Hybrid Heme Peroxidases from Rice Blast Fungus <i>Magnaporthe oryzae</i> Involved in Defence against Oxidative Stress

Abstract

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.

Keywords

• oxidative stress
• peroxidase-catalase superfamily
• <i>Magnaporthe oryzae</i>
• hybrid B heme peroxidase
• carbohydrate binding domain