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• W2124017621 abstract "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 inLeishmania 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 H2pteridines). Unlike PTR1, dihydrofolate reductase-thymidylate synthase showed weak activity with folate and no activity with pterins. Correspondingly, studies ofptr1 − 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 PTR1amplifications. We incorporate these results into a comprehensive model of pteridine metabolism and discuss its implications in chemotherapy of this important human pathogen. 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 inLeishmania 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 H2pteridines). Unlike PTR1, dihydrofolate reductase-thymidylate synthase showed weak activity with folate and no activity with pterins. Correspondingly, studies ofptr1 − 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 PTR1amplifications. We incorporate these results into a comprehensive model of pteridine metabolism and discuss its implications in chemotherapy of this important human pathogen. Leishmania are trypanosomatid protozoan parasites that infect millions of people worldwide (1Report of a WHO Expert Committee (1984) The Leishmaniasis, WHO Technical Report, Series 701, pp. 3–140..Google Scholar). Leishmaniasis takes several forms, ranging from minor or severe disfiguring cutaneous lesions to the deadly visceral form, depending upon the species and immune status of the host. Vaccines against Leishmania are not yet available, and treatment currently relies on the antiquated pentavalent antimonial compounds. These drugs are often toxic, sometimes ineffective, and their mode of action remains unknown. A better understanding of novel biochemical pathways of this primitive eukaryotic parasite clearly would be helpful in the development of selective anti-Leishmania drugs. For example, although antifolates are a mainstay in the treatment of parasitic diseases such as malaria, they have not proven clinically effective againstLeishmania (2Scott D.A. Coombs G.H. Sanderson B.E. Mol. Biochem. Parasitol. 1987; 23: 139-149Google Scholar, 3Berman J.D. Rev. Infect. Dis. 1988; 10: 560-586Google Scholar). This may reflect the fact thatLeishmania and related trypanosomatids exhibit a number of unusual features in pteridine (pterin and folate) metabolism. Improved knowledge of this pathway would likely allow the development of antifolates effective against this important disease. Leishmania and other trypanosomatids includingCrithidia are unable to synthesize the pterin moiety from GTP and thus must acquire pteridines from the host by salvage mechanisms (2Scott D.A. Coombs G.H. Sanderson B.E. Mol. Biochem. Parasitol. 1987; 23: 139-149Google Scholar, 4Nathan H.A. Hutner S.H. Levin H.L. Nature. 1956; 178: 741-743Google Scholar, 5Trager W. J. Protozool. 1969; 16: 372-375Google Scholar, 6Petrillo-Peixoto M. Beverley S.M. Antimicrob. Agents Chemother. 1987; 31: 1575-1578Google Scholar, 7Kaur K. Coons T. Emmett K. Ullman B. J. Biol. Chem. 1988; 263: 7020-7028Google Scholar, 8Beck J.T. Ullman B. Mol. Biochem. Parasitol. 1990; 43: 221-230Google Scholar, 9Beck J.T. Ullman B. Mol. Biochem. Parasitol. 1991; 49: 21-28Google Scholar, 10Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Google Scholar, 11Papadopoulou B. Roy G. Mourad W. Leblanc E. Ouellette M. J. Biol. Chem. 1994; 269: 7310-7315Google Scholar). This feature led historically to an appreciation of the pterin requirement of eukaryotes, where pterins are now known to participate as essential cofactors in hydroxylations, ether-lipid cleavage, and NO synthase (12Benkovic S.J Blakley R Folates and Pterins. 