Identification of Serratane Synthase Gene from the Fern Lycopodium clavatum

Article abstract of DOI:10.1021/acs.orglett.6b03659

Ferns are known to produce onoceroids including onoceranes and serratanes having unusual structures among triterpenes. From the fern Lycopodium clavatum, a novel onoceroid synthase gene was cloned that showed high sequence identity with a previously identified α-onocerin synthase. Functional analysis by coexpression with pre-α-onocerin synthase in yeast led to the production of tohogenol and serratenediol. The result suggested that serratanes are directly biosynthesized from pre-α-onocerin and not from α-onocerin as previously assumed.

Full text of DOI:10.1021/acs.orglett.6b03659

Letter
pubs.acs.org/OrgLett
Identification of Serratane Synthase Gene from the Fern Lycopodium
clavatum
†
†
†
‡
‡
§
Yusuke Saga, Takeshi Araki, Hiroshi Araya, Kazuki Saito, Mami Yamazaki, Hideyuki Suzuki,
and Tetsuo Kushiro*
,†
†Graduate School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan
‡Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Chuo-ku, Chiba 260-8675, Japan
§Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan
*
S Supporting Information
ABSTRACT: Ferns are known to produce onoceroids
including onoceranes and serratanes having unusual structures
among triterpenes. From the fern Lycopodium clavatum, a novel
onoceroid synthase gene was cloned that showed high
sequence identity with a previously identified α-onocerin
synthase. Functional analysis by coexpression with pre-α-
onocerin synthase in yeast led to the production of tohogenol
and serratenediol. The result suggested that serratanes are directly biosynthesized from pre-α-onocerin and not from α-onocerin
as previously assumed.
riterpenes are a large class of natural products with diverse
structures and bioactivities. To date, over 100 different
Serratane-type triterpenes possess an unusual seven-mem-
bered C-ring with seven tertiary methyl groups (Figure 1). One
methyl group is incorporated into a seven membered C-ring.
Tohogenol (5) and serratenediol (6) are the parent member8s
of serratane-type triterpenes and were found in H. serrata.
Compound 6 has been shown to induce apoptosis and inhibit
T
types of triterpene skeletons have been reported from natural
1
sources. Triterpene skeletons derive from a common
precursor, (3S)-2,3-oxidosqualene (1), via a cyclization reaction
2
catalyzed by oxidosqualene cyclases (OSCs). These skeletons
10
include sterol precursors such as lanosterol and cycloartenol as
well as precursors for various triterpenoids that are widely
distributed among plants. In addition to the well-known
triterpenes, there are several unusual types that are formed by
irregular cyclization patterns.
cell proliferation of human HL-60 leukemia cells. Lyco-
phlegmarin (7) has been isolated from H. phlegmaria and
exhibited modest growth-inhibitory activity against human
11
hepatoma cells BEL 7402.
Their biosynthesis had been proposed to involve protonation
of one of the exo-methylenes of 3 to generate a C8 tertiary
cation to which a cyclization from the neighboring exo-
The bis-cyclic triterpenes, also known as onoceroids, are
biosynthesized from squalene and (3S,22S)-2,3,22,23-dioxidos-
3
−6
qualene (2) by cyclization from both termini.
They include
12
methylene would furnish the seven membered C-ring.
compounds having onocerane and serratane skeletons and have
Indeed, protonation of one of the exo-methylenes of 3 by
been isolated from several ferns including Lycopodium
13
purely chemical means afforded serratenediol. Therefore, the
7
8
clavatum and Huperzia serrata and also from some higher
formation of serratanes was assumed to be a secondary
transformation after the formation of the onocerane skeleton.
Our recent discovery of onocerin synthase gene LCD, however,
has raised an alternative possibility in which serratanes could be
formed directly from 4 without formation of an intermediate 3
as depicted in Scheme 1. Such a scenario may require an
additional enzyme quite similar to LCD, and there is no need to
assume a hypothetical protonating enzyme that acts on 3. In
this study, we searched for a novel onoceroid synthase gene
from L. clavatum to see whether serratane formation involves
tricyclization of 4 or monocyclization of 3 and identified the
gene involved in the biosynthesis of serratanes for the first time.
9
plants. α-Onocerin (3) (onocerane-type), characterized by its
unique symmetrical structure that contains two decalin
portions, was recently demonstrated to be biosynthesized by
4
two highly unusual OSC-like enzymes in L. clavatum.
In its biosynthetic processes, first the dioxidosqualene cyclase
LCC forms the bicyclic polypodane compound pre-α-onocerin
4) with strict substrate specificity toward 2. Subsequently,
onocerin synthase LCD catalyzes the second half of the
cyclization using 4 as a substrate to produce α-onocerin
(
(
Scheme 1). Discovery of such unusual OSC-like enzymes
having substrate specificity toward 2 and 4 has prompted us to
study their recognition mechanism as well as cyclization
reaction in detail. On the other hand, however, no genetic
and molecular bases for the biosynthesis of serratanes have
been reported.
