Hydroperoxide Isomerase Research Articles

Characterization of a recombinant 7,8-linoleate diol synthase from Glomerella cingulate.

A putative diol synthase from the fungus Glomerella cingulate was cloned and expressed in Escherichia coli. The putative diol synthase from G. cingulate was purified by His-Trap affinity chromatography with a specific activity of 0.87 U mg(-1), an eightfold purification, and a yield of 28%. One unit of activity was defined as the amount of enzyme required to produce 1 μmol of 7,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid (7,8-DiHODE) per min. The purified enzyme was estimated as a 127-kDa tetramer with a molecular mass of 510 kDa by gel filtration chromatography. The enzyme converted linoleic acid to a product, identified as 7S,8S-DiHODE by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. The specific activity and catalytic efficiency (k cat/K m) of 7,8-diol synthase from G. cingulate for the conversion of fatty acid to dihydroxy fatty acid followed the order linoleic acid > α-linolenic acid > oleic acid > palmitoleic acid, indicating that the enzyme is a 7,8-linoleate diol synthase (7,8-LDS). The activity of the enzyme for the conversion of 7,8-DiHODE from linoleic acid was maximal at pH 6.5, 40 °C, and 2.5% (v/v) dimethyl sulfoxide (DMSO). Under these conditions, 7,8-LDS from G. cingulate converted 1.0 mM linoleic acid to 0.62 mM 7,8-DiHODE for 30 min, with a conversion yield of 62% (mol/mol), via 8-hydroperoxy-9,12(Z,Z)-octadecadienoic acid (8-HPODE) as an intermediate. The accumulation of 8-HPODE was due to a higher 8-dioxygenase activity in the N-terminal domain than hydroperoxide isomerase activity in the C-terminal domain.

7,8- And 5,8-linoleate diol synthases support the heterolytic scission of oxygen–oxygen bonds by different amide residues

Linoleate diol synthases (LDS) are fungal dioxygenase-cytochrome P450 fusion enzymes. They oxidize 18:2n-6 sequentially to 8R-hydroperoxylinoleic acid (8R-HPODE) and 7S,8S- or 5S,8R-dihydroxylinoleic acids (DiHODE) by intramolecular oxygen transfer. The P450 domains contain a conserved sequence, Ala-Asn-Gln-Xaa-Gln, presumably located in the I-helices. The Asn938Leu replacement of 7,8-LDS of Gaeumannomyces graminis virtually abolished and the Asn938Asp and Asn938Gln replacements reduced the hydroperoxide isomerase activity. Gln941Leu and Gln941Glu substitutions had little effects. Replacements of the homologous Asn887 and Gln890 residues of 5,8-LDS of Aspergillus fumigatus yielded the opposite results. Asn887Leu and Asn887Gln of 5,8-LDS retained 5,8-DiHODE as the main metabolite with an increased formation of 6,8- and 8,11-DiHODE, whereas Gln890Leu almost abolished the 5,8-LDS activity. Replacement of Gln890 with Glu also retained 5,8-DiHODE as the main product, but shifted oxygenation from C-5 to C-7 and C-11 and to formation of epoxyalcohols by homolytic scission of 8R-HPODE. P450 hydroxylases usually contain an “acid-alcohol” pair in the I-helices for the heterolytic scission of O2 and formation of compound I (Por+ Fe(IV)O) and water. The function of the acid-alcohol pair appears to be replaced by two different amide residues, Asn938 of 7,8-LDS and Gln890 of 5,8-LDS, for heterolysis of 8R-HPODE to generate compound I.

Expression of Fusion Proteins of Aspergillus terreus Reveals a Novel Allene Oxide Synthase

Aspergilli oxidize C18 unsaturated fatty acids by dioxygenase-cytochrome P450 fusion proteins to signal molecules involved in reproduction and host-pathogen interactions. Aspergillus terreus expresses linoleate 9R-dioxygenase (9R-DOX) and allene oxide synthase (AOS) activities in membrane fractions. The genome contains five genes (ATEG), which may code for a 9R-DOX-AOS fusion protein. The genes were cloned and expressed, but none of them oxidized 18:2n-6 to 9R-hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HPODE). ATEG_02036 transformed 9R-HPODE to an unstable allene oxide, 9(R),10-epoxy-10,12(Z)-octadecadienoic acid. A substitution in the P450 domain (C1073S) abolished AOS activity. The N964V and N964D mutants both showed markedly reduced AOS activity, suggesting that Asn(964) may facilitate homolytic cleavage of the dioxygen bond of 9R-HPODE with formation of compound II in analogy with plant AOS (CYP74) and prostacyclin synthase (CYP8A1). ATEG_03992 was identified as 5,8-linoleate diol synthase (5,8-LDS). Replacement of Asn(878) in 5,8-LDS with leucine (N878L) mainly shifted ferryl oxygen insertion from C-5 toward C-6, but replacements of Gln(881) markedly affected catalysis. The Q881L mutant virtually abolished the diol synthase activity. Replacement of Gln(881) with Asn, Glu, Asp, or Lys residues augmented the homolytic cleavage of 8R-HPODE with formation of 10-hydroxy-8(9)-epoxy-12(Z)-octadecenoic acid (erythro/threo, 1-4:1) and/or shifted ferryl oxygen insertion from C-5 toward C-11. We conclude that homolysis and heterolysis of the dioxygen bond with formation of compound II in AOS and compound I in 5,8-LDS are influenced by Asn and Gln residues, respectively, of the I-helices. AOS of A. terreus appears to have evolved independently of CYP74 but with an analogous reaction mechanism.