2. John Wiley and Sons, Inc.,, New York1985: 1-399Google Scholar, 13Tietz A. Lindberg M. Kennedy E.P. J. Biol. Chem. 1964; 239: 4081-4090Google Scholar, 14Tayeh M.A. Marletta M.A. J. Biol. Chem. 1989; 264: 19654-19658Google Scholar, 15Kwon N.S. Nathan C.F. Stuehr D.J. J. Biol. Chem. 1989; 264: 20496-20501Google Scholar). However, the pathways involved in the salvage and metabolism of pterins, and their function inLeishmania, are only beginning to emerge (9Beck J.T. Ullman B. Mol. Biochem. Parasitol. 1991; 49: 21-28Google Scholar, 10Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Google Scholar). Recently, we identified a novel pteridine reductase (PTR1) 1The abbreviations used are: PTR1, pteridine reductase 1; MTX, methotrexate; DHFR-TS, dihydrofolate reductase-thymidylate synthase; H2biopterin, dihydrobiopterin; H4biopterin, tetrahydrobiopterin; H2folate, dihydrofolate; H4folate, tetrahydrofolate; Mes, 4 morpholinethanesulfonic acid; DHPR, dihydropteridine reductase. in Leishmania (10Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Google Scholar).PTR1 (formerly hmtx r or ltdh) was originally identified as the gene responsible for methotrexate (MTX) resistance on the amplified H region in several species ofLeishmania (16Callahan H.L. Beverley S.M. J. Biol. Chem. 1992; 267: 24165-24168Google Scholar, 17Papadopoulou B. Roy G. Ouellette M. EMBO J. 1992; 11: 3601-3608Google Scholar). Sequence comparisons placed the predicted PTR1 protein in a large family of aldo-keto reductases and short chain dehydrogenases, a family including both dihydropteridine and sepiapterin reductases (16Callahan H.L. Beverley S.M. J. Biol. Chem. 1992; 267: 24165-24168Google Scholar, 17Papadopoulou B. Roy G. Ouellette M. EMBO J. 1992; 11: 3601-3608Google Scholar, 18Krozowski Z. J. Steroid Biochem. Mol. Biol. 1994; 51: 125-130Google Scholar, 19Whiteley J.M. Xuong N.H. Varughese K.I. Adv. Exp. Med. Biol. 1993; 338: 115-121Google Scholar). The ability of PTR1 to reduce pteridines such as biopterin and folate was established by genetic and biochemical approaches in our laboratory (10Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Google Scholar). First, ptr1 − null mutants specifically required H2- or H4biopterin for growth, a requirement not satisfied by H2- or H4folate. Second, partially purified recombinant PTR1 protein exhibited NADPH-dependent reductase activity with biopterin and folate and lesser activity with H2biopterin or H2folate (10Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Google Scholar). These properties placed PTR1 in a position to play a key role in the salvage of oxidized pterins. Moreover, the H2folate reductase activity of PTR1, when combined with its relative insensitivity to MTX inhibition (100 nm versus 0.1 nm for DHFR-TS; Ref. 20Meek T.D. Garvey E.P. Santi D.V. Biochemistry. 1985; 24: 678-686Google Scholar), suggested that PTR1 could compromise antifolate inhibition ofLeishmania (10Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Google Scholar). Despite the homology of PTR1 to the short chain alcohol dehydrogenase superfamily (16Callahan H.L. Beverley S.M. J. Biol. Chem. 1992; 267: 24165-24168Google Scholar, 17Papadopoulou B. Roy G. Ouellette M. EMBO J. 1992; 11: 3601-3608Google Scholar, 18Krozowski Z. J. Steroid Biochem. Mol. Biol. 1994; 51: 125-130Google Scholar), its enzymatic properties overlap those of many dihydrofolate reductases (DHFR), which is remarkable given their evolutionary divergence. The major role of DHFR is to convert H2folate to the biochemically active H4folate, a step needed for de novo synthesis of thymidylate, and in bacteria and higher eukaryotes, purine nucleotides (trypanosomatids are auxotrophic for purines). In Leishmania as well as all protozoans and plant species examined thus far, DHFR is part of a bifunctional polypeptide that also encodes thymidylate synthase (DHFR-TS; Refs. 21Garrett C.E. Coderre J.A. Meek T.D. Garvey E.P. Claman D.M. Beverley S.M. Santi D.V. Mol. Biochem. Parasitol. 