Elucidation of pseurotin biosynthetic pathway points to trans-acting C-methyltransferase: Generation of chemical diversity

Article abstract of DOI:10.1002/anie.201404804

Pseurotins comprise a family of structurally related Aspergillal natural products having interesting bioactivity. However, little is known about the biosynthetic steps involved in the formation of their complex chemical features. Systematic deletion of the pseurotin biosynthetic genes in A. fumigatus and invivo and invitro characterization of the tailoring enzymes to determine the biosynthetic intermediates, and the gene products responsible for the formation of each intermediate, are described. Thus, the main biosynthetic steps leading to the formation of pseurotinA from the predominant precursor, azaspirene, were elucidated. The study revealed the combinatorial nature of the biosynthesis of the pseurotin family of compounds and the intermediates. Most interestingly, we report the first identification of an epoxidase C-methyltransferase bifunctional fusion protein PsoF which appears to methylate the nascent polyketide backbone carbon atom in trans. A maze: Pseurotins are a family of structurally related bioactive natural products from Aspergilli. Through genetic and biochemical studies, the biosynthetic pathway for the formation of azaspirene, synerazol, and pseurotin A/D have been elucidated, and reveal the combinatorial nature of their biosyntheses. PsoF was identified as bifunctional epoxidase methyltransferase enzyme, thus providing the first example of a trans-acting polyketide C-methyltransferase.

Full text of DOI:10.1002/anie.201404804

Angewandte
Chemie
DOI: 10.1002/anie.201404804
Enzyme Catalysis
Elucidation of Pseurotin Biosynthetic Pathway Points to Trans-Acting
C-Methyltransferase: Generation of Chemical Diversity**
Yuta Tsunematsu, Manami Fukutomi, Takayoshi Saruwatari, Hiroshi Noguchi, Kinya Hotta,
Yi Tang, and Kenji Watanabe*
Abstract: Pseurotins comprise a family of structurally related
Aspergillal natural products having interesting bioactivity.
However, little is known about the biosynthetic steps involved
in the formation of their complex chemical features. Systematic
deletion of the pseurotin biosynthetic genes in A. fumigatus
and in vivo and in vitro characterization of the tailoring
enzymes to determine the biosynthetic intermediates, and the
gene products responsible for the formation of each inter-
mediate, are described. Thus, the main biosynthetic steps
leading to the formation of pseurotin A from the predominant
precursor, azaspirene, were elucidated. The study revealed the
combinatorial nature of the biosynthesis of the pseurotin
family of compounds and the intermediates. Most interestingly,
we report the first identification of an epoxidase C-methyl-
transferase bifunctional fusion protein PsoF which appears to
methylate the nascent polyketide backbone carbon atom in
trans.
identified by deletion and overexpression of the polyketide
synthase nonribosomal peptide synthetase (PKS-NRPS)
hybrid enzyme gene, psoA, in Aspergillus fumigatus
Af293.[4] PsoA was shown to be responsible for the biosyn-
thesis of the core structure of 8, that is the 1-oxa-7-azaspiro-
[4,4]non-2-ene-4,6-dione skeleton having five chiral centers.
Recently, a detailed analysis of the pseurotin biosynthetic
gene cluster describing its genetic organization was
reported.[5] However, the mechanism of pseurotin biosynthe-
sis, such as the formation of the spiro-ring core structure, still
remains undefined (Scheme 1 and Figure 1A). Another
interesting aspect of pseurotin biosynthesis is the formation
of a large number of closely related bioactive compounds,
such as azaspirene (2)[6] and synerazol (7).[7] This diversity of
pseurotin-type natural products is thought to be generated
during the post-PKS-NRPS modification steps. However, it is
often difficult to resolve the biosynthetic steps involving
multiple enzymes and complex intermediates. Here, we
carried out knockout experiments for the pseurotin biosyn-
thetic genes in the DpyrG/Dku70 strain of A. fumigatus
A1159, named AfKW1 (see the Supporting Information), and
conducted in vivo and in vitro characterization of four
modification enzymes, PsoC, PsoD, PsoE, and PsoF, to
reveal the unique mechanism involved in the biosynthesis of
the pseurotin family of natural products.
