From the Department of Pharmaceutical Biosciences, Uppsala
Biomedical Center, Uppsala University, S-751 24 Uppsala, Sweden,
and Department of Molecular Biology, Arrhenius
Laboratories, Stockholm University, S-10691 Stockholm, Sweden
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ABSTRACT |
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Linoleate diol synthase (LDS) was isolated as a
hemeprotein from the fungus Gaeumannomyces graminis. LDS
converts linoleate sequentially to 8R-hydroperoxylinoleate
(8-HPODE) through an 8-dioxygenase by insertion of molecular oxygen and
to 7S,8S-dihydroxylinoleate through a
hydroperoxide isomerase by intramolecular oxygen transfer. Light
absorption and EPR spectra of LDS indicated that the heme iron was
ferric and mainly high spin. Oxygen consumption during catalysis
started after a short time lag which was reduced by 8-HPODE. Catalysis
declined due to suicide inactivation. Stopped flow studies with LDS and
8-HPODE at 13 °C showed a rapid decrease in light absorption at 406 nm within 35 ms with a first order rate constant of 90-120
s1. Light absorption at 406 nm then increased at a rate
of ~4 s
1, whereas the absorption at 421 nm increased
after a lag time of ~5 ms at a rate of ~70 s
1. EPR
spectra at 77 K of LDS both with linoleic acid and 8-HPODE showed a
transient doublet when quenched after incubation on ice for 3 s
(major hyperfine splitting 2.3 millitesla; g = 2.005), indicating
a protein radical. The relaxation properties of the protein radical
suggested interaction with a metal center. 8-HPODE generated about
twice as much radical as linoleic acid, and the 8-HPODE-induced radical
appeared to be stable. Our results suggest that LDS may form, in
analogy with prostaglandin H synthases, ferryl intermediates and a
protein radical during catalysis.
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INTRODUCTION |
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Lipoxygenases and PGH1 synthases are two different families of dioxygenases, which oxygenate polyunsaturated fatty acids (1-3). Lipoxygenases contain a non-heme metal center (1, 3, 4). Iron lipoxygenases abstract a hydrogen from the bisallylic C-3 of the 1Z,4Z-pentadienyl group of the polyunsaturated fatty acids and insert molecular oxygen at C-1 or C-5 with formation of a cis-trans conjugated double bond (1, 3). In addition to this, a recently described manganese lipoxygenase can also oxygenate C-3 with retention of the double bonds (4, 5). PGH synthases contain heme iron and catalyze two reactions. First the double dioxygenation of arachidonic acid to PGG2 and then reduction of PGG2 to PGH2 (6). The initial step is abstraction of the 13-pro-S hydrogen from arachidonic acid and antarafacial insertion of oxygen at C-11 to generate an 11R-peroxy radical (7). The 13-pro-S hydrogen is abstracted by a protein radical (at Tyr385 of PGH synthase-1), which is formed by oxidation of the ferriheme to ferryl oxygen intermediates during reduction of PGG2 to PGH2, or during reduction of other hydroperoxides (2, 6, 8-10). PGH synthase-1 substituted with mangano protoporphyrin IX can also form prostaglandins, but the peroxidase activity is much lower than that of native PGH synthase-1 (11).
In this report we study a new fatty acid dioxygenase, LDS (12-14). This enzyme, which is a homotetramer on gel filtration with a subunit molecular mass of about 130 kDa, catalyzes two related enzyme activities, linoleate 8-dioxygenase and hydroperoxide isomerase, which will sequentially form 8-HPODE and 7,8-DiHODE (Fig. 1). The 8-dioxygenase abstracts the 8-pro-S hydrogen from linoleic acid. Molecular oxygen binds in an antarafacial way and 8R-HPODE is formed. The second enzyme activity converts 8R-HPODE into 7S,8S-dihydroxylinoleic acid. This occurs by elimination of the 7-pro-S hydrogen and intramolecular suprafacial insertion of oxygen (12, 13). LDS was isolated from the fungus Gaeumannomyces graminis as a hemeprotein with at least 2.8 heme groups per tetramer (14).
