Báo cáo khoa học: Arabidopsis thaliana CYP77A4 is the first cytochrome P450 able to catalyze the epoxidation of free fatty acids in plants potx

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Arabidopsis thaliana CYP77A4 is the first cytochromeP450 able to catalyze the epoxidation of free fattyacids in plantsVincent Sauveplane1, Sylvie Kandel2, Pierre-Edouard Kastner1,Ju¨rgen Ehlting1,Vincent Compagnon1, Danie`le Werck-Reichhart1and Franck Pinot11 Institut de Biologie Mole´culaire des Plantes, University of Strasbourg, France2 Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USAFatty acid-oxidizing enzymes have been the subject ofan increasing number of studies in all organisms, asthe products of their reactions exhibit fundamentalbiological activities [1–3]. Among these oxidases, cyto-chromes P450 play a prominent role. For example, inanimals, arachidonic acid (C20:4) is oxidized throughthe cytochrome P450 pathway, leading to the produc-tion of hydroxylated and epoxidized derivatives [4–6].The cytochrome P450 superfamily represents a highlydiversified set of heme-containing proteins found inbacteria, fungi, animals and plants [7]. In animals,members of the CYP4A gene subfamily mainly cata-lyze the formation of x- and x-1-hydroxyl derivativesof fatty acids. The regulation of some CYP4A enzymesKeywordscytochrome P450; defense; epoxide; fattyacid; plantCorrespondenceF. Pinot, IBMP-CNRS UPR 2357, Institut deBotanique, 28 rue Goethe, F-67083Strasbourg Cedex, FranceFax: +33 3 90 24 19 21Tel: +33 3 90 24 18 37E-mail: franck.pinot@ibmp-ulp.u-strasbg.fr(Received 4 September 2008, revised 20November 2008, accepted 26 November2008)doi:10.1111/j.1742-4658.2008.06819.xAn approach based on an in silico analysis predicted that CYP77A4, acytochrome P450 that so far has no identified function, might be a fattyacid-metabolizing enzyme. CYP77A4 was heterologously expressed in aSaccharomyces cerevisiae strain (WAT11) engineered for cytochrome P450expression. Lauric acid (C12:0) was converted into a mixture of hydroxy-lauric acids when incubated with microsomes from yeast expressingCYP77A4. A variety of physiological C18fatty acids were tested as poten-tial substrates. Oleic acid (cis-D9C18:1) was converted into a mixture of x-4-to x-7-hydroxyoleic acids (75%) and 9,10-epoxystearic acid (25%). Linoleicacid (cis,cis-D9,D12C18:2) was exclusively converted into 12,13-epoxyocta-deca-9-enoic acid, which was then converted into diepoxide after epoxida-tion of the D9unsaturation. Chiral analysis showed that 9,10-epoxystearicacid was a mixture of 9S ⁄ 10R and 9R ⁄ 10S in the ratio 33 : 77, whereas12,13-epoxyoctadeca-9-enoic acid presented a strong enantiomeric excess infavor of 12S ⁄ 13R, which represented 90% of the epoxide. Neither stearicacid (C18:0) nor linolelaidic acid (trans,trans- D9,D12C18:2) was metabolized,showing that CYP77A4 requires a double bond, in the cis configuration, tometabolize C18fatty acids. CYP77A4 was also able to catalyze the in vitroformation of the three mono-epoxides of a-linolenic acid (cis,cis,cis-D9,D12,D15C18:3), previously described as antifungal compounds. Epoxides gen-erated by CYP77A4 are further metabolized to the corresponding diols byepoxide hydrolases located in microsomal and cytosolic subcellular frac-tions from Arabidopsis thaliana. The concerted action of CYP77A4 withepoxide hydrolases and hydroxylases allows the production of compoundsinvolved in plant–pathogen interactions, suggesting a possible role forCYP77A4 in plant defense.AbbreviationEET, epoxyeicosatrienoic acid.FEBS Journal 276 (2009) 719–735 Journal compilation ª 2008 FEBS. No claim to original French government works 719by peroxisome proliferator-activated receptors pointsto a role in fatty acid catabolism [8]. After x-hydroxyl-ation, fatty acids can be further oxidized to diacids,which can then be eliminated by peroxisome b-oxida-tion [9]. However, investigations describing the effectof x-hydroxy fatty acids in different physiological pro-cesses [10–13] have suggested that x-hydroxylationcannot be considered only as a step leading to catabo-lism. The epoxidation of polyunsaturated fatty aciddouble bonds, particularly of arachidonic acid, hasgenerated much interest because of the biological activ-ities of the resulting metabolites [14,15]. These epoxi-dation reactions of C20:4are catalyzed by members ofthe CYP2C subfamily and by the CYP2J2 isoform[6,16,17]. Human CYP4F8 and CYP4F12 isoforms areable to epoxidize docosahexaenoic acid (C22:6) [18].In plants, fatty acids are also metabolized by cyto-chrome P450-dependent oxygenases [19], and it ispossible to distinguish x-hydroxylases and in-chainhydroxylases that attack the terminal and subtermi-nal positions, respectively. So far, the majority ofwork has addressed x-hydroxylases mainly repre-sented in CYP86 and CYP94 families [19]. Theirinvolvement in the synthesis of cutin, a protectivebiopolymer of fatty acids cross-linked by ester bonds[20], has been established [21,22]. Studies of LCR(LACERATA) and att1 (aberrant induction of typethree genes), the first Arabidopsis thaliana mutantswith alterations in the coding sequence of CYP86A8and CYP86A2, respectively, have also shown thatx-hydroxylases have key roles to play in plant devel-opment [21,22].Despite the fact that the implication of a cyto-chrome P450 in the epoxidation of a long-chain fattyacid was first demonstrated in spinach leaves morethan three decades ago [20,23], a