Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M206543200 on July 31, 2002

J. Biol. Chem., Vol. 277, Issue 41, 38245-38253, October 11, 2002
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
277/41/38245    most recent
M206543200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lye, L.-F.
Right arrow Articles by Beverley, S. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lye, L.-F.
Right arrow Articles by Beverley, S. M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Characterization of quinonoid-Dihydropteridine Reductase (QDPR) from the Lower Eukaryote Leishmania major*

Lon-Fye Lye, Mark L. Cunningham, and Stephen M. BeverleyDagger

From the Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, July 1, 2002, and in revised form, July 29, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 characterize Leishmania major quinonoid-dihydropteridine reductase (LmQDPR), the key enzyme required for regeneration and maintenance of H4biopterin pools. LmQDPR shows good homology to metazoan quinonoid-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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1). 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 (2). 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 (3-6). To accomplish this, Leishmania express a versatile pteridine salvage network, consisting of transporters with specificity for folate and biopterin (FT1 and BT1, respectively; Refs. 7-10).1 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. 11-13). The importance of folate in essential metabolic processes such as synthesis of thymidylate has been established firmly in Leishmania by pharmacological and genetic studies both in vitro and in vivo (14-16). Current data suggest that H4B is essential for growth in Leishmania (12, 17, 18) and plays a role in parasite virulence and differentiation (19). H4B has also been found to be a growth factor in Crithidia fasciculata and to effect proliferation and differentiation in various mammalian cell lines (20-22). While essential, the role(s) of H4B in Leishmania metabolism is not well understood at present. Leishmania is auxotrophic for tyrosine and trypanosomatids have been reported to lack phenylalanine hydroxylase activity (23), however, the Leishmania genome encodes a protein with strong homology to amino acid hydroxylases.3 NOS activity has been reported in Leishmania and trypanosomes (24, 25), but the Leishmania ether lipid cleavage activity utilizes NADPH rather than H4B as a cofactor (26).

In other organisms, H4B is metabolized to pterin-4alpha -carbinolamine through the action of aromatic amino acid hydroxylases or NOS, or by spontaneous oxidation (27). Two enzymes are involved in its subsequent dehydration and reduction to H4B: pterin-4alpha -carbinolamine dehydratase (PCD) (28) and quinonoid-dihydropteridine reductase (qDPR; Ref. (29), 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 (30-32). qDPR has been extensively characterized in mammalian cells, with its three-dimensional structure placing it within the family of short-chain dehydrogenases (33, 34). The qDPRs from most species show a strong dependence for NADH as a cofactor and for quinonoid-dihydrobiopterin (qH2B) as the pteridine substrate (35).


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 1.   Proposed enzymatic pathways for the consumption and regeneration of H4B in Leishmania. B, biopterin; qH2B, quinonoid-dihydrobiopterin; 4alpha -OH-H4B, pterin-4alpha -carbinolamine; and "?", predicted activity, either an unidentified enzyme or spontaneous oxidation.

Outside of metazoans there are few studies of qDPRs. Some prokaryotes exhibit qDPR activity (36) and a Pseudomonas qDPR has been characterized (37). In trypanosomatids qDPR activity has been found in crude lysates of Leishmania major and its relative Crithidia fasciculata (12, 38). Because of the importance of H4B to Leishmania metabolism and virulence, we decided to characterize the qDPR gene and enzyme from L. major (LmQDPR).

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents-- 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 (18). 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 (39).

Parasites and Transfection-- The following strains were used: L. major Friedlin V1 (MHOM/JL/80/Friedlin) and CC-1 (MHOM/IR/83/IR; Ref. 40), a null mutant CC-1 Leishmania lacking PTR1 (ptr1-) described previously (12), 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 (7). For L. major, metacyclic promastigotes were isolated from stationary phase 48-h cultures by negative selection with peanut agglutinin as described (41) and amastigotes were harvested from BALB/c mouse footpad lesions 3 weeks postinfection. Axenic culture and differentiation of L. mexicana amastigote were previously described (7). 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 (40) and clonal populations were obtained by plating on 20 µg/ml G418.

Sequencing LmQDPR-- The sequences of five random shotgun clones of L. major strain Friedlin V1 (42) spanning the QDPR repeating unit were completed (lm18b06, strain B4501; lm61b05, strain B4181; lm25d04, strain B4180; and lm78e09, strain B4254; and lm62d04, strain B4502; GenBankTM number AF523363 and AY141854). This missing ~200 nt of the 3.6-kb LmQDPR repeating 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 number AF523363). Dideoxynucleotide sequencing reactions were performed using the ABI PRISMTM BigDye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems, Foster City, CA).

Southern and Northern Analysis-- Leishmania genomic DNA was isolated from late logarithmic phase promastigotes by the LiCl method (43). T. brucei and Trypanosoma cruzi genomic DNAs were prepared by phenol extraction as described (44). 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 (45), and a PCR-derived QDPR hybridization probe (described below) was labeled with [alpha -32P]dCTP by the random-priming method (46). 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 (47) was gridded onto nylon membranes, and hybridized with the L. major QDPR full-length coding region probe. Four different cosmids containing QDPR 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 (48, 49).

Mapping the 5' Terminus of the Mature QDPR Transcript-- The 5' terminus of the mature L. major QDPR transcript was determined by reverse transcriptase-PCR (50), 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.

Trypanosome QDPR Sequencing-- A partial QDPR product 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 (GenBankTM accession 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 the Leishmania Genome Network with support by The Wellcome Trust.

Overexpression of LmQDPR in L. major-- The 690-nt LmQDPR open reading frame was amplified by PCR with Pfu polymerase (Stratagene) using primers SMB1084 (5'-gcggatccaccATGAAAAATGTACTCCTCATCG; 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 the Leishmania expression vector pXG1a (51) 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.