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Fatty acid dioxygenases with the same catalytic activity as LDS have not been described, but PGH synthases can catalyze a similar initial oxygenation. PGH synthases oxygenate 20:3n-9 at the monoallylic C-13 in an "abortive" cyclooxygenase reaction (15-17). The 8-dioxygenase of LDS catalyzes the same event at the monoallylic C-8 of linoleic acid (13). In the second step the peroxidase of PGH synthases can reduce the hydroperoxide to 13-HETrE, while the hydroperoxide isomerase of LDS converts the hydroperoxide into 7,8-DiHODE.
The reaction mechanism of LDS is unknown. We have, therefore, examined the catalytic events of LDS. We found that important features of the reaction mechanism could be delineated with linoleate and with 8-HPODE as substrates. Our major findings are that the activity of LDS is stimulated by 8-HPODE, reduced by glutathione peroxidase, and that LDS catalysis is associated with formation of ferryl oxygen intermediates and a protein (tyrosyl) radical as judged from stopped flow and EPR analysis.
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EXPERIMENTAL PROCEDURES |
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Materials--
Acetone powder of mycelia of G. graminis was prepared as described previously (14) and stored at
80 °C. [1-14C]18:2n-6 (55 Ci/mol) was
from Amersham Pharmacia Biotech (Amersham, Buckshire, UK). Precoated
TLC plates (0.25-mm Silica Gel 60A, 5 × 10 cm) were from Merck
(Darmstadt, Germany). SepPak/C18 cartridges were from
Waters (Milford, MA). The gel filtration column (Superdex 200 HR10/30)
and other media and columns for chromatography were from Amersham
Pharmacia Biotech (Uppsala, Sweden). Equipment (MiniProtean II) and
reagents for SDS-PAGE were from Bio-Rad. Bovine hemin (type 1), CHAPS,
3,3',5,5'-tetramethylbenzidine, and GSH were from Sigma. Tween 20 was
from U. S. Biochemical Corp. Tetranitromethane was from Aldrich
(Steinheim, Germany). Protein standards for SDS-PAGE were from Amersham
Pharmacia Biotech. Lyophilized GSH peroxidase (from bovine
erythrocytes; Sigma) contained 2.5% dithiothreitol, which was removed
by gel filtration (PD10, Amersham Pharmacia Biotech) before use.
8-HPODE was obtained as described previously (18).
LDS Assays-- LDS activity was assayed with 10 µM [14C]18:2n-6 essentially as described elsewhere (14). The products were separated by TLC, and the TLC plates were scanned for radioactivity (12, 14).
LDS Purification--
LDS was prepared from 5-30 g of acetone
powder by four or five chromatography steps (14) with the following
modifications. The extract of acetone powder was adjusted to 0.45 M ammonium sulfate, 0.1% Tween 20 and loaded onto the
butyl-Sepharose 4FF column (step 1). The column was washed as described
previously (14) and eluted with to 0.02 mM sodium phosphate
buffer, 1 mM GSH, 0.04% Tween 20 (pH 7.4). The fractions
with LDS activity were purified on a Zn2+-loaded column
with chelating Sepharose FF (step 2), which had been prewashed with
1-2 volumes of 25 mM sodium citrate buffer (pH 6.7) to
reduce the Zn2+ density. The column was eluted with 40 mM imidazole, 10 mM triethanolamine hydrochloride, 1 mM GSH, 0.04% Tween 20 (pH 7.4). Further
purification was achieved by gel filtration (Sephacryl S-300 HR; step
3), anion exchange chromatography (Mono Q HR 5/5; step 4), and a final
gel filtration with high performance liquid chromatography (step 5) as
described previously (14). After step 4, the 130 kDa protein band was
dominating on SDS-PAGE (cf. Fig. 4B), and after
step 5 only the 130-kDa and a weak 100-kDa band were present (14). LDS
from step 5 with a specific activity of 1-2 µmol min1
mg
1 was used for light absorption and EPR studies and for
analysis of heme binding, while the majority of the stability and
inhibitory studies were performed with LDS from step 4 with a specific
activity of at least 0.7 µmol min
1 mg
1.
