Crystal structure of a lipoxygenase from Cyanothece sp. may reveal novel features for substrate acquisition

Abstract

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.

Crystal Structure of Manganese Lipoxygenase of the Rice Blast Fungus Magnaporthe oryzae

“…The coordinating sphere is similar to coral 8R-LOX, but the metal ligands do not superimpose as neatly as those of coral 8R-LOX, sLOX-1, and 15S-LOX of P. aeruginosa (Fig. 3B) (2,16,19,20). The distances between the coordinating residues, manganese, and water are indicated in Table 2 along with a comparison with three FeLOX.…”

“…These are four structures of soybean LOX (sLOX-1, LOX-3, VLX-B, and VLX-D) (9 -13), three human LOX (15-LOX-2, 12S-LOX, and 5-LOX) (2,13,14), coral 8R-LOX and 11R-LOX (15,16), rabbit arachidonate 15-LOX-1 (17), porcine 12S-LOX (18), 15S-LOX of Pseudomonas aeruginosa (19), and linoleate 9R-LOX of Cyanotheca sp. (20). Plant and mammalian LOX consist of two domains, a relatively small eight-stranded ␤-barrel domain with homology to the PLAT (polycystin-1, lipoxygenase, ␣-toxin) domain and a larger catalytic domain of ␣-helices containing the substratebinding channel and the catalytic iron (2).…”

Kinetics of Bis‐Allylic Hydroperoxide Synthesis in the Iron‐Containing Lipoxygenase 2 from Cyanothece and the Effects of Manganese Substitution

“…6). Analysis of the products revealed that the positional specificities were the same as for linoleic acid, as FoxLOX, which is characterized as 13-LOX [10], produced mainly 13-HPODE and the 9-LOX CspLOX1 [19] produced mainly 9-HPODE (Fig. As it might be possible, that other LOX also produce 11-HPODE under these optimized conditions, we analyzed LOX from various phylogenetic groups for 11-HPODE formation using HPLC analysis.…”

“…When following the reaction of CspLOX2 at 234 nm with a spectrophotometer, two characteristic features distinguish the CspLOX2 reaction from most other LOX reactions. [19] has a comparably long lag phase (Fig. Second, the reaction comprises two linear phases, a first steep increase of absorbance and a following flat increase (Fig.…”

Crystal structure of linoleate 13R-manganese lipoxygenase in complex with an adhesion protein

“…Amino acid sequences with homology to zonadhesin occur in peptidases and few other proteins of P. pastoris. 6 Adhesion molecules of bacteria and fungi are crucial for cell-cell interaction, adherence to surrounding tissues, and the virulence of pathogens (34). Zonadhesin and several other fungal adhesins contain a ligand binding N-terminal domain of 300 residues and a C-terminal domain with repeats of Thr, Ser, and hydrophobic residues (34,35).…”