Enzymatic cyclization of dioxidosqualene to heterocyclic triterpenes

Article abstract of DOI:10.1021/ja055822g

Oxidosqualene cyclases normally produce triterpenes from 2,3-(S)-oxidosqualene (OS) but also can cyclize its minor companion (3S,22S)-2,3:22,23-dioxidosqualene (DOS). We explored DOS cyclization in plant triterpene synthesis using a recombinant lupeol synthase (LUP1) heterologously expressed in yeast. Incubation of LUP1 with 3S,22S-DOS gave epoxydammaranes epimeric at C20 and a 17,24-epoxybaccharane in a 4:2:3 ratio. The products reflected a new mechanistic paradigm for DOS cyclization. The structures were determined by NMR and GC-MS, and recent errors in the epoxydammarane literature were rectified. Some DOS metabolites are likely candidates for regulating triterpenoid biosynthesis, while others may be precursors of saponin aglycones. Our in vivo experiments in yeast generated substantial amounts of DOS metabolites in a single enzymatic step, suggesting a seminal role for the DOS shunt pathway in the evolution of saponin synthesis. Quantum mechanical calculations revealed oxonium ion intermediates, whose reactivity altered the usual mechanistic patterns of triterpene synthesis. Further analysis indicated that the side chain of the epoxydammarenyl cation intermediate is in an extended conformation. The overall results establish new roles for DOS in triterpene synthesis and exemplify how organisms can increase the diversity of secondary metabolites without constructing new enzymes. Copyright

Full text of DOI:10.1021/ja055822g

Published on Web 12/06/2005
Enzymatic Cyclization of Dioxidosqualene to Heterocyclic Triterpenes
Hui Shan, Michael J. R. Segura, William K. Wilson, Silvia Lodeiro, and Seiichi P. T. Matsuda*
Department of Biochemistry and Cell Biology and Department of Chemistry, Rice UniVersity, Houston, Texas 77005
Received August 31, 2005; E-mail: matsuda@rice.edu
Scheme 1. Enzymatic Cyclization of OS and DOS
Ginseng and other medicinal plants accumulate biologically
active epoxydammarane saponins1 at high levels (up to 5% of dry
weight1c). The aglycone precursors, such as 9b, have been proposed
to arise from tetracyclization of (3S)-2,3-oxidosqualene (OS, 1)
followed by P450 oxidations en route to the heterocyclic E ring.1c
We considered that a pathway with oxidation before cyclization
might be shorter and evolutionarily more accessible. Herein we
demonstrate that a plant triterpene synthase (LUP1) converts (3S,-
22S)-2,3:22,23-dioxidosqualene (DOS, 2)2 directly to oxacyclic
triterpenoids 8-9b in a single step (Scheme 1). The results establish
new roles for DOS in triterpene biosynthesis and illuminate substrate
folding during cyclization.
DOS is produced in limited amounts by further epoxidation of
OS and is a known substrate of several OS cyclases.3 These cyclases
all exclude the terminal olefin of OS from cyclization and convert
DOS to triterpene epoxides (cf. 3 and 6) rather than heterocycles.
In contrast, the biosynthesis of lupeol (7) from OS requires that
the terminal olefin of the dammarenyl cation (4) attack the
carbocation to form ring E. We reasoned that in the analogous DOS
pathway the distal epoxide in 5 could attack C20 to generate
heterocyclic triterpenes.4 We tested this hypothesis by incubating
the natural 3S,22S isomer of 2 (8 mg) with Arabidopsis lupeol
synthase (LUP1)5 expressed from plasmid JR1.16 in RXY6,3c
a
Scheme 2. Likely Mechanistic Pathways to 8 and 9a,b
yeast squalene epoxidase/lanosterol synthase double mutant. This
construct allowed cyclization of milligram amounts of substrate
without further metabolism toward saponins.
NMR analysis of the crude extract of RXY6[JR1.16] incubation
products showed almost complete consumption of substrate 2 and
the formation of diols 8, 9a, and 9b in a 3:4:2 ratio, together with
olefinic analogues.6 Silica gel chromatography separated 8 from
epoxydammaranes 9a and 9b, and reversed phase HPLC resolved
9a from 9b.7 Structure elucidation of these products was nontrivial
and required extensive 2D NMR and GC-MS analysis, as described
in Supporting Information. The E ring regiochemistry was deter-
mined by analyzing the GC-MS fragmentation patterns,8a and the
stereochemistry was deduced from NOE results8b and comparisons
1
with reported spectral data.1e,9 Our H and 13C NMR assignments
clarify confusion in the epoxydammarane literature, which lacks
definitive NMR data for the 20R,24S isomer 9a.9c The 20S,24S
isomer 9b is the known1e 3-epicabraleadiol, and 8 has the unusual
17,24-epoxybaccharane skeleton.10 Each product has 24S stereo-
chemistry, derived from the 22S-epoxide in 2.
