Thirty years with three-dimensional structures of lipoxygenases.

The X-ray crystal structures of soybean lipoxygenase (LOX) and rabbit 15-LOX were reported in the 1990s. Subsequent 3D structures demonstrated a conserved U-like shape of the substrate cavities as reviewed here. The 8-LOX:arachidonic acid (AA) complex showed AA bound to the substrate cavity carboxylate-out with C10 at 3.4 Å from the iron metal center. A recent cryo-electron microscopy (EM) analysis of the 12-LOX:AA complex illustrated AA in the same position as in the 8-LOX:AA complex. The 15- and 12-LOX complexes with isoenzyme-specific inhibitors/substrate mimics confirmed the U-fold. 5-LOX oxidizes AA to leukotriene A4, the first step in biosynthesis of mediators of asthma. The X-ray structure showed that the entrance to the substrate cavity was closed to AA by Phe and Tyr residues of a partly unfolded α2-helix. Recent X-ray analysis revealed that soaking with inhibitors shifted the short α2-helix to a long and continuous, which opened the substrate cavity. The α2-helix also adopted two conformations in 15-LOX. 12-LOX dimers consisted of one closed and one open subunit with an elongated α2-helix. 13C-ENDOR-MD computations of the 9-MnLOX:linoleate complex showed carboxylate-out position with C11 placed 3.4 ± 0.1 Å from the catalytic water. 3D structures have provided a solid ground for future research.

Diversity of the manganese lipoxygenase gene family - A mini-review.

Analyses of fungal genomes of escalate from biological and evolutionary investigations. The biochemical analyses of putative enzymes will inevitably lag behind and only a selection will be characterized. Plant-pathogenic fungi secrete manganese-lipoxygenases (MnLOX), which oxidize unsaturated fatty acids to hydroperoxides to support infection. Six MnLOX have been characterized so far including the 3D structures of these enzymes of the Rice blast and the Take-all fungi. The goal was to use this information to evaluate MnLOX-related gene transcripts to find informative specimens for further studies. Phylogenetic analysis, determinants of catalytic activities, and the C-terminal amino acid sequences divided 54 transcripts into three major subfamilies. The six MnLOX belonged to the same "prototype" subfamily with conserved residues in catalytic determinants and C-terminal sequences. The second subfamily retained the secretion mechanism, presumably necessary for uptake of Mn2+, but differed in catalytic determinants and by cysteine replacement of an invariant Leu residue for positioning ("clamping") of fatty acids. The third subfamily contrasted with alanine in the Gly/Ala switch for regiospecific oxidation and a minority contained unprecedented C-terminal sequences or lacked secretion signals. With these exceptions, biochemical analyses of transcripts of the three subfamilies appear to have reasonable prospects to find active enzymes.

Iron and manganese lipoxygenases of plant pathogenic fungi and their role in biosynthesis of jasmonates.

Lipoxygenases (LOX) contain catalytic iron (FeLOX), but fungi also produce LOX with catalytic manganese (MnLOX). In this review, the 3D structures and properties of fungal LOX are compared and contrasted along with their associations with pathogenicity. The 3D structures and properties of two MnLOX (Magnaporthe oryzae, Geaumannomyces graminis) and the catalysis of four additional MnLOX have provided information on the metal centre, substrate binding, oxygenation, tentative O2 channels, and biosynthesis of exclusive hydroperoxides. In addition, the genomes of other plant pathogens also code for putative MnLOX. Crystals of the 13S-FeLOX of Fusarium graminearum revealed an unusual altered geometry of the Fe ligands between mono- and dimeric structures, influenced by a wrapping sequence extension near the C-terminal of the dimers. In plants, the enzymes involved in jasmonate synthesis are well documented whereas the fungal pathway is yet to be fully elucidated. Conversion of deuterium-labelled 18:3n-3, 18:2n-6, and their 13S-hydroperoxides to jasmonates established 13S-FeLOX of F. oxysporum in the biosynthesis, while subsequent enzymes lacked sequence homologues in plants. The Rice-blast (M. oryzae) and the Take-all (G. graminis) fungi secrete MnLOX to support infection, invasive hyphal growth, and cell membrane oxidation, contributing to their devastating impact on world production of rice and wheat.

