Cloning and characterization of oxidosqualene cyclases from Kalanchoe daigremontiana: Enzymes catalyzing up to 10 rearrangement steps yielding friedelin and other triterpenoids

Article abstract of DOI:10.1074/jbc.M109.098871

The first committed step in triterpenoid biosynthesis is the cyclization of oxidosqualene to polycyclic alcohols or ketones C₃₀H₅₀O. It is catalyzed by single oxidosqualene cyclase (OSC) enzymes that can carry out varying numbers of carbocation rearrangements and, thus, generate triterpenoids with diverse carbon skeletons. OSCs from diverse plant species have been cloned and characterized, the large majority of them catalyzing relatively few rearrangement steps. It was recently predicted that special OSCs must exist that can form friedelin, the pentacyclic triterpenoid whose formation involves the maximum possible number of rearrangement steps. The goal of the present study, therefore, was to clone a friedelin synthase from Kalanchoe daigremontiana, a plant species known to accumulate this triterpenoid in its leaf surface waxes. Five OSC cDNAs were isolated, encoding proteins with 761-779 amino acids and sharing between 57.4 and 94.3% nucleotide sequence identity. Heterologous expression in yeast and GC-MS analyses showed that one of the OSCs generated the steroid cycloartenol together with minor side products, whereas the other four enzymes produced mixtures of pentacyclic triterpenoids dominated by lupeol (93%), taraxerol (60%), glutinol (66%), and friedelin (71%), respectively. The cycloartenol synthase was found expressed in all leaf tissues, whereas the lupeol, taraxerol, glutinol, and friedelin synthases were expressed only in the epidermis layers lining the upper and lower surfaces of the leaf blade. It is concluded that the function of these enzymes is to form respective triterpenoid aglycones destined to coat the leaf exterior, probably as defense compounds against pathogens or herbivores.

Full text of DOI:10.1074/jbc.M109.098871

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 39, pp. 29703–29712, September 24, 2010
2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
©
Cloning and Characterization of Oxidosqualene Cyclases
from Kalanchoe daigremontiana
ENZYMES CATALYZING UP TO 10 REARRANGEMENT STEPS YIELDING FRIEDELIN AND
□
S
*
OTHER TRITERPENOIDS
Received for publication, December 23, 2009, and in revised form, July 6, 2010 Published, JBC Papers in Press, July 7, 2010, DOI 10.1074/jbc.M109.098871
1
2
3
Zhonghua Wang, Trevor Yeats , Hong Han , and Reinhard Jetter
From the Departments of Botany and Chemistry, University of British Columbia, Vancouver,British Columbia V6T 1Z4, Canada
The first committed step in triterpenoid biosynthesis is the derivatives formed from C parent structures by side chain
3
0
cyclization of oxidosqualene to polycyclic alcohols or ketones addition/elimination (1). They are found in virtually all organ-
C H O. It is catalyzed by single oxidosqualene cyclase (OSC) isms, serving various important functions as membrane con-
30
50
enzymes that can carry out varying numbers of carbocation stituents (steroids in eukaryotes, hopanoids in prokaryotes), as
rearrangements and, thus, generate triterpenoids with diverse hormones (steroids in plants and animals), and as defense com-
carbon skeletons. OSCs from diverse plant species have been pounds (pentacyclic triterpenoids in plants). The greatest
cloned and characterized, the large majority of them catalyzing structural diversity of triterpenoids is found in higher plants,
relatively few rearrangement steps. It was recently predicted where hundreds of different backbone structures have been
that special OSCs must exist that can form friedelin, the pen- reported, with even higher diversity arising from oxidative
tacyclic triterpenoid whose formation involves the maximum modification to form various aglycones and their glycosylation
possible number of rearrangement steps. The goal of the to saponins (2).
present study, therefore, was to clone a friedelin synthase
Triterpenoid biosynthesis in eukaryotes occurs by cycliza-
from Kalanchoe daigremontiana, a plant species known to tion of epoxysqualene, leading in a single enzyme-catalyzed
accumulate this triterpenoid in its leaf surface waxes. Five reaction to an initial product C H O that can contain 1–5
3
0
50
OSC cDNAs were isolated, encoding proteins with 761–779 rings (3). Although many organisms have single product-spe-
4
amino acids and sharing between 57.4 and 94.3% nucleotide cific oxidosqualene cyclase (OSC) enzymes, plants can contain
sequence identity. Heterologous expression in yeast and arrays of cyclases competing for the same substrate to form
GC-MS analyses showed that one of the OSCs generated the diverse steroids and pentacyclic triterpenoids (2, 4, 5). This
steroid cycloartenol together with minor side products, cyclization is, thus, a branch point between primary metabo-
whereas the other four enzymes produced mixtures of penta- lism and secondary metabolism. Triterpenoid cyclization reac-
cyclic triterpenoids dominated by lupeol (93%), taraxerol tions comprise a sequence of four steps, including an initial
(
60%), glutinol (66%), and friedelin (71%), respectively. The protonation, a polyene addition cascade plus ring widening to
cycloartenol synthase was found expressed in all leaf tissues, form cycles A–E, 1,2-shifts of hydride and/or methyl groups,
whereas the lupeol, taraxerol, glutinol, and friedelin syn- and a final deprotonation (Scheme 1) (3). Substantial varia-
thases were expressed only in the epidermis layers lining the tion in carbon backbone structures is due to differences in
upper and lower surfaces of the leaf blade. It is concluded that the numbers of rearrangement steps in the third stage of the
the function of these enzymes is to form respective triterpe- reaction.
noid aglycones destined to coat the leaf exterior, probably as
defense compounds against pathogens or herbivores.
