Two phylogenetically divergent genes of the new family of dye-decolorizing peroxidases (DyPs) were found during comparison of the four DyP genes identified in the <
i>
Pleurotus ostreatus<
/i>
genome with over 200 DyP genes from other basidiomycete genomes. The heterologously expressed enzymes (<
i>
Pleos<
/i>
-DyP1 and <
i>
Pleos<
/i>
-DyP4, following the genome nomenclature) efficiently oxidize anthraquinoid dyes (such as Reactive Blue 19), which are characteristic DyP substrates, as well as low redox-potential dyes (such as 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) and substituted phenols. However, only <
i>
Pleos<
/i>
-DyP4 oxidizes the high redox-potential dye Reactive Black 5, at the same time that it displays high thermal and pH stability. Unexpectedly, both enzymes also oxidize Mn<
sup>
2+<
/sup>
to Mn<
sup>
3+<
/sup>
, albeit with very different catalytic efficiencies. <
i>
Pleos<
/i>
-DyP4 presents a Mn<
sup>
2+<
/sup>
turnover (56 s<
sup>
?1<
/sup>
) nearly in the same order of the two other Mn<
sup>
2+<
/sup>
-oxidizing peroxidase families identified in the P. ostreatus genome: manganese peroxidases (100 <
sup>
s?1<
/sup>
average turnover) and versatile peroxidases (145 s<
sup>
?1<
/sup>
average turnover), whose genes were also heterologously expressed. Oxidation of Mn<
sup>
2+<
/sup>
has been reported for an Amycolatopsis DyP (24 s<
sup>
?1<
/sup>
) and claimed for other bacterial DyPs, albeit with lower activities, but this is the first time that Mn<
sup>
2+<
/sup>
oxidation is reported for a fungal DyP. Interestingly, <
i>
Pleos<
/i>
-DyP4 (together with ligninolytic peroxidases) is detected in the secretome of <
i>
P. ostreatus<
/i>
grown on different lignocellulosic substrates. In conclusion, it is suggested that generation of Mn<
sup>
3+<
/sup>
oxidizers plays a role in the <
i>
P. ostreatus<
/i>
white-rot lifestyle since three different families of Mn<
sup>
2+<
/sup>
-oxidizing peroxidase genes are present in its genome being expressed during lignocellulose degradation.