Subcloning of the enterobactin biosynthetic gene entB: expression, purification, characterization and substrate specificity of isochorismatase
The Escherichia coli entB gene, coding for the enterobactin biosynthetic enzyme isochorismatase, has been subcloned into the multicopy plasmid pKK223-3 under the control of the tac promoter. The resulting recombinant plasmid pFR1 expresses isochorismatase amounting to over 50% of the total cellular...
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| Veröffentlicht in: | Biochemistry (Easton) 1990-02, Vol.29 (6), p.1425-1435 |
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| creator | Rusnak, Frank Liu, Jun Quinn, Nina Berchtold, Glenn A Walsh, Christopher T |
| description | The Escherichia coli entB gene, coding for the enterobactin biosynthetic enzyme isochorismatase, has been subcloned into the multicopy plasmid pKK223-3 under the control of the tac promoter. The resulting recombinant plasmid pFR1 expresses isochorismatase amounting to over 50% of the total cellular protein. The enzyme has been purified to homogeneity and a convenient assay developed. The enzyme has a Km for isochorismate of 14.7 microM and a turnover number of 600 min-1. By use of 1H NMR spectroscopy, the progress of the reaction was followed with the expected formation of 2,3-dihydro-2,3-dihydroxybenzoate product. Several substrate analogues were also utilized by the enzyme including chorismic acid, the immediate precursor to isochorismic acid in the enterobactin biosynthetic pathway. |
| doi_str_mv | 10.1021/bi00458a013 |
| format | Article |
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The resulting recombinant plasmid pFR1 expresses isochorismatase amounting to over 50% of the total cellular protein. The enzyme has been purified to homogeneity and a convenient assay developed. The enzyme has a Km for isochorismate of 14.7 microM and a turnover number of 600 min-1. By use of 1H NMR spectroscopy, the progress of the reaction was followed with the expected formation of 2,3-dihydro-2,3-dihydroxybenzoate product. Several substrate analogues were also utilized by the enzyme including chorismic acid, the immediate precursor to isochorismic acid in the enterobactin biosynthetic pathway.</description><identifier>ISSN: 0006-2960</identifier><identifier>EISSN: 1520-4995</identifier><identifier>DOI: 10.1021/bi00458a013</identifier><identifier>PMID: 2139796</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>550201 - Biochemistry- Tracer Techniques ; BACTERIA ; BARYONS ; Base Sequence ; BASIC BIOLOGICAL SCIENCES ; BENZOIC ACID ; BIOCHEMICAL REACTION KINETICS ; BIOLOGICAL PATHWAYS ; CARBOXYLIC ACIDS ; CELL CONSTITUENTS ; CLONING ; Cloning, Molecular ; DNA HYBRIDIZATION ; DNA-CLONING ; ELEMENTARY PARTICLES ; Enterobactin - metabolism ; ENZYMES ; ESCHERICHIA COLI ; Escherichia coli - enzymology ; Escherichia coli - genetics ; FERMIONS ; Gene Expression Regulation, Enzymologic ; GENES ; HADRONS ; HYBRIDIZATION ; Hydrogen-Ion Concentration ; Hydrolases - genetics ; Hydrolases - isolation & purification ; Hydrolases - metabolism ; KINETICS ; MAGNETIC RESONANCE ; Magnetic Resonance Spectroscopy ; MICROORGANISMS ; Molecular Sequence Data ; MOLECULAR STRUCTURE ; MONOCARBOXYLIC ACIDS ; NUCLEAR MAGNETIC RESONANCE ; NUCLEONS ; ORGANIC ACIDS ; ORGANIC COMPOUNDS ; OXIDOREDUCTASES ; PLASMIDS ; Promoter Regions, Genetic ; PROTONS ; PURIFICATION ; REACTION KINETICS ; RESONANCE ; Serine - analogs & derivatives ; Substrate Specificity ; SUBSTRATES</subject><ispartof>Biochemistry (Easton), 1990-02, Vol.29 (6), p.1425-1435</ispartof><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a447t-878cb9154d3fd4199e1848b940f0f4d37c21a620e87a716c5b12e1b7530d4df73</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/bi00458a013$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/bi00458a013$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>230,314,780,784,885,2765,27076,27924,27925,56738,56788</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/2139796$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://www.osti.gov/biblio/6940841$$D View this record in Osti.gov$$Hfree_for_read</backlink></links><search><creatorcontrib>Rusnak, Frank</creatorcontrib><creatorcontrib>Liu, Jun</creatorcontrib><creatorcontrib>Quinn, Nina</creatorcontrib><creatorcontrib>Berchtold, Glenn A</creatorcontrib><creatorcontrib>Walsh, Christopher T</creatorcontrib><title>Subcloning of the enterobactin biosynthetic gene entB: expression, purification, characterization and substrate specificity of isochorismatase</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>The Escherichia coli entB gene, coding for the enterobactin biosynthetic enzyme isochorismatase, has been subcloned into the multicopy plasmid pKK223-3 under the control of the tac promoter. The resulting recombinant plasmid pFR1 expresses isochorismatase amounting to over 50% of the total cellular protein. The enzyme has been purified to homogeneity and a convenient assay developed. The enzyme has a Km for isochorismate of 14.7 microM and a turnover number of 600 min-1. By use of 1H NMR spectroscopy, the progress of the reaction was followed with the expected formation of 2,3-dihydro-2,3-dihydroxybenzoate product. Several substrate analogues were also utilized by the enzyme including chorismic acid, the immediate precursor to isochorismic acid in the enterobactin biosynthetic pathway.</description><subject>550201 - Biochemistry- Tracer Techniques</subject><subject>BACTERIA</subject><subject>BARYONS</subject><subject>Base Sequence</subject><subject>BASIC BIOLOGICAL SCIENCES</subject><subject>BENZOIC ACID</subject><subject>BIOCHEMICAL REACTION KINETICS</subject><subject>BIOLOGICAL PATHWAYS</subject><subject>CARBOXYLIC ACIDS</subject><subject>CELL CONSTITUENTS</subject><subject>CLONING</subject><subject>Cloning, Molecular</subject><subject>DNA HYBRIDIZATION</subject><subject>DNA-CLONING</subject><subject>ELEMENTARY PARTICLES</subject><subject>Enterobactin - metabolism</subject><subject>ENZYMES</subject><subject>ESCHERICHIA COLI</subject><subject>Escherichia coli - enzymology</subject><subject>Escherichia coli - genetics</subject><subject>FERMIONS</subject><subject>Gene Expression Regulation, Enzymologic</subject><subject>GENES</subject><subject>HADRONS</subject><subject>HYBRIDIZATION</subject><subject>Hydrogen-Ion Concentration</subject><subject>Hydrolases - genetics</subject><subject>Hydrolases - isolation & purification</subject><subject>Hydrolases - metabolism</subject><subject>KINETICS</subject><subject>MAGNETIC RESONANCE</subject><subject>Magnetic Resonance Spectroscopy</subject><subject>MICROORGANISMS</subject><subject>Molecular Sequence Data</subject><subject>MOLECULAR STRUCTURE</subject><subject>MONOCARBOXYLIC ACIDS</subject><subject>NUCLEAR MAGNETIC RESONANCE</subject><subject>NUCLEONS</subject><subject>ORGANIC