-substrate complex is bound via a combination of electrostatic and π-stacking interactions provided by the second coordination sphere. This approach constitutes a fast and straightforward way to assemble metalloenzymes and may facilitate future optimization of the protein scaffolds via mutagenesis or directed evolution approaches.


Metal: Cu
Ligand type: Amino acid
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Max TON: 78
ee: 75
PDB: 1JTY
Notes: ---

Catalysis and Electron Transfer in De Novo Designed Helical Scaffolds

Review

Pecoraro, V.L.

Angew. Chem. Int. Ed. 2020, 59, 7678-7699, 10.1002/anie.201907502

The relationship between protein structure and function is one of the greatest puzzles within biochemistry. De novo metalloprotein design is a way to wipe the board clean and determine what is required to build in function from the ground up in an unrelated structure. This Review focuses on protein design efforts to create de novo metalloproteins within alpha-helical scaffolds. Examples of successful designs include those with carbonic anhydrase or nitrite reductase activity by incorporating a ZnHis3 or CuHis3 site, or that recapitulate the spectroscopic properties of unique electron-transfer sites in cupredoxins (CuHis2Cys) or rubredoxins (FeCys4). This work showcases the versatility of alpha helices as scaffolds for metalloprotein design and the progress that is possible through careful rational design. Our studies cover the invariance of carbonic anhydrase activity with different site positions and scaffolds, refinement of our cupredoxin models, and enhancement of nitrite reductase activity up to 1000-fold.


Notes: ---

Constructing Protein Polyhedra via Orthogonal Chemical Interactions

Tezcan, F.A.

Nature 2020, 578, 172-176, 10.1038/s41586-019-1928-2

Many proteins exist naturally as symmetrical homooligomers or homopolymers1. The emergent structural and functional properties of such protein assemblies have inspired extensive efforts in biomolecular design2,3,4,5. As synthesized by ribosomes, proteins are inherently asymmetric. Thus, they must acquire multiple surface patches that selectively associate to generate the different symmetry elements needed to form higher-order architectures1,6—a daunting task for protein design. Here we address this problem using an inorganic chemical approach, whereby multiple modes of protein–protein interactions and symmetry are simultaneously achieved by selective, ‘one-pot’ coordination of soft and hard metal ions. We show that a monomeric protein (protomer) appropriately modified with biologically inspired hydroxamate groups and zinc-binding motifs assembles through concurrent Fe3+ and Zn2+ coordination into discrete dodecameric and hexameric cages. Our cages closely resemble natural polyhedral protein architectures7,8 and are, to our knowledge, unique among designed systems9,10,11,12,13 in that they possess tightly packed shells devoid of large apertures. At the same time, they can assemble and disassemble in response to diverse stimuli, owing to their heterobimetallic construction on minimal interprotein-bonding footprints. With stoichiometries ranging from [2 Fe:9 Zn:6 protomers] to [8 Fe:21 Zn:12 protomers], these protein cages represent some of the compositionally most complex protein assemblies—or inorganic coordination complexes—obtained by design.


Metal: Fe; Zn
Ligand type: Hydroxaamate
Host protein: Cytochrome cb562
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Reaction: ---
Max TON: ---
ee: ---
PDB: BMC2
Notes: ---

Contributions of primary coordination ligands and importance of outer sphere interactions in UFsc, a de novo designed protein with high affinity for metal ions

Makhlynets, O.V.

J. Inorg. Biochem. 2020, 212, 111224, 10.1016/j.jinorgbio.2020.111224

Metalloproteins constitute nearly half of all proteins and catalyze some of the most complex chemical reactions. Recently, we reported a design of 4G-UFsc (Uno Ferro single chain), a single chain four-helical bundle with extraordinarily high (30 pM) affinity for zinc. We evaluated the contribution of different side chains to binding of Co(II), Ni(II), Zn(II) and Mn(II) using systematic mutagenesis of the amino acids that constitute the primary metal coordination and outer spheres. The binding affinity of proteins for metals was then measured using isothermal titration calorimetry. Our results show that both primary metal coordination environment and side chains in the outer sphere of UFsc are highly sensitive to even slight changes and can be adapted to binding different 3d metals, including hard-to-tightly bind metal ions such as Mn(II). The studies on the origins of tight metal binding will guide future metalloprotein design efforts.


