efficient epoxidation of unsaturated fatty acids by a ... · epoxidation of mono- and...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 265, No. 22, Issue of August 5, pp. 12887-12894.1990 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Efficient Epoxidation of Unsaturated Fatty Acids by a Hydroperoxide-dependent Oxygenase*

(Received for publication, January 26, 1990)

Elizabeth B16eS and Francis Schuberj From the Laboratoire d’Enzymologie Mol&&ire et Cell&ire (Centre National de la Recherche Scientifique URA 1182), Uniuersitt? Louis Pasteur, Znstitut de Botanique, 28 rue Goethe, 67000 Strasbourg, France and the ILaboratoire de Chimie Bioorganique (Centre National de la Recherche Scientifioue URA 1386), Facultk de Pharmacie, 74, route du Rhin, 6740iZllk;rch, France

. _

Detergent-solubilized and partially purified soybean peroxygenase was shown to actively catalyze, in the presence of alkylhydroperoxides as co-substrates, the epoxidation of mono- and polyunsaturated fatty acids such as oleic and linoleic acids. Octadecenoic acids were found to be better substrates than shorter monounsaturated fatty acids (C16:l or C14:1), but the position of their double bond (at positions 6,9, or 11) had little effect on the rates of epoxidation. The peroxygenase exhibits a strong stereospecificity since octadecenoic acids with double bonds in trans-configuration were not epoxidized at detectable rates. Oxidation of linoleic acid yielded the two positional monoepoxide isomers and, as the minor product, the diepoxide. An important regioselectivity was, however, observed in this case; i.e. the unsaturation at position 9,lO was epoxidized preferentially to that at 12,13. Oxidation of oleic acid in the presence of “O-labeled hydroperoxylinoleic acid revealed an incorporation of about 80% of the label into the epoxide ring. Products similar to those formed by the peroxygenase by epoxidation of unsaturated free fatty acids such as linoleic acid have been described as important metabolites (leukotoxins) in the defense of plants, e.g. in fungal agressions. This aspect underlines the physiological relevance of this new and potent catalytic activity of the peroxygenase.

We have recently reported the S-oxidation of alkylaryl sulfides by an unique membrane-associated oxygenase, occur- ring in higher plants, which is strictly hydroperoxide-dependent (B16e and Durst, 1986, 1987). This enzyme, solubilized from soybean microsomes and partially purified, catalyzes a highly stereoselective oxygen transfer from hydroperoxides to sulfides (B16e and Schuber, 1989). In the present study we provide evidence that this enzyme, which is closely related to the peroxygenase, a hemoprotein described earlier by Ishi- maru and Yamazaki (1977a, 1977b), is also able to epoxidize very efficiently mono- and polyunsaturated fatty acids such as oleic and linoleic acids.

Hydroperoxide-dependent epoxidations of olefins are known to be catalyzed by cytochrome P-450-dependent mixed function oxidases (MC Carthy and White, 1983) and by other hemoproteins such as hemoglobin and myoglobin (Ortiz de Montellano and Catalano, 1985; Catalan0 and Ortiz de Mon- tellano, 1987) and chloroperoxidase (Ortiz de Montellano et

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.&C. Section 1734 solely to indicate this fact.

$ To whom all correspondence should be addressed.

al., 1987). These epoxidations follow complex mechanism pathways involving ferry1 oxygen transfer to the olefin or to

In addition to its mechanism of action, this new and highly

its derived radical cation and protein-mediated co-oxidation.

potent epoxidase is of great interest because the reactions it

The balance between these different oxidative processes de- pends on the hemoprotein and on the nature of the substrates

catalyzes are of physiological relevance in higher plants. As

(Ortiz de Montellano et al., 1987; Catalan0 and Ortiz de Montellano, 1987). In addition, double bond epoxidations can

discussed before, the peroxygenase may contribute largely to

also be observed during prostaglandin H synthase turnover (Marnett, 1981; Panthananickal et al., 1983), and during

the oxidative metabolism of xenobiotics (B16e and Durst,

microsomal lipid peroxidation processes (Dix and Marnett, 1983); in these cases, however, and in contrast with the

1986, 1987). Moreover, as shown here for the first time with

foregoing reactions, the epoxidizing agents appear to be free peroxyl radicals derived from the fatty acid hydroperoxides

unsaturated fatty acids, this enzyme is also capable to parti-

(Dix and Marnett, 1981; Labhque and Marnett, 1988). In order to obtain a better understanding of the mode of action

cipate to the metabolization of endogenous substrates such as

of the soybean peroxygenase, in relationship to these afore- mentioned examples, we have addressed several key questions

linoleic acid leading to compounds structurally similar to

on the nature of the enzyme-catalyzed hydroperoxide-dependent epoxidation and on the oxidative species involved in such

metabolites which have been shown by other authors to be of

a reaction. We have determined: (i) the kinetics parameters, the regio- and stereoselectivity of the epoxidation reactions,

importance in the defense of plants (Kato et al., 1983a, 1983b;

and (ii) the origin of the oxygen atom incorporated into the epoxide. Our results indicate that the epoxidation does not occur through a free radical mechanism and establish unam-

1984). These latter aspects are also discussed.

biguously the existence for the peroxygenase of a distinct hydroperoxide-dependent epoxidase activity which involves enzyme-activated oxidative intermediate(s).

