THE JOURNAL OF Bm~oorcn~ CHEMISTRY Vo1.245, No. 15, Issueof August 10, PP. 379+3797, 1970

Printed in U.S.A.

Stereospecific Hydration of the A9 Double Bond of Oleic Acid*

(Received for publication, February 18, 1970)

WALTER G. NIEHAUS, JR.,$ A. KISIC, A. TORKELSON, D. J. BEDNARCZYK, AND G. J. SCHROEPFER, JR.§

From the Division of Biochemistry, Department of Chemistry and Chemical Engineering, University of Illinois, Urbana, Illinois 61801

SUMMARY

A soluble (105,000 x g supernatant) enzyme preparation from a pseudomonad has been obtained which catalyzes the interconversion of oleic acid snd lOn-hydroxystearic acid. Evidence compatible with a mechanism involving a hydration of the double bond is presented. The crude enzyme preparation also catalyzes the formation of Alo- frans-octadecenoic acid from either oleic acid or lOWhy- droxystearic acid. The enzyme also catalyzes the formation of lo-hydroxypahnitic acid from palmitoleic acid. The enzyme did not catalyze the formation of an olefinic acid from 9D-hydroxystearic acid.

In 1962, Wallen, Benedict, and Jackson (1) reported the iso- lation of a pseudomonad which efficiently converted added oleic acid to IO-hydroxystearic acid. Subsequent studies have shown that this reaction is characterized by notable stereospecificity. The lo-hydroxystearic acid, which is formed in this reaction, is optically active (2, 3) and has the D configuration (3). Incuba- tion of this organism with oleic acid in a medium enriched with deuterium oxide yielded lo-hydroxystearic acid containing 1 atom of stably bound deuterium (4, 5). Moreover, this deu- terium was shown to be at carbon atom 9 and in the L configura- tion (Fig. 1). These findings are consistent with a mechanism involving stereospecific addition of the elements of water to the double bond of oleic acid, analogous to the hydration of fumaric acid catalyzed by the enzyme fumarase (6). However, the pos- sibility that the over-all conversion of oleic acid to IO-hydroxy- stearic acid proceeds by an initial epoxidation of the olefin fol- lowed by a reductive opening of the epoxide could not be excluded by our previous studies with the intact organism.

We now wish to report the preparation of a soluble (105,000 x g) extract of the organism which catalyzes the reversible hydra- tion of the double bond of oleic acid. Moreover, neither DL-

c&9, lo-epoxyoctadecanoic acid nor nn-trans-9, lo-epoxyocta- decanoic acid serves as a precursor of lo-hydroxystearic acid in

* This work was supported by Grant HE-09501 from t.he Na- tional Heart Institute.

$ Recipient of a postdoctoral research fellowship from the Nnt,ional Heart Institute. Present address. Denartment of Bio- chemistry, the Pennsylvania State University, ‘University Park, Pennsylvania 16802.

$ To whom inquiries should be addressed.

COOH

I

FIG. 1. Stereochemical course of the enzymatic conversion of oleic acid to lOn-hydroxystearic acid in deuterated medium.

this enzyme system. A preliminary account of portions of this work has been published (7).’

EXPERIMENTAL PROCEDURES AND RESULTS

General Procedures-Mass spectral analyses, recording of in- frared spectra, and measurements of melting points were carried out as described previously (9). Optical rotations were measured with a Durram Jasco spectropolarimeter with a cell with a 5-cm light path. Radioactivity was assayed in a Packard liquid scin- tillation spectrometer. A Barber-Colman model 5000 gas chro- matographic unit equipped with an argon ionization detector was used for the analysis of the fatty acid methyl esters. Separation of cis and trans isomers of the methyl esters of monounsaturated fatty acids was carried out by the method of Morris (10) with plates of Silica Gel G impregnated with silver nitrate (15%) with the solvent system, pentane-ether (9O:lO). Analysis of the radioactivity of the effluent from the gas-liquid chromato- graphic columns and analysis of the radioactivity on thin layer chromatoplates of Silica Gel G were carried out as described else- where (9). In the case of chromatoplates of Silica Gel G im- pregnated with silver nitrate, the same procedure could not be applied to the analysis of radioactivity because of the occurrence of significant and variable quenching. In these cases, increments of the adsorbent of the developed chromatoplate were scraped off the plate and placed on the top of a short column of activated silicic acid. The radioactive fatty acid methyl esters were eluted from the columns with ether. After evaporation of the solvent, the residue was dissolved in the standard scintillation mixture (9) for assay of radioactivity.

Methyl esters were prepared according to the procedure of

1 A preliminary account of this work was also presented at a meeting of the American Chemical Society (8).

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Schlenk and Gellerman (11). For the precise determination of the specific activities of the various fatty acids, the methyl esters were prepared and quantitatively determined by the hydroxa- mate method of Rapport and Alonzo (12). Protein was esti- mated by the method of Lowry et al. (13).

Incubations under anaerobic conditions were carried out in a helium-filled desiccator over alkaline pyrogallol as previously described (14).