1984; 11: 257-265Google Scholar, 22Beverley S.M. Ellenberger T.E. Cordingley J.S. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2584-2588Google Scholar, 23Lazar G. Zhang H. Goodman H.M. Plant J. 1993; 3: 657-668Google Scholar). Direct comparison of the enzymatic properties of PTR1 and DHFR-TS would help in the elucidation of the salvage and metabolism of pteridines in Leishmania. Additionally, such information could establish the suitability of PTR1 and/or DHFR-TS as targets for rational Leishmania chemotherapy. Here we have purified both native and recombinant L. majorPTR1s as well as recombinant Leishmania tarentolae PTR1, and have characterized their properties including Km,Vmax, pH dependence, and inhibition by substrate and MTX. Comparisons of the wild-type and ptr1 −and dhfr-ts − knockout Leishmaniashowed that the ability to grow in diverse pterins correlated with their activity with PTR1 but not DHFR-TS, establishing PTR1 as the sole mediator of oxidized pterin salvage. Comparisons of the properties of PTR1 and DHFR-TS enzymes, and pteridine reductase activities in crudeLeishmania extracts (including those fromptr1 − and dhfr-ts −mutants), were used to establish the relative contribution of these enzymes in pteridine metabolism. With this information, we have developed a comprehensive model of the salvage and metabolism of pteridines in Leishmania. All lines of Leishmaniawere derived from L. major strain LT252 clone CC-1 and cultured in M199 medium containing 10% fetal bovine serum (24Kapler G.M. Coburn C.M. Beverley S.M. Mol. Cell. Biol. 1990; 10: 1084-1094Google Scholar). In this medium parasites grow as the promastigote form, which normally resides extracellularly within the gut of the sand fly insect vector. Null mutant Leishmania lacking DHFR-TS (dhfr-ts −) or PTR1 (ptr1 −) were created by targeted disruption of both alleles of each gene (10Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Google Scholar, 25Cruz A. Coburn C.M. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7170-7174Google Scholar). The ptr1 −mutant was grown with H2- or H4biopterin (2–4 μg/ml), and the dhfr-ts − mutant was grown with 10 μg/ml thymidine. The linesptr1 −/+PTR1 anddhfr-ts −/+DHFR-TS represent the respective null mutants transfected with plasmids pX63NEO-PTR1 (10Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Google Scholar) or pK300 (24Kapler G.M. Coburn C.M. Beverley S.M. Mol. Cell. Biol. 1990; 10: 1084-1094Google Scholar) and overexpress PTR1 and DHFR-TS, respectively (Ref. 10Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Google Scholar; this work). In some experiments cells were grown in fdM199, which is M199 medium lacking folate and thymidine and supplemented with 0.66% bovine serum albumin (U. S. Biochemical Corp.) instead of serum. Pterin supplements were H4biopterin (RBI), 6-hydroxymethylpterin, pterin, pteroic acid (Sigma), and a wide range of other pterins (Schircks Laboratories, Jona, Switzerland or from S. Kaufman, National Institutes of Health). H2neopterin was prepared from neopterin by reduction with dithionite in the presence of ascorbate (26Futterman S. J. Biol. Chem. 1957; 228: 1031-1038Google Scholar). Parasites were enumerated using a Coulter Counter (Model Zf) at the time when cultures grown in H4biopterin had reached late log phase. The initial steps of purification of recombinant L. major PTR1 have been described (10Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Google Scholar) and included expression in Escherichia coliusing the pET-3a expression vector (27Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Google Scholar), induction, cellular lysis, and purification by ammonium sulfate precipitation and DEAE-cellulose chromatography. PTR1-containing