Steric analysis of epoxyalcohol and trihydroxy derivatives of 9-hydroperoxy-linoleic acid from hematin and enzymatic synthesis

We characterize the allylic epoxyalcohols and their trihydroxy hydrolysis products generated from 9R- and 9S-hydroperoxy-octadecenoic acid (HPODE) under non-enzymatic conditions, reaction with hematin and subsequent acid hydrolysis, and enzymatic conditions, incubation with Beta vulgaris containing a hydroperoxide isomerase and epoxide hydrolase. The products were resolved by HPLC and the regio and stereo-chemistry of the transformations were determined through a combination of 1H NMR and GC–MS analysis of dimethoxypropane derivatives. Four trihydroxy isomers were identified upon mild acid hydrolysis of 9S,10S-trans-epoxy-11E-13S-hydroxyoctadecenoate: 9S,10R,13S, 9S,12R,13S, 9S,10S,13S and 9S,12S,13S-trihydroxy-octadecenoic acids, in the ratio 40:26:22:12. We also identified a prominent δ-ketol rearrangement product from the hydrolysis as mainly the 9-hydroxy-10E-13-oxo isomer. Short incubation (5 min) of 9R- and 9S-HPODE with B. vulgaris extract yielded the 9R- and 9S-hydroxy-10E-12R,13S-cis-epoxy products respectively. Longer incubation (60 min) gave one specific hydrolysis product via epoxide hydrolase, the 9R/S,12S,13S-trihydroxyoctadecenoate. These studies provide a practical approach for the isolation and characterization of allylic epoxy alcohol and trihydroxy products using a combination of HPLC, GC–MS and 1H NMR.

8R-Lipoxygenase-catalyzed synthesis of a prominent cis-epoxyalcohol from dihomo-γ-linolenic acid: a distinctive transformation compared with S-lipoxygenases

Conversion of fatty acid hydroperoxides to epoxyalcohols is a well known secondary reaction of lipoxygenases, described for S-specific lipoxygenases forming epoxyalcohols with a trans-epoxide configuration. Here we report on R-specific lipoxygenase synthesis of a cis-epoxyalcohol. Although arachidonic and dihomo-γ-linolenic acids are metabolized by extracts of the Caribbean coral Plexaura homomalla via 8R-lipoxygenase and allene oxide synthase activities, 20:3ω6 forms an additional prominent product, identified using UV, GC-MS, and NMR in comparison to synthetic standards as 8R,9S-cis-epoxy-10S-erythro-hydroxy-eicosa-11Z,14Z-dienoic acid. Both oxygens of (18)O-labeled 8R-hydroperoxide are retained in the product, indicating a hydroperoxide isomerase activity. Recombinant allene oxide synthase formed only allene epoxide from 8R-hydroperoxy-20:3ω6, whereas two different 8R-lipoxygenases selectively produced the epoxyalcohol.A biosynthetic scheme is proposed in which a partial rotation of the reacting intermediate is required to give the observed erythro epoxyalcohol product. This characteristic and the synthesis of cis-epoxy epoxyalcohol may be a feature of R-specific lipoxygenases.

Expression of 5,8-LDS of Aspergillus fumigatus and its dioxygenase domain. A comparison with 7,8-LDS, 10-dioxygenase, and cyclooxygenase

5,8-Linoleate diol synthase (5,8-LDS) of Aspergillus fumigatus was cloned, expressed, and compared with 7,8-LDS of the Take-all fungus. Replacements of Tyr and Cys in the conserved YRWH and FXXGPHXCLG sequences abolished 8 R-dioxygenase (8-DOX) and hydroperoxide isomerase activities, respectively. The predicted α-helices of LDS were aligned with α-helices of cyclooxygenase-1 (COX-1) to identify the 8-DOX domains. N-terminal expression constructs of 5,8- and 7,8-LDS (674 of 1079, and 673 of 1165 residues), containing one additional α-helix compared to cyclooxygenase-1, yielded prominent 8 R-DOX activities with apparently unchanged or slightly lower substrate affinities, respectively. Val-328 of 5,8-LDS did not influence the position of oxygenation in contrast to the homologous residues Val-349 of COX-1 and Leu-384 of 10 R-dioxygenase. We conclude that ∼675 amino acids are sufficient to support 8-DOX activity.

