- Autoxidation of linoleic acid in a strong magnetic field (9.4 T)
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Autoxidation of linoleic acid in a strong magnetic field (9.4 T) has been studied. Formation of the hydroperoxides has been monitored by the Fe(SCN)3 method, showing that the magnetic field accelerates the autoxidation of linoleic acid.
- Inotani, Masahiro,Fukuyoshi, Shuichi,Kusumi, Takenori
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Read Online
- Development and Application of a Peroxyl Radical Clock Approach for Measuring Both Hydrogen-Atom Transfer and Peroxyl Radical Addition Rate Constants
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The rate-determining step in free radical lipid peroxidation is the propagation of the peroxyl radical, where generally two types of reactions occur: (a) hydrogen-atom transfer (HAT) from a donor to the peroxyl radical; (b) peroxyl radical addition (PRA) to a C=C double bond. Peroxyl radical clocks have been used to determine the rate constants of HAT reactions (kH), but no radical clock is available to measure the rate constants of PRA reactions (kadd). In this work, we modified the analytical approach on the linoleate-based peroxyl radical clock to enable the simultaneous measurement of both kH and kadd. Compared to the original approach, this new approach involves the use of a strong reducing agent, LiAlH4, to completely reduce both HAT and PRA-derived products and the relative quantitation of total linoleate oxidation products with or without reduction. The new approach was then applied to measuring the kH and kadd values for several series of organic substrates, including para- and meta-substituted styrenes, substituted conjugated dienes, and cyclic alkenes. Furthermore, the kH and kadd values for a variety of biologically important lipids were determined for the first time, including conjugated fatty acids, sterols, coenzyme Q10, and lipophilic vitamins, such as vitamins D3 and A.
- Do, Quynh,Lee, David D.,Dinh, Andrew N.,Seguin, Ryan P.,Zhang, Rutan,Xu, Libin
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supporting information
p. 153 - 168
(2020/12/23)
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- The CYP74B and CYP74D divinyl ether synthases possess a side hydroperoxide lyase and epoxyalcohol synthase activities that are enhanced by the site-directed mutagenesis
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The CYP74 family of cytochromes P450 includes four enzymes of fatty acid hydroperoxide metabolism: allene oxide synthase (AOS), hydroperoxide lyase (HPL), divinyl ether synthase (DES), and epoxyalcohol synthase (EAS). The present work is concerned with catalytic specificities of three recombinant DESs, namely, the 9-DES (LeDES, CYP74D1) of tomato (Solanum lycopersicum), 9-DES (NtDES, CYP74D3) of tobacco (Nicotiana tabacum), and 13-DES (LuDES, CYP74B16) of flax (Linum usitatissimum), as well as their alterations upon the site-directed mutagenesis. Both LeDES and NtDES converted 9-hydroperoxides of linoleic and α?linolenic acids to divinyl ethers colneleic and colnelenic acids (respectively) with only minorities of HPL and EAS products. In contrast, LeDES and NtDES showed low efficiency towards the linoleate 13-hydroperoxide, affording only the low yield of epoxyalcohols. LuDES exhibited mainly the DES activity towards α?linolenate 13-hydroperoxide (preferred substrate), and HPL activity towards linoleate 13-hydroperoxide, respectively. In contrast, LuDES converted 9-hydroperoxides primarily to the epoxyalcohols. The F291V and A287G mutations within the I-helix groove region (SRS-4) of LuDES resulted in the loss of DES activity and the acquirement of the epoxyalcohol synthase activity. Thus, the studied enzymes exhibited the versatility of catalysis and its qualitative alterations upon the site-directed mutagenesis.
- Gorina, Svetlana S.,Grechkin, Alexander N.,Iljina, Tatiana M.,Mukhtarova, Lucia S.,Smirnova, Elena O.,Toporkova, Yana Y.
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- 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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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(Fe2+)-L·) complex, which is an intermediate in the lipoxygenase/linoleate system, tends to dissociate into LOx(Fe2+) and a linoleate allyl radical. Subsequently, LOx(Fe2+) 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-tetra-methylpiperidine 1-oxyl (TEMPO). On the contrary, water-miscible radical scavengers, 4-hydroxyl-2,2,6,6-tetramethylpiperidine 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.
- Takigawa, Yuta,Koshiishi, Ichiro
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p. 258 - 264
(2020/11/26)
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- Oxygenation reactions catalyzed by the F557V mutant of soybean lipoxygenase-1: Evidence for two orientations of substrate binding
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Plant lipoxygenases oxygenate linoleic acid to produce 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-HPOD) or 9-hydroperoxy-10E,12Z-octadecadienoic acid (9(S)-HPOD). The manner in which these enzymes bind substrates and the mechanisms by which they control regiospecificity are uncertain. Hornung et al. (Proc. Natl. Acad. Sci. USA 96 (1999) 4192–4197) have identified an important residue, corresponding to phe-557 in soybean lipoxygenase-1 (SBLO-1). These authors proposed that large residues in this position favored binding of linoleate with the carboxylate group near the surface of the enzyme (tail-first binding), resulting in formation of 13(S)-HPOD. They also proposed that smaller residues in this position facilitate binding of linoleate in a head-first manner with its carboxylate group interacting with a conserved arginine residue (arg-707 in SBLO-1), which leads to 9(S)-HPOD. In the present work, we have tested these proposals on SBLO-1. The F557V mutant produced 33% 9-HPOD (S:R = 87:13) from linoleic acid at pH 7.5, compared with 8% for the wild-type enzyme and 12% with the F557V,R707L double mutant. Experiments with 11(S)-deuteriolinoleic acid indicated that the 9(S)-HPOD produced by the F557V mutant involves removal of hydrogen from the pro-R position on C-11 of linoleic acid, as expected if 9(S)-HPOD results from binding in an orientation that is inverted relative to that leading to 13(S)-HPOD. The product distributions obtained by oxygenation of 10Z,13Z-nonadecadienoic acid and arachidonic acid by the F557V mutant support the hypothesis that ω6 oxygenation results from tail-first binding and ω10 oxygenation from head-first binding. The results demonstrate that the regiospecificity of SBLO-1 can be altered by a mutation that facilitates an alternative mode of substrate binding and adds to the body of evidence that 13(S)-HPOD arises from tail-first binding.