Purification of the Recombinant L. major qDPR-- 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 LmQDPR fusion 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. 52). A 500-ml culture was grown in L-broth medium plus ampicillin (100 µg/ml) to A600 of 0.6, at 37 °C; isopropyl-beta -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 mM NaCl, 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 mM NaCl, 250 mM imidazole). Proteins were analyzed by SDS-PAGE using a 15% acrylamide gel by standard protocols (53) and visualized by stained Coomassie Brilliant Blue.

Preparation of Crude Leishmania, T. brucei, and T. cruzi Lysates-- 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 × 109 cells/ml in 10 ml of Tris-Cl, pH 7.0, with 1 mM EDTA and a mixture of protease inhibitors as described (18). Frozen pellets of T. 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 (54) with bovine serum albumin as a standard.

Measurement of qDPR Activity and Inhibition Studies-- qDPR activity was measured at 25 °C as described (55) 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 mM H2O2, 5-320 µM DMPH4, 100 µM NADH, and 30 ng of purified qDPR, 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 (epsilon 340 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 mM Tris-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 on qDPR activity (data not shown).

Measurement of Intracellular Levels of Biopterin and H4B-- Intracellular levels of biopterin and H4B were determined as described previously (7). 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 Assay-- Ferric reductase activity was measured as described (56), 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 mM NaH2PO4, 5 mM Hepes, pH 7.4, 0.2 mM ferrozine, 50 µM ferric nitrilotriacetic acid (prepared as 1 mM ferric chloride, 4 mM nitrilotriacetic 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 (epsilon  = 27,900 M-1 cm-1). End point absorbance was measured after 60 min.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sequence Analysis of L. major QDPR

In a shotgun survey of the L. major genome, we found several recombinants whose end sequences showed homology to qDPRs of other species (42). The sequences were completed, yielding a predicted LmQDPR protein encompassing 230 amino acids, with a predicted molecular mass of 25.6 kDa. Mapping of the 5' end of the LmQDPR transcript confirmed the assignment of the start codon (described below). The predicted LmQDPR amino acid sequence was highly related to those of human, rat, Caenorhabditis elegans, and Drosophila melanogaster, showing 36-39% identity and 53-58% similarity (57-60) (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. 61-64). Additionally, distant relationships to the short-chain dehydrogenase protein family were detected, as expected for qDPRs (65, 66).


View larger version (30K):
[in this window]
[in a new window]
  		Fig. 2.   Alignment of eukaryotic qDPRs. An amino acid sequence alignment of qDPRs from L. major (this work), rat (GenBankTM accession number P11348), C. elegans (T24395), and D. melanogaster (AAF50315) was performed using the Clustal algorithm implemented in the Lasergene software (DNASTAR, Inc.). Identical amino acids in all sequences are highlighted with a gray background. The residues interacting with NADH for hydride transfers are indicated by filled circles, the residues involved in substrate binding are indicated by open circles, and the residues involved in NADH binding preference are indicated by filled triangles (61-63).

The LmQDPR Locus Contains a Tandemly Repeated Array of 8-9 Copies

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. 3A). 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, and XhoI) or twice (PstI) within the probe yielded patterns with two predominant hybridizing fragments, in each case adding to ~3.6 kb (Fig. 3A). Partial digestion with SacI confirmed the presence of tandemly repeated LmQDPR genes (Fig. 3B), as Southern blot analysis yielded a ladder of fragments, ranging up to at least five 3.6-kb repeats (Fig. 3B). Preliminary mapping of four different cosmids each bearing ~40-kb inserts of Leishmania DNA spanning the QDPR locus also confirmed the presence of the 3.6-kb tandemly repeated array ("Experimental Procedures"; data not shown). Molecular karyotype analysis of separated L. major chromosomes showed hybridization to a single chromosome of about 2000 kb (Fig. 3C).


View larger version (43K):
[in this window]
[in a new window]
  		Fig. 3.   Southern blot analysis of the genomic organization of L. major QDPR. In all panels, the hybridization probe was the LmQDPR ORF probe shown in Fig. 4A. A. Southern blot. L. major genomic DNA was completely digested with the indicated restriction enzymes, electrophoresed on a 0.8% agarose gel, blotted, and hybridized. The sizes of the molecular weight markers are shown. B, partial digestion with SacI. A standard digestion of L. major genomic DNA was initiated (0.5 unit of SacI, 7 µg of genomic DNA), and at the intervals indicated aliquots were withdrawn and the reactions stopped by adding EDTA (to 15 mM) and quick-freezing. Samples were electrophoresed on a 0.8% agarose gel, blotted, and hybridized. C, chromosomal mapping of QDPR. L. major chromosomes were resolved by pulsed field gel electrophoresis as described under "Experimental Procedures," blotted, and hybridized. The left panel shows the ethidium bromide-stained gel with S. cerevisiae chromosomal markers in kb (M), and the right panel shows the autoradiogram.

To estimate LmQDPR gene number, we compared the hybridization of the "unit" 3.6-kb fragment in the SacI, NotI, or SmaI digests to that of the more weakly hybridizing band in each digest (Fig. 3A), which we reasoned corresponded to a single copy of the gene located at the end of the tandem array. By densitometry, the ratio of the "end" to the 3.6-kb unit fragments was 1 to 8.1, 7.5, or 7.2, respectively, suggesting that the L. major QDPR locus contains 8-9 copies.

The LmQDPR Repeating Unit Contains Two Other Unrelated Genes

We identified molecular clones encompassing the entire QDPR repeating unit, determined their sequence ("Experimental Procedures"), and generated a consensus sequence for the 3541-bp QDPR repeating unit (Fig. 4A, GenBankTM accession number AF523363). In addition to QDPR, the repeating unit predicts the presence of two additional ORFs. One showed 70, 41, and 38% amino acid identity, respectively, to 20 S proteasome subunits from T. brucei (beta 7), humans, and Arabidopsis (GenBankTM accession numbers AF290945, D26600, and AF043538, respectively). The other ORF (ORF-q) comprised 112 amino acids, and did not show any relationship to other proteins in data base searches. ORF-q showed a high level of identity (86%) to a putative T. brucei ORF identified in the trypanosome genome project (data not shown), and was additionally identified by an EST from L. major amastigotes (GenBankTM accession number AA680881). While repeated gene families are common in Leishmania, ones whose repeating units include unrelated genes are relatively uncommon.