Labeled Compounds and Reference Compounds- [l-14C]-Oleic acid (6.4 mCi per mmole), purchased from New England Nu- clear, was purified by silicic acid column chromatography and, in some cases, by thin layer chromatography of the methyl ester on plates of Silica Gel G impregnated with silver nitrate (15%) with the solvent system pentane-ether (90: 10). The radiopurity was judged to be in excess of 98% on the basis of: (a) thin layer radio- chromatographic analysis of the free acid on Silica Cel G (solvent, pentane-ether-acetic acid (9O:lO: l)), (b) thin layer radiochro- matographic analysis of the methyl ester on Silica Gel G im- pregnated with silver nitrate, (c) gas-liquid radiochromato- graphic analysis of the nethyl ester on a 12.5% diethylene glycol succinate column, and (d) gas-liquid radiochromatographic analysis of the diester formed after permanganate-periodate oxi- dation (15) of the fatty acid methyl ester. [l-14C]-Oleic acid purchased from Tracerlab was similarly purified before use. In some cases, the labeled oleic acid was diluted with unlabeled oleic

’ acid prior to use in enzymatic studies. [1-%I-Elaidic acid (trans-Ag-octadecenoic acid; specific ac-

tivity, -1.6 mCi per mmole) was prepared from [l-14C]-oleic acid by a modification of the nethod of Litchfield et al. (16). [l-14C]- Oleic acid (-1 mg) was dissolved in glacial acetic acid (5 ml) and, after cooling in ice, a 20% solution of sodium nitrite (3 ml) was added dropwise. The resulting mixture was stirred at room temperature for 1 hour. After dilution with water, the fatty acids were extracted with ether. The ether solution was washed with water and dried over anhydrous magnesium sulfate. The resulting product was treated with diazomethane and the fatty acid methyl esters were subjected to chromatography on an activated silicic acid column. The nonpolar fatty acid methyl ester fraction (eluted with 5% ether in pentane) was subjected to chromatography on plates of Silica Gel G impregnated with silver nitrate. The labeled material which corresponded chromato- graphically with authentic methyl elaidate was pooled and an aliquot was analyzed for radiopurity in the same chromat,o- graphic system. Less than 1% of the radioactivity was asso- ciated chromatographically with methyl oleate. The free acid was prepared by saponification of the methyl ester.

[IO-‘4C]-Palmitoleic acid (cis-Ag-hexadecenoic acid, 12 mCi per mmole) was purchased from Le Commissariat b 1’Energie Atom- ique, Gifsur-Yvette, France. The compound, after purification by silicic acid column chromatography, showed a single radio- active component on thin layer radiochromatographic analysis on a Silica Gel G plate (solvent, pentane-ether-acetic acid (90: 10 : 1)) and on gas-liquid radiochromatographic analysis of the methyl ester on a 12.5% diethylene glycol succinate column.

[l-14C]-nL-cis-9, lo-Epoxystearic acid (specific activity, 6.4 mCi per mmole) and [l-14C]-nL-2rans-9, lo-epoxystearic acid (specific activity, -1.6 mCi per mmole) were prepared from [VC]-oleic acid and [l-14C]-elaidic acid, respectively, by treat- ment of the olefinic acid with peracetic acid under the conditions described by Julietti et al. (17) and purified by chromatography on activated silicic acid columns. The radiopurity was judged

to be in excess of 95% on the basis of thin layer radiochromato-- graphic analysis on a Silica Gel G plate with the solvent system pentane-ether-acetic acid (75:25: 1). In some cases, the labeled epoxides were diluted with carrier prior to use in the enzymatic studies.

[I-14C]-cis-AQ-Octadecen-l-01 (specific activity, -6.4 mCi per mmole) was prepared from [l-14C]-oleic acid by reduction with excess lithium aluminum hydride in ether. The labeled alcohol was purified by thin layer chromatography on Silica Gel G. The radiopurity was judged to be in excess of 99% on the basis of thin layer radiochromatographic analysis on a Silica Gel G plate (solvent, petroleum ether-ether (70 :30)).

Methyl [12,13-%-9nhydroxystearate was prepared by reduc- tion of the Al2 double bond of methyl cis-A12-9n-hydroxyocta- decenoate with tritium-labeled diimide. To a solution of methyl cis-AiQ-9nhydroxyoctadecenoate (1.253 g) in tetrahydrofuran (200 ml) was added potassium azidoformate (39 g, prepared ac- cording to the method of Thiel (18) from azodicarbonamide). Tritiated acetic acid (900 mCi, prepared by mixing tritiated water (900 mCi; 0.18 ml) with acetic acid (7 ml)) was added and the reaction mixture was stirred at room temperature for 1 hour. Additional acetic acid (16 ml) was added in increments to the stirred reaction mixture over the next hour. Water (150 ml) was added and the resulting mixture was extracted with ether. The ether extract was washed three times with water and dried over anhydrous sodium sulfate. To remove unreacted olefinic fatty acid methyl ester, the crude product was purified by treat- ment with mercuric acetate followed by column chromatography. The crude product was dissolved in anhydrous methanol (75 ml) and heated under reflux for 75 min with mercuric acetate (6 g). After evaporation of the solvent and drying under reduced pres- sure over calcium chloride, the residue was applied to an acti- vated silicic acid column in benzene (100 ml). After passing a solution of 5% ether in pentane (400 ml) through the column, the crude methyl [12, 13-aH]-9n-hydroxystearate was obtained by elution with 30% ether in pentane. The white solid (682 mg) so obtained was recrystallized two times from methanol. The specific activity was 0.59 PCi per pmole. The radiopurity was judged to be in excess of 99% on the basis of thin layer radio- chromatographic analysis on a Silica Gel G plate (solvent, pe- troleum ether-ether-acetic acid (70: 30: 1)). The methyl ester (14.7 mg, -1.9 x lo7 dpm) was dissolved in methanol (2 ml) and 1 N NaOH (0.5 ml) and allowed to stand at room temperature for 5 hours. The reaction mixture was acidified and extracted with ether. The ether extract was washed with water and dried over anhydrous sodium sulfate. The radiopurity of the isolated acid was judged to be in excess of 99 y0 on the basis of thin layer radio- chromatographic analysis on a Silica Gel G plate (solvent, pe- troleum ether-ether-acetic acid (70: 30: 1)).

