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Molecular characterization of aromatic peroxygenase from Agrocybe aegerita

Abstract

Recently, a novel group of fungal peroxidases, known as the aromatic peroxygenases (APO), has been discovered. Members of these extracellular biocatalysts produced by agaric basidiomycetes such as Agrocybe aegerita or Coprinellus radians catalyze reactions—for example, the peroxygenation of naphthalene, toluene, dibenzothiophene, or pyridine—which are actually attributed to cytochrome P450 monooxygenases. Here, for the first time, genetic information is presented on this new group of peroxide-consuming enzymes. The gene of A. aegerita peroxygenase (apo1) was identified on the level of messenger RNA and genomic DNA. The gene sequence was affirmed by peptide sequences obtained through an Edman degradation and de novo peptide sequencing of the purified enzyme. Quantitative real-time reverse transcriptase polymerase chain reaction demonstrated that the course of enzyme activity correlated well with that of mRNA signals for apo1 in A. aegerita. The full-length sequences of A. aegerita peroxygenase as well as a partial sequence of C. radians peroxygenase confirmed the enzymes’ affiliation to the heme-thiolate proteins. The sequences revealed no homology to classic peroxidases, cytochrome P450 enzymes, and only little homology (<30%) to fungal chloroperoxidase produced by the ascomycete Caldariomyces fumago (and this only in the N-terminal part of the protein comprising the heme-binding region and part of the distal heme pocket). This fact reinforces the novelty of APO proteins. On the other hand, homology retrievals in genetic databases resulted in the identification of various APO homologous genes and transcripts, particularly among the agaric fungi, indicating APO’s widespread occurrence in the fungal kingdom.

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Acknowledgments

We gratefully acknowledge the financial support from the German Environmental Foundation (Deutsche Bundesstiftung Umwelt; M.P., M.H.; grant 13225-32), the German Research Foundation (Deutsche Forschungsgemeinschaft; M.P., M.H.; grant HO1961/4-1), and the Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung; R.U., A.C., R.S., K.S., M.H.; grant 0313433D). We would like to thank Mss. U. Schneider, H. Hohlfeld, M. Brandt, and S. Schimpke for their excellent technical assistance and our colleagues E. Aranda, C. Liers, M. Kinne, M. Kluge (Inge), and R. Junek for fruitful discussions. Not least, we thank G. Gröbe for useful pieces of information on fungal cultivation.

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Correspondence to Marek J. Pecyna.

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Pecyna, M.J., Ullrich, R., Bittner, B. et al. Molecular characterization of aromatic peroxygenase from Agrocybe aegerita . Appl Microbiol Biotechnol 84, 885–897 (2009). https://doi.org/10.1007/s00253-009-2000-1

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Keywords

  • Peroxygenase
  • Chloroperoxidase
  • Cytochrome P450
  • Heme-thiolate
  • Oxygenation
  • Coprinellus