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

• 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 due to ineffective enzyme activation (MnIIMnIII) by produced hydroperoxides. Our aim was to determine whether unsaturated C16-C22 fatty acids were oxidized by MnIII-LO. MnIII-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¬glycero¬phosphatidyl¬¬choline 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 mainly oxidized 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). MnIII-LO oxidized 20:4n-6 to 15R-HPETE (~60%) and 13-HPETE (~37%) and converted 13-HPETE to 15R-HPETE. MnIII-LO G316A mainly oxygenated 16:3n-3 at positions n-7 and n-6, 19:3n-3 at n-10, n-8, and n-6, 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.

Ämnesord

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper -- Farmaceutiska vetenskaper (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine -- Pharmaceutical Sciences (hsv//eng)

Nyckelord

nonconjugated peroxyls

fatty acid oxygenation

hydroperoxide isomerase

mass spectrometry

metalloenzymes

dilinoleoyl-glycerophosphatidylcholine

thermostability

PHARMACY

FARMACI

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