The Roles of Pteridine Reductase 1 and Dihydrofolate Reductase-Thymidylate Synthase in Pteridine Metabolism in the Protozoan Parasite Leishmania major

• Published in: The Journal of biological chemistry 1997-05, Vol.272 (21), p.13883-13891
• Main Authors: Nare, B, Hardy, L W, Beverley, S M
• Format: Article
• Language: English
• Subjects: Animals; Biopterins - metabolism; Blotting, Western; Chromatography, Gel; Folic Acid - metabolism; Folic Acid Antagonists - pharmacology; Humans; Hydrogen-Ion Concentration; Kinetics; Leishmania major - metabolism; Methotrexate - pharmacology; Models, Chemical; Multienzyme Complexes - metabolism; NADP - metabolism; Oxidoreductases - metabolism; Pteridines - metabolism; Tetrahydrofolate Dehydrogenase - metabolism; Thymidylate Synthase - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.21.13883

Trypanosomatid protozoans depend upon exogenous sources of pteridines (pterins or folates) for growth. A broad spectrum pteridine reductase (PTR1) was recently identified in Leishmania major , whose sequence places it in the short chain alcohol dehydrogenase protein family although its enzymatic activities resemble dihydrofolate reductases. The properties of PTR1 suggested a role in essential pteridine salvage as well as in antifolate resistance. To prove this, we have characterized further the properties and relative roles of PTR1 and dihydrofolate reductase-thymidylate synthase in Leishmania pteridine metabolism, using purified enzymes and knockout mutants. Recombinant L. major and Leishmania tarentolae , and native L. major PTR1s, were tetramers of 30-kDa subunits and showed similar catalytic properties with pterins and folates (pH dependence, substrate inhibition with H 2 pteridines). Unlike PTR1, dihydrofolate reductase-thymidylate synthase showed weak activity with folate and no activity with pterins. Correspondingly, studies of ptr1 − and dhfr-ts − mutants implicated only PTR1 in the ability of L . major to grow on a wide array of pterins. PTR1 exhibited 2000-fold less sensitivity to inhibition by methotrexate than dihydrofolate reductase-thymidylate synthase, suggesting several mechanisms by which PTR1 may compromise antifolate inhibition in wild-type Leishmania and lines bearing PTR1 amplifications. We incorporate these results into a comprehensive model of pteridine metabolism and discuss its implications in chemotherapy of this important human pathogen.