Enantioselective Phenol Coupling by Laccases in the Biosynthesis of Fungal Dimeric Naphthopyrones

Article abstract of DOI:10.1002/anie.201903759

Biaryl compounds are ubiquitous metabolites that are often formed by dimerization through oxidative phenol coupling. Hindered rotation around the biaryl bond can cause axial chirality. In nature, dimerizations are catalyzed by oxidative enzymes such as la

Full text of DOI:10.1002/anie.201903759

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Accepted Article
Title: Enantioselective Phenol Coupling by Laccases in the
Biosynthesis of Fungal Dimeric Naphthopyrones
Authors: Michael Müller, Sebastian Obermaier, Wiebke Thiele, and
Leon Fürtges
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201903759
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Enantioselective Phenol Coupling by Laccases in the
Biosynthesis of Fungal Dimeric Naphthopyrones
Sebastian Obermaier,+ Wiebke Thiele,+ Leon Fürtges, and Michael Müller*
Dedicated to Ben Feringa and in memory of Hans Wynberg
Abstract: Biaryls are ubiquitous metabolites that are often formed by
dimerization through oxidative phenol coupling. Hindered rotation
around the biaryl bond can cause axial chirality. In nature,
dimerizations are catalyzed by oxidative enzymes such as laccases.
This class of enzymes is known for non-specific oxidase reactions
while inherent enantioselectivity is hitherto unknown. Here, we
describe four related fungal laccases that catalyze γ-naphthopyrone
dimerization in a regio- and atropselective manner. In vitro assays
revealed that three enzymes were highly P-selective (ee >95%), while
one enzyme showed a remarkable flexibility. Its selectivity for M- or
P-configured dimers varied depending on the reaction conditions. For
As they possess three possible positions for C–C bond
formation (positions 7, 9, and 10), monomeric γ-naphthopyrones
can theoretically be coupled to six regioisomeric dimers via
oxidative phenol coupling. However, exclusively 9,9′-coupled
dimers are found in ustilaginoidin producers, so the coupling was
expected to proceed in a regioselective manner. Furthermore, the
biaryl bond is axially chiral: the rotation of the naphthopyrone
moieties is sterically hindered, resulting in two stable
atropisomers. Whereas multiple chemically modified derivatives
co-exist in most producer strains, the configuration of the biaryl
axis is often the same for all congeners from one fungal strain
(Figure 1). For example, more than 20 γ-naphthopyrone
9,9′-dimers have been found in Ustilaginoidea virens, all of which
are M-configured, while for Chaetomium arcuatum only
P-configured compounds are known (Figures S1–S3).[16–19] It may
thus be concluded that the biosynthesis of these compounds
proceeds via dimerization systems that favor one regio- and
atropisomer. The complementarity of the M- and P-configured
metabolites prompted us to investigate the enzymatic systems
responsible for dimerization in ustilaginoidin biosynthesis and to
characterize their selectivity.
example,
P-ustilaginoidin A, whereas the M-atropisomer was favored at higher
concentration. These results demonstrate an inherent
a
lower enzyme concentration yielded primarily
enantioselectivity in an enzyme class that was previously thought to
comprise only non-selective oxidases.
Laccases are copper-dependent one-electron oxidases that
act on phenolic compounds, generating phenoxy radicals that
undergo subsequent coupling reactions including C–C, C–O, and
C–N bond formations. Typically, the outcome of these
downstream reactions is not controlled by the laccase, thus
leading to a mixture of regio- and stereoisomeric products.[1–4]
Recent research on polyketide dimerization in fungi led to the
discovery of regioselective laccases.[5,6] In these examples, the
polyketide monomers possess several activated positions, of
which only one is selected for coupling by a distinct laccase. In
contrast to inherent regioselectivity, enantioselectivity has only
been found when laccases were combined with other proteins.[7–
Here, we present laccases that are regioselective and also
dimerize γ-naphthopyrone monomers enantioselectively, favoring
the formation of either M- or P-atropisomers. One of the enzymes
revealed a marked flexibility, as the atropselectivity was reversed
depending on the reaction conditions.
