Su C, Oliw E H
Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala Biomedical Center, Uppsala University, S-751 24 Uppsala, Sweden.
J Biol Chem. 1998 May 22;273(21):13072-9. doi: 10.1074/jbc.273.21.13072.
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.
通过疏水相互作用、阳离子交换、凝集素亲和及尺寸排阻色谱法，从全蚀病菌禾顶囊壳的培养基中纯化出一种（13R）-亚油酸脂氧合酶，使其达到同质。纯化后的双加氧酶在300至700纳米之间没有光吸收。凝胶过滤显示，在6M尿素中其表观分子量约为135 kDa，在缓冲液中约为160 kDa。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）表明该酶大小不均一，由100至140 kDa的弥散蛋白带组成。用糖苷酶处理N-和O-连接的寡糖后，SDS-PAGE上出现一条约73 kDa的独特蛋白带。原子发射光谱表明每个酶分子含有0.5至1.0个锰原子。其等电点约为9.7，该酶在pH 5至11之间有活性，在pH 7.0时活性最佳。对于分子氧，米氏常数（Km）为30微摩尔，最大反应速度（Vmax）为10微摩尔·毫克-1·分钟-1；对于亚油酸，Km为4.4微摩尔，Vmax为8.2微摩尔·毫克-1·分钟-1，周转数为1100分钟-1。该酶氧化亚麻酸的速度比亚油酸快两倍。通过质谱鉴定，主要产物分别为13-氢过氧-（9Z,11E,15Z）-十八碳三烯酸和13-氢过氧-（9Z,11E）-十八碳二烯酸。将氢过氧化物还原后，通过手性高效液相色谱对13-羟基十八碳二烯酸甲酯进行立体分析，得到一种对映体（>95%），它与（13R,13S）-羟基十八碳二烯酸甲酯的R-立体异构体共洗脱。花生四烯酸和二高-γ-亚麻酸不是底物，而耗氧量、紫外分析和质谱分析表明γ-亚麻酸在C-11和C-13处均被氧化。该酶在60℃时仍有活性，且在6M尿素处理后仍有活性。它受到10至50微摩尔浓度的二十碳四炔酸和一种脂氧合酶抑制剂（N-（3-苯氧基肉桂基）乙酰氧肟酸）的强烈抑制，但许多其他脂氧合酶抑制剂（100微摩尔）无效。我们得出结论，去糖基化后，该酶在SDS-PAGE上的大小与哺乳动物和海洋脂氧合酶相同，但在三个方面与所有先前描述的脂氧合酶不同。它是分泌型的，形成（13R）-氢过氧-（9Z,11E）-十八碳二烯酸，且含有锰。