cytochrome P450 ableto epoxidize fatty acids is still poorly documented inplants. Biochemical studies performed with unsatu-rated analogues of lauric acid (C12:0) clearly demon-strated the existence in plants of a cytochrome P450able to epoxidize the double bonds of fatty acids. Theterminal olefin 11-dodecenoic acid is converted into11,12-epoxylauric acid by a cytochrome P450 inVicia sativa microsomes [24]. The epoxidation ofunsaturated analogues of lauric acid by cytochromeP450 was also reported in microsomes from Jerusalemartichoke [25,26], as well as in microsomes from wheat[27]. However, none of the enzymes implicated in thesereactions have been characterized and, to date, nocytochrome P450 able to epoxidize free fatty acids hasbeen identified in plants. The epoxidation of physiolog-ical substrates, such as oleic acid (cis-D9C18:1) and lino-leic acid (cis,cis-D9,D12C18:2), has been reported inVicia faba [28] and Glycine max [29]. However, thesereactions were not catalyzed by cytochrome P450, butrather by peroxygenases, which are hydroperoxide-dependent fatty acid epoxidases. Recently, studies of aperoxygenase purified from oat have demonstratedthat this enzyme is deeply buried in microsomes or inlipid droplets [30]. Lee et al. [31] identified a non-hemedi-iron enzyme, a ‘desaturase-like’ protein, able totransform linoleic acid into 12,13-epoxyoctadeca-cis-9-enoic acid (vernolic acid). This compound can makeup 50–90% of total fatty acids in seed oil of certainEuphorbiaceae, such as Euphorbia lagascae [32]. In thisplant, the enzyme involved in its production wasdescribed recently [32]. This enzyme, classified asCYP726A1, does not epoxidize free fatty acids, butfatty acids bound to phosphatidylcholine [32].A new approach, based on an in silico analysis ofpublicly available transcriptome data, has been devel-oped recently to map cytochrome P450 genes onto spe-cific metabolic pathways [33]. This analysis identifiesmetabolic genes that are co-expressed with a given baitP450 during plant development, on stress and hormonetreatment, and in mutant wild-type comparisons.Based on the functional annotation of co-expressedgenes, a metabolic pathway in which the bait P450may act is predicted. This approach suggested thatCYP77A4 could be involved in fatty acid metabolismas it is developmentally co-expressed across hundredsof biological samples with several characterizedenzymes involved in lipid metabolism. The most simi-larly expressed genes are CYP86A8 encoding a fatty acidx-hydroxylase, a putative epoxide hydrolase, severalgenes encoding enzymes involved in the synthesis offatty acids in plastids, including the stearoyl acyl carrierprotein desaturase SSI2, and the plastidic long-chainacyl-CoA synthetase LACS9 (for a complete list ofco-expressed genes, see http://www-ibmp.u-strasbg.fr/~CYPedia/CYP77A4/CoExpr_CYP77A4_Organs.html).In this work, we report the heterologous expressionand functional characterization of CYP77A4. Substratespecificity and catalytic properties were explored usingrecombinant CYP77A4 expressed in an engineeredyeast strain. Our study confirms that this enzyme is afatty acid-metabolizing enzyme. We show thatCYP77A4 is able to catalyze, in vitro, the epoxidationof physiological unsaturated fatty acids. Our work alsoshows that the epoxides generated can be furtherhydrolyzed to the corresponding diols by epoxidehydrolases present in subcellular fractions of A. thali-ana. Thus, CYP77A4 from A. thaliana, described inthis work, is the first cytochrome P450 able to catalyzefree fatty acid epoxidation, identified in plants. Itsphysiological significance remains to be establishedCYP77A4, an epoxy fatty acid-forming enzyme V. Sauveplane et al.720 FEBS Journal 276 (2009) 719–735 Journal compilation ª 2008 FEBS. No claim to original French government worksand will be assessed by future studies of A. thalianamutated in the coding sequence of CYP77A4.ResultsSelection, cloning and expression of CYP77A4An approach based on an in silico analysis predictedthat CYP77A4 could be involved in fatty acid metabo-lism [33]. The coding sequence of CYP77A4 wasamplified by PCR from a cDNA library of Arabidopsisand subsequently cloned into a yeast expression vector.The deduced protein (512 amino acids) has a calcu-lated mass of 58 134 Da and a pI of 8.71. Enzymaticcharacterization of CYP77A4 was carried out employ-ing microsomes from the yeast strain WAT11 trans-formed with the plasmid pYeDP60 [34] containing thecoding sequence of CYP77A4. WAT11 over-expressesa plant P450 reductase in order to optimize electrontransfer during catalysis and probably to increase thestability of the expressed P450. Furthermore, there areonly three cytochromes P450 encoded by the yeast gen-ome. They are either not expressed or expressed at anegligible level in the growth conditions used here, andnone is able to metabolize fatty acids, ensuring thatthe metabolism described here results from enzymaticreactions catalyzed by CYP77A4 [34]. After micro-somal membrane isolation from the CYP77A4-trans-formed yeasts, the level of expression of the enzymewas evaluated on the basis of the differential absor-bance of reduced CO-bound versus reduced micro-somes at 450 nm [35]. The CYP77A4 content of themicrosomal preparation used in our experiments was0.1 nmolÆmg)1protein (Fig. S1). No absorbance at450 nm and no enzymatic activity with the substratestested were detected in microsomes from yeast trans-formed with a void plasmid