Receptor outer membrane siderophore

While most workers report the outer membrane siderophore receptors to have molecular weights in the 75-95K range, some variation in the magnitude of these numbers may be attributed to the preparative and analytical methods as well as to the particular standards used. Since enterobactin will rapidly remove iron from ferrichrome, the transport of the latter must perforce be studied in mutants lacking the former. However, such mutants often display multiple lesions. Additionally, isogenic strains have seldom been used and variations in media and cultural conditions will further confound attempts to compare results reported from different laboratories. 

In the past few years, crystal structmes have been published of three of the seven outer-membrane-siderophore receptors from E.coli, namely of FepA,FhuA, and FecA. FepA and FhuA represent similar monomeric transmembrane proteins that are composed of 22 antiparallel B-strands of approximately 70 A height (Figme 11). The right-handed twist of the /3-strands produces an ellipticalshaped barrel with a diameter of 35 x 47 A constituting a transmembrane pore. Targe extracellular loops extend... 

In the past few years, crystal structures have been published of three of the seven outer-membrane-siderophore receptors from E.coli, namely of FepA, FhuA, and Fee FepA and FhuA represent similar monomeric... 

However, this approach still produces racemic mixtures that preclude assessing chiral recognition events taking place between siderophore mimics and their bacterial outer-membrane receptors. Since many siderophore receptors were shown to exhibit high enan-tioselectivity " " , it was anticipated that pure enantiomeric siderophore analogs would allow one to identify the preferred directionality of the helical twist about the iron for the specihc receptors. 

In times of iron deficiency, many bacteria and fungi release low molecular weight chelators called siderophores (see Iron Transport Siderophores). These molecules bind ferric iron tightly and the ferric-siderophore complexes are then transported into the cell by a system of uptake proteins. The first stage in the uptake process involves an outer membrane receptor specific to each siderophore. One of the best characterized of these receptors is FhuA, the ferrichrome uptake receptor of E. coli, and we will describe this in detail. However, though other ferric-siderophore complexes are taken up by cells, and their iron released by systems similar to those of ferrichrome, their mechanisms may vary from those of ferrichrome in some respects. FepA and FecA" are two of the outer membrane ferric-siderophore receptors that have recently been structurally characterized. 

Figure 11 Crystal structure of the OM-siderophore receptor FhuA with the bound siderophore ferrichrome (emphasized by a space filling atom presentation). The plot is based on PDB file IQFF, taken from the NCBl database using the program PYMOL. Resolution of the structure is 2.70 A, and the space group is P6i. The upper side of the figure corresponds to the extracellular side of the outer membrane. See Section 5.2 for a detailed discussion...

Figure 2 Siderophore-mediated iron-uptake systems in E. coli. Siderophore-iron complexes bind to transporter proteins located in the outer membrane (also known as OM), a barrier that is characteristic of Gram-negative bacteria. The region between the outer and inner is known as the periplasmic space. Specific carrier proteins such as FhuD transport iron fi om the outer membrane to the inner or cytosolic membrane (also known as CM). The TonB/ExbB/ExbD complex spans the inner and outer membranes and interacts with FepA, as shown, as well as all of the outer membrane receptors. The linkage that the TonB/ExbB/ExbD complex provides between the inner or cytosolic membrane to the outer membrane is thought to allow transmission of sufficient energy from the cytosol to drive siderophore-iron uptake across the outer membrane...

Siderophores. If a suitably high content of iron (e.g., 50 pM or more for E. coli) is maintained in the external medium, bacteria and other microorganisms have little problem with uptake of iron. However, when the external iron concentration is low, special compounds called siderophores are utilized to render the iron more soluble. For example, at iron concentrations below 2 pM, E. coli and other enterobacteria secrete large ammmts of enterobactin (Fig. 16-1). The stable Fe +-enterobactin complex is taken up by a transport system that involves receptors on the outer bacterial membrane. Siderophores from many bacteria have in common with enterobactin the presence of catechol (ort/zo-dihydroxybenzene) groups... 

All outer-membrane transporters (OMTs) involved in iron uptake are made up of a 22-stranded p—barrel, which is occluded by an independently folded mixed a P globular cork domain of around 160 amino acid residues. This is illustrated for a vitamin B12 receptor and the ferric siderophore receptors for citrate, enterobactin, ferrichrome, pyochelin, and pyoverdin from E. coli and P. aeruginosa in Fig. 7.6. The ferric siderophore sits on top of the cork domain, as can be seen in Fig. 7.7 for FecA. The binding of the ligand induces a conformational... 