fractions from the DEAE step were pooled and the buffer changed to 20 mm Mes, pH 6.0, by passage over PD10 columns of Sephadex G-25 (Pharmacia Biotech Inc.). Subsequent purification steps were carried out by fast protein liquid chromatography (Pharmacia). Protein was applied to an ion exchange Mono-S HR 5/5 column and eluted with a 20-min 0–0.2 m NaCl gradient at 1 ml/min. An ion exchange Mono-Q 5/5 column was also tested and found to give an equivalent purification. PTR1-containing fractions were combined, and the volume reduced to 1 ml using YM10 filters (Amicon). The concentrate was applied to a Superdex 200HR 10/30 column and eluted at a flow rate of 0.5 ml/min with 20 mm Mes, pH 6.0, containing 0.1 m NaCl. Recombinant PTR1 was purified 10-fold with overall yields of 80%. The coding region for L. tarentolae PTR1 was amplified by the polymerase chain reaction using Taqpolymerase, template DNA from the MG strain of L. tarentolae, and the primers SMB-8 (5′-ggcagatcTCAGGCCCGGGTAAGGC) and SMB-9 (5′-cgcagatctcccatATGACGACTTCTCCGA; lowercase letters indicate bases not present in PTR1), with 25 amplification cycles of 1 min at 94 °C, 1 min at 57 °C, and 2 min at 72 °C. The expected fragment was obtained, digested with NdeI andBglII, inserted into the pET-3a expression vector (Novagen), and transformed into E. coli strain BL21(DE3)/pLysS (27Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Google Scholar). The expression of L. tarentolae PTR1 was induced and the enzyme purified as described for L. major. Native PTR1 was purified from 7.5 × 1010 ptr1 −/+PTR1 L. major, in a manner similar to that used for the recombinant enzyme except that the cells were lysed by 3 cycles of freezing and thawing followed by sonication. The lysate was centrifuged at 100,000 × g for 30 min, and the supernatant was loaded onto a DEAE-cellulose column, eluted (10Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Google Scholar), and further purified as described for the recombinant enzyme. Native PTR1 was purified 200-fold and obtained in 72% yield. Purified PTR1 preparations were stored at −80 °C in the presence of 20% glycerol and 20 mm β-mercaptoethanol. The molecular weights of nondenatured PTR1s were estimated on a Sephacryl S-200 column (120 × 0.8 cm) at a flow rate of 0.5 ml/min. Three different pH values were tested using the following buffers: 20 mm Tris-HCl, pH 7.0, 20 mm NaPO4, pH 6.0, or 20 mmsodium acetate, pH 4.7, each containing 0.1 m NaCl. Molecular mass markers were β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa). Fractions were monitored at 280 nm and for PTR1 activity. Spectrophotometric pteridine reductase assays were performed at 30 °C in the presence of NADPH (usually 100 μm) and pteridines as indicated (10Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Google Scholar). The pH dependence of PTR1 activity was determined using three overlapping buffers: 20 mm sodium acetate, pH 3.6–6.0, NaPO4, pH 5.5–7.5, or Tris-HCl, pH 7.0–8.0. Radiometric assays of folate and/or H2folate reductase activities (28Rothenberg S.P. Iqbal M.P. Da Costa M. Anal. Biochem. 1980; 103: 152-156Google Scholar) were performed using 40 μm [3′,4′,7,9-3H]folate (24.1 Ci/mmol, Moravek Biochemicals), which was purified prior to use (29Ellenberger T.E. Beverley S.M. J. Biol. Chem. 1987; 262: 10053-10058Google Scholar). To test the nature of the product formed from reduction of biopterin or H2biopterin by PTR1 or DHFR-TS, a coupled assay was used (30Guroff G. Rhoads C.A. Abramowitz A. Anal. Biochem. 