Dioxygenase Activity of Epidermal Lipoxygenase-3 Unveiled: TYPICAL AND ATYPICAL FEATURES OF ITS CATALYTIC ACTIVITY WITH NATURAL AND SYNTHETIC POLYUNSATURATED FATTY ACIDS

Epidermal lipoxygenase-3 (eLOX3) exhibits hydroperoxide isomerase activity implicated in epidermal barrier formation, but its potential dioxygenase activity has remained elusive. We identified herein a synthetic fatty acid, 9E,11Z,14Z-20:3ω6, that was oxygenated by eLOX3 specifically to the 9S-hydroperoxide. Reaction showed a pronounced lag phase, which suggested that eLOX3 is deficient in its activation step. Indeed, we found that high concentrations of hydroperoxide activator (e.g. 65 μM) overcame a prolonged lag phase (>1 h) and unveiled a dioxygenase activity with arachidonic acid; the main products were the 5-, 9-, and 7-hydroperoxyeicosatetraenoic acids (HPETEs). These were R/S mixtures (ranging from ∼50:50 to 73:27), and as the bis-allylic 7-HPETE can be formed only inside the enzyme active site, the results indicate there is oxygen availability along either face of the reacting fatty acid radical. That the active site oxygen supply is limited is implied from the need for continuous re-activation, as carbon radical leakage leaves the enzyme in the unactivated ferrous state. An Ala-to-Gly mutation, known to affect the positioning of O(2) in the active site of other lipoxygenase enzymes, led to more readily activated reaction and a significant increase in the 9R- over the 5-HPETE. Activation and cycling of the ferric enzyme are thus promoted using the 9E,11Z,14Z-20:3ω6 substrate, by continuous hydroperoxide activation, or by the Ala-to-Gly mutation. We suggest that eLOX3 represents one end of a spectrum among lipoxygenases where activation is inefficient, favoring hydroperoxide isomerase cycling, with the opposite end represented by readily activated enzymes in which dioxygenase activity is prominent.

On the Role of Molecular Oxygen in Lipoxygenase Activation: COMPARISON AND CONTRAST OF EPIDERMAL LIPOXYGENASE-3 WITH SOYBEAN LIPOXYGENASE-1

The oxygenation of polyunsaturated fatty acids by lipoxygenases (LOX) is associated with a lag phase during which the resting ferrous enzyme is converted to the active ferric form by reaction with fatty acid hydroperoxide. Epidermal lipoxygenase-3 (eLOX3) is atypical in displaying hydroperoxide isomerase activity with fatty acid hydroperoxides through cycling of the ferrous enzyme. Yet eLOX3 is capable of dioxygenase activity, albeit with a long lag phase and need for high concentrations of hydroperoxide activator. Here, we show that higher O(2) concentration shortens the lag phase in eLOX3, although it reduces the rate of hydroperoxide consumption, effects also associated with an A451G mutation known to affect the disposition of molecular oxygen in the LOX active site. These observations are consistent with a role of O(2) in interrupting hydroperoxide isomerase cycling. Activation of eLOX3, A451G eLOX3, and soybean LOX-1 with 13-hydroperoxy-linoleic acid forms oxygenated end products, which we identified as 9R- and 9S-hydroperoxy-12S,13S-trans-epoxyoctadec-10E-enoic acids. We deduce that activation partly depends on reaction of O(2) with the intermediate of hydroperoxide cleavage, the epoxyallylic radical, giving an epoxyallylic peroxyl radical that does not further react with Fe(III)-OH; instead, it dissociates and leaves the enzyme in the activated free ferric state. eLOX3 differs from soybean LOX-1 in more tightly binding the epoxyallylic radical and having limited access to O(2) within the active site, leading to a deficiency in activation and a dominant hydroperoxide isomerase activity.

