FEMS Microbiology Ecology 46 (2003) 197^202
www.fems-microbiology.org
Fungal modi¢cation of the hydroxyl radical detector
coumarin-3-carboxylic acid
Andrei Iakovlev a , Anders Broberg
b
b;
, Jan Stenlid
a
a
Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden
Department of Chemistry, Swedish University of Agricultural Sciences, P.O. Box 7015, SE-750 07 Uppsala, Sweden
Received 26 March 2003; received in revised form 7 August 2003; accepted 7 August 2003
First published online 5 September 2003
Abstract
The feasibility of using coumarin-3-carboxylic acid (3-CCA) for detection of hydroxyl radicals in pure cultures of wood-decaying fungi
was tested. Fungi were incubated on a 3-CCA-containing medium. The transformation of 3-CCA to the fluorescent hydroxyl radical
detector substance 7-hydroxycoumarin-3-carboxylic acid and other compounds was studied by chromatographic and spectroscopic
techniques. During incubation of all tested fungi, a small fraction of the 3-CCA was hydroxylated to 7-hydroxycoumarin-3-carboxylic
acid and a major fraction of the 3-CCA was metabolized by fungi to 2-(2-hydroxybenzyl)malonic acid. In most cultures the concentration
of 3-CCA was below detection limit at the end of incubation. The fungal metabolism was suggested to be involved in the formation of
2-(2-hydroxybenzyl)malonic acid from 3-CCA, consequently making this method of hydroxyl radical detection less suitable to use on
cultures of wood-decaying fungi.
3 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords : Hydroxyl radical detection ; Coumarin-3-carboxylic acid ; 7-Hydroxycoumarin-3-carboxylic acid ; Fungal physiology
1. Introduction
Reactive oxygen species are produced by all aerobic
cells as by-products of normal metabolism [1] but their
overproduction may damage biological macromolecules
leading to cytotoxicity [2,3]. One of the reactive oxygen
species, the hydroxyl radical (c OH), is a strong oxidizing
agent and is primarily responsible for the cytotoxic e¡ect
of oxygen in plants, animals and microorganisms [4,5].
The involvement of c OH in normal as well as pathological
cell processes stimulates considerable interest in this radical. One type of process where c OH is believed to play an
important role is in the wood-degradation by white-rot
and brown-rot fungi [6,7]. To be able to study the mechanism behind fungal wood-degradation it is important to
have a reliable method to estimate the production of c OH.
In a previous study, Tornberg and Olsson [8] detected c OH
produced by wood-decomposing fungi. These authors used
a method based on hydroxylation of coumarin-3-carbox-
* Corresponding author. Tel. : +46 (18) 67 22 17;
Fax : +46 (18) 67 34 76.
E-mail address : [email protected] (A. Broberg).
ylic acid (3-CCA) by c OH, a reaction that produces one
major £uorescent product, 7-hydroxycoumarin-3-carboxylic acid (7-OHCCA) [9^11]. Some advantages of this
method over similar techniques of c OH detection (for review see [12]) are the possibility of real-time detection of
c OH in intact organisms and the minimal number of required manipulations.
In spite of its advantages, application of this method for
detection of c OH in some fungi may have limitations. The
compounds 3-CCA and 7-OHCCA are derivatives of coumarin, a compound produced in plants from precursors in
lignin biosynthesis [13,14]. Furthermore, the ability of
wood-decaying fungi to modify and degrade lignin and
phenolic compounds is well documented [15^17]. Consequently, wood-decaying fungi that are able either to degrade lignin (white-rot fungi) or modify it (brown-rot fungi) may a¡ect 3-CCA and/or 7-OHCCA, which may cause
an underestimation of the c OH formation. Results of the
study by Tornberg and Olsson [8] indicated that such
underestimation can vary from 10 to 30% of the control
value. Therefore, the risk of fungal modi¢cation of 3-CCA
and 7-OHCCA needs to be assessed.
