Fungal Dirigent Protein Controls the Stereoselectivity of Multicopper Oxidase-Catalyzed Phenol Coupling in Viriditoxin Biosynthesis

Article abstract of DOI:10.1021/jacs.9b03354

Paecilomyces variotii produces the antibacterial and cytotoxic (M)-viriditoxin (1) together with a trace amount of its atropisomer (P)-viriditoxin 1′. Elucidation of the biosynthesis by heterologous pathway reconstruction in Aspergillus nidulans identified the multicopper oxidase (MCO) VdtB responsible for the regioselective 6,6′-coupling of semiviriditoxin (10), which yielded 1 and 1′ at a ratio of 1:2. We further uncovered that VdtD, an α/β hydrolase-like protein lacking the catalytic serine, directs the axial chirality of the products. Using recombinant VdtB and VdtD as cell-free extracts from A. nidulans, we demonstrated that VdtD acts like a dirigent protein to control the stereoselectivity of the coupling catalyzed by VdtB to yield 1 and 1′ at a ratio of 20:1. Furthermore, we uncovered a unique Baeyer-Villiger monooxygenase (BVMO) VdtE that could transform the alkyl methylketone side chain to methyl ester against the migratory aptitude.

Full text of DOI:10.1021/jacs.9b03354

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Communication
A Fungal Dirigent Protein Controls the Stereoselectivity of Multicopper
Oxidase-Catalyzed Phenol Coupling in Viriditoxin Biosynthesis
Jinyu Hu, Hang Li, and Yit-Heng Chooi
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 02 May 2019
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A Fungal Dirigent Protein Controls the Stereoselectivity of
Multicopper Oxidase-Catalyzed Phenol Coupling in Viriditoxin
Biosynthesis
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†
†
†,
Jinyu Hu, Hang Li, Yit-Heng Chooi *
†School of Molecular Sciences, University of Western Australia, Perth, Western Australia 6009, Australia.
Supporting Information Placeholder
the close cooperation of an MCO and a berberine bridge
ABSTRACT:
Paecilomyces
variotii
produces
the
11
enzyme-like oxidase. However, it was difficult to attribute
the stereoselectivity to one or both enzymes due to the
double coupling reactions could not be separated and the
naphthol substrate is unstable.
antibacterial and cytotoxic (M)-viriditoxin (1) together with a
trace amount of its atropisomer (P)-viriditoxin 1′. Elucidation
of the biosynthesis by heterologous pathway reconstruction
in Aspergillus nidulans identified the multicopper oxidase
(
MCO) VdtB responsible for the regioselective 6,6′-coupling
of semiviriditoxin (10), which yielded 1 and 1′ at a ratio of
:2. We further uncovered that VdtD, an α/β hydrolase-like
Here, we focus on the investigation of the biosynthesis
of a bisnaphthopyrone, viriditoxin (1), with a single C-C
bridge between the two monomers. Compound 1 was first
1
12
protein lacking the catalytic serine, directs the axial chirality
of the products. Using recombinant VdtB and VdtD as cell-
free extracts from A. nidulans, we demonstrated that VdtD
acts like a dirigent protein to control the stereoselectivity of
the coupling catalyzed by VdtB to yield 1 and 1′ at a ratio of
isolated from Aspergillus viridinutans,
in which the
structure was revised to 6,6′-bisnaphtho-α- pyrone with an
R-configuration at the 6,6′ axis, which gives an (M) helical
13
conformation. Another fungus Paecilomyces variotii has
14-16
been reported to produce
1 and its derivatives.
2
0:1. Furthermore, we uncovered a unique Baeyer-Villiger
Compound 1 possesses wide spectrum antibacterial activity
17-19
monooxygenase (BVMO) VdtE that could transform the alkyl
methylketone side chain to methyl ester against the
migratory aptitude.
and cytotoxicity against different cancer cell lines.
It had
been shown to inhibit the bacterial cell division protein FtsZ
1
7, 20
under certain conditions.
1
Despite the broad interest in
and established total synthesis routes,21, 22 little was
known about the molecular basis of its biosynthesis.
