an enzymaticconversionof lipoxygenase products ... · preparation andpurification of hydroperoxides...

Plant Physiol. (1989) 91, 1280-12870032-0889/89/91/121 2/08/$01 .00/0

Received for publication June 1, 1989and in revised form July 18, 1989

An Enzymatic Conversion of Lipoxygenase Products by aHydroperoxide Lyase in Blue-Green Algae (Oscillatoria sp.)

Rivo-Hery Andrianarison, Jean-Louis Beneytout*, and Marie Tixier

Laboratoire de Biochimie, Facult6 de Pharmacie, 2, rue du Docteur Marcland, 87025 Limoges C6dex, France

ABSTRACT

An enzyme has been isolated from blue-green algae Oscilla-toria sp. which utilizes the product, 13-hydroperoxy-9,11-octa-decadienoic acid (13-HPOD), of lipoxygenase for its substrate.This enzyme, termed hydroperoxide lyase, converts the conju-gated diene 13-hydroperoxide of linoleic acid to 13-oxotrideca-9,1 -dienoic acid. The structure of the latter has been determinedby ultraviolet spectroscopy and mass spectrometry. 9-HPOD isnot a substrate for this enzyme. The hydroperoxide lyase fromOscillatoria sp. has a maximum of activity at pH 6.4 and 300C.The molecular weight of the enzyme was estimated at 56,000.The enzyme was not inhibited by BW 755C, but was inhibited bymolecules containing more than one hydroxyl group. Quercetinwas found to be the best inhibitor of the enzyme activity. Thepurified hydroperoxide lyase from Oscillatoria sp. showed anapparent Km of 7.4 micromolar and a V,,,. of 35 nanomoles perminute per milligram of protein for 13-HPOD. An enzymatic path-way for the biogenesis of oxodienoic acid from linoleic acid isproposed. This involves the sequential activity of lipoxygenaseand hydroperoxide lyase enzymes.

Lipoxygenases, which catalyze the dioxygenation of unsat-urated fatty acids to hydroperoxides with a pair of cis-transconjugated double bonds, have been found widely in plants(5, 9, 29, 37). The hydroperoxy fatty acids may then beenzymatically converted to hydroxy fatty acids (20-22), a-

or f-ketols (4, 10, 11, 42, 43), divinyl ether derivative (7),epoxy-,-y-hydroxy derivative (14, 31), trihydroxy octadecenoicacids (1), or carbonyl compounds (8, 24, 29). It has beenreported that secondary processes occurred during the oxida-tion of linoleic acid by soybean lipoxygenase, leading to thedisappearance of hydroperoxydienes, and to the formation ofanother reaction product, oxodienoic acid (12, 35, 39). Thisformation of oxodiene reveals a lipohydroperoxidase activity.We reported in our previous paper (2) the isolation of a

lipoxygenase from blue-green algae (Oscillatoria sp.). And inorder to identify the metabolic reaction in which hydroper-oxides could be involved in a lower form of plant life, we

would now like to report the isolation of an enzyme fromblue-green algae Oscillatoria sp. which catalyzes the degrada-tion of an unsaturated hydroperoxide to an oxodienoic acidderivative. The identification of this enzyme and its reactionproduct provide a new insight into the area of lipid metabo-lism in plants.

MATERIALS AND METHODS

Oscillatoria sp. was grown and maintained as previouslydescribed (2).

