Enantioselective monoterpene alcohol acetylation in Origanum, Mentha and Salvia species

Article abstract of DOI:10.1016/j.phytochem.2008.07.018

Selected plants within the Origanum, Mentha and Salvia genera, that contain significant amounts of chiral volatile alcohols and their related acetates, exhibit remarkable enantioselectivity of alcohol acetyl transferase (AAT) activity and particularly can discriminate between linalool enantiomers. Origanum dayi AAT produced almost enantiomerically pure (R)-linalyl acetate by enzymatic acetylation of racemic linalool, whereas the closely related O. majorana AAT produced a mixture of (R)- and (S)-linalyl acetate with a ratio of 6:4. V_max of O. dayi acetylation activity was 30-fold higher for (R)-linalool, whereas in O. majorana no such differences were found.

Full text of DOI:10.1016/j.phytochem.2008.07.018

Phytochemistry 69 (2008) 2565–2571
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Enantioselective monoterpene alcohol acetylation in Origanum, Mentha and
Salvia species
Olga Larkov a, Alon Zaks a, Einat Bar a, Efraim Lewinsohn a, Nativ Dudai a, Alfred M. Mayer b, Uzi Ravid a,
*
a Department of Vegetable Crops, Agricultural Research Organization (ARO), Newe Ya’ar Research Center, P.O. Box 1021, Ramat Yishay 30095, Israel
b Department of Plant and Environmental Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 May 2008
Received in revised form 18 July 2008
Available online 1 October 2008
Selected plants within the Origanum, Mentha and Salvia genera, that contain significant amounts of chiral
volatile alcohols and their related acetates, exhibit remarkable enantioselectivity of alcohol acetyl trans-
ferase (AAT) activity and particularly can discriminate between linalool enantiomers. Origanum dayi AAT
produced almost enantiomerically pure (R)-linalyl acetate by enzymatic acetylation of racemic linalool,
whereas the closely related O. majorana AAT produced a mixture of (R)- and (S)-linalyl acetate with a ratio
of 6:4. Vmax of O. dayi acetylation activity was 30-fold higher for (R)-linalool, whereas in O. majorana no
such differences were found.
Keywords:
Origanum dayi
Lamiaceae
Monoterpene biosynthesis
Enantioselective acetyl transferase
(S)-linalyl acetate
(R)-linalyl acetate
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Lamiaceae family, including six Origanum species, M. citrata and
Salvia dominica. We have also evaluated the formation of additional
Esters are important constituents of herbs, flowers and fruits
imparting unique aromas and constituting key compounds of
essential oils. The enzymatic formation of volatile esters is well
documented in the literature. These reactions are catalyzed by
alcohol acetyl transferases (AAT), which are able to transfer an acyl
moiety from a CoA thioester to an alcohol acceptor. Many genes
encoding AATs have been isolated from fruits such as apple (Soul-
eyre et al., 2005), melon (Yahyaoui et al., 2002), strawberry (Aharo-
ni et al., 2000) and flowers such as rose (Shalit et al., 2003) and
Clarkia breweri (Dudareva et al., 1998). AAT enzymatic activity
has been studied in cell-free extracts or after expression of AAT
in yeast or bacteria. Generally AATs can use a broad spectrum of
alcohols as substrates including straight and branched chain ali-
phatic and aromatic alcohols. These enzyme activities cannot effi-
ciently accept linalool or other tertiary monoterpene alcohols as
substrates. Recently, an enzyme activity able to esterify linalool
has been evaluated in Mentha citrata, a plant that produces copious
amounts of linalyl acetate (Zaks, personal communication). How-
ever, the stereoselectivity of this enzyme has not been evaluated.
In the present study we report on the enzymatic and stereose-
lective formation of linalyl acetate in eight selected species of the
tertiary acetates such as cis- and trans-sabinene hydrate acetates,
-terpinyl acetate and terpinen-4-yl acetate that are known con-
stituents of these species. Since the substrate alcohols have asym-
metric carbon atoms, the possibility of stereoselective acetate
formation was investigated.
a
2. Results and discussion
2.1. Enantiomeric distribution of chiral monoterpene alcohols and
acetates
The enantiomeric distribution of the chiral monoterpene alco-
hols (Fig. 1) and their corresponding acetates in Origanum, Salvia
and Mentha species are listed in Table 1. The results indicate differ-
ent patterns of stereoselective acetate accumulation according to
the species analyzed. The first pattern of acetate accumulation fol-
lowed an enantioselective pattern, in which both alcohol enantio-
mers were present, but only one acetate enantiomer was
accumulated. This type of enantioselective pattern was observed
for linalyl acetate in O. dayi and in O. syriacum; for trans-sabinene
hydrate acetate in O. dayi, O. vulgare and O. onites and for cis-sabin-
ene hydrate acetate in O. dayi (Table 1). In the second pattern of
enantiomeric distribution both enantiomers of the alcohols and
both of their corresponding acetates were found. This pattern
was observed for linalyl acetate in O. vulgare and in O. majorana,
and for terpinen-4-yl acetate in O. dayi and in O. vulgare. A third
Abbreviations: AAT, alcohol acetyl transferase; ee, enantiomeric excess; HS-
SPME, head space-solid phase micro extraction.