Axially chiral biaryl natural products pose unique challenges
for total synthesis due to the presence of both
regiochemistry and stereochemistry at the biaryl axis. In
nature, biaryl compounds are generated via intermolecular
oxidative phenol coupling. For fungi, phenol coupling has
OMe
OH OH
O
OH OH
O
8
O
OH OH
1
O
OH OH
MeO
(M/P)-dinapinone A
been shown to be catalyzed by P450 monooxygenases or
OMe
OH O
OH OH O
2-7
OMe
O
laccases/multicopper oxidases (MCOs).
Although P450s
O
O
O
O
have been shown to catalyze regio- and stereoselective
MeO
MeO
O
O
O
O
MeO
MeO
MeO
MeO
OMe
OMe
6
phenol coupling, such as in biosynthesis of kotanin (Figure
6
1
), MCOs are not known to be able to control the axial
O
OMe
2,
7
chirality of biaryl products.
For example, an MCO with
OMe
OH
O
OH OH O
regioselective 8, 8′ ortho-ortho coupling activity was
identified from Talaromyces pinophilus in the biosynthesis of
(P)-kotanin
(M)-elsinochrome A
(M)-viriditoxin, 1
2
Figure 1. Examples of axially chiral biaryl natural products
dinapinone A (Figure 1), albeit it is not stereoselective. In
from fungi.
higher plants, the regio- and stereoselective coupling by
MCOs are controlled by non-catalytic auxiliary proteins
The genome of P. variotii strain CBS101075 (Figure 1)
8-10
known as dirigent proteins,
but no such protein had
23
was recently sequenced.
The biosynthesis of 1 is
been uncovered in fungi to date. Recently, our group
showed that the regio- and stereoselective double coupling
of naphthols to form the two C-C bridges in fungal
perylenequinones (e.g. elsinochrome A in Figure 1) involved
hypothesized to start from a naphtho-α-pyrone synthesized
by non-reducing polyketide synthase (NR-PKS). BLASTp
24
query using the NR-PKS ElcA from the elsinochrome
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pathway, known to produce a similar naphthopyrone nor-
We next co-expressed vdtE encoding the BVMO with
vdtA/C and vdtA/C/F in A. nidulans. A. nidulans vdtA/C/E
produced the methyl ester derivative 8 and unexpectedly,
together with a hydrolyzed carboxyl derivative 7. With vdtF,
the 7/8 pair were converted to 9/10 in A. nidulans
vdtA/C/E/F (Figure 3A). The results confirmed the role of
VdtE as BVMO, but confusion remained as BVMOs have
1
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8
9
1
1
1
1
1
1
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toralactone, identified an NR-PKS protein (ID 480069) with
high similarity to ElcA in the P. variotii CBS101075 genome.
Further analysis of its flanking region revealed a putative
biosynthetic gene cluster of 9 genes (Figure 2), which
encode all tailoring enzymes predicted to be required in the
biosynthetic pathway of 1 (renamed as vdt cluster). Analysis
of the metabolite profile of P. variotii CBS101075 showed
28, 29
never been reported to possess hydrolyzing activity.
To
that it produces (M)-viriditoxin
atropisomer (P)-viriditoxin 1′ in approximately 20:1 ratio
Figure S1) suggesting the existence of regio- and stereo-
1
and traces of the
further characterize the function of VdtE, we purified VdtE as
maltose binding protein (MBP)-tagged recombinant protein
from E. coli BL21(DE3) for in vitro enzymatic assay using 5 as
substrate. NADH and NADPH were both tested as reducing
cofactors. In the presence of FAD, VdtE was able to utilize
NADPH to fully convert 5 to 8 and 6 to 10 (Figure 3B and
S4C). Thus, it is conclusive that VdtE is a methyl ester-
forming BVMO and the formation of the carboxyl
derivatives 7 and 9 and in A. nidulans is likely due to
endogenous hydrolase activity.
0
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(
selective coupling enzyme in viriditoxin biosynthesis.
