We have determined the crystal structure of dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Cryptosporidium hominis, revealing a unique linker domain containing an 11-residue ␣-helix that has extensive interactions with the opposite DHFR-TS monomer of the homodimeric enzyme. Analysis of the structure of DHFR-TS from C. hominis and of previously solved structures of DHFR-TS from Plasmodium falciparum and Leishmania major reveals that the linker domain primarily controls the relative

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... rientation of the DHFR and TS domains. Using the tertiary structure of the linker domains, we have been able to place a number of protozoa in two distinct and dissimilar structural families corresponding to two evolutionary families and provide the first structural evidence validating the use of DHFR-TS as a tool of phylogenetic classification. Furthermore, the structure of C. hominis DHFR-TS calls into question surface electrostatic channeling as the universal means of dihydrofolate transport between TS and DHFR in the bifunctional enzyme. Thymidylate synthase (TS) 1 and dihydrofolate reductase (DHFR) are essential enzymes in the cell cycle of all organisms, since they catalyze the production of dTMP, required for DNA replication. TS converts the substrate, dUMP, to dTMP by reductive methylation using the cofactor, 5,10-methylene tetrahydrofolate, and releases dihydrofolate (1). In the presence of the cofactor NADPH, DHFR reduces dihydrofolate to tetrahydrofolate. The folate cycle is completed by serine hydroxymethyl transferase, which converts tetrahydrofolate back to 5,10-methylene tetrahydrofolate. Recently, Stechmann and Cavalier-Smith (2) have addressed