Diol Synthase Research Articles

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.

Linoleate 9 R-dioxygenase and allene oxide synthase activities of Aspergillus terreus

Oxygenation of linoleic acid by Aspergillus terreus was studied with LC–MS/MS. 9( R) -Hydroperoxy-10( E),12( Z)-octadecadienoic acid (9 R-HpODE) was identified along with 10( R)-hydroxy-8( E),12( Z)-octadecadienoic acid and variable amounts of 8( R)-hydroxy-9( Z),12( Z)-octadecadienoic acid. 9 R-HpODE was formed from [11 S- 2H]18:2 n − 6 with loss of the deuterium label, suggesting antarafacial hydrogen abstraction and oxygenation. Two polar metabolites were identified as 9-hydroxy-10-oxo-12( Z)-octadecenoic acid (α-ketol) and 13-hydroxy-10-oxo-11( E)-octadecenoic acid (γ-ketol), likely formed by spontaneous hydrolysis of an unstable allene oxide, 9( R),10-epoxy-10,12( Z)-octadecadienoic acid. α-Linolenic acid and 20:2 n − 6 were oxidized to hydroperoxy fatty acids at C-9 and C-11, respectively, but α- and γ-ketols of these fatty acids could not be detected. The genome of A. terreus lacks lipoxygenases, but contains genes homologous to 5,8-linoleate diol synthases and linoleate 10 R-dioxygenases of aspergilli. Our results demonstrate that linoleate 9 R-dioxygenase linked to allene oxide synthase activities can be expressed in fungi.

Biosynthesis of a linoleic acid allylic epoxide: mechanistic comparison with its chemical synthesis and leukotriene A biosynthesis

Biosynthesis of the leukotriene A (LTA) class of epoxide is a lipoxygenase-catalyzed transformation requiring a fatty acid hydroperoxide substrate containing at least three double bonds. Here, we report on biosynthesis of a dienoic analog of LTA epoxides via a different enzymatic mechanism. Beginning with homolytic cleavage of the hydroperoxide moiety, a catalase/peroxidase-related hemoprotein from Anabaena PCC 7120, which occurs in a fusion protein with a linoleic acid 9R-lipoxygenase, dehydrates 9R-hydroperoxylinoleate to a highly unstable epoxide. Using methods we developed for isolating extremely labile compounds, we prepared and purified the epoxide and characterized its structure as 9R,10R-epoxy-octadeca-11E,13E-dienoate. This epoxide hydrolyzes to stable 9,14-diols that were reported before in linoleate autoxidation (Hamberg, M. 1983. Autoxidation of linoleic acid: Isolation and structure of four dihydroxy octadecadienoic acids. Biochim. Biophys. Acta 752: 353-356) and in incubations with the Anabaena enzyme (Lang, I., C. Göbel, A. Porzel, I. Heilmann, and I. Feussner. 2008. A lipoxygenase with linoleate diol synthase activity from Nostoc sp. PCC 7120. Biochem. J. 410: 347-357). We also prepared an equivalent epoxide from 13S-hydroperoxylinoleate using a "biomimetic" chemical method originally described for LTA(4) synthesis and showed that like LTA(4), the C18.2 epoxide conjugates readily with glutathione, a potential metabolic fate in vivo. We compare and contrast the mechanisms of LTA-type allylic epoxide synthesis by lipoxygenase, catalase/peroxidase, and chemical transformations. These findings provide new insights into the reactions of linoleic acid hydroperoxides and extend the known range of catalytic activities of catalase-related hemoproteins.

Leucine/Valine Residues Direct Oxygenation of Linoleic Acid by (10R)- and (8R)-Dioxygenases: EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF (10R)-DIOXYGENASE WITH EPOXYALCOHOL SYNTHASE ACTIVITY

