Enzymatic Formation of a Skipped Methyl-Substituted Octaprenyl Side Chain of Longestin (KS-505a): Involvement of Homo-IPP as a Common Extender Unit

Article abstract of DOI:10.1002/anie.201802116

Longestin (KS-505a), a specific inhibitor of phosphodiesterase, is a meroterpenoid that consists of a unique octacyclic terpene skeleton with branched methyl groups at unusual positions (C1 and C12). Biochemical analysis of Lon23, a methyltransferase involved in the biosynthesis of longestin, demonstrated that it methylates homoisopentenyl diphosphate (homo-IPP) to afford (3Z)-3-methyl IPP. This compound, along with IPP, is selectively accepted as extender units by Lon22, a geranylgeranyl diphosphate (GGPP) synthase homologue, to yield dimethylated GGPP (dmGGPP). The absolute configuration of dmGGPP was determined to be (4R,12R) by degradation and chiral GC analysis. These findings allowed us to propose an enzymatic sequence for key steps of the biosynthetic pathway of the unusual homoterpenoid longestin.

Full text of DOI:10.1002/anie.201802116

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Accepted Article
Title: Enzymatic formation of a skipped methyl-substituted octaprenyl
side chain of longestin (KS-505a): Involvement of homo-IPP as a
common extender unit
Authors: Taro Ozaki, Sandip S. Shinde, Lei Gao, Ryo Okuizumi,
Chengwei Liu, Yasushi Ogasawara, Xiaoguang Lei, Tohru
Dairi, Atsushi Minami, and Hideaki Oikawa
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201802116
Angew. Chem. 10.1002/ange.201802116
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10.1002/anie.201802116
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Enzymatic formation of a skipped methyl-substituted octaprenyl
side chain of longestin (KS-505a): Involvement of homo-IPP as a
common extender unit
Taro Ozaki[a]†, Sandip S. Shinde[a],[b]†, Lei Gao[a],[d], Ryo Okuizumi[a], Chengwei Liu[a], Yasushi
Ogasawara[c], Xiaoguang Lei[d], Tohru Dairi[c], Atsushi Minami* [a], and Hideaki Oikawa*[a]
Abstract: Longestin (KS-505a),
a
specific inhibitor of
octacyclic terpene skeleton, and branched methyl groups at
unusual positions (C1 and C12).[5b] Although the origin of the
branched methyl groups on the octaprenyl chain was elucidated
via [S-13CH3]-L-methionine feeding experiments, the timing of
methylation at C1 and C12 remains to be established.[6] In 2007,
the longestin biosynthetic gene cluster, consisting of 24 genes,
was identified.[7] Among three methyltransferase genes in this
cluster, lon23 was homologous (38% identical) to geranyl
diphosphate (GPP) methyltransferase,[8] which catalyzes unique
C-methylation at a vinyl position adjacent to a pyrophosphate.
This homology implies that a methyl group is introduced to IPP to
afford homo-IPP at the initial stage of longestin biosynthesis.
According to this hypothesis, Lon22, which synthesizes
geranylgeranyl diphosphate (GGPP) from dimethylallyl
diphosphate (DMAPP) and IPP, may use DMAPP, IPP, and
homo-IPP as substrates to afford methyl branched GGPP
(Scheme 1). Herein, we perform a functional analysis of
methyltransferase Lon23 and prenyltransferase Lon22, which are
involved in the biosynthesis of homoterpenoid 1.
To examine the above biosynthetic hypothesis, Lon23 was
overexpressed as a fused form of maltose binding protein in
Escherichia coli BL21(DE3), and purified using affinity
chromatography (Figure S1). Reactions of IPP in the presence of
Mg2+ and S-adenosyl-L-methionine (SAM) with recombinant
methyltransferase Lon23 were conducted, and the reaction
mixture was directly submitted to hydrolysis with acid
phosphatase (Scheme 2A). GC-MS analysis of the resultant
alcohol enabled us to detect 3Z-3-methylpenten-1-ol (2b), whose
tR and fragmentation pattern were identical to those of a synthetic
standard (Figure 1A and Figure S2). This result unequivocally
indicated that Lon23 synthesized 3Z-3-methyl IPP (Z-homo-IPP,
2a) from IPP and SAM. Recently, we found GPP
methyltransferase (MT) participated in the biosynthesis of 2-
methylisoborneol, and we obtained the MT crystal structure
complexed with GPP, sinefungin (a SAM mimic), and Mg2+.[9]
Both GPP-MT and Lon23 have a Mg2+ binding motif, which plays
a key role in recognizing prenyl diphosphate substrates. As in the
case of GPP-MT, addition of Mg2+ is essential for the Lon23
reaction.
phosphodiesterase, is a meroterpenoid that consists of a unique
octacyclic terpene skeleton with branched methyl groups at unusual
positions (C1 and C12). The biochemical analysis of Lon23, a
methyltransferase involved in the biosynthesis of longestin,
demonstrated that methylation of IPP afforded 3Z-3-methyl IPP. This
compound as well as IPP was selectively accepted as extender units
by Lon22, a geranylgeranyl diphosphate (GGPP) synthase homolog,
to yield dimethylated GGPP (dmGGPP). Absolute configuration of
dmGGPP was determined to be (4R, 12R) by degradation and chiral
GC analysis. These findings led us to propose key steps of the
biosynthetic pathway of the unusual homoterpenoid longestin.
