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.

The essential enzyme TS-DHFR from Cryptosporidium hominis undergoes an unusually rapid rate of catalysis at the conserved TS domain, facilitated by two nonconserved residues, Ala287 and Ser290, in the folate tail-binding region. Mutation of these two residues to their conserved counterparts drastically affects multiple steps of the TS catalytic cycle. We have determined the crystal structures of all three mutants (A287F, S290G, and A287F/S290G) in complex with active site ligands dUMP and CB3717. The structural data show two effects of the mutations: an increased distance between the ligands in the active site and increased flexibility of the folate ligand in the partially open enzyme state that precedes conformational change to the active catalytic state. The latter effect is able to be rescued by the mutants containing the A287F mutation. In addition, the conserved water network of TS is altered in each of the mutants. The structural results point to a role of the folate tail-binding residues in closely positioning ChTS ligands and restricting ligand flexibility in the partially open state to allow for a rapid transition to the active closed state and enhanced rate of catalysis. These results provide an explanation on how folate tail-binding residues at one end of the active site affect long-range interactions throughout the TS active site and validate these residues as targets for species-specific drug design.Thymidylate synthase (TS) 1 is an essential enzyme in all organisms, catalyzing the de novo synthesis of deoxythymidine monophosphate (dTMP) from deoxyuridine monophosphate (dUMP), and as such has long been used as a chemotherapeutic target for cancer and infections. During the step of methyl transfer to dUMP, TS utilizes the folate cofactor 5,10-methylenetetrahydrofolate (CH 2 H 4 folate), making the partially reduced dihydrofolate (H 2 folate) in the process (1). The dihydrofolate product is used by dihydrofolate reductase (DHFR), with nicotinamide adenine dinucleotide phosphate (NADPH), to produce the reduced folate, tetrahydrofolate (H 4 folate) (2). Tetrahydrofolate is used as a source of one-carbon unit transfers in multiple biological processes, including dTMP and purine nucleotide biosyn-thesis, and synthesis of glycine and methionine. Thymidylate synthase is a highly conserved enzyme across all species in overall structure on the residue level at the active site (1). † This work was supported in part by NIH Grant AI 44630 (to K.S.A.) and NIH Grant 5T32-AI 07404 (to W.E.M.). ‡ The coordinates for the structures reported in this work have been deposited in the Protein Data Bank under the following file names: A287F, 3DL5; S290G, 2OIP; A287F/S290G, 3DL6. * To whom correspondence should be addressed. Telephone: (203) While TS and DHFR are expressed as separate mono-functional enzymes in humans and most other species (1), in protozoan parasites the two catalytic activities exist as one bifunctional enzyme, residing on the same polypeptide chain (3). The thymidylate synthase domain of bifu...

In contrast to most species, including humans, having monofunctional forms of the folate biosynthetic enzymes thymidylate synthase (TS) and dihydrofolate reductase (DHFR), several pathogenic protozoal parasites, including Cryptosporidium hominus, contain a bifunctional form of the enzymes on a single polypeptide chain having both catalytic activities. The crystal structure of the bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) Cryptosporidium hominis reveals a dimer with a "crossover helix", a swap domain between DHFR domains, unique in that this helical region from one monomer makes extensive contacts with the DHFR active site of the other monomer. In this study, we used site-directed mutagenesis to probe the role of this crossover helix in DHFR catalysis. Mutations were made to the crossover helix: an "alanine face" enzyme in which the residues on the face of the helix close to the DHFR active site of the other subunit are mutated to alanine, a "glycine face" enzyme in which the same residues are mutated to glycine, and an "all alanine" helix in which all residues of the helix were mutated to alanine. These mutant enzymes were studied using a rapid transient kinetic approach. The mutations cause a dramatic decrease in the DHFR activity. The DHFR catalytic activity of the alanine face mutant enzyme is 30 s -1 , the glycine face mutant enzyme is 17 s -1 , and the all alanine helix enzyme is 16 s -1 , all substantially impaired from the wildtype DHFR activity of 152 s -1 . It is clear that loss of helix interactions results in a marked decrease in DHFR activity, supporting a role for this swap domain in DHFR catalysis. The crossover helix provides a unique structural feature of C. hominis bifunctional TS-DHFR that could be exploited as a target for species-specific non-active site inhibitors.

The bifunctional enzyme thymidylate synthase–dihydrofolate reductase (TS–DHFR) from the protozoal parasite Cryptosporidium hominis is a potential molecular target for the design of antiparasitic therapies for AIDS-related opportunistic infections. The enzyme exists as a homodimer with each monomer containing a unique swap domain known as a “crossover helix” that binds in a cleft on the adjacent DHFR active site. This crossover helix is absent in species containing monofunctional forms of DHFR such as human. An in-depth understanding of protein–protein interactions between the crossover helix and adjacent DHFR active site that might modulate enzyme integrity or function would allow for insights into rational design of species-specific allosteric inhibitors. Mutational analysis coupled with structural studies and biophysical and kinetic characterization of crossover helix mutants identifies this domain as essential for full enzyme stability and catalytic activity, and pinpoints these effects to distinct faces of the crossover helix important in protein–protein interactions. Moreover, targeting this helical protein interaction with α-helix mimetics of the crossover helix leads to selective inhibition and destabilization of the C. hominis TS–DHFR enzyme, thus validating this region as a new avenue to explore for species-specific inhibitor design.