Faculty of Agriculture, Kyushu University, 6-10-1, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan1
Author for correspondence: Hiroyuki Wariishi. Tel/Fax: +81 92 642 2993. e-mail: hirowari{at}agr.kyushu-u.ac.jp
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: brown-rot fungus, benzaldehyde, hydroxylation, lignin, metabolism
Abbreviations: HN, high nitrogen; LN, low nitrogen
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The involvement of a low-molecular-mass one-electron oxidant, such as the hydroxyl radical and Fenton reagent, has been suggested for brown-rot-type wood decay (Backa et al., 1992 ; Chandhoke et al., 1992
; Goodell et al., 1997
; Hirano et al., 1995
, 1997
; Hyde & Wood, 1997
; Koenigs, 1975
). Recently, an effective cleavage of polyether was shown to be catalysed by the brown-rot basidiomycete Gloeophyllum trabeum via extracellular one-electron oxidation caused by hydroquinone-driven Fenton reaction (Kerem et al., 1998
, 1999
). Besides a partial modification of lignin, the degradation of lignin model compounds by brown-rot fungi has been reported (Enoki et al., 1985
), but neither the detail of reaction pathways nor the fate of aromatic components has been elucidated. If the Fenton type of reaction is involved in the brown-rot fungal metabolism, the effect of the hydroxyl radical on the aromatic moiety is of great interest.
In this study, benzaldehyde was utilized as the aromatic substrate for the brown-rot basidiomycetes Tyromyces palustris and G. trabeum. Product analysis revealed the occurrence of benzylic oxidation and reduction. A site-specific hydroxylation of benzaldehyde and benzoic acid was also found to be catalysed by these brown-rot fungi. Furthermore, no inhibition was observed for the hydroxylation reactions by either mannitol or thiourea, well-known hydroxyl radical scavengers. Hydroxylated intermediates were further metabolized rapidly without the occurrence of the phenoloxidase-derived radical coupling reactions which have been observed for white-rot fungi. Finally, the effective mineralization of aromatic components by the brown-rot fungi, proof of the occurrence of ring-fission reactions, was confirmed using radioactive substrates.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chemicals.
Benzyl alcohol, benzaldehyde, benzoic acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, 3,4-dihydroxybenzaldehyde (protocatechualdehyde), 3,4-dihydroxybenzoic (protocatechuic) acid, 1,4-dihydroxybenzene (hydroquinone) and 1,2,4-trihydroxybenzene were obtained from Wako Pure Chemicals. Methyl 4-hydroxybenzoate was prepared from 4-hydroxybenzoic acid refluxed in dry methanol in the presence of catalytic H2SO4. All substrates were recrystallized or purified using a silica gel column or preparative HPLC before use. Hydrogen peroxide (30%), mannitol, thiourea and piperonyl butoxide were purchased from Wako Pure Chemicals. 1-Aminobenzotriazole was obtained from TCI. All other chemicals were reagent grade. Deionized water was obtained from Milli Q System (Millipore).
Ring-U-14C-labelled benzaldehyde was purchased from Muromachi Chemical Engineering Co.; it was oxidized by KMnO4 to obtain ring-U-14C-labelled benzoic acid. Labelled substrates were purified by preparative TLC (ethyl acetate/hexane, 1:1, v/v) before use.
Enzymes.
Laccase, manganese peroxidase and lignin peroxidase activities in culture medium were measured as 2,6-dimethoxyphenol oxidation in the absence of H2O2 and MnII, MnIIImalonate complex formation, and veratryl alcohol oxidation activities, respectively, as previously described (Hiratsuka et al., 2001 ; Ichinose et al., 1999
; Kirk & Farrell, 1987
; Wariishi et al., 1992
). Catalase was commercially obtained from Sigma.
Metabolic reactions.
After a period of incubation as described above, the substrates were added to cultures to a final concentration of 0·5 mM. Metabolic products were analysed either by HPLC after homogenization, centrifugation (2000 g; 5 min), and filtration (0·45 µm), or by GC-MS after extraction with ethyl acetate at pH 2, drying over Na2SO4, evaporation under N2, and derivatization using BSTFA/pyridine (2:1, v/v) as previously described (Valli et al., 1992 ; Wariishi et al., 1989
).
Fenton reaction was performed in 500 mM acetate buffer (pH 4·5) containing 5 µM FeSO4, 0·01% H2O2 and 0·5 mM benzoic acid. The reaction was initiated by the addition of H2O2, then performed at room temperature in the dark for 24 h.
Effect of inhibitors.
