On the substrate binding of linoleate 9-lipoxygenases.
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 AtLOX1. 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 (5S-H(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.
Andreou
Alexandra-Zoi
AZ
Department of Plant Biochemistry, Georg-August-University of Göttingen, Albrecht-von-Haller-Institute of Plant Sciences, Göttingen, Germany.
Hornung
Ellen
E
Kunze
Susan
S
Rosahl
Sabine
S
Feussner
Ivo
I
D017360:Arabidopsis / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D008084:Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D011198:Solanum tuberosum / Q000201:enzymology; D013379:Substrate Specificity
D008084:Lipoxygenase