1. Available online at www.sciencedirect.com
Plant Physiology and Biochemistry 46 (2008) 302e308
www.elsevier.com/locate/plaphy
Review
Molecular mechanism of enzymatic allene oxide cyclization in plants
Eckhard Hofmann a,*, Stephan Pollmann b
a
Biophysics, Department of Biology and Biotechnology, Ruhr-University Bochum, Universitaetsstrasse 150, D-44801 Bochum, Germany
b
Plant Physiology, Department of Biology and Biotechnology, Ruhr-University Bochum, Universitaetsstrasse 150, D-44801 Bochum, Germany
Received 14 November 2007
Available online 31 December 2007
Abstract
Jasmonates, a collective term combining both jasmonic acid (JA) and related derivatives, are ubiquitously distributed in the plant kingdom.
They are characterized as lipid-derived signal molecules which mediate a plethora of physiological functions, in particular stress responses, male
fertility, and a multitude of developmental processes. In the course of JA biosynthesis, the first oxylipin with signal character, cis-(þ)-12-oxo-
phytodienoic acid (OPDA), is produced in a cyclization reaction catalyzed by allene oxide cyclase (AOC). This enzyme-catalyzed ring closure is
of particular importance, as it warrants the enantiomeric structure at the cyclopentenone ring which in the end results in the only bioactive JA
enantiomer, cis-(þ)-JA. In this review, we focus on the structural and molecular mechanisms underlying the above mentioned cyclization re-
action. In this context, we will discuss the crystal structure of AOC2 of Arabidopsis thaliana with respect to putative binding sites of the instable
substrate, 12,13-epoxy-9(Z ),11,15(Z )-octadecatrienoic acid (12,13-EOT), as well as possible intermolecular rearrangements during the cycliza-
tion reaction.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Allene oxide cyclase; Allene oxide synthase; Jasmonate; 12-Oxo-phytodienoic acid; Oxylipins; X-ray structure
1. Introduction Jasminum grandiflorum in the early 1960s [7], it took nearly
another twenty years until the first physiological effects of
Besides brassinosteroids and oligopeptides with hormone- MeJA and the occurrence of the free acid were described
like functions, the jasmonates are among the most recently [4,33]. To date, jasmonic acid and its derivatives are associated
identified signal molecules with phytohormone properties, with diverse physiological functions. The most prominent one
and are widespread throughout a variety of different plant is the involvement of JA in wound response and pathogenesis
phyla [25]. Although the jasmonic acid methyl ester (MeJA) [8,18,37]. Additionally, jasmonates play a crucial role in re-
was demonstrated to be a constituent of the essential oil of production [9], metabolic regulation [35], and as a signal
transducer in mechanotransduction [31,38,39]. JA is also re-
quired for protection from ozone damage [26,27], and has
Abbreviations: ACS, acyl-CoA synthase; AOC, allene oxide cyclase; 12,13- a pivotal role in the production of protective secondary metab-
EOT, 12,13(S )-epoxy-9(Z ),11,15(Z )-octadecatrienoic acid; 12,13-EOD, olites in cell cultures of Eschscholtzia californica [2,3].
12,13(S )-epoxy-9(Z ),11-octadecatrienoic acid; AOS, allene oxide synthase; The pathway of jasmonic acid biosynthesis is shown in
CESG, Center for Eucaryotic Structural Genomics; CTS/PXA1, ABC trans-
porter for OPDA or OPDA-CoA import; HPOD, 13(S )-hydroperoxy-
Fig. 1. Jasmonic acid and its octadecanoid precursors are syn-
9(Z ),11(E )-octadecadienoic acid; JA, jasmonic acid; MeJA, jasmonic acid thesized from a-linolenic acid (a-LA) which is found in great
methylester; OPC-8:0, 3-oxo-2(20 (Z )-pentenyl)-cyclopentane-1-octanoic acid; extent in plastidial membranes. From there, a-LA is suggested
LA, a-linolenic acid; OPDA, 12-oxo-phytodienoic acid; OPR, 12-oxo-phytodie- to be released by the action of lipases, e.g. the phospholipase
noic acid reductase; 13(S )-HPOT, 13(S )-hydroperoxy-9(Z ),11(E ),15(Z )-octa- A1 DAD1 [19]. The subsequent oxygenation of a-LA at the
decatrienoic acid; 13-LOX, 13-lipoxygenase.
* Corresponding author. Tel.: þ49 234 32 24463; fax: þ49 234 32 14238.
