Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity

Article abstract of DOI:10.1074/jbc.M114.623348

Chrysanthemyl diphosphate synthase (CDS) is the first path-way-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1′-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a K_m value for CPP of 196 μM. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ～100 μM, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12-0.16 μg h^-1 g^-1 fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate.

Full text of DOI:10.1074/jbc.M114.623348

Plant Biology:
Chrysanthemyl Diphosphate Synthase
Operates in Planta as a Bifunctional
Enzyme with Chrysanthemol Synthase
Activity
Ting Yang, Liping Gao, Hao Hu, Geert
Stoopen, Caiyun Wang and Maarten A.
Jongsma
J. Biol. Chem. 2014, 289:36325-36335.
doi: 10.1074/jbc.M114.623348 originally published online November 5, 2014
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 52, pp. 36325–36335, December 26, 2014
© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Chrysanthemyl Diphosphate Synthase Operates in Planta as a
Bifunctional Enzyme with Chrysanthemol Synthase Activity*
Received for publication, November 4, 2014 Published, JBC Papers in Press, November 5, 2014, DOI 10.1074/jbc.M114.623348
Ting Yang‡§¶, Liping Gao‡, Hao Huʈ, Geert Stoopen‡, Caiyun Wangʈ, and Maarten A. Jongsma‡1
From ‡Business Unit PRI-Bioscience, Wageningen UR, P.O. Box 16, 6700 AA Wageningen, The Netherlands, the §Laboratory of
Entomology, Wageningen UR, P.O. Box 8031, 6700 EH Wageningen, The Netherlands, the ¶Laboratory of Plant Physiology,
Wageningen UR, P.O. Box 658, 6700 AR Wageningen, The Netherlands, and the ʈKey Laboratory for Biology of Horticultural Plants,
Ministry of Education, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, China
Background: Chrysanthemyl diphosphate synthase (CDS) is known to catalyze the formation of the irregular terpenoid,
chrysanthemyl diphosphate (CPP).
Results: CDS also catalyzes the next step from CPP to chrysanthemol.
Conclusion: CDS is actually a chrysanthemol synthase (CHS) with bifunctional enzyme activity.
Significance: Identification of CHS increases our understanding in terpene biosynthesis and paves the way for engineering
biosynthesis of the most widely used natural pesticide, pyrethrins.
Chrysanthemyl diphosphate synthase (CDS) is the first path-
way-specific enzyme in the biosynthesis of pyrethrins, the most
widely used plant-derived pesticide. CDS catalyzes c1؅-2-3
cyclopropanation reactions of two molecules of dimethylallyl
diphosphate (DMAPP) to yield chrysanthemyl diphosphate
(CPP). Three proteins are known to catalyze this cyclopropana-
tion reaction of terpene precursors. Two of them, phytoene and
squalene synthase, are bifunctional enzymes with both prenyl-
transferase and terpene synthase activity. CDS, the other mem-
ber, has been reported to perform only the prenyltransferase
step. Here we show that the NDXXD catalytic motif of CDS,
under the lower substrate conditions prevalent in plants, also
catalyzes the next step, converting CPP into chrysanthemol by
hydrolyzing the diphosphate moiety. The enzymatic hydrolysis
reaction followed conventional Michaelis-Menten kinetics,
with a Km value for CPP of 196 ␮M. For the chrysanthemol syn-
thase activity, DMAPP competed with CPP as substrate. The
DMAPP concentration required for half-maximal activity to
produce chrysanthemol was ϳ100 ␮M, and significant substrate
inhibition was observed at elevated DMAPP concentrations.
The N-terminal peptide of CDS was identified as a plastid-tar-
geting peptide. Transgenic tobacco plants overexpressing CDS
emitted chrysanthemol at a rate of 0.12–0.16 ␮g h؊1 g؊1 fresh
weight. We propose that CDS should be renamed a chrysanthe-
mol synthase utilizing DMAPP as substrate.
toxins effective against a wide range of insect species and are
applied broadly in private homes, gardens, stables, and organic
agriculture. Their low toxicity to warm-blooded animals and
high degradability under sunlight gives them environmentally
friendly properties (3).
Pyrethrins comprise a group of six closely related esters with
either chrysanthemic acid (type I esters) or pyrethric acid (type
II esters) as terpene acid moieties. Chrysanthemic and pyre-
thric acid share a common cyclopropane ring structure, but the
latter possesses a terminal carboxymethyl group. The acid moi-
eties have been shown to be predominantly derived from the
methyl-erythritol phosphate pathway (4, 5). The methyl-eryth-
ritol phosphate pathway provides two universal C5 terpene
building blocks, isopentenyl diphosphate (IPP)2 and its isomer
dimethylallyl diphosphate (DMAPP), in the plastids of plants.
Monoterpenes (C10), diterpenes (C20), and carotenoids (C40)
are synthesized via this pathway (6). The first pathway-specific
step for pyrethrin biosynthesis is the condensation of two mol-
ecules of DMAPP, yielding chrysanthemyl diphosphate (CPP)
(Fig. 1A) (7). The gene responsible for this step has been cloned,
and the recombinant enzyme has been characterized as chry-
santhemyl diphosphate synthase (CDS, EC 2.5.1.67) (8). The
genes and enzymes involved in the next steps, converting CPP
into the monoterpene alcohol, chrysanthemol, and the further
oxidation to chrysanthemic acid, have not been identified yet,
but, recently, the reactions have been shown to occur in the
glandular trichomes (9). The final step of the esterification of
chrysanthemic acid with one of the lipid alcohols is performed
by a recently cloned GDSL lipase from pyrethrum. This enzyme
transfers the chrysanthemoyl group from the CoA thioester to
pyrethrolone to produce pyrethrin I but does so in the pericarp
of the seeds and not in the glandular trichomes (9, 10).
