Manganese lipoxygenase. Purification and characterization.
A linoleic acid (13R)-lipoxygenase was purified to homogeneity from the culture medium of Gäumannomyces 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 approximately 135 kDa in 6 M urea and approximately 160 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 approximately 73 kDa on SDS-PAGE. Atomic emission spectroscopy indicated 0.5-1.0 manganese atom/enzyme molecule. The isoelectric point was approximately 9.7, and the enzyme was active between pH 5 and 11 with optimum activity at pH 7. 0. For molecular oxygen, Km was 30 microM and Vmax 10 micromol mg-1min-1; for linoleic acid, Km was 4.4 micromol, Vmax 8.2 micromol mg-1min-1, and the turnover number 1100 min-1. 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 gamma-linolenic acid was oxygenated both at C-11 and C-13. The enzyme was active at 60 degreesC and after treatment with 6 M urea. It was strongly inhibited by 10-50 microM concentrations of eicosatetraynoic acid and a lipoxygenase inhibitor (N-(3-phenoxycinnamyl)acetohydroxamic acid), but many other lipoxygenase inhibitors (100 microM) 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.
Su
C
C
Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala Biomedical Center, Uppsala University, S-751 24 Uppsala, Sweden.
Oliw
E H
EH
D001203:Ascomycota / Q000201:enzymology*; D002384:Catalysis; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008084:Lipoxygenase / Q000302:isolation & purification* / Q000378:metabolism; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D005231:Fatty Acids, Unsaturated; D016859:Lipoxygenase Inhibitors; C112608:manganese lipoxygenase; D008084:Lipoxygenase; D010100:Oxygen