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
Angew. Chem. Int. Ed. 2014, 53, 8475 –8479
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
8475