The concentration of LDS is given as monomer concentration unless stated otherwise.
Light Absorption Spectroscopy--
Light absorption was
registered with a dual beam spectrophotometer (Shimadzu UV-2101PC)
equipped with a temperature-controlled cuvette holder, and the cuvette
windows were purged with cooled air to prevent condensation. Spectra
and difference spectra were recorded at 22 °C or at 10 °C using
0.15-ml cuvettes. For freezing temperatures we used LDS with 27%
ethylene glycol, which did not interfere with LDS activity. Anaerobic
incubations at
10 °C were conducted under argon, in
argon-saturated solutions, and with 70 milliunits of glucose oxidase
(ICN Biomedicals, OH) per ml and 8 mM glucose to further
reduce the oxygen concentration. Ferric derivatives were prepared by
addition of a few grains of NaF or KCN directly to the native enzyme in
the sample cuvette (19). Ferrous deoxygenated derivatives with NO and
CO were prepared as described previously (14, 19).
EPR Analysis--
Three different batches of purified LDS (5 µM holoenzyme with a specific activity of 1-2 µmol
min1 mg
1 protein) were incubated with 0.3 mM linoleic acid or with 0.1 mM 8-HPODE in 140 µl for 3 or 45 s on ice and then quenched (in precooled
n-pentane at
105 °C). EPR spectra were recorded with an
EPR spectrometer (ESP 300, Bruker) at 10 K (with an Oxford-Instrument helium cryostat) or at 77 K (with a cold finger Dewar). Subtractions were performed using the ESP 300 software. Measurement of radical content was performed by comparing double integrals with that from
Cu2+-EDTA standard (1 mM Cu2+; 10 mM EDTA) or by using a secondary standard of protein R2
ribonucleotide reductase from Escherichia coli with a known
tyrosyl radical content. Power of half-saturation, P1/2, was
evaluated from microwave saturation curves according to Sahlin et
al. (21). For the samples, which were incubated with 8-HPODE, the
saturation curve was constructed after subtraction of a background
signal from the LDS protein at the same power. The LDS protein used in
two other incubations with linoleic acid did not have this background
signal.
Other Analyses-- SDS-PAGE was performed as described previously (14). Protein concentration was determined with bovine albumin as a standard (22). Heme peroxidase activity on SDS-PAGE with 2% SDS was assayed essentially as described elsewhere (23). Oxygen consumption was measured as described previously (14). After equilibration for 1-2 min, the reactions were initiated by addition of 18:2n-6 to give a concentration of 0.1 mM.
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RESULTS |
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LDS of Mycelia of Different Strains--
The main obstacle of this
investigation was the variable LDS activity of the mycelia. The LDS
activity varied between 0.3 and 2 nmol min1
mg
1 in low speed supernatants of mycelia, which were
prepared from different batches of G. graminis var.
gramini grown under similar conditions. Two other strains of
G. graminis (var. tritici, var. avenae) and several different liquid media were tested with
variable results, but all strains showed LDS activity.
Perturbation of Hydroperoxide Isomerase Activity-- The crude preparations of mycelia with LDS showed highly variable hydroperoxide isomerase activity. Following column chromatography, e.g. ion exchange or zinc-affinity chromatography, hydroperoxide isomerase activity was often decreased and sometimes even undetectable by TLC, whereas 8-dioxygenase activity was always present. However, storage of the enzyme for a few days on ice often restored the hydroperoxide isomerase and appeared to increase the LDS activity as well. An experiment with this result is shown in Fig. 2A. The hydroperoxide isomerase activity may thus be compromised without greatly affecting the 8-dioxygenase activity.
