Napthrene Compounds from Mycelial Fermentation Products of Marasmius berteroi.

The metabolites of the genus Marasmius are diverse, showing good research prospects for finding new bioactive molecules. In order to explore the active metabolites of the fungi Marasmius berteroi, the deep chemical investigation on the bioactive compounds from its cultures was undertaken, which led to the isolation of three new naphthalene compounds dipolynaphthalenes A-B (1,2) and naphthone C (3), as well as 12 known compounds (4-15). Compounds 1, 2, and 4 are dimeric naphthalene compounds. Their structures were elucidated by MS, 1D and 2D NMR spectroscopic data, as well as ECD calculations. Compounds 2-4 and 7 exhibited acetylcholinesterase (AChE) inhibitory activities at the concentration of 50 µg/mL with inhibition ratios of 42.74%, 44.63%, 39.50% and 51.49%, respectively. Compounds 5 and 7,8 showed weak inhibitory activities towards two tumor cell lines, with IC50 of 0.10, 0.076 and 0.058 mM (K562) and 0.13, 0.18, and 0.15 mM (SGC-7901), respectively.

Family of Papain-Like Fungal Chimerolectins with Distinct Ca2+-Dependent Activation Mechanism.

An important function of fungal lectins is to protect their host. Marasmius oreades agglutinin (MOA) is toxic to nematodes and exerts its protective effect through protease activity. Its proteolytic function is associated with a papain-like dimerization domain. The closest homologue of MOA is Polyporus squamosus lectin 1a (PSL1a). Here, we probed PSL1a for catalytic activity and confirmed that it is a calcium-dependent cysteine protease, like MOA. The X-ray crystal structures of PSL1a (1.5 Å) and MOA (1.3 Å) in complex with calcium and the irreversible cysteine protease inhibitor E-64 elucidated the structural basis for their mechanism of action. The comparison with other calcium-dependent proteases (calpains, LapG) reveals a unique metal-dependent activation mechanism relying on a calcium-induced backbone shift and intradimer cooperation. Intriguingly, the enzymes appear to use a tyrosine-gating mechanism instead of pro-peptide processing. A search for potential MOA orthologues suggests the existence of a whole new family of fungal chimerolectins with these unique features.

Optimization of Physiochemical Parameters during Bioremediation of Synthetic Dye by Marasmius cladophyllus UMAS MS8 Using Statistical Approach.

In many industrial areas such as in food, pharmaceutical, cosmetic, printing, and textile, the use of synthetic dyes has been integral with products such as azo dye, anthrax, and dyestuffs. As such, these industries produce a lot of waste by-products that could contaminate the environment. Bioremediation, therefore, has become an important emerging technology due to its cost-sustainable, effective, natural approach to cleaning up contaminated groundwater and soil via the use of microorganisms. The use of microorganisms in bioremediation requires the optimisation of parameters used in cultivating the organism. Thus the aim of the work was to assess the degradation of Remazol Brilliant Blue R (RBBR) dye on soil using Plackett-Burman design by the basidiomycete, M. cladophyllus UMAS MS8. Biodegradation analyses were carried out on a soil spiked with RBBR and supplemented with rice husk as the fungus growth enhancer. A two-level Plackett-Burman design was used to screen the medium components for the effects on the decolourization of RBBR. For the analysis, eleven variables were selected and from these four parameters, dye concentration, yeast extract concentration, inoculum size, and incubation time, were found to be most effective to degrade RBBR with up to 91% RBBR removal in soil after 15 days.

Steroid hydroxylation by basidiomycete peroxygenases: a combined experimental and computational study.

The goal of this study is the selective oxyfunctionalization of steroids under mild and environmentally friendly conditions using fungal enzymes. With this purpose, peroxygenases from three basidiomycete species were tested for the hydroxylation of a variety of steroidal compounds, using H2O2 as the only cosubstrate. Two of them are wild-type enzymes from Agrocybe aegerita and Marasmius rotula, and the third one is a recombinant enzyme from Coprinopsis cinerea. The enzymatic reactions on free and esterified sterols, steroid hydrocarbons, and ketones were monitored by gas chromatography, and the products were identified by mass spectrometry. Hydroxylation at the side chain over the steroidal rings was preferred, with the 25-hydroxyderivatives predominating. Interestingly, antiviral and other biological activities of 25-hydroxycholesterol have been reported recently (M. Blanc et al., Immunity 38:106-118, 2013, http://dx.doi.org/10.1016/j.immuni.2012.11.004). However, hydroxylation in the ring moiety and terminal hydroxylation at the side chain also was observed in some steroids, the former favored by the absence of oxygenated groups at C-3 and by the presence of conjugated double bonds in the rings. To understand the yield and selectivity differences between the different steroids, a computational study was performed using Protein Energy Landscape Exploration (PELE) software for dynamic ligand diffusion. These simulations showed that the active-site geometry and hydrophobicity favors the entrance of the steroid side chain, while the entrance of the ring is energetically penalized. Also, a direct correlation between the conversion rate and the side chain entrance ratio could be established that explains the various reaction yields observed.

A free cyanohydrin as arms and armour of Marasmius oreades.

Cyanogenic plants and fungi are widespread in nature. Although the origin of hydrocyanic acid in plants has been studied in detail, little is known about its origin in fungi. Here, we report the identification of the cyanohydrin of glyoxylic acid as the precursor of hydrocyanic acid in the fungus Marasmius oreades and several other cyanogenic fungi. Moreover, a feeding experiment revealed glycine as biosynthetic precursor of the cyanohydrin of glyoxylic acid. Thus, the cyanogenesis of M. oreades and other fungi is fundamentally different from cyanogenesis in plants.

