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Crystallization and preliminary crystallographic analysis of manganese lipoxygenase
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
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.

Place, publisher, year, edition, pages
2014. Vol. 70, 522-525 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-224330DOI: 10.1107/S2053230X14005548ISI: 000333757800027OAI: oai:DiVA.org:uu-224330DiVA: diva2:717094
Available from: 2014-05-14 Created: 2014-05-09 Last updated: 2017-12-05Bibliographically 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.Karkehabadi, Saeid

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