The Crystal Structure of Arabidopsis thaliana Allene Oxide ...which produces both allene oxides from the respective 13(S)-hydroperoxy fatty acids (18:3 and 18:2, respectively). It

The Crystal Structure of Arabidopsis thaliana Allene OxideCyclase: Insights into the Oxylipin Cyclization Reaction W

Eckhard Hofmann,a,1,2 Philipp Zerbe,b and Florian Schallerb,1,2

a Lehrstuhl für Biophysik, Ruhr-Universität Bochum, D-44780 Bochum, Germanyb Lehrstuhl für Pflanzenphysiologie, Ruhr-Universität Bochum, D-44780 Bochum, Germany

We describe the crystallization and structure elucidation of Arabidopsis thaliana allene oxide cyclase 2 (AOC2), a key enzyme

in the biosynthesis of jasmonates. In a coupled reaction with allene oxide synthase, AOC2 releases the first cyclic and bio-

logically active metabolite, 12-oxo-phytodienoic acid (OPDA). AOC2 (AT3G25770) folds into an eight-stranded antiparallel

b-barrel with a C-terminal partial helical extension. The protein forms a hydrophobic binding cavity with two distinct polar

patches. AOC2 is trimeric in crystals, in vitro and in planta. Based on the observed folding pattern, we assigned AOC2 as a low

molecular weight member of the lipocalin family with enzymatic activity in plants. We determined the binding position of the

competitive inhibitor vernolic acid (a substrate analog) in the binding pocket. Based on models for bound substrate 12,13-

epoxy-9,11,15-octadecatrienoic acid and product OPDA, we propose a reaction scheme that explains the influence of the C15

double bond on reactivity. Reaction is promoted by anchimeric assistance through a conserved Glu residue. The transition

state with a pentadienyl carbocation and an oxyanion is stabilized by a strongly bound water molecule and favorable p–p

interactions with aromatic residues in the cavity. Stereoselectivity results from steric restrictions to the necessary substrate

isomerizations imposed by the protein.

INTRODUCTION

Since the initial discovery of methyl jasmonate (MeJA) as a

secondary metabolite in essential oils of jasmine in 1962 (Demole

et al., 1962), JAs have become accepted as a new class of plant

hormone. In the early 1980s, their widespread occurrence

throughout the plant kingdom (Meyer et al., 1984) and their

growth-inhibitory (Dathe et al., 1981) and senescence-promoting

activities (Ueda and Kato, 1980) were established. It has become

increasingly clear, however, that biological activity is not limited

to JA but extends to, and even differs among, its many metab-

olites and conjugates as well as its cyclopentenone precursors

(Kramell et al., 1997; Stintzi et al., 2001). Because of the different

biological function of each member of the group of JAs, the

enzymes of JA biosynthesis and metabolism may thus have a

regulatory function in controlling the activity and relative levels of

different signaling molecules.

Research in recent years generally confirmed the Vick and

Zimmerman pathway of JA biosynthesis (the octadecanoid path-

way) and made considerable progress with respect to the bio-

chemistry of the enzymes involved as well as the molecular

organization and regulation of the pathway.

The early elucidation of the JA biosynthetic pathway and the

demonstration of the growth-inhibiting and senescence-promoting

activity were followed by the discovery that JAs are involved in

plant defense reactions, which greatly stimulated the interest in

these compounds as plant signaling molecules. A role in plant

defense was first shown by Farmer and Ryan, who demonstrated

the induction of proteinase inhibitors by MeJA and JA as part of

the defense response against herbivorous insects (Farmer and

Ryan, 1990; Farmer et al., 1991). JAs were then shown to be

active inducers of antimicrobial phytoalexins by Gundlach et al.

(1992), and subsequent work clearly established their defense

gene–inducing activity for induced resistance against insect pred-

ators and pathogens (for a review, see Reymond and Farmer,

1998; Weiler et al., 1998; Blée, 2002). Such a role was unequiv-

ocally confirmed by the analysis of mutants compromised in

either the synthesis or the perception of JA signals (for a review,

see Schaller et al., 2005).

The pathway of JA biosynthesis is shown in Figure 1. Biosyn-

thesis is believed to start with the oxygenation of free a-linolenic

acid (LA), which is converted to (9Z,11E,15Z,13S)-13-hydroper-

oxy-9,11,15-octadecatrienoic acid [13(S)-HPOT] in a reaction

catalyzed by 13-lipoxygenase. The presumed release of LA from

membrane lipids through the action of a lipase may be triggered

by local or systemic signals, such as oligogalacturonides, chitosan,

systemin (Farmer and Ryan, 1992; Mueller et al., 1993; Narváez-

Vásquez et al., 1999), or wounding (Conconi et al., 1996;

Narváez-Vásquez et al., 1999). The 13(S)-HPOT serves as a

substrate for several enzymes (Figure 1), such as divinylether

synthase, lipoxygenase, hydroperoxide reductase, epoxyalco-

holsynthase, peroxygenase, hydroperoxide lyase, or allene ox-

ide synthase (AOS) (Blée and Joyard, 1996; Vick and

Zimmerman, 1981, 1987). AOS converts 13(S)-HPOT to the

1 These authors contributed equally to this work.2 To whom correspondence should be addressed. E-mail [email protected] or [email protected]; fax 49-234-32-14748or 49-234-32-14187.The authors responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) are: EckhardHofmann ([email protected]) and Florian Schaller ([email protected]).W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.106.043984

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unstable epoxide 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic

acid (12,13-EOT), which is cyclized by allene oxide cyclase

(AOC) to the first cyclic and biologically active compound of the

pathway, 12-oxo-phytodienoic acid (OPDA). Reduction of the

10,11-double bond by an NADPH-dependent OPDA reductase

then yields 3-oxo-2(29(Z)-pentenyl)-cyclopentane-1-octanoicacid (OPC-8:0), which is believed to undergo three cycles of

b-oxidation to yield the end product of the pathway [i.e., JA with

(3R,7S)-configuration; (þ)-7-iso-JA] (Vick and Zimmerman,1984). The biosynthesis of JA appears to involve two different

compartments: the conversion of LA to OPDA is localized in the

chloroplasts (Vick and Zimmerman, 1987; Song et al., 1993;

Laudert et al., 1997), while the reduction of OPDA to OPC-8:0

(Schaller and Weiler, 1997a, 1997b; Schaller et al., 2000;

Strassner et al., 2002) and the three steps of b-oxidation (i.e.,

conversion of OPC-8:0 to JA) occur in peroxisomes (Gerhardt,

1983; Vick and Zimmerman, 1984; Li et al., 2005). Like JA,

OPDA may accumulate to substantial amounts in plant tissue

(Stelmach et al., 1998). By contrast, OPC-8:0 occurs only in trace

amounts.

Vick and Zimmerman (1981) postulated the existence of a

hydroperoxide cyclase involved in the conversion of 13(S)-HPOT

into OPDA. The allene oxide 12,13-EOT was shown to serve as

the immediate precursor of OPDA in plants (Baertschi et al.,

1988; Crombie and Morgan, 1988; Hamberg and Hughes, 1988).

Hamberg and Fahlstadius (1990) showed that this cyclization

reaction is catalyzed by AOC, a soluble enzyme in maize (Zea

mays). AOC was subsequently purified as an apparent dimer of

47 kD from maize kernels (Ziegler et al., 1997) and characterized

with respect to its substrate specificity: the enzyme accepted

12,13(S)-epoxylinolenic acid but not 12,13(S)-epoxylinoleic acid

as a substrate (Ziegler et al., 1999). This is in contrast with AOS,

which produces both allene oxides from the respective 13(S)-

hydroperoxy fatty acids (18:3 and 18:2, respectively). It thus

appears that AOC confers additional specificity to the octade-

canoid biosynthetic pathway.

