Identification and characterization of an arachidonate 11R-lipoxygenase
Identification and characterization of an arachidonate 11R-lipoxygenase
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DOI：
10.1016/j.abb.2005.10.023
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发表时间：
2006-01-01
影响因子：
3.9
通讯作者：
Järving, I
中科院分区：
文献类型：
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作者：
Mortimer, M;Järving, R;Järving, I
已在几种海洋无脊椎动物中检测到 11R-脂氧合酶 (11R-LOX) 活性，在此我们报告了该酶的首次克隆和表达。通过 RT-PCR 从软珊瑚 Gersemia fruticosa 中分离出编码 77 kDa 蛋白质的 cDNA，并在大肠杆菌中表达。重组酶与花生四烯酸一起孵育产生单一产物，通过 RP-HPLC、GC-MS 和手性相 HPLC 鉴定为 11R-氢过氧二十碳四烯酸。其他 C 18、C20 和 C22 底物也被氧化，优先在 w10 位置。值得注意的是，催化活性需要 Ca2+ 离子和膜部分。钙会影响可溶性 11R-LOX 向膜的易位，并且这种关联可通过 Ca2+ 螯合来逆转。该酶序列包含一些与哺乳动物 5-LOX 的钙激活有关的保守氨基酸，并且由于其对膜相互作用的必然要求，11R-LOX 可能因此为进一步分析脂氧合酶激活的这一方面提供新模型。 (c) 2005 Elsevier Inc. 保留所有权利。
11R-Lipoxygenase (11R-LOX) activity has been detected in several marine invertebrates, and here we report the first cloning and expression of the enzyme. The cDNA encoding a protein of 77 kDa was isolated by RT-PCR from the soft coral Gersemia fruticosa and expressed in Escherichia coli. Incubations of recombinant enzyme with arachidonic acid yielded a single product, identified by RP-HPLC, GC-MS, and chiral phase-HPLC as 11R-hydroperoxyeicosatetraenoic acid. Other C 18, C20, and C22 substrates are also oxygenated, preferentially at the w10 position. Significantly, both Ca2+ -ions and a membrane fraction are required for catalytic activity. Calcium effects translocation of the soluble 11R-LOX to the membrane and this association is reversible by Ca2+ chelation. The enzyme sequence contains some conserved amino acids implicated in calcium activation of mammalian 5-LOX, and with its obligate requirement for membrane interaction the 11R-LOX may thus provide a new model for further analysis of this aspect of lipoxygenase activation. (c) 2005 Elsevier Inc. All rights reserved.