The sequence of rearrangements can be predicted based
on the occurrence of tertiary carbocations (Scheme 1) and
has, for the early steps along the mechanistic pathway, been
confirmed by experimental evidence using inhibitor studies
Triterpenoids are a very diverse group of natural products and enzyme domain swapping/site-directed mutagenesis
with complex alicyclic structures containing C30 backbones or (6–15). The sequence of rearrangements shifts the position of
the carbocation from the E ring, where it resides at the end of
the polyene reaction, toward the A ring. The cyclization prod-
*
This work was supported by a Natural Sciences and Engineering Research
Council of Canada Special Research Opportunity Grant, by the Canada
Foundation for Innovation, and by the Canada Research Chairs Program.
S
ucts may, accordingly, be classified depending on the CϭC
double bond position and the number of rearrangement steps
and carbocationic intermediates occurring en route. Thus, ger-
manicane derivatives can be considered as the structures with
five six-membered rings with the minimum number of rear-
rangements, whereas ␤-amyrin formation involves two more
intermediates, isomultiflorenol involves another two, and glu-
tinol involves three more. Friedelin is of special interest because
□
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Tables 1 and 2.
Thenucleotidesequence(s)reportedinthispaperhasbeensubmittedtotheGen-
TM
Bank /EBI Data Bank with accession number(s) HM623868, HM623869,
HM623870, HM623871, and HM623872.
Present address: Dept. of Plant Biology, Cornell University, Ithaca, NY 14853.
Present address: Dept. of Molecular Genetics, University of Toronto,
1
2
Toronto, Ontario M5S 1A8, Canada.
To whom correspondence should be addressed: Dept. of Botany, University
3
of British Columbia, 6270 University Blvd., Vancouver, British Columbia V6T
4
1
Z4, Canada. Fax: 604-822-6089; E-mail: jetter@interchange.ubc.ca.
The abbreviation used is: OSC, oxidosqualene cyclase.
SEPTEMBER 24, 2010•VOLUME 285•NUMBER 39
JOURNAL OF BIOLOGICAL CHEMISTRY 29703

Oxidosqualene Cyclases of K. daigremontiana
it is the triterpenoid product with
the maximum number of rearrange-
ments possible. Once the entire
series of rearrangements is com-
pleted and the positive charge has
migrated back to the A ring, then
deprotonation from the hydroxyl
group rather than from a methylene
unit becomes possible. Due to
abstraction of the same proton that
was added in the initial step of the
reaction, friedelin is the only triter-
penoid ketone formed directly by
cyclization, in contrast to all of the
other C H O isomers that are
30
50
synthesized as unsaturated alcohols.
It seems likely that the same amino
acid that is donating the proton ini-
tially will accept it back after the
ring formation and rearrangement
steps have occurred. The residue
responsible for substrate protona-
tion is known to be an aspartate in
the DCTAE motif well conserved
among all OSC enzymes (16).
OSCs from many plant species
have been cloned and characterized
by heterologous expression in yeast.
Most of these enzymes were found
to be product-specific lupeol or
␤
-amyrin synthases, with only a few
enzymes also capable of synthesiz-
ing triterpenoid products further
along the rearrangement sequence
(
2). The currently known OSC cata-
lyzing the highest number of rear-
rangement steps is a product-spe-
cific enzyme from Luffa cylindrica
thatformsisomultiflorenol(17).How-
ever, corresponding OSCs synthesiz-
ing friedelin either as the sole product
or in a mixture with other triterpe-
noid products have not been de-
scribed to date, leaving the possi-
bility that this triterpenoid might
be generated by modification of
preformed pentacyclic triterpe-
nols through rearrangements (i.e.
in a two-enzyme process) rather
than by direct cyclization of oxidos-
qualene catalyzed by a single enzyme.
Corey and co-workers (18) have
recently calculated free energies of
various carbocations involved in the
rearrangement series and found
that the later rearrangement reac-
tions are slightly endergonic. There-
fore, it was concluded that the late
2
9704 JOURNAL OF BIOLOGICAL CHEMISTRY
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Oxidosqualene Cyclases of K. daigremontiana
rearrangement reactions must be coupled with earlier ones in a of the core fragments of OSCs from K. daigremontiana. PCR
single enzymatic reaction in order to occur. Based on this find- was carried out with recombinant TaqDNA polymerase (Fer-
ing, friedelin synthases are likely to be true OSC enzymes and mentas) and the following cycling conditions: 2 min at 94 °C (45 s
should be cloned and characterized as such.
at 94 °C, 60 s at 50 °C, and 90 s at 72 °C) ϫ 30 cycles, with a final
The goal of the current investigation was to identify and extension of 10 min at 72 °C. The resulting PCR products were
characterize an enzyme forming friedelin. To that end, we had separated by gel electrophoresis (1% agarose) and extracted
recently screened a number of Crassulaceae species for the using the QIAquick gel extraction kit (Qiagen). A DNA product
accumulation of friedelin because multiple occurrences of this
compound had previously been reported for this plant family.