- Hershelman, Dillon,Kahler, Kirsten M.,Price, Morgan J.,Lu, Iris,Fu,Plumeri, Patricia A.,Karaisz, Fred,Bassett, Natasha F.,Findeis, Peter M.,Clapp, Charles H.
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- Microbial Synthesis of Linoleate 9 S-Lipoxygenase Derived Plant C18 Oxylipins from C18 Polyunsaturated Fatty Acids
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Plant oxylipins, including hydroxy fatty acids, epoxy hydroxy fatty acids, and trihydroxy fatty acids, which are biosynthesized from C18 polyunsaturated fatty acids (PUFAs), are involved in pathogen-specific defense mechanisms against fungal infections. However, their quantitative biotransformation by plant enzymes has not been reported. A few bacteria produce C18 trihydroxy fatty acids, but the enzymes and pathways related to the biosynthesis of plant oxylipins in bacteria have not been reported. In this study, we first report the biotransformation of C18 PUFAs into plant C18 oxylipins by expressing linoleate 9S-lipoxygenase with and without epoxide hydrolase from the proteobacterium Myxococcus xanthus in recombinant Escherichia coli. Among the nine types of plant oxylipins, 12,13-epoxy-14-hydroxy-cis,cis-9,15-octadecadienoic acid was identified as a new compound by NMR analysis, and 9,10,11-hydroxy-cis,cis-6,12-octadecadienoic acid and 12,13,14-trihydroxy-cis,cis-9,15-octadecadienoic were suggested as new compounds by LC-MS/MS analysis. This study shows that bioactive plant oxylipins can be produced by microbial enzymes.
- An, Jung-Ung,Lee, In-Gyu,Ko, Yoon-Joo,Oh, Deok-Kun
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- Microbial Synthesis of Linoleate 9 S-Lipoxygenase Derived Plant C18 Oxylipins from C18 Polyunsaturated Fatty Acids
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Plant oxylipins, including hydroxy fatty acids, epoxy hydroxy fatty acids, and trihydroxy fatty acids, which are biosynthesized from C18 polyunsaturated fatty acids (PUFAs), are involved in pathogen-specific defense mechanisms against fungal infections. However, their quantitative biotransformation by plant enzymes has not been reported. A few bacteria produce C18 trihydroxy fatty acids, but the enzymes and pathways related to the biosynthesis of plant oxylipins in bacteria have not been reported. In this study, we first report the biotransformation of C18 PUFAs into plant C18 oxylipins by expressing linoleate 9S-lipoxygenase with and without epoxide hydrolase from the proteobacterium Myxococcus xanthus in recombinant Escherichia coli. Among the nine types of plant oxylipins, 12,13-epoxy-14-hydroxy-cis,cis-9,15-octadecadienoic acid was identified as a new compound by NMR analysis, and 9,10,11-hydroxy-cis,cis-6,12-octadecadienoic acid and 12,13,14-trihydroxy-cis,cis-9,15-octadecadienoic were suggested as new compounds by LC-MS/MS analysis. This study shows that bioactive plant oxylipins can be produced by microbial enzymes.
- An, Jung-Ung,Lee, In-Gyu,Ko, Yoon-Joo,Oh, Deok-Kun
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p. 3209 - 3219
(2019/03/26)
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- Epoxyalcohol Synthase RjEAS (CYP74A88) from the Japanese Buttercup (Ranunculus japonicus): Cloning and Characterization of Catalytic Properties
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Cytochromes P450 of the CYP74 family play a key role in the lipoxygenase cascade generating oxylipins (products of polyunsaturated fatty acid oxidation). The CYP74 family includes allene oxide synthases, hydroperoxide lyases, divinyl ether synthases, and epoxyalcohol synthases. In this work, we cloned the CYP74A88 gene from the Japanese buttercup (Ranunculus japonicus) and studied the properties of the encoded recombinant protein. The CYP74A88 enzyme specifically converts linoleic acid 9-and 13-hydroperoxides to oxiranyl carbinols 9,10-epoxy-11-hydroxy-12-octadecenoic acid and 11-hydroxy-12,13-epoxy-9-octadecenoic acid, respectively, which was confirmed by GC-MS analysis and kinetic studies. Therefore, the CYP74A88 enzyme is a specific epoxyalcohol synthase.
- Toporkova,Fatykhova,Gorina,Mukhtarova,Grechkin
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p. 171 - 180
(2019/04/01)
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- Allene Oxide Synthase Pathway in Cereal Roots: Detection of Novel Oxylipin Graminoxins
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Young roots of wheat, barley, and sorghum, as well as methyl jasmonate pretreated rice seedlings, undergo an unprecedented allene oxide synthase pathway targeted to previously unknown oxylipins 1–3. These Favorskii-type products, (4Z)-2-pentyl-4-tridecene-1,13-dioic acid (1), (2′Z)-2-(2′-octenyl)-decane-1,10-dioic acid (2), and (2′Z,5′Z)-2-(2′,5′-octadienyl)-decane-1,10-dioic acid (3), have a carboxy function at the side chain, as revealed by their MS and NMR spectral data. Compounds 1–3 were the major oxylipins detected, along with the related α-ketols. Products 1–3 were biosynthesized from (9Z,11E,13S)-13-hydroperoxy-9,11-octadecadienoic acid, (9S,10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid (9-HPOD), and (9S,10E,12Z,15Z)-9-hydroperoxy-10,12,15-octadecatrienoic acid, respectively, via the corresponding allene oxides and cyclopropanones. The data indicate that conversion of the allene oxide into the cyclopropanone is controlled by soluble cyclase. The short-lived cyclopropanones are hydrolyzed to products 1–3. The collective name “graminoxins” has been ascribed to oxylipins 1–3.