Pyrethrins are found predominantly in pyrethrum flower
heads (Tanacetum cinerariifolium) and represent the econom-
ically most important natural pesticide (1, 2). They are neuro-
* This work was supported by Technological Top Institute Green Genetics
(Stichting TTI Groene Genetica) of The Netherlands Grant 1C001RP and by
National Natural Science Foundation of China Project 31372103.
Thenucleotidesequence(s)reportedinthispaperhasbeensubmittedtotheGen-
BankTM/EBI Data Bank with accession number(s) JX913536 and JX913537.
1 To whom correspondence should be addressed: Plant Research Interna-
tional, Wageningen UR, P.O. Box 619, 6700 AP Wageningen, The
Netherlands. Tel.: 31-317-480-932; Fax: 31-317-41-80-94; E-mail:
maarten.jongsma@wur.nl.
2 The abbreviations used are: IPP, isopentenyl diphosphate; DMAPP, di-
methylallyl diphosphate; CPP, chrysanthemyl diphosphate; CDS, chrysan-
themyl diphosphate synthase; FDS, farnesyl diphosphate synthase; LPP,
lavandulyl diphosphate; GPP, geranyl diphosphate; GDS, geranyl diphos-
phate synthase; FPP, farnesyl diphosphate; SQS, squalene synthase; PTS,
phytoene synthase; FW, fresh weight; RACE, rapid amplification of cDNA
ends; MOPSO, 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid.
DECEMBER 26, 2014•VOLUME 289•NUMBER 52
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Bifunctional Irregular Terpene Synthase
CDS, the enzyme catalyzing the first pathway-specific step, vert CPP to chrysanthemol. This suggested that CDS could also
evolved from farnesyl diphosphate synthase (FDS) (8, 11, 12). It be a bifunctional enzyme catalyzing two consecutive reactions
takes two molecules of DMAPP as substrate to produce CPP as from DMAPP to CPP and then to chrysanthemol (Fig. 1A). We
major product and lavandulyl diphosphate (LPP) as minor now report the characterization of CDS to synthesize chrysan-
product (Fig. 1). Therefore, compared with FDS, the product themol from CPP and DMAPP in vitro and the confirmation of
size of CDS has shifted from C15 to C10, and the enzyme activity this claim in planta using transgenic tobacco plants.
of CDS has changed from chain elongation to cyclopropana-
EXPERIMENTAL PROCEDURES
tion. CDS has been cloned and characterized from pyrethrum
(T. cinerariifolium) (8) and sagebrush (Artemisia tridentata
Plant Materials and Chemicals—Pyrethrum plants (T. cin-
ssp. spiciformis) (13), but, recently, CDS-like genes have also erariifolium) were grown in the field in Wageningen, The Neth-
been reported from Tanacetum coccineum, Achillea asiatica, erlands. CPP and chrysanthemol were purchased from Iso-
Chrysanthemum lavandulifolium, Aster ageratoides, Helian- prenoids Lc. DMAPP was obtained from Sigma. CPP was a
thus exilis, and Helianthus annuus (12). For sagebrush, the CDS mixture of trans- and cis-racemic forms in a ratio around 9:1,
protein sequence shares 75% identity (and 96% similarity) with and so was the chrysanthemol.
the FDS protein sequence from the same species. FDS catalyzes
Cloning Full-length cDNA and Genomic DNA of CDS—To
the sequential c1Ј-4 condensation of IPP with DMAPP to form clone the full-length cDNA of CDS, the following primers were
the intermediate geranyl diphosphate (GPP, C10) and, with used in 3Ј and 5Ј RACE PCRs: TcCDS_F, 5Ј-CATCTTCTGG-
another IPP, the product farnesyl diphosphate (FPP, C15), or it ACCTCTTCAATGAG-3Ј; TcCDS_R, 5Ј-GTACTGAACAA-
catalyzes c1Ј-4 condensation of GPP with IPP to yield FPP (14). TCCGACGGTTAAG-3Ј. RACE libraries were synthesized
CDS catalyzes the c1Ј-2-3 condensation of two units of with 1 ␮g of total RNA from a pyrethrum ovary using Super-
DMAPP, yielding CPP (C10) (8), but the reported affinity of cript௡ II reverse transcriptase (Invitrogen) and the Smart RACE
DMAPP for CDS is very low, with a Km value of 600 ␮M (8) kit (Clontech). In a final step on the basis of the complete
compared with Km values for FDS of 1–20 ␮M for IPP, sequence information, primers were designed to obtain the full-
DMAPP, or GPP (15). Furthermore, DMAPP concentrations length cDNA (accession number JX913536). Eight clones were
in plastids are estimated at 30 ␮M (see “Discussion”).
sequenced. On the basis of the full-length cDNA information,
The c1Ј-2-3 cyclopropane ring structure in CPP is also found primers were designed for amplification of the genomic DNA
in the C30 and C40 terpenoids, presqualene diphosphate and corresponding to the gene with introns (accession number
prephytoene diphosphate. Presqualene diphosphate and pre- JX913537). The sequence of CDS obtained by us differs slightly
phytoene diphosphate are intermediates in reactions produc- from the one reported by Rivera et al. (8) (accession number
ing squalene and phytoene, which are precursors for biosynthe- I13995). The detailed differences are explained in the results.
sis of sterols and carotenoids. These reactions are catalyzed by The CDS sequence used in this study is the one obtained by us
squalene synthase (EC 2.5.1.21) and phytoene synthase (EC (accession number JX913536).
