Open AccessIn Vitro Biosynthetic Pathway Investigations of Neuroprotectin D1 (NPD1) and Protectin DX (PDX) by Human 12-Lipoxygenase, 15-Lipoxygenase-1, and 15-Lipoxygenase-2
Author(s) -
WanChen Tsai,
Chakrapani Kalyanaraman,
Adriana Yamaguchi,
Michael Holinstat,
Matthew P. Jacobson,
Theodore R. Holman
Publication year - 2021
Publication title -
biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.43
H-Index - 253
eISSN - 1520-4995
pISSN - 0006-2960
DOI - 10.1021/acs.biochem.0c00931
Subject(s) - lipoxygenase , enzyme kinetics , arachidonate 5 lipoxygenase , docosahexaenoic acid , arachidonic acid , chemistry , stereochemistry , biochemistry , isozyme , linoleic acid , biosynthesis , enzyme , fatty acid , polyunsaturated fatty acid , active site
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 S -hydroperoxy-4 Z ,7 Z ,10 Z ,13 Z ,15 E ,19 Z -docosahexaenoic acid (17S-HpDHA). The k cat / K M 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 k cat / K M 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 k cat / K M values ( k cat / K M × 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), 16 S ,17 S -epoxy-4 Z ,7 Z ,10 Z ,12 E ,14 E ,19 Z -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 k cat / K M values, h12-LOX was markedly less effective than h15-LOX-1 in reacting with 17S-HpDHA, with a 55-fold lower effective k cat / K M in producing 16S,17S-epoxyDHA and a 27-fold lower effective k cat / K M 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 ( K d = 5.9 μM), 12 S -hydroxy-5 Z ,8 Z ,10 E ,14 Z -eicosatetraenoic acid (12S-HETE) ( K d = 2.5 μM), and 17 S -hydroxy-13 Z ,15 E ,19 Z -docosatrienoic acid (17S-HDTA) ( K d = 1.4 μM), suggesting a possible regulatory pathway in reducing epoxide formation. Finally, 17S-HpDHA and PDX inhibited platelet aggregation, with EC 50 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.