Unlabeled oleic acid and palmitoleic acid were purchased from the Hormel Institute, Austin, Minnesota. lOn-Hydroxystearic acid was prepared as previously described (3). SD-Hydroxy- stearic acid was a sample prepared previously (3). cisdlQ-9n- Hydroxyoctadecenoic acid was isolated from strophanthus oil, a generous gift from Professor K. Bloch, and purified by silicic acid column chromatography. The methyl ester was purified by column chromatography on an activated silicic acid column. nn-ciS-9,10-Epoxystearic acid, nn-trans-9, lo-epoxystearic acid, and nn-&s-Q, lo-epoxypalmitic acid were prepared from oleic acid, elaidic acid, and palmitoleic acid, respectively, by the method of Julietti et al. (17). Unlabeled elaidic acid was prepared from oleic

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00- 201

CO-55 07 172

74 69

40-

8

f 264 E 2632f6

5 60 SO 100 120 140 160 100 200 220 240 260 260 300 - w 1 IOO- 169 s 2 EO- 201

172

60 - 07 55 74

40 - 69

264 2832, I I

60 00 100 I20 I40 I60 I60 200 220 240 260 280 300

FIG. 2. Mass spectra of methyl lo-hydroxyoctadecanoates. Bottom, authentic methyl lOn-hydroxyoctadecanoate; top, methyl 10nhydroxyoctadecanoate formed from oleic acid in presence of enzyme. All peaks in the range m/e 50 to 320 which were at least 1% of the intensity of the base peak have been plotted.

acid by the method of Litchfield et al. (16). g(lO)-Hydroxypal- mitic acid was prepared by reduction of cis-9, lo-epoxypalmitic acid under conditions described by Mack and Bickford (19) and Light, Lennarz, and Bloch (20). Dimethyl octanedioate, non- anedioate, and decanedioate were purchased from Applied Sci- ence Laboratories, Inc. (State College, Pennsylvania).

Activated silicic acid (Unisil, 100 to 200 mesh) was purchased from the Clarkson Chemical Company (Williamsport, Pennsyl- vania). Silica Gel G for thin layer chromatography was obtained from Brinkmann. The petroleum ether used in these studies had a boiling range of 30-60”.

Preparation of Bacterial E&act-The organism (NRRL-B- 2994) was grown in 15-liter carboys in a medium containing po- tassium phosphate (37 mM), ammonium chloride (40 mM), glu- cose (11 .l mM), potassium oleate (5 mrvr), and inorganic salts (MgSO4, 1.5 mM; NazMoO+ 0.02 mM; NaCl, 0.021 mM; MnCL, 0.4 mM; CaC&., 0.03 mM; FeS04, 0.04 mM). The pH of the medium was 8.0. The bacteria were harvested in early sta- tionary phase with a Sharples centrifuge. The cells were sus- pended in Tris-Cl buffer (20 InM, pH 8.0, 2 ml per g of cellular paste) containing mercaptoethanol (2 mM) and subjected to sonic oscillation with a Branson sonic probe (6 amps, loo-ml volume) for 4 min at 4”. The supernatant fraction obtained after centrifugation at 20,000 x g for 20 min was recentrifuged for 90 min at 108,000 x g. The resulting supernatant fluid was used as a source of the enzyme for this study.

Enzyme-catalyzed Formation of lOD-Hyclroxystearic Acid from Oleic Acid: Characterization of Product-[1-“Cl-Oleic acid (300 mg, 3100 cpm per pmole) was neutralized with KOH and dis- solved in water (35 ml). Bovine serum albumin (2.8 g) was added, and the pH of the resulting mixture was adjusted to 8.0 with dilute HCl. The crude enzyme (25 ml, 275 mg of protein) was added, and the resulting mixture was incubated with shaking for 5 hours at 30”. The incubation mixture was heated on a steam bath with 10% methanolic KOH for 1 hour, and, after

TABLE I Optical activity of IO-hydroxy fatty acid methyl esters

Rotations were measured in methanol. Path length was 0.5 dm. Sample I, methyl lOn-hydroxyoctadecanoate derived from incubation of pseudomonad with oleic acid; Sample II, IO-hy- droxyoctadecanoate derived from incubation of oleic acid with soluble enzyme preparation.

Specific rotation ([a] rt S.D.)

