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

Abstract

Lipoxygenases (LOX) are non-heme metal enzymes, which oxidize polyunsaturated fatty acids to hydroperoxides. All LOX belong to the same gene family, and they are widely distributed. LOX of animals, plants, and prokaryotes contain iron as the catalytic metal, whereas fungi express LOX with iron or with manganese. Little is known about metal selection by LOX and the adjustment of the redox potentials of their protein-bound catalytic metals. Thirteen three-dimensional structures of animal, plant, and prokaryotic FeLOX are available, but none of MnLOX. The MnLOX of the most important plant pathogen, the rice blast fungus Magnaporthe oryzae (Mo), was expressed in Pichia pastoris. Mo-MnLOX was deglycosylated, purified to homogeneity, and subjected to crystal screening and x-ray diffraction. The structure was solved by sulfur and manganese single wavelength anomalous dispersion to a resolution of 2.0 Å. The manganese coordinating sphere is similar to iron ligands of coral 8R-LOX and soybean LOX-1 but is not overlapping. The Asn-473 is positioned on a short loop (Asn-Gln-Gly-Glu-Pro) instead of an ␣-helix and forms hydrogen bonds with Gln-281. Comparison with FeLOX suggests that Phe-332 and Phe-525 might contribute to the unique suprafacial hydrogen abstraction and oxygenation mechanism of Mo-MnLOX by controlling oxygen access to the pentadiene radical. Modeling suggests that Arg-525 is positioned close to Arg-182 of 8R-LOX, and both residues likely tether the carboxylate group of the substrate. An oxygen channel could not be identified. We conclude that MoMnLOX illustrates a partly unique variation of the structural theme of FeLOX. Lipoxygenases (LOX)3 are iron-or manganese-containing dioxygenases that oxidize polyunsaturated fatty acids containing one or more 1Z,4Z-pentadiene units to hydroperoxides (1, 2). These hydroperoxides are precursors of signal molecules in animals, plants, and fungi. They may take part in inflammation, asthma, cancer development, and the chemical warfare between plants, fungi, and other microorganisms (3, 4). The LOX mechanism is initiated with hydrogen abstraction from a bis-allylic carbon of the 1Z,4Z-pentadiene of fatty acids. This is followed by oxygen insertion, which usually produces cis-transconjugated hydroperoxy fatty acids (1, 2). Plant, mammals, and a few prokaryotes express FeLOX, whereas both MnLOX and FeLOX occur in plant pathogenic fungi (5-8).All LOX belong to the same gene family, but plant FeLOX, mammalian FeLOX, and fungal FeLOX and MnLOX form separate subfamilies (5, 8). The prototype MnLOX is secreted by the take-all fungus of wheat, Gaeumannomyces graminis (7). The evolution of this enzyme, 13R-MnLOX, and five members of the MnLOX subfamily are illustrated in a phylogenetic tree together with pro-and eukaryotic LOX, including fungal FeLOX (Fig. 1A).The three-dimensional structures of 11 eukaryotic and two prokaryotic FeLOX are available. 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, ...

Two isoforms of lipoxygenase from mature grains of pearl millet [Pennisetum glaucum (L.) R. Br.]: purification and physico-chemico-kinetic characterization

“…Fe 3? is considered essential for LOX activity in most of the plants (Baysal and Demirodoven 2007;Wennman et al 2016). Zn 2?…”

“…Moreover, few enzymes, which are essential in the metabolism of plants, remain active even after harvesting and lead to detrimental changes in quality attributes such as colour, flavour, texture and nutritional value of its products. Lipoxygenases (LOX; linoleate oxygen oxidoreductase; EC 1.13.11.12), the enzymes found in all forms of life, catalyze the oxidation of PUFA and lipids containing a cis,cis-1,4-pentadiene structure (Wennman et al 2016). This reaction produces lipid hydroperoxides that decompose and form secondary oxidation products, which can react with chlorophylls, carotenoids, ascorbic acid, phenols, a-tocopherol, etc.…”

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

“…This loop is also present in Mo-MnLOX (Fig. 4C), but corresponds to an -helix in 8R-LOX (7,36). The oxygen of Gln-287 forms tentative hydrogen bonds to the nitrogen of Asn-482 with a distance of 2.9 Å. His-290, Val-618, and and substrates to the active site (Fig.…”

“…The 3D structure of Mo-MnLOX was reported recently (7). The sequence identity between Gg-and Mo-MnLOX is 56% (see Fig.…”

“…Methyl esters of fatty acids are not oxygenated by GgMnLOX, and the carboxyl group of C 18 fatty acids are likely tethered to the Arg residues at the entrance of the substrate channel (7). The number of double bonds reduces the flexibility of fatty acids, and a comparison of the oxygenation of 20:4n-6 and 20:5n-3 may therefore illustrate the conformation of the active sites.…”