under the same growthconditions.Metabolism of lauric acid by CYP77A4To validate the hypothesis of CYP77A4 being a fattyacid-metabolizing enzyme, we incubated radiolabeledlauric acid (C12:0) with microsomes from yeast express-ing CYP77A4. The resolution of reaction products wasperformed by directly loading the incubation mediumonto a TLC plate. Figure 1 shows the radiochromato-grams obtained after incubation in the absence(Fig. 1A) or presence (Fig. 1B–D) of NADPH. A largepeak of radioactivity was detected after 20 min of incu-bation (peak 1, Fig. 1B). It was not formed in theabsence of NADPH (Fig. 1A), with microsomes fromyeast transformed with a void plasmid (Fig. 1C) orwith boiled microsomes (Fig. 1D). Taken together,these results demonstrate the involvement of CYP77A4in the formation of this radioactive peak. Metabolitesfrom this peak were purified, derivatized and subjectedto GC ⁄ MS analysis (Experimental procedures). Themass spectrum of the derivatized metabolite 1 (Fig. S2)showed ions at m ⁄ z (relative intensity, %) values of 73(41%) [(CH3)3Si+], 75 (23%) [(CH3)2Si+=O], 117(100%), 255 (15%) (M-47) [loss of methanol from the(M-15) fragment], 271 (3%) (M-31) (loss of OCH3fromthe methyl ester), 287 (6%) (M-15) (loss of a methylfrom the trimethylsilyl group). This fragmentationpattern is characteristic of the derivative of 11-hydroxy-lauric acid (x-1) (M = 302 gÆmol)1). The mass spec-trum of derivatized metabolite 2 (Fig. S2) showed ionsat m ⁄ z (relative intensity, %) values of 73 (70%)[(CH3)3Si+], 75 (30%) [(CH3)2Si+=O], 131 (100%),255 (12%) (M-47) [loss of methanol from the (M-15)fragment], 271 (4%) (M-31) (loss of OCH3from themethyl ester), 273 (51%), 287 (2%) (M-15) (loss of amethyl from the trimethylsilyl group). This fragmen-tation pattern is characteristic of the derivative of10-hydroxylauric acid ( x-2) (M = 302 gÆmol)1). Themass spectrum of derivatized metabolite 3 (Fig. S2)showed ions at m ⁄ z (relative intensity, %) values of 73(75%) [(CH3)3Si+], 75 (31%) [(CH3)2Si+=O], 145(100%), 255 (11%) (M-47) [loss of methanol from the(M-15) fragment], 259 (59%), 271 (3%) (M-31) (loss ofOCH3from the methyl ester), 287 (2%) (M-15) (loss ofa methyl from the trimethylsilyl group). This fragmen-tation pattern is characteristic of the derivative of9-hydroxylauric acid (x-3) (M = 302 gÆmol)1). Themass spectrum of derivatized metabolite 4 (Fig. S2)showed ions at m ⁄ z (relative intensity, %) values of 73(68%) [(CH3)3Si+], 75 (28%) [(CH3)2Si+=O], 159(100%), 245 (68%), 255 (9%) (M-47) [loss of methanolfrom the (M-15) fragment], 271 (4%) (M-31) (loss ofOCH3from the methyl ester), 287 (2%) (M-15) (loss ofa methyl from the trimethylsilyl group). This fragmen-tation pattern is characteristic of the derivative of 8-hy-droxylauric acid (x-4) (M = 302 gÆmol)1). The massspectrum of derivatized metabolite 5 (Fig. S2) showedions at m ⁄ z (relative intensity, %) values of 73 (97%)[(CH3)3Si+], 75 (39%) [(CH3)2Si+=O], 173 (100%),231 (71%) 255 (11%) (M-47) [loss of methanol fromthe (M-15) fragment], 271 (4%) (M-31) (loss of OCH3from the methyl ester), 287 (5%) (M-15) (loss of amethyl from the trimethylsilyl group). This fragment-ation pattern is characteristic of the derivative of7-hydroxylauric acid (x-5) (M = 302 gÆmol)1). Theiridentification revealed that the reaction product is com-posed of a mixture of five different in-chain hydroxyl-ation products of lauric acid, which is predominantlyV. Sauveplane et al. CYP77A4, an epoxy fatty acid-forming enzymeFEBS Journal 276 (2009) 719–735 Journal compilation ª 2008 FEBS. No claim to original French government works 721hydroxylated on the x-1 position. When oxidizing lauricacid, CYP77A4 exhibits the following regioselectivity:x-1 (53%), x-2 (15%), x-3 (8%), x-4 (18%) and x-5(6%). For substrate oxidation, we determined,by kinetic studies, Km,appand Vmax,appvalues of172 ± 13 lm and 117 ± 5 nmolÆmin)1Ænmol)1P450,respectively (Fig. S3). The x-1 position of lauric acidcorresponds to carbon 11 of physiological C18fattyacids, closely located near the double bonds of oleic,linoleic and a-linolenic acids. We therefore tested thesedifferent unsaturated fatty acids as potential substrates.Metabolism of oleic acid by CYP77A4We first incubated mono-unsaturated oleic acid(C18:1). The radiochromatograms obtained after reso-lution of the reaction products on TLC are presentedin Fig. 2. Incubation was carried out in the absence(Fig. 2A) or presence (Fig. 2B–D) of NADPH. Twonew peaks of radioactivity (peak 1 and peak 2,Fig. 2B) were detected; their formation required thepresence of NADPH in the incubation. They werealso not formed on incubation with microsomes fromyeast transformed with a void plasmid (Fig. 2C) orwith boiled microsomes (Fig. 2D). Metabolites frompeak 1 were purified, derivatized and subjected toGC ⁄ MS analysis. The mass spectrum of derivatizedmetabolite 1 (Fig. S4) showed ions at m ⁄ z (relativeintensity, %) values of 73 (100%) [(CH3)3Si+], 75(58%) [(CH3)2Si+=O], 337 (9%) (M-47) [loss ofmethanol from the (M-15) fragment], 353 (2%)(M-31) (loss of OCH3from the methyl ester), 369Fig. 1. Radiochromatographic resolution by TLC of metabolites generated in incubations of lauric acid with microsomes from yeast express-ing CYP77A4. Microsomes were incubated with 100 lM [1-14C]lauric acid in the absence (A) or presence (B) of NADPH. Incubations wereperformed at 27 °C and contained 20 pmol of CYP77A4. They were stopped after 30 min by the addition of 