Recently, we solved the structure of allene oxide cyclase 2 AOC2 of Arabidopsis thaliana, which is, together with the other three AOCs, a key enzyme in the biosynthesis of jasmonates, in
Trang 1thaliana – localization and cyclization
Florian Schaller1,*,Philipp Zerbe1,*, Steffen Reinbothe1, Christiane Reinbothe2, Eckhard Hofmann3 and Stephan Pollmann1
1 Lehrstuhl fu¨r Pflanzenphysiologie, Ruhr-Universita¨t Bochum, Germany
2 Lehrstuhl fu¨r Pflanzenphysiologie, Universita¨t Bayreuth, Germany
3 Lehrstuhl fu¨r Biophysik, AG Ro¨ntgenstrukturanalyse an Proteinen, Ruhr-Universita¨t Bochum, Germany
Since the initial discovery of methyl jasmonate as a
secondary metabolite in essential oils of jasmine in
1962 [1], jasmonates have become accepted as a new
class of plant hormones In the early 1980s, their
wide-spread occurrence throughout the plant kingdom [2]
and their growth-inhibitory [3] and
senescence-promot-ing activities [4] were established, and their route of biosynthesis was elucidated [5]
Research in recent years generally confirmed the Vick and Zimmerman pathway of jasmonic acid (JA) biosynthesis (the octadecanoid pathway) and brought considerable progress with respect to the biochemistry
Keywords
12-oxo-phytodienoic acid; allene oxide
cyclase; allene oxide synthase; oxylipins;
signaling
Correspondence
S Pollmann, Lehrstuhl fu¨r
Pflanzenphysiologie, Ruhr-Universita¨t
Bochum, Germany
Fax: +49 234 32 14187
Tel: +49 234 32 24294
E-mail: stephan.pollmann@rub.de
*These authors contributed equally to this
work
(Received 12 December 2007, revised 24
January 2008, accepted 10 March 2008)
doi:10.1111/j.1742-4658.2008.06388.x
Jasmonates are derived from oxygenated fatty acids (oxylipins) via the octadecanoid pathway and are characterized by a pentacyclic ring struc-ture They have regulatory functions as signaling molecules in plant devel-opment and adaptation to environmental stress Recently, we solved the structure of allene oxide cyclase 2 (AOC2) of Arabidopsis thaliana, which
is, together with the other three AOCs, a key enzyme in the biosynthesis
of jasmonates, in that it releases the first cyclic and biologically active metabolite – 12-oxo-phytodienoic acid (OPDA) On the basis of models for the bound substrate, 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid, and the product, OPDA, we proposed that a conserved Glu promotes the reaction by anchimeric assistance According to this hypothesis, the transition state with a pentadienyl carbocation and an oxyanion is stabi-lized 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 pro-tein environment Here, site-directed mutagenesis was used to explore and verify the proposed reaction mechanism In a comparative analysis of the AOC family from A thaliana involving enzymatic characterization, in vitro import, and transient expression of AOC–enhanced green fluorescent pro-tein fusion propro-teins for analysis of subcellular targeting, we demonstrate that all four AOC isoenzymes may contribute to jasmonate biosynthesis,
as they are all located in chloroplasts and, in concert with the allene oxide synthase, they are all able to convert 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid into enantiomerically pure cis(+)-OPDA
Abbreviations
12,13-EOT, 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid; AOC, allene oxide cyclase; AOS, allene oxide synthase; EGFP, enhanced green fluorescent protein; HPOD, 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid; HPOT,
13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid; JA, jasmonic acid; OPC-8:0, 3-oxo-2[2¢(Z)-pentenyl]-cyclopentane-1-octanoic acid; OPDA, 12-oxo-phytodienoic acid; OPR, 12-oxo-phytodienoic acid reductase; RBCS, ribulose-1,5-bisphosphate carboxylase ⁄ oxygenase.