Metal: Co; Mn; Ni; Zn
Ligand type: Amino acid
Anchoring strategy: Dative
Optimization: Genetic
Reaction: ---
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Cupin Variants as a Macromolecular Ligand Library for Stereoselective Michael Addition of Nitroalkanes

Fujieda, N.; Itoh, S.

Angew. Chem. 2020, 132, 7791-7794, 10.1002/ange.202000129

Cupin superfamily proteins (TM1459) work as a macromolecular ligand framework with a double-stranded β-barrel structure ligating to a Cu ion through histidine side chains. Variegating the first coordination sphere of TM1459 revealed that H52A and H54A/H58A mutants effectively catalyzed the diastereo- and enantioselective Michael addition reaction of nitroalkanes to an α,β-unsaturated ketone. Moreover, calculated substrate docking signified C106N and F104W single-point mutations, which inverted the diastereoselectivity of H52A and further improved the stereoselectivity of H54A/H58A, respectively.


Metal: Cu
Ligand type: Amino acid
Anchoring strategy: Dative
Optimization: Chemical & genetic
Reaction: Michael addition
Max TON: 250
ee: 99
PDB: 6L2D
Notes: ---

Design and Evaluation of Artificial Hybrid Photoredox Biocatalysts

Brustad, E.M.; Nicewicz, D.A.

ChemBioChem 2020, 21, 3146-3150, 10.1002/cbic.202000362

A pair of 9-mesityl-10-phenyl acridinium (Mes−Acr+) photoredox catalysts were synthesized with an iodoacetamide handle for cysteine bioconjugation. Covalently tethering of the synthetic Mes−Acr+ cofactors with a small panel of thermostable protein scaffolds resulted in 12 new artificial enzymes. The unique chemical and structural environment of the protein hosts had a measurable effect on the photophysical properties and photocatalytic activity of the cofactors. The constructed Mes−Acr+ hybrid enzymes were found to be active photoinduced electron-transfer catalysts, controllably oxidizing a variety of aryl sulfides when irradiated with visible light, and possessed activities that correlated with the photophysical characterization data. Their catalytic performance was found to depend on multiple factors including the Mes−Acr+ cofactor, the protein scaffold, the location of cofactor immobilization, and the substrate. This work provides a framework toward adapting synthetic photoredox catalysts into artificial cofactors and includes important considerations for future bioengineering efforts.


Metal: ---
Host protein: Aspertate dehydrogenase
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Max TON: ---
ee: ---
PDB: ---
Notes: Maximum conversion is 95%; In most cases, a comparable yield or modest increase in yield was observed for the protein-bound catalyst compared to the unbound cofactor.

Metal: ---
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Max TON: ---
ee: ---
PDB: ---
Notes: Maximum conversion is 95%; In most cases, a comparable yield or modest increase in yield was observed for the protein-bound catalyst compared to the unbound cofactor.

Metal: ---
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Max TON: ---
ee: ---
PDB: ---
Notes: Maximum conversion is 95%; In most cases, a comparable yield or modest increase in yield was observed for the protein-bound catalyst compared to the unbound cofactor.

DNA‐Based Asymmetric Inverse Electron‐Demand Hetero‐Diels–Alder

Arseniyadis, S.; Campagne, J.; Smietana, M.

Chem. Eur. J. 2020, 26, 3519-3523, 10.1002/chem.202000516

While artificial cyclases hold great promise in chemical synthesis, this work presents the first example of a DNA-catalyzed inverse electron-demand hetero-Diels–Alder (IEDHDA) between dihydrofuran and various α,β-unsaturated acyl imidazoles. The resulting fused bicyclic O,O-acetals containing three contiguous stereogenic centers are obtained in high yields (up to 99 %) and excellent diastereo- (up to >99:1 dr) and enantioselectivities (up to 95 % ee) using a low catalyst loading. Most importantly, these results show that the concept of DNA-based asymmetric catalysis can be expanded to new synthetic transformations offering an efficient, sustainable, and highly selective tool for the construction of chiral building blocks.