2.5.1.32), respectively (16). Squalene synthase catalyzes the
Protein Production in E. coli and Purification—The open
condensation of two molecules of FPP to give presqualene reading frame of CDS without the putative targeting sequence
diphosphate, and subsequent reductive rearrangement of this was subcloned into the pRSET-A expression vector (Invitro-
intermediate generates squalene (17). Phytoene synthase cata- gen) fused to an amino-terminal histidine tag and expressed in
lyzes a similar reaction, taking geranylgeranyl diphosphate as E. coli BL21(AI) (forward primer, 5Ј-TTATGGATCCACTAC-
substrate to give prephytoene diphosphate as an intermediate GACATTGAGCAGCAATCTAG-3Ј; reverse primer, 5Ј-TTA-
and phytoene as a final product (16). Notably, both these terpe- TGAATTCTTACTTATGTCCCTTATACATCTTTTCC-3Ј;
noid synthases are bifunctional enzymes catalyzing consecutive restriction sites are underlined). E. coli cells were grown to an
prenylation and dephosphorylation steps (18, 19).
A600 of 0.6 and then induced with 0.02% L-arabinose at 18 °C for
Among the very large family of short-chain prenyltrans- 16 h. After harvesting, the cells were resuspended in lysis buffer
ferases and terpene synthases (C10, C15, and C20) (20–22), to containing 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, and 1.4 mM
our knowledge, only two of them were bifunctional. ␣- ␤-mercaptoethanol; disrupted by sonification (6 ϫ for 10 s);
Farnesene synthase from apple (Malus domestica) could take and centrifuged at 13,000 ϫ g for 10 min at 4 °C. His-tagged
both FPP (C15) or GPP (C10) and IPP (C5) to produce proteins were purified with nickel-nitrilotriacetic acid-agarose
␣-farnesene (C15), and both activities were associated with the (Qiagen) according to the protocol of the manufacturer. Pro-
(semi)conserved DDXXD motif (23), which is required for teins were concentrated and desalted in 50 mM Tris-HCl (pH
metal ion binding (24). Myrcene synthase from bark beetle (Ips 7.5, containing 1.4 mM ␤-mercaptoethanol) using Amicon cen-
pini) was initially identified as a GDS (25) but was later discov- trifugal filters for protein purification and concentration (Mil-
ered to take GDP to further produce myrcene (C10) (26). The lipore), checked for purity by SDS-PAGE, and quantified by the
authors predicted that both prenyltransferase and terpene syn- Bradford method using bovine serum albumin as a standard
thase activities shared the same catalytic domain on the basis of (27). The protein purity was greater than 90%.
modeling of the protein structure.
Reaction of CDS with CPP—The activity of CDS to produce
Our preliminary experiments with cell lysate of Escherichia chrysanthemol from CPP was assayed by incubating 35 ␮g of
coli expressing CDS showed that the lysate was able to produce purified enzyme in a final volume of 100 ␮l of assay buffer (pH
chrysanthemol directly from DMAPP, whereas cell lysate of 7.0) containing 15 mM MOPSO, 2 mM dithioerythreitol, 12.5%
E. coli transformed with the empty vector was not able to con- (v/v) glycerol, 1 mM MgCl2, 1 mM ascorbic acid, 0.1% (v/v)
36326 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 289•NUMBER 52•DECEMBER 26, 2014

Bifunctional Irregular Terpene Synthase
Tween 20, and various concentrations of CPP. After addition of above). The enzyme assays were conducted as described above
a 100-␮l pentane overlay, the assays were incubated for 20 h at (20-h incubation). DMAPP concentrations were tested in a
30 °C. Then the assay mixture was extracted as described pre- range of 0–600 ␮M with eight different data points. The deter-
viously (28), and the organic phase was concentrated to 200 ␮l minations were replicated three times. Terpene alcohol and
under nitrogen flow. A 1-␮l portion of the concentrated diphosphate products were analyzed by GC-MS as described
organic phase was analyzed by GC-MS using a GC (Agilent above.
7890A) equipped with an HP5 MS column (30 m ϫ 0.25 mm
Site-directed Mutagenesis—Site-directed mutagenesis was
inner diameter, 0.25 ␮m film thickness) and a mass-selective performed using a QuikChange Lightning site-directed
detector (Agilent 5975C, Hewlett Packard, Agilent Technolo- mutagenesis kit (Agilent) according to the instructions of the
gies). The injection port (splitless mode), interface, and MS manufacturer. The following oligonucleotides were synthe-
source temperatures were 250 °C, 290 °C, and 180 °C, respec- sized and used for the substitution of the residue asparagine (at
tively. The oven was programmed at an initial temperature of position 283) by aspartate or glycine, respectively: TcCDS-
45 °C for 1 min with a ramp of 15 °C minϪ1 to 280 °C and a final N283D_F, 5Ј-GGTATGTATTATCAAATTCAGGATGATT-
time of 3.5 min. Proteins extracted from E. coli lysate cells har- ATCTCGACAC-3Ј and TcCDS-N283D_R, 5Ј-GTGTCGA-
boring the empty vector were assayed with CPP as a control. For GATAATCATCCTGAATTTGATAATACATACC-3Ј (the
identification, the authentic standard of chrysanthemol was aspartate residue is underlined); TcCDS-N283G_F, 5Ј-GGTA-
run under identical conditions.