* Corresponding author. Tel.: +972 4 953 9509; fax: +972 4 983 6936.
E-mail address: uziravid@agri.gov.il (U. Ravid).
0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.07.018

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O. Larkov et al. / Phytochemistry 69 (2008) 2565–2571
strate-correlated acetate formation (Pattern 2) was observed for
terpinen-4-yl acetate (Fig. 2D).
HO
R
HO
R
HO
OH
R
S
S
S
R
S
S
R
2.2. Enzymatic acetylation activity of soluble cell-free extracts
R
S
2.2.1. Radioactive AcCoA as substrate
cis-Sabinene hydrate
trans-Sabinene hydrate
In order to clarify whether enzymatic acetylation activity is
responsible of acetate accumulation, we conducted enzymatic
acetylation reactions with [14C]AcCoA as an acetyl donor utilizing
desalted soluble cell-free extracts from the species of interest with
several alcohol substrates. The aliphatic alcohol hexanol was acet-
ylated to hexyl acetate by all of the soluble cell-free extracts
(Fig. 3). Linalyl acetate was formed only by soluble cell-free ex-
tracts from O. dayi, O. syriacum, O. majorana, O. vulgare, S. dominica
and M. citrata, which accumulated significant amounts of linalyl
acetate in their essential oils. O. ramonense, which did not accumu-
late linalyl acetate and O. dayi (clone No.2), which contained only
trace amounts of linalyl acetate (data not shown), showed negligi-
ble acetylation activity when (RS)-linalool was offered as a sub-
strate. Interestingly, only S. dominica, a plant that accumulates
HO
HO
HO
HO
H
H
R
S
R
S
Linalool
α-Terpineol
OH
OH
S
R
significant amounts of
showed -terpineol acetylation activity. The plants that lack
pinyl acetate also failed to acetylate -terpineol in vitro (Fig. 3).
a
-terpinyl acetate in its essential oil,
a
a-ter-
a
Terpinen-4-ol
Fig. 1. Tertiary monoterpene alcohol structures.
2.2.2. Chiral analyses of acetate formation
To further investigate whether the enzymatic acetylation ob-
served is specific towards a particular linalool enantiomer, we con-
ducted experiments with non-radioactive AcCoA and determined
the enantiomeric composition of the produced linalyl acetate by
chiral analyses. The results showed that when racemic linalool
was given as a substrate, soluble cell-free extracts from different
plants utilized the two linalool enantiomers at different rates
(Fig. 4). (R)-linalool was preferably converted to (R)-linalyl acetate
with enantiomeric excesses ranging from 87% (O. dayi) to 15% (O.
onites). Soluble cell-free extracts from O. dayi preferentially acety-
lated (R)-linalool with high stereoselectivity when linalool was of-
fered at various (R)/(S) ratios (Fig. 5A). In contrast, it seems that
both enantiomers are acetylated at similar rates by extracts of O.
majorana (Fig. 5B).
In order to study the differences in the rates of the acetylation
activity of O. dayi and O. majorana enzymatic preparations, we
determined the kinetic parameters for both (R)- and (S)-linalool
with saturated AcCoA and for AcCoA with saturated (R)- and (S)-
linalool. The Km values for (R)- and (S)-linalools were similar both
in O. dayi and O. majorana (0.02 and 0.03 mM for (R)- and (S)-linal-
pattern of enantiomeric distribution was found in the majority of
herbs in which only one alcohol enantiomer was present and only
one corresponding acetate (if any) was accumulated (Table 1).
Indications of possible enantioselective acetate formation in O.
dayi were obtained from analyses of the enantiomeric composition
of 58 accessions of ten populations originating in the Judean Des-
ert. The chiral GC–MS analyses of such accessions, which grew un-
der natural conditions with different soil characteristics, allowed
us to conduct correlation analyses of enantiomeric excesses of
alcohols vs. enantiomeric excesses of their corresponding acetates.
The same pattern 1 of enantioselective acetate formation observed
before (Table 1) was observed for linalyl acetate and trans-sabin-
ene hydrate acetate in the wild 58 accessions (Figs. 2A and B).
cis-Sabinene hydrate acetylation was stereoselective in 21 plant
accessions that were characterized by considerably higher total
amounts of formed acetate compared with the other 37 plant
accessions which contained negligible amounts of this acetate
and did not show the same enantioselectivity (Fig. 2C). Sub-
Table 1
Enantiomeric composition of monoterpene alcohols and their corresponding acetates in six Origanum species, M. citrata and S. dominica
Monoterpene
Species
O. dayi
O. syriacum
O. majorana
O. vulgare
O. ramonense
O. onites
S. dominica
M. citrata
Linalool
Alcohol
Acetate
Alcohol
Acetate
Alcohol
Acetate
Alcohol
Acetate
Alcohol
Acetate
Alcohol
Acetate
(R) 21 (t)a
(R) 99
(S) 88
(S) 99
(R) 10
(R) 96
(S) 22
(S) 29 (t)
(S) 10
n.d.
(R) 45
(R) 99
(R) 54
n.d.
(R) 99
n.d.
