Feedback activation of ferrous 5-lipoxygenase during leukotriene synthesis by coexisting linoleic acid

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

doi:10.1194/jlr.m700055-jlr200

Cited by 5 publications

(3 citation statements)

References 20 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 the present study, we employed soybean 15-lipoxygenase to establish the evaluation method for scavenging effi ciency against a fatty acid allyl radical on the lipoxygenase. In our previous study (Takajo et al, 2007b), we demonstrated that a nitroxyl radical can trap a fatty acid allyl radical in rabbit reticulocyte 12/15-lipoxygenase and potato 5-lipoxygenase as well as soybean 15-lipoxygenase at lower oxygen content. Namely, the fatty acid allyl radical on the enzyme is a common target for inactivation of these lipoxygenase isozymes.…”

Section: Resultsmentioning

confidence: 85%

Trapping of fatty acid allyl radicals generated in lipoxygenase reactions in biological fluids by nitroxyl radical

Takajo

Tsuchida

Yokota

et al. 2010

Biomedical Chromatography

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Nitroxyl radicals can trap fatty acid allyl radicals on ferric-lipoxygenases at lower oxygen content, which are an intermediate in the lipoxygenase reaction. In the present study, we examined whether nitroxyl radical-trapping of fatty acid allyl radicals on the enzyme proceeds in biological fluids with abundant antioxidants. The fatty acid allyl radical-nitroxyl radical adducts were quantified by HPLC with electrochemical detection (HPLC-ECD); the adducts in eluate degraded into nitroxyl radical by passing through heating coil at 100 degrees C, and then nitroxyl radical was detected by electrochemical detector. Soybean 15-lipoxygenase and nitroxyl radical (3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl, CmDeltaP) were mixed with rat serum prepared from fresh venous blood, and the solution was stood at 37 degrees C for 1 h. One volume of the solution was mixed with 5 vols of cold acetonitrile. After centrifugation, the supernatant was subjected to HPLC-ECD. Arachidonate allyl radical-CmDeltaP adducts as well as linoleate allyl radical-CmDeltaP adducts were detected in the solution, and the content of these adducts remarkably increased in the presence of phospholipase A(2). It is proved for the first time that nitroxyl radical traps fatty acid allyl radicals generated in the lipoxygenase reaction in biological fluid without competition from endogenous antioxidants.

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“…[4][5][6][7] In this pseudoperoxidase reaction, ferrous lipoxygenase donates one electron to fatty acid hydroperoxide, producing fatty acid alkoxyl radical through cleavage of the O-O bond. [8][9][10][11][12] Even though the fate of the fatty acid alkoxyl radical remains obscure, it seems reasonable to assume that the fatty acid alkoxyl radical is converted to oxo-fatty acid by one-electron oxidation. In the lipoxygenase/ polyunsaturated fatty acid system, one-electron oxidation of fatty acid alkoxyl radical is thought to be coupled with oneelectron reduction of a free radical by radical-radical dismutation (R(-H)-O• + R′• R = O + R′-H).…”

Section: Introductionmentioning

confidence: 99%

Catalytic Production of Oxo-fatty Acids by Lipoxygenases Is Mediated by the Radical–Radical Dismutation between Fatty Acid Alkoxyl Radicals and Fatty Acid Peroxyl Radicals in Fatty Acid Assembly

Takigawa

Koshiishi

2020

Chem. Pharm. Bull.

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Oxo-octadecadienoic acids (OxoODEs) act as peroxisome proliferator-activated receptor (PPAR) agonists biologically, and are known to be produced in the lipoxygenase/linoleate system. OxoODEs seem to originate from the linoleate alkoxyl radicals that are generated from (E/Z)-hydroperoxy octadecadienoic acids ((E/Z)-HpODEs) by a pseudoperoxidase reaction that is catalyzed by ferrous lipoxygenase. However, the mechanism underlying the conversion of alkoxyl radical into OxoODE remains obscure. In the present study, we confirmed that OxoODEs are produced in the lipoxygenase/linoleate system in an oxygen-dependent manner. Interestingly, we revealed a correlation between the (E/Z)-OxoODEs content and the (E/E)-HpODEs content in the system. (E/E)-HpODEs could have been derived from (E/E)-linoleate peroxyl radicals, which are generated by the reaction between a free linoleate allyl radical and an oxygen molecule. Notably, the ferrous lipoxygenase-linoleate allyl radical (LOx(Fe 2)-L•) complex, which is an intermediate in the lipoxygenase/linoleate system, tends to dissociate into LOx(Fe 2) and a linoleate allyl radical. Subsequently, LOx(Fe 2) converts (E/Z)-HpODEs to an (E/Z)-linoleate alkoxyl radical through one-electron reduction. Taken together, we propose that (E/Z)-OxoODEs and (E/E)-HpODEs are produced through radical-radical dismutation between (E/Z)-linoleate alkoxyl radical and (E/E)-linoleate peroxyl radical. Furthermore, the production of (E/Z)-OxoODEs and (E/E)-HpODEs was remarkably inhibited by a hydrophobic radical scavenger, 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO). On the contrary, water-miscible radical scavengers, 4-hydroxyl-2,2,6,6tetramethylpiperidine 1-oxyl (OH-TEMPO) and 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (CmΔP) only modestly or sparingly inhibited the production of (E/Z)-OxoODEs and (E/E)-HpODEs. These facts indicate that the radical-radical dismutation between linoleate alkoxyl radical and linoleate peroxyl radical proceeds in the interior of micelles.

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Feedback activation of ferrous 5-lipoxygenase during leukotriene synthesis by coexisting linoleic acid

Cited by 5 publications

References 20 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

Trapping of fatty acid allyl radicals generated in lipoxygenase reactions in biological fluids by nitroxyl radical

Catalytic Production of Oxo-fatty Acids by Lipoxygenases Is Mediated by the Radical–Radical Dismutation between Fatty Acid Alkoxyl Radicals and Fatty Acid Peroxyl Radicals in Fatty Acid Assembly

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