Publications

• Mutagenesis of the folC gene encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli.

  Kimlova L.J., Pyne C., Keshavjee K., Huy J., Beebakhee G., Bognar A.L.

  The folC gene of Escherichia coli, cloned in a pUC19 vector, was mutagenized by progressive deletions from both the 5' and the 3' ends and by TAB linker insertion. A number of 5'-deleted genes, which had the initiator ATG codon removed, produced a truncated gene product, in reduced amounts, from a ... >> More

  The folC gene of Escherichia coli, cloned in a pUC19 vector, was mutagenized by progressive deletions from both the 5' and the 3' ends and by TAB linker insertion. A number of 5'-deleted genes, which had the initiator ATG codon removed, produced a truncated gene product, in reduced amounts, from a secondary initiation site. The most likely position of this site at a GTG codon located 35 codons downstream of the normal start site. This product could complement the folC mutation in E. coli strain SF4 as well as a strain deleted in the folC gene. The specific activity of extracts of the mutant enzyme are 4-16% that of the wild type enzyme for the folylpolyglutamate synthetase activity and 6-19% for the dihydrofolate synthetase activity. The relative amount of protein expressed by the mutant, compared to the wild type, in maxicells was comparable to the relative specific activity, suggesting that the kcat of the mutant enzyme is similar to that of the wild type. Mutants with up to 14 amino acids deleted from the carboxy terminal could still complement the folC deletion mutant. Seven out of ten linker insertions dispersed through the coding region of the gene showed complementation of the folC mutation in strain SF4 but none of these insertion mutants were able to complement the strain containing a deleted folC gene. None of the carboxy terminal or linker insertion mutants had a specific activity greater than 0.5% that of the wild type enzyme. The dihydrofolate synthetase and folylpolyglutamate synthetase activities behaved similarly in all mutants, both retaining a large fraction of the wild type activity in the amino terminal deletions and both being very low in the carboxy terminal deletions and linker insertion mutants. These studies are consistent with a single catalytic site for the two activities catalyzed by this enzyme. << Less

  Arch. Biochem. Biophys. 284:9-16(1991) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification and properties of Lactobacillus casei folylpoly-gamma-glutamate synthetase.

  Bognar A.L., Shane B.

  Folylpolyglutamate synthetase was purified 200,000-fold from extracts of Lactobacillus casei. The homogeneous protein was a monomer of Mr = 43,000. The purified enzyme catalyzed a MgATP-dependent addition of glutamate to 5,10-methylene-tetrahydropteroylmono-, di-, and triglutamate substrates and m ... >> More

  Folylpolyglutamate synthetase was purified 200,000-fold from extracts of Lactobacillus casei. The homogeneous protein was a monomer of Mr = 43,000. The purified enzyme catalyzed a MgATP-dependent addition of glutamate to 5,10-methylene-tetrahydropteroylmono-, di-, and triglutamate substrates and metabolized (6R)-5,10-methylene-tetrahydro[3H]folate to the tetraglutamate derivative. Other folate derivatives were poor substrates or lacked activity. The specificity of the nucleotide site was wide. The magnesium salts of dATP, GTP, ITP, and UTP were effective alternate substrates for the reaction. The specificity of the glutamate binding site was very narrow. Of a wide variety of analogs tested, only L-homocysteate and 4-fluoroglutamate demonstrated affinity for the enzyme. Kinetic studies were consistent with an ordered Ter Ter mechanism with MgATP binding first to the enzyme, folate second, and glutamate last. The order of product dissociation from the enzyme was ADP, folate product, and Pi. This mechanism precludes the sequential addition of glutamate moieties to enzyme-bound folate. The Michaelis constants for (6R)-5,10-methylene-tetrahydropteroyldiglutamate, the most effective folate substrate, MgATP, and L-glutamate were 2.3 microM, 5.6 mM, and 423 microM, respectively. Adenosine 5'-(3-thio)triphosphate and beta, gamma-methylene-ATP were inhibitors of the reaction and had higher affinities for the enzyme than ATP. << Less

  J. Biol. Chem. 258:12574-12581(1983) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Folylpoly-gamma-glutamate synthetase-dihydrofolate synthetase. Cloning and high expression of the Escherichia coli folC gene and purification and properties of the gene product.

  Bognar A.L., Osborne C., Shane B., Singer S.C., Ferone R.

  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. ... >> More

  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. << Less

  J. Biol. Chem. 260:5625-5630(1985) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.