P
seurotins[1] make up a family of fungal secondary metab-
olites which exhibit wide-ranging biological activities of
medicinal importance. For example, pseurotin A (8; see
Scheme 1) was reported to inhibit monoamine oxidase[2] and
induce cell differentiation in PC12 cells.[3] Previously, the gene
cluster responsible for biosynthesizing 8 was predicted and
Sequence analysis (see the Supporting Information)
indicated that PsoF is a single polypeptide comprised of an
unusual combination of two domains, one homologous to
a methyltransferase (MT) and another to an FAD-containing
monooxygenase (FMO). Deletion of psoF abolished produc-
tion of 8, 9, 13, and 14, thus indicating the essential role of
PsoF in pseurotin biosynthesis (Figure 2i versus ii). Surpris-
ingly, the deletion also resulted in the formation of 22 (see
Table S15 and Figures S45–S48 in the Supporting Informa-
tion), a C3-desmethylated analogue of 2 (Figure 2i versus ii).
A mixture of the geometric isomer 23 and the reduced
product 24, both lacking the C3 methyl group (see Table S16
and Figures S49–S52), were also isolated from DpsoF/
AfKW1. Close analysis revealed that the MT domain of
PsoA actually exhibited residual activity as judged by
marginal formation of 4 in the DpsoF/AfKW1 strain (see
Figures S5ii and viii). These results allowed us to propose
a mechanism of how the C3 methyl group is introduced into
the pseurotin scaffold (Figure 1A). For the FMO domain of
PsoF, lack of formation of the pseurotin-type compounds 8, 9,
13, and 14 suggested its role in the formation of the 10,11-
epoxide.
[*] Dr. Y. Tsunematsu, M. Fukutomi, Dr. T. Saruwatari,
Prof. Dr. H. Noguchi, Prof. Dr. K. Watanabe
Department of Pharmaceutical Sciences
University of Shizuoka, Shizuoka 422-8526 (Japan)
E-mail: kenji55@u-shizuoka-ken.ac.jp
Prof. Dr. K. Hotta
School of Biosciences, The University of Nottingham Malaysia
Campus, Selangor 43500 (Malaysia)
Prof. Dr. Y. Tang
Department of Chemical and Biomolecular Engineering and
Department of Chemistry and Biochemistry
University of California, Los Angeles, CA 90095 (USA)
[**] This work was supported by the Japan Society for the Promotion of
Science (JSPS) through the “Funding Program for Next Generation
World-Leading Researchers”, initiated by the Council for Science
and Technology Policy (No. LS103) (K.W.), and by the Industrial
Technology Research Grant Program in 2009 (No. 09C46001a) from
New Energy and Industrial Technology Development Organization
(NEDO) of Japan (K.W.). This work was also supported in part by
The Uehara Memorial Foundation (K.W.), by the Mochida Memorial
Foundation for Medical and Pharmaceutical Research (K.W.), by The
Naito Foundation Japan (K.W.), by the Nagase Science and
Technology Foundation Japan (K.W.), and by JSPS Fellowship for
Research In Japan (K.H.).
To characterize in detail the biochemical functions of the
C-MT and FMO domains of PsoF, we cloned psoF from
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201404804.
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Scheme 1. Biosynthetic pathway for the transformation of 1 into the pseurotins 8 and 9 involving methylation and multiple oxidation steps. The
pathway enclosed within the broken-line box with thick arrows represents the proposed main pathway toward formation of 8 and 9.
mRNA and expressed it heterologously in Escherichia coli.
The C-methylation step was examined in vitro using an N-
acetylcysteamine (NAC) thioester[8] which mimics the natural
substrate, phosphopanteteine-linked 25 (see the Supporting
Information). The purified PsoF was able to catalyze meth-
ylation of 25 to form 26 (Figure 1B and Figure 3). Interest-
ingly, we did not observe the formation of the epoxidated
products of 25 or 26, thus suggesting those early intermediates
are not recognized as substrates by the FMO domain of PsoF.