Linoleate (10R)-dioxygenase (10R-DOX) of Aspergillus fumigatus was cloned and expressed in insect cells. Recombinant 10R-DOX oxidized 18:2n-6 to (10R)-hydroperoxy-8(E),12(Z)-octadecadienoic acid (10R-HPODE; approximately 90%), (8R)-hydroperoxylinoleic acid (8R-HPODE; approximately 10%), and small amounts of 12S(13R)-epoxy-(10R)-hydroxy-(8E)-octadecenoic acid. We investigated the oxygenation of 18:2n-6 at C-10 and C-8 by site-directed mutagenesis of 10R-DOX and 7,8-linoleate diol synthase (7,8-LDS), which forms approximately 98% 8R-HPODE and approximately 2% 10R-HPODE. The 10R-DOX and 7,8-LDS sequences differ in homologous positions of the presumed dioxygenation sites (Leu-384/Val-330 and Val-388/Leu-334, respectively) and at the distal site of the heme (Leu-306/Val-256). Leu-384/Val-330 influenced oxygenation, as L384V and L384A of 10R-DOX elevated the biosynthesis of 8-HPODE to 22 and 54%, respectively, as measured by liquid chromatography-tandem mass spectrometry analysis. The stereospecificity was also decreased, as L384A formed the R and S isomers of 10-HPODE and 8-HPODE in a 3:2 ratio. Residues in this position also influenced oxygenation by 7,8-LDS, as its V330L mutant augmented the formation of 10R-HPODE 3-fold. Replacement of Val-388 in 10R-DOX with leucine and phenylalanine increased the formation of 8R-HPODE to 16 and 36%, respectively, whereas L334V of 7,8-LDS was inactive. Mutation of Leu-306 with valine or alanine had little influence on the epoxyalcohol synthase activity. Our results suggest that Leu-384 and Val-388 of 10R-DOX control oxygenation of 18:2n-6 at C-10 and C-8, respectively. The two homologous positions of prostaglandin H synthase-1, Val-349 and Ser-353, are also critical for the position and stereospecificity of the cyclooxygenase reaction.

Identification of PpoA from Aspergillus nidulans as a Fusion Protein of a Fatty Acid Heme Dioxygenase/Peroxidase and a Cytochrome P450

The homothallic ascomycete Aspergillus nidulans serves as model organism for filamentous fungi because of its ability to propagate with both asexual and sexual life cycles, and fatty acid-derived substances regulate the balance between both cycles. These so-called psi (precocious sexual inducer) factors are produced by psi factor-producing oxygenases (Ppo enzymes). Bioinformatic analysis predicted the presence of two different heme domains in Ppo proteins: in the N-terminal region, a fatty acid heme dioxygenase/peroxidase domain is predicted, whereas in the C-terminal region, a P450 heme thiolate domain is predicted. To analyze the reaction catalyzed by Ppo enzymes, PpoA was expressed in Escherichia coli as an active enzyme. The protein was purified by 62-fold and identified as a homotetrameric ferric heme protein that metabolizes mono- as well as polyunsaturated C(16) and C(18) fatty acids at pH approximately 7.25. The presence of thiolate-ligated heme was confirmed on the basis of sequence alignments and the appearance of a characteristic 450 nm CO-binding spectrum. Studies on its reaction mechanism revealed that PpoA uses different heme domains to catalyze two separate reactions. Within the heme peroxidase domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic acid by abstracting a H-atom from C-8 of the fatty acid, yielding a carbon-centered radical that reacts with molecular dioxygen. In the second reaction step, 8-hydroperoxyoctadecadienoic acid is isomerized within the P450 heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. We identify PpoA as a bifunctional P450 fusion protein that uses a previously unknown reaction mechanism for forming psi factors.

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.

His-311 and Arg-559 Are Key Residues Involved in Fatty Acid Oxygenation in Pathogen-inducible Oxygenase

Pathogen-inducible oxygenase (PIOX) oxygenates fatty acids into 2R-hydroperoxides. PIOX belongs to the fatty acid alpha-dioxygenase family, which exhibits homology to cyclooxygenase enzymes (COX-1 and COX-2). Although these enzymes share common catalytic features, including the use of a tyrosine radical during catalysis, little is known about other residues involved in the dioxygenase reaction of PIOX. We generated a model of linoleic acid (LA) bound to PIOX based on computational sequence alignment and secondary structure predictions with COX-1 and experimental observations that governed the placement of carbon-2 of LA below the catalytic Tyr-379. Examination of the model identified His-311, Arg-558, and Arg-559 as potential molecular determinants of the dioxygenase reaction. Substitutions at His-311 and Arg-559 resulted in mutant constructs that retained virtually no oxygenase activity, whereas substitutions of Arg-558 caused only moderate decreases in activity. Arg-559 mutant constructs exhibited increases of greater than 140-fold in Km, whereas no substantial change in Km was observed for His-311 or Arg-558 mutant constructs. Thermal shift assays used to measure ligand binding affinity show that the binding of LA is significantly reduced in a Y379F/R559A mutant construct compared with that observed for Y379F/R558A construct. Although Oryza sativa PIOX exhibited oxygenase activity against a variety of 14-20-carbon fatty acids, the enzyme did not oxygenate substrates containing modifications at the carboxylate, carbon-1, or carbon-2. Taken together, these data suggest that Arg-559 is required for high affinity binding of substrates to PIOX, whereas His-311 is involved in optimally aligning carbon-2 below Tyr-379 for catalysis.

