dihydrofolate reductase-thymidylate synthase of Leishmania major

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Dec 24, 1985 - STEPHEN M. BEVERLEY, THOMAS E. ELLENBERGER, AND JOHN S. CORDINGLEY. Department of Pharmacology, Harvard Medical School, ...
Proc. Nati. Acad. Sci. USA Vol. 83, pp. 2584-2588, April 1986 Genetics

Primary structure of the gene encoding the bifunctional dihydrofolate reductase-thymidylate synthase of Leishmania major (chemotherapy/protein homologies/intervening sequences/gene ampliflcation/protozoan parasites)

STEPHEN M. BEVERLEY, THOMAS E. ELLENBERGER, AND JOHN S. CORDINGLEY Department of Pharmacology, Harvard Medical School, Boston, MA 02125

Communicated by George H. Hitchings, December 24, 1985

within the R region encoding the bifunctional enzyme DHFRTS of Leishmania major. MATERIALS AND METHODS Cells, Genomic, and Recombinant DNAs. The LT 252 cell line (11) and the methotrexate-resistant R1000 derivative cell line (6) were utilized as sources of genomic DNA and RNA as described (10). We have shown by DNA hybridization and serological tests that this line actually is of the species Leishmania major (L. tropica major; S.M.B. and D. McMahonPratt, unpublished data). A 4.5-kilobase (kb) Sal I fragment from phage X LTS-5 (10) was isolated and inserted into a pUC vector (12); this recombinant is named pLTS-5-S45. cDNA Library. A cDNA library of polyadenylylated mRNA from R1000 promastigotes was constructed in the bacteriophage vectors XgtlO (13) and Xgtll (14) using the protocol of T. St. John, J. Rosen, and H. Gershenfeld (personal communication), and screened with pLTS-5-S45 (15). Approximately 400,000 independent recombinants were obtained for each library. The largest recombinant insert, one of 0.6 kb in XgtlO, was inserted into M13 vectors for DNA sequencing. DNA Sequencing. DNA sequencing was performed using the dideoxynucleotide technique (16) with 3"S-labeled deoxynucleotide triphosphates (17), and Leishmania-derived DNAs were inserted into, M13 vectors (18). The entire sequence was determined on both strands.

We have determined the nucleotide sequence ABSTRACT of the dihydrofolate reductase-thymidylate synthetase (DHFRTS) gene of the protozoan parasite Leishmania major (dihydrofolate reductase, EC 1.5.1.3 and thymidylate synthase, EC 2.1.1.45). The DHFR-TS protein is encoded by a single 1560-base-pair open reading frame within genomic DNA, in contrast to vertebrate DHFRs or mouse and phage T4 TSs, which contain intervening sequences. Comparisons of the DHFR-TS sequence with DHFR and TS sequences of other organisms indicate that (i) the order of enzymatic activities within the bifunctional polypeptide chain is DHFR followed by TS, (ii) the Leishmania bifunctional DHFR-TS evolved independently and not through a phage T4-related intermediate, and (iii) the rate of evolution of both the DHFR and TS domains has not detectably changed despite the acquisition of new functional properties by the bifunctional enzyme. The Leishmania gene is 86% G+C in the third codon position, in contrast to genes of the parasite Plasmodium falciparum, which exhibit an opposite bias toward A+T. The DHFR-TS locus is encoded within a region of DNA amplified in methotrexate-resistant lines, as previously proposed.

The enzymes dihydrofolate reductase (DHFR; EC 1.5.1.3) and thymidylate synthase (TS; EC 2.1.1.45) have critical roles in intermediary metabolism and consequently are important targets for chemotherapy. Drugs used in the treatment of human tumors that inhibit DHFR and TS are methotrexate and metabolites of 5-fluorouracil, respectively (1, 2). Other antifolates such as trimethoprim and pyrimethamine have been employed in the treatment of pathogens (3, 4). In most organisms DHFR and TS exist as separate molecular entities, DHFR as a monomer of about 20 kDa and TS as a dimer made up of --35-kDa subunits (5). In contrast, in Leishmania as well as all protists examined to date, these enzymes are part of a bifunctional DHFR-TS complex, consisting of a homodimer of a 54- to 100-kDa polypeptide chain (6-8). This bifunctional enzyme exhibits distinct biochemical properties such as metabolic channeling (9) and disparate binding of methotrexate and 2'-deoxy-5-fluorouridine monophosphate (5-FdUMP) (8, 9). Detailed molecular and biochemical studies may reveal additional features that will prove useful in developing agents that may selectively inhibit the Leishmania enzyme, analogous to the selective inhibition of Plasmodium DHFR by pyrimethamine (4). Mutants of Leishmania have been obtained that overproduce the bifunctional enzyme DHFR-TS, thus facilitating its isolation and characterization (6, 9). Mutants exhibiting enzyme overproduction also exhibit amplification of extrachromosomal circular DNAs (10), one of which is correlated with enzyme overproduction (the R region). We have now determined the nucleotide sequence of the gene

