Characterization of quinonoid-Dihydropteridine Reductase (QDPR) from the Lower Eukaryote Leishmania major
2002; Elsevier BV; Volume: 277; Issue: 41 Linguagem: Inglês
10.1074/jbc.m206543200
ISSN1083-351X
AutoresLon‐Fye Lye, Mark L. Cunningham, Stephen M. Beverley,
Tópico(s)Biochemical and Molecular Research
ResumoBiopterin is required for growth of the protozoan parasite Leishmania and is salvaged from the host through the activities of a novel biopterin transporter (BT1) and broad-spectrum pteridine reductase (PTR1). Here we characterizeLeishmania major quinonoid-dihydropteridine reductase (LmQDPR), the key enzyme required for regeneration and maintenance of H4biopterin pools. LmQDPR shows good homology to metazoanquinonoid-dihydropteridine reductase and conservation of domains implicated in catalysis and regulation. Unlike other organisms, LmQDPR is encoded by a tandemly repeated array of 8–9 copies containing LmQDPR plus two other genes.QDPR mRNA and enzymatic activity were expressed at similar levels throughout the infectious cycle. The pH optima, kinetic properties, and substrate specificity of purified LmQDPR were found to be similar to that of other qDPRs, although it lacked significant activity for non-quinonoid pteridines. These and other data suggest that LmQDPR is unlikely to encode the dihydrobiopterin reductase activity (PTR2) described previously. Similarly LmQDPR is not inhibited by a series of antifolates showing anti-leishmanial activity beyond that attributable to dihydrofolate reductase or PTR1 inhibition. qDPR activity was found in crude lysates of Trypanosoma brucei and Trypanosoma cruzi, further emphasizing the importance of H4biopterin throughout this family of human parasites. Biopterin is required for growth of the protozoan parasite Leishmania and is salvaged from the host through the activities of a novel biopterin transporter (BT1) and broad-spectrum pteridine reductase (PTR1). Here we characterizeLeishmania major quinonoid-dihydropteridine reductase (LmQDPR), the key enzyme required for regeneration and maintenance of H4biopterin pools. LmQDPR shows good homology to metazoanquinonoid-dihydropteridine reductase and conservation of domains implicated in catalysis and regulation. Unlike other organisms, LmQDPR is encoded by a tandemly repeated array of 8–9 copies containing LmQDPR plus two other genes.QDPR mRNA and enzymatic activity were expressed at similar levels throughout the infectious cycle. The pH optima, kinetic properties, and substrate specificity of purified LmQDPR were found to be similar to that of other qDPRs, although it lacked significant activity for non-quinonoid pteridines. These and other data suggest that LmQDPR is unlikely to encode the dihydrobiopterin reductase activity (PTR2) described previously. Similarly LmQDPR is not inhibited by a series of antifolates showing anti-leishmanial activity beyond that attributable to dihydrofolate reductase or PTR1 inhibition. qDPR activity was found in crude lysates of Trypanosoma brucei and Trypanosoma cruzi, further emphasizing the importance of H4biopterin throughout this family of human parasites. bifunctional dihydrofolate reductase-thymidylate synthase quinonoid-dihydropteridine reductase Leishmania major qDPR gene/enzyme dihydrobiopterin tetrahydrobiopterin open reading frame pteridine reductase 1 postulated dihydrobiopterin reductase pterin-4 α-carbinolamine dehydratase dimethyl-5,6,7,8-tetrahydropterin nucleotide(s) nitric oxide synthase Leishmania are trypanosomatid protozoan parasites that infect over 15 million people in tropical and subtropical regions of the