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Applied and Environmental Microbiology, September 1999, p. 3867-3872, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Enzymatic Formation of G-Group Aflatoxins and
Biosynthetic Relationship between G- and B-Group Aflatoxins
Kimiko
Yabe,1,*
Miki
Nakamura,1 and
Takashi
Hamasaki2
National Food Research Institute, Tsukuba,
Ibaraki 305-8642,1 and Faculty of
Agriculture, Tottori University, Tottori
608-0945,2 Japan
Received 4 January 1999/Accepted 11 June 1999
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ABSTRACT |
We detected biosynthetic activity for aflatoxins G1 and
G2 in cell extracts of Aspergillus parasiticus
NIAH-26. We found that in the presence of NADPH, aflatoxins
G1 and G2 were produced from O-methylsterigmatocystin and
dihydro-O-methylsterigmatocystin, respectively. No G-group
aflatoxins were produced from aflatoxin B1, aflatoxin
B2, 5-methoxysterigmatocystin, dimethoxysterigmatocystin, or sterigmatin, confirming that B-group aflatoxins are not the precursors of G-group aflatoxins and that G- and B-group aflatoxins are
independently produced from the same substrates
(O-methylsterigmatocystin and
dihydro-O-methylsterigmatocystin). In competition
experiments in which the cell-free system was used, formation of
aflatoxin G2 from
dihydro-O-methylsterigmatocystin was suppressed when
O-methylsterigmatocystin was added to the reaction mixture,
whereas aflatoxin G1 was newly formed. This result
indicates that the same enzymes can catalyze the formation of
aflatoxins G1 and G2. Inhibition of G-group
aflatoxin formation by methyrapone, SKF-525A, or imidazole indicated
that a cytochrome P-450 monooxygenase may be involved in the formation of G-group aflatoxins. Both the microsome fraction and a cytosol protein with a native mass of 220 kDa were necessary for the formation of G-group aflatoxins. Due to instability of the microsome fraction, G-group aflatoxin formation was less stable than B-group aflatoxin formation. The ordA gene product, which may catalyze the
formation of B-group aflatoxins, also may be required for G-group
aflatoxin biosynthesis. We concluded that at least three reactions,
catalyzed by the ordA gene product, an unstable microsome
enzyme, and a 220-kDa cytosol protein, are involved in the enzymatic
formation of G-group aflatoxins from either
O-methylsterigmatocystin or dihydro-O-methylsterigmatocystin.
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INTRODUCTION |
Aflatoxin B1
(AFB1), AFB2, AFG1, and
AFG2 are toxic, carcinogenic secondary metabolites that are
produced by some strains of Aspergillus flavus,
Aspergillus parasiticus, Aspergillus nomius, and
Aspergillus tamarii (10). The biosynthetic
pathway consists of more than 18 enzyme steps from acetyl coenzyme A
(2, 4-6, 9, 25, 27, 30-35, 37). Many of the genes involved
in aflatoxin biosynthesis have been isolated, and most of them are
clustered (reviewed in references 26 and
28). The pathway leading to the formation of G-group
aflatoxins has not been determined yet because the enzyme activities
required to synthesize these compounds have not been detected in
cell-free systems.
AFB1 and AFG1 contain dihydrobisfuran rings,
and AFB2 and AFG2 contain tetrahydrobisfuran
rings. In vivo feeding experiments have shown that AFB1 and
AFG1 are produced from O-methylsterigmatocystin (OMST) and that AFB2 and AFG2 are produced from
dihydro-O-methylsterigmatocystin (DHOMST) (4, 6,
30). Also, AFB1 and AFB2 are
independently produced from OMST and DHOMST, respectively, through
common reactions in in vitro cell-free systems (4, 30).
The biosynthetic pathway(s) associated with the formation of G-group
aflatoxins has been controversial for a long time. All known G-group
aflatoxin-producing strains also produce B-group aflatoxins
(18). No mutants that produce only G-group aflatoxins have
been isolated in mutagenesis experiments performed with aflatoxigenic strains (3, 36). Low levels of conversion of radioactive AFB1 to other aflatoxins, including AFG1, have
been obtained by using a cell-free homogenate (22). These
results are consistent with the hypothesis that G-group aflatoxins may
be produced from B-group aflatoxins. However, some researchers have
reported that in feeding experiments radioactive AFB1 is
converted to other aflatoxins (16, 22), while other workers
have not observed this (11, 17). Despite these differences,
it is generally assumed that G-group aflatoxins are produced from
B-group aflatoxins by insertion of oxygen into a C-C bond through a
Baeyer-Villigar reaction (9, 27).