Received: December 8, 2016
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.6b03659
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
a
Scheme 1. Cyclization of Dioxidosqualene (2) into α-Onocerin (3), Tohogenol (5), and Serratenediol (6)
a
(
A) Proposed reaction mechanism of LCC, LCD, and LCE. (B) Previous proposal of serratenediol formation from 3.
code for another onoceroid synthase. Characteristic DCTAE
and QW motifs of OSCs were found as well, and similar to the
LCD case, a sequence around the MWCYC motif is rather
diverged from known OSCs that produce a normal type of
triterpenes. The corresponding motif of β-amyrin synthase was
previously shown to be the active site residues largely
1
4
responsible for determining the product outcome. Thus, we
considered LCE as a candidate gene for onoceroid synthase and
set out a functional study of this gene.
The full-length sequence of LCE was obtained by PCR from
cDNAs of L. clavatum. The obtained cDNA was cloned into the
yeast expression vector pESC-Ura under the control of the
GAL10 promoter to produce pESC-LCE. In addition, LCC and
LCE were simultaneously introduced into pESC-Ura to obtain
pESC-LCC and -LCE, while pESC-LCC was from our previous
4
study. These plasmids were introduced and heterologously
expressed in the Saccharomyces cerevisiae strain GIL77, a
lanosterol synthase deficient mutant that accumulates 1 within
the cells, and because of a squalene epoxidase that epoxidizes
1
5
Figure 1. Structures of representative serratane-type triterpenes.
In our previous study on the biosynthesis of 3, we performed
another terminal olefin, 2 also accumulates in these cells.
After induction by galactose and the resting cultures, the
transformant cells were harvested, extracted with hexane, and
analyzed by TLC. While the LCC expressing transformant
produced 4, both LCC and LCE expressing transformant gave
4
the RNA-seq analysis of L. clavatum. To obtain further
onoceroid synthase genes, we searched the RNA-seq data for
additional OSC homologues using known OSC genes from
plants as a query. To our delight, we found that L. clavatum
possesses another OSC family gene that might be responsible
for an onoceroid biosynthesis. This gene, which we denoted
LCE, showed moderate sequence identity (58%) to both LCD
and cycloartenol synthase LCA (51%), suggesting that this may
transgene specific spots, one with a R value similar to 3 and the
f
other more polar on TLC, both of which were absent in the
transformants expressing only LCE. These spots were further
analyzed by GC−MS (Figure 2). The major product gave a
molecular ion peak at m/z 460 which was 18 Da larger than
that of 4 (m/z 442), implicating a structure equivalent to a
B
DOI: 10.1021/acs.orglett.6b03659
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
1
6
upon GC−MS analysis. The H NMR spectrum (in CDCl )
3
of the minor product exhibited seven singlet methyl signals (δH
0
0
3
1
.664 (s, 3H), 0.764 (s, 3H), 0.796 (s, 3H), 0.826 (s, 6H),
.961 (s, 3H), 0.967 (s, 3H)), two hydroxymethine signals (δH
.185 (dd, J = 11.2, 4.8 Hz, 1H), 3.231 (dd, J = 11.5, 4.1 Hz,
H)), and an olefinic signal (δ 5.331 (m, 1H)), indicating a
H
13
presence of an olefin. The C NMR spectrum showed the
presence of two carbons bearing hydroxyl groups (δ 78.81,
C
7
9.14) and an olefin (δ 122.14, 138.18). These data were in
C
complete agreement with the literature data as well as the data
18
for the authentic sample of 6.
Therefore, LCE was demonstrated to be a novel onoceroid
synthase producing serratane-type triterpenes with 5 as the
major product together with 6 as the minor product in a ∼10:1
ratio. The structures of these LCE products suggested that the
LCE-catalyzed reaction of 4 was initiated by a proton attack on
the epoxide moiety, followed by bicyclization to give a C8
cation, a common cationic intermediate en route to 3 (Scheme
1). Subsequent cyclization from the neighboring C14−C27 exo-
methylene bridged the two decalin structures to construct a
seven-membered C-ring and gave a 6/6/7/6/6-fused pentacy-
clic C14 carbocation intermediate. This cation underwent a
nucleophilic addition of a water to produce 5, or alternatively,
deprotonation of H15 to yield 6. As with the case of LCD, LCE
preferably utilized 4 among other substrates such as 1 and 2
and catalyzed the formation of the fused cyclic structure on the
linear side chain of 4. These results indicated that in ferns,
onocerane and serratane, two of the major onoceroid, were
each individually produced by a different cyclase using 4 as a
common substrate. It is noteworthy that the serratane skeleton
was biosynthesized from a bicyclic triterpene such as 4. This
finding may disprove the previously accepted proposal that the
serratane formation involves protonation of one of the exo-
methylenes of 3 followed by a cyclization into a seven-
membered ring. Our novel finding proposed that serratane
formation did not require the formation of an intermediary 3
and was directly produced from 4.
