Benzaldehyde and its metabolic intermediates were effectively degraded by the brown-rot basidiomycetes Tyromyces palustris and Gloeophyllum trabeum. The pathway of benzaldehyde degradation was elucidated by the identification of fungal metabolites produced upon the addition of benzaldehyde and its metabolic intermediates. The oxidation and reduction occurred simultaneously, forming benzyl alcohol and benzoic acid as major products. Hydroxylation reactions, which seemed to be a key step, occurred on benzaldehyde and benzoic acid, but not on benzyl alcohol, to form corresponding 4-hydroxyl and 3,4-dihydroxyl derivatives. 1-Formyl derivatives were oxidized to 1-carboxyl derivatives at several metabolic stages. All of these reactions resulted in the formation of 3,4-dihydroxybenzoic acid. This was further metabolized via the decarboxylation reaction to yield 1,2,4-trihydroxybenzene, which may be susceptible to the ring-fission reaction. Ring-U-14 C-labelled benzaldehyde and benzoic acid were effectively mineralized, clearly indicating that the brown-rot basidiomycetes are capable of metabolizing certain aromatic compounds to CO 2 and H 2 O, despite the fact that brown-rot fungi cannot degrade polymeric lignin. Inhibitor experiments, using hydroxyl radical scavengers, catalase and cytochrome P450 inhibitors, strongly suggested that the aromatic hydroxylation reactions found in the brown-rot fungi are catalysed by intracellular enzyme(s), but not by Fenton-reactionderived hydroxyl radicals.
Under ligninolytic conditions, the white-rot basidiomycete Coriolus versicolor metabolized chloronitrofen (2, 4, 6-trichloro-4'-nitrodiphenyl ether; CNP) and nitrofen (2, 4-dichloro-4'-nitrodiphenyl ether, NIP), which constitute the largest class of commercially produced diphenyl ether herbicides. The pathway of CNP degradation was elucidated by the identification of fungal metabolites upon addition of CNP and its metabolic intermediates. The metabolic pathway was initially branched to form four metabolites--2, 4, 6-trichloro-3-hydroxy-4'-nitrodiphenyl ether, 2, 4-dichloro-6-hydroxy-4'-nitrodiphenyl ether, NIP, and 2, 4, 6-trichloro-4'-aminodiphenyl ether--indicating the involvement of hydroxylation, oxidative dechlorination, reductive dechlorination, and nitro-reduction. Of these reactions, hydroxylation was relatively major compared to the others. Extracellular ligninolytic enzymes such as lignin peroxidase, manganese peroxidase and laccase did not catalyze the oxidation of either CNP or NIP. Piperonyl butoxide, an inhibitor of cytochrome P450, suppressed fungal oxidation of CNP and NIP to their hydroxylated products. The inhibition resulted in increasing the amount of reductively dechlorinated and nitro-reduced products. These observations strongly suggest that basidiomycetes may possess a mechanism for a strict substrate recognition system and a corresponding metabolic response system to effectively degrade environmentally persistent aromatic compounds.
We have proposed the term 'protein chemotaxonomy' for molecular taxonomy based on the primary structures of common plant proteins, instead the of so-called secondary metabolites. To evaluate the effectiveness of this concept, we have carried out a series of studies on the family Solanaceae, using ferredoxin (Fd), an iron-sulfur electron-transfer protein.2) This protein was chosen for this study because it is easy to isolate and has an appropriate molecular weight for determining the primary structure. Previously, we have reported the primary structures of Fds from seven Datura plants [3][4][5][6] and other solanaceous plants 7-11) Our recent results have suggests that S. japonica is closely related taxonomically to Datura plants, and especially to D. arborea. Interestingly, both S. japonica and Datura plants contain tropane-alkaloids such as hyoscyamine, atropine, and scopolamine. It may be worthwhile to determine the relationship between pharmacologically important constituents and the amino acid sequences of Fds from these plants. These considerations led us to elucidate the amino acid sequences of Fds from Atropa belladonna and Hyoscyamus niger, which are important medicinal plants that contain tropane-alkaloids.In this report, we determined the primary structures of Fds from A. belladonna and H. niger (Solanaceae), and compared them with those of Fds from other higher plants as well as those from Datura plants and other solanaceous plants.
MATERIALS AND METHODS
Materials Atropa belladonna and Hyoscyamus nigerwere cultivated in the herb garden at Osaka University of Pharmaceutical Sciences.Isolation of Ferredoxin Each protein (15.4 or 3.0 mg) was purified from the fresh leaves (0.6 or 0.5 kg) of A. belladonna or H. niger as described previously.
3,7)Sequence Determination The amino acid sequences of the Fds were determined using a gas-phase protein sequencer with automated Edman degradation of S-carboxymethylcysteinyl (Cm) Fd and the peptides obtained by lysyl endopeptidase, trypsin, or endoproteinase Asp-N digestion. C-terminal analysis was carried out with carboxypeptidase Y.The detailed procedure and the other methods have been described previously.
3,7)Construction of a Phylogentic Tree A phylogenetic tree was constructed from the amino acid sequences (97 residues) of higher-plant Fds (30 species) using the unweighed pairgroup method with the arithmetical averages (UPGMA) method of Nei (GENETYX software, Software Development, Japan). 13) The molar absorption coefficient at 420 nm, based on the spectrum and protein determination, was 11000 M Ϫ1 cm Ϫ1 , which was similar to those of other higher-plant Fds.2,13) The biological activities and other physico-chemical properties of Ab-and HnFds will be published elsewhere, together with those of other solanaceous Fds.Sequence Determination The sequencing strategy is summarized in Fig. 1. The analytical results regarding the amino acid compositions of both Cm-Fds and the peptides obtained by enzymatic digestion, were consistent with the derived sequences. Automat...
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