Aflatoxins are produced intracellularly and then excreted. In feeding
experiments, exogenous AFB1 may not reach the aflatoxin biosynthetic site in the cell, and confirmation of AFB1
entrance into the cell is essential. However, since aflatoxins are
hydrophobic substances, distinguishing the entrance of AFB1
into a cell from nonspecific binding of AFB1 to hydrophobic
cellular components (e.g., membranes) is very difficult. Therefore, an
in vitro cell-free system for G-group aflatoxin biosynthesis is
essential for determining the biosynthetic relationship between the G-
and B-group aflatoxins. Our objectives in this study were (i) to
establish a cell-free system for biosynthesis of G-group aflatoxins,
(ii) to clarify the biosynthetic relationship between G- and B-group
aflatoxins, and (iii) to characterize the enzyme steps for G-group
aflatoxin formation. Finally, we describe a scheme for the biosynthetic pathway for the G-group aflatoxins.
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MATERIALS AND METHODS |
Microorganisms and cultures.
A. parasiticus NIAH-26, a
UV-irradiated mutant of A. parasiticus SYS-4 (= NRRL 2999),
was used in this study (36). A. parasiticus NIAH-26 induced all enzymes required to convert norsolorinic acid to
aflatoxins in YES liquid culture medium (2% yeast extract, 20%
sucrose), although it produced no aflatoxins or anthraquinone or
xanthone precursors. This mutant may be blocked in the pathway before
the formation of norsolorinic acid (30-35, 37).
Non-aflatoxin-producing wild-type strain Aspergillus oryzae
SYS-2 (= IFO 4251) also was used. The fungal strains were cultured
without agitation at 28°C for 4 days either in YES liquid medium (an
aflatoxin-inducing medium) or in YEP liquid medium (2% yeast extract,
20% peptone) (a non-aflatoxin-inducing medium) (1).
Standard metabolite samples.
We prepared OMST and DHOMST as
described previously (30). 5-Methoxysterigmatocystin
(7-hydroxy-6-10-dimethoxydifuroxanthone) and sterigmatin were isolated
by extracting mycelia of Aspergillus versicolor (Vuillemin)
Tiraboschi (13), and dimethoxysterigmatocystin (6,9,10-trimethoxy-7-hydroxydifuroxanthone) was isolated by extracting mycelia of Aspergillus multicolor (14). A
standard kit (Makor, Israel) was used to analyze the AFB1,
AFB2, AFG1, and AFG2. The sterigmatocystin derivatives were dissolved in dimethylformamide and
diluted in methanol, and their concentrations were determined from UV
absorption spectra by using the following molar absorption coefficients
(8, 13): OMST (310 nm), 16,500; DHOMST (311 nm), 17,300;
5-methoxysterigmatocystin (331 nm), 12,100; dimethoxysterigmatocystin (330 nm), 19,200; sterigmatin (324 nm), 16,900; AFB1 (362 nm), 21,800; AFB2 (363 nm), 24,000; AFG1 (362 nm), 16,100; and AFG2 (365 nm), 19,300.
Preparation of cell extract, microsome fractions, and cytosol
fractions.
The cell extract, microsome, and cytosol fractions were
prepared from mycelia of cultures grown in YES or YEP medium at 28°C for 4 days by grinding the mycelia and then centrifuging them (32,
37). Samples were frozen at 
80°C until they were used.
Enzyme assays for aflatoxin formation.
We determined assay
conditions that optimized the enzyme activity. Lecithin from eggs
(Merck, Rahway, N.J.) was dissolved in a mixture containing chloroform
and methanol and dried by rotary evaporation. After six repetitions of
solubilization and drying, the resultant lipid film was suspended in a
solution containing 20 mM Tris-HCl (pH 7.5) and 1 mM EDTA by mixing
with a Vortex mixer and sonication in order to obtain a liposome
solution with a final lecithin concentration of 5 mg/ml
(21). The resultant liposome solution was stored at 4°C.
To determine the effect of lipid form on enzyme activity, lecithin also
was suspended in water and then sonicated without evaporation; in the
resultant solution, the lipid could assume the form of a micelle
instead of a liposome. Both lipid preparations were effective for
G-group aflatoxin formation activity for at least 6 months.
