On the Substrate Binding of Linoleate 9-Lipoxygenases

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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Lipoxygenases (LOX; linoleate:oxygen oxidoreductase EC 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
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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