Special focus was on plant surface waxes, because they are
known to be principal lipophilic pools containing triterpenoid
aglycones. Kalanchoe daigremontiana leaf waxes were found to
be particularly rich in friedelin (19) and were chosen for further
studies because this plant species is easy to cultivate and to use
for molecular genetic experiments. K. daigremontiana is of fur-
ther interest because its leaf surface waxes contain three other
triterpenoids besides friedelin, raising the question whether
these different products are formed by one promiscuous or sev-
eral specific OSC enzymes. Triterpenoids accumulate mainly
during the first 30 days of leaf development, indicating that
activities of relevant triterpenoid synthases and, thus, expres-
sion of corresponding genes peak early during leaf development
of the desired length was cloned into the pGEM-T vector (Pro-
mega) and transformed into Top10 competent cells (Invitro-
gen). Plasmid DNA was purified from transformed cells using
the QIAprep spin miniprep kit (Qiagen) and sequenced. All
further PCR products mentioned below were subcloned and
sequenced by the same procedure.
The core fragment was extended in a second PCR (condi-
tions as above) using the primer BWN1S (5Ј-CARWAYR-
DDGAYGGNGGNTGGG-3Ј) and gene-specific primers
synthesized according to the obtained core sequence (supple-
mental Table 1). For 3Ј-end amplification of the open reading
frame (ORF), first strand synthesis was carried out for 1 h at
4
2 °C using 5 ␮g of total RNA, AP-primer (5Ј-GGCCA-
CGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3Ј), and
reverse transcriptase in 20 ␮l. The product served as template
in a PCR (conditions as above except annealing temperature
(
20). Based on these findings, the current study aimed at the
isolation of mRNA coding for enzymes involved in the forma-
tion of triterpenoids in K. daigremontiana, followed by cloning
of corresponding cDNAs into yeast and finally the biochemical
characterization of the enzymes overexpressed there.
5
5 °C) with the adapter primer AUAP (5Ј-GGCCACGCGTC-
GACTAGTAC-3Ј) and a specific primer (supplemental
Table 1). To obtain the 5Ј-end of the cDNA, the 5Ј-rapid ampli-
fication of cDNA ends system of Invitrogen was employed with
the two nested gene-specific antisense primers (supple-
mental Table 1).
EXPERIMENTAL PROCEDURES
Plant Growth and Triterpenoid Analysis—K. daigremonti-
ana plants were grown in standard soil under temperate green-
house conditions. Leaves were harvested, surface-extracted
The corresponding full-length cDNAs were amplified using
K. daigremontiana cDNA as a template and specific N- and
C-terminal primers (supplemental Table 1). The PCRs were
conducted with Phusion high fidelity DNA polymerase (New
England Biolabs) in a final volume of 50 ␮l (1 ␮M each primer
and 1 ␮l of cDNA) under the following conditions: 30 s at 98 °C
with CHCl to remove cuticular waxes, and then ground to
3
powder in liquid nitrogen. The powder was extracted with
CHCl , and the lipid mixture was fractionated by TLC (20 ϫ 20
3
cm, silica gel, 0.5 mm; Merck) using CHCl as the mobile phase.
3
(
10 s at 98 °C, 30 s at 55 °C, and 75 s at 72 °C) ϫ 28 cycles, 5 min
Resulting bands were stained with primuline, viewed under UV
at 72 °C. The resulting 2.3-kb PCR products were purified by gel
electrophoresis. Sequences were analyzed by NCBI BLAST
light, scratched from the plates, extracted with CHCl , and ana-
3
lyzed by GC-MS and GC-flame ionization detection for identi-
fication and quantification, respectively, of the triterpenoids
contained in the internal leaf tissues. GC conditions were used
as described below for yeast extract analyses.
(
available from the National Institutes of Health Web site) and
DNAstar software (available on the World Wide Web). Se-
quence information for the putative triterpenoid synthases has
TM
been deposited in GenBank
(accession numbers KdTAS
OSC Cloning—K. daigremontiana leaves were harvested,
immediately placed in liquid nitrogen, and ground thoroughly
with a mortar and pestle. Total RNA was extracted from the
powder using the TRIzol reagent (Invitrogen) and used for
cDNA synthesis by SuperScript II reverse transcriptase (In-
HM623868, KdGLS HM623869, KdFRS HM623870, KdLUS
HM623871, KdCAS HM623872).
