New Insights into the Conversion of Versicolorin A in the Biosynthesis of Aflatoxin B1

Article abstract of DOI:10.1021/jacs.5b06770

A crucial and enigmatic step in the complex biosynthesis of aflatoxin B₁ is the oxidative rearrangement of versicolorin A to demethylsterigmatocystin. This step is thought to proceed by an oxidation-reduction-oxidation sequence, in which the NADPH-dependent oxidoreductase AflM catalyzes the enclosed reduction step. AflM from Aspergillus parasiticus, after heterologous production in E. coli and purification, however, catalyzed the reduction of the hydroquinoid form of the starting compound versicolorin A (25% conversion) to a so far unknown product of aflatoxin biosynthesis. The asymmetric reduction of emodin hydroquinone to (R)-3,8,9,10-tetrahydroxy-6-methyl-3,4-dihydroanthracen-1(2H)-one (up to 82% for AflM) has also been observed in previous studies using MdpC from Aspergillus nidulans (monodictyphenone biosynthetic gene cluster). The first (nonenzymatic) reduction of emodin to emodin hydroquinone, for example with sodium dithionite, is obligatory for the enzymatic reduction by AflM or MdpC. These results imply an unprecedented role of AflM in the complex enzymatic network of aflatoxin biosynthesis.

Full text of DOI:10.1021/jacs.5b06770

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Communication
New Insights into the Conversion of Versicolorin
A in the Biosynthesis of Aflatoxin B1
David Conradt, Michael A Schätzle, Julian Haas, Craig A. Townsend, and Michael Müller
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b06770 • Publication Date (Web): 12 Aug 2015
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New Insights into the Conversion of Versicolorin A in the Biosynthesis
of Aflatoxin B1
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David Conradt,†,§ Michael A. Schätzle,†,§ Julian Haas,† Craig A. Townsend‡ and Michael Müller†,*
†Institut für Pharmazeutische Wissenschaften, Albert-Ludwigs-Universität Freiburg, Albertstr. 25, 79104 Freiburg, Germany
‡Department of Chemistry, The Johns Hopkins University, 3400 N. Charles St., Baltimore, Maryland, USA
Supporting Information Placeholder
hydroxyl group of 3, and Baeyer–Villiger-like oxidative cleavage
ABSTRACT: A crucial and enigmatic step in the complex
resulting in the loss of C10 and formation of the xanthone
biosynthesis of aflatoxin B1 is the oxidative rearrangement of
derivative 4. Biosynthesis studies have identified up to four genes
versicolorin A to demethylsterigmatocystin. This step is thought to
proceed by an oxidation–reduction–oxidation sequence, in which
which encode for the enzymes AflN (also denoted as VerA), AflX,
AflM and AflY.5–9 Individual gene disruption experiments have
the NADPH-dependent oxidoreductase AflM catalyzes the
shown an accumulation of 3 and a diminished production of 1.5–9
enclosed reduction step. AflM from Aspergillus parasiticus, after
heterologous production in E. coli and purification, however,
catalyzed the reduction of the hydroquinoid form of the starting
According to Henry and Townsend, the initial reaction step has
been speculated to be an epoxidation of 3 to 5 catalyzed by AflN, a
putative cytochrome P450 enzyme.4 AflX has some sequence
compound versicolorin A (25% conversion) to a so far unknown
similarity with epoxide hydrolases and thus is seemingly involved
product of aflatoxin biosynthesis. The asymmetric reduction of
in the epoxide ring opening of 5, leading to the hydroxydienone 6.7
emodin hydroquinone to (R)-3,8,9,10-tetrahydroxy-6-methyl-3,4-
dihydroanthracen-1(2H)-one (up to 82% for AflM) has also been
observed in previous studies using MdpC from Aspergillus
In the next reaction steps, a reduction supposedly catalyzed by the
NADPH-dependent oxidoreductase AflM and an elimination of
water, leading to 7, has been assumed.4,8 AflY, a putative Baeyer–
nidulans (monodictyphenone biosynthetic gene cluster). The first
Villiger oxidase, is assumed to catalyze the formation of the lactone
(nonenzymatic) reduction of emodin to emodin hydroquinone, for
8, which is probably then nonenzymatically converted into
example with sodium dithionite, is obligatory for the enzymatic
reduction by AflM or MdpC. These results imply an unprecedented
role of AflM in the complex enzymatic network of aflatoxin
xanthone 4.9 This reaction sequence of oxidation–reduction–
oxidation (Scheme 1) under preclusion of an initial reduction step
was postulated due to the failure of incorporation of other possible
biosynthesis.
