Manganese Lipoxygenase
DISCOVERY OF A BIS-ALLYLIC HYDROPEROXIDE AS PRODUCT AND INTERMEDIATE IN A LIPOXYGENASE REACTION*

Mats HambergDagger §, Chao Su, and Ernst Oliw

From the Dagger  Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm and the  Division of Biochemical Pharmacology, Department of of Pharmaceutical Biosciences, Uppsala Biomedical Center, Uppsala University, S-751 24 Uppsala, Sweden

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Linoleic acid was incubated with manganese lipoxygenase (Mn-LO) from the fungus Gäumannomyces graminis. The product consisted of (13R)-hydroperoxy-(9Z,11E)-octadecadienoic acid ((13R)-HPOD) and a new hydroperoxide, (11S)-hydroperoxy-(9Z,12Z)-octadecadienoic acid ((11S)-HPOD). Incubation of (11R)-[2H]- and (11S)-[2H]linoleic acids with Mn-LO led to the formation of hydroperoxides that largely retained and lost, respectively, the deuterium label. Conversion of the (11S)-deuteriolinoleic acid was accompanied by a primary isotope effect, which manifested itself in a strongly reduced rate of formation of hydroperoxides and in a time-dependent accumulation of deuterium in the unconverted substrate. These experiments indicated that the initial step catalyzed by Mn-LO consisted of abstraction of the pro-S hydrogen of linoleic acid to produce a linoleoyl radical. (11S)-HPOD was converted into (13R)-HPOD upon incubation with Mn-LO. The mechanism of this enzyme-catalyzed hydroperoxide rearrangement was studied in experiments carried out with 18O2 gas or 18O2-labeled hydroperoxides. Incubation of [11-18O2](11S)-HPOD with Mn-LO led to the formation of (13R)-HPOD, which retained 39-44% of the 18O label, whereas (11S)-HPOD incubated with Mn-LO under 18O2 produced (13R)-HPOD, which had incorporated 57% of 18O. Furthermore, analysis of the isotope content of (11S)-HPOD remaining unconverted in such incubations demonstrated that [11-18O2](11S)-HPOD suffered a time-dependent loss of 18O when exposed to Mn-LO, whereas (11S)-HPOD incorporated 18O when incubated with Mn-LO under 18O2. On the basis of these experiments, it was proposed that the conversion of (11S)-HPOD into (13R)-HPOD occurred in a non-concerted way by deoxygenation into a linoleoyl radical. Subsequent reoxygenation of this intermediate by dioxygen attack at C-13 produced (13R)-HPOD, whereas attack at C-11 regenerated (11S)-HPOD. The hydroperoxide rearrangement occurred by oxygen rebound, although, as demonstrated by the 18O experiments, the oxygen molecule released from (11S)-HPOD exchanged with surrounding molecular oxygen prior to its reincorporation.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Lipoxygenase-catalyzed dioxygenation of polyunsaturated fatty acids leads to the formation of reactive fatty acid hydroperoxides. Mammalian lipoxygenases can catalyze oxygenation at carbons 5, 8, 12, and 15 of their predominant substrate, i.e. arachidonic acid (1). Many plant lipoxygenases can also utilize arachidonic acid, although their most important substrates are the C-18 fatty acids linoleic acid and alpha -linolenic acid (2). Interest in lipoxygenases stems partly from the fact that fatty acid hydroperoxides can be further metabolized into biologically active oxylipins such as leukotrienes and jasmonates.

Lipoxygenases contain ferrous iron, which is oxidized into the ferric state by, e.g., hydroperoxides. The ferric form of lipoxygenases is catalytically active (3) and catalyzes the stereospecific abstraction of one hydrogen from the bis-allylic methylene group of the (1Z,4Z)-pentadiene structure of the substrate as the initial step (4). Attack by dioxygen at one of the terminal positions of the resulting pentadienyl radical results in the formation of a hydroperoxide having one pair of E/Z-conjugated double bonds. Studies of the regio- and stereochemistry of the steps occurring in the oxygenation of 8,11,14-eicosatrienoic acid by soybean lipoxygenase-1 revealed that the pro-S hydrogen was stereospecifically removed from C-13 and that dioxygen was regio- and stereospecifically inserted at C-15 to produce a (15S)-hydroperoxide (4). This finding, and results of similar studies carried out with linoleic acid (9S)-lipoxygenase from corn (5), arachidonic acid (12S)-lipoxygenases from human platelets (6) and a red alga (7), arachidonic acid (5S)-lipoxygenases from rat basophil leukemia cells and potato (8), and arachidonic acid (8S)-lipoxygenase from mouse epidermis (9) indicated the existence of an antarafacial relationship between hydrogen abstraction and oxygen insertion as a common feature of dioxygenations catalyzed by lipoxygenases. Interestingly, such a steric relationship has also been found for the dioxygenation catalyzed by an "R" lipoxygenase, i.e. (12R)-lipoxygenase from sea urchin (10), as well as for dioxygenations catalyzed by prostaglandin endoperoxide synthases I (11) and II (12), and by ferrylmyoglobin (13). Mammalian and plant lipoxygenases so far studied catalyze production of hydroperoxides that have the "S" absolute configuration. In contrast, a number of marine invertebrates, such as starfish, sea urchin, and the coral Plexaura homomalla, express lipoxygenases, which catalyze formation of "R" hydroperoxides, as demonstrated by the configuration of hydroperoxides formed by oxygenation of arachidonic acid at the C-5, C-8, C-11, and C-12 positions. One of these enzymes, arachidonic acid (8R)-lipoxygenase, was recently cloned and sequenced (14).