Functional Expression of OSC cDNAs in Yeast—The full-
length cDNAs of the putative triterpenoid synthases were
vitrogen) following standard protocols. The resulting cDNA digested with appropriate restriction enzymes and ligated into
mixture served directly as template for the following PCRs. the yeast expression vector pYES2 (Invitrogen) behind the
Degenerate oligonucleotide primers OGL3S (5Ј-TTYGGNW- GAL1 promoter. The resulting constructs were transformed
GYCARNHRTGGGAT-3Ј), OGC1S (5Ј-TAYAATGGMAGY- into the mutant yeast strain GIL77 by the LiAc/SS-DNA/PEG
CARYTVTGGGA-3Ј), and OGN3A (5Ј-GGTGGDTGGRG- method (22), and yeast expression experiments were carried
DGARAGYYA-3Ј), previously designed to target conserved out as described previously (21). In brief, cells were induced
regions in plant OSCs (21), were used here for the amplification with galactose, incubated in 0.1 M potassium phosphate-con-
SCHEME 1. Mechanism for the first committed step during biosynthesis of pentacyclic triterpenoids. The reaction starts with the protonation of oxidos-
qualene (top) and then involves a series of carbocationic intermediates that first undergo cyclization and then various rearrangements (right) and finally ends
by deprotonation into various natural products (left).
SEPTEMBER 24, 2010•VOLUME 285•NUMBER 39
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Oxidosqualene Cyclases of K. daigremontiana
taining glucose and hemin for 24 h, collected, refluxed for 5 min out with the ClustalX program Version 1.83 (23), using the
in 20% KOH, 50% EtOH, and extracted twice with 5 ml of hex- amino acid sequences of cloned and characterized plant OSCs.
ane. Both hexane solutions were combined, the solvent was A phylogenetic tree was created based on 1,000 bootstrap rep-
TM
removed under a gentle stream of N , and the residue was redis- lications with the MEGA3.1 program. The GenBank
data
2
solved in 0.2 ml of CHCl for chemical analyses.
base accession numbers of the sequences used in the analysis
Chemical Analysis of Yeast Expression Products—Yeast lipid are shown in supplemental Table 2.
extracts were either analyzed directly by GC-MS or first puri-
3
RT-PCR Analysis—The epidermal cell layers were peeled
fied by TLC and then analyzed by GC-MS and NMR. For direct from the upper and lower surfaces of K. daigremontiana leaves,
GC-MS analyses, extracts were derivatized using bis-N,O-tri- and the remaining inner tissue as well as the epidermis prepa-
methylsilyltrifluoroacetamide (in pyridine at 70 °C for 60 min) or rations were immediately frozen in liquid nitrogen, ground into
Ac O (in pyridine at 70 °C for 4 h), dried under N , and taken up powders using a mortar and pestle, and used to extract RNA
2
2
in CHCl . The qualitative composition was studied using a with TRIzol reagent (Invitrogen). The RNA samples were used
3
6
890N Network GC system (Agilent) equipped with a mass for cDNA synthesis by SuperScript II reverse transcriptase
spectrometric detector (Agilent 5973N) and an HP-1 capillary (Invitrogen) following standard protocols. Gene-specific prim-
column (Agilent, length 30 m, inner diameter 320 ␮m, 1-␮m ers were designed to amplify fragments of the five different
film thickness). 1 ␮l of each sample was injected on column into OSCs (listed in supplemental Table 1). Additionally, a 287-bp
a flow of helium gas held constant at 1.4 ml/min. The oven fragment of the actin gene was amplified as a positive control
temperature was programmed for 2 min at 50 °C, followed by a using the primers 5Ј-CGTTCTCTCCTTGTATGCCAGTG-
4
3
0 °C/min ramp to 200 °C, held at 200 °C for 2 min, increased by GTC-3Ј and 5Ј-GAGCTGCTCTTGGCAGTCTCAAGTTC-
°C/min to 320 °C, and held at 320 °C for 30 min. Triterpenoids 3Ј. PCR cycle numbers and template amounts were optimized
were identified by comparison with literature data and with to yield products in the linear range of the reaction. PCR con-
authentic compounds. Standards of friedelin, ␤-amyrin, and ditions for comparison of transcription levels for the five OCSs
lupeol were purchased from Roth. A standard of glutinol had were as follows: 25 ng of template cDNA denatured at 94 °C for
previously been isolated from K. daigremontiana wax, purified 2 min, followed by 23 cycles of 94 °C for 30 s, 55 °C for 45 s, and
by TLC as well as HPLC, and authenticated using GC-MS 72 °C for 60 s. Reactions were maintained at 72 °C for 10 min
and NMR (20). Finally, taraxerol was generated semisyntheti- before separation of PCR products by electrophoresis in a 1%
cally by lithium aluminum hydride reduction of taraxerone iso- agarose gel.
lated as previously described from the surface wax mixture on Quantitative RT-PCR Analysis—RNA samples from leaf epi-
Macaranga hypoleuca leaves and was purified by TLC and dermal cell layers were used for cDNA synthesis by SuperScript
characterized by GC-MS as acetate and TMS derivative.
II Reverse Transcriptase (Invitrogen) following standard proto-
Further structure confirmation was obtained from NMR cols. Gene-specific primers were designed to amplify fragments
analyses. To this end, compounds were purified from yeast of the four different OSCs, and the specificity of these primers
extracts using TLC as described above for plant lipid separa- was confirmed by a series of PCRs with plasmid DNA as a tem-
13
tion. C NMR spectra of the resulting fractions were recorded plate. Quantitative PCR was carried out using SYBR GreenER
at 400 MHz in CDCl solution. Chemical shifts, expressed as qPCR SuperMix Universal kit (Invitrogen). The quantitative
3
1
3
values relative to the solvent signals ␦ 77.0 (t) for C were PCRs were programmed at 95 °C for 9 min, followed by 40
recorded for two triterpenoids isolated from transgenic yeast: cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s on an MJ
compound 3 ␦ 16.41 (C-25), 18.43 (C-1), 18.63 (C-26), 19.83 MiniOpticon real-time PCR system (Bio-Rad).
