Raman Characterization of Fungal DHN and DOPA Melanin Biosynthesis Pathways

Abstract

Fungal melanins represent a resource for important breakthroughs in industry and medicine, but the characterization of their composition, synthesis, and structure is not well understood. Raman spectroscopy is a powerful tool for the elucidation of molecular composition and structure. In this work, we characterize the Raman spectra of wild-type Aspergillus fumigatus and Cryptococcus neoformans and their melanin biosynthetic mutants and provide a rough “map” of the DHN (A. fumigatus) and DOPA (C. neoformans) melanin biosynthetic pathways. We compare this map to the Raman spectral data of Aspergillus nidulans wild-type and melanin biosynthetic mutants obtained from a previous study. We find that the fully polymerized A. nidulans melanin cannot be classified according to the DOPA pathway; nor can it be solely classified according to the DHN pathway, consistent with mutational analysis and chemical inhibition studies. Our approach points the way forward for an increased understanding of, and methodology for, investigating fungal melanins.

Dihydroxynaphthalene-Based Allomelanins: A Source of Inspiration for Innovative Technological Materials

“…38 Moreover, through the comparative inspection of time-resolved spectra assisted by DFT calculations, it was possible to conclude that once the naphthoxyl radical has formed, the 2,2′-dimerization is the most favorable event, followed by a further oxidation step affording the corresponding extended quinones (Figure 4). 38 As shown by MALDI mass spectrometry and ATR-FTIR, solid-state 13 C NMR, and EPR spectroscopies, 36,40 the resulting synthetic polymer was characterized by a certain degree of redox complexity, in good agreement with what was reported in the case of eumelanins, exhibiting the typical features of the phenolic, semiquinone, and quinone moieties. In particular, the latter were clearly evident from the presence of both O−H and conjugated CO stretching bands at 3100−3600 and 1621 and 1665 cm −1 , respectively, and from the 13 C resonances at 190 and 160 ppm ascribable to conjugated ketones and aromatic C−OH carbon atoms.…”

“…38 As shown by MALDI mass spectrometry and ATR-FTIR, solid-state 13 C NMR, and EPR spectroscopies, 36,40 the resulting synthetic polymer was characterized by a certain degree of redox complexity, in good agreement with what was reported in the case of eumelanins, exhibiting the typical features of the phenolic, semiquinone, and quinone moieties. In particular, the latter were clearly evident from the presence of both O−H and conjugated CO stretching bands at 3100−3600 and 1621 and 1665 cm −1 , respectively, and from the 13 C resonances at 190 and 160 ppm ascribable to conjugated ketones and aromatic C−OH carbon atoms.…”

“…As is evident from the data reported in Table , there is no straight correlation between the type of fungal melanin and the phylum from which each fungus derives, even if 1,8-DHNmel seems to be commonly biosynthesized by fungi belonging to the Ascomycota phylum whereas basidiomycetes use the l -DOPA pathway or alternative pathways. In some cases, it has been reported that the same fungus can produce two different kinds of melanic pigments, as in the case of Aspergillus fumigatus that biosynthesizes mainly 1,8-DHN melanin and to a minor extent pyomelanin and Agaricus bisporus affording both DOPA and GHB melanins . In other cases, identification of the melanin type is still controversial as for Aspergillus niger, Aspergillus nidulans , Histoplasma capsulatum , and Paracoccidioides brasiliensis for which both the DOPA and the 1,8-DHN pathways have been proposed by independent studies …”

“…In some cases, it has been reported that the same fungus can produce two different kinds of melanic pigments, as in the case of Aspergillus f umigatus that biosynthesizes mainly 1,8-DHN melanin and to a minor extent pyomelanin 13 and Agaricus bisporus affording both DOPA and GHB melanins. 14 In other cases, identification of the melanin type is still controversial as for Aspergillus niger, 15 Aspergillus nidulans, 13 Histoplasma capsulatum, 16 and Paracoccidioides brasiliensis for which both the DOPA and the 1,8-DHN pathways have been proposed by independent studies. 17 This high uncertainty can be traced back to the many difficulties found in isolating in high yields and in pure form the intact pigment due to its strong association with the polysaccharidic matrix of cell walls and in assessing its structure due to its high insolubility.…”

Raman microscopy tracks maturity of melanin intermediates in Botrytis cinerea, a plant pathogen

“…20 Additionally, a Raman difference spectroscopy study, using a near infrared laser at 785 nm, suggested a promising alternative to sample Raman spectra in hyphal specimens: the authors discussed synthesis of DHN melanin in Aspergillus fumigatus and other saprotrophic fungi. 21…”