PTR1 - A REDUCTASE MEDIATING SALVAGE OF OXIDIZED PTERIDINES AND METHOTREXATE RESISTANCE IN THE PROTOZOAN PARASITE LEISHMANIA-MAJOR

Trypanosomatid protozoans are pterin auxotrophs, a finding noted decad es ago which heralded the discovery of key metabolic roles played by p teridines in eukaryotes. We have now identified the enzyme mediating u nconjugated pteridine salvage in the human parasite Leishmania major, PTR1 (pteridine reductase 1, formerly hmtx(r) or ltdh). PTR1 is the ge ne in the amplified H region responsible For methotrexate (MTX) resist ance, and belongs to a large family of oxidoreductases with diverse su bstrates and roles. We generated Leishmania lacking PTR1 by homologous gene targeting, and these ptr1(-) mutants required reduced biopterin (dihydro- or tetrahydrobiopterin) for growth. PTR1 purified from engin eered Escherichia coli exhibited a MTX-sensitive, NADPH-dependent biop terin reductase activity. PTR1 showed good activity with folate and si gnificant activity with dihydrofolate and dihydrobiopterin, but not wi th quinonoid dihydrobiopterin. PTR1 thus differs considerably from pre viously reported pteridine reductases of trypanosomatids and vertebrat es. Pteridine reductase activity was diminished in ptr1(-) Leishmania and was elevated in transfected parasites bearing multiple copies of P TR1; correspondingly, ptr1(-) was MTX-hypersensitive whereas the multi copy transfectant was MTX-resistant. The concordance of the biochemica l and genetic properties of PTR1 suggests that this is the primary enz yme mediating pteridine salvage. These findings suggest several possib le mechanisms for PTR1-mediated MTX resistance and should aid in the d esign of rational chemotherapy.