However, in the vdt cluster, the only candidate gene
encoding phenol coupling enzyme is vdtB encoding an
MCO, which have never been reported to be stereoselective.
Therefore, the basis for the stereoselective para-para
coupling in biosynthesis of 1 was enigmatic. Furthermore,
the presence of vdtE encoding
a
Baeyer-Villiger
monooxygenase (BVMO) suggests the methyl ester in 1
could be formed via a Baeyer-Villiger oxidation. Such
naturally occurring methyl ester-forming BVMOs are rare as
methyl esters often exist as a minor co-product to the other
A. nidulans
in vivo
A

B
in vitro
280nm
4

280nm
8
25, 26
5
6
more preferred regioisomer.
Thus, we set out to
vdtA
VdtE+5
VdtE+6
5
10
investigate the biosynthetic pathway by heterologous
reconstruction in A. nidulans accompanied by in vitro
characterization of selected enzymes either as purified
recombinant proteins or cell-free lysate from A. nidulans
vdtA/C
6
vdtA/C/F
vdtA/C/E
8 std.
expressing the corresponding proteins.
Compounds
7
obtained from the strains constructed were isolated and
characterized by MS and NMR analyses (Table S4-S18). We
employed the episomal yeast-fungal artificial chromosome
8
10 std.
9
10
6
8
min
11
(
YFAC) expression system we established previously and
27
vdtA/C/E/F
cell-free lysate assays
the A. nidulans strain LO7890. Firstly, expression of vdtA in
A. nidulans resulted in the accumulation of 4 as expected.
Following our hypothesized pathway, we then added vdtC
encoding O-methyltransferase with vdtA in A. nidulans and
the strain produces the methylated 5 (Figure 3A and 4).

280 nm
8
1'
7
1
10
11'
vdtA/C/D/E
VdtB_An
10
1
VdtB_An +
VdtD_An
vdtA/C/D/E/F
11
Next, we co-expressed vdtF encoding
a short-chain
11
11'
13
1'
dehydrogenase/reductase (SDR) with vdtA/C in A. nidulans
and resulted in the production of 6, suggesting that VdtF
acts as a stereo-specific reductase converting the pyrone 5
to dihydropyrone 6 (Figure 3A and 4).
12
1
VdtB_An +
VdtD_Ec
vdtA/B/C/E/F
11'
1
10
10
1
'
vdtA-F
min
VdtD_An
vdtA
ORF1
vdtR vdtG vdtF vdtE vdtDvdtCvdtB
6
8
6
8
min
NR-PKS
BVMO
HR-PKS
C6 transcription factor
transporter
O-methyltransferase MCO
OH OH
SDR
Figure 3. LC-DAD-MS traces for (A) heterologous
reconstruction of vdt cluster and (B) in vitro reactions of
VdtE with 5 and 6 (top) and VdtB/VdtD cell-free lysates with
10 (bottom). An, A. nidulans cell-free lysates; Ec,
recombinant VdtD purified from E. coli.
/-hydrolase-like
O

280nm
1
2
O
O
MeO
MeO
OMe
OMe
3
1'
O
O
We initially proposed that the hydrolase VdtD could
hydrolyze the methyl ester and hence its co-expression
would lead to an increase in the carboxyl acid derivatives.
Interestingly, co-expression of vdtD with vdtA/C/E resulted
in increase in the methyl ester 8 instead compared to
without vdtD, while co-expression of vdtD along with
vdtA/C/E/F resulted in loss of 9 and exclusive accumulation
OH OH
(P)-
O
1'
6
8
min
Figure 2. The vdt cluster (see Table S3) and the metabolite
profile of Paecilomyces variotii showing the production of 1
and atropisomer 1′.
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OH OH
O
acetyl-CoA +
7x malonyl-CoA
O
O
MeO
MeO
6
1/1'
(1:2)
VdtA
VdtE
VdtF
VdtB
OH OH
O
OH OH
O
OH OH
O
OH OH O
VdtB
VdtD
x 2
O
VdtC
VdtE
VdtF
OMe MeO
O
O
O
O
O
O
O
O
1/1'
20:1)
(
HO
MeO
OMe
1
1
1
1
1
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0
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0
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9
0
4
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10
OH OH
OH OH
O
endogenous
hydrolase?
endogenous
hydrolase?
