In Vitro Biosynthetic Pathway Investigations of Neuroprotectin D1 (NPD1) and Protectin DX (PDX) by Human 12-Lipoxygenase, 15-Lipoxygenase-1, and 15-Lipoxygenase-2.
In Vitro Biosynthetic Pathway Investigations of Neuroprotectin D1 (NPD1) and Protectin DX (PDX) by Human 12-Lipoxygenase, 15-Lipoxygenase-1, and 15-Lipoxygenase-2.
复制标题
DOI：
10.1021/acs.biochem.0c00931
复制
发表时间：
2021-06-08
期刊：
影响因子：
2.9
通讯作者：
Holman, Theodore R.
中科院分区：
文献类型：
--
作者：
Tsai, Wan-Chen;Kalyanaraman, Chakrapani;Yamaguchi, Adriana;Holinstat, Michael;Jacobson, Matthew P.;Holman, Theodore R.
本文观察到人血小板12-脂氧合酶[h12-LOX（ALOX 12）]、人网织红细胞15-脂氧合酶-1 [h15-LOX-1（ALOX 15）]和人上皮细胞15-脂氧合酶-2 [h15-LOX-2（ALOX 15 B）]与二十二碳六烯酸（DHA）反应生成17 S-HpDHA。h12-LOX、h15-LOX-1和h15-LOX-2与DHA的kcat/KM值分别为12、0.35和0.43 s−1 μM−1，这表明h12-LOX是三者中最有效的。这些值与花生四烯酸（AA）的相应kcat/KM值相当，分别为14、0.98和0.24 s−1 μM−1。比较它们与DHA的产物谱表明，三种LOX同工酶在不同程度上产生11 S-HpDHA、14 S-HpDHA和17 S-HpDHA，其中17 S-HpDHA仅是15-LOX同工酶的主要产物。三种同工酶对17 S-HpDHA的有效kcat/KM值（kcat/KM ×产物形成百分比）表明，h12-LOX的体外值比h15-LOX-1大2.8倍，比h15-LOX-2大1.3倍。17 S-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：protectin DX（PDX），16 S，17 S-epoxy-4 Z，7Z，10 Z，12 E，14 E，19 Z-二十二碳六烯酸（16 S，17 S-epoxyDHA），神经保护D1生物合成的关键中间体[NPD 1，也称为保护素D1（PD 1）]，11，17 S-diHDHA和16，17 S-diHDHA。然而，h15-LOX-2不与17-HpDHA反应。关于它们的有效kcat/KM值，h12-LOX在与17 S-HpDHA反应中的有效性明显低于h15-LOX-1，在产生16 S，17 S-epoxyDHA中的有效kcat/KM低55倍，在产生PDX中的有效kcat/KM低27倍。这是h15-LOX-1催化该反应的首次直接证明，并揭示了PDX和NPD 1中间体生物合成的体外途径。此外，17 S-HpDHA和h15-LOX-1的环氧化物形成通过17 S-HpDHA的变构调节受到负面影响（Kd = 5.9 μM），12 S-羟基-5Z，8 Z，10 E，14 Z-二十碳四烯酸（12 S-HETE）（Kd = 2.5 μM）和17 S-hydroxy-13 Z，15 E，19 Z-二十二碳三烯酸（17 S-HDTA）（Kd = 1.4 μM），表明可能存在减少环氧化物形成的调节途径。最后，17 S-HpDHA和PDX抑制血小板聚集，EC 50值分别约为1和3 μM。本文提供的体外结果可能有助于建议体内PDX和NPD 1中间体（即，16 S，17 S-epoxyDHA）生物合成的研究，并支持富含DHA的饮食的好处。
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 17S-hydroperoxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid (17S-HpDHA). The kcat/KM values with DHA for h12-LOX, h15-LOX-1, and h15-LOX-2 were 12, 0.35, and 0.43 s−1 μM−1, respectively, which demonstrate h12-LOX as the most efficient of the three. These values are comparable to their counterpart kcat/KM values with arachidonic acid (AA), 14, 0.98, and 0.24 s−1 μM−1, 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 kcat/KM values (kcat/KM × percent product formation) for 17S-HpDHA of the three isozymes indicate that the in vitro 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), 16S,17S-epoxy-4Z,7Z,10Z,12E,14E,19Z-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 kcat/KM values, h12-LOX was markedly less effective than h15-LOX-1 in reacting with 17S-HpDHA, with a 55-fold lower effective kcat/KM in producing 16S,17S-epoxyDHA and a 27-fold lower effective kcat/KM in generating PDX. This is the first direct demonstration of h15-LOX-1 catalyzing this reaction and reveals an in vitro 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 (Kd = 5.9 μM), 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12S-HETE) (Kd = 2.5 μM), and 17S-hydroxy-13Z,15E,19Z-docosatrienoic acid (17S-HDTA) (Kd = 1.4 μM), suggesting a possible regulatory pathway in reducing epoxide formation. Finally, 17S-HpDHA and PDX inhibited platelet aggregation, with EC50 values of approximately 1 and 3 μM, respectively. The in vitro results presented here may help advise in vivo PDX and NPD1 intermediate (i.e., 16S,17S-epoxyDHA) biosynthetic investigations and support the benefits of DHA rich diets.
登录
查看更多内容
影响因子：
5.1
作者：
Aursnes M;Tungen JE;Colas RA;Vlasakov I;Dalli J;Serhan CN;Hansen TV
通讯作者：
Hansen TV
影响因子：
2.9
作者：
Freedman C;Tran A;Tourdot BE;Kalyanaraman C;Perry S;Holinstat M;Jacobson MP;Holman TR
通讯作者：
Holman TR
影响因子：
1.9
作者：
BILD, GS;RAMADOSS, CS;AXELROD, B
通讯作者：
AXELROD, B
影响因子：
6.5
作者：
Butovich, IA
通讯作者：
Butovich, IA
影响因子：
4.8
作者：
Chen, Ping;Vericel, Evelyne;Guichardant, Michel
通讯作者：
Guichardant, Michel