title: 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 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.
authors.affiliation: Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University Biomedical Center, SE-75124 Uppsala, Sweden.
authors.forename: Anneli
authors.initials: A
authors.lastname: Wennman
authors.affiliation: Department of Chemistry, Ångström Laboratory, Uppsala University, SE-75120 Uppsala, Sweden.
authors.forename: Ann
authors.initials: A
authors.lastname: Magnuson
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Solna, Sweden.
authors.forename: Mats
authors.initials: M
authors.lastname: Hamberg
authors.affiliation: Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University Biomedical Center, SE-75124 Uppsala, Sweden.
authors.forename: Ernst H
authors.initials: EH
authors.lastname: Oliw
mesh_terms: D055162:Biocatalysis; D005227:Fatty Acids / Q000096:biosynthesis* / Q000737:chemistry*; D005670:Fusarium / Q000201:enzymology*; D008084:Lipoxygenase / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D013237:Stereoisomerism
chemical_list: D005227:Fatty Acids; C112608:manganese lipoxygenase; D008084:Lipoxygenase
keywords: Fusarium gloeosporioides; Fusarium oxysporum; Pichia pastoris; gene expression; mass spectrometry; oxygenation mechanism; oxylipins; yeast expression