Biochemical Characterization of the Oxygenation of Unsaturated Fatty Acids by the Dioxygenase and Hydroperoxide Isomerase of Pseudomonas aeruginosa 42A2

We have studied oxygenation of fatty acids by cell extract of Pseudomonas aeruginosa 42A2. Oleic acid ((9Z)-18:1) was transformed to (10S)-hydroperoxy-(8E)-octadecenoic acid ((10S)-HPOME) and to (7S,10S)-dihydroxy-(8E)-octadecenoic acid (7,10-DiHOME). Experiments under oxygen-18 showed that 7,10-DiHOME contained oxygen from air and was formed sequentially from (10S)-HPOME by isomerization. (10R)-HPOME was not isomerized. The (10S)-dioxygenase and hydroperoxide isomerase activities co-eluted on ion exchange chromatography and on gel filtration with an apparent molecular size of approximately 50 kDa. 16:1n-7, 18:2n-6, and 20:1n-11 were also oxygenated to 7,10-dihydroxy fatty acids, and (8Z)-18:1 was oxygenated to 6,9-dihydroxy-(7E)-octadecenoic acid. A series of fatty acids with the double bond positioned closer to ((6Z)-18:1, (5Z,9Z)-18:2) or more distant from the carboxyl group ((11Z)-, (13Z)-, and (15Z)-18:1) were poor substrates. The oxygenation mechanism was studied with [7S-(2)H]18:1n-9, [7R-(2)H]18:2n-6, and [8R-(2)H]18:2n-6 as substrates. The pro-R hydrogen at C-8 was lost in the biosynthesis of (10S)-HPODE, whereas the pro-S hydrogen was lost and the pro-R hydrogen was retained at C-7 during biosynthesis of the 7,10-dihydroxy metabolites. Analysis of the fatty acid composition of P. aeruginosa revealed relatively large amounts of (9E/Z)-16:1 and (11E/Z)-18:1 and only traces of 18:1n-9. We found that (11Z)-18:1 (vaccenic acid) was transformed to (11S,14S)-dihydroxy-(12E)-octadecenoic acid and to a mixture of 11- and 12-HPOME, possibly due to reverse orientation of (11Z)-18:1 at the active site compared with oleic acid. The reaction mechanism of the hydroperoxide isomerase suggests catalytic similarities to cytochrome P450.

Gene Deletion of 7,8-Linoleate Diol Synthase of the Rice Blast Fungus: STUDIES ON PATHOGENICITY, STEREOCHEMISTRY, AND OXYGENATION MECHANISMS

Linoleate diol synthases (LDS) are heme enzymes, which oxygenate 18:2n-6 sequentially to (8R)-hydroperoxylinoleic acid ((8R)-HPODE) and to (5S,8R)-dihydroxy-, (7S,8S)-dihydroxy-, or (8R,11S)-dihydroxylinoleic acids (DiHODE). The genome of the rice blast fungus, Magnaporthe oryzae, contains two genes with homology to LDS. M. oryzae oxidized 18:2n-6 to (8R)-HPODE and to (7S,8S)-DiHODE, (6S,8R)-DiHODE, and (8R,11S)-HODE. Small amounts of 10-hydroxy-(8E,12Z)-octadecadienoic acid and traces of 5,8-DiHODE were also detected by liquid chromatography-mass spectrometry. The contribution of the 7,8-LDS gene to M. oryzae pathogenicity was evaluated by replacement of the catalytic domain with hygromycin and green fluorescent protein variant (SGFP) cassettes. This genetically modified strain Delta7,8-LDS infected rice leaves and roots and formed appressoria and conidia as the native fungus. The Delta7,8-LDS mutant had lost the capacity to biosynthesize all the metabolites except small amounts of 8-hydroxylinoleic acid. Studies with stereospecifically deuterated linoleic acids showed that (8R)-HPODE was formed by abstraction of the pro-S hydrogen at C-8 and antarafacial oxygenation, whereas (7S,8S)-DiHODE and (8R,11S)-DiHODE were formed from (8R)-HPODE by suprafacial hydrogen abstraction and oxygenation at C-7 and C-11, respectively. A mac1 suppressor mutant (Delta mac1 sum1-99) of M. oryzae, which shows cAMP-independent protein kinase A activity, oxygenated 18:2n-6 to increased amounts of (10R)-HPODE and (5S,8R)-DiHODE. Expression of the 7,8-LDS gene but not of the second homologue was detected in the suppressor mutant. This suggests that PKA-mediated signaling pathway regulates the dioxygenase and hydroperoxide isomerase activities of M. oryzae.

Reaction mechanism of 5,8-linoleate diol synthase, 10 R-dioxygenase, and 8,11-hydroperoxide isomerase of Aspergillus clavatus

