Description of a Non-Canonical Mn(II)-Oxidation Site in Peroxidases
PublisherAmerican Chemical Society
Rights accessRestricted access - publisher's policy (embargoed until 2019-07-31)
European Commisision's projectEnzOx2 - New enzymatic oxidation%2Foxyfunctionalization technologies for added value bio-based products (EC-H2020-720297)
INDOX - Optimized oxidoreductases for medium and large scale industrial biotransformations (EC-FP7-613549)
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.
CitationFernández-Fueyo, E. [et al.]. Description of a Non-Canonical Mn(II)-Oxidation Site in Peroxidases. "", 31 Juliol 2018, vol. 8, p. 8386-8395.
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