EXPERIMENTAL PROCEDURES

Materials-[1-“C]Oleic acid (55 mCi/mmol) and [I-“Cllinoleic acid (59 mCi/mmol) were purchased from CEN Saclav (Gif sur Yvette, France). Cumene hidroperoxide was from Rh&s Poulenc- Agrochimie (Lyon, France) and emulphogene BC-720 from GAF (Louvres, France). The unsaturated fatty acids were obtained from Aldrich (Strasbourg, France) and iV,O-bis(trimethylsilyl)trifluoroacetamide from Pierce-Europe (The Netherlands).

Preparation of Products-The epoxides were prepared according to standard procedures by oxidation of the fatty acids with m-chloro- perbenzoic acid. After purification, either as free acids or methyl esters, by thin layer chromatography on silica gel plates (60 Fzs4;

12887

12888 Epoxidation of Unsaturated Fatty Acids

Merck) they were analyzed by GC/MS.’ [‘80]13-Hydroperoxylinoleic acid was prepared by soybean lipoxygenase-catalyzed oxidation of linoleic acid under isO2 atmosphere as already reported (Blie and Durst, 1987) and purified on silica columns as described by Dix and Marnett (1985). [‘so] content of the hydroperoxide was determined by GC/MS analysis of the product obtained after reduction by NaBH.,, methylation with etheral diazomethane, and silylation with N,O-bis(trimethylsilyl)trifluoroacetamide. The atom percent excess lRO in the labeled hydroperoxide was found to be 98% as determined from the ratio of ion intensities at m/t 313 (MI’sOl-&HI,) and 311 (M[‘60]-C,Hn), or 227 (M[‘“O]-(CH&COOCH‘,) and 225 (M[160]- (CH,)COOCH,) relative to a standard samole of derivatized 1r60113- hydroperoxylinoleic acid. l-‘?-Labeled and unlabeled 13-hydroperoxylinoleic acids were prepared as above with soybean lipoxygenase (Type IV, Sigma) under normal atmosphere. The corresponding 13- hydroxylinoleic acids were obtained by reduction of the hydroperoxides with an excess of sodium borohydride in ethanol as described by Dix and Marnett (1985).

Analytical Methods-Capillary gas chromatography analyses of the methyl esters of the monounsaturated fatty acids and their epoxide derivatives were performed on a Carlo-Erba (Fractovap 4160) chro- matograph apparatus equipped with a Spectra Physics (SP 4270) integrator. The sample was injected directly into the OV-1 coated fused silica capillary column (25 m, 0.25-mm inner diameter; Inter- chim, Montlucon, France) at 60 “C and the analysis was performed (H2, 2 ml/min) with the following temperature program: 30 “C/min to 150 “C! followed by 5 “C/min to 240 “C. For the separation of the two monoepoxides of linoleic acid methyl esters, a fused silica capillary column (30 m, 0.25-mm inner diameter) coated with DB-Wax (J. W. Scientific) was used with the temperature program: 10 “C/min to 150 “C, followed by 3 “C/min to 196’C and i “C/min to 220 “C. GC/MS was uerformed on a LKB 9000s with ionizina enerw of 18 or 70 eV. The separations were carried out on a SE-30-“(J. W. Scientific) fused silica capillary column (30 m) operated between 100 and 270 “C at the rate of 3 “C/min.

Radioactivity was read on TLC plates by a Berthold TLC linear analyzer LB 283 and the peaks integrated by a data acquisition system LB 511. Spectrophotometric measurements were obtained with a Shimadzu model MPS-2000 spectrophotometer.

Enzyme Preparorion-Microsomes were prepared from soybean (Glycine man) seedlings and carefully washed to eliminate soluble peroxidases and lipoxygenases (Blee and Schuber, 1989). The peroxygenase was solubilized with emulphogene BC-720 and purified by ion exchange chromatography as described previously (Blee and Schuber, 1989). Its activity was routinely assayed by using aniline as substrate (Blee and Durst, 1987). In the present work we indicate (Fig. 2) the elution profiles of the peroxygenase and lipoxygenase isozymes on a CM-Sepharose column. The lipoxygenase activities were measured spectrophotometrically at 234 nm (Gaillard and Chan, 1980) in 0.1 M glycine-NaOH (pH 9.0) or in 0.1 M sodium acetate (pH 5.5) buffers. Due to the instrinsic instability of the solubilized enzyme, the preparation used in this work corresponds to the purest currently avail- able. It contains about 5-6 nmol of heme/mg of protein and revealed by electrophoresis under denaturing conditions (sodium dodecyl sulfate) a major band at 34 kDa on polyacrylamide gels. Protein concentrations were determined (Bradford, 1976) using bovine serum albu- min as standard.

&oxidation Assays-Oleic acid epoxidation was routinely measured by incubating (final volume: 0.1 ml) the enzymatic fraction (1 pg of protein), 20 pM [l-i4C]oleic acid (2 X lo5 dpm), and 100 pM cumene hydroperoxide or 13-hydroperoxylinoleic acid in Buffer A, i.e. 10 mM sodium acetate buffer (pH 5.5) containing 20% (v/v) glycerol and 0.1% emulphogene. After 5 min at 25 “C, the reaction was stopped by addition of 0.1 ml of acetonitrile containing 0.2% acetic acid. Then 0.1 ml of the sample was applied to silica gel TLC plates and developed in a diethyl ether/petroleum ether (40-60 ‘C)/ formic acid (50:50:1) solvent system (System A). Radioactivity of the bands corresponding to the epoxide (RF 0.62) and to the residual oleic acid (RF 0.84) was then determined.

