Characterization of a mutation affecting the function of Escherichia coli folylpolyglutamate synthetase-dihydrofolate synthetase and further mutations produced in vitro at the same locus.
The folC gene from mutant strain SF4 was cloned into a pUC19 plasmid. Expression of the mutant gene from the lac promoter of the plasmid complemented the auxotrophy for methionine of the SF4 strain. The only difference in sequence between the mutant and wild-type genes was a G925A base change resulting in an A309T amino acid change. The mutant enzyme had a 30-fold higher Km for 10-formyltetrahydrofolate as well as a 60-fold higher Km for glutamate and a 200-fold higher Km for dihydropteroate of the dihydrofolate synthetase activity. Site-specific mutagenesis was used to substitute other amino acids at codon 309. Mutants with glycine, isoleucine, and valine substitutions at this position, when expressed from multicopy plasmids, complemented the SF4 strain. The glycine mutant had properties similar to the wild-type enzyme, whereas the isoleucine and valine mutants had properties similar to the threonine mutant, SF4. Mutant genes with arginine, glutamate, and leucine substitutions, which did not complement the SF4 strain, could complement a folC deletion strain, but produced smaller colonies on complex plates and did not grow on minimal medium. In the deletion strain, an increasing requirement for folate product supplements was observed as the folylpolyglutamate synthetase-dihydrofolate synthetase activities of the complementing mutants decreased.
Department of Microbiology, University of Toronto, Ontario, Canada.
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Keshavjee
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Pyne
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Bognar
D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D007700:Kinetics; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids
D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D010453:Peptide Synthases; C500101:folC protein, E coli; C020041:dihydrofolate synthetase; C023103:folylpolyglutamate synthetase