A methyltransferase initiates terpene cyclization in teleocidin B biosynthesis

Article abstract of DOI:10.1021/ja505224r

Teleocidin B is an indole terpenoid isolated from Streptomyces. Due to its unique chemical structure and ability to activate protein kinase C, it has attracted interest in the areas of organic chemistry and cell biology. Here, we report the identification of genes encoding enzymes for teleocidin B biosynthesis, including nonribosomal peptide synthetase (tleA), P-450 monooxygenase (tleB), prenyltransferase (tleC), and methyltransferase (tleD). The tleD gene, which is located outside of the tleABC cluster on the chromosome, was identified by transcriptional analysis and heterologous expression. Remarkably, TleD not only installs a methyl group on the geranyl moiety of the precursor but also facilitates the nucleophilic attack from the electron-rich indole to the resultant cation, to form the indole-fused six-membered ring. This is the first demonstration of a cation, generated from methylation, triggering successive terpenoid ring closure.

Full text of DOI:10.1021/ja505224r

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Communication
A methyltransferase initiates terpene cyclization in teleocidin B biosynthesis
Takayoshi Awakawa, Lihan Zhang, Toshiyuki Wakimoto, Shotaro Hoshino,
Takahiro Mori, Takuya Ito, Jun Ishikawa, Martin E. Tanner, and Ikuro Abe
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja505224r • Publication Date (Web): 03 Jul 2014
Downloaded from http://pubs.acs.org on July 7, 2014
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Journal of the American Chemical Society
A methyltransferase initiates terpene cyclization in teleocidin
B biosynthesis
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Takayoshi Awakawa,† Lihan Zhang,† Toshiyuki Wakimoto,† Shotaro Hoshino,† Takahiro Mori,†
Takuya Ito,‡ Jun Ishikawa,¶ Martin E. Tanner,§ and Ikuro Abe*,†
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† Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyoꢀku, Tokyo 113ꢀ0033, Japan.
‡ Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiroꢀcho, Tokushima 770ꢀ8514, Japan
¶ National Institute of Infectious Diseases, Shinjukuꢀku, Tokyo 162ꢀ8640, Japan
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§ Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada
Supporting Information Placeholder
However, the reaction mechanism for the formation
of its indoleꢀfused sixꢀmembered ring remained
unclear. Therefore, we set out to identify the genes
ABSTRACT: Teleocidin B is an indole terpenoid
isolated from Streptomyces. Due to its unique
chemical structure and ability to activate protein
kinase C, it has attracted interest in the areas of
organic chemistry and cell biology. Here, we report
the identification of genes encoding enzymes for
teleocidin B biosynthesis, including nonꢀribosomal
peptide synthetase (tleA), Pꢀ450 monooxygenase
encoding
the
biosynthetic
enzymes,
and
investigated their reactions in vitro, to shed light on
the later stage of the teleocidin biosynthesis.
17
16
15
H
18
14
N10
(tleB),
prenyltransferase
(tleC),
and
11
12
4a
13N
OH
H
9
8
methyltransferase (tleD). The tleD gene, which is
located outside of the tleABC cluster on the
chromosome, was identified by transcriptional
analysis and heterologous expression. Remarkably,
TleD not only installs a methyl group on the geranyl
moiety of the precursor, but also facilitates the
nucleophilic attack from the electronꢀrich indole to
the resultant cation, to form the indoleꢀfused
sixꢀmembered ring. This is the first demonstration of
a cation, generated from methylation, triggering
successive terpenoidꢀring closure.
N
O
4
N
OH
28
26
5
6
3
2
O
5
7a
27
N1
H
7
25
29
19
2
24
N
H
20
7
23
21
22
indolactam V (2)
teleocidin B (1)
Figure 1 The chemical structures of teleocidin B
and related compounds. (19R, 25S), (19S, 25R),
(19S, 25S), and (19R, 25R)ꢀteleocidin B are named
teleocidin Bꢀ1 (1a), Bꢀ2 (1b), Bꢀ3 (1c), and Bꢀ4 (1d),
respectively.