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Stability of LDS-- Extensively purified LDS was quite stable at +4 °C. About 70% of the LDS activity remained after 3 months and 30% after 6 months on ice. SDS-PAGE analysis showed that the 130-kDa protein gradually was transformed to a 100-kDa protein (and a 30-kDa protein, data not shown) during storage for 3 months at 4 °C (Fig. 2B). The 100-kDa protein was thus formed from the 130-kDa protein, which might explain why many attempts to completely separate the 130- and 100-kDa proteins have been unsuccessful (14).
The LDS was analyzed for heme peroxidase following SDS-PAGE with 2% SDS. Under these denaturing conditions, heme peroxidase was detected at the bottom of the gel and not at the position of the protein. This indicated that heme was not covalently bound to LDS.Stimulation and Inhibition of LDS-- The LDS activity was markedly increased by some detergents, e.g. by 0.04% Tween 20 or 0.5 mM CHAPS in 0.05 mM sodium phosphate buffer. Ascorbic acid (1 mM) was without effect, whereas 0.4 mM tetranitromethane, 1 mM phenol, 0.1 mM guaiacol, and phosphatidylcholine (0.4 mg/ml) were inhibitory (>50% reduction). Addition of heme did not augment the LDS activity, and heme did not appear to be incorporated into the protein. The ratio between light absorption of the enzyme at 280 nm (protein) and 406 nm (Soret region) was unchanged after separation of the added heme from LDS by gel filtration.
p-Hydroxymercuribenzoate was found to inhibit the hydroperoxide isomerase activity with a smaller effect on the total oxygenation by LDS. The hydroperoxide isomerase activity was reduced by 50% by 0.2 mM p-hydroxymercuribenzoate, whereas the total oxygenation decreased less than 10%. Corresponding figures for 0.5 mM p-hydroxymercuribenzoate were 90 and 20%, respectively (data not shown).Effects of Hydroperoxides and Glutathione Peroxidase-- Formation of 8-HPODE and 7,8-DiHODE from linoleic acid by purified LDS at 25 °C was monitored by an oxygen electrode. The reaction was started by addition of substrate. Oxygen consumption started with a time lag of ~10 s. Preincubation with 13-HPODE (6-8 µM) did not reduce this time lag, but 8-HPODE (6-8 µM) reduced it to ~3 s (Fig. 3A). 8-HPODE is a substrate of the hydroperoxide isomerase, but 13-HPODE is not (12, 18).
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Suicide Inactivation-- Oxygen consumption during sequential addition of linoleic acid and LDS is shown in Fig. 4. The first addition of linoleic acid to the LDS in the reaction vessel rapidly reduced the oxygen tension, but the second addition of substrate had little or no effect. Addition of LDS increased the oxygen consumption. We interpret the results as a substrate inactivation of LDS during catalysis. The data suggested that 1 mol of LDS (tetramer) generated ~2500 mol of product at 25 °C before inactivation.
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Light Absorption, Spin States, and Stopped Flow Analysis--
The
light absorption spectrum of purified native protein showed a distinct
absorption maximum at 406 nm () and broad absorption maxima at about
630-635 nm, 566 (
) nm, and 522 (
) nm, which suggested a ferric
hemeprotein in high spin states (14). This was confirmed by the Soret
absorption of F
and CN
derivatives, by
light absorption during acid-alkaline transition (Table
I), and by EPR (see below).
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Substrate-induced Protein Radials Studied by EPR-- EPR at 10 K of LDS without substrate confirmed the presence of mainly high spin ferric iron from the resonance at g = 6.3, while low spin iron resonances were noted at g = 2.47, 2.25, and 1.91 (Fig. 7). The heterogeneity of iron centers in three different preparations of LDS was obvious from the EPR spectra both regarding content of the ferric heme iron (g = 6.3), variable amounts of rhombic ferric ion (g = 4.3) and a "constitutive" free radical at g = 2.00 in one preparation. In the presence of linoleic acid and 8-HPODE, the shape of the ferric heme iron resonance was altered in a similar way (Fig. 7), which indicated that binding of linoleic acid and 8-HPODE to LDS changed the ligation of the heme iron. However, the most striking effect of linoleic acid and 8-HPODE on LDS was the appearance of a radical.
