Dye-decolorizing peroxidases (DyPs) represent a novel family of heme peroxidases that use H2O2 as the final electron acceptor to catalyze the oxidation of various organic compounds. A DyP from Irpex lacteus F17 (Il-DyP4, corresponding to GenBank MG209114), obtained by heterologous expression, exhibits a high catalytic efficiency for phenolic compounds and a strong decolorizing ability toward various synthetic dyes. However, the enzyme structure and the catalytic residues involved in substrate oxidation remain poorly understood. Here, we obtained a high-resolution structure (2.0 Å, PDB: 7D8M) of Il‑DyP4 with α-helices, anti-parallel β-sheets and one ferric heme cofactor sandwiched between two domains. The crystal structure of Il‑DyP4 revealed two heme access channels leading from the enzyme molecular surface to its heme region, and also showed four conserved amino acid residues forming the pocket for the conversion of hydrogen peroxide into the water molecule. In addition, we found that Trp264 and Trp380, were two important residues with different roles in Il‑DyP4, by using site-directed mutagenesis and an electron paramagnetic resonance (EPR) study. Trp264 is a noncatalytic residue that mainly is used for maintaining the normal spatial conformation of the heme region and the high-spin state of heme Fe3+ of Il‑DyP4, while Trp380 serves as the surface-exposed radical-forming residue that is closely related to the oxidation of substrates including not only bulky dyes, but also simple phenols. This study is important for better understanding the catalytic properties of fungal DyPs and their structure–function relationships.

中文翻译：

---

揭示两种重要的色氨酸残基在来自Irpex lacteus F17的染料脱色过氧化物酶中具有完全不同的作用

染料脱色过氧化物酶 (DyPs) 代表了一个新的血红素过氧化物酶家族，它们使用 H2O2 作为最终电子受体来催化各种有机化合物的氧化。通过异源表达获得的 Irpex lacteus F17（Il-DyP4，对应于 GenBank MG209114）的 DyP，对酚类化合物具有高催化效率，对各种合成染料具有很强的脱色能力。然而，酶结构和参与底物氧化的催化残基仍然知之甚少。在这里，我们获得了 Il-DyP4 的高分辨率结构（2.0 Å，PDB：7D8M），其具有 α-螺旋、反平行的 β-折叠和夹在两个结构域之间的一个铁血红素辅因子。Il-DyP4 的晶体结构揭示了从酶分子表面通向其血红素区域的两个血红素通路，并且还显示了四个保守的氨基酸残基形成了用于将过氧化氢转化为水分子的口袋。此外，我们通过定点诱变和电子顺磁共振 (EPR) 研究发现 Trp264 和 Trp380 是两个在 Il-DyP4 中具有不同作用的重要残基。Trp264 是一种非催化残基，主要用于维持 Il-DyP4 血红素区域的正常空间构象和血红素 Fe3+ 的高自旋状态，而 Trp380 作为表面暴露的自由基形成残基，与底物的氧化不仅包括大块染料，还包括简单的酚类。这项研究对于更好地理解真菌 DyPs 的催化特性及其结构 - 功能关系很重要。