- Grechkin, Alexander N.,Ogorodnikova, Anna V.,Egorova, Alevtina M.,Mukhitova, Fakhima K.,Ilyina, Tatiana M.,Khairutdinov, Bulat I.
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p. 336 - 343
(2018/06/04)
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- Replacement of two amino acids of 9R-dioxygenase-allene oxide synthase of Aspergillus niger inverts the chirality of the hydroperoxide and the allene oxide
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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.
- Sooman, Linda,Wennman, Anneli,Hamberg, Mats,Hoffmann, Inga,Oliw, Ernst H.
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p. 108 - 118
(2016/01/08)
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- Calcium modulates membrane association, positional specificity, and product distribution in dual positional specific maize lipoxygenase-1
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This study investigates how calcium modulates the properties of dual positional specific maize lipoxygenase-1, including its interaction with substrate, association with subcellular membrane and alteration of product distribution. Bioinformatic analyses identified Asp38, Glu127 and Glu201 as putative calcium binding residues and Leu37 as a flanking hydrophobic residue also potentially involved in calcium-mediated binding of the enzyme to subcellular membranes. Asp38 and Leu37 were shown to be important but not essential for calcium-mediated association of maize lipoxygenase-1 to subcellular membranes in vitro. Kinetic studies demonstrate that catalytic efficiency (Vmax/Km) shows a bell-shaped dependence on log of the molar ratio of substrate to unbound calcium. Calcium also modulates product distribution of the maize lipoxygenase-1 reaction, favoring 13-positional specificity and increasing the relative amount of (E,Z)-isomeric products. The results suggest that calcium regulates the maize lipoxygenase-1 reaction by binding to substrate, and by promoting binding of substrate to enzyme and association of maize lipoxygenase-1 to subcellular membranes.
- Cho, Kyoungwon,Han, Jihoon,Rakwal, Randeep,Han, Oksoo
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- Liquid chromatography-tandem mass spectrometry determination of human plasma 1-palmitoyl-2-hydroperoxyoctadecadienoyl-phosphatidylcholine isomers via promotion of sodium adduct formation
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Accumulation of phosphatidylcholine hydroperoxide (PCOOH), a primary oxidation product of phosphatidylcholine, in blood plasma has been observed in various pathological conditions, including atherosclerosis. In this study, we investigated the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to develop a method for accurate quantification of PCOOH (1-palmitoyl-2-hydroperoxyoctadecadienoyl-sn-glycero-3-phosphocholine, 16:0/HpODE PC), focusing on isomers such as 16:0/13-HpODE PC and 16:0/9-HpODE PC. Sodiated PCOOH ([M+Na]+, m/z 812) provided not only a known product ion (m/z 147) but also characteristic product ions (m/z 541 for 16:0/13-HpODE PC and m/z 388 for 16:0/9-HpODE PC). Thus, three multiple reaction monitorings (MRMs) could be performed. MRM (812/147) enabled determination of 16:0/HpODE PC, and MRM (812/541) and MRM (812/388) allowed specific measurement of 16:0/13-HpODE PC and 16:0/9-HpODE PC, respectively. By using this method, we could determine plasma PCOOH concentrations in healthy subjects and patients with angiographically significant stenosis. In healthy subject and patient plasma, the concentration of 16:0/HpODE PC was close to the sum of the concentrations of 16:0/13-HpODE PC and 16:0/9-HpODE PC. This finding shows that radical and/or enzymatic oxidation, rather than singlet oxygen oxidation, is recognized to cause peroxidation of PC. The newly developed LC-MS/MS method appears to be a powerful tool for developing a better understanding of in vivo lipid peroxidation and its involvement in human diseases.
- Kato, Shunji,Nakagawa, Kiyotaka,Suzuki, Yuuri,Asai, Akira,Nagao, Mototsugu,Nagashima, Kazuyuki,Oikawa, Shinichi,Miyazawa, Teruo
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- Whole-cell one-pot biosynthesis of azelaic acid
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Polymers benefit from the use of biogenic resources such as fatty acids. They enable easy access to valuable monomeric building blocks, which, in comparison to their exclusively fossil counterparts, lead to products with improved physicochemical properties. Monomers of special interest are medium-chain dicarboxylic acids, which are not easy to obtain by traditional chemical means. Previously, we established an in vitro pathway that combined a 9-lipoxygenase and a 9/13-hydroperoxide lyase, which enabled the conversion of linoleic acid via a hydroperoxy intermediate into 9-oxononanoic acid, the precursor of azelaic acid. Herein, we aimed for the further development of the multi-enzyme cascade, which included the oxidation of 9-oxononanoic acid and the establishment of a suitable whole-cell catalyst. A detailed investigation of the simultaneous in vitro reaction setup revealed that both lipoxygenase activation and the subsequent hydroperoxide lyase reaction depend on the hydroperoxide reaction intermediate. For the activation of lipoxygenase, the hydroperoxide lyase activity, therefore, has to be significantly reduced. In accordance with these observations, we established a suitable dual-expression system and we further demonstrated that endogenous E. coli redox enzymes are feasible to oxidize 9-oxononanoic acid to azelaic acid. The resulting whole-cell catalyst is, therefore, able to perform the direct bioconversion of linoleic acid into azelaic acid. The use of organic solvent as the second phase improved the overall performance of the E. coli host strain. The developed one-pot, single-step process afforded 29 mg L-1 of azelaic acid within 8 h with a substrate conversion of 34 % and a selectivity of 47 %. Tiny polymer factories: An E. coli whole-cell catalyst is developed for the bioconversion of linoleic acid to azelaic acid, an important building block for biopolymers. The catalytic machinery of the prokaryotic host is equipped with two plant enzymes, a lipoxygenase, and a hydroperoxide lyase. Together with an endogenous oxidoreductase, this three-enzyme cascade reaction catalyzes the oxidative cleavage of the fatty acid substrate.
