Fussenegger, M.; Matile, S.; Ward, T.R.

Nat. Commun. 2018, 9, 10.1038/s41467-018-04440-0

Complementing enzymes in their native environment with either homogeneous or heterogeneous catalysts is challenging due to the sea of functionalities present within a cell. To supplement these efforts, artificial metalloenzymes are drawing attention as they combine attractive features of both homogeneous catalysts and enzymes. Herein we show that such hybrid catalysts consisting of a metal cofactor, a cell-penetrating module, and a protein scaffold are taken up into HEK-293T cells where they catalyze the uncaging of a hormone. This bioorthogonal reaction causes the upregulation of a gene circuit, which in turn leads to the expression of a nanoluc-luciferase. Relying on the biotin–streptavidin technology, variation of the biotinylated ruthenium complex: the biotinylated cell-penetrating poly(disulfide) ratio can be combined with point mutations on streptavidin to optimize the catalytic uncaging of an allyl-carbamate-protected thyroid hormone triiodothyronine. These results demonstrate that artificial metalloenzymes offer highly modular tools to perform bioorthogonal catalysis in live HEK cells.

Green, A.P.; Hilvert, D.

J. Am. Chem. Soc. 2018, 140, 1535-1543, 10.1021/jacs.7b12621

Expanding the range of genetically encoded metal coordination environments accessible within tunable protein scaffolds presents excellent opportunities for the creation of metalloenzymes with augmented properties and novel activities. Here, we demonstrate that installation of a noncanonical Nδ-methyl histidine (NMH) as the proximal heme ligand in the oxygen binding protein myoglobin (Mb) leads to substantial increases in heme redox potential and promiscuous peroxidase activity. Structural characterization of this catalytically modified myoglobin variant (Mb NMH) revealed significant changes in the proximal pocket, including alterations to hydrogen-bonding interactions involving the prosthetic porphyrin cofactor. Further optimization of Mb NMH via a combination of rational modification and several rounds of laboratory evolution afforded efficient peroxidase biocatalysts within a globin fold, with activities comparable to those displayed by nature’s peroxidases.

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.

Onoda, A.; Salmain, M.

Bioorganometallic Chemistry: Applications in Drug Discovery, Biocatalysis, and Imaging 2014, 305-338, 10.1002/9783527673438.ch10

Enzymes are the catalysts of the living world. Nature has tailored proteins to catalyze an incredibly wide range of reactions with exquisite selectivity and efficiency under very mild conditions of temperature, pH, pressure, and so on. Protein engineering combined with molecular modeling techniques affords tailor‐made biocatalysts for the industrial production of chiral synthons. Nonetheless, endowing a given protein scaffold with a totally new activity remains a challenging task for the biochemist. Among the current strategies to impart proteins with unnatural activity, those dealing with the construction of artificial metalloenzymes are particularly promising. By definition, artificial metalloenzymes are hybrid catalysts resulting from the incorporation of a transition metal species within a biomacromolecular scaffold. The rationale behind this concept is to combine the wide catalytic scope of transition metal complexes with the high activity and selectivity of biocatalysts. In most of the hybrid catalysts reported so far, the roles devoted to both partners are clearly separated: the metal complex being responsible for reactivity, while the protein environment is used to induce selectivity in the chemical process. In that, artificial metalloenzymes truly resemble enzymes whose efficiency relies on both the active site and the second sphere of coordination (also called the outer coordination sphere). In this chapter, we intend to give an overview of the various anchoring strategies reported over the last decade for the controlled, site‐selective attachment of nonnative metal cofactors within protein matrices together with the activity/selectivity displayed by these hybrid enzymes.

Onoda, A.

J. Inorg. Biochem. 2016, 158, 55-61, 10.1016/j.jinorgbio.2015.12.026

A hybrid biocatalyst containing a metal terpyridine (tpy) complex within a rigid β-barrel protein nitrobindin (NB) is constructed. A tpy ligand with a maleimide group, N-[2-([2,2′:6′,2′′-terpyridin]-4′-yloxy)ethyl]maleimide (1), was covalently linked to Cys96 inside the cavity of NB to prepare a conjugate NB–1. Binding of Cu2 +, Zn2 +, or Co2 + ion to the tpy ligand in NB–1 was confirmed by UV–vis spectroscopy and ESI–TOF MS measurements. Cu2 +-bound NB–1 is found to catalyze a Diels–Alder reaction between azachalcone and cyclopentadiene in 22% yield, which is higher than that of the Cu2 +–tpy complex without the NB matrix. The results suggest that the hydrophobic cavity close to the copper active site within the NB scaffold supports the binding of the two substrates, dienophile and diene, to promote the reaction.

Song, W.J.

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.

Hayashi, T; Onoda, A.

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.

Green, A.P.; Hilvert, D.