Manganese lipoxygenase (Mn-LO)1 was purified from the fungus Gäumannomyces graminis as described in the accompanying paper (15). The protein was found to be heavily glycosylated and to contain 0.5-1 atom of manganese per molecule. The molecular mass of the native protein (~135 kDa) was comparable to that of arachidonic acid (15S)-lipoxygenase purified from the fungus Saprolegnia parasitica (145-150 kDa) (16). Interestingly, the molecular mass of the deglycosylated Mn-LO (~73 kDa) (15) was similar to those of mammalian lipoxygenases. The present paper is concerned with the stereochemistry and mechanism of the Mn-LO-catalyzed dioxygenation. In the course of the work, it was unexpectedly found that the enzyme catalyzes bis-allylic oxygenation of polyunsaturated fatty acids. Thus, oxygenation of linoleic acid resulted in the formation of 11-hydroperoxylinoleic acid, a new member of the oxylipin family of compounds.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- [1-14C]Linoleic acid was purchased from NEN Life Science Products. Dilution with unlabeled material (Nu-Chek-Prep, Elysian, MN) followed by purification by SiO2 chromatography afforded a specimen having a specific radioactivity of 5.0 kBq/µmol. (11R)-[2H]Linoleic acid (37.2% monodeuterated and 62.8% undeuterated molecules) and (11S)-[2H]linoleic acid (32.6% monodeuterated and 67.4% undeuterated molecules) were prepared by lithium aluminum deuteride reduction of the p-tolylsulfonyl derivatives of methyl (11S)- and (11R)-hydroxystearates, respectively, followed by biological desaturation of the resulting stereospecifically deuterated stearic acids as described in detail elsewhere (17, 18). The optical purities of the chiral 11-hydroxystearates were: (11R)-hydroxystearate (precursor of (11S)-[2H]linoleic acid), 98.3% R; (11S)-hydroxystearate (precursor of (11R)-[2H]linoleic acid), 95.4% S. (9S)-HPOD and (13S)-HPOD were prepared by incubation of linoleic acid with tomato lipoxygenase (19) and soybean lipoxygenase (20), respectively. The corresponding alcohols, (9S)-HOD and (13S)-HOD, were obtained from the hydroperoxides by reduction with sodium borohydride. (11R)-HOD and 11-ketolinoleic acid were prepared by incubation of linoleic acid with an enzyme preparation of the red alga Lithothamnion corallioides (21). 18O2, 99.4 atom %, was obtained from Isotec, Miamisburg, OH. Glutathione peroxidase, reduced glutathione, and soybean lipoxygenase were purchased from Sigma. Mn-LO was prepared as described in the accompanying paper (15). Solutions of the enzyme (22 µg/ml) could be stored at 4 °C for several weeks without appreciable loss of activity.

Geometrical Isomers of Methyl 11-Hydroxy-9,12-octadecadienoate-- The methyl ester of (11R,11S)-HOD (i.e. the natural (9Z,12Z)-isomer) was prepared by treatment of linoleic acid (1 mM) with myoglobin (100 µM) and cumene hydroperoxide (3 mM) at 0 °C as recently described (22). The methyl ester of (9E,12E)-(11R,11S)-HOD was prepared in an analogous way starting with (9E,12E)-octadecadienoic acid, although the poor solubility of this acid in aqueous buffer made it necessary to carry out the hydroxylation at 23 °C. A mixture of (9E,12Z)- and (9Z,12E)-octadecadienoic acids was obtained from a commercially available mixture of the four geometrical isomers of 9,12-octadecadienoic acid (Sigma) using RP-HPLC. Treatment of the acids with myoglobin/cumene hydroperoxide at 0 °C followed by esterification and isolation by RP-HPLC afforded a mixture of comparable amounts of the methyl esters of (9E,12Z)-(11R,11S)-HOD and (9Z,12E)-(11R,11S)-HOD; UV spectrum (methanol), featureless in the region 210-340 nm; FT-IR spectrum (film), absorption bands inter alia at 3200-3620 cm-1 (hydroxyl), 1742 cm-1 (ester carbonyl), and 968 cm-1 (E double bond). The four isomeric methyl 11-hydroxy-9,12-octadecadienoates were separable as their Me3Si derivatives by GLC, i.e. derivative of (11R,11S)-HOD, C-19.41; derivative of (9E,12E)-(11R,11S)-HOD, C-19.62; and derivatives of (9E,12Z)-(11R,11S)-HOD and (9Z,12E)-(11R,11S)-HOD, C-19.48 and C-19.56 (these two isomers have not yet been individually correlated with the two C-values). The mass spectra of the four isomers were similar and showed strong ions at m/z 382 (M), 311 (M - 71; loss of bullet (CH2)4-CH3), and 225 ([CH=CH-CH(OSiMe3)-CH=CH-(CH2)4-CH3]+).

Incubations-- Most incubations were carried out by stirring [1-14C]linoleic acid (50-700 µM) or [1-14C](11S)-HPOD (40-60 µM) with 0.5-2.2 µg of Mn-LO at 23 °C in 0.5-2.1 ml of one of the following buffers: 0.1 M potassium phosphate buffer, pH 7.4 (buffer A); 0.1 M sodium borate buffer, pH 10.0 (buffer B); or 10 mM triethanolamine acetate, 1 mM EDTA, 2 mM sodium azide, 0.04% Tween 20, pH 7.0 (buffer C). An aliquot of the reaction mixture was transferred to a cuvette (path length, 1 or 10 mm; temperature, 23 °C) and the reaction progress was followed by monitoring the absorbance at 235 nm versus time. Incubations under 18O2 were conducted at 23 °C in a reaction vessel connected to a high vacuum line and a supply of argon. Buffer A (2 ml) and Mn-LO were added to the reaction vessel, and the solution was de-aerated at 0 °C by five cycles of vacuum and argon purging prior to introduction of 18O2 gas and substrate. Isolation of reaction products was performed by extraction with diethyl ether at pH 5, followed by RP-HPLC.

Chemical Methods-- Preparation and purification of (-)-menthoxycarbonyl (MC) derivatives (23), and steric analysis of 9-HOD and 13-HOD (23) and of 11-HOD (21) were performed as indicated. Catalytic hydrogenation was performed with platinum catalyst (3 mg) using methanol (1 ml) as the solvent. Partial hydrogenation of the methyl ester of 11-HOD (25 µg) was carried out by stirring with 5% palladium-on-calcium carbonate (5 mg) in ethyl acetate (3 ml) under hydrogen gas for 4 min (cf. Ref. 21). Oxidative ozonolysis was carried out as described (23) using an ozone generator (model T-12) purchased from TriO3 Industries, Fort Pierce, FL.

