The biosynthetic origin of irregular monoterpenes in lavandula: Isolation and biochemical characterization of a novel cis-prenyl diphosphate synthase gene, lavandulyl diphosphate synthase

Article abstract of DOI:10.1074/jbc.M112.431171

Lavender essential oils are constituted predominantly of regular monoterpenes, for example linalool, 1,8-cineole, and camphor. However, they also contain irregular monoterpenes including lavandulol and lavandulyl acetate. Although the majority of genes responsible for the production of regular monoterpenes in lavenders are now known, enzymes (including lavandulyl diphosphate synthase (LPPS)) catalyzing the biosynthesis of irregular monoterpenes in these plants have not been described. Here, we report the isolation and functional characterization of a novel cis-prenyl diphosphate synthase cDNA, termed Lavandula x intermedia lavandulyl diphosphate synthase (LiLPPS), through a homology-based cloning strategy. The LiLPPS ORF, encoding for a 305-amino acid long protein, was expressed in Escherichia coli, and the recombinant protein was purified by nickel-nitrilotriacetic acid affinity chromatography. The approximately 34.5-kDa bacterially produced protein specifically catalyzed the head-to-middle condensation of two dimethylallyl diphosphate units to LPP in vitro with apparent K_m and k _cat values of 208 ± 12 μM and 0.1 s^-1, respectively. LiLPPS is a homodimeric enzyme with a sigmoidal saturation curve and Hill coefficient of 2.7, suggesting a positive co-operative interaction among its catalytic sites. LiLPPS could be used to modulate the production of lavandulol and its derivatives in plants through metabolic engineering.

Full text of DOI:10.1074/jbc.M112.431171

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 9, pp. 6333–6341, March 1, 2013
©
2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
The Biosynthetic Origin of Irregular Monoterpenes in
Lavandula
ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF A NOVEL cis-PRENYL
□
S
*
DIPHOSPHATE SYNTHASE GENE, LAVANDULYL DIPHOSPHATE SYNTHASE
Received for publication, October 26, 2012, and in revised form, January 6, 2013 Published, JBC Papers in Press, January 10, 2013, DOI 10.1074/jbc.M112.431171
1
2
Zerihun A. Demissie , Lauren A. E. Erland, Mark R. Rheault, and Soheil S. Mahmoud
From the Department of Biology, University of British Columbia, Kelowna, British Columbia V1V 1V7, Canada
Background: Lavandula accumulate irregular monoterpenes of unknown biosynthetic origin.
Results: We cloned a cis-prenyl diphosphate synthase (cis-PDPS) that produces precursor for irregular monoterpenes in
lavenders.
Conclusion: Unlike other plants that utilize trans-PDPSs, Lavandula employ a cis-PDPS to initiate the biosynthesis of irregular
monoterpenes.
Significance: This is the first report of the involvement of a cis-PDPS in irregular monoterpene biosynthesis.
3
Lavender essential oils are constituted predominantly of reg- phate (IPP) and dimethylallyl diphosphate (DMAPP) predom-
ular monoterpenes, for example linalool, 1,8-cineole, and cam- inantly synthesized through the 2-C-methyl-D-erythritol
phor. However, they also contain irregular monoterpenes 4-phosphate metabolic pathway in plants (1). En route to
including lavandulol and lavandulyl acetate. Although the monoterpene synthesis IPP and DMAPP, or two DMAPP units,
majority of genes responsible for the production of regular can be condensed to form structurally diverse branch point C
1
0
monoterpenes in lavenders are now known, enzymes (including precursor molecules (2). The diversity of these molecules arises
lavandulyl diphosphate synthase (LPPS)) catalyzing the biosyn- from the capability of prenyl diphosphate synthase enzymes
thesis of irregular monoterpenes in these plants have not been (PDPSs) to condense isoprenes in head-to-tail or non–head-to-
described. Here, we report the isolation and functional charac- tail orientations (3, 4) and outcomes with cis or trans geometric
terization of a novel cis-prenyl diphosphate synthase cDNA, configurations (5). For example, the head-to-tail coupling of
termed Lavandula x intermedia lavandulyl diphosphate syn- two isoprene units to geranyl diphosphate (C ; GPP) or its
1
0
thase (LiLPPS), through a homology-based cloning strategy. cisoid isomer neryl diphosphate (C ; NPP) is catalyzed by gera-
1
0
The LiLPPS ORF, encoding for a 305-amino acid long protein, nyl diphosphate synthase (GPPS) and neryl diphosphate syn-
was expressed in Escherichia coli, and the recombinant protein thase (NPPS), respectively. The non–head-to-tail condensa-
was purified by nickel-nitrilotriacetic acid affinity chromatog- tion of isoprene units to lavandulyl diphosphate (C ; LPP) and
1
0
raphy. The approximately 34.5-kDa bacterially produced pro- chrysanthemyl diphosphate (C ; CPP), on the other hand, is
1
0
tein specifically catalyzed the head-to-middle condensation of catalyzed by lavandulyl diphosphate synthase (LPPS) and chry-
two dimethylallyl diphosphate units to LPP in vitro with appar- santhemyl diphosphate synthase (CPPS), respectively (Fig. 1).
؊
1
ent K and k values of 208 ؎ 12 ␮M and 0.1 s , respectively. The final stage of monoterpene synthesis, transforming and
m
cat
LiLPPS is a homodimeric enzyme with a sigmoidal saturation elaborating the different precursor molecules to monoter-
curve and Hill coefficient of 2.7, suggesting a positive co-opera- penes, is catalyzed by another group of enzymes called mono-
tive interaction among its catalytic sites. LiLPPS could be used terpene synthases (mTPSs).
to modulate the production of lavandulol and its derivatives in
plants through metabolic engineering.
The head-to-tail condensation of IPP and DMAPP and the
enzymes catalyzing these reactions are the most common in
nature. Thus, monoterpenes derived from GPP and NPP such
as linalool, 1,8-cineole, limonene, and so forth, are widely dis-
Monoterpenes, the C10 class of the isoprenoids, are derived tributed and are referred to as “regular monoterpenes.” Subse-
from the universal terpene building blocks isopentenyl diphos- quently, their biosynthetic route is well established, and cDNAs
encoding for GPPS (6–10), NPPS (11), and mTPSs capable of
*
This work was supported through grants or in-kind contributions by the
University of British Columbia Okanagan, Investment Agriculture Founda-
tion of British Columbia, National Research Council Plant Biotechnology
Institute through the NAPGEN program, and Genome British Columbia (to
S. S. M.) and by the Natural Sciences and Engineering Research Council of
Canada (to S. S. M. and M. R. R.).
