Arabidopsis thaliana CYP77A4 is the first cytochrome P450 able to catalyze the epoxidation of free fatty acids in plants Vincent Sauveplane 1 , Sylvie Kandel 2 , Pierre-Edouard Kastner 1 ,Ju ¨ rgen Ehlting 1 , Vincent Compagnon 1 , Danie ` le Werck-Reichhart 1 and Franck Pinot 1 1 Institut de Biologie Mole ´ culaire des Plantes, University of Strasbourg, France 2 Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA Fatty acid-oxidizing enzymes have been the subject of an increasing number of studies in all organisms, as the products of their reactions exhibit fundamental biological activities [1–3]. Among these oxidases, cyto- chromes P450 play a prominent role. For example, in animals, arachidonic acid (C 20:4 ) is oxidized through the cytochrome P450 pathway, leading to the produc- tion of hydroxylated and epoxidized derivatives [4–6]. The cytochrome P450 superfamily represents a highly diversified set of heme-containing proteins found in bacteria, fungi, animals and plants [7]. In animals, members of the CYP4A gene subfamily mainly cata- lyze the formation of x- and x-1-hydroxyl derivatives of fatty acids. The regulation of some CYP4A enzymes Keywords cytochrome P450; defense; epoxide; fatty acid; plant Correspondence F. Pinot, IBMP-CNRS UPR 2357, Institut de Botanique, 28 rue Goethe, F-67083 Strasbourg Cedex, France Fax: +33 3 90 24 19 21 Tel: +33 3 90 24 18 37 E-mail: franck.pinot@ibmp-ulp.u-strasbg.fr (Received 4 September 2008, revised 20 November 2008, accepted 26 November 2008) doi:10.1111/j.1742-4658.2008.06819.x An approach based on an in silico analysis predicted that CYP77A4, a cytochrome P450 that so far has no identified function, might be a fatty acid-metabolizing enzyme. CYP77A4 was heterologously expressed in a Saccharomyces cerevisiae strain (WAT11) engineered for cytochrome P450 expression. Lauric acid (C 12:0 ) was converted into a mixture of hydroxy- lauric acids when incubated with microsomes from yeast expressing CYP77A4. A variety of physiological C 18 fatty acids were tested as poten- tial substrates. Oleic acid (cis-D 9 C 18:1 ) was converted into a mixture of x-4- to x-7-hydroxyoleic acids (75%) and 9,10-epoxystearic acid (25%). Linoleic acid (cis,cis-D 9 ,D 12 C 18:2 ) was exclusively converted into 12,13-epoxyocta- deca-9-enoic acid, which was then converted into diepoxide after epoxida- tion of the D 9 unsaturation. Chiral analysis showed that 9,10-epoxystearic acid was a mixture of 9S ⁄ 10R and 9R ⁄ 10S in the ratio 33 : 77, whereas 12,13-epoxyoctadeca-9-enoic acid presented a strong enantiomeric excess in favor of 12S ⁄ 13R, which represented 90% of the epoxide. Neither stearic acid (C 18:0 ) nor linolelaidic acid (trans,trans- D 9 ,D 12 C 18:2 ) was metabolized, showing that CYP77A4 requires a double bond, in the cis configuration, to metabolize C 18 fatty acids. CYP77A4 was also able to catalyze the in vitro formation of the three mono-epoxides of a-linolenic acid (cis,cis,cis-D 9 , D 12 ,D 15 C 18:3 ), previously described as antifungal compounds. Epoxides gen- erated by CYP77A4 are further metabolized to the corresponding diols by epoxide hydrolases located in microsomal and cytosolic subcellular frac- tions from Arabidopsis thaliana. The concerted action of CYP77A4 with epoxide hydrolases and hydroxylases allows the production of compounds involved in plant–pathogen interactions, suggesting a possible role for CYP77A4 in plant defense. Abbreviation EET, epoxyeicosatrienoic acid. FEBS Journal 276 (2009) 719–735 Journal compilation ª 2008 FEBS. No claim to original French government works 719 by peroxisome proliferator-activated receptors points to 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 effect of x-hydroxy fatty acids in different physiological pro- cesses [10–13] have suggested that x-hydroxylation cannot be considered only as a step leading to catabo- lism. The epoxidation of polyunsaturated fatty acid double bonds, particularly of arachidonic acid, has generated much interest because of the biological activ- ities of the resulting metabolites [14,15]. These epoxi- dation reactions of C 20:4 are catalyzed by members of the CYP2C subfamily and by the CYP2J2 isoform [6,16,17]. Human CYP4F8 and CYP4F12 isoforms are able to epoxidize docosahexaenoic acid (C 22:6 ) [18]. In plants, fatty acids are also metabolized by cyto- chrome P450-dependent