Trang 2of the enzymes involved, as well as the molecular
organization and regulation of the pathway [6,7]
Biosynthesis is believed to start with the oxygenation
of a-linolenic acid, which is converted to
13(S)-hydro-peroxy-9(Z),11(E),15(Z)-octadecatrienoic acid (HPOT)
in a reaction catalyzed by 13-lipoxygenase (Fig 1)
Allene oxide synthase (AOS) converts HPOT to the unstable epoxide 12,13(S)-epoxy-9(Z),11,15(Z)-octa-decatrienoic acid (12,13-EOT), which is cyclized by allene oxide cyclase (AOC) to give rise to the first cyc-lic 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 [8,9] then yields 3-oxo-2[2¢(Z)-pentenyl]-cyclopentane-1-octanoic acid (OPC-8:0), which under-goes three cycles of b-oxidation to yield the end product of the pathway, i.e JA with a (3R,7S)-configu-ration [(+)-7-iso-JA] [10,11] The biosynthesis of JA appears to involve two different compartments The conversion of LA to OPDA is localized in chloroplasts [10–13], whereas the reduction of OPDA to OPC-8:0 [14,15] and the three steps of b-oxidation, i.e conver-sion of OPC-8:0 to JA, occur in peroxisomes [11,15] (Fig 1) OPDA transport into peroxisomes may be facilitated by ion trapping or via the ABC transporter CTS⁄ PXA1 located in the peroxisomal membrane [16] Whether OPDA is transported in its free acid form or activated by thioesterification to CoA, a pro-cess that may occur in the cytosol, the endoplasmic reticulum, or in the peroxisome, triggered by the activity of specific acetyl-CoA synthetases, is yet to be demonstrated
AOC, a soluble enzyme in corn [17], was biochemi-cally purified as an apparent dimer of 47 kDa from maize kernels [18] and characterized with respect to its substrate specificity In contrast to AOS, which produces both allene oxides from the respective 13(S)-hydroperoxy fatty acids (18:3 and 18:2, respec-tively), the enzyme accepted 12,13(S)-epoxylinolenic acid but not 12,13(S)-epoxylinoleic acid as a substrate [19] It thus appeared that AOC confers additional specificity to the octadecanoid biosynthetic pathway AOC has been cloned as a single-copy gene from tomato [20] and barley [21] and as a small gene family (AOC1–4) from Arabidopsis thaliana [22]
Recently, we solved the structure of A thaliana AOC2 trimers [23] and proposed a mechanism for the enzymatically catalyzed cyclization reaction (Fig 2) In the AOC2 crystals, the competitive inhibitor (±)cis-12,13-epoxy-9(Z)-octadecenoic acid (vernolic acid) was found to be positioned inside the barrel of one mono-mer No induced fit mechanism could be observed On the basis of the resulting arrangement of protein side chains around the bound substrate analog, the following reaction mechanism for the cyclization reac-tion inside the protein was postulated (Fig 2) Subse-quent to the positioning of 12,13-EOT inside the binding pocket of AOC2, Glu23 introduces a negative charge and thus leads to delocalization of the C15
Fig 1 JA biosynthesis in A thaliana LOX2, 13-lipoxygenase
OPR3, 12-oxo-phytodienoate reductase 3; ACS, acyl-CoA
syn-thetase; ACX, acyl-CoA oxidase; MFP, multifunctional protein; KAT,
L -3-ketoacyl-CoA thiolase; CTS ⁄ PXA1, ABC transporter for OPDA or
OPDA-CoA import.
Trang 3double bond The delocalization of the positive charge between C13 and C16 promotes the opening of the oxirane (Fig 2A) and the cyclization reaction by the mechanism of anchimeric assistance The formed oxyanion is stabilized by a water molecule (water75), which is tightly bound and positioned by the forma-tion of hydrogen bonds with the protein environment (Fig 2B) Asn25, Asn53 and Ser31, together with the main chain nitrogen of Pro32, build a polar patch that stabilizes the water molecule in its position To facili-tate the subsequent pericyclic ring closure, a trans–cis isomerization around the C10–C11 bond is necessary, resulting in the formation of a nonplanar ring-like pentadienyl carbocation This carbocation might be stabilized by p–p interaction with the aromatic elec-trons of Phe51 or by Cys71 Owing to steric restric-tions inside the protein cavity, the conformational change around the C10–C11 bond from trans to cis geometry has to be accompanied by a cis–trans rota-tion around the C8–C9 bond The conformarota-tional change is further promoted by a hydrophobic effect, as
it buries more of the hydrocarbon tail of the molecule inside the cavity
In contrast to the spontaneous cyclization of 12,13-EOT, which is likely to proceed via dipolar ring closure [24], the situation is not yet clear for the enzymatic cyclization reaction Here, a classic conro-tary pericyclic ring closure, according to the Wood-ward–Hoffmann rules, seems to be more favorable The absolute stereoselectivity of the reaction is steri-cally controlled by the protein environment and allows only the formation of the cis intermediate A rotation in the opposite direction would automatically result in the opposite stereoisomer of the product Phe43, Val45, Phe85 and Tyr105, in particular, seem
to form a greasy slide that, at the same time, facili-tates and restricts the conformational change of the hydrocarbon tail
Both the spontaneous cyclization of allene oxides [25] and the AOC-catalyzed cyclization share a require-ment for the C15 double bond in the supposed anchi-meric assistance mechanism This also explains the impact of the protein environment on the stereoselec-tivity of the cyclization reaction As a result of the steric restrictions in the cavity, changes in the position
of the epoxide group will affect the binding affinities
of possible substrates
In the present study, we scrutinized the reaction mechanism proposed for AOC2, using site-directed mutagenesis and biochemical characterization of the mutant proteins Moreover, we compared all four AOC isoenzymes of A thaliana with respect to their specific activity and subcellular localization
Fig 2 Schematic overview of the proposed cyclization reaction of
A thaliana AOC2 The AOC2-catalyzed cyclization reaction involves
the opening of the oxirane ring and a subsequent conrotatory
peri-cyclic ring closure (see text for details) The fatty acid moiety of
12,13-EOT [(CH2)7COOH] is labeled as R, and possibly involved
amino acids are presented in the one-letter code following the
numbering of the recombinant protein (see Experimental
proce-dures) Adapted from [48].