Lyngbyatoxin
A
(3, Scheme 1), which is
Teleocidin B (1) is a unique indole alkaloid, with an
indolactam and a monoterpenoid moiety fused with
Cꢀ6 and Cꢀ7 of the indole (Figure 1). It was first
isolated from Streptomyces mediocidicus,1 and
consists of a mixture of four stereoisomers (Bꢀ1,
Bꢀ2, Bꢀ3, and Bꢀ4).2 1 is also known as a potent
protein kinase C activator,3 which makes it a
potentially valuable reagent in pharmaceutical and
biochemical research. Thus, the unique structures
and specific bioactivities have attracted the
structurally related to 1, was isolated from the
cyanobacterium Moorea producens (formerly
Lyngbya majuscula).7 In 2004, Edwards and
Gerwick identified the lyngbyatoxin biosynthetic
gene cluster, and characterized the reaction of an
aromatic prenyltransferase (LtxC) to attach a
geranyl group to the Cꢀ7 position of 2.8
A
nonꢀribosomal peptide synthetase (LtxA) and a
Pꢀ450 monooxygenase (LtxB) encoded in the ltx
gene cluster were also identified as biosynthetic
enzymes. Subsequently, LtxB was shown to oxidize
and cyclize methylꢀLꢀvalylꢀLꢀtryptophanol to yield 2
in vitro.9 Given the structural similarity between 1
and 3, we hypothesized that enzymatic homologs of
attention of organic chemists, leading to many
4
isolation studies2,
and total syntheses5 of its
derivatives. The biosynthetic routes to these
molecules are also interesting, and have been
investigated
by
chemical
conversion
or
LtxABC would be involved in teleocidin
B
isotopeꢀfeeding experiments.6 These studies
revealed that the (−)ꢀindolactam V (2) moiety of 1
(Figure 1) is biosynthesized from Lꢀvaline and
Lꢀtryptophan, and the monoterpenoid moiety is
derived from the nonꢀmevalonate pathway.
biosynthesis. A Blast search of the draft genome
sequence of the teleocidin B producing strain
Streptomyces blastmyceticus NBRC 12747, using
the amino acid sequence of LtxC as a query,
revealed a gene encoding a prenyltransferase with
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A)
40% amino acid identity to LtxC, which was
1d
1a
4
1
2
3
4
5
6
7
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designated as TleC. The upstream region of tleC
contained two genes, encoding a nonꢀribosomal
peptide synthetase and a Pꢀ450 monooxygenase,
which were named tleA and tleB, respectively. TleA
has the same catalytic domain structure as LtxA
with 49% identity, and TleB shares 47% identity with
LtxB. Thus, we identified a 23.2 kb contig containing
iii)
3
ii)
i)
15
20
25
30
35
t/min
B)
3
tleABC, as
a
candidate for the teleocidin
1d
1a
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4
biosynthetic gene cluster (Figure S1). On the other
hand, no genes related to a Cꢀmethyltransferase
(CꢀMT) or a terpene cyclase (TPC), which are
thought to be involved in teleocidin B biosynthesis,
were found in the region adjacent to the tle cluster.
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ii)
3
i)
35
To test whether the tle cluster is responsible for
teleocidin B production, the fosmid bearing the tle
cluster (pCCꢀtle) was introduced into Streptomyces
15
20
25
30
t/min
Figure 2 (A) LCꢀESIMS profiles of cell extracts
from transformants harboring i) the void vector, ii)
the tle cluster, and iii) the tle cluster and tleD. (B)
LCꢀESIMS profiles of the products of the in vitro
enzyme assays, in the i) absence or ii) presence of
lividans TK21. We engineered pCCꢀtle by
recombination for integration into
λ
ꢀRED
the
chromosome,10 and introduced the resultant
plasmid, pCCSTꢀtle, into the host. The compounds
that accumulated within the S.lividans/pCCSTꢀtle
cells, cultured in TP2 medium for 3 days, were
analyzed by LCꢀESIMS. As a result, compound 3,
which was not detected in the S. lividans harboring
pCCST (void vector), was newly detected in the S.
lividans/pCCSTꢀtle (Figure 2A). In accordance with
TleD, with
3
as
a
substrate. All of the
chromatograms were monitored at 280 nm.
