Intramolecular rearrangement of linolenate peroxyl radicals in lipoxygenase reactions at lower oxygen content

Takajo, Tokuko; Tsuchida, Kazunori; Murahashi, Tsuyoshi; Ueno, Koichi; Koshiishi, Ichiro

doi:10.1194/jlr.m600446-jlr200

Cited by 5 publications

(5 citation statements)

References 18 publications

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“…Radical Spin Trapping Adducts-To investigate the formation of a carbon-centered radical at C-8 (15), we used the method of Koshiishi and co-workers (33)(34)(35) to scavenge the hypothetical C-8 radical with the radical scavenger Cm⌬P. In this case, a nitroxyl radical competes with the oxygen molecule for the lipid allyl radical on the enzyme, resulting in the formation of a lipid allyl radical-Cm⌬P adduct that can be detected by mass spectrometry.…”

Section: Analysis Of Product Formation In the Presencementioning

confidence: 99%

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

Brodhun¹,

Göbel²,

Hornung³

et al. 2009

Journal of Biological Chemistry

140

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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 ϳ7.25. 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.The fungus Aspergillus nidulans (teleomorph Emericella nidulans) is a homothallic ascomycete that has a defined sexual and asexual developmental cycle. Therefore, it serves as a model system for the understanding of fungal development (1). Oxidized unsaturated fatty acids, so-called oxylipins, derived from endogenous fatty acids were found to influence the development of the asexual conidiophores and sexual cleistothecia (2-6). Moreover, they seem to regulate the secondary metabolism of the fungus (7). These substances were collectively named psi factors and are primarily a mixture of hydroxylated oleic (18:1 ⌬9Z

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Section: Analysis Of Product Formation In the Presencementioning

confidence: 99%

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

Brodhun¹,

Göbel²,

Hornung³

et al. 2009

Journal of Biological Chemistry

140

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“…In order to investigate the formation of carbon-centred fatty acid radicals during the reaction of PpoC and 18:2 9Z,12Z we used the method of Koshiishi and co-workers to scavenge the hypothetical radical with the radical scavenger Cm P (3-carbamoyl-2,2,5,5tetramethyl-3-pyrroline-N-oxyl) [35,36]. Recently, we adapted this approach for the PpoA/linoleate system and found the formation of two radical spin-trapping adducts, which we tentatively assigned as the trapped C-8 and C-10 radical adducts [14]; this finding that now proves to be consistent with our observation that PpoA is also able to form 10-HODE/10-HPODE ( Figure 9B).…”

Section: Radical Spin-trapping Adductsmentioning

confidence: 99%

PpoC from Aspergillus nidulans is a fusion protein with only one active haem

Brodhun¹,

Schneider²,

Göbel³

et al. 2010

Biochemical Journal

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In Aspergillus nidulans Ppos [psi (precocious sexual inducer)-producing oxygenases] are required for the production of so-called psi factors, compounds that control the balance between the sexual and asexual life cycle of the fungus. The genome of A. nidulans harbours three different ppo genes: ppoA, ppoB and ppoC. For all three enzymes two different haem-containing domains are predicted: a fatty acid haem peroxidase/dioxygenase domain in the N-terminal region and a P450 haem-thiolate domain in the C-terminal region. Whereas PpoA was shown to use both haem domains for its bifunctional catalytic activity (linoleic acid 8-dioxygenation and 8-hydroperoxide isomerization), we found that PpoC apparently only harbours a functional haem peroxidase/dioxygenase domain. Consequently, we observed that PpoC catalyses mainly the dioxygenation of linoleic acid (18:2Delta9Z,12Z), yielding 10-HPODE (10-hydroperoxyoctadecadienoic acid). No isomerase activity was detected. Additionally, 10-HPODE was converted at lower rates into 10-KODE (10-keto-octadecadienoic acid) and 10-HODE (10-hydroxyoctadecadienoic acid). In parallel, decomposition of 10-HPODE into 10-ODA (10-octadecynoic acid) and volatile C-8 alcohols that are, among other things, responsible for the characteristic mushroom flavour. Besides these principle differences we also found that PpoA and PpoC can convert 8-HPODE and 10-HPODE into the respective epoxy alcohols: 12,13-epoxy-8-HOME (where HOME is hydroxyoctadecenoic acid) and 12,13-epoxy-10-HOME. By using site-directed mutagenesis we demonstrated that both enzymes share a similar mechanism for the oxidation of 18:2Delta9Z,12Z; they both use a conserved tyrosine residue for catalysis and the directed oxygenation at the C-8 and C-10 is most likely controlled by conserved valine/leucine residues in the dioxygenase domain.

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“…Subsequently, the Fe 3+ in the enzyme is reduced by the unsaturated lipid, and the lipid molecule is converted to the lipid radical. The complex of LOX(Fe 2+ ) with the lipid radical (L) takes up O 2 to produce LOOH [29,30]. These reactions are represented as follows:…”

Section: Discussionmentioning

confidence: 99%

The peroxidase activity of ADM-Fe3+ cooperates with lipid peroxidation: The participation of hydroperoxide and hydroxyl radicals in the damage to proteins and DNA

Miura

2015

Chemico-Biological Interactions

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Intramolecular rearrangement of linolenate peroxyl radicals in lipoxygenase reactions at lower oxygen content

Cited by 5 publications

References 18 publications

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

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

PpoC from Aspergillus nidulans is a fusion protein with only one active haem

The peroxidase activity of ADM-Fe3+ cooperates with lipid peroxidation: The participation of hydroperoxide and hydroxyl radicals in the damage to proteins and DNA

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