An interesting aspect of the AOC reaction is the apparent

competition between the spontaneous decomposition of its

substrate, the unstable allene oxide, to form a- and g-ketols and

racemic cis-OPDA, and the enzyme-catalyzed formation of op-

tically pure (9S,13S)-OPDA [i.e., cis(þ)-OPDA], the first pathwayintermediate having the characteristic pentacyclic ring structure

of JAs (Figure 1) and biological activity. The extremely short half-

life of allene oxides (half-time

biochemical and structural studies on AOC2 because this iso-

zyme has been shown to be highly expressed in planta and was

described as being the most active of the four Arabidopsis AOCs

(Stenzel et al., 2003b).

RESULTS

Overexpression, Purification, and Crystallization

of Arabidopsis AOC2

Because of relatively weak expression levels of published con-

structs (C. Wasternack, personal communication) we cloned

differently truncated versions of AOC2 in pET 21b(þ) (Novagen/Merck His6-tag, C-terminally) and pQE30 (Qiagen His6-tag,

N-terminally) and analyzed expression levels of insoluble and

soluble AOC2. Constructs beginning at base 132 showed the

highest expression of soluble protein in both vectors and were

subsequently used.

Using SDS-PAGE analysis, we observed a strong influence of

the His6-tag location on the oligomerization state of the protein.

While AOC2 with a C-terminal His6-tag runs as a monomer with

an apparent mass of ;22 kD, the N-terminally tagged proteinshows a dominant band at a molecular mass of ;60 kD, con-sistent with an SDS-stable trimer formation. The identity of the

bands could be confirmed by immunoblot analyses using anti-

bodies raised against AOC2 or the His6-tag.

A similar pattern to that of the N-terminally tagged protein has

been observed in immunoblot analyses of native AOCs from plant

extracts (P. Zerbe and F. Schaller, unpublished data). This clearly

shows that the trimeric form is present in planta as well and is not

an artifact due to addition of the N-terminal His6-tag. Addition of

a C-terminal His6-tag seems to destabilize the trimers.

Overall Structure

The structure of Arabidopsis AOC2 has been determined by

multiwavelength anomalous dispersion with selenomethionine

(SeMet)-labeled protein crystals of the space group P212121 and

was refined to a resolution of 1.5 Å (SeMet-AOC2). Additionally,

we solved the structure of the unlabeled protein in space group

P21 at a resolution of 1.8 Å (WT-AOC2). In both space groups,

AOC2 forms closely packed trimers. In space group P21, we find

two copies of this AOC2 trimer, which superimpose with a root

mean square deviation (RMSD) of 0.33 Å for all Ca atoms. Both

align well with the single trimer found in the SeMet-AOC2 struc-

ture with RMSDs of 0.40 and 0.44 Å, respectively. Data collection

and refinement statistics are presented in Table 1.

Figure 2 shows the structure of SeMet-AOC2 as a ribbon

diagram together with a topology diagram. The prominent struc-

tural feature of AOC2 is the eight-stranded antiparallel b-barrel of

;40-Å height formed by residues 17 to 147 (numbered accord-ing to the recombinantly expressed protein). Based on the offset

observed in the hydrogen bonding pattern upon closure of the

barrel, we can assign a shear number of 10, which is a measure

for the tilt of the b-strands relative to the barrel axis (Murzin,

1994). The central cross section is slightly elliptical with axes of

;14 and 18 Å (Figure 2B). The 41 C-terminal residues cover the

bottom and one side of the barrel with two one-turn 3/10 helices

and a short a-helix connected by random coil stretches.

The starting residue of the first b-strand (Glu-17, or Glu-82 in

the unprocessed protein) corresponds to the predicted cleavage

site of the transit peptide (ChloroP; Emanuelsson et al., 1999). In

our construct, we included five additional residues, two of which

are well defined in the density. The first 14 residues, including the

N-terminal His6-tag and some vector-specific residues are dis-

ordered in the crystal structure.

The barrel forms an elongated cavity, which is lined mostly by

aromatic and hydrophobic residues and reaches ;14 Å into theprotein (Figure 2C). Three areas of the cavity surface are note-

worthy. First, the conserved Glu-23 is positioned at the very

bottom of the cavity, introducing a negative charge. Second, an

additional polar patch is formed by Ser-31, Asn-25, and Asn-53

on one side of the cavity. These three residues together with

the main chain nitrogen from Pro-32 coordinate a tightly bound

water molecule that is found in all our AOC2 structures. Third, we

found Cys-71 on the opposing wall of the cavity. These residues

are strictly conserved among all AOC sequences in the EBI

UNIRef100 database (Figure 3).

Quarternary Structure (Trimer)

In complete agreement with our biochemical data, we found

AOC2 to form trimers when crystallized (Figure 4). The barrel

axes of the monomers are tilted ;308 with respect to the trimeraxis and pack closely with their barrel walls. Trimerization buries

;2000 A2 of the barrel surface and is the only interaction in thecrystal that is identical for all copies of the molecule. Crystal

contacts cover a significantly smaller surface (800 to 900 A2 per

monomer) and are not identical for all monomers. Identical

trimerization is observed in both the WT-AOC2 and SeMet-

AOC2 crystals, which have different space groups and packing

interactions. Residues involved in trimerization are highly con-

served in known AOCs (see Supplemental Figure 1 online). The

majority of the C-terminal part of the protein is not involved in the

interaction of the monomers (Figure 4), but the C terminus (Asn-

188) together with Arg-29 forms a strong salt bridge to Glu-75 of

a neighboring monomer.

Structural Comparison with Other Proteins

According to the rules underlying the SCOP database (Murzin

et al., 1995), the AOC2 structure with an eight-stranded strictly

antiparallel b-barrel with a shear number of 10 would be either

assigned to the streptavidin fold or to a new fold due to the to-

pological differences in the orientation of the barrel. For func-

tional analysis, it is more fruitful to discuss its relation to protein

families. AOC2 certainly belongs to the superfamily of calycins

(Flower et al., 2000), which are characterized by a central

b-barrel forming a hydrophobic cavity for binding of small lipo-

philic compounds. Major members of this superfamily are the

triabins, metalloproteinase inhibitors, the fatty acid binding pro-

teins, the avidins, and the lipocalins. Of these, the lipocalins most

closely resemble the tertiary structure and fold of AOC2. Indeed,

a DALI (Holm and Sander, 1994) structural similarity search

clearly assigns AOC to the lipocalin fold, finding as the top hits

Crystal Structure of AOC2 3203

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retinol binding protein (RBP; 1AQB) and nitrophorin (1NP1) each

with a Z-score of 3.2.

The lipocalin protein family (Pervaiz and Brew, 1985, 1987;

Grzyb et al., 2006) is a large group of small extracellular proteins

with great diversity at the sequence level but mostly with either

three characteristic short conserved sequence motifs (SCR I-III;

the kernel lipocalins) or else one conserved motif (SCR I; the

outlier lipocalins). However, the crystal structures are highly

conserved and comprise a single eight-stranded continuously

hydrogen-bonded antiparallel b-barrel with an integral ligand

binding site (Figure 5). The structural basis for these SCRs is a

clustering of conserved residues around a central Trp in SCR I at

the beginning of b-strand A, which stabilizes barrel closure. Sev-

eral members of the lipocalin family bind to specific cell surface

receptors or form complexes with soluble macromolecules. Ini-

tially, lipocalins were described as transport proteins; however, it

has become clear that the lipocalins exhibit a multitude of func-

tions, with roles in retinol transport, olfaction, and pheromone

transport, prostaglandin biosynthesis, invertebrate cryptic col-

oration, and a role in the xanthophyll cycle. In addition to the

central b-barrel, the C-terminal part of the protein is usually ap-

pended by a characteristic a-helix. One end of the barrel is

closed by the N terminus that traverses the base of the barrel

(Figure 5). At the opposite end, the b-barrel is typically open to

solvent and provides access to the cavity. These binding sites of

the lipocalins may have very different shapes (e.g., with a ligand

pocket like an extended cave with lobes, a ligand pocket deep

within the hydrophobic core, with loops encapsulating the ligand,

or a binding site with a wide, funnel-like opening to the solvent)

(for a review, see Schlehuber and Skerra, 2005).