(S) 69
n.d.
(R) 20
n.d.
(S) 10
(R) 48
(R) 53
n.d.
(R) 99
(R) 99
(S) 88
n.d.
(R) 26
n.d.
(S) 99
n.s.
(S) 45
(S) 22
(R) 42
(R) 99
(R) 96
(R) 97
(S) 74
(S) 22 (t)
(R) 20
n.d.
n.d.b
n.d.
(R) 92
n.d.
(R) 96
(R) 99
(S) 57
n.d.
(S) 91
n.d.
(R) 99 (t)
(R) 99
(R) 37
(R) 99
(R) 96
(R) 98
(S) 46 (t)
n.d.
(R) 26 (t)
n.d.
(S) 99 (t)
n.s.
(R) 99 (t)
(R) 99
n.d.
n.d.
n.d.
n.d.
n.d
n.d
(R) 99
(R) 99
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
(trans)-Sabinene hydrate
(cis)-Sabinene hydrate
Terpinen-4-ol
a-Terpineol
(S) 71(t)
c
n.s.
n.d.
n.d.
n.d.
Borneol
(S) 99
n.s.
(S) 99
n.s.
(S) 99
n.s.
(S) 99
n.s.
n.d.
n.d.
Single plants were examined at two months intervals for at least 1 year. The enantiomeric composition remained constant through the year in the case of enantiomerically
pure compounds. In other cases the variability was 4% of ee.
Enantiomeric composition expressed in enantiomeric excess (ee, %).
a
(t), Low relative abundance (<1%).
n.d., Not detected (<0.05%).
n.s., Enantiomers were not separated.
b
c

O. Larkov et al. / Phytochemistry 69 (2008) 2565–2571
2567
100
1100
A
B
50
0
550
0
-50
--50
-1100
-100
-
100
-
50
0
50
100
-100
-50
0
50
100
ee.(S)-(trans)-sabinene hydrate,%
ee.(R)-linalool,%
1
0
0
0
0
0
C
D
100
5
50
0
-50
-50
-100
-100
-100
0
-100
-50
50
100
0
-50
5
0
110000
ee.(S)-(cis)-sabinene hydrate,%
ee. (S)-terpinen-4-ol, %
Fig. 2. Correlation between enantiomeric composition (expressed as enantiomeric excesses [ee], %) of alcohols and their corresponding acetates in 58 accessions of O. dayi. (A)
(R)-linalool/acetate; (B) (1S)-(trans)-sabinene hydrate/acetate; (C) (1S)-(cis)-sabinene hydrate/acetate; and (D) (S)-terpinen-4-ol/acetate. Negative numbers are shown when
the opposite enantiomer is predominant.
0.7
A
0.6
0.5
0.4
0.3
0.2
0.1
0
Plant spp.
70
B
60
50
40
30
20
10
0
i
e
a
um
ns
rat
lagre
a
dya
i
u
v
r
oen
O.
Mc.i
mojrna
Oo.nites
O.
Sd.omnic
Os.y
O.
Or.am
Fig. 3. (A) AAT activity of cell-free protein extracts from Oregano, Salvia and Mentha spp. with [14C]acetyl-CoA as acyl donor. Substrates used: hexanol (black bars), (RS)-
linalool (grey bars) and (RS)- -terpineol (white bars). Averages of two replicates + STDEV are shown. The experiment was replicated two times with similar results. The
control using AcCoA alone has been included in the calculations. (B) Ester content (%) in SPME extracts from plants used for protein extraction. Acetates shown: linalyl acetate
(grey bars) and -terpinyl acetate (white bars). (Hexyl acetate was not found).
a
a

2568
O. Larkov et al. / Phytochemistry 69 (2008) 2565–2571
2
2
O. dayi
O.syriacum
2
1
Racemic linalyl
acetate
10000
10000
10000
1
1
0
0
0
45
46
45
46
45
46
2
2
S. dominica
2
O. majorana
M. citrata
50000
5000
7000
1
1
1
0
0
0
45
46
45
46
45
46
2
O. vulgar
e
O. onites
1 2
20000
1
3500
0
0
45
46
45
46
Fig. 4. GC–MS analyses of linalyl acetate enantiomers generated in vitro by AAT activity of desalted protein extracts with racemic linalool as a substrate (1.6 lM) and non-
radioactive AcCoA as an acyl donor (0.1 mM). 1-(S)-linalyl acetate; 2-(R)-linalyl acetate. The enantiomeric excess (%) of (R)-linalyl acetate was 87 (O. dayi); 82 (O. syriacum); 56
(M. citrata); 41 (S. dominica); 22 (O. majorana); 18 (O. vulgare); 15 (O. onites). x-axis – retention time (min), y-axis – abundance of m/z 80 ion.