Aspergilli express fusion proteins of an animal haem peroxidase domain with fatty acid dioxygenase (DOX) activity (∼ 600 amino acids) and a functional or non-functional hydroperoxide isomerase/cytochrome P450 domain (∼ 500 amino acids with EXXR and GPHXCLG motifs). 5,8-Linoleate diol synthases (LDS; ppoA) and 10 R-DOX (ppoC) of A spergillus nidulans and A. fumigatus belong to this group. Our objective was to determine the oxylipins formed from linoleic acid by A. clavatus and their mechanism of biosynthesis. A. clavatus oxidized linoleic acid to (8 R)-hydroperoxylinoleic acid (8 R-HPODE), (10 R)-hydroperoxy-8( E),12( Z)-octadecadienoic acid (10 R-HPODE), and to (5 S,8 R)-dihydroxy- and (8 R,11 S)-dihydroxylinoleic acids (DiHODE) as major products. This occurred by abstraction of the pro-S hydrogen at C-8 and antarafacial dioxygenation at C-8 or at C-10 with double bond migration. 8 R-HPODE was then isomerized to 5 S,8 R-DiHODE and to 8 R,11 S-DiHODE by abstraction of the pro-S hydrogens at C-5 and C-11 of 8 R-HPODE, respectively, followed by suprafacial oxygenation. The genome of A. clavatus codes for two enzymes, which can be aligned with > 65% amino acid identity to 10 R-DOX and 5,8-LDS, respectively. The 5,8-LDS homologue likely forms and isomerizes 8 R-HPODE to 5 S,8 R-DiHODE. A third gene (ppoB) codes for a protein which carries a serine residue at the cysteine position of the P450 motif. This Cys to Ser replacement is known to abolish P450 2B4 catalysis and the hydroperoxide isomerase activity of 5,8-LDS, suggesting that ppoB of A. clavatus may not be involved in the biosynthesis of 8 R,11 S-DiHODE.

Critical amino acids for the 8(R)-dioxygenase activity of linoleate diol synthase. A comparison with cyclooxygenases

7,8-Linoleate diol synthase (7,8-LDS) of the take-all fungus and cyclooxygenases can be aligned with ∼24% amino acid identity and both form a tyrosyl radical during catalysis. 7,8-LDS was expressed in insect cells with native 8R-dioxygenase and hydroperoxide isomerase activities. We studied conserved residues of 7,8-LDS, which participate in cyclooxygenases for heme binding (His residues), hydrogen abstraction (Tyr), positioning (Tyr, Trp), and ionic binding of substrates (Arg). Site-directed mutagenesis abolished 8R-dioxygenase activities with exception of the putative distal histidine (His203Gln) and a tyrosine residue important for hydrogen bonding and substrate positioning (Tyr329Phe). The results demonstrate structural similarities between 7,8-LDS and cyclooxygenases.

Pichia expression and mutagenesis of 7,8-linoleate diol synthase change the dioxygenase and hydroperoxide isomerase

Linoleate diol synthases (LDS) are homologous 8(R)-dioxygenases with hydroperoxide isomerase activities, expressed in fungal pathogens of humanitarian importance. We report for the first time expression and site-directed mutagenesis of LDS. 7,8-LDS of the take-all fungus, expressed in Pichia pastoris, oxygenated 18:2n−6 to 8(R)-hydroperoxylinoleic acid, which was unexpectedly isomerized to 5,8(R)-dihydroxylinoleic acid (60% 5S) and to 8(R),13-dihydroxyoctadeca-9(E),11(E)-dienoic acid. The latter was likely formed via hydrolysis of an unstable intermediate, 8(R),9(S)-epoxyoctadeca-10(E),12(Z)-dienoic acid. A tyrosyl radical is formed during 7,8-LDS catalysis, and Tyr376 is the sequence homolog to Tyr385 of cyclooxygenase-1. Tyr376Phe retained hydroperoxide isomerase activity but lacked 8(R)-dioxygenase activity. The putative proximal heme ligand His379 and the N-glycosylation site at Asn216 appeared to be critical for 8(R)-dioxygenase activity, as His379Gln and Asn216Gln were inactive. Treatment with α-mannosidase to shorten N- and O-linked mannosides inhibited the hydroperoxide isomerase but not the 8(R)-dioxygenase. Our results suggest that post-translational modifications may influence the oxidation mechanism of 7,8-LDS.

Identification of 15-hydroxy-11,12-epoxyeicosatrienoic acid as a vasoactive 15-lipoxygenase metabolite in rabbit aorta

