Site-directed mutagenesis of a highly conserved aspartate in the putative 10-formyl-tetrahydrofolate binding site of yeast C1-tetrahydrofolate synthase.
C1-tetrahydrofolate (THF) synthase is a eukaryotic trifunctional protein possessing the activities 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase. Although the 10-formyl-THF synthetase reaction (a reversible ATP-dependent formylation of THF) has been studied extensively, little is known about specific residues involved in the catalytic mechanism. In this study, we have examined the role of a highly conserved aspartate residue, Asp449 of yeast cytoplasmic C1-THF synthase. Asp449 is part of a putative folate binding site found in many proteins that bind 10-formyl-THF. The corresponding aspartate has been identified as a critical catalytic residue in Escherichia coli and human GAR transformylase, which catalyzes a 10-formyl-THF-dependent formyl transfer. In order to determine if Asp449 has a similar catalytic role in the 10-formyl-THF synthetase reaction, three mutant proteins were produced by site-directed mutagenesis in which Asp449 of yeast cytoplasmic C1-THF synthase was changed to Asn, Glu, or Ala. The mutant proteins were expressed in yeast, purified, and characterized with respect to kinetic properties and enzyme stability. All three of the mutant enzymes retained substantial 10-formyl-THF synthetase activity, indicating that Asp449 is not a critical catalytic residue. However, our data suggest that it does play a role in folate binding, probably by contributing to the proper conformation of the active site. Thus, these results suggest that the 10-formyl-THF binding site differs significantly between the GAR transformylase and 10-formyl-THF synthetase families, and that the conserved aspartate plays different roles in the two enzymes.
Kirksey
T J
TJ
Department of Chemistry and Biochemistry, University of Texas at Austin 78712, USA.
Appling
D R
DR
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000235:genetics* / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005574:Formate-Tetrahydrofolate Ligase / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D019877:Hydroxymethyl and Formyl Transferases; D007700:Kinetics; D002955:Leucovorin / Q000031:analogs & derivatives* / Q000378:metabolism; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D051026:Phosphoribosylglycinamide Formyltransferase; D017354:Point Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; C010161:10-formyltetrahydropteroylglutamic acid; D001224:Aspartic Acid; C021836:formyl-methenyl-methylenetetrahydrofolate synthetase; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D019877:Hydroxymethyl and Formyl Transferases; D051026:Phosphoribosylglycinamide Formyltransferase; D000217:Acyltransferases; D000619:Aminohydrolases; D005574:Formate-Tetrahydrofolate Ligase; D002955:Leucovorin