Nonconserved Residues Ala287 and Ser290 of the Cryptosporidium hominis Thymidylate Synthase Domain Facilitate Its Rapid Rate of Catalysis^,

Abstract

Cryptosporidium hominis TS-DHFR exhibits an unusually high rate of catalysis at the TS domain, at least 10-fold greater than those of other TS enzymes. Using site-directed mutagenesis, we have mutated residues Ala287 and Ser290 in the folate-binding helix to phenylalanine and glycine, respectively, the corresponding residues in human and most other TS enzymes. Our results show that the mutant A287F, the mutant S290G, and the double mutant all have reduced affinities for methylene tetrahydrofolate and reduced rates of reaction at the TS domain. Interestingly, the S290G mutant enzyme had the lowest TS activity, with a catalytic efficiency approximately 200-fold lower than that of the wild type (WT). The rate of conformational change of the S290G mutant is approximately 80 times slower than that of WT, resulting in a change in the rate-limiting step from hydride transfer to covalent ternary complex formation. We have determined the crystal structure of ligand-bound S290G mutant enzyme, which shows that the primary effect of the mutation is an increase in the distance between the TS ligands. The kinetic and crystal structure data presented here provide the first evidence explaining the unusually fast TS rate in C. hominis.

Structural studies provide clues for analog design of specific inhibitors of Cryptosporidium hominis thymidylate synthase–dihydrofolate reductase

“…The phenyl ring of compound 1 interacts with residues I315, F433 and M519. The non-conserved and unique residue A287 interacts with the glutamate tail of the inhibitor 10 . Four hydrogen bonds stabilize compound 1 optimally in the TS active site.…”

“…In other parasite species and also human, these residues are Phe and Gly, respectively 12, 13 . These residues have been shown to be important for optimal positioning of the cofactor CH 2 H 4 F and catalysis 10 . In addition to the van der Waals interaction between the glutamate tail of compound 1 and A287, the structural differences between Ch TS and hTS can be exploited to design a parasite specific TS inhibitor.…”

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).…”

Exploring novel strategies for AIDS protozoal pathogens: α-helix mimetics targeting a key allosteric protein–protein interaction in C. hominis thymidylate synthase-dihydrofolate reductase (TS-DHFR)

“…We have previously established that Ch TS is unusually fast compared to other orthologs, one determinant of which was found to be unusual positioning of TS ligands. 25,26 It will be interesting to determine if the crossover helix has a role in TS ligand orientation, or contributes to TS catalysis by another mechanism. Mutational and structural research is currently underway to pinpoint the effect of the crossover helix on the TS domain.…”