Fatty acid dioxygenase-cytochrome P450 fusion enzymes of filamentous fungal pathogens.

Oxylipins designate oxygenated unsaturated C18 fatty acids. Many filamentous fungi pathogens contain dioxygenases (DOX) in oxylipin biosynthesis with homology to human cyclooxygenases. They contain a DOX domain, which is often fused to a functional cytochrome P450 at the C-terminal end. A Tyr radical in the DOX domain initiates dioxygenation of linoleic acid by hydrogen abstraction with formation of 8-, 9-, or 10-hydroperoxy metabolites. The P450 domains can catalyze heterolytic cleavage of 8- and 10-hydroperoxides with oxidation of the heme thiolate iron for hydroxylation at C-5, C-7, C-9, or C-11 and for epoxidation of the 12Z double bond; thus displaying linoleate diol synthase (LDS) and epoxy alcohol synthase (EAS) activities. LSD activities are present in the rice blast pathogen Magnaporthe oryzae, Botrytis cinerea causing grey mold and the black scurf pathogen Rhizoctonia solani. 10R-DOX-EAS has been found in M. oryzae and Fusarium oxysporum. The P450 domains may also catalyze homolytic cleavage of 8- and 9-hydroperoxy fatty acids and dehydration to produce epoxides with an adjacent double bond, i.e., allene oxides, thus displaying 8- and 9-DOX-allene oxide synthases (AOS). F. oxysporum, F. graminearum, and R. solani express 9S-DOX-AOS and Zymoseptoria tritici 8S-and 9R-DOX-AOS. Homologues are present in endemic human-pathogenic fungi with extensive studies in Aspergillus fumigatus, A. flavus (also a plant pathogen) as well as the genetic model A. nidulans. 8R-and 10R-DOX appear to bind fatty acids "headfirst" in the active site, whereas 9S-DOX binds them "tail first" in analogy with cyclooxygenases. The biological relevance of 8R-DOX-5,8-LDS (also designated PpoA) was first discovered in relation to sporulation of A. nidulans and recently for development and programmed hyphal branching of A. fumigatus. Gene deletion DOX-AOS homologues in F. verticillioides, A. flavus, and A. nidulans alters, inter alia, mycotoxin production, sporulation, and gene expression.

WITHDRAWN: Fatty acid dioxygenase-cytochrome P450 fusion enzymes of the top 10 fungal pathogens in molecular plant pathology and human-pathogenic fungi.

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Linoleate diol synthase related enzymes of the human pathogens Histoplasma capsulatum and Blastomyces dermatitidis.

Histoplasma capsulatum is an ascomyceteous fungus and a human lung pathogen, which is present in river valleys of the Americas and other continents. H. capsulatum and two related human pathogens, Blasmomyces dermatitidis and Paracoccidioides brasiliensis, belongs to the Ajellomycetaceae family. The genomes of all three species code for three homologous and tentative enzymes of the linoleate diol synthase (LDS) family of fusion enzymes with dioxygenase (DOX) and cytochrome P450 domains. One group aligned closely with 8R-DOX-5,8-LDS of Aspergilli, which oxidizes linoleic acid to 5S,8R-dihydroxylinoleic acid; this group was not further investigated. The second group aligned with 10R-DOX-epoxy alcohol synthase (EAS) of plant pathogens. Expression of this enzyme from B. dermatitidis revealed only 10R-DOX activities, i.e., oxidation of linoleic acid to 10R-hydroperoxy-8E,12Z-octadecadienoic acid. The third group aligned in a separate entity. Expression of these enzymes of H. capsulatum and B. dermatitidis revealed no DOX activities, but both enzymes transformed 13S-hydroperoxy-9Z,11E-octadecadienoic acid efficiently to 12(13S)epoxy-11-hydroperoxy-9Z-octadecenoic acid. Other 13-hydroperoxides of linoleic and α-linolenic acids were transformed with less efficiency and the 9-hydroperoxides of linoleic acid were not transformed. In conclusion, a novel EAS has been found in H. capsulatum and B. dermititidis with 13S-hydroperoxy-9Z,11E-octadecadienoic acid as the likely physiological substrate.

