Effect of melanin on enzymatic hydrolysis of cellulosic waste

Wood waste powder from Tectona grandis containing melanin was less susceptible to enzymatic hydrolysis than powder without melanin. About a 53% increase in saccharification was noted when melanin was removed. Melanin caused inhibition to all cellulolytic enzymes, but in different degrees. Endo-beta-1,4-glucanase and beta-glucosidase were markedly inhibited when melanin was preincubated with enzyme, while exo-beta-1,4-glucanase was severely inhibited when melanin was preincubated with substrate. The latter was found to be dependent on the contact time. The activities of endo-beta-1,4-glucanase and beta-glucosidase were noncompetitively inhibited by melanin.     

Incorporation and binding of estrogens into melanin: comparison of mushroom and mammalian tyrosinases.

The activities of mushroom and melanoma tyrosinases towards the estrogens were compared. While the fungal enzyme is capable of hydroxylating estradiol to the 2-hydroxy compound and to oxidize the latter to the quinone, the mammalian enzyme does not have this ability. With dopa as substrate and an estrogen present in the reaction mixture, both enzyme reactions yield melanin with the steroid firmly incorporated into the pigment, although with the mammalian enzyme the incorporation is small. The steroid appears to be incorporated by covalent linkage. It is suggested that the incorporation of estrogens into melanin produced by mammalian tyrosinase is via their oxidation by oxidized intermediates of the dopa to melanin transformation. Melanin itself may function as oxidant for the estrogens. Whole melanoma cells are capable of binding estrogens and incorporating small amounts into melanosomes. Similarly, fresh melanosomes in isolation can incorporate estrogens into their structure, presumably by covalent bonding to their melanin.     

Synthesis and assembly of fungal melanin

Melanin is a unique pigment with myriad functions that is found in all biological kingdoms. It is multifunctional, providing defense against environmental stresses such as ultraviolet (UV) light, oxidizing agents and ionizing radiation. Melanin contributes to the ability of fungi to survive in harsh environments. In addition, it plays a role in fungal pathogenesis. Melanin is an amorphous polymer that is produced by one of two synthetic pathways. Fungi may synthesize melanin from endogenous substrate via a 1,8-dihydroxynaphthalene (DHN) intermediate. Alternatively, some fungi produce melanin from l-3,4-dihydroxyphenylalanine (l-dopa). The detailed chemical structure of melanin is not known. However, microscopic studies show that it has an overall granular structure. In fungi, melanin granules are localized to the cell wall where they are likely cross-linked to polysaccharides. Recent studies suggest the fungal melanin may be synthesized in internal vesicles akin to mammalian melanosomes and transported to the cell wall. Potential applications of melanin take advantage of melanin's radioprotective properties and propensity to bind to a variety of substances.     

The function of melanin in the ectomycorrhizal fungus Cenococcum geophilum under water stress

Despite the ubiquity and importance of ectomycorrhizal fungi to ecosystem function, our understanding of their functional ecology remains poor. The highly melanized and common ectomycorrhizal fungus, Cenococcum geophilum, is drought tolerant and abundant in water-stressed habitats, yet the responsible functional traits have not been identified. The production of melanin, a class of complex dark polymers found in fungal cell walls, may be a key functional trait to water stress tolerance. To test this hypothesis, we devised a series of experiments determining the effect of the melanin biosynthesis inhibitor, tricyclazole, on response to osmotic and desiccation stresses. Melanin inhibition only had negative effects on growth when C. geophilum isolates were subjected to osmotic and desiccation stress (?1.7 MPa and desiccated) but not under control conditions (?0.01 MPa and non-desiccated). This suggests that melanin production is an important functional trait that contributes to water stress tolerance of this cosmopolitan ectomycorrhizal fungus.     

Isolation and Characterization of a Galactosamine Wall from Spores and Spherules of Physarum polycephalum

The myxomycete, Physarum polycephalum, can be induced under laboratory conditions to form two different hard-walled forms, spores and spherules. Characterization of both types of walls revealed only a single sugar, galactosamine. It was identified after acid hydrolysis of the isolated walls by chromatography in three solvent systems, by its positive reaction with ammoniacal silver nitrate, ninhydrin, Galactostat, and the Elson-Morgan test, and by ninhydrin degradation to lyxose. Galactosamine was present as a polymer with solubility characteristics the same as the β1-4–linked glucosamine polymer (chitosan). The walls were also found to contain about 2% protein. Spherule walls revealed a single glycoprotein on gel electrophoresis. Spore walls contained a similar protein component. The phosphate content of isolated spherule walls was 9.8%, and that of spore walls was 1.4%. Spore walls also contained about 15% melanin which was shown to be similar to fungal melanin. A novel method was used to measure the rate of mature spherule formation based on the loss of extractability of P. polycephalum natural pigment. The presence of a rare galactosamine polymer in P. polycephalum spore and spherule walls as the only carbohydrate suggests that the myxomycetes are not closely related to the fungi or the protozoa.     

