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.

Pirro, F., La Gatta, S., Arrigoni, F., Famulari, A., Maglio, O., Del Vecchio, P., et al. (2023). A De Novo-Designed Type 3 Copper Protein Tunes Catechol Substrate Recognition and Reactivity. ANGEWANDTE CHEMIE. INTERNATIONAL EDITION, 62(1 (January 2, 2023)) [10.1002/anie.202211552].

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

Arrigoni, Federica;De Gioia, Luca;Bertini, Luca;
2023

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.
Articolo in rivista - Articolo scientifico
Artificial Metalloenzymes; Phenol Oxidases; Protein Design; Substrate Selectivity; T3 di-Copper Site;
English
5-nov-2022
2023
62
1 (January 2, 2023)
e202211552
none
Pirro, F., La Gatta, S., Arrigoni, F., Famulari, A., Maglio, O., Del Vecchio, P., et al. (2023). A De Novo-Designed Type 3 Copper Protein Tunes Catechol Substrate Recognition and Reactivity. ANGEWANDTE CHEMIE. INTERNATIONAL EDITION, 62(1 (January 2, 2023)) [10.1002/anie.202211552].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/397690
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