(
C-27), 23.86 (C-7), 25.66 (C-23), 28.04 (C-2), 28.46 (C-20),
RESULTS AND DISCUSSION
2
9.17 (C-24), 30.31 (C-17), 30.57 (C-12), 32.25 (C-28), 32.30
(
C-21), 32.61 (C-30), 33.34 (C-11), 34.74 (C-29), 34.83 (C-15),
The goal of the current investigation was to identify and
3
5.06 (C-9), 35.30 (C-16), 36.24 (C-19), 38.06 (C-14), 39.17 characterize enzymes forming pentacyclic triterpenoids
(
C-22), 39.52 (C-13), 41.03 (C-4), 43.29 (C-18), 47.65 (C-8), through skeletal rearrangement series much longer than
4
9.91 (C-10), 76.54 (C-3), 122.26 (C-6), 141.84 (C-5); com- those occurring in previously known OSCs. In particular, the
pound 4 ␦ 7.04 (C-23), 14.88 (C-24),18.16 (C-25), 18.46 (C-7), synthesis of friedelin had been predicted to involve the max-
8.88 (C-27), 20.48 (C-30), 22.50 (C-1), 28.39 (C-20), 30.22 imum number of 1,2-methyl and 1,2-hydride shifts after initial
C-17), 30.73 (C-21), 32.00 (C-26), 32.31 (C-28), 32.65 (C-12), cyclization and thus requires an OSC capable of stabilizing
3.00 (C-15), 35.24 (C-29), 35.57 (C-11), 35.85 (C-16), 36.24 more high energy carbocation intermediates than any other
C-19), 37.67 (C-9), 38.52 (C-13), 39.47 (C-22), 39.92 (C-14), comparable enzymes (18). K. daigremontiana leaf surface lipids
1
(
3
(
4
5
1.52 (C-6), 41.75 (C-2), 42.36 (C-5), 43.03 (C-8), 53.33 (C-18), accumulate friedelin, together with a number of other pentacy-
8.45 (C-4), 59.71 (C-10), 213.40 (C-3). clic triterpenoids, suggesting that this species must contain a
The quantitative composition of the yeast lipid mixtures was friedelin synthase (19). It was therefore chosen as a model for
studied using capillary GC with a flame ionization detector the current study.
under the same GC conditions as above but H carrier gas at 2.0
OSC Cloning and Sequence Analysis—Previous analyses had
2
ml/min. Five independent analyses were carried out for each shown that K. daigremontiana leaves accumulate triterpenoids
transgenic yeast line.
during the first stages of expansive growth, for ϳ40 days after
Phylogenetic Analyses—Sequence alignments and phyloge- onset of leaf development (20). Here, it was further found that
netic analyses based on a neighbor-joining method were carried the major portion of these triterpenoids is accumulating in the
2
9706 JOURNAL OF BIOLOGICAL CHEMISTRY
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Oxidosqualene Cyclases of K. daigremontiana
FIGURE1. AminoacidsequencesoffiveOSCsfromK. daigremontiana. TheconservedQWmotifsthoughttostabilizeOSCstructurearehighlightedbysingle
underlines, whereas the highly conserved DCTAE motif containing the aspartate residue involved in substrate protonation is marked by a double underline.
TABLE 1
cuticular waxes coating the exterior walls of epidermal cells,
DNA sequence identities between OSCs cloned from
K. daigremontiana
with only 2% of the total amounts of glutinol and of friedelin
being present in the internal tissues of K. daigremontiana
KdGLS
%
94.3
72.5
94.3
57.4
KdFRS
%
KdTAS
%
KdLUS
%
leaves. Therefore, it was assumed that OSC expression should
be largely restricted to the epidermal cell layers in this tissue,
and they were peeled from both sides of young K. daigremonti-
ana leaves (Ͻ20 days old) to harvest mRNA enriched in target
OSC sequences. The resulting mRNA population was tran-
scribed into cDNA, which served as a template for the following
KdFRS
KdTAS
KdLUS
KdCAS
75.0
92.8
61.3
75.1
59.4
60.4
PCR-based cloning. In a first PCR experiment, degenerate five full-length sequences named KdTAS, KdGLS, KdFRS,
primers targeting the highly conserved consensus sequences KdLUS, and KdCAS, each containing one of the core segments
of published OSCs were employed to amplify the core segment of (Fig. 1).
potential OSC sequences. The PCR product mixture was sepa-
All of the cloned K. daigremontiana OCSs possess the amino
rated by gel electrophoresis, yielding one major band corre- acid motif DCTAE implicated in substrate binding (16) and the
sponding to the expected size of 750 bp that was isolated, four QW motifs characteristic of the OSC superfamily (24). The
cloned, and amplified in E. coli. Based on restriction and PCR ORFs of the K. daigremontiana OSCs range from 2,286 to 2,340
analyses, 32 bacterial colonies containing plasmids with inserts nucleotides in length, sharing between 57.4 and 94.3% with
of the expected length were selected for sequencing. Inserts each other (Table 1). The amino acid sequences of these four
with five different sequences were found, each of them repre- genes show relatively high levels of identity with known ␤-
sented between three and six times. Extended core sequences amyrin synthases from other plant species, whereas the KdCAS
were obtained by a set of PCRs. Finally, 5Ј- and 3Ј-rapid ampli- ORF has substantially higher similarity with corresponding
fication of cDNA end reactions with specific primers yielded cycloartenol synthases (Table 2).