TGTATTATCAAATTCAGGGTGATTATCTCGACAC-3Ј
To determine the apparent Km value of CDS for CPP, the and TcCDS-N283G_R, 5Ј-GTGTCGAGATAATCACCCTG-
Mg2ϩ concentration (1.0 mM) and pH value (7.0) were set at AATTTGATAATACATACC-3Ј (the glycine residue is under-
optimal levels as described previously (8), and the CPP concen- lined). The resulting mutant cDNAs were sequenced to verify
tration varied between 10 and 600 ␮M with eight different data mutations. Mutated proteins were then expressed and assayed
points. Triplicate sets of assays were performed at each CPP as described above. For the enzyme assays, the mutants were
concentration. The apparent Km value was obtained by fitting assayed with either CPP or DMAPP, and the substrate concen-
the data to the Michaelis-Menten equation using KaleidaGraph tration for CPP and DMAPP was 200 and 600 ␮M, respectively.
(Synergy Software).
CDS was used as positive control.
Reaction of CDS with DMAPP—The activity of CDS to pro-
The product detection was done slightly differently com-
duce diphosphates (CPP and LPP) and terpene alcohols (chry- pared with the other experiments because of equipment
santhemol and lavandulol) from DMAPP was assayed by incu- change. A 1-␮l portion of the organic phase was analyzed by
bating 35 ␮g of purified enzyme in a final volume of 100 ␮l of GC-MS coupled with a DSQ mass spectrometer using an Agi-
assay buffer, described above, but with 600 ␮M DMAPP instead lent 6890 Network detector (Thermo Fisher Scientific) and
of CPP. After addition of a 100-␮l pentane overlay, the assays equipped with a HP5 MS column (30 m ϫ 0.25 mm i.d., 0.25-␮l
were incubated for 96 h at 30 °C. The chrysanthemol produc- film thickness, Agilent Technologies). The oven was pro-
tion was determined by analyzing the organic phase as grammed at an initial temperature of 40 °C for 3 min with a
described above. CPP production was determined by analyzing ramp of 10 °C minϪ1 to 280 °C and final time of 2 min.
the water phase of the same reaction tube. For that, 50 ␮l of
Subcellular Localization of CDS Using GFP—The gene frag-
water phase was transferred to a fresh reaction tube and treated ment corresponding to the first 54 amino acids of CDS or the
with calf alkaline phosphatase (Sigma). Liberated compounds whole CDS gene without the stop codon were cloned into the
were extracted as described previously (8). The extracted com- binary vector pBINPLUS-1.1eGFP (29) as an in-frame protein
pounds were then analyzed by GC-MS as described above. For fusion to a GFP reporter gene using the KpnI and XbaI restric-
Km determination, assays were performed with different tion sites. These constructs were named pCDSTAR-GFP and
DMAPP concentrations from 10–600 ␮M with eight different pCDS-GFP, respectively. Tobacco plants (Nicotiana tabacum
data points. Triplicate sets of assays were performed at each Samsun) were transformed with these constructs as described
DMAPP concentration. To check non-enzymatic conversion previously (30). Transgenic tobacco mesophyll protoplasts
from CPP to chrysanthemol in this 96-h assay, control reactions were isolated according to Sheen (31). Images were taken using
without enzyme were performed with CPP concentrations a confocal laser-scanning microscope (Zeiss) with fluorescence
ranging from 2–80 ␮M. This range covered a maximum con- bandwith filters of 620–750 nm for chlorophyll imaging and
centration of 25 ␮M CPP produced by CDS under different 500- 530 nm for GFP.
DMAPP concentrations.
Generation of Transgenic Tobacco—The complete cDNA of
For identification of chrysanthemol, the authentic standard CDS was placed under the control of chrysanthemum RbcS1
of chrysanthemol was run under identical conditions. For iden- promoter, and Agrobacterium tumefaciens strain AGL-0 har-
tification of lavandulol, mass spectra of eluted peak fractions boring the binary vector was used to transform the tobacco
were compared with the published mass spectrum for lavandu- plants using protocols described previously (30). Tobacco
lol (11).
plants transformed with a vector lacking CDS were used as a
Determination of DMAPP Substrate Inhibition in the Reac- control. Transgenic plants were grown in a greenhouse at 25 Ϯ
tion of CDS with CPP—To check whether chrysanthemol pro- 2 °C under a 18/6 h light/dark photoperiod. The presence of
duction by CDS from CPP is inhibited by DMAPP, different CDS was checked by PCR. All PCR-positive plantlets of the T0
amounts of DMAPP were added into CDS enzyme assays con- transgenic line were analyzed by GC-MS to check the presence
taining 150 ␮M CPP (the apparent Km value of CPP determined of chrysanthemol in the headspace. The three plantlets with the
DECEMBER 26, 2014•VOLUME 289•NUMBER 52
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Bifunctional Irregular Terpene Synthase
FIGURE 1. Reactions catalyzed by CDS. A, schematic of the two-step reaction catalyzed by CDS. The chirality of the molecules is shown as publication (11).
Major and minor refer to the relative product quantity in each conversion. B, GC-MS chromatograms of the products of purified CDS enzyme assayed with CPP
or DMAPP. Empty vector, control assay with protein purified (by eluate volume) from E. coli cells harboring empty vector; standard, a mixture of trans- and
cis-chrysanthemol. For the assay with CPP, the assay time was 20 h. For the assay with DMAPP, the assay time was 96 h.
highest production of chrysanthemol were analyzed by LC-MS. CDS cDNA using RACE PCR. Eight 5Ј end sequences obtained
Three plantlets from the empty vector control line were also by us were quite different from the 5Ј end sequence reported
analyzed by GC-MS and LC-MS.
