Siderophores are small, high-affinity ferric iron chelators released by many microorganisms and some plants to solubilize iron. They are of great interest due to their clinical use to treat iron overload in humans, and also in relation to the development of novel antibiotics that target the biosynthetic and uptake pathways for iron in pathogens. Pathogens such as Bacillus anthracis excrete more than one type of siderophore. This is linked to increased pathogenicity. The Gram-positive soil bacterium Streptomyces coelicolor A3(2) excretes three siderophores: desferrioxamine B, desferrioxamine E and coelichelin. These displace iron from insoluble ferric hydroxides, and the resulting ferric complexes are transported into the cell via siderophore-binding proteins (lipoprotein receptors) associated with ATP-binding cassette (ABC) transporters. Previous studies showed that some of the genes in the biosynthetic clusters of the desferrioxamines (des) and coelichelin (cch) were required for efficient uptake of ferrioxamine E and ferri-coelichelin respectively and a third ABC transporter gene cluster (cdt), not associated with siderophore biosynthesis genes, was implicated in the import of ferrioxamine B. In this study, the lipoprotein receptors encoded within the des, cch and cdt clusters - DesE, CchF and CdtB – were recombinantly overproduced in E. coli and purified by immobilized metal affinity chromatography. Also, ferri-coelichelin was purified from cultures of S. coelicolor. The binding of the ferric complexes of the three cognate siderophores, as well as the xenosiderophores ferrichrome and ferrialbomycin, to the lipoprotein receptors was monitored by intrinsic fluorescence quenching. Dissociation constants of receptor-siderophore complexes were found to be in the nanomolar range, and a revised model of cognate siderophore transport in S. coelicolor was proposed. In collaboration with researchers at St. Andrews University, an X-ray crystal structure was solved for apo-DesE and DesE bound to ferrioxamine B, which demonstrated the similarity of DesE to other bacterial siderophore-binding proteins and the negligible conformational change on substrate binding. Ferrioxamine B also exhibited an unusual configuration not observed before in X-ray crystals of this ferri-siderophore. Also, a forcefield was constructed to model the structure and distortions ferric-tris-hydroxamate complexes, which could be used in the future to investigate the molecular basis of the tight and specific binding of ferri-siderophores to siderophore-binding proteins.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:524504 |
| Date | January 2009 |
| Creators | Patel, Prakash |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/2786/ |
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