Mannitol and thiourea were added to the cultures to final concentrations of 50 and 25 mM, respectively, 5 min prior to the addition of benzoic acid. Catalase (5900 U) was added to the cultures immediately prior to the addition of benzoic acid. Piperonyl butoxide and 1-aminobenzotriazole (final concentration 2·5 mM) were added to the cultures 3090 min prior to the addition of benzoic acid (Eilers et al., 1999 ; Hiratsuka et al., 2001
; Ichinose et al., 1999
). The quantitative analysis was performed as described above.
These inhibitors were added immediately prior to the addition of H2O2 when examined in the Fenton reaction system. Products were analysed using HPLC as described above after centrifugation and filtration.
Mineralization of 14C-labelled substrates.
Ring-U-14C-labelled substrates (benzaldehyde or benzoic acid) were added to cultures (104 d.p.m. per flask), which were purged with oxygen or air as described above. Evolved 14CO2 was trapped in a basic scintillation fluid as previously described (Kirk, 1975 ; Valli & Gold, 1991
) and radioactivity was measured in a Beckman LS-6500 scintillation counter. The efficiency of 14CO2 trapping after 10 min purging was greater than 98%. The residual radioactivity in flasks was determined after mixing the medium with a Beckman Ready-Solv scintillation cocktail. The counting efficiency was monitored with an automatic external standard.
Chromatography and spectrometry.
GC-MS was performed with a JMS-AM II 15 mass spectrometer (JEOL) at 70 eV fitted with a Shimadzu GC-17A gas chromatograph and a fused silica column (30-m DB-5, J & W Scientific). The GC temperature was programmed from 80 to 280 °C at 8 °C min-1.
Products were also analysed by HPLC using an STR ODS-II column (Shimadzu) with a linear gradient from 20% acetonitrile in 0·05% phosphoric acid to 100% acetonitrile in 16 min (flow rate 1·0 ml min-1). A UV detector at 210, 254 or 280 nm was used for monitoring the substrate and products. Products were identified by comparison of their retention times on GC and HPLC and of their mass fragmentation patterns with standards. Quantitative analysis was achieved by HPLC using calibration curves prepared with standards.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Time-course of metabolism
To better understand the metabolic pathway, the time-courses of the disappearance of the substrate and the formation of products were monitored using HPLC. Fig. 2 shows the metabolism of benzaldehyde and its metabolic intermediates by T. palustris in HN cultures under air. During the metabolism of benzaldehyde, benzoic acid was accumulated in the early stages of incubation and disappeared after day 2, indicating that benzoic acid was a metabolic intermediate and was further converted to 4-hydroxybenzoic acid, protocatechuic acid, hydroquinone and trihydroxybenzene (Fig. 2
). Since benzaldehyde was also directly hydroxylated to yield 4-hydroxybenzaldehyde and protocatechualdehyde (Fig. 1
), those intermediates were also exogenously added to T. palustris cultures as substrates. They were effectively oxidized to the corresponding acid, and further metabolized (Fig. 3
). Hydroquinone and trihydroxybenzene were metabolized but no formation of products was observed (Fig. 4
).
|
|
|
A comparison of substrate disappearance rates in HN and LN cultures showed that the rates were about twice as fast in the HN cultures, which was proportional to the dry weight of mycelia (data not shown). Non-specific adsorption of the substrates was estimated by feeding them to azide-treated cells. The addition of all the substrates shown in Fig. 1 to cultures of the brown-rot fungi, 10 min after the addition of 1 mM sodium azide, resulted in the quantitative recovery of the substrates, indicating that non-specific adsorption of the substrates is not significant.
Recovery of aromatic compounds (remaining substrate plus aromatic intermediates) from the cultures either 4 or 48 h after substrate addition was determined. After 4 h incubation of benzaldehyde and benzoic acid with the brown-rot fungi, 8595% recovery of aromatic components was observed; but after 48 h incubation, recovery fell to 3555%. These results strongly suggest the occurrence of ring fission by the brown-rot fungi. Purging with 100% oxygen (15 min per day) did not affect either the rate or the recovery for any of the substrates.