Trang 4Overexpression, purification, and enzymatic
characterization of AOC1–AOC4
Because of relatively weak expression levels of published
constructs (C Wasternack, unpublished results), we
cloned differently N-terminally truncated versions of
AOC2 in pET21b(+) (Novagen⁄ Merck, Nottingham,
UK; C-terminal His-tag) and pQE30 (Qiagen, Hilden,
Germany; N-terminal His-tag), respectively, and
ana-lyzed the expression levels of insoluble and soluble
AOC2 Both N-terminally and C-terminally His6-tagged
truncated versions of AOC2, beginning at amino acid 78
of the preprotein in the pET21b and the pQE30 vectors,
showed the highest expression of soluble protein (data
not shown) AOC1, AOC3 and AOC4 were expressed as
N-terminally His-tagged proteins All AOC isoforms
could be easily purified via affinity chromatography on
Ni–nitrilotriacetic acid agarose and were characterized
biochemically (Fig 3A) In a first experiment, the
neces-sity of the C15 double bond for the enzymatic
cycliza-tion was analyzed in a coupled assay that included
recombinant Arabidopsis AOS [26] Using HPOT as the
substrate, AOS produced 12,13-EOT, which, in the
absence of AOC activity, cyclized spontaneously [27]
When HPOD
[13(S)-hydroperoxy-9(Z),11(E)-octadeca-dienoic acid], lacking the C15 double bond, was used as
the substrate for AOS, only trace amounts of
cyclopen-tenones could be detected, indicating that 12,13-EOD
does not readily undergo spontaneous cyclization
[24] Also, in the coupled assay of AOS with the four
AOC isoforms, no cyclization of 12,13-EOD was
observed (data not shown) In contrast, OPDA
produc-tion was detectable, and a shift in the enantiomeric
composition towards the (+)-enantiomer occurred
when HPOT was used as substrate (Fig 3B)
Furthermore, an increase in OPDA formation and
stereoselectivity towards the cis(+)-enantiomer
occurred when methylated HPOT was used as
sub-strate (Fig 3B); this effect was most probably caused
by an improved positioning of the methylated allene
oxide within the hydrophobic barrel of the enzyme,
leading to a larger amount of substrate for the AOC
reaction Thus, the carboxylic group of HPOT plays
no essential role in the cyclization reaction
Cellular distribution of AOS and AOC1–AOC4
All four AOCs of A thaliana, as well as the
Arabidop-sis AOS, are predicted to be localized in the
chloro-plast and to contain appropriate N-terminal transit
peptides (predicted by chlorop [28]) In previously
performed immunocytochemical analyses [22], an anti-body capable of detecting AOC1–AOC4, although detecting AOC2 preferentially over the other isoen-zymes, proved the plastid localization of AOC To val-idate these data and to investigate the subcellular localization specifically for each of the four AOC
A
B
Fig 3 Substrate specificity of A thaliana AOC1–AOC4 (A) Coo-massie-stained SDS ⁄ PAGE (I) and western blot analysis using a monoclonal a-(His)5-antibody (II) of affinity-purified AOCs (1) AOC1, (2) AOC2, (3) AOC3, and (4) AOC4 (B) Ten micrograms of AOS and
5 lg of AOC were incubated in 10 m M PP i buffer (pH 7.0) for
15 min with 100 lg of HPOT (left bars) and methylated HPOT (right bars), respectively The enzymatic reaction was stopped by acidifi-cation and extraction with ethyl acetate Product formation was quantified via chiral GC-MS, and is given as relative activity as com-pared to the yield with AOC2 (1) AOS; (2) AOS and AOC1; (3) AOS and AOC2; (4) AOS and AOC3; (5) AOS and AOC4 cis-(+)-OPDA and cis-( ))-OPDA are indicated by white and gray bars, respectively.