Automated superposition of AOC2 with the two lipocalin hits

from the DALI search leads to RMSD values of 2.6 Å for 74

matched Ca for 1AQB and 3.6 Å for 100 matched Ca for 1NP1.

The superposition with RBP as an archetypal representative of

the lipocalin family is shown in Figure 5.

Stretches of superposed residues all fall within the b-strands,

therefore aligning the barrel walls well, while the loop regions

show large differences. AOC2 does not show the classical

C-terminal helix, and the barrel is not closed by the preceding

sequence but by the extended C terminus. Most striking is the

Table 1. Data Collection and Refinement Statisticsa

Data Set Peak

Inflection

Point Remote Inhibitor Wild Type

Beamline ESRF ID13.1 In house SLS PXII

Resolution (Å) 37–1.5 (1.6–1.5) 38–1.7 (1.8–1.7) 50–1.8 (2.1–1.8)

Cell parameters (a, b, c; Å)

(a, b, g; 8)

64.48, 99.95, 106.15

90 90 90

64.5, 99.8, 105.9

90 90 90

67.12, 104.59, 86.96

90 95.0 90

Space group P212121 P212121 P21Wavelength 0.97935 0.97955 0.9789 1.54 0.950

Completeness (%) 95.9 (94.2) 95.9 (94.2) 95.9 (94.2) 93.5 (89.8) 97.7 (93.8)

Multiplicity 7.8 (7.8) 7.8 (7.8) 7.8 (7.8) 4.2 (4.2) 4.3 (3.4)

Average I/sI 19.3 (5.3) 19.3 (5.3) 18.6 (4.9) 15.2 (4.2) 11.3 (4.8)

Rsym (%) 7.0 (38.8) 7.0 (39.1) 7.4 (42.7) 7.9 (34.5) 7.4 (22.0)

Rmeas (%)b 7.5 (41.6) 7.5 (41.9) 7.9 (45.8) 9.1 (39.5) 8.3 (25.8)

Rmrgd-F (%)b 7.0 (26.1) 7.0 (26.1) 7.6 (29.0) 9.5 (24.2) 8.6 (28.2)

Phasing

FOM (RESOLVE)c 0.86

Refinement

Resolution (Å) 37–1.5

(1.54–1.5)

73–1.7 (1.74–1.7) 86–1.8 (1.85–1.8)

Rcryst (%) 17.3 (18.8) 19.8 (29.7) 20.7 (43.4)

Rfree (%)d 19.1 (21.8) 23.3 (36.7) 24.8 (51.2)

Number of atoms

Protein 4194 4124 8179

Inhibitor 21

Glycerol 42 30 30

Water/ion 556 369 660/2

Number of side chains with

alternate conformations

19 6 5

Average B-factor protein (inhibitor) 13.5 12.8 (48.2) 23.8

RMSD from ideality (protein atoms)

Bonds (Å) 0.008 0.021 0.022

Angles (8) 1.224 1.838 1.757

a Data in parentheses represent values in the highest resolution bin.b For definition of Rmeas and Rmrgd-F, see Diederichs and Karplus (1997).c FOM calculated after density modification and threefold averaging in RESOLVE.d Rfree calculated from 5% of data omitted from refinement.

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different position of the termini with respect to the barrel orien-

tation. In the orientation shown in Figure 5, the origin of the closed

b-sheet is on the top for the lipocalins, whereas it is on the bottom

side for AOC2. This topological difference could be the result of a

strand exchange in which either the first or last b-strand (strand A

or strand H in lipocalin nomenclature) has been replaced by C- or

N-terminal sequence stretches, respectively. Notably, the se-

quence DLVPFTNKLY (residues 47 to 56) from AOC2 could be

automatically aligned to SCR I in pig RPB (accession number

1AQB). While we can observe a weak sequence similarity in this

stretch, the conserved Gly and Trp (residues 22 and 24, respec-

tively, in RBP) are missing in the AOC sequence. Interestingly, the

region identified in AOC2 falls within the second b-strand and

superimposes spatially with the location of SCR I in b-strand A of

the lipocalins (Figure 5).

Recently, the structure of a coral AOS domain has been

reported (Oldham et al., 2005). In soft corals, no AOC has been

described so far, so an intriguing possibility would be that coral

AOS catalyzes both epoxidation and cyclization reactions. In-

deed, coral AOS has been found to have a catalase-type fold,

which includes an eight-stranded antiparallel barrel. However,

there has been no report that the barrel fulfils a catalytic role in

cyclization in this case, and the interior is tightly packed with

protein side chains, preventing a similar substrate binding as in

lipocalins.

Substrate Specificity

To gain more information about the catalytic mechanism, we

tested 13(S)-HPOT and 13(S)-hydroperoxy-octadecadienoic acid

(13(S)-HPOD) as substrates for Arabidopsis AOC2 and AOS. We

found that with 13(S)-HPOD as substrate, only negligible amounts

of cyclic product were formed (data not shown). Since AOS

makes use of linoleic acid and linolenic acid derivatives (Siedow,

1991; Song and Brash, 1991; Pan et al., 1995; Laudert et al., 1996),

AOC2 is apparently incapable of cyclizing the corresponding

Figure 2. Structure of the AOC2 Monomer.

(A) Side view of the AOC2 monomer shown in ribbon representation, color changing from blue (N terminus) to red (C terminus).

(B) Top view, rotated by 908 toward the viewer with respect to (A). The same representation is used as in (A).

(C) Molecular surface representation of the proposed binding pocket. The orientation and color scheme are similar to (A). Also shown are the residues

discussed in the text in stick representation. Water 75 is shown as a red sphere.

(D) Topology diagram in the same color coding as in (A). b-Strands are shown as arrows (with numbers indicating the first and last residues) and helices

as small cylinders.


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epoxide, which was indicated before by studies of Ziegler et al.

(1997) on AOC from maize seeds. Therefore, the double bond at

position 15 seems to be essential for the enantioselective cycli-

zation by AOC2. This is in agreement with a proposal for the

chemical cyclization reaction, whereby the 15(Z) double bond

facilitates the opening of the oxirane ring of the allene oxide, and

the resulting C-13 carbocation is stabilized by the p electrons of

the 15(Z) double bond (Grechkin, 1994).

Substrate Binding Site and Biochemical Analysis of

Mutated AOC2 Protein

In 1990, a competitive inhibitor of the AOC of maize [(þ/�)-cis-12,13-epoxy-9(Z)-octadecenoic acid ¼ vernolic acid] was de-scribed (Hamberg and Fahlstadius, 1990). Vernolic acid is a

naturally occurring fatty acid enriched especially in the seed oil

(50 to 90% [w/w] of the total fatty acids) of several Asteraceae

genera (Gunstone, 1954; Badami and Patil, 1980) and Euphor-

biaceae species (Kleinman et al., 1965; Spitzer et al., 1996) and is

a substrate analog of 12,13-EOT only missing the C11 double

bond. We tested vernolic acid for its inhibiting effects on Arabi-

dopsis AOC2. In our coupled enzymatic assay, total activity was

downregulated by 50%, with some loss of stereoselectivity

(Figure 6).