oxygenases [19], and it is possible to distinguish x-hydroxylases and in-chain hydroxylases that attack the terminal and subtermi- nal positions, respectively. So far, the majority of work has addressed x-hydroxylases mainly repre- sented in CYP86 and CYP94 families [19]. Their involvement in the synthesis of cutin, a protective biopolymer of fatty acids cross-linked by ester bonds [20], has been established [21,22]. Studies of LCR (LACERATA) and att1 (aberrant induction of type three genes), the first Arabidopsis thaliana mutants with alterations in the coding sequence of CYP86A8 and CYP86A2, respectively, have also shown that x-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 fatty acid was first demonstrated in spinach leaves more than three decades ago [20,23], a cytochrome P450 able to epoxidize fatty acids is still poorly documented in plants. Biochemical studies performed with unsatu- rated analogues of lauric acid (C 12:0 ) clearly demon- strated the existence in plants of a cytochrome P450 able to epoxidize the double bonds of fatty acids. The terminal olefin 11-dodecenoic acid is converted into 11,12-epoxylauric acid by a cytochrome P450 in Vicia sativa microsomes [24]. The epoxidation of unsaturated analogues of lauric acid by cytochrome P450 was also reported in microsomes from Jerusalem artichoke [25,26], as well as in microsomes from wheat [27]. However, none of the enzymes implicated in these reactions have been characterized and, to date, no cytochrome P450 able to epoxidize free fatty acids has been identified in plants. The epoxidation of physiolog- ical substrates, such as oleic acid (cis-D 9 C 18:1 ) and lino- leic acid (cis,cis-D 9 ,D 12 C 18:2 ), has been reported in Vicia faba [28] and Glycine max [29]. However, these reactions were not catalyzed by cytochrome P450, but rather by peroxygenases, which are hydroperoxide- dependent fatty acid epoxidases. Recently, studies of a peroxygenase purified from oat have demonstrated that this enzyme is deeply buried in microsomes or in lipid droplets [30]. Lee et al. [31] identified a non-heme di-iron enzyme, a ‘desaturase-like’ protein, able to transform linoleic acid into 12,13-epoxyoctadeca-cis-9- enoic acid (vernolic acid). This compound can make up 50–90% of total fatty acids in seed oil of certain Euphorbiaceae, such as Euphorbia lagascae [32]. In this plant, the enzyme involved in its production was described recently [32]. This enzyme, classified as CYP726A1, does not epoxidize free fatty acids, but fatty acids bound to phosphatidylcholine [32]. A new approach, based on an in silico analysis of publicly available transcriptome data, has been devel- oped recently to map cytochrome P450 genes onto spe- cific metabolic pathways [33]. This analysis identifies metabolic genes that are co-expressed with a given bait P450 during plant development, on stress and hormone treatment, and in mutant wild-type comparisons. Based on the functional annotation of co-expressed genes, a metabolic pathway in which the bait P450 may act is predicted. This approach suggested that CYP77A4 could be involved in fatty acid metabolism as it is developmentally co-expressed across hundreds of biological samples with several characterized enzymes involved in lipid metabolism. The most simi- larly expressed genes are CYP86A8 encoding a fatty acid x-hydroxylase, a putative epoxide hydrolase, several genes encoding enzymes involved in the synthesis of fatty acids in plastids, including the stearoyl acyl carrier protein desaturase SSI2, and the plastidic long-chain acyl-CoA synthetase LACS9 (for a complete list of co-expressed genes, see http://www-ibmp.u-strasbg.fr/ ~CYPedia/CYP77A4/CoExpr_CYP77A4_Organs.html). In this work, we report the heterologous expression and functional characterization of CYP77A4. Substrate specificity and catalytic properties were explored using recombinant CYP77A4 expressed in an engineered yeast strain. Our study confirms that this enzyme is a fatty acid-metabolizing enzyme. We show that CYP77A4 is able to catalyze, in vitro, the epoxidation of physiological unsaturated fatty acids. Our work also shows that the epoxides generated can be further hydrolyzed to the corresponding diols by epoxide hydrolases present in subcellular fractions of A. thali- ana. Thus, CYP77A4 from A. thaliana, described in this work, is the first cytochrome