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 4.   The L. major QDPR repeating unit contains two other genes while the syntenic region of T. brucei lacks QDPR. A, map of the consensus L. major QDPR repeating unit. This was assembled from individual shotgun sequences as described in the text and is shown linearized arbitrarily at a BsaBI site (GenBankTM accession numbers AF523363 and AY141854). Arrows show the coding regions for ORF-q, QDPR, and the 20 S proteasome beta 7 subunit. A, "*" indicates polymorphic sites as discussed in the text. B, comparison of the organization of the QDPR locus of L. major with a syntenic region of T. brucei. Preliminary contigs spanning the LmQDPR region or its equivalent in T. brucei were prepared using information deposited in GenBankTM or generously made available by the Leishmania and T. brucei genome projects (see "Acknowledgments"). The predicted gene order is shown; these regions are syntenic except for the absence of the QDPR in T. brucei. The contigs and specific sequence information used in their assembly are available from the authors by request.

The sequences of the individual shotgun clones used to assemble the QDPR repeating unit were identical, except that there were 5 differences observed between the consensus and clone lm18b06. (GenBankTM accession no. AY141854.) One substitution was located between the QDPR and the 20 S proteasome beta 7 subunit intergenic region (marked by a asterisk in Fig. 4A). The other 4 differences were clustered and occurred in the 3' half of the predicted 20 S proteasome beta 7 protein; as a consequence, a frameshift leading to the predicted formation of a subunit with a variant C' terminus occurred (also marked by a asterisk in Fig. 4A). These data raise the possibility of microheterogeneity among the other genes encoded in the QDPR repeating unit.

Mapping the 5' End of the LmQDPR mRNA

In Leishmania and related protozoans, every mRNA contains a 39-nt "mini-exon" or "spliced leader" at its 5' end that is added by trans-splicing (67). This enabled the mapping of the LmQDPR 5' mRNA terminus by reverse transcriptase-PCR, using mini-exon and QDPR-specific primers. With an LmQDPR primer located 332 nt 3' of the presumptive initiating ATG, a single ~500-nt product was obtained. Sequence analysis of this product mapped the 5' splice acceptor to position -136 bp relative to the presumptive translation start codon (data not shown). No other ATG intervened between the trans-splice acceptor site and the initiating ATG for the LmQDPR ORF.

Overexpression of LmQDPR in L. major

The LmQDPR ORF was inserted in both orientations in the Leishmania expression vector pXG1a (51), which replicates as a multicopy episome. These constructs (as well as the empty vector) were transfected into wild-type CC-1 L. major. Assays of qDPR activity in crude lysates of these transfectants revealed 7-8-fold higher levels in the sense pXG-QDPR transfectants, when compared with the control or vector transfectants (Table I). Thus LmQDPR overexpression conferred elevated qDPR activity. Whereas the increase was less than typically seen by pXG-mediated overexpression of single copy genes (18), recall that there are 8-9 copies of LmQDPR already in the Leishmania genome (Fig. 3). No significant change in qDPR activity was seen with the antisense transfectants relative to controls (pXG-RPDQ; Table I), and the parasites grew normally.

                              
View this table:
[in this window]
[in a new window]
 
Table I
QDPR activity in crude extracts of Leishmania LmQDPR transfectants
Data presented are the mean ± S.D. from three experiments. Crude extracts of Leishmania were prepared as described under "Experimental Procedures" and the assay conditions were: 5-20 µg/ml soluble crude extract protein, DMPH4 (100 µM) and NADH (100 µM).

qDPR Expression during the Leishmania Infectious Cycle

Northern blots were used to determine LmQDPR mRNA levels throughout the Leishmania infectious cycle. A single 1.1-kb transcript was detected in all stages, with little variation during development (Fig. 5). After normalizing for total RNA loading with rRNA, the ratios of the intensities among early log, late log, and metacyclic promastigotes, and amastigotes were 1.3:1:1.8:1.4.


View larger version (42K):
[in this window]
[in a new window]
  		Fig. 5.   Expression of QDPR mRNA during the Leishmania infectious cycle. Total RNAs (5 µg) were separated on a 1.5% formaldehyde-agarose gel, transferred to GeneScreen Plus, and hybridized with the QDPR probe (Fig. 4A). The autoradiogram is shown in the upper panel with the single ~1.1-kb QDPR transcript. Molecular weight standards (kb) are shown. The lower panel shows the ethidium bromide-stained gel region containing the three rRNAs as a loading control. Samples were RNAs from early logarithmic promastigotes (lane 1), late logarithmic promastigotes (lane 2), metacyclic promastigotes (lane 3), and amastigotes (lane 4).

We measured qDPR activity in crude lysates from different growth phases or developmental stages of L. major, L. mexicana, and L. donovani (Table II). In stationary phase cultures, Leishmania promastigotes differentiate to the infective metacyclic stage that are most conveniently purified from L. major (68), although L. mexicana has the ability to differentiate to the amastigote stage in vitro ("Experimental Procedures"). In keeping with the mRNA levels (Fig. 5), there was little change in qDPR activity during the infectious cycle in L. mexicana or between log and stationary phase (Table II).

                              
View this table:
[in this window]
[in a new window]
 
Table II
qDPR activity in crude lysates of T. brucei, T. cruzi, and different Leishmania species in differing growth phases and developmental stages (nmol/min/mg)
The mean ± S.D. of two independent experiences and six replicates are shown. Crude extracts were prepared as described under "Experimental Procedures." The assays included 100 µg of soluble crude extract protein per assay, NADH (100 µM) and DMPH4 (10 µM). Whereas this concentration of substrate is below the Km for these species of qDPRs, it was necessary due to problems arising from interference we observed occurring in assays of crude extracts. Consistent with the results shown in Table I and below, preliminary studies using substrate concentrations of 320 µM DMPH4 yielded specific activities of 177, 302, and 63 nmol/min/mg protein for log phase L. major, T. brucei, and T. cruzi, respectively.

