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标题：Secretion of two novel enzymes, manganese 9S-lipoxygenase and epoxy alcohol synthase, by the rice pathogen Magnaporthe salvinii
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作者：Anneli Wennman ; Ernst H. Oliw
期刊名称：JLR Papers In Press
印刷版ISSN：0022-2275
电子版ISSN：1539-7262
出版年度：2013
卷号：54
期号：3
页码：762-775
DOI：10.1194/jlr.M033787
语种：English
出版社：American Society for Biochemistry and Molecular Biology
摘要：The mycelium of the rice stem pathogen, Magnaporthe salvinii , secreted linoleate 9 S -lipoxygenase (9 S -LOX) and epoxy alcohol synthase (EAS). The EAS rapidly transformed 9 S- hydroperoxy-octadeca-10 E ,12 Z -dienoic acid (9 S- HPODE) to threo 10 (11)-epoxy-9 S -hydroxy-12 Z -octadecenoic acid, but other hydroperoxy FAs were poor substrates. 9 S -LOX was expressed in Pichia pastoris. Recombinant 9 S -LOX oxidized 18:2n-6 directly to 9 S- HPODE, the end product, and also to two intermediates, 11 S- hydroperoxy-9 Z ,12 Z -octadecenoic acid (11 S- HPODE; ∼5%) and 13 R- hydroperoxy-9 Z ,11 E -octadecadienoic acid (13 R- HPODE; ∼1%). 11 S- and 13 R- HPODE were isomerized to 9 S- 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-10 E ,12,14 E- octadecatrienoic acid. 9 S -LOX contained catalytic manganese (Mn:protein ∼0.2:1; Mn/Fe, 1:0.05), and its sequence could be aligned with 77% identity to 13 R -LOX with catalytic manganese lipoxygenase (13 R- MnLOX) of the Take-all fungus. The Leu350Met mutant of 9 S- LOX shifted oxidation of 18:2n-6 from C-9 to C-13, and the Phe347Leu, Phe347Val, and Phe347Ala mutants of 13 R- MnLOX from C-13 to C-9. In conclusion, M. salvinii secretes 9 S -LOX with catalytic manganese along with a specific EAS. Alterations in the Sloane determinant of 9 S -LOX and 13 R -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.
关键词：lipoxygenase pathway ; metalloenzyme ; mutagenesis site specific ; hydroperoxide rearrangement ; oxylipin biosynthesis ; regiospecificity