Linolenate 9R-dioxygenase and allene oxide synthase activities of lasiodiplodia theobromae

Article abstract of DOI:10.1007/s11745-011-3622-5

Jasmonic acid (JA) is synthesized from linolenic acid (18:3n-3) by sequential action of 13-lipoxygenase, allene oxide synthase (AOS), and allene oxide cyclase. The fungus Lasiodiplodia theobromae can produce large amounts of JA and was recently reported to form the JA precursor 12-oxophytodienoic acid. The objective of our study was to characterize the fatty acid dioxygenase activities of this fungus. Two strains of L. theobromae with low JA secretion (～0.2 mg/L medium) oxygenated 18:3n- 3 to 5,8-dihydroxy-9Z,12Z,15Z- octadecatrienoic acid as well as 9R-hydroperoxy-10E,12Z,15Z-octadecatrienoic acid, which was metabolized by an AOS activity into 9-hydroxy-10-oxo-12Z,15Z- octadecadienoic acid. Analogous conversions were observed with linoleic acid (18:2n-6). Studies using [11S-²H]18:2n-6 revealed that the putative 9R-dioxygenase catalyzed stereospecific removal of the 11R hydrogen followed by suprafacial attack of dioxygen at C-9. Mycelia from these strains of L. theobromae contained 18:2n-6 as the major polyunsaturated acid but lacked 18:3n-3. A third strain with a high secretion of JA (～200 mg/L) contained 18:3n-3 as a major fatty acid and produced 5,8-dihydroxy-9Z,12Z,15Z- octadecatrienoic acid from added 18:3n-3. This strain also lacked the JA biosynthetic enzymes present in higher plants.

Full text of DOI:10.1007/s11745-011-3622-5

Lipids (2012) 47:65–73
DOI 10.1007/s11745-011-3622-5
ORIGINAL ARTICLE
Linolenate 9R-Dioxygenase and Allene Oxide Synthase Activities
of Lasiodiplodia theobromae
•
Fredrik Jerneren Felipe Eng Mats Hamberg
•
•
´
Ernst H. Oliw
Received: 19 August 2011 / Accepted: 15 September 2011 / Published online: 3 November 2011
Ó AOCS 2011
Abstract Jasmonic acid (JA) is synthesized from linole-
nic acid (18:3n-3) by sequential action of 13-lipoxygenase,
allene oxide synthase (AOS), and allene oxide cyclase. The
fungus Lasiodiplodia theobromae can produce large
amounts of JA and was recently reported to form the JA
precursor 12-oxophytodienoic acid. The objective of our
study was to characterize the fatty acid dioxygenase
activities of this fungus. Two strains of L. theobromae with
low JA secretion (*0.2 mg/L medium) oxygenated 18:3n-
3 to 5,8-dihydroxy-9Z,12Z,15Z-octadecatrienoic acid as
well as 9R-hydroperoxy-10E,12Z,15Z-octadecatrienoic
acid, which was metabolized by an AOS activity into
9-hydroxy-10-oxo-12Z,15Z-octadecadienoic acid. Analo-
gous conversions were observed with linoleic acid (18:2n-6).
Studies using [11S-2H]18:2n-6 revealed that the putative
9R-dioxygenase catalyzed stereospecific removal of the
11R hydrogen followed by suprafacial attack of dioxygen
at C-9. Mycelia from these strains of L. theobromae con-
tained 18:2n-6 as the major polyunsaturated acid but lacked
18:3n-3. A third strain with a high secretion of JA
(*200 mg/L) contained 18:3n-3 as a major fatty acid and
produced 5,8-dihydroxy-9Z,12Z,15Z-octadecatrienoic acid
from added 18:3n-3. This strain also lacked the JA bio-
synthetic enzymes present in higher plants.