world, with a further 350 million at risk (1WHO Expert Committee The Leishmaniasis. WHO, Geneva1984Google Scholar). Leishmaniasis manifests as cutaneous lesions from minor to severe, or as a visceral form that, if untreated, has a high fatality rate. Existing chemotherapies are unsatisfactory, relying upon pentavalent antimonial compounds despite considerable host toxicity and some evidence for the emergence of parasite resistance (2Grogl M. Thomason T.N. Franke E.D. Am. J. Trop. Med. Hyg. 1992; 47: 117-126Crossref PubMed Scopus (227) Google Scholar). Presently no effective vaccine against leishmaniasis is available. Leishmania have a digenetic life cycle, first residing in the gut of phlebotomine sand flies where they replicate as a procyclic promastigote. As parasites enter stationary phase they differentiate into the infectious metacyclic promastigote, which is ultimately transmitted by the bite of a sand fly. Once parasites are introduced into the mammalian host, they are taken up by macrophages where they differentiate into amastigotes. Amastigotes reside and propagate within the phagolysosome, where they induce pathology and disease. Leishmania and other trypanosomatid protozoan parasites are incapable of de novo synthesis of pteridines (folate and pterins) and must obtain them by salvage from their insect or mammalian hosts (3Beck J.T. Ullman B. Mol. Biochem. Parasitol. 1990; 43: 221-230Crossref PubMed Scopus (42) Google Scholar, 4Petrillo-Peixoto M. Beverley S.M. Antimicrob. Agents Chemother. 1987; 31: 1575-1578Crossref PubMed Google Scholar, 5Scott D.A. Coombs G.H. Sanderson B.E. Mol. Biochem. Parasitol. 1987; 23: 139-149Crossref PubMed Scopus (31) Google Scholar, 6Trager W. J. Protozool. 1969; 16: 372-375Crossref PubMed Scopus (27) Google Scholar). To accomplish this, Leishmania express a versatile pteridine salvage network, consisting of transporters with specificity for folate and biopterin (FT1 and BT1, respectively; Refs.7Cunningham M.L. Beverley S.M. Mol. Biochem. Parasitol. 2001; 113: 199-213Crossref PubMed Scopus (81) Google Scholar, 8Lemley C. Yan S. Dole V.S. Madhubala R. Cunningham M.L. Beverley S.M. Myler P.J. Stuart K.D. Mol. Biochem. Parasitol. 1999; 104: 93-105Crossref PubMed Scopus (62) Google Scholar, 9Kundig C. Haimeur A. Legare D. Papadopoulou B. Ouellette M. EMBO J. 1999; 18: 2342-2351Crossref PubMed Scopus (89) Google Scholar, 10Myler P.J. Lodes M.J. Merlin G. de Vos T. Stuart K.D. Mol. Biochem. Parasitol. 1994; 66: 11-20Crossref PubMed Scopus (30) Google Scholar). 1J. Moore and S. M. Beverley, manuscript in preparation. Following uptake, two pteridine reductases, one specific for folate (a bifunctional dihydrofolate reductase-thymidylate synthase; DHFR-TS)2 and a second with broader specificity (pteridine reductase 1 or PTR1), reduce folate and biopterin, respectively, into the active forms, tetrahydrofolate (H4folate) and tetrahydrobiopterin (H4B; Refs.11Nare B. Luba J. Hardy L.W. Beverley S. Parasitology. 1997; 114: S101-S110Crossref PubMed Google Scholar, 12Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Crossref PubMed Scopus (151) Google Scholar, 13Beverley S.M. Ellenberger T.E. Cordingley J.S. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2584-2588Crossref PubMed Scopus (110) Google Scholar). The importance of folate in essential metabolic processes such as synthesis of thymidylate has been established firmly inLeishmania by pharmacological and genetic studies bothin vitro and in vivo (14Ivanetich K.M. Santi D.V. Exp. Parasitol. 1990; 70: 367-371Crossref PubMed Scopus (38) Google Scholar, 15Cruz A. Beverley S.M. Nature. 