previously for CDS (accession number I13995, Ref. 8). We then
Volatile GC-MS and Non-volatile LC-MS Analysis of Trans- determined the genomic DNA sequence of CDS (accession
genic Tobacco—Volatiles from cut leaves were collected from number JX913537) from pyrethrum to see whether alternative
4-week-old plants. The second leaf from the top was harvested splicing could explain these differences. By comparing the
for headspace trapping. The volatiles were sampled for 2 h and cDNA and genomic DNA sequences, an intron was easily iden-
then analyzed by GC-MS as described previously (32). The tem- tified at the 5Ј end. The putative start codon proposed by Rivera
perature program of the gas chromatograph was 40 °C for 3 et al. (8) is actually in this intron region. Twenty-four nucleo-
min, rising to 280 °C at 10 °C minϪ1, and final time for 2 min. tides corresponding to the first eight amino acids of CDS
The mass spectrometer was set to scan from 35–450 m/z. The reported by Rivera et al. (8) were the intron sequence. The rest
helium flow was constant at 1.0 ml minϪ1. The ionization of the coding sequence of CDS from Rivera et al. (8) is nearly
potential was set at 70 eV. For the identification of chrysanthe- 100% the same as that obtained by us, with only 1 bp difference
mol, the authentic standard of chrysanthemol was run under and no effect on the protein sequence. We also found that one
identical conditions. For identification of lavandulol, the mass
spectrum of the eluted peak was compared with the published
mass spectrum of lavandulol (11). The third leaf from the top
was used for non-volatile analysis according to a protocol for
untargeted metabolomics of plant tissues (33) as described in
detail previously (32).
of our eight sequenced 5Ј RACE products was not correctly
spliced (data not shown). Apparently, Rivera et al. (8) cloned
this low-frequency mRNA product. The CDS used in this study
was our own clone isolated from pyrethrum ovaries (accession
number JX913536).
Enzymatic Characterization of CDS with CPP—CDS takes
DMAPP as substrate to produce CPP (8). Surprisingly, when
CPP was provided, CDS converted CPP into chrysanthemol
(Fig. 1B). The reaction followed standard Michaelis-Menten
kinetics (r ϭ 0.997) with apparent Km and kcat values of 196 Ϯ
23 ␮M and 3.3 Ϯ 0.2 ϫ 10Ϫ3 minϪ1, respectively (Fig. 2A). Chry-
santhemol was not detected in the control assay incubated with
CPP for 20 h (Fig. 1B).
Statistical analyses of GC-MS and LC-MS data were con-
ducted as reported previously (32). The processing parameters
of MetAlign for GC-MS data were set to analyze scans 1340–
16,000 (corresponding to a retention time of 2.32–28.05 min)
with a maximum amplitude of 3.5 ϫ 108. The parameters for
LC-MS data were set to analyze scans 70–2620 (corresponding
to a retention time of 1.4–49.73 min) with a maximum ampli-
tude of 35,000.
Product Analysis of CDS with DMAPP—The conversion of
CPP into chrysanthemol suggested that CDS supplied with
DMAPP should also produce chrysanthemol. However, no for-
RESULTS
Sequence Determination of Full-length cDNA of CDS—In ear- mation of chrysanthemol was detected when CDS was assayed
lier work, the start codon of pyrethrum CDS was not deter- with 2 mM DMAPP for 2 h (8) or upon longer incubation for 6,
mined (8). This motivated us to study the 5Ј end sequence of 12, and 24 h.
36328 JOURNAL OF BIOLOGICAL CHEMISTRY
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Bifunctional Irregular Terpene Synthase
FIGURE 2. GC-MS product analyses of reactions of purified CDS with CPP and/or DMAPP as substrate. A, CDS turnover rate to produce chrysanthemol
when CPP was provided as a substrate. The incubation time was 20 h. B, product analysis of CDS incubated with different concentrations of DMAPP for 96 h
measuring both the intermediate diphosphate and product alcohol. 1.Eϩ06, 1 ϫ exponent of 10 (6) ϭ 1,000,000. C, inhibition of CDS chrysanthemol synthase
activity by the substrate DMAPP. In these assays, both CPP and DMAPP were substrates, but CPP concentration was kept at 150 ␮M and DMAPP concentration
varied from 0–600 ␮M. The incubation time was 20 h. Data points with the same letter (a, b, c, or d) are not significantly different (p Ͻ 0.05). Error bars indicate
S.E. (n ϭ 3).
We then decided to prolong the assay time to 96 h and low- tion increased with DMAPP concentrations up to the tested
ered the DMAPP concentration. First we made sure that CDS maximum of 600 ␮M (Fig. 2B, top panel). To check non-enzy-
was active during the 96-h assay. CDS was incubated with 400 matic conversion of CPP into chrysanthemol, control reactions
␮M CPP, and chrysanthemol production was checked every without enzymes were performed at CPP concentrations rang-
24 h. A linear correlation was found between chrysanthemol ing from 2–80 ␮M. This range covered the CPP production in
production and assay hours with R2 ϭ 0.995 (data not shown), 96-h assays, which produced a maximum of 25 ␮M CPP with
indicating that CDS was fully active during this 96-h assay. The DMAPP. However, no chrysanthemol was detected in any of
DMAPP concentration was then lowered from 2000 to 600 ␮M, the control reactions.
which is the previously published Km value of CDS for DMAPP
The relationship between DMAPP substrate concentra-
to produce CPP (8). Fig. 1B shows that, under those modified tion and lavandulol or LPP production was very similar to
assay conditions, chrysanthemol, as well as lavandulol, were chrysanthemol and CPP (Fig. 2B, bottom panel). Lavandulol
detected in the 96-h assay. However, by incubating the same production was highest with 150 ␮M DMAPP and decreased
water phase with alkaline phosphatase, significant concentra- with higher concentrations of DMAPP, whereas LPP pro-
tions of CPP and LPP were also found in the assays. Clearly, a duction increased with DMAPP concentrations up to the
quantitative approach was necessary to establish the balance in 600 ␮M tested maximum.
