PMID:10986237
| Citation |
Sprencel, C, Cao, Z, Qi, Z, Scott, DC, Montague, MA, Ivanoff, N, Xu, J, Raymond, KM, Newton, SM and Klebba, PE (2000) Binding of ferric enterobactin by the Escherichia coli periplasmic protein FepB. J. Bacteriol. 182:5359-64 |
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| Abstract |
The periplasmic protein FepB of Escherichia coli is a component of the ferric enterobactin transport system. We overexpressed and purified the binding protein 23-fold from periplasmic extracts by ammonium sulfate precipitation and chromatographic methods, with a yield of 20%, to a final specific activity of 15,500 pmol of ferric enterobactin bound/mg. Periplasmic fluid from cells overexpressing the binding protein adsorbed catecholate ferric siderophores with high affinity: in a gel filtration chromatography assay the K(d) of the ferric enterobactin-FepB binding reaction was approximately 135 nM. Intrinsic fluorescence measurements of binding by the purified protein, which were more accurate, showed higher affinity for both ferric enterobactin (K(d) = 30 nM) and ferric enantioenterobactin (K(d) = 15 nM), the left-handed stereoisomer of the natural E. coli siderophore. Purified FepB also adsorbed the apo-siderophore, enterobactin, with comparable affinity (K(d) = 60 nM) but did not bind ferric agrobactin. Polyclonal rabbit antisera and mouse monoclonal antibodies raised against nearly homogeneous preparations of FepB specifically recognized it in solid-phase immunoassays. These sera enabled the measurement of the FepB concentration in vivo when expressed from the chromosome (4,000 copies/cell) or from multicopy plasmids (>100,000 copies/cell). Overexpression of the binding protein did not enhance the overall affinity or rate of ferric enterobactin transport, supporting the conclusion that the rate-limiting step of ferric siderophore uptake through the cell envelope is passage through the outer membrane. |
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| Keywords |
Animals; Carrier Proteins/genetics; Carrier Proteins/isolation & purification; Carrier Proteins/metabolism; Chromatography, Affinity/methods; Enterobactin/metabolism; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins; Ferric Compounds/metabolism; Gene Expression; Iron Radioisotopes/metabolism; Membrane Transport Proteins; Mice; Periplasm/metabolism; Periplasmic Proteins; Protein Binding; Rabbits; Siderophores/metabolism |
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