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The structural basis for the catalytic specificity of manganese lipoxygenases: 3D structure analysis of the lipoxygenase of Magnaporthe oryzae
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Biokemisk Farmakologi)
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 [en]
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: urn:nbn:se:uu:diva-262762ISBN: 978-91-554-9347-9 (print)OAI: oai:DiVA.org:uu-262762DiVA: diva2:855212
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
List of papers
1. Catalytic convergence of manganese and iron lipoxygenases by replacement of a single amino Acid
Open this publication in new window or tab >>Catalytic convergence of manganese and iron lipoxygenases by replacement of a single amino Acid
2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 38, 31757-31765 p.Article in journal (Refereed) Published
Abstract [en]

Lipoxygenases (LOXs) contain a hydrophobic substrate channel with the conserved Gly/Ala determinant of regio- and stereospecificity and a conserved Leu residue near the catalytic non-heme iron. Our goal was to study the importance of this region (Gly(332), Leu(336), and Phe(337)) of a lipoxygenase with catalytic manganese (13R-MnLOX). Recombinant 13R-MnLOX oxidizes 18:2n-6 and 18:3n-3 to 13R-, 11(S or R)-, and 9S-hydroperoxy metabolites (∼80-85, 15-20, and 2-3%, respectively) by suprafacial hydrogen abstraction and oxygenation. Replacement of Phe(337) with Ile changed the stereochemistry of the 13-hydroperoxy metabolites of 18:2n-6 and 18:3n-3 (from ∼100% R to 69-74% S) with little effect on regiospecificity. The abstraction of the pro-S hydrogen of 18:2n-6 was retained, suggesting antarafacial hydrogen abstraction and oxygenation. Replacement of Leu(336) with smaller hydrophobic residues (Val, Ala, and Gly) shifted the oxygenation from C-13 toward C-9 with formation of 9S- and 9R-hydroperoxy metabolites of 18:2n-6 and 18:3n-3. Replacement of Gly(332) and Leu(336) with larger hydrophobic residues (G332A and L336F) selectively augmented dehydration of 13R-hydroperoxyoctadeca-9Z,11E,15Z-trienoic acid and increased the oxidation at C-13 of 18:1n-6. We conclude that hydrophobic replacements of Leu(336) can modify the hydroperoxide configurations at C-9 with little effect on the R configuration at C-13 of the 18:2n-6 and 18:3n-3 metabolites. Replacement of Phe(337) with Ile changed the stereospecific oxidation of 18:2n-6 and 18:3n-3 with formation of 13S-hydroperoxides by hydrogen abstraction and oxygenation in analogy with soybean LOX-1.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemical Pharmacology
Identifiers
urn:nbn:se:uu:diva-183487 (URN)10.1074/jbc.M112.364331 (DOI)000309059400017 ()22822060 (PubMedID)
Funder
Knut and Alice Wallenberg Foundation, KAW 2004.0123
Available from: 2012-10-26 Created: 2012-10-26 Last updated: 2017-12-07Bibliographically approved
2. Secretion of two novel enzymes, manganese 9S-Lipoxygenase and epoxy alcohol synthase, by the rice pathogen Magnaporthe salvinii
Open this publication in new window or tab >>Secretion of two novel enzymes, manganese 9S-Lipoxygenase and epoxy alcohol synthase, by the rice pathogen Magnaporthe salvinii
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.

National Category
Medical and Health Sciences
Research subject
Biochemical Pharmacology
Identifiers
urn:nbn:se:uu:diva-188356 (URN)10.1194/jlr.M033787 (DOI)000314877000019 ()23233731 (PubMedID)
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
3. Kinetic investigation of the rate-limiting step of manganese- and iron-lipoxygenases
Open this publication in new window or tab >>Kinetic investigation of the rate-limiting step of manganese- and iron-lipoxygenases
2014 (English)In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 555, 9-15 p.Article in journal (Refereed) Published
Abstract [en]

