Mechanistic and structure-function analysis of thymidylate synthase-dihydrofolate reductase from protozoal parasites
This is an Open Access Thesis
A major advance in the field of bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) is the recent crystallization of two apicomplexan TS-DHFR enzymes, from Cryptosporidium hominis and Plasmodium falciparum, which demonstrate significant structural differences from the well-known kinetoplastid TS-DHFR structure (Leishmania major ). A principal finding of this dissertation research is that the significant structural differences between the apicomplexa and kinetoplastid bifunctional TS-DHFR enzymes translate as significant mechanistic differences between parasite classes and among parasites within the same class.An in depth mechanistic analysis of C. hominis TS-DHFR and a preliminary analysis of the P. falciparum enzyme were completed. Results with C. hominis TS-DHFR mark a paradigm shift for TS activity and substrate channeling behavior. Despite a well conserved active-site, C. hominis TS activity was found to be 10--40 fold faster than that of other TS enzymes characterized. C. hominis also represents the first bifunctional TS-DHFR enzyme for which there is clear evidence against substrate channeling, or direct transfer of dihydrofolate produced at TS to DHFR, where it serves as a substrate. Neither C. hominis nor another apicomplexan protozoa, Toxoplasma gondii, exhibit the TS-DHFR domain-domain communication observed with L. major.In the second and third parts of this dissertation, two concurrent approaches are taken to assess the putative electrostatic channeling region in L. major TS-DHFR: site-directed mutagenesis of solvent-exposed basic residues and molecular docking. In order to test the electrostatic channeling hypothesis, 12 charge reversal or charge neutralization mutants were made, with up to 6 putative channel residues changed at once. The mutants were assessed for impaired channeling using two criteria: a lag in product formation at DHFR and an increase in H2folate accumulation. Surprisingly, none of the mutations produced changes consistent with impaired channeling. Therefore, our findings do not support the electrostatic channeling hypothesis.Molecular docking was also used to target the shallow groove between the two active sites of the L. major bifunctional enzyme. One of the predicted inhibitors, eosin B, was found to inhibit both the L. major TS and DHFR catalytic activities, without competing with substrates, suggesting that the non-active site region, unique to bifunctional enzymes, represents a valid therapeutic target.