Mineralization of aromatic compounds
To further characterize the non-quantitative recovery of aromatic components (including quinones), ring-U-14C-labelled substrates were utilized. Fig. 5 shows the liberation of 14CO2 from benzaldehyde and benzoic acid by T. palustris and G. trabeum, indicating the occurrence of the mineralization of aromatic components. CO2 liberation rates were somewhat faster with G. trabeum.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Metabolic pathway and mechanisms of benzaldehyde
Benzaldehyde was mainly converted to benzyl alcohol and benzoic acid by T. palustris and G. trabeum (Figs 14
). Although the conversion of benzaldehyde to alcohol was the main pathway (Fig. 2
), benzyl alcohol was not further converted except for reoxidation back to benzaldehyde (data not shown). Thus, the oxidation of the formyl group to a carboxyl group seemed to be more important than the reduction during the metabolism. The reduction to form a hydroxymethyl group may be required to control the concentration of benzaldehyde in the fungal system because of its possible toxicity to the fungi. 4-Hydroxybenzaldehyde was converted to 4-hydroxybenzoic acid; on the other hand, no formation of 4-hydroxybenzaldehyde was observed from exogenously added 4-hydroxybenzoic acid, suggesting that the enzyme(s) involved in the conversion of benzaldehyde and benzoic acid may not catalyse that of 4-hydroxyl derivatives. 4-Hydroxybenzaldehyde and protocatechualdehyde seemed to be further metabolized via an intermediate formation of the corresponding acid (Fig. 3b
, c
).
Benzaldehyde and benzoic acid, especially benzoic acid, were effectively hydroxylated specifically at the C-4 position (Figs 1, 2
and 3a
). Hydroxylation reactions were also involved in the formation of protocatechualdehyde and protocatechuic acid. From the present data, it is strongly suggested that the hydroxylation reactions play key roles in the metabolism of benzaldehyde by the two brown-rot fungi utilized in this study.
The hydroxylation reaction is very common and essential in the microbial degradation of aromatic compounds. The white-rot fungi cause aromatic hydroxylation through peroxidase-catalysed quinone formation followed by the action of quinone reductase (Brock & Gold, 1996 ; Gold et al., 1989
; Kirk & Farrell, 1987
; Tien, 1987
; Valli & Gold, 1991
; Valli et al., 1992
), as well as through the action of cytochrome P450s (Bezalel et al., 1996
; Hiratsuka et al., 2001
; Ichinose et al., 1999
, 2002
; Masaphy et al., 1996
). It has also been reported that the hydroxyl radical might be formed by the brown-rot fungi during wood decay. A Fenton-type mechanism for hydroxyl radical generation by the brown-rot fungi has been proposed (Backa et al., 1992
; Chandhoke et al., 1992
; Goodell et al., 1997
; Hirano et al., 1995
, 1997
; Hyde & Wood, 1997
; Kerem et al., 1998
, 1999
; Koenigs, 1975
). In the present study, the addition of hydroxyl radical scavengers or catalase to cultures of brown-rot fungi during benzoic acid metabolism did not inhibit the production of 4-hydroxybenzoic acid (Table 2
). However, this observation does not exclude the formation of a hydroxyl radical by brown-rot fungi as reported in previous studies. We assume that fungal production of a hydroxyl radical might be more influential for extracellular reactions, which seems to be advantageous for polymer degradation such as carbohydrates and polyethylene oxide, as previously reported (Hirano et al., 1995
, 1997
; Kerem et al., 1998
). At this time, it is not possible to propose one definitive catalytic mechanism involved in the hydroxylation reactions observed in this study. However, the peroxidase-catalysed quinone formation can at least be eliminated, since no activities of peroxidalaccases were observed. Our preliminary observations indicated that cytochrome P450 inhibitors showed some inhibitory effect (Table 2
). The involvement of intracellular cytochrome-P450-mediated hydroxylation reactions in the metabolism of monomeric aromatic compounds by brown-rot fungi needs to be investigated. Identification of cytochrome P450 in brown-rot fungi is now being attempted.
Both brown-rot fungi yielded almost identical products from benzaldehyde. Methylation of 4-hydroxybenzoic acid was only seen with T. palustris. Except for this methylation reaction, both fungi metabolize benzaldehyde by the same mechanism. The rate of benzaldehyde metabolism was twice as fast with G. trabeum (data not shown). That is partly because G. trabeum did not accumulate the dead-end product methyl 4-hydroxybenzoate, whereas T. palustris did so.
From the comparison of disappearance rates shown in Fig. 4, hydroquinone seemed to be converted to trihydroxybenzene and further degraded. The low level of accumulation of trihydroxybenzene during benzaldehyde metabolism (Fig. 2
) suggested that this compound might be a direct substrate for the ring-fission reaction. The occurrence of the ring-fission reaction was proved by 14C-labelling experiments (Fig. 5
).
