A dye-decolorizing peroxidase (DyP) from Pleurotus ostreatus (PosDyP4) catalyzes the oxidation of Mn2+ to Mn3+, in the presence of H2O2, with an efficiency similar to the well known manganese peroxidases and versatile peroxidases from this and other white-rot fungi. PosDyP4 has been overexpressed in Escherichia coli as an active enzyme, and its crystal structure has been solved at 1.56 Å resolution. A combination of substrate diffusion simulations on the solved structure using the PELE software, electron paramagnetic resonance and site-directed mutagenesis led to identification of the residues involved in Mn2+ oxidation. The oxidation site in PosDyP4 is different to the conserved site in the other Mn-oxidizing peroxidases mentioned above, and it includes four acidic residues (three aspartates and one glutamate) located at the surface of the protein. Moreover, since the Mn2+ ion is not in direct contact with the heme propionates, a tyrosine residue participates in the electron transfer to the cofactor being the only essential individual residue for PosDyP4 oxidation of the metal ion. The four acidic residues contribute to Mn2+ binding in different extents, with the glutamate also involved in the initial electron transfer to the key tyrosine, as confirmed by the >50-fold decreased kcat after removing its side-chain carboxylic group. A second electron transfer pathway operates in PosDyP4 for the oxidation of aromatics and dyes starting at a surface tryptophan, as reported in other fungal and prokaryotic DyPs, and connecting with the final part of the Mn2+ oxidation route. Both tryptophanyl and tyrosyl radicals, potentially involved in catalysis, were detected by electron paramagnetic resonance of the native enzyme and its tryptophan-less variant, respectively.

Fernandez-Fueyo, E., Davó-Siguero, I., Almendral, D., Linde, D., Baratto, M.C., Pogni, R., et al. (2018). Description of a non-canonical Mn(II)-oxidation site in peroxidases. ACS CATALYSIS, 8(9), 8386-8395 [10.1021/acscatal.8b02306].

Description of a non-canonical Mn(II)-oxidation site in peroxidases

Baratto, Maria Camilla;Pogni, Rebecca;
2018-01-01

Abstract

A dye-decolorizing peroxidase (DyP) from Pleurotus ostreatus (PosDyP4) catalyzes the oxidation of Mn2+ to Mn3+, in the presence of H2O2, with an efficiency similar to the well known manganese peroxidases and versatile peroxidases from this and other white-rot fungi. PosDyP4 has been overexpressed in Escherichia coli as an active enzyme, and its crystal structure has been solved at 1.56 Å resolution. A combination of substrate diffusion simulations on the solved structure using the PELE software, electron paramagnetic resonance and site-directed mutagenesis led to identification of the residues involved in Mn2+ oxidation. The oxidation site in PosDyP4 is different to the conserved site in the other Mn-oxidizing peroxidases mentioned above, and it includes four acidic residues (three aspartates and one glutamate) located at the surface of the protein. Moreover, since the Mn2+ ion is not in direct contact with the heme propionates, a tyrosine residue participates in the electron transfer to the cofactor being the only essential individual residue for PosDyP4 oxidation of the metal ion. The four acidic residues contribute to Mn2+ binding in different extents, with the glutamate also involved in the initial electron transfer to the key tyrosine, as confirmed by the >50-fold decreased kcat after removing its side-chain carboxylic group. A second electron transfer pathway operates in PosDyP4 for the oxidation of aromatics and dyes starting at a surface tryptophan, as reported in other fungal and prokaryotic DyPs, and connecting with the final part of the Mn2+ oxidation route. Both tryptophanyl and tyrosyl radicals, potentially involved in catalysis, were detected by electron paramagnetic resonance of the native enzyme and its tryptophan-less variant, respectively.
2018
Fernandez-Fueyo, E., Davó-Siguero, I., Almendral, D., Linde, D., Baratto, M.C., Pogni, R., et al. (2018). Description of a non-canonical Mn(II)-oxidation site in peroxidases. ACS CATALYSIS, 8(9), 8386-8395 [10.1021/acscatal.8b02306].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11365/1058978