Purification of Oscillatoria sp. Hydroperoxide Lyase

All the steps were performed below 4°C. One L of cellculture suspension was harvested during the log phase ofgrowth and homogenized with 200 mL of deoxygenated (2)66 ,uM K-phosphate (pH 7) for 15 min using an Ultra Turraxhomogenizer (3000 rpm) because Oscillatoria cells are diffi-cult to break open. Then a nonionic detergent Brij 99 (0.1 %)(33) was added to the extraction buffer followed by furtherstirring for 48 h under anaerobic conditions. After extraction,the mixture was filtered through double layers of gauze toremove cell debris. The resulting solution was clarified bycentrifugation at 4700g for 60 min. Crystallized ammoniumsulfate was added slowly under stirring to 30% saturation.After 1 h, the precipitate was centrifuged (1000g; 15 min) anddiscarded. The supernatant was dialyzed overnight against100 volumes of 0.01 M Tris-acetate buffer (pH 7.2). Afterconcentration with PEG 6000, the sample which was namedS30 fraction was applied to a DEAE-Trisacryl (IBF France)column (25 x 40 cm) as described in previous paper (2). Theactive fractions were pooled and dialyzed overnight against20 mm Tris-acetate buffer (pH 7.2). After concentration withPEG 6000, the dialysate was subjected to a Sephadex G-150(4 x 46 cm) column equilibrated with 66 ,uM K-phosphate(pH 7). The enzyme was eluted with the same buffer. Theactive fractions were pooled, concentrated and used for sub-sequent studies.

Preparation and Purification of Hydroperoxides

13-HPOD' was prepared by incubating linoleic acid (All-tech. Associates, Inc.) with soybean lipoxygenase type 1 at pH9 (16). 9-HPOD was obtained by the simple method ofMatthew et al. (32) using tomato extracts. Lipoxygenase prod-ucts were isolated by acidification and extraction using diethylether and were separated from their positional isomers by SP-HPLC (2). All hydroperoxides were stored in ethanol at-20°C.

Assay of Hydroperoxide Lyase Activity

Hydroperoxide lyase activity was determined by measuringthe loss of absorbance at 235 nm in a recording Perkin-ElmerLambda 5 spectrophotometer equipped with a constant tem-perature cell holder at 30°C. The reaction mixture contained1 mL of citrate-phosphate-borate/HCl buffer (pH 6.4), 19

'Abbreviations: 1 3-HPOD, 1 3-hydroperoxy-9, 1 l-octadecadienoicacid; 9-HPOD, 9-hydroperoxy-10,12-octadecadienoic acid; SP-HPLC, straight phase HPLC; NDGA, nordihydroguaiaretic acid.

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GREEN ALGAE HYDROPEROXIDE LYASE

units of enzyme extract, and 33 tM hydroperoxide in theorder given. One unit of enzyme was defined as the amountof enzyme that permitted the loss of 1 nmol of unsaturatedhydroperoxide per min at 30°C. The specific activity wasexpressed as the number of units per mg of protein. Theprotein concentration was determined according to Lowry etal. (28) using BSA as a standard. A molar extinction coeffi-cient of23,600 m' . cm-' (13) was used to convert absorbanceat 235 nm readings to moles of hydroperoxides.

Molecular Weight

The mol wt of the enzyme was estimated by gel filtrationon a Sephadex G- 150 (Pharmacia Fine Chemicals) column (4x 46 cm) equilibrated with 0.2 M K-phosphate (pH 7). Markerproteins were BSA (Mr 67,000), peroxidase (Mr 40,000), lac-tate dehydrogenase (Mr 140,000), and aldolase (Mr 161,000).

Synthesis, Purification, and Identification of ReactionProducts

To characterize the products of the enzyme reaction, thecontents of the reaction system were increased 20 times. Thereaction was carried out in a test tube containing 20 mL ofcitrate-phosphate-borate/HCl buffer (pH 6.4), 380 units ofenzyme, and 33 ,uM of 13-HPOD at 30°C under aerobicconditions. A blank was run under the identical conditionsexcluding the enzyme. At the end of 18 min, the reactionproduct was isolated by acidification and extraction usingdiethyl ether (3 x 30 ml). To purify the reaction product,SP-HPLC was performed with a LDC multiple deliverysystem model CM 4000 equipped with a detector model SM4000. Then the organic extract was applied to a ,uPorasil (10,um) Waters column (3.9 x 30 cm) and eluted with hexane/

4

*4 800-

0Na 400 -v

v

A

0I-0. 4 N

0

0N

v

-O. 2 mv

m4m¢.

