Manganese lipoxygenase of F. oxysporum and the structural basis for biosynthesis of distinct 11-hydroperoxy stereoisomers

• The biosynthesis of jasmonates in plants is initiated by 13S-lipoxygenase (LOX), but details of jasmonate biosynthesis by fungi, including Fusarium oxysporum, are unknown. The genome of F. oxysporum codes for linoleate 13S-LOX (Fox-LOX) and for F. oxysporum manganese LOX (Fo-MnLOX), an uncharacterized homolog of 13R-MnLOX of Gaeumannomyces graminis. We expressed Fo-MnLOX and compared its properties to Cg-MnLOX from Colletotrichum gloeosporioides. Electron paramagnetic resonance and metal analysis showed that Fo-MnLOX contained catalytic Mn. Fo-MnLOX oxidized 18:2n-6 mainly to 11 R-hydroperoxyoctadecadienoic acid (HPODE), 13S-HPODE, and 9(S/R)-HPODE, whereas Cg-MnLOX produced 9S-, 11S-, and 13R-HPODE with high stereoselectivity. The 11-hydroperoxides did not undergo the rapid beta-fragmentation earlier observed with 13R-MnLOX. Oxidation of [11S-H-2] 18:2n-6 by Cg-MnLOX was accompanied by loss of deuterium and a large kinetic isotope effect (>30). The Fo-MnLOX-catalyzed oxidation occurred with retention of the H-2-label. Fo-MnLOX also oxidized 1-lineoyl-2-hydroxy-glycero3- phosphatidylcholine. The predicted active site of all MnLOXs contains Phe except for Ser(348) in this position of Fo-MnLOX. The Ser348Phe mutant of Fo-MnLOX oxidized 18: 2n-6 to the same major products as Cg-MnLOX.Jlr Our results suggest that Fo-MnLOX, with support of Ser(348), binds 18:2n-6 so that the pro R rather than the proShydrogen at C-11 interacts with the metal center, but retains the suprafacial oxygenation mechanism observed in other MnLOXs.

Ämnesord

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinsk bioteknologi -- Medicinsk bioteknologi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Medical Biotechnology -- Medical Biotechnology (hsv//eng)

Nyckelord

Fusarium gloeosporioides

gene expression

oxygenation mechanism

oxylipins

Pichia pastoris

yeast expression

mass spectrometry

Fusarium oxysporum

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