Cryptococcus neoformans melanization incorporates multiple catecholamines to produce polytypic melanin

Melanin is a major virulence factor in pathogenic fungi that enhances the ability of fungal cells to resist immune clearance. Cryptococcus neoformans is an important human pathogenic fungus that synthesizes melanin from exogenous tissue catecholamine precursors during infection, but the type of melanin made in cryptococcal meningoencephalitis is unknown. We analyzed the efficacy of various catecholamines found in brain tissue in supporting melanization using animal brain tissue and synthetic catecholamine mixtures reflecting brain tissue proportions. Solid-state NMR spectra of the melanin pigment produced from such mixtures yielded more melanin than expected if only the preferred constituent dopamine had been incorporated, suggesting uptake of additional catecholamines. Probing the biosynthesis of melanin using radiolabeled catecholamines revealed that C. neoformans melanization simultaneously incorporated more than one catecholamine, implying that the pigment was polytypic in nature. Nonetheless, melanin derived from individual or mixed catecholamines had comparable ability to protect C. neoformans against ultraviolet light and oxidants. Our results indicate that melanin produced during infection differs depending on the catecholamine composition of tissue and that melanin pigment synthesized in vivo is likely to accrue from the polymerization of a mixture of precursors. From a practical standpoint, our results strongly suggest that using dopamine as a polymerization precursor is capable of producing melanin pigment comparable to that produced during infection. On a more fundamental level, our findings uncover additional structural complexity for natural cryptococcal melanin by demonstrating that pigment produced during human infection is likely to be composed of polymerized moieties derived from chemically different precursors.     

Unusual kinetic behavior of Endothia parasitica protease in hydrolysis of small peptides

Endothia parasitica protease hydrolyzes l-leucyl-l-leucine amide and l-leucyl-l-phenylalanine amide at the peptide bond. l-Phenylalanyl-l-leticine amide, N-carbobenzoxy-l-leucyl-l-phenylalanine amide, N-carbobenzoxy-l-leucyl-l-pheml-alanine, N-carbobenzoxy-l-phenylalanyl-l-valine amide, and l-leucyl-β-naphthyl-amide are not hydrolyzed. In contrast to the kinetics of hydrolysis of casein and oxidized B-chain of insulin and activation of trypsinogen by Endothia parasitica protease which are normal, reaction progress curves for hydrolysis of l-leucyl-l-leucine amide and l-leucyl-l-phenylalanine amide are sigrnoidal. Initially, the reaction rates were of the order of 0.5–2.5% of the maximum rates eventually attained. With increasing time of incubation the reaction rates became faster and faster until maximum rates were achieved. This abnormal behavior was not eliminated by recrystallization of substrate or by incubation of enzyme alone or with products of the reaction prior to addition of substrate. Addition of a new aliquot of substrate, vizl-leucyl-l-leucine amide, to the reaction prior to complete hydrolysis of all of a previous aliquot of the same substrate, or reactions containing a mixture of oxidized B chain of insulin and l-leucyl-l-leucine amide, gave normal reaction progress curves. The duration of abnormal behavior before a maximum rate was attained was a function of enzyme concentration and temperature but not of substrate concentration even though substrate was in less than saturating amounts. The reaction data follow second-order autocatalytic kinetics with respect to enzyme concentration. It is proposed that most of the enzyme is in an inactive form in absence of substrate but is rapidly converted to the active form on combination with a good substrate such as trypsinogen, casein, or oxidized B chain of insulin. However, with a poor substrate such as l-leucyl-l-leucine amide, conversion to active enzyme is mediated through formation of an active enzyme-inactive enzyme complex followed by combination with substrate and hydrolysis.     