For functional analysis of the FMO domain, initially 2 was
subjected to an in vitro assay with the recombinant PsoF. The
assay revealed that PsoF was completely unreactive to 2.
Since the presence of an epoxide moiety in synerazol (7; see
Table S7 and Figure S22) hinted that 7 could be formed by the
activity of an FMO, we subjected the mixture of geometric
isomers 4 and 6 to PsoFas substrates in an in vitro reaction. In
this reaction, PsoF converted the substrates into four
products: the epoxide-containing 7 and the spontaneously
hydrolyzed epoxide products, 8, 9, and 18 (Figure 4). The
chemical structures of all compounds, 4 (Table S5 and
Figures S14–S17), 6 (Table S6 and Figure S18–S21), 7, 8
(Table S8 and Figure S23), 9 (Table S9 and Figure S24–S27),
and the new compound 18 (Table S13 and Figure S40–S43)
13E-containing products in the wild-type strain (Figure 2i)
suggests that 13Z-configured compounds are likely to be the
true substrate of PsoF. Furthermore, formation of epoxidized
or hydroxylated products from the 11Z-containing compound
6 was not observed in the reaction. Thus, the preferred
substrate of PsoF is considered to be 5, an 11E,13Z-
configured isomer. Based on the chemical structures of the
products 8 and 9, we propose that those products are formed
from 5 through epoxidation catalyzed by the FMO domain of
PsoF. After epoxidation of 5 by PsoF to form 7, 7 can be
attacked by a water molecule nonenzymatically at two
different positions to yield two diol products, 8 and 9, through
a SN2 and SN2’ reaction, respectively (Scheme 2). For the
formation of 8, the water molecule attacks 7 at C11 from the
opposite face of the epoxide in an antiperiplanar manner to
yield the 10S,11S-diol of 8. In contrast, 9 is formed by the
water molecule attacking 7 at C13. Subsequent allyl migration
and epoxide opening yields the diol product. Taken together,
these results show PsoF to be a unique bifunctional enzyme
catalyzing two different types of reactions at completely
separate steps of the pseurotin biosynthetic pathway.
Next, we examined psoC and psoD, which are predicted to
code for a MT and a cytochrome P450, respectively. To
understand the function of PsoC, we analyzed the mutant
strain, DpsoC/AfKW1. The UV traces from HPLC analysis of
the DpsoC/AfKW1 culture extract showed accumulation of
the O8-unmethylated products 13 (Table S10 and Figure S28–
S31) and 14, with loss of 8 and 9 (Figure 2i versus iii). This
1
were elucidated by HRESIMS, and H NMR and 13C NMR
spectroscopy (see the Supporting Information). In vitro
transformation of 4 into 18 by PsoF indicated that the FMO
domain is capable of accepting the 13E-containing compound
as its substrate. However, a complete lack of formation of any
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Figure 2. HPLC traces of metabolic extracts from the cultures of
various A. fumigatus A1159-based deletion strains to identify the genes
responsible for the biosynthetic steps involved in the formation of 8
during the pseurotin biosynthesis. All deletion strains were prepared in
AfKW1 by replacing the target gene with pyrG by homologous
recombination. All cultures were grown in MYG medium. All HPLC
traces were monitored at l=280 nm. Extract of the culture of i) AfKW1
harboring pKW20088 for the expression of pyrG as a wild-type control,
ii) DpsoF strain, iii) DpsoC strain, iv) DpsoD strain, and v) DpsoE strain.
IS=internal standard, which is the N-Boc-l-tryptophan methyl ester
(Boc-Trp-OMe).
Figure 3. HPLC analysis of 26 biosynthesis using purified PsoF. HPLC
traces of the i) control reaction without PsoF and ii) in vitro reaction
with PsoF, thus affording the methylated product 26 after 1 h at 308C
with 0.2 mm of substrate, which was a keto–enol equilibrated mixture
of 25 and its geometric isomers; 1 mm SAM and 0.1m NaCl in 0.1m
Tris-HCl (pH 7.4). All HPLC traces were monitored at l=280 nm.