Linoleic acid epoxidation was measured by incubating [1-“Cllino- leic acid (100 pM, 4 X lo5 dpm) in the presence of 100 pM cumene hydroperoxide or 13-hydroperoxylinoleic acid and peroxygenase fraction (5 pg of protein) under the same conditions as above. After 20 min at 25 “C, the mixture was acidified (pH 3-4) with HCl, extracted twice with 2 volumes of ethyl ether, and evaporated to dryness under

i The abbreviation used is: GC/MS, gas chromatography/mass spectrometry.

a stream of argon. After formation of the methyl esters with diazomethane, the reaction products were chromatographed on TLC developed in hexane/ethyl acetate (85:15). In addition to the methyl ester of the starting material (RF 0.72), two radioactive bands were detected which co-migrated with authentic monoepoxides (RF 0.55) and diepoxide (RF 0.35) methyl esters of linoleic acid. These bands were scrapped off and analyzed by GC and GC/MS as described above. The relative proportions of the two positional isomers of the monoepoxides, i.e. 12,13-epoxy-9-octadecenoate and 9,10-epoxy-12- octadecenoate, were obtained by GC using a carbowax-type capillary column. Under the chromatographic conditions given above, their average retention times were, respectively, 49.6 and 49.9 min. These monoepoxides were identified by GC/MS using the same column; their mass spectra were found identical to that of authentic standards and were in agreement with previously published data (Kleiman and Spencer, 1973; Ozawa et aZ., i986).

Euoxidation of mvristoleic. oalmitoleic. nalmitelaidic. oetrosilinic. . - *

and elaidic acids were measured by incubating 200 pM of each acid in the presence of enzyme (1 pg of protein) and 200 pM cumene hydroperoxide in Buffer A for 15 min at 25 ‘C. The reaction was stopped by acidification of the medium to pH 3-4, and the mixture was extracted immediately with ethyl ether (3 x 1 volume). After formation of the methyl esters with diazomethane, the samples were analyzed by GC as described above. Identity of each epoxide was confirmed by GC/MS.

Epoxidation of Oleic Acid in the Presence of fsO]13-Hydroperoxy- linoleic Acid-Freshly prepared peroxygenase (3-ml final volume, Buffer A, 0.15 mg of protein) was incubated in the presence of 2.6 mM oleic acid. The reaction was initiated by 3 mM [‘s0]13-hydroperoxylinoleic acid (in 200 ~1 of methanol). After 15 min at 25 ‘C, the reaction was stopped by 4 N HCl (pH 2-3) and the mixture extracted with diethyl ether. The extracts, dried over sodium sulfate, were evaporated with a stream of argon. Half of the extract was methylated by diazomethane and the residue was applied on TLC plates which were developed in hexane/ethyl acetate (85:15). The band which co- migrated with authentic 9,10-epoxyoctadecanoate methyl ester (RF 0.53) was eluted by ether. The isotope composition of the epoxide was determined by GC/MS as described above using the ratio of ion intensities of the fragments at 157 (M[‘“O]-(CH,),COOCH,) and 155 (M[‘“O]-(CH&COOCH,) or 201 (M[“O]-(CH&ZH,) and 199 (M[“O]-(CH&CH,). From the other half [‘so] 13-hydroxylinoleic acid was isolated and analyzed by GC/MS according to procedures described below.

Enzymatic TFUIWfOFmUtiOn of 13(S)-Hydroperoxy-[l -‘4C]linokic acid--The hydroperoxide (200 pM, 2.7 X 10” cpm) was incubated with 200 WM oleic acid and peroxygenase (0.5 rg of protein) in Buffer A (0.1 ml of final volume). After 20 min at 25 “C, the mixture was extracted twice with 2 volumes of ethyl acetate. The extracts were then concentrated under argon and analyzed by TLC using System A. The compound which co-migrated with authentic 13-hydroxylinoleic acid (RF 0.47) was scrapped off the plate eluted with ethyl acetate. After treatment with etheral diazomethane and derivatiza- tion with N,O-bis(trimethylsilyl)trifluoroacetamide, the resulting compound was analyzed by GC/MS (see above).

Stoichiometry of the Epoxidution Reaction-In parallel reactions either 13(S)-hydroperoxy-[1-“Cllinoleic acid (70 pM, 2.1 X 10s dpm) and oleic acid (70 pM) or 13(S)-hydroperoxylinoleic acid (70 pM) and [1-r4C]oleic acid (70 pM; 2 X lo6 dpm) were incubated in Buffer A (O.l-ml final volume) in the presence of peroxygenase (2.4 pg of protein). After 3 min at 25 “C!, the reaction mixtures were worked-up as above and the products separated by TLC using ethyl ether/ hexane/formic acid (70:30:1) as eluent. The relative proportions of 13-hydroxylinoleic acid and 9,10-epoxyoctadecanoate formed were obtained from the radio-thin layer chromatograms.

Determination of the Kinetic Parameters-Peroxygenase (0.5 rg of protein) in Buffer A (final volume: 0.1 ml) was incubated for 5 min at 25 “C with the following substrate concentrations: (i) [1-i4C]oleic acid (3-75 NM; 8 data points) and cumene hydroperoxyde (100 PM), (ii) 13-hydroperoxylinoleic acid (lo-250 pM; 8 data points) and [l- i4C]oleic acid (50 PM), and (iii) cumene hydroperoxyde (l-100 pM; 5 data points) and [l-‘4C]oleic acid (50 @M). After work-up (see above), reaction progress was estimated from radio-thin layer chromatograms of the reaction products and the kinetic parameters were determined by use of a nonlinear regression program.