The aim of the present study was to investigate the
feasibility of using the 3-CCA-based method for detection
0168-6496 / 03 / $22.00 3 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/S0168-6496(03)00213-7
FEMSEC 1574 7-10-03
198
A. Iakovlev et al. / FEMS Microbiology Ecology 46 (2003) 197^202
of c OH in pure cultures of wood-decaying fungi. This was
achieved by incubating fungi with the c OH detector
3-CCA and analyzing if the reporter substance 7-OHCCA
was formed during the incubation. Furthermore, it was
investigated if other products were formed either from
3-CCA or 7-OHCCA during the incubation.
2. Materials and methods
2.1. Chemicals
3-CCA, 7-OHCCA, and N,O-bis(trimethylsilyl)acetamide were bought from Sigma-Aldrich (Stockholm, Sweden), Molecular Probes (Leiden, Netherlands), and Pierce
(Rockford, IL, USA) respectively.
2.2. Organisms and culture conditions
The fungi Antrodia heteromorpha (Fr.) Donk, Antrodia
vaillantii (DC. : Fr.) Ryv., Antrodiella citrinella Niemela«
and Ryvarden, Coniophora arida (Fr.) Karst., Coniophora
puteana (Schum.: Fr.) Karst., Heterobasidion annosum
(Fr.) Bref., Junghuhnia collabens (Fr.) Vesterholt, Laetiporus sulphureus (Fr.) Murr., Phlebia centrifuga P. Karst.,
Phlebiopsis gigantea (Fr.: Fr.) Julich and Resinicium bicolor (Alb. and Schwein.: Fr.) Parmasto were obtained
from the culture collection of the Department of Forest
Mycology and Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden. All fungi were grown on
Hagem agar medium [18] containing 3-CCA (0.1 mM) [8]
in 5-cm diameter Petri dishes at 21‡C in darkness (10^
20 cultures of each fungus). P. centrifuga was also incubated in liquid Hagem medium containing 3-CCA (0.2 mM)
at 21‡C in darkness (static culture). P. centrifuga, P. gigantea and C. arida were also incubated in liquid Hagem
medium containing 7-OHCCA (2 WM) at 21‡C in darkness
(static cultures).
2.3. Analysis by high-performance liquid chromatography
(HPLC)
Samples from liquid cultures were taken after 10 day
incubation with fungi. Following centrifugation (10 min
at 11 000Ug) the samples were transferred to vials for
analysis by HPLC. Samples (1U1 cm) from the Petri
dish cultures were extracted twice with 1 ml 50% aqueous
methanol after 10 day incubation with fungi. Following
centrifugation (10 min at 11 000Ug) and drying in a vacuum centrifuge, the Petri dish samples were dissolved in
100 Wl 50% aqueous methanol and transferred to HPLC
vials for analysis. HPLC analysis was performed on a
reversed phase column (Discovery0 , 4.6U150 mm; Supelco, Bellefonte, PA, USA) coupled to an analytical HPLC
system (AS-2000A autosampler, L-6200A pump, L-4000
UV detector; Merck-Hitachi, Germany). An elution gra-
dient was formed with acetonitrile and 10 mM phosphate
bu¡er at pH 2.8 (10^20% acetonitrile in 10 min followed
by 20^90% acetonitrile in 10 min) and the £ow rate was
1 ml min31 . The eluate was monitored for UV absorption
at 210 or 350 nm. Solutions of commercial 3-CCA and
7-OHCCA as well as the isolated 3-CCA derivative (see
Section 2.4) were used as reference material. Relative
quanti¢cation was achieved by comparison of the peak
areas of 3-CCA and 7-OHCCA with the peak area of
the 3-CCA derivative. Di¡erences in response factors between the substances were corrected for by multiplying the
peak area of the 3-CCA derivative with 1.15 as determined
by analysis of solutions of known concentrations.
