• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Nov 22, 1994; 91(24): 11442–11446.

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 decades ago which heralded the discovery of key metabolic roles played by pteridines in eukaryotes. We have now identified the enzyme mediating unconjugated pteridine salvage in the human parasite Leishmania major, PTR1 (pteridine reductase 1, formerly hmtxr or ltdh). PTR1 is the gene in the amplified H region responsible for methotrexate (MTX) resistance, and belongs to a large family of oxidoreductases with diverse substrates 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 engineered Escherichia coli exhibited a MTX-sensitive, NADPH-dependent biopterin reductase activity. PTR1 showed good activity with folate and significant activity with dihydrofolate and dihydrobiopterin, but not with quinonoid dihydrobiopterin. PTR1 thus differs considerably from previously reported pteridine reductases of trypanosomatids and vertebrates. Pteridine reductase activity was diminished in ptr1- Leishmania and was elevated in transfected parasites bearing multiple copies of PTR1; correspondingly, ptr1- was MTX-hypersensitive whereas the multicopy transfectant was MTX-resistant. The concordance of the biochemical and genetic properties of PTR1 suggests that this is the primary enzyme mediating pteridine salvage. These findings suggest several possible mechanisms for PTR1-mediated MTX resistance and should aid in the design of rational chemotherapy.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.3M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • HUTNER SH, LEVIN HL, NATHAN HA. Independent requirements for crithidia factor and folic acid in a trypanosomid flagellate. Nature. 1956 Oct 6;178(4536):741–742. [PubMed]
  • Tayeh MA, Marletta MA. Macrophage oxidation of L-arginine to nitric oxide, nitrite, and nitrate. Tetrahydrobiopterin is required as a cofactor. J Biol Chem. 1989 Nov 25;264(33):19654–19658. [PubMed]
  • Kwon NS, Nathan CF, Stuehr DJ. Reduced biopterin as a cofactor in the generation of nitrogen oxides by murine macrophages. J Biol Chem. 1989 Dec 5;264(34):20496–20501. [PubMed]
  • Beck JT, Ullman B. Nutritional requirements of wild-type and folate transport-deficient Leishmania donovani for pterins and folates. Mol Biochem Parasitol. 1990 Dec;43(2):221–230. [PubMed]
  • Beck JT, Ullman B. Biopterin conversion to reduced folates by Leishmania donovani promastigotes. Mol Biochem Parasitol. 1991 Nov;49(1):21–28. [PubMed]
  • Kaur K, Coons T, Emmett K, Ullman B. Methotrexate-resistant Leishmania donovani genetically deficient in the folate-methotrexate transporter. J Biol Chem. 1988 May 25;263(15):7020–7028. [PubMed]
  • Peixoto MP, Beverley SM. In vitro activity of sulfonamides and sulfones against Leishmania major promastigotes. Antimicrob Agents Chemother. 1987 Oct;31(10):1575–1578. [PMC free article] [PubMed]
  • Ferone R, Roland S. Dihydrofolate reductase: thymidylate synthase, a bifunctional polypeptide from Crithidia fasciculata. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5802–5806. [PMC free article] [PubMed]
  • Coderre JA, Beverley SM, Schimke RT, Santi DV. Overproduction of a bifunctional thymidylate synthetase-dihydrofolate reductase and DNA amplification in methotrexate-resistant Leishmania tropica. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2132–2136. [PMC free article] [PubMed]
  • Beverley SM, Ellenberger TE, Cordingley JS. Primary structure of the gene encoding the bifunctional dihydrofolate reductase-thymidylate synthase of Leishmania major. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2584–2588. [PMC free article] [PubMed]
  • Grumont R, Washtien WL, Caput D, Santi DV. Bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania tropica: sequence homology with the corresponding monofunctional proteins. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5387–5391. [PMC free article] [PubMed]
  • Callahan HL, Beverley SM. A member of the aldoketo reductase family confers methotrexate resistance in Leishmania. J Biol Chem. 1992 Dec 5;267(34):24165–24168. [PubMed]
  • Papadopoulou B, Roy G, Ouellette M. A novel antifolate resistance gene on the amplified H circle of Leishmania. EMBO J. 1992 Oct;11(10):3601–3608. [PMC free article] [PubMed]
  • Whiteley JM, Xuong NH, Varughese KI. Is dihydropteridine reductase an anomalous dihydrofolate reductase, a flavin-like enzyme, or a short-chain dehydrogenase? Adv Exp Med Biol. 1993;338:115–121. [PubMed]
