This project continues to focus on dihydrofolate reductase, a critical enzyme in the metabolism of the B-vitamin folic acid (a pteridine), and beta-carotene, a precursor of vitamin A. The maintenance of folic acid at the fully reduced level, tetrahydrofolate, by dihydrofolate reductase is essential to cellular survival. Anti-folate drugs which target this enzyme continue to be widely used in cancer treatment and an increasing number of autoimmune diseases. Beta-carotene is currently receiving considerable attention as a biological antioxidant, particularly with respect to cardiovascular disease. The enzymatic reduction of other pteridines is also catalyzed by dihydrofolate reductase, although with decreased efficiency. Of particular biological interest is the reduction of biopterin, the cofactor for the enzymes that hydroxylate tyrosine, phenylalanine and tryptophan. The enzyme that specifically reduces biopterin is pteridine reductase. However, we have shown that dihydrofolate reductase can reduce dihydrobiopterin from 3% in beef liver to 50% in chicken liver, as compared with the rate of dihydrofolate reduction. Furthermore, biopterin is tightly bound to dihydrofolate reductase. The chicken liver dihydrofolate reductase has been crystallized not only as a binary and ternary complex with substrates and inhibitors, we now report the crystallization and x-ray analysis of a complex with biopterin and NADP, a ternary complex with both substrate and cofactor. X-ray analysis of this structure has shed further light on a possible mechanism of substrate protonation and on the possible catalytic function of the p- aminobenzoylglutamate moiety of folate substrates. In continuing studies of carotene metabolism, the effect of copper deficiency on the conversion of beta-carotene to vitamin A (retinol) was measured. The rationale was that the enzyme postulated to convert carotene to retinol in the intestinal wall is a dioxygenase containing copper. Copper-deficient rats were found to store retinol as efficiently as copper-adequate rats. When beta-carotene was fed to copper-deficient rats, liver storage of retinol, a measure of carotene absorption and conversion, was not different from controls. These results bring into question the possible role of a dioxygenase in carotene conversion to retinol.

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