P450 able to catalyze free fatty acid epoxidation, identified in plants. Its physiological significance remains to be established CYP77A4, 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 works and will be assessed by future studies of A. thaliana mutated in the coding sequence of CYP77A4. Results Selection, cloning and expression of CYP77A4 An approach based on an in silico analysis predicted that CYP77A4 could be involved in fatty acid metabo- lism [33]. The coding sequence of CYP77A4 was amplified by PCR from a cDNA library of Arabidopsis and 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. Enzymatic characterization of CYP77A4 was carried out employ- ing microsomes from the yeast strain WAT11 trans- formed with the plasmid pYeDP60 [34] containing the coding sequence of CYP77A4. WAT11 over-expresses a plant P450 reductase in order to optimize electron transfer during catalysis and probably to increase the stability of the expressed P450. Furthermore, there are only three cytochromes P450 encoded by the yeast gen- ome. They are either not expressed or expressed at a negligible level in the growth conditions used here, and none is able to metabolize fatty acids, ensuring that the metabolism described here results from enzymatic reactions catalyzed by CYP77A4 [34]. After micro- somal membrane isolation from the CYP77A4-trans- formed yeasts, the level of expression of the enzyme was 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 the microsomal preparation used in our experiments was 0.1 nmolÆmg )1 protein (Fig. S1). No absorbance at 450 nm and no enzymatic activity with the substrates tested were detected in microsomes from yeast trans- formed with a void plasmid under the same growth conditions. Metabolism of lauric acid by CYP77A4 To validate the hypothesis of CYP77A4 being a fatty acid-metabolizing enzyme, we incubated radiolabeled lauric acid (C 12:0 ) with microsomes from yeast express- ing CYP77A4. The resolution of reaction products was performed by directly loading the incubation medium onto 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 large peak of radioactivity was detected after 20 min of incu- bation (peak 1, Fig. 1B). It was not formed in the absence of NADPH (Fig. 1A), with microsomes from yeast transformed with a void plasmid (Fig. 1C) or with boiled microsomes (Fig. 1D). Taken together, these results demonstrate the involvement of CYP77A4 in the formation of this radioactive peak. Metabolites from this peak were purified, derivatized and subjected to GC ⁄ MS analysis (Experimental procedures). The mass spectrum of the derivatized metabolite 1 (Fig. S2) showed ions at m ⁄ z (relative intensity, %) values of 73 (41%) [(CH 3 ) 3 Si + ], 75 (23%) [(CH 3 ) 2 Si + =O], 117 (100%), 255 (15%) (M-47) [loss of methanol from the (M-15) fragment], 271 (3%) (M-31) (loss of OCH 3 from the methyl ester), 287 (6%) (M-15) (loss of a methyl from the trimethylsilyl group). This fragmentation pattern 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 ions at m ⁄ z (relative intensity, %) values of 73 (70%) [(CH 3 ) 3 Si + ], 75 (30%) [(CH 3 ) 2 Si + =O], 131 (100%), 255 (12%) (M-47) [loss of methanol from the (M-15) fragment], 271 (4%) (M-31) (loss of OCH 3 from the methyl ester), 273 (51%), 287 (2%) (M-15) (loss of a methyl from the trimethylsilyl group). This fragmen- tation pattern is characteristic of the derivative of 10-hydroxylauric acid ( x-2) (M = 302 gÆmol )1 ). The mass spectrum of derivatized metabolite 3 (Fig. S2) showed ions at m ⁄ z (relative intensity, %) values of 73 (75%) [(CH 3 ) 3 Si + ], 75 (31%) [(CH 3 ) 2 Si + =O], 145 (100%), 255 (11%) (M-47) [loss of methanol from the (M-15) fragment], 259 (59%), 271 (3%) (M-31) (loss of OCH 3 from the methyl ester), 287 (2%) (M-15) (loss of a methyl from the trimethylsilyl group). This fragmen- tation pattern is characteristic of the derivative of 9-hydroxylauric acid (x-3) (M = 302 gÆmol )1 ). The mass spectrum of derivatized metabolite 4 (Fig. S2) showed ions at m ⁄ z (relative intensity, %) values of 73 (68%) [(CH 3 ) 3 Si + ], 75 (28%) [(CH 3 ) 2 Si + =O], 159 (100%), 245 (68%), 255 (9%) (M-47) [loss of methanol from the (M-15) fragment], 271 (4%) (M-31) (loss of OCH 3 from the methyl ester), 287 (2%) (M-15) (loss of a 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 mass spectrum of derivatized metabolite 