A

-150 v

0

00

NI

z

u

0 24 4bS 7X2 905

PRACT ION NOUMBER

Figure 1. Elution profile of Oscillatoria hydroperoxide lyase fromDEAE-Trisacryl column (25 x 400 mm). Column was equilibratedwith 0.02 M Tris-acetate buffer (pH 7.2) and elution conducted by 0to 0.15 M sodium acetate linear gradient (* *) at a flow rate of 1mL/min. Fractions of 6 mL were collected and proteins were moni-tored at 280 nm (-U). Activity was measured by monitoring thedecrease in optical density at 235 nm at pH 6.4 in citrate-phosphate-borate/HCI buffer at 300C (A A).

ethanol/acetic acid (98:1.9:0.1, v/v) at a flow rate of 2 mL/min. Absorbance was monitored at 284 nm.The product of the hydroperoxide lyase reaction purified

by SP-HPLC was then converted into methyl-ester with di-azomethane and its UV spectra in methanol was recorded.The UV spectra in methanol of the methyl ester derivative ofthe reaction product was also recorded after reduction toalcohol by NaBH4. The hydroxy-acid methyl ester was ana-lyzed by GC-MS after silylation of hydroxyl group to tri-methylsilane by bis(trimethylsilyl)trifluoroacetamide.

Volatile hydrocarbon products were characterized by gaschromatographic analysis. A 100 mL aliquot of S30 fractionwas blended aerobically for 45 min with 900 mL of deoxy-genated 66 AM K-phosphate (pH 7) containing 50 ,uM of 13-HPOD. The reaction products were isolated by extractionusing chloroform (3 x 100 mL) and dried with anhydrousCaCl2. After filtration, the chloroform was removed undervacuum and replaced by 10 AL of hexane.

Volatile compounds were analyzed by injection onto acarbowax 20 M column (25 m x 0.25 mm i.d.), temperatureprogrammed from 35 to 55C at 1°C per min and comparisonswere made with the retention behavior of known standards.

RESULTS AND DISCUSSION

Purification of Oscillatoria Hydroperoxide Lyase

Figure 1 shows the elution profile of Oscillatoria hydroper-oxide lyase from DEAE-Trisacryl column. The active frac-tions were pooled, concentrated and dialyzed as described in"Materials and Methods." The dialysate was subjected to aSephadex G- 150 column and the enzyme was eluted as shownin Figure 2A.

Table I summarizes purification stages. Recovery factor istaken as the remaining amount of total protein relative tothat of crude extract, while the purification factor is taken asthe increase in specific activity of the enzyme relative to thatof the crude extract. The three purification steps resulted in a45-fold enzyme purification.

Molecular Weight

The mol wt determined as described in "Materials andMethods" was estimated at 56,000 (Fig. 2B). This is less thanthe mol wt of Oscillatoria lipoxygenase which was estimatedat 124,000 (2) but close to those of hydroperoxide lyase fromChlorella pyrenoidosa (38) which was estimated at 48,000.This result suggests that the soluble hydroperoxide lyase andlipoxygenase activities from Oscillatoria are not contained onthe same protein or protein complex.

Effects of pH

In order to study the effects ofpH on the enzyme activity,the reactions were initiated by addition of 33 jM 13-HPODto 19 units of purified enzyme in 1 mL of buffer at 30°C. Onebuffer was used: citrate-phosphate-borate/HCl for pH 3.2 to8.5. The pH dependency of the enzyme activity showed a

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ANDRIANARISON ET AL.

symmetric curve with maximal activity at pH 6.4 as shownin Figure 3. This is similar to the optimum pH of the hydro-peroxide cleavage system from cucumber fruits (8).