- Otte, Konrad B.,Kittelberger, Jens,Kirtz, Marko,Nestl, Bettina M.,Hauer, Bernhard
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p. 1003 - 1009
(2014/05/06)
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- LOXPsa1, the first recombinant lipoxygenase from a basidiomycete fungus
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A dioxygenase from the edible basidiomycete Pleurotus sapidus, originally researched because of its distinct ability to convert the sequiterpene (+)-valencene to the valuable grapefruit aroma (+)-nootkatone, was identified as a potent lipoxygenase (LOXsu
- Plagemann, Ina,Zelena, Katerina,Arendt, Philipp,Ringel, Peter D.,Krings, Ulrich,Berger, Ralf G.
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- Physcomitrella patens has lipoxygenases for both eicosanoid and octadecanoid pathways
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Mosses have substantial amounts of long chain C20 polyunsaturated fatty acids, such as arachidonic and eicosapentaenoic acid, in addition to the shorter chain C18 α-linolenic and linoleic acids, which are typical substrates of lipoxygenases in flowering p
- Anterola, Aldwin,G?bel, Cornelia,Hornung, Ellen,Sellhorn, George,Feussner, Ivo,Grimes, Howard
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experimental part
p. 40 - 52
(2009/07/11)
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- Rate constants for peroxidation of polyunsaturated fatty acids and sterols in solution and in liposomes
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Rate constants for autoxidation propagation of several unsaturated lipids in benzene solution at 37°C and in phosphatidylcholine liposomes were determined by a linoleate radical clock. This radical clock is based on competition between hydrogen atom abstraction by an intermediate peroxyl radical derived from linoleic acid that leads to a trans,cis-conjugated hydroxyoctadecadienoic product and β-fragmentation of the same peroxyl that gives the trans,trans-product hydroxyoctadecadienoic acid. Rate constants determined by this approach in solution relative to linoleic acid (k p) 62 M-1 s-1) were: arachidonic acid (k p = 197 ± 13 M-1 s-1), eicosapentaenoic acid (kp = 249 ± 16 M-1 s-1), docosahexaenoic acid (kp = 334 ± 37 M-1 s -1), cholesterol (kp= 11 ± 2 M-1 s -1), and 7-dehydrocholesterol (kp = 2260 ± 40 M-1 s-1). Free radical oxidations of multilamellar and unilamellar liposomes of various mixtures of glycerophosphatidylcholine molecular species were also carried out. In some experiments, cholesterol or 7-dehydrocholesterol was incorporated into the lipid mixture undergoing oxidation. A phosphatidylcholine bearing a linoleate ester at sn-2 was a component of each liposome peroxidation reaction and the ratio of trans,cis/trans,trans (t,c/t,t)-conjugated diene oxidation products formed from this phospholipid was determined for each oxidation reaction. This t,c/t,t-product ratio from linoleate was used to "clock" liposome constituents as hydrogen atom donors in the lipid bilayer. Application of this lipid bilayer radical clock gives relative autoxidation propagation rate constants of arachidonate (20:4), eicosapentaenoate (20:5), docosahexaenoate (22:6), and 7-dehydrocholesterol to be 115 ± 7, 145 ± 8, 172 ± 13, and 832 ± 86, respectively, a reactivity trend that parallels the one in solution. We also conclude from the liposome oxidations that linoleate peroxyl radicals at different positions on the eighteen-carbon chain (at C-9 and C-13) have different kinetic properties. This is in contrast to the results of solution oxidations of linoleate in which the C-9 and C-13 peroxyl radicals have similar reactivities. We suggest that peroxyl radical β-scission depends on solvent polarity and the polarity of the local environment of peroxyl radicals in liposomal oxidations depends on the position of the peroxyl radical on the 18-carbon chain.
- Xu, Libin,Davis, Todd A.,Porter, Ned A.
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experimental part
p. 13037 - 13044
(2009/12/08)
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- Characterisation of lipoxygenase isoforms from olive callus cultures
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Two lipoxygenase isoforms from olive callus cultures were separated from each other. Acetone powders were made to stabilise activity and remove lipids. Separation was then achieved by salt precipitation and ion-exchange chromatography. Both isoforms had comparable activity with linoleic and α-linolenic acid substrates, a basic pH optimum and had molecular masses of around 95 kDa. The callus extracts preferentially formed the 13-hydroperoxy products, in keeping with the pattern of volatile derivatives found in olive tissues and oils derived therefrom.
- Williams, Mark,Harwood, John L.