Epoxidation of Unsaturated Fatty Acids 12889

RESULTS

Hydroperoxide-dependent Epoxidation of Unsaturated Fatty Acids by Peroxygenuse-Incubation of [1-14C]oleic acid with the detergent-solubilized and partially purified soybean peroxygenase, in the presence of 13(S)-hydroperoxylinoleic acid, led to the rapid formation of a single new radioactive product. On TLC it was more polar than the substrate and co-migrated with the corresponding epoxide (Fig. IA). If cumene hydroperoxide was used instead of the fatty acid hydroperoxide, the same reaction product was obtained. After isolation, this product was converted to the methyl ester with diazomethane and analyzed by GC/MS. The mass spectrum obtained was identical to that of authentic 9,10-epoxyoctadecanoate methyl ester, i.e. each spectrum featured a molecular ion at m/t 312 and prominent ions at 155, base peak (M-157, loss of . (CH&COOCH3), 199 (M-113, loss of . (CH&CHJ, 281 (M- 31, loss of .0CH3), and 294 (M-18, loss of H,O). This is in agreement with data published earlier (Ryhage and Sten- hagen, 1960; Kleinman and Spencer, 1973; Fahlstadius, 1988). Importantly, the formation of this epoxide was completely abolished when hydroperoxide was omitted from the incubation medium (Fig. 1B) or, in the presence of hydroperoxides, by using a boiled enzyme preparation (Fig. 1C). Moreover, when the enzymatic reaction was performed in the presence of antioxidants such as butylated hydroxyanisole or butylated hydroxytoluene (10 PM) no inhibition of the epoxidation of

FA

\, 1

0 20

b distance from origin ccm) 20

FIG. 1. Radio-thin layer chromatograms of the reaction products of [1-“Cloleic acid incubated with soybean peroxygenase. Labeled oleic acid (20 pM) was incubated in Buffer A (final volume, 0.1 ml) for 10 min at 26 “C with partially purified peroxygenase (1 pg) in the presence (A) or absence (B) of 13-hydroperoxylinoleic acid (100 PM). The reaction products were isolated as indicated in the text and chromatographed on silica TLC plates using ethyl ether/ petroleum ether (40-60 ‘Q/formic acid (50:50:1, v/v/v) as eluent. Radioactive peaks correspond to oleic acid (a) and to authentic standards of 9,10-epoxy oleic acid (b). C, control experiment performed as in A but with boiled enzyme.

oleic acid could be observed (not shown). It seems therefore that the epoxidation reaction of oleic acid demonstrated here cannot be explained, a priori, by a chemical reaction between the hydroperoxide and the double-bond catalyzed by trace metals (Gardner, 1989) or hemin present in the enzyme fraction (Dix and Marnett, 1985; Labeque and Marnett, 1988), but rather involves the catalytic activity of a hydroperoxide- dependent oxidase.

The fate of the epoxidation co-substrate, i.e. 13(S)-[1-‘4C] hydroperoxylinoleic acid, was investigated. During the enzymatic epoxidation of oleic acid, the hydroperoxide was pre- ponderantly converted into a product which co-migrated with a 13-hydroxylinoleic acid standard and into several other products (see below). The identity of the major reaction product was confirmed by GC/MS analysis after its conversion into a methyl ester and silylation. The stoichiometry of the epoxidation catalyzed by the peroxygenase was also determined. Under experimental conditions using similar concentrations of oleic acid and 13(S)-hydroperoxylinoleic acid, about 1.7 hydroperoxides were consumed per epoxide formed; moreover 13-hydroxylinoleic acid accounted for about 65% of the products derived from the hydroperoxide (i.e. the product ratio of 13-hydroxylinoleic acid/9,10-epoxyoctadecanoic acid was 1.08 + 0.08 (n = 4)). The full structural characterization of the minor products is presently under investigation. Pre- liminary results indicate that they are isomers of epoxidized hydro(pero)xylinoleic acid, suggesting that the hydroperoxide can compete with oleic acid as substrate of the peroxygenase oxidizing complex.’ At this level it is worth mentioning that similar transformations of fatty acid hydroperoxides into their corresponding epoxy alcohols have already been described by Hamberg et al. (1986) in the fungus Saprolegnia parasitica.

Characterization of the Epoxidase Enzymatic Fraction- Independently from the peroxygenase catalyzed epoxidation reactions of unsaturated fatty acids as studied here, it must be kept in mind that hydroperoxides can also serve as substrates in co-oxidation reactions. These reactions can be enzymatic, e.g. co-oxidations catalyzed by lipoxygenases (Chan, 1971; Belvedere et al., 1983) or nonenzymatic, e.g. initiated by transition metals or heme complexes (Gardner, 1989; see “Discussion”). In crude systems such as microsomes, such side reactions could constitute serious limitations in the study of the mechanism of peroxygenase-catalyzed epoxidations. Since our work rests largely on kinetic measurements and on the isolation, chemical analysis, and identification of the reaction products, it was of great importance to have access to an enzyme preparation which largely obliterates such complicating factors. This was reached by solubilizing the peroxygenase with non-ionic detergents and by a purification step on a cation-exchange chromatography column (B16e and Schuber, 1989).

