Abstract
North Pacific krill (Euphausia pacifica) contain 8R-hydroxy-eicosapentaenoic acid (8R-HEPE), 8R-hydroxy-eicosatetraenoic acid (8R-HETE) and 10R-hydroxy-docosahexaenoic acid (10R-HDHA). These findings indicate that E. pacifica must possess an R type lipoxygenase, although no such enzyme has been identified in krill. We analyzed E. pacifica cDNA sequence using next generation sequencing and identified two lipoxygenase genes (PK-LOX1 and 2). PK-LOX1 and PK-LOX2 encode proteins of 691 and 686 amino acids, respectively. Recombinant PK-LOX1 was generated in Sf9 cells using a baculovirus expression system. PK-LOX1 metabolizes eicosapentaenoic acid (EPA) to 8R-HEPE, arachidonic acid (ARA) to 8R-HETE and docosahexaenoic acid (DHA) to 10R-HDHA. Moreover, PK-LOX1 had higher activity for EPA than ARA and DHA. In addition, PK-LOX1 also metabolizes 17S-HDHA to 10R,17S-dihydroxy-docosahexaenoic acid (10R,17S-DiHDHA). PK-LOX1 is a novel lipoxygenase that acts as an 8R-lipoxygenase for EPA and 10R-lipoxygenase for DHA and 17S-HDHA. Our findings show PK-LOX1 facilitates the enzymatic production of hydroxy fatty acids, which are of value to the healthcare sector.
Highlights
Abbreviations LOX Lipoxygenase EPA Eicosapentaenoic acid DHA Docosahexaenoic acid arachidonic acid (ARA) Arachidonic acid HEPE Hydroxy-eicosapentaenoic acid HDHA Hydroxy-docosahexaenoic acid HETE Hydroxy-eicosatetraenoic acid MeOH Methanol polyunsaturated fatty acids (PUFAs) Polyunsaturated fatty acid LC/QTOFMS Liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry
Three types of 10,17-DiHDHA: 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Zhexaenoic acid, 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid and 10S,17S-dihydroxydocosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid were identified from human leukocytes and mouse exudates. 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid is a major type in human leucocyte and defined PD128
The biosynthesis of these three 10,17-DiHDHA compounds starts from DHA, which is acted upon by ALOX15 to generate Protectin D1 (PD1) by enzymatic epoxidation. 10,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Zhexaenoic acid is generated by double oxidation28. 10S,17S-dihydroxydocosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid (PDX) can be produced from DHA by soybean 15-lipoxygenase[29,30]
Summary
Abbreviations LOX Lipoxygenase EPA Eicosapentaenoic acid DHA Docosahexaenoic acid ARA Arachidonic acid HEPE Hydroxy-eicosapentaenoic acid HDHA Hydroxy-docosahexaenoic acid HETE Hydroxy-eicosatetraenoic acid MeOH Methanol PUFA Polyunsaturated fatty acid LC/QTOFMS Liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry. Lipoxygenases (LOXs) are non-heme iron-containing dioxygenases that catalyze the dioxygenation of polyunsaturated fatty acids (PUFAs)[1,2,3,4]. We showed that 8-HEPE could be produced enzymatically from EPA in E. pacifica[17,18] These observations suggest that E. pacifica has a lipoxygenase that metabolizes EPA to 8R-HEPE, ARA to 8R-HETE and DHA to 10R-HDHA. 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid is a major type in human leucocyte and defined PD128. The biosynthesis of these three 10,17-DiHDHA compounds starts from DHA, which is acted upon by ALOX15 to generate PD1 by enzymatic epoxidation. 10R,17S-DiHDHA produced by double dioxygenation (10R,17Sdihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid) has equipotent activity to PD1 and PDX in terms of blocking neutrophil infiltration and inhibiting platelet aggregation, r espectively[31,33] PDX shows higher activity for inhibiting platelet aggregation than PD132,33. 10R,17S-DiHDHA produced by double dioxygenation (10R,17Sdihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid) has equipotent activity to PD1 and PDX in terms of blocking neutrophil infiltration and inhibiting platelet aggregation, r espectively[31,33]
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