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DISCUSSION |
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We have investigated the catalytic properties of LDS. A catalytic model of LDS must accommodate all experimental observations and be consistent with the catalytic mechanisms of related heme enzymes. We will first recapitulate important properties of LDS and compare LDS with PGH synthase-1 and peroxidases.
LDS forms two main products from linoleic acid and molecular oxygen, viz., 8-HPODE and 7,8-DiHODE, whereas 8-HODE is formed in small amounts. Purification of LDS suggests that the two enzyme activities are present in a homotetramer with 130-kDa subunits. Different preparations of LDS varied in hydroperoxide isomerase activity, which could be due to partial inactivation of the hydroperoxide isomerase by column chromatography. This suggests that hydroperoxide isomerase is more sensitive to perturbations of protein structure than the 8-dioxygenase activity.
PGH synthase-1 and LDS are both inhibited by GSH peroxidase in the presence of GSH and activated by fatty acid hydroperoxides (6), although LDS might be preferentially activated by 8-HPODE. Both enzymes are also subject to suicide inactivation. An interesting similarity is that both enzymes can cleave the C-H bond of a monoallylic carbon. PGH synthases can oxygenate C-13 of 20:3n-9, whereas LDS can oxygenate C-8 of linoleic acid and C-8 of oleic acid (12, 15-17). There are also obvious differences between the enzymes. LDS has hydroperoxide isomerase, whereas PGH synthase-1 has peroxidase activity. The ferryl oxygen intermediates I and II of PGH synthase-1 are formed during the peroxidase-catalyzed reduction of PGG2 to PGH2 and during regeneration of ferric PGH synthase-1 with reducing equivalents (8, 9). The hydroperoxide isomerase of LDS does not need reducing equivalents. With oleic acid as a substrate, molecular oxygen can be inserted at C-8, the oxygen-oxygen bound cleaved and one atom of oxygen inserted either at C-7 to yield 7,8-dihydroxyoleic acid (12) or at the 9-10 double bond to yield 8-hydroxy-9(10)-epoxystearate.2 These observations suggest an interesting analogy between LDS and peroxidases. It is well known that compound I of peroxidases can catalyze epoxidation of double bonds, lipid peroxidation, oxidation of bisallylic carbons, and even hydroxylation of unsaturated carbon chains (10, 30).
Spectroscopy showed that LDS contained mainly high spin ferriheme.
Difference spectra showed that linoleic acid caused spectral changes,
which were similar to those of type I substrates of cytochrome P450
(Fig. 5), but linoleic acid had little effect on the spin state (Fig.
7). Importantly, linoleic acid and 8-HPODE increased the light
absorption at 412 nm, which might be related to the formation of the
protein radical discussed below. Stopped flow also showed that the
Soret absorption at 406 nm decayed rapidly within 10-20 ms after
mixing with 8-HPODE, and then increased at a much lower rate. As the
light absorption at 406 nm decreased, the light absorption increased at
421 nm after a lag time of 5 ms. These kinetic traces suggest but do
not prove that ferryl intermediates are formed. We know from stopped
flow and other analyses of horse radish peroxidase and PGH synthases
that similar kinetic traces can be recorded, which are due to a
transient ferryl oxygen intermediate and a porphyrin -cation radical
(compound I or intermediate I), which is converted to a second
intermediate, most likely a ferryl oxygen and a protein radical
(compound II or intermediate II), after a short time lag (9, 10, 31). The formation of a protein radical during LDS catalysis would support
the formation of compound II.
