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

Zobrazit více v PubMed

Baker A., Lin C.-C., Lett C., Karpinska B., Wright M.H., Foyer C.H. Catalase: A critical node in the regulation of cell fate. Free Radic. Biol. Med. 2023;199:56–66. doi: 10.1016/j.freeradbiomed.2023.02.009. PubMed DOI

Veitch N.C. Horseradish peroxidase: A modern view of a classic enzyme. Phytochemistry. 2004;65:249–259. doi: 10.1016/j.phytochem.2003.10.022. PubMed DOI

Ivancich A., Donald L.J., Villanueva J., Wiseman B., Fita I., Loewen P.C. Spectroscopic and Kinetic Investigation of the Reactions of Peroxyacetic Acid with Burkholderia pseudomallei Catalase-Peroxidase, KatG. Biochemistry. 2013;52:7271–7282. doi: 10.1021/bi400963j. PubMed DOI

Savelli B., Li Q., Webber M., Jemmat A.M., Robitaille A., Zamocky M., Mathé C., Dunand C. RedoxiBase: A database for ROS homeostasis regulated proteins. Redox Biol. 2019;26:101247. doi: 10.1016/j.redox.2019.101247. PubMed DOI PMC

Zamocky M., Furtmüller P.G., Bellei M., Battistuzzi G., Stadlmann J., Vlasits J., Obinger C. Intracellular catalase/peroxidase from the phytopathogenic rice blast fungus Magnaporthe grisea: Expression analysis and biochemical characterization of the recombinant protein. Biochem. J. 2009;418:443–451. doi: 10.1042/BJ20081478. PubMed DOI

Chelikani P., Fita I., Loewen P.C. Diversity of structures and properties among catalases. Cell. Mol. Life Sci. 2004;61:192–208. doi: 10.1007/s00018-003-3206-5. PubMed DOI PMC

Passardi F., Bakalovic N., Teixeira F.K., Margis-Pinheiro M., Penel C., Dunand C. Prokaryotic origins of the non-animal peroxidase superfamily and organelle-mediated transmission to eukaryotes. Genomics. 2007;89:567–579. doi: 10.1016/j.ygeno.2007.01.006. PubMed DOI

Passardi F., Zamocky M., Favet J., Jakopitsch C., Penel C., Obinger C., Dunand C. Phylogenetic distribution of catalase-peroxidases: Are there patches of order in chaos? Gene. 2007;397:101–113. doi: 10.1016/j.gene.2007.04.016. PubMed DOI

Zámocký M., Furtmüller P.G., Obinger C. Two distinct groups of fungal catalase/peroxidases. Biochem. Soc. Trans. 2009;37:772–777. doi: 10.1042/BST0370772. PubMed DOI PMC

Jakopitsch C., Kolarich D., Petutschnig G., Furtmüller P.G., Obinger C. Distal side tryptophan, tyrosine and methionine in catalase-peroxidases are covalently linked in solution. FEBS Lett. 2003;552:135–140. doi: 10.1016/S0014-5793(03)00901-3. PubMed DOI

Zámocký M., García-Fernández Q., Gasselhuber B., Jakopitsch C., Furtmüller P.G., Loewen P.C., Fita I., Obinger C., Carpena X. High Conformational Stability of Secreted Eukaryotic Catalase-peroxidases. J. Biol. Chem. 2012;287:32254–32262. doi: 10.1074/jbc.M112.384271. PubMed DOI PMC

Wingfield P.T. Protein Precipitation Using Ammonium Sulfate. Curr. Protoc. Protein Sci. 1998;13:A.3F.1–A.3F.8. doi: 10.1002/0471140864.psa03fs13. PubMed DOI PMC

Magnusson A.O., Szekrenyi A., Joosten H., Finnigan J., Charnock S., Fessner W. nanoDSF as screening tool for enzyme libraries and biotechnology development. FEBS J. 2019;286:184–204. doi: 10.1111/febs.14696. PubMed DOI PMC

Michalski A., Damoc E., Lange O., Denisov E., Nolting D., Müller M., Viner R., Schwartz J., Remes P., Belford M., et al. Ultra High Resolution Linear Ion Trap Orbitrap Mass Spectrometer (Orbitrap Elite) Facilitates Top Down LC MS/MS and Versatile Peptide Fragmentation Modes. Mol. Cell. Proteom. 2012;11:O111.013698. doi: 10.1074/mcp.O111.013698. PubMed DOI PMC

Cox J., Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 2008;26:1367–1372. doi: 10.1038/nbt.1511. PubMed DOI

Barr D., Aust S. On the Mechanism of Peroxidase-Catalyzed Oxygen Production. Arch. Biochem. Biophys. 1993;303:377–382. doi: 10.1006/abbi.1993.1298. PubMed DOI

Mutsuda M., Ishikawa T., Takeda T., Shigeoka S. The catalase-peroxidase of Synechococcus PCC 7942: Purification, nucleotide sequence analysis and expression in Escherichia coli. Biochem. J. 1996;316:251–257. doi: 10.1042/bj3160251. PubMed DOI PMC

Rodríguez-López J., Bañón-Arnao M., Martinez-Ortiz F., Tudela J., Acosta M., Varón R., García-Cánovas F. Catalytic oxidation of 2,4,5-trihydroxyphenylalanine by tyrosinase: Identification and evolution of intermediates. Biochim. Biophys. Acta (BBA)-Protein Struct. Mol. Enzym. 1992;1160:221–228. doi: 10.1016/0167-4838(92)90011-2. PubMed DOI

