Andreou, A Z and Hornung, E and Kunze, S and et al, .
(2009)
On the Substrate Binding of Linoleate 9-Lipoxygenases.
Lipids, 44.
pp. 207-215.
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Abstract
Lipoxygenases (LOX; linoleate:oxygen oxidoreductase
EC 1.13.11.12) consist of a class of enzymes that
catalyze the regio- and stereo specific dioxygenation of
polyunsaturated fatty acids. Here we characterize two proteins
that belong to the less studied class of 9-LOXs,
Solanum tuberosum StLOX1 and Arabidopsis thaliana At-
LOX1. The proteins were recombinantly expressed in E. coli
and the product specificity of the enzymes was tested against
different fatty acid substrates. Both enzymes showed high
specificity against all tested C18 fatty acids and produced
(9S)-hydroperoxides. However, incubation of the C20 fatty
acid arachidonic acid with AtLOX1 gave a mixture of
racemic hydroperoxides. On the other hand, with StLOX1
we observed the formation of a mixture of products among
which the (5S)-hydroperoxy eicosatetraenoic acid (5SH(
P)ETE) was the most abundant. Esterified fatty acids were
no substrates. We used site directed mutagenesis to modify a
conserved valine residue in the active site of StLOX1 and
examine the importance of space within the active site,
which has been shown to play a role in determining the
positional specificity. The Val576Phe mutant still catalyzed the formation of (9S)-hydroperoxides with C18 fatty acids,while it exhibited altered specificity against arachidonic acid and produced mainly (11S)-H(P)ETE. These data confirm the model that in case of linoleate 9-LOX binding of the substrate takes place with the carboxyl-group first.
| Item Type: |
Article
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| Additional Information: |
The authors are grateful to Dr. Cornelia Go¨bel,
Go¨ttingen, for her expert technical support. A.A. is supported by the
International master/Ph.D. program Molecular Biology and the Max
Planck Research School Molecular Biology (Go¨ttingen). The project
is funded by IRTG 1422 Metal Sites in Biomolecules: Structures,
Regulation and Mechanisms. We thank the ABRC for providing with
the cDNA encoding AtLOX1.
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which permits
any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited. |
| Uncontrolled Keywords: |
Lipid peroxidation; Oxylipin formation ; Solanum tuberosum |
| Author Affiliation: |
Department of Plant Biochemistry,
Georg-August-University of Go¨ttingen,
Albrecht-von-Haller-Institute of Plant Sciences,
Justus-von-Liebig-Weg 11, 37085 Go¨ttingen, Germany |
| Subjects: |
Crop Improvement > Genetics/Genomics |
| Divisions: |
General |
| Depositing User: |
Mr Siva Shankar
|
| Date Deposited: |
11 Apr 2012 08:06 |
| Last Modified: |
11 Apr 2012 08:07 |
| Official URL: |
http://dx.doi.org/10.1007/s11745-008-3264-4 |
| URI: |
http://eprints.icrisat.ac.in/id/eprint/4638 |
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