National Biomedical Resource for
Advanced ESR Spectroscopy

Portal to Lipoxygenase Active Site Revealed by Paramagnetic Distance Geometry

Lipoxygenase enzymes include members able to specifically target oxygen to different sites on arachidonate, and other polyunsaturated acyl chains. The diverse roles of lipoxygenases in development and inflammation originate in the specificity of this oxygenation. A question of how acyl chain oxidation is governed by structure arises because sequence and structure are highly conserved in this enzyme family. Determining where the polar and methyl ends of a substrate dock in lipoxygenases is critical to elucidating specificity. Pulsed dipolar EPR spectroscopy (PDS) experiments were designed to determine distances between selected points on the structure of the major isoform of soybean seed lipoxygenase (SBL1) and a point on the polar end of a lysooleoyllecithin substrate analog. The points are nitroxide spin labels. A low resolution "paramagnetic" structure of SBL1, with spin labels replacing natural side chains at five sites, was determined by optimizing the fit of measured PDS and calculated distances between ten doubly spin labeled constructs. Distances between the spin of the lysolecithin polar end and each single spin site on SBL1 were obtained. With these fifteen distances, a triangulation grid, constructed by distance geometry, localizes the polar end, and spin, of the lysolecithin in the SBL1 structure. The nearest neighbors for the lipid spin are atoms of residues K260 and residues in a loop: E236 and V237 and I257. This corresponds to a "tail first" insertion of the lysolecithin into SBL1 as is shown in the Figure below left (A) and its enlargement below right (B). Mutation of another nearby residue, E256A, relieves the high pH requirement for enzyme activity of SBL1 and allows lipid binding at pH 7.2.

Publication: B.J. Gaffney, M.D. Bradshaw, S. Frausto, F. Wu, J.H. Freed, and P. Borbat, Biophys. J., 103, 2134-2144 (2012); PMCID: PMC3512035.

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