2.4. Isolation of the 3-CCA derivative
The supernatant (900 ml) from a 10-day-old culture
(containing 0.2 mM 3-CCA) of P. centrifuga (pH set to
2.8 by the addition of 6 M HCl) was extracted on two
solid phase extraction (SPE) columns (Isolute, C18, 10 g;
International Sorbent Technology Ltd., UK) pre-conditioned with 200 ml acetonitrile and 200 ml phosphate bu¡er (10 mM, pH 2.8). Following sample loading, the SPE
columns were washed with 200 ml water (pH 2.8) and
eluted with 80 ml 30% acetonitrile in water. The acetonitrile was removed under reduced pressure and the residual
water solution was freeze-dried. The 3-CCA derivative was
subsequently isolated on a reversed phase HPLC column
(Discovery0 , 21.2U100 mm; Supelco, Bellefonte, PA,
USA) on a preparative HPLC system (Gilson 305 and
306 pumps, Gilson 811 mixer, Gilson 118 UV detector;
Gilson, Villiers-le-Bel, France). Three 1-ml portions (in
50% aqueous methanol) of the SPE sample containing
the 3-CCA derivative were manually injected and the column was eluted with acetonitrile and 10 mM phosphate
bu¡er at pH 2.8 (10^30% acetonitrile in 20 min followed
by 30^90% acetonitrile in 10 min at 10 ml min31 ). The
eluate was monitored at 210 nm and fractions were manually collected. The fractions containing the 3-CCA derivative were pooled and the acetonitrile was removed under
reduced pressure. The pooled fractions were desalted on a
SPE column (Isolute, C18, 1 g) as described above, but
eluted with methanol. The isolated product in methanol
was dried under a stream of compressed air.
2.5. Characterization of the 3-CCA derivative
The 3-CCA derivative was characterized by nuclear
magnetic resonance spectroscopy (NMR) at 400 MHz
and 600 MHz (Bruker DRX-400 and DRX-600 NMR
spectrometers equipped with 5-mm probe heads; Bruker,
Germany), and gas chromatography-mass spectrometry
(GC-MS) (HP5890 GC, HP5970 MSD ; Hewlett-Packard,
Palo-Alto, CA, USA). Methanol-d4 was used as NMR
solvent and data from various one- and two-dimensional
NMR experiments were recorded, including 1 H^1 H corre-
FEMSEC 1574 7-10-03
A. Iakovlev et al. / FEMS Microbiology Ecology 46 (2003) 197^202
lation spectroscopy, 1 H^13 C heteronuclear multiple quantum coherence and 1 H^13 C heteronuclear multiple bond
correlation experiments (NMR pulse programs were supplied by Bruker). 1 H chemical shifts were referenced to the
residual CD2 HOD signal at NH 3.31 and 13 C chemical
shifts (from 1 H^13 C heteronuclear multiple quantum coherence and 1 H^13 C heteronuclear multiple bond correlation experiments) were referenced to the residual
CD2 HOD signal at NC 49.15. The NMR data were recorded at 30‡C. NMR analysis was also performed on
crude samples from culture supernatants after freeze-drying and these samples were studied in D2 O.
GC-MS was performed on trimethylsilylated samples of
the 3-CCA derivative. Trimethylsilylation was achieved by
treating a small amount of a dry sample with N,O-bis(trimethylsilyl)acetamide (60 Wl) in acetonitrile (40 Wl) for
20 min at room temperature. After addition of 100 Wl
acetonitrile, the sample was analyzed by GC-MS (30 m
U0.25 mm, BP5, 0.25 Wm; SGE Ltd, UK). The injector
was kept at 240‡C and the GC-MS interface at 260‡C. A
temperature program (5 min at 90‡C followed by 90^
240‡C at 5‡ min31 ) was employed for the analysis. The
sample (1 Wl) was injected in split mode and helium was
used as carrier gas (column £ow 1 ml min31 ).