Fungal oxylipins direct programmed developmental switches in filamentous fungi.

Filamentous fungi differentiate along complex developmental programs directed by abiotic and biotic signals. Currently, intrinsic signals that govern fungal development remain largely unknown. Here we show that an endogenously produced and secreted fungal oxylipin, 5,8-diHODE, induces fungal cellular differentiation, including lateral branching in pathogenic Aspergillus fumigatus and Aspergillus flavus, and appressorium formation in the rice blast pathogen Magnaporthe grisea. The Aspergillus branching response is specific to a subset of oxylipins and is signaled through G-protein coupled receptors. RNA-Seq profiling shows differential expression of many transcription factors in response to 5,8-diHODE. Screening of null mutants of 33 of those transcription factors identifies three transcriptional regulators that appear to mediate the Aspergillus branching response; one of the mutants is locked in a hypo-branching phenotype, while the other two mutants display a hyper-branching phenotype. Our work reveals an endogenous signal that triggers crucial developmental processes in filamentous fungi, and opens new avenues for research on the morphogenesis of filamentous fungi.

Charge migration fragmentation in the negative ion mode of cyclopentenone and cyclopentanone intermediates in the biosynthesis of jasmonates.

RATIONALE: Jasmonates are formed from 12-oxo-10,15(Z)-phytodienoic acid (12-OPDA) in plants and also from 12-oxo-10-phytoenoic acid (12-OPEA) in fungi. Collision-induced dissociation (CID) of [M-H]- generates characteristic product anions at m/z 165 [C11 H17 O]- . Our goal was to investigate the structure and mode of formation of this anion by CID of 12-OPDA, 12-OPEA, and 12-oxophytonoic acid (12-OPA). METHODS: We investigated the CID of the [M-H]- , [M-H-CO2 ]- , and [M-H-H2 O]- anions using electrospray ionization and MS/MS analysis of 12-OPDA, 12-OPEA, and 12-OPA, and compared the results with the data obtained with the corresponding compounds labeled with 2 H at C-6 and C-7 and with structural and side chain analogs. RESULTS: CID of [6,6,7,7-2 H4 ]12-OPEA and [6,6-2 H2 ]12-OPDA ([M-H]- and [M-H-CO2 ]- ) showed that one or two 2 H atoms were transferred to anions at m/z 165 as judged by the signal intensities of m/z 165 + 1 or 165 + 2, respectively. CID of [6,6-2 H2 ]- and [6,6,7,7-2 H4 ]-12-OPA ([M-H]- and [M-H-CO2 ]- ) yielded the loss of H2 from the cyclopentanone and displayed the transfer of one 2 H atom in analogy to 12-OPEA. In contrast, CID of [6,6,7,7-2 H4 ]12-OPEA and [6,6,7,7-2 H4 ]12-OPA [M-H-H2 O]- demonstrated the transfer of two 2 H atoms (m/z 165 + 2). All spectra obtained by CID of [6,6,7,7-2 H4 ]12-OPDA and [6,6,7,7-2 H4 ]12-oxo-9(13),15(Z)-phytodienoic acid showed that one or two additional 2 H atoms could be transferred to this anion at m/z 167 of [6,6-2 H2 ]12-OPDA due to isotope scrambling. CONCLUSIONS: CID of 12-OPDA and 12-OPEA generates cyclopentanone enolate anions at m/z 165 by charge-driven hydride transfer as a common mechanism and by bond cleavage between C-7 and C-8 of the carboxyl side chains with either gain or loss of a hydrogen atom.

Biosynthesis of Jasmonates from Linoleic Acid by the Fungus Fusarium oxysporum. Evidence for a Novel Allene Oxide Cyclase.

