Bifunctional triterpene/sesquarterpene cyclase: Tetraprenyl-β- curcumene cyclase is also squalene cyclase in bacillus megaterium

Article abstract of DOI:10.1021/ja2060319

This study demonstrates that a tetraprenyl-β-curcumene cyclase, which was originally identified as a sesquarterpene cyclase that converts a head-to-tail type of monocycle to a pentacycle, also cyclizes a tail-to-tail type of linear squalene into a bicyclic triterpenol, 8α-hydroxypolypoda- 13,17,21-triene. The 8α-hydroxypolypoda-13,17,21-triene was found to be a natural triterpene from B. megaterium. It was also demonstrated that cyclizations of both tetraprenyl-β-curcumene and squalene occurred with a purified B. megaterium TC homologue in the same reaction mixture. These results suggest that the tetraprenyl-β-curcumene cyclase is bifunctional, cyclizing both tetraprenyl-β-curcumene and squalene in vivo. This is the first report describing a bifunctional terpene cyclase, which biosynthesizes two classes of cyclic terpenes with different numbers of carbons as natural products in the organism.

Full text of DOI:10.1021/ja2060319

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Bifunctional Triterpene/Sesquarterpene Cyclase:
Tetraprenyl-β-curcumene Cyclase Is Also Squalene Cyclase in
Bacillus megaterium
Tsutomu Sato,† Hiroko Hoshino,† Satoru Yoshida,† Mami Nakajima,‡ and Tsutomu Hoshino†
†Department of Applied Biological Chemistry, Faculty of Agriculture, and Graduate School of Science and Technology,
Niigata University, Ikarashi 2-8050, Nishi-ku, Niigata 950-2181, Japan
‡Center for Instrumental Analysis, Niigata University, Ikarashi 2-8050, Nishi-ku, Niigata 950-2181, Japan
S Supporting Information
b
Scheme 1. Proposed Pathway for the Biosynthesis of the
Sesquarterpenes in B. subtilisa
ABSTRACT: This study demonstrates that a tetraprenyl-β-
curcumene cyclase, which was originally identified as a
sesquarterpene cyclase that converts a head-to-tail type of
monocycle to a pentacycle, also cyclizes a tail-to-tail type of
linear squalene into a bicyclic triterpenol, 8α-hydroxypoly-
poda-13,17,21-triene. The 8α-hydroxypolypoda-13,17,21-
trienewas foundtobeanaturaltriterpenefromB. megaterium.
It was also demonstrated that cyclizations of both tetra-
prenyl-β-curcumene and squalene occurred with a purified
B. megaterium TC homologue in the same reaction mixture.
These results suggest that the tetraprenyl-β-curcumene
cyclase is bifunctional, cyclizing both tetraprenyl-β-curcu-
mene and squalene in vivo. This is the first report describing
a bifunctional terpene cyclase, which biosynthesizes two
classes of cyclic terpenes with different numbers of carbons
as natural products in the organism.
a The nonenzymatic synthetic pathways of 1 f 2 and 3 f 4 have
previously been described by Takigawa et al.7
recent biosynthetic study on the C35 terpenes from Bacillus
A
subtilis identified a novel terpene cyclase, tetraprenyl-β-
curcumene synthase (TS), which converted heptaprenyl dipho-
sphate to tetraprenyl-β-curcumene (1) (Scheme 1) and had no
sequence homology with any of the known terpene cyclases.1
Since this result was the first to demonstrate on both the gene
and enzyme levels that C35 terpenes were biosynthesized via the
cyclization of the linear C35 isoprenoid, we proposed a novel
family name for these C35 terpenes: sesquarterpenes.1 An enzy-
matic reaction involving tetraprenyl-β-curcumene cyclase (TC),
which cyclizes 1 into C35 terpenol (3), has also been demonstra-
ted in vitro (Scheme 1).1 The primary structure of the B. subtilis
TC is similar to that of the squalene-hopene cyclase (SHC) and
has two characteristic motifs conserved in the SHCs: the DXDDTA
and QW motifs.2 The SHC catalyzes the conversion of squalene
(C30, 8) to pentacyclic triterpenes, hopene (9) and hopanol (10)
(Scheme 2), and has been investigated in depth to elucidate the
common catalytic mechanism among the triterpene cyclase family
members.2 This was achieved by determining crystal structures,
using site-directed mutagenesis, and analyzing products bio-
synthesized from substrate analogues.2 Despite the similarity of
the TC to the SHC, Bosak et al. reported that cell lysates of E. coli
overexpressing B. subtilis TC could not react with 8.3 However, it
was possible that the overexpressed enzyme was not functional,
because no enzymatic activity was confirmed using any substrate.
