10-Formyltetrahydrofolate dehydrogenase (FDH) regulates 10- formyltetrahydrofolate (10-formylTHF) and tetrahydrofolate (THF) pools by converting 10-formylTHF to THF and CO2 in an NADP-dependent dehydrogenase reaction or to THR and formate in an NADP-independent hydrolase reaction. FDH may regulate purine biosynthesis by controlling the level of 10- formylTHF. The applicant's group showed that NAP and IMP activate FDH suggesting the existence of a regulatory nucleotide-binding site. FDH also plays an essential role in metabolizing formate by removing it as CO2, thus protecting the cell from formate intoxication. The enzyme is a natural fusion of two unrelated proteins in which the amino-terminal domain bears the substrate-binding site and hydrolase catalytic center while the catalytic machinery of the aldehyde dehydrogenase homologous carboxyl-terminal domain is used as the catalytic center in the dehydrogenase reaction. Thus, FDH has two catalytic centers but one substrate-binding site. A 100-residue intermediate domain is required to bring the two functional domains together to intermediate domain is required to bring the two functional domains together to catalyze the dehydrogenase reaction. The applicant suggest that the intermediate domain of FDH is flexible and allows conformational changes which alter the orientation of the amino- an carboxyl-terminal conformational changes which alter the orientation of the amino-an carboxyl-terminal domains thus regulating enzyme activity. In vitro, FDH proposes that 2-ME be directly involved in the mechanism of enzyme catalysis Specific aims include: 1) to map the FDH folate binding, and to resolve the crystal structure of the amino-terminal domain of FDH; 2) to study the mechanism of activation of FDH by nucleotide including identification and characterization of the regulatory site; 3) to study the mechanism of activation of FDH by 2-ME and to search for naturally occurring compounds which can replace 2-ME inside the cell; and 4) to study the role of the intermediate domain of FDH in enzyme function. Site-directed mutagenesis, expression of truncated forms of FDH, affinity labeling, assay of enzyme activity, binding studies, and crystallographic methods will be used to achieve the goals of the project. The well-known relationship between folate deficiency and megaloblastic anemia a well as the recently discovered association with the risk of vascular disease together with the importance of folate in prevention of neural-tube defects make these studies particularly relevant.
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