Keywords Botryodiplodia theobromae Á Cytochrome
P450 Á 9R-HPODE Á Heme peroxidase Á Jasmonic acid Á
Oxygenation mechanism
Abbreviations
AOS
Allene oxide synthase
Chiral phase
CP
P450
Cytochrome P450
DiHODE
DOX
Dihydroxyoctadecadienoic acid
Dioxygenase
HHDTrE
HPHDTrE
HOME
HPODE
HPOTrE
JA
Hydroxyhexadecatrienoic acid
Hydroperoxyhexadecatrienoic acid
Hydroxyoctadecenoic acid
Hydroperoxyoctadecadienoic acid
Hydroperoxyoctadecatrienoic acid
(-)-Jasmonic acid
JAs
Jasmonates
(?)-7-iso-JA 3R,7S-JA
LDS
LOX
MO
Linoleate diol synthase
Lipoxygenase
Methyloxime
´
F. Jerneren Á E. H. Oliw (&)
NP
Normal phase
Department of Pharmaceutical Biosciences,
Uppsala Biomedical Center, Uppsala University,
P.O. Box 591, 751 24 Uppsala, Sweden
e-mail: Ernst.Oliw@farmbio.uu.se
OPDA
RP
Oxophytodienoic acid
Reversed phase
F. Eng
Cuban Research Institute on Sugar Cane Byproducts,
Via Blanca and Carretera Central 804,
P.O. Box 4026, Havana, Cuba
Introduction
Jasmonic acid (JA), an important signal molecule in plant
defense and development, is present throughout the plant
kingdom as the free acid, the methyl ester, the hydroxyl
M. Hamberg
Department of Medical Biochemistry and Biophysics,
Karolinska Institutet, 171 77 Stockholm, Sweden
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Lipids (2012) 47:65–73
derivatives, or amino acid conjugates [1–3]. The tropical and
subtropical plant pathogen Lasiodiplodia theobromae (syno-
nym Botryodiplodia theobromae) secretes JA [4, 5] and some
strainscould even be used for commercial JA production[5, 6].
JA is formed in plants sequentially from a-linolenic acid
(18:3n-3) or hexadecatrienoic acid (16:3n-3) by 13S-
lipoxygenase (13S-LOX), allene oxide synthase (AOS),
allene oxide cyclase, 12-oxophytodienoate reductase, and
three steps of b-oxidation [1–3, 7]. Key intermediates are
12-oxophytodienoic acid (12-OPDA) and dinor-12-OPDA
from 18:3n-3 to 16:3n-3, respectively. JAs have a wide array
of biological activities in plants (plant defense, stress adap-
tation and development) and can inhibit aflatoxin production
and delay spore germination of Aspergillus flavus [8]. A
recent report also suggests that JA could be of medical value
as a growth suppressor of human cancer cells [9].
U[13C]18:2n-6 (98%), 18:3n-3 (99%), c-linolenic acid
(18:3n-6; 99%), and 9Z,12Z-eicosadienoic acid (20:2n-6;
99%) were from Lipidox, Sigma and Larodan, and stored as
stock solutions (50–100 mM) in ethanol at -20 °C. HPLC
solvents were from VWR. [11S-2H]18:2n-6 ([95% 2H) was
prepared as described [20, 21]. 9-Hydroxy-10-oxo-10,
12Z-octadecadienoic acid was from Lipidox (Stockholm,
Sweden). 11S- and 11R-hydroperoxy-7Z,9E,13Z-hexadec-
atrienoic acids (11-HPHDTrE), 9S-hydroperoxy-10E,
12Z-octadecadienoic acid (9S-HPODE), 13R-hydroperoxy-
9Z,11E-octadecadienoic acid (13R-HPODE), 13R-hydro-
peroxy-9Z,11E,15Z-octadecatrienoic acid (13R-HPOTrE),
and 13S-HPODE were prepared with LOXs (tomato fruit
[22], recombinant manganese LOX (Mn-LOX) [23], and
soybean LOX-1 (Lipoxidase, Sigma)). 11S- and 11R-
HOME(12Z) were prepared with PGH synthase-1 and Mn-
LOX [24, 25]. A racemic mixture of HPODE was obtained
by photo oxidation with methylene blue [26]. The
O-methyloxime (MO) derivative of [5,5,7-2H3](-)-JA used
as an internal standard during quantification of JA was
synthesized by treatment of (-)-JA with Na2CO3 in D2O
followed by preparation and isolation of the MO derivative.
Solvents for HPLC and other chemicals were of analytical
grade and obtained from VWR and Sigma-Aldrich.
L. theobromae (CBS 117454; CBS 122127) were from CBS
Fungal Biodiversity Center (Baarn, Delft, The Netherlands).
The B. theobromae strain 2334 was isolated in Cuba, char-
acterized and grown as described [19], and it will be referred
to as L. theobromae strain 2334.
A biosynthetic pathway from 18:3 to 16:3 to JAs, essen-
tially as described in plants, seems likely in fungi [6, 10], and
plants and fungi form JAs with identical absolute configura-
tion [5, 11]. JAs and the methyl ester of a key intermediate,
12-OPDA, were identified as a metabolite of 18:3n-3 in the
culture medium of L. theobromae [10]. However, whether JA
and 12-OPDA are formed by the same enzymes as in plants is
unknown. LOXs are ubiquitous in plants, but only few LOXs
of fungi have been identified and characterized. Unfortu-
nately, the genome of L. theobromae has not yet been
sequenced. Hydroperoxy fatty acids can be formed in fungi
also by heme containing dioxygenases, e.g., linoleate diol
synthases (LDS) and other oxygenases of the dioxygenase-
cytochrome P450 (DOX-CYP) family of fusion proteins [12–
16]. For comparison, Aspergillus terreus was recently found
to express linoleic acid 9R-dioxygenase (9R-DOX) and AOS
activities [17]. This 9R-DOX activity could be due to a heme
containing dioxygenase, as LOX genes have not been iden-
tified in the genome of A. terreus [17].
