A heme peroxidase of the ascomyceteous lichen Leptogium saturninum oxidizes high-redox potential substrates Christiane Liers a,⇑ , René Ullrich a , Martin Hofrichter a , Farida V. Minibayeva b , Richard P. Beckett c a Unit of Environmental Biotechnology, International Graduate School of Zittau, Markt 23, 02763 Zittau, Germany b Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, PO Box 30, Kazan 420111, Russia c School of Biological and Conservation Sciences, University of KwaZulu-Natal, Private Bag X01, Pietermaritzburg, Scottsville 3209, South Africa article info Article history: Received 31 August 2011 Accepted 21 October 2011 Available online 28 October 2011 Keywords: Lichen Ascomycetes Heme peroxidase Non-phenolic aromatics Phenols abstract Lichens belonging to the order Peltigerales display strong activity of multi-copper oxidases (e.g. tyrosi- nase) as well as heme-containing peroxidases. The lichen peroxidase was purified to homogeneity from the thallus of Leptogium saturninum (LsaPOX) by fast protein liquid chromatography and then partially characterized. The oligomeric protein occurs as both 79 kDa dimeric and 42 kDa monomeric forms, and displayed broad substrate specificity. In addition to an ability to oxidize classic peroxidase substrates (e.g. 2,6- dimethoxyphenol), the enzyme could convert recalcitrant compounds such as synthetic dyes (e.g. Azure B and Reactive Blue 5), 4-nitrophenol and non-phenolic methoxylated aromatics (e.g. veratryl alcohol). Comparing LsaPOX with a basidiomycete dye-decolorizing (DyP)-type peroxidase from Auricularia auric- ula-judae showed that the lichen enzyme has a high-redox potential, with oxidation capabilities ranging between those of known plant and fungal peroxidases. Internal peptide fragments show homology (up to 60%) with putative proteins from free-living ascomycetes (e.g. Penicillium marneffei and Neosartorya fisc- heri), but not to sequences of algal or cyanobacterial peptides or to known fungal, bacterial or plant per- oxidases. LsaPOX is the first heme peroxidase purified from an ascomyceteous lichen that may help the organism to successfully exploit the extreme micro-environments in which they often grow. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction Numerous ascomycete fungi (comprising ca. 20% of all fungi and 40% of the ascomycetes) live in symbiosis with one or two pho- tosynthetic partners, which can be cyanobacterial (e.g. Nostoc sp.) and/or green algae (e.g. Coccomyxa sp.) (Kirk et al., 2008). Only lim- ited information is available on the oxidoreductases from liche- nized fungi, but recently it has been shown that some species can display high activities of the multi-copper oxidases laccase and tyrosinase (Laufer et al., 2006a,b, 2009; Zavarzina and Zavarzin, 2006; Lisov et al., 2007). These enzymes probably play more than one role in lichen biol- ogy, mainly as a result of their ability to metabolize reactive oxy- gen species (ROS) (Beckett et al., 2005). First, drawing analogies from free-living fungi (Ruiz-Duenas and Martinez, 2010), extracel- lular ROS generated by these enzymes may assist in the breakdown of cellulose, allowing lichens a measure of saprophytic existence. Second, they may be involved in the stress-induced extracellular production of ROS, or ‘‘oxidative burst’’ (Mika et al., 2004). This burst may deter potential pathogens, induce melanin synthesis and/or strengthen cell walls. Third, oxidoreductases may be in- volved in the regulation of the levels of stress-induced intracellular ROS to prevent their harmful effects to the lichen (Kranner et al., 2003; Weissman et al., 2005; Beckett and Minibayeva, 2007). Therefore, it seems likely that the roles of oxidoreductases in lichen biology, as for other organisms, will be found to include both the production and detoxification of ROS. Although heme peroxidases have been reported from almost all organisms, perhaps surprisingly, no unequivocal reports exist for the occurrence of these enzymes in lichens. Heme peroxidases use hydrogen peroxide (H 2 O 2 ) or organic hydroperoxides (R–OOH) as electron accepting co-substrates to oxidize a variety of organic compounds (Welinder, 1992). Enzymes that contain proximal histidine as the fifth ligand to the heme iron can be classified into two main superfamilies: the peroxidase–cyclooxygenase super- family, formerly known as animal or mammalian peroxidases and the non-animal peroxidases, also designated as the plant and microbial peroxidase or peroxidase–catalase superfamily (Hofrichter et al., 2010; Zámocky ´ and Obinger, 2010). The later superfamily comprises three main classes. Class I contains intracel- lular non-animal peroxidases, class II comprises secreted fungal 1087-1845/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2011.10.004 ⇑ Corresponding author. Fax: +49 3583 612734. E-mail address: liers@ihi-zittau.de (C. Liers). Fungal Genetics and Biology 48 (2011) 1139–1145 Contents lists available at SciVerse ScienceDirect Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi