Pterin and Folate Reduction by theLeishmania tarentolaeH Locus Short-Chain Dehydrogenase/Reductase PTR1

Overproduction of the short-chain dehydrogenase/reductase PTR1 confers resistance to the dihydrofolate reductase inhibitor methotrexate in the protozoan parasiteLeishmania.Genetic analysis has previously implicated PTR1 in pterin and folate metabolism. PTR1 was purified from a fusion protein expressed inEscherichia coli.Purified PTR1 exhibits NADPH-dependent biopterin, dihydrobiopterin, folate, and dihydrofolate reductase activities. The highest activity was found with the most oxidized pterins. The active protein was found to be a tetramer as demonstrated by gel-filtration chromatography. Kinetic constants (Km), as determined by double-reciprocal plots, were calculated for NADPH and for several of PTR1ʹs substrates. The PTR1 ofLeishmania tarentolaehad aKmof 16.9 μmfor the cofactor NADPH andKmvalues ranging from 3.5 to 85 μmfor the various substrates. The dissociation constant (KD), as determined by fluorescence titration, for NADPH was estimated to be 130 μm. The biochemical characterization of this important and novel enzyme involved in folate and pterin metabolism ofLeishmaniashould be useful for structure–function analysis and for developing specific inhibitors against this putative important chemotherapeutic target.