Biosynthesis of lipid mediators including the leukotrienes, lipoxins, eoxins, resolvins, and protectins entails the lipoxygenase (LOX) catalyzed synthesis of an allylic epoxide intermediate, designated as leukotriene A4 (LTA4) and LTA analogues, and heretofore considered too unstable for direct structural characterization. In this dissertation I developed methods involving biphasic reaction conditions for the LOX-catalyzed synthesis of LTA epoxides and their structural analysis by NMR. As proof of concept, human 15-LOX-1 was shown to convert 15S-hydroperoxyeicosatetraenoic acid (15S-HPETE) to the LTA analogue, 14,15-LTA4 (thus identifying eoxin A4). Using this methodology I then showed that recombinant Arabidopsis AtLOX1, an arachidonate 5-LOX, converts 5S-HPETE to the trans epoxide LTA4, and 5R-HPETE to the cis epoxide 5-epi-LTA4. The results are reconciled with a mechanism based on a dual role of the LOX non-heme iron in LTA epoxide biosynthesis. The same methodology was used to structurally characterize the LTA-related epoxides from omega-3 fatty acids, including identification of the proposed epoxide intermediate in protectin biosynthesis.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-08232013-110456 |
| Date | 19 September 2013 |
| Creators | Jin, Jing |
| Contributors | Claus Schneider, Alan R. Brash, Sean S. Davies, David L. Hachey, Ned A. Porter |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-08232013-110456/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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