Fujieda, N.; Itoh, S.

Chem. - Asian J. 2012, 7, 1203-1207, 10.1002/asia.201101014

Teaching metalloenzymes new tricks: An artificial type III dicopper oxidase has been developed using a hydrolytic enzyme, metallo‐β‐lactamase, as a metal‐binding platform. The triple mutant D88G/S185H/P224G redesigned by computer‐assisted structural analysis showed spectroscopic features similar to those of type III copper proteins and exhibited a high catalytic activity in the oxidation of catechols under aerobic conditions.

Bäckvall, J.E.; Diéguez, M.; Pàmies, O.

Adv. Synth. Catal. 2015, 357, 1567-1586, 10.1002/adsc.201500290

Artificial metalloenzymes combine the excellent selective recognition/binding properties of enzymes with transition metal catalysts, and therefore many asymmetric transformations can benefit from these entities. The search for new successful strategies in the construction of metal‐enzyme hybrid catalysts has therefore become a very active area of research. This review discusses all the developed strategies and the latest advances in the synthesis and application in asymmetric catalysis of artificial metalloenzymes with future directions for their design, synthesis and application (Sections 2–4). Finally, advice is presented (to the non‐specialist) on how to prepare and use artificial metalloenzymes (Section 5).

Fujieda, N.; Itoh, S.

J. Am. Chem. Soc. 2017, 139, 5149-5155, 10.1021/jacs.7b00675

Thermally stable TM1459 cupin superfamily protein from Thermotoga maritima was repurposed as an osmium (Os) peroxygenase by metal-substitution strategy employing the metal-binding promiscuity. This novel artificial metalloenzyme bears a datively bound Os ion supported by the 4-histidine motif. The well-defined Os center is responsible for not only the catalytic activity but also the thermodynamic stability of the protein folding, leading to the robust biocatalyst (Tm ≈ 120 °C). The spectroscopic analysis and atomic resolution X-ray crystal structures of Os-bound TM1459 revealed two types of donor sets to Os center with octahedral coordination geometry. One includes trans-dioxide, OH, and mer-three histidine imidazoles (O3N3 donor set), whereas another one has four histidine imidazoles plus OH and water molecule in a cis position (O2N4 donor set). The Os-bound TM1459 having the latter donor set (O2N4 donor set) was evaluated as a peroxygenase, which was able to catalyze cis-dihydroxylation of several alkenes efficiently. With the low catalyst loading (0.01% mol), up to 9100 turnover number was achieved for the dihydroxylation of 2-methoxy-6-vinyl-naphthalene (50 mM) using an equivalent of H2O2 as oxidant at 70 °C for 12 h. When octene isomers were dihydroxylated in a preparative scale for 5 h (2% mol cat.), the terminal alkene octene isomers was converted to the corresponding diols in a higher yield as compared with the internal alkenes. The result indicates that the protein scaffold can control the regioselectivity by the steric hindrance. This protein scaffold enhances the efficiency of the reaction by suppressing disproportionation of H2O2 on Os reaction center. Moreover, upon a simple site-directed mutagenesis, the catalytic activity was enhanced by about 3-fold, indicating that Os-TM1459 is evolvable nascent osmium peroxygenase.

Gras, E.; Hureau, C.

Coord. Chem. Rev. 2016, 308, 445-459, 10.1016/j.ccr.2015.05.011

Artificial metalloenzymes, with their high selectivity and specificity combined with a wide scope of reactivity and substrates, constitute an original approach for catalyst development. Different strategies have been proposed for their elaboration, proceeding from modification of natural enzymes using bioengineering methods to de novo protein design. Another bio-inspired methodology for the development of hybrid catalysts consists in the incorporation of coordination complexes into biomolecules, with the aim to upgrade their catalytic abilities. In these systems, the reaction performed by the naked catalyst is modulated by the well-defined structure of the host biomolecule. This conveys added value to the catalyst, such as enantioselectivity or chemoselectivity. DNA, apo-enzymes, proteins and peptides have been engaged in this approach, affording a wide diversity of reactivities and substrates. The resulting systems can then be improved by combined chemical and bioengineering optimization, allowing access to powerful catalysts. Because this approach can virtually be applied to any biomolecule or coordination complex, the elaboration of bio-based hybrid catalysts seems promising for advance in catalysis.

Fujieda, N.; Itoh, S.

Angew. Chem. 2020, 132, 7791-7794, 10.1002/ange.202000129

Cupin superfamily proteins (TM1459) work as a macromolecular ligand framework with a double-stranded β-barrel structure ligating to a Cu ion through histidine side chains. Variegating the first coordination sphere of TM1459 revealed that H52A and H54A/H58A mutants effectively catalyzed the diastereo- and enantioselective Michael addition reaction of nitroalkanes to an α,β-unsaturated ketone. Moreover, calculated substrate docking signified C106N and F104W single-point mutations, which inverted the diastereoselectivity of H52A and further improved the stereoselectivity of H54A/H58A, respectively.