3 publications

3 publications

Artificial Dicopper Oxidase: Rational Reprogramming of Bacterial Metallo- b-lactamase into a Catechol Oxidase

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.


Metal: Cu
Ligand type: Amino acid
Host protein: β-lactamase
Anchoring strategy: Dative
Optimization: Genetic
Reaction: Catechol oxidation
Max TON: ---
ee: ---
PDB: 2FU7
Notes: ---

A Well-Defined Osmium–Cupin Complex: Hyperstable Artificial Osmium Peroxygenase

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.


Metal: Os
Ligand type: Amino acid
Host protein: TM1459 cupin
Anchoring strategy: Metal substitution
Optimization: Genetic
Reaction: Dihydroxylation
Max TON: 45
ee: ---
PDB: 5WSE
Notes: Exclusively cis dihydroxylation product obtained

Metal: Os
Ligand type: Amino acid
Host protein: TM1459 cupin
Anchoring strategy: Metal substitution
Optimization: Genetic
Reaction: Dihydroxylation
Max TON: 45
ee: ---
PDB: 5WSF
Notes: Exclusively cis dihydroxylation product obtained

Enzyme Repurposing of a Hydrolase as an Emergent Peroxidase Upon Metal Binding

Fujieda, N.; Ward, T.R.

Chem. Sci. 2015, 6, 4060-4065, 10.1039/c5sc01065a

Adding a metal cofactor to a protein bearing a latent metal binding site endows the macromolecule with nascent catalytic activity.


Metal: Cu
Ligand type: Amino acid
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 35
ee: ---
PDB: ---
Notes: ---