Unveiling the mechanism of dye-decolorizing peroxidase (DyP): unique anthraquinone-directed catalysis exposes a...

Dye-decolorizing peroxidases (DyPs) are heme-containing peroxidases that catalyze the reduction of H₂O₂ to water, accompanied by the oxidation of diverse substrates, including lignin-related phenolic and non-phenolic compounds, as well as a wide range of synthetic dyes. These enzymes exhibit a marked preference for decolorizing recalcitrant anthraquinone dyes, setting them apart from other peroxidases; however, the molecular mechanism underlying this preference remains to be elucidated. This study explores the catalytic profile of recombinant Il-DyP4, a dye-decolorizing peroxidase from Irpex lacteus F17, to clarify the molecular basis of its substrate preference. We found that Il-DyP4 exhibits remarkable H₂O₂ tolerance in the oxidation of anthraquinone dyes, maintaining approximately 50% activity even at H₂O₂ concentrations above 20 mM, while its activity toward phenolic substrates decreases by 50% at only 0.6 mM H₂O₂ concentration. These results demonstrate the distinct capability of Il-DyP4 to catalyze anthraquinone dyes at high H₂O₂. Analysis of the intermediates of Reactive Blue 19 transformation showed that this catalytic ability is linked to the bifunctional activity of Il-DyP4: the enzyme couples peroxidative oxidation with hydrolytic cleavage to rapidly scavenge H₂O₂, thereby averting self-inactivation of the enzyme. In contrast, guaiacol oxidation by Il-DyP4 followed the canonical peroxidase route, proceeding exclusively via C-C radical coupling to yield oligomeric products. These findings provide a mechanistic rationale for the intrinsic preference of DyPs for anthraquinone-based dyes and highlight Il-DyP4 as a resilient, high-performance biocatalyst for remediating dye-contaminated wastewater.IMPORTANCEHere, we reveal that the long-sought specificity of dye-decolorizing peroxidase (DyP) for anthraquinone dyes arises from a bifunctional mechanism orchestrated by Il-DyP4, encompassing both peroxidase-mediated oxidation and hydrolytic cleavage. Elucidating this dual chemistry not only redefines the catalytic behavior of the DyP family but also provides a mechanistic cornerstone for engineering robust biocatalysts to eliminate recalcitrant dye pollutants in industrial wastewater.