PMID:2985605

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.

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Annotations

Gene product	Qualifier	GO ID	GO term name	Evidence Code	with/from	Aspect	Notes	Status
FolC		GO:0005737	cytoplasm	IDA: Inferred from Direct Assay		C	Cloned and overexpressed FolC was purified from the supernatant after subcellular fractionation.	complete
FolC		GO:0046656	folic acid biosynthetic process	IDA: Inferred from Direct Assay		P	FolC is bifunctional, including carrying dihydrofolate synthetase activity, which is necessary for folate production (Table 3).	complete
FolC		GO:0009257	10-formyltetrahydrofolate biosynthetic process	IDA: Inferred from Direct Assay		P	Folylpolyflutamate synthetase activity is required to convert folates into tetrahydrofolylpolyglutamate.	complete
FolC		GO:0008841	dihydrofolate synthase activity	IDA: Inferred from Direct Assay		F	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.	complete
FolC		GO:0004326	tetrahydrofolylpolyglutamate synthase activity	IDA: Inferred from Direct Assay		F	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.	complete
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