Binding of ferric enterobactin by the Escherichia coli periplasmic protein FepB.
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.
Sprencel
C
C
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, USA.
Cao
Z
Z
Qi
Z
Z
Scott
D C
DC
Montague
M A
MA
Ivanoff
N
N
Xu
J
J
Raymond
K M
KM
Newton
S M
SM
Klebba
P E
PE
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D004758:Enterobactin / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005290:Ferric Compounds / Q000378:metabolism*; D015870:Gene Expression; D007504:Iron Radioisotopes / Q000378:metabolism; D026901:Membrane Transport Proteins; D051379:Mice; D019897:Periplasm / Q000378:metabolism; D033903:Periplasmic Proteins; D011485:Protein Binding; D011817:Rabbits; D017262:Siderophores / Q000378:metabolism
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D005290:Ferric Compounds; D007504:Iron Radioisotopes; D026901:Membrane Transport Proteins; D033903:Periplasmic Proteins; D017262:Siderophores; C061266:fepB protein, E coli; D004758:Enterobactin