Characterization of a metalloenzyme from a wild mushroom, Tricholoma saponaceum.
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Two kinds of metalloendopeptidases from the fruiting bodies of Tricholoma saponaceum (TSMEP1 and TSMEP2) have been purified, and TSMEP1 has been characterized based on their fibrinolytic activity. The enzymes have the same N-terminal amino acid sequence, Ala-Leu-Tyr-Val-Gly-X-Ser-Pro-X-Gln-Gln-Ser-Leu-Leu-Val, but slightly different molecular weights of 18,147 and 17,947, as measured by matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. The N-terminal sequence do not match with any known protein or open reading frame. TSMEP1 hydrolyzes fibrinogen as well as fibrin, but does not show any proteolytic activity for other blood proteins such as thrombin, human albumin, human IgG, hemoglobin, or urokinase. The enzyme hydrolyzes both A alpha and B beta subunits of human fibrinogen with equal efficiency but didn't show any reactivity for the gamma form of human fibrinogen. The enzymatic activity is strongly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzymes are metalloproteases. No inhibition was found with phenylmethylsulfonyl fluoride (PMSF), L-trans-epoxysuccinyl leucylamido-(4-guanidino)-butane (E-64), pepstatin and 2-mercaptoethanol. The activity of the purified enzyme was increased by Mg2+, Fe2+, Zn2+, and Co2+, and slightly decreased by Ca2+, but the enzyme activity was dramatically decreased by Cu2+, and totally inhibited by Hg2+. It has broad substrate specificity for synthetic peptides, and keep the high activity from pH 7.5 to 9, suggesting that the purified enzyme was a basic protease. The enzyme was stable up to 30 degrees C and the maximum fibrinolytic activity was at 55 degrees C. (+info)
Purification and characterization of an aminopeptidase from the edible basidiomycete Grifola frondosa.
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An aminopeptidase was purified 178-fold from an extract of Grifola frondosa by ammonium sulfate precipitation and a series of column chromatographies on phenyl-Toyopearl, Sephadex G-25, and Mono-Q. The molecular mass of the enzyme was estimated to be 27 kDa and 30 kDa by gel filtration and SDS-PAGE, respectively. The enzyme had an optimum pH of 8.5 and was stable between pH 6.0 and pH 10.5, and it also had a high level of heat stability. The enzyme was inactivated by EDTA and o-phenanthroline, and it was also strongly inhibited by bestatin, but no inhibitory effect of DFP was observed. The enzyme preferentially hydrolyzed peptides containing hydrophobic residues in the N-terminal position. (+info)
Isolation and characterization of a symbiosis-regulated ras from the ectomycorrhizal fungus Laccaria bicolor.
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Ectomycorrhizae formed by the symbiotic interaction between ectomycorrhizal fungi and plant roots play a key role in maintaining and improving the health of a wide range of plants. Mycorrhizal initiation, development, and functional maintenance involve morphological changes that are mediated by activation and suppression of several fungal and plant genes. We identified a gene, Lbras, in the ectomycorrhizal fungus Laccaria bicolor that belongs to the ras family of genes, which has been shown in other systems to be associated with signaling pathways controlling cell growth and proliferation. The Lbras cDNA complemented ras2 function in Saccharomyces cerevisiae and had the ability to transform mammalian cells. Expression of Lbras, present as a single copy in the genome, was dependent upon interaction with host roots. Northern analysis showed that expression was detectable in L bicolor 48 h after interaction as well as in the established mycorrhizal tissue. Phylogenetic analysis with other Ras proteins showed that Lbras is related most closely to Aras of Aspergillus nidulans. (+info)
Effect of captopril on mushroom tyrosinase activity in vitro.
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The study presented here demonstrates that the antihypertensive drug captopril ([2S]-N-[3-mercapto-2-methylpropionyl]-L-proline) is an irreversible non-competitive inhibitor and an irreversible competitive inhibitor of the monophenolase and diphenolase activities of mushroom tyrosinase when L-tyrosine and L-DOPA were assayed spectrophotometrically in vitro, respectively. Captopril was rendered unstable by tyrosinase catalysis because of the interaction between the enzymatic-generated product (o-quinone) and captopril to give rise to a colourless conjugate. Therefore, captopril was able to prevent melanin formation. The spectrophotometric recordings of the inhibition of tyrosinase by captopril were characterised by the presence of a lag period prior to the attainment of an inhibited steady state rate. The lag period corresponded to the time in which captopril was reacting with the enzymatically generated o-quinone. Increasing captopril concentrations provoked longer lag periods as well as a concomitant decrease in the tyrosinase activity. Both lag period and steady state rate were dependent of captopril, substrate and tyrosinase concentrations. The inhibition of both monophenolase and diphenolase activities of tyrosinase by captopril showed positive kinetic co-operativity which arose from the protection of both substrate and o-quinone against inhibition by captopril. Inhibition experiments carried out using a latent mushroom tyrosinase demonstrated that captopril only bound the enzyme at its active site. The presence of copper ions only partially prevented but not reverted mushroom tyrosinase inhibition. This could be due to the formation of both copper-captopril complex and disulphide interchange reactions between captopril and cysteine rich domains at the active site of the enzyme. (+info)
New sterols and triterpenoids from four edible mushrooms.
