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

Anneli Wennman,Ann Magnuson,Mats Hamberg,Ernst H Oliw

doi:10.1194/jlr.m060178

Anneli Wennman, Ann Magnuson + Show 2 more

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https://doi.org/10.1194/jlr.m060178

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Abstract

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 (FoxLOX) 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 11R-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 β-fragmentation earlier observed with 13R-MnLOX. Oxidation of [11S-(2)H]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 (2)H-label. Fo-MnLOX also oxidized 1-lineoyl-2-hydroxy-glycero-3-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. Our results suggest that Fo-MnLOX, with support of Ser(348), binds 18:2n-6 so that the proR rather than the proS hydrogen at C-11 interacts with the metal center, but retains the suprafacial oxygenation mechanism observed in other MnLOXs.

Highlights

The biosynthesis of jasmonates in plants is initiated by 13S-lipoxygenase (LOX), but details of jasmonate biosynthesis by fungi, including Fusarium oxysporum, are unknown
Fatty acids were dissolved in ethanol and stored in stock solutions (50–100 mM) at Ϫ20°C. 13S-hydroperoxyoctadecadienoic acid (HPODE), 13RHPODE, 13R-hydroperoxyoctadecatrienoic acid (HPOTrE), 9SHPODE, and 9S-HPOTrE were obtained by biosynthesis and purified by reversed phase (RP)-HPLC or by open-column silicic acid chromatography (SepPak/SiO2)
We confirmed that Cg-manganese lipoxygenase (MnLOX) formed 11-HPOTrE, which was transformed by 13R-MnLOX to 13-HPOTrE

Summary

Introduction

The biosynthesis of jasmonates in plants is initiated by 13S-lipoxygenase (LOX), but details of jasmonate biosynthesis by fungi, including Fusarium oxysporum, are unknown. Electron paramagnetic resonance and metal analysis showed that FoMnLOX contained catalytic Mn. Fo-MnLOX oxidized 18:2n-6 mainly to 11R-hydroperoxyoctadecadienoic acid (HPODE), 13S-HPODE, and 9(S/R)-HPODE, whereas Cg-MnLOX produced 9S-, 11S-, and 13R-HPODE with high stereoselectivity. Oxidation of [11S-2H]18:2n-6 by Cg-MnLOX was accompanied by loss of deuterium and a large kinetic isotope effect (>30). The predicted active site of all MnLOXs contains Phe except for Ser348 in this position of FoMnLOX. The Ser348Phe mutant of Fo-MnLOX oxidized 18:2n-6 to the same major products as Cg-MnLOX. Our results suggest that Fo-MnLOX, with support of Ser348, binds 18:2n-6 so that the proR rather than the proS hydrogen at C-11 interacts with the metal center, but retains the suprafacial oxygenation mechanism observed in other MnLOXs.— Wennman, A., A. Manganese lipoxygenase of F. oxysporum and the structural basis for biosynthesis of distinct 11-hydroperoxy stereoisomers.

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