On the Substrate Binding of Linoleate 9‐Lipoxygenases

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 (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.

An Iron 13S-Lipoxygenase with an α-Linolenic Acid Specific Hydroperoxidase Activity from Fusarium oxysporum

“…These three analogous positions are occupied by space filling amino acids in FoxLOX: Trp-400 (indicated as Bo 1 in Figure 2), Phe-466 (indicated as Sl in Figure 2), and Leu-657 (indicated as Bo 2 in Figure 2). (ii) In plants, on the other hand positional specificity has been suggested to be mainly determined by the orientation of the fatty acid within the active site [9], [37]. According to this model, 13-lipoxygenation occurs when the substrate enters the active pocket with its methyl end first.…”

“…In analogy to the triad concept derived from mammalian LOXes, the penetration depth of this binding mode is determined by the same space filling residues described by Sloane and Borngräber [42], [43]. Then again, an inverse substrate orientation has been proposed for 9-lipoxygenation, according to which the substrate enters the active site with its carboxy-terminus first [37]. The negative charge of the carboxy terminus is then stabilized by a positively charged Arg residue, which in 13-LOXes is masked by bulky amino acids in the active site [44].…”

“…Separation of the positional hydroxyl isomers was performed using straight phase (SP)-HPLC. A Zorbax SIL column (2.1×150 mm, 5 µm particle size; Agilent) and a solvent system consisting of n-hexane/2-propanol/trifluoroacetic acid (100/1/0.02, v/v/v) was used for this analysis as described in [37]. The separation of stereo isomers via chiral phase (CP)-HPLC was performed on a Chiracel OD-H column (2.1×150 mm, 5 µm particle size; Baker) with a solvent system consisting of n-hexane/isopropanol/trifluoroacetic acid (100/5/0.1, v/v/v).…”

A multifunctional lipoxygenase from Pyropia haitanensis— The cloned and functioned complex eukaryotic algae oxylipin pathway enzyme

“…The biosynthesis of oxylipins involves lipoxygenases (LOXs) and several enzyme members of the cytochrome P450 family, designated CYP74, which includes allene oxide synthase (AOS), hydroperoxide lyase (HPL), and divinyl ether synthase [6]. In most plant species, LOXs are encoded by gene families, comprising a number of isozymes, which differ in the substrate oxygenation position and substrate specificity [7]. However, in the lower organism, the oxylipin pathway is strikingly simple and its multifunctional properties have been increasingly reported [8][9][10][11][12].…”

A Bisallylic Mini-lipoxygenase from Cyanobacterium Cyanothece sp. That Has an Iron as Cofactor

“…The same mode of binding has been described for 13S-LOXs (53, 54), 9R-LOXs (23,44), and Mn-LOX (51). On the other hand, a number of linoleate 9S-LOX and arachidonate 8S-and 5S-LOX cannot metabolize such substrates (55)(56)(57) and have been thought to bind fatty acids with the carboxylic end buried in the active site pocket. The production of the bisallylic product from esterified substrate has also been reported for Mn-LOX (51).…”