Mushroom worker's lung resulting from indoor cultivation of Pleurotus osteatus.
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Indoor cultivation of oyster mushroom Pleurotus osteatus lead to an outbreak of extrinsic allergic alveolitis in two workers. High titer of indirect fluorescent antibody and positive precipitins against basidiospores of P. osteatus were demonstrated in sera of the patients. Mushroom workers should protect themselves from the basidiospores, being aware of their pathogenicity. (+info)
Description of a versatile peroxidase involved in the natural degradation of lignin that has both manganese peroxidase and lignin peroxidase substrate interaction sites.
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Two major peroxidases are secreted by the fungus Pleurotus eryngii in lignocellulose cultures. One is similar to Phanerochaete chrysosporium manganese-dependent peroxidase. The second protein (PS1), although catalyzing the oxidation of Mn2+ to Mn3+ by H2O2, differs from the above enzymes by its manganese-independent activity enabling it to oxidize substituted phenols and synthetic dyes, as well as the lignin peroxidase (LiP) substrate veratryl alcohol. This is by a mechanism similar to that reported for LiP, as evidenced by p-dimethoxybenzene oxidation yielding benzoquinone. The apparent kinetic constants showed high activity on Mn2+, but methoxyhydroquinone was the natural substrate with the highest enzyme affinity (this and other phenolic substrates are not efficiently oxidized by the P. chrysosporium peroxidases). A three-dimensional model was built using crystal models from four fungal peroxidase as templates. The model suggests high structural affinity of this versatile peroxidase with LiP but shows a putative Mn2+ binding site near the internal heme propionate, involving Glu36, Glu40, and Asp181. A specific substrate interaction site for Mn2+ is supported by kinetic data showing noncompetitive inhibition with other peroxidase substrates. Moreover, residues reported as involved in LiP interaction with veratryl alcohol and other aromatic substrates are present in peroxidase PS1 such as His82 at the heme-channel opening, which is remarkably similar to that of P. chrysosporium LiP, and Trp170 at the protein surface. These residues could be involved in two different hypothetical long range electron transfer pathways from substrate (His82-Ala83-Asn84-His47-heme and Trp170-Leu171-heme) similar to those postulated for LiP. (+info)
Molecular cloning of aryl-alcohol oxidase from the fungus Pleurotus eryngii, an enzyme involved in lignin degradation.
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Aryl-alcohol oxidase (AAO), an extracellular enzyme characteristic of fungi from the genus Pleurotus, constitutes a source for H2O2 required in lignin biodegradation. The gene aao has been cloned, sequenced and characterized for the first time in Pleurotus eryngii. Both cDNA and genomic libraries were screened with probes obtained by PCR using as primers oligonucleotides corresponding to the N-terminus and internal sequences of AAO. DNA sequences from positive clones showed a unique open reading frame of 1779 nucleotides interrupted by 12 introns. The conceptual translation of the protein agrees with the partial amino acid sequences obtained from protein sequencing. A search for proteins with related amino-acid sequences revealed that glucose oxidase from Aspergillus niger has 33% identity and 51% similarity. A comparison with other oxidoreductases showed common motifs in both N- and C-terminal regions corresponding, respectively, to the FAD-binding region and the enzyme active site. However, AAO probably has structural differences with other oxidases, as deduced from its unique ability to generate H2O2 from the oxidation of aromatic alcohols. (+info)
Protein and gene structure of a blue laccase from Pleurotus ostreatus1.
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A new laccase isoenzyme (POXA1b, where POX is phenol oxidase), produced by Pleurotus ostreatus in cultures supplemented with copper sulphate, has been purified and fully characterized. The main characteristics of this protein (molecular mass in native and denaturing conditions, pI and catalytic properties) are almost identical to the previously studied laccase POXA1w. However, POXA1b contains four copper atoms per molecule instead of one copper, two zinc and one iron atom per molecule of POXA1w. Furthermore, POXA1b shows an unusually high stability at alkaline pH. The gene and cDNA coding for POXA1b have been cloned and sequenced. The gene coding sequence contains 1599 bp, interrupted by 15 introns. Comparison of the structure of the poxa1b gene with the two previously studied P. ostreatus laccase genes (pox1 and poxc) suggests that these genes belong to two different subfamilies. The amino acid sequence of POXA1b deduced from the cDNA sequence has been almost completely verified by means of matrix-assisted laser desorption ionization MS. It has been demonstrated that three out of six putative glycosylation sites are post-translationally modified and the structure of the bound glycosidic moieties has been determined, whereas two other putative glycosylation sites are unmodified. (+info)
Spectral characterization and chemical modification of FMN-containing ascorbyl free-radical reductase from Pleurotus ostreatus.