All these data allow us to propose the reaction pathways of benzaldehyde by the brown-rot fungi (Fig. 6). The oxidation and reduction at the benzylic position occur simultaneously, forming benzyl alcohol and benzoic acid as major products. Hydroxylation reactions, which seem to be a key step, occur on benzaldehyde and benzoic acid, but not on benzyl alcohol, to form corresponding 4-hydroxyl and 3,4-dihydroxyl derivatives. 1-Formyl derivatives are oxidized to 1-carboxyl derivatives at several metabolic stages. All these reactions result in the formation of 3,4-dihydroxybenzoic acid. This is further metabolized via the decarboxylation reaction to yield 1,2,4-trihydroxybenzene, which may be susceptible to the ring-fission reaction. It may be too early to make broad generalizations about the reaction mechanism of the brown-rot fungi; however, at least the two fungi used in this study share the same mechanism.
|
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bavendamm, W. (1928). Originalabhandlungen. Über das Vorkommen und den Nachweis von Oxydasen bei Holzzerstörenden Pilzen. Z Pflanzenkr Pflanzenschuz 38, 257-276.
Bezalel, L., Hadar, Y., Fu, P. P., Freeman, J. P. & Cerniglia, C. (1996). Initial oxidation products in the metabolism of pyrene, anthracene, fluorene, and dibenzothiophene by the white rot fungus Pleurotus ostreatus. Appl Environ Microbiol 62, 2554-2559.[Abstract]
Brock, B. J. & Gold, M. H. (1996). 1,4-Benzoquinone reductase from the basidiomycete Phanerochaete chrysosporium: spectral and kinetic analysis. Arch Biochem Biophys 331, 31-40.[Medline]
Brown, W., Cowling, E. B. & Falkehag, S. I. (1968). Molecular size distributions of lignins liberated enzymatically from wood. Sven Papperstidning 22, 811-821.
Chandhoke, V., Goodell, B., Jellison, J. & Fekete, F. A. (1992). Oxidation of 2-keto-4-thiomethylbutyric acid (KTBA) by iron-binding compounds produced by the wood-decaying fungus Gloeophyllum trabeum. FEMS Microbiol Lett 90, 263-266.
Crawford, R. L. (1981). Lignin Biodegradation and Transformation. New York: Wiley-Interscience.
Dsouza, T. M., Boominathan, K. & Reddy, C. A. (1996). Isolation of laccase gene-specific sequences from white rot and brown rot fungi by PCR. Appl Environ Microbiol 62, 3739-3744.
Eilers, A., Rüngeling, E., Stündl, U. M. & Gottschalk, G. (1999). Metabolism of 2,4,6-trinitrotoluene by the white-rot fungus Bjerkandera adusta DSM 3375 depends on cytochrome P-450. Appl Microbiol Biotechnol 53, 75-80.
Enoki, A., Takahashi, M., Tanaka, H. & Fuse, G. (1985). Degradation of lignin-related and wood components by white-rot and brown-rot fungi. Mokuzai Gakkaishi 31, 397-408.
Eriksson, K.-E. L., Blanchette, R. A. & Ander, P. (1990). Microbial and Enzymatic Degradation of Wood and Wood Components. Berlin: Springer.
Franklin, M. R. (1972). Inhibition of hepatic oxidative xenobiotic metabolism by piperonyl butoxide. Biochem Pharmacol 21, 3287-3299.[Medline]
Gold, M. H., Wariishi, H. & Valli, K. (1989). Extracellular peroxidases involved in lignin degradation by the white rot basidiomycete Phanerochaete chrysosporium. In Biocatalysis in Agricultural Biotechnology (ACS Symposium Series vol. 389), pp. 127140. Edited by J. R. Whitaker. & P. E. Sonnet. Washington DC: American Chemical Society.
Goodell, B., Jellison, J., Liu, J., Daniel, G., Paszczynski, A., Fekete, F., Krishnamurthy, S., Jun, L. & Xu, G. (1997). Low molecular weight chelators and phenolic compounds isolated from wood decay fungi and their role in the fungal biodegradation of wood. J Biotechnol 53, 133-162.
Hirano, T., Tanaka, H. & Enoki, A. (1995). Extracellular substance from the brown-rot basidiomycete Tyromyces palustris that reduces molecular oxygen to hydroxyl radicals and ferric iron to ferrous iron. Mokuzai Gakkaishi 41, 334-341.
Hirano, T., Tanaka, H. & Enoki, A. (1997). Relationship between production of hydroxyl radicals and degradation of wood by the brown-rot fungus, Tyromyces palustris. Holzforschung 51, 389-395.
Hiratsuka, N., Wariishi, H. & Tanaka, H. (2001). Degradation of diphenyl ether herbicides by the lignin-degrading basidiomycete Coriolus versicolor. Appl Microbiol Biotechnol 57, 563-571.[Medline]
Hyde, S. M. & Wood, P. (1997). A mechanism for production of hydroxyl radicals by the brown-rot fungus Coniophora puteana: Fe(III) reduction by cellobiose dehydrogenase and Fe(II) oxidation at a distance from the hyphae. Microbiology 143, 259-266.
