Jasmonates constitute a family of lipid-derived signaling molecules that are abundant in higher plants. The biosynthetic pathway leading to plant jasmonates is initiated by 13-lipoxygenase-catalyzed oxygenation of α-linolenic acid into its 13-hydroperoxide derivative. A number of plant pathogenic fungi (e.g. Fusarium oxysporum) are also capable of producing jasmonates, however, by a yet unknown biosynthetic pathway. In a search for lipoxygenase in F. oxysporum, a reverse genetic approach was used and one of two from the genome predicted lipoxygenases (FoxLOX) was cloned. The enzyme was heterologously expressed in E. coli, purified via affinity chromatography, and its reaction mechanism characterized. FoxLOX was found to be a non-heme iron lipoxygenase, which oxidizes C18-polyunsaturated fatty acids to 13S-hydroperoxy derivatives by an antarafacial reaction mechanism where the bis-allylic hydrogen abstraction is the rate-limiting step. With α-linolenic acid as substrate FoxLOX was found to exhibit a multifunctional activity, because the hydroperoxy derivatives formed are further converted to dihydroxy-, keto-, and epoxy alcohol derivatives.

This article is available online at http://www.jlr.org PUFA oxygenation is a process that leads to the formation of bioactive lipid compounds with a diversity of biological functions in plants and animals ( 1, 2 ). Oxidation of PUFAs may be catalyzed by two major classes of enzymes, cyclooxygenases or ␣ -dioxygenases and lipoxygenases (LOXs) ( 3 ). Of the two, LOXs are nonheme iron-containing dioxygenases that are widely found in higher plants and animals, but have also been detected in some corals, mosses, fungi, and a number of bacteria ( 4, 5 ). Members of the LOX family catalyze the regio-and stereospecifi c oxygenation of PUFAs with one or more (1 Z ,4 Z )-pentadiene moieties leading to the formation of hydroperoxy PUFAs ( 6 ). LOX hydroperoxide products are precursors of important signaling compounds such as aldehydes and jasmonates in plants and leukotrienes, resolvins, and lipoxins in mammals ( 3 ). These signaling molecules play an important role in wound and defense responses as well as in aspects of plant development ( 7 ), while in mammals they function in infl ammation, asthma, and the development of atherosclerosis and cancer ( 1 ). Other than in higher organisms, very little is still known regarding the overall function of LOX products in prokaryotes and fungi ( 4, 5 ).As the regio-and stereospecifi city of the LOX reaction has an infl uence on the biological function of the product, many studies have focused on the molecular basis of this specifi city. In the case of arachidonic acid [20:4(n-6)], which is a typical mammalian LOX substrate, several Abstract In eukaryotes, oxidized PUFAs, so-called oxylipins, are vital signaling molecules. The fi rst 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 identifi ed an N-terminal helical extension, reminiscent of the long ␣ -helical insertion in Pseudomonas aeruginosa LOX. In liposome fl otation studies, this helical extension, rather than the ␤ -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-fi rst 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 9 R -and 11 R -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.

Lipoxygenases (LOX) catalyze the regio- and stereospecific insertion of dioxygen into polyunsaturated fatty acids. While the catalytic metal of LOX is typically a non-heme iron, some fungal LOX contain manganese as catalytic metal (MnLOX). In general, LOX insert dioxygen at C9 or C13 of linoleic acid leading to the formation of conjugated hydroperoxides. MnLOX (EC 1.13.11.45), however, catalyze the oxygen insertion also at C11, resulting in bis-allylic hydroperoxides. Interestingly, the iron-containing CspLOX2 (EC 1.13.11.B6) from Cyanothece PCC8801 also produces bis-allylic hydroperoxides. What role the catalytic metal plays and how this unusual reaction is catalyzed by either MnLOX or CspLOX2 is not understood. Our findings suggest that only iron is the catalytically active metal in CspLOX2. The enzyme loses its catalytic activity almost completely when iron is substituted with manganese, suggesting that the catalytic metal is not interchangeable. Using kinetic and spectroscopic approaches, we further found that first a mixture of bis-allylic and conjugated hydroperoxy products is formed. This is followed by the isomerization of the bis-allylic product to conjugated products at a slower rate. These results suggest that MnLOX and CspLOX2 share a very similar reaction mechanism and that LOX with a Fe or Mn cofactor have the potential to form bis-allylic products. Therefore, steric factors are probably responsible for this unusual specificity. As CspLOX2 is the LOX with the highest proportion of the bis-allylic product known so far, it will be an ideal candidate for further structural analysis to understand the molecular basis of the formation of bis-allylic hydroperoxides.

The biological function of lipoxygenases depends on the regio and stereo specific formation of fatty acid-derived hydroperoxides and different concepts exist to explain the mechanism that directs dioxygen to a specific carbon atom within the substrate. Here, we report the 1.8 Å resolution crystal structure of a cyanobacterial lipoxygenase that produces bis-allylic hydroperoxides (CspLOX2). Site directed mutagenesis experiments combined with computational approaches reveal that residues around the active site direct dioxygen to a preferred carbon atom and stereo configuration in the substrate fatty acid. Modulating the cavity volume around the pentadiene system of linoleic acid shifted the product formation towards 9S-, 9R-, 13S- or 13R-hydroperoxides in correlation with the site of mutation, thus decreasing the amount of the bis-allylic 11R-hydroperoxide. Decreasing the channel size of a 9R-lipoxygenase (CspLOX1) on the other hand could in turn induce formation of the bis-allylic 11R-hydroperoxide. Together this study suggests that an active site clamp fixing the pentadiene system of the substrate together with steric shielding controls the stereo and regio specific positioning of dioxygen at all positions of the reacting pentadiene system of substrate fatty acids.

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