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
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GREEN ALGAE HYDROPEROXIDE LYASE
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
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ANDRIANARISON ET AL.
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.
< (A-A( -* >
0. 06
0. 03
U
e
0
v
¢
m0
0. 00
0
Tr IME mli )
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GREEN ALGAE HYDROPEROXIDE LYASE
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 >
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ANDRIANARISON ET AL.
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
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GREEN ALGAE HYDROPEROXIDE LYASE
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
18. Hatanaka A, Kajiwara T, Sekiya J (1976) Biosynthesis of trans-2-hexenal in chloroplasts from Thea sinensis. Phytochemistry15: 1125-1126
19. Hatanaka A, Sekiya J, Kajihara T (1977) Enzyme system cata-lyzing formation of cis-3-hexenal and n-hexanal from linolenicand linoleic acids in Japanese silver (Farfugium japonicumKitamura) leaves. Plant Cell Physiol 18: 107-116
20. Heimann W, Dresen P, Klaiber V (1973) Formation anddecomposition of linoleic acid hydroperoxides in cereals.Quantitative determination of the reaction products. Z Le-bensm Unters Forsch 153: 1-5
21. Heimann W, Dresen P, Schreier P (1973) The lipoxygenase-"lipoperoxidase" system in cereals. Isolation of two proteincomplexes with lipoxygenase and linoleic acid hydroperoxideactivities from oats and soybeans. Z Lebensm Unters Forsch152: 147-151
22. Heimann W, Reinartz F, Schreier P (1972) Lipoxygenase-"lipo-peroxidase" system in cereals. III. On the kinetics of enzymatic
degradation of linoleic acid hydroperoxides. Helv Chim Acta55: 2257-2262
23. Hurt GB, Axelrod B (1977) Characterization of two isoenzymesof lipoxygenase from bush beans. Plant Physiol 59: 695-700
24. Jadhav S, Singh B, Salunkle DK (1972) Metabolism of unsatu-rated fatty acids in tomato fruit: linoleic and linolenic acids asprecursors of hexanal. Plant Cell Physiol 13: 449-459
25. Jolivet P, Bergeron E (1988) Production of hydroperoxides andoxoenes during the oxidation of linoleic and linolenic acids bywheat foliar lipoxygenase. Plant Physiol Biochem 26: 55-63
26. Kajiwara T, Oadake Y, Hatanaka A (1975) Synthesis of 3Z-nonenal and 3Z,6Z-nonadienal. Plant Physiol Biochem 39:1617-1621
27. Kunh H, Salzmann-Reinhardt V, Ludwig P, Ponicke K, ScheweI, Rapoport S (1986) The stoichiometry of oxygen uptake andconjugated diene formation during the oxygenation of linoleicacid by the pure reticulocyte lipoxygenase. Evidence for aerobichydroperoxidase activity. Biochim Biophys Acta 876: 187-193
28. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Proteinmeasurements with the Folin phenol reagent. J Biol Chem193: 265-267
29. De Lumen BO, Stone EJ, Kazeniac SJ, Forsythe RH (1978)Formation of volatile flavor compounds in green beans fromlinoleic and linolenic acids. Journal of Food Science 43:698-708
30. Mack AJ, Peterman TK, Siedow JN (1987) Lipoxygenases iso-zymes in higher plants: biochemical properties and physiolog-ical role. Isozymes Cuff Top Biol Med Res 13: 127-154
31. Mann DL, Morrison WR (1975) The effects of ingredients onthe oxidation of linoleic acid by lipoxygenase in bread doughs.J Sci Food Agric 26: 493-505
32. Matthew JA, Chan HWS, Galliard T (1977) A simple methodfor the preparation of pure 9-D-hydroperoxide of linoleic acidand methyl linoleate based on the positional specificity oflipoxygenase in tomato fruits. Lipids 12: 324-326
33. Mulliez E, Leblanc JP, Rigaud M, Chottard JC (1987) 5-Lipox-ygenase from potato tubers. Improved purification and phys-iochemical characteristics. Biochim Biophys Acta 916: 13-23
34. Nicolas J, Drapron R (1981) Les lipoxygenases vegetales. Etatactuel de nos connaissances. I. Aspects biochimiques. Sci Ali-ments 1: 91-168
35. Regdel D, Schewe T, Rapoport SM (1985) Enzymatic propertiesof the lipoxygenase from pea seeds. Biomed Biochim Acta 44:1411-1428
36. Veldink GA, Vliegenthart JFG, Boldingh J (1970) Oxygen trans-fer in the enzymatic conversion of '8O-labelled linoleic acidhydroperoxide into the 12-keto- 13-hydroxy-octadec-cis-9-enoic acid. FEBS Lett 7: 188-190
37. Vick BA, Zimmerman DC (1987) Oxidative systems for modifi-cation of fatty acids: the lipoxygenase pathway. Biochem Plants9: 53-90
38. Vick BA, Zimmerman DC (1989) Metabolism of fatty acid hy-droperoxides by Chlorella pyrenoidosa. Plant Physiol 90: 125-132
39. Vioque E, Holman RT (1962) Characterization of the ketodienesformed in the oxidation of linoleate by lipoxidase. ArchBiochem Biophys 99: 522-528
40. Vliegenthart JFG (1979) Enzymatic and nonenzymatic oxida-tion of polyunsaturated fatty acids. Chem Ind 241-251
41. Wurzenberger M, Grosch W (1984) Stereochemistry of the cleav-age of the 13-hydroperoxide isomer of linoleic acid to l-octen-3-ol by a hydroperoxide lyase from mushrooms (Psalliotabispora). Biochim Biophys Acta 795: 163-165
42. Zimmerman DC (1966) A new product of linoleic acid oxidationby a flaxseed enzyme. Biochem Biophys Res Commun 23:398-402
43. Zimmerman DC, Vick BA (1970) Hydroperoxide isomerase.Plant Physiol 46: 445-453
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