Publications

• Secretion of two novel enzymes, manganese 9S-lipoxygenase and epoxy alcohol synthase, by the rice pathogen Magnaporthe salvinii.

  Wennman A., Oliw E.H.

  The mycelium of the rice stem pathogen, Magnaporthe salvinii, secreted linoleate 9S-lipoxygenase (9S-LOX) and epoxy alcohol synthase (EAS). The EAS rapidly transformed 9S-hydroperoxy-octadeca-10E,12Z-dienoic acid (9S-HPODE) to threo 10 (11)-epoxy-9S-hydroxy-12Z-octadecenoic acid, but other hydrope ... >> More

  The mycelium of the rice stem pathogen, Magnaporthe salvinii, secreted linoleate 9S-lipoxygenase (9S-LOX) and epoxy alcohol synthase (EAS). The EAS rapidly transformed 9S-hydroperoxy-octadeca-10E,12Z-dienoic acid (9S-HPODE) to threo 10 (11)-epoxy-9S-hydroxy-12Z-octadecenoic acid, but other hydroperoxy FAs were poor substrates. 9S-LOX was expressed in Pichia pastoris. Recombinant 9S-LOX oxidized 18:2n-6 directly to 9S-HPODE, the end product, and also to two intermediates, 11S-hydroperoxy-9Z,12Z-octadecenoic acid (11S-HPODE; ∼5%) and 13R-hydroperoxy-9Z,11E-octadecadienoic acid (13R-HPODE; ∼1%). 11S- and 13R-HPODE were isomerized to 9S-HPODE, probably after oxidation to peroxyl radicals, β-fragmentation, and oxygen insertion at C-9. The 18:3n-3 was oxidized at C-9, C-11, and C-13, and to 9,16-dihydroxy-10E,12,14E-octadecatrienoic acid. 9S-LOX contained catalytic manganese (Mn:protein ∼0.2:1; Mn/Fe, 1:0.05), and its sequence could be aligned with 77% identity to 13R-LOX with catalytic manganese lipoxygenase (13R-MnLOX) of the Take-all fungus. The Leu350Met mutant of 9S-LOX shifted oxidation of 18:2n-6 from C-9 to C-13, and the Phe347Leu, Phe347Val, and Phe347Ala mutants of 13R-MnLOX from C-13 to C-9. In conclusion, M. salvinii secretes 9S-LOX with catalytic manganese along with a specific EAS. Alterations in the Sloane determinant of 9S-LOX and 13R-MnLOX with larger and smaller hydrophobic residues interconverted the regiospecific oxidation of 18:2n-6, presumably by altering the substrate position in relation to oxygen insertion. << Less

  J. Lipid Res. 54:762-775(2013) [PubMed] [EuropePMC]

  This publication is cited by 12 other entries.

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

  Wennman A., Jerneren F., Magnuson A., Oliw E.H.

  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 ... >> More

  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 β-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. << Less

  Arch. Biochem. Biophys. 583:87-95(2015) [PubMed] [EuropePMC]

  This publication is cited by 8 other entries.

• Manganese lipoxygenase. Purification and characterization.

  Su C., Oliw E.H.

  A linoleic acid (13R)-lipoxygenase was purified to homogeneity from the culture medium of Gäumannomyces graminis, the take-all fungus, by hydrophobic interaction, cation exchange, lectin affinity, and size-exclusion chromatography. The purified dioxygenase lacked light absorption between 300 and 7 ... >> More