20 lL of acetonitrile (containing0.2% acetic acid) and directly spotted onto TLC. Peak S, lauric acid; peak 1, mixture of 11-, 10-, 9-, 8- and 7-hydroxylauric acids. Experimentsin (C) and (D) were performed as in (B), but with microsomes from yeast transformed with a void plasmid (C) or with boiled microsomes(D). The structures of the metabolites are described in (E).CYP77A4, an epoxy fatty acid-forming enzyme V. Sauveplane et al.722 FEBS Journal 276 (2009) 719–735 Journal compilation ª 2008 FEBS. No claim to original French government works(3%) (M-15) (loss of a methyl from the trimethylsilylgroup) and 384 (2%) (M). The mass spectrum alsoshowed ions at 159 (63%) and 327 (7%), resultingfrom cleavage on both sides of the hydroxyl functioncarrying the trimethylsilyl group. This fragmentationpattern is characteristic of the derivative of 14-hy-droxyoleic acid (x-4) (M = 384 gÆmol)1). The massspectrum of derivatized metabolite 2 (Fig. S4) showedions at m ⁄ z (relative intensity, %) values of 73(100%) [(CH3)3Si+], 75 (50%) [(CH3)2Si+=O], 337(11%) (M-47) [loss of methanol from the (M-15)fragment], 369 (3%) (M-15) (loss of a methyl fromthe trimethylsilyl group). The mass spectrum alsoshowed ions at 173 (34%) and 313 (18%), resultingfrom cleavage on both sides of the hydroxyl functioncarrying the trimethylsilyl group. This fragmentationpattern is characteristic of the derivative of 13-hy-droxyoleic acid (x-5) ( M = 384 gÆmol)1). The massspectrum of derivatized metabolite 3 (Fig. S4) showedions at m ⁄ z (relative intensity, %) values of 73 (48%)[(CH3)3Si+], 75 (12%) [(CH3)2Si+=O], 337 (3%) (M-47) [loss of methanol from the (M-15) fragment], 353(1%) (M-31) (loss of OCH3from the methyl ester),369 (0.5%) (M-15) (loss of a methyl from the trim-ethylsilyl group). The mass spectrum also showed ionsat 187 (100%) and 299 (4%), resulting from cleavageon both sides of the hydroxyl function carrying thetrimethylsilyl group. This fragmentation pattern ischaracteristic of the derivative of 12-hydroxyoleic acid(x-6) (M = 384 gÆmol)1). The mass spectrum ofderivatized metabolite 4 (Fig. S4) showed ions at m ⁄ z(relative intensity, %) values of 73 (35%) [(CH3)3Si+],75 (14%) [(CH3)2Si+=O], 337 (3%) (M-47) [lossof methanol from the (M-15) fragment], 353 (1%)(M-31) (loss of OCH3from the methyl ester), 369(1%) (M-15) (loss of a methyl from the trimethylsilylgroup) and 384 (0.5%) (M). The mass spectrum alsoshowed ions at 201 (1%) and 285 (100%), resultingFig. 2. Radiochromatographic resolution by TLC of metabolites generated in incubations of oleic acid with microsomes from yeast express-ing CYP77A4. Microsomes were incubated with 100 lM [1-14C]oleic acid in the absence (A) or presence (B) of NADPH. Incubations wereperformed at 27 °C and contained 20 pmol of CYP77A4. They were stopped after 30 min by the addition of 20 lL of acetonitrile (containing0.2% acetic acid) and directly spotted onto TLC. Peak S, oleic acid; peak 1, mixture of 14-, 13-, 12- and 11-hydroxyoleic acids; peak 2, 9,10-epoxystearic acid. Experiments in (C) and (D) were performed as in (B), but with microsomes from yeast transformed with a void plasmid(C) or with boiled microsomes (D). The structures of the metabolites are described in (E).V. Sauveplane et al. CYP77A4, an epoxy fatty acid-forming enzymeFEBS Journal 276 (2009) 719–735 Journal compilation ª 2008 FEBS. No claim to original French government works 723from cleavage on both sides of the hydroxyl functioncarrying the trimethylsilyl group. This fragmentationpattern is characteristic of the derivative of 11-hy-droxyoleic acid (x-7) (M = 384 gÆmol)1). The identifi-cation of metabolites from peak 1 by GC ⁄ MS afterpurification and derivatization revealed thatCYP77A4 hydroxylates oleic acid with the followingregioselectivity: x-7 (58%), x-6 and x-5 (30%), x-4(12%). The metabolite from peak 2 displayed theTLC mobility expected for 9,10-epoxystearic acid,and was indeed identified as 9,10-epoxystearic acid byGC ⁄ MS analysis (Fig. S4). For substrate oxidation,we determined, by kinetic studies, Km,appand Vmax,appvalues of 84 ± 23 lm and 26 ± 5 nmolÆmin)1Ænmol)1P450, respectively (Fig. S3). We determined the ste-reochemistry of this epoxide after purification andanalysis by HPLC using a chiral column. The radio-chromatogram of Fig. 3 shows that it is a mixture ofthe two enantiomers, 9S ⁄ 10R and 9R ⁄ 10S, in theratio 33 : 77, respectively.Metabolism of linoleic acid by CYP77A4Figure 4 shows the radioactivity profiles obtained afterincubation of linoleic acid (C18:2) with microsomesfrom yeast expressing CYP77A4. The addition ofNADPH to the incubation medium led to the forma-Fig. 3. Radiochromatographic resolution by HPLC of the enantio-mers of 9,10-epoxystearic acid produced by CYP77A4. (A) Afterincubation of oleic acid with microsomes from yeast expressingCYP77A4, the 9,10-epoxystearic produced (peak 2, Fig. 2B) waspurified and subjected to chiral HPLC analysis with hexane ⁄ propan-2-ol ⁄ acetic acid (99.7 : 0.2 : 0.1, v ⁄ v ⁄ v) at a flow rate of 0.8mLÆmin)1. (B) Structures of the enantiomers.Fig. 4. Radiochromatographic resolution by TLC of metabolites gen-erated in incubations of linoleic acid with microsomes from yeastexpressing CYP77A4. Microsomes were incubated with 100 lM[1-14C]linoleic acid in the absence (A) or presence (B) of NADPH.Incubations were performed at 27 °C and contained 20 pmol ofCYP77A4. They were stopped after 30 min by the addition of 20 lLof acetonitrile (containing 0.2% acetic acid) and directly spotted ontoTLC. Peak S, linoleic acid; peak 1, 9,10:12,13-diepoxyoctadecanoicacid; peak 2, 12,13-epoxyoctadeca-9-enoic acid. Experiments in (C)and (D) were performed as in (B), but with microsomes from yeasttransformed with a void plasmid (C) or with boiled microsomes (D).The structures of the metabolites are described in (E).CYP77A4, an epoxy fatty acid-forming enzyme V. Sauveplane et al.724 FEBS Journal 276 (2009) 719–735 Journal compilation ª 2008 FEBS. No claim to original French government workstion of a major radioactive peak (peak 2, Fig. 4B)which was not present in the absence of NADPH(Fig. 4A). It results from a reaction catalyzed byCYP77A4, as it was not formed when the microsomeswere from yeast transformed with a void plasmid(Fig. 4C) or were boiled (Fig. 4D). This peak containsonly one metabolite, which was identified by GC ⁄ MSanalysis (Fig. S5) after purification reaction in acidicmethanol and derivatization as 12,13-epoxyoctadeca-9-enoic acid (vernolic acid), resulting from the epoxi-dation of the D12double bond. The kinetic parametersfrom the reaction of substrate oxidation are Km,app=61±3 lm and Vmax,app= 13 ± 0.3 nmolÆmin)1Ænmol)1P450 (Fig. S3). Stereochemistry studies presented inFig. 5 show that CYP77A4 possesses a strong enantio-specificity: the epoxide formed is a mixture of 12S ⁄ 13Rand 12R ⁄ 13S in the ratio 90 : 10, thus presenting astrong enantiomeric excess in favor of the 12S ⁄ 13Rconformation. The metabolite from the minor peak(peak 1, Fig. 4B) was identified by GC ⁄ MS (Fig. S5)as 9,10:12,13-diepoxyoctadecanoic acid after puri-fication and derivatization. CYP77A4 was also ableto catalyze its formation in incubations with purified12,13-epoxyoctadeca-9-enoic acid (data not shown).Metabolism of a-linolenic acid by CYP77A4The incubation of a-linolenic acid (C18:3) with micro-somes from yeast expressing CYP77A4 led to theformation of one major radioactive peak, as shown onthe radiochromatogram in Fig. 6 (peak 2, Fig. 6B). Itresults from a reaction catalyzed by CYP77A4, as itrequires the presence of NADPH and is not formedwith microsomes from yeast transformed with a voidplasmid (Fig. 6C) or on incubation with boiled micro-somes (Fig. 6D). The shape of this peak suggests thatit contains more than one metabolite. After purifica-tion, acidic treatment and derivatization, GC ⁄ MSanalysis showed that it was indeed a mixture of thethree epoxide derivatives of a-linolenic acid.The mass spectrum of derivatized metabolite 1(Fig. S6) showed ions at m ⁄ z (relative intensity, %)values of 73 (100%) [(CH3)3Si+], 75 (14%)[(CH3)2Si+=O], 439 (4%) (M-31) (loss of OCH3fromthe methyl ester), 455 (1%) (M-15) (loss of a methylfrom the trimethylsilyl group), 470 (0.5%) (M). Themass spectrum also showed ions at 171 (40%) and 299(44%), resulting from the cleavage between twohydroxyls carrying the trimethylsilyl group generatedby hydrolysis in perchloric acid. This fragmentationpattern is characteristic of the derivative after acidichydrolysis of 12,13-epoxyoctadeca-9,15-dienoic acid(M = 470 gÆmol)1) which represents 87% of themetabolites. The mass spectrum of derivatized meta-bolite 2 (Fig. S6) showed ions at m ⁄ z (relative inten-sity, %) values of 73 (100%) [(CH3)3Si+], 75 (17%)[(CH3)2Si+=O], 439 (2%) (M-31) (loss of OCH3fromthe methyl ester), 455 (0.5%) (M-15) (loss of a methylfrom the trimethylsilyl group), 470 (1%) (M). Themass spectrum also showed ions at 211 (11%) and 259(81%), resulting from the cleavage between twohydroxyls carrying the trimethylsilyl group generatedby hydrolysis in perchloric acid. This fragmentationpattern is characteristic of the derivative after acidichydrolysis of 9,10-epoxyoctadeca-12,15-dienoic acid(M = 470 gÆmol)1) which represents 7% of the meta-bolites. The mass spectrum of derivatized metabolite 3(Fig. S6) showed ions at m ⁄ z (relative intensity, %)values of 73 (100%) [(CH3)3Si+], 75 (21%)[(CH3)2Si+=O], 439 (3%) (M-31) (loss of OCH3fromthe methyl ester), 455 (0.5%) (M-15) (loss of a methylfrom the trimethylsilyl group), 470 (4%) (M). Themass spectrum also showed ions at 131 (67%) and 339(20%), resulting from the cleavage between twohydroxyls carrying the trimethylsilyl group generatedby hydrolysis in perchloric acid. This fragmentationpattern is characteristic of the derivative after acidichydrolysis of 15,16-epoxyoctadeca-9,12-dienoic acidFig. 5. Radiochromatographic resolution by HPLC of the enantio-mers of 12,13-epoxyoctadeca-9-enoic acid produced by CYP77A4.(A) After incubation of linoleic acid with microsomes from yeastexpressing CYP77A4, the 12,13-epoxyoctadeca-9-enoic acidproduced (peak 2, Fig. 4B) was purified, methylated and subjectedto chiral HPLC analysis with 100% heptane at a flow rate of0.5 mLÆmin)1. (B) Structures of the enantiomers.V. Sauveplane et al. CYP77A4, an epoxy fatty acid-forming enzymeFEBS Journal 276 (2009) 719–735 Journal compilation ª 2008 FEBS. No claim to original French government works 725(M = 470 gÆmol)1) which represents 6% of the meta-bolites. Kinetic parameters from the reaction ofsubstrate oxidation are Km,app=29±4lm andVmax,app= 38 ± 2 nmolÆmin)1Ænmol)1P450 (Fig. S3).Metabolites from the minor peak (peak 1, Fig. 6B)have not been identified.Fig. 7. Radiochromatographic resolution by TLC of metabolite gen-erated in the incubation of 12,13-epoxyoctadeca-9-enoic acid withmicrosomes or cytosol from A. thaliana. Microsomes (350 lg pro-tein) or cytosol (600 lg protein) from A. thaliana was incubatedwith 100 lM of 12,13-epoxyoctadeca-9-enoic acid for 20 min at27 °C. Incubation was stopped by the addition of 20 lL of acetoni-trile (containing 0.2% acetic acid) and directly spotted onto TLC. (A)Experiment performed with microsomes. (B) Experiment performedwith boiled microsomes. (C) Experiment performed with cytosol.