1967; 21: 8-273Google Scholar) where the synthesis of H4biopterin is linked to the hydroxylation of [4-3H]Phe (27 Ci/mmol, Amersham Corp.) by mammalian phenylalanine hydroxylase (Sigma). After incubation for 30 min at 25 °C, the [3H]Tyr formed was iodinated, the sample was passed over a Dowex 50 column, and the tritiated water was quantified by scintillation counting. The kinetic parameters Km and Vmax for the pteridine substrates were measured in a spectrophotometric assay with 100 μm NADPH as described previously (10Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Google Scholar). Extinction coefficients used for various pteridines were determined spectrophotometrically, and PTR1 activity was calculated based on the decrease in absorbance of both NADPH and the pteridine substrates. Kinetic data for oxidized pteridines were evaluated by fitting to the Michaelis-Menten equation by nonlinear regression (Hyper Version 1.02A; J.S. Eastby, Liverpool, UK). Both H2folate and H2biopterin showed substrate inhibition at concentrations above 5 and 10 μm, respectively, and for these,Km, Vmax, andKi (for substrate) values were evaluated using graphical plots and the general equation for substrate inhibition (31Cleland W. Methods Enzymol. 1979; 63: 501-513Google Scholar). For inhibition studies, PTR1 was incubated with MTX and NADPH and the reaction initiated with the pteridine substrate (40 μmfolate, 100 μm biopterin, 10 μmH2biopterin, or 5 μm H2folate). Inhibition was examined at several concentrations of enzyme, and the data were analyzed using a method for tight binding inhibitors to obtain Ki (32Cha S. Biochem. Pharmacol. 1975; 24: 2177-2185Google Scholar). Recombinant DHFR-TS fromL. major was purified from adhfr − E. coli strain (33Howell E.E. Foster P.G. Foster L.M. J. Bacteriol. 1988; 170: 3040-3045Google Scholar) bearing the expression plasmid 02CLSA-4 (34Knighton D.R. Kan C.C. Howland E. Janson C.A. Hostomska Z. Welsh K.M. Matthews D.A. Nat. Struct. Biol. 1994; 1: 186-194Google Scholar). Cells were lysed by two cycles through a French press (15,000 p.s.i.), and DHFR-TS was purified by binding and elution from a MTX-Sepharose column (Sigma) (34Knighton D.R. Kan C.C. Howland E. Janson C.A. Hostomska Z. Welsh K.M. Matthews D.A. Nat. Struct. Biol. 1994; 1: 186-194Google Scholar, 35Coderre J.A. Beverley S.M. Schimke R.T. Santi D.V. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2132-2136Google Scholar). The eluate was concentrated using YM10 membrane filters (Amicon) and loaded onto a Sephacryl S-200 column (120 × 0.8 cm). Electrophoretically homogeneous enzyme was eluted with 50 mm Tris·HCl, 0.1m NaCl at a flow rate of 0.5 ml/min, desalted over PD10 columns of Sephadex G-25 (Pharmacia), and stored at −80 °C in the presence of 10% glycerol. Polyclonal antiserum against PTR1 was elicited in New Zealand White rabbits using 200 μg of L. major PTR1 in Freund's complete adjuvant (Sigma) in the primary immunization. The rabbits were boosted 5 times with 100 μg PTR1 each in incomplete Freund's adjuvant at 3-week intervals, and serum was obtained after the last bleeding. For immunoblots, purified PTR1 and crude Leishmania extracts were separated on a 12.5% SDS-polyacrylamide gel (36Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar) and electrophoretically transferred onto Millipore polyvinylidene difluoride membranes (37Towbin H. Staehlin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Google Scholar) using a semi-dry blot apparatus (Owl Scientific). Blots were incubated with antiserum to PTR1 (1:1000), and binding was detected using either horseradish peroxidase-conjugated goat anti-rabbit antibody (1:3000) and chemiluminescence (Amersham Corp.) or alkaline phosphatase-conjugated goat anti-rabbit antibody and developed with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. Late logarithmic phase promastigotes were collected by centrifugation, washed twice with phosphate-buffered saline (138 mm NaCl, 2.7 mmKCl, 10 mm Na2HPO4, and 1.8 mm KH2PO4), and resuspended (3 × 109/ml) in phosphate-buffered saline supplemented with 1 mm EDTA and a mixture of protease inhibitors suggested by Meek et al. (20Meek T.D. Garvey E.P. Santi D.V. Biochemistry. 