Synthesis and Applications of Stereospecifically 3H-Labeled Arachidonic Acids as Mechanistic Probes for Lipoxygenase and Cyclooxygenase Catalysis1

Schneider, Claus; Boeglin, William E.; Lai, Sheng; K., Jin; Brash, Alan R.

doi:10.1006/abio.2000.4670

Cited by 14 publications

(7 citation statements)

References 31 publications

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“…Incubation with Stereospecifically Labeled Linoleic Acids-The stereospecifically labeled 11R-3 H and 11S-3 H linoleic acids were synthesized previously as described (25). The tritium-labeled linoleates were mixed with [ 14 C]LA, which served as an internal standard for measurement of tritium retention.…”

Section: Methodsmentioning

confidence: 99%

On the Relationships of Substrate Orientation, Hydrogen Abstraction, and Product Stereochemistry in Single and Double Dioxygenations by Soybean Lipoxygenase-1 and Its Ala542Gly Mutant

Coffa

Imber

Maguire

et al. 2005

Journal of Biological Chemistry

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111

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Recent findings associate the control of stereochemistry in lipoxygenase (LOX) catalysis with a conserved active site alanine for S configuration hydroperoxide products, or a corresponding glycine for R stereoconfiguration. To further elucidate the mechanistic basis for this stereocontrol we compared the stereoselectivity of the initiating hydrogen abstraction in soybean LOX-1 and an Ala542Gly mutant that converts linoleic acid to both 13S and 9R configuration hydroperoxide products. Using 11R-3 Hand 11S-3 H-labeled linoleic acid substrates to examine the initial hydrogen abstraction, we found that all the primary hydroperoxide products were formed with an identical and highly stereoselective pro-S hydrogen abstraction from C-11 of the substrate (97-99% pro-S-selective). This strongly suggests that 9R and 13S oxygenations occur with the same binding orientation of substrate in the active site, and as the equivalent 9R and 13S products were formed from a bulky ester derivative (1-palmitoyl-2-linoleoylphosphatidylcholine), one can infer that the orientation is tail-first. Both the EPR spectrum and the reaction kinetics were altered by the R product-inducing Ala-Gly mutation, indicating a substantial influence of this Ala-Gly substitution extending to the environment of the active site iron. To examine also the reversed orientation of substrate binding, we studied oxygenation of the 15S-hydroperoxide of arachidonic acid by the Ala542Gly mutant soybean LOX-1. In addition to the usual 5S,15S-and 8S,15S-dihydroperoxides, a new product was formed and identified by high-performance liquid chromatography, UV, gas chromatography-mass spectrometry, and NMR as 9R,15S-dihydroperoxyeicosa-5Z,7E,11Z,13E-tetraenoic acid, the R configuration "partner" of the normal 5S,15S product. This provides evidence that both tail-first and carboxylate end-first binding of substrate can be associated with S or R partnerships in product formation in the same active site.

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Section: Methodsmentioning

confidence: 99%

On the Relationships of Substrate Orientation, Hydrogen Abstraction, and Product Stereochemistry in Single and Double Dioxygenations by Soybean Lipoxygenase-1 and Its Ala542Gly Mutant

Coffa

Imber

Maguire

et al. 2005

Journal of Biological Chemistry

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111

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“…Experiments with Stereospecifically Labeled H]Linoleic Acids-In previous studies we prepared 3 H-labeled linoleic acids with a pro-R or pro-S tritium label on the bis-allylic 11-carbon, each mixed with [1-14 C]linoleic acid, which served as internal standard for assessment of tritium loss or retention in the transformation to lipoxygenase products (26 C]linoleic acid) were monitored by UV spectrophotometry; aliquots (1 ml) were removed at three time points and added to 2 volumes of ice-cold ethanol. The reaction products were reduced by adding 50 l of 10 mg/ml NaBH 4 in ethanol, and subsequently acidified to pH 5 and extracted using a Waters Oasis C18 cartridge.…”

Section: Methodsmentioning

confidence: 99%

A 49-kDa Mini-lipoxygenase from Anabaena sp. PCC 7120 Retains Catalytically Complete Functionality