100
A
B
100
50
50
0
0
-50
-100
-50
-100
-100
-50
0
50
100
-100
-50
0
50
100
ee.(R)-linalool, %
ee.(R)-linalool, %
Fig. 5. Correlation between enantiomeric excesses of the substrate (R)-linalool and in vitro produced (R)-linalyl acetate. (A) O. dayi cell-free protein extract; (B) O. majorana
cell-free protein extract.
ool in O. dayi and 0.18 and 0.05 mM in O. majorana for (R)- and (S)-
linalool, respectively) at a fixed AcCoA concentration of 0.7 mM
(Table 2). The Vmax value for (R)- linalool in O. dayi cell-free extracts
was much higher compared to (S)-linalool (3.96 and
0.14 nmol minÀ1 mg proteinÀ1, respectively). In contrast, no differ-
ences in Vmax of (R)- and (S)-linalool were observed in O. majorana
(0.66 and 0.51 nmol minÀ1 mg proteinÀ1, respectively). Values of
the Km’s for both linalool enantiomers at acetyl CoA saturation
are lower than the Km for acetyl CoA when alcohol substrates are
saturated (0.02 and 0.03 mM for (R)- and (S)-linalool, respectively,
comparing to 0.8 and 0.6 mM). In spite of the very similar Km val-
ues observed for both linalool enantiomers, large differences were
seen between (R)- and (S)-linalool enantiomers in terms of Vmax
values. When (R)-linalool was the alcohol substrate, whether at
saturation concentrations or not, the Vmax observed was approxi-
mately 30-fold higher compared to that observed when (S)-linalool
was used as a substrate. The lack of differences in Km and in Vmax
values in O. majorana may confirm the non-selective model of
linalyl acetate formation observed in enzymatic reactions. How-
ever, we cannot exclude the possibility that O. majorana possesses
more than one activity, each specific for a certain enantiomer and
the values we measure reflect an average of the kinetic parameters
of each activity. Indeed since we did not use a purified enzyme
preparation it is likely that enzyme extract contained a number
of AAT activities. The differences in Vmax when linalool and AcCoA
were used either at saturating or non-saturating concentrations
could be due to various reasons including inhibition by high sub-
strate concentration. Such differences have been reported for
example by Souleyre et al. (2005).
To further evaluate the substrate specificity of the enzymes stud-
ied we tested additional chiral alcohol substrates. The primary ali-
phatic alcohol 2-methyl butanol was readily acetylated in this
reaction by both O. dayi and O. majorana enzymatic preparations.
No preference towards a specific enantiomer of 2-methyl butanol

O. Larkov et al. / Phytochemistry 69 (2008) 2565–2571
2569
Table 2
Kinetic parameters of AAT activities in cell-free extracts of O. dayi and O. majorana
Protein
source
Co-substrate S1 (variable
concentration)
Co-substrate S2 (saturating
concentration)
Km (mM)
Vmax (nmol minÀ1 mg
Vmax/Km (10À6 L minÀ1 mg
proteinÀ1
)
proteinÀ1
)
O. dayi
(R)-linalool
(S)-linalool
AcCoA
AcCoA
AcCoA
(R)-linalool
(S)-linalool
AcCoA
AcCoA
(R)-linalool
(S)-linalool
0.02
0.03
0.80
0.60
0.18
0.05
0.06
0.16
3.96
0.14
0.90
0.03
0.66
0.51
0.12
0.18
198.0
4.7
1.1
0.1
3.7
10.2
2.0
1.1
AcCoA
O. majorana
(R)-linalool
(S)-linalool
AcCoA
AcCoA
AAT activity was measured using non-radioactive assay and various concentrations of alcohol substrates (0.1
various concentrations of AcCoA (0.02–1 mM) at saturated concentration of alcohols (0.3 mM).
lM to 0.3 mM) at saturated concentration of AcCoA (0.6 mM) or
Table 3
acetyl transferase from Pichia kluyveri bacteria was capable of
enantioselective conversion of racemic citronellol to (S)-citronellyl
acetate with an E-value of more than 20 (Oda et al., 1999).
Enantiomeric composition of acetates produced by cell-free extracts with different
racemic alcohol substrates
Racemic alcohol substrate
2.2.3. Enantiomeric specificity of AAT
Enantiomeric
ratio of produced
acetate (%)
2-
Methyl
butanol
Lavandullol Neomenthol Isopulegol Borneol
Our findings are of interest because known alcohol acetyl trans-
ferases involved in volatile ester biosynthesis in flowers and fruits
have been reported to exhibit a broad substrate specificity, capable
of accepting a wide range of alcohol substrates (Souleyre et al.,
2005; Shalit et al., 2001; Beekwilder et al., 2004; D‘Auria, 2006;
Gutermanetal., 2006). Thelimitedrangeof estersfoundintheplants
was attributed to the lack of availability of the endogenous sub-
strates. Our work also shows that simple aliphatic alcohols hexanol
and 2-methyl butanol were acetylated with ease by soluble cell-free
extracts. However, specificity was observed when more complex
(R) (S) (R)
(S)
(R)
(S)
(R)
(S)
(R) (S)
O. dayi
O. majorana
50 50 50
50 50 n.d.
50
n.d.
