A 5‑lipoxygenase-specific sequence motif impedes enzyme activity and confers dependence on a partner protein.
A 5‑lipoxygenase-specific sequence motif impedes enzyme activity and confers dependence on a partner protein.
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5α脂氧合酶特异性序列基序会阻碍酶活性并赋予其对伙伴蛋白的依赖性。
DOI：
10.1016/j.bbalip.2018.09.011
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Newcomer,MarciaE
中科院分区：
文献类型：
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作者：
Schexnaydre,ErinE;Gerstmeier,Jana;Garscha,Ulrike;Jordan,PaulM;Werz,Oliver;Newcomer,MarciaE
白三烯（LT）是炎症反应的脂质介质，在哮喘和动脉粥样硬化等疾病中发挥关键作用。前体白三烯A4（LTA4）是由花生四烯酸（AA）通过5-脂氧合酶（5- lox）（一种膜相关酶）在5-脂氧合酶激活蛋白（FLAP）（一种核跨膜蛋白）的帮助下合成的。在脂氧合酶中，c端主链羧酸盐是非血红素铁的配体，因此是催化中心的一部分。我们研究了富含赖氨酸的序列（KKK653-655）在5-LOX特有的c端上游20个氨基酸的作用，该序列可能取代铁配位球中的主链羧酸盐。在没有和存在FLAP的情况下，将kkk653 - 655被ENL取代的5-LOX突变体转染到HEK293细胞。该突变体在受刺激的HEK细胞中产生的5-LOX产物水平比野生型5-LOX高约20倍。与FLAP共表达的酶导致检测到的5-LOX产物相等，野生型5-LOX产物水平升高，而突变酶的5-LOX产物水平降低。这些数据表明，KKK基序限制了5-LOX的活性，而这种减弱的活性必须通过FLAP作为有效LT生物合成的伴侣蛋白来补偿。
Leukotrienes (LT) are lipid mediators of the inflammatory response that play key roles in diseases such as asthma and atherosclerosis. The precursor leukotriene A4(LTA4) is synthesized from arachidonic acid (AA) by 5‑lipoxygenase (5-LOX), a membrane-associated enzyme, with the help of 5‑lipoxygenase-activating protein (FLAP), a nuclear transmembrane protein. In lipoxygenases the main chain carboxylate of the C-terminus is a ligand for the non-heme iron and thus part of the catalytic center. We investigated the role of a lysine-rich sequence (KKK653–655) 20 amino acids upstream of the C-terminus unique to 5-LOX that might displace the main-chain carboxylate in the iron coordination sphere. A 5-LOX mutant in which KKK653–655is replaced by ENL was transfected into HEK293 cells in the absence and presence of FLAP. This mutant gave ~20-fold higher 5-LOX product levels in stimulated HEK cells relative to the wild-type 5-LOX. Co-expression of the enzymes with FLAP led to an equalization of 5-LOX products detected, with wild-type 5-LOX product levels increased and those from the mutant enzyme decreased. These data suggest that the KKK motif limits 5-LOX activity and that this attenuated activity must be compensated by the presence of FLAP as a partner protein for effective LT biosynthesis.