To determine the location of the active site and to gain insight

into possible substrate binding modes, we soaked SeMet-AOC2

crystals with vernolic acid and solved the structure of the com-

plex at 1.7-Å resolution (for details, see Methods and Table 1). As

expected by the structural similarity to the lipocalins, we found

vernolic acid bound in the hydrophobic barrel cavity (Figure 7A).

In the observed binding mode, the charged carboxylic head

group is located outside the cavity on the protein surface, while

the more hydrophobic part of the molecule with the oxirane is

buried deep in the barrel. The molecule is in the extended con-

formation, which is not directly compatible with the cyclization

reaction. The dominant molecular interactions with the protein

are shown in Figure 7B. Overall the protein contributes mostly

aromatic and hydrophobic residues to binding. The only polar/

charged contacts are with Glu-23 close to the C15 double bond

and with the tightly bound water molecule toward the oxirane.

Figure 3. Sequence Alignment of Known AOCs.

Representation of secondary structure elements and numbering is based on the SeMet-AOC2 structure. Asterisks mark residues modeled with

alternate conformations. The following AOC2 sequences are listed (accession numbers in parentheses): Arabidopsis, AOC2_AT (Q9LS02), AOC1_AT

(Q9LS03), AOC3_AT (Q9LS01), and AOC4_AT (Q93ZC5); Pisum sativum, AOC_PS (Q3LI84); Medicago truncatula, AOCa_MT (Q711Q9) and AOCb_MT

(Q599T8); Oryza sativa, AOC_OS (Q8L6H4); Nicotiana tabacum, AOC_NT (Q711R1); Hordeum vulgare, AOC_HV (Q711R0); Zea mays, AOC_ZM

(Q6RW09); Humulus lupulus, AOC1_HL (Q68IP7); AOC4_HL (Q68IP6); Bruguiera sexangula, MAN_BS (Q9AYT8); Lycopersicon esculentum, AOC_LE

(Q9LEG5); S. tuberosum, AOC_ST (Q8H1X5); Physcomitrella patens, AOCa _PP (Q8GS38) and AOCb_PP (Q8H0N6). Figure was produced using the

ESPript server (Gouet et al., 1999).

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Based on the observed protein–inhibitor interactions, we de-

cided to investigate the influence of single amino acid substitutions

on the activity of AOC2. Phe-85 seems to contribute to binding of

the substrate by forming a hydrophobic slide, which at the same

time restricts the conformational freedom of the substrate during

cyclization and thereby could render the reaction stereoselec-

tive. With PCR-based mutagenesis, we exchanged Phe-85 with

Leu (F85L) and Ala (F85A). Both mutants can be purified in similar

amounts as the wild-type protein and show no overall folding

defect as judged by circular dichroism (data not shown). The

F85L mutant was successfully crystallized and showed no

structural deviations from the wild-type protein. In the coupled

AOS/AOC2 activity test, the F85L mutant showed a moderately

reduced total activity and a slight loss of stereoselectivity (Figure

6). For the F85A mutant, this result was even more pronounced:

here, total activity was reduced ;50%, and the stereoselectivitywas reduced to a ratio of 1:4. As this mutant was not as highly

purified as the other proteins, the absolute activity values have to

be treated with some caution.

DISCUSSION

Comparison with Other Arabidopsis AOC Structures

During the preparation of this manuscript, the Center for Eukary-

otic Structural Genomics consortium (Madison, WI) deposited

the coordinates of ligand-free AOC2 (CESG-AOC2, entry 1Z8K)

and ligand-free AOC1 (CESG-AOC1, entry 1ZVC) to the Protein

Data Bank (G.E. Wesenberg, G.N. Phillips Jr., E. Bitto, C.A.

Bingman, and S.T.M. Allard, unpublished data). Although there is

no publication yet on these structures, it is interesting to compare

our results with the available coordinates.

CESG-AOC2 has been solved using SeMet-labeled protein

and was refined at 1.7-Å resolution in the same orthorhombic

space group as our SeMet-AOC2 structure. Both models super-

impose very well with an RMSD of only 0.31 Å for 173 Ca atoms.

A stereoview of the superposition is shown in Figure 8. Closer

inspection does not reveal any significant structural differences;

specifically, the trimerization and the tightly bound water molecule

inside the cavity are also found in the CESG-AOC2 structure.

More interesting is the comparison with CESG-AOC1. The

structure has been solved at a resolution of 1.8 Å in a hexagonal

space group. In these crystals, the same mode of trimerization is

observed for the protein, with the trimer axis now corresponding

to a crystallographic symmetry. Again, we find the same tightly

bound water molecule in the proposed active site of the enzyme.

Sequence comparison to AOC2 shows a total of 10 mutations

Figure 4. Trimeric Form of Arabidopsis AOC2.

Ribbon representation of the AOC2 trimer. Residues 15 to 147 are shown

in gray, and residues 148 to 188 are colored red. The view is along the

trimer axis.

Figure 5. Structural Superposition of AOC2 and RBP.

Stereo diagram of pig RBP (Protein Data Bank accession code 1AQB) with the bound ligand retinol superposed with AOC2. RBP is drawn as a yellow

ribbon, and the bound retinol is shown in van der Waals representation in purple and red. AOC2 is shown as a gray ribbon. To illustrate the discussed

strand exchange, N-terminal portions of both proteins are colored blue (residues preceding the first b-strand in RBP and in the first b-strand in AOC2).

C-terminal stretches of the proteins are colored maroon (including the last b-strand for RBP) and red (after the last residue in the barrel for AOC2). For

clarity, the last 29 residues of RBP have been omitted, which include the conserved lipocalin helix mentioned in the text.


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(depicted in stick model in Figure 8), most of which can be

classified as conservative exchanges, while in three cases a

charge reversal occurs. Introduction of a positive charge at

position 42 (Met to Arg) in the first turn is counterbalanced by the

exchange of Lys-81 to Asn, which is located on the neighboring

turn after the third b-strand. While we can observe a slight

displacement of the main chain in that region, this is likely to be a

result of different crystal packing, as Leu-41 and Met-42 are

involved in crystal contacts in the case of AOC2. The preceding

three residues (corresponding to residues 39 to 41 in AOC2) have

not been modeled in CESG-AOC1, probably due to disorder.

Another exchange involving a reversal in local charge is located

on the opposite side of the protein at residue 178 due to the

exchange of Glu to Lys.

None of the changes seem to be of potential relevance for the

ligand binding site. Indeed, we found no significant differences in

either total activity, stereo, or substrate specificity for all four

AOC isoforms (AOC1-4) in our activity assays (P. Zerbe, V.

Effenberger, and F. Schaller, unpublished data). This is in con-

trast with the observation of Stenzel et al. (2003b), who reported

AOC2 having the highest activity but without quantifying this.

A differential expression pattern of AOCs is clearly established.

Upon wounding and application of jasmonic acid, expression of

AOC2 is upregulated to a higher degree than that of AOC1

(Stenzel et al., 2003b), whereas during senescence, a stronger

upregulation of expression has been observed for AOC1 than for

AOC2 (He et al., 2002). The other two isoforms, AOC3 and AOC4,

seem not to be as strongly upregulated or sometimes even

downregulated. The small structural differences, which mainly

involve a change in the surface potential pattern between AOC1

and AOC2, might therefore represent a subtle optimization for

interactions of these proteins with different partners either in

different cellular locations or physiological states of the plant. For

a complete understanding of these regulatory processes, more

data on the expression profiles for all four enzymes in planta are

necessary. Meanwhile, the structure elucidation of AOC3, whose

crystallization has already been indicated by the CESG in the

structural genomics target database (http://targetdb.pdb.org/),

might shed some more light on the structural variations between

the isoforms.