Trang 5isoforms, we carried out in vitro import studies in
con-junction with transient expression assays using
enhanced green fluorescent protein (EGFP)
technol-ogy First, 35S-labeled precursor (p)AOC1–pAOC4
were synthesized from corresponding cDNAs and
imported into isolated Arabidopsis leaf mesophyll
chlo-roplasts Figure 4 shows that [35S]pAOC2 and all of
the other 35S-labeled AOC isoforms were faithfully
taken up by chloroplasts and processed to mature size
The lack of salt extractability of the imported proteins
revealed a membrane localization in all four cases
(Fig 4E–G) Similarly,35S-labeled precursor AOS was
also imported into leaf mesophyll chloroplasts and
processed to mature size, and accumulated in a
salt-resistant form in total membranes (Fig 4A–D)
Second, DNAs encoding full-length AOC1–AOC4
precursors fused to EGFP were transformed into
Arabidopsisleaf epidermis cells and coexpressed with a
construct encoding the first 32 amino acids of the
small subunit of ribulose-1,5-bisphosphate
carboxyl-ase⁄ oxygenase (RBCS) fused to the DsRed protein,
selected as an internal control Transient expression
and subsequent confocal laser scanning microscopy
clearly showed a plastid colocalization of all four
AOC–EGFP fusion proteins as well as of an AOS–
EGFP chimeric protein with the RBCS–DsRed protein
(Fig 5) Together, these results confirmed and
extended findings for AOS (AF230372) of tomato [29]
as well as AOC3 and AOC4 [7] of Arabidopsis, and
showed that plastids have the ability to import all
AOC preproteins (pAOC1–pAOC4) as well as pAOS
Substrate-binding site and biochemical analysis
of mutated AOC2
The barrel part of AOC2 forms an elongated cavity
that is lined mostly by aromatic and hydrophobic
resi-dues and reaches about 14 A˚ into the protein [23] In
particular, Val49, Phe43, Phe51, Phe85 and Tyr105 are
part of this interior hydrophobic pocket, forming a
greasy slide, and these residues, at the same time,
impose the necessary conformational specificity to the
hydrocarbon tail (Fig 6) In addition to these
hydro-phobic residues, the conserved Glu23 is positioned at
the very bottom of the cavity, introducing a negative
charge to initiate the opening of the oxirane ring, and
the subsequent formation of the classic pentadienyl
cation after substrate binding An additional polar
patch is formed by Ser31, Asn25 and Asn53 on one
side of the cavity These three residues, together with
the main chain nitrogen from Pro32, are in
appropri-ate positions for coordinating a tightly bound wappropri-ater
molecule, which is found in all solved AOC2
struc-tures At last we find Cys71 on the opposite wall of the cavity, also being in a favorable position to stabi-lize the pentadienyl cation These residues are strictly conserved among all AOC sequences in the EBI UNIRef100 database
A B
C D
E F
G
Fig 4 In vitro plastid import of AOS and AOC1-4 35 S[Met]-labeled pAOS and pAOC1–pAOC4 were synthesized from respective cDNAs by coupled transcription ⁄ translation in a wheat germ lysate and imported into isolated Arabidopsis leaf mesophyll chloroplasts After 15 min, intact plastids were reisolated on Percoll Unimported precursors were degraded by thermolysin (Thl), and plastid protein was extracted and resolved by 10–20% polyacrylamide gradient gels containing SDS (A) Precursor (P) and mature (m) AOS levels
in intact chloroplasts; TP, translation product (B) Salt extraction of [ 35 S]AOS from total membranes recovered after import from lysed chloroplasts with either 1 M NaCl or 0.1 M Na2CO3 (pH 11), followed by centrifugation of the assays to obtain respective membrane (M) and supernatant (S) fractions (C,D) Time course of [ 35 S]pAOC2 plastid import P, precursor AOC2; m, mature AOC2 (E, F) Plastid import of [35S]pAOC1, [35S]pAOC2, [35S]pAOC3 and [ 35 S]pAOC4 Precursor (P) and mature (m) AOC1–AOC4 protein lev-els are shown for reisolated, intact chloroplasts treated with (+) or without ( )) Thl (G) Salt extraction of total membranes containing imported [35S]AOC1–AOC4 with 0.1 M Na 2 CO 3 (pH 11) After treat-ment, the assays were centrifuged AOC1–AOC4 were detected in the resulting membrane and supernatant (Sups) fractions by SDS ⁄ PAGE and autoradiography.