1985; 24: 678-686Google Scholar). Cells were lysed by freeze thawing and sonication and the extracts clarified by centrifugation at 15,000 × g for 30 min. Previously we reported upon the partial purification of L. major PTR1, expressed in engineeredE. coli (10Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Google Scholar). Inclusion of two additional steps (ion exchange and gel filtration chromatography) yielded preparations that were electrophoretically homogeneous, even when the gel was overloaded (Fig. 1 A, lanes 3–5). We also overexpressed and purified native PTR1 from L. major parasites and recombinant L. tarentolae PTR1. The recombinant and native PTR1s behaved similarly during purification and exhibited similar mobilities upon SDS-polyacrylamide gel electrophoresis (Fig. 1 A, lanes 5–7). The apparent subunit molecular masses were 30 kDa (10Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Google Scholar, 11Papadopoulou B. Roy G. Mourad W. Leblanc E. Ouellette M. J. Biol. Chem. 1994; 269: 7310-7315Google Scholar, 16Callahan H.L. Beverley S.M. J. Biol. Chem. 1992; 267: 24165-24168Google Scholar, 17Papadopoulou B. Roy G. Ouellette M. EMBO J. 1992; 11: 3601-3608Google Scholar). Western blot analysis with a polyclonal antiserum to recombinantL. major PTR1 detected a 30-kDa protein in wild-typeL. major extracts whose size was identical to that of purified PTR1s (Fig. 1 B, lanes 2–4). This protein was absent in the ptr1 − L. majordeletion mutant obtained previously by gene targeting (Fig. 1 B, lane 1) (10Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Google Scholar) and was expressed at approximately 100-fold higher levels in the L.major line overexpressing PTR1 (Fig.1 B, lane 3; note that 100-fold less protein was loaded inlane 3). In wild-type cells, PTR1 constituted about 0.01% of the total cellular protein. By gel filtration chromatography, the apparent molecular mass of PTR1 was estimated to be 116 and 117 kDa for the recombinant and nativeL. major enzymes, respectively (not shown). Similar values were obtained at pH values of 4.7, 6.0, and 7.0 (data not shown). We infer that PTR1 is a tetramer of identical 30-kDa subunits and that significant alterations in molecular shape are not associated with differences in the pH dependence of folate versus biopterin reduction (below). Previous studies of partially purified PTR1 showed it to have two pH optima, one of about 4.7 for biopterin and H2biopterin and one of about 6.0 for folate and H2folate (10Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Google Scholar). Studies of the homogeneous L. major and purified L. tarentolae PTR1s have refined and extended these initial findings. At the optimum pH for each substrate, PTR1 activity with oxidized biopterin and folate exhibited standard Michaelis-Menten kinetics (Fig.2). However, H2biopterin and H2folate showed substrate inhibition at concentrations above 10 and 5 μm, respectively (Fig. 2).Vmax values with H2biopterin and H2folate were derived from analyses that included considerations of substrate inhibition (31Cleland W. Methods Enzymol. 1979; 63: 501-513Google Scholar) and yielded values that were at least 50% that of the corresponding oxidized pteridines (TableI). Previously, only substrate concentrations of 100 μm were tested (10Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Google Scholar), which led to a 3–4-fold underestimate of the rate of reduction of H2pteridines by PTR1. H2neopterin and H2sepiapterin also showed substrate inhibition, whereas l- andd-biopterin, l- and d-neopterin, 6-hydroxymethylpterin, l- andd-monapterin, 6-formylpterin, and 6,7-dimethylpterin showed standard Michaelis-Menten kinetics (data not shown). This suggests that substrate inhibition was a general feature of PTR1 activity, but only with H2pteridines.Table IKinetic parameters for Leishmania PTR1sPteridine substratepH1-aKinetic parameters were determined in 20 mm each of sodium acetate, pH 4.7, or sodium phosphate, pH 6.0 or 7.0 (as appropriate substrates were fixed at 100 μm NADPH; 100 μm biopterin; 40 μm H2folate; 10 μmH2biopterin; 5 μm H4folate).Km(pteridine)Km(NADPH)Ki1-bKi for pteridine substrates that exhibit inhibition of enzymatic activity.