2.6. Irradiation of 3-CCA and 7-OHCCA
Samples of 3-CCA (50 WM) and 7-OHCCA (50 WM) in
80 ml of either liquid Hagem medium or phosphate bu¡er
(20 mM, pH 5.6, room temperature) were ¢lter sterilized
and irradiated in two di¡erent experiments to 40 and 80
Gy Q 5% with 6-MV Q-rays from a GE linear accelerator
(Saturne 42F). The samples were irradiated with two opposite beams to obtain a homogenous dose distribution.
The possible production of the 3-CCA and 7-OHCCA
derivatives was followed by analytical HPLC as described
above.
199
Table 2
Electron impact mass spectrometric data for the trimethylsilyl derivative
of HBMA
m/z
Abundance (%)a
Assignment
426
411
336
321
308
293
219
30
29
9
10
42
35
52
Mvcþ
M^c CH3 vþ
M^(CH3 )3 SiOHvcþ
M^c CH3 ^(CH3 )3 SiOHvþ
^
^
^
a
Relative to m/z 73 (Si(CH3 )3 vcþ ).
3. Results
When supernatants from liquid cultures of P. centrifuga,
originally containing 0.2 mM 3-CCA, were analyzed by
HPLC with detection at 350 nm, only 3-CCA and 7-OHCCA (elution time 13.2 min and 11.1 min, respectively) were
observed in the samples. The 3-CCA and 7-OHCCA concentrations were both approximately 0.01 mM after 10 day
incubation with the fungus. When HPLC analysis of the
same samples was performed with detection at 210 nm,
another major compound (elution time 9.5 min) was detected. This compound was subsequently isolated by SPE
and preparative HPLC. NMR data (Table 1) in combination with mass spectrometric data from GC-MS (Table 2)
showed that this compound was 2-(2-hydroxybenzyl)malonic acid (HBMA, Fig. 1) [19]. NMR analysis of P. centrifuga crude culture samples originally containing 0.1 mM
3-CCA demonstrated that approximately 90% of the
3-CCA was converted to HBMA during the incubation.
HPLC analysis of samples from other cultures of wooddecaying fungi (in static agar cultures) showed that 3-CCA
was modi¢ed to HBMA during the incubation in all cultures, as exempli¢ed in Fig. 2. For the di¡erent fungal
species 3-CCA was below the detection limit in 58^100%
of the cultures (10^20 cultures of each fungus), at the end
of the incubation period (Table 3). The average ratio between 3-CCA and HBMA at the end of the incubation
was in the range 0^0.19 (Table 3). The compound
Table 1
NMR data for HBMA in methanol-d4 at 30‡C (numbering according to
Fig. 1)
Nucleus
Na
Scalar couplings (Hz)
H-2
3.78
3
H-4a/b
3.12
H-7
H-8
H-9
H-10
6.74
7.02
6.70
7.07
3
J2;4 7.5
J7;8 8.0, 4 J7;9 1.2
J8;9 7.5, 4 J8;10 1.5
3
J9;10 7.5
3
Nucleus
Na
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
173.3
52.5
173.3
31.3
125.8
156.5
116.0
128.8
120.4
131.7
a
Chemical shifts (N) were referenced to the signal of residual CD2 HOD
(NH 3.31 and NC 49.15). 13 C chemical shifts were extracted from 1 H^13 C
heteronuclear multiple quantum coherence experiments and 1 H^13 C heteronuclear multiple bond correlation experiments.
Fig. 1. The structures of 3-CCA and 7-OHCCA and a proposed scheme
for the formation of HBMA from 3-CCA.
FEMSEC 1574 7-10-03
200
A. Iakovlev et al. / FEMS Microbiology Ecology 46 (2003) 197^202
products from these compounds were observed. Several
products were found to be formed during the irradiation
of 3-CCA; of these 7-OHCCA was found to be formed at
the highest concentration. The irradiation of 7-OHCCA
resulted in the production of at least two di¡erent doubly
hydroxylated compounds as determined by one-dimensional 1 H NMR data (preliminary data, not presented).