Trang 6Fig 5 Detection of chimeric fluorophores by confocal laser scanning microscopy Chimeric fusion proteins were transformed into A thaliana using biolistic transformation Left row: AOC–EGFP fluorescence (500–530 nm) Middle row: RBCS–DsRed fluorescence (575–605 nm) Right row: superimposition of the GFP channel and the DsRed channel.
Trang 7The contribution of Phe85 to substrate positioning
has been shown previously [23] Substitution of Phe85
with either Ala or Leu resulted in moderately reduced
reactivity and stereoselectivity of the reaction (Table 1
[23]) To further characterize the importance of the
hydrophobic environment inside the binding pocket
of AOC2, and to assess the role of the amino acids
forming the polar patch, we generated point mutants
of AOC2 and analyzed their ability to catalyze the
cyclization reaction Except for P32V and N53L, all
of the different mutants could be affinity purified to
homogeneity in similar amounts as the wild-type CD
spectroscopic analyses excluded overall folding defects
(supplementary Figs S1 and S2) Any changes in
enzymatic activity are thus caused by the effects of
the single amino acid substitutions With respect to
the importance of the hydrophobic protein
environ-ment inside the barrel, we analyzed the relevance of
Val49, Phe43 and Phe51, in addition to the previously
characterized Phe85 Single point mutations of Phe43
to Tyr, Val49 to Phe and Phe51 to Ala did not
reduce AOC2 activity In contrast, the V49F
muta-tion augmented the enzymatic activity of the protein (Table 1)
Mutation of Glu23 to Ala resulted in a complete loss of the enzymatic activity of AOC2 (Table 1) As the CD spectra of the mutant did not show significant alterations as compared to the wild-type protein, loss
of function can be attributed specifically to the point mutation The data support the proposed role for Glu23, which is to introduce a negative charge into the binding pocket, leading to a delocalization of the C15 double bond towards the oxirane, which promotes its opening and the cyclization reaction by the mechanism
of anchimeric assistance (Fig 2A)
According to the proposed reaction scheme [23], the oxyanion in the transition state is stabilized by a water molecule (water75) (Fig 2B) This water molecule is assumed to be tightly bound by either Ser31, Asn25,
or Asn53, together with Pro32 [23] Mutational experi-ments on these four amino acids clearly demonstrate that Pro32 and Asn25, which were substituted by Val and Leu, respectively, are essential for the enzymatic activity of AOC2 Ser31 (S31A) and Asn53 (N53L), on
Fig 6 Ligplot sketch showing the molecular interactions of AOC2
and the bound inhibitor vernolic acid Contacts with the conserved
water, water75, are shown as blue dashed lines with the
corre-sponding distances (A ˚ ); hydrophobic contacts are represented by
opposing gray spoked arcs The influence of the negative charge of
Glu23 is represented as a red arrow Adapted from [23].
Table 1 Analysis of point mutants of AOC2 of A thaliana The results are presented as relative amount of total OPDA formed, and the specific formation of the cis(+)-enantiomer as compared to the yield with AOC2 Standard deviations have been calculated on the basis of three independent triplicate measurements Discrepan-cies in the CD spectropolarimetric analyses as compared to the wild-type are marked as ‘ )’ Consistency between wild-type and mutant CD spectra are indicated by ‘+’.