All three compounds showed psuedomolecular
ions at m/z = 451 consistent with 1. After a large
scale fermentation and intensive spectroscopic
analyses, 1d was identified as teleocidin Bꢀ4, by
1
the HRꢀMS and H NMR spectra, and the rotation
value in the literature,7 3 was identified as
lyngbyatoxin A (Supplementary data). This result
suggested that the enzymes encoded in the tle
cluster are responsible for the production of 3, but
not for the methylation or the cyclization of the
geranyl moiety required for the biosynthesis of
teleocidin B. Therefore, we anticipated that one or
more genes encoding the enzyme(s) that catalyze
the Cꢀmethylation and/or cyclization of the geranyl
moiety of lyngbyatoxin A are located outside of the
tle cluster.
comparing its Hꢀ and 13Cꢀ NMR, HRꢀMS spectra,
1
and optical rotation value (supplementary data) with
data from the literature.13,14 The H and 13C NMR
1
spectra of 1a were similar to those of 1d, except for
a characteristic difference observed in the 13C
chemical shift at Cꢀ20: 26.9 ppm for 1a, and 21.5
ppm for 1d. This difference is probably attributable
to a γꢀgauche effect between Cꢀ20 and the
substituent at Cꢀ25.2 Thus, 1a was determined to be
teleocidin Bꢀ1, which is the Cꢀ25 epimer of 1d.
Compound
4
was
identified
as
We searched for the genes encoding a putative
CꢀMT responsible for the methylation of 3 in the
draft genome sequence of S. blastmyceticus, and
found nine candidate CꢀMT genes. Since an
RTꢀPCR analysis revealed that three of them were
not coꢀtranscribed with tleC (Figure S3), they were
excluded from the candidates. Each of the six
remaining CꢀMT genes was introduced into S.
lividans harboring the tle cluster. The LCꢀESIMS
analyses revealed that three compounds (1a, 1d,
and 4) (Scheme 1) were newly detected only in the
cellꢀextract of the transformant that harbors tleD,
which shares moderate homology with other CꢀMTs
that catalyze the Cꢀmethylation of the prenyl group,
such as the sterol 24ꢀCꢀMT from Magnaporthe
oryzae11 and the geranyl diphosphate (GPP)
2ꢀCꢀMT from Streptomyces coelicolor A3(2)12
(Figure 2A).
desꢀOꢀmethylꢀolivoretin C, by comparing its 1Hꢀ and
13Cꢀ NMR, HRꢀMS spectra, and optical rotation
value (supplementary data) with the published data
of olivoretin C.15 These data demonstrated that the
TleDꢀmediated methylation of the geranyl moiety of
3 resulted in the formation of three cyclized
products: 1a, 1d, and 4. In the process of the assay,
we also identified a MT that catalyzes the
Oꢀmethylation of the Cꢀ14 hydroxyl group of 3
(Supplementary information). Interestingly, like tleD,
this gene is also located outside of the tle cluster.
To understand the catalytic function of TleD, we
performed in vitro enzyme reactions, using purified
Hisꢀtagged recombinant TleD with 3 as a substrate.
Our results confirmed that TleD alone successfully
afforded the cyclized products 1a, 1d and 4 (Figure
2B). This finding clearly ruled out the involvement of
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other enzymes in the cyclization reaction, and labeling experiment. The TleC enzyme reaction with
[6ꢀ2H]ꢀGPP and 2 afforded [25ꢀ2H]ꢀ3, which was
then used as a substrate for the TleD reaction.