Wave length

w

589 500 436 400 365 313 280 250

Sample I cc, 9.57)

-0.18 & 0.01” -0.13 f 0.03” -0.28 + 0.01 -0.19 i 0.02 -0.41 & 0.01 -0.52 zk 0.03 -0.45 rt 0.01 -0.58 * 0.02 -0.58 III 0.01 -0.67 zk 0.02 -0.86 j, 0.01 -0.83 xk 0.04 -1.17 f 0.02 -1.33 f 0.07 -1.57 f 0.09 -1.76 f 0.19

acidification to pH 1 with dilute HCl, the mixture was extracted several times with ether. The ether solution was dried over anhydrous magnesium sulfate, and, after evaporation of the sol- vent, the residue was applied to an activated silicic acid column. The nonpolar fatty acids were eluted with 10% ether in pentane, and the hydroxy fatty acid fraction was eluted with 50% ether in pentane. The latter material was treated with diazomethane and the resulting methyl ester was recrystallized from acetone- water, yielding 71 mg of melting point 55-56’ (undepressed upon mixture melting point with authentic methyl lOn-hydroxy- octadecanoate). The specific activity was 3290 cpm per pmole, indicating the absence of any dilution of the enzymatically formed product with IO-hydroxystearic acid in the enzyme prep- aration. Upon thin layer chromatographic analysis on a Silica Gel G plate (solvent, pentane-ether-acetic acid (80:20: l)), the

product showed a single spot which corresponded to the mobility of authentic methyl IOn-hydroxyoctadecanoate. Gas-liquid chromatographic analysis on a 5% diethylene glycol succinate column and on a 3.8% SE-30 column showed a single component with the same mobility as the authentic compound. The infra- red spectrum and the mass spectrum (Fig. 2) were essentially the same as authentic methyl 1Onhydroxyoctadecanoate. A major peak in the spectrum of this compound is that at m/e 201 repre- senting the fragment HOCH-(CHt)-COOCH3 (5, 21). The absence of analogous peaks at m/e 187 and m/e 215 exclude the presence of significant ( > 1%) contamination with methyl 9-hy- droxyoctadecanoate and 11-hydroxyoctadecanoate, respectively. The optical rotation of the methyl ester was measured at several wave lengths and was essentially the same as that of authentic methyl 10nhydroxyoctadecanoate (Table I).

A sample of [lJ4C]-lo-hydroxystearic acid (specific activity, -33 PCi per mg) was prepared by incubation of [1-i4C]-oleic acid with the bacterial extract. After purification by silicic acid col- umn chromatography, the radiopurity of the hydroxy fatty acid

was judged to be in excess of 99 y. on the basis of thin layer chro- matographic analysis on a Silica Gel G plate.

Thin Layer Radiochromatographic Assay of Conversion of Oleic Acid to iO-Hydroxgocladecanoic Acid-The following procedure was adopted as a simple assay of the conversion of oleic acid to

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TABLE II Enzymatic conversion of oleic acid to IO-hydroxyoctadecanoic acid

Incubation conditions: 3.55 pmoles of [1-“Cl-oleic acid (in 0.1 ml of albumin solution), 0.2 ml of enzyme extract (2.4 mg of pro- tein), 1.0 ml of Tris buffer (0.02 M, pH 8.0), 1 hour at 30”.

Radioactivity associa~~hchromatographicaIly”

IllCllbatiOIl

IO-Hydroxycydtadecanoic

I Oleic acid

%

Aerobic 93 7

93 7

Anaerobic a7 13 90 10

Boiled enzyme <O.l >99

0 Thin layer radiochromatographic analysis on a Silica Gel G

-1

FIG. 3. Enzymatic conversion of oleate to IO-hydroxystearate

IO-hydroxyoctadecanoic acid. [IX]-Oleic acid was diluted with unlabeled oleic acid, neutralized with dilute aqueous KOH, and mixed with bovine serum albumin, and the pH of the result- ing solution (10 mg of oleic acid per ml, 85 mg of albumin per ml) was adjusted to pH 8. The solution of the substrate was incu- bat.ed with varying amounts of the enzyme in Tris-Cl buffer (0.02 M, pH 8) at 30’. The incubations were terminated by the addition of hot 15% ethanolic KOH. After heating for 3 hours on a steam bath, the incubation mixtures were acidified to pH 1, and the fatty acids were extracted with ether. Aliquots of the ether extracts were applied to Silica Gel G thin layer plates along with unlabeled oleic acid and IO-hydroxystearic acid. After development of the plates (solvent, pentane-ether-acetic acid (90:15:1)), l-cm increments of the silica gel were scraped into counting vials with a device similar to that described by Snyder (22) and assayed for radioactivity as described elsewhere (9). Fig. 3 shows the amounts of lo-hydroxystearic acid formed when 3.55 pmoles of the [V4C]-oleic acid were incubated with varying amounts of the bacterial extract for 60 min at 30’. The total volume was 1.2 ml. The amount of IO-hydroxystearate formed was a linear function of the amount of enzyme protein.

The reaction proceeded under anaerobic conditions. Boiled enzyme controls were negative (Table II).

Enzyme-catalyzed Formation of IO-Hydroxyhexadecanoic Acid from cis-Ag-Hexaclecenoic Acid-[ 10-14C]-Palmitoleic acid (5 x 10’ cpm) in ethanol (0.1 ml) was incubated with the enzyme prep- aration (0.1 ml, 2 mg of protein) in Tris-Cl buffer (1.0 ml, pH 8) for 1 hour at 30” as described above. The fatty acids were iso- lated from the incubation medium and a portion of this material was subjected to thin layer radiochromatographic analysis on a Silica Gel G plate as described above. Approximately 20% of the radioactivity was associated chromatographically with carrier (9( IO) -hydroxyhexadecanoic acid). The remainder of the radio- activity had the same mobility as authentic A~-cis-hexadecenoic acid. Another portion of the fatty acids recovered from the incubation medium was treated with diazomethane, and the re- sulting methyl esters were subjected to gas-liquid radiochromato- graphic analysis on a column of 5% diethylene glycol succinate on acid-washed Chromosorb W (60 to 80 mesh). The column temperature was 170” and the inlet pressure was 12 psi. The effluent from the column was collected and assayed for radio- activity as described elsewhere (9). The resulting chromatogram is shown in Fig. 4. Approximately 18% of the recovered radio- activity was associated chromatographically with methyl 9(10)-

plate.

