## 4 Discussion
maydis_ H+ ATPase, mutation of any of these genes did not affect the proton pumping or internal pH changes [31]. Fungal melanin biosynthesis hinges on two primary pathways: the 1,8-dihydroxy naphthalene intermediate (DHN-pathway) and the L-3,4-dihydroxyphenylalanine (L-DOPA pathway) [47, 48]. Both pathways are widespread across bacterial and fungal king-doms [40]. Typically, bacteria and basidiomycetous fungi prefer the L-DOPA pathway, while ascomycetous fungi, and even some non-microscopic counterparts like _Tuber_ spp., favor the DHN-pathway for melanin production [34]. However, _U. maydis_ sets itself apart by showcasing an unconventional melanin production pathway [18]. Researchers have identified a gene cluster associated with melanin production in this fungus. This cluster comprises five pivotal enzymes, including three polyketide synthases (pks3, pks4, and pks5), a cytochrome p540 monooxygenase, and a protein akin to versicolor synthase B (vbs1). The deletion of this gene cluster indicated that _U. maydis_ employs an alternative mechanism for polyketide synthesis that ultimately leads to the formation of DHN-type melanin. This is in stark contrast to ascomycetes like _Aspergillus flavus_, _A. niger_, and _A. tamarii_, which primarily employ the L-DOPA pathway for melanin synthesis, or others like _A. tubingensis_ and _A. terreus_, which opt for the DHN pathway [40]. On the other hand, _Cryptococcus neoformans_ utilizes the L-DOPA pathway, akin to specific bacteria and mammals [49; 50]. Generally, microbial melanin synthesis predominantly follows the L-DOPA pathway, utilizing tyrosine as a precursor. Most Ascomycata favor the DHN melanin pathway, while basidiomycetes produce L-DOPA melanin [50]. Interestingly, _U. maydis_'s ability to coexist with both melanin synthesis pathways, akin to _Aspergillus_ spp., adds to the intrigue [40]. Our results underscore that a significant portion of the accumulated melanin in _U. maydis_ originates from either tyrosine or L-DOPA as the substrate. Tyrosinases (EC 1.14.18.1) have been isolated and characterized from plants, animals, and microorganisms [51]. The enzyme catalyzes two types of reactions: (a) as monophechnase, hydroxylating monophenchols (e.g., L-tyrosine) to O-diphenylnels (e.g., L-DOPA), and (b) as diphenolase, oxidizing O-diphenylnels to O-DOPAquinone [51]. The subsequent reactions need two molecules of O-DOPAquinone to generate L-DOPA and dopochrome molecules. In most cases, the synthesis of melanin depends on the L-DOPA pathway, in which tyrosinase is one of the key enzymes [51]. To examine if the melanin biosynthesis in _U. maydis_ follows this pathway, we measured the tyrosine activity in wild-type and mutant cells grown for 72 h in the crude extract. Our results indicated that the highest activity of tyrosinase was found in the \(\Delta\)PMA1 strain, which also has the melanin accumulated with a preference for tyrosine substrate. This difference is obtained regardless of the culture medium (YPD or MM). It has been reported that tyrosinase activity requires metal ions to reach activity [52; 53]. We explored the effect of different ions (Mg\({}^{2+}\), Cu\({}^{2+}\), Zn\({}^{2+}\), Co\({}^{2+}\), and Fe\({}^{2+}\)). Remarkably, a noticeable difference in the tyrosinase activity was found when tyrosine or L-DOPA were used as a substrate. Metal ions also activate the tyrosinase activity in both substrates, but the higher activation was obtained with tyrosine Cu\({}^{+2}\), Co\({}^{2+}\), and Zn\({}^{2+}\), which were found to be good cofactors for the _U. maydis_ enzyme. The majority of the tyrosinases accept Cu\({}^{2+}\) and Zn\({}^{2+}\) as cofactors. However, the effect of Co\({}^{2+}\) was unexpected because, for most of the tyrosinases, this metal ion does not function as an essential activator. Finally, the melanized cells carrying the point mutation had a higher capacity for the removal of methylene blue. The dye was inside the cells, and the melanin may have been saturated. The use of a chitosan-based biosorbent with immobilized microfungal spores of _U.