120A

I

A965

;

,2

v

40

,2 24

6 4

V_ <mrl >

Figure 2. (A) Elution profile of Oscillatoria hydroperoxide lyase fromSephadex G-1 50 column (4 x 46 cm). Column was equilibrated with66 Mm K-phosphate (pH 7). Fractions of 3 mL were collected andassayed for proteins (E-E) at 280 nm and hydroperoxide lyaseactivity ( (B) Calibration curve for determination of the molec-ular weight of the Oscillatoria hydroperoxide lyase with Sephadex G-150. The logarithmic values of the molecular weights of four standardmarker proteins were plotted against their mobilities on the molecularsieve chromatography in a column (4 x 46 cm) packed with SephadexG-150.

Ka, = (Ks, - K0)/(Kt -K)

Kd = elution volume; Kt = total volume determined with acetone; K0= exclusion volume determined with Dextran blue (mol wt 800,000).

Effects of Temperature

The activity of the enzyme is temperature dependent. Thepurified enzyme solution was treated for 10 min at differenttemperatures. After cooling down in ice, the hydroperoxidelyase activity was measured in standard conditions. Thisparticulate enzyme system was fully active at 30°C (Fig. 4). Asimilar result has been reported by Jadhav et al. (24) that anincrease in activity of hydroperoxide decomposing enzyme intomato fruit was noticed with the increase in temperature upto 30°C. Fifty per cent ofthe remaining activity of Oscillatoriahydroperoxide lyase was obtained at about 53°C. The enzymeactivity was abolished by a 15 min treatment at 75C.

Oxygen Nonrequirement of Enzyme Activity

The formation of oxoenes as secondary oxidation productshas been reported until now from linoleic acid (3, 12, 23, 27,36, 39, 40). These oxoenes would be produced preferentiallyunder anaerobic conditions or under conditions with higherconcentration of linoleic acid and limiting oxygen supply. Anexception is wheat foliar (25) in which secondary-consumingprocesses were involved in aerobic conditions to produceoxodienoic acid.Replacement of air with nitrogen in the incubation vessel

did not reduce the increase of absorbance at 284.4 nm in thecase of Oscillatoria hydroperoxide lyase (Fig. 5).

Nature of Substrate for Hydroperoxide Lyase Enzyme

In the presence of the Oscillatoria hydroperoxide lyase, theloss of absorbance at 234 nm occurred only with 13-HPODas substrate (Fig. 6). In this case, a non-negligible part ofhydroperoxides was converted to a reaction product whichhad maximum ofabsorbance at 284.4 nm in the assay system.9-HPOD was not a substrate for the Oscillatoria hydroper-oxide lyase (data not shown).The hydroperoxide cleavage enzyme from cucumber fruit

attacks both 9- and 13-hydroperoxide isomers with equalfacility (8). Although lipoxygenase from tomato fruits formspredominantly 9-hydroperoxides from linoleic and linolenicacids (32), the cleavage enzyme from tomato does not attackthis positional isomer, but rather is specific for the 1 3-hydro-peroxide isomer (6).

Effects of Known Lipoxygenase Inhibitors on OscillatoriaHydroperoxide Lyase Activity

As shown in Table II, hydroperoxide lyase activity fromOscillatoria sp. cells was slightly inhibited by esculetin,NDGA, and caffeic acid. On the other hand, quercetin seemsto be the more potent inhibitor, while BW 755C does notexhibit an inhibitor effect for Oscillatoria hydroperoxidelyase. All assays were conducted as described in "Materialsand Methods." If necessary, inhibitors were predissolved inethanol, whereby the final concentration of the latter did notexceed 1% (v/v) in the assay system. Appropriate blanks (heatdenaturated enzyme) and controls (ethanolic buffer instead

1282 Plant Physiol. Vol. 91, 1989




Table I. Purification Procedure of Hydroperoxide Lyase from Oscillatoria sp.Steps 1, 11, 111, and IV represent, respectively, the crude extract, the S30 fraction, the active fractions

from DEAE-Trisacryl eluate and the active fractions from Sephadex G-1 50 eluate.