C-13 position is catalyzed by 13-lipoxygenase [1]. The resulting
E-mail address: eckhard.hofmann@bph.ruhr-uni-bochum.de (E. 13-hydroperoxide, 13(S )-hydroperoxy-9(Z ),11(E ),15(Z )-
Hofmann). octadecatrienoic acid (13-HPOT), is further dehydrated with
0981-9428/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.plaphy.2007.12.007

2. E. Hofmann, S. Pollmann / Plant Physiology and Biochemistry 46 (2008) 302e308 303
the help of allene oxide synthase [29,30], providing the unstable
intermediate, 12,13-epoxy-9(Z ),11,15(Z )-octadecatrienoic
acid (12,13-EOT). Allene oxide cyclase [14,32] catalyzes the
reaction within the octadecanoid pathway which guarantees
enantiomeric specificity, by converting 12,13-EOT to 12-
oxo-10,15(Z )-phytodienoic acid (OPDA). OPDA is then trans-
ferred from the chloroplast to the peroxisome where it is further
metabolized by reduction of the D10-double bond catalyzed
by oxo-phytodienoic acid reductase [28], yielding 3-
oxo-2(20 (Z )-pentenyl)-cyclopentane-1-octanoic acid (OPC-
8:0). Due to radiotracer experiments [36], it is generally agreed
that OPC-8:0 undergoes three consecutive cycles of b-oxidation
which results in the production of bioactive JA with (3R,7S )-
configuration, i.e. (þ)-7-iso-JA.
Allene oxide cyclase (AOC) has been described for the first
time from Zea mays [14,41], followed by the cloning of the
corresponding genes from tomato [42], Arabidopsis [32], and
barley [24]. While in tomato AOC is encoded as a single
gene, in A. thaliana four isogenes can be found, which most
likely evolved from one ancestral isoform by gene duplication
events. With respect to functional differences of the four isoen-
zymes, it has been shown that especially AOC2 mRNA accumu-
lates in the case of local as well as systemic wound response,
whereas AOC1 mRNA seems to be preferentially transcribed
in systemic wound response. By reason that allene oxide syn-
thase (AOS) transcription is also systemically induced after
wounding [20,21], a specific interaction of AOS and AOC1
might be supposable in systemically responding leaves. Further
evidence for functional differences of the AOCs is emphasized
by the occurrence of dinor-oxo-phytodienoic acid (dnOPDA)
which is synthesized from hexadecatrienoic acid. Possibly,
in this context, the isoenzymes possess diverse substrate
specificities [32]. Unfortunately, investigations of either en-
zyme kinetic or substrate specificity of the AOCs have been
hampered by the instability of their substrate. So far, all activity
assays utilized a coupled test system, determining the combined
activity of both AOS and AOC.
The AOCs from Arabidopsis contain a predicted plastidial
target sequence which facilitates the import of the enzymes
into the chloroplast. Functional import of AOC into the chloro-
plast, investigated by immunocytochemical means, has already
been described [32]. However, differentiation between the indi-
vidual isoforms was not possible, suggesting that a more de-
tailed examination of the import of the single isoforms of
AOC is needed. Intriguingly, the expression of Arabidopsis
AOCs has been shown for all plant organs, including roots
[6]. This finding is in contrast to that obtained from tomato
where AOC expression is described to be restricted to floral
Fig. 1. Pathway of jasmonic acid biosynthesis in plants. Intermediates are ab- organs and vascular bundles [15]. However, there are still
breviated as: 13-HPOT, 13(S )-hydroperoxy-9(Z ),11(E ),15(Z )-octadecatrie-
many open questions which mark challenges for future work.
noic acid; 12,13-EOT, 12,13(S )-epoxy-9(Z ),11,15(Z )-octadecatrienoic acid;
OPDA, cis-(þ)-12-oxo-phytodienoic acid; OPDA-CoA, cis-(þ)-12-oxo-phyto- One of the most urgent topics, after unraveling the molecular
dienoic acid-coenzyme A; OPC8:0, 3-oxo-2(20 (Z )-pentenyl)-cyclopentane-1- mechanism of AOC catalyzed 12,13-EOT cyclization, is the
octanoic acid. The enzymes are indicated as: LIP, lipase; LOX, lipoxygenase; elucidation of the functional interconnection of AOS and
AOS, allene oxide synthase; AOC, allene oxide cyclase; OPR, oxo-phytodie- AOC. Although a covalent interaction of AOS and AOC has
noic acid reductase; CTS/PXA1, comatose, ABC transporter for OPDA or
been described as unnecessary [40], the close vicinity of the
OPDA-CoA import; ACS, acyl-CoA synthase.
two proteins seems to enhance their combined activity
(P. Zerbe, personal communication).