Structural Relation to the Lipocalin Family

While a database search with the PROSITE lipocalin signature

motif results in 113 hits from plants, there are to our knowledge

only four examples of plant lipocalins explicitly assigned in the

literature: the small temperature-induced lipocalin TIL (Charron

et al., 2002), the two xanthophyll cycle enzymes violaxanthin

deepoxidase (VDE) and zeaxanthin epoxidase (ZEP) (Burgos

et al., 1998), and the recently identified chloroplast lipocalin CHL

(Charron et al., 2005). The last three proteins are located in the

chloroplast. While all four proteins share homology in the struc-

turally conserved regions, especially in SCR I with the invariant

Gly and Trp present, homology in SCR II and III is much weaker,

suggesting that these plant proteins would fall in the class of

outlier lipocalins (Burgos et al., 1998). The other hits produced by

the automated search are either false positive hits or the clas-

sification as lipocalins has not yet been supported by analysis of

functional data.

As AOC2 can be assigned structurally to the lipocalin fold, this

would allow placement in one of several protein families showing

this general folding pattern or even setting AOC2 aside as a new

structural motif on its own. Considering the observed topology of

the barrel and functional similarities, we propose to consider

AOC2 as a distant member of the lipocalin family in plants. The

most relevant differences are discussed below.

The different placement of the termini in the superposition is

striking (Figure 5). The barrel organization can be described right-

or left-handed in the following way: if the thumb points upwards

in the direction of the first b-strand, the bent fingers of the re-

spective hand should indicate the sequential organization of the

successive b-strands. By this definition, all lipocalin family mem-

bers known so far (to our knowledge, this extends to all structures

assigned to the lipocalin fold) form a left-handed barrel, while the

AOC2 calyx is right-handed. In both cases, possible ligand

binding sites are inside the palm and are accessible from above.

As mentioned before, N- or C-terminal strand insertion with

concomitant strand removal on the opposite end would auto-

matically convert right- to left-handed barrels and vice versa.

Along these lines, the first b-strand in AOC2 (blue in Figure 5)

could have been formed by the insertion of the longer N-terminal

part of the classical lipocalins (also colored blue) into the barrel.

This would push b-strand H of the lipocalins (shown in maroon)

out of the continuous b-sheet. The resulting differences in the

structural organization would reduce the central role of the

tryptophane in SCR I, and a loss of similarity in the SCRs would

be obvious. Indeed, the observed weak similarity to SCR I in the

second b-strand could be indicative of such an early process.

Figure 6. Enzymatic Activity of AOC2 Mutants and AOC2 with the

Inhibitor Vernolic Acid.

For determination of enzymatic activity, 5 mg of AOS and 10 mg of AOC2

were mixed in 1 mL of 10 mM PPi buffer, pH 7.0. After addition of 100 mg

of HPOT as substrate, samples were incubated at room temperature

for 15 min. Synthesized OPDA was extracted with ethyl acetate and

analyzed via gas chromatography–mass spectrometry (GC-MS) as

described in Methods. Shown are the results of the coupled enzymatic

assay for AOS alone (1), AOS and AOC2 (2), AOS and AOC2 in presence

of 160 mM inhibitor vernolic acid (3), and the mutants F85L (4) and F85A

(5). The results are presented as the relative amount of synthesized

OPDA compared with the yield with AOC2. The gray bars show total

synthesized OPDA, whereas the black and white bars represent the yield

of (þ)- and (�)-enantiomer, respectively. Error bars represent SD basedon three independent triplicate measurements (1, 2, 4, and 5) or a

duplicate measurement (3).

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Figure 7. Structure of AOC2 with Bound Inhibitor Vernolic Acid.

(A) In the stereoview, monomer B is shown in gray ribbon representation, vernolic acid (VA) as blue stick model, and Water75 as red sphere. Residues

directly involved in interactions with either the inhibitor or Water75 are depicted in yellow stick representation and labeled. Phe-85 is colored red. Omit

difference density for vernolic acid (see Methods) at 2.4 s is drawn as red chicken wire. The view is rotated ;908 around the vertical with respect toFigure 2A.

(B) Ligplot sketch showing the molecular interactions between the protein and the bound inhibitor vernolic acid. Contacts to the conserved Water75 are

shown in green dashed lines with distances, and hydrophobic contacts are represented by opposing red spoked arcs. The interaction of Glu-23 is

represented as a yellow arc. Vernolic acid is shown in purple.


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This divergence in structure from the classical fold is not

unexpected. Investigation of the exon-intron structure of lip-

ocalins clearly places the plant lipocalins together with Dictos-

telium lipocalins into a gene structure–related group distinct from

lipocalins from bacteria, insects, or animals (Sanchez et al.,

2003). Since the early acquisition of the ancestor gene, it has

been duplicated and adapted to different tasks. While TIL still can

be counted as a genuine lipocalin, VDE evolved to be catalytically

active in deepoxidation and acquired additions to the central

barrel structure. With a size of ;350 amino acids, VDEs aremuch larger than the typical lipocalin monomer, which consists

of 160 to 180 amino acids (Flower, 1994; Flower et al., 1995;

Burgos et al., 1998). The same is true for ZEP, for which threading

methods don’t even predict a barrel structure. Therefore, the

peculiar architecture of these proteins raised doubt as to whether

they truly belong to the lipocalin family (Ganfornina et al., 2000;

Salier, 2000).

Up to now, not enough representatives of plant lipocalin genes

have been identified to decide whether the clade of plant

lipocalins does indeed show a much higher variability as the

better-characterized lipocalins from bacteria, insects, or ani-

mals.

On the other hand, one cannot exclude the possibility that we

in fact do observe an example of convergent evolution. This is an

ongoing debate given the very low sequence homology among

lipocalins and especially for the outlier lipocalins, for which only

sequence similarity in SCR I is detectable anyway (Grzyb et al.,

2006).

Oligomerization of AOC2

In former investigations, AOC2 has been characterized to form

dimers based on size exclusion chromatography (Ziegler et al.,

1997), but several lines of evidence point toward a trimer as the

physiological relevant oligomerization state of the protein. First,

we can detect trimers of recombinant AOC2 or AOC2 from plant

extracts in SDS page or immunoblots. Secondly, we observe

very large buried surface areas upon trimerization that include

three salt bridges involving the terminal carboxy group and

several strong hydrogen bonds. This is in agreement with the loss

of trimer formation for the construct with the C-terminal His-tag,

which would likely interfere with the formation of the salt bridges.

Taken together, this pattern is indicative of a stable protein–

protein interaction. Importantly, residues involved in these interac-

tions are predominantly conserved in all known AOC sequences.

Additionally, while both wild-type AOC2 and the CESG-AOC1

from Arabidopsis crystallize in different space groups and have

therefore a modified crystal packing organization, in both cases

the same mode of trimerization is observed.

AOC from maize has been shown to have an apparent molec-

ular mass of 47 kD in size exclusion chromatography and was

discussed as a dimer (Ziegler et al., 1997). We found a similar

behavior for AOC2 of Arabidopsis (data not shown), but based

on our additional structural and biochemical data, we attribute

this discrepancy of 25% to the calculated molecular weight of a

trimer to an atypical retardation of this protein on the size

exclusion matrix.

We therefore expect the trimeric form to be the physiological

relevant oligomerization state for AOCs of Arabidopsis and maize.

Nevertheless, this SDS-stable trimer formation seems not to be

absolutely required, since both the C- and N-terminally tagged

proteins show similar enzymatic activities. However, trimer for-

mation might improve overall protein stability.