1990; 348: 171-173Crossref PubMed Scopus (193) Google Scholar, 16Titus R.G. Gueiros-Filho F.J. de Freitas L.A. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10267-10271Crossref PubMed Scopus (195) Google Scholar). Current data suggest that H4B is essential for growth inLeishmania (12Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Crossref PubMed Scopus (151) Google Scholar, 17Papadopoulou B. Roy G. Mourad W. Leblanc E. Ouellette M. J. Biol. Chem. 1994; 269: 7310-7315Abstract Full Text PDF PubMed Google Scholar, 18Nare B. Hardy L.W. Beverley S.M. J. Biol. Chem. 1997; 272: 13883-13891Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) and plays a role in parasite virulence and differentiation (19Cunningham M.L. Titus R.G. Turco S.J. Beverley S.M. Science. 2001; 292: 285-287Crossref PubMed Scopus (108) Google Scholar). H4B has also been found to be a growth factor in Crithidia fasciculata and to effect proliferation and differentiation in various mammalian cell lines (20Iwai K. Bunno M. Kobashi M. Suzuki T. Biochim. Biophys. Acta. 1976; 444: 618-622Crossref PubMed Scopus (15) Google Scholar, 21Kerler F. Ziegler I. Schmid C. Bacher A. Exp. Cell Res. 1990; 189: 151-156Crossref PubMed Scopus (24) Google Scholar, 22Golderer G. Werner E.R. Leitner S. Grobner P. Werner-Felmayer G. Genes Dev. 2001; 15: 1299-1309Crossref PubMed Scopus (70) Google Scholar). While essential, the role(s) of H4B inLeishmania metabolism is not well understood at present.Leishmania is auxotrophic for tyrosine and trypanosomatids have been reported to lack phenylalanine hydroxylase activity (23Kaufman S. Proc. Natl. Acad. Sci. U. S. A. 1963; 50: 1085-1093Crossref PubMed Scopus (347) Google Scholar), however, the Leishmania genome encodes a protein with strong homology to amino acid hydroxylases. 3L.-F. Lye, M. L. Cunningham, and S. M. Beverley, unpublished data. NOS activity has been reported in Leishmania and trypanosomes (24Basu N.K. Kole L. Ghosh A. Das P.K. FEMS Microbiol. Lett. 1997; 156: 43-47Crossref PubMed Scopus (40) Google Scholar, 25Paveto C. Pereira C. Espinosa J. Montagna A.E. Farber M. Esteva M. Flawia M.M. Torres H.N. J. Biol. Chem. 1995; 270: 16576-16579Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), but the Leishmania ether lipid cleavage activity utilizes NADPH rather than H4B as a cofactor (26Ma D. Beverley S.M. Turco S.J. Biochem. Biophys. Res. Commun. 1996; 227: 885-889Crossref PubMed Scopus (14) Google Scholar). In other organisms, H4B is metabolized to pterin-4α-carbinolamine through the action of aromatic amino acid hydroxylases or NOS, or by spontaneous oxidation (27Nagatsu T. Ichinose H. Mol. Neurobiol. 1999; 19: 79-96Crossref PubMed Scopus (62) Google Scholar). Two enzymes are involved in its subsequent dehydration and reduction to H4B: pterin-4α-carbinolamine dehydratase (PCD) (28Hauer C.R. Rebrin I. Thony B. Neuheiser F. Curtius H.C. Hunziker P. Blau N. Ghisla S. Heizmann C.W. J. Biol. Chem. 1993; 268: 4828-4831Abstract Full Text PDF PubMed Google Scholar) andquinonoid-dihydropteridine reductase (qDPR; Ref. (29Kaufman S. Tetrahydrobiopterin: Basic Biochemistry and Role in Human Disease. Johns Hopkins University Press, Baltimore1997Google Scholar), respectively (Fig. 1). Regeneration of H4B allows organisms to efficiently use biopterin cofactor in metabolism, and in humans qDPR deficiency is the second most common cause of hyperphenylalanemia (30Dianzani I. de Sanctis L. Smooker P.M. Gough T.J. Alliaudi C. Brusco A. Spada M. Blau N. Dobos M. Zhang H.P. Yang N. Ponzone A. Armarego W.L. Cotton R.G. Hum. Mutat. 