Expression in E. coli and Enzymatic Characterization of Recombinant LmQDPR

LmQDPR was expressed in E. coli as an N-terminal His-tagged fusion protein, under the control of an inducible T7 RNA polymerase system. After addition of isopropyl-1-thio-beta -D-galactoside, a major protein band of about 28 kDa was observed following SDS-PAGE (Fig. 6A, lane 3), in agreement with the predicted size of the fusion protein. The recombinant His-tagged LmQDPR was purified by chromatography on nickel-agarose, yielding an apparently homogeneous protein (Fig. 6A, lane 4) with typical recoveries of ~0.2 mg of LmQDPR/500-ml bacterial culture. This preparation was active and its catalytic properties are summarized in Table III and below.


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 6.   Expression and characterization of recombinant L. major QDPR. A, purification of LmQDPR from E. coli. Samples were electrophoresed through a 15% SDS-polyacrylamide gel, and the gel was stained with Coomassie Brilliant Blue. Lane 1, molecular mass markers (in kDa); lane 2, crude extract of uninduced E. coli; lane 3, E. coli lysate after isopropyl-1-thio-beta -D-galactoside induction; and lane 4, protein recovered after Ni2+-nitrilotriacetic acid-agarose chromatography. The arrow indicates His6-QDPR. B, pH profile. qDPR activity assays were performed with recombinant L. major QDPR (30 ng of purified recombinant protein/ml, 100 µM NADH, 100 µM DMPH4) using two overlapping buffers at the indicated pH. open circle , 50 mM sodium phosphate, and , 50 mM Tris-HCl. The values shown are the average of three independent experiments ± S.D. C, substrate dependence of qDPR activity. The activity of purified recombinant LmQDPR was assayed at the indicated concentrations of qDMPH2.

                              
View this table:
[in this window]
[in a new window]
 
Table III
Enzymatic parameters of L. major and other qDPRs

Enzyme Activity-- The specific activity when assayed with the substrate qDMPH2 was 2550 ± 145 µmol/min/mg (Fig. 6C). This was greater than that seen previously with rat and human qDPRs (300 and 450 µmol/min/mg, respectively; Ref. 34).

pH Optimum-- Two overlapping buffers were used to assay qDPR activity over a pH range from 4.8 to 11.1, using saturating levels of NADH (100 µM) and qDMPH2 (100 µM). Activity was maximal at pH 7.2 and dropped at more acid pH values, pH 4.8, and more gradually at more alkaline pH values (Fig. 6B). The pH optimum was similar to that of purified qDPRs from rat and Pseudomomas species (37, 62).

Kinetic Parameters-- We determined a Km for qDMPH2 of 36.5 ± 7.1 µM and for NADH of 23.1 ± 3.8 µM (Table III). These values are comparable with those reported previously for rat and human (11-13 µM for NADH and 27-41 µM for qDMPH2) (64, 69).

Substrate and Cofactor Specificity-- LmQDPR showed high specificity for the quinonoid substrates, as the activity for H2B was barely detectable (31.1 ± 0.8 nmol/min/mg) and about 66,000-fold less than obtained with quinonoid pteridine substrates. The specific activities when assayed with the substrate qDMPH2 for NADH and NADPH were 1630 ± 12 and 10.2 ± 0.9 µmol/min/mg, respectively, yielding a substrate preference for NADH of ~160-fold.

Ferric Reductase Activity-- Recently it was reported that mammalian qDPRs possess a pteridine-independent NADH-dependent ferric reductase activity (56). LmQDPR had detectable ferric reductase activity, although its specific activity was 308-fold less than that of the ferric reductase of Mycobacterium paratuberculosis (0.037 versus 11.4 nmol/min/mg, respectively; Table III) (70).

LmQDPR Does Not Provide H2biopterin Reductase Activity in Vivo

Previous studies of ptr1- mutants suggested that Leishmania possesses a second activity capable of the reduction of H2B to H4B, provisionally termed PTR2 (12, 18, 19). While LmQDPR had little H2B reductase activity, potentially even a low level of activity could lead to significant production of H4B over long periods of time in vivo. To test this idea, we introduced the pXG-QDPR construct into ptr1- null mutants, as the absence of PTR1-dependent H2B reductase activity would increase the ability to detect changes in H4B formation. Crude lysates from the ptr1-/pXG-QDPR transfectants showed elevated qDPR activity, comparable with that of the wild-type pXG-QDPR transfectants (data not shown; Table I). Parasites were then grown in folate-deficient M199 medium with H2B as the sole exogenous pteridine source, and pteridine levels were determined during the logarithmic phase of the 4th passage by a high performance liquid chromatography-based method (7, 19). As seen previously, wild-type and vector control transfectant Leishmania contained ~2 nmol of biopterin/109 cells, of which 82% was present as H4B, although the ptr1- mutant contains lower levels of biopterin, only 62% of which was H4B (Table IV). Despite expression of high levels of LmQDPR, and growth in H2B containing media, the ptr1-/pXG-QDPR transfectants showed no elevation in total cellular biopterin or H4B levels, compared with the ptr1- mutant or control transfectants (Table IV). These findings suggest that LmQDPR is not the source of H2B reductase activity in vivo.

                              
View this table:
[in this window]
[in a new window]
 
Table IV
Biopterin levels in QDPR-overexpressing L. major
Cells were grown in folate-deficient M199 (18) supplemented with 2 µg/ml H2B and collected in log phase. Total and H4-biopterin were determined as described under "Experimental Procedures" from 6 replicates (mean ± S.D.).

Tests of Candidate QDPR Inhibitors

To date, only weak qDPR inhibitors have been identified (71, 72). Previously, we tested a collection of pteridine analogs for activity against Leishmania and purified pteridine reductases (39); several showed good activity against Leishmania, but in a PTR1 and DHFR-thymidylate synthase-independent manner. In contrast to their ability to inhibit Leishmania growth and the reductases significantly below 1 µM, these compounds showed minimal inhibition of LmQDPR, at best 60% inhibition with compounds 35, 36, 66, and 70 when tested at 10 µM (Fig. 7). These data suggest that it is unlikely that LmQDPR is their target in vivo.


