arabidopsis isochorismate synthase functional in pathogen

Arabidopsis Isochorismate Synthase Functional inPathogen-induced Salicylate Biosynthesis ExhibitsProperties Consistent with a Role in Diverse Stress Responses*□S

Received for publication, May 31, 2006, and in revised form, December 19, 2006 Published, JBC Papers in Press, December 26, 2006, DOI 10.1074/jbc.M605193200

Marcus A. Strawn‡, Sharon K. Marr‡, Kentaro Inoue§, Noriko Inada‡1, Chloe Zubieta¶, and Mary C. Wildermuth‡2

From the ‡Department of Plant and Microbial Biology, University of California at Berkeley, Berkeley, California 94720-3102,the §Department of Plant Sciences, University of California at Davis, Davis, California 95616-8780, and the ¶Joint Center forStructural Genomics, Stanford University, Menlo Park, California 94025

Salicylic acid (SA) is a phytohormonebest known for its role inplant defense. It is synthesized in response to diverse pathogensand responsible for the large scale transcriptional induction ofdefense-related genes and the establishment of systemicacquired resistance. Surprisingly, given its importance in plantdefense, anunderstanding of the underlying enzymology is lack-ing. In Arabidopsis thaliana, the pathogen-induced accumula-tion of SA requires isochorismate synthase (AtICS1). Here, weshow that AtICS1 is a plastid-localized, stromal protein usingchloroplast import assays and immunolocalization. AtICS1 actsas a monofunctional isochorismate synthase (ICS), catalyzingthe conversion of chorismate to isochorismate (IC) in a reactionthat operates near equilibrium (Keq ! 0.89). It does not convertchorismate directly to SA (via an IC intermediate) as doesYersinia enterocolitica Irp9. Using an irreversible coupled spec-trophotometric assay, we found that AtICS1 exhibits an appar-ent Km of 41.5 "M and kcat ! 38.7 min#1 for chorismate. Thisaffinity for chorismate would allow it to successfully competewith other pathogen-induced, chorismate-utilizing enzymes.Furthermore, the biochemical properties of AtICS1 indicate itsactivity is not regulated by light-dependent changes in stromalpH, Mg2$, or redox and that it is remarkably active at 4 °C con-sistent with a role for SA in cold-tolerant growth. Finally, ouranalyses support plastidic synthesis of stress-induced SA with therequirement for one or more additional enzymes responsible forthe conversion of IC to SA, because non-enzymatic conversion ofIC to SA under physiological conditions was negligible.

Chorismate partitioning and utilization are highly regulatedas chorismate is used by bacteria, fungi, and plants to synthesizeboth primarymetabolites such as the aromatic amino acids anddiverse specialized products (1) (see Fig. 1). Isochorismate syn-

thases (EC 5.4.99.6) catalyze the reversible conversion of cho-rismate to IC,3 thereby controlling chorismate partitioning toIC-derived products. Although synthesis and regulation of thebacterial IC pathways have been investigated, there is limitedknowledge regarding these enzymes, their products, and theirfunctional roles in plants. In bacteria, ICS enzymes have beenassociated with either the synthesis of low molecular weightFe3! chelators (siderophores) or menaquinones that functionas electron acceptors. For example, Escherichia coli containstwo ICS genes located in distinct operons encoding proteinswith distinct biochemical properties: EntC is expressed underiron-limited conditions and associated with siderophore bio-synthesis, and MenF is required for menaquinone biosynthesisand expressed under anaerobic conditions where it plays anessential role in anaerobic electron transport (2–5). The initialsteps in bacterial siderophore production from IC involve thesynthesis of salicylic acid (2-hydroxybenzoic acid) or 2,3-dihy-droxybenzoic acid; these compounds are then incorporatedinto siderophores such as pyochelin (Pseudomonas aeruginosa(6)) or enterobactin (E. coli (7)), respectively. In P. aeruginosa,ICS (PchA) and isochorismate pyruvate lyase (PchB) arerequired for the synthesis of SA fromchorismate (6), whereasY.enterocolitica Irp9, a bifunctional SA synthase (SAS) (8, 9),directly converts chorismate to SA via an isochorismate inter-mediate. Importantly, the monofunctional ICS enzyme EcoEntC and the bifunctional SAS Yec Irp9 exhibit a high degree ofstructural similarity and a highly conserved active site suggest-ing that they are evolutionarily related (10). On the otherhand, as shown in Fig. 1, bacterial synthesis of menaquinonesoccurs via the intermediates ortho-succinyl benzoate and1,4-dihydroxy-2-naphthoate (NA) (11). Although bi- (ormulti-) functional bacterial ICS enzymes associated withsynthesis of ortho-succinyl benzoate have not been reported,eubacterial genomes contain conserved colocalized MenF(ICS) and MenD (2-succinyl-6-hydroxy-2,4-cyclohexa-diene-1-carboxylate synthase) genes (12) to channel IC tothe menaquinone pathway (e.g. Ref. 2).

* This work was supported using startup funds provided by the University ofCalifornia at Berkeley. The costs of publication of this article were defrayedin part by the payment of page charges. This article must therefore behereby marked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. 1S and Table 1S.

1 Present address: Dept. of Biological Sciences, Nara Institute of Science andTechnology, 8916-5 Takayama, Ikoma-shi, Nara 630-0101, Japan.

2 To whom correspondence should be addressed: Dept. of Plant and Micro-bial Biology, 111 Koshland Hall, University of California at Berkeley, Berke-ley, CA 94720-3102. Tel.: 510-643-4861; Fax: 510-642-4995; E-mail:[email protected].

3 The abbreviations used are: IC, isochorismate; ICS, isochorismate synthase;SA, salicylic acid; SAS, SA synthase; PhQ, phylloquinone; SAG, SA conju-gate; HPLC, high-performance liquid chromatography; BSA, bovine serumalbumin; PBS, phosphate-buffered saline; DTT, dithiothreitol; IPL, isocho-rismate pyruvate lyase; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS,3-(N-morpholino)propanesulfonic acid; CHES, 2-(cyclohexylamino)eth-anesulfonic acid; CAPS, 2-(cyclohexylamino)propanesulfonic acid; Pae,pseudomonas aeruginosa.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 8, pp. 5919 –5933, February 23, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

FEBRUARY 23, 2007 • VOLUME 282 • NUMBER 8 JOURNAL OF BIOLOGICAL CHEMISTRY 5919

at University of California, Berkeley on February 26, 2007 www.jbc.org

Downloaded from

http://www.jbc.org/cgi/content/full/M605193200/DC1Supplemental Material can be found at:

Plants have long been postulated to produce essential andinduced IC-derived products similar tomenaquinone via ortho-succinyl benzoate and 1,4-dihydroxy-2-naphthoate (13) (Fig.1). For example, phylloquinone (PhQ), a substituted 1,4-naph-thoquinone with an 18-carbon saturated phytyl tail, functionsas an electron acceptor in the photosystem I complex (14).Anthraquinones and naphthoquinones are typically elicitor-in-duced colored compounds, reported to have antioxidant, anti-

microbial, and cytotoxic activities (15, 16).More recently, it wasfound that, similar to bacteria, plants can also produce SA (17)and 2,3-dihydroxybenzoic acid (18) from IC, although there hasbeen no evidence supporting plant production of siderophoresincorporating SA or 2,3-dihydroxybenzoic acid.Plant ICS enzymes have been predicted to be plastid-local-

ized based on 1) the presence of a predicted choroplast transitsequence (e.g. Refs. 17 and 19) and 2) the fact that the substrate

FIGURE 1. Position of ICS in chorismate metabolism in plants and bacteria. Chorismate-derived products include primary metabolites such as the aromaticamino acids, and numerous specialized metabolites, including siderophores and phenazines in bacteria and phenylpropanoids, indole alkaloids, and anthra-quinones in plants. Known chorismate-utilizing enzymes of plant and/or bacterial origin are shown in red: AS, anthranilate synthase; ADCS, 4-amino-4-deoxychorismate synthase; CM, chorismate mutase; CPL, chorismate pyruvate lyase. In Y. enterocolitica, SA is synthesized directly from chorismate by Irp9 anSAS, whereas, in P. aeruginosa, the isochorismate pyruvate lyase PchB is required to convert isochorismate to SA and pyruvate (not shown). IPL (in blue),isochorismate pyruvate lyase.

AtICS1 Functional in Stress-induced Salicylate Biosynthesis

5920 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 8 • FEBRUARY 23, 2007


Downloaded from

chorismate is synthesized in the plastid and largely localized tothe plastid (1, 20, 21); however, experimental evidence support-ing these predictions has been lacking. Past biochemical char-acterizations of plant ICS enzymes utilized ICS (partially) puri-fied from elicited cell suspension cultures ofGaliummollugo L(22, 23), Rubia tinctorum (24), or Catharanthus roseus (19),because these systems provided sufficient activity, protein, andproduct. Elicited cell suspension cultures have also been used toinvestigate the regulation of IC-derived products such asanthraquinones produced byMorinda citrifolia (25). However,these species were not genetically tractable, and thus detailedknowledge of the genes and enzymes involved in the synthesisof IC-derived plant products has been limited. Using the genet-ic/genomic model plant A. thaliana, we provided the firstgenetic evidence for an operational ICS pathway in plants, SAbiosynthesis via AtICS1 (17), and genetic evidence for PhQ bio-synthesis from IC has now been reported (12).Here, we focus on the enzymology of AtICS1, a gene/enzyme