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Ascorbyl free-radical reductase was purified 1143-fold with an overall yield of 9.9% from the cytosolic fraction of Pleurotus ostreatus. The native enzyme had a molecular mass of 127 kDa and SDS/PAGE revealed that the enzyme consists of two subunits, each with a molecular mass of 62 kDa. The enzyme utilized only NADH as an electron donor. The enzyme was highly specific for ascorbyl free radical as an electron acceptor and capable of catalysing the reduction of ferricyanide and 2,6-dichloroindophenol as artificial electron acceptors. The apparent K(m) values of the enzyme towards NADH and ascorbyl free radical were 35+/-0.22 and 2.1+/-0.03 microM, respectively. The catalytic mechanism of this enzyme is of Ping Pong type. The enzyme contained FMN as a prosthetic group and showed the characteristic absorption spectrum ascribed to the charge-transfer interaction of thiolate anion with FMN. The enzyme contained eight cysteine residues per monomer and was inactivated more rapidly by mercurials than by thiol-alkylating reagents. Kinetic analysis of the inactivation process revealed that the enzyme had 1 mol of thiol group/mol of subunit in the active site with a pK(a) of 6.9. The modification of the thiol group of the enzyme caused the loss of charge-transfer absorbance centred at 540 nm and blocked the electron-transfer process from NADH to FMN. The modification of lysine, arginine and histidine residues led to the loss of its activity. Unlike the active enzyme, the fluorescence quenching of NADH was not observed in the lysine-modified enzyme, which implies that lysine residues can participate in the interaction with NADH. (+info)
Molecular karyotype of the white rot fungus Pleurotus ostreatus.
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The white rot fungus Pleurotus ostreatus is an edible basidiomycete with increasing agricultural and biotechnological importance. Genetic manipulation and breeding of this organism are restricted because of the lack of knowledge about its genomic structure. In this study, we analyzed the genomic constitution of P. ostreatus by using pulsed-field gel electrophoresis optimized for the separation of its chromosomes. We have determined that it contains 11 pairs of chromosomes with sizes ranging from 1.4 to 4.7 Mbp. In addition to chromosome separation, the use of single-copy DNA probes allowed us to resolve the ambiguities caused by chromosome comigration. When the two nuclei present in the dikaryon were separated by protoplasting, analysis of their karyotypes revealed length polymorphisms affecting various chromosomes. This is, to our knowledge, the clearest chromosome separation available for this species. (+info)
Regulation of peroxidase transcript levels in liquid cultures of the ligninolytic fungus Pleurotus eryngii.
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A versatile peroxidase able to oxidize Mn(2+) as well as phenolic and nonphenolic aromatic compounds is produced in peptone-containing liquid cultures of Pleurotus eryngii encoded by the gene mnpl. The regulation of its transcript levels was investigated by Northern blotting of total RNA. High-peroxidase transcripts and activity were found in cultures grown in glucose-peptone medium, whereas only basal levels were detected in glucose-ammonium medium. The addition of more than 25 microM Mn(2+) to the former medium did not result in detectable peroxidase transcripts or activity. Potential regulators were also added to isolated mycelium. In this way, it was shown that high transcript levels (in peroxidase-expressing mycelium) were maintained on peptone, whereas expression was not induced in short-term incubation experiments. Similar results were obtained with Mn(2+) ions. Strong induction of mnpl expression was caused by exogenous H(2)O(2) or by continuous H(2)O(2) generation during redox cycling of menadione. By the use of the latter system in the presence of Fe(3+), which catalyzes the reduction of H(2)O(2) to hydroxyl radical, it was shown for the first time that the presence of this strong oxidant causes a rapid increase of the transcripts of a ligninolytic peroxidase. In conclusion, peptone and Mn(2+) affect the levels of transcripts of this versatile peroxidase in culture, and reduced oxygen species induce short-term expression in isolated mycelium, probably via a stress response mechanism. (+info)
Heterologous expression of Pleurotus eryngii peroxidase confirms its ability to oxidize Mn(2+) and different aromatic substrates.
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A versatile ligninolytic peroxidase has been cloned from Pleurotus eryngii and its allelic variant MnPL2 expressed in Aspergillus nidulans, with properties similar to those of the mature enzyme from P. eryngii. These include the ability to oxidize Mn(2+) and aromatic substrates, confirming that this is a new peroxidase type sharing catalytic properties of lignin peroxidase and manganese peroxidase. (+info)