Fusarium oxysporum f. sp. tulipae (FOT) secretes (+)-7-iso-jasmonoyl-(S)-isoleucine ((+)-JA-Ile) to the growth medium together with about 10 times less 9,10-dihydro-(+)-7-iso-JA-Ile. Plants and fungi form (+)-JA-Ile from 18:3n-3 via 12-oxophytodienoic acid (12-OPDA), which is formed sequentially by 13S-lipoxygenase, allene oxide synthase (AOS), and allene oxide cyclase (AOC). Plant AOC does not accept linoleic acid (18:2n-6)-derived allene oxides and dihydrojasmonates are not commonly found in plants. This raises the question whether 18:2n-6 serves as the precursor of 9,10-dihydro-JA-Ile in Fusarium, or whether the latter arises by a putative reductase activity operating on the n-3 double bond of (+)-JA-Ile or one of its precursors. Incubation of pentadeuterated (d5 ) 18:3n-3 with mycelia led to the formation of d5 -(+)-JA-Ile whereas d5 -9,10-dihydro-JA-Ile was not detectable. In contrast, d5 -9,10-dihydro-(+)-JA-Ile was produced following incubation of [17,17,18,18,18-2 H5 ]linoleic acid (d5 -18:2n-6). Furthermore, 9(S),13(S)-12-oxophytoenoic acid, the 15,16-dihydro analog of 12-OPDA, was formed upon incubation of unlabeled or d5 -18:2n-6. Appearance of the α-ketol, 12-oxo-13-hydroxy-9-octadecenoic acid following incubation of unlabeled or [13 C18 ]-labeled 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid confirmed the involvement of AOS and the biosynthesis of the allene oxide 12,13(S)-epoxy-9,11-octadecadienoic acid. The lack of conversion of this allene oxide by AOC in higher plants necessitates the conclusion that the fungal AOC is distinct from the corresponding plant enzyme.

Polyunsaturated C18 fatty acids derivatized with Gly and Ile as an additional tool for studies of the catalytic evolution of fungal 8- and 9-dioxygenases.

The fungal linoleate diol synthase (LDS) family contains over twenty characterized 8-, 9-, and 10-dioxygenases (DOX), usually fused to catalytically competent cytochromes P450. Crystal structures are not available, but indirect evidence suggests that linoleic acid enters the active site of 8R-DOX-LDS headfirst and enters 9S-DOX-allene oxide synthase (AOS) with the ω-end (tail) first. Fatty acids derivatized with amino acids can conceivably be used to study oxidation in tail first position by enzymes, which bind natural fatty acids headfirst. The results might reveal catalytic similarities of homologous enzymes. 8R-DOX-5,8-LDS oxidize 18:2n-6-Ile and 18:2n-6-Gly in tail first position to 9S-hydroperoxy metabolites, albeit with less position and stereo specificity than 9S-DOX-AOS. The oxygenation mechanism of 9S-DOX-AOS with antarafacial hydrogen abstraction at C-11 and oxygen insertion at C-9 was also retained. Two homologues, 8R-DOX-7,8-LDS and 8R-DOX-AOS, oxidized 18:2n-6-Ile and 18:2n-6-Gly at C-9, suggesting a conserved feature of 8R-DOX domains. 9R-DOX-AOS, with 54% sequence identity to 9S-DOX-AOS, did not oxidize the derivatized C18 fatty acids. 9Z,12Z-16:2, two carbon shorter than 18:n-6 from the ω-end, was rapidly metabolized to an α-ketol, but 7Z,10Z-16:2 was not a substrate. An unsaturated carbon chain from C-1 to C-8 was apparently more important than the configuration at the ω-end. 8R-DOX-LDS and 9R-DOX-AOS may thus bind 18:2n-6 in the same orientation. The oxidation of 18:2n-6 in straight or reverse head-to-tail positions illustrates evolutionary traits between 8- and 9-DOX domains. Fatty acids derivatized with amino acids provide a complementary tool for the analysis of evolution of enzymes.

Biosynthesis of Oxylipins by Rhizoctonia solani with Allene Oxide and Oleate 8S,9S-Diol Synthase Activities.

