Explaining an Unusually Fast Parasitic Enzyme: Folate Tail-Binding Residues Dictate Substrate Positioning and Catalysis in Cryptosporidium hominis Thymidylate Synthase

Abstract

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...

First Three-Dimensional Structure of Toxoplasma gondii Thymidylate Synthase–Dihydrofolate Reductase: Insights for Catalysis, Interdomain Interactions, and Substrate Channeling

“…The DHFR k chem for Tg TS-DHFR is approximately three- to four-fold faster than that Ch TS-DHFR whereas Ch TS-DHFR has an unusually fast TS reaction due to the presence of non-conserved residues at the TS active site. (35) A fast TS rate could lead to a buildup of H 2 F and conversely, a slower rate of DHFR catalysis would require more time to bind and be converted to product leading to an apparent lag time between the two reactions. Therefore, the buildup of H 2 F for Ch TS-DHFR in the bifunctional reaction might be due to a fast TS rate of chemistry and a slower rate of DHFR catalysis.…”

Mg2+ Binds to the Surface of Thymidylate Synthase and Affects Hydride Transfer at the Interior Active Site

“…69 Mutations on those two residues affected the positioning and flexibility of the cofactor, and this effect propagated into the active site and reduced k cat of the TSase domain. 70 …”

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

“…Though most residues in Ch TS active site are conserved, there are two residues (A287 and S290) which are unique to Ch TS. 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 .…”