Review
Molecular mechanism of enzymatic allene oxide cyclization in plants
Eckhard Hofmann
a,
*, Stephan Pollmann
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 2007Available 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, malefertility, 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 isof particular importance, as it warrants the enantiomeric structure at the cyclopentenone ring which in the end results in the only bioactive JAenantiomer,
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 instablesubstrate, 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
Besides brassinosteroids and oligopeptides with hormone-like functions, the jasmonates are among the most recentlyidentified signal molecules with phytohormone properties,and are widespread throughout a variety of different plantphyla[25]. Although the jasmonic acid methyl ester (MeJA)was demonstrated to be a constituent of the essential oil of
Jasminum grandiflorum
in the early 1960s[7], it took nearlyanother twenty years until the first physiological effects of MeJA and the occurrence of the free acid were described[4,33]. To date, jasmonic acid and its derivatives are associatedwith diverse physiological functions. The most prominent oneis the involvement of JA in wound response and pathogenesis[8,18,37]. Additionally, jasmonates play a crucial role in re-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 hasa pivotal role in the production of protective secondary metab-olites in cell cultures of
Eschscholtzia californica
[2,3].The pathway of jasmonic acid biosynthesis is shown inFig. 1. Jasmonic acid and its octadecanoid precursors are syn-thesized from
a
-linolenic acid (
a
-LA) which is found in greatextent in plastidial membranes. From there,
a
-LA is suggestedto be released by the action of lipases, e.g. the phospholipaseA
1
a
S
)-hydroperoxy-9(
Z
),11(
E
),15(
Z
)-octadecatrienoic acid (13-HPOT), is further dehydrated with
Abbreviations:
ACS, acyl-CoA synthase; AOC, allene oxide cyclase; 12,13-EOT, 12,13(
S
)-epoxy-9(
Z
),11,15(
Z
)-octadecatrienoic acid; 12,13-EOD,12,13(
S
)-epoxy-9(
Z
),11-octadecatrienoic acid; AOS, allene oxide synthase;CESG, Center for Eucaryotic Structural Genomics; CTS/PXA1, ABC trans-porter for OPDA or OPDA-CoA import; HPOD, 13(
S
)-hydroperoxy-9(
Z
),11(
E
)-octadecadienoic acid; JA, jasmonic acid; MeJA, jasmonic acidmethylester; OPC-8:0, 3-oxo-2(2
0
(
Z
)-pentenyl)-cyclopentane-1-octanoic acid;LA,
a
-linolenicacid;OPDA,12-oxo-phytodienoicacid;OPR,12-oxo-phytodie-noic acid reductase; 13(
S
)-HPOT, 13(
S
)-hydroperoxy-9(
Z
),11(
E
),15(
Z
)-octa-decatrienoic acid; 13-LOX, 13-lipoxygenase.* Corresponding author. Tel.:
þ
49 234 32 24463; fax:
þ
49 234 32 14238.
E-mail address:
Ó
2007 Elsevier Masson SAS. All rights reserved.doi:10.1016/j.plaphy.2007.12.007
Available online at www.sciencedirect.com
Plant Physiology and Biochemistry 46 (2008) 302
e
Z
),11,15(
Z
)-octadecatrienoicacid (12,13-EOT). Allene oxide cyclase[14,32]catalyzes thereaction within the octadecanoid pathway which guaranteesenantiomeric 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 peroxisomewhere it is furthermetabolized by reduction of the
D
10
0
(
Z
)-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0). Due to radiotracer experiments[36], it is generally agreedthatOPC-8:0undergoesthreeconsecutivecyclesof
b
-oxidationwhich results in the production of bioactive JA with (3
R
,7
S
)-configuration, i.e. (
þ
)-7-iso-JA.Allene oxide cyclase (AOC) has been described for the firsttime from
Zea mays
Arabidopsis
A. thaliana
four isogenes can be found, which mostlikely evolved from one ancestral isoform by gene duplicationevents. With respect to functional differences of the four isoen-zymes,ithasbeenshownthatespecially
AOC2
mRNAaccumu-lates in the case of local as well as systemic wound response,whereas
AOC1
mRNA seems to be preferentially transcribedin systemic wound response. By reason that allene oxide syn-thase (AOS) transcription is also systemically induced afterwounding[20,21], a specific interaction of AOS and AOC1might be supposable in systemically responding leaves. Furtherevidence for functional differences of the AOCs is emphasizedby the occurrence of dinor-oxo-phytodienoic acid (dnOPDA)which is synthesized from hexadecatrienoic acid. Possibly,in this context, the isoenzymes possess diverse substratespecificities[32]. Unfortunately, investigations of either en-zyme kinetic or substrate specificity of the AOCs have beenhampered by the instability of their substrate. So far, all activityassaysutilizedacoupledtestsystem,determiningthecombinedactivity of both AOS and AOC.The AOCs from
Arabidopsis
contain a predicted plastidialtarget sequence which facilitates the import of the enzymesinto the chloroplast. Functional import of AOC into the chloro-plast, investigated by immunocytochemical means, has alreadybeen 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 tomatowhere AOC expression is described to be restricted to floralorgans and vascular bundles[15]. However, there are stillmany open questions which mark challenges for future work.One of the most urgent topics, after unraveling the molecularmechanism of AOC catalyzed 12,13-EOT cyclization, is theelucidation of the functional interconnection of AOS andAOC. Although a covalent interaction of AOS and AOC hasbeen described as unnecessary[40], the close vicinity of thetwo proteins seems to enhance their combined activity(P. Zerbe, personal communication).
