PMID:2139796: Difference between revisions

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<span style = 'font-size: large'> '''Rusnak, F, Liu, J, Quinn, N, Berchtold, GA and Walsh, CT'''  (1990) Subcloning of the enterobactin biosynthetic gene entB: expression, purification, characterization, and substrate specificity of isochorismatase.''Biochemistry'' '''29''':1425-35</span>
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'''Rusnak, F, Liu, J, Quinn, N, Berchtold, GA and Walsh, CT'''  (1990) Subcloning of the enterobactin biosynthetic gene entB: expression, purification, characterization, and substrate specificity of isochorismatase. ''Biochemistry'' '''29''':1425-35
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!align=left  |Abstract
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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.
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[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=2139796 PubMed]


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|{{TableHeadingColor}} |'''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.
!align=left  |Keywords
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Base Sequence; Cloning, Molecular; Enterobactin/metabolism; Escherichia coli/enzymology; Escherichia coli/genetics; Gene Expression Regulation, Enzymologic; Hydrogen-Ion Concentration; Hydrolases/genetics; Hydrolases/isolation & purification; Hydrolases/metabolism; Kinetics; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Molecular Structure; Plasmids; Promoter Regions, Genetic; Serine/analogs & derivatives; Substrate Specificity
|{{TableHeadingColor}} nowrap |'''Links''' ||  [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=2139796 PubMed]
 
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Latest revision as of 04:38, 15 September 2013

Citation

Rusnak, F, Liu, J, Quinn, N, Berchtold, GA and Walsh, CT (1990) Subcloning of the enterobactin biosynthetic gene entB: expression, purification, characterization, and substrate specificity of isochorismatase. Biochemistry 29:1425-35

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.

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Keywords

Base Sequence; Cloning, Molecular; Enterobactin/metabolism; Escherichia coli/enzymology; Escherichia coli/genetics; Gene Expression Regulation, Enzymologic; Hydrogen-Ion Concentration; Hydrolases/genetics; Hydrolases/isolation & purification; Hydrolases/metabolism; Kinetics; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Molecular Structure; Plasmids; Promoter Regions, Genetic; Serine/analogs & derivatives; Substrate Specificity

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