A general method for generation and analysis of defined mutations in enzymes involved in a tetrahydrofolate-interconversion pathway.
In eukaryotes, 10-formyltetrahydrofolate (THF) synthetase, 5,10-methenyl-THF cyclohydrolase and 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. These reactions are generally catalyzed by three separate monofunctional enzymes in prokaryotic cells. In this report a general method for the generation, detection and analysis of specific mutations affecting the catalytic activity of any of the reactions catalyzed by C1-THF synthase or its monofunctional counterparts is described. The method relies on plasmid-borne expression of genes in strains of the yeast Saccharomyces cerevisiae that are missing one or more of the activities of C1-THF synthase. Specific segments of the gene are subjected in vitro to random mutagenesis, the mutant genes expressed in yeast and screened by phenotype for inactivating mutations. Plasmids encoding mutant enzymes are recovered for sequence analysis. One-step purification of C1-THF synthase from the yeast expression system is demonstrated. The feasibility and versatility of the method is shown with the yeast ADE3 gene encoding the cytoplasmic C1-THF synthase and the gene encoding the monofunctional 10-formyl-THF synthetase from Clostridium acidiurici.
Barlowe
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CK
Clayton Foundation Biochemical Institute, University of Texas, Austin 78712.
Appling
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DR
D000619:Aminohydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015139:Blotting, Southern; D002846:Chromatography, Affinity; D005574:Formate-Tetrahydrofolate Ligase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005796:Genes; D005800:Genes, Fungal; D008025:Ligases / Q000235:genetics*; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009097:Multienzyme Complexes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics*; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013763:Tetrahydrofolates / Q000378:metabolism*
D009097:Multienzyme Complexes; D013763:Tetrahydrofolates; C021836:formyl-methenyl-methylenetetrahydrofolate synthetase; D010088:Oxidoreductases; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D000619:Aminohydrolases; D008025:Ligases; D005574:Formate-Tetrahydrofolate Ligase