71
23
29
77
99
1
50 50
n.d. n.d. 50 50
was observed (Table 3). The enantiomers of the primary monoter-
pene alcohol lavandullol were also acetylated by O. dayi soluble
preparations at similar ratios. Therefore, O. dayi AAT could not differ-
entiate betweenprimaryalcoholenantiomerswith a hydroxylgroup
monoterpenols such as a-terpineol or linalool were used as a sub-
strates. In this work we also show that the availability of AAT may
limit the formation of monoterpene acetates in the Lamiaceae. Sub-
strate availability may also have a crucial role in determining the
composition of acetates in the essential oil. Many monoterpene syn-
thases are reported as stereospecific enzymes providing enantiome-
rically pure substrates for subsequent acetylation and thus
determining the stereochemistry of the acetylation (Köllner et al.,
2004). Linalool is formed from the achiral precursor geranyl diphos-
phate by various linalool synthases (LS) that are highly stereospe-
cific. (R)-linalool synthases were identified in M. citrata (Crowell
et al., 2002), Ocimum basilicum (Iijima et al., 2004) and Lavandula
angustifilia (Landmann et al., 2007). (S)-linalool synthases were
identified in C. breweri (Pichersky et al., 1995), Cereus peruvianus
(Sitrit et al., 2004) and Arabidopsis thaliana (Aharoni et al., 2003).
in a-position to the chiral center. Similarly, other highly enantiose-
lective enzymes, such as lipases, do not necessarily show any enanti-
oselectivity toward primary alcohols. Few cases of primary alcohol
enanioselective acetylation have been described (Oda et al., 1999).
O. dayi and O. majorana enzymatic preparations acetylated the
secondary alcohol neomenthol (Table 3). Neomenthyl acetate with
an enantiomeric ratio of 77 (S):23 (R) was produced from racemic
neomenthol by an O. dayi enzymatic preparation, whereas an O.
majorana enzymatic preparation acetylated racemic neomenthol
to a mixture of neomenthyl acetate with the almost reverse enan-
tiomeric ratio of 27 (S): 73 (R). Racemic isopulegol was acetylated
to an enantiomerically pure (R)-isopulegyl acetate by O. dayi AAT,
but we were unable to obtain a product using O. majorana ATT.
O. dayi and O. majorana enzymatic preparations acetylated the sec-
ondary alcohol borneol (Table 3). When two pure borneol enantio-
mers were used as substrates separately, similar amounts of bornyl
acetate enantiomers were obtained. We therefore assume that
AATs of O. dayi and O. majorana either have no preference for nat-
urally occurring (S)-borneol or that two separate AAT’s are present
in this plant. These results are different from the results of the acet-
yl transferase activity of M.x piperita (Croteau and Hooper, 1978).
They reported that both neomenthol, menthol and isomenthol
enantiomers were equally acetylated by acetyl transferase prepa-
rations of M.x piperita.
Our results indicate that some plants within Origanum, Mentha
and Salvia genera exhibit remarkable enantioselectivity of alcohol
acetyl transferase activity and can particularly discriminate be-
tween linalool enantiomers. This enantioselectivity was observed
by chiral analyses of the essential oil of these plants and demon-
strated in enzymatic reactions (Table 1, Figs. 4 and 5). Few stereo-
selective acetyltransferases have been reported to date.
Salutaridinol 7-O-acetyltransferase from Papaver somniferum, an
enzyme involved in alkaloid biosynthesis, showed a high stereose-
lectivity toward salutaridionol and did not accept its diastereomer
7-epi-salutaridinol as a substrate (Lenz and Zenk, 1995). Alcohol
3. Concluding remarks
Enzymatic reactions utilizing chiral alcohol substrates showed
remarkable stereoselectivity towards the (R)-linalool enantiomer
in O. dayi. This enantioselective pattern was observed in O. dayi
plant extracts when these were examined by chiral GC analyses.
Both enantiomers of linalool and cis- and trans-sabinene hydrate
were present but only one acetate enantiomer was accumulated.
The AAT stereoselectivities in various plants of the Lamiaceae fam-
ily were found to be different.
4. Experimental
4.1. Plant material
Leaves of Origanum spp., M. citrata and S. dominica (Table 4) were
harvested from a collection of live material maintained at Newe
Ya’ar Research Center in Israel. Large experimental plots were kept
under field conditions and selected plants were transferred to the

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O. Larkov et al. / Phytochemistry 69 (2008) 2565–2571
Table 4
Origins of the investigated plants
Accession
Genus/section
Origin
Four selected clones from a wild population in the Judean
Main monoterpene acetates detected
O. dayi Post
Origanum/
(trans)-Sabinene hydrate acetate (cis)-sabinene hydrate acetate
linalyl acetate (Dudai et al., 2003 ;Amzallag et al., 2005)
(cis)-Sabinene hydrate acetate (Larkov et al., 2005) linalyl acetate
Campanulaticalyx Desert , Israel 58 accessions collected in the wild
Origanum/
Majorana
O. syriacum L.
ssp.
Selected clone from Newe Ya‘ar, Israel
syriacum
O. majorana L.
Origanum/
Majorana
Origanum/
Origanum
Origanum/
A commercial variety provided by CN Seeds Company, UK
Selected clone from a wild population near Kalamaki, Greece
(cis)-Sabinene hydrate acetate (Larkov et al., 2005) linalyl acetate
O. vulgare L.
ssp. vulgare
O. ramonense
Danin
linalyl acetate
Selected clone from a wild population in the Ramon Heights , (cis)-Sabinene hydrate acetate (Larkov et al., 2005)
Campanulaticalyx Israel
O. onites L.
Origanum/
Majorana
Salvia
Selected clone from a wild population near Kalamaki , Crete,
Greece
Selected clone from seeds collected in a wild population on the Linalyl acetate,
Gilboa mountains.
