Catalase-peroxidases (KatGs) are unique bifunctional oxidoreductases that contain heme in their active centers allowing both the peroxidatic and catalatic reaction modes. These originally bacterial enzymes are broadly distributed among various fungi allowing them to cope with reactive oxygen species present in the environment or inside the cells. We used various biophysical, biochemical, and bioinformatics methods to investigate differences between catalase-peroxidases originating in thermophilic and mesophilic fungi from different habitats. Our results indicate that the architecture of the active center with a specific post-translational modification is highly similar in mesophilic and thermophilic KatG and also the peroxidatic acitivity with ABTS, guaiacol, and L-DOPA. However, only the thermophilic variant CthedisKatG reveals increased manganese peroxidase activity at elevated temperatures. The catalatic activity releasing molecular oxygen is comparable between CthedisKatG and mesophilic MagKatG1 over a broad temperature range. Two constructed point mutations in the active center were performed selectively blocking the formation of described post-translational modification in the active center. They exhibited a total loss of catalatic activity and changes in the peroxidatic activity. Our results indicate the capacity of bifunctional heme enzymes in the variable reactivity for potential biotech applications.

Department of Glycobiology Institute of Chemistry Slovak Academy of Sciences Dúbravská Cesta 9 84538 Bratislava Slovakia

Department of Information Systems Faculty of Information Technology Brno University of Technology 61200 Brno Czech Republic

Department of Inorganic Chemistry Faculty of Natural Sciences Comenius University in Bratislava Mlynská Dolina Ilkovičova 6 84215 Bratislava Slovakia

Institute of Molecular Biology Slovak Academy of Sciences Dúbravská Cesta 21 84551 Bratislava Slovakia

International Clinical Research Centre St Anne's University Hospital Brno 65691 Brno Czech Republic

Loschmidt Laboratories Department of Experimental Biology and RECETOX Faculty of Science Masaryk University 61137 Brno Czech Republic

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