EPR demonstrated that a protein radical was indeed formed upon incubation of LDS with linoleic acid and with 8-HPODE. LDS and linoleic acid generated a doublet hyperfine pattern at 3 and 45 s. The relaxation properties of the protein radical indicated that the radical was located near the heme group. However, a radical at g = 2.00 could also be a substrate radical, but we think this is unlikely for several reasons. Although LDS and linoleic acid likely can form a carbon-centered free radical, it will rapidly react with molecular oxygen to a peroxyl free radical. The former is characterized by a singlet (g = 2.004), while the latter is characterized by a singlet (or a doublet with major hyperfine splitting 0.38 mT) at g = 2.014, which were both detected by continuous flow EPR (32). These signals are clearly different from those we observed. In addition, it seems unlikely that LDS and 8-HPODE can form a peroxyl free radical. The fact that LDS incubated with 8-HPODE or with linoleic acid for 3 and 45 s on ice generated radicals with seemingly identical features suggests that these radicals could be due to one radicalized amino acid residue. The relaxation properties of the two radicals were also found to be similar. The main difference was that 8-HPODE induced a higher concentration of the radical than did linoleic acid in the same experiment. This difference is likely substrate-dependent, reflecting the reaction scheme described below.
Protein radicals have been described in many biological systems. Tyrosyl radicals are found in aerobic ribonucleotide reductase, e.g. Tyr122 in E. coli ribonucleotide reductase (Fig. 8, inset) (29), photosystem II (33), PGH synthases (2), and bovine liver catalase (34). Glycyl radicals are found in the pyruvate formate-lyase enzyme system (35), the anaerobic ribonucleotide reductases from E. coli (36) and from the bacteriophage T4 (Fig. 8, inset) (37). Transient tryptophan radicals are found in cytochrome c peroxidase (38) and in ribonucleotide reductase of E. coli with mutated protein R2 (Y122F) upon formation of the dinuclear iron center (39). Cysteine radicals have been proposed as reaction intermediates during ribonucleotide reductase catalysis (29). Likely candidates for the protein radical of LDS could be tyrosine, tryptophan, glycine, and cysteine amino acid residues.
The main splitting in the EPR spectrum of a protein radical is often
due to coupling of the unpaired electron to one or both of the
-methylene hydrogens of the radicalized amino acid. In case of an
amino acid with an aromatic ring, the spin density will be delocalized
over the ring system. For tyrosine, a large portion is found at C-1,
which will couple to the
-protons (one proton in E. coli
ribonucleotide reductase and two protons in photosystem II). The ring
protons at C-3 and C-5 of tyrosine are almost equivalent and will give
rise to hyperfine splitting of ~0.6 mT (40-42). For tryptophan
radicals that are neutral and deprotonated a large spin density is
found at C-3 of the ring and will couple to the
-methylene protons.
A doublet splitting can be observed when only one of the
-protons
couples to the spin density at C-3 and a quartet splitting when both
-hydrogens couple with equal strength (39). The couplings observed
for protein glycyl radicals are characterized by a major doublet with
hyperfine splitting constant of 1.4-1.5 mT and a g value of 2.004. Cysteine radicals have not been observed unless covalently coupled to a
reactant in a suicidal reaction (43) or ligated to a metal (44).
The observed features of the LDS doublet intermediate seem most similar to tyrosyl radicals (Fig. 8, inset). Both the g values, the magnitude of the hyperfine splitting, and also the poorly resolved 0.6-mT splitting, which could be ascribed to the C-3 and C-5 ring protons, are similar in tyrosyl radicals and in the radical of LDS. Furthermore, PGH synthase-1 forms a radical at Tyr385, which is assumed to be of catalytic importance. This assignment is based on the three-dimensional structures of PGH synthases (2) and on the EPR spectra of the radical (8, 27). Conclusive identification of the radicalized amino acid as a catalytically important tyrosine residue of LDS will require isotope labeling of recombinant LDS, mutation of tyrosine residues to other amino acids, and the three-dimensional structure of LDS.