Thus, we retested the cyclization of 8 with the enzymatic reac-
tion system using recombinant TC from B. subtilis, which was
shown to convert 1 to 3. The reaction of recombinant SHC from
Alicyclobacillus acidocaldarius with 1 was also analyzed and com-
pared with the reaction of the TC with 1. As a result, we found an
additional function for TC, which also produces bicyclic triter-
pene from 8 in B. megaterium cells. To date, no bifunctional ter-
pene cyclase, which biosynthesizes two classes of cyclic terpenes
with different numbers of carbons as natural products in the
organism, has yet been reported. It is also noteworthy that the
bifunctional triterpene/sesquarterpene cyclase constructs two
different cyclic scaffolds—the 6/6/6/6-fused tetracyclic and 6/6-
fused bicyclic ring systems—from 1 and 8, respectively.
The substrates 1 and 8 were separately incubated with the TC,
which was purified by the methods described in our previous
report,1 and the reaction products were analyzed by GC-MS
(Figure 1). It was confirmed that the prepared TC could cyclize
Received: June 29, 2011
Published: October 07, 2011
r
2011 American Chemical Society
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Scheme 2. Pathway for the Biosynthesis of the Hopene (9)
and the Hopanol (10)
its natural substrate 1 into its usual product 3 (Figure 1A), sug-
gesting that the purified enzymes were functional. The com-
pound 4 and its thermally dehydrated products, which were
detected in the chromatogram of Figure 1A, are synthesized
by the nonenzymatic reaction of 3 (Scheme 1).1,4 We revealed
that the TC produced a significant GC-MS peak of 11 from 8
(Figure 1C). In order to determine the structure of 11, 20 mg of 8
was incubated with cell-free extracts prepared from E. coli BL21
(DE3) cells overexpressing recombinant TC as described
before.1 n-Hexane extracts from the reaction mixture were puri-
fied by silica gel column chromatography and SiO2 HPLC to
yield 3.7 mg of pure 11. Using NMR (1H, 13C, DEPT, COSY,
HOHAHA, NOESY, HMQC, and HMBC) and MS (ESI and
EI), the compound 11 was shown to be 8α-hydroxypolypoda-
13,17,21-triene (Scheme 3),5 which is found in the fern Poly-
podium niponicum. The molecular formula of 11 was determined
to be C30H52O on the basis of the HR-ESI-MS results. In the 1H
NMR spectrum, three olefinic protons (δH 5.56, 5.45, 5.37, each
1H, t, J = 6.9 or 7.0) and four allylic methyl protons (δH 1.84,
1.81, 1.75, 1.69, each 3H, s) were found, indicating that three
double bonds remained unchanged. The remaining four methyl
protons appeared at higher field (δH 1.18, 0.97, 0.88, 0.82, each
3H, s), suggesting that four tertiary methyl groups were in the
cyclized bicyclic ring. The relative stereochemistry of 11 was
determined by observing the following NOE: H-5/H-9, Me-23/
Me-25, and Me-25/Me-26. The specific optical rotation
([α]D = À1.8°) was nearly identical with the published value
([α]D = À0.9°),5 suggesting the absolute configuration of 11
shown in Scheme 3. Thus, it was clear that the TC produces
bicyclic 11 by the cyclization of 8. The recombinant TC prepared
by Bosak et al. may have no enzymatic activity toward substrates
1 and 8.3
Figure 1. GC-MS total ion chromatogram, used to analyze the enzy-
matic activityof purifiedTC andSHC with thesubstrates 1 and8, andthe
n-hexane extract from B. megaterium cells: (A) reaction mixture after the
incubation of substrate 1 with the TC; (B) reaction mixture after
the incubation of substrate 1 with the SHC; (C) reaction mixture after
