Single-stranded DNA binding activity of C1-tetrahydrofolate synthase enzymes.
In eukaryotes C1-5,6,7,8-tetrahydrofolate (THF) synthase is a trifunctional enzyme that catalyzes the interconversion of reduced forms of folate to supply activated one-carbon units required for a variety of metabolic pathways. The enzymatic activities include 10-formyl-THF synthetase (EC 6.3.4.3), 5,10-methenyl-THF cyclohydrolase (EC 3.5.4.9), and 5,10-methylene-THF dehydrogenase (EC 1.5.1.5). In bacteria separate, monofunctional or bifunctional polypeptides catalyze the same reactions. We have purified C1-THF synthase from the fission yeast Schizosaccharomyces pombe and found its physical and enzymatic properties similar to those of other eukaryotic C1-THF synthase enzymes. Unexpectedly, the S. pombe enzyme bound strongly (Keq = 100 pM) to single-stranded DNA, but not to double-stranded DNA or to RNA. The binding was sequence-independent, apparently not cooperative, and not detectably inhibited by C1-THF synthase substrates or cofactors. Trifunctional cytoplasmic enzyme from Saccharomyces cerevisiae and monofunctional (synthetase) enzyme from Clostridium acidiurici also bound tightly to single-stranded DNA, while bifunctional (dehydrogenase and cyclohydrolase) enzyme from Escherichia coli did not, suggesting that single-stranded DNA binding is a conserved function of the synthetase domain of C1-THF synthase enzymes.
Wahls
W P
WP
Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
Song
J M
JM
Smith
G R
GR
D000619:Aminohydrolases / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001667:Binding, Competitive; D002244:Carbon / Q000378:metabolism; D003013:Clostridium / Q000201:enzymology; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005574:Formate-Tetrahydrofolate Ligase / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012568:Schizosaccharomyces / Q000201:enzymology; D013045:Species Specificity
D004277:DNA, Single-Stranded; D009097:Multienzyme Complexes; C021836:formyl-methenyl-methylenetetrahydrofolate synthetase; D002244:Carbon; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D000619:Aminohydrolases; D005574:Formate-Tetrahydrofolate Ligase