Tezcan, F.A.

Science 2014, 346, 1525-1528, 10.1126/science.1259680

The generation of new enzymatic activities has mainly relied on repurposing the interiors of preexisting protein folds because of the challenge in designing functional, three-dimensional protein structures from first principles. Here we report an artificial metallo-β-lactamase, constructed via the self-assembly of a structurally and functionally unrelated, monomeric redox protein into a tetrameric assembly that possesses catalytic zinc sites in its interfaces. The designed metallo-β-lactamase is functional in the Escherichia coli periplasm and enables the bacteria to survive treatment with ampicillin. In vivo screening of libraries has yielded a variant that displays a catalytic proficiency [(kcat/Km)/kuncat] for ampicillin hydrolysis of 2.3 × 106 and features the emergence of a highly mobile loop near the active site, a key component of natural β-lactamases to enable substrate interactions.

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.

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.

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.

Tezcan, F.A.

Acc. Chem. Res. 2019, 52, 345-355, 10.1021/acs.accounts.8b00617

Nature puts to use only a small fraction of metal ions in the periodic table. Yet, when incorporated into protein scaffolds, this limited set of metal ions carry out innumerable cellular functions and execute essential biochemical transformations such as photochemical H2O oxidation, O2 or CO2 reduction, and N2 fixation, highlighting the outsized importance of metalloproteins in biology. Not surprisingly, elucidating the intricate interplay between metal ions and protein structures has been the focus of extensive structural and mechanistic scrutiny over the last several decades. As a result of such top-down efforts, we have gained a reasonably detailed understanding of how metal ions shape protein structures and how protein structures in turn influence metal reactivity. It is fair to say that we now have some idea–and in some cases, a good idea–about how most known metalloproteins function and we possess enough insight to quickly assess the modus operandi of newly discovered ones. However, translating this knowledge into an ability to construct functional metalloproteins from scratch represents a challenge at a whole different level: it is one thing to know how an automobile works; it is another to build one. In our quest to build new metalloproteins, we have taken an original approach in which folded, monomeric proteins are used as ligands or synthons for building supramolecular complexes through metal-mediated self-assembly (MDPSA, Metal-Directed Protein Self-Assembly). The interfaces in the resulting protein superstructures are subsequently tailored with covalent, noncovalent, or additional metal-coordination interactions for stabilization and incorporation of new functionalities (MeTIR, Metal Templated Interface Redesign). In an earlier Account, we had described the proof-of-principle studies for MDPSA and MeTIR, using a four-helix bundle, heme protein cytochrome cb562 (cyt cb562), as a model building block. By the end of those studies, we were able to demonstrate that a tetrameric, Zn-directed cyt cb562 complex (Zn4:M14) could be stabilized through computationally prescribed noncovalent interactions inserted into the nascent protein–protein interfaces. In this Account, we first describe the rationale and motivation for our particular metalloprotein engineering strategy and a brief summary of our earlier work. We then describe the next steps in the “evolution” of bioinorganic complexity on the Zn4:M14 scaffold, namely, (a) the generation of a self-standing protein assembly that can stably and selectively bind metal ions, (b) the creation of reactive metal centers within the protein assembly, and (c) the coupling of metal coordination and reactivity to external stimuli through allosteric effects.

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.

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.

Tezcan, F.A.

Curr. Opin. Chem. Biol. 2014, 19, 42-49, 10.1016/j.cbpa.2013.12.013

Metal ions are frequently found in natural protein–protein interfaces, where they stabilize quaternary or supramolecular protein structures, mediate transient protein–protein interactions, and serve as catalytic centers. Paralleling these natural roles, coordination chemistry of metal ions is being increasingly utilized in creative ways toward engineering and controlling the assembly of functional supramolecular peptide and protein architectures. Here we provide a brief overview of this emerging branch of metalloprotein/peptide engineering and highlight a few select examples from the recent literature that best capture the diversity and future potential of approaches that are being developed.

Tezcan, F.A.

Methods Enzymol. 2016, 10.1016/bs.mie.2016.05.009

Owing to their central roles in cellular signaling, construction, and biochemistry, protein–protein interactions (PPIs) and protein self-assembly have become a major focus of molecular design and synthetic biology. In order to circumvent the complexity of constructing extensive noncovalent interfaces, which are typically involved in natural PPIs and protein self-assembly, we have developed two design strategies, metal-directed protein self-assembly (MDPSA) and metal-templated interface redesign (MeTIR). These strategies, inspired by both the proposed evolutionary roles of metals and their prevalence in natural PPIs, take advantage of the favorable properties of metal coordination (bonding strength, directionality, and reversibility) to guide protein self-assembly with minimal design and engineering. Using a small, monomeric protein (cytochrome cb562) as a model building block, we employed MDPSA and MeTIR to create a diverse array of functional supramolecular architectures which range from structurally tunable oligomers to metalloprotein complexes that can properly self-assemble in living cells into novel metalloenzymes. The design principles and strategies outlined herein should be readily applicable to other protein systems with the goal of creating new PPIs and protein assemblies with structures and functions not yet produced by natural evolution.

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.