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experimental part
p. 2532 - 2538
(2009/04/04)
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- Properties of a mini 9R-lipoxygenase from Nostoc sp. PCC 7120 and its mutant forms
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Lipoxygenases (LOXs) consist of a class of enzymes that catalyze the regio- and stereospecific dioxygenation of polyunsaturated fatty acids. Current reports propose that a conserved glycine residue in the active site of R-lipoxygenases and an alanine residue at the corresponding position in S-lipoxygenases play a crucial role in determining the stereochemistry of the product. Recently, a bifunctional lipoxygenase with a linoleate diol synthase activity from Nostoc sp. PCC7120 with R stereospecificity and the so far unique feature of carrying an alanine instead of the conserved glycine in the position of the sequence determinant for chiral specificity was identified. The recombinant carboxy-terminal domain was purified after expression in Escherichia coli. The ability of the enzyme to use linoleic acid esterified to a bulky phosphatidylcholine molecule as a substrate suggested a tail-fist binding orientation of the substrate. Site directed mutagenesis of the alanine to glycine did not cause alterations in the stereospecificity of the products, while mutation of the alanine to valine or isoleucine modified both regio- and enantioselectivity of the enzyme. Kinetic measurements revealed that substitution of Ala by Gly or Val did not significantly influence the reaction characteristics, while the A162I mutant showed a reduced vmax. Based on the mutagenesis data obtained, we suggest that the existing model for stereocontrol of the lipoxygenase reaction may be expanded to include enzymes that seem to have in general a smaller amino acid in R and a bulkier one in S lipoxygenases at the position that controls stereospecificity.
- Andreou, Alexandra-Zoi,Vanko, Marian,Bezakova, Lydia,Feussner, Ivo
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p. 1832 - 1837
(2008/09/20)
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- Identification of an amino acid determinant of pH regiospecificity in a seed lipoxygenase from Momordica charantia
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Lipoxygenases (LOX) form a heterogeneous family of lipid peroxidizing enzymes, which catalyze specific dioxygenation of polyunsaturated fatty acids. According to their positional specificity of linoleic acid oxygenation plant LOX have been classified into linoleate 9- and linoleate 13-LOX and recent reports identified a critical valine at the active site of 9-LOX. In contrast, more bulky phenylalanine or histidine residues were found at this position in 13-LOX. We have recently cloned a LOX-isoform from Momordica charantia and multiple amino acid alignments indicated the existence of a glutamine (Gln599) at the position were 13-LOX usually carry histidine or phenylalanine residues. Analyzing the pH-dependence of the positional specificity of linoleic acid oxygenation we observed that at pH-values higher than 7.5 this enzyme constitutes a linoleate 13-LOX whereas at lower pH, 9-H(P)ODE was the major reaction product. Site-directed mutagenesis of glutamine 599 to histidine (Gln599His) converted the enzyme to a pure 13-LOX. These data confirm previous observation suggesting that reaction specificity of certain LOX-isoforms is not an absolute enzyme property but may be impacted by reaction conditions such as pH of the reaction mixture. We extended this concept by identifying glutamine 599 as sequence determinant for such pH-dependence of the reaction specificity. Although the biological relevance for this alteration switch remains to be investigated it is of particular interest that it occurs at near physiological conditions in the pH-range between 7 and 8.
- Hornung, Ellen,Kunze, Susan,Liavonchanka, Alena,Zimmermann, Grit,Kuehn, Diana,Fritsche, Kathrin,Renz, Andreas,Kuehn, Hartmut,Feussner, Ivo
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scheme or table
p. 2774 - 2780
(2009/04/10)
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- Detection of divinyl ether synthase in Lily-of-the-Valley (Convallaria majalis) roots
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Incubations of linoleic acid with cell-free preparations from Lily-of-the-Valley (Convallaria majalis L., Ruscaceae) roots revealed the presence of 13-lipoxygenase and divinyl ether synthase (DES) activities. Exogenous linoleic acid was metabolized predominantly into (9Z,11E,1′E)-12-(1′-hexenyloxy)-9,11-dodecadienoic (etheroleic) acid. Its identification was confirmed by the data of ultraviolet spectroscopy, mass spectra, 1H NMR, COSY, catalytic hydrogenation. The isomeric divinyl ether (8E,1′E,3′Z)-12-(1′,3′-nonadienyloxy)-8-nonenoic (colneleic) acid was detected as a minor product. Incubations with linoleic acid hydroperoxides revealed that 13-hydroperoxide was a preferential substrate, while the 9-hydroperoxide was utilized with lesser efficiency.
- Ogorodnikova, Anna V.,Latypova, Larisa R.,Mukhitova, Fahima K.,Mukhtarova, Lucia S.,Grechkin, Alexander N.
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experimental part
p. 2793 - 2798
(2009/04/11)
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- Regiochemical and stereochemical evidence for enzyme-initiated catalysis in dual positional specific maize lipoxygenase-1
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Dual positional specific maize lipoxygenase-1 catalyzed the formation of racemic mixtures of four possible regioisomers and was strongly inhibited by the radical scavenger, 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinoxy radical. Molecular modeling studies indicated that the oxygen-binding cavity is segregated from the substrate-binding cavity. The data suggest that a bis-allylic radical reaction intermediate is generated enzymatically, released from the enzyme active site, and subsequently oxygenated outside of the enzyme active site by a nonenzymatic mechanism.
