Many aerobic microorganisms produce siderophores which are secreted into the environment. The siderophores chelate Fe3+ specifically. The complexation constants are very high (10 22-10/50). The complexes are too large (M.W. 800-1500) to be transported by porins. For competitive reasons microorganisms produce often more than one siderophore, and the structures of approximately 100 siderophores are known. Also, for better competition, bacteria produce more than one uptake system for ferric siderophores. For instance, under iron deficient conditions, E. coli has an uptake system for ferric enterobactin, the siderophore E. coli produces, consisting of FepA (in the outer membrane), FepB, C, D, and G in the periplasm and inner membrane. However, E. coli also has an uptake system for ferrichrome which it does not produce. This uptake system uses FhuA in the outer membrane and associated Fhu proteins in the periplasm and inner membrane. E. coli also has a citrate inducible uptake system for ferric citrate which uses FecA in the O.M. It is our plan, in cooperation with Dr. Deisenhofer, to determine the crystal structures of FepA, FhuA and their complexes with Fe enterobactin and ferrichrome, respectively, as well as FecA. All these proteins have homology but are different from any known protein. Native data sets (2.8 angstroms) have been taken for FepA, diffraction-quality crystals are available for Se-Met-FepA and both FepA-Fe(ent) and FhuA-ferrichrome complexes have been crystallized. The proteins require detergents also for crystallization. The O.M. of gram negative bacteria has no energy source. TonB, anchored in the I.M., is known to interact with FepA and FhuA. We hypothesize in this proposal a detailed mechanism for transport which can be probed by the structure determinations and the planned binding experiments which are described. The specificity of transport resides in the O.M. proteins, FepA and FhuA. This specificity will be probed: a) by point mutations in the protein (primarily FepA); b) by using structurally similar siderophores (primarily for FhuA); and c) by changing the Fe3+ in the complexes for similar ions (GA3+, Al3+ and In3+). This will be done with binding experiments and difference Fouriers. The intent of the proposal is to understand, in detail, the specific transport of large molecules across a membrane.
| Piyamongkol, Sirivipa; Ma, Yong M; Kong, Xiao L et al. (2010) Amido-3-hydroxypyridin-4-ones as iron(III) ligands. Chemistry 16:6374-81 |
