Manganese Lipoxygenase

Abstract

A linoleic acid (13R)-lipoxygenase was purified to homogeneity from the culture medium of Gä umannomyces graminis, the take-all fungus, by hydrophobic interaction, cation exchange, lectin affinity, and size-exclusion chromatography. The purified dioxygenase lacked light absorption between 300 and 700 nm. Gel filtration indicated an apparent molecular mass of ϳ135 kDa in 6 M urea and ϳ160 kDa in buffer. SDS-polyacrylamide gel electrophoresis (PAGE) showed that the enzyme was heterogeneous in size and consisted of diffuse protein bands of 100 -140 kDa. Treatment with glycosidases for N-and O-linked oligosaccharides yielded a distinct protein of ϳ73 kDa on SDS-PAGE. Atomic emission spectroscopy indicated 0.5-1.0 manganese atom/enzyme molecule. The isoelectric point was ϳ9.7, and the enzyme was active between pH 5 and 11 with optimum activity at pH 7.0. For molecular oxygen, K m was 30 M and V max 10 mol mg . The enzyme oxidized linolenic acid twice as fast as linoleic acid. The main products were identified by mass spectrometry as 13-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic and 13-hydroperoxy-(9Z,11E)-octadecadienoic acids, respectively. After reduction of the hydroperoxide, steric analysis of methyl 13-hydroxyoctadecadienoate by chiral high performance liquid chromatography yielded one enantiomer (>95%), which co-eluted with the R-stereoisomer of methyl (13R,13S)-hydroxyoctadecadienoate. Arachidonic and dihomogammalinolenic acids were not substrates, while oxygen consumption, UV analysis, and mass spectrometric analysis indicated that ␥-linolenic acid was oxygenated both at C-11 and C-13. The enzyme was active at 60°C and after treatment with 6 M urea. It was strongly inhibited by 10 -50 M concentrations of eicosatetraynoic acid and a lipoxygenase inhibitor (N-(3-phenoxycinnamyl)acetohydroxamic acid), but many other lipoxygenase inhibitors (100 M) were without effect. We conclude that, after deglycosylation, the enzyme has the same size on SDS-PAGE as mammalian and marine lipoxygenases, but it differs from all previously described lipoxygenases in three ways. It is secreted, it forms (13R)-hydroperoxy-(9Z,11E)-octadecadienoic acid, and it contains manganese.

Multifrequency pulsed EPR studies of biologically relevant manganese(II) complexes

“…(1a) (1b) Other examples of such mononuclear redox-active enzymes include the manganese peroxidase responsible for lignin degradation by white-rot fungus [10][11][12]; a unique Mndependent form of lipoxygenase [13][14][15][16]; oxalate decarboxylase [17,18]; as well as an extradiol catechol dioxygenase [19][20][21]. …”

“…Many Mn-dependent enzymes take advantage o f the rich redox chemistry available to the metal, accessing the +2, +3, +4, and perhaps even the +5 oxidation states during their turnover. For example, Mn-superoxide dismutase (MnSOD), which detoxifies the cell of the superoxide radical , cycles between the Mn(II) and Mn(III) oxidation states via the ping-pong type mechanism shown below [5][6][7][8][9].(1a) (1b) Other examples of such mononuclear redox-active enzymes include the manganese peroxidase responsible for lignin degradation by white-rot fungus [10][11][12]; a unique Mndependent form of lipoxygenase [13][14][15][16]; oxalate decarboxylase [17,18]; as well as an extradiol catechol dioxygenase [19][20][21]. …”

Molecular Characterization of a Lipoxygenase from the Basidiomycete MushroomPleurotus ostreatus

“…Genomic DNA was digested with HindIII, and was self-ligated overnight at 16 C with a DNA Ligation Kit (Takara Bio). PCR and inverse PCR were performed using Ex Taq DNA polymerase (Takara Bio) and genomic DNA and the self-ligated DNA respectively as template.…”

“…[10][11][12][13] Fungal LOXs have been characterized in molds such as Fusarium proliferatum 14) and mushrooms such as Morchella esculenta. 15) Among these, a unique LOX (Mn-LOX), which contains Mn as the catalytic metal, has been well studied in a take-all fungus, Gaeumannomyces graminis, 16) but information on the molecular structure and biological function of fungal LOXs is limited. To our knowledge, the only reports on fungal LOX genes are those on the G. graminis Mn-LOX-Gga gene 17) and the Aspergillus flavus AfLOX gene.…”

A Protein Radical and Ferryl Intermediates Are Generated by Linoleate Diol Synthase, a Ferric Hemeprotein with Dioxygenase and Hydroperoxide Isomerase Activities

“…Lipoxygenases contain a non-heme metal center (1,3,4). Iron lipoxygenases abstract a hydrogen from the bisallylic C-3 of the 1Z,4Z-pentadienyl group of the polyunsaturated fatty acids and insert molecular oxygen at C-1 or C-5 with formation of a cis-trans conjugated double bond (1,3).…”