1998; 12: 267-273Crossref PubMed Scopus (39) Google Scholar, 31Smooker P.M. Cotton R.G. Hum. Mutat. 1995; 5: 279-284Crossref PubMed Scopus (15) Google Scholar, 32Blau N. Barnes I. Dhondt J.L. J. Inherit. Metab. Dis. 1996; 19: 8-14Crossref PubMed Scopus (113) Google Scholar).qDPR has been extensively characterized in mammalian cells, with its three-dimensional structure placing it within the family of short-chain dehydrogenases (33Varughese K.I. Skinner M.M. Whiteley J.M. Matthews D.A. Xuong N.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6080-6084Crossref PubMed Scopus (149) Google Scholar, 34Su Y. Varughese K.I. Xuong N.H. Bray T.L. Roche D.J. Whiteley J.M. J. Biol. Chem. 1993; 268: 26836-26841Abstract Full Text PDF PubMed Google Scholar). The qDPRs from most species show a strong dependence for NADH as a cofactor and forquinonoid-dihydrobiopterin (qH2B) as the pteridine substrate (35Chang C.F. Bray T. Varughese K.I. Whiteley J.M. Adv. Exp. Med. Biol. 1999; 463: 403-410Crossref PubMed Scopus (2) Google Scholar). Outside of metazoans there are few studies of qDPRs. Some prokaryotes exhibit qDPR activity (36Vasudevan S.G. Shaw D.C. Armarego W.L. Biochem. J. 1988; 255: 581-588PubMed Google Scholar) and aPseudomonas qDPR has been characterized (37Williams C.D. Dickens G. Letendre C.H. Guroff G. Haines C. Shiota T. J. Bacteriol. 1976; 127: 1197-1207Crossref PubMed Google Scholar). In trypanosomatids qDPR activity has been found in crude lysates of Leishmania major and its relative Crithidia fasciculata (12Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Crossref PubMed Scopus (151) Google Scholar, 38Hirayama K. Nakanisi N. Sueoka T. Katoh S. Yamada S. Biochim. Biophys. Acta. 1980; 612: 337-343Crossref PubMed Scopus (10) Google Scholar). Because of the importance of H4B to Leishmania metabolism and virulence, we decided to characterize the qDPR gene and enzyme from L. major (LmQDPR). Biopterin and H2B were purchased from Schircks Laboratories (Jona, Switzerland). 6,7-Dimethyl-5,6,7,8-tetrahydropterin (DMPH4), NADPH, NADH, and horseradish peroxidase were purchased from Sigma. Folate-deficient medium was custom manufactured by Invitrogen and is identical to M199 except that it lacks folate and thymidine (18Nare B. Hardy L.W. Beverley S.M. J. Biol. Chem. 1997; 272: 13883-13891Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). All other reagents were of analytical grade. Several pteridine analogs were tested for inhibition of LmQDPR activity whose structures, provenance, and usage were described previously (39Hardy L.W. Matthews W. Nare B. Beverley S.M. Exp. Parasitol. 1997; 87: 158-170Crossref PubMed Google Scholar). The following strains were used: L. major Friedlin V1 (MHOM/JL/80/Friedlin) and CC-1 (MHOM/IR/83/IR; Ref. 40Kapler G.M. Coburn C.M. Beverley S.M. Mol. Cell. Biol. 1990; 10: 1084-1094Crossref PubMed Scopus (359) Google Scholar), a null mutant CC-1 Leishmanialacking PTR1 (ptr1−) described previously (12Bello A.R. Nare B. Freedman D. Hardy L. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11442-11446Crossref PubMed Scopus (151) Google Scholar),Leishmania donovani Sudanese strain 1S2D (MHOM/S.D./00/1S-2D), and Leishmania mexicana(MNYC/BZ/62/M379). Wild-type promastigotes were maintained by serial passage in M199 medium supplemented with 10% fetal calf serum at 26 °C; ptr1− parasite media additionally contained 2 μg/ml H2B (7Cunningham M.L. Beverley S.M. Mol. Biochem. Parasitol. 2001; 113: 199-213Crossref PubMed Scopus (81) Google Scholar). For L. major, metacyclic promastigotes were isolated from stationary phase 48-h cultures by negative selection with peanut agglutinin as described (41da Silva R. Sacks D.L. Infect. Immun. 