the incubation of substrate 8 with the TC; (D) reaction mixture after the
incubation of substrate 8 with the SHC; (E) n-hexane extract from the
B. megaterium cells.
Scheme 3. Cyclization of 1 and 8 Catalyzed by the TC and
the SHCa
a This study revealed that the 8α-hydroxypolypoda-13,17,21-triene (11)
from 8 was formed by the TC and that no product from 1 was bio-
synthesized by the SHC.
B. subtilis and several other Bacillus species, such as B. amylo-
liquefaciens, B. atrophaeus, B. cellulosilyticus, B. pumilus, and B.
megaterium, contain homologues of the squalene synthase (SS)
or phytoene synthase (PS) that catalyze the tail-to-tail condensa-
tion of two farnesyl diphosphates to 8 (Scheme 2) or two geranyl-
geranyl diphosphates to phytoene (C40) (Table 1).6 However,
the production of 8 and phytoene in the Bacillus species, except
for B. subtilis,7 has never been analyzed. It is possible that 8 is
formed by the SS or PS homologues and 11 is produced from 8
by the TC homologue in the Bacillus cells. In order to find the
producer of both sesquarterpenes (1À4) andtriterpenes(8and 11),
six species shown in Table 1 were cultured in sporulation medium
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Table 1. Genes and Homologues of the Terpene Synthases and the Production of the Terpenes in Bacillus Species Cultured in
Sporulation Mediuma
genes and homologues
TC
production of sesquarterpenesc
production of triterpenes
species
TS
SS or PS
1 and 2
3 and 4
8
11
d
B. subtilis
B. atrophaeus
B. amyloliquefaciens BAMF_2833
BSU30500
BSU19320
BSU10810
+
+
+
+
+
+
+
À
À
À
À
À
+
À
À
À
À
À
+
BATR1942_13035 BATR1942_08330 BATR1942_03015
À
+
BAMF_2003
Bcell_4159
BAMF_1165
B. cellulosilyticus
B. pumilus
Bcell_3320
BPUM_2682
BMD_4842
Bcell_3989
À
+
BPUM_1858
BPUM_1012
BMD_0659,b BMD_3864b
B. megaterium
BMD_2134
+
a Legend: +, detected; À, not detected. b B. megaterium has two SS/PS homologues. c The production of sesquarterpenes in B. subtilis,
B. amyloliquefaciens, and B. pumilus was reported in ref 1. d Although the production of 8 in B. subtilis cultured in sporulation and vegetative medium
could not be detected, its production in biofilm-inducing and the pigment-producing medium was confirmed.
and the lipid fractions analyzed by GC-MS. The triterpenes 8 and
11, which have the same retention times and mass spectrum
profiles as the authentic samples, were successfully found in the
lipids of B. megaterium cells in addition to the sesquarterpenes
(1À4) (Figure 1E, Figure S4 (Supporting Information), and
Table 1). The result is unique to our study, in that 11 has been
found in bacteria. In contrast, the other five species produced
only the sesquarterpenes (1 and 2 or 1À4) (Table 1). Since only
one TC (or SHC) homologue exists in B. megaterium (Table 1),
this TC would be bifunctional, producing 3 and 11 from 1 and 8
in vivo, respectively. It has recently been reported that the SS/PS
homologue from B. subtilis (BSU10810 or YisP) converted FPP
to 8 in vitro and was crucial for the formation of biofilms.8 In
addition, it has been speculated that 8 was converted to an
unidentified pigment.8 Thus, we also analyzed the lipid fractions
of B. subtilis cultured in the biofilm-inducing and the pigment-
producing media. Although a trace amount of 8 (<2% less than
that produced by B. megaterium) could be detected from both
cultures, that of 11 could not be confirmed. This suggests that
the SS was expressed under the biofilm-inducing and pigment-
producing conditions, but the TC was not. The B. subtilis TC is
probably not bifunctional in vivo.