Products 8, 9a, and 9b are formed via oxonium ion intermediates
10, 11, and 12 (Scheme 2). The exothermicity of oxonium ion for-
mation leads irreversibly from 5 to 11 or 12 if side chain rotation
brings the distal epoxide oxygen into proximity of the C20 cation.11
Migration of the positive charge from carbon to oxygen disables
the usual mechanistic processes12 of triterpene synthesis, which are
guided by activation of certain bonds through hyperconjugation with
the carbocation 2p orbital. DFT calculations predicted an activation
enthalpy of >16 kcal/mol for the conversion of 11 to 10.11 Thus,
8 is formed only when D ring expansion is faster than side chain
folding.
The above mechanistic analysis indicates that the side chain of
5 is mobile and not prefolded for E ring formation.13 A partially
extended side chain conformation, as inferred for squalene-hopene
cyclase,14 allows the side chain to slide through the active site tunnel
as the substrate contracts during tetracyclization. Assuming that the
distal epoxide does not perturb enzyme-substrate interactions, the
9
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J. AM. CHEM. SOC. 2005, 127, 18008-18009
10.1021/ja055822g CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Waller, G. R., Yamasaki, K., Eds.; Plenum Press: New York, 1996; pp
129-149. (d) Tran, Q. L.; Adnyana, I. K.; Tezuka, Y.; Nagaoka, T.; Tran,
Q. K.; Kadota, S. J. Nat. Prod. 2001, 64, 456-461. (e) Fu, L.; Zhang, S.;
Li, N.; Wang, J.; Zhao, M.; Sakai, J.; Hasegawa, T.; Mitsui, T.; Kataoka,
T.; Oka, S.; Kiuchi, M.; Hirose K.; Ando, M. J. Nat. Prod. 2005, 68,
198-206. (f) Betancor, C.; Freire, R.; Hernandez, R.; Suarez, E.; Cortes,
M.; Prange, T.; Pascard, C. J. Chem. Soc., Perkin Trans. 1 1983, 1119-
1126.
side chain of the dammarenyl cation (4) in lupeol synthesis should
also have rotational mobility and a partially extended conformation.
Oxacyclic triterpenes, such as 8-9b, are presumably generated
by all eukaryotes having pentacyclic triterpene synthases15 since
DOS is ubiquitous as a minor byproduct of OS biosynthesis. Low
physiological concentrations of 8 and 9a are suggested by their
rarity in natural product isolations and by the generally limited
metabolic flux through the DOS shunt pathway. Variations in trace
levels of 24,25-epoxycholesterol, a DOS metabolite, indirectly
monitor enzyme activities and are used to regulate transcription in
mammalian cholesterol homeostasis.2a,e,16 Levels of diols 8 and 9a
also reflect epoxidase/cyclase activity and may similarly serve as
regulators of triterpenoid synthesis.
Diol 9b may be produced in nature at much higher levels than
8 or 9a. Epoxydammarane and dammarenediol saponins commonly
occur together and generally have a 20S configuration. A dam-
marenediol synthase could make 3â,20S-dammarenediol from OS
and the 20S,24S-epoxydammarane 9b from DOS. The active site
of dammarenediol synthases, unlike that of LUP1, evidently
obstructs the re face of C20 to exclude E ring formation, and this
would block formation of 8 and 9a. DOS cyclization might also
produce 24R-epoxydammaranes in dammarenediol synthases, as
indicated in the conversion of 5 to 9c.17 However, our isolation of
epoxydammaranes as DOS metabolites does not preclude their
origin from other pathways. For example, cycloartenol analogues
of epoxydammaranes18 must arise from post-cyclization oxidation
because DOS cyclization cannot generate both cyclopropyl and
heterocyclic rings. Similar oxidation by P450s and other oxidases
may have evolved to become the major biosynthetic route to
epoxydammaranes in many plants.1c,19 The best current evidence
for the DOS pathway in secondary metabolism is the isolation of
17,24-epoxybaccharanes20a and olefinic epoxydammaranes,20b struc-
tures that are unlikely products of P450 pathways.
To model how readily oxacycles could arise in vivo, we
expressed JR1.16 in the yeast lanosterol synthase mutant SMY8.
