Lipoxygenase enzymes catalyze the first step in one of the major pathways to signalling molecules derived from arachidonic acid. A theme emerging from the medical perspective on different human lipoxygenases is one of both positive and negative influences on human health from these molecules. The overall focus of this proposal is to understand how the active site cavity of lipoxygenase domains interact with fatty acids in catalytically relevant states. The approaches developed will have broader applications to inhibition of lipoxygenase and other pharmaceutical target enzymes having lipid substrates, and to design of NMR structural studies based on paramagnetic relaxation. Here, electron paramagnetic resonance (EPR) spectroscopy experiments are proposed to map interactions of substrate/product analogs with the active site cavity of a lipoxygenase. The prototypical lipoxygenase, soybean isoform 1, LOX-1, is the subject of most experiments.
The specific aims are: .
Specific Aim 1 - A grid of distances between spins in doubly-spin labeled LOX-1 will be established by pulsed dipolar EPR spectroscopy (PDS). Positions of introduced spin labels within the structure will be determined by triangulation and modeling, to facilitate subsequent protein-ligand distance determinations.
Specific Aim 2 - The location of the doxyl spin of spin label stearates (DSAs), inhibitors of lipoxygenase, will be determined by EPR distance measurements between DSAs and covalently spin labeled LOX-1 grid sites.
Specific Aim 3 - A hypothesis that fatty acid hydroperoxides (activators) and the substrate interact differently with the lipoxygenase cavity will be examined using spin label nitroxide esters at the polar ends of substrate and product analogs. Each of the spin label esters proposed in this section will be prepared in two versions: with an oleic acyl chain for EPR distance measurements and with a linoleoyl chain for analysis of enzymatic products.
Specific Aim 4 - Fatty acid binding to the LOX-1 mutant, A542T, that is inactive for turnover, will be characterized with EPR studies of ferric iron in the mutant and spin-labeled fatty acid localization.
Specific Aim 5 - Similar approaches will be taken to map fatty acid interactions with the coral allene oxide synthase (cAOS) cavity.

Public Health Relevance

In humans, lipoxygenase isoforms are tissue specific and are related to pathological and house-keeping cell functions and inhibitors must be selective. The work proposed will develop substrate analogs that include a versatile spectroscopic functionality that can be used to examine how and where an inhibitor resides in the protein structure. Lipoxygenases are important in other organisms also and the methods could be widely applied to understand non-mammalian signalling pathways as well. The type of medium-resolution EPR structure obtained here can provide essential background for higher resolution NMR structure studies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM065268-08
Application #
8204959
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Smith, Ward
Project Start
2003-04-10
Project End
2014-12-31
Budget Start
2012-01-01
Budget End
2014-12-31
Support Year
8
Fiscal Year
2012
Total Cost
$199,130
Indirect Cost
$77,317
Name
Florida State University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
790877419
City
Tallahassee
State
FL
Country
United States
Zip Code
32306
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