Isoprenoids usually consist of multiple numbers of isoprene units
(C5). However, exceptional cases with extra carbon atoms are
occasionally found in nature. These unusual isoprenoids include
steroids, whose side chains
are
homologated by
methyltransferase[1], and floral homoterpenes[2], which are
degraded by cytochrome P450 catalyzing the oxidative scission
of nerolidol and geranyllinalool. The homoterpenes faranal, a trail
pheromone of pharaoh ant, and juvenile hormones, have unusual
ethyl substituents and a branched methyl group on their isoprene
skeletons.[3] These compounds are proposed to be derived from
homomevalonate via homoisopentenyl diphosphate (homo-
IPP).[3,4] However, the enzymes responsible for forming homo-IPP
and converting it to a polyprenyl chain have yet to be reported.
Longestin (KS-505a, 1) from Streptomyces argenteolus is a
specific inhibitor of phosphodiesterase which could be a potential
drug lead, and also showed antitrypanosomal activity.[5] This
unusual meroterpenoid has unique structural features, such as an
[a]
Dr. T. Ozaki, R. Okuizumi, Dr. C. Liu, Dr. A. Minami, Prof. Dr. H.
Oikawa
Division of Chemistry, Graduate School of Science, Hokkaido
University, Sapporo, Hokkaido 060-0810 (Japan)
E-mail: aminami@sci.hokudai.ac.jp
hoik@sci.hokudai.ac.jp
[b]
[c]
[d]
Dr. S. S. Shinde
Organic Chemistry Division, CSIR-National Chemical Laboratory,
Dr. Homi Bhabha Road, Pashan, Pune-411008, India
Dr. Y. Ogasawara, Prof. Dr. T. Dairi
Graduate School of Engineering, Hokkaido University
Sapporo, Hokkaido 060-8628 (Japan)
Subsequently, a N-terminal hexahistidine-tagged Lon22 was
overexpressed in Escherichia coli M15 [pREP4], and purified as
previously reported (Figure S1) [7]. We studied the enzymatic
reaction of the recombinant Lon22, which was previously found
to catalyze the conversion of DMAPP and IPP to GGPP.[7] To
examine the catalytic activity of Lon22, we conducted an
enzymatic reaction with Lon22 using starter unit DMAPP, two
extender units IPP and Z-homo-IPP 2a (Scheme 2B). After
L. Gao, Prof. Dr. X. Lei
College of Chemistry and Molecular Engineering, Peking University,
Haidian District, Beijing 100871, China
†These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201802116
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Scheme 1. Proposed biosynthetic pathway of longestin (1). Enzymatic construction of dimethyloctaprenyl diphosphate (dmOPP) proposed in this study is enclosed
in solid squares.
enzymatic hydrolysis of the product, GC-MS analysis of the
reaction mixture revealed a new peak that had a molecular ion at
m/z 318 corresponding to that of dimethylated geranylgeraniol 4b
(Figure 1B, Figure S3A). In the reactions with GPP and farnesyl
diphosphate (FPP) as starter units, we observed the same
product with a molecular ion at m/z 304 that corresponded to the
monomethylated product 5 (Figure 1B, Figure S3BC). In the
absence of 2a, geranylgeraniol 6 was produced as reported
(Figure 1B, Figure S3D).[7] These observations indicated that
Lon22 selectively incorporated two or one Z-homo-IPP in the
above reactions, although equal concentrations of IPP and Z-
homo-IPP were added to the reaction mixture. Considering the
chain length of the starter units in each reaction, the C6 units
derived from 2a were located at the second and fourth positions
in 4a. To determine the structure of reaction product 4b as 4,12-
dimethylgeranylgeraniol, we used COSY, HSQC, HMBC, and
NOESY NMR analyses (Scheme 1, Figure S4, Table S1). The
absolute configuration of 4b was determined as follows.
Scheme 2. (A) Methylation of IPP catalyzed by Lon23, and enzymatic
hydrolysis of the product. (B) Selective prenylation catalyzed by Lon22 with
various starter units, and enzymatic hydrolysis of the products. (C) Ozonolysis
of 4b.
