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 of inflammation. Their biosynthesis has been proposed to proceed through sequential oxidations of docosahexaenoic acid (DHA) by lipoxygenase enzymes, specifically by h5-LOX performing the first oxidation to 7S-HDHA followed by h12-LOX oxidation to form 7S,14S-diHDHA or h15-LOX-1 oxidation to form RvD5. In this work, we determine that the oxidation of 7S-HpDHA to 7S,14S-diHDHA can be carried out 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 7S,14S-diHDHA was unexpected. This result raised questions regarding biosynthesis of RvD5. Strikingly, we find that h15-LOX-2 oxygenates 7S-HDHA almost exclusively at C17 to form RvD5, with faster kinetics than that of h15-LOX-1. We also determine that the reactions of h5-LOX with 14S-HpDHA and 17S-HpDHA are kinetically slow, suggesting these may be minor biosynthetic routes in vivo.

We determine that altered positional specificity of h15-LOX-1 extends to 5S-HETE and show that 15-LOX-2 plays a greater role in generating 5S,15S-diHETE, while h15-LOX-1 primarily generates the non-canonical product, 5S,12S-diHETE.

Previous work has indicated numerous active-site amino acids that influence regiospecificity of h15-LOX-1 with AA. We extend this work to oxylipins and show that the depth of the active site as determined by I417 and F352 strongly affects the regiospecificity with 7S-HDHA and 5S-HETE, with F352W almost entirely reversing the altered positional specificity seen in wildtype 15-LOX-1.

Our results may extend beyond the resolution of inflammation. Specifically, we show that both 7S,14S-diHDHA and RvD5 have anti-aggregation properties with platelets at low micro-molar concentrations, which could directly regulate clot resolution. 15-(S)hydroxyeicosatrienoic acid (15-HETrE), generated by reaction of the omega-6 polyunsaturated fatty acid dihomo-γ-linolenic acid (DGLA) with h15-LOX-1, is known to inhibit platelet activation through an unknown mechanism. To investigate the basis of this inhibition, human platelets were treated with 15-HETrE and platelet aggregation was assayed by various methods. 15-HETrE was shown to inhibit platelet activation through activation of PPARβ and inhibition of 12-lipoxygenase (12-LOX).

The research presented in this dissertation focuses on two distinct areas of 15-lipoxygenase biology: screening and identification of inhibitors toward human 15-lipoxygenase enzymes and biological and biochemical characterization of a 15-lipoxygenase enzyme (p15-LOX) from the opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa). Two novel, selective inhibitors are reported for human 15-lipoxygenase-2 (15-LOX-2). The reported compounds were micromolar active with competitive and mixed type inhibition. They will serve as chemical tools for future studies to more deeply probe the role of 15-LOX-2 in atherosclerosis, cancer, and for general elucidation of 15-LOX-2 function in tissues. In addition, the binding affinities for five nanomolar active human 15-lipoxygenase-1 inhibitors were determined by a novel mass spectrometric assay with the ability to measure binding affinities well below the current limit of quantitation for the traditional UV assay. The assay also represents the first of its kind capable of assessing allosteric effect in a high throughput, 96-well format, opening the possibility of screening compound libraries to identify allosteric effectors to lipoxygenase (LOX) enzymes.

Finally, a number of biochemical and biological properties of the LOX enzyme expressed by P. aeruginosa were determined. The most actively metabolized substrate is AA and K_cat far exceeds human homologues at low pH. The enzyme is active with a variety of polyunsaturated fatty acid substrates, but phospholipids are not metabolized well compared to human LOX enzymes. Selective inhibitors to human LOX isozymes do not significantly inhibit p15-LOX. Expression of LOX during P. aeruginosa infection increases observed cytokines from several cell types. Together these studies support the hypothesis that this enzyme is capable of disrupting host immunity, but do not strongly support expression of p15-LOX solely for this purpose. Subsequent studies are needed to further elucidate the biological function of p15-LOX.

The research contained in this dissertation examines the kinetic, mechanistic, and structural basis of human lipoxygenase (LOX) catalysis. LOX are found throughout the plant and animals kingdoms, and in humans LOX are involved in inflammatory signaling. Through this involvement in the inflammatory process and the ability to oxidize membrane bound polyunsaturated fatty acids (PUFA), LOX have been implicated in a variety of cancers and disease states. The impact of substrate saturation on platelet 12-LOX kinetics and mechanism was investigated in Chapter 2. The platelet signaling pathways and implications on platelet aggregation of these substrates were determined. Additionally, the novel allosteric effect of 15S-HpEPE on 12-LOX mechanism was probed. Chapter 3 scrutinized a previously discovered lipoxygenase activator. This dissertation works shows that the activator is LOX isoform specific, with only h15-LOX-1 being kinetically activated in the presence of the compound. Furthermore, the activator was found to activate the catalysis of shorter PUFA substrates, AA and LA, but not that of the longer SPM precursor, DHA. The implications of this finding has consequences on the potential therapeutic development of the activating compound. The effect on enzyme kinetics and mechanism was assayed, and a V-type activation was observed. Chapter 4 builds off of a previous discovery in our lab that demonstrated the altered positional specificity of h15-LOX-1 when reacting with the metabolites of 5-LOX. In this chapter, site-directed mutagenesis was performed on key active site residues, with the kinetic and mechanistic implications of those residues determined experimentally. Additionally, the consequence of the altered positional specificity with regards to the biosynthesis of the potent SPM, Resolvin E4, was examined.