Chem. - Eur. J. 2018, 24, 11821-11830, 10.1002/chem.201800975

Nature employs a limited number of genetically encoded, metal‐coordinating residues to create metalloenzymes with diverse structures and functions. Engineered components of the cellular translation machinery can now be exploited to encode non‐canonical ligands with user‐defined electronic and structural properties. This ability to install “chemically programmed” ligands into proteins can provide powerful chemical probes of metalloenzyme mechanism and presents excellent opportunities to create metalloprotein catalysts with augmented properties and novel activities. In this Concept article, we provide an overview of several recent studies describing the creation of engineered metalloenzymes with interesting catalytic properties, and reveal how characterization of these systems has advanced our understanding of nature's bioinorganic mechanisms. We also highlight how powerful laboratory evolution protocols can be readily adapted to allow optimization of metalloenzymes with non‐canonical ligands. This approach combines beneficial features of small molecule and protein catalysis by allowing the installation of a greater variety of local metal coordination environments into evolvable protein scaffolds, and holds great promise for the future creation of powerful metalloprotein catalysts for a host of synthetically valuable transformations.

Song, W.J.; Tezcan, F.A.

J. Am. Chem. Soc. 2017, 139, 16772-16779, 10.1021/jacs.7b08981

We describe the design and evolution of catalytic hydrolase activity on a supramolecular protein scaffold, Zn4:C96RIDC14, which was constructed from cytochrome cb562 building blocks via a metal-templating strategy. Previously, we reported that Zn4:C96RIDC14 could be tailored with tripodal (His/His/Glu), unsaturated Zn coordination motifs in its interfaces to generate a variant termed Zn8:A104AB34, which in turn displayed catalytic activity for the hydrolysis of activated esters and β-lactam antibiotics. Zn8:A104AB34 was subsequently subjected to directed evolution via an in vivo selection strategy, leading to a variant Zn8:A104/G57AB34 which displayed enzyme-like Michaelis–Menten behavior for ampicillin hydrolysis. A criterion for the evolutionary utility or designability of a new protein structure is its ability to accommodate different active sites. With this in mind, we examined whether Zn4:C96RIDC14 could be tailored with alternative Zn coordination sites that could similarly display evolvable catalytic activities. We report here a detailed structural and functional characterization of new variant Zn8:AB54, which houses similar, unsaturated Zn coordination sites to those in Zn8:A104/G57AB34, but in completely different microenvironments. Zn8:AB54 displays Michaelis–Menten behavior for ampicillin hydrolysis without any optimization. Yet, the subsequent directed evolution of Zn8:AB54 revealed limited catalytic improvement, which we ascribed to the local protein rigidity surrounding the Zn centers and the lack of evolvable loop structures nearby. The relaxation of local rigidity via the elimination of adjacent disulfide linkages led to a considerable structural transformation with a concomitant improvement in β-lactamase activity. Our findings reaffirm previous observations that the delicate balance between protein flexibility and stability is crucial for enzyme design and evolution.

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.

Onoda, A.

ACS Catal. 2014, 4, 2645-2648, 10.1021/cs500392e

The hydrogen-evolving diiron complex, (μ-S)2Fe2(CO)6 with a tethered maleimide moiety was synthesized and covalently embedded within the cavity of a rigid β-barrel protein matrix by coupling a maleimide moiety to a cysteine residue within the β-barrel. The (μ-S)2Fe2(CO)6 core within the cavity was characterized by UV–vis absorption and a characteristic CO vibration determined by IR measurements. The diiron complex embedded within the cavity retains the necessary catalytic activity (TON up to 130 for 6 h) to evolve H2 via a photocatalytic cycle with a Ru photosensitizer in a solution of 100 mM ascorbate and 50 mM Tris/HCl at pH 4.0 and 25 °C.

Song, W.J.

Chem. Commun. 2020, 56, 9586-9599, 10.1039/d0cc03137b

By combining synthetic catalysts and biochemical tools, numerous artificial metalloenzymes have been designed to exhibit high catalytic activity and selectivity in diverse chemical transformations. Out of the nearly infinite number of discovered or characterised proteins, however, only a handful of proteins have been employed as scaffolds for artificial metalloenzymes, implying that specific proteins are preferred owing to their native structural, functional, or biochemical properties. In the present review, we extract and group the biochemical and structural properties of proteins that are advantageous in the design of artificial metalloenzymes; protein stability, pre-existing metal centre, native binding affinity for small molecules, confined and empty space, well-defined secondary structure, and native cellular location. The desirable properties highlight proteins as the key players in the design of metal-dependent biocatalysts. We also propose rarely considered, yet promising, proteins that could be versatile and unique scaffolds for novel metalloenzymes.

Song, W.J.

Chem. Sci. 2021, 12, 5091-5101, 10.1039/d0sc06823c

Directed evolution has provided us with great opportunities and prospects in the synthesis of tailor-made proteins. It, however, often requires at least mid to high throughput screening, necessitating more effective strategies for laboratory evolution. We herein demonstrate that protein symmetry can be a versatile criterion for searching for promising hotspots for the directed evolution of de novo oligomeric enzymes. The randomization of symmetry-related residues located at the rotational axes of artificial metallo-β-lactamase yields drastic effects on catalytic activities, whereas that of non-symmetry-related, yet, proximal residues to the active site results in negligible perturbations. Structural and biochemical analysis of the positive hits indicates that seemingly trivial mutations at symmetry-related spots yield significant alterations in overall structures, metal-coordination geometry, and chemical environments of active sites. Our work implicates that numerous artificially designed and natural oligomeric proteins might have evolutionary advantages of propagating beneficial mutations using their global symmetry.