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Secretion of two novel enzymes, manganese 9S-Lipoxygenase and epoxy alcohol synthase, by the rice pathogen Magnaporthe salvinii
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (biokemisk farmakologi)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (biokemisk farmakologi)
2013 (English)In: Journal of Lipid Research, ISSN 0022-2275, E-ISSN 1539-7262, Vol. 54, no 3, 762-775 p.Article in journal (Refereed) Published
Abstract [en]

The mycelium of the rice stem pathogen, Magnaporthe salvinii, secreted linoleate 9S-lipoxygenase (9S-LOX) and epoxy alcohol synthase (EAS). The EAS rapidly transformed 9S-hydroperoxy-octadeca-10E,12Z-dienoic acid (9S-HPODE) to threo 10(11)-epoxy-9S-hydroxy-12Z-octadecenoic acid, but other hydroperoxy fatty acids were poor substrates. 9S-LOX was expressed in Pichia pastoris. Recombinant 9S-LOX oxidized 18:2n-6 directly to 9S-HPODE, the end product, and also to two intermediates, 11S-hydroperoxy-9Z,12Z-octadecenoic acid (11S-HPODE; ~5%) and 13R-hydroperoxy-9Z,11E-octadecenoic acid (13R-HPODE; ~1%). 11S- and 13R-HPODE were isomerized to 9S-HPODE, likely after oxidation to peroxyl radicals, β-fragmentation, and oxygen insertion at C-9. 18:3n-3 was oxidized at C-9, C-11, and C-13, and to 9,16-dihydroxy-10E,12,14E-octadecatrienoic acid. 9S-LOX contained catalytic manganese (Mn:protein ≥ 0.2:1; Mn/Fe, 1:0.05), and its sequence could be aligned with 77% identity to 13R-LOX with catalytic manganese (13R-MnLOX) of the Take-all fungus. The Leu350Met mutant of 9S-LOX shifted oxidation of 18:2n-6 from C-9 to C-13, and the Phe347Leu, Phe347Val, and Phe347Ala mutants of 13R-MnLOX from C-13 to C-9. In conclusion, M. salvinii secretes 9S-LOX with catalytic manganese along with a specific EAS. Alterations in the Sloane determinant of 9S-LOX and 13R-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.

Place, publisher, year, edition, pages
2013. Vol. 54, no 3, 762-775 p.
National Category
Medical and Health Sciences
Research subject
Biochemical Pharmacology
Identifiers
URN: urn:nbn:se:uu:diva-188356DOI: 10.1194/jlr.M033787ISI: 000314877000019PubMedID: 23233731OAI: oai:DiVA.org:uu-188356DiVA: diva2:577699
Funder
Swedish Research Council, 06523Knut and Alice Wallenberg Foundation, 2004.0123
Available from: 2012-12-16 Created: 2012-12-16 Last updated: 2017-12-06Bibliographically approved
In thesis
1. The structural basis for the catalytic specificity of manganese lipoxygenases: 3D structure analysis of the lipoxygenase of Magnaporthe oryzae
Open this publication in new window or tab >>The structural basis for the catalytic specificity of manganese lipoxygenases: 3D structure analysis of the lipoxygenase of Magnaporthe oryzae
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Lipoxygenases (LOX) catalyze regio- and stereospecific oxygenation of polyunsaturated fatty acids to hydroperoxides. These hydroperoxides are further metabolized to leukotrienes and lipoxins in mammals, and are involved in asthma and inflammation. LOX of animals and plants contain iron as catalytic metal (FeLOX). Filamentous fungi use both FeLOX, and manganese containing LOX (MnLOX). The role of LOX in fungi is still not known. This thesis focuses on expression of novel MnLOX, analyses of their reaction mechanism and products by HPLC-MS/MS, protein crystallization and analysis of the first MnLOX structure.  

MnLOX from G. graminis, M. salvinii, M. oryzae, F. oxysporum and C. gloeosporioides were expressed in Pichia pastoris, purified and characterized by HPLC-MS/MS. All MnLOX catalyzes suprafacial hydrogen abstraction and oxygen insertion. Replacement of one Ile to Phe in the active site of MnLOX of G. graminis could switch the mechanism from suprafacial to mainly antarafacial. MnLOX of F. oxysporum was interesting since it catalyzes oxygenation of linoleic acid to 11R- instead of the more common 11S-hydroperoxides. This feature could be attributed to a single Ser/Phe exchange in the active site.  

We found that Gg-MnLOX utilizes hydrogen tunneling in the reaction mechanism, but was slightly more temperature dependent than soybean FeLOX. It is an intriguing question why some fungal LOX use manganese and not iron as catalytic metal and whether the large redox potential of Mn2+/Mn3+ (1.5 V) can be tuned close to that of Fe2+/Fe3+ (0.77 V) for redox cycling and catalysis.

We present crystallization conditions for two MnLOX, and the 2.07 Å crystal structure of MnLOX from M. oryzae, solved using sulfur and manganese single anomalous dispersion (SAD). The structure reveals a similar metal coordinating sphere as FeLOX but the metal ligand Asn473 was positioned on a short loop instead of a helix and formed interactions with a conserved Gln. This feature could be essential for the use of manganese as catalytic metal in LOX. We found three Phe residues that likely facilitate the suprafacial hydrogen abstraction and oxygen insertion for MnLOX.

These findings provide new insight into the unique reaction mechanism of MnLOX.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 62 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 204
Keyword
oxylipin, lipoxygenase, crystal structure, crystallography, HPLC, mass spectrometry, yeast, site-directed mutagenesis, fungi
National Category
Structural Biology Biochemistry and Molecular Biology
Research subject
Biochemical Pharmacology
Identifiers
urn:nbn:se:uu:diva-262762 (URN)978-91-554-9347-9 (ISBN)
Public defence
2015-11-06, A1:107a, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2015-10-16 Created: 2015-09-19 Last updated: 2015-10-27

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Wennman, AnneliOliw, Ernst H

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