A careful fractionation of detergent-solubilized soybean microsomes by chromatography on a CM-Sepharose column allowed the preparation of a peroxygenase fraction devoid of active or denatured cytochrome P-450 isozymes (Blee and Schuber, 1989). Moreover, as shown here (Fig. 2), this purification step gives also a clear-cut separation of the peroxygenase from the solubilized membrane-bound lipoxygenases (active at pH 9.0 or 5.5) which are eluted at much higher ionic strengths. Fig. 2 shows the perfect co-purification of the epoxidase and sulfoxidase activities in this chromatography. Finally, this cation-exchange step is also important since it eliminates most free fatty acids and neutral and negatively charged (phospho)lipids from the peroxygenase preparation.

We have studied further the involvement of a hemoprotein

* E. Blee, unpublished data.


sWmM NoCl

300 mM

Fractions FIG. 2. Separation of peroxygenase and lipoxygenase ac-

tivities by chromatography on a CM-Sepharose column. The fraction (20 ml, 50 mg of protein) obtained by solubilization of soybean microsomes with emulphogene BC-720 (see text for details) was adsorbed on a CM-Sepharose column (2 x 12 cm) previously equilibrated with a 10 mM sodium acetate buffer (pH 5.5) containing 0.8% emulphogene and 20% glycerol. After unadsorbed material has been washed out with Buffer A, elution was performed first with a gradient O-300 mM NaCl, then 500 mM NaCl in Buffer A and fractions of 3 ml were collected at the flow rate of 30 ml/h. Lipoxygenase activities determined at pH 5.5 and 9.0 are expressed in nanomoles/ min. Peroxygenase was determined for its sulfoxidase (a) and epoxidase activities (A), expressed in nanomoles/min.

in the oxidative processes catalyzed by the peroxygenase. Confirming the results obtained previously (Blee and Schuber, 1989), the enzyme preparation was shown to contain a hemoprotein. In Fig. 3, A and B, are represented the fluorescence spectrum of the protoheme, the light absorbance spectra of the oxidized and reduced forms of the peroxygenase fraction and of its pyridine-ferrohemoprotein complex. Addition to the peroxygenase of cumene hydroperoxyde (Fig. 3C, inset), and to a lesser extent 13(S)-hydroperoxylinoleic acid (not shown), resulted in a gradual decrease of the Soret band at 407 nm. Since it is well known that, in the absence of substrates, the prosthetic heme of cytochrome P-450 and of other hemoproteins is readily degraded in vitro by alkyl hydroperoxides (Ortiz de Montellano, 1986, and references therein), it was of importance to correlate such an effect with the decline of the peroxygenase hydroperoxide-supported epoxidation reaction. Fig. 3C shows the perfect parallelism between these two phenomena, i.e. both the decrease in the absorbance at 407 nm and enzyme inactivation follow pseudo-first order kinetics with similar half-lives (about 4 min). It was noted before (Blbe and Durst, 1987) that under turnover conditions, i.e. in the presence of substrate, the enzyme degradation is slowed down.

Characteristics of the Epoxidation Reaction Catalyzed by Perorygenase-As found classically with other oxidases which are inactivated under turnover conditions, the formation of the epoxide of oleic acid, catalyzed by the partially purified soybean peroxygenase, was linear with time only for short periods, e.g. about 5 min under standard conditions with a reaction progress of 20% (using 5 pM linoleic acid). Under similar experimental conditions, initial rates were linearly related to protein concentrations up to 1 kg. The epoxidation activity was maximal around pH 6.0 and revealed Michaelis- Menten-type kinetics with an apparent K,,, of 20 + 3 pM for oleic acid, when using cumene hydroperoxyde as co-substrate, and V,,, of 1.02 pmol. mini. mg-i. Similar classical plots were observed with 13(S)-hydroperoxylinoleic acid and cumene hydroperoxide, their apparent Km being, respectively, 24 f 8 and 63 + 20 pM.

Specificity of the Epoxidase: Oxidation of 9-ci.s- Unsaturated

560 580 600 620 640 660 680 70; wavelength (nrn,

00 nr n:

45’0 wavelengt

0.

550 h trim)

650

1 I 4 I

2 6 10 14

Time (min)

FIG. 3. Spectral characteristics of the peroxygenase and inactivation of the enzyme by cumene hydroperoxide. A, fluorescence (emission) spectrum of the protoheme of peroxygenase (solid line). The purified preparation was treated with base and oxalic acid according to Sassa and Kappas (1977) and the spectrum recorded with an excitation wavelength of 400 nm. Dashed line, hemin under ’ identical conditions. B, light absorption spectra of peroxygenase. The purified preparation (0.23 mg/ml) was in Buffer A (see text). Solid line, absolute absorption spectrum; dashed line, difference spectrum (dithionite-reduced minus oxidized). Inset, difference spectrum (dithionite-reduced minus oxidized) in the presence of 20% pyridine

Epoxidation of Unsaturated Fatty Acids

TABLE I

Effect of the chain length of 9,lOwzsaturated fatty acids on the rate of eporidation catalyzed by the peroxygenase

The fatty acids (200 FM) were incubated with solubilized and partially purified peroxygenase as detailed under “Experimental Pro- cedures.”

Fatty acids Relative rates of epoxide formation”

5% Cl&l 100 C16:l 56 C14:l 14

’ The 100% value corresponds to an epoxidation rate of 36 nmol/ min.

TABLE II

Influence of the location and the stereochemistry of the double bond on the rate of eponidation of Cl8 monounsaturated fatty acids

catalyzed by the peroxygenase The fatty acids (200 pM) were incubated with solubilized and

partially purified peroxygenase as detailed under “Experimental Pro- cedures.”

