Expression and characterization of manganese lipoxygenase of the rice blast fungus reveals prominent sequential lipoxygenation of α-linolenic acid
2015 (English)In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 583, 87-95 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2015. Vol. 583, 87-95 p.
Fusarium gloeosporioides, gene expression, oxygenation mechanism, oxylipins, Pichia pastoris, yeast expression, mass spectrometry, Fusarium oxysporum
IdentifiersURN: urn:nbn:se:uu:diva-262345DOI: 10.1016/j.abb.2015.07.014ISI: 000361644400011PubMedID: 26264916OAI: oai:DiVA.org:uu-262345DiVA: diva2:853452
FunderSwedish Research Council, 03X-06523Knut and Alice Wallenberg Foundation, KAW 2004.0123Swedish Energy Agency