Mercury in the fairy-ring of Gymnopus erythropus (Pers.) and Marasmius dryophilus (Bull.) P. Karst. mushrooms from the Gongga Mountain, Eastern Tibetan Plateau.

Gongga Mountain or Minya Konka, like the Himalayan Dimension Mountains, has its own microclimate and a 'circum-polar' climate and hence is sensitive to contamination by persistent pollutants that are trapped by cold temperature and wet precipitation. Elemental mercury (Hg) as vapour easy diffuses into the atmosphere and the rate of Hg deposition from global fallout is dependent on locally ambient temperature and precipitation. We investigated the accumulation and distribution of total Hg in two species of mushrooms, Gymnopus erythropus and Marasmius dryophilus, which grew on Gongga Mountain. The fruiting bodies were collected at a height of 2946m above see level. Both species efficiently accumulated Hg. The median values for caps of M. dryophilus and G. erythropus were 1.168 and 3.078, and for stipes 0.573 and 1.636mg/kg dry matter, respectively, and in the beneath litter and soil were 0.13 and 0.15mg/kg dry matter. The Hg contents of the caps of M. dryophilus and the beneath litter and soils from pristine Himalayan forest of 1.168, 0.132 and 0.116mg/kg dry matter (respectively) is high compared to values reported for similar species and soils from background areas in Poland -0.58-0.70 and 0.047-0.048mg/kg dry matter. The absence of industrial activities, urbanization and Hg ore deposits at Gongga Mountain suggests that long-range atmospheric transport and subsequent deposition is the major source of elevated Hg observed in the mushrooms, litter and surface layer of soils in the outskirts of Gongga Mountain maritime glacier that has a peak of 7556m above sea level.

Characterization and cloning of GNA-like lectin from the mushroom Marasmius oreades.

A new mannose-recognizing lectin (MOL) was purified on an asialofetuin-column from fruiting bodies of Marasmius oreades grown in Japan. The lectin (MOA) from the fruiting bodies of the same fungi is well known to be a ribosome-inactivating type lectin that recognizes blood-group B sugar. However, in our preliminary investigation, MOA was not found in Japanese fruiting bodies of M. oreades, and instead, MOL was isolated. Gel filtration showed MOL is a homodimer noncovalently associated with two subunits of 13 kDa. The N-terminal sequence of MOL was blocked. The sequence of MOL was determined by cloning from cDNA and by protein sequencing of enzyme-digested peptides. The sequence shows mannose-binding motifs of bulb-type mannose-binding lectins from plants, and similarity to the sequences. Analyses of sugar-binding specificity by hemagglutination inhibition revealed the preference of MOL toward mannose and thyroglobulin, but asialofetuin was the strongest inhibitor of glycoproteins tested. Furthermore, glycan-array analysis showed that the specificity pattern of MOL was different from those of typical mannose-specific lectins. MOL preferred complex-type N-glycans rather than high-mannose N-glycans.

Nematotoxicity of Marasmius oreades agglutinin (MOA) depends on glycolipid binding and cysteine protease activity.

Fruiting body lectins have been proposed to act as effector proteins in the defense of fungi against parasites and predators. The Marasmius oreades agglutinin (MOA) is a Galα1,3Gal/GalNAc-specific lectin from the fairy ring mushroom that consists of an N-terminal ricin B-type lectin domain and a C-terminal dimerization domain. The latter domain shows structural similarity to catalytically active proteins, suggesting that, in addition to its carbohydrate-binding activity, MOA has an enzymatic function. Here, we demonstrate toxicity of MOA toward the model nematode Caenorhabditis elegans. This toxicity depends on binding of MOA to glycosphingolipids of the worm via its lectin domain. We show further that MOA has cysteine protease activity and demonstrate a critical role of this catalytic function in MOA-mediated nematotoxicity. The proteolytic activity of MOA was dependent on high Ca(2+) concentrations and favored by slightly alkaline pH, suggesting that these conditions trigger activation of the toxin at the target location. Our results suggest that MOA is a fungal toxin with intriguing similarities to bacterial binary toxins and has a protective function against fungivorous soil nematodes.

Alkylphenol oxidation with a laccase from a white-rot fungus: effects of culture induction and of ABTS used as a mediator.

We investigated the potential of the laccase from the white-rot fungus Marasmius quercophilus to transform certain alkylphenols (p-nonylphenol, p-octylphenol and p-t-octylphenol). We tested the reactivity of this enzyme under different conditions: in liquid cultures and using the partially purified laccase with and without 2,2'-azino-bis-3-ehtylbenzothiazoline-6-sulfonicacid (ABTS) as a mediator. The percentage of p-t-octylphenol disappearance in liquid cultures was 69.0 ± 1.5% and 81 ± 5% after a 8-d or 15-d incubation, respectively, with p-nonylphenol, these percentages were 62 ± 4% and 91 ± 6% and with p-octylphenol 37 ± 3% and 65 ± 1% after a 15-d and a 21-d incubations, respectively. Induced pre-cultures were also used to inoculate the liquid cultures to enhance p-octylphenol transformation: the percentages of disappearance were 91.0 ± 0.5% and 97 ± 1% after a 8-d and a 15-d incubation, respectively. Mass spectrometry analysis showed that the products of oxidation of p-octylphenol were dimers with a mass of 411 m/z. Furthermore, we identified a purple compound (m/z 476) formed when ABTS was added to the reaction medium with the purified laccase. This result confirms that, in complex environments such as soils or litters where many molecules can interact with the enzyme substrate or the product of oxidation, laccase activities and those of other phenoloxidases should not be measured with ABTS.