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.

Properties of a mini 9 R-lipoxygenase from Nostoc sp. PCC 7120 and its mutant forms

Lipoxygenases (LOXs) consist of a class of enzymes that catalyze the regio- and stereospecific dioxygenation of polyunsaturated fatty acids. Current reports propose that a conserved glycine residue in the active site of R-lipoxygenases and an alanine residue at the corresponding position in S-lipoxygenases play a crucial role in determining the stereochemistry of the product. Recently, a bifunctional lipoxygenase with a linoleate diol synthase activity from Nostoc sp. PCC7120 with R stereospecificity and the so far unique feature of carrying an alanine instead of the conserved glycine in the position of the sequence determinant for chiral specificity was identified. The recombinant carboxy-terminal domain was purified after expression in Escherichia coli. The ability of the enzyme to use linoleic acid esterified to a bulky phosphatidylcholine molecule as a substrate suggested a tail-fist binding orientation of the substrate. Site directed mutagenesis of the alanine to glycine did not cause alterations in the stereospecificity of the products, while mutation of the alanine to valine or isoleucine modified both regio- and enantioselectivity of the enzyme. Kinetic measurements revealed that substitution of Ala by Gly or Val did not significantly influence the reaction characteristics, while the A162I mutant showed a reduced v max. Based on the mutagenesis data obtained, we suggest that the existing model for stereocontrol of the lipoxygenase reaction may be expanded to include enzymes that seem to have in general a smaller amino acid in R and a bulkier one in S lipoxygenases at the position that controls stereospecificity.

A lipoxygenase with linoleate diol synthase activity from Nostoc sp. PCC 7120

The dioxygenation of PUFAs (polyunsaturated fatty acids) in plants is mainly catalysed by members of the LOX (lipoxygenase) enzyme family. LOX products may be further metabolized, and are known as signalling substances in plant development and in responses to wounding and pathogen attack. In contrast with the situation in eukaryotes, information on the relevance of lipid peroxide metabolism in prokaryotic organisms is scarce. Therefore, we aimed to analyse LOXs and oxylipin patterns of cyanobacterial origin. A search of the genomic sequence of the cyanobacterium Nostoc sp. PCC 7120 suggested an open reading frame encoding a putative LOX named NspLOX that harboured an N-terminal extension. Individual analysis of recombinant C-terminal domain revealed enzymatic activity as a linoleate (9R)-LOX. Analysis of the full-length NspLOX protein, however, revealed linoleate diol synthase activity, generating (10E,12E)-9,14-dihydroxy-10,12-octadecadienoic acid as the main product from LA (linoleic acid) and (10E,12E,14E)-9,16-dihydroxy-10,12,14-octadecatrienoic acid as the main product from ALA (alpha-LA) substrates respectively, with ALA as preferred substrate. The enzyme exhibited a broad pH optimum between pH 7 and pH 10. Soluble extracts of Nostoc sp. contain more 9-LOX-derived hydroperoxides in sonified than in non-sonified cells, but products of full-length NspLOX were not detectable under the conditions used. As no other LOX-like sequence was identified in the genome of Nostoc sp. PCC 7120, the results presented suggest that (9R)-LOX-derived oxylipins may represent the endogenous products of NspLOX. Based on the biochemical results of NspLOX, we suggest that this bifunctional enzyme may represent a more ancient way to control the intracellular amount of oxylipins in this cyanobacterium.

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.

Linoleate diol synthase of the rice blast fungus Magnaporthe grisea.