required for pathogen-induced SA biosynthesis (17). SA is bestknown for its role as a key regulator of plant defense againstpathogens. It is synthesized and accumulates primarily as a glu-cose conjugate (SAG) in response to viral, bacterial, and fungalpathogens (26) and is required for the induction of hundreds ofdefense-related genes (e.g. pathogenesis-related PR1), and theestablishment of local and systemic acquired resistanceresponses (26, 27). InA. thaliana, null mutations in the AtICS1gene abrogated induced SA and SAG accumulation and associ-ated defensive responses (17, 28, 29). Over the past few years, amore general role for SA as a mediator of diverse stressresponses has been emerging. For example, inArabidopsis SA issynthesized in response to abiotic stresses such as UV-C (30),ozone (31), cold (32), and heat (33, 34) and has been shown toplay a role in stress-induced developmental transitions, includ-ing flowering (35) and senescence (36). Although AtICS1 hasbeen confirmed to be involved inmany of these processes, it hasonly been implicated in others, such as cold-tolerant growth.Despite the importance of SA, our understanding of the

underlying enzymology is very limited. Here, we have under-taken experiments to fill this knowledge gap and present thefirst biochemical characterization of a plant ICS enzymeinvolved in SA biosynthesis (AtICS1), including quantitativeassessment of its kinetic parameters and subcellular localiza-tion. This is particularly critical, because past biochemical stud-ies did not measure nor account for the fact that ICS enzymestypically operate near equilibrium. Here we address this revers-ibility directly and provide the first accurate and detailedkinetic and thermodynamic properties for a plant ICS. Detailedknowledge of the nature of the ICS reaction, and its affinity forchorismate, as well as its subcellular localization, allows us to 1)examine positive selection for monofunctional ICS versusbifunctional SAS in higher plants, 2) assess the influence ofenvironmental factors such as light and temperature onAtICS1activity, SA biosynthesis, and function, and 3) understand con-trols over chorismate partitioning and utilization. A. thalianacontains multiple genes encoding the chorismate-utilizingenzymes chorismate mutase (including AtCM1, AtCM2, andAtCM3 (37, 38)), anthranilate synthase (e.g. ! subunit: AtASA1and AtASA2; " subunit AtASB1 and AtASB2 (39)), and ICS

(AtICS1 and AtICS2 (17)). Although some of these genes areconstitutively expressed, a subset, including AtICS1, AtCM1,andAtASA1, is induced in response to pathogen treatment (e.g.Refs. 17, 37–39); therefore, knowledge of the biochemical prop-erties of AtICS1 allows one to assess its ability to successfullycompete for available chorismate. Because it is estimated that20% of carbon fixed by plants flows through the shikimate path-way to chorismate under normal growth conditions with thebulk of this fixed carbon utilized in the synthesis of specializedmetabolites (40), detailed knowledge of chorismate partition-ing and utilization is essential to our understanding of plantfitness.EXPERIMENTAL PROCEDURES

Materials and General Protocols

All specialty reagents and chemicals were obtained from Sig-ma-Aldrich unless otherwise specified. HPLC-grade solvents(EMDBiosciences) were employed in the HPLC analyses. Cho-rismic acid (Sigma C-1761, #80% purity) was used in all assayswith the following exception: barium chorismate (SigmaC-1259, 60–80% purity) was used to assess recombinantAtICS1 activity during overexpression and purification and fordetermining the temperature-dependence of AtICS1 activity.For selection and growth of transformed cells (describedbelow): pBAD33 derivatives were selectedwith 30$g/ml chlor-amphenicol; pET-28 derivatives were selected with 50 $g/mlkanamycin; and pME3368 was selected with 100 $g/ml ampi-cillin. Commonly utilized protein and molecular biologicalreagents and protocols were prepared/performed as described in(41). Independent replicate experiments were performed for allexperiments described under “Results,” with similar findings.

Chloroplast Import Assay

Radiolabeled precursors were prepared using the TNT" T7Coupled Reticulocyte Lysate System (Promega) and L-[35S]me-thionine (Amersham Biosciences) with pSM156–29 (full-length AtICS1 cDNA (AY056055) cloned into pCR-Blunt II-TOPO) or plasmids containing Toc75 (42) or Hsp93 (43) ascontrols. Chloroplasts were isolated from 12-day-old pea seed-lings as described previously (44). Import reactions were per-formed as in a previous study (42). In brief, chloroplasts (12.5$g of chlorophyll) were incubated with radiolabeled precursorin a final volume of 50 $l of import buffer (50 mMHepes-KOH,330 mM sorbitol, pH 8.0) with 3 mM Mg-ATP at room temper-ature in the light for 20min. Intact chloroplastswere re-isolatedby 40% Percoll and washed once with the import buffer. Forprotease treatment, the intact chloroplasts that contained theimported proteins were resuspended into 100 $l of the importbuffer with or without 1.25 $g of trypsin and incubated on icefor 30 min in the dark; trypsin inhibitor (1.25 $g/100 $l ofimport buffer) was then added, and intact chloroplasts werere-isolated as above. The chloroplasts were then resuspendedwith hypotonic lysis buffer (10 mMHepes-KOH, pH 8.0, 10 mMMgCl2) and centrifuged at 16,000 " g at 4 °C for 10 min toobtain the soluble andmembrane fractions. To precipitate pro-tein in the soluble fraction, 4" volume of cold acetone wasadded, followed by incubation for 1 h at #20 °C, and centrifu-gation at 16,000" g at 4 °C for 20min. Each fraction was resus-




Downloaded from

pended in sample buffer and analyzed by SDS-PAGE, followedby fluorography.

Immunolocalization of AtICS1

Arabidopsis AtICS1-V5 ics1 Transgenic Line—The A. thali-ana mutant eds16–1 (ics1–2) was stably transformed withAtICS1C-terminalV5-hexaHis under control of the nativeAtICS1 promoter. Agrobacterium-mediated transformationwas performed using pCAMBIA3301 with 35S promoter, andGUS reporter was removed and replaced with the AtICS12.5-kb native promoter and genomic sequence with a V5-hexa-His (Invitrogen) C-terminal tag. Glufosinate-tolerant selectionwas performed using Finale, and surviving plants were selfedand tested for resistance and presence of the insert (assessedusing PCR). The AtICS1-V5 ics1 transgenic line (SM156) ishomozygous, and induced SA accumulation and PR1 expres-sion are restored.Leaf Digestion and Slide Preparation—Mature leaves of

4-week-old Arabidopsis Col-0, eds16–1 (ics1–2), andAtICS1-V5 ics1 transgenics (SM156) were inoculated withPseudomonas syringae pv. maculicola ES4326 at A600 $ 0.002or with 10 mM MgCl2 (using separate plants) as previouslydescribed (45). At 9 h post inoculation, leaves were excised,dissected into %5-mm pieces using a sharp razor blade, andimmediately transferred to enzyme solution (100mMMES, 10%BSA, 0.8 M Mannitol, 1 M KCl, 10 mM CaCl2, 1% w/v cellulase(Lot# 8901, Karlan), 0.26% w/v macerozyme (Lot# 2038, Kar-lan)) to digest cell walls. After vacuum infiltration for 10 min,dissected leaves were incubated at 37 °C for 1 h, rinsed withMSB (0.5 M sucrose, 30 mM PIPES, pH 6.8, 10 mM EGTA, 5 mMMgCl2), and fixed with 3% paraformaldehyde in MSB at roomtemperature for 30 min. Meanwhile, poly-L-lysine-coated glassslides were prepared by immersing slides in 10% poly-L-lysine,0.01% Triton X-100 solution for 10 min and air-drying.Digested leaf material was rinsed twice with MSB, placed onpoly-L-lysine-coated glass slides, and covered with a coverslip.Digested leaf material (e.g. protoplasts) was squashed by gentlytapping the coverslip several times with the end of a pencil. Thecoverslip was removed, and the slides were air-dried.Immunoreaction—The immunoreaction protocol was mod-

ified from a previous study (46). Slides (prepared above) wereimmersed in 0.05%Triton X-100 in 1" PBS, incubated at roomtemperature for 15 min, and rinsed by filling chamber with 1"PBS and incubating at room temperature for 10 min. Slideswere then placed in a moisture chamber (wetted filter paperplaced in a Petri dish) for blocking and immunoreaction. Forblocking, each squashed cell area was covered with 40 $l ofTBSA (5%BSA in 1"PBSwith 0.01%Tween 20), and incubatedat 37 °C for 20 min. 10 $l of 1/100 dilution (in TBSA) mouseanti-V5 antibody (final concentration 1/500, Invitrogen) wasadded to the blocking solution. The moisture chamber wassealed, covered with aluminum, and incubated at 4 °C over-night. Slides were then rinsed with 1% BSA in PBS at roomtemperature for 15 min, placed in a moisture chamber, andincubatedwith 40$l of 5%BSA in PBS at 37 °C for 30min. 10$lof 2° Ab (anti-mouse goat antibody conjugated to Alexa 488fluorophore (Invitrogen) diluted in TBSA) was added (finalconcentration, 1/1000), and slides were incubated in amoisture

chamber at 37 °C for 1 h. After washing with 1" PBS at roomtemperature for 15 min, material on glass slides was counter-stained with 20 $l of 1 $g/ml 4&,6-diamidino-2-phenylindole(Sigma) diluted from 200 $g/ml stock in TAN buffer (0.5 mMEDTA, 20 mM Tris, pH 7.5, 1.2 mM spermidine, 0.05% v/v2-mercaptoethanol) to visualize double-stranded DNA andmounted with a drop of ProLong Antifade (Invitrogen). Pic-tures were taken with Axiophot 381 (Zeiss) with: excitationband pass 450–490 and emission long pass 520 to visualizeAlexa 488 fluorophore, excitation band pass 365/20 and emis-sion long pass 397 to observe 4&,6-diamidino-2-phenylindolestaining, and excitation band pass 535–555 and emission longpass 590 to visualize chlorophyll autofluorescence.