To detect enzyme activity, we incubated cell extracts (containing 1.3 mg of protein per ml) in a reaction mixture containing 90 mM potassium
phosphate (pH 7.5), 10% (vol/vol) glycerol, each substrate at a
concentration of 80 µM, 4 mM NADPH, 0.9 mg of bovine serum albumin
(BSA) per ml, and 0.5 mg of liposome per ml. The cell extract was first
mixed with BSA and liposome, and the reaction was started by adding the
other constituents. The final volume of the reaction mixture was 50 µl. Reactions were routinely carried out at 24°C for 40 min and
then terminated by adding 75 µl of water-saturated chloroform and
mixing the preparation with a Vortex mixer. After centrifugation at
10,000 × g for 1 min, an aliquot of the lower chloroform
layer was injected directly into a model LC-6A high-performance liquid
chromatograph (HPLC) (Shimadzu, Kyoto, Japan) equipped with a silica
gel HPLC column (4.6 by 150 mm; Shim-pack CLC-SIL; Shimadzu) and a
guard column (4 by 10 mm; Shim-pack G-Sil). The solvent system
consisted of toluene, ethyl acetate, formic acid, and methanol
(198:15:4:3, vol/vol/vol/vol). The fluorescence intensities of the
aflatoxins were monitored with a Shimadzu model RF-535 HPLC
fluorescence monitor (excitation wavelength, 365 nm; emission
wavelength, 425 nm) by using a flow rate of 1 ml/min at room
temperature. The retention times of AFB1, AFB2,
AFG1, and AFG2 were compared with the retention
times of standard metabolite samples.
Microsome fractions were mixed with or without the cytosol fraction
(final concentration, 0.36 mg of protein/ml) in the presence of BSA and
liposome in order to localize enzyme activity. Enzyme reaction was
started by adding the reaction mixture containing the other constituents.
To determine the molecular mass of the cytosolic factor used for
AFG
1 formation, we prepared the cytosol fraction (6 mg of
protein; 0.4 ml) by concentrating the fraction to 80% saturation
with
ammonium sulfate and then suspending it in a 20 mM Tris-HCl
(pH 7.5)
solution. The resultant solution was loaded onto an Ultrogel
AcA 34 column (1.25 by 56 cm; Amersham Pharmacia Biotech AB, Uppsala,
Sweden)
that had previously been equilibrated with a solution
containing 20 mM
Tris-HCl (pH 7.5). After 0.5-ml fractions were
collected, a 70-µl
aliquot from each fraction was mixed with the
microsome fraction (5 µl; final concentration, 0.84 mg/ml) and
then with a mixture
containing lipid and BSA. The reaction was
then started by adding a
reaction mixture containing the other
constituents described above. The
final volume of the reaction
mixture was 100 µl. The reaction was
carried out at 24°C for 40
min, and the amounts of AFB
1
and AFG
1 were
measured.
To determine the stability of enzyme activity, the cell extracts were
incubated with or without BSA (2.3 mg/ml) and lipid
(1.25 mg/ml) for
various times at 24°C. The enzyme activity that
remained was
determined by adding a reaction mixture containing
the other
constituents described above and then incubating the
preparation at
24°C for 40
min.
To determine which fraction (microsome or cytosol) resulted in the
instability of G-group aflatoxin formation activity, one
fraction was
incubated at 24°C for 60 min, and then the other
fraction was added.
The aflatoxin biosynthesis enzyme activities
were then measured. The
final concentrations of microsomes and
cytosol were 1.9 and 0.36 mg/ml,
respectively.
Characterization of enzyme activity.
Methyrapone
(2-methyl-1,2-di-3-pyridyl-1-propanone), SKF-525A [Proadifen;
-phenyl--propylbenzeneacetic acid 2-(diethylamino)ethyl ester],
and imidazole were purchased from Sigma Chemical Co. (St. Louis, Mo.).
Ethoxyquin (6-methoxy-1,2-dihydro-2,2,4-trimethylquinoline) was
purchased from ICN Pharmaceuticals, Inc. (Costa Mesa, Calif.). For gel
filtration we used an Ultrogel AcA 34 column, and the following
molecular mass standards (Amersham Pharmacia Biotech AB) were used to
calibrate the molecular mass measurements: dextran blue (2,000 kDa),
ferritin (450 kDa), catalase (240 kDa), aldolase (158 kDa), BSA (68 kDa), egg albumin (45 kDa), and cytochrome c (12.5 kDa).