O
O
O
O
O
O
O
O
OH OH
O
OH OH
O
x 2
MeO
MeO
OMe MeO
VdtB
11/11'
MeO
O
O
O
O
(1:2)
MeO
OH MeO
OH
7
9
OH OH
O
O
OH OH
O
O
(
M)-2
(M)-3
OH OH
OH OH
OH OH
O
OH OH O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
MeO
MeO
OH
OH
MeO
MeO
OH
OH
MeO
MeO
OH
MeO
HO
OH
OH
O
O
OH OH
O
OH OH
O
OH OH
O
OH OH
O
M)-11
(P)-11'
(P)-12
(P)-13
(
Figure 4. Elucidated biosynthetic pathway and structures of bisnaphthopyrone compounds isolated (box). * indicates
proposed structure for 13.
of the methyl ester 10 (Figure 3A). This observation of the
shifts toward the methyl ester derivatives in the presence of
VdtD (Figure 3A) raised the question about the function of
the putative hydrolase VdtD in viriditoxin biosynthesis. The
results suggest that instead of functioning as a hydrolase,
VdtD possesses an opposite role - it protects the methyl
ester from being hydrolyzed by the endogenous hydrolases,
perhaps via binding to the compounds. Analysis of VdtD
protein sequence identified a mutation in the catalytic triad
by the endogenoushydrolase activity in the A. nidulans host
and the absence of VdtD, which has been implicated above
to have protective effects on the methyl esters against
hydrolysis. Lastly, we showed that addition of ORF1
encoding
a highly-reducing (HR-PKS) resulted in no
changes in the metabolic profile (Figure S1B), indicating that
it is not involved in the biosynthesis of 1.
To corroborate the function of VdtB and VdtD, we
attempted to characterize the proteins in vitro. Our attempts
to express the proteins in E. coli was unsuccessful for VdtB.
Given the success in the recent study using cell-free extract
30
Ser-(Glu/Asp)-His, in which the important serine residue
was mutated to aspartate (Figure S5).
2
Next, vdtB encoding the MCO was co-expressed with
other vdt genes. Co-expression of vdtA-F in A. nidulans
reconstituted the production of 1/1′ at a 20:1 ratio similar to
P. variotii. However, in the absence of vdtD encoding the
to reconstitute the activity of a fungal MCO, we took a
similar approach. Cell-free extracts containing VdtB and
VdtD were prepared separately from A. nidulans expressing
the corresponding gene. Recombinant VdtD from E. coli was
also included in the assays. The napthopyrone methyl ester
10 was used as the substrate. As shown in Figure 3B, VdtB
alone slightly favored the production of 1′ over 1 at a ratio
of 2:1. When 10 was assayed together with VdtB and VdtD
cell lysates, 1 was exclusively produced with a trace amount
of the corresponding (M) carboxyl derivative 11. Likewise,
when the carboxylic acid monomer 9 was tested, VdtD
altered the stereoselectivity of the oxidative coupling to
favor the (M) 11 over (P) 11′, but less drastically (Figure
S4B). The MBP-tagged VdtD from E. coli had no influence on
the stereoselectivity of the VdtB-catalyzed coupling,
suggesting either that the recombinant protein was
non-catalytic
hydrolase-like
protein,
A.
nidulans
vdtA/B/C/E/F produced multiple new peaks with UV-vis
spectra similar to bisnathopyrones along with traces of 1
and 1′ (Figure 3A and S2). Among them, the major products
that we were able to isolate are a pair of atropisomeric
carboxyl bisnaphthopyrone derivatives 11 and 11′ and two
heterodimers 12 and 13 in (P) conformation (Figure 4). It
appears that in the absence of VdtD, VdtB lost the
stereoselectivity and to a certain extent, the substrate
fidelity as well. The presence of 13 also suggests that the
methylation by VdtC could occur after VdtE and VdtF-
catalyzed reactions. The carboxylic acids could be explained
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incorrectly folded or post-translational modification is
there are other mechanisms for controlling the
stereoselectivity of MCOs in fungi. This study expands the
enzymatic tools for phenol oxidative coupling and Baeyer-
Villiger oxidation.