Cloning, Expression, and Purification of Recombinant AtICS1

The AtICS1 coding region (without the chloroplast transitsequence) was PCR-amplified from A. thaliana ecotypeColumbia-O cDNA isolated from induced leaves (forwardprimer: 5&-atcgtcgacccatatgaatggttgtgatgga-3&; reverse primer:5&-atcgtcgactcaattaatcgcctgtagaga-3&) and inserted as an NdeI/SalI fragment into the NdeI/XhoI sites of pET-28c (Novagen).The resulting construct, pSM157-16, contains an N-terminalhistidine tag fused to amino acid 48 of the ICS1 coding region.Construction of the recombinant precursor protein (contain-ing the chloroplast transit sequence) expression vector used 5&-atcgtcgacccatatggcttcacttcaattttc-3& as the forward primer; theresulting construct, pSM159-18, contains an N-terminal histi-dine tag fused to the start codon. The pSM157-16 andpSM159-18 AtICS1 coding sequences were confirmed to beidentical to AtICS1 sequence AY056055 (At1g74710.1).E. coliRosetta2 (DE3) cells (Novagen)were transformedwith

these plasmids. Crude cell extracts were prepared from a 2 literculture of transformed cells in TB media containing 0.2% glu-cose, 50 $g/ml kanamycin, and 30 $g/ml chloramphenicol.Cultures were grown at 37 °C to mid-log phase, 0.2 mM isopro-pyl-"-D-thiogalactopyranoside was added to induce His-ICS1synthesis, and cells were harvested after 18 h at 21 °C ('30 gwet weight) and resuspended in 150 ml of buffer A (20 mMsodiumphosphate buffer, pH7.4, 500mM sodiumchloride, 10%glycerol) containing 1 mM dithiothreitol (DTT), 2 mM phenyl-methanesulfonyl fluoride, 5 $M leupeptin, 10 $g/ml DNase,and 1% Triton X-100. The cells, which could be stored at#20 °C, were lysed by two passages through a French press at18,000 p.s.i. Following centrifugation, the supernatant was fil-tered by syringe through anHPFMillex-HV 0.45-$m filter unitattached in series to a Millex-AP prefilter (Millipore). Filtratewas applied to a 1-ml HisTrap HP nickel affinity column(Amersham Biosciences) at 1 ml/min by use of an AKTA fastprotein liquid chromatography system (Amersham Bio-sciences). After washing column with 10 ml of buffer A, His-AtICS1 was eluted with 40 ml of a linear gradient from 0% to15% at 1.0 ml/min of buffer A containing 500 mM imidazole.Fractions containing ICS activity were pooled and concen-trated using an Amicon Ultra-15 (10-kDa molecular mass cut-off) ultrafiltration device (Millipore) to a final volume of 900$l.The pooled solution was applied to a HiPrep 16/60 SephacrylS-200 High Resolution gel filtration column (Amersham Bio-sciences) previously equilibratedwith 200ml of buffer B (50mM




Downloaded from

sodiumphosphate buffer, pH8.0, 150mM sodiumchloride, 10%glycerol, and 1 mMDTT). The enzyme was eluted at a flow rateof 1.0 ml/min as a broad peak with 60 ml of buffer B. Fractionscontaining ICS activity were pooled and concentrated as beforeto a final volume of 2 ml. The concentrate was dialyzed over-night into buffer C (100 mM Tris buffer, pH 7.7, 10% glycerol,and 1 mM DTT) with or without 10 mM MgCl2 as desired. Theprotein was aliquoted and stored at #80 °C. All further charac-terization was performed on the mature AtICS1 (without thechloroplast transit sequence) recombinant-purified protein.

Cloning and Overexpression of His-PchB

The P. aeruginosa PchB coding region was PCR-amplified(forward primer: 5&-atcgagctcagaaggagtacatatgatgaaaactcccg-aag-3&; reverse primer: 5&-atctctagatcaggcgacgccgcgct-3&) frompME3368 (6) and cloned into the SacI/PstI sites of pBAD33(47). PchB was then subcloned into the NdeI/HindIII sites ofpET-28a (Novagen). The resulting construct, pSM147-1, con-tained an N-terminal histidine tag and 135 nucleotides of thePchA coding region downstream of the PchB stop codon.Crude cell extracts were prepared from a 2-liter culture of

E. coli Rosetta2 (DE3) cells transformed with pSM147-1 growninTBmedia containing 0.2% glucose, 50$g/ml kanamycin, and30 $g/ml chloramphenicol. Cultures were grown at 37 °C tomid-log phase, 1 mM isopropyl-"-D-thiogalactopyranoside wasadded to induce His-PchB synthesis, and cells were harvestedafter 4 h ('25 g wet weight) and stored overnight at #20 °C.His-PchB was then purified using nickel-nitrilotriacetic acidHis-Bind Resin (Novagen) according to the manufacturer’sdirections. Aliquots of the purified recombinant PchB protein(54 mg/ml in 100 mM Tris, pH 7.7, 10% glycerol, 1 mM DTT)were stored at #80 °C.

Determination of Protein Concentration

Protein concentrations were determined by the method ofBradford modified for use in a 96-well plate format with Coo-massie BlueG-250 (EMBiosciences) and analyzed using a Spec-tramax Plus microplate spectrophotometer (MolecularDevices) with bovine serum albumin as the standard.

Molecular Mass Estimation

The subunit molecular mass of AtICS1 was estimated bySDS-PAGE in a 10% gel with Precision Plus unstained proteinstandards (Bio-Rad). The native molecular mass was estimatedby gel filtration chromatography on a HiPrep 16/60 SephacrylS-200 High Resolution column in buffer B (above) at 1.0ml/min, using thyroglobulin (670 kDa), %-globulin (158 kDa),ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B12 (1.35kDa) asmarkers (Bio-RadGel Filtration Standards). The appar-ent molecular mass of AtICS1 was determined from a plot ofthe elution volumes against the logarithm of the molecularmasses.

ICS Activity Assays

HPLC ICS Activity Assay—This assay was modified from aprevious study (19). 40$l of substrate solution (2mM chorismicacid in buffer D: 100 mM Tris, pH 7.7, 10% glycerol, 10 mMMgCl2, 1 mM DTT) was added to 40 $l of a 220 $g/ml solution

of AtICS1 in buffer D, incubated for 60min at 30 °C, and imme-diately filtered through a 0.2-$mMillex-LG syringe filter (Mil-lipore), and a 50-$l aliquot was injected into a Shimadzu SCL-10AVP series HPLC system equipped with a ShimadzuSPD-10AVP photodiode array detector and a ShimadzuRF-10AXL fluorescence detector. A 5-$m, 15-cm " 4.6-mminner diameter Supelcosil LC-ABZPlus column (Supelco) pre-ceded by a LC-ABZPlus guard column was maintained at 27 °Cand previously equilibrated in 15% acetonitrile with 25 mMpotassium phosphate buffer, pH 2.5, at a flow rate of 1.0 ml/min.The elution program began with an isocratic flow of 15% acetoni-trile with 25 mM potassium phosphate buffer, pH 2.5, for 1 min,followed by a linear increase to 20% acetonitrile over 7 min. Priorto injecting subsequent samples, a linear decrease to 15% acetoni-trile over 2 min was undertaken, followed by re-equilibration at15%acetonitrile for at least 5min.Under these conditions, isocho-rismic acid eluted (A280) at'3.4 min and chorismic acid (A280) at4.2min. The calibration curve for chorismic acid is as follows: y$0.00386319x# 11.2600withR2( 0.999,where x$ area units andy$ chorismic acid in nanograms.Coupled HPLC Assay for ICS Activity—This assay was mod-

ified from a previous study (48). 437.5$l of a solution of AtICS1(3.8$g/ml) and PchB in excess (218$g/ml) in buffer E (100mMpotassium phosphate buffer, pH 7.0, 15 mM MgCl2, 10% glyc-erol, and 1 mM DTT) was mixed with 62.5 $l of substrate solu-tion (4mM chorismic acid in buffer E) and incubated for 60minat 30 °C. The reaction was filtered as above, and a 50-$l aliquotwas injected into the HPLC system with column describedabove. The elution program beganwith an isocratic flow of 15%acetonitrilewith 25mMpotassiumphosphate buffer, pH2.5, for1 min, followed by a linear increase to 20% acetonitrile over 5min, isocratic flow at 20% for 10 min, a linear increase from 20to 55% acetonitrile over 17.5 min, a linear increase from 55 to66%over 5min, and an isocratic flow at 66% for 1.5min. Prior toinjecting subsequent samples, a linear decrease to 15% acetoni-trile over 5min was undertaken, followed by re-equilibration at15% acetonitrile for at least 5min. This program allowed for thedetection of isochorismic acid (A280, 3.7 min), chorismic acid(A280, 4.9 min), and salicylic acid (ex 305 nm/em 407 nm, 22.9min). The calibration curve was: y $ 0.000231406x # 2.81054with R2 $ 0.999, where x $ area units, y $ ng SA. Salicylic acid(S-5922, Sigma ultrapure) was used for calibrations.Coupled Spectrophotometric Assay For ICS Activity—The

ICS reaction rate was measured by coupling excess amounts ofisochorismate pyruvate lyase (recombinant PchB) and lacticdehydrogenase (SigmaL-1254), which catalyzes theNADH-de-pendent conversion of pyruvate to lactate. Dilution series ofAtICS1 were performed to establish the linear range for thisassay (to '25 $g/ml recombinant, purified AtICS1) and ourstandard assay conditions (10 $g/ml recombinant purifiedAtICS1). Velocity was linear with time (following an initial lag)for(15minwith 10$g/mlAtICS1.Unless otherwise indicated,the 200 $l per well assay volume contained 0.2 mM NADH,0.833 $g/ml L-lactic dehydrogenase, 32.0 $g/ml PchB, 10.0$g/ml AtICS1, and 2 mM chorismic acid in buffer D. The reac-tion was initiated by addition of the chorismic acid to the reac-tion mixture in a 96-well plate preheated to 30 °C and analyzedusing a Spectramax Plus microplate spectrophotometer




Downloaded from

(Molecular Devices). The change in absorbance at 340 nm wasmeasured in each well in increments of 30 or 60 s and moni-tored at least 30 min. The initial reaction rate in each well wasassessed for a 5- to 10-min period'7min after initiation of thereaction based onmanual confirmation of linear range and cal-culated using least squares fitting of each curve. An extinctioncoefficient for NADH of 6220 M#1 cm#1 was used for conver-sion of these values to units of micromolar/min.

