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Manganese lipoxygenase of F. oxysporum and the structural basis for biosynthesis of distinct 11-hydroperoxy stereoisomers
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
2015 (English)In: Journal of Lipid Research, ISSN 0022-2275, E-ISSN 1539-7262, Vol. 56, no 8, 1606-1615 p.Article in journal (Refereed) Published
Abstract [en]

The biosynthesis of jasmonates in plants is initiated by 13S-lipoxygenase (LOX), but details of jasmonate biosynthesis by fungi, including Fusarium oxysporum, are unknown. The genome of F. oxysporum codes for linoleate 13S-LOX (Fox-LOX) and for F. oxysporum manganese LOX (Fo-MnLOX), an uncharacterized homolog of 13R-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 18:2n-6 mainly to 11 R-hydroperoxyoctadecadienoic acid (HPODE), 13S-HPODE, and 9(S/R)-HPODE, whereas Cg-MnLOX produced 9S-, 11S-, and 13R-HPODE with high stereoselectivity. The 11-hydroperoxides did not undergo the rapid beta-fragmentation earlier observed with 13R-MnLOX. Oxidation of [11S-H-2] 18:2n-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 H-2-label. Fo-MnLOX also oxidized 1-lineoyl-2-hydroxy-glycero3- phosphatidylcholine. The predicted active site of all MnLOXs contains Phe except for Ser(348) in this position of Fo-MnLOX. The Ser348Phe mutant of Fo-MnLOX oxidized 18: 2n-6 to the same major products as Cg-MnLOX.Jlr Our results suggest that Fo-MnLOX, with support of Ser(348), binds 18:2n-6 so that the pro R rather than the proShydrogen at C-11 interacts with the metal center, but retains the suprafacial oxygenation mechanism observed in other MnLOXs.

Place, publisher, year, edition, pages
2015. Vol. 56, no 8, 1606-1615 p.
Keyword [en]
Fusarium gloeosporioides, gene expression, oxygenation mechanism, oxylipins, Pichia pastoris, yeast expression, mass spectrometry, Fusarium oxysporum
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:uu:diva-260821DOI: 10.1194/jlr.M060178ISI: 000358666000022PubMedID: 26113537OAI: oai:DiVA.org:uu-260821DiVA: diva2:849253
Funder
Swedish Research Council, 03X-06523Knut and Alice Wallenberg Foundation, KAW 2004.0123
Available from: 2015-08-27 Created: 2015-08-25 Last updated: 2017-12-04Bibliographically 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, AnneliMagnuson, AnnOliw, Ernst H.

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