植物病原真菌黑色素与致病性关系的研究进展

黑色素是一类生物聚合分子的总称,不同来源的黑色素种类不同,其中报道较多的是DOPA黑色素和DHN黑色素。DOPA黑色素和DHN黑色素具有相似的理化性质但其合成底物和途径不同。DHN黑色素在植物病原直菌中广泛存在,与病原菌致病能力密切相关。病原菌侵染寄主时黑色素沉积存附着胞细胞壁的内层,防止了形成膨压的溶质渗透到细胞外,产生很大的机械压力,保证病原菌侵入寄主。结合作者的研究结果综述了黑色素的种类、性质及黑色素与病原菌致病性关系等方面的研究进展。     

Pentaketide metabolites of melanin synthesis in the dematiaceous fungus Wangiella dermatitidis

Melanin synthesis in the dematiaceous, polymorphic hyphomycete Wangiella dermatitidis, a human pathogen, was investigated by biochemical and physiological techniques. Mutants with a decrease or loss in melanin synthesis were induced and isolated. Melanin precursors were obtained from the mutants, purified, and then identified by comparison with authentic compounds from Verticillium dahliae. Isolation of scytalone, vermelone, flaviolin, and 1,8-dihydroxynaphthalene from the mutants of Wangiella dermatitidis, and cross-feeding of the mutants with those of Verticillium dahliae indicated that melanin synthesis in this organism took place by the pentaketide pathway. Melanin that formed in cell walls of an albino mutant treated with scytalone was identical in appearance to that in cell walls of the wild-type strain. This also suggested that pentaketide synthesis of melanin occurred in the fungus.     

Preparation and Characterization of <Emphasis Type="Italic">Alphitobius diaperinus</Emphasis> Melanin

Melanin with a high antioxidant and sorption activity comparable to that of synthetic dioxyphenylalanine (DOPA)-melanin was isolated from the biomass of the darkling beetle Alphitobius diaperinus. The pigment was extracted with a solution of potassium hydroxide, followed by precipitation with concentrated hydrochloric acid and hydrolysis of the resulting precipitate with the same acid. The electron paramagnetic resonance (EPR) signal of melanin was characteristic of eumelanins with a spin concentration of 4.9 × 10¹⁷ spin per 1 g of dry weight. The melanin concentration that induced 50% inhibition of peroxidation was 9.2 μg/mL (the analogous concentration of DOPA-melanin was 8.0 μg/mL). The maximum of methylene-blue binding to the beetle melanin was 700 mg of dye per 1 g of dry weight of the preparation. The lipid-free melanin preparation exhibited antiradical activity.     

Glycohydrolase contamination of commercial enzymes frequently used in the preparation of fungal cell walls

Commercial enzyme preparations frequently used in the preparation of fungal cell walls, viz., proteases, a lipase, and a phosphatase, were examined for the presence of contaminating glycohydrolase activity, since such activity could result not only in the removal of cytoplasmic constituents but also in the removal of portions of the wall itself. Glucosidase activities were detected in a protease of fungal origin, in a lipase from wheat germ, and in a phosphatase from potatoes. Additionally, two commercial protease preparations from Streptomyces griseus contained β-1,3-glucanase activity in significant amounts, a third contained trace amounts of the glucanase, but a fourth was totally free of glycohydrolase activity. The protease preparations from S. griseus released laminaribiose from yeast-phase cell walls of Histoplasma capsulatum chemotypes I and II, but only trace amounts of glucose were released. One protease was examined more closely and was found to be optimally active on laminarin at pH 5.5 and 50°C. It was also highly active on the same substrate at pH 8.0 and 37°C, however. A protease preparation from Aspergillus oryzae released glucose from the yeast-phase cell walls of H. capsulatum chemotypes I and II as well as from cell walls of Blastomyces dermatitidis, suggesting that the preparation contained both α- and β-glucanases.     

Sensitivity of Aspergillus nidulans to the Cellulose Synthase Inhibitor Dichlobenil: Insights from Wall-Related Genes’ Expression and Ultrastructural Hyphal Morphologies

The fungal cell wall constitutes an important target for the development of antifungal drugs, because of its central role in morphogenesis, development and determination of fungal-specific molecular features. Fungal walls are characterized by a network of interconnected glycoproteins and polysaccharides, namely α-, β-glucans and chitin. Cell walls promptly and dynamically respond to environmental stimuli by a signaling mechanism, which triggers, among other responses, modulations in wall biosynthetic genes’ expression. Despite the absence of cellulose in the wall of the model filamentous fungus Aspergillus nidulans, we found in this study that fungal growth, spore germination and morphology are affected by the addition of the cellulose synthase inhibitor dichlobenil. Expression analysis of selected genes putatively involved in cell wall biosynthesis, carried out at different time points of drug exposure (i.e. 0, 1, 3, 6 and 24 h), revealed increased expression for the putative mixed linkage β-1,3;1,4 glucan synthase celA together with the β-1,3-glucan synthase fksA and the Rho-related GTPase rhoA. We also compared these data with the response to Congo Red, a known plant/fungal drug affecting both chitin and cellulose biosynthesis. The two drugs exerted different effects at the cell wall level, as shown by gene expression analysis and the ultrastructural features observed through atomic force microscopy and scanning electron microscopy. Although the concentration of dichlobenil required to affect growth of A. nidulans is approximately 10-fold higher than that required to inhibit plant cellulose biosynthesis, our work for the first time demonstrates that a cellulose biosynthesis inhibitor affects fungal growth, changes fungal morphology and expression of genes connected to fungal cell wall biosynthesis.     

