Hybrid B heme peroxidases are recently discovered unique oxidoreductases present solely in the fungal kingdom. We have investigated two typical representatives from Magnaporthe oryzae—one of the most dangerous phytopathogens known as a causal agent of the rice blast disease. First, we focused on native expression of two detected hyBpox paralogs by the means of reverse-transcription quantitative real-time PCR. Our results indicate a 7-fold induction of the MohyBpox1 transcript in a medium with H₂O₂ and a 3-fold induction in a medium with peroxyacetic acid. For the MohyBpox2 paralog the induction patterns were up to 12-fold and 6.7-fold, respectively. We have successfully expressed the shorter gene, MohyBpox1, heterologously in Pichia pastoris for detailed characterization. Observed biochemical and biophysical properties of the highly purified protein reveal that a typical HyBPOX is significantly different from previously investigated APx-CcP hybrids. This newly discovered secretory peroxidase reveals a Soret maximum at 407 nm, Q bands at 532 and 568 nm, CT band at 625 nm and a purity number of 1.48. Electron paramagnetic resonance (EPR) analysis suggests a mixture of high and low spin species in the ferric state dependent on calcium contents. Steady-state kinetic data reveal the highest peroxidase activity with ABTS, 5-aminosalycilate and efficient oxidation of tyrosine. MoHyBPOX1 as a fusion protein consists of two domains. The longer conserved N-terminal peroxidase domain is connected with a shorter C-terminal domain containing a carbohydrate binding motif of type CBM21. We demonstrate the capacity of MoHyBPOX1 to bind soluble starch efficiently. Potential involvement of hybrid peroxidases in the pathogenicity of M. oryzae is discussed.

中文翻译：

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来自稻瘟病真菌稻瘟病菌的混合血红素过氧化物酶参与防御氧化应激。

混合 B 血红素过氧化物酶是最近发现的仅存在于真菌界的独特氧化还原酶。我们调查了稻瘟病菌的两个典型代表——一种最危险的植物病原体，被称为稻瘟病的病原体。首先，我们通过逆转录定量实时 PCR 关注两个检测到的hyBpox旁系同源物的天然表达。我们的结果表明MohyBpox1转录物在 H ₂ O ₂培养基中的诱导 7 倍和在过氧乙酸培养基中的 3 倍诱导。对于MohyBpox2paralog 诱导模式分别高达 12 倍和 6.7 倍。我们已经成功地在毕赤酵母中异源表达了较短的基因MohyBpox1进行详细表征。观察到的高度纯化的蛋白质的生化和生物物理特性表明，典型的 HyBPOX 与先前研究的 APx-CcP 杂种有显着不同。这种新发现的分泌过氧化物酶在 407 nm 处显示 Soret 最大值，在 532 和 568 nm 处显示 Q 带，在 625 nm 处显示 CT 带，纯度数为 1.48。电子顺磁共振 (EPR) 分析表明，铁态的高自旋和低自旋物种的混合物取决于钙含量。稳态动力学数据显示 ABTS、5-氨基水杨酸和酪氨酸的有效氧化具有最高的过氧化物酶活性。MoHyBPOX1 作为一种融合蛋白由两个结构域组成。较长保守的 N 末端过氧化物酶结构域与较短的 C 末端结构域连接，该结构域包含 CBM21 型碳水化合物结合基序。我们证明了 MoHyBPOX1 有效结合可溶性淀粉的能力。杂合过氧化物酶在致病性中的潜在参与讨论了M. oryzae。