ACCARC

Engineering of an Artificial Capsidic Enzyme for Aqueous Dirhodium Catalysis

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 Obiettivo del progetto (Objective)

'Artificial metalloenzymes are expected to bring together the best of the two worlds of homogeneous and enzyme catalysis, combining broad substrate scope with high activity and reaction selectivity under mild conditions. Herein we propose to construct an artificial metalloenzyme based on a dirhodium active site in the capsid scaffold of the protein ferritin. Catalytically competent dirhodium compounds are derived from dirhodium tetraacetate, which possesses fourfold symmetry, with the ligands symmetrically arranged around the equator of the rhodium dimer. The transition metal core is active in a wide range of reactions including cyclopropanation, C-H activation and O-H insertion. These transformations play an important role in the synthesis of natural products, pharmaceuticals, and other industrially relevant targets. We will exploit the fourfold-symmetric pores of the capsidic protein ferritin to construct a dirhodium binding site. A ferritin mutant will be produced with four glutamate residues pointing in the channel lumen, suitable as ligands for the rhodium dimer. After derivatization with dirhodium the artificial capsid will be employed as a catalyst in organometallic reactions, such as the cyclopropanation of diaza carbonyl compounds with olefins in aqueous solution. The catalytic properties of this first dirhodium enzyme will be fine-tuned by the highly modular secondary ligand environment of the protein. The dirhodium binding site will also be introduced at the inner mouth of the fourfold channel, resulting in an active site inside the capsid. High local substrate concentrations and the presence of a second, complementary reaction center inside the capsid might allow multi-enzyme cascade catalysis, thus paving the way toward artificial nanoreactors with tailored properties.'

Introduzione (Teaser)

Compartmentalisation allows nature to combine otherwise incompatible biochemical processes. Inspired by nature, scientists seek to create nano-scale encapsulation systems to enable drug delivery, catalysis and bioimaging.

Descrizione progetto (Article)

Current encapsulation systems such as droplets, liposomes or polymersomes produce structures with sizes ranging from tens of nanometres to a few hundred micrometres. Natural capsids, cage-like protein structures developed by some viruses, present an important alternative for compartmentalisation. Certain proteins also have internal pores of well-defined shape and symmetry that can be used as nanocompartments.

Directed mutagenesis allows controlling the properties of the individual protein and/or the structure of the resulting capsid. Altering the charge of amino acids, or 'supercharging' the protein, confers new surface properties to it while maintaining protein functionality. One of the important features of supercharged proteins is their enhanced thermal stability.

The project 'Engineering of an artificial capsidic enzyme for aqueous dirhodium catalysis' (ACCARC) was dedicated to the engineering of ferritin to become a functional nanocompartment. Ferritin is a globular protein complex with symmetrical internal cavities serving as iron storage in both prokaryotes and eukaryotes. Using computational design, the outside surface of ferritin was reengineered to produce a supercharged nanocage carrying the positive charge. The resulting supercharged ferritin was thermostable and could bind efficiently to negatively charged surfaces.

Scientists used the engineered ferritin nanocage to synthesise iron-oxide nanoparticles. This enabled monitoring of the spatial localisation of the nanocages in transfection experiments. It was found that supercharged ferritin is readily incorporated into cells of a human cancer cell model.

Next, supercharged ferritin was assembled inside a larger redesigned protein nanocage, AaLS-13. AaLS-13 is mutant of Aquifex aeolicus lumazine synthase, a capsid-forming enzyme engineered to have negative charge to carry positively charged protein cargo. By tuning the electrostatic interactions between supercharged ferritin and AaLS-13, the scientists obtained nested structures.

These Matryoshka-type structures contained several iron-loaded, supercharged ferritin nanocages encapsulated within the larger cage. The project opened up new possibilities towards construction of nano-sized structures such as artificial organelles or microcompartments.