5 (Fig. S2) showed ions at m ⁄ z (relative intensity, %) values of 73 (97%) [(CH 3 ) 3 Si + ], 75 (39%) [(CH 3 ) 2 Si + =O], 173 (100%), 231 (71%) 255 (11%) (M-47) [loss of methanol from the (M-15) fragment], 271 (4%) (M-31) (loss of OCH 3 from the methyl ester), 287 (5%) (M-15) (loss of a methyl from the trimethylsilyl group). This fragment- ation pattern is characteristic of the derivative of 7-hydroxylauric acid (x-5) (M = 302 gÆmol )1 ). Their identification revealed that the reaction product is com- posed of a mixture of five different in-chain hydroxyl- ation products of lauric acid, which is predominantly V. Sauveplane et al. CYP77A4, an epoxy fatty acid-forming enzyme FEBS Journal 276 (2009) 719–735 Journal compilation ª 2008 FEBS. No claim to original French government works 721 hydroxylated on the x-1 position. When oxidizing lauric acid, 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, K m,app and V max,app values of 172 ± 13 lm and 117 ± 5 nmolÆmin )1 Ænmol )1 P450, respectively (Fig. S3). The x-1 position of lauric acid corresponds to carbon 11 of physiological C 18 fatty acids, closely located near the double bonds of oleic, linoleic and a-linolenic acids. We therefore tested these different unsaturated fatty acids as potential substrates. Metabolism of oleic acid by CYP77A4 We first incubated mono-unsaturated oleic acid (C 18:1 ). The radiochromatograms obtained after reso- lution of the reaction products on TLC are presented in Fig. 2. Incubation was carried out in the absence (Fig. 2A) or presence (Fig. 2B–D) of NADPH. Two new peaks of radioactivity (peak 1 and peak 2, Fig. 2B) were detected; their formation required the presence of NADPH in the incubation. They were also not formed on incubation with microsomes from yeast transformed with a void plasmid (Fig. 2C) or with boiled microsomes (Fig. 2D). Metabolites from peak 1 were purified, derivatized and subjected to GC ⁄ MS analysis. The mass spectrum of derivatized metabolite 1 (Fig. S4) showed ions at m ⁄ z (relative intensity, %) values of 73 (100%) [(CH 3 ) 3 Si + ], 75 (58%) [(CH 3 ) 2 Si + =O], 337 (9%) (M-47) [loss of methanol from the (M-15) fragment], 353 (2%) (M-31) (loss of OCH 3 from the methyl ester), 369 Fig. 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 l M [1- 14 C]lauric acid in the absence (A) or presence (B) of NADPH. Incubations were performed at 27 °C and contained 20 pmol of CYP77A4. They were stopped after 30 min by the addition of 20 lL of acetonitrile (containing 0.2% acetic acid) and directly spotted onto TLC. Peak S, lauric acid; peak 1, mixture of 11-, 10-, 9-, 8- and 7-hydroxylauric acids. 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). 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 trimethylsilyl group) and 384 (2%) (M). The mass spectrum also showed ions at 159 (63%) and 327 (7%), resulting from cleavage on both sides of the hydroxyl function carrying the trimethylsilyl group. This fragmentation pattern is characteristic of the derivative of 14-hy- droxyoleic acid (x-4) (M = 384 gÆmol )1 ). The mass spectrum of derivatized metabolite 2 (Fig. S4) showed ions at m ⁄ z (relative intensity, %) values of 73 (100%) [(CH 3 ) 3 Si + ], 75 (50%) [(CH 3 ) 2 Si + =O], 337 (11%) (M-47) [loss of methanol from the (M-15) fragment], 369 (3%) (M-15) (loss of a methyl from the trimethylsilyl group). The mass spectrum also showed ions at 173 (34%) and 313 (18%), resulting from cleavage on both sides of the hydroxyl function carrying the trimethylsilyl group. This fragmentation pattern is characteristic of the derivative of 13-hy- droxyoleic acid (x-5) ( M = 384 gÆmol )1 ). The mass spectrum of derivatized metabolite 3 (Fig. S4) showed ions at m ⁄ z (relative intensity, %) values of 73 (48%) [(CH 3 ) 3 Si + ], 75 (12%) [(CH 3 ) 2 Si + =O], 337 (3%) (M- 47) [loss of methanol from the (M-15) fragment], 353 (1%) (M-31) (loss of OCH 3 from the methyl ester), 369 (0.5%) (M-15) (loss of a methyl from the trim- ethylsilyl group). The mass spectrum also showed ions at 187 (100%) and 299 (4%), resulting from cleavage on both sides of the hydroxyl function carrying the trimethylsilyl group. This fragmentation pattern is characteristic of the derivative of 12-hydroxyoleic acid (x-6) (M = 384 gÆmol )1 ). The mass spectrum of derivatized metabolite 4 (Fig. S4) showed ions at m ⁄ z (relative intensity, %) values of 73 (35%) [(CH 3 ) 3 Si + ], 75 (14%) [(CH 3 ) 2 Si + =O], 337 (3%) (M-47) [loss of methanol from the (M-15) fragment], 353 (1%) (M-31) (loss of OCH 3 from the methyl ester), 369 (1%) (M-15) (loss of a methyl from the trimethylsilyl group) and 384 (0.5%) (M). The mass spectrum also showed ions at 201 (1%) and 285 (100%), resulting Fig. 