What is the origin of the LDS radical? It seems likely that it is related to oxidation of the ferriheme and to the catalytic activity of LDS for several reasons. The relaxation properties of the radical indicated that it was located in the vicinity of the metal center. Exogenous 8-HPODE stimulates LDS activity, is metabolized to 7,8-DiHODE by the hydroperoxide isomerase (18), and generates the protein radical. Furthermore, stopped flow experiments with LDS and 8-HPODE showed transient spectral changes in the Soret region with at least two different rate constants, suggesting that ferryl intermediates similar to compounds I and II of peroxidases might be generated. Finally, work in progress suggests that the primary structure of LDS can be related to peroxidases, albeit distantly. A 200-amino acid long sequence of LDS can be aligned with 25% identity with a peroxidase, including 31% identity over a 70-amino acid sequence.3
Our observations and the apparent analogy with PGH synthase-1 and
peroxidases (2, 10) suggest the following catalytic mechanism of LDS
(Fig. 9). The initiating step of
catalysis could be the reduction of 8-HPODE to 8-HODE, or the reduction
of other hydroperoxides, with formation of ferryl oxygen with a
porphyrin -cation (compound I). The latter is rapidly reduced to
compound II with one electron, and a protein (tyrosyl) radical is
formed. The protein (tyrosyl) radical could initiate the LDS cycle: (i) the 8-pro-S hydrogen of linoleate is eliminated by the
protein radical, (ii) molecular oxygen reacts with the alkyl radical, (iii) 8R-peroxylinoleate is formed and it could form 8-HPODE
either by abstracting a hydrogen from the tyrosyl residue (and
regenerating the tyrosyl radical) or by receiving an electron from
other electron donors, (iv) the hydroperoxide oxygen-oxygen bound is
cleaved, (v) ferryl oxygen and 8-HODE are formed, and (vi) the ferryl
oxygen is inserted at C-7 of 8-HODE, 7,8-DiHODE is released from LDS, and ferriheme is formed. This hypothetical reaction mechanism of LDS
can be described as a branched chain reaction (45, 46), which is
loosely coupled.
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G. graminis causes a root disease of wheat with the ominous name "take-all" (47). The biological function of LDS is unknown, but 8-HODE has been identified as a sporulation hormone in Aspergillus nidulans (48, 49). LDS may therefore have an important physiological function. The reaction mechanism of LDS makes it of scientific interest in its own right. Only PGH synthases, which are of eminent pharmacological importance, are known to oxygenate polyunsaturated fatty acids by a related mechanism, and few, if any, hemeproteins can isomerize fatty acid hydroperoxides in the same way as does LDS.
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ACKNOWLEDGEMENT |
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We thank Dr. H. E. Nilsson of the Swedish Agricultural University for generous advice.
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FOOTNOTES |
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* This work was supported by the Swedish Medical Research Council (6523), Magn. Bergvalls Stiftelse (to E. O., C. S, and M. S.) and the Swedish Cancer Society (to Britt-Marie Sjöberg for M. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Presented in part at the Fourth International Congress on Essential Fatty Acids and Eicosanoids, Edinburgh, Scotland, July 20-24, 1997, and at the 5th International Conference on Eicosanoids and Other Bioactive Lipids in Cancer, Inflammation, and Related Diseases, La Jolla, CA, September 17-20, 1997.
§ To whom correspondence should be addressed: Dept. of Pharmaceutical Biosciences, Uppsala Biomedical Center, P. O. Box 591, S-751 24 Uppsala, Sweden. Tel.: 46 18 471 44 55; Fax: 46 18 55 29 36; E-mail: Ernst.Oliw{at}bmc.uu.se.
The abbreviations used are: PG, prostaglandin; CHAPS, 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonate; 7, 8-DiHODE, 7S,8S-dihydroxyoctadeca-9Z,12Z-dienoic acid13-HETrE, 13R-hydroxy-5Z,8Z,11Z-eicosatrienoic acid8-H(P)ODE, 8R-hydro(pero)xy-octadeca-9Z,12Z-dienoic acid13-HPODE, 13S-hydroperoxy-octadeca-9Z,11E-dienoic acidLDS, linoleate diol synthasePAGE, polyacrylamide gel electrophoresisP1/2, power of half-saturationmW, milliwattmT, millitesla.
2 E. H. Oliw and C. Su, unpublished observation.
3 L. Hörnsten, C. Su, U. Hellman, A. E. Osbourn, and E. H. Oliw, unpublished observation.
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REFERENCES |
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