5

A

i’->-+iq7- T=- ~ IO 20 30 40

Time (minutes)

FIG. 4. Gas-liquid radiochromatogram of products of incuba- tion of [10-14C]-palmitoleic acid with bacterial enzyme. A, methyl palmitoleate; B, methyl oleate; C, methyl 9(10)-hydroxypalmitate.

hydroxyhexadecanoate; the remainder emerged from the column with authentic methyl palmitoleate.

To obtain sufficient material for analysis by infrared and mass spectroscopy, an incubation of the enzyme with larger quantities of the substrate was carried out. Ag-cis-Hexadecenoic acid (20 mg) was incubated for 3 hours at 30” with the enzyme prep- aration (5 ml, 100 mg of protein in Tris-Cl buffer (5 ml, pH 8.0)) as described in the case of the large scale incubation of the enzyme with oleic acid. The acids recovered from the incubation me- dium were separated into nonpolar fatty acids and hydroxy fatty acids by chromatography on an activated silicic acid col- umn as described above. The hydroxy fatty acids (-5 mg) were treated with diazomethane and the resulting methyl esters were subjected to preparative gas-liquid chromatography on an SE-30 column to separate the desired product, methyl hydroxyhexadec- anoate, from small amounts of the corresponding &-hydroxy fatty acid endogenous in the crude enzyme preparation. Ap- proximately 1.2 mg of methyl hydroxyhexadecanoate were thus obtained which was judged to be pure (98%) on the basis of gas- liquid chromatographic analysis on an SE-30 column and thin layer chromatographic analysis on a Silica Gel G plate. The infrared spectrum showed absorption at -3500 cm-l as a result of O-H stretching. The mass spectrum of the methyl ester is

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?2 40

s d 20 a

0 60 60 100 120 140 160 160 200 220 240 260 260

m/e

FIG. 5. Mass spectrum of methyl lo-hydroxyhexadecanoate derived from the action of the enzyme on cisdg-hexadecenoic acid. All peaks in the range m/e 50 to 300 which were at least 1% of the intensity of the base peak have been plotted.

shown in Fig. 5. The fragmentation pattern of the isolated hydroxy fatty acid methyl ester upon electron impact is compati- ble with the structure methyl IO-hydroxyhexadecanoate. The mass spectrum of authentic methyl IO-hydroxyhexadecanoate has not been published previously. However, a large number of other hydroxy fatty acid methyl esters have been studied in con- siderable detail (5, 21, 23-25). Based on the results of these studies, the following major ions (and their tentative assign- ments) are expected in the mass spectrum of methyl lo-hydroxy- hexadecanoate: m/e 286 (M), m/e 268 (M-HzO), m/e 255 (M-OCHa), m/e 236 (M-H=JO--CH~OH), m/e 201 (+HOCH -(CH2)-COOCHa), m/e 172 (+(CHz)-COOCHs + H), and m/e 169 (201-CH30H). These peaks are present in the mass spectrum of the isolated hydroxy fatty acid methyl ester (Fig. 5).

The enzyme extract also catalyzed the conversion of linoleic acid (cis, cis-Ags12-octadecadienoic acid) to cis-A12-lOn-hydroxy- octadecenoic acid (26).

Lack of Enzymatic Action on Elaidic Acid (trans-A@-Octadece- noic Acid-[lJ4C]-Elaidic acid (1 x lo6 cpm) in 95% ethanol (0.1 ml) was incubated with the enzyme extract (2 mg of protein, 0.2 ml) in Tris-Cl buffer (1.0 ml, pH 8.0) for 30 min as described above. After saponification and acidification to pH 1, the fatty acids were extracted with ether and the amounts of radioactivity associated chromatographically with authentic lo-hydroxy- stearic acid and oleic acid (same chromatographic mobility as elaidic acid in this system) were assayed by thin layer radio- chromatography on Silica Gel G plates (solvent, pentane-ether- acetic acid (85: 15:2)). In two separate experiments, less than 0.2% of the recovered radioactivity was associated chromato- graphically with the hydroxy fatty acid. Under the same ex- perimental conditions (with the same enzyme preparation), oleic acid was converted to lo-hydroxystearic acid to the extent of 20%.

Lack of Enzyme Action on So-Hydroxysteatic Acid--[la, 13-aH]- SD-Hydroxystearic acid (1.4 mg, -1.8 x lo6 cpm) was dissolved in a solution of KOH (0.33 ml, 0.425 mg per ml) and incubated with the enzyme preparation (5 ml, 9.4 mg of protein per ml, pH 8.1) for 2) hours at 10”. Methanolic KOH (5 ml, 10%) was added and the resulting mixture was heated on a steam bath for 30 min. After acidification to pH 1, the mixture was extracted with ether. The ether extract was washed with water and dried over anhydrous sodium sulfate. An aliquot of the ether extract was applied to a Silica Gel G plate, After development (solvent, petroleum ether-ether-acetic acid (70:30:1)) of the plate, the distribution of radioactivity was determined as previously de-

TABLE III Conversion of lo-hydroxyoctadecanoic acid to oleic acid and lack of

conversion of cis- or trans-9,10-epoxyoctadecanoic acid to oleic

acid or IO-hydroxyoctadecanoic acid Incubation conditions: 3 to 18 mpmoles of substrate in 0.1 ml of

95% ethanol, 1.0 ml of Tris buffer (0.02 M, pH 8.0), 0.1 ml of en- zyme extract (1.2 mg of protein), 2 hours at 30”.