Metal: Cu
Ligand type: Cu(dmbipy)(NO3)2
Host protein: DNA
Anchoring strategy: Supramolecular
Optimization: Chemical
Max TON: 3.33
ee: 95
PDB: ---
Notes: ---

Electrochemical Characterization of the Artificial Metalloenzyme Papain-[(η6-arene)Ru(1,10-phenanthroline)Cl]+

Hromadová, M.

J. Electroanal. Chem. 2020, 859, 113882, 10.1016/j.jelechem.2020.113882

Electrochemical properties were studied for [(η6-arene)Ru(1,10-phenanthroline)Cl]Cl (arene = C6H5(CH2)2NHCOCH2Cl) organometallic complex 1, protein Papain PAP and its conjugate with organometallic complex 1-PAP. The latter can serve as an artificial metalloenzyme with catalytic activity in transfer hydrogenation. This work demonstrates that AC voltammetry and electrochemical impedance spectroscopy can be used as fast tools to screen the catalytic ability of 1-PAP electrochemically by studies of the catalytic hydrogen evolution reaction (HER). Proteins are known to catalyze this process, but we have shown that additional HER signal associated with the catalytic activity of 1 is observed for its conjugate with Papain 1-PAP.


Metal: Ru
Ligand type: Cp*; Phenanthroline
Host protein: Papain (PAP)
Anchoring strategy: Covalent
Optimization: ---
Reaction: H2 evolution
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Enabling Protein-Hosted Organocatalytic Transformations

Review

Luk, L.Y.P.

RSC Adv. 2020, 10, 16147-16161, 10.1039/d0ra01526a

In this review, the development of organocatalytic artificial enzymes will be discussed. This area of protein engineering research has underlying importance, as it enhances the biocompatibility of organocatalysis for applications in chemical and synthetic biology research whilst expanding the catalytic repertoire of enzymes. The approaches towards the preparation of organocatalytic artificial enzymes, techniques used to improve their performance (selectivity and reactivity) as well as examples of their applications are presented. Challenges and opportunities are also discussed.


Notes: ---

Enantioselective Olefin Cyclopropanation with G-Quadruplex DNA-Based Biocatalysts

Li, C.

ACS Catal. 2020, 10, 6561-6567, 10.1021/acscatal.0c01203

Developing high-performance DNA-based biocatalysts for desired stereoselective syntheses remains a formidable challenge. Here, we report promising DNA-based catalysts comprised of G-quadruplex (G4) and Fe porphyrin for asymmetric olefin cyclopropanation. After the G4-based catalysts are optimized by several rounds of site mutation, their catalytic enantioselectivities achieve +81% and −86% enantiomeric excess (eetrans) at a turnover number (TON) as high as 500. The Fe porphyrin, binding upon the 5′,3′-end G-quartet, constitutes the active center for olefin cyclopropanation via an iron porphyrin carbene intermediate. The findings provide an opportunity for generating high-value chiral cyclopropane blocks via G4 biocatalysts and shed light on the potential of DNA as protein enzymes for catalysis.


Metal: Fe
Ligand type: Porphyrin
Host protein: DNA
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Cyclopropanation
Max TON: 500
ee: 86
PDB: ---
Notes: ---

Enhanced Photocatalytic Hydrogen Production by Hybrid Streptavidin‐Diiron Catalysts