In order to obtain further evidence on the bifunctionality of
B. megaterium TC, the B. megaterium tc gene was introduced
into the pColdTF vector, the recombinant B. megaterium TC was
expressed in E. coli, and the activity cyclizing both 1 and 8,
separately, was confirmed by using purified B. megaterium TC
(Figure S5 and S6 (Supporting Information)). Furthermore, the
products biosynthesized by incubating the B. megaterium TC
with a mixture of the substrates 1 and 8 were analyzed by GC-
MS. Since the amount of 8 was about 4-fold larger than that of 1 +
2 in B. megaterium cells (Figure 1E), concentrations of 1 and 8
were prepared as 40 μM/160 μM for 1/8. The products 3 and 11
were successfully detected in the reaction mixture (Figure S6).
Therefore, it was demonstrated that cyclizations of both tetra-
prenyl-β-curcumene and squalene occurred with a purified
B. megaterium TC homologue in the same reaction mixture. It
has recently been reported that changing the pH of the reaction
dramatically changes the product profile of the sesquiterpene
cyclase.9 In contrast, the product selectivity of the B. megaterium
TC was not influenced by the surrounding pH (Figures S7 and S8
(Supporting Information)). Ferns produce 11 in addition to other
triterpenes. Although several squalene cyclases have been iso-
lated from the fern,10 the enzyme that forms 11 remains unknown.
This is therefore the first report describing the 8α-hydroxypoly-
poda-13,17,21-triene (11) synthase.
When it was cultured in sporulation medium, B. megaterium
produced the TC products 3, 4, and 11 in addition to the TC
substrates 1, 2, and8(Figure 1E), while the vegetative B. megaterium
cells accumulated only the TC substrates 1, 2, and 8. This result is
similar to the productivities of the sesquarterpenes in B. subtilis;
the TC products 3 and 4 were only produced under the sporula-
tion conditions.3 Studies on B. subtilis have demonstrated that the
expression of tc depends on the sporulation-specific RNA poly-
merase subunit σE that controls the expression of a number of
genes during sporulation and that the intracellular localization of
the TC was also sporulation specific.3 Thus, the B. megaterium tc
gene may also be expressed under sporulation conditions. It has
been proposed that sporulenes, the thermally dehydrated com-
pounds from 4, increase the resistance of spores to reactive
oxygen species.3 The compound 11 may therefore have a unique
physiological function that acts only in the B. megaterium spore
and not in other Bacillus species.
The TC family, one of which is the bifunctional triterpene/
sesquarterpene cyclase in B. megaterium, constructs different
cyclic scaffolds—6/6/6/6-fused tetracycle (3) and 6/6-fused
bicycle (11)—from a head-to-tail type of 1 and a tail-to-tail type
of 8, respectively (Scheme 3). The cyclization of 1 into the 6/6/
6/6-fused 3, catalyzed by the TC, proceeds only via tertiary
carbocations, whereas the cyclization of 8 into any compound
containing a 6/6/6/6-fused ring, such as 9 and 10, accompanies
two unstable secondary carbocations (anti-Markovnikov closure)
at C-14 and C-18 (Scheme 3). It has been proposed that an
aromatic π electron in Phe601 from the A. acidocaldarius SHC
functions to stabilize the secondary carbocations through a
cationÀπ interaction at C-14 and C-18 in 8.2,11 The correspond-
ing residue for Phe601 in the SHC is Leu597 in the B. subtilis TC.
Thus, the lack of stabilization at C-14 and C-18 may be one of the
reasons the cyclization of 8 is quenched at the bicyclic inter-
mediate stage. However, this hypothesis is incomplete in that it
does not account for the cyclization mechanism, because F601A
SHC produced no bicyclic compound other than the 6/6/5-
fused tricycles, 6/6/6/5-fused tetracycles, and 6/6/6/6/5-fused
pentacycles.2,11 The methyl groups in 1 and 8 are found in
different positions, at C-14 and C-18 in 1 versus C-15 and C-19 in 8
(Scheme 3). These distinct methyl positions may promote different
binding conformations of 1/8 in the active site cavity in the TC,
resulting in the different numbers of rings in the terpene compounds.