Chromatographic and Instrumental Methods-- RP-HPLC was performed with a column of Nucleosil 100-5 C18 (250 × 4.6 mm) purchased from Macherey-Nagel (Düren, Germany). The solvent system used consisted of acetonitrile/water/2 M hydrochloric acid (60:40:0.02, v/v/v). Straight phase high performance liquid chromatography was carried out with a column of Nucleosil 50-5 (200 × 4.6 mm) and a solvent system of 2-propanol/hexane (1:99, v/v). The absorbance (217 nm) and radioactivity of high performance liquid chromatography effluents were determined on-line using a Spectromonitor III ultraviolet detector (Laboratory Data Control, Riviera Beach, FL) and a liquid scintillation counter (IN/US Systems, Tampa, FL), respectively. GLC was performed with a Hewlett-Packard (Avondale, PA) model 5890 gas chromatograph equipped with a methyl silicone capillary column (length, 25 m; film thickness, 0.33 µm). Helium at a flow rate of 25 cm/s was used as the carrier gas. Retention times were converted into C-values using standards of saturated fatty acid methyl esters (24). GC-MS was carried out with a Hewlett-Packard model 5970B mass selective detector connected to a Hewlett-Packard model 5890 gas chromatograph. LC-MS was performed as described in the accompanying paper (15). Ultraviolet absorption as a function of wavelength or time was recorded with a Hitachi (Tokyo, Japan) model U-2000 UV-visible spectrophotometer. Infrared spectrometry was carried out using a Perkin-Elmer model 1650 FT-IR spectrophotometer. Radioactivity was determined with a Packard Tri-Carb model 4450 liquid scintillation counter (Packard Instruments, Downer's Grove, IL).

    RESULTS
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Procedures
Results
Discussion
References

Oxidation of Linoleic Acid by Manganese Lipoxygenase

Isolation of Reaction Products of Linoleic Acid-- [1-14C]Linoleic acid (350 µM) was stirred for 30 min with Mn-LO (1.5 µg) in buffer A (2.1 ml) at 23 °C. Material isolated by extraction with diethyl ether was subjected to RP-HPLC radiochromatography. As seen in Fig. 1, three peaks of radioactivity appeared. The least polar material (29%; 81.1 ml of effluent) was identical to [1-14C]linoleic acid remaining unconverted. Compound A (12%; 14.1 ml of effluent) was intermediate in polarity to references of authentic (11R,11S)-HOD (12.1 ml of effluent) and (13S)-HOD (15.4 ml of effluent). Compound B (59%; 17.0 ml of effluent) cochromatographed with authentic (13S)-HPOD.


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Fig. 1.   RP-HPLC radiochromatogram of reaction product formed from linoleic acid incubated with Mn-LO. [1-14C]Linoleic acid (350 µM) was treated with Mn-LO (1.5 µg) in 2.1 ml of buffer C at 23 °C for 30 min, and the reaction product was isolated by extraction with diethyl ether. Solvent system, acetonitrile/water/2 M hydrochloric acid (60:40:0.02, v/v/v); flow rate 1 ml/min. A, compound A; B, compound B; C, linoleic acid; BHT, 2,6-di-tert-butyl-4-methylphenol, antioxidant added at a level of 10 ppm to the diethyl ether used for extraction.

Identification of Compound B-- Analysis of compound B by LC-MS demonstrated a peak (10.4 min effluent) giving prominent ions at m/z 311 (M - 1; loss of H), 295 (M - 17; loss of OH), and 293 (M - (1 + 18); loss of H plus H2O). When compound B was dissolved in D2O/CD3OD (1:1, v/v), the - 1 ion shifted to m/z 312. An aliquot of compound B was reduced by treatment with sodium borohydride. Analysis of the esterified product by straight phase high performance liquid chromatography produced the methyl esters of 13-HOD (97.1%; 11.6 ml of effluent) and 9-HOD (2.9%; 14.7 ml of effluent) as judged by their chromatographic behaviors, UV spectrometry, and mass spectrometry. The two hydroxyoctadecadienoates were converted into their MC derivatives and subjected to oxidative ozonolysis. Analysis of the esterified product by GLC demonstrated the formation of dimethyl azelate as well as the MC derivative of methyl 2-hydroxyheptanoate (R/S, 97.5:2.5) from the derivative of 13-HOD. The derivative of 9-HOD afforded the MC derivative of dimethyl 2-hydroxysebacate (R/S, 37.0:63.0). These experiments confirmed the results of the accompanying paper (15), which identified the major hydroperoxide produced from linoleic acid as (13R)-HPOD. In addition, the analyses showed that the Mn-LO-catalyzed oxygenation of linoleic acid resulted in small amounts of the following hydroperoxyoctadecadienoate isomers: (13S)-HPOD (2.4%), (9R)-HPOD (1.1%), and (9S)-HPOD (1.8%).