Protein
Purity (%)
Activity analysis
CD spectra Total OPDA
(%)
cis-(+)-OPDA (%)
S31A ⁄ N53L > 90 93.6 ± 11.6 84.9 ± 1.4 +
Trang 8the other hand, seemed to be of minor importance for
AOC2 functionality (Table 1) As the N53L mutant
could not be expressed as soluble protein, it was
puri-fied from inclusion bodies and successfully refolded
(supplementary Fig S3) The S31A⁄ N53L double
mutant exhibited almost wild-type activity (Table 1),
confirming that neither amino acid is involved in water
stabilization The mutation P32V resulted in complete
loss of stereoselectivity of AOC2, whereas the residual
activity of total racemic OPDA formation was still
higher than in the AOS control reaction, reflecting
AOC-independent, autocatalytic cyclization of
12,13-EOT However, this protein could not be purified to
apparent homogeneity, and the CD spectrum showed
slight alterations relative to wild-type AOC2, so the
absolute activity values have to be viewed with
cau-tion In the N25L mutant, stereoselectivity was
influ-enced to a minor degree, but the capability for total
OPDA formation was decreased as compared to the
P32V mutant This could be due to the different nature
of the side chains of Asn and Leu and steric
con-straints imposed by the two methyl groups of Leu on
the substrate The finding that the location of the
epoxy group in the 12,13-position of vernolic acid, an
inhibitor of AOC (see below), is essential for binding
to AOC from corn [18] is consistent with the observed
binding position in corn AOC [23] Any shift of the
epoxy group would inhibit the formation of the
hydro-gen bond to the conserved water molecule
We next tested the effect of
(±)cis-12,13-epoxy-9(Z)-octadecenoic acid (vernolic acid) on AOC1–AOC4
and the respective AOC2 mutant proteins
Intrigu-ingly, the total activity of wild-type AOC1 and AOC2
was inhibited by 40% by 0.64 lm vernolic acid, with
a slight loss of stereoselectivity, whereas AOC3 and
AOC4 were only marginally inhibited The sensitivity
of the AOC2 L27R mutant towards vernolic acid
was not enhanced as compared to wild-type AOC2
(Table 2)
Discussion
Substrate-binding site and biochemical analysis
of mutated AOC2 protein
In the present study, a structure–function analysis was performed for AOC2 of A thaliana According to the proposed reaction mechanism of cyclization for the pericyclic ring closure, a conformational change around the C10–C11 bond from trans to cis geometry
is necessary, producing a nonpolar ring-like pentadie-nyl carbocation (Fig 2B) It was previously assumed that Phe51 would be positioned to stabilize this carbo-cation by p–p interaction Here we have demonstrated that Phe51 is not involved in carbocation stabilization, because the F51A mutant shows wild-type-like AOC2 activity, which is reflected by slightly enhanced total OPDA formation and wild-type-like stereoselectivity Alternatively, Cys71 had been postulated to be in a favorable position to stabilize the positive carbocation
We mutated Cys71 to Ala, Ser and Tyr to assess the importance of the sulfhydryl group of Cys The muta-tion of Cys71 to Ser and Ala resulted in fully active proteins Only the mutation to Tyr led to nearly com-plete loss of enzymatic activity, which is most likely explained by steric hindrance invoked by the bulky phenyl ring system
In the proposed catalytic cavity, Phe51, in conjunc-tion with Phe43, Phe85 and Val49, is supposed to form
a greasy slide that helps to coordinate the hydrocarbon tail of the substrate, 12,13-EOT Except for Phe85, none
of these amino acids appeared to be essential for enzy-matic activity Rather, the sum of the hydrophobic resi-dues located within the protein’s catalytic cavity must contribute to the stereoselectivity and cyclization mech-anism In fact, only the mutation of Phe85 to Leu or Ala significantly reduced enzymatic activity (Table 1)
In the coupled AOS⁄ AOC2 activity test, the F85L mutant showed moderately reduced total activity and
Table 2 Studies on the inhibitory effect of vernolic acid on the Arabidopsis AOC The results are presented as relative amount of total OPDA formed, and the specific formation of the cis(+)-enantiomer as compared to the yield with AOC2 in the absence of the inhibitor Stan-dard deviations have been calculated on the basis of two independent duplicate measurements.
Total OPDA (%) cis-(+)-OPDA (%) Total OPDA (%) cis-(+)-OPDA (%) Total OPDA (%) cis-(+)-OPDA (%)
Trang 9a slight loss of stereoselectivity (Table 1) For the
F85A mutant, this result was even more pronounced:
here, total activity was reduced by about 50%, and the
stereoselectivity was reduced to a ratio of 1 : 4 [23] As
this mutant could not be as highly purified as the other
proteins, the absolute activity values have to be treated
with caution Consistent with the proposed function of
Glu23 in the delocalization of the C15 double bond
towards the oxirane (Fig 2A), this residue was found
to be essential for enzymatic activity: substitution of
