| 1. |
- Hoffmann, Inga, 1984-, et al.
(författare)
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Expression of 5,8-LDS of Aspergillus fumigatus and its dioxygenase domain : a comparison with 7,8-LDS, 10-dioxygenase, and cyclooxygenase
- 2011
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 506:2, s. 216-222
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Tidskriftsartikel (refereegranskat)abstract
- 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 8R-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 8R-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 10R-dioxygenase. We conclude that ∼675 amino acids are sufficient to support 8-DOX activity.
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| 2. |
- Oliw, Ernst H, 1948-, et al.
(författare)
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Biosynthesis of Jasmonates from Linoleic Acid by the Fungus Fusarium oxysporum. Evidence for a Novel Allene Oxide Cyclase
- 2019
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Ingår i: Lipids. - : Wiley. - 0024-4201 .- 1558-9307. ; 54:9, s. 543-556
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Tidskriftsartikel (refereegranskat)abstract
- 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 [17,17,18,18,18‐2H5]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 [13C18]‐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.
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| 3. |
- Oliw, Ernst H, 1948-, et al.
(författare)
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Charge migration fragmentation in the negative ion mode of cyclopentenone and cyclopentanone intermediates in the biosynthesis of jasmonates
- 2020
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Ingår i: Rapid Communications in Mass Spectrometry. - : WILEY. - 0951-4198 .- 1097-0231. ; 34:8
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Tidskriftsartikel (refereegranskat)abstract
- 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 [C11H17O](-). 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-H2O](-) 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 H-2 at C-6 and C-7 and with structural and side chain analogs.Results: CID of [6,6,7,7-H-2(4)]12-OPEA and [6,6-H-2(2)]12-OPDA ([M-H](-) and [M-H-CO2](-)) showed that one or two H-2 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-H-2(2)]- and [6,6,7,7-H-2(4)]-12-OPA ([M-H](-) and [M-H-CO2](-)) yielded the loss of H-2 from the cyclopentanone and displayed the transfer of one H-2 atom in analogy to 12-OPEA. In contrast, CID of [6,6,7,7-H-2(4)]12-OPEA and [6,6,7,7-H-2(4)]12-OPA [M-H-H2O](-) demonstrated the transfer of two H-2 atoms (m/z 165 + 2). All spectra obtained by CID of [6,6,7,7-H-2(4)]12-OPDA and [6,6,7,7-H-2(4)]12-oxo-9(13),15(Z)-phytodienoic acid showed that one or two additional H-2 atoms could be transferred to this anion at m/z 167 of [6,6-H-2(2)]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.
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| 4. |
- Oliw, Ernst H., 1948-
(författare)
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Diversity of the manganese lipoxygenase gene family - A mini-review
- 2022
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Ingår i: Fungal Genetics and Biology. - : Elsevier. - 1087-1845 .- 1096-0937. ; 163
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Forskningsöversikt (refereegranskat)abstract
- 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 Cterminal 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.
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| 5. |
- Oliw, Ernst H., 1948-
(författare)
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Fatty acid dioxygenase-cytochrome P450 fusion enzymes of filamentous fungal pathogens
- 2021
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Ingår i: Fungal Genetics and Biology. - : Elsevier. - 1087-1845 .- 1096-0937. ; 157
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Tidskriftsartikel (refereegranskat)abstract
- 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-DOXAOS. 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 DOXAOS homologues in F. verticillioides, A. flavus, and A. nidulans alters, inter alia, mycotoxin production, sporulation, and gene expression.
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| 6. |
- Oliw, Ernst H., 1948-
(författare)
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Iron and manganese lipoxygenases of plant pathogenic fungi and their role in biosynthesis of jasmonates
- 2022
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier. - 0003-9861 .- 1096-0384. ; 722
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Forskningsöversikt (refereegranskat)abstract
- 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 O-2 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.
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| 7. |
- Oliw, Ernst H., 1948-
(författare)
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Linoleate diol synthase related enzymes of the human pathogens Histoplasma capsulatum and Blastomyces dermatitidis
- 2020
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 696
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Oliw, Ernst H., 1948-, et al.
(författare)
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Manganese lipoxygenase oxidizes bis-allylic hydroperoxides and octadecenoic acids by different mechanisms
- 2011
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Ingår i: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. - : Elsevier BV. - 1388-1981 .- 1879-2618. ; 1811:3, s. 138-147
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Tidskriftsartikel (refereegranskat)abstract
- Manganese lipoxygenase (MnLOX) oxidizes (11R)-hydroperoxylinolenic acid (11R-HpOTrE) to a peroxyl radical. Our aim was to compare the enzymatic oxidation of 11R-HpOTrE and octadecenoic acids with LOO-H and allylic C-H bond dissociation enthalpies of ~88 and ~87kcal/mol. Mn(III)LOX oxidized (11Z)-, (12Z)-, and (13Z)-18:1 to hydroperoxides with R configuration, but this occurred at insignificant rates (<1%) compared to 11R-HpOTrE. We next examined whether transitional metals could mimic this oxidation. Ce(4+) and Mn(3+) transformed 11R-HpOTrE to hydroperoxides at C-9 and C-13 via oxidation to a peroxyl radical at C-11, whereas Fe(3+) was a poor catalyst. Our results suggest that MnLOX oxidizes bis-allylic hydroperoxides to peroxyl radicals in analogy with Ce(4+) and Mn(3+). The enzymatic oxidation likely occurs by proton-coupled electron transfer of the electron from the hydroperoxide anion to Mn(III) and H(+) to the catalytic base, Mn(III)OH(-). Hydroperoxides abolish the kinetic lag times of MnLOX and FeLOX by oxidation of their metal centers, but 11R-HpOTrE was isomerized by MnLOX to (13R)-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic acid (13R-HpOTrE) with a kinetic lag time. This lag time could be explained by two competing transformations, dehydration of 11R-HpOTrE to 11-ketolinolenic acid and oxidation of 11R-HpOTrE to peroxyl radical; the reaction rate then increases as 13R-HpOTrE oxidizes MnLOX with subsequent formation of two epoxyalcohols. We conclude that oxidation of octadecenoic acids and bis-allylic hydroperoxides occurs by different mechanisms, which likely reflect the nature of the hydrogen bonds, steric factors, and the redox potential of the Mn(III) center.
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| 9. |
- Oliw, Ernst H, 1948-
(författare)
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Product specificity of fungal 8R- and 9S-dioxygenases of the peroxidase-cyclooxygenase superfamily with amino acid derivatized polyenoic fatty acids
- 2018
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Ingår i: Archives of Biochemistry and Biophysics. - : ELSEVIER SCIENCE INC. - 0003-9861 .- 1096-0384. ; 640, s. 93-101
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Tidskriftsartikel (refereegranskat)abstract
- 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 alpha-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.
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| 10. |
- Oliw, Ernst H., 1948-
(författare)
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Thirty years with three-dimensional structures of lipoxygenases
- 2024
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier. - 0003-9861 .- 1096-0384. ; 752
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Forskningsöversikt (refereegranskat)abstract
- 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.
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