1987; 55: 2802-2806Crossref PubMed Google Scholar) and amastigotes were harvested from BALB/c mouse footpad lesions 3 weeks postinfection. Axenic culture and differentiation ofL. mexicana amastigote were previously described (7Cunningham M.L. Beverley S.M. Mol. Biochem. Parasitol. 2001; 113: 199-213Crossref PubMed Scopus (81) Google Scholar). The procyclic Trypanosoma brucei cell line YTAT 1.1 (a gift from E. Ullu, Yale University) was propagated in Cunningham's SM medium supplemented with 10% heat inactivated fetal calf serum. Methods for DNA transfection of Leishmania by transfection were previously described (40Kapler G.M. Coburn C.M. Beverley S.M. Mol. Cell. Biol. 1990; 10: 1084-1094Crossref PubMed Scopus (359) Google Scholar) and clonal populations were obtained by plating on 20 μg/ml G418. The sequences of five random shotgun clones of L. major strain Friedlin V1 (42Akopyants N.S. Clifton S.W. Martin J. Pape D. Wylie T., Li, L. Kissinger J.C. Roos D.S. Beverley S.M. Mol. Biochem. Parasitol. 2001; 113: 337-340Crossref PubMed Scopus (39) Google Scholar) spanning theQDPR repeating unit were completed (lm18b06, strain B4501; lm61b05, strain B4181; lm25d04, strain B4180; and lm78e09, strain B4254; and lm62d04, strain B4502; GenBankTM number AF523363and AY141854). This missing ∼200 nt of the 3.6-kb LmQDPRrepeating unit was amplified by PCR using primers SMB1465 (5′-AACATTGAGCGGCAGAGGATGT) and SMB1466 (5′-TGATGGTGCGGCACTCGCGGTA), with DNA from cosmid 1c15-2 (strain B4259). The PCR product was A-tailed and cloned into pGEMTM-T Easy (Promega, Madison, WI) and sequenced (strain B4506, GenBankTM accession numberAF523363). Dideoxynucleotide sequencing reactions were performed using the ABI PRISMTM BigDye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems, Foster City, CA). Leishmaniagenomic DNA was isolated from late logarithmic phase promastigotes by the LiCl method (43Medina-Acosta E. Cross G.A. Mol. Biochem. Parasitol. 1993; 59: 327-329Crossref PubMed Scopus (234) Google Scholar). T. brucei and Trypanosoma cruzi genomic DNAs were prepared by phenol extraction as described (44Beverley S.M. Coderre J.A. Santi D.V. Schimke R.T. Cell. 1984; 38: 431-439Abstract Full Text PDF PubMed Scopus (165) Google Scholar). Total RNA was isolated from early and late logarithmic phase promastigotes, metacyclic cells, and lesion amastigotes by using the phenol/guanidine isothyocyanate reagent TRIzolTM(Invitrogen) according to the manufacturer's instructions. Southern and Northern blots were performed following standard procedures (45Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 7.39-7.52Google Scholar), and a PCR-derived QDPR hybridization probe (described below) was labeled with [α-32P]dCTP by the random-priming method (46Feinberg A.P. Vogelstein B. Anal. Biochem. 1983; 132: 6-13Crossref PubMed Scopus (16653) Google Scholar). Quantitation was performed with a laser densitometer (Molecular Dynamics with ImageQuantTM version 3.0; Molecular Dynamics). A L. major Friedlin V1 cosmid library prepared in the shuttle vector cLHYG (47Ryan K.A. Dasgupta S. Beverley S.M. Gene (Amst.). 1993; 131: 145-150Crossref PubMed Scopus (81) Google Scholar) was gridded onto nylon membranes, and hybridized with the L. major QDPRfull-length coding region probe. Four different cosmids containingQDPR were obtained (c1e16-1, strain B4257; c1c15-2, strain B4259; c7c21-2, strain B4260; and c12d12-3, strain B4264). Chromosomes were prepared and separated by pulsed field electrophoresis with a Bio-Rad Chef Mapper as described (48Beverley S.M. Nucleic Acids Res. 1988; 16: 925-939Crossref PubMed Scopus (135) Google Scholar, 49Chu G. Vollrath D. Davis R.W. Science. 