Structure of Compound A-- The retention time of compound A upon LC-MS analysis was 8.3 min, and the mass spectrum showed prominent ions at 311 (M - 1; loss of H), 295 (M - 17; loss of OH), and 293 (M - (1 + 18); loss of H plus H2O). This result indicated that compound A was a hydroperoxyoctadecadienoic acid (molecular weight, 312). The UV spectrum of compound A was featureless (Fig. 2) demonstrating the absence of, e.g., conjugated double bonds. Treatment of compound A with a small amount of perchloric acid resulted in a rapid appearance of UV absorption bands at 259, 268, and 279 nm (Fig. 2). Analysis of the esterified reaction product by GLC and GC-MS demonstrated the presence of geometrical isomers of methyl 8,10,12- and 9,11,13-octadecatrienoates as judged by their molecular weight (292) and retention times (C-19.00 (21%), C-19.13 (21%), C-19.42 (28%), and C-19.46 (30%)). The fact that a virtually identical mixture of geometrical isomers of methyl 8,10,12- and 9,11,13-octadecatrienoates was formed upon acid treatment of the methyl ester of (11R)-HOD (21) suggested that compound A was a derivative of linoleic acid substituted at the bis-allylic position (C-11). Treatment of compound A with sodium borohydride followed by esterification afforded the methyl ester of 11-HOD as judged by GLC and GC-MS. The retention time corresponded to C-19.41, a value identical to that observed for the Me3Si derivative of the methyl ester of authentic 11-HOD but different from the values recorded for the Me3Si derivatives of the methyl esters of (9E,12Z)-11-HOD and (9Z,12E)-11-HOD (C-19.48/C-19.56) and (9E,12E)-11-HOD (C-19.62). The mass spectrum of the reduced product obtained from compound A was identical to that of the Me3Si derivative of the methyl ester of 11-HOD and showed prominent ions at m/z 382 (M, 38%), 311 (M - 71; loss of bullet (CH2)4-CH3, 32%), 253 (9%), and 225 ([CH=CH-CH(OSiMe3)-CH=CH-(CH2)4-CH3]+, 44%). Reduction of compound A with sodium borodeuteride afforded 11-HOD without significant incorporation of deuterium (less than 0.5%), thus excluding the possibility of a 11-keto group in compound A. Catalytic hydrogenation of compound A using platinum as catalyst resulted in the formation of a 1:1 mixture of 11-hydroxyoctadecanoate and 11-ketooctadecanoate as shown by GC-MS analysis using the authentic compounds as references. On the basis of these data, compound A was identified as a 11-hydroperoxy derivative of linoleic acid.


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Fig. 2.   Acid-catalyzed elimination of hydrogen peroxide from (11S)-HPOD. (11S)-HPOD (15 µg) was added to acetonitrile (1 ml; 23 °C) in a cuvette having 10 mm path length. UV spectra were recorded before (solid line) and 10 min after (dashed line) addition of 2 µl of 70% (w/w) perchloric acid.

Partial hydrogenation of compound A followed by oxidative ozonolysis performed on the MC derivative was used to determine the double bond positions and the configuration of C-11. Analysis of the esterified ozonolysis product by GLC and GC-MS demonstrated the presence of the MC derivatives of methyl (2S)-hydroxynonanoate (less than 2% of the (2R)-hydroxynonanoate; fragment originating in the MC derivative of methyl 11-hydroxy-9-octadecenoate) and of dimethyl (2R)-hydroxydodecane-1,12-dioate (less than 2% of the (2S)-hydroxydodecanedioate; fragment originating in the MC derivative of methyl 11-hydroxy-12-octadecenoate). This experiment thus established that the two double bonds of compound A were localized in the Delta 9 and Delta 12 positions, and that the absolute configuration of the alcohol group at C-11 was "S." The data presented demonstrated that compound A was identical to (11S)-hydroperoxy-(9Z,12Z)-octadecadienoic acid ((11S)-HPOD).

Conversion of (11S)-HPOD into (13R)-HPOD

Formation of (11S)-HPOD and (13R)-HPOD as a Function of Time and Substrate Concentration-- Linoleic acid was incubated with Mn-LO and the product compositions at different times of incubation and varying substrate concentrations were determined by RP-HPLC (cf. Fig. 1). Because of the different extinction coefficients of (13R)-HPOD, (11S)-HPOD, and linoleic acid at the wavelength used for detection (217 nm), the peak areas had to be divided with relative response factors in order to obtain the molar composition of reactions products. These relative response factors, i.e. 0.0732 (linoleic acid), 0.578 ((11S)-HPOD), and 1 ((13R)-HPOD, were calculated by performing repeated injections of mixtures containing the three 14C-labeled compounds onto the high performance liquid chromatography column and separately determining the peak areas of aborbance at 217 nm and the radioactivity present in the corresponding effluents.

As seen in Fig. 3, when a relatively low concentration of linoleic acid (175 µM) was incubated with Mn-LO, (11S)-HPOD was transient in appearance. The maximum level was observed at 10-20 min of incubation, and at 30 min only a trace amount of the hydroperoxide was detectable. In contrast, when the linoleic acid concentration was increased to 526 µM, the amounts of (11S)-HPOD continued to increase during the entire incubation period of 30 min. Interestingly, analyses by RP-HPLC of products from experiments, where (11S)-HPOD had been generated from linoleic acid and then allowed to disappear, did not show any new peak(s) of UV absorption ascribable to the further conversion of (11S)-HPOD. It was conceivable that (11S)-HPOD had been converted into non-UV-absorbing compound(s); however, analysis by RP-HPLC radiochromatography of reaction products generated from [1-14C]linoleic acid showed the presence of (13R)-HPOD as the single labeled product. These results suggested that (11S)-HPOD was convertible to (13R)-HPOD in the presence of Mn-LO, and that this conversion was slowed down in the presence of high concentrations of linoleic acid.


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Fig. 3.   Time courses of formation of (11S)-HPOD and (13R)-HPOD. Panel A, linoleic acid (175 µM) in buffer C (2.1 ml) was treated with Mn-LO (1.1 µg) at 23 °C. The absorbance at 235 nm was measured using a cuvette having 1 mm path length. Aliquots (0.65 ml) were removed at 6.5, 15, and 30 min of incubation and subjected to RP-HPLC for determination of the molar ratio of (11S)-HPOD/(13R)-HPOD (Q). The amounts of (13R)-HPOD (open circle ) were calculated from the absorbance values using epsilon  = 26,000, and the amounts of (11S)-HPOD (bullet ) were obtained from the amounts of (13R)-HPOD multiplied with Q. A second addition of enzyme (0.5 µg) was made, as indicated by arrow. Panel B, same as A, but conducted with 526 µM linoleic acid. Aliquots for RP-HPLC analysis were removed at 7, 14, and 30 min. A second addition of enzyme (0.8 µg) was made, as indicated by arrow.