- Jang, Sungkuk,Huon, Thavrak,Kim, Keumhwa,Um, Eunji,Han, Oksoo
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p. 3113 - 3116
(2008/02/10)
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- Identification and functional analyses of two cDNAs that encode fatty acid 9-/13-hydroperoxide lyase (CYP74C) in rice
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Fatty acid hydroperoxide lyase (HPL), a member of cytochrome P450 (CYP74), produces aldehydes and oxoacids involved in plant defensive reactions. In monocots, HPL that cleaves 13-hydroperoxides of fatty acids has been reported, but HPL that cleaves 9-hydroperoxides is still unknown. To find this type of HPL, in silico screening of candidate cDNA clones and subsequent functional analyses of recombinant proteins were performed. We found that AK105964 and AK107161 (Genbank accession numbers), cDNAs previously annotated as allene oxide synthase (AOS) in rice, are distinctively grouped from AOS and 13-HPL. Recombinant proteins of these cDNAs produced in Escherichia. coli cleaved both 9- and 13-hydroperoxide of linoleic and linolenic into aldehydes, while having only a trace level of AOS activity and no divinyl ether synthase activity. Hence we designated AK105964 and AK107161 OsHPL1 and OsHPL2 respectively. They are the first CYP74C family cDNAs to be found in monocots.
- Kuroda, Hisao,Oshima, Toshiyuki,Kaneda, Hirotaka,Takashio, Masachika
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p. 1545 - 1554
(2008/02/03)
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- Characterization of 9-fatty acid hydroperoxide lyase-like activity in germinating barley seeds that transforms 9(S)-hydroperoxy-10(E),12(Z)- octadecadienoic acid into 2(E)-nonenal
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Previously, we reported that 2(E)-nonenal, having a low flavor threshold (0.1 ppb) and known as the major contributor to a cardboard flavor (stale flavor) in stored beer, is produced by lipoxygenase-1 and a newly found factor named 9-fatty acid hydroperoxide lyase-like (9-HPL-like) activity in malt. To assess the involvement of 9-HPL-like activity in beer staling, we compared the values of the wort nonenal potential, an index for predicting the staleness of beer, with the lipoxygenase and 9-HPL-like activity of 20 commercial malts. There was a significant correlation between the malt 9-HPL-like activity and the values of wort nonenal potential (r = 0.53, P 0.05), while the correlation between malt lipoxygenase activity and the wort nonenal potential was statistically insignificant. Analysis of the partially purified 9-HPL-like activity from embryos of germinating barley seeds indicated that 9-HPL-like activity consisted of fatty acid hydroperoxide lyase and 3Z:2E isomerase.
- Kuroda, Hisao,Kojima, Hidetoshi,Kaneda, Hirotaka,Takashio, Masachika
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p. 1661 - 1668
(2008/02/03)
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- Mechanism of lipid peroxidation photosensitized by tiaprofenic acid: Product studies using linoleic acid and 1,4-Cyclohexadienes as model substrates
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A careful study of the linoleic acid hydroperoxide (LOOH) profile obtained upon peroxidation of linoleic acid (LA) photosensitized by tiaprofenic acid (TPA) and analogous ketones has been undertaken to distinguish between type-I and type-II photoperoxidation mechanisms. 1,4-Cyclohexadiene and 1,2-dimethylcyclohexa-2,5-diene-carboxylic acid (CHDCA) have also been used as models for LA since they also have double allylic systems. Coir-radiation of LA with TPA and decarboxytiaprofenic acid (DTPA) in acetonitrile and micellar media produced significant amounts of conjugated dienic LOOH. The cis,trans to trans,trans ratio depended on the irradiation time; thus, this parameter is an ambiguous tool for mechanistic assignment. An interesting finding was the decrease of the LOOH level after long irradiation times in mixtures photooxidized by DTPA, which is attributed to quenching of the DTPA triplet by the generated dienic LOOH. High-performance liquid chromatography analyses confirmed that the main pathway operating in photodynamic lipid peroxidation sensitized by (D)TPA is a type-I mechanism. However, product studies using CHDCA have clearly shown that a type-II mechanism is also operating and might contribute to the overall photooxidation process in a significant way.
- Samadi, Abdelouahid,Martinez, Luis A.,Miranda, Miguel A.,Morera, Isabel M.
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p. 359 - 365
(2007/10/03)
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- Antioxidant activities of major components of γ-oryzanol from rice bran using a linoleic acid model
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The change in hydroperoxides of linoleic acid incubated with constant micro air flow at 37°C was used to evaluate the antioxidant activities of three major components of γ-oryzanol from rice bran (cycloartenyl ferulate, 24-methylene cycloartanyl ferulate, and campesteryl ferulate) compared with α-tocopherol and ferulic acid. The four hydroperoxide isomers of linoleic acid, 9-hydroperoxy-10-trans, 12-cis-octadecadienoic acid [9HPODE(t,c)], 9-hydroperoxy-10-trans,12-trans-octadecadienoic acid, 13-hydroperoxy-9-cis, 11-trans-octadecadienoic acid [13HPODE(c,t)], and 13-hydroperoxy-9-trans, 11-trans-octadecadienoic acid, were measured using normal-phase high-performance liquid chromatography with an ultraviolet detector. The three components of γ-oryzanol evidenced significant antioxidant activity when they were mixed with linoleic acid in a molar ratio of 1:100 and 1:250 but not in a molar ratio of 1:500 (P 0.05). α-Tocopherol and ferulic acid also demonstrated significant antioxidant activity at all three molar ratios (P 0.05). The highest molar ratio (1:100) of α-tocopherol, however, caused greater levels of 9HPODE(t,c) and 13HPODE(c,t) than the other two less concentrated treatments.