Arachidonic acid (AA) causes endothelium-dependent smooth muscle hyperpolarizations and relaxations that are mediated by a 15-lipoxygenase-I (15-LO-I) metabolite, 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA). We propose that AA is metabolized sequentially by 15-LO-I and hydroperoxide isomerase to an unidentified hydroxyepoxyeicosatrienoic acid (HEETA), which is hydrolyzed by a soluble epoxide hydrolase (sEH) to 11,12,15-THETA. After incubation of aorta with 14C-labeled AA, metabolites were extracted and the HEETAs were resolved by performing HPLC. Mass spectrometric analyses identified 15-Hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA). Incubation of aortic incubates with methanol and acetic acid trapped the acid-sensitive 15-H-11,12-EETA as methoxydihydroxyeicosatrienoic acids (MDHEs) (367 m/z, M-H). Pretreatment of the aortic tissue with the sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA; 10(-6) M) increased the formation of 15-H-11,12-EETA, measured as MDHEs. Thus 15-H-11,12-EETA is an acid- and sEH-sensitive precursor of 11,12,15-THETA. Aortic homogenates and endothelial cells contain a 57-kDa protein corresponding to the rabbit sEH. In preconstricted aortic rings, AA (10(-7)-10(-4) M) and acetylcholine (10(-9)-10(-6) M) caused concentration-related relaxations that were enhanced by pretreatment with AUDA. These enhanced relaxations were inhibited by increasing extracellular [K(+)] from 4.8 to 20 mM. AA (3 x 10(-6) M) induced cell membrane hyperpolarization (from -31.0 +/- 1 to -46.8 +/- 2 mV) in aortic strips with an intact endothelium, which was enhanced by AUDA. These results indicate that 15-H-11,12-EETA is produced by the aorta, hydrolyzed by sEH to 11,12,15-THETA, and mediates relaxations by membrane hyperpolarization. 15-H-11,12-EETA represents an endothelium-derived hyperpolarizing factor.

11(R),12(S),15(S)-trihydroxyeicosa-5(Z),8(Z),13(E)-trienoic acid: an endothelium-derived 15-lipoxygenase metabolite that relaxes rabbit aorta

Previous studies indicate that 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA), an endothelium-derived hyperpolarizing factor in the rabbit aorta, mediates a portion of the relaxation response to acetylcholine by sequential metabolism of arachidonic acid by 15-lipoxygenase, hydroperoxide isomerase, and epoxide hydrolase. To determine the stereochemical configuration of the endothelial 11,12,15-THETA, its activity and chromatographic migration were compared with activity and migration of eight chemically synthesized stereoisomers of 11,12,15(S)-THETA. Of the eight isomers, only 11(R),12(S),15(S)-trihydroxyeicosa-5(Z),8(Z),13(E)-trienoic acid comigrated with the biological 11,12,15-THETA on reverse- and normal-phase HPLC and gas chromatography. The same THETA isomer (10(-7)-10(-4) M) relaxed the rabbit aorta in a concentration-related manner (maximum relaxation = 69 +/- 5%). These relaxations were blocked by apamin (10(-7) M), an inhibitor of small-conductance Ca2+-activated K+ channels. In comparison, 11(S),12(R),15(S),5(Z),8(Z),13(E)-THETA (10(-4) M) relaxed the aorta by 22%. The other six stereoisomers were inactive in this assay. With use of the whole cell patch-clamp technique, it was shown that 10(-4) M 11(R),12(S),15(S),5(Z),8(Z),13(E)-THETA increased outward K+ current in isolated aortic smooth muscle cells by 119 +/- 36% at +60 mV, whereas 10(-4) M 11(R),12(R),15(S),5(Z),8(Z),13(E)-THETA increased outward K+ current by only 20 +/- 2%. The 11(R),12(S),15(S),5(Z),8(Z),13(E)-THETA-stimulated increase in K+ current was blocked by pretreatment with apamin. These studies suggest that 11(R),12(S),15(S)-trihydroxyeicosa-5(Z),8(Z),13(E)-trienoic acid is the active stereoisomer produced by the rabbit aorta. It relaxes smooth muscle by activating K+ channels. The specific structural and stereochemical requirements for K+ channel activation suggest that a specific binding site or receptor of 11,12,15-THETA is involved in these actions.

Identification of Dioxygenases Required for Aspergillus Development

Aspergillus sp. contain ppoA, ppoB, and ppoC genes, which code for fatty acid oxygenases with homology to fungal linoleate 7,8-diol synthases (7,8-LDS) and cyclooxygenases. Our objective was to identify these enzymes, as ppo gene replacements show critical developmental aberrancies in sporulation and pathogenicity in the human pathogen Aspergillus fumigatus and the genetic model Aspergillus nidulans. The PpoAs of A. fumigatus and A. nidulans were identified as (8R)-dioxygenases with hydroperoxide isomerase activity, designated 5,8-LDS. 5,8-LDS transformed 18:2n-6 to (8R)-hydroperoxyoctadecadienoic acid ((8R)-HPODE) and (5S,8R)-dihydroxy-9Z,12Z-octadecadienoic acid ((5S,8R)-DiHODE). We also detected 8,11-LDS in A. fumigatus and (10R)-dioxygenases in both Aspergilli. The diol synthases oxidized [(8R)-(2)H]18:2n-6 to (8R)-HPODE with retention of the deuterium label, suggesting antarafacial hydrogen abstraction and insertion of molecular oxygen. Experiments with stereospecifically deuterated 18:2n-6 showed that (8R)-HPODE was isomerized by 5,8- and 8,11-LDS to (5S,8R)-DiHODE and to (8R,11S)-dihydroxy-9Z,12Z-octadecadienoic acid, respectively, by suprafacial hydrogen abstraction and oxygen insertion at C-5 and C-11. PpoCs were identified as (10R)-dioxygenases, which catalyzed abstraction of the pro-S hydrogen at C-8 of 18:2n-6, double bond migration, and antafacial insertion of molecular oxygen with formation of (10R)-hydroxy-8E,12Z-hydroperoxyoctadecadienoic acid ((10R)-HPODE). Deletion of ppoA led to prominent reduction of (8R)-H(P)ODE and complete loss of (5S,8R)-DiHODE biosynthesis, whereas biosynthesis of (10R)-HPODE was unaffected. Deletion of ppoC caused biosynthesis of traces of racemic 10-HODE but did not affect the biosynthesis of other oxylipins. We conclude that ppoA of Aspergillus sp. may code for 5,8-LDS with catalytic similarities to 7,8-LDS and ppoC for linoleate (10R)-dioxygenases. Identification of these oxygenases and their products will provide tools for analyzing the biological impact of oxylipin biosynthesis in Aspergilli.