Oxylipin biosynthesis by fungi is catalyzed by both the lipoxygenase (LOX) family and the linoleate diol synthase (LDS) family of the peroxidase-cyclooxygenase superfamily. Rhizoctonia solani, a pathogenic fungus, infects staple crops such as potato and rice. The genome predicts three genes with 9-13 introns, which code for tentative dioxygenase (DOX)-cytochrome P450 fusion enzymes of the LDS family, and one gene, which might code for a 13-LOX. The objective was to determine whether mycelia or nitrogen powder of mycelia oxidized unsaturated C18 fatty acids to LDS- or LOX-related metabolites. Mycelia converted 18:2n-6 to 8R-hydroxy-9Z,12Z-octadecadienoic acid and to an α-ketol, 9S-hydroxy-10-oxo-12Z-octadecenoic acid. In addition to these metabolites, nitrogen powder of mycelia oxidized 18:2n-6 to 9S-hydroperoxy-10E, 12Z-octadecadienoic, and 13S-hydroperoxy-9Z,11E-octadecadienoic acids; the latter was likely formed by the predicted 13-LOX. 18:1n-9 was transformed into 8S-hydroperoxy-9Z-octadecenoic and into 8S,9S-dihydroxy-10E-octadecenoic acids, indicating the expression of 8,9-diol synthase. The allene oxide, 9S(10)epoxy-10,12Z-octadecadienoic acid, is unstable and decomposes rapidly to the α-ketol above, indicating biosynthesis by 9S-DOX-allene oxide synthase. This allene oxide and α-ketol are also formed by potato stolons, which illustrates catalytic similarities between the plant host and fungal pathogen.

Product specificity of fungal 8R- and 9S-dioxygenases of the peroxidase-cyclooxygenase superfamily with amino acid derivatized polyenoic fatty acids.

Pathogenic fungi express fatty acid dioxygenases (DOX) fused to cytochromes P450 with diol or allene oxide synthase activities. The orientation of the fatty acids in the active sites of DOX was investigated with amino acid conjugates of 18:3n-3 and 18:2n-6. 9S-DOX-allene oxide synthase (AOS) oxidized the Gly, Ile, and Trp derivatives at C-9, which suggests that these conjugates enter the substrate recognition site with the omega end in analogy with fatty acids bound to cyclooxygenases and coral 8R-lipoxygenase (8R-LOX). In contrast, 7,8-diol synthases (7,8-LDS), 5,8-LDS, and 8R-DOX-AOS oxidized the Gly conjugates in most case only to small amounts of metabolites, but with retention of hydrogen abstraction at C-8 and relatively minor hydrogen abstraction at C-11. The Ile and Trp conjugates were not oxidized at C-8, and often insignificantly at C-9/C-13. The 8-DOX domains of these enzymes likely position the carboxyl group of substrates at the end of the active site in analogy with plant α-DOX and 9-LOX. Tyr radicals of the 9S-DOX and 8R-DOX domains catalyze antarafacial hydrogen abstraction and oxygen insertion in 18:3n-3. This occurs by abstraction of the proR and proS hydrogens at C-11 and C-8, respectively, in agreement with different "head to tail" orientation in the active site.

An allene oxide and 12-oxophytodienoic acid are key intermediates in jasmonic acid biosynthesis by Fusarium oxysporum.

Fungi can produce jasmonic acid (JA) and its isoleucine conjugate in large quantities, but little is known about the biosynthesis. Plants form JA from 18:3n-3 by 13S-lipoxygenase (LOX), allene oxide synthase, and allene oxide cyclase. Shaking cultures of Fusarium oxysporum f. sp. tulipae released over 200 mg of jasmonates per liter. Nitrogen powder of the mycelia expressed 10R-dioxygenase-epoxy alcohol synthase activities, which was confirmed by comparison with the recombinant enzyme. The 13S-LOX of F. oxysporum could not be detected in the cell-free preparations. Incubation of mycelia in phosphate buffer with [17,17,18,18,18-2H5]18:3n-3 led to biosynthesis of a [2H5]12-oxo-13-hydroxy-9Z,15Z-octadecadienoic acid (α-ketol), [2H5]12-oxo-10,15Z-phytodienoic acid (12-OPDA), and [2H5]13-keto- and [2H5]13S-hydroxyoctadecatrienoic acids. The α-ketol consisted of 90% of the 13R stereoisomer, suggesting its formation by nonenzymatic hydrolysis of an allene oxide with 13S configuration. Labeled and unlabeled 12-OPDA were observed following incubation with 0.1 mM [2H5]18:3n-3 in a ratio from 0.4:1 up to 47:1 by mycelia of liquid cultures of different ages, whereas 10 times higher concentration of [2H5]13S-hydroperoxyoctadecatrienoic acid was required to detect biosynthesis of [2H5]12-OPDA. The allene oxide is likely formed by a cytochrome P450 or catalase-related hydroperoxidase. We conclude that F. oxysporum, like plants, forms jasmonates with an allene oxide and 12-OPDA as intermediates.