Metabolism of (11S)-HPOD by Mn-LO-- [1-14C](11S)-HPOD (60 µg) was obtained by incubations of a high concentration of [1-14C]linoleic acid (701 µM) with Mn-LO (2.2 µg) followed by isolation by RP-HPLC. That no degradation of the collected hydroperoxide had taken place was verified by analysis of an aliquot of the specimen by RP-HPLC radiochromatography. Treatment of (11S)-HPOD (52 µM) with Mn-LO resulted in the formation of (13R)-HPOD as judged by RP-HPLC radiochromatography (cf. Fig. 1). The identity of the hydroperoxide produced from (11S)-HPOD as (13R)-HPOD was based on its UV spectrum (solvent methanol; lambda max = 234 nm), its conversion into 13-HOD upon reduction with sodium borohydride, its conversion into 13-hydroxystearate upon reduction followed by catalytic hydrogenation, and on results of its chemical degradation by oxidative ozonolysis. As was the case with (13R)-HPOD isolated following incubation of linoleic acid with Mn-LO, the (13R)-HPOD produced from (11S)-HPOD was accompanied by small amounts of 9-hydroperoxyoctadecadienoates, i.e. (9R)-HPOD (1.2%) and (9S)-HPOD (1.7%).

Conversion of (11S)-HPOD into (13R)-HPOD was conveniently monitored by recording the UV absorbance at 235 nm versus time (Fig. 4). At the concentrations used, the maximum rate of formation of (13R)-HPOD from (11S)-HPOD, i.e. 18.3 nmol min-1 µg-1, was ~50% greater than that of formation of (13R)-HPOD from linoleic acid, i.e. 12.1 nmol min-1 µg-1. Interestingly, a slight lag phase was consistently observed when (11S)-HPOD was used as the substrate (Fig. 4).


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Fig. 4.   Time courses of formation of (13R)-HPOD from (11S)-HPOD (A) and linoleic acid (B). A, (11S)-HPOD (52 µM) in buffer C (1.05 ml) was treated with Mn-LO (1.1 µg) at 23 °C. The absorbance at 235 nm was measured using a cuvette having 10 mm path length. The maximum rate of increase of the absorbance at 235 nm was 0.50 absorbance unit/min corresponding to a rate of formation of (13R)-HPOD equal to 18.3 nmol min-1 µg-1. B, linoleic acid (53 µM) in buffer C (1.05 ml) was treated with Mn-LO (1.1 µg) at 23 °C and the absorbance was measured as described in A. The maximum rate of increase of the absorbance at 235 nm was 0.33 absorbance unit/min corresponding to a rate of formation of (13R)-HPOD equal to 12.1 nmol min-1 µg-1.

Incubations in the Presence of Glutathione Peroxidase-- Glutathione peroxidase and reduced glutathione catalyze reduction of fatty acid hydroperoxides into hydroxy acids. Trapping of hydroperoxides by this enzyme has been used in previous studies to confirm the existence of fatty acid hydroperoxides as intermediates in the biosynthesis of epoxy-hydroxy acids (25) and dihydroxy acids (26, 7). If the Mn-LO-catalyzed formation of (13R)-HPOD from linoleic acid took place by the sequence linoleic acid right-arrow (11S)-HPOD right-arrow (13R)-HPOD, inclusion of glutathione peroxidase and glutathione would be expected to result in high yields of 11-HOD, the reduction product of (11S)-HPOD, and in low yields of 13-HOD, the reduction product of (13R)-HPOD. In a typical experiment, a mixture of linoleic acid (224 µM), glutathione peroxidase (3 units) and reduced glutathione (3 mM) in 0.8 ml of buffer C was treated with Mn-LO (0.86 µg) at 23 °C. Spectrophotometric assay of the reaction mixture showed that the rate of formation of 13-H(P)OD was 2.1 nmol min-1 µg-1, a rate considerably lower than that observed in a control incubation carried out in the absence of glutathione peroxidase and reduced glutathione, i.e. 9.8 nmol min-1 µg-1. This reduced rate of formation of (13R)-HPOD in the presence of glutathione peroxidase was not due to a selective interference with formation of this hydroperoxide but to partial inhibition of linoleic acid oxygenation. Thus, analysis of the reaction product (20 min of incubation) by RP-HPLC showed that unconverted linoleic acid was the main component (75%). The remaining part of the product was due to 13-HOD (21%; formed by reduction of (13R)-HPOD) and a small percentage of 11-HOD (4%; formed by reduction of (11S)-HPOD).

Incubations of (11S)-HPOD with Soybean Lipoxygenase-- In order to test the possibility that (11S)-HPOD served as a substrate for soybean lipoxygenase, the hydroperoxide (48 µM) in 1 ml of buffer A was treated with soybean lipoxygenase (750 units) at 23 °C. No increase in the absorbance at 235 nm could be detected (Fig. 5). In another experiment, soybean lipoxygenase (600 units) in 1 ml of buffer B was treated with (11S)-HPOD (3 or 5 µM) for 2 min. Subsequently, linoleic acid (73 µM) was added. Measurement of the absorbance at 235 nm showed that the rate of conversion of linoleic acid by soybean lipoxygenase treated with (11S)-HPOD was virtually identical to that of untreated lipoxygenase (Fig. 5).


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Fig. 5.   Tests of (11S)-HPOD as substrate or inhibitor of soybean lipoxygenase. (11S)-HPOD (48 µM) was dissolved in buffer A (1 ml) at 23 °C, and soybean lipoxygenase (750 units) was added at time indicated by arrow. The absorbance at 235 nm was followed versus time (black-square). (11S)-HPOD (3 µM) in buffer B (1 ml) was preincubated with soybean lipoxygenase (600 units) at 23 °C for 2 min. Linoleic acid (73 µM) was added, and the absorbance at 235 nm was monitored (bullet ). As a control, linoleic acid (73 µM) in buffer B (1 ml) was treated with soybean lipoxygenase (600 units) (open circle ).

Isotope Experiments

18O2 Experiments-- The mechanism of formation of hydroperoxides catalyzed by Mn-LO was studied in experiments where linoleic acid, (11S)-HPOD, or (13R)-HPOD were incubated with Mn-LO in the presence of 18O2. The products were isolated by RP-HPLC (cf. Fig. 1), and aliquots were reduced with sodium borohydride, esterified, and analyzed by GC-MS as the Me3Si derivatives. In other experiments, [11-18O2](11S)-HPOD and [13-18O2](13R)-HPOD were biosynthesized from linoleic acid and reincubated with Mn-LO under ambient air.

