Thu, Aug 05, 2021:On Demand
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.
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.