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Kinetic investigation of the rate-limiting step of manganese- and iron-lipoxygenases
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: 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. 

Place, publisher, year, edition, pages
2014. Vol. 555, 9-15 p.
Keyword [en]
Arrhenius plot, Deuterium kinetic isotope effect, Hydrogen abstraction, Site-directed mutagenesis, Hydrogen tunneling
National Category
Pharmaceutical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-229705DOI: 10.1016/j.abb.2014.05.014ISI: 000338824100002PubMedID: 24857825OAI: oai:DiVA.org:uu-229705DiVA: diva2:738549
Available from: 2014-08-18 Created: 2014-08-12 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, AnneliKarkehabadi, SaeidOliw, Ernst H.

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