Inducible expression of 15-lipoxygenase-2 and 8-lipoxygenase inhibits cell growth via common signaling pathways

Human 15-lipoxygenase (LOX)-2 and mouse 8-LOX represent orthologous members of the LOX family but display different positional specificities and tissue distribution. To study the functional role of 15-LOX-2 and 8-LOX in keratinocytes, an inducible Tet-On gene expression system was established in the premalignant mouse keratinocyte cell line 308. Doxycycline (dox)-induced expression of enzymatically active 15-LOX-2 and 8-LOX led to an inhibition of cell growth that was associated with an inhibition of DNA synthesis, as shown by a 15-46% reduction of 5-bromo-2-deoxy-uridine (BrdU) incorporation. The inhibitory effects were increased in the presence of exogenous arachidonic acid. In contrast, addition of linoleic acid or the LOX inhibitor baicalein reversed the growth-inhibitory effects. Treatment of the cells with 15-hydroxyeicosatetraenoic acid (HETE) or 8-HETE resulted in a similar inhibition of BrdU incorporation, whereas 13-hydroxyoctadecadienoic acid (HODE) and 9-HODE, in contrast, had no effects. Dox-induced keratinocytes showed increased levels of reactive oxygen species (ROS). The antioxidant N-acetyl-L-cysteine and a specific inhibitor of p38 mitogen-activated protein kinase, but not of extracellular signal-regulated kinase 1/2 or c-Jun N-terminal kinase/stress-activated kinases, completely abolished the LOX-induced growth inhibition, indicating a critical role of ROS and p38. Our data suggest that 15-LOX-2 and 8-LOX, although displaying different positional specificity, may use common signaling pathways to induce growth inhibition in premalignant epithelial cells.

A G316A Mutation of Manganese Lipoxygenase Augments Hydroperoxide Isomerase Activity: MECHANISM OF BIOSYNTHESIS OF EPOXYALCOHOLS

Lipoxygenases with R stereospecificity have a conserved Gly residue, whereas (S)-lipoxygenases have an Ala residue. Site-directed mutagenesis has shown that these residues control position and S/R stereospecificity of oxygenation. Recombinant Mn-LO was expressed in Pichia pastoris, and its conserved Gly-316 residue was mutated to Ala, Ser, Val, and Thr. The G316A mutant was catalytically active. We compared the catalytic properties of Mn-LO and the G316A mutant with 17:3n-3, 18:2n-6, 18:3n-3, and 19:3n-3 as substrates. Increasing the fatty acid chain length from C17 to C19 shifted the oxygenation by Mn-LO from the n-6 toward the n-8 carbon. The G316A mutant increased the oxygenation at the n-8 carbon of 17:3n-3 and at the n-10 carbon of the C17 and C18 fatty acids (from 1-2% to 7-11%). The most striking effect of the G316A mutant was a 2-, 7-, and 15-fold increase in transformation of the n-6 hydroperoxides of 19:3n-3, 18:3n-3, and 17:3n-3, respectively, to keto fatty acids and epoxyalcohols. The n-3 double bond was essential. An experiment under an oxygen-18 atmosphere showed that both oxygen atoms were retained in the epoxyalcohols. (R)-Hydroperoxides at n-6 of C17:3, 18:3, and 19:3 were transformed 5 times faster than S stereoisomers. The G316A mutant converted (13R)-hydroperoxylinolenic acid to 13-ketolinolenic acid (with an apparent K(m) of 0.01 mm) and to epoxyalcohols (viz. erythro- and threo-11-hydroxy-(12R,13R)-epoxy-(9Z,15Z)-octadecadienoic acids and one of the corresponding cis-epoxides as major products). A reducing lipoxygenase inhibitor stimulated the hydroperoxide isomerase activity, whereas a suicide-type lipoxygenase inhibitor reduced this activity. The n-3 double bond also appeared to influence the anaerobic formation of epoxyalcohols by Mn-LO, since 18:2n-6 and 18:3n-3 yielded different profiles of epoxyalcohols. Our results suggest that the G316A mutant augmented the hydroperoxide isomerase activity by positioning the hydroperoxy group at the n-6 carbon of n-3 fatty acids closer to the reduced catalytic metal.

