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

Abstract

The biosynthesis of jasmonates in plants is initiated by 13 S -lipoxygenase (LOX), but details of jasmonate biosynthesis by fungi, including Fusarium oxysporum , are unknown. The genome of F. oxysporum codes for linoleate 13 S -LOX (Fox- LOX) and for F. oxysporum manganese LOX (Fo-MnLOX), an uncharacterized homolog of 13 R -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 18:2 n -6 mainly to 11 R- hydroperoxyoctadecadienoic acid (HPODE), 13 S -HPODE, and 9( S/R )-HPODE, whereas Cg-MnLOX produced 9 S -, 11 S -, and 13 R -HPODE with high stereoselectivity. The 11-hydroperoxides did not undergo the rapid β - fragmentation earlier observed with 13 R -MnLOX. Oxidation of [11 S - 2 H]18:2 n -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 Mn- LOXs contains Phe except for Ser 348 in this position of Fo- MnLOX. The Ser48Phe mutant of Fo-MnLOX oxidized 18:2 n -6 to the same major products as Cg-MnLOX. Our results suggest that Fo-MnLOX, with support of Ser 348 , binds 18:2 n -6 so that the pro R rather than the pro S hydrogen at C-11 interacts with the metal center, but retains the suprafacial oxygenation mechanism observed in other MnLOXs.