Source: Dissertation Abstracts International, Volume: 74-11(E), Section: B.

Adviser: Karen S. Anderson.

The parasitic protozoan Toxoplasma gondii is the cause of the infection known as toxoplasmosis. Some current treatments for toxoplasmosis target the dimeric, bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR); however, these treatments often have complications from toxicity and the development of drug resistance. Thus, a mechanistic characterization of TS-DHFR is an important step towards discovering novel therapeutics with increased potency and selectivity. This dissertation elucidates several key structure-function elements of TS-DHFR and provides a rationale for the design of allosteric inhibitors.

Bifunctional TS-DHFR plays an essential role in DNA synthesis and is unique to several species of protozoans, including T. gondii. Previous studies of this enzyme from other species revealed that some have the ability to channel dihydrofolate directly from the TS site, where it is formed as a product, to the DHFR site, where it is used as a substrate. Using pre-steady state kinetics and analysis of TS-DHFR from several species, we confirm that TS-DHFR from T. gondii is capable of channeling and we identify the mechanism by which channeling takes place.

An interesting feature of T. gondii is the large linker region that joins the TS and DHFR, which are expressed on a single polypeptide chain. This linker of about 69 amino acids extends from the DHFR in one monomer and contacts the DHFR of the other monomer in the form of a crossover helix. The linker then joins with the TS domain at the TS junctional region. Mutational analysis reported here demonstrates that interactions formed by the crossover helix and the junctional region are important in maintaining optimal activity of both active sites, further advancing our understanding of interdomain communication.

Finally, using fluorescence spectroscopy, we demonstrate that T. gondii TS undergoes conformational switching between active and inactive states. To take advantage of this, we designed peptides that target the dimer interface between the TS domains of bifunctional T. gondii TS-DHFR by mimicking beta-strands at the interface. We show that the peptides bind to the apo-dUMP enzyme to inhibit both the TS and distal DHFR with selectivity over the human enzymes. These experiments reveal the potential of allosteric inhibition of apo-TS, specifically at the TS-TS interface, as a target for species-specific therapeutics for treating parasitic infection and overcoming drug resistance.