3. 304 E. Hofmann, S. Pollmann / Plant Physiology and Biochemistry 46 (2008) 302e308
2. Structures of AOC2 looking along the threefold axis; in Fig. 3 the monomer is
shown.
The structure of AOC2 from Arabidopsis thaliana has been The main structural feature of AOC2 is the central 8-
determined by X-ray crystallography independently in two stranded antiparallel b-barrel. It has a slightly elliptical cross
different labs. Due to these efforts five different structures ˚
section with axes of about 14 and 18 A and walls of height be-
are available from the protein databank. Selenomethionine tween 11 and 30 A ˚ [16]. The barrel is not filled completely by
labeled protein has been crystallized in orthorhombic space side chain atoms but rather forms an elongated hydrophobic
˚ ˚
groups and solved to a resolution of 1.7 A and 1.5 A by the cavity reaching deep into the protein. Notably the highest re-
Center for Eucaryotic Structural Genomics (CESG) (1Z8K, gions of the wall (strands 3e5) which show the most extended
Wesenberg et al., unpublished) and by a group from the h-bonding network and are therefore expected to be the most
Ruhr University Bochum (2BRJ, [16]), respectively. The inde- stable areas of the structure interact with the neighboring
pendently determined structures superpose extremely well monomers in the trimer interface. This trimerization interface
with an overall root mean square deviation of only 0.31 A ˚ ˚
covers roughly 2000 A2 of a monomer surface [16]. While the
for 173 Ca atoms [16]. In both crystal packings one stable barrel is formed by residues 17e147, the remaining 41 C-
trimer of AOC2 is observed per asymmetric unit. As a control terminal residues (colored salmon in Fig. 3) form a mixture
the Bochum group also solved the structure of the unlabelled of helical and random coil structures, which cover the bottom
˚
protein in a monoclinic spacegroup at 1.8 A with two trimers of the barrel and the sides not involved in trimerization. The
in the asymmetric unit (2GIN, [16]). Of functional importance first 16 N-terminal residues are not visible in the structure
is the result of soaking experiments of orthorhombic crystals due to disorder. They consist of the engineered His6-tag with
with a competitive inhibitor, which led to the coordinates of
this molecule inside the proposed catalytic site of the enzyme
(2DIO, [16]). Reevaluation of the original data of the CESG
with an improved refinement protocol resulted in the deposi-
tion of the fifth coordinate set (2Q4I, [22]).
In the following review we will use the highest resolution
coordinates 2BRJ to introduce the overall architecture of the
enzyme. As noted above, in all crystal forms observed so
far, AOC2 was found to form a trimeric quarternary struc-
ture. In Fig. 2 this trimer is shown in ribbon representation
Fig. 2. Structure of AOC2 from Arabidopsis thaliana. Shown is the complete
trimer found in the asymmetric unit of the crystal (accession code 2BRJ). Each Fig. 3. The AOC2 monomer. Shown is the protein in ribbon representation
monomer is individually colored from blue (N-terminus) to red (C-terminus) (2BRJ, chain A). The C-terminal residues 148e188 are colored in salmon.
and labeled with the chain ID (A,B,C). The position of the threefold non- The view is rotated about 90 with respect to Fig. 2. The b-strands of the barrel
crystallographic axis is marked by the black triangle. Figure produced with are labeled S1eS8, the termini of the model are labeled N and C.
Pymol [5]. Figure produced with Pymol [5].

4. E. Hofmann, S. Pollmann / Plant Physiology and Biochemistry 46 (2008) 302e308 305
some additional linker residues and the first two residues after O O
the predicted signal peptidase processing site. The equivalent
residues are missing in all structures in the database so far, re- O O
gardless of the expression constructs used. O O
12,13-EOT 12,13-EOD
Based on topological arguments and on the results of
a DALI-search [17], AOC has been tentatively assigned to
be a member of the lipocalin family [10,16]. Intriguingly, par-
allels exist both in the overall architecture and the substrate
class bound in the central cavity. Most notably prostaglandin O
D synthase is an enzyme involved in the prostanoid synthesis AOC2
in mammals and has been found to share a lipocalin fold [34]. O
Nevertheless key sequence features of lipocalins [10] are miss-
O
ing in the AOC2 structure and the observed similarity might 12,13-Epoxyoctadecadienoic acid
well be a result of convergent evolution instead of a common
O
ancestor [16]. Based on sequence data, the four other known
O
lipocalins from plants have been classified to be members of
O
a divergent subclass of lipocalins termed outlier lipocalins
[13]. Therefore the proposal should be critically reevaluated OPDA
once more lipocalins from plants are structurally known.