Steps Volume Protein Lyase Rove Spcific PurifictionActivity vey Activity Prfcto

ml mg units % U/mg fold

1 150 129 114,000 100 884 111 180 100 106,000 77.4 1,060 1.2

III 18 3.55 16,200 2.75 4,550 5IV 18 0.046 1,820 0.036 39,600 45

>4H4

HU4

044

H

UQ4

100

50

H

Hu4

4

4E

0H

U

3 4 5 6 7

43. 4

Figure 3. pH dependency of the enzyme activity.

of test drugs) were run through the same procedure. The meanof the activity was calculated from four separate observations.

Esculetin, NDGA, caffeic acid, and quercetin have at leasta pyrocatechol group in their molecules. Judging from our

data shown in Table II, it is suggested that phenolic hydroxylgroups are necessary for hydroperoxide lyase inhibition.

Kinetic Studies

We demonstrated that hydroperoxide lyase activity ofOscillatoria at pH 6.4 at 30°C obeys normal Michaelis-Menten kinetics (Fig. 7). The Lineweaver-Burk plot of Vi'versus [ 3-HPOD]' was linear (data not shown) and apparentKm values of approximately 7.4 AiM were obtained for 13-HPOD. The purified hydroperoxide lyase from Oscillatoriashowed a Vmax of 35 nmol/min/mg protein for 13-HPOD atpH 6.4 at 30°C.

Reaction Products

Identification of 13-Oxotrideca-9, 1 1-Dienoic Acid

The enzyme conversion of 1 3-HPOD led to a polar metab-olite, named product X (Fig. 8A). Its retention time on SP-HPLC was very short. The methyl ester derivative of Xshowed absorption bands at 203.6 and 280 nm in methanol(Fig. 8B). Besides, absorption near 280 nm was characteristicof a conjugated dienone chromophore (39).

20 30 40 50 60 70 80

TEMIB:ERATUR1E 'C

Figure 4. Effects of temperature on the enzyme activity.

Presence of conjugated dienone was further confirmed bythe UV spectra of the methyl ester derivative of X afterreduction of the carbonyl group to alcohol by NaBH4, inwhich the absorption maxima at 280 nm was shifted to 232nm (Fig. 8B). This can be explained by the fact that conjugateddienone was converted into conjugated diene monohydroxidegroup as shown in Figure 9. The mass spectrum ofthe methylester, trimethylsilyl ether derivative ofX was characterized bya molecular ion (M+) at m/z 312 and by diagnostic ions atm/z 103 [(CH3)3Si-O-CH2], 156 [(CH3)3Si-O-CH2-CH==CH-CH=CH + H], 157 [CH2(CH2)6-COOCH3], 210[CH==CH-CH==CH-(CH2)7-COOCH3 + H].From these results, it appeared that Oscillatoria lyase cata-

lyzed the cleavage of 13-HPOD to produce an oxodienoicacid, the 1 3-oxotrideca-9, 1 1-dienoic acid.

Identification of Pentanol

Figure 9 shows the gas chromatographic analysis of volatilecompounds from the reaction of the S30 fraction with 13-HPOD substrate.The retention time of the lyase product corresponded with

that of pentanol on a 25 m Carbowax 20 M capillary column.Pentanol production was reduced by 98% upon treatment ofthe enzyme at 80°C for 15 min.A possible pathway for forming oxodienoic acid and pen-

tanol from linoleic acid is shown in Figure 10. It is proposedthat the hydroperoxide lyase induces a scission of the HOO-

