The enzymes of the 10-formyl-tetrahydrofolate synthetic pathway are found exclusively in the cytosol of the trypanosomatid parasite Leishmania major

In most organisms 10-formyl-tetrahydrofolate (10-CHO-THF) participates in the synthesis of purines in the cytosol and formylation of mitochondrial initiator methionyl-tRNA(Met). Here we studied 10-CHO-THF biosynthesis in the protozoan parasite Leishmania major, a purine auxotroph. Two distinct synthetic enzymes are known, a bifunctional methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (DHCH) or formyl-tetrahydrofolate ligase (FTL), and phylogenomic profiling revealed considerable diversity for these in trypanosomatids. All species surveyed contain a DHCH1, which was shown recently to be essential in L. major. A second DHCH2 occurred only in L infantum, L mexicana and T cruzi, and as a pseudogene in L major. DHCH2s bear N-terminal extensions and we showed a LiDHCH2-GFP fusion was targeted to the mitochondrion. FTLs were found in all species except Trypanosoma brucei. L. major ftl(-) null mutants were phenotypically normal in growth, differentiation, animal infectivity and sensitivity to a panel of pteridine analogs, but grew more slowly when starved for serine or glycine, as expected for amino acids that are substrates in C1-folate metabolism. Cell fractionation and western blotting showed that both L major DHCH1 and FTL were localized to the cytosol and not the mitochondrion. These localization data predict that in L major cytosolic 10-formyl-tetrahydrofolate must be transported into the mitochondrion to support methionyl-tRNA(Met) formylation. The retention in all the trypanosomatids of at least one enzyme involved in 10-formyl-tetrahydrofolate biosynthesis, and the essentiality of this metabolite in L major, suggests that this pathway represents a promising new area for chemotherapeutic attack in these parasites. (C) 2009 Elsevier B.V. All rights reserved.