S
This article contains supplemental Figs. S1–S3 and Table S1.
Supported by the Pacific Century Graduate Scholarships from the province
of British Columbia through the Ministry of Advanced Education.
To whom correspondence should be addressed: Dept. of Biology, University
of British Columbia, 3333 University Way, Kelowna, BC V1V 1V7, Canada.
E-mail: soheil.mahmoud@ubc.ca.
3
The abbreviations used are: IPP, isopentenyl diphosphate; CIP, calf intestinal
alkaline phosphatase; CPP, chrysanthemyl diphosphate; CPPS, CPP syn-
thase; dDPPS, dehydrodolichol diphosphate synthase; DMAPP, dimethy-
lallyl diphosphate; EO, essential oil; EST, expressed sequence tag; FPP,
farnesyl diphosphate; FPPS, FPP synthase; GGPPS, geranylgeranyl diphos-
phate synthase; GPP, geranyl diphosphate; GPPS, GPP synthase; LiCINS, L. x
intermedia 1,8-cineole synthase; LiLPPS, L. x intermedia LPPS; LPP, lavandu-
lyl diphosphate; LPPS, LPP synthase; mTPS, monoterpene synthase; NPP,
neryl diphosphate; NPPS, NPP synthase; PDP, prenyl diphosphate; PDPS,
PDP synthase.
□
1
2
MARCH 1, 2013•VOLUME 288•NUMBER 9
JOURNAL OF BIOLOGICAL CHEMISTRY 6333

The Biosynthesis of Irregular Monoterpenes in Lavenders
FIGURE 1. Schematic representation of IPP and DMAPP coupling reactions catalyzed by typical trans- and cis-PDPS family members. The newly
identified enzyme and the reaction it catalyzed are circled (adapted from Thulasiram et al. (3)). CPP, chrysanthemyl diphosphate; CPPS, chrysanthemyl
diphosphatesynthase;DMAPP, dimethylallyldiphosphate;IPP, isopentenyldiphosphate;GPP, geranyldiphosphate;GPPS, geranyldiphosphatesynthase;LPP,
lavandulyl diphosphate; LiLPPS, L. x intermedia lavandulyl diphosphate synthase; LinS, linalool synthase; NPP, neryl diphosphate; and NPPS, neryl diphosphate
synthase.
transforming GPP and/or NPP into core monoterpenes have pests and are subsequently used in artificial pheromone prepa-
been described from diverse plant species (12, 13). GPPS, a rations to disrupt the mating behaviors of economically impor-
trans-PDPS, and NPPS, a cis-PDPS, elongate the linear prenyl tant pests (23, 24). Yet, little is known about the biosynthetic
chain by coupling DMAPP with IPP through a chain elongation pathways leading to these monoterpenes and their derivatives.
(
c1Ј- 4) reaction to generate GPP and NPP, respectively (3). Only two cDNAs encoding for CPPSs, both trans-PDPS family
Despite catalyzing the same reaction, however, GPPSs and members, have been isolated and functionally characterized
NPPS are more distantly related to one another than they are to from Chrysanthemum cinerariaefolium (20) and Artemisia tri-
PDPSs of similar geometric outcomes. GPPSs share high dentate ssp. spiciformis (25) so far. CPPSs catalyze predomi-
sequence similarity with trans-PDPSs catalyzing the higher nantly the cyclopropanation (c1Ј-2-3) reaction in which the
order terpene precursors and are distinguished by two aspar- non–head-to-tail condensation of two DMAPP molecules gen-
tate-rich conserved motifs, DDX D and (N/D)DXXD. These erates the pyrethrin branch point intermediate, CPP. In addi-
2–4
motifs serve as a substrate and divalent metal ion cofactor, often tion to their major product, the A. tridentate CPPS and its chi-
2
ϩ
Mg , binding sites for the carbocation rearrangement-medi- meric derivatives were found to catalyze the non–head-to-tail
ated condensation reactions (2, 5). A third conserved motif, the coupling of two DMAPP molecules through branching (c1Ј-2)
CXXXC motif (where X is any hydrophobic residue), is present to generate LPP (3, 4). LPP is the branch point precursor of
in heterodimeric PDPSs (14). Heterodimeric PDPSs, like the monoterpenes with head-to-middle condensed PDP back-
GPPSs cloned from Mentha x piperita (8), Salvia miltiorrhiza bones, such as lavandulol and lavandulyl acetate (19). It is also
(
15) etc., are enzymes constituted of two subunits (large and the source of the lavandulyl side group of sophoraflavanone G
small) which interact through the CXXXC conserved motif to in Sophora flavescens Ait (26) that determines their antitumor
be catalytically active. NPPS and other cis-PDPSs, on the other (27) and phospholipase C␥1 inhibition properties (28). To our
hand, do not necessarily retain the above motifs but share five knowledge a wild type LPPS gene has not been described from
conserved regions designated as Regions I–V. In particular, the plants or other organisms.
aspartate residue in Region IV and the glutamate residue of
Region V are catalytically essential (16, 17).
Essential oils (EOs) of the genus Lavandula (lavenders) are
constituted primarily of a few “regular” monoterpenes and their
PDPSs catalyzing non–head-to-tail coupling reactions, and derivatives. For example, the economically important EOs
monoterpenes derived from LPP and CPP such as lavandulol derived from L. angustifolia and L. x intermedia species, con-
and pyrethrins, respectively, are encountered less frequently in tain large amounts of linalool, linalool acetate, and 1,8-cineole.