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 l M [1- 14 C]oleic acid in the absence (A) or presence (B) of NADPH. Incubations were performed at 27 °C and contained 20 pmol of CYP77A4. They were stopped after 30 min by the addition of 20 lL of acetonitrile (containing 0.2% 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 enzyme FEBS Journal 276 (2009) 719–735 Journal compilation ª 2008 FEBS. No claim to original French government works 723 from cleavage on both sides of the hydroxyl function carrying the trimethylsilyl group. This fragmentation pattern 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 after purification and derivatization revealed that CYP77A4 hydroxylates oleic acid with the following regioselectivity: x-7 (58%), x-6 and x-5 (30%), x-4 (12%). The metabolite from peak 2 displayed the TLC mobility expected for 9,10-epoxystearic acid, and was indeed identified as 9,10-epoxystearic acid by GC ⁄ MS analysis (Fig. S4). For substrate oxidation, we determined, by kinetic studies, K m,app and V max,app values of 84 ± 23 lm and 26 ± 5 nmolÆmin )1 Ænmol )1 P450, respectively (Fig. S3). We determined the ste- reochemistry of this epoxide after purification and analysis by HPLC using a chiral column. The radio- chromatogram of Fig. 3 shows that it is a mixture of the two enantiomers, 9S ⁄ 10R and 9R ⁄ 10S, in the ratio 33 : 77, respectively. Metabolism of linoleic acid by CYP77A4 Figure 4 shows the radioactivity profiles obtained after incubation of linoleic acid (C 18:2 ) with microsomes from yeast expressing CYP77A4. The addition of NADPH 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) After incubation of oleic acid with microsomes from yeast expressing CYP77A4, the 9,10-epoxystearic produced (peak 2, Fig. 2B) was purified 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 0.8 mLÆmin )1 . (B) Structures of the enantiomers. Fig. 4. Radiochromatographic resolution by TLC of metabolites gen- erated in incubations of linoleic acid with microsomes from yeast expressing CYP77A4. Microsomes were incubated with 100 l M [1- 14 C]linoleic acid in the absence (A) or presence (B) of NADPH. Incubations were performed at 27 °C and contained 20 pmol of CYP77A4. They were stopped after 30 min by the addition of 20 lL of acetonitrile (containing 0.2% acetic acid) and directly spotted onto TLC. Peak S, linoleic acid; peak 1, 9,10:12,13-diepoxyoctadecanoic acid; peak 2, 12,13-epoxyoctadeca-9-enoic 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). 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 works tion 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 by CYP77A4, as it was not formed when the microsomes were from yeast transformed with a void plasmid (Fig. 4C) or were boiled (Fig. 4D). This peak contains only one metabolite, which was identified by GC ⁄ MS analysis (Fig. S5) after purification reaction in acidic methanol and derivatization as 12,13-epoxyoctadeca- 9-enoic acid (vernolic acid), resulting from the epoxi- dation of the D 12 double bond. The kinetic parameters from the reaction of substrate oxidation are K m,app = 61±3 lm and V max,app = 13 ± 0.3 nmolÆmin )1 Ænmol )1 P450 (Fig. S3). Stereochemistry studies presented in Fig. 5 show that CYP77A4 possesses a strong enantio- specificity: the epoxide formed is a mixture of 12S ⁄ 13R and 12R ⁄ 13S in the ratio 90 : 10, thus presenting a strong enantiomeric excess in favor of the 12S ⁄ 13R conformation. 