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 act...
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description | 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. |
doi_str_mv | 10.1128/JB.182.19.5359-5364.2000 |
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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.</description><identifier>ISSN: 0021-9193</identifier><identifier>EISSN: 1098-5530</identifier><identifier>DOI: 10.1128/JB.182.19.5359-5364.2000</identifier><identifier>PMID: 10986237</identifier><identifier>CODEN: JOBAAY</identifier><language>eng</language><publisher>United States: American Society for Microbiology</publisher><subject>Animals ; Bacteria ; Bacteriology ; Biochemistry ; Carrier Proteins - genetics ; Carrier Proteins - isolation & purification ; Carrier Proteins - metabolism ; Cell Surfaces ; Chromatography, Affinity - methods ; Enterobactin - metabolism ; Escherichia coli ; Escherichia coli - genetics ; Escherichia coli - metabolism ; Escherichia coli Proteins ; FepB protein ; Ferric Compounds - metabolism ; ferric enterobactin ; Gene Expression ; Iron Radioisotopes - metabolism ; Membrane Transport Proteins ; Mice ; Periplasm - metabolism ; Periplasmic Proteins ; Protein Binding ; Proteins ; Rabbits ; siderophores ; Siderophores - metabolism</subject><ispartof>Journal of bacteriology, 2000-10, Vol.182 (19), p.5359-5364</ispartof><rights>Copyright American Society for Microbiology Oct 2000</rights><rights>Copyright © 2000, American Society for Microbiology 2000</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c423t-5905746f77d7a9fd28b500a5f60477bd4085bb06281320a660cb60d7314f6f033</citedby><cites>FETCH-LOGICAL-c423t-5905746f77d7a9fd28b500a5f60477bd4085bb06281320a660cb60d7314f6f033</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC110977/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC110977/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10986237$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Sprencel, C</creatorcontrib><creatorcontrib>Cao, Z</creatorcontrib><creatorcontrib>Qi, Z</creatorcontrib><creatorcontrib>Scott, D C</creatorcontrib><creatorcontrib>Montague, M A</creatorcontrib><creatorcontrib>Ivanoff, N</creatorcontrib><creatorcontrib>Xu, J</creatorcontrib><creatorcontrib>Raymond, K M</creatorcontrib><creatorcontrib>Newton, S M</creatorcontrib><creatorcontrib>Klebba, P E</creatorcontrib><title>Binding of ferric enterobactin by the Escherichia coli periplasmic protein FepB</title><title>Journal of bacteriology</title><addtitle>J Bacteriol</addtitle><description>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.</description><subject>Animals</subject><subject>Bacteria</subject><subject>Bacteriology</subject><subject>Biochemistry</subject><subject>Carrier Proteins - genetics</subject><subject>Carrier Proteins - isolation & purification</subject><subject>Carrier Proteins - metabolism</subject><subject>Cell Surfaces</subject><subject>Chromatography, Affinity - methods</subject><subject>Enterobactin - metabolism</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli - metabolism</subject><subject>Escherichia coli Proteins</subject><subject>FepB protein</subject><subject>Ferric Compounds - metabolism</subject><subject>ferric enterobactin</subject><subject>Gene Expression</subject><subject>Iron Radioisotopes - metabolism</subject><subject>Membrane Transport Proteins</subject><subject>Mice</subject><subject>Periplasm - metabolism</subject><subject>Periplasmic Proteins</subject><subject>Protein Binding</subject><subject>Proteins</subject><subject>Rabbits</subject><subject>siderophores</subject><subject>Siderophores - metabolism</subject><issn>0021-9193</issn><issn>1098-5530</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2000</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkcFu1DAURa2Kqh0Kv4AsFuwSnp9jO1mwYKoWqCp1A2vLceyOq0wc7AxS_x6HqVBh05VlvXOtd30IoQxqxrD9eLOtWYs162rBRVcJLpsaAeCEbBh0bSUEh1dkA4Cs6ljHz8nrnB8AWNMIPCPnKySRqw2524ZpCNM9jZ56l1Kw1E2LS7E3dgkT7R_psnP0KtudK8NdMNTGMdC53ObR5H0JzCkurrDXbt6-IafejNm9fTovyI_rq--XX6vbuy_fLj_fVrZBvlSiA6Ea6ZUalOn8gG0vAIzwEhql-qGBVvQ9SGwZRzBSgu0lDIqzxksPnF-QT8d350O_d4MtSycz6jmFvUmPOpqg_51MYafv4y_NSnelSv7DUz7FnweXF70P2bpxNJOLh6wVolCA_EWQqRaVQCjg-__Ah3hIU_kEjajgT5ECtUfIpphzcv7vxgz0qlbfbHVRq1mnV7V6VatXtSX67nnjZ8GjS_4b8WCe0w</recordid><startdate>20001001</startdate><enddate>20001001</enddate><creator>Sprencel, C</creator><creator>Cao, Z</creator><creator>Qi, Z</creator><creator>Scott, D