Martinez M.J., Ruiz-Duenas F.J., Guillen F., Martinez A.T. Purification and Catalytic Properties of Two Manganese Peroxidase Isoenzymes from Pleurotus eryngii. JBIC J. Biol. Inorg. Chem. 1996;237:424–432. doi: 10.1111/j.1432-1033.1996.0424k.x. PubMed DOI

Hildebrandt A.G., Roots I. Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent formation and breakdown of hydrogen peroxide during mixed function oxidation reactions in liver microsomes. Arch. Biochem. Biophys. 1975;171:385–397. doi: 10.1016/0003-9861(75)90047-8. PubMed DOI

Hekkelman M.L., de Vries I., Joosten R.P., Perrakis A. AlphaFill: Enriching AlphaFold models with ligands and cofactors. Nat. Methods. 2023;20:205–213. doi: 10.1038/s41592-022-01685-y. PubMed DOI PMC

Jumper J., Evans R., Pritzel A., Green T., Figurnov M., Ronneberger O., Tunyasuvunakool K., Bates R., Žídek A., Potapenko A., et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596:583–589. doi: 10.1038/s41586-021-03819-2. PubMed DOI PMC

Drozdetskiy A., Cole C., Procter J., Barton G.J. JPred4: A protein secondary structure prediction server. Nucleic Acids Res. 2015;43:W389–W394. doi: 10.1093/nar/gkv332. PubMed DOI PMC

Bhattacharya D., Nowotny J., Cao R., Cheng J. 3Drefine: An interactive web server for efficient protein structure refinement. Nucleic Acids Res. 2016;44:W406–W409. doi: 10.1093/nar/gkw336. PubMed DOI PMC

Stourac J., Vavra O., Kokkonen P., Filipovic J., Pinto G., Brezovsky J., Damborsky J., Bednar D. Caver Web 1.0: Identification of tunnels and channels in proteins and analysis of ligand transport. Nucleic Acids Res. 2019;47:W414–W422. doi: 10.1093/nar/gkz378. PubMed DOI PMC

Chovancova E., Pavelka A., Benes P., Strnad O., Brezovsky J., Kozlikova B., Gora A., Sustr V., Klvana M., Medek P., et al. CAVER 3.0: A Tool for the Analysis of Transport Pathways in Dynamic Protein Structures. PLOS Comput. Biol. 2012;8:e1002708. doi: 10.1371/journal.pcbi.1002708. PubMed DOI PMC

Vavra O., Filipovic J., Plhak J., Bednar D., Marques S.M., Brezovsky J., Stourac J., Matyska L., Damborsky J. CaverDock: A molecular docking-based tool to analyse ligand transport through protein tunnels and channels. Bioinformatics. 2019;35:4986–4993. doi: 10.1093/bioinformatics/btz386. PubMed DOI

Yuan F., Yin S., Xu Y., Xiang L., Wang H., Li Z., Fan K., Pan G. The Richness and Diversity of Catalases in Bacteria. Front. Microbiol. 2021;12:645477. doi: 10.3389/fmicb.2021.645477. PubMed DOI PMC

Ślesak I., Kula M., Ślesak H., Miszalski Z., Strzałka K. How to define obligatory anaerobiosis? An evolutionary view on the antioxidant response system and the early stages of the evolution of life on Earth. Free. Radic. Biol. Med. 2019;140:61–73. doi: 10.1016/j.freeradbiomed.2019.03.004. PubMed DOI

Salar R. Thermophilic Fungi: Basic Concepts and Biotechnological Applications. CRC Press; Boca Raton, FL, USA: 2018.

Gold M.H., Youngs H.L., Gelpke M.D. Manganese peroxidase. Met. Ions Biol. Syst. 2000;37:559–586. PubMed

Colin J., Wiseman B., Switala J., Loewen P.C., Ivancich A. Distinct Role of Specific Tryptophans in Facilitating Electron Transfer or as [Fe(IV)=O Trp•] Intermediates in the Peroxidase Reaction of Bulkholderia pseudomallei Catalase-Peroxidase: A Multifrequency EPR Spectroscopy Investigation. J. Am. Chem. Soc. 2009;131:8557–8563. doi: 10.1021/ja901402v. PubMed DOI

Casella L., Monzani E., Nicolis S. In: Potential Applications of Peroxidases in the Fine Chemical Industries BT–Biocatalysis Based on Heme Peroxidases: Peroxidases as Potential Industrial Biocatalysts. Torres E., Ayala M., editors. Springer; Berlin/Heidelberg, Germany: 2010. pp. 111–153.

Farkas Z., Puškárová A., Šišková A.O., Poljovka A., Zámocký M., Vadkerti E., Urík M., Farkas B., Bučková M., Kraková L., et al. Evaluation of enzymatic stamp removal strategies on handmade (cellulose-based) and machine-made (lignin-containing) papers. Int. J. Biol. Macromol. 2023;242:124599. doi: 10.1016/j.ijbiomac.2023.124599. PubMed DOI

Zámocký M., Kamlárová A., Maresch D., Chovanová K., Harichová J., Furtmüller P.G. Hybrid Heme Peroxidases from Rice Blast Fungus Magnaporthe oryzae Involved in Defence against Oxidative Stress. Antioxidants. 2020;9:655. doi: 10.3390/antiox9080655. PubMed DOI PMC

Zámocký M., Musil M., Danchenko M., Ferianc P., Chovanová K., Baráth P., Poljovka A., Bednář D. Deep Insights into the Specific Evolution of Fungal Hybrid B Heme Peroxidases. Biology. 2022;11:459. doi: 10.3390/biology11030459. PubMed DOI PMC

Zámocký M., Harichová J. Evolution of Heme Peroxygenases: Ancient Roots and Later Evolved Branches. Antioxidants. 2022;11:1011. doi: 10.3390/antiox11051011. PubMed DOI PMC