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Title

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

AuthorsFernández-Fueyo, Elena ; Davó-Siguero, Irene; Almendral, David; Linde, Dolores ; Baratto, M. Camila; Pogni, Rebecca; Romero, Antonio ; Guallar, Victor; Martínez, Ángel T.
KeywordsMn-oxidation site
Dye-decolorizing peroxidase
Crystal structure
Ligand diffusion
PELE,
Electron transfer
QM/MM calculations
EPR spectroscopy
Issue Date31-Jul-2018
PublisherAmerican Chemical Society
CitationACS Catal 8386–8395 (2018)
AbstractA 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.
Description30 p.-9 fig.-2 tab.-10 fig. supl.-3 tab. supl.
Publisher version (URL)https://doi.org/10.1021/acscatal.8b02306
URIhttp://hdl.handle.net/10261/169307
DOI10.1021/acscatal.8b02306
E-ISSN2155-5435
Appears in Collections:(CIB) Artículos
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