nature (18–20). Irregular monoterpenes, like pyrethrins, are Consequently, these oils are industrially utilized as ingredients
the major ingredients in leading botanical and EPA-certified of various cosmetic and antiseptic products (29). The biosyn-
insecticides (21, 22). Lavandulol and its ester derivative lavan- thetic pathway leading to these monoterpenes from IPP and
dulyl acetate were identified in pheromones of major insect DMAPP has been defined both experimentally (30–32) and
6
334 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 288•NUMBER 9•MARCH 1, 2013

The Biosynthesis of Irregular Monoterpenes in Lavenders
TABLE 1
Oligonucleotides used in this study
Primers
Sequences
LiLPPS set I
F: 5Ј-CACTCATATGGCATTCCTGCAGTTACC-3Ј
R1: 5Ј-CTCTTCATGAATTCCTTTTCGTTCACCG-3Ј
F: 5Ј-CACTCATATGGCATTCCTGCAGTTACC-3Ј
R2: 5Ј-AATTCTCTTCATGAATTCCTTTTCGTTCACCG-3Ј
LiLPPS set II
through in silico analysis of Lavandula EST databases (30, 31, directions. The putative LiLPPS ORF was amplified with the
3). In this regard, four cDNAs encoding for mTPSs that trans- full-length cloning primer sets (Table 1) and iProof™ High-Fi-
form GPP and NPP into linalool, 1,8-cineole, limonene, and delity DNA Polymerase (Bio-Rad). The PCR program used was
3
␤
-phellandrene in vitro have been cloned from L. angustifolia 95 °C for 5 min, followed by 37 cycles of 95 °C for 1 min, 58 °C
and L. x intermedia species (30–32). However, lavender EOs, for 30 s, and 72 °C for 1 min, and a 5-min final extension at
particularly L. x intermedia species, also contain the irregular 72 °C. The amplified fragments were cloned into the NdeI/
monoterpene lavandulol and its ester derivative lavandulyl ace- EcoRI sites of the pET41b(ϩ) expression vector and expressed
tate (34) whose biosynthetic origin has not yet been investi-
gated. Here, we report the isolation and functional character-
ization of a novel cis-PDPS cDNA encoding for LPPS, an
enzyme that condenses two DMAPP molecules to generate the
lavandulol branch point precursor LPP in vitro, from a L. x
intermedia oil gland cDNA library.
in Escherichia coli BL21(DE3) strain (EMD Chemicals, Darm-
stadt, Germany) following previously described procedures (30,
3
1). Except for the bind buffer that was slightly modified (0.5 M
NaCl, 50 mM KH PO , pH 8.0) the procedure described in the
2
4
aforementioned papers was followed to enrich the expressed
protein. Sequence information for LiLPPS was deposited in the
NCBI public data bank with the accession number JX985358.
LiLPPS Product Assay and EO Constituent Identification—
Initial in vitro enzyme activity assays were performed in a
EXPERIMENTAL PROCEDURES
LiLPPS Candidate Selection—Our group recently reported
the construction of a cDNA library and its corresponding anno-
tated expressed sequence tag (EST) database from L. x interme-
dia cv. Grosso secretory cells of oil glands, tissues specialized
for EO biosynthesis and secretion (31). All PDPS homologs in
the database were retrieved by searching the EST database
using the strings “diphosphate synthase” and “pyrophosphate
synthase.” We then synchronized the search results and manu-
ally excluded EST homologs known to be involved in regular
terpene biosynthesis including GPPS, trans-farnesyl diphos-
phate synthase (trans-FPPS), and trans-geranylgeranyl diphos-
phate synthase (trans-GGPPS), and PDPS homologs that are
not involved in terpene biosynthesis. This led us to acquire a
novel cis-PDPS homolog contig that was later determined to be
L. x intermedia lavandulyl diphosphate synthase (LiLPPS). The
transcriptional expression pattern of this contig and previously
described mTPSs throughout L. x intermedia cv. Grosso flower
developmental stages was assessed by microarray analysis using
the Agilent oligonucleotide-based microarray technology,
through services provided by the University Health Network
Microarray Centre (Toronto, ON, Canada). The three floral
developmental stages were unopened buds, anthesis, and
mature flowers in which 30% of the buds were in bloom (for
photographic description see Ref. 33). After validating the
microarray data using standard PCR, we selected the contig for
further detailed analysis.
5
00-␮l reaction volume containing the assay buffer (50 mM
Tris-HCl, 5% glycerol, 1 mM MnCl , 1 mM MgCl , pH 8.0), 1 mM
2
2
DTT, 40 ␮M IPP, and 40 ␮M DMAPP (Echelon, Salt Lake City,
UT), and 2.5–50 ␮g of LiLPPS. After a 2-h incubation at 30 °C,
the reaction mix was heated at Ͼ80 °C for 10 min and kept on
ice for 2 min. Then 30 units of calf intestinal alkaline phospha-
tase (CIP; New England Biolabs) were added to the reaction
mix, overlaid by 500 ␮l of pentane and incubated overnight at
3
2.5 °C to hydrolyze the prenyl product. GPP and NPP stand-
ards (Echelon) were also hydrolyzed in the same reaction mix
and conditions, as a control of the hydrolysis reaction. The
reaction was stopped by vigorous vortexing followed by flash
freezing in liquid nitrogen and stored in a Ϫ80 °C freezer
until analyzed. Assay product identification and quantifica-
tion, and EO extraction and quantification were performed
by gas chromatography-mass spectrometry (GC-MS) following
procedures described previously (32, 35). Purified protein
extract of E. coli BL21(DE3) strain transformed with empty
pET41b(ϩ) expression vector, i.e. without insert, was also
assayed under the same conditions as a negative control.
Biochemical Assay—Biochemical characterization of LiLPPS
was performed following the procedure described above with
slight modifications: the reaction volume was reduced to 125
␮
l, 2.5 ␮g of LiLPPS was used, and assays were incubated at
3
3
7 °C for 1 h after adding CIP. Six temperature levels (25, 27.5,
0, 32.5, 35, and 37.5 °C) were tested to determine LiLPPS opti-
Cloning, Protein Expression, and Enrichment of LiLPPS—
Glandular trichome secretory cells were isolated from mature
flowers of L. x intermedia cv. Grosso plants grown at the Uni-
mum temperature, and the optimum pH was determined using
versity of British Columbia, Okanagan campus lavender field MES and MOPS buffers at pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5.
following a previously described modified glass bead abrasion The kinetic properties of LiLPPS were deduced from LPP accu-
method (31). Total RNA was extracted from 100 mg of the mulation data in assays with increasing DMAPP concentra-
tissue using an RNA extraction kit (OMEGA bio-tek) and tions (6.25, 12.5, 25, 50, 100, 150, 200, 300, 450, and 600 ␮M) at
reverse transcribed in a reaction containing the oligo(dT) the optimal conditions. Prism software version 5.0d (GraphPad
primer (Fisher Scientific) and M-MuLV reverse transcriptase Software, Inc., La Jolla, CA) was used to fit the data into a sig-
enzyme (New England Biolabs) following the manufacturer’s moidal substrate concentration-dependent enzyme response
MARCH 1, 2013•VOLUME 288•NUMBER 9
JOURNAL OF BIOLOGICAL CHEMISTRY 6335

The Biosynthesis of Irregular Monoterpenes in Lavenders
x
x
x
curve (y ϭ a*b /(c ϩ b )), and to calculate the V , K , and composition in this tissue. However, given the fact that GPP is
max
m
k
values. Substrate specificity of the enzyme was determined the preferred substrate for regular monoterpenes (11, 36) and
cat
from the type and amount of reactants consumed versus prod- typical sesquiterpenes synthesized from cis-FPP (17) were not
uct accumulated in assays containing a constant IPP concentra- detected in the L. x intermedia EO (supplemental Fig. S1), the
tion (150 ␮M) combined with various DMAPP levels (6.25–200 identification of NPPS and cis-FPPS homologs was unforeseen.
