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Journal of Bacteriology, October 2000, p. 5359-5364, Vol. 182, No. 19
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Binding of Ferric Enterobactin by the Escherichia coli Periplasmic Protein FepB

Cathy Sprencel,1 Zhenghua Cao,1 Zengbiao Qi,1 Daniel C. Scott,1 Marjorie A. Montague,1 Nora Ivanoff,1 Jide Xu,2 Kenneth M. Raymond,2 Salete M. C. Newton,1 and Phillip E. Klebba1,*

Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019,1 and Department of Chemistry, University of California, Berkeley, California 947202

Received 25 April 2000/Accepted 6 July 2000

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 Kd 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 (Kd = 30 nM) and ferric enantioenterobactin (Kd = 15 nM), the left-handed stereoisomer of the natural E. coli siderophore. Purified FepB also adsorbed the apo-siderophore, enterobactin, with comparable affinity (Kd = 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.

* Corresponding author. Mailing address: Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019. Phone: (405) 325-4969. Fax: (405) 325-6111. E-mail: peklebba{at}ou.edu.

Journal of Bacteriology, October 2000, p. 5359-5364, Vol. 182, No. 19
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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