Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, California 95064, United States.
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, San Francisco, California 94158, United States.
Biochemistry. 2021 Jun 8;60(22):1741-1754. doi: 10.1021/acs.biochem.0c00931. Epub 2021 May 24.
In this paper, human platelet 12-lipoxygenase [h12-LOX (ALOX12)], human reticulocyte 15-lipoxygenase-1 [h15-LOX-1 (ALOX15)], and human epithelial 15-lipoxygenase-2 [h15-LOX-2 (ALOX15B)] were observed to react with docosahexaenoic acid (DHA) and produce 17-hydroperoxy-4,7,10,13,15,19-docosahexaenoic acid (17S-HpDHA). The / values with DHA for h12-LOX, h15-LOX-1, and h15-LOX-2 were 12, 0.35, and 0.43 s μM, respectively, which demonstrate h12-LOX as the most efficient of the three. These values are comparable to their counterpart / values with arachidonic acid (AA), 14, 0.98, and 0.24 s μM, respectively. Comparison of their product profiles with DHA demonstrates that the three LOX isozymes produce 11S-HpDHA, 14S-HpDHA, and 17S-HpDHA, to varying degrees, with 17S-HpDHA being the majority product only for the 15-LOX isozymes. The effective / values (/ × percent product formation) for 17S-HpDHA of the three isozymes indicate that the value of h12-LOX was 2.8-fold greater than that of h15-LOX-1 and 1.3-fold greater than that of h15-LOX-2. 17S-HpDHA was an effective substrate for h12-LOX and h15-LOX-1, with four products being observed under reducing conditions: protectin DX (PDX), 16,17-epoxy-4,7,10,12,14,19-docosahexaenoic acid (16S,17S-epoxyDHA), the key intermediate in neuroprotection D1 biosynthesis [NPD1, also known as protectin D1 (PD1)], 11,17S-diHDHA, and 16,17S-diHDHA. However, h15-LOX-2 did not react with 17-HpDHA. With respect to their effective / values, h12-LOX was markedly less effective than h15-LOX-1 in reacting with 17S-HpDHA, with a 55-fold lower effective / in producing 16S,17S-epoxyDHA and a 27-fold lower effective / in generating PDX. This is the first direct demonstration of h15-LOX-1 catalyzing this reaction and reveals an pathway for PDX and NPD1 intermediate biosynthesis. In addition, epoxide formation from 17S-HpDHA and h15-LOX-1 was negatively affected via allosteric regulation by 17S-HpDHA ( = 5.9 μM), 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12S-HETE) ( = 2.5 μM), and 17-hydroxy-13,15,19-docosatrienoic acid (17S-HDTA) ( = 1.4 μM), suggesting a possible regulatory pathway in reducing epoxide formation. Finally, 17S-HpDHA and PDX inhibited platelet aggregation, with EC values of approximately 1 and 3 μM, respectively. The results presented here may help advise PDX and NPD1 intermediate (i.e., 16S,17S-epoxyDHA) biosynthetic investigations and support the benefits of DHA rich diets.
在本文中，观察到人类血小板 12-脂氧合酶 [h12-LOX (ALOX12)]、人类网织红细胞 15-脂氧合酶-1 [h15-LOX-1 (ALOX15)] 和人类上皮细胞 15-脂氧合酶-2 [h15-LOX-2 (ALOX15B)] 与二十二碳六烯酸 (DHA) 反应生成 17-羟基-4,7,10,13,15,19-二十二碳六烯酸 (17S-HpDHA)。h12-LOX、h15-LOX-1 和 h15-LOX-2 与 DHA 的 / 值分别为 12、0.35 和 0.43 s μM，表明 h12-LOX 是三者中最有效的一种。这些值与它们与花生四烯酸 (AA) 的对应 / 值 14、0.98 和 0.24 s μM 相当。与 DHA 的产物谱比较表明，三种 LOX 同工酶在不同程度上产生 11S-HpDHA、14S-HpDHA 和 17S-HpDHA，只有 15-LOX 同工酶的主要产物是 17S-HpDHA。三种同工酶 17S-HpDHA 的有效 / 值 (/ × 产物形成百分比) 表明 h12-LOX 的 / 值比 h15-LOX-1 高 2.8 倍，比 h15-LOX-2 高 1.3 倍。17S-HpDHA 是 h12-LOX 和 h15-LOX-1 的有效底物，在还原条件下观察到四种产物：保护素 DX (PDX)、16,17-环氧-4,7,10,12,14,19-二十二碳六烯酸 (16S,17S-环氧DHA)、神经保护 D1 生物合成的关键中间体[NPD1，也称为保护素 D1 (PD1)]、11,17S-二 HDHA 和 16,17S-二 HDHA。然而，h15-LOX-2 不与 17-HpDHA 反应。就其有效 / 值而言，h12-LOX 与 17S-HpDHA 反应的有效 / 值明显低于 h15-LOX-1，生成 16S,17S-环氧 DHA 的有效 / 值低 55 倍，生成 PDX 的有效 / 值低 27 倍。这是首次直接证明 h15-LOX-1 催化该反应，并揭示了 PDX 和 NPD1 中间体生物合成的途径。此外，17S-HpDHA 和 h15-LOX-1 的环氧化物形成受到 17S-HpDHA ( = 5.9 μM)、12-羟基-5,8,10,14-二十碳四烯酸 (12S-HETE) ( = 2.5 μM) 和 17-羟基-13,15,19-二十二碳三烯酸 (17S-HDTA) ( = 1.4 μM) 的别构调节的负面影响，表明存在可能的调节途径来减少环氧化物的形成。最后，17S-HpDHA 和 PDX 抑制血小板聚集，EC 值分别约为 1 和 3 μM。这里呈现的结果可能有助于指导 PDX 和 NPD1 中间体 (即 16S,17S-环氧 DHA) 生物合成的研究，并支持富含 DHA 的饮食的益处。