Determination of Keq Using 1H NMR

Our protocol is similar to that previously used with E. coliEntC (4). The spectrum of the equilibrium mixture of choris-mate and IC was acquired as follows: 500 $l of 220 $g/mlAtICS1 exchanged into D2O buffer containing 50 mM potas-siumphosphate, pD 7.5, 5mMMgCl2was incubatedwith 500$lof chorismic acid solution (2 mM in D2O buffer) at 30 °C for 60min. (HPLC analyses of successive aliquots confirmed the reac-tionwas in equilibrium.)NMR spectra for a 900-$l aliquotwererecorded using a 500-MHz Bruker DRX-500 spectrometer.Spectrawere scanned every 8 s for a total period of 9 h each. Theratio between chorismate and IC was determined by integra-tion of the two peaks most downfield in the spectrum (the C-2protons). The chorismate spectra were obtained with choris-mic acid in the above D2O buffer.

Km Determination

The coupled spectrophotometric ICS assay was modified byusing working chorismic acid concentrations of 1.5 mM, 1.0mM, 750$M, 500$M, 400$M, 300$M, 250$M, 225$M, 200$M,180$M, 160$M, 133$M, 100$M, 80$M, 40$M, and 20$M. Thereaction was initiated by the addition of chorismic acid. Reac-tions without AtICS1were used as blanks. As per standard pro-tocol, triplicate samples were run for each condition. Initialvelocity data were fitted to the equation of Hanes to determinekinetic parameters (49). To determine the effective chorismateconcentrations in the above assay, we assessed the conversionof chorismate to prephenate via PchB in parallel. In brief, asmodified from a previous study (38), standard coupled reac-tions in the 96-well plate were treated with acid to convertprephenate to phenyl pyruvate after the reaction progressed for0, 5, 10, 15, or 20min, followed by neutralization with base, andmeasurement at A320. Prephenate standard curves were run inparallel for quantification. Chorismate utilization via PchB wasestimated as equal on a molar basis to prephenate.

Effect of Mg2$ on ICS Activity

The coupled spectrophotometric ICS assay was modified byusing buffer C (no Mg2!) supplemented with MgCl2 at the fol-lowing working concentrations: 15 mM, 10 mM, 5 mM, 3 mM, 2mM, 1mM, 800 $M, 650 $M, 500 $M, 200 $M, 100 $M, 80 $M, 50$M, 20$M, 10$M, 5$M, and 0$M. Reactionswere performed intriplicate, and reactions without AtICS1 were used as blanks.Note that neither isochorismate pyruvate lyase (IPL) (50) norlactic dehydrogenase activities were impacted by Mg2!. Initialvelocity data were fitted to the equation of Hanes to determinekinetic parameters (49).

Effect of Other Metals on ICS Activity

The coupled spectrophotometric assay wasmodified by sub-stituting other divalentmetals forMg2!. As above, buffer Cwasused and a working concentration of 10 mM, 1 mM, and 0.1 mMof each of the following divalent metals was substituted: CaCl2,BaCl2, MnCl2, ZnCl2, and CdCl2. Each reaction was performedin triplicate. Reactions without AtICS1 were used as blanks.Reactions containing no added metal ion were also included.IPL activity is reported to be unaffected by divalent cations(48). However, lactic dehydrogenase (Sigma L-1254, rabbitmuscle) activity may be inhibited by heavy metals such asHg2! and Pb2! (Sigma-Aldrich Technical Service). Therefore,we repeated the metal experiments using 1 mM of the divalentmetal ion and theHPLC ICS assay, which directly measures theconversion of chorismate to IC. For metals with significantabsorbance at 340 nm, we exclusively employed the HPLC ICSassay. Buffer C was substituted for buffer D, and the reactionswere supplemented with each of the following metals: FeCl2,FeCl3, CoCl2, NiCl2, or CuCl2. Working concentrations of 10mM, 1 mM, and 0.1 mM of each were used in the reactions.

pH Profile of ICS Activity

The HPLC assay for ICS activity was modified by changingthe pH of the reaction buffer. 40 $l of 100 mM buffer solutioncontaining 220 $g/ml AtICS1 was mixed with 40 $l of a 2 mMsolution of chorismic acid. Buffer solutions utilized 100 mMMES at pH 5.0, 5.5, 6.0, and 6.5;MOPS at pH 7.0 and 7.5; Tris atpH 7.5, 7.7, 8.0, and 8.5; CHES at pH 9.0, 9.5, and 10.0; or CAPSat pH 11.0. Reactions were performed in triplicate with resultsreported for the formation of IC. Reactions without AtICS1were used as controls (to be subtracted from results withenzyme), although we observed no non-enzymatic productionof isochorismate.

Temperature Profile of ICS Activity

The HPLC assay for ICS activity was modified by changingthe temperature atwhich the reactionwas incubated. Reactionswere performed in triplicate incubating for 60 min at each ofthe following temperatures: 4 °C, 15 °C, 23 °C, 30 °C, 37 °C,44 °C, 51 °C, 60 °C, and 70 °C. Results are reported for the for-mation of isochorismate. Non-enzymatic production of IC wasnot observed. At 51 °C and above, there was some decomposi-tion of chorismate (to products other than IC); however, [cho-rismate] was not limiting.

Non-enzymatic Synthesis of SA from Isochorismate

IC was produced enzymatically by conversion of chorismateto isochorismate via ICS (purified recombinant Eco EntC) andpurified as in a previous study (51). ICwas incubated in solution(37 $M IC, pH 7.5, 3 mM Mg2!, 23 °C) for 1 h, 3 h, and 6 h, andchorismate, IC, and SA were analyzed by HPLC (using methodof coupled HPLC ICS assay above). Incubations were per-formed in triplicatewith the conversion rate of IC to SA (micro-molar SA/h) as the slope of SA production with time (y $0.036x ! 1.40; R2 $ 0.999).




Downloaded from

Homology Modeling of ICS Enzymes

The primary sequences for A. thaliana AtICS1 (AAL17715)and Y. enterocolitica Irp9 (CAB46570) were aligned with Clust-alW (52) using the default parameters. Due to poor alignmentof the N-terminal portion of the proteins, residues 1–198 and1–92 of AtICS1 and Yec Irp9, respectively, were not used inmodel generation. Note that AtICS1 contains an N-terminalchloroplast transit sequence of'45 amino acids as predicted byChloroP (53) and confirmed via chloroplast import assays (Fig.2). Homology modeling was performed with Modeler 8v2 (54)using the Yec Irp9 crystal structure in complex with its reactionproducts SA and pyruvate (Protein Data Bank identificationcode 2FN1) (10). Five models were generated, and the lowestenergy model was selected. The AtICS1 model and Yec Irp9were superimposed in COOT (55). All figures were generatedwith PyMOL.4 Homology modeling of additional ICS enzymes,including Mycobacterium tuberculosis MbtI (CAE55483), PaePchA (CAA57969), Eco EntC (POAEJ2), AtICS2 (NP_173321),C. roseus ICS (CAA06837), and Capsicum annum ICS(AAW66457) was performed in a similar manner.

RESULTS

AtICS1 Is Plastid-localized—We performed chloroplastimport assays to determine whether AtICS1 is capable of beingimported into the chloroplast and where within the chloroplastit resides. Immunolocalization experiments using epitope-tagged AtICS1 transgenics expressed under control of theAtICS1 native promoter were then undertaken to assess

whether AtICS1 is plastid-localized in planta in response topathogens. We found radiolabeled AtICS1 precursor protein('62 kDa) was imported into the chloroplast and processed toits mature form ('58 kDa) (Fig. 2A). Chloroplasts containingthe imported proteinwere then exposed to trypsin treatment todistinguish stromal proteins from those residing in the inter-membrane space. Mature AtICS1 was trypsin-resistant indica-tive of a stromal protein. Parallel assays were performed usingToc75 and Hsp93 as controls for sensitivity and lack of sensi-tivity to trypsin digestion, respectively. Consistent with previ-ous results (42), Toc75, a chloroplast outer envelope protein,was recovered in the membrane fraction (pellet) and was sus-ceptible to trypsin treatment (Fig. 2B), whereas Hsp93, a stro-mal protein, was recovered in the soluble fraction and wastrypsin insensitive (data not shown).Furthermore, AtICS1 was detected in plastids of pathogen-

induced mature leaves of Arabidopsis AtICS1-V5 ics1 trans-genics (SM156) by immunofluorescence (Fig. 3). For these ics1transgenics, AtICS1-V5 expressed under control of the nativeAtICS1 promoter restored pathogen-induced SA biosynthesis,and induction of SA-dependent PR1 expression.5 The size,shape, chlorophyll autofluorescence, and 4&,6-diamidino-2-phenylindole-staining of double-stranded DNA of the plastidshown in Fig. 3 are hallmarks of mesophyll chloroplasts. Fluo-rescence of the Alexa 488 fluorophore (conjugated to the 2°antibody) was observed in chloroplasts of the transgenic lineSM156 but not in wild-type Columbia-O or ics1 plants (notshown). Alexa 488 fluorescence was also absent in chloroplasts

4 W. L. DeLano (2002) PyMOL, DeLano Scientific, San Carlos, CA. 5 S. K. Marr and M. C. Wildermuth, unpublished data.

FIGURE 2. AtICS1 is targeted to the chloroplast stroma and processed toits mature form. The 35S-radiolabeled AtICS1 (A) and Toc75 (B) precursorproteins (tl, 10% input) were incubated with intact chloroplasts under theimport condition. The chloroplasts were re-isolated and analyzed directly(imp), or incubated in import buffer without (#) or with (!) trypsin on ice inthe dark for 30 min followed by the addition of trypsin inhibitor and hypo-tonic lysis before being fractionated into the supernatant (s) and the pellet (p)by centrifugation. Radiolabeled proteins were separated by SDS-PAGE andvisualized by fluorography. AtICS1 precursor protein (indicated with an aster-isk) was processed to its mature form (mICS1). The precursor to the outerenvelope protein Toc75 (indicated with an asterisk) was processed to an inter-mediate (i75) and mature (m75) form.

