The broad objectives of this proposal are to understand the metabolic role of two abundant folate-binding proteins we discovered in rat liver cytosol. We have identified them as enzymes having unusual properties. The first of these is 10-formyltetrahydrofolate dehydrogenase (10- FTHFDH) which is isolated carrying tightly bound 10- formyltetrahydrofolate (10-HCO-THF). Its function is believed to be the conversion of any 10-formyltetrahydrofolate (10-HCO-THF) not needed for de novo purine synthesis back to tetrahydrofolate (THF). NEUT2 mice have no 10-FTHFDH but appear to have a normal phenotype except that breeding homozygous pairs produce viable litters only after a long time, if at all. We believe this is due to the greatly reduced pool of THF in these animals which is just enough to permit normal growth, but not enough for successful pregnancy. One of our specific aims is to examine the effects of folate deficiency or supplementation on these NEUT2 mice. We have cloned the cDNA for 10-FTHFDH and showed that each monomer of the tetrameric protein has one domain similar to enzymes that transfer the 10-formyl group from 10-HCO-THF and a second domain similar to aldehyde dehydrogenase. 10-HCO-THF catalyzes two reactions: the NADP- dependent formation of CO2 and the NADP-independent hydrolysis of 10- HCO-THF to formate and THF. Covalent labeling shows 2 moles of folate bound per mole of monomer. In order to understand how these observations can be explained in terms of a putative structure of the enzyme, we have as specific aims to characterize the folate binding site of 10-FTHFDH; to investigate the catalytic role of the tightly bound folate in 10-FTHFDH; to express and characterize the functional domain of 10-FTHFDH; and to crystallize recombinant 1O-FTHFDH in preparation for X-ray diffraction studies. The second of these folate binding proteins with unusual enzymatic properties is glycine N-methyltransferase (GNMT). This is inhibited by 5-methyltetrahydrofolate and is believed to regulate the ratio of S- adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) and thereby modulate cellular methylation reactions. Using immunohistochemistry we have found GNMT in the nuclei of developing spermatocytes of the seminiferous tubules of the rat. This is a site of active DNA methylation suggesting that DNA methylation may be regulated by GNMT and folate in the developing sperm. Another of our specific aims is to characterize this form of GNMT. The relationship between folate nutriture and the incidence of neural- tube defects makes the studies proposed here particularly relevant.
Showing the most recent 10 out of 56 publications
