Studies of Leishmania major Pteridine Reductase 1, a Novel Short Chain Dehydrogenase

Pteridine reductase 1 (PTR1) is an NADPH dependent reductase that catalyzes the reduction of several pterins and folates. The gene encoding this enzyme was originally identified in Leishmania based on its abilty to provide resistance to the drug methotrexate (MTX). The DNA and amno acid sequences are known , and overproducing strains of Escherichia coli are available. PTR1 has been previously shown to be required for the salvage of oxidized pteridines (folate, biopterin, and others). Since Leishmania are folate and pterin auxotrophes, PTR1 is a possible target for novel anti-folate drugs for the treatment of leishmaniasis. PTR1 catalyzes the transfer of hydride from NADPH to the 2-amno-4-oxopteridine ring system yielding 7, 8-dihydropteridines, and to the pteridine ring system of , 8-dihydropteridines yielding 5, 6, 7 , 8-tetrahydropteridines. PTR1 shows a pH dependent substrate specificity. At pH 4.6 the specific activity of PTR1 is highest with pterins, while at pH 6.0 the specific activity of PTR1 was highest with folates. The sequence of PTR1 is only 20-30% homologous to the sequences of members of the short chain dehydrogenase/reductase enzyme famly. Although this is typical for members of this enzyme famly, it does not allow for unambiguous classification in this famly. In fact, when the DNA sequence of PTRI was first determned, PTR1 was classified as an aldoketo reductase. To classify PTRI definitively, further biochemical characterization was required. To provide this information, the work described here was undertaken: (i) the stereochemical and kinetic course of PTR1 was determned; (ii) residues important in catalysis and ligand binding were identified; and (iii) conditions for the crystallization of PTRI were developed. The stereochemistry of hydride transfer The use of eH)-folate, showed that the ultimate product ofPTRl was 5, 6, tetrahydrofolate. 4R-eH)-NADPH and 4S-eH)-NADPH were synthesized enzymatically and used as the cofactor for the reduction of folate. PTRI was coupled to thymidylate synthase (TS), and tritium from 4S-eH)-NADPH was transferred to thymidylate. Therefore, the pro-S hydride of NADPH was transferred to the si face of dihydrofolate (DHF; see figure 11). The transfer of the pro-S hydride indicates that PTRI is a B-side dehydrogenase which is consistent with its membership in the short chain dehydrogenase