Incubation of linoleic acid under 18O2 led to the formation of (11S)-HPOD and (13R)-HPOD that had incorporated 18O2 (Table I). Incubations of [11-18O2](11S)-HPOD with Mn-LO led to the formation of (13R)-HPOD that retained 39-44% of the 18O2 label. In addition, 9-HPOD, a minor hydroperoxide of Mn-LO catalysis, was labeled at the same level (incubations 2 and 4, Table I). In another experiment, (11S)-HPOD was incubated with Mn-LO under 18O2. As seen (incubation 5, Table I), this led to the formation of (13R)-HPOD that had incorporated a significant amount of 18O2. Furthermore, analysis of the 18O content of (11S)-HPOD remaining not converted in incubations of [11-18O2](11S)-HPOD conducted under air, and in an incubation of (11S)-HPOD under an atmosphere of 18O2, demonstrated a time-dependent exchange of the hydroperoxide oxygens with O2 (incubations 4 and 5, Table I). Such oxygen exchange was not observed when (13R)-HPOD was treated with Mn-LO (incubation 6, Table I).

                              
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Table I
18O experiments performed on conversions catalyzed by manganese-lipoxygenase

Incubations of Stereospecifically Deuterated Linoleic Acids-- (11S)-HPOD and (13R)-HPOD biosynthesized from (11R)-[2H]- and (11S)-[2H]linoleic acids were isolated by RP-HPLC and their isotope content determined by GC-MS after reduction, esterification, and conversion into the Me3Si derivatives. As seen in Table II, hydroperoxides produced from (11R)-[2H]linoleic acid retained most of the deuterium label, whereas hydroperoxides generated from (11S)-[2H]linoleic acid lost most of the label. These results demonstrated that the hydrogen abstracted from the C-11 methylene group by Mn-LO had the pro-S configuration. The isotope contents of linoleic acid remaining unconverted in incubations of (11R)-[2H]- and (11S)-[2H]linoleic acids were also determined. As seen (Table II), incubation of (11S)-[2H]linoleic acid was accompanied by a time-dependent enrichment of deuterium in unconverted linoleic acid. The presence of a kinetic isotope effect in the enzyme-catalyzed abstraction of the (11S) deuterium indicated by this experiment also manifested itself in the time course of formation of (13R)-HPOD from (11S)-[2H]linoleic acid measured spectrophotometrically. As seen in Fig. 6, production of (13R)-HPOD from (11S)-[2H]linoleic acid (32.6% deuterated and 67.4% undeuterated molecules) occurred by a biphasic time course. This was explainable if it is assumed that undeuterated linoleic acid present in the mixture incubated was mainly oxygenated in the early phase of the incubation (segment A in Fig. 6), whereas the gradually accumulating deuterated substrate was oxygenated at a much slower rate during the later phase (segments B and C in Fig. 6). Estimates of the rates of conversion of undeuterated and deuterated molecules could be made from the slopes of segments A-C, i.e. 7.9 nmol min-1 µg-1 for segment A (roughly corresponding to oxygenation of undeuterated linoleic acid), and 0.4-0.5 nmol min-1 µg-1 for segments B and C (roughly corresponding to oxygenation of deuterated linoleic acid). The magnitude of the kinetic isotope effect estimated from these rates was kH/kD = 15-22. The reported value of the isotope effect in the soybean lipoxygenase-catalyzed oxygenation of linoleic acid dideuterated at C-11 (94-95% dideuterated molecules) is kH/kD = 8-9 (27, 28); however, much larger values have been reported recently (29-31).

                              
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Table II
2H experiments performed on conversions catalyzed by manganese-lipoxygenase


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Fig. 6.   Rates of formation of (13R)-HPOD from deuterated linoleic acids. (11R)-[2H]Linoleic acid (411 µM) in buffer A (1.05 ml) was treated with Mn-LO (1.5 µg) at 23 °C. An aliquot was transferred to a cuvette having 1 mm path length, and the absorbance at 235 nm was monitored versus time (open circle ). Aliquots (0.3 ml) were removed at 3.5, 11.5, and 16 min of incubation and subjected to RP-HPLC. Hydroperoxides (3.5-min sample) and linoleic acid were collected, derivatized, and subjected to GC-MS analysis for determination of isotope content. (11S)-[2H]Linoleic acid (321 µM) was treated with Mn-LO (1.5 µg) in buffer A (1.05 ml) at 23 °C. An aliquot was transferred to a cuvette having 1 mm path length, and the absorbance at 235 nm was monitored versus time (bullet ). Aliquots (0.3 ml) were removed at 4.5, 15, and 49 min of incubation. Products were isolated by RP-HPLC, derivatized, and subjected to GC-MS analysis. Arrow indicates addition of additional Mn-LO (1.5 µg). The estimated rates of formation of (13R)-HPOD in time segments A, B, and C were 7.9, 0.5, and 0.4 nmol min-1 µg-1, respectively.

An interesting finding in the experiments with the stereospecifically deuterated linoleic acids was the slightly different deuterium contents of (11S)-HPOD and (13R)-HPOD biosynthesized from (11R)-[2H]linoleic acid (Table II). The optical purity of the (11S)-hydroxystearate used to prepare the (11R)-[2H]linoleic acid was 95.4% (18); therefore, the percentage retentions of deuterium observed for (11S)-HPOD (98%) and (13R)-HPOD (86%) were somewhat higher and lower, respectively, compared with the theoretical value (~95%). A secondary isotope effect has been described for the oxygenation of (10R)-[3H]arachidonic acid catalyzed by human platelet 12-lipoxygenase (32), and it is tempting to speculate that the slightly different extent of labeling of the two hydroperoxides formed from (11R)-[2H]linoleic acid in the presence of Mn-LO is due to a secondary isotope effect in the conversion of [11-2H](11S)-HPOD into [11-2H](13R)-HPOD (cf. Fig. 7).