Fig. 4. Structural formulas of productive substrate 12,13-EOT, not cyclizable
The finding that AOC2 forms trimers in the crystal struc- analogs 12,13-EOD and the competitive inhibitor vernolic acid (12,13 epox-
tures led to a reevaluation of biochemical data. For corn yoctadecadienoic acid) together with the product OPDA.
AOC a dimeric form had been proposed based on size exclu-
sion chromatography [41]. Similar results have been reported
for AOC2 [16], but have been interpreted as being compatible distance to the conserved water molecule W75. In the complex
with trimers. The observation of SDS-stable trimers both in structure no induced fit of the protein is observed (Fig. 5A).
plant extracts and in purified enzyme led to the conclusion Based on the structural evidence for the binding of vernolic
that the retardation on the size exclusion column might be acid, both substrate 12,13-EOT and product OPDA were mod-
higher than expected by the molecular weight. A more system- eled into the binding pocket to facilitate discussion of possible
atic biochemical analysis is underway in our laboratory but reaction mechanisms (Fig. 5B) [16]. On this basis the follow-
has not yet resulted in clear evidence for the oligomeric state ing scheme was postulated: 12,13-EOT binds with the u-end
in solution (S. Pollmann, unpublished results). into the hydrophobic pocket of AOC2. The epoxide oxygen
is coordinated by a conserved water molecule, which is in
3. The reaction mechanism turn tightly bound by the surrounding protein residues P32,
S31, N25 and N53. The carboxylic moiety of 12,13-EOT rea-
Analysis of the AOC2 structure already suggests that the ches out of the pocket to the surface of the protein. Cyclization
active site is located inside the barrel cavity. While it is mostly is initiated by the delocalization of the C15 double bond which
lined by hydrophobic and aromatic residues, three patches of in turn is triggered by the essential E23 (Fig. 6A). This leads to
more polar residues are noteworthy. One patch is formed by the epoxide opening by anchimeric assistance. The importance
a proline (P32), serine (S31), and two asparagine residues of the C15 double bond for enzyme-controlled cyclization has
(N25, N53) which coordinate a water molecule found in all been demonstrated by several studies and would be rational-
available AOC structures so far. On the opposite side of the ized by this proposal. After epoxide opening the resulting oxy-
barrel wall a cysteine (C71) is located. Finally at the bottom anion is readily stabilized by the conserved water. Hofmann
of the barrel a glutamate residue (E23) is found. All of these et al. [16] proposed the formation of a classical pentadienyl
residues are strictly conserved in known AOC sequences. cation. To reach a productive conformation a transecis isom-
To obtain experimental evidence for the location of the ac- erization around the C10eC11 bond has to be assumed, which
tive site the competitive inhibitor vernolic acid has been used could be driven by the hydrophobic effect due to a better burial
(Fig. 4). It differs from the substrate EOT by the absence of the of the hydrocarbon chain in the pocket. The resulting non-
C11eC12 double bond and can therefore not undergo the cy- planar dienyl-like pentadienyl cation would be stabilized by
clization reaction. Vernolic acid has been shown to be a potent pep interactions of nearby aromatic residues; also the con-
inhibitor of the corn AOC [41], but the inhibitory effect is not served C71 was postulated to stabilize the delocalized positive
as pronounced in the case of Arabidopsis AOC1-4 (P. Zerbe, charge (Fig. 6B). The final step of the reaction would be a con-
personal communication). rotary pericyclic ring closure along the lines of the Wood-
In the structure of AOC2 in complex with vernolic acid wardeHoffmann rules (Fig. 6C). Stereoselectivity would be
(2DIO; Fig. 5A), the inhibitor is bound with the acyl chain mainly achieved by the geometrical control of the transecis
buried deep inside the pocket. The carboxylic moiety is not isomerization by the hydrophobic parts of the pocket.
tightly bound, but rather flexible on the protein surface. The While the proposed mechanism builds upon the concept of
epoxy group of vernolic acid is found in hydrogen-bonding anchimeric assistance proposed for the oxylipin cyclization by

5. 306 E. Hofmann, S. Pollmann / Plant Physiology and Biochemistry 46 (2008) 302e308
Fig. 5. The AOC2 binding pocket. (A) Inhibitor vernolic acid (VA, salmon stick model) bound in the barrel cavity. Shown are both the ligand-free (2BRJ, grey) and
˚
the ligand-bound (2DIO, yellow) structure superposed. Residues closer than 4 A to either vernolic acid or the conserved water molecule W75 (red sphere) are
shown as sticks. (B) Substrate EOT (green) and reaction product OPDA (purple) modeled into the binding pocket of AOC2. The conserved water molecule
W75 is shown as a red sphere, surrounding sidechains are shown as sticks and labeled. Figure produced with Pymol [5].