FIGURE 3. AtICS1 is detected in plastids of pathogen-infected matureleaves by immunofluorescence. AtICS1 is detected in plastids of PsmES4326-infected mature leaves of AtICS1-V5 ics1 transgenics (SM156) but notin Col-O wild-type Arabidopsis plants. Cell walls of excised leaves weredigested, and the resulting leaf fragments (e.g. protoplasts) were gentlysquashed to facilitate the immunoreaction. Incubation without primaryanti-V5 Ab (mouse) was included as a control. Excitation and emission filtersets allow for visualization of secondary antibody conjugated to Alexa 488fluorophore (green), chlorophyll autofluorescence (red), and 4&,6-diamidino-2-phenylindole (DAPI) staining of double-stranded DNA (blue). A single plas-tid is shown. Bar, 10 $m.




Downloaded from

of SM156 transgenics when the primary anti-V5 antibody wasexcluded from the immunoreaction.AtICS1 Exhibits ICS Activity Not SA Synthase Activity—In

bacteria, the synthesis of SA via IC occurs either via a bifunc-tional SAS (e.g. Yec Irp9 (9)) or an enzyme complex consistingof ICS and IPL genes coexpressed and present in cis (e.g. PaePchBA (6, 48)). ICS and SAS enzymes share similar globalstructure and highly conserved active sites (9, 10); therefore, itwas important for us to determine whether AtICS1 exhibitsmonofunctional ICS activity or bifunctional SAS activity.Because AtICS1 expression and function are correlated withinduced SA accumulation (see the introduction and Refs. 57and 58) and IC-pathways likely derive from bacterial endosym-biosis (e.g. Ref. 12), we thought it quite possible that AtICS1could act as a bifunctional SAS.To assess AtICS1 biochemical activity, we overexpressed

N-terminal hexaHis-tagged mature AtICS1 (without the chlo-roplast transit sequence) in E. coli and purified the solubleinduced protein (see supplemental data). For a typical prepara-tion, AtICS1 was purified 69-fold from induced cell extracts tonear homogeneity. The specific activity of the recombinantpurified AtICS1 was 241 nmol min#1 mg#1 (4,010 picokatalsmg#1). All further biochemical characterization (below) wasperformed on the purified, recombinant mature AtICS1enzyme.

Our HPLC analyses showed that AtICS1 converts choris-mate to IC (Fig. 4A). Isochorismate was confirmed by 1HNMR.Salicylic acid was not detected as a product of this reaction (Fig.4B, top). The addition of recombinant Pae PchB (an IPL) to theAtICS1 reaction did result in the production of SA (Fig. 4B,bottom). Therefore, AtICS1 functions as a traditional mono-functional ICS and not a bifunctional SAS like Irp9 (9).AtICS1 Is an Active Monomer—To determine whether

AtICS1 likely exists as a monomer, dimer, or other multimer,we estimated the molecular mass of the recombinant purifiedenzyme by fast protein liquid chromatography using a cali-brated Sephacryl S-200 gel filtration column. Active AtICS1was estimated to have a molecular mass of 57.5 kDa. SDS-PAGE performed on eluted fractions indicated that the ICSmonomer (estimated to be '59 kDa, supplemental data) wasexclusively enriched in the fractions with ICS activity.Therefore, similar to the bacterial ICS proteins Pae PchA(48) and Eco EntC (4), AtICS1 appears to function as a mon-omer, whereas the SAS Yec Irp9 appears to function as adimer (9).AtICS1 Catalyzes a Reversible Reaction and Exhibits an

Apparent Km of 41.5 $M for Chorismate—The chorismate-iso-chorismate interconversion catalyzed by monofunctional bac-terial ICS enzymes has been shown to be reversible, favoringchorismate (3, 4, 48). However, past work on plant ICSs had not

FIGURE 4. AtICS1 exhibits ICS activity. A, chorismate (CA) is converted to isochorismate (IC) in the presence of enzyme (lower chromatogram) but not in the noenzyme control (above). An HPLC ICS assay was employed. B, salicylic acid (SA) is not a product of the ICS reaction (top chromatogram). SA is observed whenAtICS1 and Pae PchB, an IPL that converts IC to SA, are both present (bottom). A coupled HPLC ICS assay was utilized. Fluorescence was measured at ex 305/em407 nm.




Downloaded from

examined the reversibility of the reaction and had not consid-ered this reversibility when apparent Km values were deter-mined (19, 23, 24). This is critical as the previously employedICS equilibrium assays are not suitable for obtaining accuratekinetic parameters for enzymes such as ICS that operate nearequilibrium (59). Therefore, for AtICS1 we experimentallydetermined Keq and calculated quantitative kinetic parametersusing a coupled irreversible continuous assay similar to thoseemployed in the characterization of Pae PchA (48) and EcoEntC (4).To determine the equilibrium constant (Keq) for recombi-

nantAtICS1, we followed the conversion of chorismate to IC byHPLC and obtained 1H NMR spectra for the equilibrium mix-ture (Fig. 5). The ratio between chorismate and IC at equilib-rium was calculated from the integration of the unique olefinicprotons in each compound (i.e. the C-2 protons) as shown inFig. 5B. We obtained a Keq for AtICS1 of 0.89 ) 0.02, showingthat a plant ICS (AtICS1) does operate near equilibrium, withthe reaction slightly favoring chorismate.To calculate the apparent Km of AtICS1 for chorismate, we

developed a coupled assay in which IC (produced via AtICS1) isconverted irreversibly to SA and pyruvate by the IPL Pae PchB(in excess), similar to the protocol used to assess the catalyticproperties of PchA (48). To facilitate kinetic measurements,lactate dehydrogenase is included (in excess) to convert pyru-vate to lactate in an NADH-dependent reaction, and thedecrease in A340 associated with the conversion of NADH toNAD! is followed by spectroscopy. We found AtICS1 exhib-ited standard Michaelis-Menten kinetics for chorismate. Thecorresponding Hanes plot yielded an apparentKm $ 84.2) 3.9$M for chorismate, withVmax $ 5.84) 1.14$Mmin#1 and kcat $34.7) 6.8min#1. However, PchB can also utilize chorismate asa substrate (producing prephenate) but with lower affinity forchorismate (Km $ 150 $M) than for isochorismate (Km $ 14$M) (50). Because the affinity of PchB for chorismate at optimalconditions (pH 7.0, 37 °C) is similar to that of AtICS1 (above,assessed at pH 7.7, 30 °C), we wanted to determine whetherPchBwas reducing the effective concentration of chorismate inour assay. Therefore, we assessed effective chorismate concen-trations for ourKm experiment bymeasuring prephenate accu-mulation in the coupled spectrophotometric ICS assay in par-allel. We found significant conversion of chorismate toprephenate at concentrations below 200 $M chorismate. Theadjusted kinetic parameters calculated using the effective cho-rismate concentrations were: Km $ 41.5 $M, Vmax $ 6.50 $Mmin#1, and kcat$ 38.7min#1. TheKm ofAtICS1 for chorismateis '10-fold lower than past reports for plant ICS enzymes,which employed inaccurate equilibrium assays (19, 23, 24) and'10-fold higher than bacterial enzymes involved in SA synthe-sis from chorismate via IC (Pae PchA (48) and YecIrp9 (9)).Catalytic Properties of AtICS1—Weassessed the dependence

of AtICS1 activity on Mg2!, an array of other divalent cations,and Fe3!. The presence of Mg2! was found to be an absoluteand specific requirement for AtICS1 activity, similar to otherplant and bacterial ICS enzymes (e.g. Refs. 4, 19, 23, 48, and 60).This requirement is consistent with the dominant proposedreaction mechanism for ICS: regiospecific 1,5-SN2* addition ofthe nucleophile water to C2 of chorismate via a Mg2!-bound

transition state with concomitant loss of the hydroxyl group atC4 (61, 62). A typical saturation curve was obtained for AtICS1activity as a function of Mg2! concentration (assessed with 2mM chorismate) with saturation occurring at ' 2 mM Mg2!

(Fig. 6A), and a Km of 193 $M for Mg2! was obtained. Incuba-tion with other divalent cations, including Mn2!, Co2!, Ni2!,Ca2!, Zn2!, Ba2!, Cu2!, Cd2!, and Fe2!, and with Fe3! at 0.1,1, or 10 mM, did not result in significant AtICS1 activity.

The pH optimum for recombinant AtICS1 was determinedusing the range of pH 5–11. As shown in Fig. 6B, AtICS1 has abroad pH range of maximal activity from 7 to 9.5, with optimalAtICS1 activity from pH 7.5 to 8. A similar range of maximalactivity was reported for other plant ICS enzymes (19, 23, 24),although values at each pH were not provided; pH optima forbacterial ICS enzymes of 7.0 (48) and 7.5–8.0 (5) have beenreported.The temperature dependence of AtICS1 activity was

assessed from 4 to 70 °Cwithmaximal activity at room temper-ature (23 °C) and no activity at 70 °C (Fig. 6C). AtICS1 exhibiteda surprisingly broad range of activity with(75%maximal activ-ity from 4 to 44 °C, and (90% of maximal activity from 4 to37 °C. Extensive temperature profiles of plant ICS enzymes hadnot been previously reported (19, 23, 24), although similaroptima had been found (e.g. G. mollugo (23)). Where reported,bacterial ICS enzymes exhibited optimal activity at 37 °C (e.g.Ref. 5).Non-enzymatic Synthesis of SA from Isochorismate Is

Negligible—Non-enzymatic conversion of IC to SA ('25% con-version, 100 °C, 10min) has long been reported (60, 63). There-fore, using our findings (above) regarding the subcellular local-ization and biochemical properties of AtICS1, we directlyexamined the potential for non-enzymatic formation of SAfrom IC (produced via AtICS1) under physiological conditions.We first calculated a reasonable plastidic IC pool. We assumedthat AtICS1 operates near its Km (41.5 $M for chorismate);therefore, the plastidic [chorismate] is taken to be 41.5 $M, anda plastidic ICpool of 37$Mwas estimated (Keq$ 0.89).We thenassessed non-enzymatic conversion of IC to SA using condi-tions consistent with the chloroplast stroma (37 $M IC, pH 7.5,3 mMMg2!, 23 °C). Very little non-enzymatic conversion of ICto SAwas observed:%0.1% IC converted to SA/h (“Experimen-tal Procedures”). This conversion rate is consistent with arecent IC thermal decomposition study (see calculation at30 °C, pH 7, 10mMMg2! (64)). Furthermore, we did not detecta substantial SA signature in 1H NMR spectra of AtICS1 inequilibrium ([IC] '0.47 mM, pH 7.5, 5 mM Mg2!, 22 °C)acquired over a 9-h period (Fig. 5, data not shown).