SEPTEMBER 24, 2010•VOLUME 285•NUMBER 39
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Oxidosqualene Cyclases of K. daigremontiana
TABLE 2
Sequence identities between OSCs cloned from K. daigremontiana and selected OSCs previously described in the literature
Amino acid sequence similarity with K. daigremontiana OSCs
(
amino acid number in parentheses)
Plant species
Major product
Accession number
KdGLS (767)
KdFRS (761)
KdTAS (779)
KdLUS (765)
KdCAS (764)
%
%
%
%
%
B. platyphylla
P. ginseng
␤-Amyrin
AB055512
AB014057
AB009030
AB263203
AB206469
AB181246
AB009029
76.5
74.6
75.8
75.2
75.3
58.0
57.0
76.7
74.9
75.9
75.9
75.7
58.1
58.2
84.7
82.3
80.6
80.6
80.7
58.7
59.2
78.6
75.8
77.5
77.1
77.0
59.7
58.6
57.5
57.3
57.4
56.4
57.6
79.8
79.2
␤-Amyrin
P. ginseng
␤-Amyrin
Rhizophora stylosa
Euphorbia tirucalli
Lathyrus japonicus
P. ginseng
Multifunctional
␤-Amyrin
Cycloartenol
Cycloartenol
is more closely related to the
-amyrin synthases BPY, PNY, and
␤
PNY2 from Betula platyphylla and
Panax ginseng. Each of the four
OSCs from K. daigremontiana shares
at least 76% sequence identity with
one or more OSCs reported in the
literature to date (Table 2). Finally,
KdCAS was found to group together
with the cycloartenol synthases
from various other species (Fig. 2).
Functional cDNA Expression in
Yeast—In order to biochemically
characterize the putative OSCs from
K. daigremontiana, their full-length
ORFs were heterologously expressed
in Saccharomyces cerevisiae. The
yeast mutant GIL77 (gal2 hem3-6
erg7 ura3-167) was used for this
purpose because it is deficient in
lanosterol synthase and therefore
accumulates high concentrations of
2
,3-oxidosqualene that can serve as
substrate for OSC-catalyzed cycli-
zation (25). However, growth of this
strain requires exogenous ergos-
terol, which must be added to the
medium and is consequently de-
tected in triterpenoid analyses to-
gether with the products of the
transgenic OSCs. All five K. daigre-
montiana cDNAs were subcloned
into the yeast expression vector
pYES2, and the resulting constructs
were transformed into GIL77. In
parallel, the empty vector was trans-
formed into the yeast mutant to
serve as a negative control. Three
FIGURE 2. Phylogenetic analysis comparing the new OSC cDNAs cloned from K. daigremontiana with independent lines were selected
previously known OSCs from other plant species.
from each transformation experi-
ment and were further grown in the
presence of ergosterol, induced with galactose, and extracted.
The extracts were acetylated and analyzed by GC-MS in order
Phylogenetic analyses using the neighbor-joining method
ClustalX) (23) showed that KdFRS, KdGLS, KdLUS, and
(
KdTAS all cluster very closely together within a clade of OSC
enzymes previously characterized as ␤-amyrin synthases (Fig. to identify triterpenoids, whereas GC-flame ionization detec-
3
). Although the first three enzymes from K. daigremontiana tion was used to assess relative quantities of the compounds. No
form a new branch within this cluster of OSC enzymes, KdTAS differences between parallel lines were found, and therefore
2
9708 JOURNAL OF BIOLOGICAL CHEMISTRY
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Oxidosqualene Cyclases of K. daigremontiana
FIGURE 3. Mass spectra of pentacyclic triterpenoids. Compounds 1–5, formed by yeast strains heterologously expressing various OSCs from K. daigremon-
tiana (left), have fragmentation patterns identical with those of authentic standards (right). For the triterpenoid alcohols 1–3 and 5 as well as the corresponding
standards, spectra of acetates are shown.
results are shown here for only one line harboring each OSC identified as the triterpenoid ketone friedelin based on direct
construct.
comparison with a commercial standard of this compound and
1
13
All six yeast transformants were found to contain two com-
pounds in common, which were identified as ergosterol and
H NMR and C NMR analysis.