␮
M) under the optimized conditions. We also tested LPP as a In addition, the results of our transcript-profiling experiment
potential substrate for L. x intermedia 1,8-cineole synthase indicated that the transcriptional expression of this EST paral-
LiCINS), an enzyme that primarily produced 1,8-cineole from leled those of other lavender mTPSs (31) and was developmen-
(
both GPP and NPP (31).
tally regulated in L. x intermedia flowers (data not shown).
Phylogenetic Relationship Analysis—LiLPPS was aligned Therefore, we decided to further investigate this contig which
with cis-PDPSs isolated from tomato using the default param- was later determined to be LiLPPS. The ORF of LiLPPS was
eters of the ClustalW alignment tool available at the EBI plat- expressed in E. coli BL21(DE3) cells for further analysis.
form. The phylogenetic relation of LiLPPS with other PDPSs
The ORF of LiLPPS is 918 nucleotides long encoding a 305-
was constructed using the Jukes-Cantor genetic distance model amino acid protein that includes a short N-terminal plastidial
and UPGMA tree building method of Geneious Tree Builder transit peptide (30 amino acids) and shares 66.4 and 66%
module (Geneious 5.0.3 software, Auckland, New Zealand).
sequence similarity with NPPS and cis-FPPS of tomato, respec-
tively. Like many other cis-PDPSs, LiLPPS lacks the two aspar-
RESULTS
tate rich trans-PDPS signature motifs DDX
D and
2
–4
Lavandula x intermedia cv. Grosso EO Composition— (N/D)DXXD but maintains all five semiconserved regions
GC-MS analysis of EO distilled from L. x intermedia cv. Grosso (I–V) that define the cis-PDPSs (Fig. 2). In particular, the cata-
floral tissues profiled the following terpenes in their respective lytically important aspartate and glutamate residues of Regions
abundance order: linalool, linalool acetate, 1,8-cineole, cam- IV and V were conserved in LiLPPS at 201 and 276 positions
phor, isoborneol, lavandulyl acetate, ␣-terpineol, ␣-bisabolol, from the N-terminal, respectively (Fig. 2). Multiple alignment
␣
-cadinol, ␤-ocimen, ␤-caryophyllene, lavandulol, limonene, of LiLPPS and its splice variant, both at the nucleotide and
␦
-carene, myrcene, geraniol, nerol, neryl acetate, and a few amino acid level, revealed that the splice variant lacks a stretch
other minor products (supplemental Fig. S1). Except for the of 22 amino acids in the middle of its ORF that included a
irregular monoterpenes lavandulol and lavandulyl acetate, portion of the conserved region III (Fig. 2). Repeated attempts
which are derived from LPP, the remaining EO mono- and ses- to amplify the splice variant from reverse transcribed total
quiterpene constituents arise from the corresponding linear mRNA isolated from L. x intermedia glandular secretory cells
precursors GPP and trans-FPP, respectively.
failed possibly due to the low abundance of the corresponding
PDPS Candidate Selection and Sequence Analysis—Search- cDNA. It is also possible that the splice variant singleton was an
ing our EST library using the string pyrophosphate synthase as artifact that resulted from sequence assembly errors during
a query identified 11 different prenyltransferase homolog uni- database construction. Therefore, only the full-length contig
genes whereas the string diphosphate synthase identified 31. was considered for further investigation.
Duplicate search results and prenyltransferases with no known
Cloning, Expression, and Functional Characterization of
involvement in terpene biosynthesis (e.g. ribose phosphate LiLPPS—The approximately 34.5-kDa recombinant LiLPPS
pyrophosphokinase and cytidine diphosphate diacylglycerol was successfully expressed in bacterial cells and enriched using
synthase) were disregarded to consolidate the search results. the nickel-nitrilotriacetic acid-agarose affinity chromatogra-
The final synchronized search result identified 25 unigenes that phy system. Incubation of the purified recombinant protein
were homologous to known PDPSs involved in terpene biosyn- with IPP and DMAPP followed by the hydrolysis of the assay
thesis (supplemental Table S1). From these sequences 12 were mix by CIP resulted in the production of lavandulol as the only
homologous to PDPSs involved in the 2-C-methyl-D-erythritol product (Fig. 3A). The identity of the product was determined
4
-phosphate pathway, eight were homologous to trans-PDPSs by comparing its mass spectrum and retention time with those
(
GPPSs, FPPSs, GGPPSs), and one was homologous to bornyl of an authentic lavandulol standard (Sigma) (Fig. 3B). Given
diphosphate synthase. The remaining four candidates were that the alkaline CIP-mediated hydrolysis of LPP is known to
homologous to cis-PDPSs. Two of these homologs corre- yield lavandulol (3, 4, 19), we concluded that LiLPPS catalyzes
sponded to dehydrodolichol diphosphate synthase (dDPPS) the synthesis of LPP from isoprene units. In this respect, the
genes, which catalyzes the prenyl chain elongation reaction to hydrolysis of GPP and NPP with CIP under the same conditions
produce the polyprenyl backbone of dolichol. The other unige- produced geraniol and nerol, respectively, as anticipated.
nes, one of which belonged to a contig with 18 EST members Hydrolysis of the control assay mixes contained only the prenyl
and the other was a singleton, exhibited significant homology to alcohol derivatives of IPP (buten-1-ol, Ͻ3-methyl-3) and
NPPS (FJ797956) and cis-FPPS (ACJ38408) from cultivated DMAPP (buten-1-ol, Ͻ3-methyl-2) (Fig. S2, A and B).