Chem. Eur. J. 2020, 26, 6240-6246, 10.1002/chem.202000204

Hybrid protein–organometallic catalysts are being explored for selective catalysis of a number of reactions, because they utilize the complementary strengths of proteins and of organometallic complex. Herein, we present an artificial hydrogenase, StrepH2, built by incorporating a biotinylated [Fe–Fe] hydrogenase organometallic mimic within streptavidin. This strategy takes advantage of the remarkable strength and specificity of biotin-streptavidin recognition, which drives quantitative incorporation of the biotinylated diironhexacarbonyl center into streptavidin, as confirmed by UV/Vis spectroscopy and X-ray crystallography. FTIR spectra of StrepH2 show characteristic peaks at shift values indicative of interactions between the catalyst and the protein scaffold. StrepH2 catalyzes proton reduction to hydrogen in aqueous media during photo- and electrocatalysis. Under photocatalytic conditions, the protein-embedded catalyst shows enhanced efficiency and prolonged activity compared to the isolated catalyst. Transient absorption spectroscopy data suggest a mechanism for the observed increase in activity underpinned by an observed longer lifetime for the catalytic species FeIFe0 when incorporated within streptavidin compared to the biotinylated catalyst in solution.


Metal: Fe
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: ---
Reaction: H2 evolution
Max TON: 47.63 (± 3.16)
ee: ---
PDB: 5VCQ
Notes: Photocatalytic activity, expressed as TON, for ArM is about 8 times higher than that of the biotinylated cofactor.The increase in TON is largely due to increased lifetime of the catalytically competent intermediate, FeIFe0 core when embeded inside streptavidin.

Heteromeric Three-Stranded Coiled Coils Designed Using a Pb(ii)(Cys)3 Template Mediated Strategy

Pecoraro, V.L.

Nat. Chem. 2020, 12, 405-411, 10.1038/s41557-020-0423-6

Three-stranded coiled coils are peptide structures constructed from amphipathic heptad repeats. Here we show that it is possible to form pure heterotrimeric three-stranded coiled coils by combining three distinct characteristics: (1) a cysteine sulfur layer for metal coordination, (2) a thiophilic, trigonal pyramidal metalloid (Pb(ii)) that binds to these sulfurs and (3) an adjacent layer of reduced steric bulk generating a cavity where water can hydrogen bond to the cysteine sulfur atoms. Cysteine substitution in an a site yields Pb(ii)A2B heterotrimers, while d sites provide pure Pb(ii)C2D or Pb(ii)CD2 scaffolds. Altering the metal from Pb(ii) to Hg(ii) or shifting the relative position of the sterically less demanding layer removes heterotrimer specificity. Because only two of the eight or ten hydrophobic layers are perturbed, catalytic sites can be introduced at other regions of the scaffold. A Zn(ii)(histidine)3(H2O) centre can be incorporated at a remote location without perturbing the heterotrimer selectivity, suggesting a unique strategy to prepare dissymmetric catalytic sites within self-assembling de novo-designed proteins.


Metal: Pb; Zn
Ligand type: Amino acid
Host protein: De novo-designed protein
Anchoring strategy: ---
Optimization: ---
Reaction: Ester hydrolysis
Max TON: ---
ee: ---
PDB: ---
Notes: PDB: 6EGP, 6MCD

Highly Efficient Cyclic Dinucleotide Based Artificial Metalloribozymes for Enantioselective Friedel–Crafts Reactions in Water

Chen, Y.; Wang, C.

Angew. Chem. Int. Ed. 2020, 59, 3444-3449, 10.1002/anie.201912962

The diverse secondary structures of nucleic acids are emerging as attractive chiral scaffolds to construct artificial metalloenzymes (ArMs) for enantioselective catalysis. DNA‐based ArMs containing duplex and G‐quadruplex scaffolds have been widely investigated, yet RNA‐based ArMs are scarce. Here we report that a cyclic dinucleotide of c‐di‐AMP and Cu2+ ions assemble into an artificial metalloribozyme (c‐di‐AMP⋅Cu2+) that enables catalysis of enantioselective Friedel–Crafts reactions in aqueous media with high reactivity and excellent enantioselectivity of up to 97 % ee. The assembly of c‐di‐AMP⋅Cu2+ gives rise to a 20‐fold rate acceleration compared to Cu2+ ions. Based on various biophysical techniques and density function theory (DFT) calculations, a fine coordination structure of c‐di‐AMP⋅Cu2+ metalloribozyme is suggested in which two c‐di‐AMP form a dimer scaffold and the Cu2+ ion is located in the center of an adenine‐adenine plane through binding to two N7 nitrogen atoms and one phosphate oxygen atom.


