Eryanni-Levin Suzy, Khatib Soliman, Levy-Rosenzvig Reut, Tamir Snait, Szuchman-Sapir Andrea
Laboratory of Human Health and Nutrition Sciences, MIGAL-Galilee Research Institute, Ltd., Kiryat Shmona, Israel; Tel-Hai College, Upper Galilee, Israel.
Laboratory of Oxidative Stress and Human Diseases, MIGAL-Galilee Research Institute, Ltd., Kiryat Shmona, Israel; Tel-Hai College, Upper Galilee, Israel.
Biochim Biophys Acta. 2015 Sep;1851(9):1118-22. doi: 10.1016/j.bbalip.2015.04.008. Epub 2015 May 6.
Paraoxonase 1 (PON1) is an antiatherogenic high density lipoprotein-associated lactonase. Recent findings revealed that PON1 knockout mice have low blood pressure, which is negatively correlated with the level of 5,6-epoxyeicosatrienoic acid (5,6-EET), a cytochrome P450 -derived arachidonic acid metabolite. 5,6-EET is an endothelium-derived hyperpolarizing factor that causes arterial dilation. Under physiological conditions, 5,6-EET is unstable, transforming to its δ-lactone (5,6-δ-DHTL) that evades the degradation by soluble epoxide hydrolase (sEH), arguing for the existence of yet another enzyme that is responsible specifically for its hydrolysis. We therefore hypothesized that PON1 degrades the 5,6-δ-DHTL, and this specific PON1 lactonase activity thus decreases endothelial vasodilatation. The aim of the present study was to investigate the PON1-5,6-δ-DHTL relationship. A liquid chromatography mass spectrometry based method for 5,6-EET derivatives identification was developed. Tracking the lactonization of 5,6-EET in a physiological solution revealed that 5,6-EET was fully converted into 5,6-δ-DHTL. Incubation of 5,6-δ-DHTL with rePON1 resulted in 85.1±3.4% degradation of the substrate to 5,6 dihydroxytrienoic acid (5,6-DHET), while only 12.0±8.7% hydrolysis was detected in the absence of PON1. Accordingly, the levels of 5,6-DHTL were found to be significantly higher in the PON1KO mice than in the wild type mice. Kinetic analysis revealed values of Vmax=0.021±0.01μM/s and Km=150.99±62.1μM. Calculation of the docking energy suggested possible interaction of the 5,6-δ-DHTL in the catalytic region of PON1 with free energy of-5.57 Kcal/mol, preferentially for the (S) enantiomer. These findings demonstrate that 5,6-δ-DHTL is a PON1 substrate and imply that the 5,6-EET vasodilation effect may be impaired by PON1.
对氧磷酶1（PON1）是一种与抗动脉粥样硬化高密度脂蛋白相关的内酯酶。最近的研究发现，敲除PON1基因的小鼠血压较低，这与细胞色素P450衍生的花生四烯酸代谢产物5,6-环氧二十碳三烯酸（5,6-EET）的水平呈负相关。5,6-EET是一种内皮源性超极化因子，可引起动脉扩张。在生理条件下，5,6-EET不稳定，会转化为其δ-内酯（5,6-δ-DHTL），从而避免被可溶性环氧化物水解酶（sEH）降解，这表明存在另一种专门负责其水解的酶。因此，我们推测PON1可降解5,6-δ-DHTL，这种特定的PON1内酯酶活性会降低内皮血管舒张功能。本研究的目的是探讨PON1与5,6-δ-DHTL之间的关系。我们开发了一种基于液相色谱质谱法的5,6-EET衍生物鉴定方法。追踪生理溶液中5,6-EET的内酯化过程发现，5,6-EET完全转化为5,6-δ-DHTL。将5,6-δ-DHTL与重组PON1一起孵育，结果显示底物有85.1±3.4%降解为5,6-二羟基二十碳三烯酸（5,6-DHET），而在没有PON1的情况下，仅检测到12.0±8.7% 的水解。因此，发现PON1基因敲除小鼠中5,6-DHTL的水平显著高于野生型小鼠。动力学分析显示Vmax值为0.021±0.01μM/s，Km值为150.99±62.1μM。对接能的计算表明，5,6-δ-DHTL在PON1催化区域可能与（S）对映体优先相互作用，自由能为-5.57千卡/摩尔。这些发现表明5,6-δ-DHTL是PON1的底物，并暗示PON1可能会损害5,6-EET的血管舒张作用。