1986; 234: 1582-1585Crossref PubMed Scopus (1093) Google Scholar). The 5′ terminus of the mature L. major QDPR transcript was determined by reverse transcriptase-PCR (50Wang Y. Dimitrov K. Garrity L.K. Sazer S. Beverley S.M. Mol. Biochem. Parasitol. 1998; 96: 139-150Crossref PubMed Scopus (33) Google Scholar), with primers specific for the L. major spliced leader sequence (SMB936: 5′-ACCGCTATATAAGTATCAGTTCTGTACTTTA) and the QDPR coding region (SMB 1036: 5′-TTCACCCTGCGTACTGAACACAT; the 1st base is located 332 nt downstream of the LmQDPR ATG). Complementary DNA was made from 5 μg of stationary promastigote RNA primed with oligo(dT) by Superscript II reverse transcriptase (Invitrogen) prior to PCR amplification, and the PCR product was purified and sequenced directly. A partial QDPRproduct was obtained by PCR amplification using primers based upon EST sequences (GenBankTM accession number AL390114; SMB 1609, 5′-ATGGCCCAAAAGAGCGGATTGG; SMB1610, 5′-CTGCCGGCTTGCACCCTGGCCA); it was inserted into pGEMTM-T Easy and sequenced (strain B4586; GenBankTM accession number AF523371). From comparisons with the LmQDPR repeat and flanking regions, we assembled a preliminary contig for the syntenic T. brucei region (see legend to Fig. 4); as this assembly exhibited several gaps, we designed PCR primers to obtain the sequence of these (GenBankTMaccession numbers AF523369 and AF523370). The contig sequences are available from the authors on request. Sequence data for L. major was obtained from The Sanger Institute website at www.sanger.ac.uk/Projects/L_major and was accomplished as part of theLeishmania Genome Network with support by The Wellcome Trust. The 690-ntLmQDPR open reading frame was amplified by PCR withPfu polymerase (Stratagene) using primers SMB1084 (5′-gcggatcc accATGAAAAATGTACTCCTCATCG; the underlined sequence corresponds to a BamHI site and the bold nucleotides correspond to a "Kozak" sequence), SMB1085 (5′-cgggatcCTACACAATAAAACGCGTCTT), and 50 ng of template DNA (clone lm61b05). This PCR product was also used as a probe for Southern and Northern blot hybridization. The amplified DNA fragment was digested and cloned into the BamHI site of theLeishmania expression vector pXG1a (51Ha D.S. Schwarz J.K. Turco S.J. Beverley S.M. Mol. Biochem. Parasitol. 1996; 77: 57-64Crossref PubMed Scopus (221) Google Scholar) in both orientations; the QDPR sequences were confirmed by sequencing. The resulting constructs (sense and antisense constructs pXG-QDPR and pXG-RPDQ, respectively; strains B4102 and B4103), were transfected into Leishmania. pET-15b DNA (Novagen, Madison, WI) was digested with NdeI, blunt-ended with T4 DNA polymerase, and ligated to the BamHI fragment from pXG-QDPR (also blunt-ended), yielding pET-QDPR (strain B4184; confirmed by sequencing). This provided a LmQDPRfusion construct bearing an N-terminal His tag. For protein expression, pET-QDPR was transformed into Escherichia coli strain BLR(DE3) pLys-S (strain B4244; Ref. 52Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6003) Google Scholar). A 500-ml culture was grown in L-broth medium plus ampicillin (100 μg/ml) toA 600 of 0.6, at 37 °C; isopropyl-β-d-thiogalactoside was added to a 1 mm final concentration and the culture was further incubated at 37 °C for another 4 h. Cells were harvested and resuspended in 25 ml of phosphate-buffered saline (150 mmNaCl, 16 mm Na2HPO4, 4 mm NaH2PO4, pH 7.3), lysed by sonication, and the debris was precipitated by centrifugation at 4,000 × g for 