View larger version (33K):
[in this window]
[in a new window]
  		Fig. 7.   Tests of pteridine analogs for inhibition of purified recombinant LmQDPR. qDPR activity was assayed in the presence of the indicated inhibitors whose structures can be found in Ref. 39. The results are expressed as percentage reduction activity with respect to a control without inhibition. The mean ± S.D. from three experiments are shown.

PCD and QDPR Homologs and QDPR Activity in Trypanosomes

The presence of qDPR in Leishmania suggested that PCD, the first enzyme of the H4B regeneration cycle, should be present as well. A probable hit was found in the unannotated L. major sequence data base, and good candidates had been deposited previously for T. brucei (GenBankTM accession numbers T26730 and AL485250) and T. cruzi (GenBankTM accession number AI110297). In contrast, searches of the T. brucei genome with qDPRs of Leishmania or other species failed to identify a convincing hit, whereas a candidate qDPR EST was found in T. cruzi (GenBankTM accession numbers AA676052 and AF523371). In T. brucei we were able to identify homologs of the two other genes present in the Leishmania QDPR repeat (20 S proteasome beta 7 subunit and ORF-q), and from a combination of genomic project information as well as additional sequencing we were able to assemble a preliminary contig spanning this region. Unlike Leishmania, the 20 S proteasome beta 7 subunit and ORF-q genes appeared to be single copy, in agreement with previous studies of this locus (73). Comparisons with a preliminary contig of the L. major QDPR array and flanking regions with that of T. brucei showed considerable synteny, except for a gap occurring in T. brucei located where QDPR resided in Leishmania (Fig. 4B and data not shown). Southern blot data suggested that the T. cruzi QDPR may also be single copy (data not shown).

The above data raised the question of whether T. brucei possessed a QDPR gene. Because the genomes of neither T. brucei nor T. cruzi are completed as yet, we asked whether trypanosomes possessed qDPR activity. Lysates from log phase procyclic stage T. brucei and epimastigote stage T. cruzi showed good activity, with T. brucei being the highest (Table II). Preliminary studies suggested that the Km for qDMPH2 of the trypanosomes was about 2-3-fold higher than that of recombinant LmQDPR (data not shown). Little developmental or growth phase regulation was observed in the three species of Leishmania or T. cruzi, in contrast to the 14-fold decreasing activity between log and stationary phases of T. brucei promastigotes (Table II).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nutritional and gene knockout studies have shown that H4B is essential for growth of L. major in vitro (12, 18) and that the levels of H4B are regulated and affect the ability of the parasite to differentiate into the infective metacyclic stage (7, 19). H4B levels are maintained primarily by regeneration of H4B, which in mammals requires two enzymes, PCD and qDPR. In this report, we have shown that Leishmania possesses a qDPR whose sequence and enzymatic properties closely resemble those of mammalian qDPRs (Fig 2, Table III). qDPR activity was found at high levels throughout the parasite infectious cycle, confirming that Leishmania possesses an efficient H4B regeneration system akin to those of other organisms.

Northern blot and enzyme activity assays in three different Leishmania species show that QDPR is expressed constitutively throughout the infectious cycle (Fig. 5, Table II), as are the DHFR and PTR1 reductases required for activation of pteridines to the tetrahydro level (7). This is in keeping with the importance of H4B as a cofactor required for diverse aspects of parasite growth, differentiation, and virulence, raising the possibility that inhibition of parasite qDPR might be a potential target for chemotherapy. Therefore, we attempted to decrease L. major qDPR levels through expression of an antisense construct, however, this was unsuccessful (Table I) and the parasites grew normally. We also tested a panel of pteridine analogs that had previously been shown to inhibit Leishmania growth through PTR1- and DHFR-independent mechanisms (39), however, these were ineffective against LmQDPR (Fig. 7). As of yet, no strong inhibitors of qDPR in any species have been described (72). Thus, whereas we expect inhibition of qDPR to have important consequences to Leishmania growth, further studies involving gene inactivation or pharmacological inhibition will be required to formally establish this.

One unexpected finding was that LmQDPR is encoded as a tandemly repeated array bearing 8-9 copies of a unit encoding LmQDPR, an unidentified protein (ORF-q), and a 20 S proteasome beta 7 subunit (Figs. 3 and 4). While repeated gene families are common in Leishmania and trypanosomes, it is less common for the repeating units to contain unrelated genes. Because qDPR is encoded by a single copy gene in most species including T. cruzi (data not shown), it seems likely that QDPR underwent amplification in the lineage leading to Leishmania. Gene amplification in response to laboratory selective pressures or occurring spontaneously has been widely observed in Leishmania, especially for genes involved in pteridine metabolism (18, 74). While amplifications typically encompass contiguous regions bearing dozens of genes on circular or linear episomes, chromosomally integrated tandem repeats have been observed (74, 75).

These data invite speculations about the forces leading to amplification of QDPR in Leishmania. Potentially, it could be associated with a need for increased H4B levels accompanying adaptation of Leishmania to sand flies or inside of macrophages, as pteridine levels in these environments may be sufficiently limiting so as to force parasites to make the most efficient use of biopterin through regeneration. Another possibility comes from the finding that LmQDPR, like that of other species, exhibits ferric reductase activity (Table III). Iron levels frequently are limiting for the growth of pathogens, and ferric reductase has been shown to play an important role in iron acquisition in some species (76). However, the ferric reductase activity of recombinant LmQDPR is low (Table III). From its specific activity in crude extracts we calculate that the total ferric reductase activity conferred by Leishmania QDPR would be less than 0.002% of that found in M. paratuberculosis, where this activity has been found to contribute to virulence (70), thus casting doubt on this scenario in Leishmania. It is also possible that the driving force for amplification may not be directed at LmQDPR at all, but instead could arise from pressures involving the 20 S proteasome beta 7 subunit or ORF-q, both of which are highly conserved in trypanosomatids.