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Four edible mushrooms, Panellus serotinus, Lepista nuda, Tricholoma matsutake and Naematoloma sublateritium, have been investigated chemically. Two new sterols, 5alpha,9alpha-epidioxy-(22E)-ergosta-7,22-diene-3beta,6alpha-diol (1) and 5alpha,9alpha-epidioxy-(22E)-ergosta-7,22-diene-3beta,6beta-diol (2), have been isolated from Panellus serotinus. Compound 2 was also isolated from Lepista nuda. A new sterol, 3beta,5alpha,9alpha,14beta-tetrahydroxy-(22E)-ergosta-7,22-dien-6-one (3), and compound 2 have been isolated from Tricholoma matsutake. Three new triterpenoids, sublateriols A-C (4-6), have been isolated from Naematoloma sublateritium. The structures of the new compounds were elucidated on the basis of their spectral data. (+info)
Molecular cloning, characterization and regulation by cadmium of a superoxide dismutase from the ectomycorrhizal fungus Paxillus involutus.
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The gene encoding a superoxide dismutase (PiSOD) was cloned by suppressive subtractive hybridization from cDNA library of the ectomycorrhizal fungus, Paxillus involutus, grown under cadmium-stress conditions. The encoded protein was presumed to be localized in the peroxisomes because it contained a C-terminal peroxisomal localization peptide (SKL) and lacked an N-terminal mitochondrial transit peptide. Complementation of an Escherichia coli SOD null strain that is unable to grow in the presence of paraquat or cadmium indicated that cloned Pisod encoded a functional superoxide dismutase. Sensitivity of PiSOD activity to H2O2 but not KCN, and sequence homologies to other SODs strongly suggest that it is a manganese-containing superoxide dismutase. Monitoring PiSOD transcript, immunoreactive polypeptide and superoxide dismutase activity following cadmium stress suggests that the principal level of control is post-translational. This is, to our knowledge, the first insight in the characterization of molecular events that take place in an ectomycorrhizal fungus during exposure to heavy metals. (+info)
Proposal of Mycetocola gen. nov. in the family Microbacteriaceae and three new species, Mycetocola saprophilus sp. nov., Mycetocola tolaasinivorans sp. nov. and Mycetocola lacteus sp. nov., isolated from cultivated mushroom, Pleurotus ostreatus.
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The taxonomic positions of 10 tolaasin-detoxifying bacteria, which were isolated from the cultivated mushroom Pleurotus ostreatus, were investigated. These strains are Gram-positive, obligately aerobic, non-sporulating and irregular rod-shaped bacteria. They have the following characteristics: the major menaquinone is MK-10, the DNA G+C content ranges from 64 to 65 mol%, the diamino acid in the cell wall is lysine and the muramic acid in the peptidoglycan is an acetyl type. The major fatty acids are anteiso-C15:0 and anteiso-C17:0. On the basis of morphological, physiological and chemotaxonomic characteristics, together with DNA-DNA reassociation values and 16S rRNA gene sequence comparison data, the new genus Mycetocola gen. nov. is proposed for these bacteria in the family Microbacteriaceae and three new species are also proposed: Mycetocola saprophilus sp. nov. (type strain CM-01T = IFO 16274T = MAFF 211324T = NRRL B-24119T), Mycetocola tolaasinivorans sp. nov. (type strain CM-05T = IFO 16277T = MAFF 211325T = NRRL B-24120T) and Mycetocola lacteus sp. nov. (type strain CM-10T = IFO 16278T = MAFF 211326T = NRRL B-24121T). The type species of the genus is Mycetocola saprophilus sp. nov. (+info)
Analysis and interpretation of the action mechanism of mushroom tyrosinase on monophenols and diphenols generating highly unstable o-quinones.
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Tyrosinase can act on monophenols because of the mixture of met- (E(m)) and oxy-tyrosinase (E(ox)) which exists in the native form of the enzyme. The latter form is active on monophenols, while the former is not. However, the kinetics are complicated because monophenols can bind to both enzyme forms. This situation becomes even more complex since the products of the enzymatic reaction, the o-quinones, are unstable and continue evolving to generate o-diphenols in the medium. In the case of substrates such as L-tyrosine, tyrosinase generates very unstable o-quinones, in which a process of cyclation and subsequent oxidation-reduction generates o-diphenol through non-enzymatic reactions. However, the release of o-diphenol through the action of the enzyme on the monophenol contributes to the concentration of o-diphenol in the first pseudo-steady-state [D(0)](ss). Hence, the system reaches an initial pseudo-steady state when t-->0 and undergoes a transition phase (lag period) until a final steady state is reached when the concentration of o-diphenol in the medium reaches the concentration of the final steady state [D(f)](ss). These results can be explained by taking into account the kinetic and structural mechanism of the enzyme. In this, tyrosinase hydroxylates the monophenols to o-diphenols, generating an intermediate, E(m)D, which may oxidise the o-diphenol or release it directly to the medium. We surmise that the intermediate generated during the action of E(ox) on monophenols, E(m)D, has axial and equatorial bonds between the o-diphenol and copper atoms of the active site. Since the orbitals are not coplanar, the concerted oxidation-reduction reaction cannot occur. Instead, a bond, probably that of C-4, is broken to achieve coplanarity, producing a more labile intermediate that will then release the o-diphenol to the medium or reunite it diaxially, involving oxidation to o-quinone. The non-enzymatic evolution of the o-quinone would generate the o-diphenol ([D(f)](ss)) necessary for the final steady state to be reached after the lag period. (+info)