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Fig. 7.   Mechanism proposed for oxygenation of linoleic acid catalyzed by Mn-LO. In the initial step, Mn-LO abstracts the pro-S hydrogen from C-11 of linoleic acid. The resulting linoleoyl radical is reversibly oxygenated at C-11 to produce an (11S)-peroxy radical that can be further converted into (11S)-HPOD. Alternatively, oxygen attack at the C-13 position of the linoleoyl radical results in irreversible formation of (13R)-HPOD. Free radical conversions are shown, although it is conceivable that heterolytic reactions may partly be involved (cf. Ref. 37). R1, (CH2)7-COOH; R2, (CH2)4-CH3.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Mn-LO purified from the fungus G. graminis as described in the accompanying paper (15) catalyzes conversion of linoleic acid into (13R)-HPOD as the major product. This transformation consisted of dioxygenation of a fatty acid possessing a (1Z,4Z)-pentadiene moiety into a fatty acid hydroperoxide having a 1-hydroperoxy-(2E,4Z)-pentadiene structure, thus satisfying the requirements for classifying Mn-LO as a lipoxygenase enzyme. The aim of the present study was originally to determine the stereochemistry of the biosynthesis of (13R)-HPOD using stereospecifically deuterated linoleic acids. In the course of this work, it became apparent that Mn-LO not only catalyzes transformation of linoleic acid into (13R)-HPOD but also other reactions, which are not characteristic of traditional lipoxygenases.

Experiments with linoleic acids labeled with 2H in the (11R) and (11S) positions demonstrated that Mn-LO, like soybean lipoxygenase-1, catalyzed abstraction of the pro-S hydrogen from C-11 of linoleic acid (Table II). As was found previously for soybean lipoxygenase (4, 27-31), a pronounced primary isotope effect resulting in accumulation of 2H in the unconverted substrate was noted in these experiments (Table II, Fig. 6). This result indicated that the first step of the Mn-LO-catalyzed oxygenation, like that of the soybean lipoxygenase-catalyzed oxygenation, consisted of hydrogen abstraction from the bis-allylic methylene group to produce a pentadienyl moiety. The overall steric course of formation of (13R)-HPOD from linoleic acid in the presence of Mn-LO consisted of hydrogen abstraction and oxygen insertion occurring in a suprafacial way (Fig. 7). This was in contrast to the antarafacial stereochemistry repeatedly observed for oxygenations catalyzed by soybean lipoxygenase and other lipoxygenases (4-10).

Analysis of the hydroperoxide product isolated following incubation of linoleic acid with Mn-LO demonstrated the presence of a second, less abundant component in addition to (13R)-HPOD (compound A, Fig. 1). The structure of this compound was determined by ultraviolet spectroscopy and mass spectrometry and by chemical methods. An important clue to the structure was provided by the finding that the hydroperoxy group of compound A was rapidly eliminated upon acid treatment to provide a mixture of 8,10,12- and 9,11,13-octadecatrienoates (Fig. 2). This type of conversion had earlier been observed with 11-HOD (21, 22), thus indicating that compound A was a bis-allylic hydroperoxide. Reduction of compound A by treatment with sodium borohydride afforded 11-HOD. The geometry of the two double bonds of this product was rigorously established as Z,Z by comparison with authentic 11-HOD and with chemically prepared (9E,12Z)-, (9Z,12E)-, and (9E,12E)-11-HOD. Partial hydrogenation of the methyl ester of 11-HOD derived from compound A followed by treatment with (-)-menthoxycarbonyl chloride and oxidative ozonolysis resulted in the formation of chiral fragments whose structures localized the double bonds of compound A to the Delta 9 and Delta 12 positions and demonstrated that the absolute configuration of C-11 was "S." The experiments thus allowed compound A to be formulated as (11S)-hydroperoxy-(9Z,12Z)-octadecadienoic acid ((11S)-HPOD). This bis-allylic hydroperoxide was a new oxylipin, although a geometrical isomer of the compound, i.e. methyl (11R,11S)-hydroperoxy-(9Z,12E)-octadecadienoate, had been prepared by a singlet oxygen reaction (results referred to in Ref. 33). Furthermore, a more unsaturated analog, i.e. 11-hydroperoxyoctadec-12-en-9-ynoic acid, was recently isolated following incubation of the lipoxygenase inhibitor octadec-(12Z)-en-9-ynoic acid with soybean lipoxygenase (34). Biosynthesis of (11S)-HPOD catalyzed by Mn-LO was accompanied by selective loss of the pro-S hydrogen from the carbon dioxygenated (Table II) and thus proceeded with retention of absolute configuration (Fig. 7).

The proportion between (11S)-HPOD and (13R)-HPOD depended on the time of incubation and the substrate concentration (Fig. 3). Short times of incubation of high substrate concentrations gave the highest yields of (11S)-HPOD and a ratio (11S)-HPOD/(13R)-HPOD equal to 0.31. As shown by RP-HPLC radiochromatographic analysis, the only product present in incubations where (11S)-HPOD had been allowed to disappear was (13R)-HPOD, thus suggesting that (11S)-HPOD was converted into (13R)-HPOD in the presence of Mn-LO. This could be directly demonstrated in experiments where (11S)-HPOD was incubated with Mn-LO and the ultraviolet absorbance due to (13R)-HPOD was monitored versus time. The time course of formation of (13R)-HPOD showed a characteristic sigmoidal shape because of the presence of a lag phase (Fig. 4). The maximum rate of formation of (13R)-HPOD from (11S)-HPOD was ~50% greater than that of formation of (13R)-HPOD from linoleic acid. Conversion of (11S)-HPOD into (13R)-HPOD was suppressed in the presence of high concentrations of linoleic acid (Fig. 3), indicating that (11S)-HPOD and linoleic acid competed for the same catalytic site of Mn-LO. The fact that (11S)-HPOD was converted into (13R)-HPOD in the presence of Mn-LO raised the question whether (11S)-HPOD served as an obligatory intermediate in the formation of (13R)-HPOD. The enzyme glutathione peroxidase, which traps fatty acid hydroperoxides as the corresponding hydroxy compounds, has been successfully utilized to prove the existence of hydroperoxides as intermediates in the biosynthesis of various oxylipins (7, 25, 26). Inclusion of glutathione peroxidase and reduced glutathione in incubations of linoleic acid with Mn-LO resulted in a decreased rate of formation of (13R)-H(P)OD; however, this was not caused by trapping of (11S)-HPOD but by partial inhibition of oxygenation of linoleic acid. Importantly, the ratio of the reduction products, (11S)-HOD/(13R)-HOD, did not increase in the presence of glutathione peroxidase, thus disfavoring the hypothetical sequence linoleic acid right-arrow (11S)-HPOD right-arrow (13R)-HPOD.