Purification and site-directed mutagenesis of linoleate 9S-dioxygenase-allene oxide synthase of Fusarium oxysporum confirms the oxygenation mechanism.

Plants and fungi form jasmonic acid from α-linolenic acid. The first two steps of biosynthesis in plants occur by sequential transformation by 13S-lipoxygenase and allene oxide synthase (AOS). The biosynthesis in fungi may follow this classical scheme, but the only fungal AOS discovered so far are cytochromes P450 (CYP) fused to 8- and 9-dioxygenases (DOX). In the present report, we purified recombinant 9S-DOX-AOS of Fusarium oxysporum from cell lysate by cobalt affinity chromatography to near homogeneity and studied key residues by site-directed mutagenesis. Sequence homology with 8R-DOX-linoleate diol synthases (8R-DOX-LDS) suggested that Tyr414 catalyzes hydrogen abstraction and that Cys1051 forms the heme thiolate ligand. Site-directed mutagenesis (Tyr414Phe; Cys1051Ser) led to loss of 9S-DOX and 9S-AOS activities, respectively, but other important residues in the CYP parts of 5,8- and 7,8-LDS or 9R-AOS were not conserved. The UV-visible spectrum of 9S-DOX-AOS showed a Soret band at 409 nm, which shifted to 413 nm in the Cys1051Ser mutant. The 9S-AOS of the Tyr414Phe mutant transformed 9S-hydroperoxides of α-linolenic and linoleic acids to allene oxides/α-ketols, but it did not transform 13-hydroperoxides. We conclude that 9S- and 8R-DOX catalyze hydrogen abstraction at C-11 and C-8, respectively, by homologous Tyr residues.

A new class of fatty acid allene oxide formed by the DOX-P450 fusion proteins of human and plant pathogenic fungi, C. immitis and Z. tritici.

Linoleate dioxygenase-cytochrome P450 (DOX-CYP) fusion enzymes are common in pathogenic fungi. The DOX domains form hydroperoxy metabolites of 18:2n-6, which can be transformed by the CYP domains to 1,2- or 1,4-diols, epoxy alcohols, or to allene oxides. We have characterized two novel allene oxide synthases (AOSs), namely, recombinant 8R-DOX-AOS of Coccidioides immitis (causing valley fever) and 8S-DOX-AOS of Zymoseptoria tritici (causing septoria tritici blotch of wheat). The 8R-DOX-AOS oxidized 18:2n-6 sequentially to 8R-hydroperoxy-9Z,12Z-octadecadienoic acid (8R-HPODE) and to an allene oxide, 8R(9)-epoxy-9,12Z-octadecadienoic acid, as judged from the accumulation of the α-ketol, 8S-hydroxy-9-oxo-12Z-octadecenoic acid. The 8S-DOX-AOS of Z. tritici transformed 18:2n-6 sequentially to 8S-HPODE and to an α-ketol, 8R-hydroxy-9-oxo-12Z-octadecenoic acid, likely formed by hydrolysis of 8S(9)-epoxy-9,12Z-octadecadienoic acid. The 8S-DOX-AOS oxidized [8R-(2)H]18:2n-6 to 8S-HPODE with retention of the (2)H-label, suggesting suprafacial hydrogen abstraction and oxygenation in contrast to 8R-DOX-AOS. Both enzymes oxidized 18:1n-9 and 18:3n-3 to α-ketols, but the catalysis of the 8R- and 8S-AOS domains differed. 8R-DOX-AOS transformed 9R-HPODE to epoxy alcohols, but 8S-DOX-AOS converted 9S-HPODE to an α-ketol (9-hydroxy-10-oxo-12Z-octadecenoic acid) and epoxy alcohols in a ratio of ∼1:2. Whereas all fatty acid allene oxides described so far have a conjugated diene impinging on the epoxide, the allene oxides formed by 8-DOX-AOS are unconjugated.