Substrate

[I-WI-lo-Hydroxyoctadecanoic acid

[I-‘%]-lo-Hydroxyoctadecanoic acid, boiled 99.5 <0.5 enzyme extract 99.7 <0.3

[l-‘4C]-Oleic acid 77 82

<I <I

<0.3 <0.3

<0.2 <0.2

23 18

[I-WI-Oleic acid, boiled enzyme extract

[1-I%]-DL-C~S-9, lo-Epoxyoctadecanoic acid

[l-14C]-DL-trans-9, lo-Epoxyoctadecanoic acid

lecovered radioactivity associated chromato-

graphically” with

D-Hydroxy- :$gd;;id Oleic acid

%

85 15” 80 14 90 10

1 -

99 99

<0.2 <0.2

<0.2 <0.2

a Thin layer radiochromatographic analysis on a Silica Gel G plate.

b Subsequent detailed analysis of this material indicated that 95% of this material represented &-As-octadecenoic acid and 5% represented tralzs-A1O-octadecenoic acid.

scribed. No significant conversion ( <0.7 %) of the Sn-hydroxy- stearate to oleate was observed. Under essentially the same conditions (with the same enzyme preparation) [lJ4C]-lOn-hy- droxystearic acid was converted to material with the chromato- graphic mobility of oleic acid (12.3% conversion) and [1J4C]- oleic acid was converted to material with the chromatographic mobility of IO-hydroxystearic acid (90% conversion).

Lack of Enzyme Action on Oleyl Alcohol (cisdg-Ocladecen-l-01) -[l-14C]-Oleyl alcohol (2.45 x lo5 cpm) in dioxane (0.1 ml) was incubated with the bacterial enzyme (5 mg of protein, 0.5 ml) in Tris-Cl buffer (0.3 ml, pH 8.0) for 30 min at 10”. At the end of the incubation the mixture was acidified to pH 1 with 2 N HCl. Methanol (0.5 ml) was added and the resulting mixture was ex- tracted three times with ether (5-ml portions). The ether ex- tract was dried over anhydrous sodium sulfate and an aliquot was applied to a Silica Gel G plate. After development (solvent, ether-petroleum ether (70:30)) the distribution of radioactivity on the plate was determined as described previously. No sig- nificant incorporation ( <0.6%) of the label of [l-14C]-oleyl alco- hol into material showing the chromatographic behavior of 1, lo- octadecane-diol was observed. Upon incubation of [I-14C]-oleic acid (0.5 pmole) with the same enzyme preparation under the same conditions, efficient conversion (51 To) to 10.hydroxystearic acid was observed.

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Issue of August 10, 1970 Niehaus, Kisic, Torkelson, Bednarczyk, and Xchroepfer 3795

Possible Reaction Mechanism: Possible Intermediate of Epoxy- octadecanoic Acid in Conversion of Oleic Acid to lo-Hydroxy- octadecanoic Acid--The results of our previous studies with the intact organism are compatible with a mechanism involving a stereospecific addition of the elements of water across the double bond (Fig. 1). However, the possibility that the over-all con- version of oleic acid to lo-hydroxyoctaclecanoic acid proceeds by way of an initial epoxidation of the olefin followed by a reductive opening of the epoxide ring cannot be excluded by our previous findings with the intact organism. To test this possibility [ lJ4C]-nL-cis-9, 10.epoxyoctaclecanoic acid and [l-14C]-nL-trans- 9, lo-epoxyoctadecanoic acid were incubated with the enzyme preparation. No conversion to lo-hydroxyoctadecanoic acid or oleic acid was observed (Table III). However, as noted in the accompanying paper (27), the epoxy acids were enzymatically hydrated, yielding 9, lo-dihydroxyoctadecanoic acids.

Reversibility of Conversion of Oleic Acid to IO-Hydroxyoctadeca- noic Acid and Formation of trans-ArQ-Octadecetic Acid-The re- sults of incubations of [1-WI-lo-hyclroxyoctaclecanoic acid (Table III) indicated the reversible nature of the reaction. In one experiment 86% of the recovered radioactivity cochromato- graphed with lo-hyclroxyoctadecanoic acid on thin layer chro- matographic analysis on a Silica Gel G plate. The remaining I4 y0 of the recovered radioactivity cochromatographecl with authentic oleic acid. The latter material was applied to an activated silicic acid column and elutecl with 10% ether in pen- tane. The recovered material was methylated with diazo- methane. The methyl ester was subjected to thin layer raclio- chromatographic analysis on a Silica Gel G plate. Over 99% of the radioactivity was associated chromatographically with au- thentic methyl oleate. On gas-liquid radiochromatographic analysis on a diethylene glycol succinate column, over 95% of the radioactivity showed the same retention time as the methyl oleate standard. The labeled unsaturated methyl ester was oxi- dized withpermanganate-periodateand assayed, in the form of the methyl esters, by gas-liquid radiochromatography on a &ethylene glycol succinate column. Approximately 3, 90, and 6% of the recovered radioactivity was associated chromatographically with authentic &methyl octaneclioate, &methyl nonaneclioate, and dimethyl decanedioate, respectively. On thin layer radio- chromatographic analysis of the unsaturated methyl ester on a Silica Gel G-silver nitrate plate approximately 95% of the re- covered radioactivity cochromatographecl with authentic methyl oleate. Approximately 5% of the recovered radioactivity showed the same chromatographic mobility as authentic methyl elaidate. This combination of findings indicates that oleic acid was formed from the labeled lo-hydroxyoctadecanoic acid. In addition, these findings suggest the possible formation of a small amount of trans-A10-octadecenoic acid.