Linalyl acetate
S. dominica L.
M. citrata L.
a-terpinyl acetate (Ravid and Putievsky, 1985)
Mentha
Plants grown at the Newe Ya’ar herbs collection
Linalyl acetate (Ravid et al., 1994)
greenhouse. O. ramonense, that cannot survive under open field con-
ditions, and O. majorana were cultivated in a greenhouse.
Additionally 58 O. dayi plant accessions from 10 wild popula-
tions originated in the Judean desert in Israel were investigated.
The plants were collected and extracted as described by Amzallag
et al. (2005) and analyzed by chiral chromatography.
4.3.2. Hydrodistillation
Samples of at least 250 g of fresh plant material were hydrodis-
tilled for 1.5 h in a modified Clevenger apparatus. The essential oil
was cooled and separated from the water.
4.3.3. Solid-phase microextraction (SPME)
Fresh plant material or enzyme preparations were placed in
sealed vials. The volatiles were extracted from the headspace using
4.2. Chemicals
a 65 lm PDMS/DVB fiber [polydimethylsiloxane/divinylbenzene],
(R)-linalool ex. bois de rose (enantioneric purity 99%, R.C. Treatt
& Co., England), (S)-linalool (enantioneric purity 88%, ex. coriander
oil prepared by flash chromatography as described by Larkov et al.,
2005) and ( )-linalool (Roth Chemical Co., Karlsruhe, Germany);
(1R,3R,4S)-(À)-isopulegol (Aldrich) and (1S,3S,4R)-(+)-isopulegol
(Aldrich); (S)-(À)-2-methyl-1-butanol (Aldrich) and ( )-2-methyl-
1-butanol (Aldrich); ( )-lavandulol (Roth), ( )-neomenthol (Fluka),
(R)-borneol (Fluka), (S)-borneol (Fluka), (À)-terpineol (Fluka), (+)-
terpineol (Fluka), (+)/(À)-terpinen-4-ol (60/40) (Aldrich) were uti-
lized as substrates.
(R)-(À)-linalyl acetate (enantioneric purity 99%, R.C.Treatt & Co.,
ex petitgrain) and ( )-linalyl acetate (Fluka); (S)-(À)-2-methyl-1-
butyl acetate (Aldrich); ( )-lavandulyl acetate (Fluka), (1S)-(+)-
neomenthyl acetate (Fluka), (1R)-(À)-neomenthyl acetate (Fluka)
were used as standards.
(Supelco, PA, USA)] for 30 min. Desorbtion time was 5 min. The
injections were performed with an autosampler (a GC PAL or Combi
PAL systems, CTC Analytics) or manually.
4.4. Chiral chromatography
Linalyl acetate enantiomers were separated on a FS-Lipodex E
(Macherey-Nagel,Germany)
cyclodextrin
column
(25 m Â
0.25 mm  0.25 m) using an Agilent GC-MSD system (CA, USA).
l
Injector and transfer line temperatures were 230 °C. The following
temperature program was used: 50 °C for 20 min, 50–70 °C at 1 °C/
min; 70–180 °C at 5 °C/min; carrier gas was He at constant pres-
sure 8 psi, linear velocity 44 cm/s. Enantiomeric ratio or excess
were calculated using peak height.
Other chiral separations were performed on an Rt-bDEXsm (Res-
tek, PA, USA) cyclodextrin column (30 m  0.25 mm Â
a-Nerpinyl acetate and terpinen-4-yl acetate were not commer-
cially available. They were obtained by acetylation with Ac2O/pyridine
0.25 lm) on a Hewlett-Packard GCD gas chromatograph apparatus.
(Larkov et al., 2005). When only minute amounts were needed, a fast
on-fiber acetylation procedure was followed. Alcohol (1 ll) was
placed in a 2 ml autosampler vial. A SPME fiber was inserted into
the headspace to adsorb alcohols for 10 min at ambient temperature.
The fiber with the adsorbed alcohol was inserted into a second 2 ml
The injector and transfer line temperatures were 230 °C. Mass range
m/z 41–350. Carrier gas was He at a constant flow of 0.8 ml/min, lin-
ear velocity 32 cm/s. The temperature program was as follows: 50 °C
for 1 min, 50–200 °C at 1.5 °C/min. In someplant extracts(1R,4R,5S)-
(cis)-sabinene hydrate interfered with (S)-linalool and we used
either a temperature program of 50 °C for1 min, 50–200 °C at
20 °C/min or the chiral column Hydrodex-b-TBDAc (Macherey-Na-
vial containing Ac2O (10 ll) and pyridine (10 ll) for 10 min. The fiber
with the newly formed acetates and the residual alcohols was inserted
into the GC injection port for analyses. The conversion rate was 5–20%
and depended on the type of acetylated alcohol.
gel) (25 m  0.25 mm  0.25
lm) with a program of 50 °C for
20 min, 50–70 °C at 1 °C/min; 70–180 °C at 5 °C/min; carrier gas
was He at a constant pressure of 8 psi, linear velocity 44 cm/s. Split-
less or split modes with split ratios 5–50 were used. For separation of
lavandullol/lavandullyl acetate enantiomers we used a program of
50 °C for 20 min, 50–200 °C at 3 °C/min.
