De novo metalloprotein design is a remarkable approach to shape protein scaffolds toward specific functions. Here, we report the design and characterization of Due Rame 1 (DR1), a de novo designed protein housing a di-copper site and mimicking the Type 3 (T3) copper-containing polyphenol oxidases (PPOs). To achieve this goal, we hierarchically designed the first and the second di-metal coordination spheres to engineer the di-copper site into a simple four-helix bundle scaffold. Spectroscopic, thermodynamic, and functional characterization revealed that DR1 recapitulates the T3 copper site, supporting different copper redox states, and being active in the O2 -dependent oxidation of catechols to o-quinones. Careful design of the residues lining the substrate access site endows DR1 with substrate recognition, as revealed by Hammet analysis and computational studies on substituted catechols. This study represents a premier example in the construction of a functional T3 copper site into a designed four-helix bundle protein.

A De Novo-Designed Type 3 Copper Protein Tunes Catechol Substrate Recognition and Reactivity

Famulari, Antonino;Chiesa, Mario;
2023-01-01

Abstract

De novo metalloprotein design is a remarkable approach to shape protein scaffolds toward specific functions. Here, we report the design and characterization of Due Rame 1 (DR1), a de novo designed protein housing a di-copper site and mimicking the Type 3 (T3) copper-containing polyphenol oxidases (PPOs). To achieve this goal, we hierarchically designed the first and the second di-metal coordination spheres to engineer the di-copper site into a simple four-helix bundle scaffold. Spectroscopic, thermodynamic, and functional characterization revealed that DR1 recapitulates the T3 copper site, supporting different copper redox states, and being active in the O2 -dependent oxidation of catechols to o-quinones. Careful design of the residues lining the substrate access site endows DR1 with substrate recognition, as revealed by Hammet analysis and computational studies on substituted catechols. This study represents a premier example in the construction of a functional T3 copper site into a designed four-helix bundle protein.
2023
62
1
1
10
https://onlinelibrary.wiley.com/doi/10.1002/anie.202211552
Artificial Metalloenzymes; Phenol Oxidases; Protein Design; Substrate Selectivity; T3 di-Copper Site
Pirro, Fabio; La Gatta, Salvatore; Arrigoni, Federica; Famulari, Antonino; Maglio, Ornella; Del Vecchio, Pompea; Chiesa, Mario; De Gioia, Luca; Bertini, Luca; Chino, Marco; Nastri, Flavia; Lombardi, Angela
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/1882080
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