The doubly hydroxylated compounds were not detected
in any of the fungal cultures.
4. Discussion
Fig. 2. Chromatograms from HPLC analyses of A. heteromorpha (Fr.)
Donk, cultivated on Hagem agar medium containing 0.1 mM 3-CCA.
A: Analysis of 50% aqueous methanol extract. B: Analysis of 50%
aqueous methanol extract after addition of 3-CCA and 7-OHCCA. For
experimental details see Section 2.
3-CCA was also found to be hydroxylated to 7-OHCCA
in cultures of all the tested fungi. The average ratio between 7-OHCCA and HBMA was in the interval 0.09^
0.50 (Table 3). The concentrations of 3-CCA and 7-OHCCA varied extensively between cultures of the same species
of wood-decaying fungi (Table 3). No distinction could be
made between brown-rot and white-rot fungi based on
3-CCA and 7-OHCCA concentrations (Table 3). The control samples (medium with 3-CCA but without fungi) did
not contain detectable amounts of HBMA. HPLC analysis
of samples from fungal cultures originally containing only
7-OHCCA showed no di¡erence in concentrations of this
compound before and after the 10 day incubation.
To test if HBMA could be formed from 3-CCA or
7-OHCCA by the action of c OH alone and if 7-OHCCA
could be hydroxylated by c OH, solutions of 3-CCA and
7-OHCCA were irradiated with Q-rays that produce c OH
[4]. HPLC analyses of these samples showed no formation
of HBMA from either 3-CCA or 7-OHCCA, but other
The 3-CCA-based method for c OH detection has previously been suggested to have excellent potential for use in
biological systems [9,11]. An obvious prerequisite for such
use is that 3-CCA must be hydroxylated by c OH to
7-OHCCA in the biological system studied. This was
found to be valid for cultures of wood-decaying fungi,
since 3-CCA was hydroxylated to 7-OHCCA in cultures
of all the fungi studied in the present investigation. Other
prerequisites are that 3-CCA and 7-OHCCA must not be
modi¢ed by the metabolism of the studied organism and
that hydroxylation of 7-OHCCA by c OH does not occur
to a large extent. No fungal modi¢cation of 7-OHCCA or
hydroxylation of 7-OHCCA by c OH was detected during
this study. 3-CCA was, however, modi¢ed to HBMA by
cultures of all studied wood-decaying fungi. The modi¢cation is not likely to be the result of normal lignin-degrading processes since both white-rot fungi, known to degrade
lignin, and brown-rot fungi, without lignin-degrading
capacity, modi¢ed 3-CCA. The fungal modi¢cation of
3-CCA to HBMA can be problematic when using this
method for c OH detection, as will be discussed below.
For this method to be reliable in the detection of c OH,
there must be a su⁄ciently high concentration of 3-CCA
present at all times during the incubation, to e⁄ciently
trap most of the c OH formed. Should the 3-CCA concen-
Table 3
Concentrations of 3-CCA and 7-OHCCA relative to HBMA in cultures of wood-decaying fungi after 10 day incubation with 0.1 mM 3-CCA
Fungusa
Rot-typeb
3-CCAc
Antrodia heteromorpha (12)
Antrodia vaillantii (12)
Antrodiella citrinella (17)
Coniophora arida (12)
Coniophora puteana (20)
Heterobasidion annosum (12)
Junghuhnia collabens (12)
Laetiporus sulphureus (18)
Phlebia centrifuga (16)
Phlebiopsis gigantea (17)
Resinicium bicolor (10)
b
b
w
b
b
w
w
b
w
w
w
0.03 (0.07,
n.d.d
0.09 (0.20,
0.13 (0.22,
0.07 (0.14,
0.05 (0.11,
0.03 (0.11,
0.19 (0.35,
0.02 (0.06,
n.d.