While not the focus of this work, more limited data with trypanosomes showed that these parasites also possess an efficient H4B regeneration system. Cell-free lysates derived from the insect stages of T. brucei and T. cruzi revealed that these parasites had substantial qDPR activity, with T. brucei being the highest and T. cruzi being lowest. While a T. brucei QDPR gene has not been found, a partial sequence for the T. cruzi QDPR was identified. Additionally, we were able to identify candidate PCD genes in the emerging genome project data bases for all three trypanosomatid species.

In summary, our studies of LmQDPR gene structure and enzymatic activity in Leishmania (as well as more limited data in trypanosomes) show that these parasites possess a potent system for regenerating H4B, in common with other eukaryotes. This further serves to emphasize the importance of H4B metabolism in these organisms, although as yet the precise role of H4B remains to be established. Because H4B has been shown to be required for growth of several species of Leishmania in vitro and in vivo (12, 17, 18), our studies suggest that parasite qDPRs may prove to be useful targets for chemotherapy in the future.

    ACKNOWLEDGEMENTS

We thank N. Akopyants and N. El-Sayed for providing the shotgun sequence clones studied here and other advice, E. Ullu for T. brucei cultures, M. Pereira for T. cruzi epimastigotes, B. Nare for advice on qDPR assays, and the Sanger Institute Websites, TIGR, the Leishmania Genome Network, the National Institutes of Health, and the Wellcome Trust for access to preliminary L. major and T. brucei genome sequence data. We thank D. Dobson, K. Robinson, and K. Zhang for helpful discussions and comments on the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants AI21903 and AI29646 (to S. M. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF523363, AY141854, AF523371, AF523369, and AF523370.

Dagger To whom correspondence should be addressed: Dept. of Molecular Microbiology, Campus Box 8230, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-747-2630; Fax: 314-747-2634; E-mail: beverley@borcim.wustl.edu.