the production of chrysanthemyl and lavandulyl diphosphates
Chrysanthemol Production with CPP Is Inhibited by
and alcohols. For this, a series of DMAPP concentrations rang- DMAPP—The above experiments suggested that higher con-
ing from 10–600 ␮M were assayed with CDS for 96 h, and both centrations of DMAPP substrate caused inhibition of chrysan-
terpene alcohol and phosphate production was determined themol production. We therefore assayed CDS activity with
(Fig. 2B). Chrysanthemol production increased with DMAPP both CPP and DMAPP as substrates in the same assay tube. In
concentration up to 300 ␮M (Fig. 2B, top panel). Further all assays, CPP concentration was kept constant at 150 ␮M
increase of DMAPP concentration led to a decrease of chrysan- (around the Km value of CDS for CPP), whereas DMAPP con-
themol but a strong further increase of CPP production. More centrations varied from 0–600 ␮M. In general, addition of
chrysanthemol than CPP was detected, with DMAPP concen- DMAPP to the assays caused a decrease of chrysanthemol pro-
trations ranging from 40–150 ␮M (Fig. 2B, top panel). The duction, and this effect was significant from 75 ␮M upward (Fig.
DMAPP concentration required for half-maximal activity to 2C). Chrysanthemol production was not inhibited when 10–40
produce chrysanthemol was ϳ100 ␮M, and, at that concentra- ␮M DMAPP was added.
tion on a molar basis, at least twice as much chrysanthemol was
Subcellular Localization of CDS—The N-terminal sequence
formed compared with CPP. On the other hand, CPP produc- of CDS was predicted to serve as a targeting signal to plastids
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Bifunctional Irregular Terpene Synthase
FIGURE 4. Shown are GC-MS chromatograms obtained by dynamic head-
space trapping of cut leaves of empty vector control (A) and CDS-expressing
(B) tobacco plants.
FIGURE 3. Subcellular localization of CDS using transient expression of
GFP fusions in tobacco protoplasts. CDS_TAR-GFP, the putative CDS target-
ing signal (first 54 amino acids) fused to GFP. CDS-GFP, the complete CDS
fused to GFP.
Sequence Alignment of CDS and Related Bifunctional
Enzymes—To indicate amino acid sequences potentially re-
sponsible for hydrolysis activity in CDS, we compared the
amino acid sequence among CDS, FDS, and the other two
bifunctional short-chain terpene synthases known so far,
myrcene synthase from bark beetle (I. pini) (IpMS) and
farnesene synthase from apple (M. domestica) (MdFS) (Fig. 6).
For FDS, CDS, and IpMS, there are two conserved DDXXD
domains, whereas MdFS only contains one. However, the
sequence for the second aspartate-rich motif in CDS contains
the rare mutation NDXXD, with asparagine replacing the first
aspartate of the DDXXD motif. Interestingly, this Asp 3 Asn
replacement also occurs in IpMS, SQS, and PTS. For SQS and
PTS it is the only “conserved” domain with short-chain terpene
synthases (Fig. 6).
(8). To test the prediction, we analyzed the targeting with gene
fusions to GFP. The gene fragment corresponding to the first 54
amino acids or the whole cDNA without the stop codon were
fused to GFP and transferred to tobacco (N. tabacum) proto-
plasts. Transient expression of the fused GFP in tobacco cells
showed that both the N-terminal signal peptide and the com-
plete CDS protein targeted GFP to plastids (Fig. 3).
Headspace Emissions of Transgenic Tobacco Overexpressing
CDS—To verify the function of CDS in vivo, its cDNA was
cloned under the regulation of the chrysanthemum RbcS1 pro-
moter into a binary vector and used to transform tobacco
plants. Strong emissions of chrysanthemol and lower emissions
of lavandulol, similar to the in vitro ratios of volatile products of
CDS, were detected in the headspace of transgenic tobacco
plants expressing CDS (n ϭ 9) but not in control tobacco plants
(n ϭ 3), which were transformed with the empty vector lacking
Mutagenesis of the Asparagine Abolishes Terpene Synthase
and Prenyltransferase Activities—To evaluate the importance
of the asparagine in the aspartate-rich motif mentioned above,
we generated two site-directed mutants of CDS, N283D, and
N283G, replacing the asparagine with either aspartate or gly-
cine. Both the terpene synthase and prenyltransferase activities
of CDS were completely lost in both mutants. Neither CPP nor
chrysanthemol were detected in the enzyme assays when the
mutants were used (Fig. 7).
CDS (Fig. 4). Chrysanthemol was emitted at levels of 0.12–0.16
Ϫ1 Ϫ1
␮g h
g
FW. Transgenic tobacco plants were shorter and
leaf color was lighter compared with empty vector control
plants.
Non-volatile Profile of Transgenic Tobacco Overexpressing
CDS—To study whether there is also accumulation of non-vol-
atile derivative(s) of chrysanthemol and/or lavandulol, we ana-
lyzed the non-volatile metabolites in transgenic (n ϭ 3) and
control (n ϭ 3) plants using a non-targeted approach (Fig. 5A).
DISCUSSION
Although pyrethrins represent the economically most
The newly produced compound that was most abundant, as important and widely used natural pesticide, not much is
determined by UV absorption (220 nm) resulting from the dou- known about their biosynthesis at the genetic or enzymatic lev-
ble bond of the compounds, showed up as m/z of 803.3498 and els. Only the genes involved in the first and last step of the
eluted at a retention time of 45.93 min. In control plants, this biosynthetic pathway have been cloned (8, 10). CDS has been
compound was not detected. The non-volatile product was reported to catalyze the first committed step of pyrethrin bio-
putatively identified as chrysanthemol conjugated to malonyl synthesis by joining two molecules of DMAPP to produce CPP
glucose by comparing its mass spectrum to that of geranoyl-6- (8), but the recently evolved enzyme also has roles in other
O-malonyl-␤-D-glucopyranoside, which was identified by composite plant species that do not produce pyrethrins (12, 13).