DISCUSSION

Herein, we presented the first experimental evidence that afunctional plant ICS enzyme, AtICS1, is localized to the plastid.We determinedKeq for AtICS1 (Keq $ 0.89), establishing that aplant ICS enzyme operates very near equilibrium.We then pro-vided the first kinetic data (Km, Vmax, and kcat) for a plant ICSusing an irreversible coupled continuous assay similar to thatused to obtain accurate kinetic data for bacterial ICS enzymes(e.g. Ref. 4). Previously, Km (but not kcat) values for plant ICSenzymes have been reported (19, 23, 24); however, these studies




Downloaded from

FIGURE 5. 1H NMR spectra of chorismate (A) and the chorismate/isochorismate equilibrium mixture in the presence of AtICS1 (B). The vinylic regions (5.0–6.75ppm) of the NMR spectra are displayed. The four vinyl protons 2, 5, 6, and 11 for chorismate and 2&, 3&, 4&, and 11& for isochorismate are indicated on each structure, andthe corresponding NMR peaks are labeled. The equilibrium constant calculation used the integration of the two most downfield proton peaks (2& and 2) for deter-mining the ratio of chorismate to isochorismate.




Downloaded from

employed equilibrium assays, which are not suitable for obtain-ing accurate kinetic parameters for enzymes such as ICS thatoperate near equilibrium (59). AtICS1 is required for theinduced synthesis of SA from chorismate (17). Therefore,

below, we use our detailed biochemical characterization andsubcellular localization of AtICS1 to provide insights into SAbiosynthesis and function as well as chorismate partitioningand utilization.AtICS1 Is a Monofunctional ICS, Not a Bifunctional SA

Synthase—BecauseAtICS1 expression is highly correlated withSA accumulation (see the introduction and Refs. 57 and 58), wethought it quite possible that AtICS1 could act as a bifunctionalSAS. However, we found that AtICS1 does not directly convertchorismate to SA but acts as a monofunctional ICS similar tomost bacterial ICS proteins (Fig. 4). Residues essential to SASIPL activity have yet to be determined (9, 10, 65); therefore, weperformed homology modeling of known monofunctional ICSenzymes involved in SA biosynthesis (AtICS1 and Pae PchA)and the recently confirmed SASM. tuberculosisMbtI (66) usingthe crystal structure of the SAS Yec Irp9 in complex with itsreaction products SA and pyruvate (10). As expected, ourhomology modeling of AtICS1 with Yec Irp9 indicates that theactive site is highly conserved (Fig. 7). Comparison of afore-mentioned active sites leads us to speculate that Thr-348 iscritical for IPL activity with potential hydrogen bondingbetween Thr-348 and the 2-hydroxyl group of SA (or the simi-larly positioned 6-hydroxyl group of isochorismate). Yec Irp9inhibitor studies using IC mimics also support this postulation(65). Thr-348 is conserved in Irp9 and MbtI but replaced withAla in AtICS1 and PchA. In addition, AtICS2 (the other pre-dicted functional Arabidopsis ICS), other plant ICS proteins,and Eco EntC and MenF also contain an Ala at this position. Itwill be interesting to see whether site-directed mutagenesis ofAtICS1 can convert it to an SA synthase.Given that mono- and bifunctional ICS enzymes exhibit a

high degree of overall structural similarity and a highly con-served active site inwhich few residues are likely responsible forICS versus SAS activity (Fig. 7) (10), we would expect to observepositive selection for eithermonofunctional ICSor bifunctionalSAS enzymes in plants. To date, onlymonofunctional plant ICSactivities have been reported (herein andRefs. 19, 23, and 24). Itshould be noted, however, that previous studies characterizingplant ICS enzymes did not examine ICS enzymes associatedwith SA biosynthesis and did not specifically look for a productother than IC. Very recently, PHYLLO, a fusion of a non-func-tional, truncated 5& ICS and three full-length individual eubac-terial genes involved in bacterial menaquinone biosynthesis,was reported to be required for PhQ production inArabidopsis(12). The architecture of the fused PHYLLO locus is conservedin the nuclear genomes of plants and green algae, with genefission and inactivation of the ICS module of PHYLLO occur-ring in higher plants. Therefore, whereas green algae encode amultifunctional enzyme involved in the conversion of choris-mate to a naphthoquinone/PhQ/anthraquinone intermediate(such as 1,4-dihydroxy-2-napthoate), higher plants require anindependent ICS for initial production of IC from chorismate(12) (see Fig. 1) providing strong support for positive selectionfor multiple monofunctional ICS enzymes in plants.What would be the advantage of monofunctional plant ICS

enzymes? First, because monofunctional ICS enzymes catalyzethe reversible conversion of chorismate to IC, expression of amonofunctional ICS would not drain chorismate from other

FIGURE 6. Dependence of AtICS1 activity on Mg2$ (A), pH (B), and temper-ature (C). Each assay was performed in triplicate as described under “Exper-imental Procedures.”




Downloaded from

plastid-localized chorismate-utilizing pathways such as aro-matic acid biosynthesis or phenylpropanoid production. This isa valid concern because our kinetic measurements suggest thatAtICS1 could potentially compete for chorismate with consti-tutively expressed, chorismate-utilizing enzymes associatedwith aromatic amino acid biosynthesis (e.g. Ref. 38). Further-more, constitutive expression of a PchBA SAS fusion protein inArabidopsis resulted both in a (20-fold increase in SA levelsand in severe dwarfism/infertility (20). The severity of thedwarfism/infertility phenotype compared withmutants consti-tutively overexpressing SA is most likely due to the channelingof chorismate to SA at the expense of other essential choris-mate- and IC-derived products (e.g. aromatic amino acids andPhQ, respectively), because the affinity of PchA for chorismate(48) is 10-fold higher than that of AtICS1.Second, expression ofmultiplemonofunctional ICS enzymes

could allow for IC to be channeled to different products (e.g. SA,induced naphthoquinones or anthraquinones, PhQ) dependingupon coexpression of downstream enzymes (see Fig. 1). Forexample, although AtICS1 expression and function is associ-atedwith induced SA biosynthesis, it may also contribute to thesynthesis of other IC-derived products (e.g. Refs. 12 and 67).Indeed, the recent examination of PhQ biosynthesis found thatonly the ics1ics2 double mutant but not single ics mutantsresulted in a PhQ-deficient phenotype (12) suggesting thateither both Arabidopsis ICS enzymes contribute IC for PhQbiosynthesis in wild-type plants or that one ICS may com-pensate for a deficiency in the other. In the case of SA bio-synthesis, the Arabidopsis ics1 single mutant was sufficientto abolish pathogen-induced SA accumulation; however,low level endogenous SA production was unaffected (17, 28).As AtICS2 is not significantly induced in response to patho-gens, its expression may be insufficient to functionally comple-ment induced SA biosynthesis; alternatively, AtICS2 mayexhibit a lower affinity for chorismate than AtICS1 and be lesssuccessful in competing with other pathogen-induced choris-mate-utilizing enzymes for chorismate. To determine whetherAtICS1 can channel isochorismate to different products, we areexamining whether AtICS1 can form complexes with different

enzyme partners depending uponthe developmental stage or biotic/abiotic stressor.Third, multiple monofunctional

ICS enzymes could provide partialfunctional redundancy for therobust synthesis of essential com-pounds such as PhQ from IC. Geneduplication is a common means offacilitating the robustness of meta-bolic pathways (68, 69). Robustnessis typically defined as a measure ofthe ability of a biochemical networkto withstand perturbations. Indeed,as mentioned above, the ics1ics2doublemutant exclusively displayedthe phyllo phenotype: no PhQ,5–15% of wild-type photosystem Iactivity, bleached leaves, and seed-

ling lethality (12).AtICS1 Activity and SA Biosynthesis as a Function of Light

and Temperature—A number of SA-dependent defense-re-lated processes are light-dependent (70); however, a detailedmechanistic understanding of this light dependence is lacking.Using chloroplast import studies and immunolocalization, wedetermined that AtICS1 is localized to the chloroplast stroma(Fig. 2) and is induced in response to pathogens in mesophyllchloroplasts of infected mature leaf tissue (Fig. 3). Light-de-pendent changes in pH, Mg2!, and redox status may regulatethe activity of enzymes localized to the plastid stroma (e.g. Ref.71). Therefore, we were interested in determining whetherAtICS1 activity would be dramatically impacted by thesechanges. We found no evidence for substantial regulation ofAtICS1 activity associated with reported light-dependentchanges in stromal pH (from 7 (dark) to 8 (light)) and Mg2!