Glu23 with Ala resulted in complete loss of activity
The intermediate oxyanion of the cyclization reaction
was proposed to be stabilized by a tightly bound water
molecule (Fig 2B) Two amino acids previously
impli-cated in water binding, i.e Pro32 and Asn25, were
found to be essential for the enzymatic activity of
AOC2, presumably by providing a water scaffold to
guide oxylipin cyclization for the enantioselective
for-mation of OPDA Taking into account the high degree
of sequence conservation among AOCs of Arabidopsis
and other plant species, and considering the lack of
sub-stantial differences in their enzymatic activity, our
find-ings implicate a general oxylipin cyclization mechanism
Substrate-binding site of AOC2 of Arabidopsis in
comparison to the AOC binding site of corn
Vernolic acid is a substrate analog of 12,13-EOT
lack-ing the C11 double bond, and is a strong inhibitor of
corn AOC but not of Arabidopsis AOC2 The binding
mechanism of the corn enzyme supposes an interaction
between Arg27, which is absent from the Arabidopsis
enzyme and replaced by a Leu, and the carboxylic acid
group of the inhibitor Arg27 in corn AOC is in a
favorable position to form a strong salt bridge to the
inhibitor [22] Its lack in case of the Arabidopsis
enzyme could explain why the AOC2 crystal structure
soaked with vernolic acid did not provide a defined
electron density indicative of strong interactions for
the first five carbon units of the inhibitor [23]
There-fore, we tested vernolic acid and its methyl ester for
their inhibitory activity on A thaliana AOS, AOC1–
AOC4, and the L27R AOC2 mutant (Table 2) As
compared to the AOC of corn, the Arabidopsis AOCs
displayed a much lower affinity for the inhibitor, even
at high inhibitor concentrations (Table 2) Suprisingly,
the substitution of Leu27 by Arg in Arabidopsis AOC2
did not result in enhanced vernolic acid sensitivity,
arguing against the proposed role for Arg in the
bind-ing of the inhibitor’s carboxylate moiety
Likewise, the increased stereoselectivity of the
reac-tion, when using methylated HPOT as substrate, which
is at variance with findings of Ziegler et al [22], seems
to disprove a role for the inhibitor’s carboxylate moi-ety in enzyme binding Consistent with previous work,
we conclude that the epoxy group in the 12,13-posi-tion, in conjunction with other, yet to be identified, structural elements of the inhibitor, may play the strongest role in inhibitor binding to the enzyme [18,19] However, further effort is needed to resolve the issue of vernolic acid binding to AOC
Comparison of AOC1–AOC4 and their physiological functions
The amino acid sequence of mature AOCs is highly similar, with all amino acids postulated to be involved
in the cyclization reaction being conserved [23], and their three-dimensional structures, including the sub-strate-binding pocket, are almost identical The simi-larity in structure is consistent with the observed similarity with respect to their biochemical proper-ties All Arabidopsis AOCs convert the allene oxide resulting from HPOT with similar efficiency, and none
of the isoforms accepts the HPOD-derived epoxide as
a substrate, supporting the importance of the double bond at position C15 for both the spontaneous and the enzyme-catalyzed cyclization reactions [24,25] Moreover, the increased formation of cis(+)-OPDA with methylated HPOT as substrate demonstrates that the carboxylic moiety of 12,13-EOT is not essential for the cyclization reaction This finding is consistent with the disordered state of the carboxylate tail of vernolic acid in the structure of the AOC2–inhibitor complex (accession code 2DIO) The enhanced activity is due to the improved positioning of the epoxide within the hydrophobic barrel of AOC rather than to enhanced stability of the substrate, as we have observed no con-centration dependency This finding is further under-scored by the high rate of the catalyzed HPOT conversion, combined with a moderate stability of HPOT in aqueous solutions; collectively, these factors eclipse the aspect of substrate stability in this particu-lar context The simiparticu-larity of Arabidopsis AOCs is not limited to structure and activity but extends to subcel-lular localization All four isoforms were localized to plastids (Fig 5), where they are potentially involved in
JA biosynthesis Confocal laser scanning microscopy revealed a plastid localization also for EGFP-tagged Arabidopsis AOS (Fig 5), making an interaction with all AOC isoforms in planta possible These findings are consistent with a recent study showing a plastid locali-zation for AOS and AOC of Solanum tuberosum Potato AOS was found to be associated with thylakoid membranes, whereas AOC was identified as a predomi-nantly soluble protein in the plastid stroma, with only
Trang 10a minor thylakoid association [30] This is somewhat
contradictory to the situation found for A thaliana, as
AOS and AOC2 were shown to be part of the
Arabid-opsisinner envelope membrane (Fig 4) However, our
