Biosynthetic Pathway Investigations of Neuroprotectin D1 (NPD1) and Protectin DX (PDX) by Human 12-Lipoxygenase, 15-Lipoxygenase-1, and 15-Lipoxygenase-2.

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Wan-Chen Tsai, Chakrapani Kalyanaraman, Adriana Yamaguchi, Michael Holinstat, Matthew P Jacobson, Theodore R Holman

Author Information

Wan-Chen Tsai: Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, California 95064, United States.
Chakrapani Kalyanaraman: Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, San Francisco, California 94158, United States.
Adriana Yamaguchi: Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109, United States.
Michael Holinstat: Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109, United States.
Matthew P Jacobson: Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, San Francisco, California 94158, United States. ORCID
Theodore R Holman: Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, California 95064, United States. ORCID

PMID: 34029049 DOI: 10.1021/acs.biochem.0c00931

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.

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R01 GM105671/NIGMS NIH HHS
R01 HG011405/NHGRI NIH HHS
R35 GM131835/NIGMS NIH HHS

Allosteric Regulation

Arachidonate 12-Lipoxygenase

Arachidonate 15-Lipoxygenase

Arachidonic Acid

Arachidonic Acids

Biosynthetic Pathways

Blood Platelets

Docosahexaenoic Acids

Humans

Lipoxygenase

Lipoxygenases

10,17-dihydroxydocosa-4,7,11,13,15,19-hexaenoic acid

17-hydro-(peroxy)-docosahexaenoic acid