(from 1–3 mM to 3–6 mM) (14). Although we did not directlyexamine the potential for redox regulation of AtICS1, webelieve redox regulation of AtICS1 is unlikely. First, the AtICS1protein does not contain cysteine residues within sufficient dis-tance to form a disulfide bridge, based on homology modelingwith Irp9 (above). Second, DTT was not required to obtainactive recombinant mature AtICS1. DTT (or a similar thiolreductant) is typically required throughout isolation and puri-fication protocols to retain the activity of reduced redox-acti-vated enzymes (e.g. Ref. 72). Taken together, our findings sug-gest that the light-dependent regulation of AtICS1 activity dueto changes in stromal pH, Mg2!, and/or redox status do notsignificantly contribute to the light dependence observed forSA-dependent defense responses. This supports a previousreport that the observed light dependence of PR1 expressionoccurs downstream of SA (70).Initially, we were surprised by the broad temperature

range for AtICS1 activity ((90% of maximal activity from 4to 37 °C, Fig. 6C). However, from a defense perspective, itcould benefit the plant to synthesize SA and induce plantdefense responses over a wide range of naturally occurringtemperatures (73). Furthermore, a more general role for SAas a mediator of temperature-dependent stress is emerging

FIGURE 7. Homology modeling of AtICS1 using the crystal structure of Y. enterocolitica Irp9. A, AtICS1(green) overlaid on the Yec Irp9 crystal structure (gray) in complex with its reaction products SA and pyruvatecolored by atom with carbons in cyan and oxygens in red (PDB code 2FN1) (10). Mg2! is shown as a magentasphere. Insertion regions of AtICS1 are colored in red. B, enlarged view of the active site with Irp9 side chains ingray and AtICS1 side chains in green. Irp9 residues are labeled with one-letter amino acid code and number forposition in protein with AtICS1 residues given in parentheses. The active site of Irp9 is completely conserved inMtu MbtI (CAE55483). AtICS1 (AAL17715), Pae PchA (CAA57969), and Eco EntC (POAEJ2) contain an Ala atThr-348 (marked with arrow). Thr-348 is predicted to H-bond with the 2-OH of SA. See “Experimental Proce-dures” for additional details.




Downloaded from

(e.g. Refs. 32 and 34). A. thaliana Col-O accumulates SA('30 $g/g fresh weight/SAG) in response to extendedgrowth at chilling temperatures (5 °C) (32) at levels similar tostrong induction by pathogens. Because AtICS1 is active atthese temperatures in planta, it suggests that SA made viaAtICS plays a role in cold acclimation or cold-tolerantgrowth. Arabidopsis is a chilling-resistant species, able tofully mature and produce seed at 4 °C. It will be interesting toascertain whether ICS enzymes isolated from chilling-sensi-tive plant species are also highly active at 4 °C and to explorethe potential selection for and mechanism of this cold-adapted catalysis. For example, enzymatic rates can be dra-matically enhanced over non-enzymatic rates (kcat/knon) withdecreasing temperature (e.g.Ref. 74).TheextentofAtICS1activityand SAG accumulation at 4 °C (or 5 °C) also suggests that SA (andSAG in particular) may play an unexplored role in cold-tolerantgrowth.AtICS1 Can Compete with Other Stress-induced, Plastid-

localized Chorismate-utilizing Enzymes—Using our coupledirreversible continuous assay, we determined that matureAtICS1 has an apparentKm of 41.5 $M for chorismate. This iscritical as it now appears that a plant ICS, AtICS1, can com-pete with other plastid-localized chorismate-utilizingenzymes for chorismate (see Fig. 1). For example, AtICS1and AtASA1 are induced in response to bacterial pathogens(17, 39) and defense-related products of these pathways,camalexin (via anthranilate) and SA (via IC), are detected inparallel.5 AtASA1 (At5g05730) has an apparent Km for cho-rismate in the range of 21 $M (75) to 180 $M (76) and thuscould theoretically compete with AtICS1 for available cho-rismate. In contrast, past comparison of apparent Km valuesfor chorismate of elicitor-induced C. roseus ICS isoforms(558 and 319 $M (19)) with AS (67 $M (77)) did not favor ICS.AtCM1 (At3g29200) is also induced in response to bacterialpathogens (37, 38). AtCM1 has a reported apparent Km of 2.9mM for chorismate (38); however, it is possible that this Kmfor chorismate is artificially high due to the presence of thechloroplast transit sequence (38). Alternatively, becausephenylpropanoids typically dominate other chorismate-de-rived pathogen-induced products, this higher Km may bephysiologically relevant, resulting in increased flux onlywhen sufficient chorismate is available. In addition, bothAtASA1 and AtCM1 activities are allosterically modulatedby the aromatic amino acids (37, 38, 76); whereas, there hasbeen no evidence for allosteric regulation of a plant or bac-terial ICS enzyme (19, 48). Therefore, should these enzymesbe expressed in the same cells, it appears that AtICS1 couldsuccessfully direct available chorismate to the production ofSA. Indeed, we found that overexpression of AtICS1 undercontrol of its native promoter resulted in increased stress-induced SA accumulation suggesting that the amount ofAtICS1 and not available chorismate limits induced SA syn-thesis.5 The affinity of AtICS1 for chorismate also suggeststhe possibility that it can compete with a subset of choris-mate-utilizing enzymes involved in “constitutive” aromaticamino acid and folate production. For example, AtICS1could successfully compete for chorismate with all threecharacterized A. thaliana chorismate mutases (37, 38),

whereas the recently characterized Arabidopsis aminode-oxychorismate synthase exhibits (10-fold higher affinity forchorismate than does AtICS1 (78).Stress-induced SA Biosynthesis from IC Is Likely Enzymatic

and Plastidic—The localization ofmonofunctional AtICS1 andthus IC production to the plastid strongly suggests stress-in-duced SA biosynthesis is plastidic. Results from two overex-pression studies support this hypothesis. First, studies withtransgenic tobacco plants overexpressing bacterial monofunc-tional ICS and IPL enzymes targeted to either the plastid orcytosol singly or in combination found that only transgenicplants in which both ICS and IPL were targeted to the plastidexhibited an SA overexpression functional phenotype: SAGlevels similar to local tobaccomosaic virus-infected leaves, con-stitutive expression of pathogenesis-related genes, and a reduc-tion in tobaccomosaic virus-induced lesion size (56). Second, inArabidopsis, an SA synthase PchBA fusion protein was consti-tutively expressed in either the plastid or the cytosol (20). Heretoo, a true SA overexpression phenotype (SAG '20 $g/g freshweight, PR1 expression) was only observed when the fusionprotein was targeted to the plastid, implying that SA synthesisoccurs in the plastid in Arabidopsis.How then is stress-induced SA made from IC? The evi-

dence supports enzymatic synthesis of SA from isochoris-mate. We found non-enzymatic conversion of IC to SAunder physiological conditions to be negligible. Further-more, total SA accumulates to levels similar to those inducedby pathogen inArabidopsis grown at 5 °C (32), a temperatureat which non-enzymatic conversion of IC to SA should beinsignificant. In P. aeruginosa, a direct comparison of theenzymatic (kcat Pae PchB (50)) and non-enzymatic (64) ratesof SA formation from IC indicates that the enzyme acceler-ates SA formation '4 " 105-fold (64). Does Arabidopsiscontain a gene encoding a protein with IPL activity similar toPae PchB? Pae PchB encodes a small 101-amino acid proteinthat appears to have evolved from a chorismate mutase andretains residual CM activity with 10-fold lower affinity forchorismate than for IC (50). Arabidopsis contains threegenes encoding proteins with confirmed chorismate mutaseactivity (AtCM1 At3g29200, AtCM2 At5g10870, and AtCM3At1g69370 (37, 38)) and the putative chorismate mutaseAt3g07630. All but AtCM2 are predicted to be plastid-local-ized and are possible IPL candidates, although there are alsoother possible routes to SA from IC.In conclusion, AtICS1 is required for the pathogen-induced

accumulation of SA, a key phytohormone mediating plantresponse to pathogens, abiotic stress, and stress-induced devel-opmental transitions. The biochemical properties of AtICS1described herein suggest 1) its activity is not regulated by light-dependent changes in the chloroplast stroma, 2) it can effec-tively compete with other stress-induced and “constitutive”plastidic chorismate-utilizing enzymes, and 3) it is active atboth low and high physiologically relevant temperatures sup-porting its proposed role mediating temperature-dependentstress. Finally, we provide evidence in support of positive selec-tion for monofunctional ICS enzymes in plants and argue thatstress-induced SA biosynthesis occurs in the plastid andrequires AtICS1 and one or more additional enzymes.




Downloaded from

Acknowledgments—We thank Dr. C. Reimmann (Universite deLausanne) for supplying pME3368, and Dr. B. Buchanan andA. M. Jones (University of California at Berkeley) and Dr. A. The-ologis (Plant Gene Expression Center-U. S. Dept. of Agriculture/University of California at Berkeley) for review of the manuscript.

REFERENCES1. Herrmann, K. M., and Weaver, L. M. (1999) Annu. Rev. Plant Physiol.

Plant Mol. Biol. 50, 473–5032. Buss, K., Muller, R., Dahm, C., Gaitatzis, N., Skrzypczak-Pietraszek, E.,

Lohmann, S., Gassen, M., and Leistner, E. (2001) Biochim. Biophys. Acta1522, 151–157

3. Dahm, C., Muller, R., Schulte, G., Schmidt, K., and Leistner, E. (1998)Biochim. Biophys. Acta 1425, 377–386

4. Liu, J., Quinn, N., Berchtold, G. A., and Walsh, C. T. (1990) Biochemistry29, 1417–1425

5. Daruwala, R., Bhattacharyya, D. K., Kwon, O., and Meganathan, R. (1997)J. Bacteriol. 179, 3133–3138

6. Serino, L., Reimmann, C., Baur, H., Beyeler, M., Visca, P., and Haas, D.(1995)Mol. Gen. Genet. 249, 217–228

7. Liu, J., Duncan, K., and Walsh, C. T. (1989) J. Bacteriol. 171, 791–7988. Pelludat, C., Brem, D., and Heesemann, J. (2003) J. Bacteriol. 185,

5648–56539. Kerbarh, O., Ciulli, A., Howard, N. I., and Abell, C. (2005) J. Bacteriol. 187,

5061–506610. Kerbarh, O., Chirgadze, D. Y., Blundell, T. L., and Abell, C. (2006) J. Mol.

