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 than 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.

This work has been funded by the H2020 BBI-JU (www.bbi-europe.eu) project EnzOx2 (BBI-PPP-2015-2-720297; www. enzox2.eu) and the FP7 project INDOX (KBBE-2013-7-613549; www.indoxproject.eu), together with the BIO2014-56388-R (NOESIS), BFU2016-77835-R, CTQ2016-79138-R, and (BIO2017-86559-R (GENOBIOREF) projects of the Spanish Ministry of Economy, Industry and Competitiveness, cofinanced by FEDER funds. The sequencing of P. ostreatus genome at JGI was supported by the DOE Office of Science, under Contract No. DE-AC02-05CH11231. We thank the staff of the ID30B beamline at ESRF (Grenoble, France) for assistance. CSGI (Florence, Italy) FFABR 2017 (Legge 232/ 2016 Comma 295, and Legge di Bilancio 2017) and MIUR (Dipartimento di Eccellenza, 2018-2022) grants are acknowledged for financial support.

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