4.3. Extraction of volatiles
4.3.1. Solvent extraction
Solvent extraction was carried out as described by Larkov et al.
(2005). Fresh young leaves were extracted for 2 h by gentle shaking
with tert-butyl methyl ether (MTBE) (5 ml per gram of fresh
weight) at room temperature. The extracts were purified by pass-
ing them through a Pasteur pipette containing anhydrous Na2SO4
and Silica gel 60 (230–400 mesh, Merck) to dry the sample and
to remove polar substances of high molecular weight that might
interfere with the GC–MS analyses.
4.5. Enzyme extraction
Fresh plant samples (1 g of 2–3 young leaf pairs) were ground
with a pestle in a chilled mortar in liquid nitrogen, 0.5 g sand
and 0.1 g PVPP insoluble until a uniform powder was obtained.
Ice-cold extraction buffer (1:5 w/v), consisting of 50 mM bis-
Tris-propane pH 6.9, 10% (v/v) glycerol, 10 mM dithiothreitol

O. Larkov et al. / Phytochemistry 69 (2008) 2565–2571
2571
Aharoni, A., Keizer, L.C.P., Bouwmeester, H.J., Sun, Z., Alvarez-Huerta, M., Verhoeven,
H.A., Blaas, J., van Houwelingen, A.M.M.L., De Vos, R.C.H., van der Voet, H.,
Jansen, R.C., Guis, M., Mol, J., Davis, R.W., Schena, M., van Tunen, A.J., O’Connell,
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Alonso, W.R., Rajaonarivony, J.I.M., Gershenzon, J., Croteau, R., 1992. Purification of
4S-limonene synthase, a monoterpene cyclase from the glandular trichomes of
peppermint (Mentha x piperita) and spearmint (Mentha spicata). J. Biol. Chem.
267, 7582–7587.
(DTT), 5 mM Na2S2O5 and 1% (w/v) PVP-40 was added (buffer A).
The slurry was centrifuged at 14,000g for 15 min at 4 °C. The super-
natant (2 ml) was next passed through a P-6 column (1 Â 10 cm)
(Bio-Rad, Bio-Gel desalting gel 90–180 lm, Bio-Rad, Germany) to
remove interfering low-molecular-weight compounds. We used
buffer B (buffer A without PVP-40, pH 7.5) for column equilibration
and protein elution. Protein concentration in 1 ml fractions was de-
tected by the Bradford protein assay (Bradford, 1976) utilizing bo-
Amzallag, G.N., Larkov, O., Ben Hur, M., Dudai, N., 2005. Soil microvariations as a
source of variability in the wild: the case of secondary metabolism in Origanum
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vine serum albumin as
a standard. GC–MS analyses were
performed to ensure that interfering essential oil compounds were
removed. Fractions were kept at À20 °C for further analysis.
Mentha citrata enzyme preparations were obtained from young
leaves peltate glandular trichomes and isolated as previously de-
scribed (Alonso et al., 1992). Isolated glandular trichomes were
ground with buffer A, and the trichome extract was desalted on
P6 column as described above.
Beekwilder, J., Alvarez-Huerta, M., Neef, E., Verstappen, F.W.A., Bouwmeester, H.J.,
Aharoni, A., 2004. Functional characterization of enzymes forming volatile
esters from strawberry and banana. Plant Physiol. 135, 1865–1878.
Bradford, M.M., 1976.
A rapid and sensitive method for the quantitation of
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Anal. Biochem. 72, 248–254.
Croteau, R., Hooper, C.L., 1978. Metabolism of monoterpenes. Acetylation of (À)-
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4.6. Enzyme assay
D’Auria, J.C., 2006. Acyltransferases in plants: a good time to be BAHD. Curr. Opin.
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4.6.1. Radioactive assay
Dudai, N., Larkov, O., Chaimovitsh, D., Lewinsohn, E., Freiman, L., Ravid, U., 2003.
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benzylalcohol acetyltransferase – an enzyme involved in floral scent production
in Clarkia breweri. Plant J. 14, 297–304.
Guterman, I., Masci, T., Chen, X., Negre, F., Pichersky, E., Dudareva, N., Weiss,
D., Vainstein, A., 2006. Generation of phenylpropanoid pathway-derived
volatiles in transgenic plants: rose alcohol acetyltransferase produces
phenylethyl acetate and benzyl acetate in petunia flowers. Plant Mol.
Biol. 60, 555–563.
Iijima, Y., Davidovich-Rikanati, R., Fridman, E., Gang, D.R., Bar, E., Lewinsohn, E.,
Pichersky, E., 2004. The biochemical and molecular basis for the divergent
patterns in the biosynthesis of terpenes and phenylpropenes in the peltate
glands of three cultivars of basil. Plant Physiol. 136, 3724–3736.