The mechanism of the conversion of (11S)-HPOD into (13R)-HPOD was studied by 18O experiments (Table I). In one set of incubations, linoleic acid was treated with Mn-LO under 18O2 and the hydroperoxides formed were isolated by RP-HPLC. Aliquots were reduced and analyzed by GC-MS as the methyl ester/Me3Si derivatives. As expected, (11S)-HPOD and (13R)-HPOD had both incorporated 18O. Reincubation of [11-18O2](11S)-HPOD with Mn-LO resulted in the formation of (13R)-HPOD that retained 39-44% of the 18O label. In a complementary experiment, incubation of (11S)-HPOD under 18O2 led to the formation of (13R)-HPOD that had incorporated 57% 18O. The findings that (13R)-HPOD only partially retained the 18O label when formed from [11-18O2](11S)-HPOD, and that the hydroperoxide had partially incorporated 18O when biosynthesized from (11S)-HPOD under 18O2, showed that the oxygen molecule migrating from C-11 to C-13 during the hydroperoxide rearrangement was subject to exchange with surrounding molecular oxygen. This fact, in turn, necessitated that the conversion of (11S)-HPOD into (13R)-HPOD took place in a stepwise way involving a deoxygenated intermediate. Interestingly, the oxygen exchange also manifested itself in the isotope content of (11S)-HPOD remaining not converted. As seen in Table I, there was a time-dependent loss of 18O from [11-18O2](11S)-HPOD incubated with Mn-LO under ambient air, and an incorporation of 18O from 18O2 into (11S)-HPOD incubated under 18O2. These results gave further support for the existence of a deoxygenated intermediate and additionally indicated that this intermediate could be reversibly oxygenated into (11S)-HPOD. Fig. 7 shows the mechanism proposed for the Mn-LO-catalyzed oxygenation of linoleic acid. The initial step consisted of abstraction of the pro-S hydrogen from C-11 to produce a linoleoyl radical. This intermediate was reversibly converted into (11S)-HPOD via the corresponding (11S)-peroxy radical, or irreversibly converted into (13R)-HPOD via the corresponding (13R)-peroxy radical. Chemical studies on fatty acid autoxidation have demonstrated that conversion of carbon-centered fatty acid radicals into peroxy radicals occurs by reversible binding of dioxygen (35). Furthermore, non-enzymatic free radical rearrangements of fatty acid hydroperoxides have been described (33, 36). In a study of rearrangement of hydroperoxides derived from linoleic acid (36), the methyl ester of (9S)-HPOD (incorrectly referred to as (9R)-HPOD in Ref. 36) was found to yield a mixture of the E,Z- and E,E-isomers of 9- and 13-HPOD methyl esters when kept in hexane solution under O2. When the reaction was performed under 18O2, partial incorporation of 18O into the hydroperoxides was observed. Although the 18O experiments of the present study demonstrated the existence of a non-concerted pathway involving a deoxygenated intermediate for the transformation of (11S)-HPOD into (13R)-HPOD, they did not exclude the possibility of an additional mechanism contributing to the formation of (13R)-HPOD, i.e. a direct conversion of the (11S)-peroxy radical into the (13R)-peroxy radical by concerted transfer of the peroxy radical oxygen from C-11 to C-13. Studies of the viability of such a contributing pathway are under way.

It is uncertain whether the mechanism proposed for Mn-LO involving a bis-allylic peroxy radical and a bis-allylic hydroperoxide has any relevance for other lipoxygenases such as soybean lipoxygenase. 11-Hydroperoxyoctadec-12-en-9-ynoic acid has been isolated as one of several products formed from an acetylenic inhibitor, octadec-(12Z)-en-9-ynoic acid, upon incubation with soybean lipoxygenase (34). However, despite extensive studies of soybean lipoxygenase-catalyzed oxygenations, formation of bis-allylic hydroperoxides from polyunsaturated fatty acids has never been reported. In the present study, (11S)-HPOD was tested as a substrate for soybean lipoxygenase with negative result. The possible roles of the corresponding (11S)-peroxy radical, and of (11R)-HPOD or its corresponding peroxy radical, as intermediates in soybean lipoxygenase catalysis remains to be examined.

    ACKNOWLEDGEMENT

We gratefully acknowledge the expert technical assistance of G. Hamberg.

    FOOTNOTES

* This work was supported by Swedish Medical Research Council Projects 03X-5170 and 6523 and by a grant from Vesical AB.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 46-8-728-7640; Fax: 46-8-736-0439; E-mail: mats.hamberg{at}mbb.ki.se.

1 The abbreviations used are: Mn-LO, manganese lipoxygenase; 9-H(P)OD, 9-hydro(pero)xy-(10E,12Z)-octadecadienoic acid; 11-H(P)OD, 11-hydro(pero)xy-(9Z,12Z)-octadecadienoic acid; 13-H(P)OD; 13-hydro(pero)xy-(9Z,11E)-octadecadienoic acid; GLC, gas-liquid chromatography; GC-MS, gas-liquid chromatography-mass spectrometry; FT-IR, Fourier transform-infrared; LC-MS, liquid chromatography-mass spectrometry; MC, (-)-menthoxycarbonyl; Me3Si, trimethylsilyl; RP-HPLC, reversed phase high performance liquid chromatography.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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