observations corroborate previous findings [31,32]
Although complex formation of Arabidopsis AOS and
AOC is not obligatory for cis(+)-OPDA formation
in vitro [26], both the enhanced enzymatic activity
when the polypeptides come into close vicinity and the
plastid colocalization of both emzymes suggest novel
means and regulatory mechanisms for controlling the
flow of metabolites through the Vick and Zimmerman
pathway in planta
Considering the similarity of Arabidopsis AOCs with
respect to structure, catalytic activity, and subcellular
localization, the question arises as to what the specific
functions of individual isoforms may be All enzymes
are likely to perform the same reaction in the JA
bio-synthetic pathway, and different functionalities of
the Arabidopsis AOC isoforms may thus result from
developmental and⁄ or tissue-specific expression of the
individual genes Indeed, the expression of AOC genes
was shown to be transiently and differentially
upregu-lated upon wounding, both locally and systemically,
and is induced by JA treatment [22] Furthermore,
the recent analysis of transgenic lines carrying the
GUS reporter gene under the control of the individual
AOC promoters revealed nonredundant promoter
activities in different tissues and during distinct stages
of development [7] (C Delker, unpublished results)
The characterization of the specific role of each of
the AOC isozymes concomitant with JA production
during plant growth and development and in response
to stress will thus require the detailed analysis of
loss-of-function mutants for each of the AOC genes in
Arabidopsis
Experimental procedures
Protein expression and purification
A truncated version of AOC2 from A thaliana was cloned
into the vector pQE30 (Qiagen) using standard protocols
according to [33] or [34], to yield a fusion protein
(20.2 kDa) in which the first 77 amino acids (the predicted
transit peptide for chloroplast targeting) are replaced by a
His6-tag (MRGSHHHHHHRS) A second construct was
generated in pET21b(+) (Novagen), in which the His6-tag
is linked to the C-terminus of the truncated protein
Simi-larly, truncated versions of AOC1, AOC3 and AOC4 were
amplified by PCR with the full-length cDNA (see below) as
the template These fragments were cloned into the BamHI
and SalI restriction sites of pQE30 The constructs were
transformed into Escherichia coli strains M15 and BL21AI, respectively Cells were grown in 2YT medium, and expres-sion was induced at D600 nm0.5–0.6 by addition of 0.2 mm isopropyl thio-b-d-galactoside or 0.2% arabinose, respec-tively Bacteria harboring the pQE30 constructs were har-vested after an induction time of about 5 h at 37C and
220 r.p.m Bacteria with the pET21b construct were induced for an additional 30–40 h at 25C Cells were lysed by soni-cation, and the fusion proteins were purified by affinity chromatography (Ni–nitrilotriacetic acid; Qiagen) and con-centrated by ultrafiltration (Centricon concentrators,
5000 Da cutoff; Millipore, Billerica, MA, USA) The yield
of purified proteins exceeded 10 mgÆL)1culture If not sta-ted otherwise, the mutasta-ted proteins of AOC2 were expressed
in the pQE30 vector under the same conditions as the native AOC2 Primers were as follows: AOC1, 5¢-TATGGATCCC CAAGCAAAGTTCAAGAACTG-3¢, and 5¢-TATGTCGA CGACTAATTTTATTCACTAATT-3¢; AOC3, 5¢-TATGG ATCCCCAAGTAAGATCCAAGAACTA-3¢, and 5¢-TATG TCGACTAAACAGCTAATTACTTAATT-3¢; and AOC4, 5¢-TATGGATCCCCAACTAAGATCCAAGAGCTT-3¢, and 5¢-TATGTCGACACAAAGATTTAGATTTCAATT-3¢ PCR conditions comprised 30 cycles of 94C for 30 min,
54C for 45 min, and 72 C for 100 min
Recombinant AOS [35] was expressed according to Oh & Murofushi [36] in TB medium after induction by addition
of 0.4 mm isopropyl thio-b-d-galactoside at a D600 nm of 0.5–0.6 Cells were sedimented after 20 h at 16C and
150 r.p.m., and lysed by sonication, and recombinant AOS was purified as described by Zerbe et al [26]
Purification of inclusion bodies and refolding The N53L mutant of AOC2 failed to express as soluble protein but accumulated in inclusion bodies, which were purified from bacterial lysates; the recombinant protein was subsequently refolded using the iFOLD refolding system (Invitrogen) The denatured protein was purified via affinity chromatography on Ni–nitrilotriacetic acid agarose and allowed to refold at room temperature during dialysis against 50 mm Tris (pH 7.5), 250 mm NaCl, 12.5 mm b-cyclodextrine, 1 mm Tris(2-carboxyethyl)-phosphine hydrochloride, and 0.5 m l-Arg
Gel electrophoresis and protein immunoblotting Denaturing gel electrophoresis was performed according to [37] The discontinuous systems consisted of 4% stacking gels and 12.5% resolving gels Protein blotting onto nitro-cellulose was carried out electrophoretically overnight (4C, 60 mA) as described by Towbin et al [38] Immun-odetection followed standard procedures [39], with either goat anti-(rabbit IgG)-conjugated or goat anti-(mouse IgG)-conjugated alkaline phosphatase as the secondary