  A linoleic acid (13R)-lipoxygenase was purified to homogeneity from the culture medium of Gäumannomyces graminis, the take-all fungus, by hydrophobic interaction, cation exchange, lectin affinity, and size-exclusion chromatography. The purified dioxygenase lacked light absorption between 300 and 700 nm. Gel filtration indicated an apparent molecular mass of approximately 135 kDa in 6 M urea and approximately 160 kDa in buffer. SDS-polyacrylamide gel electrophoresis (PAGE) showed that the enzyme was heterogeneous in size and consisted of diffuse protein bands of 100-140 kDa. Treatment with glycosidases for N- and O-linked oligosaccharides yielded a distinct protein of approximately 73 kDa on SDS-PAGE. Atomic emission spectroscopy indicated 0.5-1.0 manganese atom/enzyme molecule. The isoelectric point was approximately 9.7, and the enzyme was active between pH 5 and 11 with optimum activity at pH 7. 0. For molecular oxygen, Km was 30 microM and Vmax 10 micromol mg-1min-1; for linoleic acid, Km was 4.4 micromol, Vmax 8.2 micromol mg-1min-1, and the turnover number 1100 min-1. The enzyme oxidized linolenic acid twice as fast as linoleic acid. The main products were identified by mass spectrometry as 13-hydroperoxy-(9Z,11E, 15Z)-octadecatrienoic and 13-hydroperoxy-(9Z,11E)-octadecadienoic acids, respectively. After reduction of the hydroperoxide, steric analysis of methyl 13-hydroxyoctadecadienoate by chiral high performance liquid chromatography yielded one enantiomer (>95%), which co-eluted with the R-stereoisomer of methyl (13R, 13S)-hydroxyoctadecadienoate. Arachidonic and dihomogammalinolenic acids were not substrates, while oxygen consumption, UV analysis, and mass spectrometric analysis indicated that gamma-linolenic acid was oxygenated both at C-11 and C-13. The enzyme was active at 60 degreesC and after treatment with 6 M urea. It was strongly inhibited by 10-50 microM concentrations of eicosatetraynoic acid and a lipoxygenase inhibitor (N-(3-phenoxycinnamyl)acetohydroxamic acid), but many other lipoxygenase inhibitors (100 microM) were without effect. We conclude that, after deglycosylation, the enzyme has the same size on SDS-PAGE as mammalian and marine lipoxygenases, but it differs from all previously described lipoxygenases in three ways. It is secreted, it forms (13R)-hydroperoxy-(9Z, 11E)-octadecadienoic acid, and it contains manganese. << Less

  J. Biol. Chem. 273:13072-13079(1998) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

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

  Oliw E.H., Su C., Skogstrom T., Benthin G.

  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, an ... >> More

  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. << Less

  Lipids 33:843-852(1998) [PubMed] [EuropePMC]

• Manganese lipoxygenase. Discovery of a bis-allylic hydroperoxide as product and intermediate in a lipoxygenase reaction.

  Hamberg M., Su C., Oliw E.

  Linoleic acid was incubated with manganese lipoxygenase (Mn-LO) from the fungus Gäumannomyces graminis. The product consisted of (13R)-hydroperoxy-(9Z,11E)-octadecadienoic acid ((13R)-HPOD) and a new hydroperoxide, (11S)-hydroperoxy-(9Z,12Z)-octadecadienoic acid ((11S)-HPOD). Incubation of (11R)-[ ... >> More

  Linoleic acid was incubated with manganese lipoxygenase (Mn-LO) from the fungus Gäumannomyces graminis. The product consisted of (13R)-hydroperoxy-(9Z,11E)-octadecadienoic acid ((13R)-HPOD) and a new hydroperoxide, (11S)-hydroperoxy-(9Z,12Z)-octadecadienoic acid ((11S)-HPOD). Incubation of (11R)-[2H]- and (11S)-[2H]linoleic acids with Mn-LO led to the formation of hydroperoxides that largely retained and lost, respectively, the deuterium label. Conversion of the (11S)-deuteriolinoleic acid was accompanied by a primary isotope effect, which manifested itself in a strongly reduced rate of formation of hydroperoxides and in a time-dependent accumulation of deuterium in the unconverted substrate. These experiments indicated that the initial step catalyzed by Mn-LO consisted of abstraction of the pro-S hydrogen of linoleic acid to produce a linoleoyl radical. (11S)-HPOD was converted into (13R)-HPOD upon incubation with Mn-LO. The mechanism of this enzyme-catalyzed hydroperoxide rearrangement was studied in experiments carried out with 18O2 gas or 18O2-labeled hydroperoxides. Incubation of [11-18O2](11S)-HPOD with Mn-LO led to the formation of (13R)-HPOD, which retained 39-44% of the 18O label, whereas (11S)-HPOD incubated with Mn-LO under 18O2 produced (13R)-HPOD, which had incorporated 57% of 18O. Furthermore, analysis of the isotope content of (11S)-HPOD remaining unconverted in such incubations demonstrated that [11-18O2](11S)-HPOD suffered a time-dependent loss of 18O when exposed to Mn-LO, whereas (11S)-HPOD incorporated 18O when incubated with Mn-LO under 18O2. On the basis of these experiments, it was proposed that the conversion of (11S)-HPOD into (13R)-HPOD occurred in a non-concerted way by deoxygenation into a linoleoyl radical. Subsequent reoxygenation of this intermediate by dioxygen attack at C-13 produced (13R)-HPOD, whereas attack at C-11 regenerated (11S)-HPOD. The hydroperoxide rearrangement occurred by oxygen rebound, although, as demonstrated by the 18O experiments, the oxygen molecule released from (11S)-HPOD exchanged with surrounding molecular oxygen prior to its reincorporation. << Less

  J Biol Chem 273:13080-13088(1998) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.