Figure 1. Modular organization of the uncharacterized iterative PKS-
NRPS encoded by psoA (12.3 kb) and C-methylation activity of PsoF.
A) Proposed biosynthetic pathway for the formation of 8 and 22. The
iterative PKS-NRPS is shown with its predicted core domains. Path-
ways toward formation of 8 versus 22, depending on the presence of
PsoF, are shown. B) C methylation of the polyketide backbone core by
the MT domain of PsoF. Functional analysis was performed with
a chemically synthesized NAC thioester 25, which was converted into
a methylated product 26 by PsoF. A=adenylation, ACP=acyl carrier
protein, AT=acyltransferase, C=condensation, DH=dehydratase,
KR=ketoreductase, KS=ketosynthase, MT=methyltransferase,
MT*=partially active methyltransferase, R=reductase, SAM=S-ade-
nosyl-l-methionine, T=thiolation.
result indicated that PsoC likely performs O methylation of
the hydroxy group of C8 in 3 to form 4 (Scheme 1). To
characterize the O-methylation activity of PsoC in detail, we
examined recombinant PsoC in vitro using 13 as a substrate.
Interestingly, PsoC did not convert 13 into 8. This result
indicates that PsoC does not tolerate modifications to the
diene side chain of the azaspirene skeleton as its substrate. To
investigate the role of PsoD, we prepared another mutant
strain, DpsoD/AfKW1. HPLC analysis of the extract showed
an accumulation of 16 (Table S11 and Figures S32–S35) and
17 (Table S12 and Figures S36–S39) and a loss of 8 and 9
(Figure 2i versus iv), thus indicating that this cytochro-
me P450 is responsible for installing the ketone group at
C17 (Scheme 1). To confirm the activity of PsoD, in vivo
Figure 4. In vitro characterization of PsoF for the formation of 7, 8, 9,
and 18. All HPLC traces were monitored at l=280 nm. Analysis of
in vitro reaction of PsoF with a mixture of the geometric isomers 4 and
6 as substrate. i) Negative control using heat-inactivated PsoF.
ii) Epoxide intermediates were formed from 4 and 6 by PsoF after 1 h
at 308C. iii) the three products 8, 9, and 18 were formed from the
substrates by PsoF at 308C after 10 h. Authentic references of iv) 9,
v) 8, vi) 18, and vii) 7 are also shown.
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predicted to be formed immediately before the PsoF-cata-
lyzed epoxidation step. As discussed earlier, the preferred
substrate for PsoF is predicted to be 13Z-configured com-
pound. Thus, accumulation of 4 and 6 in the DpsoE strain, and
previous reports of isomerase activity of certain GSTs,[9]
suggest that PsoE may be involved in the trans-to-cis isomer-
ization of the C13 olefin. As shown in the in vitro assay of
PsoF (Figure 4ii and iii), 18 is formed by C10,C11 epoxidation
by PsoF at low efficiency as 4 accumulates in the DpsoE cell.
In contrast, formation of 8 in this mutant strain can be
explained by the low level of isomerization of the C13 olefin
converting 4 into 5. This isomerization was also observed in
the in vitro assay of PsoF, where a small amount of 8 was
formed in the absence of the 13Z-containing substrate
(Figure 4iii). As to the stereoisomerism at C11, concomitant
accumulation of the 11E- and 11Z-configured geometric
isomers, 22 and 23, respectively, in the DpsoF/AfKW1 strain
and similar accumulation of 4 and 6 upon disruption of
another gene, psoE, suggest that cis/trans isomerization of the
C11 olefin is non-enzymatic. We also observed the formation
of 24 in the DpsoF strain and cephalimysin A (19)[10] in the
DpsoB strain (Figure S6, Table S14, and Figure S44). Those
compounds appear to be formed by reduction by a nonspecific
oxidoreductase, since no other oxidoreductase gene is found
in or near the gene cluster.
Scheme 2. Proposed reaction mechanism of diol formation (8 and 9)
by either SN2 or SN2’ reaction from the substrate 7, which is formed
using PsoF.