Mass spectrometry of the products from the
reaction using [25ꢀ2H]ꢀ3 gave an m/z value of 452,
which revealed that all three products, 1a, 1d and 4,
retained the deuterium atom (Figure S17). In the 1H
NMR spectrum of Dꢀlabeled 1d, the multiplet signal
corresponding to Hꢀ26 was no longer detected, and
the two doublet methyl signals corresponding to
Hꢀ27 and Hꢀ28 were observed as a singlet (Figure
S19). These data provided convincing evidence that
the deuterium atom at Cꢀ25 is shifted to Cꢀ26, and
confirmed the 1,2ꢀhydride shift in the cationic
intermediate. The kcat and Km values for the
formation of Dꢀlabeled 1a, 1d and 4 were
comparable to those in the reaction using unlabeled
3: the kcat and Km values for the formation of
Dꢀlabeled 4 were 0.41 minꢀ1 and 2.4 ꢁM, while those
for the formation of Dꢀlabeled 1a were 0.14 minꢀ1
and 3.7 ꢁM, and for the formation of Dꢀlabeled 1d
were 0.88 minꢀ1 and 2.4 ꢁM. These results excluded
the deuterium isotope effect during the
methylation/cyclization reaction, and confirmed that
the 1,2ꢀhydride shift in the cationic intermediate is
not the rateꢀlimiting step.
1
2
3
4
5
6
7
8
suggested that a cation generated by methylation
initiated the cyclization of the geranyl moiety
(Scheme 1). Next, to further characterize the
enzyme reaction, we measured the steadyꢀstate
kinetic parameters of TleD. The kcat and Km values
for the formation of 4 were 0.37 minꢀ1 and 2.7 ꢁM,
while those for the formation of 1a were 0.17 minꢀ1
and 3.4 ꢁM, and for the formation of 1d were 0.84
minꢀ1 and 3.2 ꢁM. These values are comparable to
those of other MTs,16 thus indicating that TleD
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accepts
3 as a genuine substrate, and is
responsible for the biosynthesis of teleocidin B. To
confirm the native role of TleD in the biosynthesis,
gene deletion study is required. Otherwise, we
cannot rule out the possibility of having other genes
to perform the same reaction, or the possibility for
TleD to be involved in other pathways. Interestingly,
TleD also accepted 14ꢀOꢀmethyllyngbyatoxin A, but
with less efficiency (Figure S15).
Considering that both the TleDꢀcatalyzed
methylation and subsequent nucleophilic attack
take place on the same carbon, we supposed that
the Cꢀ25 cationic intermediate should be formed
prior to cyclization, presumably via a 1,2ꢀhydride
shift (Scheme 1). To confirm the 1,2ꢀhydride shift in
the proposed pathway, we performed a deuterium
Scheme 1 Proposed mechanism of TleDꢀcatalyzed reaction.
H
H
H
H
N
N
N
N
N
OH
N
OH
N
OH
N
OH
O
O
O
O
1,2ꢀhydride shift
Reꢀface attack
TleD
a
N
H
N
H
N
H
b
N
H
26
25
H
H
lyngbyatoxin A (3)
[CH3]
Siꢀface attack
pathaa
pathab
H
N
H
H
N
N
N
OH
N
OH
N
O
OH
O
O
N
H
N
H
N
H
1a
4
1d
Although the previous feeding experiments
suggested that 3 was a precursor of 1,6b the details
of the reaction mechanism had not been reported.
Our in vitro study clearly illustrated that 3 is the
direct precursor of 1a, 1d and 4, and the reaction is
catalyzed by one enzyme TleD. Moreover, the
deuterium labeling experiment corroborated the
biosynthetic pathway originally proposed by Irie et
al (Scheme 1).6b First, TleD installs a methyl group
at Cꢀ25, and the hydride at Cꢀ25 migrates to Cꢀ26.
The resultant cation at Cꢀ25 is then attacked by the
intermediate. Finally, the CꢀC bond, which consists
of the spiroꢀring, migrates via path a, to afford 1d. A
similar spiroꢀring compound was reported as the
product in the PictetꢀSpengler reaction of
Nbꢀhydroxytryptamines and cysteinals,17 thus
supporting this proposed reaction mechanism.
