Kinetic Characterization of Bifunctional Thymidylate Synthase-Dihydrofolate Reductase (TS-DHFR) from Cryptosporidium hominis

Abstract

This study presents a kinetic characterization of the recently crystallized bifunctional thymidylate synthasedihydrofolate reductase (TS-DHFR) enzyme from the apicomplexa parasite, Cryptosporidium hominis. Our study focuses on determination of the C. hominis TS-DHFR kinetic mechanism, substrate channeling behavior, and domain-domain communication. Unexpectedly, the unique mechanistic features of C. hominis TS-DHFR involve the highly conserved TS domain. At 45 s ؊1 , C. hominis TS activity is 10 -40-fold faster than other TS enzymes studied and a new kinetic mechanism was required to simulate C. hominis TS behavior. A large accumulation of dihydrofolate produced at TS and a lag in product formation at DHFR were observed. These observations make C. hominis TS-DHFR the first bifunctional TS-DHFR enzyme studied for which there is clear evidence against dihydrofolate substrate channeling. Furthermore, whereas with Leishmania major TS-DHFR there are multiple lines of evidence for domain-domain communication (ligand binding at one active site affecting activity of the other enzyme), no such effects were observed with C. hominis TS-DHFR.Protozoal parasites such as Cryptosporidium hominis, Plasmodium falciparum, Toxoplasma gondii, and Leishmania major are unusual in that their thymidylate synthase (TS) 1 and dihydrofolate reductase (DHFR) enzymes exist on the same polypeptide as part of a single bifunctional TS-DHFR enzyme.2 TS and DHFR are critical enzymes and established drug targets. TS represents the only means of de novo synthesis of 2Ј-deoxythymidylate (dTMP) for DNA synthesis, via reductive methylation of 2-deoxyuridate (dUMP) with methylene tetrahydrofolate (CH 2 H 4 folate), producing dihydrofolate (H 2 folate) in the process. DHFR catalyzes the reduction of H 2 folate by NADPH to generate tetrahydrofolate (H 4 folate), used for one carbon unit transfer reactions in several biochemical processes, including thymidylate, purine, and amino acid biosynthesis.Almost a decade of research on bifunctional TS-DHFR from protozoal parasites has relied on the only available TS-DHFR crystal structure, that from the kinetoplastid protozoa L. major (1). The recent solution of the crystal structures of TS-DHFR enzymes from two clinically relevant apicomplexan protozoa, P. falciparum and C. hominis, however, has revealed unanticipated deviations from the kinetoplastid model (2, 3). An obvious next question is whether these significant structural differences translate as significant mechanistic differences between parasite classes. We sought to address this question through a detailed kinetic characterization of TS-DHFR from C. hominis, again yielding surprising results.A major conclusion of the recent solution of the apicomplexan TS-DHFR structures is that there exist two families of bifunctional TS-DHFR structures: a short linker family with an Nterminal tail, as in the kinetoplastids (a class that includes Leishmania and the trypanosomes); and a long linker family with a donated or crossover helix, as in the apicomplexan paras...

Electrostatic Channeling in P. falciparum DHFR-TS: Brownian Dynamics and Smoluchowski Modeling

“…Because of the structural similarity between L. major DHFR-TS and other protozoan DHFR-TS enzymes, we hypothesized that these other enzymes may also support significant electrostatic channeling of dihydrofolate. However, despite multiple kinetic experiments investigating substrate channeling in this system, it has been found that the structurally similar DHFR-TS enzyme from C. hominis does not exhibit any measurable substrate channeling (12). This experimental result suggests that different protozoan DHFR-TS enzymes may exhibit varying efficiency of substrate channeling.…”

“…Although our main focus is on the electrostatic channeling of dihydrofolate in P. falciparum DHFR-TS, to see if this inefficient channeling observed in P. falciparum DHFR-TS is exceptional or general in the family of protozoan DHFR-TS enzymes, we include one more protozoan species, C. hominis, of the bifunctional DHFR-TS enzyme family that has been the focus of experimental kinetic studies (12), and we calculate the dihydrofolate channeling efficiency to compare the channeling efficiency between three species of protozoan DHFR-TS enzymes (P. falciparum, L. major, and C. hominis). It is known that L. major DHFR-TS exhibits substantial dihydrofolate channeling with some predictions of R80% transfer efficiency (6), whereas dihydrofolate channeling in C. hominis DHFR-TS is known to be minimal or nonexistent (12).…”

Inhibitor-bound complexes of dihydrofolate reductase-thymidylate synthase fromBabesia bovis

“…These residues lie at one end of the cofactor-binding site near the solvent-exposed glutamate tail of mTHF and other folate-like molecules when bound. Alanine and serine at these positions are associated with higher TS reaction rates, as observed for DHFR-TS from C. hominis relative to L. major and those of other species (Atreya & Anderson, 2004;Doan et al, 2007).…”

Genomic Distance between Thymidylate Synthase and Dihydrofolate Reductase Genes Does Not Correlate With Phylogenetic Evolution in Bacteria

“…Those sequences were analyzed by multiple alignment have presented lack of similarity with each other (data not shown), with exception of species of the same genus. This, in part, could explain the absence of homology found between junctional peptides of bifunctional DHFR-TS in protozoa (Atreya and Anderson, 2004;Miles et al, 1999;Trujillo et al, 1997).…”

“…TS and DHFR are enzymes that work together in successive reactions of folate synthesis pathway. These proteins exist, in some protozoans, as bifunctional enzymes, which are important in channeling the substrate between TS and DHFR active sites (Atreya and Anderson, 2004;Miles et al, 1999;Trujillo et al, 1997). These genes are neighbors in some bacterial genomes, whereas they are located millions of base pairs apart (and even in opposite DNA strands) in others.…”