(D) Experiment performed with boiled cytosol. Peak S, 12,13-epoxy-octadeca-9-enoic acid; peak 1, 12,13-dihydroxyoctadeca-9-enoicacid. The structure of the metabolite is described in (E).Fig. 6. Radiochromatographic resolution by TLC of metabolites gen-erated in incubations of a-linolenic acid with microsomes from yeastexpressing CYP77A4. Microsomes were incubated with 100 lm[1-14C]a-linolenic acid in the absence (A) or presence (B) of NADPH.Incubations were performed at 27 °C and contained 20 pmol ofCYP77A4. They were stopped after 30 min by the addition of 20 lLof acetonitrile (containing 0.2% acetic acid) and directly spotted ontoTLC. Peak S, a-linolenic acid; peak 1, non-identified; peak 2, mixtureof 12,13-epoxyoctadeca-9,15-dienoic, 9,10-epoxyoctadeca-12,15-die-noic and 15,16-epoxyoctadeca-9,12-dienoic acids. Experiments in(C) and (D) were performed as in (B), but with microsomes fromyeast transformed with a void plasmid (C) or with boiled micro-somes (D). The structures of the metabolites are described in (E).CYP77A4, an epoxy fatty acid-forming enzyme V. Sauveplane et al.726 FEBS Journal 276 (2009) 719–735 Journal compilation ª 2008 FEBS. No claim to original French government worksRequirements for CYP77A4 activityTo check the importance of the double bond forCYP77A4 activity, we tested as potential substratesstearic acid (C18:0), which is saturated, and linolelaidicacid, which is linoleic acid containing trans doublebonds. No metabolites were detected on TLC afterincubation of radiolabeled C18:0with microsomes fromyeast expressing CYP77A4 (data not shown). To testlinolelaidic acid, which is not available radiolabeled,we performed two experiments. In the first experiment,we incubated radiolabeled linoleic acid with micro-somes in the presence of an increasing concentrationof unlabeled linolelaidic acid, and did not detect anyinhibition of epoxidation of linoleic acid (data notshown). In a second experiment, we ran GC ⁄ MS anal-ysis after the incubation of linolelaidic acid with yeastmicrosomes expressing CYP77A4, and did not detectany metabolite (data not shown). Together, this showsthat CYP77A4 requires the presence of unsaturationto metabolize C18fatty acids; furthermore, unsatura-tion must be in the cis configuration.Hydrolysis of vernolic acid in microsomes andcytosol from A. thalianaIn order to test whether the metabolites generated byCYP77A4 were end products or could be substrates ofother enzymatic systems (i.e. epoxide hydrolase) fromA. thaliana, we purified vernolic acid that was producedby CYP77A4 (peak 2, Fig. 4B). This epoxide was sub-sequently incubated with microsomes isolated fromA. thaliana seedlings. The results are presented in Fig. 7.A peak of radioactivity (peak 1, Fig. 7A) was detectedafter resolving the products of reaction on TLC. Nometabolite was formed if the microsomes were boiledbefore incubation (Fig. 7B). This demonstrates the enzy-matic origin of the metabolite from peak 1. The massspectrum of this derivatized metabolite (Fig. S7) showedions at m⁄ z (relative intensity, %) values of 73 (100%)[(CH3)3Si+], 75 (17%) [(CH3)2Si+=O], 457 (2%)(M-15) (loss of a methyl from the trimethylsilyl group).The mass spectrum also showed ions at 173 (40%) and299 (8%), resulting from the cleavage between twohydroxyls carrying the trimethylsilyl group. This frag-mentation pattern is characteristic of the derivative of12,13-dihydroxyoctadeca-9-enoic acid (M = 472 gÆmol)1). The same results were obtained when incubationwas carried out with the cytosolic fraction of A. thaliana(Fig. 7C). Together, these experiments show that vernol-ic acid produced by CYP77A4 can be converted to thecorresponding diol by microsomal and cytosolic epoxidehydrolase (Fig. 8). These epoxide hydrolases can alsoconvert epoxides from C18:3into the corresponding diols(data not shown).DiscussionA new approach, based on an in silico analysis of pub-licly available transcriptome data (http://www-ibmp.u-strasbg.fr/~CYPedia/), has been developed recentlyfor the mapping of cytochromes P450 onto specificmetabolic pathways based on large-scale co-expressionanalysis [33]. This approach showed that CYP77A4 wasco-regulated across 167 developmental samples (cover-ing more than 400 publicly available Affymetrix micro-array data sets) with a set of enzymes implicated in fattyacid metabolism. Although co-expression correlationswere relatively low compared with other co-expressedgenes acting in a common pathway [33], with Pearsoncorrelation coefficients not exceeding 0.75, it was strik-ing that the top eight co-expressed genes with CYP77A4have been functionally characterized as being involvedin fatty acid metabolism (http://www-ibmp.u-strasbg.fr/~CYPedia/CYP77A4/CoExpr_CYP77A4_Organs.html).We thus found it worthwhile to test experimentally thehypothesis generated by this bioinformatic approachand to elucidate the physiological role of CYP77A4,also because no function has been reported for membersbelonging to this cytochrome P450 family to date.Heterologous expression of CYP77A4 in an engineeredstrain of yeast, and incubations of a diverse set of fattyacids with yeast microsomes, allowed us to confirm thecapacity of this newly characterized P450 to metabolizefatty acids, highlighting the predictive power of thein silico co-expression analysis. Based on phylogeneticreconstructions [36], CYP77A4 belongs to the CYP71Fig. 8. Conversion of linoleic acid to 12,13-dihydroxyoctadeca-9-enoic acid by CYP77A4 and epoxide hydrolases from A. thaliana.