  • Hirayama K, Nakanisi N, Sueoka T, Katoh S, Yamada S. Dihydropteridine reductase and tetrahydropterin in Crithidia fasciculata cells. Biochim Biophys Acta. 1980 Apr 11;612(2):337–343. [PubMed]
  • Baker ME. Human placental 17 beta-hydroxysteroid dehydrogenase is homologous to NodG protein of Rhizobium meliloti. Mol Endocrinol. 1989 May;3(5):881–884. [PubMed]
  • Baker ME. A common ancestor for Candida tropicalis and dehydrogenases that synthesize antibiotics and steroids. FASEB J. 1990 Sep;4(12):3028–3032. [PubMed]
  • Kapler GM, Coburn CM, Beverley SM. Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression. Mol Cell Biol. 1990 Mar;10(3):1084–1094. [PMC free article] [PubMed]
  • Cruz A, Coburn CM, Beverley SM. Double targeted gene replacement for creating null mutants. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7170–7174. [PMC free article] [PubMed]
  • Freedman DJ, Beverley SM. Two more independent selectable markers for stable transfection of Leishmania. Mol Biochem Parasitol. 1993 Nov;62(1):37–44. [PubMed]
  • Cruz A, Beverley SM. Gene replacement in parasitic protozoa. Nature. 1990 Nov 8;348(6297):171–173. [PubMed]
  • Cruz AK, Titus R, Beverley SM. Plasticity in chromosome number and testing of essential genes in Leishmania by targeting. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1599–1603. [PMC free article] [PubMed]
  • Beverley SM. Estimation of circular DNA size using gamma-irradiation and pulsed-field gel electrophoresis. Anal Biochem. 1989 Feb 15;177(1):110–114. [PubMed]
  • LeBowitz JH, Coburn CM, Beverley SM. Simultaneous transient expression assays of the trypanosomatid parasite Leishmania using beta-galactosidase and beta-glucuronidase as reporter enzymes. Gene. 1991 Jul 15;103(1):119–123. [PubMed]
  • Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Davis MD, Kaufman S, Milstien S. The auto-oxidation of tetrahydrobiopterin. Eur J Biochem. 1988 Apr 15;173(2):345–351. [PubMed]
  • Beverley SM, Coderre JA, Santi DV, Schimke RT. Unstable DNA amplifications in methotrexate-resistant Leishmania consist of extrachromosomal circles which relocalize during stabilization. Cell. 1984 Sep;38(2):431–439. [PubMed]
  • Papadopoulou B, Roy G, Mourad W, Leblanc E, Ouellette M. Changes in folate and pterin metabolism after disruption of the Leishmania H locus short chain dehydrogenase gene. J Biol Chem. 1994 Mar 11;269(10):7310–7315. [PubMed]
  • Ellenberger TE, Beverley SM. Multiple drug resistance and conservative amplification of the H region in Leishmania major. J Biol Chem. 1989 Sep 5;264(25):15094–15103. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Greco WR, Hakala MT. Evaluation of methods for estimating the dissociation constant of tight binding enzyme inhibitors. J Biol Chem. 1979 Dec 10;254(23):12104–12109. [PubMed]
  • Nichol CA, Smith GK, Duch DS. Biosynthesis and metabolism of tetrahydrobiopterin and molybdopterin. Annu Rev Biochem. 1985;54:729–764. [PubMed]
  • Meek TD, Garvey EP, Santi DV. Purification and characterization of the bifunctional thymidylate synthetase-dihydrofolate reductase from methotrexate-resistant Leishmania tropica. Biochemistry. 1985 Jan 29;24(3):678–686. [PubMed]
  • White JC. Reversal of methotrexate binding to dihydrofolate reductase by dihydrofolate. Studies with pure enzyme and computer modeling using network thermodynamics. J Biol Chem. 1979 Nov 10;254(21):10889–10895. [PubMed]
  • Ellenberger TE, Beverley SM. Biochemistry and regulation of folate and methotrexate transport in Leishmania major. J Biol Chem. 1987 Jul 25;262(21):10053–10058. [PubMed]
  • Ellenberger TE, Beverley SM. Reductions in methotrexate and folate influx in methotrexate-resistant lines of Leishmania major are independent of R or H region amplification. J Biol Chem. 1987 Oct 5;262(28):13501–13506. [PubMed]
  • Santi DV, Nolan P, Shane B. Folylpolyglutamates in Leishmania major. Biochem Biophys Res Commun. 1987 Aug 14;146(3):1089–1092. [PubMed]
  • Ellenberger TE, Wright JE, Rosowsky A, Beverley SM. Wild-type and drug-resistant Leishmania major hydrolyze methotrexate to N-10-methyl-4-deoxy-4-aminopteroate without accumulation of methotrexate polyglutamates. J Biol Chem. 1989 Sep 25;264(27):15960–15966. [PubMed]
  • Gutteridge WE, McCormack JJ, Jr, Jaffe JJ. Presence and properties of dihydrofolate reductases within the genus Crithidia. Biochim Biophys Acta. 1969 May 27;178(3):453–458. [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...