1 283

100

0




Figure 5. (A) Reaction rate curves of hydroper-oxide lyase showing the oxygen non-require-ment of the enzyme activity. Activity was meas-ured by monitoring the increase in absorbanceat 284.4 nm to the forming oxodienoic acid atpH 6.4 in citrate-phosphate-borate/HCI buffer at300C in presence of air (A A) or nitrogen(* *). (B) Spectra of oxodienoic acid formationin presence of air. The figure shows the UVspectra from zero (the top of the figure) to 18min (the lower part of the figure). The cycle timewas 12 s.

group with formation of an HO-ion (or HO-radical) which isadded to the carbon-12. The cleavage of the bond betweencarbon- 12 and -13 and the formation of the carbonyl group

at carbon 13 (Fig. 10) conclude the reaction sequence to yieldpentanol and 10-oxotrideca-9,11-dienoic acid. Thus, we

thought that the cis-9,trans- 11 double bond geometry remainsunchanged from the precursor 1 3-HPOD. A similary pathwaywas proposed by Wurzenberger and Grosch (41) to explainthe cleavage of 1 3-HPOD by hydroperoxide lyase frommushrooms.Comparing Oscillatoria hydroperoxide lyase with other

higher plant hydroperoxide cleavage enzymes, some differ-ences are noteworthy. In some plants, cis-3-nonenal andtrans-2-nonenal are produced from linoleic acid (15, 17, 26).Only C6-aldehydes are produced in tea (18) and Farfungiumjaponicum leaves ( 19). This may imply that the lipoxygenase-hydroperoxide lyase enzyme systems in F. japonicum and tealeaves attack the double bond at C- 12, while the enzyme

system in cucumber fruits attacks mainly the double bond atC-9. From our results we can assume that the lipoxygenase-hydroperoxide lyase enzyme system from Oscillatoria sp. also

attacked the C-12 double bond, but cleavage occurred be-tween C-13,14 rather than C-12,13 as it does in tea and F.japonicum leaves.

Recently, Vick and Zimmerman (38) showed that Chlorellaextracts possessed a hydroperoxide lyase activity. Comparinghydroperoxide lyase from Oscillatoria and Chlorella, some

significant similarities are noteworthy. The two enzymes pro-duced the same 1 3-oxotrideca-9, 1 1-dienoic acid and they wereactive over a broad pH range from pH 6 to 8.The C-S carbon product of Oscillatoria hydroperoxide lyase

reaction differed from those reported of Chlorella hydroper-oxide lyase reaction. Thus we thought that the cleavage of 13-HPOD and the formation of oxodienoic acid were due to an

intramolecular displacement of oxygen from the hydroper-oxide group to lead to a heterolytic scission in C13-C14 (Fig.

1. 00,

0. OC

34. 0 n

200 320

WAVELENGTH C rxm>

Figure 6. Spectra of hydroperoxide decomposition by Oscillatoriahydroperoxide lyase from zero to 18 min. The absorbance at 234 nmdecrease, while the absorbance at 284.4 nm increase showing theoxodienoic acid formation.

10). Nevertheless, this hypothetical mechanism remains to befurther investigated.

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0. 06

0. 03

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¢

m0

0. 00

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Tr IME mli )

1284 Plant Physiol. Vol. 91, 1989




Table II. Effects of Various Inhibitors on Oscillatoria HydroperoxideLyase Activity

Enzyme (19 units) was preincubated with inhibitors at two concen-trations (respectively 54 or 0.1 Mm) for 10 min in citrate-phosphate-borate/HCI buffer (pH 6.4) (1 ml) and the reaction was started up byaddition of 13-HPOD (33 AM). (Lyase activity was measured by theinitial velocity.) Initial velocity measured was directly proportional tothe activity in incubation mixture. All assays were performed at 300Cunder aerobic conditions.