Mycelia of two strains of Magnaporthe grisea, Guy 11 and TH3, were incubated with linoleic acid, and the metabolites were isolated and identified by GC-MS and LC-MS. The two main metabolites were identified as 8-hydroxylinoleic and 7,8-dihydroxylinoleic acids, and the former was further oxidized by n-2 and by n-3 hydroxylation to 8,16- and 8,17-dihydroxylinoleic acids. Lipoxygenase metabolites of linoleic acid could not be detected. The sequence of the genome of M. grisea has been released from the Whitehead Institute; it contains a gene with close homology to the linoleate diol synthase gene of the take-all fungus Gaeumannomyces graminis. Both genes appear to have the same organization, with four exons and three short introns, and the intron-exon borders were determined by reverse-transcription PCR and sequencing. The linoleate diol synthase precursor of G. graminis consists of 978 amino acids, whereas the putative diol synthase precursor of M. grisea contains 987 amino acids. The diol synthases of G. graminis and M. grisea can be aligned with 65% identical and 78% positive amino acid residues, and catalytically important amino acid residues were conserved.

α-Dioxygenase, a new enzyme in fatty acid metabolism

Infection of tobacco leaves with bacteria resulted in the accumulation of a 75-kDa oxygenase. This enzyme and a homologous one from Arabidopsis were expressed in insect cells and their catalytic properties were studied. Linolenic acid incubated with preparations containing the recombinant enzymes was converted into 2( R)-hydroperoxylinolenic acid, an unstable oxylipin which underwent spontaneous degradation into a mixture of 8( Z), 11( Z), 14( Z)-heptadecatrienal, 2( R)-hydroxylinolenic acid, and 8( Z), 11( Z), 14( Z)-heptadecatrienoic acid. Studies with stereospecifically deuterated linolenic acid demonstrated that the enzymatic α-dioxygenation proceeded with retention of absolute configuration. The α-dioxygenases from tobacco and Arabidopsis showed homology to mammalian prostaglandin endoperoxide synthases-1 and -2 and to linoleate diol synthase from certain fungi. His-388 and His-207 (heme-binding), Gln-203 (charge-stabilizing), and Tyr-385 (hydrogen-abstracting) of ovine endoperoxide synthase-1 were conserved in the α-dioxygenases, and mutation studies of the Arabidopsis enzyme showed that their replacement with other amino acids resulted in loss of catalytic activity. The α-dioxygenase activity in tobacco leaves increased from three- to fourfold upon mechanical wounding, about fourfold in response to infection by Pseudomonas syringae pv syringae (incompatible strain), and from six- to eightfold in response to P. syringae pv tabaci (compatible strain).

Identification of genes expressed during cercosporin biosynthesisin Cercospora zeae-maydis

Gray leaf spot, caused by the fungus Cercospora zeae-maydis, is the most destructive foliar disease of maize in the United States. However, little is known about the biochemical and molecular events of pathogenesis. C. zeae-maydis produces cercosporin, a phytotoxin shown to be a virulence factor in diseases caused by other Cercospora species. To identify genes involved in cercosporin biosynthesis and ultimately determine the role of cercosporin in disease development, we constructed a cDNA subtraction library of C. zeae-maydis by suppression subtractive hybridization. Poly (A) + RNA isolated from cultures grown in a cercosporin-suppressing medium was subtracted from poly (A) + RNA isolated from cultures grown in a cercosporin-inducing medium, resulting in transcripts that are specific to the cercosporin-producing culture. Analyses of 768 sequences in this cDNA subtraction library revealed 197 cDNAs with high similarity to genes in the GenBank and Saccharomyces Genome Database, and these genes were grouped into nine categories based on predicted functions of the encoded proteins. Northern analysis of seven selected clones with predicted functions in fatty acid metabolism (fatty acid synthase, oleate Δ-12 desaturase, and linoleate diol synthase) and secondary metabolism [cytochrome P450 oxidoreductase, cytochrome P450 monooxygenase, dihydrogeodin (phenol) oxidase, and coproporphyrinogen oxidase] indicated that those genes were expressed in cercosporin-inducing conditions. Analysis of expression kinetics confirmed that those genes are expressed concomitantly with cercosporin accumulation. Published by Elsevier Science Ltd.

A gene that encodes a product with similarity to dioxygenases is highly expressed in teliospores of Ustilago maydis.