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 able to catalyze its formation in incubations with purified 12,13-epoxyoctadeca-9-enoic acid (data not shown). Metabolism of a-linolenic acid by CYP77A4 The incubation of a-linolenic acid (C 18:3 ) with micro- somes from yeast expressing CYP77A4 led to the formation of one major radioactive peak, as shown on the radiochromatogram in Fig. 6 (peak 2, Fig. 6B). It results from a reaction catalyzed by CYP77A4, as it requires the presence of NADPH and is not formed with microsomes from yeast transformed with a void plasmid (Fig. 6C) or on incubation with boiled micro- somes (Fig. 6D). The shape of this peak suggests that it contains more than one metabolite. After purifica- tion, acidic treatment and derivatization, GC ⁄ MS analysis showed that it was indeed a mixture of the three 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%) [(CH 3 ) 3 Si + ], 75 (14%) [(CH 3 ) 2 Si + =O], 439 (4%) (M-31) (loss of OCH 3 from the methyl ester), 455 (1%) (M-15) (loss of a methyl from the trimethylsilyl group), 470 (0.5%) (M). The mass spectrum also showed ions at 171 (40%) and 299 (44%), resulting from the cleavage between two hydroxyls carrying the trimethylsilyl group generated by hydrolysis in perchloric acid. This fragmentation pattern is characteristic of the derivative after acidic hydrolysis of 12,13-epoxyoctadeca-9,15-dienoic acid (M = 470 gÆmol )1 ) which represents 87% of the metabolites. The mass spectrum of derivatized meta- bolite 2 (Fig. S6) showed ions at m ⁄ z (relative inten- sity, %) values of 73 (100%) [(CH 3 ) 3 Si + ], 75 (17%) [(CH 3 ) 2 Si + =O], 439 (2%) (M-31) (loss of OCH 3 from the methyl ester), 455 (0.5%) (M-15) (loss of a methyl from the trimethylsilyl group), 470 (1%) (M). The mass spectrum also showed ions at 211 (11%) and 259 (81%), resulting from the cleavage between two hydroxyls carrying the trimethylsilyl group generated by hydrolysis in perchloric acid. This fragmentation pattern is characteristic of the derivative after acidic hydrolysis 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%) [(CH 3 ) 3 Si + ], 75 (21%) [(CH 3 ) 2 Si + =O], 439 (3%) (M-31) (loss of OCH 3 from the methyl ester), 455 (0.5%) (M-15) (loss of a methyl from the trimethylsilyl group), 470 (4%) (M). The mass spectrum also showed ions at 131 (67%) and 339 (20%), resulting from the cleavage between two hydroxyls carrying the trimethylsilyl group generated by hydrolysis in perchloric acid. This fragmentation pattern is characteristic of the derivative after acidic hydrolysis of 15,16-epoxyoctadeca-9,12-dienoic acid Fig. 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 yeast expressing CYP77A4, the 12,13-epoxyoctadeca-9-enoic acid produced (peak 2, Fig. 4B) was purified, methylated and subjected to chiral HPLC analysis with 100% heptane at a flow rate of 0.5 mLÆmin )1 . (B) Structures of the enantiomers. V. Sauveplane et al. CYP77A4, an epoxy fatty acid-forming enzyme FEBS 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 of substrate oxidation are K m,app =29±4lm and V max,app = 38 ± 2 nmolÆmin )1 Ænmol )1 P450 (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 with microsomes or cytosol from A. thaliana. Microsomes (350 lg pro- tein) or cytosol (600 lg protein) from A. thaliana was incubated with 100 l M of 12,13-epoxyoctadeca-9-enoic acid for 20 min at 27 °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 performed with 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-enoic acid. 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 yeast expressing CYP77A4. Microsomes were incubated with 100 lm [1- 14 C]a-linolenic acid in the absence (A) or presence (B) of NADPH. Incubations were performed at 27 °C and contained 20 pmol of CYP77A4. They were stopped after 30 min by the addition of 20 lL of acetonitrile (containing 0.2% acetic acid) and directly spotted onto TLC. Peak S, a-linolenic acid; peak 1, non-identified; peak 2, mixture of 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 from yeast 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 works Requirements for CYP77A4 activity To check the importance of the double bond for CYP77A4 activity, we tested as potential substrates stearic acid (C 18:0 ), which is saturated, and linolelaidic acid, which is linoleic acid containing trans double bonds. No metabolites were detected on TLC after incubation of radiolabeled C 18:0 with microsomes from yeast expressing CYP77A4 (data not shown). To test linolelaidic 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 concentration of unlabeled linolelaidic acid, and did not detect any inhibition of epoxidation of linoleic acid (data not shown). In a second experiment, we ran GC ⁄ MS anal- ysis after the incubation of linolelaidic acid with yeast microsomes expressing CYP77A4, and did not detect any metabolite (data not shown). Together, this shows that CYP77A4 requires the presence of