For heat treatment of the cytosol fraction, the cytosol was kept at
90°C for 5 min and then cooled. The protein concentration
was
determined by using the Bradford method (
7) and BSA as
the
standard.
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RESULTS |
Detection of enzymatic activity for G-group aflatoxin
formation.
When the cell extract of A. parasiticus
NIAH-26 was incubated with OMST in the presence of NADPH at 24°C for
40 min, AFG1 and AFB1 were formed (Fig.
1A). When DHOMST instead of OMST was added to the reaction mixture, AFG2 and AFB2
were produced. The amounts of AFB1 and AFG1
produced from OMST continued to increase for at least 40 min (Fig. 1B).
When OMST was used as a substrate, the apparent
Km values of OMST and NADPH for AFG1
formation were 1.8 and 154 µM, respectively.
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FIG. 1.
Enzymatic formation of G- and B-group aflatoxins. (A)
Cell extract was incubated with OMST (left) or DHOMST (right) in the
presence of NADPH, and a chloroform extract of the reaction mixture was
then analyzed by silica gel HPLC. The retention times were 8.3 min for
AFB1, 12.7 min for AFG1, 10.7 min for
AFB2, and 17.1 min for AFG2. (B) Cell extract
was incubated with OMST at 24°C for various times in the presence of
liposome and BSA, and AFG1 ( ) and AFB1 ( ) were
produced. The error bars indicate the differences in the duplicate
experiments.
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Because the enzyme activity for G-group aflatoxin formation appeared to
be weak, approximately 0.6% of the enzyme activity
for B-group
aflatoxin formation, we attempted to optimize the
activities by
supplying substances generally assumed to be useful
for activating or
stabilizing enzymes (e.g., BSA, lipids, and
polyols) (
15,
20,
29). When an extract was mixed with various
concentrations (0 to
1.8 mg/ml) of BSA and liposome and then the
reaction was started by
adding substrate and other constituents,
the enzyme activity was
enhanced (Fig.
2). Addition of only lipid
(0.5 mg/ml) significantly increased the enzyme activity for G-group
aflatoxin formation, and addition of both lipid (0.5 mg/ml) and
BSA
(0.9 mg/ml) resulted in the maximum enzyme activity. We also
found that
enzyme activity was significantly greater when the
lipid-BSA mixture
was added first to the cell extract, before
the other constituents were
added, than when the reactions were
started by adding a single solution
containing all of the constituents.
In similar experiments, we found
that the micelle form of the
lipid instead of the liposome form
resulted in a similar enhancement
effect, although the liposome form
was routinely used. In contrast,
increasing the glycerol concentration
from 10 to 25% did not affect
the enzyme activity, although more than
20% glycerol is generally
considered a stabilizer for cytochrome P-450
monooxygenase activity
(
19).
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FIG. 2.
Activation of aflatoxin production with BSA and
liposome. Cell extract was added to mixtures containing various
concentrations of BSA with () or without () liposome (0.5 mg/ml),
and then the reactions were started by adding the other constituents,
including OMST and NADPH. After incubation at 24°C for 40 min, the
amount of AFG1 produced was measured. The error bars
indicate the differences in the duplicate experiments.
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Independent formation of G- and B-group aflatoxins.
We
examined the enzyme activities for G-group aflatoxin formation when
various metabolites were used as substrates. When 9 µM OMST was added
to the reaction mixture, AFG1 (0.20 ± 0.011 pmol/mg
of protein/min) but not AFG2 was produced, and when the same concentration of DHOMST was added, only AFG2
(0.17 ± 0.014 pmol/mg/min) was produced. However, neither
AFG1 nor AFG2 was produced from either 85 µM
5-methoxysterigmatocystin or 87 µM sterigmatin. A trivial amount of
AFG1 (0.009 ± 0.007 pmol/mg/min) was produced when 77 µM dimethoxysterigmatocystin was added, which may have been due to
contamination of the dimethoxysterigmatocystin by a small amount OMST.
In the same experiments, neither AFG1 nor AFG2
was produced from either 9 µM AFB1 or 9 µM
AFB2, nor did the reverse reaction from G-group aflatoxins
to B-group aflatoxins occur.
