Microbial manganese(II) oxidation catalyzed by multicopper oxidases and FAD oxidoreductases in filamentous Ascomycete fungi

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Carolyn A. Zeiner, Department of Biology, University of St. Thomas, Saint Paul, MN, Samuel O. Purvine and Ljiljana Paša-Tolić, Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, Erika M. Zink, Biological Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, Si Wu, Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, Dominique L. Chaput, Biosciences, Geoffrey Pope Building, University of Exeter, Exeter, United Kingdom, Cara M. Santelli, Department of Earth and Environmental Sciences, University of Minnesota, Minneapolis, MN, Colleen M. Hansel, Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, MA

Background/Question/Methods
Manganese (Mn) (III/IV) oxide minerals are ubiquitous in the environment, and they can be formed by both abiotic and microbially-mediated processes. Due to their small particle size, large surface area, and high oxidative capacity, Mn oxides can impact a variety of biogeochemical processes, including degradation of recalcitrant organic compounds, remediation of contaminated soil and water, and cycling of trace metals. Thus, isolation of Mn(II)-oxidizing microorganisms and elucidation of the underlying mechanisms has the potential to aid in large-scale environmental preservation efforts. While white-rot Basidiomycete fungi oxidize Mn(II) using laccases and Mn peroxidases in association with lignocellulose degradation, Mn(II) oxidation mechanisms in filamentous Ascomycete fungi, a ubiquitous and cosmopolitan yet understudied group, remain poorly understood. In addition, a physiological role for Mn(II) oxidation in these organisms remains elusive. We hypothesized that extracellular proteins produced by Ascomycetes can directly catalyze Mn(II) oxidation in the secretome, that Mn(II)-oxidizing proteins vary by species, and that these proteins are distinct from those used by Basidiomycetes. To test this, we grew 3 phylogenetically diverse species of filamentous Ascomycetes in liquid culture, harvested their secretomes, and analyzed them for Mn(II)-oxidizing proteins using a combination of chemical and enzyme activity assays, gel electrophoresis, and bulk mass spectrometry.
Results/Conclusions
Our results show that Mn(II) oxidative capacity in the secretomes of 3 Ascomycete fungi is dictated by species-specific extracellular enzymes. To our surprise, we identified multiple redox-active proteins in Mn(II)-oxidizing gel bands in each species. Moreover, we revealed the presence of both copper-based and FAD-based Mn(II) oxidation mechanisms in all 3 species, demonstrating mechanistic redundancy. Specifically, we identified candidate Mn(II)-oxidizing enzymes as tyrosinase and glyoxal oxidase in Stagonospora sp. SRC1lsM3a, bilirubin oxidase in Stagonospora sp. and Paraconiothyrium sporulosum AP3s5-JAC2a, and GMC oxidoreductase in all 3 species, including Pyrenochaeta sp. DS3sAY3a. To support these identifications, we showed strong inhibition of Mn(II) oxidative capacity by the metal chelator o-phenanthroline and the FAD inhibitor diphenylene iodonium (DPI) in all 3 species. Protein identifications were also supported by multiple sequence alignment with known Ascomycota enzymes and enzyme-specific inhibition assays. Our results provide a complement to previous research on microbial Mn(II) oxidation mechanisms, extending work on multicopper oxidases to potentially include Mn(II)-oxidizing FAD oxidoreductases. The diversity of the candidate Mn(II)-oxidizing enzymes we identified suggests that the ability of fungal secretomes to oxidize Mn(II) may be more widespread than previously thought.