ACIDS</subject><subject>ORGANIC COMPOUNDS</subject><subject>OXIDOREDUCTASES</subject><subject>PLASMIDS</subject><subject>Promoter Regions, Genetic</subject><subject>PROTONS</subject><subject>PURIFICATION</subject><subject>REACTION KINETICS</subject><subject>RESONANCE</subject><subject>Serine - analogs & derivatives</subject><subject>Substrate Specificity</subject><subject>SUBSTRATES</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1990</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNptkUuP0zAUhS0EGsrAijWSxQIWELATJ07Y0REvacRDUxBiY9nOzdRDawdfR5ryI_jNuE01YsHKuud8PtfyIeQhZy84K_lL4xgTdasZr26RBa9LVoiuq2-TBWOsKcquYXfJPcSrPAomxQk5KXnVya5ZkD8Xk7Gb4J2_pGGgaQ0UfIIYjLbJeWpcwJ3PcnKWXoI_2MtXFK7HCIgu-Od0nKIbnNXpMNm1jvkuRPf7oFDte4qTwRR1Aooj2D3t0m6_0GGw6xAdbnXSCPfJnUFvEB4cz1Py9e2b1dn74vzTuw9nr88LLYRMRStbazpei74aesG7DngrWtMJNrAhi9KWXDclg1ZqyRtbG14CN7KuWC_6QVan5PGcGzA5hfk1YNc2eA82qSbntIJn6MkMjTH8mgCT2jq0sNloD2FCJTvZcM5ZBp_NoI0BMcKgxui2Ou4UZ2pfkfqnokw_OsZOZgv9DXvsJPvF7DtMcH1j6_hTNbKStVp9vlDNj9Xyy7fvS_Ux809nXltUV2GKPv_cfzf_BfOTqm8</recordid><startdate>19900213</startdate><enddate>19900213</enddate><creator>Rusnak, Frank</creator><creator>Liu, Jun</creator><creator>Quinn, Nina</creator><creator>Berchtold, Glenn A</creator><creator>Walsh, Christopher T</creator><general>American Chemical Society</general><scope>BSCLL</scope><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>7X8</scope><scope>OTOTI</scope></search><sort><creationdate>19900213</creationdate><title>Subcloning of the enterobactin biosynthetic gene entB: expression, purification, characterization and substrate specificity of isochorismatase</title><author>Rusnak, Frank ; Liu, Jun ; Quinn, Nina ; Berchtold, Glenn A ; Walsh, Christopher T</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a447t-878cb9154d3fd4199e1848b940f0f4d37c21a620e87a716c5b12e1b7530d4df73</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1990</creationdate><topic>550201 - Biochemistry- Tracer Techniques</topic><topic>BACTERIA</topic><topic>BARYONS</topic><topic>Base Sequence</topic><topic>BASIC BIOLOGICAL SCIENCES</topic><topic>BENZOIC ACID</topic><topic>BIOCHEMICAL REACTION KINETICS</topic><topic>BIOLOGICAL PATHWAYS</topic><topic>CARBOXYLIC ACIDS</topic><topic>CELL CONSTITUENTS</topic><topic>CLONING</topic><topic>Cloning, Molecular</topic><topic>DNA HYBRIDIZATION</topic><topic>DNA-CLONING</topic><topic>ELEMENTARY PARTICLES</topic><topic>Enterobactin - metabolism</topic><topic>ENZYMES</topic><topic>ESCHERICHIA COLI</topic><topic>Escherichia coli - enzymology</topic><topic>Escherichia coli - genetics</topic><topic>FERMIONS</topic><topic>Gene Expression Regulation, Enzymologic</topic><topic>GENES</topic><topic>HADRONS</topic><topic>HYBRIDIZATION</topic><topic>Hydrogen-Ion Concentration</topic><topic>Hydrolases - genetics</topic><topic>Hydrolases - isolation & purification</topic><topic>Hydrolases - metabolism</topic><topic>KINETICS</topic><topic>MAGNETIC RESONANCE</topic><topic>Magnetic Resonance Spectroscopy</topic><topic>MICROORGANISMS</topic><topic>Molecular Sequence Data</topic><topic>MOLECULAR STRUCTURE</topic><topic>MONOCARBOXYLIC ACIDS</topic><topic>NUCLEAR MAGNETIC RESONANCE</topic><topic>NUCLEONS</topic><topic>ORGANIC ACIDS</topic><topic>ORGANIC COMPOUNDS</topic><topic>OXIDOREDUCTASES</topic><topic>PLASMIDS</topic><topic>Promoter Regions, Genetic</topic><topic>PROTONS</topic><topic>PURIFICATION</topic><topic>REACTION