(A) Linoleic acid. (B) 12,13-Epoxyoctadeca-9-enoic acid. (C) 12,13-Dihydroxyoctadeca-9-enoic acid.V. Sauveplane et al. CYP77A4, an epoxy fatty acid-forming enzymeFEBS Journal 276 (2009) 719–735 Journal compilation ª 2008 FEBS. No claim to original French government works 727clan and, within this clan, forms a basal clade with theCYP89, CYP753 and CYP752 families, none of whichhas been functionally characterized. In contrast, mostfunctionally characterized plant fatty acid hydroxylasesbelong to the divergent CYP86 clan (including CYP86and CYP94 families). Both the CYP71 and CYP86 clansappear to have evolved independently within the greenplant lineage, as they are evolutionary separated by fam-ilies that pre-date land plant evolution [36]. Thus, afunction of CYP77A4 as a fatty acid-metabolizingenzyme would not have been predicted based on phylo-genetic reconstructions, again highlighting the power ofthe co-expression analysis approach, which is indepen-dent of sequence or structural similarities.On the model substrate lauric acid, CYP77A4hydroxylated predominantly the x-1 carbon, which cor-responds to a carbon in the environment of unsaturationin oleic (C18:1), linoleic (C18:2) and a-linolenic (C18:3)acids, the common physiological C18fatty acids inplants. We therefore assayed these compounds as poten-tial substrates and demonstrated that CYP77A4 wasable to produce, in vitro, epoxide derivatives of thesefatty acids. Investigations on the members of the CYP2family in animals have previously demonstrated that theregioselectivity and enantioselectivity of epoxidation arecytochrome P450 dependent [37,38]. For CYP77A4, therequirement of unsaturation, in the cis configuration,together with the regioselectivity and enantioselectivityobserved, probably reflect steric constraints on the sub-strate in the active site. The fact that C18:2is epoxidizedfirst exclusively on the D12unsaturated position, withstrong enantiomeric excess (the epoxide formed is a mix-ture of 12S ⁄ 13R 12R ⁄ 13S in the ratio 90 : 10), showsthat it is probably hindered in the active site, suggestingthat it could be a physiological substrate.In animals, epoxides of arachidonic acid (C20:4),formed by epoxidases (mainly belonging to the CYP2family), are well documented. This is mainly a resultof the large array of biological effects attributed toepoxyeicosatrienoic acids (EETs). For example, activa-tion of CYP epoxidases in endothelial cells is a keystep in vasodilatation events [14]. EETs also play amajor role in cell proliferation and angiogenesis viathe activation of an epidermal growth factor [39–41].Over-expression of CYP2C9 and exogenous applica-tion of EETs to cultured endothelial cells are associ-ated with angiogenesis [41,42]. CYP2C and CYP2J2have also been shown to be expressed in differenttumor tissues [43,44]. Epoxides of fatty acids are lessdescribed in plants, and only a few biological activitieshave been attributed to them. The discovery of suchactivities in plants might help to understand the physi-ological role of CYP77A4. This lack of data couldexplain the small amount of information availabletoday concerning the ability of plant enzymes to gener-ate epoxides of fatty acids, despite the fact that thistype of reaction was described for the first time morethan three decades ago [23]. Thus, the discovery ofCYP77A4 carrying such activity opens the door notonly for detailed biochemical characterizations, butalso for an understanding of the physiological role ofepoxides of fatty acids in plants.In addition to cytochromes P450, two distinct typesof plant enzyme, unrelated to cytochrome P450, withepoxidase activity, have been described. The first, aperoxygenase, was reported in Vicia faba [28] and Gly-cine max [29]. This type of enzyme uses hydroperox-ides as cofactors to catalyze the epoxidation of fattyacids. The second, described by Lee et al. [31], is anon-heme di-iron enzyme, also named ‘desaturase-like’enzyme. It thus appears that fatty acid epoxidation inplants can be facilitated by evolutionarily divergentsets of enzymes, further suggesting a pivotal role ofthese epoxides or derivatives thereof.CYP77A4, described in this work, catalyzed the oxy-gen incorporation into double bonds of oleic (C18:1),linoleic (C18:2) and a-linolenic (C18:3) acids, but did notmetabolize saturated stearic acid (C18:0). Furthermore,it did not metabolize linolelaidic acid, which is thehomolog of linoleic acid possessing two trans doublebonds, not commonly found in natural fatty acids.These observations suggest that the physiological func-tion of CYP77A4 could be epoxidation of unsaturatedC18fatty acids. This hypothesis is supported by in silicoco-expression analysis, showing that CYP77A4 isco-regulated with a stearoyl acyl carrier protein desat-urase and a putative epoxide hydrolase [33]. Cahoonet al. [32], in E. lagascae seed, identified a cytochromeP450, classified as CYP726A1, able to convert linoleicacid into 12,13-epoxyoctadeca-9-enoic acid (vernolicacid). CYP77A4 differs from this enzyme; indeed, itmetabolizes free fatty