(pteridine)VmaxKi (MTX)μ mμ mμ mμmol/min/mgnmRecombinant L. major PTR1Biopterin4.7*12.2 ± 1.613.2 ± 0.6NI1-cNI − PTR1 activity is not inhibited by substrate at maximum concentrations used for kinetic analysis (40–100 μm). * indicates optimum pH for each substrate. ND, not determined.1.2 ± 0.330.7 ± 5.76.019.8 ± 2.5NDNI 1-cNI − PTR1 activity is not inhibited by substrate at maximum concentrations used for kinetic analysis (40–100 μm). * indicates optimum pH for each substrate. ND, not determined.0.66 ± 0.07102 ± 257.039.9 ± 5.9NDNI 1-cNI − PTR1 activity is not inhibited by substrate at maximum concentrations used for kinetic analysis (40–100 μm). * indicates optimum pH for each substrate. ND, not determined.0.21 ± 0.03276 ± 61H2biopterin4.7*7.6 ± 2.814.5 ± 1.814.5 ± 0.90.87 ± 0.258.3 ± 156.05.6 ± 1.7ND21.2 ± 3.50.50 ± 0.0988 ± 207.05.4 ± 2.3ND18.2 ± 2.70.23 ± 0.04342 ± 110Folate4.71.6 ± 0.3NDNI 1-cNI − PTR1 activity is not inhibited by substrate at maximum concentrations used for kinetic analysis (40–100 μm). * indicates optimum pH for each substrate. ND, not determined.0.32 ± 0.05200 ± 236.0*2.6 ± 0.412.2 ± 0.9NI 1-cNI − PTR1 activity is not inhibited by substrate at maximum concentrations used for kinetic analysis (40–100 μm). * indicates optimum pH for each substrate. ND, not determined.0.56 ± 0.2265 ± 287.08.5 ± 3.4NDNI 1-cNI − PTR1 activity is not inhibited by substrate at maximum concentrations used for kinetic analysis (40–100 μm). * indicates optimum pH for each substrate. ND, not determined.0.29 ± 0.04801 ± 172H2folate4.76.1 ± 1.0ND13.1 ± 1.30.22 ± 0.06176 ± 376.0*3.4 ± 0.214.2 ± 1.213.5 ± 0.90.38 ± 0.07191 ± 507.05.4 ± 1.2ND11.2 ± 2.50.25 ± 0.04509 ± 185Native L. major PTR1Biopterin4.7*10.1 ± 1.411.6 ± 1.1NI 1-cNI − PTR1 activity is not inhibited by substrate at maximum concentrations used for kinetic analysis (40–100 μm). * indicates optimum pH for each substrate. ND, not determined.0.55 ± 0.126.3 ± 4.7Folate6.0*2.4 ± 0.313.5 ± 2.6NI 1-cNI − PTR1 activity is not inhibited by substrate at maximum concentrations used for kinetic analysis (40–100 μm). * indicates optimum pH for each substrate. ND, not determined.0.28 ± 0.1NDRecombinant L. tarentolae PTR1Biopterin4.7*10.9 ± 2.512.3 ± 1.7NI 1-cNI − PTR1 activity is not inhibited by substrate at maximum concentrations used for kinetic analysis (40–100 μm). * indicates optimum pH for each substrate. ND, not determined.0.98 ± 0.128.3 ± 8H2biopterin4.7*8.5 ± 2.49.35 ± 4.821.1 ± 2.70.62 ± 0.162.5 ± 22Folate6.0*1.9 ± 0.314.6 ± 1.1NI 1-cNI − PTR1 activity is not inhibited by substrate at maximum concentrations used for kinetic analysis (40–100 μm). * indicates optimum pH for each substrate. ND, not determined.0.46 ± 0.1248 ± 26H2folate6.0*6.7 ± 1.612.0 ± 5.521.5 ± 3.20.23 ± 0.02210 ± 29Results are the average of 2–4 determinations presented with standard deviations.1-a Kinetic parameters were determined in 20 mm each of sodium acetate, pH 4.7, or sodium phosphate, pH 6.0 or 7.0 (as appropriate substrates were fixed at 100 μm NADPH; 100 μm biopterin; 40 μm H2folate; 10 μmH2biopterin; 5 μm H4folate).1-b Ki for pteridine substrates that exhibit inhibition of enzymatic activity.1-c NI − PTR1 activity is not inhibited by substrate at maximum concentrations used for kinetic analysis (40–100 μm). * indicates optimum pH f" @default.