Figure 2. GC−MS analysis of each enzymatic product: (i) standard
sample of 6, (ii) hexane extracts of samples expressing both LCC and
LCE, (iii) LCC, (iv) LCE, (v) empty vector. Compounds 5 and 6
were only seen in (ii).
hydrated 4. On the other hand, the minor product gave a
molecular ion peak at m/z 442 that was equivalent to 4. In
addition, the characteristic peaks at m/z 207 and 189 due to
retro Diels−Alder fragmentation of the D-ring were observed
1
6
for this compound.
To clarify the structures of these products, from a large-scale
culture (4 L), these products were purified by silica gel column
to yield 10 mg of the major product and 1 mg of the minor
1
product and then subjected to NMR analyses. The H NMR
spectrum (in CDCl ) of the major product showed the
3
presence of seven singlet methyls (δ 0.751 (s, 3H), 0.781 (s,
H
3
3
1
H), 0.786 (s, 3H), 0.932 (s, 6H), 0.946 (s, 3H), 0.973 (s,
H)) and two hydroxymethine protons (δH 3.195 (dd, (J =
1.5, 4.5 Hz, 1H), 3.202 (dd, J = 11.0, 5.0 Hz, 1H). The 13
C
NMR spectrum showed 30 carbon signals in which three of
them bear a hydroxyl group (δ 75.64, 78.76, 78.85). Two of
C
them were methines while the other was a quaternary carbon.
Among serratane-type triterpenes, 5 possesses a structure that
agrees with this data. The literature data for 5 were reported
With both onocerin and serratane synthases in hand, we can
now study the difference in the catalytic mechanism of the two
enzymes. As mentioned, the two enzymes catalyze the same
cyclization cascade up to the C8 cation intermediate, from
which an immediate deprotonation will give rise to onocerane
while an additional cyclization from the neighboring exo-
methylene will result in the formation of serratane skeleton.
What factor of these enzymes is responsible for the difference
between these reaction paths remains to be elucidated. In
addition, the LCD-catalyzed cyclization into 3 is terminated by
deprotonation, while the LCE-catalyzed reaction is terminated
by water addition. The difference of termination mechanism
between LCD and LCE is intriguing. We speculate that LCE
may have an additional space around C−D rings in its active
site cavity, which allows a solvent water molecule to enter.
Mutational studies would clarify such questions and elucidate
the molecular basis for the formation of two of the most
unusual triterpenes found in nature.
Our studies also pointed out that an evolutionary divergence
of OSC-like genes such as LCD and LCE was responsible for a
production of diverse onoceroid structures in ferns. While the
LCC gene would produce the bicyclic triterpene 4, a
combination of LCC and LCD would produce onoceranes,
while that of LCC and LCE would produce serratanes. Such a
switch in production of these onoceroids can be controlled by
an expression of either gene in the cells. Here we also
17
with pyridine-d as a solvent, while the data for diacetylated 5
5
8
were reported with CDCl . Therefore, our sample of the major
3
product was acetylated and analyzed by NMR, which showed
complete agreement with the literature values. We also
1
13
measured H and C NMR in pyridine-d as solvent, which
5
1
7
showed complete agreement with the literature data.
To further clarify the structure including the stereochemistry
of the tertiary hydroxyl group at C14, the diacetylated sample
was subjected to dehydration conditions using SOCl . Under
2
such conditions, 5 having a β-hydroxyl group at C14 would
result in production of serratenediol bearing an olefin between
C14−C15, while the α-epimer would give a mixture of
serratenediol and its olefinic isomer isoserratenediol having
8
an olefin between C13−C14. The configuration of the
hydroxyl group may result in such a difference since trans-
elimination toward C13 to produce isoserratenediol is only
possible with the α-configuration. Our result after GC−MS
analysis showed almost exclusive production of diacetylated
serratenediol, which agreed well with the results from the
literature and further confirmed that our major product was
8
indeed 5.
On the other hand, the minor product showed identical
retention time and a MS spectrum with an authentic sample of
C
DOI: 10.1021/acs.orglett.6b03659
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
demonstrated that such a selection of onoceroid production
could be performed on a yeast platform by choosing the
combination of genes to be expressed. It should be noted that
an isolation of serratane-type triterpenes have not been
reported from L. clavatum yet. Our results indicated that this
fern does have the ability to produce serratanes. It would be
interesting to see if other ferns that are known to produce
serratanes possess a gene similar to LCE.
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ASSOCIATED CONTENT
Supporting Information
(
3
(
2
■
*
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.6b03659.
(
Experimental details, supplementary figures, and spectral
data including EIMS and NMR (PDF)
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: kushiro@meiji.ac.jp.
ORCID
Tetsuo Kushiro: 0000-0002-7598-4791
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Prof. Emeritus Ushio Sankawa and Prof. Emeritus
Yutaka Ebizuka of the University of Tokyo for providing the
authentic sample of 6, Ms. Sayaka Shinpo and Dr. Michimi
Nakamura from Kazusa DNA Research Institute for technical
support for RNAseq analysis, and Dr. Takahisa Nakane from
Showa Pharmaceutical University for measuring optical
rotations. We are grateful to Meiji University for financial
support.
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DOI: 10.1021/acs.orglett.6b03659
Org. Lett. XXXX, XXX, XXX−XXX