NMR in geraniol synthase-expressing maize (Fig. 5B and Ref. Interestingly, our in vitro and in vivo results demonstrate that
32).
CDS also catalyzes the subsequent conversion of CPP to chry-
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Bifunctional Irregular Terpene Synthase
FIGURE 5. LC-MS analysis of leaf extracts of empty vector and CDS-expressing tobacco plants. A, negative mode LC-MS chromatograms of aqueous
methanol extract of leaves of an empty vector tobacco plant (a) and a CDS-expressing tobacco plant (b). The most significantly different compound eluting at
45.93 min is indicated in the box, and it was only detected in CDS-expressing tobacco plants but not in empty vector control plants. B, the MS spectrum of this
significantly different compound (a) and the inset (b) show the schematic of its collision-induced fragmentation. The compound was putatively identified as
chrysanthemol conjugated to malonyl glucose.
santhemol, which furthers our understanding of the monoter- gent, Tween 20, to promote the release of the hydrophobic
pene branch of pyrethrin biosynthesis that occurs in pyrethrum product; a low substrate concentration, common to plants; and
glandular trichomes (9).
a long incubation time to observe products in this notoriously
CDS was active with DMAPP and CPP as substrates. In both slow reaction in vitro. In former assays, the lack of an organic
cases, the production of the monoterpene alcohols chrysanthe- layer to capture the volatile product may have prevented
mol and lavandulol was detected. Rivera et al. (8) reported ear- detecting chrysanthemol formation. The standard presence of
lier that CPP and LPP were the final products of CDS. We used Tween 20 in our assays may have increased the CDS activity to
a typical protocol for the activity analysis of terpene synthases, produce chrysanthemol, as reported for SQS and PTS. For SQS,
which produce volatile and hydrophobic products (28, 34), Tween 80 stimulated its activity 10- to 20-fold, and Tween 20
whereas earlier protocols were on the basis of the activity anal- showed similar stimulating effects (36). PTS also requires a
ysis of diphosphate synthases, which produce non-volatile and detergent for maximal activity because the presence of Tween
hydrophilic products (13, 35). Although we used similar reac- 80 has been reported to result in almost 20,000-fold higher
tion conditions, the combination of the following factors in our activity compared with PTS activities reported previously (19).
Ϫ3
assays could contribute to the observed differences: a pentane
Although the turnover from CPP (3.3 ϫ 10 minϪ1) or
overlay to capture the volatile alcohol; the addition of deter- DMAPP (ϳ 1 ϫ 10Ϫ4 minϪ1) to chrysanthemol was highly inef-
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Bifunctional Irregular Terpene Synthase
FIGURE 6. Alignment of chrysanthemol synthase with two other bifunctional short-chain terpene synthases and FDS, the ancestor of CHS. AaFDS, FDS
from sagebrush (Artemisia annua), which is also in the Asteraceae family as pyrethrum (T. cinerariifolium); TcCHS, bifunctional chryanthemyl diphosphate/
chrysanthemol synthase from pyrethrum; IpMS, bifunctional geranyl diphosphate/myrcene synthase from bark beetle (I. pini); MdFS, bifunctional FDS/
farnesene synthase from apple (M. domestica). The boxes indicate the conserved DDXXD domain of prenyltransferases and terpene synthases. SQS and PTS
have very limited similarity to short-chain prenyltransferases or terpene synthases, with only the motif GXXXQXXDDXXD at the second DDXXD region in FDS
partially conserved (DD ϭ Ni) (8).
ficient in the in vitro assays, the production of chrysanthemol nous cofactor that potentiates CDS activity (38, 39). Our exper-
by CDS was highly efficient in vivo. Chrysanthemol was emitted iments and existing literature seem to best support the latter
from leaves of transgenic tobacco overexpressing CDS (Fig. 4) possibility for the reasons given below.
at rates of 120–160 ng hϪ1 gϪ1 FW. This rate was on the high
First, any free aspecific phosphatase in the chloroplast
end of the emission rates of other overexpressed terpenes in stroma would be expected to also lead to a hydrolysis of
tobacco leaves. In tobacco plants overexpressing three mono- DMAPP, GPP, and geranylgeranyl diphosphate. The fact that
terpene synthases (CaMV35S promoter), the total level of addi- plants possess pools of these substrates freely accessible to
tional monoterpenes emitted from leaves reached up to 30 ng other enzymes and do not have nonspecific production/emis-
Ϫ1 Ϫ1
h
g
FW (37), and plants overexpressing patchoulol syn- sion of prenol, geraniol, or geranylgeraniol suggests that such
thase emitted patchoulol at levels of 50–100 ng hϪ1 gϪ1 FW (6). phosphatases are normally not in the same cellular compart-
The high efficiency of chrysanthemol production in plants ment with prenylphosphate synthases and their products.
suggests the possible presence of an endogenous phosphatase
Second, our in vitro results showed that the in vivo precursor
that efficiently hydrolyzes CPP or the presence of an endoge- diphosphate concentrations of DMAPP in plants are too low to
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Bifunctional Irregular Terpene Synthase
FIGURE7. ReactionscatalyzedbyCDSanditsmutantsCDS-N283DandCDS-N283G. A, GC-MSchromatogramsoftheproductsofpurifiedenzymesassayed
with CPP. B, GC-MSchromatogramsofthe productsinthe organic phase (for chrysanthemol production) of theassayswhen DMAPP was provided assubstrate.