acids, whereas CYP726A1 meta-bolizes fatty acids incorporated into phosphatidylcho-line [32,45]. The physiological role of CYP77A4 isunlikely to be the production of fatty acid epoxides foraccumulation in seeds as, unlike E. lagascae and plantsbelonging to the Aesteraceae genera, such as Crepispalaestina, A. thaliana does not store fatty acid epox-ides. Cytochrome P450-dependent fatty acid oxidases inplants have been mainly investigated with regard tocutin synthesis [19]. Cutin consists of a biopolymer offatty acids belonging to the protective envelope ofplants: the cuticle [20]. Epoxides of fatty acids may rep-resent up to 60% of cutin monomers [46,47]. Cutin anal-ysis of A. thaliana has been performed recently [48], and18-hydroxy-9,10-epoxystearic acid was shown to beCYP77A4, an epoxy fatty acid-forming enzyme V. Sauveplane et al.728 FEBS Journal 276 (2009) 719–735 Journal compilation ª 2008 FEBS. No claim to original French government works[...]... al present in cutin CYP77A4 could account for its formation by introducing the oxygen between carbon 9 and 10 of oleic acid before incorporation of the monomer into the cutin In this context, it is interesting to note that inhibition studies allowed LeQueu et al [49] to demonstrate the involvement of a peroxygenase in the formation of cutin of corn The three epoxide derivatives from a-linolenic acid,... also the first report describing a cytochrome P450 which can catalyze the epoxidation of free fatty acids in plants Plants are sessile organisms and therefore rely on a battery of defense chemicals for survival To produce these chemical defenses, they have developed a complex metabolic network using the diversified catalytic properties of cytochrome P450 enzymes [36] Lipid metabolism is a major player in. .. that CYP77A4 can catalyze the in vitro production of the di-epoxide derivative of linoleic acid Interestingly, biochemical studies performed with mouse liver microsomes showed that this di-epoxide, produced during the oxidation of linoleic acid by cytochrome P450, was then converted to cyclic tetrahydrofurans after hydrolysis by epoxide hydrolases [66] In analogy, it would be very interesting to investigate... of hydroxylauric acids produced by CYP77A4 Fig S3 Lineweaver–Burk reciprocal plot of oxidation of fatty acids by CYP77A4 Fig S4 Fragmentation patterns of the derivatives of hydroxyoleic and epoxystearic acids produced by CYP77A4 Fig S5 Fragmentation patterns of the derivatives of the epoxides of linoleic acid produced by CYP77A4 Fig S6 Fragmentation patterns of the derivatives of the epoxides of linolenic... substrates were dissolved in ethanol which was evaporated before the addition of microsomes into the glass tube Resolubilization of the substrates was confirmed by measuring the radioactivity of the incubation media The enzymatic activities of CYP77A4 from transformed yeast or Arabidopsis microsomes were determined by following the formation rate of the metabolites The standard assay (0.1 mL) contained 20 mm... investigate the cyclization of di-epoxide derivatives of linoleic acid after hydrolysis by plant epoxide hydrolases, because the resulting tetrahydrofurans could represent a novel class of plant oxylipins In conclusion, we have described the first biochemical characterization of a member of the CYP77 family, and have shown that CYP77A4 is capable of epoxidizing, in vitro, unsaturated C18 fatty acids This is. .. by CYP77A4, have been shown to confer resistance of rice against rice blast disease [50] This indicates a possible involvement of CYP77A4 in plant defense events As discussed below, diol derivatives of fatty acids also participate in plant defense The presence of an epoxide hydrolase in A thaliana was first reported by Kiyosue et al [51] Furthermore, a putative epoxide hydrolase is co-expressed with CYP77A4. .. analysis, which revealed the presence of five metabolites The mass spectra are given in Fig S2 The regioselectivity of CYP77A4 was determined on the basis of the peak area of each metabolite detected by GC Metabolites of oleic acid For the analysis of the products generated by recombinant CYP77A4 on incubation with oleic acid, the metabolite of peak 2 (Fig 2B) was eluted from silica with 10 mL of diethyl... After the addition of adenine nucleotides on each side of the PCR product by an additional step with Taq polymerase (10 min at 72 °C), the purified PCR product was cloned into PCRII TOPO vector (Invitrogen, Carlsbad, CA, USA), and transferred to the pYeDP60 vector using the BamHI and KpnI restriction sites The sequence was verified by DNA sequencing after the cloning step in the PCRII TOPO vector Heterologous... Epoxyoctadecanoic acids in plant cutins and suberins Phytochemistry 12, 1721–1735 Matzke K & Riederer M (1990) The composition of the cutin of the caryopses and leaves of Triticum aestivum L Planta 182, 461–466 Bonaventure G, Beisson F, Ohlrogge J & Pollard M (2004) Analysis of the aliphatic monomer composition of polyesters associated with Arabidopsis epidermis: occurrence of octadeca-cis-6,cis-9-diene-1,18-dioate . Arabidopsis thaliana CYP77A4 is the first cytochrome P450 able to catalyze the epoxidation of free fatty acids in plants Vincent Sauveplane1,. understanding of the physiological role of epoxides of fatty acids in plants. In addition to cytochromes P450, two distinct types of plant enzyme, unrelated to cytochrome
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