Cl&l Relative rates of epoxide formation”

7% 64s 100 94s 97 9-trans 0

ll-cis 96 ll-truns 0

a The 100% value corresponds to an epoxidation rate of 37 nmol/ min.

Fatty Acids of Various Chain Lengths-Oleic acid exhibits a cis-double bond at position 9,lO of the carbon chain. Keeping the unsaturation at the same position but shortening the length of the alkyl substituent proved increasingly unfavora- ble to the epoxidation (Table I). It should be noted here that the methyl ester of oleic acid was also an excellent substrate of the epoxidase; i.e. the V,,,,, ratio was 2.2 in favor of the ester indicating that a free carboxylic group does not seem obligatory for the positioning of the substrate in the active site.

Regio- and Stereoselectivity of the Epoxidase-Moving the single c&double bond of octadecenoic acid to positions 6 or 11 did not affect notably the epoxidation rates (Table II). The soybean epoxidase exhibited, however, a strong stereoselectivity since no epoxidation could be observed with the unsaturated fatty acids having the double bond in trans-configuration, e.g. elaidic and trans-vaccenic acids. Such a lack of epoxidation could result from (i) an intrinsic inability of peroxygenase to oxidize trans-double bonds or (and) (ii) the occurrence of a steric hindrance which forbids for such trans- unsaturated fatty acids a productive binding in the active site. We have therefore investigated if elaidic acid could compete for the epoxidation of [‘4C]oleic acid. The results revealed an

(v/v) in 0.1 N NaOH. C, inactivation of peroxygenase (m) and decrease of the Soret band (V) as a function of time at 25 “C. The reaction mixture contained purified peroxygenase (0.24 mg of protein in 1 ml of Buffer A, see text) and cumene hydroperoxide (1 mM). The decay of the Soret band is expressed as the absorbance decrease at 407 nm compared to the total observed decrease in A,,,, i.e. (A, - Am)/(A, - A,) with A, and A,, respectively, the initial and final (end point) absorbances. At times indicated, residual peroxygenase activity was determined on aliquots using oleic acid as substrate (see text). The loss of enzyme activity is expressed as the percent of residual activity compared to total activity. Inset, repetitive scanning of the absolute spectrum of peroxygenase obtained on addition of cumene hydroperoxide (1 mM).

absence of inhibition by elaidic acid, e.g. less than 5% decrease in oleic acid epoxidation rate was observed in the presence of a 20-fold molar excess (400 PM) of the trans-fatty acid. This suggests that double bonds with trans-configuration do not reach the active site of the enzyme, at least when incorporated in long chain fatty acids.

Origin of the Oxygen Atom Introduced into the Epoxide-In order to test the oxygen transfer mechanism, we have prepared 13(S)-hydroperoxylinoleic acid labeled with lRO which was then used as hydroperoxide co-substrate in the epoxidation of oleic acid catalyzed by the peroxygenase. Determina- tion by GC/MS of the isotope composition of 9,10-epoxyoctadecanoic acid, revealed an 80% incorporation of the hydroperoxide “0 label into the epoxide ring. This result suggests that a direct oxygen transfer between the hydroperoxide and the double bond of the fatty acid is prevalent in this epoxidation reaction. Moreover, the “0 content of 13-hydroxylinoleic acid isolated after the epoxidation reaction was found identical to that of the starting hydroperoxide. This would exclude the occurrence of an exchange mechanism between the “0 of hydroperoxylinoleic acid and, e.g. atmospheric O2 as noted previously by Dix and Marnett (1985) in reactions involving peroxyl radicals.

Epoxidation of Linoleic Acid-So far we have demonstrated an epoxidation of monounsaturated fatty acids, such as oleic acid, which are useful model compounds for mechanistic studies. However, from a physiological point of view, linoleic acid is of greater interest, since its oxidized derivatives have been found to be involved in plant defense mechanisms (Kato et al., 1983). Accordingly, we have incubated [1-14C]linoleic acid in the presence of cumene hydroperoxide and soybean peroxygenase. Epoxidation occurred readily, i.e. under similar experimental conditions (100 PM unsaturated fatty acid) the rate of substrate disappearance was 2-fold higher for linoleic acid than for oleic acid. The formation of three compounds was observed: the two monoepoxides (9,10-epoxy-12-octadecenoate and its isomer 12,13-epoxy-9-octadecenoate) and the diepoxide (9,10- and 12,13-diepoxyoctadecanoate). After isolation by TLC, the diepoxide was transformed into its methyl ester and identified by GC/MS. Its mass spectrum (Fig. 4) presented the following fragmentation pattern: molecular ion at m/z 326 and a base peak at m/z 155 (187-32; 9,lO cleavage then loss of CHSOH). Prominent ions were also seen at 84 (12,13 cleavage and loss of 0), 69 (84-15, loss of .CHB), 308 (M-18), 295 (M-31, loss of .0CH3), and 277 (M-31-18). The diepoxide constitutes the minor reaction product, e.g. in a reaction with 100 pM initial concentrations of both linoleic and 13-hydroperoxylinoleic acids, it accounted for about 16% of the products formed after a 45% reaction progress. The methyl esters of the two monoepoxides were best separated by GC, using a carbowax-type capillary column, and were identified by their fragmentation patterns in MS (see “Ex- perimental Procedures”). Interestingly, an important regioselectivity was observed in the formation of the monoepoxides, i.e. 9,10-epoxy-12-octadecenoate represented more than 72% of the monoepoxides formed. Thus, at least with linoleic acid, the oxidation of the cis-double bond at position 9,lO is somewhat prevalent over that at position 12,13.