(
Solanum lycopersicum) (11) and wild tomato (Solanum habro-
Substrate Specificity and Kinetic Properties of LiLPPS—
chaites) (17), respectively. The singleton was later determined When LiLPPS was incubated with IPP alone, detectable
to be a splice variant of the contig and was disregarded.
amounts of lavandulol or any other terpene prenyl alcohols
The trans-PDPS and dDPPS homologs were anticipated to be were not identified after the alkaline hydrolysis step. We also
present in a database derived from L. x intermedia secretory observed that assay reactions containing 5 ␮M each IPP and
cells because they catalyze reactions that are consistent with EO DMAPP, or 5 ␮M DMAPP alone produced equivalent amounts
6
336 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 288•NUMBER 9•MARCH 1, 2013

The Biosynthesis of Irregular Monoterpenes in Lavenders
FIGURE 2. Multiple alignment of LiLPPS with NPPS and cis-FPPS of tomato. Bars indicate the five conserved regions (I–V), and the aspartate and glutamate
residues in Regions IV and V, respectively, are boxed. Identical amino acid residues are represented by asterisks, conserved amino acid substitutions are
represented by a semicolon, and semiconserved amino acid substitutions are represented by a period. LiLPPS (JX985358), L. x intermedia lavandulyl diphos-
phate synthase; zFPS_SOLHA (B8XA40.1), Z,Z-farnesyl diphosphate synthase from (S. habrochaites) and NPPS (NP_001234633.1), neryl diphosphate synthase
from (S. lycopersicum).
of LPP. Furthermore, lowering the concentration of DMAPP in
the assay resulted in a parallel reduction in LPP production,
although reducing the concentration or excluding IPP from the
assay had no effect. When LiLPPS was incubated with a con-
stant IPP concentration (150 ␮M) combined with an increasing
DMAPP amount (6.25–200 ␮M), the amount of LPP accumu-
2
lated correlated (R ϭ 0.99; p Ͻ 0.0001) with the amount of
DMAPP consumed whereas the IPP consumption remained
close to zero at all combinations (Fig. 4).
The optimum temperature and pH of LiLPPS were found to
be 30 °C and 8.0 (supplemental Fig. S3, A and B), respectively.
Unlike other PDPSs cloned from related plant species, the sub-
strate concentration-dependent saturation curve of LiLPPS did
not follow the standard Michaelis-Menten kinetics for single
substrate enzymes. Instead, the amount of LPP accumulated in
response to increasing DMAPP concentration levels (6.25–600
␮
M) fitted a sigmoidal saturation curve (Fig. 5), typical of two
substrate enzymes. The nonlinear regression equation V ϭ
o
H
H
H
V
*[S] /(k ϩ [S] ) was used to calculate the K and V
max
m
m
max
of LiLPPS which were determined to be 208 Ϯ 12 ␮M and 448 Ϯ
2 pmol/min, respectively. The Hill coefficient (H) value of
LiLPPS was 2.7 Ϯ 0.3 whereas the k (V /[E]) and catalytic
2
cat
max
Ϫ1
efficiency (k /K ) of the enzyme were calculated as 0.1 s
cat
m
Ϫ10 Ϫ1 Ϫ1
and 5 ϫ 10
M
s
, respectively.
LPP as a Substrate for Regular mTPSs—mTPSs involved in
the biosynthesis of regular monoterpenes in lavenders accept
both GPP and NPP as in vitro substrates. We thus examined the
ability of the recombinant LiCINS to utilize LPP as a substrate
in standard assay reactions. As anticipated, LiCINS, which pro-
duced 1,8-cineole as a major product upon incubation with
GPP and NPP (32), did not produce detectable quantities of a
product from LPP.
FIGURE 3. GC chromatogram and mass spectrum of LiLPPS product from
DMAPP after calf intestinal alkaline phosphatase hydrolysis (A) and
authentic lavandulol standard (B). Peaks corresponds to: IPP prenyl alcohol
(1), DMAPP prenyl alcohol (2), and lavandulol (3).
MARCH 1, 2013•VOLUME 288•NUMBER 9
JOURNAL OF BIOLOGICAL CHEMISTRY 6337

The Biosynthesis of Irregular Monoterpenes in Lavenders
2
FIGURE 4. LPP accumulation versus DMAPP and IPP consumption (R ؍ 0.99, p < 0.0001, n ؍ 2). Error bars indicate S.D.
The cis and trans PDPS family members often share higher
sequence similarity with each other irrespective of the genetic
relatedness among donor organisms (5, 16). This is contrary to
some other enzymes involved in terpene biosynthesis, particu-
larly terpene synthases. Typically, terpene synthases of a given
species are more related to one another than to those of dis-
tantly related species, even if they catalyze the same reaction.
For example ␤-phellandrene synthase of L. angustifolia shares
4
8 and 47% identity with linalool and limonene synthases of the
same species, respectively. However, the gene only shares 31
and 25% similarity with ␤-phellandrene synthases of grand fir
and tomato, respectively (30). In this study, we observed that L.
x intermedia trans-PDPS homologs (GPPS, FPPS, and GGPPS)
share a higher sequence similarity among each other and with
other trans-PDPSs cloned from genetically unrelated species
than they do with LiLPPS. LiLPPS is closely related to NPPS and
cis-FPPS of cultivated (S. lycopersicum) (11) and wild (S. habro-
chaites) (17) tomato, respectively. The phylogenetic tree pre-
sented in Fig. 6 clearly suggests that LiLPPS together with NPPS
and cis-FPPS diverged very early from trans-PDPSs.
FIGURE 5. Kinetic properties of LiLPPS at increasing DMAPP concentra-
tions. The nonlinear regression equation used to fit LPP accumulation
H
H
H
against DMAPP concentrations was V ϭ Vmax*[S] /(k
ϩ [S] ) (n ϭ 2). Error
mm
bars indicate S.D.
DISCUSSION
Like those of many other plants, the EOs of lavenders are
dominated by regular monoterpenes synthesized through the
head-to-tail condensation of isoprene units. It was therefore
not surprising that the EST database derived from L. x interme-
dia secretory cells, tissues specialized for EO biosynthesis, con-
tained a substantial number of ESTs corresponding to enzymes
involved in the biosynthesis of regular monoterpenes (supple-
mental Table S1). Given that L. x intermedia plants also accu-
mulate irregular monoterpenes, particularly the LPP deriva-
tives lavandulol and lavandulyl acetate (19) (supplemental Fig.
S1), it was also not surprising to find ESTs related to irregular
monoterpene biosynthesis, including the cloned LiLPPS, in our
LiLPPS catalyzes the head-to-middle condensation of two
DMAPP molecules to synthesize the linear lavandulol branch
point precursor, LPP (C ). As demonstrated by Thulasiram et
10
al. (3, 4), LPP synthesis proceeds via rearrangement of the dou-
ble bond position to create the highly reactive carbocation
ϩ
ϩ
intermediate lavandulyl cation (L ). L will eventually be
transformed to LPP after a mandatory proton loss (Fig. 1).