Inhibitors [Inhibitor] Mean Percent of Activity

pmol % ± SD

BW 755C 54 100.00 ± 0.120.1 100.00 ± 0.09

Esculetin 54 90.60 ± 0.150.1 100.00±0.10

Quercetin 54 0.50 ± 0.010.1 24.10± 0.02

NDGA 54 84.70 ± 0.080.1 100.00 ± 0.10

Caffeic acid 54 79.41 ± 0.130.1 94.60 ± 0.10

Io

(0

0

(0Co

I~~~~~~~~~~~~~~~~~~I I I I I

0 210 1- f~0

0

1U0

wv

H

H

10-- 10--

Figure 7. Effect of 13-HPOD concentration on the initial velocity ofhydroperoxide decomposition by Oscillatoria hydroperoxide lyase.Initial velocity was measured by monitoring the decrease in opticaldensity at 235 nm at pH 6.4 at 300C. Information concerning theenzyme assay is given in the text.

0.24

m

.4

m0 0.12

co

4

0. 00

TI: (m<x)200 320

WAVE:LENGTH C< m>

Figure 8. (A) SP-HPLC analysis of reaction product obtained by the incubation of 13-HPOD (33 MM) with 19 units of Oscillatoria hydroperoxide

lyase as described in "Materials and Methods." (B) UV spectra of the methyl ester of the product X before ( ) and after (-----) NaBH4

reduction.

A

x

1 285

2

I

0

E 1L -3E-C:OD-) < lxc3I m,3l >




In many plants, the presence of lipoxygenase seems topredetermine lipid hydroperoxide formation especially if cel-lular integrity is disrupted mechanically. Despite the widedistribution of lipoxygenase among a range of plant families,no physical role for hydroperoxide formation has yet beendiscovered. Owing to the presumed toxicity of hydroperoxidesfor living cells, the production of these compounds cannot bea goal in itself. Various plants appear to differ in their routesto metabolize hydroperoxides: e.g. a- and y-ketols (flax, corn,alfalfa, barley), unsaturated ethers (potato), mono-, di- andtri-hydroxy acids (wheat, peas), oxodiene (Oscillatoria sp.),

(J)<cz fatty acid dimers (soybean) and alkanes (soybean, peanuts)can be formed. Evidently, no universal principle is operativein the conversion of hydroperoxides, which makes it hard tobelieve that a comprehensive theory can be given comprising

0 the biological function of the different metabolites. However,P4common to all reactions is the fact that the polyunsaturatedfatty acids and oxygen are metabolized. Then, in our opinion,the bioenergetic aspects would prevail and the metaboliteswould be of secondary importance. This does not exclude a

o co possible role for some metabolites.u l ll ll l The location of lipoxygenase and hydroperoxide lyase in

lower forms of plant life as Oscillatoria sp. and Chlorellapyrenoidosa is of particular interest in relation to metabolicfunctions of these enzymes.

Note Added in Proof

Reference No. 2 appeared in the September 1989 issue ofPlant Physiology, 91: 367-372, titled "Properties of a Lipox-

< > ygenase in Green Algae (Oscillatoria sp.)." In a notice pub-lished in the November 1989 issue of Plant Physiology, 91:

0o 2 4 <5 8 1238, the title was changed to read "Properties of a Lipoxy-JMI laU21S genase in Blue-Green Algae (Oscillatoria sp.)." Likewise, in

the text of that article, all references to "green" algae werechanged to read "blue-green" algae.

Figure 9. Gas chromatographic analysis of the Oscillatoria sp. hy- LITERATURE CITEDdroperoxide lyase reaction on a Carbowax 20 M capillary column. (1) 1. Baur C, Grosch W (1977) Investigation of the taste of di-, tri-n-Alcohol standards; (2) analysis showing pentanol formation from and tetra-hydroxy fatty acids. Z Lebensm Unters Forsch 165:the hydroperoxide lyase reaction with 13-HPOD; H, hexane. 82-84

TRAPSORXAT10N OP 13-HPOD INTOCAIRN0NNYL CO3EOUNDS 8Y OSCIXLATORIA B

HYDROPBIROXYDB LYASE. B

Figure 10. (A) Proposed schematic pathways IILLIA 8YDIDPERXIDE LY

for the decomposition of 1 3-HPOD to 1 3-oxotri- NRdeca-9,1 1 -dienoic acid. (B) Mechanism pro- A\ACH-OH +posed to explain the action of hydroperoxide OHC j COO} Rlyase on 1 3-HPOD. R = -(CH2)6-COOH; R' =-(CH2)2 CH3.

