Folate metabolic pathways in Leishmania

Abstract

Trypanosomatid parasitic protozoans of the genus Leishmania are autotrophic for both folate and unconjugated pteridines. Leishmania salvage these metabolites from their mammalian hosts and insect vectors through multiple transporters. Within the parasite, folates are reduced by a bifunctional DHFR (dihydrofolate reductase)-TS (thymidylate synthase) and by a novel PTR1 (pteridine reductase 1), which reduces both folates and unconjugated pteridines. PTR1 can act as a metabolic bypass of DHFR inhibition, reducing the effectiveness of existing antifolate drugs. Leishmania possess a reduced set of folate-dependent metabolic reactions and can salvage many of the key products of folate metabolism from their hosts. For example, they lack purine synthesis, which normally requires 10-formyltetrahydrofolate, and instead rely on a network of purine salvage enzymes. Leishmania elaborate at least three pathways for the synthesis of the key metabolite 5,10-methylene-tetrahydrofolate, required for the synthesis of thymidylate, and for 10-formyltetrahydrofolate, whose presumptive function is for methionyl-tRNAMet formylation required for mitochondrial protein synthesis. Genetic studies have shown that the synthesis of methionine using 5-methyltetrahydrofolate is dispensable, as is the activity of the glycine cleavage complex, probably due to redundancy with serine hydroxymethyltransferase. Although not always essential, the loss of several folate metabolic enzymes results in attenuation or loss of virulence in animal models, and a null DHFR-TS mutant has been used to induce protective immunity. The folate metabolic pathway provides numerous opportunities for targeted chemotherapy, with strong potential for 'repurposing' of compounds developed originally for treatment of human cancers or other infectious agents.

Figure 1. Folates and pterins in Leishmania and other organisms

The structures of folic acid (upper left) and biopterin (lower left), a representative unconjugated pterin, are shown. Folates can be further modified by additional γ-glutamyl residues. Leishmania (upper right) are auxotrophic for both pteridines and must salvage and activate them, using both DHFR and pteridine reductase to form H₄F and pteridine reductase to form tetrahydrobiopterin (H₄B). In contrast, humans (lower right) can synthesize biopterin starting from GTP, but must salvage folate. Other patterns are seen in evolution; E. coli can synthesize both folates and unconjugated pteridines, and the malaria parasite Plasmodium can synthesize folates.

Figure 2. Acquisition and activation of pteridines: PTR1 as a metabolic bypass of DHFR

Folate uptake is mediated by several transporters, including FT1 and BT1, and is subsequently reduced to H₂F and H₄F by DHFR. PTR1 also reduces folates and can bypass DHFR inhibition as it is less susceptible to many antifolates, including methotrexate. H₄F is converted into a variety of C1-folate cofactors (Figure 3), including methylene-tetrahydrofolate (CH₂=H₄F), by the action of SHMT or the GCC. Although most C1-folate-utilizing enzymes maintain the H₄F reduction state, TS uniquely causes oxidation back to H₂F. In Leishmania and trypanosomes, DHFR and TS are fused into a single bifunctional protein. Biopterin is salvaged by BT1 and reduced by PTR1 to dihydrobiopterin (H₂B) and then H₄B; DHFR is unable to carry out these reductions. H₄B is consumed by phenylalanine hydroxylase (PAH) and probably other reactions as yet unknown. Pterin-dependent hydroxylases yield pterin-4a-carbinolamine, which is recycled by pterin-4a-carbinolamine dehydratase (PCD) to quinoid-dihydrobiopterin (q-H₂B), and then to H₄B through the action of quinoid-dihydrobiopterin reductase (QDPR). This research was originally published in The Journal of Biological Chemistry. Nare, B., Hardy, L.W. and Beverley, S.M. The roles of pteridine reductase 1 and dihydrofolate reductase-thymidylate synthase in pteridine metabolism in the protozoan parasite Leishmania major. J. Biol. Chem. 1997; 272: 13883–13891. c the American Society for Biochemistry and Molecular Biology.

Figure 3. Compartmentalization of folate metabolism in Leishmania

Folate metabolites are in light blue and major metabolites produced by folate-dependent enzymes are in yellow. Enzymes are in italicized capitals, with the relevant activity of bifunctional enzymes depicted in a larger font. Enzyme abbreviations and full enzymatic reactions are given in Table 1. Broken arrows depict intracellular transport steps inferred from the requirements and localizations of the known enzymes. The metabolites shown are 5,10-methylene-tetrahydrofolate (CH₂=H₄F), 5-methyltetrahydrofolate (CH₃-H₄F), 5-formyltetrahydrofolate (5-CHO-H₄F), 10-formyltetrahydrofolate (10-CHO-H₄F), 5,10-methenyltetrahydrofolate (−CH=H₄F), methionine (Met), thymidine monophosphate (TMP), glycine (Gly), serine (Ser) and formylated initiator methionyl-tRNA^Met (fMet-tRNA). cSHMT, cytosolic SHMT; mSHMT, mitochondrial SHMT.