Active Site and Reaction Mechanism

Based on the structural similarity of AOC2 to the lipocalins, we

propose that the active site is located inside the barrel cavity.

Figure 8. Structures of AOC2, CESG-AOC2, and CESG-AOC1

Shown is a stereoview of the Ca trace of AOC2 (in gray), CESG-AOC2 (accession code 1Z8K; in red), and CESG-AOC1 (accession code 1ZVC; in blue).

The 10 residues of AOC2 that differ in the AOC1 sequence are depicted in yellow stick representation.

3210 The Plant Cell

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While most lipocalins just bind small hydrophobic substrates in

the interior, very few are known to be enzymatically active. One ex-

ample is noteworthy: human b-trace or prostaglandinsynthase-D

catalyzes the conversion of prostaglandin H2 to prostaglandin

D2. As in the case of AOC2, the substrate is unstable in water and

catalysis in a protected environment seems to be favorable.

Here, the active site Cys is located in the barrel interior (Nagata

et al., 1991).

Indeed, in AOC2 we find the competitive inhibitor vernolic acid

to be bound inside the barrel of one monomer. We don’t observe

any induced fit mechanism, as the protein scaffold superposes

with an RMSD of 0.33 Å for all 174 Ca atoms with SeMet-AOC2.

Even on the level of side chains only marginal differences are

present both in comparison to SeMet-AOC2 and to the other two

monomers without bound inhibitor.

Surprisingly, the carboxylic end of the inhibitor does not seem

to be tightly bound to the protein, as there is no clear electron

density for the first five carbon units. This is in contrast with the

observation of Ziegler and coworkers that methylation of this

group abolishes the inhibitory effect for AOC from maize (Ziegler

et al., 1997). One possible explanation is the exchange of Leu-27

of AOC2 to Arg in maize AOC, which would be positioned to form

a strong salt bridge to the inhibitor in this protein. Another

possibility would be that the carboxylic moiety is involved in the

interaction with AOS for both proteins. Because we don’t have

data on the influence of methylation on inhibition of AOC2, a

decision between both cases is not possible yet. Work is in

progress to obtain comparable data on both proteins with the

different forms of the inhibitor.

The finding that the location of the epoxy group in the 12,13-

position of vernolic acid is essential for binding to AOC from

maize (Ziegler et al., 1997) is consistent with the observed bind-

ing position in our structure. Any shift of the epoxy group would

inhibit the formation of the hydrogen bond to the conserved

water molecule.

To allow a discussion of possible reaction mechanisms, we

tentatively modeled 12,13-EOT (substrate) and OPDA (product)

molecules into the pocket in the same binding mode as vernolic

acid (Figure 9A). Based on the resulting arrangement of protein

side chains around the bound substrate, we postulate the

following reaction mechanism for the cyclization reaction inside

the protein (Figure 10).

After binding into the pocket, the opening of the oxirane ring

and the subsequent formation of the classical pentadienyl cation

will be controlled by the protein environment. The influence of the

negative charge of Glu-23 leads to a delocalization of the C15

double bond toward the oxirane, which promotes its opening and

the cyclization reaction by the mechanism of anchimeric assis-

tance (Figure 10A). Formation of the oxyanion is stabilized by the

tightly bound water molecule Water75. For the subsequent peri-

cyclic ring closure, a conformational change around the C10-C11

bond from trans to cis geometry is necessary, producing a non-

planar ring-like pentadienyl carbocation (Figure 10B). Phe-51 is

positioned to stabilize this by p–p interaction. Additionally, Cys-71

is in a favorable position to stabilize the positive carbocation. This

trans-cis isomerization around C10-C11 has to be accompanied

by a cis-trans rotation around the C8-C9 bond due to the steric

restrictions in the protein cavity. Additionally, the conformational

change is promoted by the hydrophobic effect, as it buries more of

the hydrocarbon tail of the molecule inside the cavity.

The proposed reaction scheme assumes as a final step a

classical conrotary pericyclic ring closure, according to the

Woodward-Hoffmann rules (Figure 10C). The absolute stereo-

selectivity of the reaction follows from the steric restrictions by

the protein environment on the formation of the cis intermediate.

Phe-85, Val-45, and Tyr-105 especially seem to form a greasy

slide, which at the same time facilitates and restricts the confor-

mational change of the hydrocarbon tail. Indeed, the F85L and

F85A mutants show a reduced reactivity and stereoselectivity of

the reaction. The latter effect would be explained by the removal

Figure 9. Substrate and Product Modeled into the Binding Pocket of AOC2 and Comparison with PGHS-1.

(A) Shown are 12,13-EOT and OPDA as green and blue sticks, respectively. The protein is shown in a similar representation as in Figure 7. Also drawn is

the molecular protein surface colored according to atom types. Possible hydrogen bonds between substrate and protein are shown as dashed green

lines.

(B) View into the cyclooxygenase active site of PGHS-1 from sheep (Ovis aris) (accession number 1DIY) with bound substrate arachidonic acid (AA). A

similar representation is chosen as compared with (A). Also shown are the residues discussed in the text.


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of some of the steric restrictions on the trans-cis rotation around

C10-C11. A rotation in the opposite direction would automati-

cally result in the opposite stereoisomer of the product.

Our proposed mechanism explains the importance of the

C15 double bond for reactivity along the lines of the proposal of

Grechkin (1994) by the mechanism of anchimeric assistance. It

also rationalizes the impact of the protein environment on the

stereoselectivity of the cyclization reaction. It is clear by the

packing in the cavity that changes in the position of the epoxide

group will affect binding affinities of possible substrates.

A similar biosynthesis of cyclic hormones can be found in

mammals during formation of prostanoids, which are structurally

similar to JAs. One of the enzymes involved is the Lipocalin-type

prostaglandin D synthase, which has been shown to be an ex-

ample for an enzymatically active lipocalin (Urade and Hayaishi,

2000). The enzyme responsible for the first committed step in

prostanoid catalysis from the linear precursor is prostaglandin

endoperoxide synthase (PGHS). Due to its medical relevance as

target for anti-inflammatory drugs like aspirin, it has been very

well characterized. PGHS catalyzes both the cyclization of

arachidonic acid to prostaglandin G2 and the successive per-

oxidation of PGG2 to prostaglandin H2. While PGHS shows a

completely different overall architecture than AOC, the functional

homology makes a comparison of the active sites worthwhile.

PGHS is a dimeric monotopic membrane protein with a

molecular mass of ;70 kD (monomer), which is located in theendoplasmic reticulum and nuclear envelopes. Besides an

N-terminal EGF domain, the protein is mainly a-helical and has

two distinct active sites for the two catalyzed reaction steps

(Picot et al., 1994; for a review, see Garavito et al., 2002). In

the cyclooxygenase active site, arachidonic acid binds with the

carboxylic end, forming salt bridges to a conserved Arg residue.

The rest of the molecule is tightly packed into an L-shaped cavity

lined mainly with hydrophobic and aromatic residues, as found in

the AOC2 binding pocket (Figure 9B). The catalytically active Tyr

is positioned at the kink of the cavity for abstraction of the 13proS

hydrogen, the initiating step of the reaction. Interestingly, the

only other polar residue is situated on the opposite side of the

polycarbon chain, similar to the position of Cys-71 in the case of

AOC2. This has been shown to be critical in determining the

stereochemistry of the product (Schneider et al., 2002). It has

been found that the packing at the v-end of the substrate

molecule is very important for maintaining the catalytic selectivity

and activity (Rowlinson et al., 1999; Malkowski et al., 2001). While

increasing the size of residues in this region can abolish activity

due to steric inhibition of binding, mutations increasing the vol-

ume of the binding pocket mostly diminish activity, as optimal

positioning of substrate is not enforced. It has been proposed

that the carboxylic end stays fixed during catalysis in PGHS,

while the v-end has to move closer to allow for the substantial

conformational changes necessary for ring closure (Garavito

et al., 2002). In the case of AOC2 from Arabidopsis, a movement

of the carboxyl end of the substrate along our proposed reaction

scheme seems to be more likely. While the relative flexibility of

the carboxy group of vernolic acid might not represent the

situation for the substrate 12,13-EOT in planta, there are no

positively charged residues appropriately placed to form such a

strong salt linkage as observed in the case of PGHS.

