Probing the Role of Parasite-specific Distant Structural Regions on Communication and Catalysis in the Bifunctional Thymidylate Synthase- Dihydrofolate Reductase from Plasmodium falciparum

摘要

Plasmodium falciparum thymidylate synthase-dihydrofolate reductase (TS-DHFR) is an essential enzyme in nucleotide biosynthesis, and a validated molecular drug target in malaria. Because P. falciparum TS and DHFR are highly homologous to their human counterparts, existing active-site antifolate drugs can have dose-limiting toxicities. In humans, TS and DHFR are two separate proteins. In P. falciparum, however, TS-DHFR is bifunctional, with both TS and DHFR active sites on a single polypeptide chain of the enzyme. Consequently, P. falciparum TS-DHFR contains unique distant or ‘non-active’ regions which might modulate catalysis: 1) an N-terminal tail; and 2) a ‘linker’ region tethering DHFR to TS, and encoding a ‘crossover helix’ that forms critical electrostatic interactions with the DHFR active site. The role of these non-active sites in the bifunctional P. falciparum TS-DHFR is unknown. We report the first in-depth, pre-steady state, kinetic characterization of the full-length, WT P. falciparum TS-DHFR enzyme, and probe the role of distant, non-active regions through mutational analysis. We show that the overall rate-limiting step in the WT P. falciparum TS-DHFR enzyme is TS catalysis. We further show that if TS is in an ‘activated’ (liganded) conformation, the DHFR rate is 2-fold activated, from 60 s⁻¹ to 130 s⁻¹ in the WT enzyme. The TS rate is also reciprocally activated by ~1.5-fold if DHFR is in an activated, ligand-bound conformation. Mutations to the linker region affect neither catalytic rate nor domain-domain communication. Deletion of the N-terminal tail – although in a location remote to the active site - decreases DHFR single and the bifunctional TS-DHFR rate by a factor of 2. The two-fold activation of the DHFR rate by TS ligands remains intact, although even the activated N-terminal mutant has just half the DHFR activity of the WT enzyme. However, the reciprocal communication – between TS active site and DHFR ligands - is impaired in N-terminal mutants. Surprisingly, deletion of the analogous N-terminal tail in Leishmania major TS-DHFR causes a 3-fold enhancement of the DHFR rate from ~14 s⁻¹ to ~40 s⁻¹. In summary, our results demonstrate a complex interplay of domain-domain communication and non-active site modulation of catalysis in P. falciparum TS-DHFR. Furthermore, each parasitic TS-DHFR is activated by unique mechanisms, modulated by their non-active site regions. Finally, our studies suggest the N-terminal tail of P. falciparum TS-DHFR is a highly selective, novel target for potential antifolate development in malaria.