Fungal Growth
The two CBS strains were grown on agar slants (15 g agar/
2 g malt extract/0.75 g NaNO3/0.35 g MgSO4Á7H2O/5 g
sucrose) at 27 °C for 3–4 days and then stored at ?4 °C.
For analysis, a piece of the agar slants of L. theobromae
were grown in a modified Czapek-Dox medium (per liter:
2 g KH2PO4, 0.6 g MgSO4Á7H2O, 0.3 g KCl, 7.5 g
NaNO3, 50 g sucrose, 1 ml trace metals ([27]); pH was
adjusted to 5.5 with KOH) at 27 °C without shaking for
10–12 days. The L. theobromae strain 2334 was grown in
malt extract agar slant at 30 °C for 3 days and then stored
at 4 °C; for analysis, a piece of the agar slants of this
fungus was grown in a medium previously described [19].
The mycelia of the strains were harvested by filtration. The
filtrate was assayed for JAs, whereas the mycelia were
washed with saline, blotted dry, weighed, grinded in liquid
nitrogen and stored at -80 °C until analysis.
The aim of the present study was to investigate the
oxidation and further transformation of polyunsaturated
fatty acids by L. theobromae. This fungus is a devastating
plant pathogen, and its access from many fungal collection
centers is restricted for environmental safety reasons. The
few strains, which produce JA in large amounts [18], are
also guarded by commercial interests. In this study, we
used two strains of L. theobromae from the CBS Fungal
Biodiversity Center, and a non-commercial strain with
prominent JA biosynthetic capacity [19].
Materials and Methods
Materials
Assay of Enzyme Activity
16:3n-3 (99%), oleic acid (18:1n-9; 99%), 12Z-octadece-
noic acid (18:1n-6; 99%), linoleic acid (18:2n-6; 99%),
The nitrogen powder was homogenized (glass-Teflon, 10
passes; ?4 °C) in 10–20 vols. (w/v) of 0.1 mM KH2PO4
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Lipids (2012) 47:65–73
67
buffer (pH 7.4)/2 mM EDTA/0.04% Tween-20, and cen-
trifuged at 15,0009g (10 min, ?4 °C). The supernatant
was used immediately for studies of enzyme activities.
Typically, an aliquot (0.25–0.5 ml) was incubated with
80–100 lM of fatty acids or fatty acid hydroperoxides for
30–40 min on ice. The incubation was terminated with
ethanol (4 vols.) and centrifuged. The supernatant was
diluted with water and extracted (SepPak/C18; Waters), as
described [21, 28]. Triphenylphosphine was used to reduce
hydroperoxy fatty acids to alcohols. The subcellular dis-
tributions of enzyme activities were determined by differ-
ential centrifugations (15,0009g, 10 min; ?4 °C;
100,0009g; ?4 °C; 60 min) and assay of enzyme activities
in the high speed supernatant and the microsomal fractions.
of 2 mL and treated with 8 mL of a methanolic solution of
30 mM methoxyamine hydrochloride. The mixture was
vortexed and kept at room temperature for 15 h. The
O-methyloxime (MO) derivative of [5,5,7-2H3](-)-JA
(3 lg) was added as an internal standard, and the material
obtained following extraction with diethyl ether was dis-
solved in chloroform/2-propanol (2:1, v/v) and applied to a
Supelclean LC-NH2 solid phase extraction cartridge
(Supelco, Bellefonte, PA). Elution with diethyl ether/acetic
acid (98:2, v/v) afforded the JA-MO derivative, which was
methyl-esterified by treatment with diazomethane and
subjected to GC–MS analysis. For this purpose, a Hewlett-
Packard model 5970B mass selective detector connected to
a Hewlett-Packard model 5890 gas-chromatograph was
used. The instrument was operated in the selected ion
monitoring mode using the ions m/z 253 (unlabeled JA-MO
methyl ester) and 256 (deuterium-labeled JA-MO methyl
ester). Amounts of (-)-JA and (?)-7-iso-JA were calcu-
lated from areas of the m/z 253 peaks due to these com-
pounds and from the area of the m/z 256 peak due to the
deuterated standard.
LC–MS/MS
Reversed phase-HPLC (RP-HPLC) with MS/MS analysis
was performed with a Surveyor MS pump (ThermoFisher),
a manual or an automatic injector (Surveyor; Thermo-
Fisher), and with an octadecyl silica column (5 lm;
150 9 2.1 mm; Phenomenex), which was eluted at 0.3 ml/
min with methanol/water/acetic acid, 750/250/0.05 or
800/200/0.05. The effluent was subjected to electrospray
ionization (ESI) in a linear ion trap mass spectrometer
(LTQ, ThermoFisher) with analysis of negative ions. In
some experiments, we also analyzed the effluent by UV
absorption (photodiode array detector, path length 5 cm;
Surveyor PDA plus, ThermoFisher). The heated transfer
capillary was set at 315 °C and the ion isolation width
usually at 1.5 (and at 5 in the first selection for MS3
analysis of hydroperoxides [26]). The collision energy was
set at 35 (arbitrary scale) and the tube lens at 90–120 V.
For analysis of products formed from [2H1]18:2n-6, we
used an isolation width of 6 for the carboxylate anion.
Normal phase HPLC (NP-HPLC) and chiral phase-HPLC
(CP-HPLC) were performed as described in [17, 28]. Steric
analysis of 10-HODE was performed with a Reprosil Chiral-
NR column (8 lm; 250 9 2 mm; eluted with isopropanol/
hexane/acetic acid, 3/97/0.01). The enantiomers of 9- and
13-HODE, 9-HOTRE, HOME, HHDTrE and a-ketols were
resolved with Reprosil Chiral AM (5 lm; 250 9 2 mm;
eluted with methanol/hexane/acetic acid or ethanol/hexane/
acetic acid, 5/95/0.01, at 0.15–0.2 ml/min) [28]. The effluent
was mixed with isopropanol/water, 60/40, from a second
pump (Surveyor MS) [28] and subjected to ESI with MS/MS
analysis of carboxylate anions.
The fatty acid composition of L. theobromae was
determined by GC–MS analysis after hydrolysis as
described [29]. The fatty acid methyl esters were separated
on a Supelcowax-10 Capillary GC Column (30 m, film
thickness 0.25 lm, carrier gas helium), and the column
temperature was raised from 150 °C at 5 °C/min.
Results
Fatty Acid Composition
We analyzed the fatty acid composition of L. theobromae
strains CBS 122127 and 2334.
The four main fatty acids in the mycelium of CBS
122127 were 16:0 (37%), 18:2 (25%), 18:1 (24%), and
18:0 (14%). Traces of 20:0 were detected, but not the two
expected precursor fatty acids of JA biosynthesis, 18:3n-3
and 16:3n-3. In contrast, the mycelium of L. theobromae
strain 2334 contained 18:3n-3 as a major fatty acid, as
illustrated in Fig. 1 along with the major fatty acids also
found in CBS122127. 16:3n-3 was not detected.
Jasmonic Acid Production
Both commercial strains produced JAs, but (-)-JA and
(?)-JA could only be detected in the growth medium in
low concentrations [\0.2 lg/mL; e.g., 57 ng/mL (-)-JA,
109 ng/mL (?)-7-iso-JA (3R,7S-JA)]. In contrast,
L. theobromae strain 2334 formed JAs in large amounts,
about 200 lg/mL (e.g., 196 lg/mL (-)-JA and 13 lg/mL
(?)-7-iso-JA) in agreement with previous reports [18]. In
Quantitative Determination of (-)-JA and (?)-7-iso-JA
and Fatty Acid Analysis
The medium (0.05–2 mL) of the growing cultures of
L. theobromae was diluted with water to make a total volume
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Lipids (2012) 47:65–73
18:2
18:3
16:0
18:1
(-)-JA
18:0
20:0
14
6
8
10
12
Retention Time (min)
Fig. 1 GC–MS analysis of the fatty acid composition of L. theobro-
mae strain 2334 with prominent JA biosynthesis. The fatty acids were
analyzed as methyl ester derivatives. The commercial strain CBS
122127 lacked 18:3n-3, contained the other fatty acids of strain 2334,
and formed only relatively small amounts of JA (0.2 vs 200 mg/L)
another experiment, media collected after growth of strain
2234 for 10 and 12 days were found to contain 216 lg/
mL and 178 lg/mL, respectively, of (-)-JA plus (?)-7-
iso-JA.
Oxylipin Biosynthesis by Subcellular Fractions
Subcellular fractions (10,0009g) of all three strains con-
tained 5,8-LDS activity as judged from 8-hydro(per)oxy
metabolites and 5,8-diols formed from 18:2n-6, [U-13C]
18:2n-6, and 18:3n-3. Unexpectedly, subcellular fractions
of the strain 2334 with prominent secretion of JA did not
produce significant amounts of other oxylipins than
8-hydro(per)oxy metabolites and 5,8-diols, whereas the
two commercial strains appeared to oxygenate fatty acids
to these and to additional metabolites.
Fig. 2 LC–MS/MS analysis of oxidation of 18:3n-3 by subcellular
fractions of L. theobromae. a RP-HPLC–MS analysis of metabolites
formed by CBS 117454. TIC from MS/MS analysis of m/z 309 and 293.
The two major peaks contained 5,8-DiHOTrE and an a-ketol, as
indicated. Small amounts of H(p)OTrE were also detected. Strain CBS
122127 yielded a similar pattern. The inset chromatogram shows that
the S and R stereoisomers of a-ketols were formed from hydrolysis of
the allene oxide in a *10:1 ratio (CP-HPLC–MS/MS). b MS/MS
spectrum (m/z 309 ? full scan) of the a-ketol derived from hydrolysis
of an allene oxide. The insert shows formation of the even numbered
signal at m/z 200, which presumably is due to a radical anion [17]
18:3n-3 and 16:3n-3 18:3n-3 was oxidized by nitrogen
powder of the two L. theobromae CBS strains to 5,8-Di-
HOTrE and to an a-ketol, 9-hydroxy-10-keto-12Z,15Z-oc-
tadecadienoic acid, as shown in Fig. 2a. The ESI–MS/MS
spectrum of the a-ketol (m/z 309 ? full scan) is shown in
Fig. 2b. A characteristic ion with mass of even number was
noted at m/z 200, likely a radical ion, which is also present
in the corresponding spectrum of 9-hydroxy-10-oxo-12Z-
octadecenoic acid [17]. Significant amounts of the corre-
sponding c-ketol could neither be detected by RP-HPLC
nor NP-HPLC. The a-ketol was likely formed from an
allene oxide, 9,10-epoxy-10,12Z,15Z-octadecatrienoic
acid. Hydrolysis of this allene oxide was expected to
mainly form the S stereoisomer, and this was confirmed
(insert in Fig. 2a).
16:3n-3 was oxidized to approximately equal amounts
of 10-HHDTrE and 11-HHDTrE (Fig. 3). A steric analysis
by CP-HPLC showed that 10R-HHDTrE and 11S-HHDTrE
were mainly formed (Fig. 3b, c). It seems likely that the
10R- and 11S-DOX activities are due to the linolenate 9R-
DOX activity. Chain shortening of 18:3n-3 by two carbons
thus changed the oxygenation from C-9 to C-10, and led to
biosynthesis 11S-HPHDTrE after hydrogen abstraction at
C-9. 11S-HPHDTrE is a precursor of JA in plants [2], but it
was not transformed by AOS of L. theobromae.
18:2n-6, 18:3n-6, and 20:2n-6 18:2n-6 was transformed
to a major polar metabolite, which eluted after 7 min
during LC–MS/MS analysis (Fig. 4a). [U-13C]Linoleic
acid was oxidized in the same way. The mass spectrum of
the main metabolite was identical with that of an a-ketol,
9-hydroxy-10-oxo-12Z-octadecenoic acid [17]. Small
The MS/MS spectrum of 5,8-DiHOTrE was as reported
[30]. The less polar metabolites were identified as a mix-
ture of 8-H(P)OTrE, 10-H(P)OTrE, and 9-H(P)OTrE
(mainly 9R). Although 13-HPOTrE was not detected, we
investigated whether 13S- or 13R-HpOTrE were substrates
of the AOS activity, but this was not the case.
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Lipids (2012) 47:65–73
69
A100
TIC
50
100
50
m/z 295
m/z 311
8- and 9-
HODE
100
50
0
γ
-ketol
0
4
8
12
16
20
Retention Time (min)
2H
B
-
OOC
HO
171
100
80
60
40
20
0
278
ms2
277
172
196
.
80
240
280
120
160
200
m/z
Fig. 4 LC–MS/MS analysis of oxidation of 18:2n-6 by subcellular
fractions of L. theobromae (CBS 117454). a RP-HPLC–MS analysis
showed that the major products were a- and c-ketols and 8- and
9-HODE. b MS/MS analysis (m/z 292–298) ? full scan) of 9-HODE
from an incubation with [11S-2H]18:2n-6 (95% 2H). The latter was
diluted with endogenous 18:2n-6 in the incubation. The signals at
m/z 277 and 278 of this mass spectrum, mainly due to loss of water
from the carboxyl group, showing that the deuterium label was
retained, as m/z 277 and 278 were present in the same relative
intensities as in 8-HODE of this experiment
Fig. 3 Analysis of oxidation of 16:3n-3 by subcellular fractions of
L. theobromae. The products formed by strain CBS 122127 were
analyzed by RP- and CP-HPLC with ESI–MS/MS after reduction to
alcohols with triphenylphosphine. a RP-HPLC. The first eluting major
peak contained 10-HHDTrE (marked 10-HO-16:3) and the second
peak II 11-HHDTrE (marked 11-HO-16:3). b CP-HPLC–MS analysis
of 11-HHDTrE (bottom chromatogram) with aid of an 11R-HHDTrE
standard (top chromatogram; the 11R stereoisomer was produced
with Mn-LOX [32]). c CP-HPLC–MS analysis of 10-HHDTrE
(bottom chromatogram) with aid of the 10S stereoisomer (prepared
with Mn-LOX [32])
fraction, which are in agreement with the distribution of
plant LOX and AOS (CYP74) [2].
Other metabolites were 5,8-DiHODE, 8-H(P)ODE, and
small amounts of 10-H(P)ODE and 13-H(P)ODE (*5% of
9-H(P)ODE). Steric analyses showed that 10-HODE was
almost racemic (*60% S), whereas 13-HODE was mainly
formed with S configuration (data not shown).
amounts of the c-ketol, 13-hydroxy-10-oxo-11E-octadece-
noic acid, was detected after 5 min (Fig. 4a); MS/MS and
MS/MS/MS spectra as reported [17].The two ketols were
apparently formed from 9R-HPODE via hydrolysis of an
unstable allene oxide, 9,10-epoxy-10,12Z-octadecadienoic
acid [17]. Steric analysis of the a-ketol supported this
mechanism of biosynthesis from 9R-HPODE, as CP-HPLC
showed that it consisted mainly of the 9S stereoisomer (cf.
insert in Fig. 2a). The 9R-DOX activity was present in the
soluble fraction and the AOS activity in the microsomal
The oxidation of 18:2n-6 was investigated with
[11S-2H]18:2n-6 (95% 2H). RP-HPLC with MS and MS/MS
analysis of 9R-HODE showed that this deuterium label was
retained (Fig. 4b), suggesting that the 9R-DOX of L. theo-
bromae catalyzes suprafacial hydrogen abstraction at C-11
relative to the oxygenation at C-9. The apparent deuterium
content, as judged from the ion intensities at m/z 277 and 288
of both 8- and 9-HODE, was 60%, due to dilution with
endogenous 18:2n-6. The deuterium label was also retained in
13S-HODE, a minor metabolite in comparison with 9-HODE.
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The 9R-DOX activity also transformed 18:3n-6 to
A 100
TIC
9-HPOTrE(n-6) as the main metabolite, but further trans-
formation to an a-ketol could not be detected. This fatty
acid was not oxygenated by 5,8-LDS in agreement with
previous reports [31]. Significant formation of 13-HO-
TrE(n-6) was also detected, as judged from the signal
intensities of characteristic ions [m/z 169 (80%; 9-HO-
TrE(n-6)] and m/z 193 [20%, 13-HOTrE(n-6)].
50
100
50
ms2 297
8-HOME-
(9Z)
20:2n-6 was oxidized at C-11 and C-15, but these
hydroperoxides were not further converted to a-ketols.
18:1n-9 and 18:1n-6 Oleic acid was exclusively oxi-
dized by L. theobromae to 5,8-DiHOME and 8-H(P)OME,
as shown in Fig. 5a. We could not detect signs of hydrogen
abstraction at C-11, as judged from insignificant formation
of 9-HOME(10E) and 11-HOME(9Z). This suggested that
18:1n-9 was not oxidized by the 9R-DOX activity.
100
50
0
ms2
313
5,8-DiHOME
0
4
8
12
Retention Time
(min)
The 9R-DOX activity might conceivably abstract the
hydrogen at C-11 of 18:1n-6 with formation of 11S-
HOME(12Z) or 13S-HOME(11E), whereas LDS enzymes
mainly oxidize 18:1 with formation of stereoisomers with
R configuration [25]. 13S-HOME(11E) was not detected.
As shown in Fig. 5b, the formation of 11S-HOME(12Z)
was only 10–15% of the biosynthesis of 11R-HOME(12Z)
and could be attributed to oxidation by 5,8-LDS [24, 25].
In conclusion, 18:1n-9 and 18:1n-6 did not appear to be
oxidized to a significant extent by the 9R-DOX activity.
13S-HPHTrE and 11S-HPHDTrE—these hydroperox-
ides were not transformed by the AOS activity.
B 100
ms2 297
m/z 199
11R-HOME-
(12Z)
50
0
100
ms2 297
11S-
HOME-
m/z 199
(12Z)
50
Discussion
0
Tsukada et al. [10] demonstrated that JA is formed in
L. theobromae from 18:3n-3 [10]. In these experiments, the
fungus was grown for 10 days with 13C-labeled sodium
acetate or with [2H6]18:3n-3, and found to secrete signifi-
cant amounts of [13C]JA, [2H5]JA, and [2H5]-iso-JA,
respectively. These authors also isolated 12-OPDA (methyl
ester), a key intermediate in the biosynthesis of JA in
higher plants. Interestingly, NMR analysis of the deuter-
ated 12-OPDA indicated a stereochemical difference
between the fungal and plant cyclopentenone reductase
reactions. This result suggested that fungal JA biosynthesis
could have evolved independently from plant biosynthesis
with both similarities and differences.
100
ms2 297
11R
m/z 199
50
0
11S
0
8
16
24
32
40
Retention Time
(min)
Fig. 5 LC–MS analysis of products formed during oxygenation of
18:1n-9 and 18:1n-6 by subcellular fractions of L. theobromae. a RP-
HPLC analysis of oxidation of 18:1n-9 by strain CBS 122127. The
two main products were 5,8-DiHOME and 8-HOME. b CP-HPLC
analysis of oxidation of 18:1n-6–11-HOME(12Z) with aid of
standards; the top chromatogram showing elution of 11R-HOME
(prepared with Mn-LOX), the middle chromatogram elution of 11S-
HOME (prepared with COX-1). The bottom chromatogram showing
separation of products formed by L. theobromae. The latter thus
mainly formed the 11R stereoisomer, and this metabolite can be
attributed to the 5,8-LDS activity [25]
We did not find evidence for the presence in L. theo-
bromae of the JA biosynthetic enzymes present in higher
plants. Instead, we report that mycelia of two commercial
strains of L. theobromae express prominent linolenate 9R-
DOX and AOS activities after growth for about 10 days.
These strains only secreted small amounts of JA, and we
therefore also investigated the strain 2334 with large
123

Lipids (2012) 47:65–73
71
A
HOOC
A
9R-DOX
18:3n-3
13S (~5%)
HS
HS
HR
9R-DOX
13
3+
9R (~95%)
9
HOOC
HOOC
HOOC
HOO
HO
AOS
-
B
[Mn OH ]LOX
HR
HS
HR
O
O
11S (~20%)
13R (~80%)
Hydrolysis
13
9S (~2-3%)
9
α
-Ketol
Fig. 7 A schematic presentation of the hydrogen abstraction mech-
anisms of 9R-DOX and Mn-LOX. a Mechanism of hydrogen
abstraction of 18:2n-6 and direction of oxygenation by the 9R-DOX
activity. b Mechanism of hydrogen abstraction of 18:2n-6 and
direction of oxygenation by Mn-LOX. If the substrate could be
oxidized by Mn-LOX in the reverse orientation, the products and the
oxygenation mechanism would be similar to that of the 9R-DOX
activity
B
HOOC
16:3n-3
10R/11S-DOX
R
R
OOH
HOO
10R-HPHDTrE
11S-HPHDTrE
present in microsomal fraction. Both fungi therefore likely
express microsomal AOS of the cytochrome P450 family,
but the 9R-DOX activities of L. theobromae and A. terreus
could be due to different classes of dioxygenases, i.e., a
heme-containing dioxygenase in case of A. terreus and a
lipoxygenase in case of L. theobromae. The latter
hypothesis is based on the following observations.
Fig. 6 Overview of the transformation of 18:3n-3 and 16:3n-3 to cis–
trans conjugated hydroperoxides and an allene oxide by L. theobro-
mae. a Oxidation of 18:3n-3. The soluble 9R-DOX activities are
likely due to a lipoxygenase and the microsomal AOS activities to
P450. b Oxidation of 16:3n-3. These metabolites are also formed by
Mn-LOX but with the opposite chirality, and presumably formed
by the same enzyme with oxidizes 18:2n-6 and 18:3n-3 to
9R-hydroperoxy metabolites
First, c-linolenic acid (18:3n-6) was oxidized to
9-HPOTrE(n-6) as a prominent metabolite by L. theobro-
mae. This fatty acid is readily oxidized by many plant
LOX, but it is not oxidized by a series of fungal DOX-CYP
fusion proteins, e.g., 5,8-LDS, 7,8-LDS, and 10R-DOX,
and not by the 9R-DOX of A. terreus [17]. Second, LOXs
oxygenate 18:1 only slowly [24], and we could not attribute
the oxidation of 18:1n-6 or 18:1n-9 to the 9R-DOX activity
of L. theobromae. Thirdly, we were struck by the similar-
ities of Mn-LOX and 9R-DOX.
capacity to secrete JA. This strain contained 18:3n-3 and
secreted large amounts of JA at time points (10 and
12 days).
We found that 18:3n-3 and 18:2n-6 were both oxygen-
ated to 9R-hydroperoxides, which were further transformed
to allene oxides. The oxidation of 18:3n-3 by this pathway
is summarized in Fig. 6a. The 9R-DOX activity showed
broad substrate specificity, and also oxidized 16:3n-3,
18:3n-6, and 20:2n-6.