intermediates, such as 6-deoxy-3, and o-carboxybenzophenone
derivatives of 3.4,10,11
Chiang et al. have proposed a similar pathway for the
biotransformation of emodin (9) to monodictyphenone (10) based
on the high gene sequence identities of the monodictyphenone gene
cluster of Aspergillus nidulans with the aflatoxin gene cluster;
Aflatoxin B1 (AFB1, 1), a naturally occurring mycotoxin,
produced by Aspergillus flavus, A. parasiticus and A. nomius, is
one of the most potent carcinogens known.1 The biosynthesis of 1
consists of a complex sequence starting with the formation of the
polyketide synthase (PKS) product norsolorinic acid anthrone (2),
which is subsequently converted in several enzymatic reactions
into 1.2,3 A central reaction sequence in this pathway is the
conversion of versicolorin A (3) into demethylsterigmatocystin
(DMST, 4) (Scheme 1).4 This sequence includes loss of the C6
however, they could not identify
a
homolog of AflN.12
Nevertheless, other studies have indicated a different biosynthetic
pathway via chrysophanol (11) and without the need of an
epoxidation (Scheme 2).13–15
Scheme 1. Putative conversion of versicolorin A (3) into DMST (4).5–9
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dithionite was used. This result implies that E. coli possesses
enzymes that are able to catalyze the first reduction step towards
the two tautomers of emodin hydroquinone, 12 and 13 (Scheme 2).
Scheme 2. Formation of the two tautomers of emodin
hydroquinone (12/13) and enzyme-catalyzed reduction with
MdpC-his or AflM-his.13–15
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Accordingly, these results strengthen our hypothesis regarding
possible reduction of the tautomers of versicolorin A hydroquinone
(15/16) by AflM in the biosynthesis of aflatoxin B1 (1). Analogous
to the reduction of 9, we tested 3 or rather its hydroquinone as a
putative substrate of AflM (see Supporting Information). As
expected, we observed a dearomatization leading to (1'R,2'S,6R)-
1,6,9,10-tetrahydroxy-2',5,6,7-tetrahydroanthra[3,2-b]furo[2',1'-
d]furan-8(1'H)-one (17, Scheme 3). The conversion (25%) into 17
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was ascertained by H NMR analysis of the crude product. The
structure of 17 was confirmed by total correlated spectroscopic
experiments. The absolute configuration at C1' and C2' is according
to the absolute configuration of substrate 3.18 C6 was predicted to
have the (R) configuration, assuming a similar reaction mechanism
as for compound 14. NMR spectrum shows only one diastereomer
of 17, which implies a stereospecific enzyme reaction.
Scheme 3. Conversion of versicolorin A (3) into 17 with
Na2S2O4 and AflM-his.
