The broad objectives of this proposal are to understand the metabolic role and enzymatic mechanism of one of the most abundant folate enzymes, 10-formyltetrahydrofolate dehydrogenase (FDH). FDH converts 10-formyltetrahydrofolate to tetrahydrofolate in an NADP-dependent dehydrogenase reaction or in an NADP-independent hydrolase reaction thus regulating two of the major folate pools. It has been also proposed that the enzyme serves as an intracellular folate depot protecting folate coenzymes from oxidative degradation. The enzyme is a natural fusion of two unrelated proteins. The amino-terminal domain bears the folate-binding site and functions as a hydrolase. The aldehyde dehydrogenase like carboxyl-terminal domain works as the catalytic tool in the dehydrogenase reaction when the two domains are combined in one polypeptide. A hundred residue intermediate domain is a linker between the two functional domains required to bring them together to catalyze the dehydrogenase reaction. It is hypothesized that the hydrolase reaction of FDH although by itself is not of physiological significance, is an important and essential part of the FDH dehydrogenase mechanism. The FDH dehydrogenase mechanism is a combination of two sequential reactions, the hydrolase and aldehyde dehydrogenase. During the dehydrogenase reaction transfer of an intermediate product from the hydrolase domain of FDH to the aldehyde dehydrogenase domain takes place. The intermediate domain is crucial to bring two functional domains in correct orientation to allow the transfer. Another part of this project is based on the hypothesis that one of the major roles of FDH is to regulate de novo purine biosynthesis by controlling 10-formyltetrahydrofolate levels. The recent findings that FDH is highly down-regulated in carcinogenesis, apparently due to increased demand of cancer cells for purines, make the protein an important potential target in anticancer chemotherapy. The following specific aims are proposed to test the hypotheses. (1) To determine the role of the intermediate domain in the enzyme mechanism. (2) To characterize the folate binding site and to evaluate the hydrolase mechanism of FDH. (3) To crystallize and to resolve the crystal structure of the FDH individual domains and the full- length protein. (4) To elucidate the role of FDH in cellular metabolism. Site-directed mutagenesis and protein design approaches, enzyme activity assays, binding studies, crystallographic and immunochemical methods, mammalian cell expression, antisense oligonucleotide techniques, purine and folate assays will be used to achieve the goals of the project. The well known role of folate in prevention of megaloblastic anemia, vascular disease, neural tube birth defects and cancer make these studies particularly relevant.
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