Mutations associated with a congenital form of ichthyosis (NCIE) inactivate the epidermal lipoxygenases 12 R-LOX and eLOX3

Non-bullous congenital ichthyosiform erythroderma (NCIE) is one of the main clinical forms of ichthyosis. Genetic studies indicated that 12 R-lipoxygenase (12 R-LOX) or epidermal lipoxygenase-3 (eLOX3) was mutated in six families affected by NCIE [F. Jobard, C. Lefèvre, A. Karaduman, C. Blanchet-Bardon, S. Emre, J. Weissenbach, M. Özgüc, M. Lathrop, J.F. Prud'homme, J. Fischer, Lipoxygenase-3 ( ALOXE3) and 12( R)-lipoxygenase ( ALOX12B) are mutated in non-bullous congenital ichthyosiform erythroderma (NCIE) linked to chromosome 17p13.1, Hum. Mol. Genet. 11 (2002) 107–113.], but the impact of these mutations on LOX function has not been defined. To explore this, we overexpressed the wild-type or mutated enzymes in E. coli and COS7 cells and then analyzed the essential catalytic properties. We showed recently that human eLOX3 is a hydroperoxide isomerase (hepoxilin synthase) that converts the product of 12 R-LOX, 12 R-hydroperoxyeicosatetraenoic acid (12 R-HPETE) to a specific epoxyalcohol. Using incubations with [ 14C]-labeled substrates and HPLC analyses, we found that the naturally occurring mutations totally eliminate the lipoxygenase activity of 12 R-LOX and the hydroperoxide isomerase activity of eLOX3. We further demonstrate that the 12 R-LOX/eLOX3-derived 8 R-hydroxy-11 R,12 R-epoxide is converted by an epoxide hydrolase in COS7 cells and in human keratinocytes to a single isomer of 8,11,12-trihydroxyeicosa-5,9,14-trienoic acid. Taken together, the results support the hypothesis that 12 R-LOX, eLOX3, and perhaps an epoxide hydrolase function together in the normal process of skin differentiation, and that the loss of function mutations are the basis of the LOX-dependent form of NCIE.

Rabbit aorta converts 15-HPETE to trihydroxyeicosatrienoic acids: potential role of cytochrome P450

Previous work showed that rabbit aorta metabolizes arachidonic acid via 15-lipoxygenase to 15-hydroperoxyeicosatetraenoic acid (15-HPETE), which undergoes an enzymatic rearrangement to 11-hydroxy-14,15-epoxyeicosatrienoic acid (11-H-14,15-EETA) and 15-hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA). Hydrolysis of the epoxy group results in the formation of 11,14,15- and 11,12,15-trihydroxyeicosatrienoic acids (THETAs). Endothelial cells have several heme-containing enzymes including cytochromes P450 (CYP), nitric oxide synthase (eNOS), and prostacyclin (PGI 2) synthase that catalyze the rearrangement of 15-HPETE to HEETAs. Incubation of arachidonic acid and 15-lipoxygenase, or 15-HPETE with rabbit aortic microsomes or rat liver microsomes, a rich source of CYP, resulted in the formation of a product that comigrated with THETAs and HEETAs on HPLC. Immunoblot analysis showed the presence of CYP2C8 and CYP2J2 in aortic tissue and when CYP2J2 or CYP2C8 was incubated with arachidonic acid and 15-lipoxygenase, the major products were 11,12,15- and 11,14,15-THETAs. Incubation of purified hematin, CYP2C11, eNOS or PGI 2 synthase enzymes with arachidonic acid and 15-lipoxygenase produced a different pattern of metabolites from rabbit aortic microsomes. Clotrimazole, a non-specific CYP inhibitor, and ebastine and terfenadone, specific CYP2J2 inhibitors, blocked the ability of aortic microsomes to produce THETAs while specific inhibitors of PGI 2 synthase, eNOS or CYP2C8/2C9 had no effect on THETA production. We suggest that a CYP, possibly CYP2J2, may function as the hydroperoxide isomerase converting 15-HPETE to HEETAs in rabbit vascular tissue. Further hydrolysis of the epoxy group of the HEETAs results in the formation of 11,12,15- and 11,14,15-THETAs. The HEETAs and THETAs are both vasodilators and may function as important regulators of vascular tone.