To clarify the biosynthetic pathway of 1 in detail, the catalytic
function of the involved enzymes, previously identified through
gene disruption studies, needs to be investigated.14 Concerning the
monodictyphenone gene cluster, the function of MdpC, a sequence
homolog of AflM, has been elucidated in chemoenzymatic
assays.15 Two tautomeric forms of emodin hydroquinone (12/13)
were observed in solution after incubation of emodin (9) with
sodium dithionite. Subsequent enzymatic reduction by the
NADPH-dependent oxidoreductase MdpC gave (R)-3,8,9,10-
tetrahydroxy-6-methyl-3,4-dihydroanthracen-1(2H)-one (14) in
58% yield (Scheme 2).15 Due to the strong sequence similarity
between MdpC and AflM from A. parasiticus (67% amino acid
identity), we concluded that AflM might be involved in an
analogous transformation.15
In order to confirm the activity of AflM in the conversion of
anthrahydroquinones in general, the codon-optimized N-terminally
His-tagged aflM was cloned into pET19b, overexpressed in E. coli
BL21, and the obtained protein purified by Ni-NTA affinity
chromatography (see Supporting Information). To check for the
supposed AflM-catalyzed reduction of 12/13, the anthraquinone 9
was incubated with sodium dithionite and purified AflM-his, using
glucose dehydrogenase/D-glucose as an NADPH regeneration
system. The reaction mixture was stirred for 24 hours at room
temperature under nitrogen atmosphere to avoid prompt back-
oxidation to 9. The conversion of 9 via 12/13 into 14 (up to 82%)
In summary, we have shown that AflM from the aflatoxin B1
biosynthetic gene cluster is active in converting the tautomers of
emodin hydroquinone (12/13) and versicolorin A hydroquinone
(15/16) into the 3-hydroxy-3,4-dihydroanthracen-1(2H)-one
derivatives 14 and 17, respectively. AflM, as well as MdpC,
specifically accept the tautomers of hydroquinone as a substrate,
but not the anthraquinone itself.15 Moreover, 17 is most likely the
intermediate in the biosynthesis of 6-deoxy-3 in accord with
similarities to the biosynthesis of chrysophanol (11, Scheme 2).
This result contrasts with the previously postulated biosynthesis
of 6-deoxy-3 and 1, for which an initial reduction of 3 has been
precluded (as shown in Scheme 1). The potential roles of the
tautomers of versicolorin A hydroquinone (15/16) and the AflM
product 17 in aflatoxin formation remain to be determined.
Nevertheless, our results afford a chemically plausible alternative
to account for the extensive deuterium incorporation observed at
carbons 9, 10 and 11 in DMST (4) from D2O and NADPD,11 and
provide a basis to re-evaluate the sequence of reactions that relate
the anthraquinone versicolorin A (3) to the xanthone DMST (4).
1
was established by H NMR analysis. Purification by automated
flash column chromatography yielded 20% (37 µmol) of pure 14 as
an orange solid. The absolute configuration was determined as (R)
by comparing the VCD spectrum of 14 with a calculated VCD
spectrum and the CD spectrum of 14 with those of similar
compounds (see Supporting Information).16 Moreover, during
workup we observed the formation of chrysophanol (11, 8%), most
probably by a nonenzymatic elimination of water and oxidation
with atmospheric oxygen. Incubation of 9 with AflM-his in the
absence of sodium dithionite resulted in no conversion. Thus, these
results emphasize the role of hydroquinone tautomers in
biosynthesis as previously described for the enzymatic reduction
with MdpC.15,17 Interestingly, conversion (up to 34%) of 9 into 14
occurred when AflM-his cell-free extract in the absence of sodium
These findings lead to
a consistent picture of aromatic
deoxygenations extending from chrysophanol (11, Scheme 2) to 6-
deoxy-3 (Scheme 3).14,15,19 Although 6-deoxy-3 has failed to
support aflatoxin biosynthesis, keto tautomers or oxidized
derivatives of dihydroanthracenone 17 capable of Baeyer-Villiger
oxidation can be visualized to undergo cleavage, reclosure and
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Anderson, J. A.; Lin, B. K.; Williams, H. J.; Scott, A. I. J. Am.
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1
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that parallels related pathways to fungal metabolites as
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ASSOCIATED CONTENT
Supporting Information
Franck, B.; Bringmann, G.; Flohr, G. Angew. Chem. Int. Ed.
Engl. 1980, 19 (6), 460.
Experimental details and characterization data. This material is
available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
* michael.mueller@pharmazie.uni-freiburg.de
Author Contributions
§These authors contributed equally.
Notes
Financial support of this work by the Deutsche
Forschungsgemeinschaft (IRTG 1038) is gratefully acknowledged.
ACKNOWLEDGMENT
We thank Sascha Ferlaino for measurement of the NMR spectra
and Dr. Steffen Lüdeke for help with the VCD measurements and
interpretation.
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