unsaturation to metabolize C 18 fatty acids; furthermore, unsatura- tion must be in the cis configuration. Hydrolysis of vernolic acid in microsomes and cytosol from A. thaliana In order to test whether the metabolites generated by CYP77A4 were end products or could be substrates of other enzymatic systems (i.e. epoxide hydrolase) from A. thaliana, we purified vernolic acid that was produced by CYP77A4 (peak 2, Fig. 4B). This epoxide was sub- sequently incubated with microsomes isolated from A. thaliana seedlings. The results are presented in Fig. 7. A peak of radioactivity (peak 1, Fig. 7A) was detected after resolving the products of reaction on TLC. No metabolite was formed if the microsomes were boiled before incubation (Fig. 7B). This demonstrates the enzy- matic origin of the metabolite from peak 1. The mass spectrum of this derivatized metabolite (Fig. S7) showed ions at m⁄ z (relative intensity, %) values of 73 (100%) [(CH 3 ) 3 Si + ], 75 (17%) [(CH 3 ) 2 Si + =O], 457 (2%) (M-15) (loss of a methyl from the trimethylsilyl group). The mass spectrum also showed ions at 173 (40%) and 299 (8%), resulting from the cleavage between two hydroxyls carrying the trimethylsilyl group. This frag- mentation pattern is characteristic of the derivative of 12,13-dihydroxyoctadeca-9-enoic acid (M = 472 gÆ mol )1 ). The same results were obtained when incubation was 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 the corresponding diol by microsomal and cytosolic epoxide hydrolase (Fig. 8). These epoxide hydrolases can also convert epoxides from C 18:3 into the corresponding diols (data not shown). Discussion A new approach, based on an in silico analysis of pub- licly available transcriptome data (http://www-ibmp. u-strasbg.fr/~CYPedia/), has been developed recently for the mapping of cytochromes P450 onto specific metabolic pathways based on large-scale co-expression analysis [33]. This approach showed that CYP77A4 was co-regulated across 167 developmental samples (cover- ing more than 400 publicly available Affymetrix micro- array data sets) with a set of enzymes implicated in fatty acid metabolism. Although co-expression correlations were relatively low compared with other co-expressed genes acting in a common pathway [33], with Pearson correlation coefficients not exceeding 0.75, it was strik- ing that the top eight co-expressed genes with CYP77A4 have been functionally characterized as being involved in fatty acid metabolism (http://www-ibmp.u-strasbg.fr/ ~CYPedia/CYP77A4/CoExpr_CYP77A4_Organs.html). We thus found it worthwhile to test experimentally the hypothesis generated by this bioinformatic approach and to elucidate the physiological role of CYP77A4, also because no function has been reported for members belonging to this cytochrome P450 family to date. Heterologous expression of CYP77A4 in an engineered strain of yeast, and incubations of a diverse set of fatty acids with yeast microsomes, allowed us to confirm the capacity of this newly characterized P450 to metabolize fatty acids, highlighting the predictive power of the in silico co-expression analysis. Based on phylogenetic reconstructions [36], CYP77A4 belongs to the CYP71 Fig. 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 enzyme FEBS Journal 276 (2009) 719–735 Journal compilation ª 2008 FEBS. No claim to original French government works 727 clan and, within this clan, forms a basal clade with the CYP89, CYP753 and CYP752 families, none of which has been functionally characterized. In contrast, most functionally characterized plant fatty acid hydroxylases belong to the divergent CYP86 clan (including CYP86 and CYP94 families). Both the CYP71 and CYP86 clans appear to have evolved independently within the green plant lineage, as they are evolutionary separated by fam- ilies that pre-date land plant evolution [36]. Thus, a function of CYP77A4 as a fatty acid-metabolizing enzyme would not have been predicted based on phylo- genetic reconstructions, again highlighting the power of the co-expression analysis approach, which is indepen- dent of sequence or structural similarities. On the model substrate lauric acid, CYP77A4 hydroxylated predominantly the x-1 carbon, which cor- responds to a carbon in the environment of unsaturation in oleic (C 18:1 ), linoleic (C 18:2 ) and a-linolenic (C 18:3 ) acids, the common physiological C 18 fatty