Grechkin and coworkers [11], and assumes therefore a similar 4. Comparison with other AOC structures
reaction initiation, it differs in the assumed mechanism for
ring closure. In solution a strong dependency of the cyclization A new structure of AOC2 has been deposited recently by the
reaction of 12,13-EOT on the pH has been observed [12], CESG (2Q4I, [22]). Here the original crystallographic data
which cannot be easily explained by a classical pericyclic have been reevaluated with an ensemble refinement method.
ring closure. Instead Grechkin et al. [12] propose a dipolar an- AOC2 has been one of 50 structures used in this methodolog-
nulation mechanism, which includes an intermediate with ical study and is not separately discussed in the paper. In the
a carbanion located at C-9 and a carbocation located at C- deposited coordinates 8 different conformers have been refined
13. This concept is needed to accommodate the observed in parallel to allow a better representation of the inherent
pH-dependency and has been generally accepted. flexibility in the protein. The rationale behind the method is
For the enzyme-controlled reaction the situation is slightly the observation that this kind of model fits the experimental
different. In the rather tight hydrophobic pocket no free sol- data better and should therefore be more informative than the
vent molecules are available to act as stabilizing counter- classical model with isotropic temperature factors for single
charges for intermediate ionic states in the course of the atoms modeling the inherent disorder. Discussion of this ap-
reaction. Instead stabilization has to be facilitated by protein proach is beyond the scope of this review, but a brief analysis
side chains or tightly bound water molecules. So far the of the ensemble model confirms the rigid nature of the barrel,
structural evidence does not present a likely candidate to sta- while the exterior loops show a somewhat higher flexibility.
bilize the proposed carbanion localized at C-9. Therefore the Most notably the backbone and side chains of residues around
more classical scheme with a delocalized charged intermedi- the active site do not show any significant shifts in the different
ate seems to be more favorable. In addition the scheme members of the ensemble.
would be in agreement with a theoretical study on vinyl In the target list of the CESG all four isoforms of AOC have
allene oxides, suggesting a stepwise mechanism including been included (http://targetdb.pdb.org/). In addition to the
strong enantiofacial torque selectivity enforced by the protein structures of AOC2 mentioned above, the structure of AOC1
[23]. ˚
has also been solved at a resolution of 1.8 A and deposited in
Nevertheless, one has to bear in mind that there is no real the PDB (1ZVC). Interestingly, AOC1 also crystallizes as a tri-
structure of the substrateeenzyme complex, which might mer, but in a completely different crystallographic environ-
well show detailed structural features in favor of the annula- ment. This supports the hypothesis of the trimer being
tion mechanism. In addition analysis of point mutations in a relevant aggregation state of AOC1 and AOC2 in vivo. A
the active site area might allow discrimination between the comparison between the two structures showed no significant
two concepts and will clarify the situation. Clearly, more structural deviations [16]. Both proteins are very similar and
work is needed for a full understanding of the reaction differ only in 10 positions in sequence, most of which represent
mechanism. conservative changes (Fig. 7). None of these mutations are

6. E. Hofmann, S. Pollmann / Plant Physiology and Biochemistry 46 (2008) 302e308 307
Fig. 7. Superposition of AOC1 (1ZVC) and AOC2 (2BRJ). Both proteins are
shown in ribbon representation (AOC2 in gray, AOC1 in blue). The sidechains
of residues of AOC2 which are mutated in AOC1 are shown as stick models
and labeled. Figure produced with Pymol [5].
expect any large structural differences to AOC1 or AOC2,
the comparison will improve our understanding of the critical
regions of the enzyme.
Acknowledgements
We thank Dr Florian Schaller and Dr Philipp Zerbe for their
discussions and help with figure preparation. This work was
Fig. 6. Proposed cyclization reaction mechanism. Substrate, product and inter- funded by grants from the Deutsche Forschungsgemeinschaft
mediates are shown as structural formulas, important amino acids are shown as
(DFG), Bonn SCHA939 and HO2600 for EH, SFB480, project
rendered sticks. For details see text.
A-10 for SP.
found to be located close to the active site, which is consistent
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