Fig. 1. Pathway of jasmonic acid biosynthesis in plants. Intermediates are ab-breviated as: 13-HPOT, 13(
S
)-hydroperoxy-9(
Z
),11(
E
),15(
Z
)-octadecatrie-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-dienoic acid-coenzyme A; OPC8:0, 3-oxo-2(2
0
(
Z
)-pentenyl)-cyclopentane-1-octanoic acid. The enzymes are indicated as: LIP, lipase; LOX, lipoxygenase;AOS, allene oxide synthase; AOC, allene oxide cyclase; OPR, oxo-phytodie-noic acid reductase; CTS/PXA1, comatose, ABC transporter for OPDA orOPDA-CoA import; ACS, acyl-CoA synthase.303
E. Hofmann, S. Pollmann / Plant Physiology and Biochemistry 46 (2008) 302
e
308
2. Structures of AOC2
The structure of AOC2 from
Arabidopsis thaliana
has beendetermined by X-ray crystallography independently in twodifferent labs. Due to these efforts five different structuresare available from the protein databank. Selenomethioninelabeled protein has been crystallized in orthorhombic spacegroups and solved to a resolution of 1.7 A˚and 1.5 A˚by theCenter for Eucaryotic Structural Genomics (CESG) (1Z8K,Wesenberg et al., unpublished) and by a group from theRuhr University Bochum (2BRJ,[16]), respectively. The inde-pendently determined structures superpose extremely wellwith an overall root mean square deviation of only 0.31 A˚for 173 C
a
atoms[16]. In both crystal packings one stabletrimer of AOC2 is observed per asymmetric unit. As a controlthe Bochum group also solved the structure of the unlabelledprotein in a monoclinic spacegroup at 1.8 A˚with two trimersin the asymmetric unit (2GIN,[16]). Of functional importanceis the result of soaking experiments of orthorhombic crystalswith 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 CESGwith 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 resolutioncoordinates 2BRJ to introduce the overall architecture of theenzyme. As noted above, in all crystal forms observed sofar, AOC2 was found to form a trimeric quarternary struc-ture. InFig. 2this trimer is shown in ribbon representationlooking along the threefold axis; inFig. 3the monomer isshown.The main structural feature of AOC2 is the central 8-stranded antiparallel
b
-barrel. It has a slightly elliptical crosssection with axes of about 14 and 18 A˚and walls of height be-tween 11 and 30 A˚[16]. The barrel is not filled completely byside chain atoms but rather forms an elongated hydrophobiccavity reaching deep into the protein. Notably the highest re-gions of the wall (strands 3
e
5) which show the most extendedh-bonding network and are therefore expected to be the moststable areas of the structure interact with the neighboringmonomers in the trimer interface. This trimerization interfacecovers roughly 2000 A˚
2
e
147, the remaining 41 C-terminal residues (colored salmon inFig. 3) form a mixtureof helical and random coil structures, which cover the bottomof the barrel and the sides not involved in trimerization. Thefirst 16 N-terminal residues are not visible in the structuredue to disorder. They consist of the engineered His
6
-tag with
Fig. 2. Structure of AOC2 from
Arabidopsis thaliana
. Shown is the completetrimer found in the asymmetric unit of the crystal (accession code 2BRJ). Eachmonomer is individually colored from blue (N-terminus) to red (C-terminus)and labeled with the chain ID (A,B,C). The position of the threefold non-crystallographic axis is marked by the black triangle. Figure produced withPymol[5].Fig. 3. The AOC2 monomer. Shown is the protein in ribbon representation(2BRJ, chain A). The C-terminal residues 148
e
188 are colored in salmon.The view is rotated about 90
b
-strands of the barrelare labeled S1
e
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