Köllner, T.G., Schnee, C., Gershenzon, J., Degenhardt, J., 2004. The variability of
sesquiterpenes emitted from two Zea mays cultivars is controlled by allelic
variation of two terpene synthase genes encoding stereoselective multiple
product enzymes. Plant Cell 16, 1115–1131.
Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K.-H., Schwab, W., 2007.
Cloning and functional characterization of three terpene synthases from
lavender (Lavandula angustifolia). Arch. Biochem. Biophys. 465, 417–429.
Larkov, O., Dunkelblum, E., Zada, A., Lewinsohn, E., Freiman, L., Dudai, N., Ravid, U.,
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Small-scale assays were performed by mixing 20
salted cell-free extract, 0.33 mM alcohol substrate, and 23
(7.8 Ci mol-1 [14C]acetyl-CoA, Amersham) into a final volume
of 100 L of assay buffer B. The assays were incubated for 30 min
lL of a de-
l
m
l
l
l
at 30 °C. One milliliter of hexane was added to each tube, which
was then vigorously vortexed and spun for 1 min at 5000g to sep-
arate phases. 0.8 ml of the upper hexane layer, containing the new-
ly formed radiolabeled alcohol acetate esters, was placed in a
scintillation vial containing 3 ml of Ultimagold non-aqueous scin-
tillation fluid (Packard Bioscience, The Netherlands). Radioactivity
was counted by Tri Carb 2800TR liquid scintillation counter (Per-
kin–Elmer, The Netherlands). Boiled enzyme extracts and reaction
with no enzymes were used as controls. Enzyme activity was cal-
culated based on the specific activity of the substrate and using
appropriate correction factors for the counting efficiency of the
scintillation machine (Shalit et al., 2001).
4.6.2. GC–MS assay
The assay was performed in 2 ml vials at 30 °C and containing
Lenz, R., Zenk, M.H., 1995. Acetyl coenzyme A: salutaredinol-7-O-acetyltransferase
from Papaver somniferum plant cell cultures. The enzyme catalyzing the
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31096.
Oda, S., Sugai, T., Ohta, H., 1999. Optical resolution of racemic citronellol via a
double coupling system in an interface bioreactor. J. Biosci. Bioeng. 87, 473–
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Pichersky, E., Lewinsohn, E., Croteau, R., 1995. Purification and characterization of S-
linalool synthase, an enzyme involved in the production of floral scent in Clarkia
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Ravid, U., Putievsky, E., Katzir, E., 1994. Chiral GC analysis of enantiomerically pure
(R)-(À)-linalyl acetate in some Lamiaceae, myrtle and petitgrain essential oils.
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Shalit, M., Guterman, I., Volpin, H., Bar, E., Tamari, T., Menda, N., Adam, Z., Zamir, D.,
Vainstein, A., Weiss, D., Pichersky, E., Lewinsohn, E., 2003. Volatile ester
formation in roses. Identification of an acetyl-coenzyme A. Geraniol/citronellol
acetyltransferase in developing rose petals. Plant Physiol. 131, 1–9.
Shalit, M., Katzir, N., Tadmor, Y., Larkov, O., Burger, Y., Shalekhet, F., Lastochkin, E.,
Ravid, U., Amar, O., Edelstein, M., Karchi, Z., Lewinsohn, E., 2001. Acetyl-CoA:
alcohol acetyltransferase activity and aroma formation in ripening melon fruits.
J. Agric. Food Chem. 49, 794–799.
40
non-radioactive acetyl CoA (0.1 mM) and 5
alcohol (1.6 M) in sample buffer B (pH 7.5) in a total volume of
300 l. We used alcohol solutions in n-hexane at 0.5 M and then
diluted them with buffer B. After 30 min 100 l satr. CaCl2 was
ll desalted supernatant (containing 20–60
lg protein), 40 ll
ll of the appropriate
l
l
l
added to stop the reaction. Products were analyzed by GC–MS
using automated or manual HS-SPME as described above. Appro-
priate controls included the omission of substrate, omission of en-
zyme extract and the use of heat-inactivated enzyme extracts.
Comparative alcohol substrate preference assays were per-
formed as described above using non-radioactive acetyl CoA. Enan-
tiomerically pure (R)-linalool and (S)-linalool (88% enantiomeric
purity) were used. All reactions were done in duplicates. Linalyl
acetate concentration was calculated by external standard calibra-
tion. A calibration curve for linalyl acetate was obtained within the
range of 0.02–0.6 lM and was linear with R = 0.993. The Vmax and
Km were calculated from non-linear regressions of the Michaelis–
Menten plot using GraphPad Prism version 5.00 (GraphPad Soft-
ware, San Diego, California, USA).
Sitrit, Y., Ninio, R., Bar, E., Golan, E., Larkov, O., Ravid, U., Lewinsohn, E., 2004. S-
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Yahyaoui, F.E.L., Wongs-Aree, C., Latché, A., Hackett, R., Grierson, D., Pech, J.-C.,
2002. Molecular and biochemical characteristics of a gene encoding an alcohol
acyl-transferase involved in the generation of aroma volatile esters during
melon ripening. Eur. J. Biochem. 269, 2359–2366.
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