Metal: Cu
Ligand type: RNA
Host protein: RNA
Anchoring strategy: Dative
Optimization: Chemical
Max TON: 20
ee: 97
PDB: ---
Notes: ---

Intracellular Reactions Promoted by Bis(histidine) Miniproteins Stapled Using Palladium(II) Complexes

Mascareñas, J.L.

Angew. Chem. Int. Ed. 2020, 59, 9149-9154, 10.1002/anie.202002032

The generation of catalytically active metalloproteins inside living mammalian cells is a major research challenge at the interface between catalysis and cell biology. Herein we demonstrate that basic domains of bZIP transcription factors, mutated to include two histidine residues at i and i+4 positions, react with palladium(II) sources to generate catalytically active, stapled pallado-miniproteins. The resulting constrained peptides are efficiently internalized into living mammalian cells, where they perform palladium-promoted depropargylation reactions without cellular fixation. Control experiments confirm the requirement of the peptide scaffolding and the palladium staple for attaining the intracellular reactivity.


Metal: Pd
Ligand type: Amino acid
Anchoring strategy: Dative
Optimization: Genetic
Reaction: Depropargylation
Max TON: ---
ee: ---
PDB: ---
Notes: Whole cell catalysis

Iron-porphyrin Catalyzed Carbene Transfer Reactions – an Evolution fro Biomimetic Catalysis towards Chemistry-inspired Non-natural Reactivities of Enzymes

Koenigs, R.M.; Weissenborn, M.J.

ChemCatChem 2020, 10.1002/cctc.201901565

Bioinspired, synthetic porphyrin complexes are important catalysts in organic synthesis and play a pivotal role in efficient carbene transfer reactions. The advances in this research area stimulated recent, “chemo‐inspired” developments in biocatalysis. Today, both synthetic iron complexes and enzymes play an important role to conduct carbene transfer reactions. The advances and potential developments in both research areas are discussed in this concept article.


Metal: Fe
Ligand type: Porphyrin
Host protein: ---
Anchoring strategy: ---
Optimization: Chemical & genetic
Reaction: Carbene insertion
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Key Structural Motifs Balance Metal Binding and Oxidative Reactivity in a Heterobimetallic Mn/Fe Protein

Shafaat, H.S.

J. Am. Chem. Soc. 2020, 142, 5338-5354, 10.1021/jacs.0c00333

Heterobimetallic Mn/Fe proteins represent a new cofactor paradigm in bioinorganic chemistry and pose countless outstanding questions. The assembly of the active site defies common chemical convention by contradicting the Irving–Williams series, while the scope of reactivity remains unexplored. In this work, the assembly and C–H bond activation process in the Mn/Fe R2-like ligand-binding oxidase (R2lox) protein is investigated using a suite of biophysical techniques, including time-resolved optical spectroscopy, global kinetic modeling, X-ray crystallography, electron paramagnetic resonance spectroscopy, protein electrochemistry, and mass spectrometry. Selective metal binding is found to be under thermodynamic control, with the binding sites within the apo-protein exhibiting greater MnII affinity than FeII affinity. The comprehensive analysis of structure and reactivity of wild-type R2lox and targeted primary and secondary sphere mutants indicate that the efficiency of C–H bond activation directly correlates with the Mn/Fe cofactor reduction potentials and is inversely related to divalent metal binding affinity. These findings suggest the R2lox active site is precisely tuned for achieving both selective heterobimetallic binding and high levels of reactivity and offer a mechanism to examine the means by which proteins achieve appropriate metal incorporation.


