Catalase-peroxidases from phytopathogenic fungi

Catalase-peroxidases (KatGs), unique bifunctional heme oxidoreductases exhibiting both peroxidase and catalase activity, belong to the superfamily of bacterial, fungal and plant heme peroxidases. Neither structure-function relationships nor the physiological role of eukaryotic KatGs are known. A role in overcoming oxidative plant defense is in discussion. Here, from genomic analysis of recently sequenced fungal genomes four novel katG genes of the phytopathogens Gibberella zeae and Magnaporthe grisea were selected for cloning and heterologous expression to understand their native expression, cellular localization and structure-function relationships. These putative katG genes code for yet unknown catalase-peroxidases and present two different KatG types of fungal origin. Additionally, in the case of Gibberella zeae katG01 a rare fusion of katG gene with a region coding for cytochrome P450-like domain was detected thus forming a putative unique trifunctional heme containing oxidoreductase. The mode of native expression of the two katG paralogs in response to growth and stress conditions will be investigated with quantitative real-time PCR in both ascomycetes. Heterologous expression in appropriate host and purification of the recombinant enzymes will be followed by investigation of basic kinetic and spectral features in order to eludicate differences between the isoforms and generally between prokaryotic and eukaryotic enzymes and to select the most interesting candidates for structural (protein crystallization) and multi- mixing stopped-flow analysis. These transient-state kinetic analyses will unveil the role of fungal KatG-typical redox intermediates in their bifunctional activities as well as the functionality of the cytochrome P450 domain both in the putative trifunctional Gibberella zeae katG01&cytP450 multidomain protein and in the single domain form. In KatG saturation mutagenesis in selected hot spots essential for the catalase and peroxidase function will be performed to determine the peculiarities of fungal KatGs that differ them from the well studied bacterial counterparts. Understanding the structure, function and native expression of phytopathogenic catalase-peroxidases can help to unveil the mechanisms of the interactions between the plant host and its fungal pathogen thus contributing significantly to the global struggle against massive damages caused by the two fungi on cultural crops.

Catalase-peroxidases (KatGs), unique bifunctional heme oxidoreductases exhibiting both peroxidase and catalase activity, belong to the superfamily of bacterial, fungal and plant heme peroxidases. Neither structure-function relationships nor the physiological role of eukaryotic KatGs are known. A role in overcoming oxidative plant defense is in discussion. Here, from genomic analysis of recently sequenced fungal genomes four novel katG genes of the phytopathogens Gibberella zeae and Magnaporthe grisea were selected for cloning and heterologous expression to understand their native expression, cellular localization and structure-function relationships. These putative katG genes code for yet unknown catalase-peroxidases and present two different KatG types of fungal origin. Additionally, in the case of Gibberella zeae katG01 a rare fusion of katG gene with a region coding for cytochrome P450-like domain was detected thus forming a putative unique trifunctional heme containing oxidoreductase. The mode of native expression of the two katG paralogs in response to growth and stress conditions will be investigated with quantitative real-time PCR in both ascomycetes. Heterologous expression in appropriate host and purification of the recombinant enzymes will be followed by investigation of basic kinetic and spectral features in order to eludicate differences between the isoforms and generally between prokaryotic and eukaryotic enzymes and to select the most interesting candidates for structural (protein crystallization) and multi-mixing stopped-flow analysis. These transient-state kinetic analyses will unveil the role of fungal KatG-typical redox intermediates in their bifunctional activities as well as the functionality of the cytochrome P450 domain both in the putative trifunctional Gibberella zeae katG01&cytP450 multidomain protein and in the single domain form. In KatG saturation mutagenesis in selected hot spots essential for the catalase and peroxidase function will be performed to determine the peculiarities of fungal KatGs that differ them from the well studied bacterial counterparts. Understanding the structure, function and native expression of phytopathogenic catalase-peroxidases can help to unveil the mechanisms of the interactions between the plant host and its fungal pathogen thus contributing significantly to the global struggle against massive damages caused by the two fungi on cultural crops.