Abstract
In eukaryotes, oxidized PUFAs, so-called oxylipins, are vital signaling molecules. The first step in their biosynthesis may be catalyzed by a lipoxygenase (LOX), which forms hydroperoxides by introducing dioxygen into PUFAs. Here we characterized CspLOX1, a phylogenetically distant LOX family member from Cyanothece sp. PCC 8801 and determined its crystal structure. In addition to the classical two domains found in plant, animal, and coral LOXs, we identified an N-terminal helical extension, reminiscent of the long α-helical insertion in Pseudomonas aeruginosa LOX. In liposome flotation studies, this helical extension, rather than the β-barrel domain, was crucial for a membrane binding function. Additionally, CspLOX1 could oxygenate 1,2-diarachidonyl-sn-glycero-3-phosphocholine, suggesting that the enzyme may act directly on membranes and that fatty acids bind to the active site in a tail-first orientation. This binding mode is further supported by the fact that CspLOX1 catalyzed oxygenation at the n-10 position of both linoleic and arachidonic acid, resulting in 9R- and 11R-hydroperoxides, respectively. Together these results reveal unifying structural features of LOXs and their function. While the core of the active site is important for lipoxygenation and thus highly conserved, peripheral domains functioning in membrane and substrate binding are more variable.
Highlights
In eukaryotes, oxidized PUFAs, so-called oxylipins, are vital signaling molecules
The only PUFA that was found in Cyanothece sp
Cyanothece sp. lipoxygenase 1 (CspLOX1) is placed at a very distant position from other characterized LOXs and may form a rare group of LOX enzymes found in few cyanobacteria together with the LOX domain of the Nostoc punctiforme 9R-LOX (Fig. 2)
Summary
In eukaryotes, oxidized PUFAs, so-called oxylipins, are vital signaling molecules. The first step in their biosynthesis may be catalyzed by a lipoxygenase (LOX), which forms hydroperoxides by introducing dioxygen into PUFAs. This binding mode is further supported by the fact that CspLOX1 catalyzed oxygenation at the n-10 position of both linoleic and arachidonic acid, resulting in 9R- and 11R-hydroperoxides, respectively Together these results reveal unifying structural features of LOXs and their function. While most LOXs bind their substrate with the methyl end first, the 9S-lipoxygenation products of plant LOXs [10, 11], as well as the specificities of 5S-, 12R- and 8S-LOX in mammals, were explained with the reversed orientation of the substrate [12] (Fig. 1B) Another position in the active site, referred to as the Gly/Ala switch, may function as a switch for the regiospecificity of the reaction [13].
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