Published, JBC Papers in Press, July 31, 2002, DOI 10.1074/jbc.M206543200

1 J. Moore and S. M. Beverley, manuscript in preparation.

3 L.-F. Lye, M. L. Cunningham, and S. M. Beverley, unpublished data.

    ABBREVIATIONS

The abbreviations used are: DHFR-TS, bifunctional dihydrofolate reductase-thymidylate synthase; qDPR, quinonoid-dihydropteridine reductase; LmQDPR/LmQDPR, Leishmania major qDPR gene/enzyme; H2B, dihydrobiopterin; H4B, tetrahydrobiopterin; ORF, open reading frame; PTR1, pteridine reductase 1; PTR2, postulated dihydrobiopterin reductase; PCD, pterin-4 alpha -carbinolamine dehydratase; DMPH4, dimethyl-5,6,7,8-tetrahydropterin; nt, nucleotide(s); NOS, nitric oxide synthase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. WHO Expert Committee. (1984) The Leishmaniasis , WHO, Geneva
2. Grogl, M., Thomason, T. N., and Franke, E. D. (1992) Am. J. Trop. Med. Hyg. 47, 117-126[Abstract/Free Full Text]
3. Beck, J. T., and Ullman, B. (1990) Mol. Biochem. Parasitol. 43, 221-230[CrossRef][Medline] [Order article via Infotrieve]
4. Petrillo-Peixoto, M., and Beverley, S. M. (1987) Antimicrob. Agents Chemother. 31, 1575-1578[Abstract/Free Full Text]
5. Scott, D. A., Coombs, G. H., and Sanderson, B. E. (1987) Mol. Biochem. Parasitol. 23, 139-149[CrossRef][Medline] [Order article via Infotrieve]
6. Trager, W. (1969) J. Protozool. 16, 372-375[Medline] [Order article via Infotrieve]
7. Cunningham, M. L., and Beverley, S. M. (2001) Mol. Biochem. Parasitol. 113, 199-213[CrossRef][Medline] [Order article via Infotrieve]
8. Lemley, C., Yan, S., Dole, V. S., Madhubala, R., Cunningham, M. L., Beverley, S. M., Myler, P. J., and Stuart, K. D. (1999) Mol. Biochem. Parasitol. 104, 93-105[CrossRef][Medline] [Order article via Infotrieve]
9. Kundig, C., Haimeur, A., Legare, D., Papadopoulou, B., and Ouellette, M. (1999) EMBO J. 18, 2342-2351[CrossRef][Medline] [Order article via Infotrieve]
10. Myler, P. J., Lodes, M. J., Merlin, G., de Vos, T., and Stuart, K. D. (1994) Mol. Biochem. Parasitol. 66, 11-20[CrossRef][Medline] [Order article via Infotrieve]
11. Nare, B., Luba, J., Hardy, L. W., and Beverley, S. (1997) Parasitology 114, S101-110
12. Bello, A. R., Nare, B., Freedman, D., Hardy, L., and Beverley, S. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11442-11446[Abstract/Free Full Text]
13. Beverley, S. M., Ellenberger, T. E., and Cordingley, J. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2584-2588[Abstract/Free Full Text]
14. Ivanetich, K. M., and Santi, D. V. (1990) Exp. Parasitol. 70, 367-371[CrossRef][Medline] [Order article via Infotrieve]
15. Cruz, A., and Beverley, S. M. (1990) Nature 348, 171-173[CrossRef][Medline] [Order article via Infotrieve]
16. Titus, R. G., Gueiros-Filho, F. J., de Freitas, L. A., and Beverley, S. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10267-10271[Abstract/Free Full Text]
17. Papadopoulou, B., Roy, G., Mourad, W., Leblanc, E., and Ouellette, M. (1994) J. Biol. Chem. 269, 7310-7315[Abstract/Free Full Text]
18. Nare, B., Hardy, L. W., and Beverley, S. M. (1997) J. Biol. Chem. 272, 13883-13891[Abstract/Free Full Text]
19. Cunningham, M. L., Titus, R. G., Turco, S. J., and Beverley, S. M. (2001) Science 292, 285-287[Abstract/Free Full Text]
20. Iwai, K., Bunno, M., Kobashi, M., and Suzuki, T. (1976) Biochim. Biophys. Acta 444, 618-622[Medline] [Order article via Infotrieve]
21. Kerler, F., Ziegler, I., Schmid, C., and Bacher, A. (1990) Exp. Cell Res. 189, 151-156[CrossRef][Medline] [Order article via Infotrieve]
22. Golderer, G., Werner, E. R., Leitner, S., Grobner, P., and Werner-Felmayer, G. (2001) Genes Dev. 15, 1299-1309[Abstract/Free Full Text]
23. Kaufman, S. (1963) Proc. Natl. Acad. Sci. U. S. A. 50, 1085-1093[Free Full Text]
24. Basu, N. K., Kole, L., Ghosh, A., and Das, P. K. (1997) FEMS Microbiol. Lett. 156, 43-47[Medline] [Order article via Infotrieve]
25. Paveto, C., Pereira, C., Espinosa, J., Montagna, A. E., Farber, M., Esteva, M., Flawia, M. M., and Torres, H. N. (1995) J. Biol. Chem. 270, 16576-16579[Abstract/Free Full Text]
26. Ma, D., Beverley, S. M., and Turco, S. J. (1996) Biochem. Biophys. Res. Commun. 227, 885-889[CrossRef][Medline] [Order article via Infotrieve]
27. Nagatsu, T., and Ichinose, H. (1999) Mol. Neurobiol. 19, 79-96[CrossRef][Medline] [Order article via Infotrieve]
28. Hauer, C. R., Rebrin, I., Thony, B., Neuheiser, F., Curtius, H. C., Hunziker, P., Blau, N., Ghisla, S., and Heizmann, C. W. (1993) J. Biol. Chem. 268, 4828-4831[Abstract/Free Full Text]
29. Kaufman, S. (1997) Tetrahydrobiopterin: Basic Biochemistry and Role in Human Disease , Johns Hopkins University Press, Baltimore
30. Dianzani, 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., and Cotton, R. G. (1998) Hum. Mutat. 12, 267-273[CrossRef][Medline] [Order article via Infotrieve]
31. Smooker, P. M., and Cotton, R. G. (1995) Hum. Mutat. 5, 279-284[CrossRef][Medline] [Order article via Infotrieve]
32. Blau, N., Barnes, I., and Dhondt, J. L. (1996) J. Inherit. Metab. Dis. 19, 8-14[Medline] [Order article via Infotrieve]
33. Varughese, K. I., Skinner, M. M., Whiteley, J. M., Matthews, D. A., and Xuong, N. H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6080-6084[Abstract/Free Full Text]
34. Su, Y., Varughese, K. I., Xuong, N. H., Bray, T. L., Roche, D. J., and Whiteley, J. M. (1993) J. Biol. Chem. 268, 26836-26841[Abstract/Free Full Text]
35. Chang, C. F., Bray, T., Varughese, K. I., and Whiteley, J. M. (1999) Adv. Exp. Med. Biol. 463, 403-410[Medline] [Order article via Infotrieve]
36. Vasudevan, S. G., Shaw, D. C., and Armarego, W. L. (1988) Biochem. J. 255, 581-588[Medline] [Order article via Infotrieve]
37. Williams, C. D., Dickens, G., Letendre, C. H., Guroff, G., Haines, C., and Shiota, T. (1976) J. Bacteriol. 127, 1197-1207[Abstract/Free Full Text]
38. Hirayama, K., Nakanisi, N., Sueoka, T., Katoh, S., and Yamada, S. (1980) Biochim. Biophys. Acta 612, 337-343[Medline] [Order article via Infotrieve]
39. Hardy, L. W., Matthews, W., Nare, B., and Beverley, S. M. (1997) Exp. Parasitol. 87, 158-170[Medline] [Order article via Infotrieve]
40. Kapler, G. M., Coburn, C. M., and Beverley, S. M. (1990) Mol. Cell. Biol. 10, 1084-1094[Abstract/Free Full Text]
41. da Silva, R., and Sacks, D. L. (1987) Infect. Immun. 55, 2802-2806[Abstract/Free Full Text]
42. Akopyants, N. S., Clifton, S. W., Martin, J., Pape, D., Wylie, T., Li, L., Kissinger, J. C., Roos, D. S., and Beverley, S. M. (2001) Mol. Biochem. Parasitol. 113, 337-340[CrossRef][Medline] [Order article via Infotrieve]
43. Medina-Acosta, E., and Cross, G. A. (1993) Mol. Biochem. Parasitol. 59, 327-329[CrossRef][Medline] [Order article via Infotrieve]
44. Beverley, S. M., Coderre, J. A., Santi, D. V., and Schimke, R. T. (1984) Cell 38, 431-439[CrossRef][Medline] [Order article via Infotrieve]
45. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Vol. 1, pp. 7.39-7.52 and 9.31-9.58, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
46. Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13[CrossRef][Medline] [Order article via Infotrieve]
47. Ryan, K. A., Dasgupta, S., and Beverley, S. M. (1993) Gene (Amst.) 131, 145-150[CrossRef][Medline] [Order article via Infotrieve]
48. Beverley, S. M. (1988) Nucleic Acids Res. 16, 925-939[Abstract/Free Full Text]
49. Chu, G., Vollrath, D., and Davis, R. W. (1986) Science 234, 1582-1585[Abstract/Free Full Text]
50. Wang, Y., Dimitrov, K., Garrity, L. K., Sazer, S., and Beverley, S. M. (1998) Mol. Biochem. Parasitol. 96, 139-150[CrossRef][Medline] [Order article via Infotrieve]
51. Ha, D. S., Schwarz, J. K., Turco, S. J., and Beverley, S. M. (1996) Mol. Biochem. Parasitol. 77, 57-64[CrossRef][Medline] [Order article via Infotrieve]
52. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Methods Enzymol. 185, 60-89[Medline] [Order article via Infotrieve]
53. Coligan, J. E., Dunn, B. M., Speicher, D. W., and Wingfield, P. T. (1997) in Current Protocols in Protein Science, Electrophoresis (Chanda, V. B. C., ed), 3rd Ed., Vol. 1 , pp. 10.1.1-10.1.11, John Wiley & Sons, Inc., New York
54. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
55. Firgaira, F. A., Cotton, R. G., and Danks, D. M. (1981) Biochem. J. 197, 31-43[Medline] [Order article via Infotrieve]
56. Lee, P. L., Halloran, C., Cross, A. R., and Beutler, E. (2000) Biochem. Biophys. Res. Commun. 271, 788-795[CrossRef][Medline] [Order article via Infotrieve]
57. Lockyer, J., Cook, R. G., Milstien, S., Kaufman, S., Woo, S. L., and Ledley, F. D. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 3329-3333[Abstract/Free Full Text]
58. Shahbaz, M., Hoch, J. A., Trach, K. A., Hural, J. A., Webber, S., and Whiteley, J. M. (1987) J. Biol. Chem. 262, 16412-16416[Abstract/Free Full Text]
59. Wilson, R., Ainscough, R., Anderson, K., Baynes, C., Berks, M., Bonfield, J., Burton, J., Connell, M., Copsey, T., Cooper, J., et al.. (1994) Nature 368, 32-38[CrossRef][Medline] [Order article via Infotrieve]
60. Park, D., Park, S., and Yim, J. (2000) Biochim. Biophys. Acta 1492, 247-251[Medline] [Order article via Infotrieve]
61. Varughese, K. I., Xuong, N. H., Kiefer, P. M., Matthews, D. A., and Whiteley, J. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5582-5586[Abstract/Free Full Text]
62. Kiefer, P. M., Varughese, K. I., Su, Y., Xuong, N. H., Chang, C. F., Gupta, P., Bray, T., and Whiteley, J. M. (1996) J. Biol. Chem. 271, 3437-3444[Abstract/Free Full Text]
63. Grimshaw, C. E., Matthews, D. A., Varughese, K. I., Skinner, M., Xuong, N. H., Bray, T., Hoch, J., and Whiteley, J. M. (1992) J. Biol. Chem. 267, 15334-15339[Abstract/Free Full Text]
64. Kiefer, P. M., Grimshaw, C. E., and Whiteley, J. M. (1997) Biochemistry 36, 9438-9445[Medline] [Order article via Infotrieve]
65. Callahan, H. L., and Beverley, S. M. (1992) J. Biol. Chem. 267, 24165-24168[Abstract/Free Full Text]
66. Whiteley, J. M., Xuong, N. H., and Varughese, K. I. (1993) Adv. Exp. Med. Biol. 338, 115-121[Medline] [Order article via Infotrieve]
67. Stiles, J. K., Hicock, P. I., Shah, P. H., and Meade, J. C. (1999) Ann. Trop. Med. Parasitol. 93, 781-807[CrossRef][Medline] [Order article via Infotrieve]
68. Sacks, D. L., and Perkins, P. V. (1984) Science 223, 1417-1419[Abstract/Free Full Text]
69. Smooker, P. M., Howells, D. W., and Cotton, R. G. (1993) Biochemistry 32, 6443-6449[CrossRef][Medline] [Order article via Infotrieve]
70. Homuth, M., Valentin-Weigand, P., Rohde, M., and Gerlach, G. F. (1998) Infect. Immun. 66, 710-716[Abstract/Free Full Text]
71. Werner, E. R., Pitters, E., Schmidt, K., Wachter, H., Werner-Felmayer, G., and Mayer, B. (1996) Biochem. J. 320, 193-196[Medline] [Order article via Infotrieve]
72. Werner, E. R., Habisch, H. J., Gorren, A. C., Schmidt, K., Canevari, L., Werner-Felmayer, G., and Mayer, B. (2000) Biochem. J. 348, 579-583[Medline] [Order article via Infotrieve]
73. Huang, L., Jacob, R. J., Pegg, S. C., Baldwin, M. A., Wang, C. C., Burlingame, A. L., and Babbitt, P. C. (2001) J. Biol. Chem. 276, 28327-28339[Abstract/Free Full Text]
74. Ouellette, M., Haimeur, A., Leblanc, E., Grondin, K., Legare, D., Kundig, C., and Papadopoulou, B. (1996) Trop. Med. Intl. Health 1, A33-A34
75. Beverley, S. M. (1991) Annu. Rev. Microbiol. 45, 417-444[CrossRef][Medline] [Order article via Infotrieve]
76. Timmerman, M. M., and Woods, J. P. (1999) Infect. Immun. 67, 6403-6408[Abstract/Free Full Text]