DISCUSSION

In the present work we have demonstrated that peroxygenase, solubilized and partially purified from soybean microsomes, in addition to hydroperoxide-dependent hydroxylation (Ishimaru and Yamazaki, 1977a) and sulfoxidation (Blee and Durst, 1986, 1987) reactions, also catalyzes epoxidation of unsaturated fatty acids such as oleic and linoleic acids. Due


FIG. 4. Mass spectrum of methyl- ‘8 9,10:12,13-diepoxyoctadecanoate. 8 The compound obtained by oxidation of 2 linoleic acid by the peroxygenase was analyzed by GC/MS (electron energy: 20

‘- o ~II ,r

eV) as described under “Experimental Procedures.”

.z

f

0 CJ

C

to the large diversity of reactions accessible to alkylhydroperoxides, especially those derived from unsaturated fatty acids, it was of importance to first characterize the nature of the epoxidizing species involved in the peroxygenase catalyzed reactions. Epoxidation of double bonds can be observed with metal-ox0 complexes or with peroxyl radicals formed by reaction of hydroperoxides with free transition metals or heme complexes. For example, it is well documented that homolytic cleavage of the hydroperoxide bond in hydroperoxylinoleic acid produces alkoxyl radicals which, after rearrangement into allylic radicals and reaction with 02, can generate free peroxyl radicals (Dix and Marnett, 1981; Labeque and Marnett, 1988; Gardner, 1989). These peroxyl radicals are known epoxidizing agents of nonaromatic double bonds (Labbque and Marnett, 1988, and references therein). Epoxidation of double bonds by prostaglandin H synthase was suggested to involve such a process (Panthananickal et al., 1983). Therefore a key ques- tion was whether the epoxidations of unsaturated fatty acids observed in the present work resulted from a reaction of the double bonds with such free peroxyl radical or according to an enzyme-catalyzed process, i.e. involving oxidative species in the active site of the peroxygenase according to one of the mechanisms described by Ortiz de Montellano and co-workers for other hemoproteins (see e.g. Ortiz de Montellano et al., 1987; Catalan0 and Ortiz de Montellano, 1987).

The results obtained here in the epoxidation of unsaturated fatty acids are in favor of a peroxygenase-catalyzed reaction. Under standard experimental conditions which, in the presence of 13-hydroperoxylinoleic acid, permitted an efficient enzymatic epoxidation of oleic acid the following observations were made. (i) Denaturation of the enzyme totally abolished the reaction. In addition, no epoxidation could be observed with hematin (0.5 pM) as described by Dix et al. (1985), for allylic fatty acid hydroperoxide-dependent epoxidation of 7,8- dihydroxy-7,8-dihydrobenzo[a]pyrene. The antioxidants butylated hydroxyanisole and butylated hydroxytoluene, which were shown by these authors to almost completely inhibit at

55

: 69 8 155 c +- 187

1 concentrations as low as 10 pM this latter epoxidation by the free radicals generated from hydroperoxylinoleic acid by hematin, had no effect on the rate of oleic acid epoxidation by the peroxygenase. (ii) No enzyme-catalyzed epoxidation was detected when hydroperoxides were omitted from the incubation mixture. Furthermore, we have found that peroxygenase can promote with similar efficiency epoxidation of oleic acid either with hydroperoxylinoleic acid or cumene hydroperoxyde. (iii) The epoxidation reaction shows a very high specificity for &-unsaturated fatty acids. Such a stereoselectivity is not expected in reactions with free peroxyl radicals derived either from hydroperoxylinoleic acid or cumene hydroperoxide. Moreover, since free peroxyl radicals are mostly produced by chain reactions, no fixed stoichiometry is antic- ipated between the formation of epoxide and hydroperoxides added to the reaction (see e.g. Marnett et al., 1979). This was not the case for the peroxygenase-catalyzed epoxidation of oleic acid, e.g. Michaelis-Menten-type kinetics were observed when hydroperoxylinoleic acid or cumene hydroperoxide were used as variable substrates. Moreover, in the peroxygenase- catalyzed epoxidation of oleic acid the product ratio of 13- hydroxylinoleic acid/9,10-epoxyoctadecanoic acid was close to one. (iv) The principal source of the epoxide oxygen (80%) introduced during the oxidation of oleic acid by [‘sO]13- hydroperoxylinoleic acid catalyzed by peroxygenase is the hydroperoxide. Taken together, these results cannot be explained according to a free peroxyl radical-based mechanism but are rather consistent with the occurrence of a heme-iron- activated oxidative species in the active site pocket of the peroxygenase. Investigations are presently undertaken in our laboratory to determine the molecular mechanism(s) of the epoxidase activity of the peroxygenase.

In higher plants, besides lipoxygenase-catalyzed formation of epoxy alcohols from hydroperoxy fatty acids (Garssen et al., 1976) and the rare exception of the FAD-dependent oxidation of squalene into 2,3-oxidosqualene (Ono et al., 1982), most double bond epoxidations described so far are catalyzed