Involvement of the common enzyme(s) for formation of
AFG1 and AFG2.
To determine the
relationship between AFG1 formation and AFG2
formation, we examined the enzyme activities by using reaction mixtures
that contained 4.6 µM DHOMST and different concentrations of OMST
(Fig. 3). Although AFG2 was
produced from DHOMST, the amount formed decreased as the OMST
concentration increased. Adding high concentrations of OMST
(concentrations higher than 10 µM) led to exclusive formation of
AFG1. These results indicate that the same enzyme(s) may
catalyze the formation of AFG1 and AFG2.
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FIG. 3.
Competition between OMST and DHOMST for enzyme during
G-group aflatoxin formation. A cell extract was incubated with 4.6 µM
DHOMST and various concentrations of OMST at 24°C for 40 min, and
then the amounts of AFG1 () and AFG2 ()
were measured.
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Requirement for both the microsome fraction and the 220-kDa protein
for G-group aflatoxin production.
We measured the G-group
aflatoxin formation activity by using either the microsome fraction or
the cytosol fraction or a combination of both fractions (Fig.
4A). Significant amounts of
AFG1 were not formed in the reaction mixture containing
OMST plus cytosol, and the OMST-microsome reaction mixture produced
only a trace amount of AFG1. When both the cytosol and
microsome fractions were present, enzyme activity was markedly enhanced
and dependent on the microsome concentration. Preincubation of the
cytosol at 90°C for 5 min eliminated the enhancement effect during
G-group aflatoxin formation (data not shown). These results indicate
that both the cytosol fraction and the microsome fraction are required for production of AFG1.
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FIG. 4.
Requirement for both cytosol and microsome fractions
during AFG1 formation. (A) Various concentrations of the
microsome fraction were incubated with OMST in the presence () or
absence () of the cytosol fraction. (B) Concentrated cytosol
fraction was applied to an Ultrogel AcA 34 gel filtration column, and
the column effluent was supplemented with the microsome fraction. Then
the AFG1 formation activity () was measured. The
absorbance at 280 nm is shown for reference (dotted line). The
positions of molecular mass standards (in kilodaltons) are indicated,
and the volume of each fraction was 0.5 ml. OD280, optical density at
280 nm.
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Neither aflatoxin formation activity nor enhancement effects were
detected with either cytosol fractions or microsome fractions
prepared
from cultures grown in YEP medium or from
A. oryzae SYS-2
that had been cultured in YES medium. Instead, the cytosol fractions
of
cultures grown in YEP medium partially suppressed AFG
1
formation
activity of either the microsome fraction or the cytosol
fraction
prepared from
A. parasiticus NIAH-26 cultured in
YES medium (data
not
shown).
We examined the activation factor in the cytosol fraction by
fractionating it through an Ultrogel AcA 34 gel filtration column
and
then incubating each fraction with OMST in a reaction mixture
containing the microsome fraction. AFG
1 formation activity
(Fig.
4B) was increased threefold by the addition of the fraction that
corresponded to a molecular weight of about 220,000. This fraction
did
not correspond to the peak fraction containing protein, indicating
that
the enhancing activity of this fraction was not caused by
an
as-yet-unidentified stabilizing effect associated with a high
protein
concentration. These results indicate that at least two
kinds of
enzymes, one a microsome enzyme and the other a 220-kDa
soluble
protein, are necessary for the formation of AFG
1 from
OMST.
Characterization and comparison of G-group aflatoxin formation
activity and B-group aflatoxin formation activity.
Formation of
AFG1 and AFB1 was decreased by 3 mM
methyrapone, SKF-525A, or imidazole (Table
1), indicating that the formation of
AFB1 and the formation of AFG1 were dependent
on cytochrome P-450 oxygenase activity. Also, preferential inhibition
of AFG1 formation but not AFB1 formation by
ethoxyquin, which was detected in cultures of aflatoxigenic strains
(12), was observed in the cell-free system.
When the cell extract was incubated with or without liposome and BSA at
24°C for various times, the level of AFG
1 production
(Fig.
5) decreased to less than 50% of
the original level after
15 min and to less than 20% of the original
level after 30 min,
irrespective of the presence of lipid and BSA. The
AFB
1 formation
activity was more stable than the
AFG
1 formation activity was
and was about 70% of the
original activity after 30 min of incubation.