Cultures of SMY8[JR1.16] accumulated 8, 9a, and 9b at a level of
2-7% of the OS products.21 This experimental system evidently
made considerable DOS available to LUP1 for oxacycle formation.22
The crude squalene epoxidase/oxidosqualene cyclase systems that
first evolved probably also generated substantial amounts of
oxacycles. The oxacyclic triterpenoids produced by these unopti-
mized early systems may have provided aglycones for saponin
synthesis until an efficient cluster of P450s evolved. This could
explain how natural selection began assembling saponin synthesis,
a multistep process in which the individual components lack
biological activity. The use of alternative substrates23 exemplifies
one of several strategies used by plants and fungi to increase the
diversity of secondary metabolites.24 The genetic foundation for
artificial and native metabolic engineering may be broader than is
evident from natural products surveys and genomic analyses.
(2) (a) Spencer, T. A. Acc. Chem. Res. 1994, 27, 83-90. (b) Field, R. B.;
Holmlund, C. E. Arch. Biochem. Biophys. 1977, 180, 465-471. (c) Lorenz,
R. T.; Parks, L. W. J. Bacteriol. 1987, 169, 3707-3711. (d) Bai, M.;
Xiao, X.; Prestwich, G. D. Biochem. Biophys. Res. Commun. 1992, 185,
323-329. (e) Rowe, A. H.; Argmann, C. A.; Edwards, J. Y.; Sawyez, C. G.;
Morand, O. H.; Hegele, R. A.; Huff, M. W Circ. Res. 2003, 93, 717-
725.
(3) (a) Corey, E. J.; Gross, S. K. J. Am. Chem. Soc. 1967, 89, 4561-4562.
A lanosterol synthase prefers DOS over OS: (b) Boutaud, O.; Dolis, D.;
Schuber, F. Biochem. Biophys. Res. Commun. 1992, 188, 898-904. A
dammarenyl-type cyclase also readily accepts DOS: (c) Fazio, G. C.;
Xu, R.; Matsuda, S. P. T. J. Am. Chem. Soc. 2004, 126, 5678-5679.
(4) Heterocyclic triterpenes have never been reported from DOS but are
evidently formed in marine organisms by reaction of epoxides with a
cationic intermediate: (a) Valentine, J. C.; McDonald, F. E.; Neiwert, W. A.;
Hardcastle, K. I. J. Am. Chem. Soc. 2005, 127, 4586-4587. (b) Carter-
Franklin, J. N.; Butler, A. J. Am. Chem. Soc. 2005, 127, 15060-15061.
(c) Connolly, J. D.; Hill, R. A. Nat. Prod. Rep. 2003, 20, 640-659.
(5) (a) Herrera, J. B.; Bartel, B.; Wilson, W. K.; Matsuda, S. P. T.
Phytochemistry 1998, 49, 1905-1911. (b) Segura, M. J. R.; Meyer, M.
M.; Matsuda, S. P. T. Org. Lett. 2000, 2, 2257-2259.
(6) Identification of terminal olefins from NMR spectra was tentative owing
to their small amounts and coelution with other substances. The favored
quenching of oxonium ions 10-12 by attack of water rather than by
deprotonation is discussed in Supporting Information.
(7) Silica gel chromatography gave 8 (1.5 mg, Rf 0.31, 1:1 MTBE/hexane)
and a mixture of 9a and 9b (3 mg, Rf 0.37). Reversed phase HPLC was
done with a mobile phase of 9:1 MeOH/H2O.
(8) (a) GC-MS fragmentation of mono- and bis-TMS derivatives indicated
hydroxyl at C25 and an ether linkage at C24. An abundant ion at m/z 383
in 8 pointed to a stable ring skeleton derived from neutral losses of
TMSOH and the hydroxyisopropyl group. Epoxydammaranes 9a and 9b
coeluted on GC and had identical mass spectra. Their base peak at m/z
143 is characteristic of epoxydammaranes,1a and ion m/z 383 excluded
the possibility of a six-membered E ring. (b) NOE experiments indicated
that the C20 methyl and H24 proton are located on the same side of the
E ring plane in 9a and on different sides in 9b.
(9) (a) Hisham, A.; Ajitha Bai, M. D.; Fumimoto, Y.; Hara, N.; Shimada, H.
Magn. Reson. Chem. 1996, 34, 146-150. (b) Tanaka, R.; Masuda, K.;
Matsunaga, S. Phytochemistry 1993, 32, 472-74. (c) Reported NMR data
are critically reviewed with structure revisions in Supporting Information.