植物病原真菌1,8-间苯二酚黑色素研究进展

黑色素是一种广泛分布于生物体中的酚类聚合物疏水色素,分为1,8-间苯二酚(1,8-dihydroxynaphthalene,DHN)黑色素和3,4-二羟基苯丙氨酸(3,4-dihydroxyphenylalanine,L-DOPA)黑色素两种,其中DHN黑色素多存在于子囊菌门的植物病原真菌中。基因组和转录组技术的发展及功能基因组研究的深入,使DHN黑色素合成途径上关键基因在不同病原真菌中被鉴定,而且黑色素与真菌抗逆、发育和致病的关系受到越来越多的关注。本文阐述了DHN黑色素合成途径及其在真菌抗辐射与抗极端温度中的作用,以及黑色素对真菌侵染和细胞发育的影响,旨在加深人们对黑色素介导真菌与环境和寄主协同进化的认识,这对黑色素的基础研究和开发利用具有重要意义。     

Role of Melanin in Release of Extracellular Enzymes and Selection of Aggressive Isolates of Bipolaris sorokiniana in Barley

Eighteen barley isolates of Bipolaris sorokiniana belonging to wild and clonal type of black, mixed and white subpopulations were quantitatively assayed for their melanin content and aggressiveness with respect to production of some of the extracellular enzymes such as cellulase, pectinase, amylase and protease. Cellulase and pectinase constituted major portion of the enzymes recovered from the black, mixed and white isolates. Enzyme production and aggressiveness were relatively higher in melanin devoid or low melanin isolates. The melanin deficient isolates were also differentiated from black and mixed isolates on the basis of variation in internal transcribed spacer region of the ribosomal DNA. Higher enzyme productions positively correlated with area under disease progress curve (AUDPC) and lesion development. Melanin content was negatively correlated with extracellular enzymes and aggressiveness of the isolates. Based on melanin content, lesion size, AUDPC and extracellular enzymes, the isolates were grouped in two major clusters (I and II) with further division of cluster II into two sub-clusters (II-A and II-B). The results appears to indicate a possible role of melanin in release of extracellular enzymes and hence in evolution and selection of aggressive isolates of B. sorokiniana in barley.     

Melanin and HIV in sub-Saharan Africa

HIV is common in sub-Saharan Africa. Sexually transmitted bacterial and fungal infections increase the chance of HIV infection. Melanin can prevent the penetration of skin and mucus membranes by microorganisms, and soluble melanin can inhibit HIV replication. We suggest that melanin may reduce the incidence of HIV infection through venereally acquired skin lesions, thus reducing the risk of sero-conversion and slow the progress to AIDS. Indigenous sub-Saharan peoples are highly melanized, but there is pigment variation between populations. We show that skin reflectance, a negative correlate of melanin, is positively associated with adult rate of HIV in sub-Saharan countries. There is no such relationship in populations outside sub-Saharan Africa. We suggest that melanin concentration in black people may correlate with resistance to HIV infection.     

Metal loading and enzymatic degradation of fungal cell walls and chitin

The capacity of chitin (from crab shells) and of fungal cell walls from Trichoderma harzianum to accumulate zinc, cadmium and mercury was studied as well as the effects of adsorbed metals on the enzymatic hydrolysis by Novozym 234 of the two substrates. The total adsorbing capacity with respect to these metals was estimated to be at least 10 mmol kg^–1 chitin (dry weight) and 50 mmol kg^–1 fungal cell walls (dry weight), respectively, at pH 6.1. Enzymatic digestion of fungal cell walls preloaded with mercury and cadmium was significantly reduced, while zinc did not cause any significant inhibition. The effect of metal complexation by chitin on the enzymatic digestion was not as pronounced as for fungal cell walls. This could reflect the fact that chitin sorbed a lower total amount of metals. The inhibitory effect of metals on the enzymatic hydrolysis was caused by the association of the metals with the two substrates and not by the presence of free metals in solution.     