In this study, we combined targeted gene deletion,
heterologous in vivo bioconversion, and an in vitro assay to
identify the main pathway leading to the formation of
pseurotins through the formation of azaspirene and synerazol
(Scheme 1). Through the study, we identified eight of the
intermediates formed and the enzymes responsible for the
oxidation and methylation steps taken during the trans-
formation of 2 into 8. PsoF is unique in that its gene is
encoded separately from the pseurotin biosynthetic gene
cluster and embedded within the unrelated fumagillin bio-
synthetic gene cluster.[5] However, even more unexpected was
that PsoF predominantly performed an apparent trans C-
methylation of the backbone of the growing polyketide chain,
with the MT domain of the PKS-NRPS enzyme PsoA
maintaining residual methyltransferase activity. Furthermore,
PsoF turned out to be a bifunctional enzyme, where its FMO
domain catalyzes the stereospecific epoxidation of the C11,12
olefin. Presumed spontaneous conversion of the epoxide into
a diol by either an SN2 or SN2’ reaction leads to the formation
of pseurotin A (8) or D (9), respectively, as well as 13, 14, 16,
and 17. The in vitro assay of PsoF with the NAC thioester of
25 showed that PsoF catalyzed the C-methylation to form 26
(Figure 3). However, 26 was converted into neither the
epoxide nor diol product. This result suggests that PsoF
performs the C3 methylation prior to epoxidation, and the
FMO domain requires extension of the polyketide chain with
a phenylalanine residue and possibly spiro-ring formation for
activity. While CTB3 from Cercospora nicotianae, involved in
the cercosporin biosynthesis, was also reported to be a poten-
tial bifunctional enzyme predicted to have an N-terminal, O-
MT, and C-terminal FMO-like domain,[11] PsoF is the first
enzyme of the kind whose activity has been characterized in
detail. While bifunctional enzymes often catalyze two con-
secutive steps of a metabolic pathway,[12] PsoF represents an
Figure 5. In vivo characterization of the cytochrome P450 PsoD for the
formation of 3. UV trace at l=280 nm from HPLC analysis of the
conversion of 2 into 3 by PsoD. i) Saccharomyces cerevisiae BY4705
without psoD as a negative control. ii) S. cerevisiae BY4705 harboring
the expression vector carrying the psoD gene. Cultures were incubated
at 308C for 3 h after feeding 2 to the strains.
conversion of 2, fed exogenously to the culture of Saccha-
romyces cerevisiae expressing the psoD gene (Figure 5), as
well as the in vitro assay for the same conversion (Figure S12),
were conducted. The observed conversion of 2 into 3
confirmed the idea that psoD codes for an oxidoreductase
which is responsible for the oxidation of C17 in 2 and, by
extension, the oxidation of 10. Lack of formation of O8-
methylated products in the DpsoD/AfKW1 mutant indicates
that PsoC requires the presence of the C17 ketone group in its
substrate for activity. This finding places the PsoD-catalyzed
step upstream of the PsoC-catalyzed step in the overall
pseurotin biosynthetic pathway. In addition, inactivity of
PsoD against 16 and 17 (Figure S57) indicates that, like PsoC,
PsoD is also sensitive to modifications to the diene side chain.
Lastly, based on the proposed activities of PsoC and PsoD, we
predict that 22, isolated from the DpsoF strain, is derived from
21, the analogue of 2 which is lacking the C3 methyl group
because of the absence of PsoF.
Characterization of the disruption mutants and hetero-
logously produced enzymes also identified multiple analogues
arising from cis/trans isomerization of the diene portion of the
azaspirene core. Disruption of psoE, a predicted glutathione
S-transferase (GST) gene, resulted in a dramatic loss of 8, new
formation of 18, and a significant accumulation of 4 and 6 as
compared to that of the wild-type control (Figure 2v versus i).
The compounds 4 and 6 are both 13E isomers, and they are
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of accepting more than one compound as their substrate.
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Received: April 29, 2014
Published online: June 18, 2014
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Keywords: biosynthesis · enzymes · enzyme catalysis ·
natural products · polyketides
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