2 publications

2 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: ---

Computational Redesign of a Mononuclear Zinc Metalloenzyme for Organophosphate Hydrolysis

Baker, D.

Nat. Chem. Biol. 2012, 8, 294-300, 10.1038/NChemBio.777

The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency (kcat/Km) of ∼104 M−1 s−1 after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzes the hydrolysis of the RP isomer of a coumarinyl analog of the nerve agent cyclosarin, and it shows marked substrate selectivity for coumarinyl leaving groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities.


Metal: Zn
Ligand type: Amino acid
Anchoring strategy: Dative
Optimization: Genetic
Max TON: >140
ee: ---
PDB: 3T1G
Notes: kcat/KM ≈ 104 M-1*s-1