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Plant Physiol.Home page
V. Naponelli, A. Noiriel, M. J. Ziemak, S. M. Beverley, L.-F. Lye, A. M. Plume, J. R. Botella, K. Loizeau, S. Ravanel, F. Rebeille, et al.
Phylogenomic and Functional Analysis of Pterin-4a-Carbinolamine Dehydratase Family (COG2154) Proteins in Plants and Microorganisms
Plant Physiology, April 1, 2008; 146(4): 1515 - 1527.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. J. Vickers, G. Orsomando, R. D. de la Garza, D. A. Scott, S. O. Kang, A. D. Hanson, and S. M. Beverley
Biochemical and Genetic Analysis of Methylenetetrahydrofolate Reductase in Leishmania Metabolism and Virulence
J. Biol. Chem., December 15, 2006; 281(50): 38150 - 38158.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
V. Wilquet, M. Van de Casteele, D. Gigot, C. Legrain, and N. Glansdorff
Dihydropteridine Reductase as an Alternative to Dihydrofolate Reductase for Synthesis of Tetrahydrofolate in Thermus thermophilus
J. Bacteriol., January 15, 2004; 186(2): 351 - 355.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
277/41/38245    most recent
M206543200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lye, L.-F.
Right arrow Articles by Beverley, S. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lye, L.-F.
Right arrow Articles by Beverley, S. M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement