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Unprecedented LipoxygenaseHydroperoxide Lyase Pathways in the Moss Physcomitrella patens.

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Communications
Bioorganic Chemistry
Unprecedented Lipoxygenase/Hydroperoxide
Lyase Pathways in the Moss Physcomitrella
patens**
Thomas Wichard, Cornelia Gbel, Ivo Feussner, and
Georg Pohnert*
Lipoxygenase (LOX) pathways are involved in the production of important signal and defensive metabolites in mammals, higher plants, and algae.[1–4] In these pathways molecular
oxygen is introduced into a polyunsaturated fatty acid to form
an intermediate hydroperoxide, which may then be cleaved to
give shorter chain-length oxygenated products. Interestingly,
different principles of transformations have been identified.
While plants use almost exclusively C18 fatty acids for the
production of oxylipins,[1] algae and animals rely predominantly on the transformation of C20 fatty acids.[3, 5] In animals
cleavage of the intermediate hydroperoxy fatty acids is
achieved by a dual function of LOXes, while plants and
algae rely often on hydroperoxide lyases (HPLs) to produce
shorter chain oxylipins.[1–3] Due to their central importance,
LOX/HPL pathways have been well investigated in these
organisms, but nearly nothing is known about related transformations in mosses. Since mosses are located phylogenetically between higher plants and algae, the biosynthetic
pathways are of special interest.
Certain mosses are known to release unbranched unsaturated C8 and C9 alcohols and aldehydes, but until now the
pathways to and function of these metabolites have not been
addressed.[6, 7] We report here that the moss Physcomitrella
patens releases volatile oxylipins upon tissue damage. These
include (E)-non-2-enal (4) known from higher plants, which
has a cucumberlike odor, (R)-1-octen-3-ol (1) (94 % ee),[8] a
major aroma compound from mushrooms, (E)-oct-2-en-1-ol
(2), and (E)-oct-2-enal (3; Figure 1). In mushrooms (R)-1 is
derived from linoleic acid, which is transformed by a 10-LOX
and a lyase activity to give (E)-10-oxodec-8-enoic acid as a
second fragment.[9–11] In higher plants linoleic acid is also a
precursor for the generation of (E)-non-2-enal (4) and (Z)non-3-enal (8). There, a 9-LOX and a 9-HPL produce the C9
aldehyde 8 and 9-oxononanoic acid.[2, 12]
[*] T. Wichard, Dr. G. Pohnert
Max-Planck-Institut fr Chemische !kologie
Hans-Kn%ll-Strasse 8, 07745 Jena (Germany)
Fax: (+ 49) 3641-571-256
E-mail: pohnert@ice.mpg.de
Dr. C. G%bel, Prof. Dr. I. Feussner
Georg-August-Universit>t G%ttingen
Albrecht-von-Haller-Institut fr Pflanzenwissenschaften
Justus-von-Liebig-Weg 11, 37077 G%ttingen (Germany)
[**] We thank Prof. Dr. W. Boland for his support during the preparation
of this work. The DFG (G.P.) and the EC (I.F.) are acknowledged for
funding.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
158
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/anie.200460686
Angew. Chem. Int. Ed. 2005, 44, 158 –161
Angewandte
Chemie
To further investigate the mechanism of fatty acid transformation in P. patens we identified the fragments generated
from 6 in addition to 1–4. The HPLC-MS chromatograms
obtained from P. patens preparations before and after tissue
damage were used to identify two dominant metabolites
derived from fatty acids. The UV spectrum (lmax = 279 nm) of
the less polar metabolite is characteristic for an a,b,g,dunsaturated carbonyl compound, and the mass spectrum
suggested an oxidized C12 carboxylic acid (Figure 2). These
Figure 1. a) GC/MS of the SPM-extract of volatiles from P. patens;
b) same as a) from the wounded moss; c) same as b) in the presence
of 200 mg arachidonic acid (5)/ ~ 100 mg P. patens. IS = internal standard 2-decanone.
In our investigations on the biosynthesis of the volatile
oxylipins from P. patens we found that external application of
linoleic or a-linolenic acid did not result in significantly
increased production of 1–4 or related higher unsaturated
metabolites. Moreover, neither 10-oxodec-8-enoic acid nor 9oxononanoic acid, metabolites formed from analogous pathways in plants and mushrooms, were detected. These results
imply that mosses rely on other precursors for the generation
of 1–4. In contrast to higher plants, P. patens is also rich in
arachidonic acid (5),[13] which could, judging from the position
of the double bonds, also be a possible precursor for these
volatile oxylipins. Indeed, externally applied arachidonic acid
(5) was transformed with high efficiency into 1–4. This could
be shown impressively by administration of [D8]arachidonic
acid to P. patens cell preparations, which resulted in deuterated 1 (> 400 % labeled), 2, 3, and 4 (> 1100 % labeled)
(Figure 1). This surprising fact—arachidonic acid (5) has not
been identified as a precursor for 1–4 in any other organisms—prompted us to investigate the pathways in more detail.
Trapping experiments with dimethyl sulfide revealed that an
arachidonate 12-LOX is involved in the transformations.
Using this reagent, intermediate hydroperoxy fatty acids are
transformed into the corresponding alcohols, which are not
further metabolized by HPL.[3] In intact moss we found no
endogenous hydroxylated fatty acids after reduction; however, after only a few seconds after tissue damage, (S)-12hydroxyarachidonic acid ((S)-12-HETE, 83 % ee), (S)-15HETE (56 % ee), and minor amounts of nearly racemic 11HETE were detected. The related 11-hydroperoxide, 11HPETE, could also be a precursor for the C9 volatiles, but
since it is found only in minor amounts as a racemate, it is
most likely an autoxidation product of arachidonic acid and
not involved in enzymatic pathways. In the presence of 5 the
amount of 12-hydroperoxyeicosatetraenoic acid (12-HPETE,
6) increased drastically within 30 seconds after wounding and
decreased over the next 16 minutes—the time required for
the formation of 1–4 and oxo-acids (see the Supporting
Information). Accordingly, (S)-12-HPETE, which arises from
the LOX-mediated oxidation of arachidonic acid (5), is an
intermediate in the biosynthesis of 1–4.
Angew. Chem. Int. Ed. 2005, 44, 158 –161
Figure 2. Top: LC/MS of wounded P. patens in the presence of 200 mg
arachidonic acid (5)/ ~ 100 mg P. patens. The inserts show the UVspectra of the metabolites 7 and 10, respectively. Bottom: Negative
ionization mass spectra for the unlabeled (above) and labeled (below)
oxo-acids after transformation of 5 or [D8]-5.
data matched those of (5Z,8Z,10E)-12-oxododeca-5,8,10trienoic acid (12-ODTE, 7) and co-injection with synthetic
7[14] finally proved the structure. The other metabolites could
be identified as 11-oxoundeca-5,9-dienoic acid (11-OUDE,
10).[15]
These assigned structures are in accordance with biosynthetic considerations on the generation of the oxylipins shown
in Scheme 1. The identified structures suggest that 5 is
transformed without prior degradation by, for example, boxidation, into the hydroperoxide 6, which then undergoes
cleavage to give the oxo-acids and volatiles. This biosynthetic
path was further supported by incubations of damaged moss
with [D8]arachidonic acid. Mass spectra of 6, 7 and 10 showed
mass shifts of + 8, + 6 and + 5, respectively, when the
labeled acid was present (see Figure 2 and the Supporting
www.angewandte.org
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
159
Communications
Experimental Section
Scheme 1. Proposed arachidonate 12-LOX/HPL pathways in P. patens.
After administration of [D8]-arachidonic acid (5) [D8]-HPETE (6) can be
trapped as intermediate. [D2]-C8-fragments and [D6]-7 as well as [D3]-4
and [D5]-10 are formed in the subsequent reactions. All 8 deuterium
labels of 5 are thus found in the respective intermediates and break
down products.
General: Cultures of P. patens were obtained from Prof. Dr. Ralf
Reski, Freiburg, and were cultivated as described.[22] Preparation of
samples for MS analysis: A 5-mL aliquot of a dense culture was left to
settle, and the medium was removed by pipetting. The resulting pellet
with a wet weight of 100–200 mg was resuspended in 1 mL of fresh
medium and used directly or treated with 20 mL of 10 mg mL 1
solutions of fatty acids in EtOH. The suspension was sonicated and
cooled in an ice bath for 2 min. For SPME analysis the vial was sealed
with a Teflon septum, and a polydimethyldiloxane-coated fiber
(Supelco, Taufkirchen, Germany) was inserted for 15 min in the gas
phase. Analysis by GC/MS was performed as described.[14] Commercially available reference compounds were used for the identification
of 1–4. For LC/MS analysis, the moss preparation was treated 30 s or
16 min after sonication with the same volume of MeOH. The sample
was centrifuged for 5 min (12 000 rpm), and the supernatant was used
directly for analysis on an Agilent HP1100/Finnigan LCQ.
The intermediate hydroperoxy fatty acids were trapped by
addition of a 10 % solution of dimethyl sulfide in methanol to
preparations previously treated with arachidonic acid (5). The ee
value was determined by a known method.[23] For calculation, means
of measurements after 30 s, 2 min, 8 min, 15 min, and 30 min were
used.
Received: May 17, 2004
Revised: July 30, 2004
.
Keywords: biosynthesis · fatty acids · mass spectrometry ·
oxylipins · UV/Vis spectroscopy
Information). The corresponding high degree of labeling of 6
and the shorter chain oxylipins indicates that 5 is the
precursor of all the oxylipins shown in Scheme 1.
These results show that P. patens employs hitherto
unknown pathways for the production of known LOX/HPL
products. The biosynthesis of 1, 2, and 4 is initiated by a 12LOX, which provides 6 as a substrate for HPL or other fatty
acid cleaving enzymes. Further transformation of 6 results in
the C8 metabolites 1 and 2 together with 7 as the second
fragment (Scheme 1). Interestingly, 7 has been detected
previously in arachidonic acid stimulated human platelets.[16]
It acts as an agonist towards human neutrophils, presumably
interacting with the leukotriene B4 binding site.[17] Compound
7 is also known as deleterious metabolite from diatoms,[18, 19]
but there (1E,3E)-octa-1,3-diene results as the second fragment from a 12HPETE.[14] In P. patens 6 can also be transformed into (Z)-non-3-enal (8) and (5Z,8Z)-11-oxoundeca5,8-dienoic acid (9), presumably by an HPL. In analogy to
well-known transformations in higher plants, these intermediates may be transformed quickly by a 3Z:2E-enal isomerase
to give 4 and 10 (Scheme 1).[12, 20]
Our findings show that hitherto unknown LOX pathways
are involved in the biosynthesis of a multitude of oxylipins in
P. patens. The moss produces metabolites typical for animals,
plants, algae, and mushrooms by new transformations of
arachidonic acid, combining in a unique way metabolic
themes from all these organisms. Mosses are known to be
highly resistant to herbivores and pathogens, and in higher
plants this type of resistance is often mediated by lipoxygenases.[21] We are particularly interested in determining if and
how the newly identified biosynthetic pathways contribute to
the production of putative signal or defensive metabolites
involved in this remarkable resistance.
160
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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[15] The mass spectrum of 10 is in accordance with an oxidised C-11
fatty acid and the UV-maximum at 230 nm suggests the presence
of an a,b-unsaturated aldehyde (Figure 2). This structure was
further supported by GC/MS investigation of the derivatized
extract of the moss (Supporting Information).
[16] W. C. Glasgow, T. M. Harris, A. R. Brash, J. Biol. Chem. 1986,
261, 200 – 204.
www.angewandte.org
Angew. Chem. Int. Ed. 2005, 44, 158 –161
Angewandte
Chemie
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[19] G. Pohnert, O. Lumineau, A. Cueff, S. Adolph, C. Cordevant, M.
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[22] T. Girke, U. Schmidt, R. Reski, E. Heinz, Plant J. 1998, 15, 39 –
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[23] A. R. Brash, D. J. Hawkins, Methods Enzymol. 1990, 187, 187 –
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2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
161
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unprecedented, lipoxygenasehydroperoxide, patens, lyase, pathways, physcomitrella, moss
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