LiLPPS catalyzes these reactions with an apparent K and k
m
cat
Ϫ1
of 208 Ϯ 12 ␮M and 0.1 s , respectively. These catalytic prop-
erties are within the range of previously reported cis-PDPSs
Ϫ1
database. However, it was somewhat unexpected to find a cis- including NPPS, which has a K
and kcat of 177 ␮M and 0.2 s
,
m
PDPS in L. x intermedia glandular trichomes because (i) all respectively (11). The K
and kcat values of the aromatic pre-
m
Ϫ1
known Lamiaceae PDPSs (6–10), including those of Lavandula nyltransferase cloned from fungi were 325 ␮M and 0.03 s
,
(
30–32), are of the trans-PDPS type. (ii) The only reported respectively (39). Unlike other PDPSs, LiLPPS displayed a sig-
PDPSs with confirmed role in irregular monoterpene biosyn- moidal saturation curve (Fig. 5). This is a typical feature of
thesis, CPPSs (3, 4, 20), also belong to the trans-PDPSs family. enzymes with multiple substrates, in which the binding of the
The previously reported cis-PDPSs, NPPS and cis-FPPS, cata- first substrate affects the affinity of the enzyme for the second
lyzed the biosynthesis of regular (and not irregular) mono- and substrate through conformational changes or stabilization of
sesquiterpene precursors, respectively, in tomato glandular the active pocket environment (40). LiLPPS, like any other typ-
trichomes.
ical PDPS enzyme, has binding sites for a divalent metal ion
6
338 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 288•NUMBER 9•MARCH 1, 2013

The Biosynthesis of Irregular Monoterpenes in Lavenders
FIGURE6. Phylogeneticrelationshipandclassificationofprenyltransferases. Prenyltransferaseswithinthesameclassshareaminimumof50%aminoacid
identity. The trans-PDPS family members are boxed with a broken line, cis-PDPS family members are circled with a solid line, and the prenyltransferase described
in this article is boxed with a solid line. The scale bar represents amino acid substitutions per site, and numbers represent the branch support values in
percentage. Accession numbers of prenyltransferases used to generate the phylogenetic tree are: AsCPPS, AY308478.1; AtdDPPS1, NP_565551.1; AtdDPPS4,
NP_568883.1; AtuDPPS, AAM67372.1; cis-SlFPPS, B8XA40.1; cis-SlPDPS6, AFW98430.1; LiLPPS, JX985358; lsu-MpGPPS, AAF08793.1; lsu-SmGPPS, AEZ55681.1;
MpFPPS, AAK63847.1; MtdDPPS, XP_003615624.1; PkGPPS, AAW66658.1; SmFPPS, ABV08819.1; SmGPPS, ACR19637.1; SlGPPS, NP_001234302.1; SlNPPS,
NP_001234633.1; ssu-AmGPPS, AAS82859.1; ssu-LiGPPS, JX985359; ssu-MpGPPS, AAF08792.1; ssu-SmGPPS, AEZ55678.1; and TcCPPS, HQ235057.1.
cofactor and two substrates (5, 16). PDPSs accepting IPP and dues involved in DMAPP recognition and c1Ј-2 coupling reac-
DMAPP (e.g. GPPS or NPPS) can be saturated with one of the tion catalysis are yet to be determined. Our results, however,
substrates and forced to follow Michaelis-Menten kinetics typ- suggest that the active sites of LiLPPS selectively bind two
ical of single substrate enzymes for the other substrate. Because DMAPP (and not IPP) units and position them in such a way
DMAPP was the only substrate for LiLPPS, saturating one site that the first carbon atom of one unit is in close proximity to the
was not an option. Thus, plotting LiLPPS product (LPP) accu- second carbon of the other to facilitate the c1Ј-2 bond forma-
mulation against increasing substrate (DMAPP) concentra- tion. One possibility is that LiLPPS preferentially recognizes
tions assumed a sigmoidal saturation curve with a Hill coeffi- DMAPP by identifying its double bond position. It is also pos-
cient (H) value of 2.7. The positive Hill coefficient value (2.7) sible that the position of the double bond in IPP is not favorable
indicates a positive co-operativity among the binding pockets. for head-to-middle condensation.
If LPP was a condensation result of IPP and DMAPP, an
With Ͼ55,000 members, isoprenoids are the most structur-
equimolar consumption of the two isoprenes would be ally and stereochemically diverse biochemical compounds
expected in our assays. However, LPP production was inde- known to mankind (42). Much of this diversity has been attrib-
pendent of IPP and required only DMAPP. In our assays, a uted to the astounding mechanistic heterogeneity and promis-
reduction in DMAPP supply resulted in an equivalent reduc- cuity of terpene synthases. PDPSs, cis or trans, also play a major
tion in LPP accumulation. However, reducing the concentra- role in the structural diversity of isoprenoids by providing inter-
tion or excluding IPP from the reaction did not alter LPP pro- mediate precursor molecules of varying chain length (C , C ,
1
0
15
duction. This result was also confirmed when IPP was provided C , C , etc.) destined for different isoprenoid groups. In addi-
2
0
30
at a constant high concentration level (150 ␮M) while the tion, PDPSs generate the structurally distinct C precursor
1
0
amount of DMAPP was increased progressively (6.25–200 ␮M), molecules GPP/NPP, LPP, CPP, maconelliyl diphosphate (C )
1
0
and the reaction was allowed to run until LPP synthesis seized or planococcyl diphosphate (C ) by simply changing the posi-
1
0
or DMAPP molecules were nearly consumed. At all IPP and tion of the carbon-carbon bond (3,4, 20). These linear precur-
DMAPP concentration combinations assayed, IPP consump- sors are then elaborated upon by mTPSs to create various
tion remained very close to 0, whereas that of DMAPP monoterpenes (42, 43). In Lavandula, GPP and LPP are the
increased in parallel to the amount of LPP synthesized (Fig. 4). linear precursors for the biosynthesis of regular and irregular
This outcome indicated that, like the CPPS enzymes reported monoterpenes, respectively (30–32).
by Thulasiram et al. (4) and Rivera et al. (20), LiLPPS utilizes
In conclusion, through the identification and functional
DMAPP as the only substrate to synthesize LPP. The structural characterization of LiLPPS, a novel cis-PDPS, we have eluci-
features of LiLPPS underlying this catalytic property or resi- dated the biosynthetic origin of irregular monoterpene constit-
MARCH 1, 2013•VOLUME 288•NUMBER 9
JOURNAL OF BIOLOGICAL CHEMISTRY 6339

The Biosynthesis of Irregular Monoterpenes in Lavenders
uents of Lavandula EOs. To our knowledge, this is the first 16. Kharel, Y., and Koyama, T. (2003) Molecular analysis of cis-prenyl chain
elongating enzymes. Nat. Prod. Rep. 20, 111–118
report of a cis-PDPS involved in the biosynthesis of irregular
1
7. Sallaud, C., Rontein, D., Onillon, S., Jabès, F., Duffé, P., Giacalone, C.,
Thoraval, S., Escoffier, C., Herbette, G., Leonhardt, N., Causse, M., and
Tissier, A. (2009) A novel pathway for sesquiterpene biosynthesis from
Z,Z-farnesyl pyrophosphate in the wild tomato Solanum habrochaites.
