Mortimer Monika, Järving Reet, Brash Alan R, Samel Nigulas, Järving Ivar
Department of Chemistry, Tallinn University of Technology, Akadeemia tee 15, Tallinn 12618, Estonia.
Arch Biochem Biophys. 2006 Jan 1;445(1):147-55. doi: 10.1016/j.abb.2005.10.023. Epub 2005 Nov 15.
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 77kDa 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 C18, C20, and C22 substrates are also oxygenated, preferentially at the omega10 position. Significantly, both Ca(2+)-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 Ca(2+) 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.
在几种海洋无脊椎动物中检测到了11R-脂氧合酶（11R-LOX）活性，在此我们报告该酶的首次克隆和表达。通过RT-PCR从软珊瑚丛生肉芝中分离出编码77kDa蛋白质的cDNA，并在大肠杆菌中表达。重组酶与花生四烯酸孵育产生单一产物，通过反相高效液相色谱（RP-HPLC）、气相色谱-质谱联用（GC-MS）和手性相高效液相色谱（chiral phase-HPLC）鉴定为11R-氢过氧化二十碳四烯酸。其他C18、C20和C22底物也被氧化，优先在ω10位。重要的是，催化活性需要Ca(2+)离子和膜组分。钙影响可溶性11R-LOX向膜的转运，这种结合可通过Ca(2+)螯合逆转。该酶序列包含一些与哺乳动物5-LOX钙激活有关的保守氨基酸，并且由于其对膜相互作用的严格要求，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一个新模型。
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