Ar. Bello et al., PTR1 - A REDUCTASE MEDIATING SALVAGE OF OXIDIZED PTERIDINES AND METHOTREXATE RESISTANCE IN THE PROTOZOAN PARASITE LEISHMANIA-MAJOR, Proceedings of the National Academy of Sciences of the United Statesof America, 91(24), 1994, pp. 11442-11446
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.