Domain structure of rat 10-formyltetrahydrofolate dehydrogenase. Resolution of the amino-terminal domain as 10-formyltetrahydrofolate hydrolase.
We expressed the NH2-terminal domain of the multidomain, multifunctional enzyme, 10-formyltetrahydrofolate dehydrogenase (FDH), using a baculovirus expression system in insect cells. Expression of the 203-amino acid NH2-terminal domain (residues 1-203), which is 24-30% identical to a group of glycinamide ribonucleotide transformylases (EC 2.1.2.2), resulted in the appearance of insoluble recombinant protein apparently due to incorrect folding. The longer NH2-terminal recombinant protein (residues 1-310), which shares 32% identity with Escherichia coli L-methionyl-tRNA formyltransferase (EC 2.1.2.9), was expressed as a soluble protein. During expression, this protein was released from cells to the culture medium and was purified from the culture medium by 5-formyltetrahydrofolate-Sepharose affinity chromatography followed by chromatography on a Mono-Q column. We found that the purified NH2-terminal domain bears a folate binding site, possesses 10-formyltetrahydrofolate hydrolase activity, and exists as a monomer. Titration of tryptophan fluorescence showed that native FDH bound both the substrate of the reaction, 10-formyl-5, 8-dideazafolate, and the product of the reaction, 5,8-dideazafolate, with the same affinities as its NH2-terminal domain did and that both proteins bound the substrate with a 50-fold higher affinity than the product. Neither the NH2-terminal domain nor its mixture with the previously purified COOH-terminal domain had 10-formyltetrahydrofolate dehydrogenase activity. Formation of complexes between the COOH- and NH2-terminal domains also was not observed. We conclude that the 10-formyltetrahydrofolate dehydrogenase activity of FDH is a result of the action of the aldehyde dehydrogenase catalytic center residing in the COOH-terminal domain on the substrate bound in the NH2-terminal domain and that the intermediate domain is necessary to bring the two functional domains together in the correct orientation.
Krupenko
S A
SA
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Wagner
C
C
Cook
R J
RJ
D000581:Amidohydrolases / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology; D008623:Mercaptoethanol / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry*; D051381:Rats; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D000099295:Formyltetrahydrofolate Dehydrogenase
D000581:Amidohydrolases; D029968:Escherichia coli Proteins; D008623:Mercaptoethanol; D000587:Oxidoreductases Acting on CH-NH Group Donors; D000099295:Formyltetrahydrofolate Dehydrogenase; C056575:formyltetrahydrofolate deformylase protein, rat; C484016:purU protein, E coli