acids in plants. We therefore assayed these compounds as poten- tial substrates and demonstrated that CYP77A4 was able to produce, in vitro, epoxide derivatives of these fatty acids. Investigations on the members of the CYP2 family in animals have previously demonstrated that the regioselectivity and enantioselectivity of epoxidation are cytochrome P450 dependent [37,38]. For CYP77A4, the requirement of unsaturation, in the cis configuration, together with the regioselectivity and enantioselectivity observed, probably reflect steric constraints on the sub- strate in the active site. The fact that C 18:2 is epoxidized first exclusively on the D 12 unsaturated position, with strong enantiomeric excess (the epoxide formed is a mix- ture of 12S ⁄ 13R 12R ⁄ 13S in the ratio 90 : 10), shows that it is probably hindered in the active site, suggesting that it could be a physiological substrate. In animals, epoxides of arachidonic acid (C 20:4 ), formed by epoxidases (mainly belonging to the CYP2 family), are well documented. This is mainly a result of the large array of biological effects attributed to epoxyeicosatrienoic acids (EETs). For example, activa- tion of CYP epoxidases in endothelial cells is a key step in vasodilatation events [14]. EETs also play a major role in cell proliferation and angiogenesis via the 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 CYP2J2 have also been shown to be expressed in different tumor tissues [43,44]. Epoxides of fatty acids are less described in plants, and only a few biological activities have been attributed to them. The discovery of such activities in plants might help to understand the physi- ological role of CYP77A4. This lack of data could explain the small amount of information available today concerning the ability of plant enzymes to gener- ate epoxides of fatty acids, despite the fact that this type of reaction was described for the first time more than three decades ago [23]. Thus, the discovery of CYP77A4 carrying such activity opens the door not only for detailed biochemical characterizations, but also for an 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 P450, with epoxidase activity, have been described. The first, a peroxygenase, 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 fatty acids. The second, described by Lee et al. [31], is a non-heme di-iron enzyme, also named ‘desaturase-like’ enzyme. It thus appears that fatty acid epoxidation in plants can be facilitated by evolutionarily divergent sets of enzymes, further suggesting a pivotal role of these epoxides or derivatives thereof. CYP77A4, described in this work, catalyzed the oxy- gen incorporation into double bonds of oleic (C 18:1 ), linoleic (C 18:2 ) and a-linolenic (C 18:3 ) acids, but did not metabolize saturated stearic acid (C 18:0 ). Furthermore, it did not metabolize linolelaidic acid, which is the homolog of linoleic acid possessing two trans double bonds, not commonly found in natural fatty acids. These observations suggest that the physiological func- tion of CYP77A4 could be epoxidation of unsaturated C 18 fatty acids. This hypothesis is supported by in silico co-expression analysis, showing that CYP77A4 is co-regulated with a stearoyl acyl carrier protein desat- urase and a putative epoxide hydrolase [33]. Cahoon et al. [32], in E. lagascae seed, identified a cytochrome P450, classified as CYP726A1, able to convert linoleic acid into 12,13-epoxyoctadeca-9-enoic acid (vernolic acid). CYP77A4 differs from this enzyme; indeed, it metabolizes free fatty acids, whereas CYP726A1 meta- bolizes fatty acids incorporated into phosphatidylcho- line [32,45]. The physiological role of CYP77A4 is unlikely to be the production of fatty acid epoxides for accumulation in seeds as, unlike E. lagascae and plants belonging to the Aesteraceae genera, such as Crepis palaestina, A. thaliana does not store fatty acid epox- ides. Cytochrome P450-dependent fatty acid oxidases in plants have been mainly investigated with regard to cutin synthesis [19]. Cutin consists of a biopolymer of fatty acids belonging to the protective envelope of plants: 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], and 18-hydroxy-9,10-epoxystearic acid was shown to be CYP77A4, 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 Sauveplane 1 ,. 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