Metal: Fe; Mn
Ligand type: Amino acid
Anchoring strategy: Metal substitution
Optimization: ---
Reaction: C-H activation
Max TON: ---
ee: ---
PDB: ---
Notes: PDB: 6QK0, 6QJV, 6QK2, 6QK1

Metal-Mediated Protein Assembly Using a Genetically Incorporated Metal-Chelating Amino Acid

Kim, H.M.; Lee, H.S.

Biomacromolecules 2020, 21, 5021-5028, 10.1021/acs.biomac.0c01194

Many natural proteins function in oligomeric forms, which are critical for their sophisticated functions. The construction of protein assemblies has great potential for biosensors, enzyme catalysis, and biomedical applications. In designing protein assemblies, a critical process is to create protein–protein interaction (PPI) networks at defined sites of a target protein. Although a few methods are available for this purpose, most of them are dependent on existing PPIs of natural proteins to some extent. In this report, a metal-chelating amino acid, 2,2′-bipyridylalanine (BPA), was genetically introduced into defined sites of a monomeric protein and used to form protein oligomers. Depending on the number of BPAs introduced into the protein and the species of metal ions (Ni2+ and Cu2+), dimers or oligomers with different oligomerization patterns were formed by complexation with a metal ion. Oligomer sizes could also be controlled by incorporating two BPAs at different locations with varied angles to the center of the protein. When three BPAs were introduced, the monomeric protein formed a large complex with Ni2+. In addition, when Cu2+ was used for complex formation with the protein containing two BPAs, a linear complex was formed. The method proposed in this report is technically simple and generally applicable to various proteins with interesting functions. Therefore, this method would be useful for the design and construction of functional protein assemblies.


Metal: Cu; Ni
Ligand type: Bipyridine
Anchoring strategy: Dative
Optimization: ---
Reaction: ---
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Methane Generation and Reductive Debromination of Benzylic Position by Reconstituted Myoglobin Containing Nickel Tetradehydrocorrin as a Model of Methyl-coenzyme M Reductase

Hayashi, T; Oohora, K.

Inorg. Chem. 2020, 59, 11995-12004, 10.1021/acs.inorgchem.0c00901

Methyl-coenzyme M reductase (MCR), which contains the nickel hydrocorphinoid cofactor F430, is responsible for biological methane generation under anaerobic conditions via a reaction mechanism which has not been completely elucidated. In this work, myoglobin reconstituted with an artificial cofactor, nickel(I) tetradehydrocorrin (NiI(TDHC)), is used as a protein-based functional model for MCR. The reconstituted protein, rMb(NiI(TDHC)), is found to react with methyl donors such as methyl p-toluenesulfonate and trimethylsulfonium iodide with methane evolution observed in aqueous media containing dithionite. Moreover, rMb(NiI(TDHC)) is found to convert benzyl bromide derivatives to reductively debrominated products without homocoupling products. The reactivity increases in the order of primary > secondary > tertiary benzylic carbons, indicating steric effects on the reaction of the nickel center with the benzylic carbon in the initial step. In addition, Hammett plots using a series of para-substituted benzyl bromides exhibit enhancement of the reactivity with introduction of electron-withdrawing substituents, as shown by the positive slope against polar substituent constants. These results suggest a nucleophilic SN2-type reaction of the Ni(I) species with the benzylic carbon to provide an organonickel species as an intermediate. The reaction in D2O buffer at pD 7.0 causes a complete isotope shift of the product by +1 mass unit, supporting our proposal that protonation of the organonickel intermediate occurs during product formation. Although the turnover numbers are limited due to inactivation of the cofactor by side reactions, the present findings will contribute to elucidating the reaction mechanism of MCR-catalyzed methane generation from activated methyl sources and dehalogenation.


Metal: Ni
Ligand type: Tetradehydrocorrin
Host protein: Myoglobin (Mb)
Anchoring strategy: Supramolecular
Optimization: Chemical
Reaction: Methane generation
Max TON: 1.61
ee: ---
PDB: ---
Notes: ---

Metal: Co
Ligand type: Tetradehydrocorrin
Host protein: Myoglobin (Mb)
Anchoring strategy: Supramolecular
Optimization: Chemical
Max TON: 0.25
ee: ---
PDB: ---
Notes: ---

Molecular Design and Regulation of Metalloenzyme Activities through Two Novel Approaches: Ferritin and P450s

Review

Watanabe, Y.

