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标题：Manganese lipoxygenase of F. oxysporum and the structural basis for biosynthesis of distinct 11-hydroperoxy stereoisomers
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作者：Anneli Wennman ; Ann Magnuson ; Mats Hamberg 等
期刊名称：JLR Papers In Press
印刷版ISSN：0022-2275
电子版ISSN：1539-7262
出版年度：2015
卷号：56
期号：8
页码：1606-1615
DOI：10.1194/jlr.M060178
语种：English
出版社：American Society for Biochemistry and Molecular Biology
摘要：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 (FoxLOX) 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 -²H]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 ²H-label. Fo-MnLOX also oxidized 1-lineoyl-2-hydroxy-glycero-3-phosphatidylcholine. The predicted active site of all MnLOXs contains Phe except for Ser³⁴⁸ in this position of Fo-MnLOX. The Ser348Phe 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 Ser³⁴⁸, 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.
关键词：Fusarium gloeosporioides ; gene expression ; oxygenation mechanism ; oxylipins ; Pichia pastoris ; yeast expression ; mass spectrometry ; Fusarium oxysporum