1
2
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9
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1
1
1
1
1
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5
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required for its activity. The in vitro data clearly
demonstrated the role of VdtB as a 6,6′ regioselective
coupling MCO and that VdtD controls the stereoselectivity
in the formation of (M)-1.
ASSOCIATED CONTENT
Supporting Information
In summary, we have uncovered a unique methyl ester-
forming BVMO and a novel non-catalytic auxiliary protein
that controls the stereoselectivity of MCO-catalyzed phenol
coupling. The exclusive methyl ester formation catalyzed by
VdtE is intriguing, as the formation of methyl ester required
the migration of the less nucleophilic carbon during the
Criegee rearrangement, against the migratory aptitude
Experimental
spectroscopic
details,
supplementary
figures
and
data.
0
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0
The Supporting Information is available free of charge on
the ACS Publications website.
25, 26, 28
(
Figure S6A).
This is the first report of a naturally-
occurring methyl ester-forming BVMO. Recently, directed
evolution has been used to alter the regioselectivity of a
BVMO TmCHMO to generate methyl ester,3 in which the
reversal in regioselectivity was attributed to the change in
conformation of the Criegee intermediate in the binding
pocket. A unique fungal BVMO capable of inserting oxygen
on both sides of a carbonyl to form a carbonate has also
been discovered.3 Together, they highlight the catalytic
prowess of fungal BVMOs.
AUTHOR INFORMATION
Corresponding Author:
1
* yitheng.chooi@uwa.edu.au
Notes
The authors declare no competing financial interests.
2
ACKNOWLEDGMENT
Y-HC is an Australian Research Council (ARC) Future Fellow.
JH and HL are supported by Australian International
Postgraduate Research Scholarships. NMR and HR-MS were
performed at the UWA Centre for Microscopy,
Characterization and Analysis (CMCA). Special thanks to
A/Prof. Alexander Idnurm for providing the P. variotii
CBS101075 and to Dr Andrew Piggott for helpful discussion.
More significantly, we uncovered that VdtD exerts
control over the stereoselectivity of the 6,6′ regioselective
coupling catalyzed by the MCO VdtB. While preparing this
manuscript
for
submission,
another
group
has
independently demonstrated the regioselective coupling
activity of the VdtB homolog (Av-VirL) from another
33
producer of 1 A. virinidutans. Using cell-free extract and a
non-native substrate semivioxanthin, the corresponding
dimer was produced but with a higher ratio of the incorrect
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36-38
the stereochemistry of Diels-Alder reactions,
which are considered as Diels-Alderases.
some of
In contrast,
39, 40
(5).
Gil Girol, C.; Fisch, K. M.; Heinekamp, T.; Günther, S.;
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VdtD belongs to the α/β hydrolase family and is related to
fungal carboxyesterases/lipases (Figure S5). Structural and
protein interaction studies will be required to understand
the mechanism of the stereocontrol. To our knowledge,
VdtD is the first dirigent protein reported in fungi that
directs the stereoselectivity of an MCO. As gene encoding
such α/β hydrolase-like dirigent protein is not co-located
with MCO gene in some of the fungal biosynthetic gene
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33
clusters encoding biaryl compounds, it is possible that
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Journal of the American Chemical Society
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Insert Table of Contents artwork here
VdtE: Baeyer-Villiger monooxygenase
VdtB: multicopper oxidase
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VdtD: hydrolase-like dirigent protein
OH OH
O
VdtE
OH OH O
O
O
O
O
VdtB
VdtD
O
O
O
O
2 X OH OH
O
VdtB
O
O
O
O
O
O
O
(
P)
O
O
(M)
O
O
O
1/1'
10
1/1'
OH OH
1'
O
(1:2 ratio)
(20:1 ratio)
OH OH O
1
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