The outer-membrane receptors exhibit two functional states. In an unenergized conformation the siderophore binds to the receptor, but is not transported through the membrane. The energized state of the receptors requires a periplasmic protein, named TonB, which serves as a coupling device between the cytoplasmic membrane and the outer-membrane receptors. It is assumed that the above mentioned... 

Soft rot spreading depends on the efficiency of the iron uptake pathway mediated by the siderophore chrysobactin. Biosynthesis of the ferrichrysobactin outer membrane receptor (Fct) and of the chrysobactin precursor, i.e. the activated form of 2,3-dihydroxybenzoic acid, are encoded by an operon,/cr ebsCEBA [3]. 

The Fur protein regulates iron uptake systems in many Gram-negative bacteria. The striking phenotype of the first fur mutants isolated was the overexpression of the outer membrane receptors for siderophore iron transport. In addition, excretion of siderophores under iron-rich growth conditions was observed in these mutants, indicating that the biosynthesis of siderophores is also regulated by Fur. 

The first of the haem uptake systems to be characterized at molecular level was that of Yersinia enterolitica, which closely resembles a typical siderophore uptake system (Stojiljkovic and Hantke, 1992, 1994), including a TonB-dependent outer membrane receptor for haem, a periplasmic binding protein, and a cytoplasmic membrane transport system. There also seems to be a protein that degrades haem and liberates haem iron within the cell. TonB-dependent outer membrane receptor proteins for haem have been cloned and sequenced from Shigella dysenteriae and E. coli (Mills and Payne, 1995 Torres and Payne, 1997), while in Vibrio cholera two genes are required for haem utilization, one an outer membrane receptor a second which may have a TonB-like function (Henderson and Payne, 1994). 

Specific receptors for siderophores and vitamin B12 have been identified in the OM of Gram-negative bacteria. The translocation of these ligands across the outer membrane follows an energy-dependent mechanism and also involves the TonB, ExbB, ExbD proteins anchored in the cytoplasmic membrane. Biochemical and genetic data indicate that these proteins form a functional unit (the Ton complex), which couples the outer membrane receptor-mediated... 

As mentioned above, transport of siderophores across the cytoplasmic membrane is less specific than the translocation through the outer membrane. In E. coli three different outer membrane proteins (among them FepA the receptor for enterobactin produced by most E. coli strains) recognise siderophores of the catechol type (enterobactin and structurally related compounds), while only one ABC system is needed for the passage into the cytosol. Likewise, OM receptors FhuA, FhuE, and Iut are needed to transport a number of different ferric hydroxamates, whereas the FhuBCD proteins accept a variety of hydroxamate type ligands such as albomycin, ferrichrome, coprogen, aerobactin, shizokinen, rhodotorulic acid, and ferrioxamine B [165,171], For the vast majority of systems, the substrate specificity has not been elucidated, but it can be assumed that many siderophore ABC permeases might be able to transport several different but structurally related substrates. 

Figure 7.2 Schematic representation of siderophore-mediated iron uptake in E. coli. The TonB-ExbB-ExbD complex energizes and interacts with all the outer membrane receptors, not just FepA. (From Andrews et al., 2003. Reproduced with permission from Blackwell Publishing Ltd.)...

Hantke K, Nicholson G. Rabsch W, Winkelmann G (2003) Salmochelins, Siderophores of Salmonella enterica and Uropathogenic Escherichia coli Strains, are Recognized by the Outer Membrane Receptor Iron. Proc Natl Acad Sci USA 100 3677 Harada K, Tomita K, Fuji K, Masuda K, Mikami Y, Yazawa K, Komaki H (2004) Isolation and Structural Characterization of Siderophores, Madurastatins, Produced by a Pathogenic Actinomadura madurae. J Antibiot 57 125... 

When deficient in iron, bacteria and fungi produce and excrete to the extracellular medium low molecular weight, specific iron-carrier molecules, called siderophores. These siderophores bind ferric ions, to form soluble complexes. The complexed ferric ions are transported into the cell through high-affinity and energy-dependent receptor proteins located on the outer membrane. In Gram-negative bacteria, such as E. coli, the most studied system, siderophore-iron complexes are transported initially to the periplasm. 

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