Here, we find two completely different structures showing the

same binding principles. A hydrophobic mold binds the sub-

strate exactly in the right position to a single catalytically critical

polar site without apparent induced fit during binding. While the

exact reaction schemes are very different, the hydrophobic mold

in both cases also ensures the stereospecificity of the reaction.

As the comparison with PGHS shows, residues in the vicinity of

the substrate binding site are probably highly optimized to con-

trol the reaction in the cavity. Structural and functional analyses

of additional point mutants will help to clarify the individual

influence of the key amino acids in the binding pocket on the

proposed mechanism and the stereoselectivity of the reaction.

Figure 10. Schematic Overview of the Proposed Cyclization Reaction.

The AOC2 catalyzed cyclization reaction involves the opening of the

oxirane ring and a subsequent conrotary pericyclic ring closure (see text

for details). The fatty acid moiety of 12,13-EOT [(CH2)7COOH] is labeled

as R, and the schematic binding pocket of AOC2 is displayed in gray.

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METHODS

Protein Expression and Purification

A truncated version of AOC2 from Arabidopsis thaliana has been cloned

into the vector pQE30 (Qiagen) and pET21b (Novagen/Merck) to yield

a fusion protein (20.2 kD) in which the first 77 amino acids (the pre-

dicted transit signal) have been replaced by a His6-tag (sequence

MRGSHHHHHHRS) or in which the His6-tag has been coupled with the

C terminus of the truncated protein. The resulting constructs were

transformed into Escherichia coli strain M15. Cells were grown in 2YT

media and induced at an OD of 0.5 to 0.6 with addition of 0.2 mM

isopropylthio-b-galactoside and 0.2% arabinose, respectively. E. coli

with the pQE30 construct were harvested after an induction time of ;5 hat 378C and 220 rpm. Bacteria with the pET21b construct were induced

for a further 30 to 40 h at 258C. Cells were broken by ultrasonication, and

the fusion protein was purified by affinity chromatography (Ni-NTA;

Qiagen) and concentrated with Centricon concentrators (5000 D cutoff;

Millipore). The yield of purified protein exceeded 10 mg/L culture.

SeMet-labeled N-terminally His6-tagged AOC2 was produced by

growth of M15 cells in M9 minimal medium supplemented with SeMet

as described (Van Duyne et al., 1993). The mutated proteins of AOC2

were expressed in the pQE vector under the same conditions as the

native AOC2.

AOS (pQE30-AOS; Laudert et al., 1996) was expressed and purified in

accordance with Oh and Murofushi (2002). Protein expression was

induced at an OD600 of ;0.5 by addition of isopropylthio-b-galactosideto a final concentration of 0.4 mM and further incubation at 168C and 150

rpm overnight. Cells were broken by ultrasonication, and recombinant

AOS was purified via Ni-NTA agarose chromatography (Qiagen) by

adding Triton X-100 up to a final concentration of 0.1% (v/v) to the

recommended buffers.

Generation of Mutant Proteins of AOC2

The generation of AOC2 mutants was done via PCR according to the

sequential PCR step method of the current protocols in molecular

biology. Phe at position 85 was exchanged to Ala (primers: 59-GAAAGGT-

GAAAGAGCCGAAGCTACTTATA-39and 59-TATAAGTAGCTTCGGCTC-

TTTCACCTTTC-39) and Leu (primers: 59-GAAAGGTGAAAGACTCGAA-

GCTACTTATA-39and 59-TATAAGTAGCTTCGAGTCTTTCACCTTTC-39).

The N- and C-terminal primers were 59-TATGGATCCCCAAGCAAAGTT-

CAAGAACTG-39 (BamHI restriction site underlined) and 59-TATGTC-

GACTTAGTTGGTATAGTTACTTATAAC-39 (SalI restriction site under-

lined). The mutated cDNA was cloned afterwards into the expression

vector pQE30, and the protein was expressed under standard conditions.

Gel Electrophoresis and Protein Immunoblotting

Denaturing gel electrophoresis was performed according to Laemmli

(1970). The discontinuous systems consisted of 4% stacking gels and

12.5% resolving gels. Protein blotting onto nitrocellulose was done

electrophoretically overnight (48C, 64 mA) as described by Towbin et al.

(1979). Immunodetection followed standard procedures (Parets-Soler

et al., 1990) with goat anti-rabbit immunoglobin-conjugated alkaline

phosphatase and the enzyme substrates 4-nitrotetrazolium blue and

5-bromo-4-chloro-3-indolyl phosphate.

Determination of Total Enzymatic Activity via

Chiral Capillary GC-MS

For activity analysis, 5 to 20 mg of purified AOS and AOCs (weight-based

ratio 1:2) were added to 1 mL of 10 mM PPi buffer, pH 7.0. The reaction

was started by addition of 100 mg of HPOT. After incubation for 15 min at

room temperature, the reaction was stopped by acidifying with 1 M HCl to

pH 3.0. The synthesized OPDA was extracted twice with 2 volumes of

ethyl acetate and taken to dryness. For GC-MS analysis, 1 nmol of 2[H]5-

OPDA was added before extraction as an internal standard. Dried

samples were incubated in 250 mL of 0.1 M KOH for 45 min at room

temperature to convert the synthesized OPDA into trans-configuration.

After a second extraction step, OPDA was dissolved in methanol and

methylated with ethereal diazomethane (Hamberg and Fahlstadius,

1990). The dried fractions were then redissolved in 50 to 100 mL of

chloroform, and 1 mL of the sample was injected into a Varian GC 3400

gas chromatograph in splitless mode in direct connection to a MAT

Magnum ion trap mass spectrometer (Finnigan) using a chemical ioniza-

tion mode with methanol as reactant gas. Separations of the (þ)- and the(�)-enantiomer of OPDA were made on a b-Dex120 column (30 m 3 0.25mm 3 0.15 mm stationary-phase thickness) coated with 20% permethyl-

b-cyclodextrin in SPB-35 (Supelco) with He carrier gas at 1 mL/min (gas

prepressure of 80 kPa) using the following temperature program: injector

temperature of 2208C, 1 min isothermally at 508C, with 108C/min up to

1908C, 75 min isothermally at 1908C, with 38C/min up to 2208C, 15 min

isothermally at 2208C, and transfer line temperature 2208C. For details,

see Laudert et al. (1997) and Schaller et al. (1998). Quantitation of AOC

enzymatic activity was based on (þ)-OPDA-to-(�)-OPDA ratios derivedfrom integration of total ion current traces.

Crystallization

Initial crystallization screening for AOC2 was performed using vapor

diffusion against the Wizard I and II kits from Emerald BioSystems. Protein

crystals were found with condition WI-28, which was subsequently

optimized. Using 14 to 16% PEG-8000, 6% tert-butanol, and 0.1 M

MES, pH 5.0 to 6.0, as reservoir solution in either hanging- or sitting-drop

setups (4 mg/mL protein concentration), we were able to grow crystals

(space group P21) up to a size of 250 3 150 3 50 mm, which diffract to

;1.7-Å resolution.AOC2-SeMet crystals (space group P212121) were grown from reser-

voir solutions containing 10% PEG-3350, 200 mM NaCl, and 100 mM

phosphate-citrate, pH 4.2, with a protein concentration of 13.5 mg/mL.