0.15 (0.32,
a
7-OHCCAc
83%)
82%)
58%)
80%)
67%)
92%)
89%)
83%)
80%)
0.16
0.25
0.50
0.13
0.16
0.34
0.14
0.20
0.38
0.18
0.09
(0.10,
(0.07,
(0.23,
(0.14,
(0.16,
(0.27,
(0.25,
(0.26,
(0.28,
(0.19,
(0.13,
17%)
0%)
6%)
42%)
30%)
0%)
42%)
22%)
11%)
29%)
60%)
Number of cultures within parentheses.
b
Brown-rot : b; white-rot : w.
c
The concentrations are expressed relative to HBMA with standard deviation and fraction of samples below detection limit within parentheses.
d
n.d. : not detected.
FEMSEC 1574 7-10-03
A. Iakovlev et al. / FEMS Microbiology Ecology 46 (2003) 197^202
tration be lowered to below a critical level, this can introduce errors in the estimation of the c OH formation. In the
present study the 3-CCA concentration was below detection limit in many cultures after 10 days of incubation
(Table 3). The decrease in 3-CCA concentration depends
on the hydroxylation by c OH and more importantly on the
fungal modi¢cation of 3-CCA to HBMA. To be able to
use this method on cultures of wood-decaying fungi, the
decrease in 3-CCA concentration must be kept under control. One simple solution would be to use higher initial
3-CCA concentration in the medium. However, tests of
fungal tolerance of 3-CCA indicate a fungistatic e¡ect of
elevated 3-CCA concentrations [8] that is in agreement
with the known antimicrobial activity of coumarins
[20,21]. Alternatively, the 7-OHCCA £uorescence can be
measured after a short incubation time, before a large
proportion of the 3-CCA has been transformed to
HBMA. Other hypothetical solutions would be to adjust
the incubation conditions to minimize the formation of
HBMA or to use 3-CCA derivatives that are not modi¢ed
by the fungal metabolism and react in a predictable way
with c OH.
Taking the above precautions it should be possible to
use 3-CCA for detecting c OH release in early stage interactions between wood decay fungi. In our study we included pathogenic decay fungi, e.g. H. annosum and
L. sulphureus, some typical early stage non-combative decay fungi, e.g. A. heteromorpha and P. centrifuga, early
stage combative species, e.g. R. bicolor and P. gigantea,
as well as late stage combative species, e.g. J. collabens. All
of these were able to oxidize 3-CCA through c OH. It
would thus be possible to study spatial patterns of c OH
which are thought to play a key role in interactions between di¡erent successional stages of wood decay fungi
[22].
HBMA was not formed in the control cultures (medium
with 3-CCA but without fungi) and treatment with c OH
alone could not cause the formation of HBMA from
3-CCA. These ¢ndings suggest that the fungal metabolism
is involved in the formation of HBMA from 3-CCA. Furthermore, since the formation of HBMA was found in
cultures of fungi of di¡erent species and rot-types, the
modi¢cation appears to involve a mechanism common
to many wood-decaying fungi. The compound HBMA
has previously been synthesized [19], but it has never
been shown to be formed in a biological system. The formation of HBMA from 3-CCA involves reduction of the
C-3/C-4 double bond of 3-CCA as well as hydrolysis of
the lactone ring (Fig. 1). The reduction of the double bond
presumably requires the involvement of the fungal metabolism, whereas the hydrolysis of the lactone ring either can
be enzymatic or non-enzymatic. A similar modi¢cation of
coumarin has previously been found to occur in shoots of
white sweet clover (Melilotus alba Desr.) [23,24]. In this
plant, coumarin is transformed by enzymes to 3-(2-hydroxyphenyl)propanoic acid [23,24] through the same ba-
201
sic reaction steps required for the formation of HBMA
from 3-CCA (Fig. 1). Thus, in the modi¢cation of
3-CCA to HBMA, demonstrated in the current study, a
similar enzymatic system could be involved. The hydrolysis of the lactone of 3,4-dihydrocoumarin has been found
to be catalyzed by enzymes of fungal [25] and bacterial
[26,27] origin, demonstrating that enzymatic activity hydrolyzing aromatic lactones is present in many microorganisms. Hydrolysis of lactone rings can also be non-enzymatic processes, depending on factors such as pH and
the structure of the lactone (e.g. [28]).