Crystal structure of linoleate 13R-manganese lipoxygenase in complex with an adhesion protein.

The crystal structure of 13R-manganese lipoxygenase (MnLOX) of Gaeumannomyces graminis (Gg) in complex with zonadhesin of Pichia pastoris was solved by molecular replacement. Zonadhesin contains ß-strands in two subdomains. A comparison of Gg-MnLOX with the 9S-MnLOX of Magnaporthe oryzae (Mo) shows that the protein fold and the geometry of the metal ligands are conserved. The U-shaped active sites differ mainly due to hydrophobic residues of the substrate channel. The volumes and two hydrophobic side pockets near the catalytic base may sanction oxygenation at C-13 and C-9, respectively. Gly-332 of Gg-MnLOX is positioned in the substrate channel between the entrance and the metal center. Replacements with larger residues could restrict oxygen and substrate to reach the active site. C18 fatty acids are likely positioned with C-11 between Mn(2+)OH2 and Leu-336 for hydrogen abstraction and with one side of the 12Z double bond shielded by Phe-337 to prevent antarafacial oxygenation at C-13 and C-11. Phe-347 is positioned at the end of the substrate channel and replacement with smaller residues can position C18 fatty acids for oxygenation at C-9. Gg-MnLOX does not catalyze the sequential lipoxygenation of n-3 fatty acids in contrast to Mo-MnLOX, which illustrates the different configurations of their substrate channels.

Crystal Structure of Manganese Lipoxygenase of the Rice Blast Fungus Magnaporthe oryzae.

Lipoxygenases (LOX) are non-heme metal enzymes, which oxidize polyunsaturated fatty acids to hydroperoxides. All LOX belong to the same gene family, and they are widely distributed. LOX of animals, plants, and prokaryotes contain iron as the catalytic metal, whereas fungi express LOX with iron or with manganese. Little is known about metal selection by LOX and the adjustment of the redox potentials of their protein-bound catalytic metals. Thirteen three-dimensional structures of animal, plant, and prokaryotic FeLOX are available, but none of MnLOX. The MnLOX of the most important plant pathogen, the rice blast fungusMagnaporthe oryzae(Mo), was expressed inPichia pastoris.Mo-MnLOX was deglycosylated, purified to homogeneity, and subjected to crystal screening and x-ray diffraction. The structure was solved by sulfur and manganese single wavelength anomalous dispersion to a resolution of 2.0 Å. The manganese coordinating sphere is similar to iron ligands of coral 8R-LOX and soybean LOX-1 but is not overlapping. The Asn-473 is positioned on a short loop (Asn-Gln-Gly-Glu-Pro) instead of an α-helix and forms hydrogen bonds with Gln-281. Comparison with FeLOX suggests that Phe-332 and Phe-525 might contribute to the unique suprafacial hydrogen abstraction and oxygenation mechanism of Mo-MnLOX by controlling oxygen access to the pentadiene radical. Modeling suggests that Arg-525 is positioned close to Arg-182 of 8R-LOX, and both residues likely tether the carboxylate group of the substrate. An oxygen channel could not be identified. We conclude that Mo-MnLOX illustrates a partly unique variation of the structural theme of FeLOX.

Replacement of two amino acids of 9R-dioxygenase-allene oxide synthase of Aspergillus niger inverts the chirality of the hydroperoxide and the allene oxide.

The genome of Aspergillus niger codes for a fusion protein (EHA25900), which can be aligned with ~50% sequence identity to 9S-dioxygenase (DOX)-allene oxide synthase (AOS) of Fusarium oxysporum, homologues of the Fusarium and Colletotrichum complexes and with over 62% sequence identity to homologues of Aspergilli, including (DOX)-9R-AOS of Aspergillus terreus. The aims were to characterize the enzymatic activities of EHA25900 and to identify crucial amino acids for the stereospecificity. Recombinant EHA25900 oxidized 18:2n-6 sequentially to 9R-hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HPODE) and to a 9R(10)-allene oxide. 9S- and 9R-DOX-AOS catalyze abstraction of the pro-R hydrogen at C-11, but the direction of oxygen insertion differs. A comparison between twelve 9-DOX domains of 9S- and 9R-DOX-AOS revealed conserved amino acid differences, which could contribute to the chirality of products. The Gly616Ile replacement of 9R-DOX-AOS (A. niger) increased the biosynthesis of 9S-HPODE and the 9S(10)-allene oxide, whereas the Phe627Leu replacement led to biosynthesis of 9S-HPODE and the 9S(10)-allene oxide as main products. The double mutant (Gly616Ile, Phe627Leu) formed over 90% of the 9S stereoisomer of HPODE. 9S-HPODE was formed by antarafacial hydrogen abstraction and oxygen insertion, i.e., the original H-abstraction was retained but the product chirality was altered. We conclude that 9R-DOX-AOS can be altered to 9S-DOX-AOS by replacement of two amino acids (Gly616Ile, Phe627Leu) in the DOX domain.