Zheng¹,

Boeglin²,

Schneider

et al. 2008

Journal of Biological Chemistry

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Anabaena sp. PCC 7120 is one of the few prokaryotes harboring a lipoxygenase (LOX) gene. The sequence resides in an open reading frame encoding a fusion protein of a catalase-like hemoprotein with an unusually short LOX (ϳ49 kDa) at the C terminus. The recombinant mini-LOX contains a non-heme iron in the active site and is highly active with linoleic and ␣-linolenic acids (which occur naturally in Anabaena) giving the respective 9R-hydroperoxides, the mirror image of the 9S-LOX products of plants. Using stereospecifically labeled [11-3 H]linoleic acids we show that reaction is catalyzed via a typical antarafacial relationship of initial hydrogen abstraction and oxygenation. The mini-LOX oxygenated C 16 /C 18:2 -phosphatidylcholine with 9R specificity, suggesting a "tail first" mode of fatty acid binding. Site-directed mutagenesis of an active site Ala (Ala 215 ), typically conserved as Gly in R-LOX, revealed that substitution with Gly retained 9R specificity, whereas the larger Val substitution switched oxygenation to 13S, implying that Ala 215 represents the functional equivalent of the Gly in other R-LOX. Metabolism studies using a synthetic fatty acid with extended double bond conjugation, 9E,11Z,14Z-20:36, showed that the mini-LOX can control oxygenation two positions further along the fatty acid carbon chain. We conclude that the mini-LOX, despite lacking the ␤-barrel domain and much additional sequence, is catalytically complete. Interestingly, animal and plant LOX, which undoubtedly share a common ancestor, are related in sequence only in the catalytic domain; it is possible that the prokaryotic LOX represents a common link and that the ␤-barrel domain was then acquired independently in the animal and plant kingdoms. Lipoxygenases (LOX)3 oxygenate polyunsaturated fatty acids to specific hydroperoxide derivatives (1, 2). The enzymes occur widely in the eukaryotic world, being ubiquitous in plants and fungi, common in primitive animals such as corals and hydra, and again ubiquitous in higher animals (3, 4). The lipoxygenase proteins are comprised of a polypeptide chain of 75-80 kDa in animals, 94 -104 kDa in plants, and harbor a single non-heme iron in the active site. As shown by x-ray crystallography, the animal and plant LOX enzymes share the same overall topology, with the extra ϳ20 -30 kDa in the plant LOX being accounted for by extra loops between the otherwise well conserved sequences (5-8). Lipoxygenases have a two-domain structure, an N-terminal C2-like or ␤-barrel domain, and the larger, mainly ␣-helical, catalytic domain that holds the iron in the active site. In the catalytic domain the LOX enzymes share five well conserved amino acid ligands to the iron, four histidines (or three His and one Asn or Ser) and the carboxylate group of the very C-terminal amino acid, usually Ile. Analysis of the structure-function of LOX enzymes suggests that the ␤-barrel domain plays a role in membrane binding and/or substrate acquisition (9, 10), whereas the catalytic domain is involved in controlling the reactio...

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“…Subsequent elongation to the required chain-length can conveniently be performed using anodic coupling (the so-called Kolbe synthesis; see ref [93]). Chiral HPLC separation of racemic p -toluenesulfonate derivatives of hydroxystearates labeled with tritium at the chiral carbon followed by elimination of the tosylate by LiAlH 4 reduction offers an additional possibility to generate stereospecifically labeled stearates [39]. …”

Section: Synthesis Of Stereospecifically Labeled Fatty Acidsmentioning

confidence: 99%

“…The proportion of unsaturated fatty acids in Tetrahymena is temperature-dependent; lower temperatures (20–25°C) favor formation of γ-linolenate. Saprolegnia parasitica converts added stearate mainly to linoleate, arachidonate, and 5,8,11,14,17-eicosapentaenoate [94]; a reversed-phase HPLC chromatogram of the profile of fatty acids is illustrated in Schneider et al [39]. The green alga Chlorella vulgaris has been used to prepare linoleate from stearate [10].…”

Section: Synthesis Of Stereospecifically Labeled Fatty Acidsmentioning

confidence: 99%

Applications of Stereospecifically‐Labeled Fatty Acids in Oxygenase and Desaturase Biochemistry

Brash

Schneider

Hámberg

2011

Lipids

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Oxygenation and desaturation reactions are inherently associated with the abstraction of a hydrogen from the fatty acid substrate. Since the first published application in 1965, stereospecific placement of a labeled hydrogen isotope (deuterium or tritium) at the reacting carbons has proven a highly effective strategy for investigating the chemical mechanisms catalyzed by lipoxygenases, hemoprotein fatty acid dioxygenases including cyclooxygenases, cytochromes P450, and also the desaturases and isomerases. This review presents a synopsis of all published studies through 2010 on the synthesis and use of stereospecifically labeled fatty acids (70 references), and highlights some of the mechanistic insights gained by application of stereospecifically labeled fatty acids.

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Synthesis and Applications of Stereospecifically 3H-Labeled Arachidonic Acids as Mechanistic Probes for Lipoxygenase and Cyclooxygenase Catalysis1

Cited by 14 publications

References 31 publications

On the Relationships of Substrate Orientation, Hydrogen Abstraction, and Product Stereochemistry in Single and Double Dioxygenations by Soybean Lipoxygenase-1 and Its Ala542Gly Mutant

On the Relationships of Substrate Orientation, Hydrogen Abstraction, and Product Stereochemistry in Single and Double Dioxygenations by Soybean Lipoxygenase-1 and Its Ala542Gly Mutant

A 49-kDa Mini-lipoxygenase from Anabaena sp. PCC 7120 Retains Catalytically Complete Functionality

Applications of Stereospecifically‐Labeled Fatty Acids in Oxygenase and Desaturase Biochemistry

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