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.

Nolte, R.J.M.

Nat. Chem. 2013, 5, 945-951, 10.1038/NCHEM.1752

In processive catalysis, a catalyst binds to a substrate and remains bound as it performs several consecutive reactions, as exemplified by DNA polymerases. Processivity is essential in nature and is often mediated by a clamp-like structure that physically tethers the catalyst to its (polymeric) template. In the case of the bacteriophage T4 replisome, a dedicated clamp protein acts as a processivity mediator by encircling DNA and subsequently recruiting its polymerase. Here we use this DNA-binding protein to construct a biohybrid catalyst. Conjugation of the clamp protein to a chemical catalyst with sequence-specific oxidation behaviour formed a catalytic clamp that can be loaded onto a DNA plasmid. The catalytic activity of the biohybrid catalyst was visualized using a procedure based on an atomic force microscopy method that detects and spatially locates oxidized sites in DNA. Varying the experimental conditions enabled switching between processive and distributive catalysis and influencing the sliding direction of this rotaxane-like catalyst.

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.

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.

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.

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.

Birnbaum, E.R.; Darnall, D.W.

J. Biol. Chem. 1970, n/a

The rate of activation of the conversion of trypsinogen to trypsin has been found to be greatly accelerated by the neodymium(III) ion. The similarity of this process to the calcium(II) ion activation suggests that both metal ions bind at identical sites in trypsinogen. The rate of activation in the presence of the neodymium ion is much greater than that of the calcium ion, probably reflecting the increased stability constant of the neodymium-protein complex. In contrast to the calcium ion, however, neodymium(III) can be scrutinized by a variety of spectral and magnetic techniques which should reveal information concerning the calcium ion binding sites in proteins. Since the chemistry and the range of sires of the rare earth metal ions are so similar to that of the calcium ion, it is suggested that generally these ions should make good replacement ions for probing the calcium ion binding sites of proteins and enzymes.

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.