10 min. Five ml of the supernatant was loaded onto 1 ml of the Ni2+-nitriloacetic acid resin affinity column (Qiagen), which was previously equilibrated with 10 ml of wash buffer (50 mm NaH2PO4, pH 8.0, 300 mm NaCl, 20 mm imidazole). The recombinant protein was eluted with 2 ml of elution buffer (50 mm NaH2PO4, 300 mmNaCl, 250 mm imidazole). Proteins were analyzed by SDS-PAGE using a 15% acrylamide gel by standard protocols (53Coligan J.E. Dunn B.M. Speicher D.W. Wingfield P.T. Chanda V.B.C. 3rd Ed. Current Protocols in Protein Science, Electrophoresis. 1. John Wiley & Sons, Inc., New York1997: 10.1.1-10.1.11Google Scholar) and visualized by stained Coomassie Brilliant Blue. Leishmania promastigotes and T. brucei procyclics were collected by centrifugation at 1,250 × g for 10 min at 4 °C, washed twice with phosphate-buffered saline, and resuspended at 2 × 109cells/ml in 10 ml of Tris-Cl, pH 7.0, with 1 mm EDTA and a mixture of protease inhibitors as described (18Nare B. Hardy L.W. Beverley S.M. J. Biol. Chem. 1997; 272: 13883-13891Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Frozen pellets ofT. cruzi epimastigotes (Silvio strain) were generously provided by M. Pereira (Tufts University School of Medicine). Cells were lysed by three rounds of freeze thawing and sonication, and the extracts clarified by centrifugation at 15,000 × g for 30 min at 4 °C. Protein concentrations were determined by the Bradford method (54Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216202) Google Scholar) with bovine serum albumin as a standard. qDPR activity was measured at 25 °C as described (55Firgaira F.A. Cotton R.G. Danks D.M. Biochem. J. 1981; 197: 31-43Crossref PubMed Scopus (40) Google Scholar) using the quinonoid form of 6,7-dimethyl-H2-pterin (qDMPH2). Because quinonoid pteridines are very unstable, they are continuously provided by the horseradish peroxidase-catalyzed oxidation of DMPH4 in this assay. The standard reaction mixture contained 50 mm Tris-HCl, pH 7.2, 20 μg of horseradish peroxidase, 0.9 mmH2O2, 5–320 μmDMPH4, 100 μm NADH, and 30 ng of purifiedqDPR, unless otherwise indicated. Experimentally, all components except DMPH4 were incubated for 1 min prior to initiation of reaction by addition of DMPH4. NADH consumption was measured by absorbance at 340 nm in a Beckman DU-640 spectrophotometer. The initial rates were obtained from the rate of decrease of absorbence at 340 nm (ε340 for NADH or NADPH is 6200 m−1 cm−1). The pH dependence was determined using two overlapping buffers: 50 mm sodium phosphate, pH 4.8–8.8, and 50 mmTris-HCl, pH 7.0–11.1. Inhibitor studies were performed with 10 μm inhibitor and 100 μm DMPH4 and 100 μm NADH. All inhibitors except compound 66 were preincubated at 25 °C for 1 min in the presence of the complete reaction mixture containing 30 ng of purified LmQDPR, after which DMPH4 was added to start the reaction. With compound 66, addition of purified protein prior to DMPH4 resulted in a decrease in absorbance; thus, purified LmQDPR was first added together with substrate, after which compound 66 was added. Water-insoluble compounds were dissolved in dimethyl sulfoxide (Me2SO); in the final assay, the Me2SO concentration did not exceed 0.05%, a value that had no effect onqDPR activity (data not shown). Intracellular levels of biopterin and H4B were determined as described previously (7Cunningham M.L. Beverley S.M. Mol. Biochem. Parasitol. 