Quantitative analyses were carried out on five independent
2
,3-oxidosqualene. In addition, the extracts from yeast harbor- parallels of each transgenic yeast line. They showed that all of
ing K. daigremontiana cDNAs contained several compounds the lines expressing putative OSC cDNAs contained 2,3-ox-
not present in the control, most of them also differing between idosqualene at markedly lower concentrations than the empty
the lines expressing different OSC candidate cDNAs. The vector control (Fig. 4). Yeast heterologously expressing KdTAS
structures of all major compounds were elucidated by compar- cDNA produced a mixture of taraxerol (60 Ϯ 1%; mean Ϯ S.D.,
ison of mass spectral characteristics with those of authentic n ϭ 5) and ␤-amyrin (40% Ϯ 1%), whereas expression of KdGLS
standards (Fig. 3) and of GC elution behavior (Fig. 4). Most of cDNA prompted the formation of glutinol (67 Ϯ 2%) together
the triterpenoids in the yeast extracts were found to contain with friedelin (14 Ϯ 1%), ␤-amyrin (11 Ϯ 0.3%), and taraxerol
hydroxyl functions and could therefore be derivatized in order (8 Ϯ 0.2%). Expression of the plasmid pYES2-KdFRS resulted in
to improve chromatographic resolution and spectral quality. the formation of friedelin (72 Ϯ 1%), accompanied by smaller
Acetate esters and trimethylsilyl ethers were generated for all amounts of ␤-amyrin (18 Ϯ 0.1%) and taraxerol (10 Ϯ 1%).
these triterpenoid alcohols, and both derivatives together were Lines harboring KdLUS cDNA produced a mixture of lupeol
used to assign structures. Lupeol and ␤-amyrin were identified (93 Ϯ 2%) and ␤-amyrin (7 Ϯ 2%). The products formed by the
by comparison with commercial standards, whereas glutinol four OSCs together account for all of the major triterpenoids
was identified by comparison with a standard that had been found in the cuticular wax on K. daigremontiana leaves (19, 20).
extracted from K. daigremontiana leaf wax and authenticated
The relative activity of the four OSC genes characterized
1
13
by H NMR and C NMR analysis (20). The structure of tarax- here can be estimated based on our GC analyses of the trans-
erol was assigned by comparing MS data for the acetate ester genic yeast extracts (Fig. 4). Because exactly the same amounts
and TMS ether with those reported in the literature and found of ergosterol had been added to all cultures and samples had
for the corresponding triterpenoid isolated from M. hypoleuca been taken and worked up in the same way, the steroid can
leaf wax (26). Finally, one compound in the yeast extracts was serve as an internal standard to estimate the amounts of triter-
SEPTEMBER 24, 2010•VOLUME 285•NUMBER 39
JOURNAL OF BIOLOGICAL CHEMISTRY 29709

Oxidosqualene Cyclases of K. daigremontiana
of the five OSCs. It was found that
each primer pair allowed amplifica-
tion of its target OSC sequence frag-
ment but not of the other four OSCs
(
data not shown). Total RNA was
isolated from leaf inner tissue as
well as the abaxial and adaxial epi-
dermal layers of K. daigremontiana
leaves, and the RT-PCR analysis
using these RNA samples showed
that the KdCAS enzyme is expressed
equally in all tissues within the leaf
(Fig. 5), in accordance with its likely
role in membrane steroid produc-
tion of all cells. In contrast, the
KdTAS, KdFRS, KdGLS, and
KdLUS enzymes are expressed only
in the epidermal cells on both sides
of the leaf and not in internal leaf
tissues (Fig. 5). These results are in
agreement with the accumulation
of the triterpenoid products to high
concentrations in the cuticular
waxes on both leaf surfaces. Our
results show that the major biologi-
cal function of all four genes is in
formation of triterpenoids targeted
to the plant surface, where they
probably play important roles in
protecting the tissue from herbi-
vores and pathogens.
OSC Expression Levels in the
K. daigremontiana Leaf—Although
the above results show that the four
OSCs are involved in the formation
of cuticular triterpenoids in the
K. daigremontiana leaf, it remains
to be elucidated how much each
enzyme contributes to the product
mixture. However, the enzyme ac-
tivities within the tissue cannot be
determined directly due to the low
solubility of substrates and prod-
FIGURE4. Gaschromatographicanalysisoftriterpenoidsinyeaststrainsharboringthevectorsindicated
in the various panels. Triterpenoid alcohols were transformed into acetates prior to GC analysis. A and B were
present in all yeast samples and were identified as epoxysqualene and ergosterol, respectively. Compounds
1
–5 showed retention behavior similar to those of authentic standards of taraxerol, ␤-amyrin, glutinol, friede- ucts. In order to assess the relative
lin, and lupeol, respectively.
contribution of the four OSCs, the
relative expression levels of the cor-
penoid products formed. Hence, it can be concluded that responding genes were assessed using quantitative RT-PCR. It
KdLUS was the most active enzyme when tested in yeast, fol- was found that the expression levels were highest for KdLUS,
lowed by KdFRS and KdGLS, whereas KdTAS was found to be intermediate for KdTAS, and lowest for KdGLS and KdFRS
a much less active OSC. Finally, extracts of GIL77 yeast cells (Fig. 6).