Both enzymes oxidize 16:3n-3 at the n-7 (C-10) and the
n-6 carbons (C-11), albeit with different chirality [32]. The
oxidation at the n-7 position is not a common feature of
LOXs, but was recently reported by the LOX domain of a
fusion protein of a cyanobacterium [33]. Oxygenation by 9R-
DOX and Mn-LOX differ with regard to regiochemical and
stereochemical specificity, but both enzymes catalyze su-
prafacial hydrogen abstraction and oxygenation [34], as
outlined in Fig. 7. It is tempting to speculate that 9R-DOX
activity of L. theobromae could be due to a LOX homologue
of Mn-LOX with reverse substrate orientation in the active
site (cf. Fig. 7). DOX-CYP fusions proteins and PGH syn-
thases catalyze antarafacial hydrogen abstraction [24],
whereas the 9R-DOX activity of A. terreus also is suprafacial
[17]. The main difference between the two fungal 9R-DOX
activities resides in the oxidation of 18:3n-3.
16:3n-3 can be converted to JA in plants, and the first
step is biosynthesis of 11S-HPHDTrE [3]. This metabolite
was formed by L. theobromae. 16:3n-3 was oxidized to
approximately equal amounts of 10R-HHDTrE and 11S-
HHDTrE (Fig. 6b), however, since we could not detect
formation of a-ketols it is apparent that these hydroper-
oxides were not substrates of AOS. The oxidation of 16:3n-
3 is summarized in Fig. 6b.
9R-DOX and AOS activities have also been reported in
A. terreus [17]. The genome of A. terreus appears to lack
LOXs, whereas there is no information of the genome of
L. theobromae. At first, it seemed likely that these two
fungi could form 9R-HPODE and allene oxides by
homologous enzymes. The 9R-DOX activity was present in
the soluble fraction, whereas the AOS activity was mainly
123

72
Lipids (2012) 47:65–73
¨
14. Brodhun F, Gobel C, Hornung E, Feussner I (2009) Identification
of PpoA from Aspergillus nidulans as a fusion protein of a fatty
acid heme dioxygenase/peroxidase and a cytochrome P450.
J Biol Chem 284:11792–11805
The AOS activity of L. theobromae appeared to be
specific for 9R-HPODE and 9R-HPOTrE, since we could
not detect significant formation of a-ketols from 16:3n-3,
18:3n-6, 20:2n-6, 13S- or 13R-HpOTrE. The substrate
specificity of AOS of A. terreus is restricted to 9R-HPODE.
In contrast, AOS (CYP74A) of plants are specific for 9S- or
13S-hydroperoxides [35, 36].
¨
15. Brodhun F, Schneider S, Gobel C, Hornung E, Feussner I (2010)
PpoC from Aspergillus nidulans is a fusion protein with one
active heme. J Biochem 425:553–565
16. Garscha U, Oliw EH (2009) Leucine/valine residues direct oxy-
genation of linoleic acid by (10R)- and (8R)-dioxygenases:
expression and site-directed mutagenesis of (10R)-dioxygenase
with epoxyalcohol synthase activity. J Biol Chem 284:13755–
13765
In summary, we report that L. theobromae efficiently
oxidized 18:2n-6 and 18:3n-3 sequentially to 9R-hydro-
peroxides and to unstable allene oxides. The AOS activity
is microsomal and probably catalyzed by a homologue of
CYP74, whereas the soluble 9R-DOX activity might be
due to a LOX. It appears that the genome sequence of
L. theobromae will be needed to elucidate the detailed
mechanism of JA formation in this fungus.
´
17. Jerneren F, Hoffmann I, Oliw EH (2010) Linoleate 9R-dioxy-
genase and allene oxide synthase activities of Aspergillus terreus.
Arch Biochem Biophys 495:67–73 (Erratum 2010; 500; 210)
18. Dhandhukia PC, Thakkar VR (2008) Response surface method-
ology to optimize the nutritional parameters for enhanced pro-
duction of jasmonic acid by Lasiodiplodia theobromae. J Appl
Microbiol 105:636–643
19. Eng F, Gutierrez-Rojas M, Favela-Torres E (1998) Culture con-
ditions for jasmonic acid and biomass production by Botryodip-
lodia theobromae in submerged fermentation. Process Biochem
33:715–720
20. Hamberg M, Zhang LY, Brodowsky ID, Oliw EH (1994)
Sequential oxygenation of linoleic acid in the fungus Gaeu-
mannomyces graminis: stereochemistry of dioxygenase and
hydroperoxide isomerase reactions. Arch Biochem Biophys
309:77–80
Acknowledgments Supported by VR Medicine (03X-06523) and
by The Knut and Alice Wallenberg Foundation (KAW 2004.123).
F Eng was supported by a project of Dr Mariano Gutierrez-Rojas from
UAM Campus Iztapalapa (Mexico). Presented at the Third European
Workshop on Lipid Mediators, Pasteur Institute, Paris June 3–4, 2010.
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