Thymidylate Synthetase as a Chemotherapeutic Target in the Treatment of Avian Coccidiosis*

Abstract

SYNOPSIS. Thymidylate synthetase (E.C.2.1.1.45) has been demonstrated in unsporulated oocysts of Eimeria tenella. The properties of this enzyme have also been investigated in Tetrahymena pyriformis, as a protozoan model, and 7‐day‐old chick embryo, as a host model. The enzymes from E. tenella and chick embryo were inhibited by all concentrations of MnCl2 and MgCl2 tested. Tetrahymena pyriformis thymidylate synthetase was stimulated by low concentrations of both these cations but was inhibited by high concentrations. Subsequent data refer to chick embryo, E. tenella and T. pyriformis respectively: the apparent Km was 5.89 μM, 5.94 μM, and 0.53 M for the substrate dUMP: and 5.13 μM, 1.10 μM and 4.65 μM, respectively for the cofactor N5N10‐methylenetetrahydrofolate. The pH optimum for the enzyme from both chick embryo and T. pyriformis was 8.0, with Tris‐HCl buffer; activity of E. tenella thymidylate synthetase was still increasing at pH 8.2. The E. tenella enzyme was found to have a molecular weight of 4.6–4.9 × 105 daltons. The effects of nucleotides, inhibitors, and the omission of assay components on each enzyme are presented. Thymidylate synthetase from E. tenella is not greatly different from that of chick embryo, but does not resemble the enzyme from T. pyriformis. A case for using thymidylate synthetase as a chemotherapeutic target in the treatment of Eimeria infections remains. Indeed Eimeria may be considered as a model for infections caused by other protozoan parasites, such as Toxoplasma and Plasmodium, provided that suitable inhibitors can be found that are not toxic to the host.

Purine and pyrimidine metabolism in parasitic protozoa

“…Regeneration of tetrahydrofolate occurs in a reaction catalysed by dihydrofolate reductase (DHFR) 73 with NADPH as the electron donor. TS and DHFR exist as a bifunctional protein in the Trypanosomatidae [277,280], Plasmodium species [281,282] and Eimeria tenella [280,283,284], whereas the enzymes from bacteria, yeast and mammalian cells are distinct and readily separable [285]. It has been reported that Leishmania major promastigotes selected for resistance to the DHFR inhibitor methotrexate or the TS inhibitor 10-propargyl-5,8-dideazofolate overproduce the bifunctional TS-DHFR and show two distinct and uniform amplified regions of DNA [286][287][288][289].…”

Recent developments in coccidian biology; where do we go from here?

“…This is unfortunate since the oocyst is basically a dormant stage, and the activities of many enzymes of interest would be expected to be low. Thus Wang (1978) was unable to detect thymidylate synthetase in crude extracts of unsporulated oocysts of E. tenella, although we have been able to detect and characterize the enzyme using more sensitive techniques (Coles, Swoboda & Ryley, 1979). Interesting investigations have been carried out on the biochemical basis of sporulation and of catabolic systems operational in the sporozoite, but it is the growing intracellular parasite which is potentially of prime interest.…”

Isolation of second-generation schizonts of avian coccidia and their use in biochemical investigations

“…Differences in the enzyme from these sources are reported. Dihydrofolate reductase has been detected in large quantities in the oocysts of E. tenella (Wang, Stotish & Poe, 1975) and thymidylate synthetase has been investigated (Coles, Swoboda & Ryley, 1979). SHMT is the third enzyme of the cycle (Gutteridge & Coombs, 1977) which is shown in Fig.…”