Séminaire Salvatore La Gatta

21/09/2023 - 11:30 - Salle Jacques Sénez
Salvatore La Gatta, Department of Chemistry, University of Michigan, USA

De novo designed copper-containing metalloenzymes for oxidative chemistry, mimicking Lytic Polysaccharide MonoOxygenases (LPMOs)

Copper-containing metalloenzymes are a significant group of proteins found in nature, responsible for catalyzing a wide range of reactions.[1] Among several approaches to studying copper metalloenzymes, de novo protein design represents a valuable strategy to transplant the target copper-binding site in simpler and possibly more stable small-sized scaffolds.[2] In this talk, I will present our work on the design, characterization, and functional studies of three different de novo designed copper-containing metalloenzymes, and that was done during my PhD Thesis work at the Department of Chemical Sciences (Prof. Angela Lombardi’s Lab), University of Naples Federico II, Naples, Italy.
DR1 (Due Rame, in Italian) is a newly designed protein that mimics polyphenol oxidases and contains a Type 3 di-copper site. The first and second di-metal coordination spheres were hierarchically engineered in order to nest the di-copper site into a simpler scaffold made of a four-helix bundle. DR1 recapitulates the Type 3 di-copper site, supporting several copper redox states and being active in the O2-dependent oxidation of catechols to o-quinones, according to spectroscopic, thermodynamic, and functional analysis.[3] We showed that protein design allows the transplantation of the peculiar Type 2 copper site of LPMOs (Fig. 1a), known as the Histidine Brace (Fig. 1b), into the de novo designed scaffolds dHisB and miniLPMO (Fig. 1c) differing significantly from the canonical natural fold. These helical-bundle Cu miniproteins allowed unraveling, for the first time, the specific contribution to the EPR spectrum of the different protonation states of the Histidine Brace coordination over a wide range of pH, not always attainable with natural proteins. Significantly, these small proteins activate O2 or H2O2 in a similar way to their natural counterparts. In conclusion, our handcrafted metalloproteins contain the essential elements of copper active sites in natural enzymes, representing a milestone in the development of synthetic metalloenzymes for the degradation and conversion of biomass into second-generation fuels.

[1] E.I. Solomon, D. E. Heppner, E.M. Johnston, J.W. Ginsbach, J. Cirera, M. Qayyum, M.T. Kieber-Emmons, C.H. Kjaergaard, R.G. Hadt, L. Tian, Chem. Rev. 2014, 114, 3659–3853.
[2] F. Nastri, D. D’Alonzo, L. Leone, G. Zambrano, V. Pavone, A. Lombardi, Trends Biochem. Sci. 2019, 44, 1022–1040.
[3] F. Pirro, S. La Gatta, F. Arrigoni, A. Famulari, O. Maglio, P. Del Vecchio, M. Chiesa, L. De Gioia, L. Bertini, M. Chino, F. Nastri, A. Lombardi, Angew. Chem. Int. Ed. 2023, 62, e202211552.

Inv.: Anabella IVANCICH

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