BCSJ 2020, 93, 379-392, 10.1246/bcsj.20190305

We have developed two novel approaches for the construction of artificial metalloenzymes showing either unique catalytic activities or substrate specificity. The first example is the use of a hollow cage of apo-ferritin as a reaction vessel for hydrogenation of olefins, Suzuki-Miyaura C-C coupling and phenylacetylene polymerization by employing Pd0 nano-clusters, Pd2+(η3-C3H5) complexes and Rh1+(nbd) (nbd = norbornadiene) complexes introduced in the hollow cage, respectively. The second approach is the use of “decoy molecules” to change substrate specificity of P450s, allowing epoxidation and hydroxylation activities toward nonnative organic substrates in P450SPα, P450BSβ and P450BM3 without the mutation of any amino acid. Finally, the decoy strategy has been applied to an in vivo system of P450, i.e., the use of P450BM3 expressed in the whole cell of E. coli to oxidize benzene to phenol.


Notes: ---

Molecular Modeling for Artificial Metalloenzyme Design and Optimization

Review

Maréchal, J.-D.

Acc. Chem. Res. 2020, 53, 896-905, 10.1021/acs.accounts.0c00031

Artificial metalloenzymes (ArMs) are obtained by inserting homogeneous catalysts into biological scaffolds and are among the most promising strategies in the quest for new-to-nature biocatalysts. The quality of their design strongly depends on how three partners interact: the biological host, the “artificial cofactor,” and the substrate. However, structural characterization of functional artificial metalloenzymes by X-ray or NMR is often partial, elusive, or absent. How the cofactor binds to the protein, how the receptor reorganizes upon the binding of the cofactor and the substrate, and which are the binding mode(s) of the substrate for the reaction to proceed are key questions that are frequently unresolved yet crucial for ArM design. Such questions may eventually be solved by molecular modeling but require a step change beyond the current state-of-the-art methodologies. Here, we summarize our efforts in the study of ArMs, presenting both the development of computational strategies and their application. We first focus on our integrative computational framework that incorporates a variety of methods such as protein–ligand docking, classical molecular dynamics (MD), and pure quantum mechanical (QM) methods, which, when properly combined, are able to depict questions that range from host–cofactor binding predictions to simulations of entire catalytic mechanisms. We also pay particular attention to the protein–ligand docking strategies that we have developed to accurately predict the binding of transition metal-containing molecules to proteins. While this aspect is fundamental to many bioinorganic fields beyond ArMs, it has been disregarded from the molecular modeling landscape until very recently. Next we describe how to apply this computational framework to particular ArMs including systems previously characterized experimentally as well as others where computation served to guide the design. We start with the prediction of the interactions between homogeneous catalysts and biological hosts. Protein–ligand docking is pivotal at that stage, but it needs to be combined with QM/MM or MD approaches when the binding of the cofactor implies significant conformational changes of the protein or involve changes of the electronic state of the metal. Then, we summarize molecular modeling studies aimed at identifying cofactor–substrate arrangements inside the ArM active pocket that are consistent with its reactivity. These calculations stand on “Theozyme”-like dockings, MD-refined or not, which provide molecular rationale of the catalytic profiles of the artificial systems. In the third section, we present case studies to decode the entire catalytic mechanism of two ArMs: (1) an iridium based asymmetric transfer hydrogenase obtained by insertion of Noyori’s catalyst into streptavidin and (2) a metallohydrolase achieved by including a receptor. Transition states, second coordination sphere effects, as well as motions of the cofactors are identified as drivers of the enantiomeric profiles. Finally, we report computer-aided designs of ArMs to guide experiments toward chemical and mutational changes that improve their activity and/or enantioselective profiles and expand toward future directions.


Notes: ---