Metabolic role of cytoplasmic isozymes of 5,10-methylenetetrahydrofolate dehydrogenase in Saccharomyces cerevisiae

Biochemistry. 1996 Mar 5;35(9):3122-32. doi: 10.1021/bi952713d.


Saccharomyces cerevisiae possesses two cytosolic 5,10-methylenetetrahydrofolate (CH2-THF) dehydrogenases that differ in their redox cofactor specificity: an NAD-dependent dehydrogenase encoded by the MTD1 gene and an NADP-dependent activity as part of the trifunctional C1-THF synthase encoded by the ADE3 gene. The experiments described here were designed to define the metabolic roles of the NAD- and NADP-dependent CH2-THF dehydrogenases in one-carbon interconversions and de novo purine biosynthesis. Growth studies showed that the NAD-dependent CH2-THF dehydrogenase is interchangeable with the NADP-dependent CH2-THF dehydrogenase when flow of one-carbon units is in the oxidative direction but that it does not participate significantly when flux is in the reductive direction. 13C NMR experiments with [2-13C]glycine and unlabeled formate confirmed the latter conclusion. Direct measurements of cellular folate coenzyme levels revealed substantial levels of 10-formyl-THF (CHO-THF), the one-carbon donor used in purine synthesis, in the purine-requiring ade3 deletion strain. Thus, CHO-THF is necessary but not sufficient for de novo purine synthesis in yeast. Disruption of the MTD1 gene in this strain resulted in undetectable CHO-THF, indicating that the NAD-dependent CH2-THF dehydrogenase was responsible for CHO-THF production in the ade3 deletion strain. Finally, we examined the ability of wild-type and catalytically-inactive domains of the cytoplasmic C1-THF synthase to complement the adenine auxotrophy of the ade3 deletion strain. Both the dehydrogenase/cyclohydrolase (D/C) domain and the synthetase domain could functionally replace the full-length protein, but, at least for the D/C domain, complementation was not dependent on catalytic activity. These results reveal a catalytic role for the NAD-dependent CH2-THF dehydrogenase in the oxidation of cytoplasmic one-carbon units and indicate that the cytoplasmic C1-THF synthase plays both catalytic and noncatalytic roles in de novo purine biosynthesis in yeast.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • CHO Cells
  • Cricetinae
  • Cytosol / enzymology
  • Genotype
  • Glycine / metabolism
  • Isoenzymes / biosynthesis
  • Isoenzymes / chemistry
  • Isoenzymes / metabolism*
  • Kinetics
  • Liver / enzymology
  • Magnetic Resonance Spectroscopy
  • Methylenetetrahydrofolate Dehydrogenase (NADP) / biosynthesis
  • Methylenetetrahydrofolate Dehydrogenase (NADP) / chemistry
  • Methylenetetrahydrofolate Dehydrogenase (NADP) / metabolism*
  • Models, Biological
  • NAD / metabolism
  • NADP / metabolism
  • Phenotype
  • Plasmids
  • Rats
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / growth & development
  • Species Specificity


  • Isoenzymes
  • Recombinant Proteins
  • NAD
  • NADP
  • Methylenetetrahydrofolate Dehydrogenase (NADP)
  • Glycine