Ichinose, H., Wariishi, H. & Tanaka, H. (1999). Bioconversion of recalcitrant 4-methyldibenzothiophene to water-extractable products using lignin-degrading basidiomycete Coriolus versicolor. Biotechnol Prog 15, 706-714.[Medline]
Ichinose, H., Wariishi, H. & Tanaka, H. (2002). Identification and characterization of novel cytochrome P450 genes from the white-rot fungus Coriolus versicolor. Appl Microbiol Biotechnol 58, 97-105.[Medline]
Kerem, Z., Bao, W. & Hammel, K. (1998). Rapid polyether cleavage via extracellular one-electron oxidation by a brown-rot basidiomycete. Proc Natl Acad Sci USA 95, 10373-10377.
Kerem, Z., Jensen, K. A. & Hammel, K. (1999). Biodegradative mechanism of the brown rot basidiomycete Gloeophyllum trabeum: evidence for an extracellular hydroquinone-driven Fenton reaction. FEBS Lett 446, 49-54.[Medline]
Kirk, T. K. (1975). Effects of a brown-rot fungus, Lenzites trabea, on lignin in spruce wood. Holzforschung 29, 99-107.
Kirk, T. K. & Farrell, R. L. (1987). Enzymatic combustion: the microbial degradation of lignin. Annu Rev Microbiol 41, 465-505.[Medline]
Kirk, T. K. & Kelman, A. (1965). Lignin degradation as related to the phenoloxidases of selected wood-decaying basidiomycetes. Phytopathology 55, 739-745.
Kirk, T. K., Schultz, E., Connors, W. J., Lorenz, L. F. & Zeikus, J. G. (1978). Influence of culture parameters on lignin metabolism by Phanerochaete chrysosporium. Arch Microbiol 117, 277-285.
Koenigs, J. W. (1975). Hydrogen peroxide and iron: a microbial cellulolytic system. Biotechnol Bioeng Symp 5, 151-159.[Medline]
Masaphy, S., Levanon, D., Henis, Y., Venkateswarlu, K. & Kelly, S. L. (1996). Microsomal and cytosolic cytochrome P450 mediated benzo(a)pyrene hydroxylation in Pleurotus pulmonarius. Biotechnol Lett 17, 967-974.
Murray, M. & Reidy, G. F. (1990). Selectivity in the inhibition of mammalian cytochromes P-450 by chemical agents. Pharmacol Rev 42, 85-101.[Medline]
Ortiz de Montellano, P. R. & Mathews, J. M. (1981). Autocatalytic alkylation of the cytochrome P-450 prosthetic haem group by 1-aminobenzotriazole. Isolation of an NN-bridged benzyne-protoporphyrin IX adduct. Biochem J 195, 761-764.[Medline]
Paszczynski, A., Crawford, R., Funk, D. & Goodell, B. (1999). De novo synthesis of 4,5-dimethoxycatechol and 2,5-dimethoxyhydroquinone by the brown rot fungus Gloeophyllum trabeum. Appl Environ Microbiol 65, 674-679.
Sundman, V. & Näse, L. (1971). A simple plate test for direct visualization of biological lignin degradation. Papperi ja Puu 53, 67-71.
Tien, M. (1987). Properties of ligninase from Phanerochaete chrysosporium and their possible applications. CRC Crit Rev Microbiol 15, 141-168.
Valli, K. & Gold, M. H. (1991). Degradation of 2,4-dichlorophenol by the lignin-degrading fungus Phanerochaete chrysosporium. J Bacteriol 173, 345-352.[Medline]
Valli, K., Wariishi, H. & Gold, M. H. (1992). Degradation of 2,7-dichlorodibenzo-p-dioxin by the white-rot basidiomycete, Phanerochaete chrysosporium. J Bacteriol 174, 2131-2137.[Abstract]
Wariishi, H., Valli, K. & Gold, M. H. (1989). Oxidative cleavage of a phenolic diarylpropane lignin model dimer by manganese peroxidase from Phanerochaete chrysosporium. Biochemistry 28, 6017-6023.
Wariishi, H., Valli, K. & Gold, M. H. (1992). Manganese(II) oxidation by manganese peroxidase from the basidiomycete Phanerochaete chrysosporium: kinetic mechanism and role of chelators. J Biol Chem 267, 23688-23695.
Zabel, R. A. & Morrell, J. J. (1992). Wood Microbiology: Decay and its Prevention. San Diego: Academic Press.
Received 30 November 2001;
revised 18 February 2002;
accepted 7 March 2002.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |