Structural stability of soybean lipoxygenase-1 in solution as probed by small angle X-ray scattering.
Soybean lipoxygenase-1 (LOX-1) is used widely as a model for studying the structural and functional properties of the homologous family of lipoxygenases. The crystallographic structure revealed that LOX-1 is organized in a beta-sheet N-terminal domain and a larger, mostly helical, C-terminal domain. Here, we describe the overall structural characterization of native unliganded LOX-1 in solution, using small angle X-ray scattering (SAXS). We show that the scattering pattern of the unliganded enzyme in solution does not display any significant difference compared with that calculated from the crystal structure, and that models of the overall shape of the protein calculated ab initio from the SAXS pattern provide a close envelope to the crystal structure. These data, demonstrating that LOX-1 has a compact structure also in solution, rule out any major motional flexibility of the LOX-1 molecule in aqueous solutions. In addition we show that eicosatetraynoic acid, an irreversible inhibitor of lipoxygenase used to mimic the effect of substrate binding, does not alter the overall conformation of LOX-1 nor its ability to bind to membranes. In contrast, the addition of glycerol (to 5%, v/v) causes an increase in the binding of the enzyme to membranes without altering its catalytic efficiency towards linoleic acid nor its SAXS pattern, suggesting that the global conformation of the enzyme is unaffected. Therefore, the compact structure determined in the crystal appears to be essentially preserved in these various solution conditions. During the preparation of this article, a paper by M. Hammel and co-workers showed instead a sharp difference between crystal and solution conformations of rabbit 15-LOX-1. The possible cause of this difference might be the presence of oligomers in the rabbit lipoxygenase preparations.
Dainese
Enrico
E
Department of Biomedical Sciences, University of Teramo, Piazza Aldo Moro 45, 64100 Teramo, Italy.
Sabatucci
Annalaura
A
van Zadelhoff
Guus
G
Angelucci
Clotilde Beatrice
CB
Vachette
Patrice
P
Veldink
Gerrit A
GA
Agrò
Alessandro Finazzi
AF
Maccarrone
Mauro
M
D015117:5,8,11,14-Eicosatetraynoic Acid / Q000378:metabolism; D003198:Computer Simulation; D004795:Enzyme Stability / Q000502:physiology*; D005990:Glycerol / Q000378:metabolism; D008084:Lipoxygenase / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D013025:Glycine max / Q000201:enzymology*; D014961:X-Ray Diffraction
D015117:5,8,11,14-Eicosatetraynoic Acid; D008084:Lipoxygenase; D005990:Glycerol