- Xu,Samuel Godber
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p. 645 - 649
(2007/10/03)
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- Retinoic acid-dependent stimulation of 2,2'-azobis(2-amidinopropane)- initiated autoxidation of linoleic acid in sodium dodecyl sulfate micelles: A novel prooxidant effect of retinoic acid
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(E)-Retinoic acid (RA) was shown to stimulate the rate of 2,2'-azobis(2- amidinopropane) (AAPH)-initiated autoxidation of linoleic acid (18:2) in sodium dodecyl sulfate (SDS) micelles. RA-dependent stimulation of 18:2 autoxidation was characterized by enhanced rates of dioxygen uptake which were linear with retinoid concentration. In contrast, 5,6-epoxy-RA, a major oxidation product of RA, failed to affect the rate of dioxygen consumption at all concentrations tested. RA was also shown to stimulate peroxyl radical- dependent oxidation of styrene to the corresponding oxirane when styrene was included in the micellar system as a molecular probe. Furthermore, unequivocal evidence of RA-dependent stimulation of 18:2 autoxidation was obtained by relative quantitation of 13-hydroxy-(9Z, 11E)-octadecadienoic acid (13-HODE) plus 9-hydroxy-(10E,12Z)-octadecadienoic acid (9-HODE) production. In addition, enhanced carboncentered radical formation was demonstrated in the presence of RA by EPR spectroscopy using α-(4-pyridyl 1- oxide)-N-tert-butylnitrone (4-POBN) as a spin trap. Analysis and quantitation of RA oxidation products indicated that RA was oxidized to one primary product, 5,6-epoxy-RA, which was identified on the basis of cochromatography with synthetic standard (in a reverse-phase HPLC system), electronic absorption spectroscopy, and positive chemical ionization mass spectrometry of the corresponding methyl ester. Other minor oxidation products were also detected but not characterized. In contrast, reaction mixtures devoid of 18:2 failed to demonstrate significant retinoid oxidation. Mechanisms are proposed to account for the prooxidant effects of RA in this system.
- Freyaldenhoven, Mary Ann,Lehman, Paul A.,Franz, Thomas J.,Lloyd, Roger V.,Samokyszyn, Victor M.
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p. 102 - 110
(2007/10/03)
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- 9-Hydroxy-10,12-octadecadienoic acid (9-HODE) and 13-hydroxy-9,11-octadecadienoic acid (13-HODE): Excellent markers for lipid peroxidation
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Various conditions for conversion of (9S,10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid (9S-HPODE) and (13S,9Z,10E)-13-hydroperoxy-9,11-octadecadienoic acid (13S-HPODE) into the corresponding hydroxy acids, (9S,10E,12Z)-9-hydroxy-10,12-octadecadienoic acid (9S-HODE) and (13S,9Z,10E)-13-hydroxy-9,11-octadecadienoic acid (13S-HODE), were investigated in vitro. 9S-HODE and 13S-HODE were subjected to lipid peroxidation under various conditions: oxidation was carried out in air only, and in air/Fe2+/ascorbate, air/H2O2/Fe2+, air/Fe2+, and air/Fe3+. In contrast to the corresponding hydroperoxides (9S-HPODE and 13S-HPODE), 9-HODE and 13-HODE proved to be stable in all these oxidation experiments. Unexpectedly, hydroxy compounds obtained by reduction of hydroperoxides derived from arachidonic acid were not attacked by air/Fe2+/ascorbate or air/Fe2+. Thus, for instance, (15S,5Z,8Z,11Z,13E)-15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) remained unchanged in spite of possessing the structural prerequisites for attack by radicals, i.e. a CH2-group located between two double bonds. Consequently, metal-induced air oxidation reactions of these systems seem to be restricted to hydroperoxides of unsaturated acids (LOOH) and not to corresponding hydroxy compounds (LOH). The reported experiments explain why hydroxy derivatives of unsaturated acids, especially 9-HODE and 13-HODE, are enriched in naturally occurring lipid peroxidation (LPO) processes to a greater extent than any other LPO product and why they are nearly ideal markers for LPO.
- Spiteller, Peter,Spiteller, Gerhard
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p. 131 - 139
(2007/10/03)
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- Chemo-Enzymatic Synthesis of Methyl 9(S)-HODE (Dimorphecolic Acid Methyl Ester) and Methyl 9(S)-HOTE Catalysed by Barley Seed Lipoxygenase
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The straightforward chemo-enzymatic synthesis of methyl 9(S)-HODE (dimorphecolic acid methyl ester) and methyl 9(S)-HOTE, from linoleic and α-linolenic acids, using barley seeds lipoxygenase under oxygen pressure and at high substrate concentration, is described.
- Martini, Dominique,Buono, Gerard,Montillet, Jean-Luc,Iacazio, Gilles
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p. 1489 - 1492
(2007/10/03)
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- High-Performance Liquid Chromatographic Analysis of the Products of Linoleic Acid Oxidation Catalyzed by Pea (Pisum sativum) Seed Lipoxygenases
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An HPLC method is discussed for the analysis of the products formed by the pea (Pisum sativum) lipoxygenase catalyzed oxidation of linoleic acid.The results demonstrate the feasibility of analyzing all of the hydroperoxides, hydroxides, and keto fatty acids in a single chromatographic step and show that it will be possible to analyze the product profile from the lipoxygenase activity contained in a portion of a seed, which will permit the remainder of the seed to be grown on for subsequent generations.The chemical structures of the products have been identified by HPLC analysis and GC-MS. Keywords: Lipoxygenase; hydroperoxides; chromatography
- Wu, Zecai,Robinson, David S.,Domoney, Claire,Casey, Rod
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p. 337 - 342
(2007/10/02)
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- Auto-growth inhibitory substance from the fresh-water cyanobacterium Phormidium tenue
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An extract of the cyanobacterium P. tenue showed a significant inhibitory effect on its own growth. Bioassay-directed fractionation has led to the identification of the auto-growth inhibitory substance as a mixture of fatty acids. Unsaturated fatty acids such as linoleic and linolenic acids appear to be predominantly responsible for the auto-growth inhibitory effect.