Discovery of a Novel Linoleate Dioxygenase of Fusarium oxysporum and Linoleate Diol Synthase of Colletotrichum graminicola.

Fungal pathogens constitute serious threats for many forms of life. The pathogenic fungi Fusarium and Colletotrichum and their formae speciales (f. spp.) infect many types of crops with severe consequences and Fusarium oxysporum can also induce keratitis and allergic conditions in humans. These fungi code for homologues of dioxygenase-cytochrome P450 (DOX-CYP) fusion proteins of the animal heme peroxidase (cyclooxygenase) superfamily. The objective was to characterize the enzymatic activities of the DOX-CYP homologue of Colletotrichum graminicola (EFQ34869) and the DOX homologue of F. oxysporum (EGU79548). The former oxidized oleic and linoleic acids in analogy with 7,8-linoleate diol synthases (LDSs), but with the additional biosynthesis of 8,11-dihydroxylinoleic acid. The latter metabolized fatty acids to hydroperoxides with broad substrate specificity. It oxidized 20:4n-6 and 18:2n-6 to hydroperoxides with an R configuration at the (n-10) positions, and other n-6 fatty acids in the same way. [11S-(2)H]18:2n-6 was oxidized with retention and [11R-(2)H]18:2n-6 with loss of deuterium, suggesting suprafacial hydrogen abstraction and oxygen insertion. Fatty acids of the n-3 series were oxidized less efficiently and often to hydroperoxides with an R configuration at both (n-10) and (n-7) positions. The enzyme spans 1426 amino acids with about 825 residues in the N-terminal domain with DOX homology and 600 residues at the C-terminal domain without homology to other enzymes. We conclude that fungal oxylipins can be formed by two novel subfamilies of cyclooxygenase-related DOX.

Expression and characterization of manganese lipoxygenase of the rice blast fungus reveals prominent sequential lipoxygenation of α-linolenic acid.

Magnaporthe oryzae causes rice blast disease and has become a model organism of fungal infections. M. oryzae can oxygenate fatty acids by 7,8-linoleate diol synthase, 10R-dioxygenase-epoxy alcohol synthase, and by a putative manganese lipoxygenase (Mo-MnLOX). The latter two are transcribed during infection. The open reading frame of Mo-MnLOX was deduced from genome and cDNA analysis. Recombinant Mo-MnLOX was expressed in Pichia pastoris and purified to homogeneity. The enzyme contained protein-bound Mn and oxidized 18:2n-6 and 18:3n-3 to 9S-, 11-, and 13R-hydroperoxy metabolites by suprafacial hydrogen abstraction and oxygenation. The 11-hydroperoxides were subject to ß-fragmentation with formation of 9S- and 13R-hydroperoxy fatty acids. Oxygen consumption indicated apparent kcat values of 2.8 s(-1) (18:2n-6) and 3.9 s(-1) (18:3n-3), and UV analysis yielded apparent Km values of 8 and 12 µM, respectively, for biosynthesis of cis-trans conjugated metabolites. 9S-Hydroperoxy-10E,12Z,15Z-octadecatrienoic acid was rapidly further oxidized to a triene, 9S,16S-dihydroperoxy-10E,12Z,14E-octadecatrienoic acid. In conclusion, we have expressed, purified and characterized a new MnLOX from M. oryzae. The pathogen likely secretes Mo-MnLOX and phospholipases to generate oxylipins and to oxidize lipid membranes of rice cells and the cuticle.