9]
Specifically, so-called dirigent proteins are able to mediate
enantioselective coupling of radicals produced by a laccase.[10]
In this study, we focused on a group of laccases that catalyzes
coupling reactions in the biosynthesis of ustilaginoidins, dimeric
γ-naphthopyrones isolated from different fungal species.[11–15] The
archetype is ustilaginoidin A (1), which is composed of two
identical γ-naphthopyrone moieties (Figure 1). The other
congeners differ from ustilaginoidin A (1) by Δ2 double bond
saturation, methylation at position 3, or a combination of both as
in chaetochromin A (2).
Figure 1. Ustilaginoidin A (1) from Ustilaginoidea virens with unmodified, achiral
naphthopyrone moieties, naturally produced in M-configuration, and
chaetochromin A (2) from Chaetomium arcuatum, produced in P-configuration.
To identify the ustilaginoidin biosynthetic genes, we
postulated a biosynthetic pathway as depicted in Scheme 1A
(inferred from aurofusarin biosynthesis in Fusarium
graminearum).[20] It included three enzymes: a non-reducing
polyketide synthase (PKS), a dehydratase, and a laccase that
acts as the coupling enzyme. By genome analysis of the
ustilaginoidin producer U. virens, we identified a biosynthetic
gene cluster (BGC) comprising genes for all these three expected
enzymes.[21] We also found very similar putative BGCs in the
S. Obermaier,+ W. Thiele,+ Dr. L. Fürtges, Prof. Dr. M. Müller*
Institut für Pharmazeutische Wissenschaften
Albert-Ludwigs-Universität Freiburg
Albertstrasse 25, 79104 Freiburg (Germany)
E-mail: michael.mueller@pharmazie.uni-freiburg.de
These authors contributed equally to this work.
[*]
[+]
Supporting information for this article is given via a link at the end of
the document.
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genomes of Chaetomium olivicolor and Thermothelomyces
(Myceliophthora) thermophila.[22,23] C. olivicolor is closely related
to the chaetochromin producer C. arcuatum, while Thermo-
thelomyces species have not been observed to produce
ustilaginoidins as yet. In all three investigated organisms, the
BGCs are well-defined by a near-syntenic region of exactly six
genes (Scheme 1B). In addition to the expected genes noted
above, each BGC contains predicted genes coding for a methyl-
transferase with a phosphopantetheine attachment site, an efflux
pump, and a protein with a phospholipid methyltransferase
domain. Additionally, a shortened version of the above-mentioned
BGCs was found in the genome of Aschersonia paraphysata. This
gene cluster differs from the others in lacking homologs to the two
genes with predicted methyltransferase domains.
product (see Supporting Information). For the biosynthesis of
chaetochromin A (2), it is assumed that the saturation of the Δ2
double bond takes place before dimerization.[24] It is probably
catalyzed by an external reductase, as no candidate gene was
identified within the BGC.
To substantiate the proposed ustilaginoidin biosynthetic
pathway, the product of the U. virens polyketide synthase UstP
was characterized. For this, ustP was heterologously expressed
in Aspergillus aculeatus; the recombinant fungus produced a
yellow-green pigment, which was isolated. HPLC and NMR
analysis revealed that it was YWA1 (3), confirming our hypothesis
regarding the first step in the biosynthetic pathway (Figure 2A).
Figure 2. HPLC chromatograms. A) Extract of Aspergillus aculeatus ustP,
producing YWA1 (3) and traces of norrubrofusarin (5). B) The same extract after
treatment with hydrochloric acid to obtain 5. C) In vitro coupling of 5 with UstL
to yield ustilaginoidin A (1).
The essential step in the biosynthetic pathway is the phenol-
coupling reaction leading to the dimeric ustilaginoidin scaffold.
