title: Secretion of two novel enzymes, manganese 9S-lipoxygenase and epoxy alcohol synthase, by the rice pathogen Magnaporthe salvinii.
abstract: The mycelium of the rice stem pathogen, Magnaporthe salvinii, secreted linoleate 9S-lipoxygenase (9S-LOX) and epoxy alcohol synthase (EAS). The EAS rapidly transformed 9S-hydroperoxy-octadeca-10E,12Z-dienoic acid (9S-HPODE) to threo 10 (11)-epoxy-9S-hydroxy-12Z-octadecenoic acid, but other hydroperoxy FAs were poor substrates. 9S-LOX was expressed in Pichia pastoris. Recombinant 9S-LOX oxidized 18:2n-6 directly to 9S-HPODE, the end product, and also to two intermediates, 11S-hydroperoxy-9Z,12Z-octadecenoic acid (11S-HPODE; ∼5%) and 13R-hydroperoxy-9Z,11E-octadecadienoic acid (13R-HPODE; ∼1%). 11S- and 13R-HPODE were isomerized to 9S-HPODE, probably after oxidation to peroxyl radicals, β-fragmentation, and oxygen insertion at C-9. The 18:3n-3 was oxidized at C-9, C-11, and C-13, and to 9,16-dihydroxy-10E,12,14E-octadecatrienoic acid. 9S-LOX contained catalytic manganese (Mn:protein ∼0.2:1; Mn/Fe, 1:0.05), and its sequence could be aligned with 77% identity to 13R-LOX with catalytic manganese lipoxygenase (13R-MnLOX) of the Take-all fungus. The Leu350Met mutant of 9S-LOX shifted oxidation of 18:2n-6 from C-9 to C-13, and the Phe347Leu, Phe347Val, and Phe347Ala mutants of 13R-MnLOX from C-13 to C-9. In conclusion, M. salvinii secretes 9S-LOX with catalytic manganese along with a specific EAS. Alterations in the Sloane determinant of 9S-LOX and 13R-MnLOX with larger and smaller hydrophobic residues interconverted the regiospecific oxidation of 18:2n-6, presumably by altering the substrate position in relation to oxygen insertion.
authors.affiliation: Department of Pharmaceutical Biosciences, Uppsala University, Biomedical Center, SE-751 24 Uppsala, Sweden.
authors.forename: Anneli
authors.initials: A
authors.lastname: Wennman
authors.affiliation: Department of Pharmaceutical Biosciences, Uppsala University, Biomedical Center, SE-751 24 Uppsala, Sweden.
authors.forename: Ernst H
authors.initials: EH
authors.lastname: Oliw
mesh_terms: D005656:Fungal Proteins / Q000378:metabolism*; D008084:Lipoxygenase / Q000378:metabolism*; D020082:Magnaporthe / Q000201:enzymology*; D012275:Oryza / Q000382:microbiology*; D054883:Oxylipins / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D054883:Oxylipins; C112608:manganese lipoxygenase; D008084:Lipoxygenase