The presence of lipid and
BSA appeared to slightly stabilize both
activities, although an
increase in the glycerol concentration
up to 34% (final concentration)
did not stabilize the activities
(data not shown).
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FIG. 5.
Stability of aflatoxin formation activity. Cell extracts
were incubated with ( and  ) or without ( and  ) lipid and
BSA at 24°C for various times, and then the enzyme activities for
AFG1 formation ( and ) and for AFB1
formation ( and ) were determined. The activities without
preincubation (at zero time) were 0.19 pmol of AFG1/mg/min
(), 0.23 pmol of AFG1/mg/min (), 31 pmol of
AFB1/mg/min (), and 30 pmol of AFB1/mg/min
(). AF, aflatoxin.
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We determined which fraction (cytosol or microsome) contributed to the
instability of the enzyme activity by independently
incubating the
fractions at 24°C for 60 min and then adding the
other fraction. We
found that preincubation of the microsome fraction
significantly
decreased the activity to 9.5% of the maximum activity,
whereas
preincubation of the cytosol fraction only slightly decreased
the
activity to 98% of the maximum activity. These results indicate
that
the microsome enzyme may be the cause of the instability
observed in
G-group aflatoxin
biosynthesis.
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DISCUSSION |
We detected enzyme activity for G-group aflatoxin formation by
using a cell extract and a combination of cytosol and microsome fractions of A. parasiticus NIAH-26. Addition of exogenous
lipid and BSA increased G-group aflatoxin formation, but the amount of
AFG1 formed was always less than 1.5% of the amount of
AFB1 formed, even in the presence of liposome and BSA. In
contrast to this differential activity in cell-free systems, A. parasiticus NRRL 2999, the wild-type progenitor of NIAH-26,
produced similar amounts of both G- and B-group aflatoxins. Also,
A. parasiticus NIAH-26 could convert either
sterigmatocystin, dihydrosterigmatocystin, OMST, or DHOMST to
significant amounts of G-group aflatoxins that corresponded to the
amounts of B-group aflatoxins obtained in previous feeding experiments
(30). The relative decrease in G-group aflatoxin
biosynthesis may indicate that the enzymes for G-group aflatoxin
formation are relatively sensitive to the methods used to prepare the
cell-free fractions, whereas the enzymes for B-group aflatoxin
formation are much less sensitive to the process. Furthermore, G-group
aflatoxin biosynthesis requires both cytosol and microsome fractions
(Fig. 4), and if only one of these fractions was used for examining
G-group aflatoxin biosynthesis, no (or only a slight amount of) G-group
aflatoxin was detected. These findings may at least partially explain
why the enzyme activity for G-group aflatoxin biosynthesis was not
detected previously.
In this study, we obtained data supporting the hypothesis that G-group
aflatoxins and B-group aflatoxins are formed independently. AFG1 and AFG2 were produced from OMST and
DHOMST, respectively, whereas neither AFG1 nor
AFG2 was produced from AFB1 or AFB2
in the cell-free systems used. These results indicate that B-group aflatoxins are not the precursors of G-group aflatoxins and that G- and
B-group aflatoxins are independently produced from the same substrate
(OMST for AFG1 and AFB1 and DHOMST for
AFG2 and AFB2) through different pathways from
a common branching intermediate. Although the branch point is not
known, at least one intermediate following OMST or DHOMST seems to be
involved (see below).
Recently, the ord1 and ordA genes were isolated
from A. flavus (24) and A. parasiticus
(38), respectively, and were found to be required for the
conversion of OMST to AFB1. Yu et al. (38) reported that complementation of A. parasiticus SRRC 2043, an OMST-accumulating strain, with the ordA gene restores the
ability to produce AFB1, AFB2,
AFG1, and AFG2. However, in a yeast expression system ordA could convert AFB1 to OMST but not
to AFG1, and Yu et al. suggested that at least one
additional enzyme in addition to the ordA gene product may
be needed for the formation of G-group aflatoxins; our results are
consistent with this hypothesis. Both the ordA gene and the
ord1 gene encode cytochrome P-450 type monooxygenases (based
on their DNA sequences). Our finding that biosynthesis of aflatoxins
requires NADPH and is inhibited by monooxygenase inhibitors, such as
SKF-525A, is consistent with these reports. This similarity indicates
that the same enzyme may be involved in both biosynthetic pathways.
Moreover, ordA may be a microsome enzyme, since B-group
aflatoxin biosynthetic activity is limited to the microsome fraction
(4, 29a).