KINETICS</topic><topic>RESONANCE</topic><topic>Serine - analogs & derivatives</topic><topic>Substrate Specificity</topic><topic>SUBSTRATES</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Rusnak, Frank</creatorcontrib><creatorcontrib>Liu, Jun</creatorcontrib><creatorcontrib>Quinn, Nina</creatorcontrib><creatorcontrib>Berchtold, Glenn A</creatorcontrib><creatorcontrib>Walsh, Christopher T</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>OSTI.GOV</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Rusnak, Frank</au><au>Liu, Jun</au><au>Quinn, Nina</au><au>Berchtold, Glenn A</au><au>Walsh, Christopher T</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Subcloning of the enterobactin biosynthetic gene entB: expression, purification, characterization and substrate specificity of isochorismatase</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>1990-02-13</date><risdate>1990</risdate><volume>29</volume><issue>6</issue><spage>1425</spage><epage>1435</epage><pages>1425-1435</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>The Escherichia coli entB gene, coding for the enterobactin biosynthetic enzyme isochorismatase, has been subcloned into the multicopy plasmid pKK223-3 under the control of the tac promoter. The resulting recombinant plasmid pFR1 expresses isochorismatase amounting to over 50% of the total cellular protein. The enzyme has been purified to homogeneity and a convenient assay developed. The enzyme has a Km for isochorismate of 14.7 microM and a turnover number of 600 min-1. By use of 1H NMR spectroscopy, the progress of the reaction was followed with the expected formation of 2,3-dihydro-2,3-dihydroxybenzoate product. Several substrate analogues were also utilized by the enzyme including chorismic acid, the immediate precursor to isochorismic acid in the enterobactin biosynthetic pathway.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>2139796</pmid><doi>10.1021/bi00458a013</doi><tpages>11</tpages></addata></record> |
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| ispartof | Biochemistry (Easton), 1990-02, Vol.29 (6), p.1425-1435 |
| issn | 0006-2960 1520-4995 |
| language | eng |
| recordid | cdi_osti_scitechconnect_6940841 |
| source | MEDLINE; American Chemical Society Journals |
| subjects | 550201 - Biochemistry- Tracer Techniques BACTERIA BARYONS Base Sequence BASIC BIOLOGICAL SCIENCES BENZOIC ACID BIOCHEMICAL REACTION KINETICS BIOLOGICAL PATHWAYS CARBOXYLIC ACIDS CELL CONSTITUENTS CLONING Cloning, Molecular DNA HYBRIDIZATION DNA-CLONING ELEMENTARY PARTICLES Enterobactin - metabolism ENZYMES ESCHERICHIA COLI Escherichia coli - enzymology Escherichia coli - genetics FERMIONS Gene Expression Regulation, Enzymologic GENES HADRONS HYBRIDIZATION Hydrogen-Ion Concentration Hydrolases - genetics Hydrolases - isolation & purification Hydrolases - metabolism KINETICS MAGNETIC RESONANCE Magnetic Resonance Spectroscopy MICROORGANISMS Molecular Sequence Data MOLECULAR STRUCTURE MONOCARBOXYLIC ACIDS NUCLEAR MAGNETIC RESONANCE NUCLEONS ORGANIC ACIDS ORGANIC COMPOUNDS OXIDOREDUCTASES PLASMIDS Promoter Regions, Genetic PROTONS PURIFICATION REACTION KINETICS RESONANCE Serine - analogs & derivatives Substrate Specificity SUBSTRATES |
| title | Subcloning of the enterobactin biosynthetic gene entB: expression, purification, characterization and substrate specificity of isochorismatase |
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