Further studies were carried out in order to characterize the products of the reaction more fully. [lJ4C]-Oleic acid (90 PCi, 14 pmoles) was dissolved in a dilute solution of KOH (pH 8.0) and incubated with 5 ml of the enzyme extract (100 mg of protein) for 4 hours at 30”. The reaction mixture was acidified with dilute HCl and extracted several times with ether. The ether extract was washed with water and dried over anhydrous magnesium sulfate. Thin layer racliochromatographic analysis on a Silica Gel G plate showed 84% conversion to lo-hyclroxyoctadecanoic acid. The fatty acids were applied to an activated silicic acid column. The unsaturated fatty acids were elutecl with 10% ether in pentane and the hydroxy fatty acid was elutecl with 50%

ether in pentane. The racliopurity of the isolated [1-14C]-10- hyclroxyoctadecanoic acid was judged to be in excess of 98% on the basis of thin layer radiochromatographic analyses of the free acid and the methyl ester (Silica Gel G plates) and gas-liquid (3.8% SE-30 column) radiochromatographic analysis of the methyl ester.

The labeled IO-hydroxyoctaclecanoic acid (1.1 x 10s cpm) was dissolved in 6 ml of KOH (pH 8.0) and incubated with 5 ml of the enzyme extract (100 mg of protein) for 4 hours at 30”. The re- action mixture was heated with methanolic KOH, cooled, acidi- fied with dilute HCI, and extracted repeatedly with ether (99.5% recovery of the incubated radioactivity). The ether layer was washed with water and dried over anhydrous magnesium sulfate. An aliquot was subjected to thin layer radiochromatographic analysis on a Silica Gel G plate. Approximately 23% of the radioactivity was associated with oleic acid and the remainder of the radioactivity was associated with lo-hyclroxyoctadecanoic acid. The unsaturated fatty acids were isolated by chromatog- raphy on an activated silicic acid column as described previously. Thin layer radiochromatographic analysis of the unsaturated fatty acids showed that over 99% of the radioactivity was asso- ciated with authentic oleic acid. The methyl esters were pre- pared by treatment with diazomethane. A portion of the ma- terial was analyzed by thin layer radiochromatography on a Silica Gel G-silver nitrate plate. Approximately 69% of the radioactivity cochromatographecl with authentic methyl elaidate. The remainder of the radioactivity chromatographed with au- thentic methyl oleate. The labeled monounsaturated fatty acid methyl esters were separated into cis and trans fractions by pre- parative thin layer chromatography on plates of Silica Gel G- silver nitrate with a mixture of pentane and ether (9O:lO) as the developing solvent. The isolated cis- and tram-unsaturated fatty acid methyl esters were shown to be at least 95% pure by radiochromatography with the same method. The isolated labeled cis- and trans-fatty acid methyl esters cochromatographecl with authentic methyl oleate on gas-liquid chromatography on a 12.5% &ethylene glycol succinate column.

The labeled &-unsaturated fatty acid methyl ester sample was subjected to permanganate-periodate oxidation (15). After treatment with diazomethane and the addition of authentic samples of unlabeled climethyl octaneclioate, climethyl nonane- clioate, and climethyl clecaneclioate, the resulting labeled di- methyl esters were analyzed by gas-liquid radiochromatography. The results (Table IV) are compatible with a localization of the double bond in the &-unsaturated fatty acid at the A9-position.

TABLE IV

Gas-liquid radiochromatographic analyses of distribution of radio- activity following permanganate-periodate oxidation of

unsaturated fatty acids

Radioactivity associated chromatographically with

Dimethyl octanedioate nonanedioate

I I

Dimethyl Dimethyl decanedioate

cis-Monounsaturated fatty acid 5 methyl ester 17

trann-Monounsaturated fatty acid methyl ester

I % 94 1 82 1

3 97 1 99

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3796 Enzymatic Hydration of Oleic Acid Vol. 245, No. 15

6 12 16 24 30 36

TIME (min)

FIG. 6. Time course of the distribution of radioactivity upon incubation of Il-‘4Cl-oleic acid with the bacterial enzyme nrenara- tion. o-d, oleic acid; O-O, lo-hydroxystearic acid; A---& trans-A1o-octadecenoic acid.

The labeled truns-unsaturated fatty acid sample was analyzed similarly. The results (Table IV) are compatible with a locali- zation of the double bond in the trans-unsaturated fatty acid at t.he AiO-position.