Electron transfer properties of melanin.

Melanin synthesized from mushroom tyrosinase and 3,4-dihydroxyphenylalanine has been shown to oxidize NADH and NADPH, reduce ferricyanide, oxidized forms of cytochrome c and dichlorophenolindophenol, and catalyze the coupled oxidation of NADH and reduction of ferricyanide. Kinetic studies involving the determination of initial velocity at various concentrations of substrates and product inhibition measurements have been carried out on the NADH-ferricyanide-melanin reaction. The results are consistent with a ping-pong mechanism in which one product is formed prior to the reaction of melanin with the second substrate involving the reversible oxidation and reduction of melanin during the reaction. It may be concluded that melanin is capable of acting as an electron transfer agent in several reduction-oxidation systems.     

Cryptococcus neoformans Can Utilize the Bacterial Melanin Precursor Homogentisic Acid for Fungal Melanogenesis

Cryptococcus neoformans melanizes in the environment and in mammalian tissues, but the process of melanization in either venue is mysterious given that this microbe produces melanin only from exogenous substrates. Understanding the process of melanization is important because melanization is believed to protect against various stresses in the environment, including UV radiation, and pigment production is associated with virulence. Melanization in C. neoformans requires the availability of diphenolic precursors. In contrast, many bacteria synthesize melanin from homogentisic acid (HGA). We report that C. neoformans strains representing all four serotypes can produce a brown pigment from HGA. The brown pigment was acid resistant and had the electron paramagnetic resonance spectrum of a stable free radical, qualities that identified it as a melanin. Melanin “ghost”-like particles obtained from pigmented C. neoformans cells were hydrophobic, fluorescent under a variety of irradiation wavelengths, negatively charged, insoluble in organic solvents and alcohols, resistant to degradation by strong acids, and vulnerable to bleaching. HGA melanization was laccase dependent and repressed by high concentrations of glucose. The ability of C. neoformans to utilize a bacterial melanin precursor compound suggests a new substrate source for melanization in the environment.     

Growth conditions influence melanization of Brazilian clinical Sporothrix schenckii isolates

Sporothrix schenckii is known to produce DHN melanin on both conidial and yeast cells, however little information is available regarding the factors inducing fungal melanization. We evaluated whether culture conditions influenced melanization of 25 Brazilian S. schenckii strains and one control strain (ATCC 10212). Tested conditions included different media, pH, temperature, incubation time, glucose concentrations, and presence or absence of tricyclazole or L-DOPA. Melanization was reduced on Sabouraud compared to defined chemical medium. The majority of strains produced small amounts of melanin at 37 °C and none melanized at basic pH. Increased glucose concentrations did not inhibit melanization, rather increasing glucose enhanced pigment production in 27% of strains. Melanin synthesis was also enhanced by the addition of L-DOPA and its addition to medium with tricyclazole, an inhibitor of melanin synthesis, resulted in fungal melanization, including hyphal melanin production. Our results suggest that different S. schenckii strains have distinct control of melanization and that this fungus can use phenolic compounds to enhance melanization in vitro.     

Elucidation of Substrate Specificity in Aspergillus nidulans UDP-Galactose-4-Epimerase

The frequency of invasive fungal infections has rapidly increased in recent years. Current clinical treatments are experiencing decreased potency due to severe host toxicity and the emergence of fungal drug resistance. As such, new targets and their corresponding synthetic pathways need to be explored for drug development purposes. In this context, galactofuranose residues, which are employed in fungal cell wall construction, but are notably absent in animals, represent an appealing target. Herein we present the structural and biochemical characterization of UDP-galactose-4-epimerase from Aspergillus nidulans which produces the precursor UDP-galactopyranose required for galactofuranose synthesis. Examination of the structural model revealed both NAD⁺ and UDP-glucopyranose were bound within the active site cleft in a near identical fashion to that found in the Human epimerase. Mutational studies on the conserved catalytic motif support a similar mechanism to that established for the Human counterpart is likely operational within the A. nidulans epimerase. While the K _m and k _cat for the enzyme were determined to be 0.11 mM and 12.8 s^-1, respectively, a single point mutation, namely L320C, activated the enzyme towards larger N-acetylated substrates. Docking studies designed to probe active site affinity corroborate the experimentally determined activity profiles and support the kinetic inhibition results.