C, GC-MS chromatograms of the products in the water phase (for CPP production) of the assays when DMAPP was provided as substrate.
generate free CPP as a main product. CDS favored the synthesis tase because the lysate from empty vector cells did not convert
of chrysanthemol over CPP when the DMAPP concentrations CPP into chrysanthemol under the same assay conditions. This
were lower than 300 ␮M (Fig. 2B). This low DMAPP concentra- suggests that the enzyme loses activity or an activity-promoting
tion range is most likely the range encountered by CDS in vivo. component during purification. Copurified bands are not visi-
DMAPP concentrations in leaves have been determined for ble on SDS-PAGE gels, however (data not shown). We then
several plant species. Compared with the isoprene-emitting tested the effects of adding protein (BSA), all known mineral
species cottonwood (40) and oak (41), leaf DMAPP concentra- cofactors (Murashige and Skoog salts), and NADPH to the
tions in non-emitting species, such as Arabidopsis, are 6–10 enzyme assays (18), but the activity of purified CDS was not
times lower (42). Considering that tobacco is also a non-emit- improved.
ting species (43), leaf DMAPP concentrations in tobacco are
CDS shares several characteristics with the long-chain ter-
expected be similar to those in Arabidopsis at an estimated pene synthases SQS and PTS, which both catalyze intermolec-
Ϫ1
concentration of around 10 pmol mg FW (42). Then, the ular c1Ј-2-3 cyclopropanation reactions. All three synthases are
DMAPP level in tobacco plastids, the cell compartment to bifunctional enzymes, functioning as prenyltransferases in the
which CDS is targeted (Fig. 3 and Ref. 13), can be estimated to first reaction and as terpene synthases in the second reaction.
Ϫ1
be around 30 ␮M, assuming a regression coefficient of 1 g ml
However, the cyclopropane ring structure remains in the final
for leaf fresh weight to leaf volume (44), a chloroplast volume of product of CDS but not in those of SQS or PTS. The overall
20% of total cell volume in mature leaf cells (45), and 60% of enzyme activities of all three enzymes are inhibited by high
DMAPP to be found in the chloroplast (46).
substrate concentrations but the production of the intermedi-
Third, inactive GDSs have been shown before to be activated ate diphosphates is not. For example, the production of squa-
by the formation of a heterodimer with geranylgeranyldiphos- lene by SQS has been shown to be inhibited by FPP concentra-
pate synthase (39, 47, 48). The small subunits are usually inac- tions higher than 100 ␮M (18, 36), but the production of the
tive in the in vitro assays, and the big subunits are either inactive intermediate presqualene diphosphate is not inhibited by high
(35, 49) or function as well as homodimeric geranylgeranyl FPP concentrations (18). Similarly, PTS has been reported to be
diphosphate synthases (39, 47, 48). Two of four geranylgeranyl significantly inhibited at geranylgeranyl diphosphate concen-
diphosphate synthase-like enzymes of tobacco could interact in trations above 100 ␮M (19), although each of the separate reac-
tobacco with the small subunit from snapdragon (Antirrhinum tions of PTS showed Michaelis-Menten behavior (16). In our
majus) (48). Therefore, we propose that CDS may possibly also experiments, DMAPP concentrations of higher than 300 ␮M
form heterodimers with geranylgeranyl diphosphate synthase- reduced total chrysanthemol production in favor of CPP (Fig.
like large subunits of tobacco (and pyrethrum) that significantly 2B, top panel), whereas lavandulol production was reduced by
promote its activity.
DMAPP at concentrations of higher than 150 ␮M (Fig. 2B, bot-
Fourth, our tests on bacterial lysates also suggested the pos- tom panel). CPP production was not inhibited by DMAPP con-
sible presence of a cofactor. The enzyme activity of the lysate centrations of up to of 600 ␮M (Fig. 2B, top), in good agreement
was about 10- to 40-fold higher than that of purified CDS, but with earlier data (8). This indicates that DMAPP inhibits the
this activity could not be explained by a nonspecific phospha- overall CDS activity by inhibiting the second but not the first
DECEMBER 26, 2014•VOLUME 289•NUMBER 52
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Bifunctional Irregular Terpene Synthase
reaction. This was confirmed by providing both DMAPP and an evolutionary bottleneck if two enzymes are needed simulta-
CPP to CDS, resulting in a significant reduction of chrysanthe- neously to generate a useful product like chrysanthemol. The
mol production when DMAPP concentration was higher than bifunctional activity solves this issue and serves the needs of a
75 ␮M (Fig. 2C). These results suggest that DMAPP and CPP are plant that has not yet evolved a secondary purpose for CPP
converted in the same active site. This was subsequently con- substrates.
firmed by the loss of both enzyme activities after mutagenesis of
the NDXXD motif into either DDXXD or GDXXD. The bifunc-
tional AaFDS from apple similarly uses the same active site for
both activities, as demonstrated by the loss of both enzyme
activities when the first and last aspartate of DDXXD was
mutated into ADXXA/D (23).
Acknowledgments—We thank Prof. Willem J. H. van Berkel, Prof.
Marcel Dicke, and Prof. Harro J. Bouwmeester for critical and instruc-
tive comments on the manuscript.
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The alignment of amino acid sequences of FDS, CDS, and the
other bifunctional terpene synthases (Fig. 6) showed that the
Asp 3 Asn replacement in the second aspartate-rich motif in
CDS is also present in another short-chain bifunctional
enzyme, myrcene synthase, as well as in SQS and PTS. This
replacement has been suggested to be not essential for cyclo-
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molecular cyclopropanation of the distal double bond in gera-
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loss of both the CPP and chrysanthemol synthase activities.
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