Altered specificity of 15-LOX-1 in the biosynthesis of 7S,14S-diHDHA implicates 15-LOX-2 in biosynthesis of resolvin D5

Perry, S. C.; Kalyanaraman, C.; Tourdot, B. E.; Conrad, W. S.; Akinkugbe, O.; Freedman, J. C.; Holinstat, M.; Jacobson, M. P.; Holman, T. R.

doi:10.1101/2020.03.25.008573

bioRxiv · 10.1101/2020.03.25.008573

Altered specificity of 15-LOX-1 in the biosynthesis of 7S,14S-diHDHA implicates 15-LOX-2 in biosynthesis of resolvin D5

Perry, S. C.·Kalyanaraman, C.·Tourdot, B. E.·Conrad, W. S.·Akinkugbe, O.·Freedman, J. C.·Holinstat, M.·Jacobson, M. P.·Holman, T. R.

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Abstract

The oxylipins, 7S,14S-diHDHA and 7S,17S-diHDHA (RvD5), have been found in macrophages exudates and are believed to function as specialized pro-resolving mediators (SPMs). Their biosynthesis is thought to proceed through sequential oxidations of docosahexaenoic acid (DHA) by lipoxygenase enzymes, specifically by h5-LOX first to 7S-HDHA, followed by h12-LOX to form 7S,14S-diHDHA or h15-LOX-1 to form 7S,17S-diHDHA (RvD5). In this work, we determined that oxidation of 7S-HpDHA to 7S,14S-diHDHA can be performed by either h12-LOX or h15-LOX-1, with similar kinetics. The oxidation at C14 of DHA by h12-LOX was expected, but the non-canonical reaction of h15-LOX-1 to make primarily 7S,14S-diHDHA was unexpected. Computer modeling suggests the alcohol on C7 of 7S-HDHA hydrogen bonds with the backbone carbonyl of I399, forcing the hydrogen abstraction from C12 to oxygenate on C14, and not C17. This result raised questions regarding synthesis of 7S,17S-diHDHA (RvD5). Strikingly, we find h15-LOX-2 oxygenates 7S-HDHA almost exclusively at C17, forming RvD5 with faster kinetics than h15-LOX-1. The presence of h15-LOX-2 in neutrophils and macrophages, suggests it may have a greater role in biosynthesizing SPMs than previously thought. We also determined that the reactions of h5-LOX with 14S-HpDHA and 17S-HpDHA are kinetically slow compared to DHA, suggesting these may be minor biosynthetic routes in-vivo. Additionally, we show that 7S,14S-diHDHA and RvD5 have anti-aggregation properties with platelets at low micro-molar potencies, which could directly regulate clot resolution.

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Perry, S. C., Kalyanaraman, C., Tourdot, B. E., Conrad, W. S., Akinkugbe, O., Freedman, J. C., Holinstat, M., Jacobson, M. P., Holman, T. R.. 2020-03-25. Altered specificity of 15-LOX-1 in the biosynthesis of 7S,14S-diHDHA implicates 15-LOX-2 in biosynthesis of resolvin D5. https://doi.org/10.1101/2020.03.25.008573

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