Expression and characterization of manganese lipoxygenase of the rice blast fungus reveals prominent sequential lipoxygenation of α-linolenic acid

• Published in: Archives of biochemistry and biophysics Vol. 583; pp. 87 - 95
• Main Authors: Wennman, Anneli, Jernerén, Fredrik, Magnuson, Ann, Oliw, Ernst H.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.10.2015
• Subjects: alcohols; alpha-linolenic acid; alpha-Linolenic Acid - metabolism; biosynthesis; blast disease; Chromatography, Liquid; complementary DNA; Electrophoresis, Polyacrylamide Gel; fungi; Fusarium gloeosporioides; Fusarium oxysporum; Gene expression; genome; hydrogen; Komagataella pastoris; linoleate 13S-lipoxygenase; Lipoxygenase - isolation & purification; Lipoxygenase - metabolism; Magnaporthe - enzymology; Magnaporthe oryzae; manganese; mass spectrometry; metabolites; open reading frames; Oryza - microbiology; Oxidation-Reduction; oxygen consumption; Oxygenation mechanism; Oxylipins; pathogens; phospholipases; Pichia pastoris; rice; Surface Plasmon Resonance; Yeast expression; Mo; NP; LT; H(P)OTrE; H(P)ODE; Pichia pastoris; Oxylipins; sLOX-1; 18:3n-3; Yeast expression; 12Z-octadecadienoic acid;11S-H(P)ODE; 9S-H(P)OTrE; 11R-H(P)OTrE; MnLOX; Magnaporthe oryzae; Gene expression; CP; TIC; 18:2n-6; LC-MS; LOX; Oxygenation mechanism; 9S-H(P)ODE; 13R-H(P)OTrE; RP; 13R-H(P)ODE
• ISSN: 0003-9861; 1096-0384; 1096-0384
• DOI: 10.1016/j.abb.2015.07.014

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. •The lipoxygenase (LOX) of Magnaporthe oryzae was partially cloned and expressed.•The LOX was purified to homogeneity and contained catalytic manganese.•18:2n-6 was oxidized to 9S-, 11S-, and 13R-hydroperoxy metabolites.•Metabolites were formed by suprafacial hydrogen abstraction and oxygenation.•18:3n-3 was sequentially oxidized to 9S,16S-dihydroperoxy compound as end product.