2001; 113: 199-213Crossref PubMed Scopus (81) Google Scholar). Briefly, log phase promastigotes were isolated from the growth medium by centrifugation through a dibutylphthalate cushion. Cell pellets were subjected to either acid or alkaline oxidation and separated by high performance liquid chromatography. Under acidic conditions biopterin, H2B, and H4B form biopterin, while under alkaline conditions biopterin and H2B form biopterin, whereas H4B forms pterin. Ferric reductase activity was measured as described (56Lee P.L. Halloran C. Cross A.R. Beutler E. Biochem. Biophys. Res. Commun. 2000; 271: 788-795Crossref PubMed Scopus (11) Google Scholar), with some modifications. Recombinant LmQDPR protein or sheep liver dihydropteridine reductase (Sigma) were added to a 250-μl reaction mixture containing 140 mm NaCl, 5 mm KCl, 1 mm CaCl2, 1 mm MgCl2, 5 mmNaH2PO4, 5 mm Hepes, pH 7.4, 0.2 mm ferrozine, 50 μm ferric nitrilotriacetic acid (prepared as 1 mm ferric chloride, 4 mmnitrilotriacetic acid, 100 mm HCl, then the pH was adjusted to pH 6.8), with or without 100 μm NADH, in a microtiter plate. The rate of formation of NADH-dependent ferrozine complex was followed spectrophotometrically at 575 nm in a Molecular Devices Thermomax microtiter plate reader at 26 °C (ε = 27,900 m−1 cm−1). End point absorbance was measured after 60 min. In a shotgun survey of the L. major genome, we found several recombinants whose end sequences showed homology toqDPRs of other species (42Akopyants N.S. Clifton S.W. Martin J. Pape D. Wylie T., Li, L. Kissinger J.C. Roos D.S. Beverley S.M. Mol. Biochem. Parasitol. 2001; 113: 337-340Crossref PubMed Scopus (39) Google Scholar). 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Acta. 2000; 1492: 247-251Crossref PubMed Scopus (5) Google Scholar) (Fig.2). Significantly, key residues implicated by structural or mutational studies in substrate and cofactor binding in other qDPRs were conserved in the predicted LmQDPR. These included the Tyr-(Xaa)3-Lys NADH binding motif (positions 138–42, marked by solid circles in Fig. 2), Asp-32, the residue implicated in preferential binding of NADH (marked by a triangle in Fig. 2), and two residues implicated in binding of quinonoid dihydropteridine (open circles in Fig. 1; Refs. 61Varughese K.I. Xuong N.H. Kiefer P.M. Matthews D.A. Whiteley J.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5582-5586Crossref PubMed Scopus (65) Google Scholar, 62Kiefer P.M. Varughese K.I., Su, Y. Xuong N.H. Chang C.F. Gupta P. Bray T. Whiteley J.M. J. Biol. Chem. 1996; 271: 3437-3444Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 63Grimshaw C.E. Matthews D.A. Varughese K.I. Skinner M. Xuong N.H. Bray T. Hoch J. Whiteley J.M. J. Biol. Chem. 1992; 267: 15334-15339Abstract Full Text PDF PubMed Google Scholar, 64Kiefer P.M. Grimshaw C.E. Whiteley J.M. Biochemistry. 1997; 36: 9438-9445Crossref PubMed Scopus (6) Google Scholar). Additionally, distant relationships to the short-chain dehydrogenase protein family were detected, as expected for qDPRs (65Callahan H.L. Beverley S.M. J. Biol. Chem. 1992; 267: 24165-24168Abstract Full Text PDF PubMed Google Scholar, 66Whiteley J.M. Xuong N.H. Varughese K.I. Adv. Exp. Med. Biol. 1993; 338: 115-121Crossref PubMed Scopus (12) Google Scholar). With an LmQDPR ORF probe and digestion with enzymes such as SmaI, NotI, or SacI that do not cut within this probe, Southern blot analysis revealed strong hybridization to a band of 3.6 kb in each digest, as well as weaker hybridization to another band (Fig.3 A). This suggested the possibility that LmQDPR was organized as head-to-tail tandemly repeated genes. Consistent with this, Southern blot analysis using enzymes cutting once (AvaII, NaeI, andXhoI) or twice (PstI) within
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