• W2124017621 created "2016-06-24" @default.
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• W2124017621 date "1997-05-01" @default.
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• W2124017621 title "The Roles of Pteridine Reductase 1 and Dihydrofolate Reductase-Thymidylate Synthase in Pteridine Metabolism in the Protozoan Parasite Leishmania major" @default.
• W2124017621 cites W137993082 @default.
• W2124017621 cites W145884660 @default.
• W2124017621 cites W1504926718 @default.
• W2124017621 cites W1510112285 @default.
• W2124017621 cites W1517621505 @default.
• W2124017621 cites W1533126574 @default.
• W2124017621 cites W168773424 @default.
• W2124017621 cites W1873199501 @default.
• W2124017621 cites W193909774 @default.
• W2124017621 cites W1971283163 @default.
• W2124017621 cites W1971548200 @default.
• W2124017621 cites W1983111672 @default.
• W2124017621 cites W1989550386 @default.
• W2124017621 cites W1997123695 @default.
• W2124017621 cites W1999809189 @default.
• W2124017621 cites W2000355934 @default.
• W2124017621 cites W2000972006 @default.
• W2124017621 cites W2017350383 @default.
• W2124017621 cites W2018101979 @default.
• W2124017621 cites W2020444920 @default.
• W2124017621 cites W2024585751 @default.
• W2124017621 cites W2025047925 @default.
• W2124017621 cites W2042007171 @default.
• W2124017621 cites W2042652437 @default.
• W2124017621 cites W2045799225 @default.
• W2124017621 cites W2049768129 @default.
• W2124017621 cites W2051012950 @default.
• W2124017621 cites W2056022007 @default.
• W2124017621 cites W2062369787 @default.
• W2124017621 cites W2068114623 @default.
• W2124017621 cites W2071867579 @default.
• W2124017621 cites W2072607746 @default.
• W2124017621 cites W2072790705 @default.
• W2124017621 cites W2083224849 @default.
• W2124017621 cites W2085397927 @default.
• W2124017621 cites W2086707894 @default.
• W2124017621 cites W2095458479 @default.
• W2124017621 cites W2100837269 @default.
• W2124017621 cites W2101108802 @default.
• W2124017621 cites W2109364945 @default.
• W2124017621 cites W2114236987 @default.
• W2124017621 cites W2115928361 @default.
• W2124017621 cites W2135877083 @default.
• W2124017621 cites W2161709854 @default.
• W2124017621 cites W2170773647 @default.
• W2124017621 cites W2177086111 @default.
• W2124017621 cites W2321445076 @default.
• W2124017621 cites W2335433921 @default.
• W2124017621 cites W2347206875 @default.
• W2124017621 cites W36714077 @default.
• W2124017621 cites W4240205969 @default.
• W2124017621 cites W71482712 @default.
• W2124017621 cites W970978831 @default.
• W2124017621 cites W2399220599 @default.
• W2124017621 doi "https://doi.org/10.1074/jbc.272.21.13883" @default.
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