OHC f_ / COOHTNO1IsC.jN,N\ /COOCH_

O0HCCN COOCHo

HOH: _ COOCHo-

,&BBTFA

1 286 Plant Physiol. Vol. 91, 1989




2. Beneytout JL, Andrianarison RH, Rakotoarisoa Z, Tixier M(1989) Properties of a lipoxygenase in blue-green algae (Oscil-latoria sp.). Plant Physiol 91: 367-372

3. Borthakur A, Ramados CS (1986) Aerobic formation of keto-diene from linoleic aciu catalyzed by one to two forms oflipoxygenase isolated from bengal gram (Cicer arietinum). JAgric Food Chem 34: 1016-1018

4. Daood H, Blacs PA (1986) Evidence for the presence of lipoxy-genase and hydroperoxide decomposing enzyme in red pepperseeds. Acta Aliment 4: 307-318

5. Galliard T, Chan HWS (1980) Lipoxygenase. In PK Stumpf, EEConn, eds, Biochemistry of Plants, Vol 4, Academic Press,New York, pp 131-161

6. Galliard T, Matthew JA (1977) Lipoxygenase mediated cleavageof fatty acids to carbonyl fragments in tomato fruits. Phyto-chemistry 16: 339-343

7. Galliard T, Phillips DR (1972) Enzymatic conversion of linoleicacid into 9-(1,3-nonadienoxy)-8-nonenoic acid, a novel unsat-urated ether derivative isolated from homogenates of Solanumtuberosum tubers. Biochemistry 129: 743-753

8. Galliard T, Phillips DR, Reynolds J (1976) The formation of cis-3-nonenal, trans-2-nonenal and hexanal from linoleic acidhydroperoxide isomers by a hydroperoxide cleavage enzymesystem in cucumber (Cucumis sativus) fruits. Biochim BiophysActa 181-192

9. Gardner HW (1988) Lipoxygenase pathway in cereals. Adv Cer-eal Sci Technol 3: 161-215

10. Gardner HW (1970) Sequential enzymes of linoleic acid oxida-tion in corn germ. Lipoxygenase and linoleate hydroperoxideisomerase. J Lipid Res 11: 311-321

11. Gardner HW (1975) Isolation of a pure isomer of linoleic acidhydroperoxide. Lipids 10: 248-252

12. Garssen GJ, Vliegenthart JFG, Boldingh J (1971) An anaerobicreaction between lipoxygenase, linoleic acid and its hydroper-oxides. Biochem J 122: 327-332

13. Gibian MJ, Vandenberg P (1987) Product yield in oxygenationof linoleate by soybean lipoxygenase: the value of the molarextinction coefficient in the spectrophotometric assay. AnalBiochem 163: 343-349

14. Graveland A (1970) Modification of the course of the reactionbetween wheat flour lipoxygenase and linoleic acid due toadsorption of lipoxygenase on glutenin. Biochem Biophys ResCommun 41: 427-434

15. Grosch W, Schwarz JM (1971) Linoleic and linolenic acid asprecursors of the cucumber flavor. Lipids 6: 351-352

16. Hamberg M (1971) Steric analysis of hydroperoxides formed bylipoxygenase oxygenation of linoleic acid. Anal Biochem 43:515-526

17. Hatanaka A, Kajiwara T, Harada T (1975) Biosynthetic pathwayof cucumber alcohol: trans-2,cis-6-nonadienol via cis-3,cis-6-nonadienal. Phytochemistry 14: 2589-2592

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