They typically reach a size of 250 3 200 3 100 mm and diffract to better

than 1.3-Å resolution. Crystals grew at 188C in ;1 week.For soaking experiments, AOC2-SeMet crystals were transferred for

1 to 2 d into appropriate mother liquor saturated with inhibitor.

X-Ray Diffraction Data Collection

Data collection was performed at 100K on flash-frozen crystals at the

Swiss Light Source (Villigen, Switzerland) at beamline PXII using a MAR

CCD detector. Cryoprotection was achieved by briefly soaking the

crystals in paraffin oil or in mother liquor supplemented with glycerol.

For SeMet-AOC2 crystals, MAD data at the Se edge were collected at the

European Synchroton Radiation Facility (Grenoble, France) on beamline

ID13.1 at 100K using a MAR CCD detector. Data of inhibitor-soaked

SeMet-AOC2 crystals were collected in house at 100K on a Bruker FR591

rotating anode with Montel multilayer optics and a MAR345dtb detector

system.

Data were processed using the XDS package (Kabsch, 1993). Statistics

of the different data sets are shown in Table 1.

Structure Solution and Refinement

SeMet-AOC2

Self-rotation functions indicated the presence of a threefold noncrystallo-

graphic symmetry. Search for the Se substructure and initial phasing

were done with the programs of the SHELX package (Sheldrick and


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Schneider, 1997) using the hkl2map interface (Pape and Schneider, 2004)

with data to 2-Å resolution. SHELXD identified four major peaks, two of

which in retrospect correspond to two copies of SeMet42. The other two

are a double peak corresponding to the third copy of SeMet42. This

solution showed correlation coefficients of 41 for all data and 32 for weak

data. Phasing with SHELXE using data to 3 Å clearly identified the correct

hand and already resulted in a well-interpretable map. Fifty-eight cycles

of density modification using an estimated solvent content of 0.4 resulted

in density contrast of 0.73 (0.38), connectivity of 0.95 (0.91), and a

pseudo-free correlation coefficient of 83.2 (63.3) (values for the wrong

hand in parentheses).

Density modification (using the threefold symmetry), phase extension,

and auto building in RESOLVE (Terwilliger, 2000) could assign 387 of the

expected 564 (3 3 188) residues, including side chains. An additional 44

residues were placed without side chains.

For refinement, the peak wavelength data set was used. Five percent of

the data were randomly assigned to the Rfree test set and omitted from

refinement.

The resulting model was further improved in several cycles of refine-

ment, automated water placement, and manual rebuilding using pro-

grams of the CCP4 package (Collaborative Computational Project,

Number 4, 1994) and ARP/wARP (Cohen et al., 2004).

The final model contains three protein monomers composed of resi-

dues 15 to 188. The first 14 residues, including the His6-tag, could not be

placed due to missing density. Based on clear density, a total of 19

residues had to be modeled with alternate conformations. Additionally,

three glycerol and 556 water molecules were included. Part of the

experimental electron density map after RESOLVE with the final model

is shown in Supplemental Figure 2 online.

WT-AOC2

The structure of WT-AOC2 was solved using the refined coordinates of

SeMet-AOC2 after removal of solvent and alternate conformations and

the mutation of SeMet to Met as a search model. Two trimers were found

in the asymmetric unit of the monoclinic unit cell. After several rounds of

refinement in REFMAC (Murshudov et al., 1997) and manual rebuilding

and water picking in COOT (Emsley and Cowtan, 2004), the final model

consists of 1036 amino acids, five glycerol molecules, two sodium ions,

and 660 water molecules. Gln-102 had to be modeled in alternate

conformations in five of the six copies of AOC2 in the asymmetric unit.

Weak NCS restraints were used throughout the refinement.

SeMet-AOC2 Inhibitor

For analysis of the inhibitor data, one copy of the 2BRJ model without

alternate conformations and solvent molecules was placed in the unit cell.

After several rounds of refinement and water picking, extended residual

density in monomer B was fitted with a model of the inhibitor vernolic acid.

The initial atomic coordinates and the necessary geometry definitions

were created using the PRODRG server (Schuettelkopf and van Aalten,

2004). At the end of the refinement, the occupancy of the inhibitor was set

to 0.25, 0.5, 0.75, and 1 and the structure refined using identical pro-

tocols. With full occupancy, the best R-factors were reached. While we

observed clear density for the majority of the inhibitor, the carboxylic end

up to C5 did not show clear density due to disorder (Figure 7A). The shape

of the resulting difference density together with the higher flexibility of the

inhibitor in comparison to the surrounding protein atoms also points to

different minor binding modes of the inhibitor (possibly also the second

enantiomer), which were not modeled.

Elongated residual density in monomer C was judged too weak to

safely allow positioning of the inhibitor. In monomer A, the entrance to the

binding site is completely blocked by Leu-41 from a crystallographically

related molecule. Therefore, binding of inhibitor during soaking in this

monomer is prohibited. Weak NCS restraints were used in the initial

stages of the refinement but were released after inclusion of the inhibitor.

For preparation of the omit maps, vernolic acid was removed from the

final model and random positional shifts (average RMSD 0.12 Å) were

applied to the remaining coordinates to remove model bias. After another

round of refinement with REFMAC, an mFo-DFc difference omit map was

calculated.

Figure Preparation

Figures were generated using Pymol (http://www.pymol.org). To allow

coloring of residues by conservation, an alignment of 22 different AOCs

found with a National Center for Biotechnology Information BLAST search

in the UNIREF100 database was analyzed using the ESPript server (Gouet

et al., 1999). In the resulting coordinate file, the similarity score has been

assigned to the B-factor column, ranging from 100 (fully conserved) to 0

(no conservation). This range was assigned to the color range red to blue

for figure generation.

Superposition of molecules was either performed in COOT (Emsley and

Cowtan, 2004) or using the SuperPose server (Maiti et al., 2004).

Accession Numbers

Coordinates and structure factors can be found in the Protein Data Bank

under accession numbers 2BRJ, 2GIN, and 2DIO for the AOC2-SeMet,

WT-AOC2, and AOC2-SeMet inhibitor structure, respectively.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Conservation of the Trimer Interface.

Supplemental Figure 2. MAD-Solvent-Flattened Electron Density of

AOC2.

ACKNOWLEDGMENTS

We thank C. Kötting for helpful discussions, K. Diederichs and D.R.

Madden for comments on the manuscript, and E.W. Weiler for continu-

ing support and interest in the work. Part of this work was performed at

the Swiss Light Source, Beamline X10SA, Paul Scherrer Institute

(Villigen, Switzerland) and at the European Synchroton Radiation Facil-

ity, Beamline ID13.1 (Grenoble, France). We would especially like to

acknowledge the help of the respective beamline staff and of our

colleagues from the Max Planck Institutes for Medical Research

(Heidelberg) and Molecular Physiology (Dortmund) during data collec-

tion. We also acknowledge financial support from the Deutsche For-

schungsgemeinschaft by Grants SFB480-C6 (E.H.), HO2600 (E.H.), and

SCHA939 (F.S.).

Received May 10, 2006; revised August 4, 2006; accepted October 17,

2006; published November 3, 2006.

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Documents

The Crystal Structure of Arabidopsis thaliana Allene Oxide ...which produces both allene oxides from the respective 13(S)-hydroperoxy fatty acids (18:3 and 18:2, respectively). It