Our results revealed that there are differences between the G-group
aflatoxin biosynthetic activity and the B-group aflatoxin biosynthetic
activity, because the microsome enzyme fraction involved in G-group
aflatoxin formation was less stable than the microsome enzyme fraction
involved in B-group aflatoxin formation (Fig. 5). The evidence which
indicates that an additional unstable microsome enzyme may be required
for G-group aflatoxin formation includes (i) the fact that an
ordA product is the sole requirement for AFB1
formation (38), (ii) the fact that the enzyme for B-group aflatoxin biosynthesis is more stable than the enzyme for
AFG1 biosynthesis (Fig. 5), and (iii) the fact that the
unstable enzyme is in the microsome fraction. Therefore, at least three
enzymes apparently are required for conversion of OMST to
AFG1 and conversion of DHOMST to AFG2 (Fig.
6); these enzymes are a microsome
ordA gene product, an unstable microsome protein, and a
220-kDa cytosol protein. Although B-group aflatoxins may be the final
products of the reactions catalyzed by the ordA product, at
least two reactions catalyzed by the ordA product may be
included in the pathway from OMST to AFB1 or the
pathway from DHOMST to AFB2. A nonenzymatic reaction,
as well as the enzymatic reaction(s) catalyzed by the ordA gene product, might also occur. Furthermore, a
transient intermediate in the successive reactions may function as the
branch point for the formation of G-group and B-group aflatoxins and may be converted to G-group aflatoxins through two reactions catalyzed by the unstable microsome enzyme and the cytosol enzyme. We hypothesize that the preferential inhibition of G-group aflatoxin biosynthesis by
ethoxyquin (13) is due to this compound's effect on one of the latter two reaction steps that appear to be unique to G-group aflatoxin biosynthesis. The estimated molecular mass of the native cytosol protein was about 220 kDa, and this protein may be an aggregate
form under the low-salt conditions which we used in the gel filtration
procedure.
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FIG. 6.
Metabolic pathways for formation of AFG1
from OMST and formation of AFG2 from DHOMST. At least three
enzyme reactions may be commonly present in both pathways. Reaction 1 may be catalyzed by an ordA gene product, which is microsome
cytochrome P-450 oxygenase. A transient intermediate formed during this
pathway may be subsequently converted to another substance in reaction
2 and finally converted to the final product, AFG1 or
AFG2, in reaction 3. Reactions 2 and 3 may be catalyzed by
an unstable microsome enzyme and a 220-kDa cytosol protein. However,
the order of the three enzyme activities was not determined in this
study.
|
|
The biosynthesis of G-group aflatoxins was sensitive to conditions,
such as buffer, ionic strength, and ethoxyquin, that suggest that
physically and chemically unstable intermediates may be part of the
biosynthetic pathway. The next step in our research will involve
determining the intermediates involved in these pathways.
Neither G-group aflatoxin biosynthesis nor enhancement of activity due
to the microsome and cytosol fractions was detected in the cell-free
systems prepared from the strain that was cultured in
non-aflatoxin-inducible YEP medium or from the nonaflatoxigenic organism A. oryzae. Similar results have been observed for
other enzymes involved in aflatoxin biosynthesis (23, 30, 31, 33,
37), and these results suggested that G-group aflatoxin biosynthesis may be regulated at the transcriptional level by mechanism(s) common to the other enzymes (26). Cloning and
characterization of the genes related to G-group aflatoxin formation
remain to be studied.
This study brings to an end the long controversy about G-group
aflatoxin biosynthesis. It is generally accepted that strains of
A. parasiticus produce both G- and B-group aflatoxins, while A. flavus strains produce only B-group aflatoxins
(18). The absence of G-group aflatoxin biosynthesis in
A. flavus may soon be explained on the basis of biochemical
as well as molecular-biological evidence.
| |
ACKNOWLEDGMENTS |
This work was supported in part by grant-in-aid BDP-99-VI-1-3
(Bio Design Program) from the Ministry of Agriculture, Forestry and Fisheries.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: National Food
Research Institute, Tsukuba, Ibaraki 305-8642, Japan. Phone:
0298-38-8050. Fax: 0298-38-7996. E-mail:
yabek{at}nfri.affrc.go.jp.
| |
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Applied and Environmental Microbiology, September 1999, p. 3867-3872, Vol. 65, No. 9
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