The complementary M- and P-selective coupling systems in
U. virens and C. arcuatum allowed us to investigate the
stereochemistry of this key reaction. A stereochemical control is
conceivable by two different modes: an inherent selectivity of the
coupling enzyme or an unknown additional factor such as a
dirigent protein. To clarify this, the four laccase genes were
expressed in Aspergillus niger and the enzymes were tested in
vitro. Norrubrofusarin (5) was used as substrate because it was
anticipated to be the direct precursor of ustilaginoidin A (1).
Substrate 5 could be prepared in a biomimetic approach by
dehydration of YWA1 (3) obtained from the recombinant PKS
UstP (Figure 2B). Furthermore, norrubrofusarin (5) has the
advantage that it is achiral and cannot induce stereoselectivity in
the coupling reaction.
Scheme 1. A) Proposed biosynthetic pathway of the ustilaginoidins. PKS:
polyketide synthase; MT: methyltransferase. B) Biosynthetic gene clusters. The
capital letter of the gene name and the color correspond to the predicted
function or domains: M – methyltransferase with phosphopantetheine binding
site (orange);
L – phenol-coupling laccase (blue); E – phospholipid
methyltransferase domain (gray); T – efflux transporter (pink); Z – dehydratase
(yellow); P – polyketide synthase (red).
Based on the genes in the BGCs, we postulate a biosynthetic
pathway as follows, using U. virens as the example.[24] The PKS
UstP produces the γ-naphthopyrone precursor YWA1 (3), which
is followed by a UstZ-catalyzed dehydration reaction to yield nor-
rubrofusarin (5).[25] A key enzyme in the biosynthetic pathway is
the laccase UstL, which dimerizes two norrubrofusarin molecules
by oxidative phenol coupling. For the biosynthesis of
3-methylustilaginoidin derivatives such as chaetochromin A (2),
we propose that a methylated derivative (4) of YWA1 (3) is
required. The C-methylation is considered to be catalyzed by
UstM, whose phosphopantetheine attachment site indicates that
it acts on the growing polyketide chain before release of the
In an initial experiment, both culture supernatant and lysate
(cell-free extract) of A. niger ustL expression cultures were used
to assay enzymatic activity. Despite the predicted extracellular
localization of the protein, no activity was found in the culture
supernatant. For the assay of the lysate, however, the HPLC-MS
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chromatogram revealed a peak corresponding to the m/z of the
ionized dimer ustilaginoidin A (1), which was not present in the
negative controls (Figure S4). Consequently, lysate was utilized
for the subsequent in vitro assays, which were carried out in
sodium citrate buffer at 25 °C. Norrubrofusarin (5) dissolved in
dimethyl sulfoxide (DMSO) was added. All four laccases
produced the same new compound.
Unexpectedly, the ee values of ustilaginoidin A (1) from UstL-
catalyzed transformations varied between individual experiments,
which led us to test the influence of different reaction conditions
on the stereochemistry for all enzymes. We probed the influence
of an elevated reaction temperature, to 40 °C, as well as an
increased concentration of the co-solvent DMSO. The three
enzymes CheL, MytL, and AshL were P-selective (71–99% ee)
under all tested conditions. The fourth enzyme, UstL, for which
strict M-selectivity was expected initially, exhibited a pronounced
dependence on the reaction conditions. Both at 40 °C and at the
increased DMSO concentration, ustilaginoidin A (1) was
produced with an excess of the P-atropisomer (8% and 39% ee,
respectively). The M-atropisomer was only favored under the
original assay conditions at 25 °C (21% ee, Figure S6).
To determine the structure of the new product, enzymatic
conversions were performed on
a milligram scale. Nor-
rubrofusarin (5) was quantitatively converted by successive
addition of laccase-containing lysate. The ethyl acetate extract of
the reaction mixture was used without further purification to
acquire 1H, 13C, and 2D NMR spectra, which matched published
spectra of ustilaginoidin A (1).[26] Both HPLC and NMR analysis
indicated that the enzymatic conversion yielded only one product
and was thus 9,9'-regioselective (Figures 2C and S8–S11).