Plant Cell 21, 301–317
monoterpenes. In addition, LiLPPS is the first wild type gene
that catalyzes the unusual head-to-middle condensation of two
DMAPP molecules to synthesize LPP (C ) as its primary func-
10
tion. The elucidation of this pathway enables researchers to
further investigate the biosynthesis of irregular monoterpenes
in lavenders and other plants. Further, the cloned gene could be
used to modulate the accumulation of lavandulol, lavandulyl
acetate, and prenylated metabolites with a lavandulyl group to a
desired level through metabolic engineering (44).
1
1
8. Dewick, P. M. (2009) Medicinal Natural Products: A Biosynthetic Ap-
proach, 2nd Ed., John Wiley and Sons, West Sussex, England
9. Epstein, W. W., Klobus, M. A., and Edison, A. S. (1991) Irregular mono-
terpene constituents of Artemisia tridentata cana: the isolation, charac-
terization, and synthesis of two new chrysanthemyl derivatives. J. Org.
Chem. 56, 4451–4456
2
0. Rivera, S. B., Swedlund, B. D., King, G. J., Bell, R. N., Hussey, C. E., Jr.,
Shattuck-Eidens, D. M., Wrobel, W. M., Peiser, G. D., and Poulter, C. D.
Acknowledgment—We thank Mariana Galata for help with manu-
script preparation.
(
2001) Chrysanthemyl diphosphate synthase: isolation of the gene and
characterization of the recombinant non–head-to-tail monoterpene syn-
thase from Chrysanthemum cinerariaefolium. Proc. Natl. Acad. Sci. U.S.A.
9
8, 4373–4378
REFERENCES
2
1. Matsuda, K., Kikuta, Y., Haba, A., Nakayama, K., Katsuda, Y., Hatanaka,
A., and Komai, K. (2005) Biosynthesis of pyrethrin I in seedlings of Chrys-
anthemum cinerariaefolium. Phytochemistry 66, 1529–1535
1
. Croteau, R., Kutchan, T., and Lewis, N. (2000) in Biochemistry and Molec-
ular Biology of Plants (Gruissem, W., and Jones, R., eds) pp. 1250–1318,
American Society of Plant Biologists, Rockville, MD
22. Matsuda, K. (2012) Pyrethrin biosynthesis and its regulation in Chrysan-
themum cinerariaefolium. Top. Curr. Chem. 314, 73–81
2
. Walsh, C. T. (2007) Revealing coupling patterns in isoprenoid alkylation
biocatalysis. ACS Chem. Biol. 2, 296–298
23. Franco, J. C., Zada, A., and Mendel, Z. (2009) in Biorational Control of
Arthropod Pests: Application and Resistance Management (Ishaaya, I., and
Horowitz, A. R., eds) pp 233–278, Springer, New York
3
. Thulasiram, H. V., Erickson, H. K., and Poulter, C. D. (2008) A common
mechanism for branching, cyclopropanation, and cyclobutanation reac-
tions in the isoprenoid biosynthetic pathway. J. Am. Chem. Soc. 130, 24. Walton, V. M., Daane, K. M., Bentley, W. J., Millar, J. G., Larsen, T. E., and
1
966–1971
Malakar-Kuenen, R. (2006) Pheromone-based mating disruption of
Planococcus ficus (Hemiptera: Pseudococcidae) in California vineyards. J.
Econ. Entomol. 99, 1280–1290
4
. Thulasiram, H. V., Erickson, H. K., and Poulter, C. D. (2007) Chimeras of
two isoprenoid synthases catalyze all four coupling reactions in isoprenoid
biosynthesis. Science 316, 73–76
25. Hemmerlin, A., Rivera, S. B., Erickson, H. K., and Poulter, C. D. (2003)
Enzymes encoded by the farnesyl diphosphate synthase gene family in the
big sagebrush Artemisia tridentata ssp. spiciformis. J. Biol. Chem. 278,
32132–32140
5
. Liang, P. H., Ko, T. P., and Wang, A. H. (2002) Structure, mechanism and
function of prenyltransferases. Eur. J. Biochem. 269, 3339–3354
. Bouvier, F., Suire, C., d’Harlingue, A., Backhaus, R. A., and Camara, B.
6
(
2000) Molecular cloning of geranyl diphosphate synthase and compart- 26. Zhao, P., Inoue, K., Kouno, I., and Yamamoto, H. (2003) Characterization
mentation of monoterpene synthesis in plant cells. Plant J. 24, 241–252
. Burke, C., and Croteau, R. (2002) Geranyl diphosphate synthase from
Abies grandis: cDNA isolation, functional expression, and characteriza-
tion. Arch. Biochem. Biophys. 405, 130–136
of Leachianone G 2Љ-dimethylallyltransferase, a novel prenyl side-chain
elongation enzyme for the formation of the lavandulyl group of sophora-
flavanone G in Sophora flavescens Ait. cell suspension cultures. Plant
Physiol. 133, 1306–1313
7
8
9
. Burke, C. C., Wildung, M. R., and Croteau, R. (1999) Geranyl diphosphate 27. Ko, W. G., Kang, T. H., Kim, N. Y., Lee, S. J., Kim, Y. C., Ko, G. I., Ryu, S. Y.,
synthase: cloning, expression, and characterization of this prenyltrans-
ferase as a heterodimer. Proc. Natl. Acad. Sci. U.S.A. 96, 13062–13067
and Lee, B. H. (2000) Lavandulylflavonoids: a new class of in vitro apop-
togenic agents from Sophora flavescens. Toxicol. In Vitro 14, 429–433
. Schmidt, A., and Gershenzon, J. (2008) Cloning and characterization of 28. Lee, H. S., Ko, H. R., Ryu, S. Y., Oh, W. K., Kim, B. Y., Ahn, S. C., Mheen,
two different types of geranyl diphosphate synthases from Norway spruce
T. I., and Ahn, J. S. (1997) Inhibition of phospholipase C␥1 by the preny-
lated flavonoids from Sophora flavescens. Planta Med. 63, 266–268
29. Woronuk, G., Demissie, Z., Rheault, M., and Mahmoud, S. (2011) Biosyn-
thesis and therapeutic properties of Lavandula essential oil constituents.
