Doctor of Philosophy (PhD)
Evidence suggests that 15-lipoxygenase-2 (15-LOX-2) plays an important role in the progression of atherosclerosis. Due to the medical need to discover isoform specific inhibitors of 15-LOX-2, a novel human enzyme, our goal was to determine the 3-dimensional structure. Using bacterial expression, we were able to obtain a usable quantity of enzyme with a sufficient amount of the catalytic iron bound to the enzyme, however it was sensitive to cleavage by proteases. This led to a ratio of roughly 1:1 of cleaved and uncleaved protein after the final step of purification. Solving the crystal structure revealed the reason for the protease sensitivity. In the polycistin-1 lipoxygenase alpha-toxin (PLAT) domain of the protein there is a large loop composed of nonpolar amino acids, which is flanked by calcium ions. The next step was to determine the purpose of calcium binding. Analytical size exclusion chromatography (SEC) experiments revealed that when calcium binding sites were mutated, the enzyme could not bind the synthetic phospholipid bilayer mimic, nanodiscs, which indicated that calcium is necessary for membrane binding. In the crystal structure, the active site of 15-LOX-2 contained a detergent molecule, C8E4, which was necessary for crystallization. This detergent molecule was later identified as a competitive inhibitor and its size and flexibility make it comparable to the enzyme’s natural substrate arachidonic acid (AA). Comparison of the structure of 15-LOX-2 with the stable 5-LOX structure suggested that the 5-LOX active site is occluded by the broken α2-helix and must open for substrate acquisition. This structure will provide information relevant to the design of isoform specific inhibitors which will aid in determining the function of 15-LOX-2 and could result in the development of therapeutic agents to protect against atherosclerotic plaque development.
Document Availability at the Time of Submission
Release the entire work immediately for access worldwide.
Kobe, Matthew James, "Structural and Kinetic Investigations of 15-Lipoxygenase-2" (2014). LSU Doctoral Dissertations. 2220.