PMID:2985605: Difference between revisions

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<span style = 'font-size: large'> '''Bognar, AL, Osborne, C, Shane, B, Singer, SC and Ferone, R'''  (1985) Folylpoly-gamma-glutamate synthetase-dihydrofolate synthetase. Cloning and high expression of the Escherichia coli folC gene and purification and properties of the gene product.''J. Biol. Chem.'' '''260''':5625-30</span>
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!align=left  |Citation
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'''Bognar, AL, Osborne, C, Shane, B, Singer, SC and Ferone, R'''  (1985) Folylpoly-gamma-glutamate synthetase-dihydrofolate synthetase. Cloning and high expression of the Escherichia coli folC gene and purification and properties of the gene product. ''J. Biol. Chem.'' '''260''':5625-30
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!align=left  |Abstract
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The Escherichia coli gene for folylpolyglutamate synthetase-dihydrofolate synthetase was localized to plasmids pLC22-45, 24-31, and 28-44 of the Clarke-Carbon E. coli colony bank (Clarke, L., and Carbon, J. (1976) Cell 9, 91-99) by screening the bank by replica mating with an E. coli folC mutant. The folC gene was subcloned from pLC22-45 and inserted into a high copy number plasmid containing the lambda replication control region under the control of the temperature-sensitive cI857 repressor and into a high expression plasmid containing the lambda PL promoter and the cI857 repressor. The folC structural gene was located on a 1.52-kilobase PvuI fragment, sufficient to code for a protein of maximum Mr 55,000. E. coli transformants containing the recombinant plasmids, when induced by culturing at 42 degrees C, had folylpolyglutamate synthetase and dihydrofolate synthetase levels that were 100- to 400-fold higher than in wild type strains and which represented up to 4% of the soluble cell protein. The E. coli folylpolyglutamate synthetase-dihydrofolate synthetase has been purified to homogeneity from the transformants. Both activities are catalyzed by a single protein of Mr 47,000. Some kinetic properties of the enzymes and a new spectrophotometric method for assaying dihydrofolate synthetase activity are described.
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[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=2985605 PubMed]


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|{{TableHeadingColor}} |'''Abstract''' || The Escherichia coli gene for folylpolyglutamate synthetase-dihydrofolate synthetase was localized to plasmids pLC22-45, 24-31, and 28-44 of the Clarke-Carbon E. coli colony bank (Clarke, L., and Carbon, J. (1976) Cell 9, 91-99) by screening the bank by replica mating with an E. coli folC mutant. The folC gene was subcloned from pLC22-45 and inserted into a high copy number plasmid containing the lambda replication control region under the control of the temperature-sensitive cI857 repressor and into a high expression plasmid containing the lambda PL promoter and the cI857 repressor. The folC structural gene was located on a 1.52-kilobase PvuI fragment, sufficient to code for a protein of maximum Mr 55,000. E. coli transformants containing the recombinant plasmids, when induced by culturing at 42 degrees C, had folylpolyglutamate synthetase and dihydrofolate synthetase levels that were 100- to 400-fold higher than in wild type strains and which represented up to 4% of the soluble cell protein. The E. coli folylpolyglutamate synthetase-dihydrofolate synthetase has been purified to homogeneity from the transformants. Both activities are catalyzed by a single protein of Mr 47,000. Some kinetic properties of the enzymes and a new spectrophotometric method for assaying dihydrofolate synthetase activity are described.
!align=left  |Keywords
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Cloning, Molecular; DNA Restriction Enzymes/metabolism; DNA, Bacterial/analysis; Electrophoresis, Polyacrylamide Gel; Escherichia coli/genetics; Escherichia coli Proteins; Gene Expression Regulation; Kinetics; Molecular Weight; Multienzyme Complexes; Peptide Synthases/genetics; Plasmids
|{{TableHeadingColor}} nowrap |'''Links''' ||  [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=2985605 PubMed]
 
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==Significance {{HelpIcon|Help:Literature}} ==
==Significance {{HelpIcon|Help:Literature}} ==
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==Annotations {{HelpIcon|Help:Literature}}==
==Annotations {{HelpIcon|Help:Literature}}==
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!|Gene product!!Qualifier!!GO ID!!GO term name!!Evidence Code!!with/from!!Aspect!!Notes!!Status
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FolC
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GO:0005737
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cytoplasm
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IDA: Inferred from Direct Assay
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C
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Cloned and overexpressed FolC was purified from the supernatant after subcellular fractionation.
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complete
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FolC
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GO:0046656
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folic acid biosynthetic process
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IDA: Inferred from Direct Assay
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P
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FolC is bifunctional, including carrying dihydrofolate synthetase activity, which is necessary for folate production (Table 3).
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complete
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FolC
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GO:0009257
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10-formyltetrahydrofolate biosynthetic process
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IDA: Inferred from Direct Assay
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P
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Folylpolyflutamate synthetase activity is required to convert folates into tetrahydrofolylpolyglutamate.
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complete
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FolC
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GO:0008841
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dihydrofolate synthase activity
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IDA: Inferred from Direct Assay
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F
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Table 3 shows the apparent Km value for the dihydrofolate synthetase activity of the bifunctional enzyme, FolC, as being 3900 uM for the substrate glutamate.
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complete
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FolC
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GO:0004326
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tetrahydrofolylpolyglutamate synthase activity
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IDA: Inferred from Direct Assay
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F
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As reported in Table 3, FolC is a bifunctional enzyme that catalyzes the synthesis of polyglutamate compounds with an apparent Km of 333 uM for glutamate. 
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complete
 
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==EcoliWiki Links {{HelpIcon|Help:Literature}} ==
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Latest revision as of 04:45, 15 September 2013

Citation

Bognar, AL, Osborne, C, Shane, B, Singer, SC and Ferone, R (1985) Folylpoly-gamma-glutamate synthetase-dihydrofolate synthetase. Cloning and high expression of the Escherichia coli folC gene and purification and properties of the gene product. J. Biol. Chem. 260:5625-30

Abstract

The Escherichia coli gene for folylpolyglutamate synthetase-dihydrofolate synthetase was localized to plasmids pLC22-45, 24-31, and 28-44 of the Clarke-Carbon E. coli colony bank (Clarke, L., and Carbon, J. (1976) Cell 9, 91-99) by screening the bank by replica mating with an E. coli folC mutant. The folC gene was subcloned from pLC22-45 and inserted into a high copy number plasmid containing the lambda replication control region under the control of the temperature-sensitive cI857 repressor and into a high expression plasmid containing the lambda PL promoter and the cI857 repressor. The folC structural gene was located on a 1.52-kilobase PvuI fragment, sufficient to code for a protein of maximum Mr 55,000. E. coli transformants containing the recombinant plasmids, when induced by culturing at 42 degrees C, had folylpolyglutamate synthetase and dihydrofolate synthetase levels that were 100- to 400-fold higher than in wild type strains and which represented up to 4% of the soluble cell protein. The E. coli folylpolyglutamate synthetase-dihydrofolate synthetase has been purified to homogeneity from the transformants. Both activities are catalyzed by a single protein of Mr 47,000. Some kinetic properties of the enzymes and a new spectrophotometric method for assaying dihydrofolate synthetase activity are described.

Links

PubMed

Keywords

Cloning, Molecular; DNA Restriction Enzymes/metabolism; DNA, Bacterial/analysis; Electrophoresis, Polyacrylamide Gel; Escherichia coli/genetics; Escherichia coli Proteins; Gene Expression Regulation; Kinetics; Molecular Weight; Multienzyme Complexes; Peptide Synthases/genetics; Plasmids

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