Furthermore, the initial experiments indicated that the protein
concentration might influence the atropselectivity of UstL. To test
this, the amount of lysate in the total reaction volume was varied.
Five dilutions were tested for all four enzymes, corresponding to
a 250-fold difference between the highest and the lowest
concentration. The latter was chosen so that the activity was still
detectable. Again, the three enzymes CheL, MytL, and AshL
showed almost complete P-selectivity (84–99% ee). A distinct
decrease in selectivity was only observed for the lowest lysate
concentration (Figure 3B).
Having established that laccases are the coupling enzymes,
we focused on the stereochemistry of the biaryl axis. Based on
the configuration of ustilaginoidins isolated from the respective
source organisms, we expected that UstL would yield an
M-configured dimer, while CheL should give the P-product.
Circular dichroism (CD) spectroscopy was used to determine the
stereochemistry of the ustilaginoidin A samples from enzymatic
conversions.[16] For a subset of samples, the enantiomeric excess
(ee) was quantified by chiral-phase HPLC after derivatization
(Figure S5). This data was then used to calibrate CD signals of
non-derivatized samples to approximate their ee values.
UstL, however, produced both atropisomers in varying
amounts. Each concentration step resulted in an incremental
decrease of the P-atropisomer leading to a reversal from P- to
M-selectivity (Figure 3B). With the lowest concentration of UstL,
an ee of 32% for the P-atropisomer was determined, while higher
concentrations of UstL gave an ee of around 55% for the
M-atropisomer. These experiments were repeated with enriched
enzyme obtained by fractionated acetone precipitation. The
results were congruent with those of the lysate assays. Again, a
dependence of the atropselectivity on the enzyme concentration
was observed, substantiating the findings on the flexibility of UstL
(Figure S7).
The chiroptical data confirmed the expectation for the M- and
P-selective laccases UstL and CheL, respectively. The two
laccases MytL and AshL, for which we had no information about
a natural product, were both P-selective (Figure 3A). As both M-
and P-atropselectivities were observed using different enzymes
under otherwise identical conditions, an unidentified factor from
the heterologous host inducing the regio- and stereoselectivity
can be excluded.
Figure 3. A) Example CD spectra of ustilaginoidin A (1) samples from laccase-catalyzed coupling. Spectra are normalized to a UV absorbance of 1.0 at 288 nm.
B) Direct CD measurement of ee values of ustilaginoidin A formed by in vitro phenol coupling with the laccases AshL, CheL, MytL, and UstL. The amount of lysate
was varied in a total reaction volume of 251 µL.
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Until now, enantioselective phenol-coupling reactions have
been mainly associated with cytochrome P450 enzymes. Even in
biosynthetic pathways of other dimeric γ-naphthopyrones similar
to ustilaginoidins, cytochrome P450 enzymes catalyze the
dimerization reactions.[27] However, the group of laccases
described here possesses an unprecedented selectivity toward
the formation of the biaryl bond, making this a remarkable
example of convergent evolution.
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Acknowledgements
Funded by the Deutsche Forschungsgemeinschaft (DFG,
German Research Foundation) – 235777276. We thank the
NRRL (USA), TBRC (Thailand), and NARO (Japan) culture
collections for supplying the fungal strains.
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Keywords: atropisomerism • dimeric polyketides •
enantioselectivity • oxidoreductases • phenol coupling
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Entry for the Table of Contents (Please choose one layout)
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Lonesome laccases: Several fungi
selectively produce atropisomers of
biarylic polyketides. Having identified
laccases as the dimerizing enzymes,
we demonstrated their inherent
enantioselectivity, favoring either the
M- or P-atropisomer. One enzyme
even reversed its atropselectivity
upon changed reaction conditions.
Contrary to previous assumptions,
our findings show that some
Sebastian Obermaier, Wiebke Thiele,
Leon Fürtges, Michael Müller*
Page No. – Page No.
Enantioselective Phenol Coupling
by Laccases in the Biosynthesis of
Fungal Dimeric Naphthopyrones
laccases can be atropselective
without the help of dirigent proteins.
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