The phytopathogenic basidiomycete Ustilago maydis produces sexual teliospores only after infection of its host plant, maize. To investigate the process of spore formation, we have isolated Ssp1, a protein that is abundantly expressed in mature teliospores. The corresponding gene, ssp1, is expressed at low levels in haploid sporidia; however, transcriptional levels are drastically induced in mature teliospores. Transcriptional regulation of ssp1 involves positive and negative promoter elements, and is subject to control by the cAMP signaling cascade and histone deacetylase-mediated repression. Ssp1 shows similarity to linoleate diol synthase, a fatty acid dioxygenase from the fungus Gaeumannomyces graminis. In agreement with this presumed function, Ssp1 is localized on lipid bodies in germinating teliospores, suggesting a role in the mobilization of storage lipids.

Linoleate diol synthase and pgh synthases — A new gene family of fatty acid heme dioxygenases?

Linoleate diol synthase and pgh synthases — A new gene family of fatty acid heme dioxygenases?

Cloning of Linoleate Diol Synthase Reveals Homology with Prostaglandin H Synthases

Linoleate diol synthase is a homotetrameric ferric hemeprotein, which catalyzes dioxygenation of linoleic acid to (8R)-hydroperoxylinoleate and isomerization of the hydroperoxide to (7S,8S)-dihydroxylinoleate. Ferryl intermediates and a tyrosyl radical are formed in the reaction. Linoleate diol synthase was digested with endoproteinase Lys-C, and internal peptides were sequenced. The sequence information was used for reverse transcription-polymerase chain reaction analysis, and a cDNA probe was obtained. Northern blot analysis of linoleate diol synthase suggested a 3.7-kilobase pair (kb) mRNA. A full-length clone of the linoleate diol synthase gene was obtained by screening of a genomic lambda-ZAP II library of the fungus Gaeumannomyces graminis. The 5'-untranslated region contained CAAT- and TATA-like boxes. The gene contained three short introns and spanned over 3.2-kb. The deduced open reading frame consisted of 2.9-kb, which corresponded to 978 amino acids and a molecular subunit mass of 108,000. Data base analysis with the gapped BLAST algorithm showed that 391 residues of linoleate diol synthase was 23-24% identical and 36-37% positive with the catalytic domain of mammalian prostaglandin H (PGH) synthase-2. Based on homology with PGH synthases, the proximal heme ligand of linoleate diol synthase was tentatively identified as His-379 and the important tyrosine for catalysis as residue 376 (apparent consensus EFNXXXYXWH). The distal heme ligand was tentatively identified as His-203 (apparent consensus THXXFXT). We conclude from catalytic and structural similarities that linoleate diol synthase and PGH synthases likely share common ancestry and may belong to a gene family of fatty acid heme dioxygenases.

Analysis of novel hydroperoxides and other metabolites of oleic, linoleic, and linolenic acids by liquid chromatography-mass spectrometry with ion trap MSn.

Linoleate is oxygenated by manganese-lipoxygenase (Mn-LO) to 11S-hydroperoxylinoleic acid and 13R-hydroperoxyoctadeca-9Z,11E-dienoic acid, whereas linoleate diol synthase (LDS) converts linoleate sequentially to 8R-hydroperoxylinoleate, through an 8-dioxygenase by insertion of molecular oxygen, and to 7S,8S-dihydroxylinoleate, through a hydroperoxide isomerase by intramolecular oxygen transfer. We have used liquid chromatography-mass spectrometry (LC-MS) with an ion trap mass spectrometer to study the MSn mass spectra of the main metabolites of oleic, linoleic, alpha-linolenic and gamma-linolenic acids, which are formed by Mn-LO and by LDS. The enzymes were purified from the culture broth (Mn-LO) and mycelium (LDS) of the fungus Gaeumannomyces graminis. MS3 analysis of hydroperoxides and MS2 analysis of dihydroxy- and monohydroxy metabolites yielded many fragments with information on the position of oxygenated carbons. Mn-LO oxygenated C-11 and C-13 of 18:2n-6, 18:3n-3, and 18:3n-6 in a ratio of approximately 1:1-3 at high substrate concentrations. 8-Hydroxy-9(10)epoxystearate was identified as a novel metabolite of LDS and oleic acid by LC-MS and by gas chromatography-MS. We conclude that LC-MS with MSn is a convenient tool for detection and identification of hydroperoxy fatty acids and other metabolites of these enzymes.