2008 molecular mechanism of enzymatic allene oxide cyclization in plants

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    Molecular mechanism of enzymatic

    Eckhard Hofmann a,*,a Biophysics, Department of Biology and Biotechnology, Ruhr-Univers

    b Plant Physiology, Department of Biology and Biotechnology, Ruhr-Univ



    nda p

    and are widespread throughout a variety of different plant is the involvement of JA in wound response and pathogenesis

    A1 DAD1 [19]. The subsequent oxygenation of a-LA at theC-13 position is catalyzed by 13-lipoxygenase [1]. The resulting13-hydroperoxide, 13(S )-hydroperoxy-9(Z ),11(E ),15(Z )-octadecatrienoic acid (13-HPOT), is further dehydrated with

    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.: 49 234 32 24463; fax: 49 234 32 14238.E-mail address: eckhard.hofmann@bph.ruhr-uni-bochum.de (E.


    Available online at www.sciencedirect.com

    Plant Physiology and Biochemisphyla [25]. Although the jasmonic acid methyl ester (MeJA)was demonstrated to be a constituent of the essential oil of

    [8,18,37]. Additionally, jasmonates play a crucial role in re-production [9], metabolic regulation [35], and as a signaltransducer 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 phospholipase

    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 acid

    methylester; OPC-8:0, 3-oxo-2(20(Z )-pentenyl)-cyclopentane-1-octanoic acid;LA,a-linolenic acid;OPDA, 12-oxo-phytodienoic acid;OPR, 12-oxo-phytodie-identified signal molecules with phytohormone properties,

    like functions, the jasmonates are among the most recently [4,33]. To date, jasmonic acid and its derivatives are associated

    with diverse physiological functions. The most prominent onefertility, and a multitude of developmental processes. In the course of JA biosynthesis, the first oxylipin with signal character, cis-()-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-()-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. 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-

    Jasminum grandiflorum in the early 1960s [7], it took nearlyanother twenty years until the first physiological effects ofMeJA and the occurrence of the free acid were describedReceived 14

    Available online


    Jasmonates, a collective term combining both jasmonic acid (JA) aThey are characterized as lipid-derived signal molecules which mediate0981-9428/$ - see front matter 2007 Elsevier Masson SAS. All rights reserved.doi:10.1016/j.plaphy.2007.12.007w

    allene oxide cyclization in plants

    Stephan Pollmann b

    ity Bochum, Universitaetsstrasse 150, D-44801 Bochum, Germanyersity Bochum, Universitaetsstrasse 150, D-44801 Bochum, Germany

    ember 2007

    December 2007

    related derivatives, are ubiquitously distributed in the plant kingdom.lethora of physiological functions, in particular stress responses, male

    try 46 (2008) 302e308www.elsevier.com/locate/plaphy

  • 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-()-12-oxo-phytodienoic acid; OPDA-CoA, cis-()-12-oxo-phyto-dienoic acid-coenzyme A; OPC8:0, 3-oxo-2(20(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 or

    OPDA-CoA import; ACS, acyl-CoA synthase.

    E. Hofmann, S. Pollmann / Plant Physiologythe help of allene oxide synthase [29,30], providing the unstableintermediate, 12,13-epoxy-9(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 peroxisome where it is furthermetabolized by reduction of the D10-double bond catalyzedby 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 agreedthat OPC-8:0 undergoes three consecutive cycles of b-oxidationwhich results in the production of bioactive JA with (3R,7S )-configuration, i.e. ()-7-iso-JA.

    Allene oxide cyclase (AOC) has been described for the firsttime from Zea mays [14,41], followed by the cloning of thecorresponding genes from tomato [42], Arabidopsis [32], andbarley [24]. While in tomato AOC is encoded as a singlegene, in 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, it has been shown that especially AOC2mRNA accumu-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 activityassays utilized a coupled test system, determining the combinedactivity 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 ofAOC is needed. Intriguingly, the expression of ArabidopsisAOCs 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 the

    303and Biochemistry 46 (2008) 302e308two proteins seems to enhance their combined activity(P. Zerbe, personal communication).

  • 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 Afor 173 Ca 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 ofthis molecule inside the proposed catalytic site of the enzyme(2DIO, [16]). Reevaluation of the original data of the CESGwith a