Biol. 357, 524–53411. Muller, R., Dahm, C., Schulte, G., and Leistner, E. (1996) FEBS Lett. 378,

131–13412. Gross, J., Cho, W. K., Lezhneva, L., Falk, J., Krupinska, K., Shinozaki, K.,

Seki, M., Herrmann, R. G., and Meurer, J. (2006) J. Biol. Chem. 281,17189–17196

13. Dosselaere, F., and Vanderleyden, J. (2001) Crit. Rev. Microbiol. 27,75–131

14. Buchanan, B. B., Gruissem, W., and Jones, R. L. (2000) Biochemistry &Molecular Biology of Plants, American Society of Plant Physiologists,Rockville, MD

15. Polya, G. (2003) Biochemical Targets of Plant Bioactive Compounds,Taylor & Francis Ltd., London

16. Han, Y. S., Heijden, R., Lefeber, A. W., Erkelens, C., and Verpoorte, R.(2002) Phytochemistry 59, 45–55

17. Wildermuth, M. C., Dewdney, J., Wu, G., and Ausubel, F. M. (2001)Nature 414, 562–565

18. Muljono, R. A. B., Scheffer, J. J. C., and Verpoorte, R. (2002) Plant Physiol.Biochem. 40, 231–234

19. van Tegelen, L. J., Moreno, P. R., Croes, A. F., Verpoorte, R., andWullems,G. J. (1999) Plant Physiol. 119, 705–712

20. Mauch, F., Mauch-Mani, B., Gaille, C., Kull, B., Haas, D., and Reimmann,C. (2001) Plant J. 25, 67–77

21. Viitanen, P. V., Devine, A. L., Khan,M. S., Deuel, D. L., VanDyk, D. E., andDaniell, H. (2004) Plant Physiol. 136, 4048–4060

22. Leduc, C., Ruhnau, P., and Leistner, E. (1991) Plant Cell Rep. 10,334–337

23. Leduc, C., Birgel, I., Muller, R., and Leistner, E. (1997) Planta 202,206–210

24. van Tegelen, L. J. P., Bongaerts, R. J. M., Croes, A. F., Verpoorte, R., andWullems, G. J. (1999) Phytochemistry 51, 263–269

25. Stalman, M., Koskamp, A. M., Luderer, R., Vernooy, J. H., Wind, J. C.,Wullems, G. J., and Croes, A. F. (2003) J. Plant Physiol. 160, 607–614

26. Dempsey, D. M. A., Shah, J., and Klessig, D. F. (1999) Crit. Rev. Plant Sci.18, 547–575

27. Durrant, W. E., and Dong, X. (2004)Annu. Rev. Phytopathol. 42, 185–20928. Dewdney, J., Reuber, T. L., Wildermuth, M. C., Devoto, A., Cui, J., Stutius,

L. M., Drummond, E. P., and Ausubel, F. M. (2000) Plant J 24, 205–21829. Nawrath, C., and Metraux, J. P. (1999) Plant Cell 11, 1393–140430. Nawrath, C., Heck, S., Parinthawong, N., and Metraux, J. P. (2002) Plant

Cell 14, 275–28631. Sharma, Y. K., Leon, J., Raskin, I., and Davis, K. R. (1996) Proc. Natl. Acad.

Sci. U. S. A. 93, 5099–510432. Scott, I. M., Clarke, S. M.,Wood, J. E., andMur, L. A. (2004) Plant Physiol.

135, 1040–104933. Clarke, S. M., Mur, L. A., Wood, J. E., and Scott, I. M. (2004) Plant J. 38,

432–44734. Larkindale, J., Hall, J. D., Knight, M. R., and Vierling, E. (2005) Plant

Physiol. 138, 882–89735. Martinez, C., Pons, E., Prats, G., and Leon, J. (2004) Plant J. 37, 209–21736. Morris, K., MacKerness, S. A., Page, T., John, C. F., Murphy, A. M., Carr,

J. P., and Buchanan-Wollaston, V. (2000) Plant J. 23, 677–68537. Eberhard, J., Ehrler, T. T., Epple, P., Felix, G., Raesecke, H. R., Amrhein, N.,

and Schmid, J. (1996) Plant J. 10, 815–82138. Mobley, E. M., Kunkel, B. N., and Keith, B. (1999) Gene (Amst.) 240,

115–12339. Niyogi, K. K., and Fink, G. R. (1992) Plant Cell 4, 721–73340. Herrmann, K. M. (1995) Plant Cell 7, 907–91941. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G.,

Smith, J. A., and Struhl, K. (eds) (2005) Current Protocols in MolecularBiology, John Wiley & Sonsi, Inc.

42. Inoue, K., and Keegstra, K. (2003) Plant J. 34, 661–66943. Constan, D., Froehlich, J. E., Rangarajan, S., and Keegstra, K. (2004) Plant

Physiol. 136, 3605–361544. Bruce, B. D., Perry, S., Froehlich, J., andKeegstra, K. (1994)PlantMol. Biol.

Man. J1, 1–1545. Volko, S. M., Boller, T., and Ausubel, F. M. (1998) Genetics 149, 537–54846. Mori, T., Kuroiwa, H., Higashiyama, T., and Kuroiwa, T. (2003) Biochem.

Biophys. Res. Commun. 306, 564–56947. Guzman, L.M., Belin, D., Carson,M. J., and Beckwith, J. (1995) J. Bacteriol.

177, 4121–413048. Gaille, C., Reimmann, C., and Haas, D. (2003) J. Biol. Chem. 278,

16893–1689849. Rudolph, F. B., and Fromm, H. J. (1979)Methods Enzymol. 63, 138–15950. Gaille, C., Kast, P., and Haas, D. (2002) J. Biol. Chem. 277, 21768–2177551. Rusnak, F., Liu, J., Quinn, N., Berchtold, G. A., and Walsh, C. T. (1990)

Biochemistry 29, 1425–143552. Thompson, J. D., Higgins, D.G., andGibson, T. J. (1994)Nucleic Acids Res.

22, 4673–468053. Emanuelsson, O., Nielsen, H., and von Heijne, G. (1999) Protein Sci. 8,

978–98454. Sali, A., and Blundell, T. L. (1993) J. Mol. Biol. 234, 779–81555. Emsley, P., and Cowtan, K. (2004) Acta Crystallogr. Sect. D Biol. Crystallogr.

60, 2126–213256. Verberne, M. C., Verpoorte, R., Bol, J. F., Mercado-Blanco, J., and

Linthorst, H. J. (2000) Nat. Biotechnol. 18, 779–78357. Zimmermann, P., Hirsch-Hoffmann, M., Hennig, L., and Gruissem, W.

(2004) Plant Physiol. 136, 2621–263258. Craigon, D. J., James, N., Okyere, J., Higgins, J., Jotham, J., and May, S.

(2004) Nucleic Acids Res. 32, D575–D577 (database issue)59. Cornish-Bowden, A. (1979) Fundamentals of Enzyme Kinetics, pp. 39–59,

Butterworth & Co, London60. Poulsen, C., van der Heijden, R., and Verpoorte, R. (1991) Phytochemistry

30, 2873–287661. Kozlowski, M. C., Tom, N. J., Seto, C. T., Sefler, A. M., and Bartlett, P. A.

(1995) J. Am. Chem. Soc. 117, 2128–214062. Walsh, C. T., Liu, J., Rusnak, F., and Sakaitani, M. (1990) Chem. Rev. 90,

1105–112963. Young, I. G., Batterham,T. J., andGibson, F. (1969)Biochim. Biophys. Acta

177, 389–40064. DeClue, M. S., Baldridge, K. K., Kast, P., and Hilvert, D. (2006) J. Am.

Chem. Soc. 128, 2043–205165. Payne, R. J., Kerbarh, O., Miguel, R. N., Abell, A. D., and Abell, C. (2005)

Org. Biomol. Chem. 3, 1825–182766. Harrison, A. J., Yu, M., Gardenborg, T., Middleditch, M., Ramsay, R. J.,

Baker, E. N., and Lott, J. S. (2006) J. Bacteriol. 188, 6081–609167. Brodersen, P.,Malinovsky, F. G., Hematy, K., Newman,M.A., andMundy,

J. (2005) Plant Physiol. 138, 1037–1045




Downloaded from

68. Gu, Z., Steinmetz, L. M., Gu, X., Scharfe, C., Davis, R. W., and Li, W. H.(2003) Nature 421, 63–66

69. Chen, B. S., Wang, Y. C., Wu, W. S., and Li, W. H. (2005) Bioinformatics21, 2698–2705

70. Genoud, T., Buchala, A. J., Chua, N. H., and Metraux, J. P. (2002) Plant J.31, 87–95

71. Fall, R., and Wildermuth, M. C. (1998) J. Geophys. Res. 103,25,599–25,609

72. Entus, R., Poling, M., and Herrmann, K. M. (2002) Plant Physiol. 129,1866–1871

73. Al-Shehbaz, I. A., and O’Kane, J., S.L. (2002) in The Arabidopsis Book,

(Somerville, C., Meyerowitz, E., Dangl, J. L., and Stitt, M., eds) Amer-ican Society of Plant Biologists, Rockville, MD

74. Wolfenden, R., Snider, M., Ridgway, C., andMiller, B. (1999) J. Am. Chem.Soc. 121, 7419–7420

75. Li, J., and Last, R. L. (1996) Plant Physiol. 110, 51–5976. Bernasconi, P.,Walters, E.W.,Woodworth, A. R., Siehl, D. L., Stone, T. E.,

and Subramanian, M. V. (1994) Plant Physiol. 106, 353–35877. Poulsen, C., Bongaerts, R. J., andVerpoorte, R. (1993) Eur. J. Biochem. 212,

431–44078. Sahr, T., Ravanel, S., Basset, G., Nichols, B. P., Hanson, A. D., and Rebeille,

F. (2006) Biochem. J. 396, 157–162




Downloaded from

arabidopsis isochorismate synthase functional in pathogen

Documents