During the spiroꢀring formation in the pathway to 1,
the facial selectivity of the nucleophilic attack to the
Cꢀ25 cationic intermediate determines the Cꢀ25
stereochemistry of 1. Since the two bulky
substituents, the isopropyl and vinyl groups, should
be on the same face of the fiveꢀmembered ring
nucleophilic Cꢀ7, to afford
a
spiroꢀfused
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abei@mol.f.uꢀtokyo.ac.jp
intermediate to afford 1a, the facile and preferred
production of 1d over 1a is presumably due to steric
factors. Furthermore, 4 could be generated from the
same spiroꢀring intermediate, via path b. The
production ratio of 1a : 1d : 4 is 0.5 : 2.3 : 1.0 (both
in the in vitro and in vivo assay), indicating that path
a is favored. Finally, it should be noted that no
byproducts derived from deprotonation or
hydroxylation of the Cꢀ25 cationic intermediate were
detected, which suggested that the 1,2ꢀhydride shift
and the cyclization reaction take place in a
concerted manner.
1
2
3
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Notes
The authors declare no competing interest.
ACKNOWLEDGMENTS
We thank Prof. H. Onaka (Univ. Tokyo), and Prof. E.
Takano (Univ. Manchester) for kindly providing the
expression vectors. We also thank Prof. H. Ikeda
(Kitasato Univ.) for useful advice to this work. This
work was supported by GrantsꢀinꢀAid for Scientific
Research from the MEXT, Japan.
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REFERENCES
TleD shares 16.5%, 28.3%, and 33.9% amino acid
sequence identities with the GPP 2ꢀCꢀMT from S.
coelicolor A3(2),12 the sterol 24ꢀCꢀMT from M.
oryzae,11 and the rebeccamycin sugar 4'ꢀOꢀMT
RebM from Lechevalieria aerocolonigenes,16
respectively. Sequence comparisons revealed that
TleD has the consensus GXGXG motif (Figure
S20), which is a conserved SAMꢀbinding site in
MTs. Considering that TleD possesses Y200,
corresponding to F222 of GPPMT, which is
proposed to stabilize the cationic intermediate
through a cationꢀ interaction in the deprotonation
of the intermediate,12b TleD may facilitate the
methylation/cyclization reaction by a similar cation
stabilization mechanism. However, we cannot
totally exclude a possibility that TleD simply
methylates the substrate while the remaining
reactions are spontaneous. To further understand
the intimate structural details of the enzyme
reaction, an Xꢀray crystallographic study of TleD is
now in progress.
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In conclusion, we identified a suite of genes
involved in teleocidin B biosynthesis. Notably, TleD
is the first MT capable of triggering terpene
cyclization. Similar to the Class I TPCs,18 that
generate a carbocation by ionizing the allylic
diphosphate, TleD also generates a carbocation
that electrophilically adds to the aromatic ring and
then undergoes a subsequent alkyl migration.
However, the TleDꢀcatalyzed reaction differs from
those of the Class I TPCs, in that methylation
initiates cyclization. Our study illustrates the first
representative case of a new mode of terpene
cyclization, thus broadening the scope of TPC
chemistry.
(12) a) Komatsu, M.; Tsuda, M.; Omura, S.; Oikawa, H.; Ikeda,
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ASSOCIATED CONTENT
Supporting Information.
Experimental details and spectral data. This material is
available free of charge via the Internet at
http://pubs.acs.org.
(16) Singh, S.; McCoy, J. G.; Zhang, C.; Bingman, C. A.;
Phillips, G. N.; Thorson, J. S. J. Biol. Chem. 2008, 283, 22628.
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AUTHOR INFORMATION
Corresponding Author
(18) Christianson, D. W. Chem. Rev. 2006, 106, 3412.
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H
N
H
N
H
N
N
OH
N
OH
N
OH
O
O
O
MT
N
H
N
H
N
H
9
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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49
50
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lyngbyatoxin A
]
[CH3
H
N
H
N
N
OH
N
OH
O
O
N
H
N
H
teleocidin Bꢀ4
ACS Paragon Plus Environment