Ozonolysis (i, O3, -78°C; ii. H2O2, CH3CO2H) of 4b and the
subsequent methylation with CH2N2 gave a crude reaction
product, which was directly analyzed by GC-MS on a chiral
stationary phase (Scheme 2C, Figure 2). The GC-MS profile of
the degradation product of 4b presented one major peak at 27.1
min, suggesting that the two methyl groups had the same
configuration. Comparing this profile with those of synthetic
standards (RS)-7[10] and (R)-7 which was synthesized from methyl
In 1977, Ogura and co-workers reported that pig liver FPP
synthase could accept both Z-homo-IPP 2a and E-isomer 3a, and
convert them into dimethyl FPP with branched methyl groups in
(R)-3-methylsuccinate in two steps (Supporting information), we
found that the retention time of the degradation product was
identical to that of R-enantiomer 7, which indicated that 4b has
the (4R, 12R)-configuration.
the R- and S-configurations, respectively.[11] The absolute
configuration of dimethyl GGPP 4a was identical to that of
dimethyl FPP synthesized from 2a, suggesting that Lon22 has an
active site that can accommodate IPP/homo-IPP in similar
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10.1002/anie.201802116
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geometries to those in the pig liver FPP synthase. An intriguing
feature of Lon22 catalysis is that Lon22 can use two different
extender units selectively in the same active site. Similar selective
prenylations would be involved in the biosynthesis of other
homoterpenes such as insect pheromones, insect hormones, and
(bis)homoscalaranes isolated from the marine sponges (Figure
S5).[3,12] However, their biosynthesis has only been examined by
the feeding of radiolabeled compounds in the case of juvenile
hormones.[4] This study is the first to report and analyze in vitro
preparations of homo-IPP and homooligoprenyl diphosphate
using purified enzymes, providing a basis for the future
examination of selective prenylations.
Establishing these transformations in longestin biosynthesis
enables us to speculate the second stage (three steps catalyzed
by Lon13, Lon14, Lon15) to involve octaprenylation, epoxidation,
and an intriguing cyclization of the putative substrate 8 forming an
octacyclic system in a single step (Scheme 1). To examine the
reaction mechanism of this unusual cyclization with Lon15, we are
currently working on cloning and functional analysis of the
remaining genes.
Acknowledgements
This work was financially supported in part by Grants-in-Aid for
Scientific Research from the Ministry of Education, Culture,
Sports, Science and Technology, Japan (JSPS KAKENHI Grants
(15H01835 (H.O.), 16H06446 (A.M.), 17H05425 (T.O.)). The
authors thank Mr. Koichi Kawakami, Mr. Satoshi Moriya, and
Takasago International Co. for kindly analyzing chiral GC, and
Prof. Dr. K. Monde and Dr. T. Taniguchi for preliminary chiral
separation of the synthetic compounds. A JSPS PD-fellowship
(JP10F00032) for S.S.S. is gratefully acknowledged. Financial
support from NNSFC (21661140001, 21561142002, and
21625201 to X.L.), and the JSPS A3 Foresight Program are
gratefully acknowledged.
Keywords: homoterpenoid •longestin • biosynthesis •
prenyltransferase • methyltransferase
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W. Liu; Vol. Ed.: C. A. Townsend, Y. Ebizuka), Springer, Tokyo, 2010, pp.
755-787.
Figure 1. (A) GC-MS profile of the Lon23-catalyzed reaction products compared
with authentic samples. Chromatograms were extracted at m/z 100. (B) GC-MS
profile of the Lon22-catalyzed reaction products with various starter units.
Chromatograms were extracted at m/z 69.
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In the previous study, Lon12 was found to catalyze the
synthesis of octaprenyl diphosphate (OPP) from GGPP, which
was synthesized by Lon22.[7] In addition to these observations,
our results presented in this paper established the first stage
(three steps catalyzed by Lon23, Lon22, Lon12), which involved
intriguing enzymatic reactions for the highly unusual methylation
and selective introduction of the extender units IPP and homo-IPP.
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10.1002/anie.201802116
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Entry for the Table of Contents (Please choose one layout)
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Enzymatic formation of dimethyl
geranylgeranyl diphosphate
(dmGGPP), a key intermediate in the
biosynthesis of the unusual
T. Ozaki, S. S. Shinde, L. Gao, R.
Okuizumi, C. Liu, Y. Ogasawara, X. Lei,
T. Dairi, A. Minami*, H. Oikawa*
Page No. – Page No.
homoterpenoid longestin (KS-505a),
was reported. Lon23 catalyzes the
methylation of IPP to yield 3Z-3-
methyl IPP (Z-homo-IPP), which is
used as an extender unit in Lon22-
catalyzed prenylation. These findings
set the stage for the elucidation of the
later steps of the biosynthesis,
including an unusual cyclization to
construct the octacyclic terpene
skeleton.
Enzymatic formation of skipped
methyl-substituted octaprenyl side
chain of longestin (KS-505a):
Involvement of homo-IPP as a
common extender unit.
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