C</creator><creator>Montague, M A</creator><creator>Ivanoff, N</creator><creator>Xu, J</creator><creator>Raymond, K M</creator><creator>Newton, S M</creator><creator>Klebba, P E</creator><general>American Society for Microbiology</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20001001</creationdate><title>Binding of ferric enterobactin by the Escherichia coli periplasmic protein FepB</title><author>Sprencel, C ; Cao, Z ; Qi, Z ; Scott, D C ; Montague, M A ; Ivanoff, N ; Xu, J ; Raymond, K M ; Newton, S M ; Klebba, P E</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c423t-5905746f77d7a9fd28b500a5f60477bd4085bb06281320a660cb60d7314f6f033</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2000</creationdate><topic>Animals</topic><topic>Bacteria</topic><topic>Bacteriology</topic><topic>Biochemistry</topic><topic>Carrier Proteins - genetics</topic><topic>Carrier Proteins - isolation & purification</topic><topic>Carrier Proteins - metabolism</topic><topic>Cell Surfaces</topic><topic>Chromatography, Affinity - methods</topic><topic>Enterobactin - metabolism</topic><topic>Escherichia coli</topic><topic>Escherichia coli - genetics</topic><topic>Escherichia coli - metabolism</topic><topic>Escherichia coli Proteins</topic><topic>FepB protein</topic><topic>Ferric Compounds - metabolism</topic><topic>ferric enterobactin</topic><topic>Gene Expression</topic><topic>Iron Radioisotopes - metabolism</topic><topic>Membrane Transport Proteins</topic><topic>Mice</topic><topic>Periplasm - metabolism</topic><topic>Periplasmic Proteins</topic><topic>Protein Binding</topic><topic>Proteins</topic><topic>Rabbits</topic><topic>siderophores</topic><topic>Siderophores - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Sprencel, C</creatorcontrib><creatorcontrib>Cao, Z</creatorcontrib><creatorcontrib>Qi, Z</creatorcontrib><creatorcontrib>Scott, D C</creatorcontrib><creatorcontrib>Montague, M A</creatorcontrib><creatorcontrib>Ivanoff, N</creatorcontrib><creatorcontrib>Xu, J</creatorcontrib><creatorcontrib>Raymond, K M</creatorcontrib><creatorcontrib>Newton, S M</creatorcontrib><creatorcontrib>Klebba, P E</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Journal of bacteriology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Sprencel, C</au><au>Cao, Z</au><au>Qi, Z</au><au>Scott, D C</au><au>Montague, M A</au><au>Ivanoff, N</au><au>Xu, J</au><au>Raymond, K M</au><au>Newton, S M</au><au>Klebba, P E</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Binding of ferric enterobactin by the Escherichia coli periplasmic protein FepB</atitle><jtitle>Journal of bacteriology</jtitle><addtitle>J Bacteriol</addtitle><date>2000-10-01</date><risdate>2000</risdate><volume>182</volume><issue>19</issue><spage>5359</spage><epage>5364</epage><pages>5359-5364</pages><issn>0021-9193</issn><eissn>1098-5530</eissn><coden>JOBAAY</coden><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.</abstract><cop>United States</cop><pub>American Society for Microbiology</pub><pmid>10986237</pmid><doi>10.1128/JB.182.19.5359-5364.2000</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Animals Bacteria Bacteriology Biochemistry Carrier Proteins - genetics Carrier Proteins - isolation & purification Carrier Proteins - metabolism Cell Surfaces Chromatography, Affinity - methods Enterobactin - metabolism Escherichia coli Escherichia coli - genetics Escherichia coli - metabolism Escherichia coli Proteins FepB protein Ferric Compounds - metabolism ferric enterobactin Gene Expression Iron Radioisotopes - metabolism Membrane Transport Proteins Mice Periplasm - metabolism Periplasmic Proteins Protein Binding Proteins Rabbits siderophores Siderophores - metabolism |
title | Binding of ferric enterobactin by the Escherichia coli periplasmic protein FepB |
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