- Yamada,Murakami,Morimoto,Sakakibara
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p. 1863 - 1865
(2007/10/02)
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- An Isotopic Study (2H and 18O) of the Enzymatic Conversion of Linoleic Acid into Colneleic Acid with Carbon Chain Fracture: the Origin of Shorter Chain Aldehydes
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Contrary to earlier reports, the divinyl 9-ether oxygen of colneleic acid is shown by experiment with 18O2 to originate from oxygen, not water.Using -9(S)-hydroperoxyoctadeca-10(E),12(Z)-dienoic acid, made enzymatically from synthetic linoleic acid, it is found that the distribution of deuterium as determined by NMR and mass spectrometry in the fractured carbon chain of colneic acid formed by potato enzyme, is consistent with the intervention of an epoxy carbonium ion intermediate.Though divinyl acids such as colneleic and colnelenic acid give the expected shorter chain aldehydes on treatment with aqueous acid, it is likely that the latter are formed in most plants by trapping of a monovinyl oxonium ion rather than by rehydration of colneleic and colnelenic acid.
- Crombie, Leslie,Morgan, David O.,Smith, Elisabeth H.
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p. 567 - 575
(2007/10/02)
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- PURIFICATION AND PROPERTIES OF LIPOXYGENASE IN MARCHANTIA POLYMORPHA CULTURED CELLS
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Lipoxygenase activity has been detected in cultured cells of a bryophyte, Marchantia polymorpha (liverwort).The activity was enhanced in the stationary phase.Most of the activity occurred in the cytosolic fraction.The lipoxygenase was purified to homogeneity by ammonium sulphate fractionation and hydrophobic, ion-exchange, and gel filtration chromatography.From the SDS-polyacrylamide gel electrophoresis, the Mr of the lipoxygenase was estimated as 109600.The pH optimum was ca 9.0 and the pI was 4.8.This enzyme formed mainly 13-(S)-hydroperoxy-(9Z,11E)-octadecadienoic acid from linoleic acid.It showed twice the activity for γ-linolenic acid than for linoleic acid. Key Word Index - Marchantia polymorpha; Marchantiaceae; liverwort; purification; lipoxygenase.
- Matsui, Kenji,Narahara, Hiroko,Kajiwara, Tadahiko,Hatanaka, Akikazu
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p. 1499 - 1502
(2007/10/02)
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- Stereospecific Removal of the pro-R Hydrogen at C-8 of (9S)-Hydroperoxyoctadecadienoic Acid in the Biosynthesis of Colneleic Acid
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The stereochemistry of hydrogen removal in the conversion of (9S)-hydroperoxyoctadeca-(10E,12Z)-dienoic acid (2) into colneleic acid (4) was studied. -(2) and -(2) were incubated with the 105 000g particle fraction of potato homogenate and the colneleic acid formed was isolated.Mass spectrometric analysis demonstrated that colneleic acid which was biosynthesized from the -hydroperoxide was largely devoid of deuterium, whereas colneleic acid produced from the -hydroperoxide retained most of the deuterium.Accordingly, there is a selective removal of the pro-R hydrogen at C-8 in the biosynthesis of colneleic acid from (9S)-hydroperoxyoctadeca-(10E,12Z)-dienoic acid.
- Fahlstadius, Per,Hamberg, Mats
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p. 2027 - 2030
(2007/10/02)
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- BIOMIMETIC TOTAL SYNTHESIS OF COLNELEIC ACID AND ITS FUNCTION AS A LIPOXYGENASE INHIBITOR
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A biomimetic synthesis of colneleic acid (2) from 9(S)-hydroperoxy-10(E),12(Z)octadecadienoic acid (1) is reported.The lipoxygenase of potato which converts linoleic acid to 1 was found to be strongly inhibited by acid 2 (Ki=8μM).
- Corey, E. J.,Nagata, Ryu,Wright, Stephen W.
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p. 4917 - 4920
(2007/10/02)
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- Unified Mechanism for Polyunsaturated Fatty Acid Autooxidation. Competition of Peroxy Radical Hydrogen Atom Abstraction, β-Scission, and Cyclization
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The autooxidation of linoleic (18:2) and arachidonic (20:4) acids with several cosubstrates was investigated.Cumene, tetralin, 1,4-cyclohexadiene, and 9,10-dihydroanthracene in benzene were used as cosubstrates for the oxidation of linoleic acid.The distribution of products, trans,cis diene hydroperoxides and trans,trans diene hydroperoxides, was dependent on the ability of cosubstrates to donate hydrogen atoms to linoleate peroxy radicals.Arachidonic acid was oxidized in mixtures of benzene/1,4-cyclohexadiene with linoleic acid internal standard.Product distribution of six hydroperoxyeicosatetraenoic acids (HPETE) derived from arachidonic acid was established at different concentrations of 1,4-cyclohexadiene in the solvent mixture.A kinetic expression is derived that is useful in describing polyunsaturated fatty acid oxidation product mixtures.By the use of this kinetic derivation, the rate of cyclization of peroxy free radicals derived from arachidonic acid was determined.
- Porter, Ned A.,Lehman, Laura S.,Weber, Bruce A.,Smith, Karl J.
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p. 6447 - 6455
(2007/10/02)
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- Autoxidation of Polyunsaturated Lipids. Factors Controlling the Stereochemistry of Product Hydroperoxides
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The mechanism of the autoxidation of linoleic acid and phospholipid esters of this acid was investigated.The products of autoxidation,13-hydroperoxy-9-cis,11-trans-octadecadienoic (4), 13-hydroperoxy-9-trans,11-trans-octadecadienoic (5), 9-hydroperoxy-10-
- Porter, Ned A.,Weber, Bruce A.,Weenen, Hugo,Khan, Jamil A.
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p. 5597 - 5601
(2007/10/02)
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