In conclusion, the ¢nding that 7-OHCCA was present in
cultures of all tested wood-decaying fungi after 10 days of
incubation with 3-CCA indicates that this method for assaying the c OH formation in principle should be possible
to use on wood-decaying fungi. A severe drawback of the
method is the fungal modi¢cation of the hydroxyl radical
detector 3-CCA to HBMA which may result in underestimation of the c OH formation.
Acknowledgements
The Foundation for Strategic Environmental Research
(MISTRA) supported this work. Dr. Anders Montelius
and Dr. Ulf Isacsson from the Department of Oncology,
Radiology and Clinical Immunology, Uppsala University
Hospital, are acknowledged for performing Q-irradiation
of the samples.
References
[1] Nathan, C. and Shiloh, M.U. (2000) Reactive oxygen and nitrogen
intermediates in the relationship between mammalian hosts and microbial pathogens. Proc. Natl. Acad. Sci. USA 97, 8841^8848.
[2] Mates, J.M., Aledo, J.C., Perez-Gomez, C., Del Valle, A.E. and
Segura, J.M. (2000) Interrelationship between oxidative damage
and antioxidant enzyme activities : an easy and rapid experimental
approach. Biochem. Educ. 28, 93^95.
[3] Schweikert, C., Liszkay, A. and Schopfer, P. (2000) Scission of polysaccharides by peroxidase-generated hydroxyl radicals. Phytochemistry 53, 565^570.
[4] Halliwell, B. and Gutteridge, J.M.C. (1989) Free Radicals in Biology
and Medicine. Clarendon Press, Oxford.
[5] Chen, S. and Schopfer, P. (1999) Hydroxyl-radical production in
physiological reactions. A novel function of peroxidase. Eur. J. Biochem. 260, 726^735.
[6] Schlosser, D., Fahr, K., Karl, W. and Wetzstein, H.-G. (2000) Hydroxylated metabolites of 2, 4-dichlorophenol imply a Fenton-type
reaction in Gloeophyllum striatum. Appl. Environ. Microbiol. 66,
2479^2483.
[7] Tanaka, H., Itakura, S. and Enoki, A. (1999) Hydroxyl radical generation by an extracellular low-molecular-weight substance and phenol oxidase activity during wood degradation by the white-rot basidiomycete Trametes versicolor. J. Biotechnol. 75, 57^70.
[8] Tornberg, K. and Olsson, S. (2002) Detection of hydroxyl radicals
produced by wood-decomposing fungi. FEMS Microbiol. Ecol. 40,
13^20.
[9] Makrigiorgos, G.M., Baranowska-Kortylewicz, J., Bump, E., Sahu,
FEMSEC 1574 7-10-03
202
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
A. Iakovlev et al. / FEMS Microbiology Ecology 46 (2003) 197^202
S.K., Berman, R.M. and Kassis, A.I. (1993) A method for detection
of hydroxyl radicals in the vicinity of biomolecules using radiationinduced £uorescence of coumarin. Int. J. Radiat. Biol. 63, 445^458.
Collins, A.K., Makrigiorgos, G.M. and Svensson, G.K. (1994) Coumarin chemical dosimeter for radiation therapy. Med. Phys. 21,
1741^1747.
Manevich, Y., Held, K.D. and Biaglow, J.E. (1997) Coumarin-3-carboxylic acid as a detector for hydroxyl radicals generated chemically
and by gamma radiation. Radiat. Res. 148, 580^591.