functionally expressing KdCAS cDNA were found to contain
Taking the gene expression levels together with the relative
cycloartenol together with some unidentified cyclization prod- activities of the OSCs (determined in yeast; see above), it seems
ucts (data not shown). likely that KdLUS should have the highest absolute activity in
OSC Expression Patterns in the K. daigremontiana Leaf— the K. daigremontiana leaf. The other three OSC activities are
To investigate whether OSC transcription is tissue-specific, predicted to be lower than KdLUS and similar to each other
RT-PCR analyses were carried out. Five pairs of PCR primers because higher relative activities of KdGLS and KdFRS might be
were designed based on the OSC sequences and first tested for compensated for by the higher expression level of KdTAS.
gene specificity using plasmids harboring full-length cDNAs These predicted activity levels of the four OSCs do not match
2
9710 JOURNAL OF BIOLOGICAL CHEMISTRY
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Oxidosqualene Cyclases of K. daigremontiana
to have catalytic site features stabilizing the maximum possible
cationic intermediates. Interestingly, a second OSC from
K. daigremontiana was found to form predominantly glutinol,
the triterpenoid product generated immediately before friede-
lin along the rearrangement cascade. Both enzymes share very
high sequence identity, and thus the small differences in amino
acid structures between them can now be used to identify
amino acids that may be involved in determining product spec-
ificities for either friedelin or glutinol.
7
9
127
172
278
350
423
Residues Ala , Ser , Gln , His , Lys , and Phe in
the sequence of KdFRS stand out because they differ between
the friedelin synthase and most other OSCs and because they
involve aromatic amino acids either in KdFRS or in the other
enzymes. Aromatic residues are thought to be important for the
cyclization mechanism because they can stabilize the cationic
intermediates. Further amino acids of interest in KdFRS are
3
23
733
FIGURE 5. RT-PCR analysis of the expression patterns of five OSC genes in
K. daigremontiana leaves. The four enzymes generating pentacyclic triter-
penoids were found expressed only in the epidermis layers of the leaves,
Ile
and Thr
because they differ in this enzyme from all
103
other OSCs, including KdGLS. Finally, amino acids His
,
2
49
297
497
367
370
whereas the cycloartenol synthase was equally expressed in all tissues within Leu , Gln , Phe , Thr , and Thr
not only differ
the leaf.
between KdFRS and other OSCs, but they are also predicted to
line the active site or to be located near it, according to three-
dimensional structure modeling (data not shown) based on the
crystal structure available for human lanosterol synthase (27).
The role of the amino acids listed above in OSC product
specificity is presently being investigated using site-directed
mutagenesis experiments. To identify further residues possibly
involved in the cyclization mechanism, domain-swapping
experiments are also currently being carried out.
CONCLUSION
FIGURE 6. Quantitative RT-PCR analysis of the four OSC genes in K. dai-
gremontiana leaves generating pentacyclic triterpenoids. The relative
expression levels were determined in whole, still growing leaves and normal-
ized (n ϭ 3, S.D. (error bars)). Lupeol synthase was found to be expressed most
actively, taraxerol synthase at an intermediate level, and glutinol as well as
friedelin synthase least of the genes.
Altogether, five different OSCs from one plant species have
been cloned and fully characterized by heterologous expression
in yeast. Very diverse triterpenoids were identified as products
of the enzymes, ranging from taraxerol over glutinol to friede-
lin. This is the first report on the sequence and biochemical
characteristics of such a friedelin synthase, confirming the pre-
diction that this late triterpenoid product is indeed formed in
one sequence of catalytic steps. In comparison with friedelin
and glutinol, taraxerol is a relatively early product of the rear-
rangement series, and the OSC forming this product must be
stabilizing fewer cationic intermediates than the others. This is
also the first report describing a taraxerol synthase, now pro-
viding the opportunity to perform further comparative studies
of the amino acid structures determining earlier rearrangement
steps in the sequence.
the triterpenoid profile in the cuticular waxes of K. daigremon-
tiana leaves, which had been reported to contain ϳ62% glutinol
(
and its derivatives), 20% friedelin, 13% taraxerol, and 5%
␤
-amyrin, whereas lupeol had not been detected at all. It should
be noted that germanicol had been described for the leaf wax,
but further analyses using authentic standard as described here
have now confirmed that the true structure of the compound is
taraxerol both in the wax and in the yeast product.
The discrepancy between our biochemical data and the wax
triterpenoid profile might be due to one or more of the follow-
ing reasons. 1) The apparent OSC activities in yeast might be
Acknowledgments—We thank Bangjun Wang and Ortwin Guhling
misleading because of artifacts in the heterologous environ- for technical help and fruitful discussions and Dr. Y. Ebizuka (Tokyo)
ment, 2) large portions of the triterpenoid products formed by for providing the yeast strain GIL77.
the OSCs in the plant might be transformed and/or sequestered
in inner leaf tissues and therefore might not be reflected in
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9712 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 39•SEPTEMBER 24, 2010

Plant Biology:
Cloning and Characterization of
Oxidosqualene Cyclases from Kalanchoe
daigremontiana : ENZYMES
CATALYZING UP TO 10
REARRANGEMENT STEPS YIELDING
FRIEDELIN AND OTHER
TRITERPENOIDS
Zhonghua Wang, Trevor Yeats, Hong Han
and Reinhard Jetter
J. Biol. Chem. 2010, 285:29703-29712.
doi: 10.1074/jbc.M109.098871 originally published online July 7, 2010
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