(10) The lack of epimerization at C17 is discussed in Supporting Information.
(11) Side chain rotation and oxonium ion formation were modeled by DFT
methods using Gaussian software: Frisch, M. J.; et al. Gaussian 03,
revision C.02; Gaussian, Inc.: Wallingford, CT, 2003. Bonds from oxygen
to C20, C24, and C25 in 11 were 1.59, 1.50, and 1.59 Å (B3LYP/6-31G*
geometry). In the absence of enzymatic effects, activation enthalpies for
side chain rotation or D ring expansion from 5 were ca. 5 versus 16 kcal/
mol for D ring expansion from 11; see Supporting Information.
(12) (a) Wendt, K. U.; Schulz, G. E.; Corey, E. J.; Liu, D. R. Angew. Chem.,
Int. Ed. 2000, 39, 2812-2833. (b) Xu, R.; Fazio, G. C.; Matsuda, S. P.
T. Phytochemistry 2004, 65, 261-291.
(13) Hopene-type folding in LUP1 is unlikely, and lupeol-type folding would
produce only 9a. See Supporting Information for further details.
(14) Reinert, D. J.; Balliano, G.; Schulz, G. E. Chem. Biol. 2004, 11, 121-
126.
(15) Isomers of 8-9b would be produced by eukaryotic cyclases that make
hopane or protostane pentacycles; see Supporting Information.
(16) (a) Wong, J.; Quinn, C. M.; Brown, A. J. Arterioscler. Thromb. Vasc.
Biol. 2004, 24, 2365-2371. (b) Huff, M. W.; Telford, D. E. Trends
Pharmacol. Sci. 2005 26, 335-340.
(17) 24R-Epoxydammaranes can also arise during sample processing by
autoxidation of (20S)-dammar-24-ene-3â,20-diol, as occurs in the drying
of varnishes containing dammar resin triterpenes: van der Doelen, G.
A.; Boon, J. J. J. Photochem. Photobiol. A: Chem. 2000, 134, 45-57.
(18) Matsubara, C.; de Vivar, A. R. Phytochemistry 1985, 24, 613-615.
(19) Epoxydammarane biosynthesis: (a) Harrison, D. M. Nat. Prod. Rep. 1990,
7, 459-484. (b) Jung, J. D.; Park, H.-W.; Hahn, Y.; Hur, C.-G.; In, D.
S.; Chung, H.-J.; Liu, J. R.; Choi, D.-W. Plant Cell Rep. 2003, 22, 224-
230.
Acknowledgment. We thank the Robert A. Welch Foundation
(C-1323), the NSF (MCB-0209769), and the Herman Frasch
Foundation for funding.
Supporting Information Available: Complete refs 11 and 20a;
details of substrate preparation, enzymatic cyclization, molecular
modeling, NMR signal assignments, and GC-MS and NMR spectra
of 8, 9a, and 9b (PDF). This material is available free of charge via
the Internet at http://pubs.acs.org.
(20) (a) Hwang, B. Y.; et al. J. Org. Chem. 2004, 69, 3350-3358. Erratum
6156. (b) Aalbersberg, W.; Singh, Y. Phytochemistry 1991, 30, 921-
926.
(21) (a) Details are given in Supporting Information. Unlike RXY6[JR1.16],
SMY8[JR1.16] contains squalene epoxidase and thus produces OS. (b)
Traces of 8 can be seen in the published NMR spectrum of lupanediol.5b
(22) Possible factors affecting the relative levels of DOS and OS and the
availability of DOS to cyclases are discussed in Supporting Information.
(23) (a) Xiong, Q.; Zhu, X.; Wilson, W. K.; Ganesan, A.; Matsuda, S. P. T. J.
Am. Chem. Soc. 2003, 125, 9002-9003. (b) Hoshino, T.; Kumai, Y.; Kudo,
I.; Nakano S.; Ohashi, S. Org. Biomol. Chem. 2004, 2, 2650-2657.
(24) Firn, R. D.; Jones, C. G. Nat. Prod. Rep. 2003, 20, 382-391.
References
(1) (a) Tanaka, O.; Yahara, S. Phytochemistry 1978, 17, 1353-1358. (b) Zou,
K.; Zhu, S.; Tohda, C.; Cai, S.; Komatsu, K. J. Nat. Prod. 2002, 65, 346-
351. (c) Duc, N. M.; Kasai, R.; Ohtani, K.; Ito, A.; Yamasaki, K.; Nham,
N. T.; Tanaka, O. In Saponins Used in Traditional and Modern Medicine;
JA055822G
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