by cytochrome P-450-dependent enzymes. For example, the involvement of such an enzyme in the oxidation of 1%hy- droxyoleic acid by a particular fraction of spinach has been suggested (Croteau and Kolattukudy, 1975). This activity, which was shown to require NADPH but also ATP and CoASH, oxidized exclusively &double bonds and moreover seems unable to epoxidize unhydroxylated free oleic acid. More recently, 9-dodecenoic acid has been found to be converted into 9,10-epoxylauric acid by a microsomal fraction from Jerusalem artichoke tubers according to a cytochrome P-450-dependent mechanism (Salaun et al., 1989). This epoxidation does not discriminate between cis- and trawdouble bonds but is characterized by its high regioselectivity, i.e. position 9 is largely favored and in the absence of a double bond, a 9-hydroxylation reaction is observed. These results are in sharp contrast with those obtained in the present study with the soybean peroxygenase. This enzyme exhibits with octadecenoic acids a lack of regioselectivity, i.e. it epoxidizes with similar efficiency the double bond at positions 6, 9, or 11. The peroxygenase, however, shows a high stereoselectivity since it does not accept as substrates Cl8 monounsaturated fatty acids with double bonds in trans-configuration. This finding gives interesting insight into the structure of the active site of this enzyme which most probably controls the access of substrates to the oxidative species. The inability of trans- 9,10-octadecenoic acid (elaidic acid) to competitively inhibit the epoxidation of oleic acid (cis-9,10-octadecenoic acid) re- flects such steric limitations. One can speculate that a cis- double bond, which allows a “hairpin’‘-type conformation of the unsaturated fatty acid, facilitates a proper positioning of the *-orbitals with respect to the oxidant. It seems reasonable to assume that hydroperoxides and the substrates to be epoxidized have access to the heme of the peroxygenase through the same channel, therefore the &-double bond in hydroperoxylinoleic acid should also play a similar role for the hydroperoxide bond with respect to the heme iron. However, since this enzyme accepts equally well as co-substrates fatty acid hydroperoxides and sterically hindered hydroperoxides such as cumene hydroperoxide, it seems that its binding region in the vicinity of the heme group, if selective, is nevertheless quite sizeable. In this respect the soybean peroxygenase res- sembles more cytochrome P-450 isozymes than peroxidases.

Polyunsaturated fatty acid hydroperoxides are well known key intermediates in the biosynthesis of the plant hormones, e.g. traumatin (Zimmerman and Coudron, 1979) and jasmonic acid (Vick and Zimmerman, 1983, 1984), and of fragrant aldehydes (Hatanaka et al., 1987). We show here, another possible metabolic fate for these hydroperoxides which con- sists in their reduction by the peroxygenase to their corresponding alcohols. Consequently this enzyme, in the absence of other known systems which in plants could fulfill the role of glutathione peroxidase, might be involved in the catabolism of hydroperoxides. In addition, Kato et al. (1984) have reported that free hydroxyacids which can be derived from linoleic acid hydroperoxides, such as 13-hydroxy-9-c&11- trans-octadecadienoic and 9-hydroxy-lo-tran.s,12-cis-octadecadienoic acids, are inhibitors of the spore germination and germ tube growth of rice blast fungus. Thus, besides its protective role against the deleterious effects of fatty acid hydroperoxides in ho, which are generated enzymatically by lipoxygenases or nonenzymatically, e.g. under conditions of stress induced by fungal pathogens agression (Peever and Higgins, 1989, and references therein), the peroxygenase may transform these compounds into derivatives involved in the defense against fungal infections. Importantly, this activity is not restricted to soybean but was also detected in many other

plants such as pea, rice, maize, and potato tuber. An efficient reduction of hydroperoxides by the peroxygen-

ase requires the presence of oxidizable co-substrates. We have shown here that free unsaturated fatty acids can fulfill such a role, the enzyme isolated from soybean, catalyzing very effectively their transformation into the corresponding epoxides. As indicated above, enzyme systems which have been described in literature to catalyze, in uitro, similar reactions with endogenous metabolites are very rare in plants and belong to the cytochrome P-450 mixed-function oxidases. It is noteworthy, however, that compared to such enzymes, the epoxidase-specific activity of the soybean peroxygenase is, in comparable reactions, about 2 to 3 orders of magnitude higher. Importantly, the epoxy-acid derivatives such as 9,10-epoxy- 12-octadecenoic acid (coronaric acid) and 12,13-epoxy-9-octadecenoic acid (vernolic acid) which are structurally similar to the compounds shown here to be produced from linoleic acid by the action of the peroxygenase are leukotoxins (Osawa et al., 1986). These compounds, which have been reported recently to be produced by mammalian leukocytes incubated with linoleic acid (Hayakawa et al., 1986), are uncoupling agents in mitochondrial respiration and cause relaxation of rat stomach smooth muscle (Osawa et al., 1989). Leukotoxins have also been identified in higher plants and are considered, along with their reduced (i.e. monohydroxyls) and hydrolyzed (i.e. diols) derivatives, to constitute an important family of plant-defense substances (Kato et al., 1983a, 1983b; 1984). However, to date no enzyme system(s) involved in the biosynthesis of leukotoxin or leukotoxin-like compounds have been characterized. It appears therefore that the peroxygenase could be an excellent candidate to fulfill such a role in plants, its catalytic efficiency being compatible with a rapid onset of defense mechanisms. In this respect it is of interest that this enzyme presents some specificity for Cl8 unsaturated fatty acids (Table I), the major constituents of plant cells. At this point it might be relevant to indicate that the lipoxygenase activity, the enzyme responsible for the formation of fatty acid hydroperoxides, was shown to be stimulated following incubation with pathogens or treatment with elicitors (Lupu et al., 1980; Ocampo et al., 1986; Fournier et al., 1986; Hilde- brand et al., 1989).

Acknowledgments-We thank G. Teller for mass spectrometry. We are grateful to Dr. A. Van Dorsselaer and L. Menguy for their help for gas-liquid chromatography determinations.

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