Backa, S., Jansbo, K. and Reitberger, T. (1997) Detection of hydroxyl radicals by a chemiluminescence method - a critical review. Holzforschung 51, 557^564.
Baucher, M., Monties, B., Van Montagu, M. and Boerjan, W. (1998)
Biosynthesis and genetic engineering of lignin. Crit. Rev. Plant Sci.
17, 125^197.
Boudet, A.-M. (1998) A new view of ligni¢cation. Trends Plant Sci. 3,
67^71.
Blanchette, R.A. (1995) Degradation of the lignocellulose complex in
wood. Can. J. Bot. 73, S999^S1010.
Schoemaker, H.E., Tuor, U., Muheim, A., Schmidt, H.W.H. and
Leisola, M.S.A. (1991) White-rot degradation of lignin and xenobiotics. In: Biodegradation: Natural and Synthetic materials (Betts,
W.B., Ed.), pp. 157^174. Springer, Bedford.
Leonowicz, A., Matuszewska, A., Luterek, J., Ziegenhagen, D., Wasilewska, M., Cho, N.-S., Hofrichter, M. and Rogalski, J. (1999)
Biodegradation of lignin by white rot fungi. Fungal Genet. Biol.
27, 175^185.
Stenlid, J. (1985) Population structure of Heterobasidion annosum as
determined by somatic incompatibility, sexual incompatibility, and
isozyme patterns. Can. J. Bot. 63, 2268^2273.
Merijan, A. and Gardner, P.D. (1965) Quinone methides. Base-cata-
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
lyzed condensation reactions of hydroxybenzyl alcohols and ethers.
J. Org. Chem. 30, 3965^3967.
Urdangarin, C., Regente, M.-C., Jorrin, J. and de la Canal, L. (1999)
Sun£ower coumarin phytoalexins inhibit the growth of the virulent
pathogen Sclerotinia sclerotiorum. J. Phytopathol. 147, 441^443.
Ojala, T., Remes, S., Haansuu, P., Vuorela, H., Hiltunen, R., Haahtela, K. and Vuorela, P. (2000) Antimicrobial activity of some coumarin containing herbal plants growing in Finland. J. Ethnopharmacol. 73, 299^305.
Rayner, A.D.M. (1997) Degrees of Freedom. Living in Dynamic
Boundaries, 312 pp. Imperial College Press, London.
Kosuge, T. and Conn, E.E. (1959) The metabolism of aromatic compounds in higher plants. I. Coumarin and o-coumaric acid. J. Biol.
Chem. 234, 2133^2137.
Kosuge, T. and Conn, E.E. (1962) The metabolism of aromatic compounds in higher plantsV. Puri¢cation and properties of dihydrocoumarin hydrolase of Melilotus alba. J. Biol. Chem. 237, 1653^1656.
Shimizu, S., Kataoka, M., Shimizu, K., Hirakata, M., Sakamoto, K.
and Yamada, H. (1992) Puri¢cation and characterisation of a novel
lactonohydrolase, catalysing the hydrolysis of aldonate lactones and
aromatic lactones, from Fusarium oxysporum. Eur. J. Biochem. 209,
383^390.
Casellas, M., Grifoll, M., Bayona, J.M. and Solanas, A.M. (1997)
New metabolites in the degradation of £uorene by Arthrobacter sp.
strain F101. Appl. Environ. Microbiol. 63, 819^826.
Kataoka, M., Honda, K. and Shimizu, S. (2000) 3,4-Dihydrocoumarin hydrolase with haloperoxidase activity from Acinetobacter calcoaceticus F46. Eur. J. Biochem. 267, 3^10.
Andersson, L. and Kenne, L. (2000) Lactones of methyl 3-O-[(R)and (S)-1-carboxyethyl]-K-D-gluco-, galacto-, and manno-pyranosides. Carbohydr. Res. 329, 257^268.
FEMSEC 1574 7-10-03