Expression and characterization of manganese lipoxygenase of the rice blast fungus reveals prominent sequential lipoxygenation of α-linolenic acid.
Magnaporthe oryzae causes rice blast disease and has become a model organism of fungal infections. M. oryzae can oxygenate fatty acids by 7,8-linoleate diol synthase, 10R-dioxygenase-epoxy alcohol synthase, and by a putative manganese lipoxygenase (Mo-MnLOX). The latter two are transcribed during infection. The open reading frame of Mo-MnLOX was deduced from genome and cDNA analysis. Recombinant Mo-MnLOX was expressed in Pichia pastoris and purified to homogeneity. The enzyme contained protein-bound Mn and oxidized 18:2n-6 and 18:3n-3 to 9S-, 11-, and 13R-hydroperoxy metabolites by suprafacial hydrogen abstraction and oxygenation. The 11-hydroperoxides were subject to β-fragmentation with formation of 9S- and 13R-hydroperoxy fatty acids. Oxygen consumption indicated apparent kcat values of 2.8 s(-1) (18:2n-6) and 3.9 s(-1) (18:3n-3), and UV analysis yielded apparent Km values of 8 and 12 μM, respectively, for biosynthesis of cis-trans conjugated metabolites. 9S-Hydroperoxy-10E,12Z,15Z-octadecatrienoic acid was rapidly further oxidized to a triene, 9S,16S-dihydroperoxy-10E,12Z,14E-octadecatrienoic acid. In conclusion, we have expressed, purified and characterized a new MnLOX from M. oryzae. The pathogen likely secretes Mo-MnLOX and phospholipases to generate oxylipins and to oxidize lipid membranes of rice cells and the cuticle.
Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University, Biomedical Center, SE-751 24 Uppsala, Sweden. Electronic address: anneli.wennman@farmbio.uu.se.
Anneli
A
Wennman
Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University, Biomedical Center, SE-751 24 Uppsala, Sweden.
Fredrik
F
Jernerén
Department of Chemistry-Ångström Laboratory, Uppsala University, SE-75120 Uppsala, Sweden.
Ann
A
Magnuson
Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University, Biomedical Center, SE-751 24 Uppsala, Sweden.
Ernst H
EH
Oliw
D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008084:Lipoxygenase / Q000302:isolation & purification / Q000378:metabolism*; D020082:Magnaporthe / Q000201:enzymology*; D012275:Oryza / Q000382:microbiology*; D010084:Oxidation-Reduction; D020349:Surface Plasmon Resonance; D017962:alpha-Linolenic Acid / Q000378:metabolism*
D017962:alpha-Linolenic Acid; C112608:manganese lipoxygenase; D008084:Lipoxygenase
Gene expression; Magnaporthe oryzae; Oxygenation mechanism; Oxylipins; Pichia pastoris; Yeast expression