Binding of Ferric Enterobactin by theEscherichia coliPeriplasmic Protein FepB

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 enterobactinFepB 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.

Identification of the Periplasmic Cobalamin-Binding Protein BtuF ofEscherichia coli

“…Hydroxamate binding to FhuD was measured by quenching of protein tryptophan fluorescence and ranged from 300 to 400 nM for coprogen and aerobactin to 1 M for ferrichrome and to around 40 M for ferrioxamines (31). The affinity constants for binding of ferric enterobactin to FepB ranged from 135 nM when assayed by gel filtration to 30 nM when assayed by quenching of tryptophan fluorescence (39). The structures of the periplasmic iron-binding protein Hit from Haemophilus influenzae (8) and the ferrichrome-binding protein FhuD from E. coli (10) have been determined.…”

Enterobactin: An archetype for microbial iron transport

“…This transport mechanism of ferric siderophores seems widely conserved and includes: (i) binding of the substrate by the periplasmic binding protein, (ii) interaction of the binding protein with the cytoplasmic membrane spanning protein, and (iii) hydrolysis of ATP via a membrane bound protein to transport the ferric siderophore across the cytoplasmic membrane. Although the structure of the periplasmic enterobactin binding protein, FepB, has not yet been determined, recent binding studies suggest that this protein is far more specific for enterobactin (K d ϭ 30 nM) (64) than the similar catecholate siderophores vibriobactin (65) or agrobactin (64). Once carried across the periplasm by FepB, FeEnt is delivered to cytoplasmic transmembrane proteins (FepD and FepG).…”

High-Throughput Screening Assay for Inhibitors of TonB-Dependent Iron Transport

“…The TonB C-terminus adsorbs to a conserved site known as the TonB box at the N-terminus of TonB-dependent transporters (TBDTs) such as FepA, and this association is the basis of energy transfer. 4 After FeEnt passes into the periplasm, it binds to FepB, 5 which delivers it to the FepCDG ABC transporter 6 in the IM, which hydrolyzes ATP as it transports FeEnt into the cytoplasm.…”