Lipoxygenases (LOX) oxidize polyunsaturated fatty acids to hydroperoxides, which are generated by proton coupled electron transfer to the metal center with (FeOH-)-O-III or (MnOH-)-O-III. Hydrogen abstraction by (FeOH-)-O-III of soybean LOX-1 (sLOX-1) is associated with a large deuterium kinetic isotope effect (D-KIE). Our goal was to compare the D-KIE and other kinetic parameters at different temperatures of sLOX-1 with 13R-LOX with catalytic manganese (13R-MnLOX). The reaction rate and the D-KIE of sLOX-1 with unlabeled and [11-H-2(2)]18:2n-6 were almost temperature independent with an apparent D-KIE of similar to 56 at 30 degrees C, which is in agreement with previous studies. In contrast, the reaction rate of 13R-MnLOX increased 7-fold with temperature (8-50 degrees C), and the apparent D-KIE decreased linearly from similar to 38 at 8 degrees C to similar to 20 at 50 degrees C. The kinetic lag phase of 13R-MnLOX was consistently extended at low temperatures. The Phe337Ile mutant of 13R-MnLOX, which catalyzes antarafacial hydrogen abstraction and oxygenation in analogy with sLOX-1, retained the large D-KIE and its temperature-dependent reaction rate. The kinetic differences between 13R-MnLOX and sLOX-1 may be due to protein dynamics, hydrogen donor-acceptor distances, and to the metal ligands, which may not equalize the 0.7 V-gap between the redox potentials of the free metals. 

Keyword
Arrhenius plot, Deuterium kinetic isotope effect, Hydrogen abstraction, Site-directed mutagenesis, Hydrogen tunneling
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-229705 (URN)10.1016/j.abb.2014.05.014 (DOI)000338824100002 ()24857825 (PubMedID)
Available from: 2014-08-18 Created: 2014-08-12 Last updated: 2017-12-05Bibliographically approved
4. Expression and characterization of manganese lipoxygenase of the rice blast fungus reveals prominent sequential lipoxygenation of α-linolenic acid
Open this publication in new window or tab >>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
Abstract [en]

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.

Keyword
Fusarium gloeosporioides, gene expression, oxygenation mechanism, oxylipins, Pichia pastoris, yeast expression, mass spectrometry, Fusarium oxysporum
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-262345 (URN)10.1016/j.abb.2015.07.014 (DOI)000361644400011 ()26264916 (PubMedID)
Funder
Swedish Research Council, 03X-06523Knut and Alice Wallenberg Foundation, KAW 2004.0123Swedish Energy Agency
Available from: 2015-09-14 Created: 2015-09-14 Last updated: 2017-12-04Bibliographically approved
5. Manganese lipoxygenase of F. oxysporum and the structural basis for biosynthesis of distinct 11-hydroperoxy stereoisomers
Open this publication in new window or tab >>Manganese lipoxygenase of F. oxysporum and the structural basis for biosynthesis of distinct 11-hydroperoxy stereoisomers
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.

Keyword
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:nbn:se:uu:diva-260821 (URN)10.1194/jlr.M060178 (DOI)000358666000022 ()26113537 (PubMedID)
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
6. Crystallization and preliminary crystallographic analysis of manganese lipoxygenase
Open this publication in new window or tab >>Crystallization and preliminary crystallographic analysis of manganese lipoxygenase
2014 (English)In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 70, 522-525 p.Article in journal (Refereed) Published
Abstract [en]

Lipoxygenases constitute a family of nonhaem metal enzymes with catalytic iron or, occasionally, catalytic manganese. Lipoxygenases oxidize polyunsaturated fatty acids with position specificity and stereospecificity to hydroperoxides, which contribute to inflammation and the development of cancer. Little is known about the structural differences between lipoxygenases with Fe or Mn and the metal-selection mechanism. A Pichia pastoris expression system was used for the production of the manganese lipoxygenase of the take-all fungus of wheat, Gaeumannomyces graminis. The active enzyme was treated with alpha-mannosidase, purified to apparent homogeneity and subjected to crystal screening and X-ray diffraction. The crystals diffracted to 2.6 angstrom resolution and belonged to space group C2, with unit-cell parameters a = 226.6, b = 50.6, c = 177.92 angstrom, beta = 91.70 degrees.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-224330 (URN)10.1107/S2053230X14005548 (DOI)000333757800027 ()
Available from: 2014-05-14 Created: 2014-05-09 Last updated: 2017-12-05Bibliographically approved
7. Crystal structure of manganese lipoxygenase of the rice blast fungus Magnaporthe oryzae
Open this publication in new window or tab >>Crystal structure of manganese lipoxygenase of the rice blast fungus Magnaporthe oryzae
(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-262761 (URN)
Available from: 2015-09-19 Created: 2015-09-19 Last updated: 2015-10-27

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