The enzymatic formation of the labeled truns-acid could also be detected when [1-14C]-oleate was used as the substrate. Fig. 6 shows the distribution of label in lo-hydroxyoctadecanoate, trans-A10-octadecenoate, and oleate at varying times upon incu- bation of [1-%I-oleate (2.83 pmoles) with 20 ml of the enzyme extract (370 mg of protein) and Tris-Cl buffer (90 ml, 0.02 M) at 10”. The final pH was 8.3. Little formation of the trans.acid was observed under these conditions.

The activity leading to the formation of labeled trans-ArO- octadecenoate from either labeled oleate or labeled lo-hydroxy- octadecanoate was highly variable. In most cases, this activity was very low relative to the conversion of oleate to lo-hydroxy- octadecanoate. In many cases no formation of trans.Ai”-octa- decenoate could be detected. These findings suggest that the formation of the trans-acid might be due to the presence of a sec- ond enzyme in the crude soluble enzyme extract.

DISCUSSION

Previous work with the intact organism established that the conversion of oleic acid to lo-hydroxystearic acid by a pseudo- monad (1) was stereospecific (2, 3). The 10.hydroxystearic acid was found to be optically active (2,3) and was shown to have the D configuration (3). Incubation of the organism with oleic acid in a medium enriched with deuterium oxide yielded lo-hydroxy- stearic acid containing 1 atom of stably bound deuterium which was shown to be localized at carbon atom 9 in the L configuration. While these findings are compatible with a mechanism involving a stereospecific hydration of the double bond, the possibility that the over-all conversion of oleic acid to lo-hydroxystearic acid proceeds by an initial epoxidation followed by a reductive open- ing of the epoxide could not be excluded by our previous findings. It should be noted that Vioque and Maza (28) have reported the isolation of an optically active lo-hydroxystearic acid from sulfur olive oil and suggested its possible origin from trans-9, lo-epoxy- stearic acid, which also occurs in this oil (29). Knoche (30) has

also suggested the possible intermediate role of an epoxide in the over-all conversion of oleic acid to IO-hydroxystearic acid. The successful preparation of a soluble extract of the organism for use in enzymatic studies permitted clarification of this problem. The possibility of an intermediary epoxy fatty acid in the over-all conversion of oleic acid to IO-hydroxystearic acid is rendered extremely unlikely by several observations made in this study. First, the enzymatic conversion of oleate to lo-hydroxystearate was observed to proceed under anaerobic conditions, a feature not characteristic of enzymatic epoxidations of olefins. Second, neither the nn-&s-Q, lo-epoxystearate nor the nn-trans-epoxy- stearate served as precursor of either oleate or IO-hydroxystearate under the conditions studied. These findings, coupled with the observations reported previously, are compatible with a mecha- nism involving a stereospecific addition of the elements of water across the double bond of oleic acid.

The stereospecific nature of the conversion of the oleate to lo- hydroxystearate observed with the intact organism was confirmed in the case of the soluble enzyme preparation. Within the limits of detection (-1%) the only hydroxy fatty acid formed is the lo- hydroxy compound. Moreover, the isolated IO-hydroxystearic acid is in the form of the methyl ester, levorotatory in methanol, and hence the absolute configuration of the hydroxyl function is D. The soluble enzyme was specific for the cis configuration of the Cis-Ag-olefinic acid since the truns isomer (elaidic acid) of oleic acid was not hydrated by the enzyme. Although the range of specificity of the enzyme has not been exhaustively studied, the enzyme does hydrate the AQis double bonds of palmitoleic acid and linoleic acid, yielding the corresponding lo-hydroxy fatty acids. The enzyme did not catalyze the formation of an olefinic acid from SD-hydroxystearic acid.

In addition to the catalysis of the reversible interconversion of lOn-hydroxystearic acid and oleic acid, the enzyme also catalyzes the formation of AlO-truns-octadecenoic acid from added HOD- hydroxystearic acid or oleic acid. Whether the formation of the AlO-&ens-acid is due to the presence of a second enzyme in the crude soluble enzyme extract or is due to the same enzyme which catalyzes the reversible interconversion of 10nhydroxystearate and oleate is not known. Further experiments are in progress to clarify this point.

This report constitutes a description of one of the few examples of an enzymatic reaction which includes the introduction of an isolated double bond by a mechanism involving the dehydration of an alcohol. The demonstration of this specific type of reaction was previously sought (without success) in experiments designed to investigate the possible role of 9- or lo-hydroxystearate in the over-all enzymatic conversion of stearate to oleate in the yeast Saccharomyces cerevisiae (20), the slime mold Dictyostelium discoideum (31)) and Euglena gracilis (32). The results of stud- ies with the intact pseudomonad of the conversion of oleate to lo-hydroxystearate indicate the stereospecific addition of solvent hydrogen at carbon atom 9 in the L configuration (4, 5). Al- though not directly tested in the soluble enzyme extract, the re- verse reaction, the conversion of lo-hydroxystearate to oleate, presumably involves stereospecific removal of the same hydrogen from carbon atom 9. It is important to note that this is in con- trast to the results of studies of the stereochemistry of the hydro- gen removal from carbon atom 9 in the over-all enzymatic con- version of stearate to oleate by Corynebacterium diphtheriae (2,3). In the latter case, the hydrogen removed from carbon atom 9 is that in the D configuration.

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Schroepfer, Jr.Walter G. Niehaus, Jr., A. Kisic, A. Torkelson, D. J. Bednarczyk and G. J.

Double Bond of Oleic Acid9∆Stereospecific Hydration of the

1970, 245:3790-3797.J. Biol. Chem. 

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