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Papers In Press, published online ahead of print April 1, 2007
J. Lipid Res., doi:10.1194/jlr.M600505-JLR200

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Journal of Lipid Research, Vol. 48, 890-903, April 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

On the singular, dual, and multiple positional specificity of manganese lipoxygenase and its G316A mutant

Mirela Cristea and Ernst H. Oliw¹

Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University Biomedical Center, SE-751 24 Uppsala, Sweden

Published, JLR Papers in Press, January 26, 2007.

¹ To whom correspondence should be addressed. e-mail: ernst.oliw{at}farmbio.uu.se

Abstract manganese lipoxygenase (Mn-LO) oxygenates 18:3n-3 and 18:2n-6 to bis-allylic 11S-hydroperoxy fatty acids, which are converted to 13R-hydroperoxy fatty acids. Other unsaturated C₁₆-C₂₂ fatty acids, except 17:3n-3, are poor substrates, possibly because of ineffective enzyme activation (Mn^IIMn^III) by the produced hydroperoxides. Our aim was to determine whether unsaturated C₁₆-C₂₂ fatty acids were oxidized by Mn^III-LO. Mn^III-LO oxidized C₁₆, C₁₉, C₂₀, and C₂₂ n-3 and n-6 fatty acids. The carbon chain length influenced the position of hydrogen abstraction (n-8, n-5) and oxygen insertion at the terminal or the penultimate 1Z,4Z-pentadienes. Dilinoleoyl-glycerophosphatidylcholine was oxidized by Mn-LO, in agreement with a "tail-first" model. 16:3n-3 was oxidized at the bis-allylic n-5 carbon and at positions n-3, n-7, and n-6. Long fatty acids, 19:3n-3, 20:3n-3, 20:4n-6, 22:5n-3, and 22:5n-6, were oxidized mainly at the n-6 and the bis-allylic n-8 positions (in ratios of 3:2). The bis-allylic hydroperoxides accumulated with one exception, 13-hydroperoxyeicosatetraenoic acid (13-HPETE). Mn^III-LO oxidized 20:4n-6 to 15R-HPETE (60%) and 13-HPETE (37%) and converted 13-HPETE to 15R-HPETE. Mn^III-LO G316A oxygenated mainly 16:3n-3 at positions n-7 and n-6, 19:3n-3 at n-10, n-8, and n-6, and 20:3n-3 at n-10 and n-8. We conclude that Mn-LO likely binds fatty acids tail-first and oxygenates many C₁₆, C₁₈, C₂₀, and C₂₂ fatty acids to significant amounts of bis-allylic hydroperoxides.

Supplementary key words nonconjugated peroxyls • fatty acid oxygenation • hydroperoxide isomerase • mass spectrometry • metalloenzymes • dilinoleoyl-glycerophosphatidylcholine • thermostability

Abbreviations: CP, chiral phase; GPC, glycerophosphatidylcholine; HETE, hydroxyeicosatetraenoic acid; HHTrE, hydroxyhexadecatrienoic acid; HNTrE, hydroxynonadecatrienoic acid; HPETE, hydroperoxyeicosatetraenoic acid; HPHTrE, hydroperoxyhexadecatrienoic acid; HPNTrE, hydroperoxynonadecatrienoic acid; HPODE, hydroperoxyoctadecadienoic acid; HPOTrE, hydroperoxyoctadecatrienoic acid; KETE, ketoeicosatetraenoic acid; KETrE, ketoeicosatrienoic acid; KHTrE, ketohexadecatrienoic acid; KNTrE, ketononadecatrienoic acid; LOX, lipoxygenase; Mn-LO, manganese lipoxygenase; NP, normal-phase; RP, reverse-phase; sLO, soybean lipoxygenase; TPP, triphenylphosphine; UV, ultraviolet

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