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The substrates 1 and 8 were also incubated separately with the
SHC, which was purified by the methods described in our previous
report (Figure 1).12 The SHC, which cyclized the natural sub-
strates 8into their usual products 9and 10 (Figure 1D and Scheme 2),
biosynthesized no product from 1 (Figure 1B and Scheme 3).
Compound 2, which was detected in the chromatogram displayed
in Figure 1B, was synthesized by the autoxidation of 1 (Scheme1).1,4
The nonreactivity of 1 with the SHC can be interpreted as the
repulsive interaction between the active site cavity in the SHC,
and the methyl groups at C-14 and C-18 in 1 may be preventing
the terminal double bond of 1 from being located to take part in
the initial protonation. This assumption is consistent with our
previous reports describing that the introduction of a methyl
group at C-14 on the squalene backbone interrupted the cycliza-
tion of 8 by the SHC.13 Studies on the bifunctional catalytic
mechanism of the TC using a 3D structure of the TC, site-
directed mutageneses, and analyses of products biosynthesized
from substrate analogues, which can be compared with similar
studies on the SHC, will be attractive projects for the future.
In conclusion, this study has demonstrated that the TC, which
was originally identified as a sesquarterpene cyclase converting
the head-to-tail type of monocyclic 1 into the pentacyclic 3
(Scheme 1), also cyclized the tail-to-tail type of linear triterpene 8
into the bicyclic 11 (Scheme 3). The compound 11 was found to
be a natural triterpene from the B. megaterium cells in addition to
3, and it was demonstrated that cyclizations of both 1 and 8
occurred with a purified B. megaterium TC homologue in the
same reaction mixture. These results suggest that the TC would
be bifunctional, cyclizing both 1 and 8 in vivo. Although two
nearly identical nerolidol/linalool synthases are known to be
bifunctional terpene synthases, these enzymes are not cyclases
and biosynthesize two linear compounds, nerolidol and linalool,
from farnesyl diphosphate (C15) and geranyl diphosphate (C10),
respectively, as natural products in the plant.14 In addition, it has
been reported that the many terpene cyclases have broad
substrate specificities, and other classes of terpenes have been
created in vitro.9,13b,15 However, the bifunctionality of these
terpene cyclases in vivo has not been hypothesized or demon-
strated.9,13b,15 Thus, no bifunctional terpene cyclase, which bio-
synthesizes two classes of cyclic terpenes with different numbers
of carbons as natural products in vivo, has been reported to
date.2,16 Combinational studies determining enzymatic products
biosynthesized from unusual substrates and the analysis of
genome sequences will lead to the further discovery of additional
functions of these enzymes in the organism and will also reveal
additional novel natural terpenoids functioning in their producers.
Furthermore, it has been reported that many kinds of mutated SHCs
create various natural and unnatural triprepenes from 8 in addition to
9 and 10.2 However, compound 11 has never been formed by them.
This study has also revealed that the SHC was not able to utilize 1as a
substrate (Scheme 3). The TC should prove to be a promising tool
for the production of novel terpenes from the various substrates that
are able to react with the SHC and those that cannot. The finding of
novel bifunctional terpene cyclase will help us to expand our
understanding of the terpenoids and to create novel compounds.
’ AUTHOR INFORMATION
Corresponding Author
satot@agr.niigata-u.ac.jp.
’ ACKNOWLEDGMENT
This work was supported by a Grant-in-Aid for Young
Scientists (B) from the Japan Society for Promotion of Science
(JSPS) (21780109 and 23780114 to T.S.) and the Agricultural
Chemical Research Foundation (T.S.). We are grateful to
Dr. Masahiro Fujihashi of Kyoto University for helpful discussions.
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’ ASSOCIATED CONTENT
S
Supporting Information. Text, figures, and tables giving
b
experimental details, sequence data of enzymes, MS spectra, and
NMR spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
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