Planta Med. 77, 7–15
(
Picea abies). Phytochemistry 69, 49–57
1
0. Tholl, D., Kish, C. M., Orlova, I., Sherman, D., Gershenzon, J., Pichersky,
E., and Dudareva, N. (2004) Formation of monoterpenes in Antirrhinum
majus and Clarkia breweri flowers involves heterodimeric geranyl diphos-
phate synthases. Plant Cell 16, 977–992
30. Demissie, Z. A., Sarker, L. S., and Mahmoud, S. S. (2011) Cloning and
functional characterization of ␤-phellandrene synthase from Lavandula
angustifolia. Planta 233, 685–696
1
1. Schilmiller, A. L., Schauvinhold, I., Larson, M., Xu, R., Charbonneau, A. L.,
Schmidt, A., Wilkerson, C., Last, R. L., and Pichersky, E. (2009) Monoter-
penes in the glandular trichomes of tomato are synthesized from a neryl
diphosphate precursor rather than geranyl diphosphate. Proc. Natl. Acad.
Sci. U.S.A. 106, 10865–10870
31. Demissie, Z. A., Cella, M. A., Sarker, L. S., Thompson, T. J., Rheault, M. R.,
and Mahmoud, S. S. (2012) Cloning, functional characterization and
genomic organization of 1,8-cineole synthases from Lavandula. Plant
Mol. Biol. 79, 393–411
1
2. Chen, F., Tholl, D., Bohlmann, J., and Pichersky, E. (2011) The family of
terpene synthases in plants: a mid-size family of genes for specialized
metabolism that is highly diversified throughout the kingdom. Plant J. 66,
32. Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K. H., and Schwab,
W. (2007) Cloning and functional characterization of three terpene syn-
thases from lavender (Lavandula angustifolia). Arch. Biochem. Biophys
465, 417–429
2
12–229
1
1
1
3. Degenhardt, J., Köllner, T. G., and Gershenzon, J. (2009) Monoterpene
and sesquiterpene synthases and the origin of terpene skeletal diversity in 33. Lane, A., Boecklemann, A., Woronuk, G. N., Sarker, L., and Mahmoud,
plants. Phytochemistry 70, 1621–1637
S. S. (2010) in Lavender: the Genus Lavandula (Lis-Balchin, M., ed) pp.
4. Wang, G., and Dixon, R. A. (2009) Heterodimeric geranyl(geranyl)diphos-
117–123, Taylor & Francis, London
phate synthase from hop (Humulus lupulus) and the evolution of monot- 34. Lis-Balchin, M. (2002) Lavender essential oil: standardisation, ISO, adul-
erpene biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 106, 9914–9919
teration and its detection using GC, enantiomeric columns and bioactiv-
5. Ma, Y., Yuan, L., Wu, B., Li, X., Chen, S., and Lu, S. (2012) Genome-wide
ity. Lavender: The Genus Lavandula 117–123
identification and characterization of novel genes involved in terpenoid 35. Falk, L., Biswas, K., Boeckelmann, A., Lane, A., and Mahmoud, S. S. (2009)
biosynthesis in Salvia miltiorrhiza. J. Exp. Bot. 63, 2809–2823
An efficient method for the micropropagation of lavenders: regeneration
6
340 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 288•NUMBER 9•MARCH 1, 2013

The Biosynthesis of Irregular Monoterpenes in Lavenders
of a unique mutant. J. Essent. Oil Res. 21, 225–228
A. H. (2010) Structure of a heterotetrameric geranyl pyrophosphate syn-
3
6. Croteau, R., and Karp, F. (1979) Biosynthesis of monoterpenes: prelimi-
nary characterization of bornyl pyrophosphate synthetase from sage
thase from mint (Mentha piperita) reveals intersubunit regulation. Plant
Cell 22, 454–467
(
Salvia officinalis) and demonstration that geranyl pyrophosphate is the 42. Christianson, D. W. (2008) Unearthing the roots of the terpenome. Curr.
preferred substrate for cyclization. Arch. Biochem. Biophys. 198, 512–522
7. Deleted in proof
Opin. Chem. Biol. 12, 141–150
3
3
3
43. Gao, Y., Honzatko, R. B., and Peters, R. J. (2012) Terpenoid synthase struc-
tures: a so far incomplete view of complex catalysis. Nat. Prod. Rep. 29,
1153–1175
8. Deleted in proof
9. Haug-Schifferdecker, E., Arican, D., Brückner, R., and Heide, L. (2010) A
new group of aromatic prenyltransferases in fungi, catalyzing a 2,7-dihy- 44. Sugiyama, A., Linley, P. J., Sasaki, K., Kumano, T., Yamamoto, H., Shitan,
droxynaphthalene 3-dimethylallyltranferase reaction. J. Biol. Chem. 285,
N., Ohara, K., Takanashi, K., Harada, E., Hasegawa, H., Terakawa T., Ku-
zuyama, T., and Yazaki, K. (2011) Metabolic engineering for the produc-
tion of prenylated polyphenols in transgenic legume plants using bacterial
and plant prenyltransferases. Metab. Eng. 13, 629–637
1
6487–16494
4
4
0. Deleted in proof
1. Chang, T. H., Hsieh, F. L., Ko, T. P., Teng, K. H., Liang, P. H., and Wang,
MARCH 1, 2013•VOLUME 288•NUMBER 9
JOURNAL OF BIOLOGICAL CHEMISTRY 6341

Plant Biology:
The Biosynthetic Origin of Irregular
Monoterpenes in Lavandula: ISOLATION
AND BIOCHEMICAL
CHARACTERIZATION OF A NOVEL
cis-PRENYL DIPHOSPHATE SYNTHASE
GENE, LAVANDULYL DIPHOSPHATE
SYNTHASE
Zerihun A. Demissie, Lauren A. E. Erland,
Mark R. Rheault and Soheil S. Mahmoud
J. Biol. Chem. 2013, 288:6333-6341.
doi: 10.1074/jbc.M112.431171 originally published online January 10, 2013
Access the most updated version of this article at doi: 10.1074/jbc.M112.431171
Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites.
Alerts:
•
•
When this article is cited
When a correction for this article is posted
Click here to choose from all of JBC's e-mail alerts
Supplemental material:
http://www.jbc.org/content/suppl/2013/01/10/M112.431171.DC1.html
This article cites 36 references, 12 of which can be accessed free at
http://www.jbc.org/content/288/9/6333.full.html#ref-list-1