Although catalytic mechanisms in natural enzymes are well understood, achieving the diverse palette of reaction chemistries in re-engineered native proteins has proved challenging. Wholesale modification of natural enzymes is potentially compromised by their intrinsic complexity, which often obscures the underlying principles governing biocatalytic efficiency. The maquette approach can circumvent this complexity by combining a robust de novo designed chassis with a design process that avoids atomistic mimicry of natural proteins. Here, we apply this method to the construction of a highly efficient, promiscuous, and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H2O2. The maquette exhibits kinetics that match and even surpass those of certain natural peroxidases, retains its activity at elevated temperature and in the presence of organic solvents, and provides a simple platform for interrogating catalytic intermediates common to natural heme-containing enzymes.

By constructing an in vivo-assembled, catalytically proficient peroxidase, C45, we have recently demonstrated the catalytic potential of simple, de novo-designed heme proteins. Here, we show that C45’s enzymatic activity extends to the efficient and stereoselective intermolecular transfer of carbenes to olefins, heterocycles, aldehydes, and amines. Not only is this a report of carbene transferase activity in a completely de novo protein, but also of enzyme-catalyzed ring expansion of aromatic heterocycles via carbene transfer by any enzyme.

By constructing an in vivo assembled, catalytically proficient peroxidase, C45, we have recently demonstrated the catalytic potential of simple, de novo-designed heme proteins. Here we show that C45's enzymatic activity extends to the efficient and stereoselective intermolecular transfer of carbenes to olefins, heterocycles, aldehydes and amines. Not only is this the first report of carbene transferase activity in a de novo protein, but also of enzyme-catalyzed ring expansion of aromatic heterocycles via carbene transfer by any enzyme.Despite the significant advances in protein design, there still remain few examples of de novo enzymes that both approach the catalytic efficiencies of their natural counterparts and are of potential use in an industrial or biological context [1][2][3][4][5][6][7] . This reflects the inherent complexities experienced in the biomolecular design process, where approaches are principally focussed on either atomistically-precise redesign of natural proteins to stabilise reaction transition states 1-3 or imprinting the intrinsic chemical reactivity of cofactors or metal ions on simple, generic protein scaffolds 4-7 ; both can be significantly enhanced by implementing powerful, yet randomised directed evolution strategies to hone and optimise incipient function 8,9 . While the latter approach often results in de novo proteins that lack a singular structure 4,10,11 , the incorporation of functionally versatile cofactors, such as heme, is proven to facilitate the design and construction of de novo proteins and enzymes that recapitulate the function of natural heme-containing proteins in stable, simple and highly mutable tetrahelical chassis (termed maquettes) [12][13][14] . Since the maquettes are designed from first principles, they lack any natural evolutionary history and the associated functional interdependency that is associated with natural protein scaffolds can be largely circumvented 12 .We have recently reported the design and construction of a hyperthermostable maquette, C45, that is wholly assembled in vivo, hijacking the natural E. coli cytochrome c maturation system to covalently append heme onto the protein backbone (Fig. 1a) 4 . The covalently-linked heme C of C45 is axially ligated by a histidine side chain at the proximal site and it is likely that a water molecule occupies the distal site, analogous to the ligation state of natural heme-containing peroxidases 15 and metmyoglobin 16 . Not only does C45 retain the reversible oxygen binding capability of its ancestral maquettes 4,12,13 , but it functions as a promiscuous and catalytically proficient peroxidase, catalyzing the oxidation of small molecules, redox proteins and the oxidative dehalogenation of halogenated phenols with kinetic parameters that match and even surpass those of natural peroxidases 4 .