RESULTS Sequence of the DHFR-TS Region. Analysis of mRNAs encoded by the amplified R region of the methotrexateresistant R1000 line of Leishmania (6) has indicated that a 3.2-kb polyadenylylated mRNA encodes the bifunctional DHFR-TS (refs. 19 and 20; G. Kapler and S.M.B., unpublished results). This region is entirely contained within a 4.5-kb Sal I fragment (Fig. 1), which is also amplified within the R region of the methotrexate-resistant R1000 line (6, 10). By restriction enzyme mapping, this region of DNA appears to be identical in the wild-type and R1000 lines (10), as is the DHFR-TS protein synthesized as judged by biochemical criteria (6, 9). DNA sequencing of pLTS-5-S45 reveals a single large open reading frame, beginning at the ATG initiation codon numbered as base 1 and proceeding for 1560 bp (Figs. 1 and 2). This ATG is the first between the long open reading frame and the 5' side of the mRNA, as revealed by S1 analysis (unpublished data). The derived amino acid sequence of this protein includes the peptide Met-Asp-Leu-Gly-Pro-Val-Tyr-Gly-Phe-Gln-Trp-Arg-His-Phe-Xaa-Ala-Asp-Tyr-Lys-Xaa-Phe-Glu-Ala-Asn-Tyr-Xaa-Gly-Glu that differs by one amino acid from an internal peptide of the DHFR-TS of a 5,8-dideaza-10-propargyl folate-resistant line of Leishmania

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviations: DHFR, dihydrofolate reductase; TS, thymidylate

synthase; 5-FdUMP, 2'-deoxy-5-fluorouridine monophosphate; bp, base pair(s); kb, kilobase(s).

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S

A

P

P R

I

Is

B

T

P

I ^ DHFR

TS

cDNA

-4

AAAA

FIG. 1. Analysis of the DHFR-TS gene of Leishmania maor. (A) Restriction map of the 4.5-kb Sal I fragment located within the R region of Leishmania major. The orientation of this map is opposite to that shown in figure 1B of ref. 10. The symbols correspond to restriction enzyme cleavage sites as follows: P, Pvu II; R, EcoRI; B,

Bgl II; T, Sst I; S, Sal I. The region of DNA sequenced was between the EcoRI site and a Sph I site (not shown) approximately 300 bp on the 3' side of the Sst I site. The location of the 0.6-kb cDNA clone is also indicated. (B) Summary of features of the DHFR-TS gene. The location of the single large open reading frame of 1560 bp within the mRNA is indicated by the thick region. The 22-amino acid hydrophilic leader sequence is shown as a solid region, and the location of the DHFR and TS domains are taken from the alignments in Fig. 3. The location of the poly(A) tract of the mRNA as revealed by sequence analysis of the cDNA is also shown.

(21). (The histidine at position 13 within the peptide was identified as glycine by these authors. DNA sequencing of both strands in this region unambiguously predicts histidine.) The predicted DHFR-TS has a molecular weight of 58,684, somewhat larger than the 57-kDa subunit molecular size reported for Leishmania and other trypanosomatids (6-8). This may reflect the accuracy of gel electrophoresis methods for estimating protein molecular weights. Alternatively, the DHFR-TS may be processed in vivo to a smaller final product. Alignment of the Leishmania DHFR-TS with DHFRs of diverse origin indicates that the Leishmania gene bears a hydrophilic amino-terminal extension of 22 amino acids (Fig. 3). The amino terminus of the mature DHFR-TS has been reported to be blocked (9, 21), and the precise amino terminus of the DHFR-TS is currently unknown. Another 1040 bp of sequence follow the open reading frame, at which point the sequence of the cDNA and genomic sequences diverge, corresponding to the poly(A) tract of the mRNA. The region between the long open reading frame and the poly(A) tract contains no long open reading frames. As reported previously for genes of the related parasite Trypanosoma brucei, we can find no eukaryotic consensus poly(A) addition signal (AATAAA) prior to the poly(A) tract (32, 33). Furthermore, we cannot find a consensus homology in this region with the corresponding regions in other trypanosome genes (33, 34). Comparison of the Predicted Leishmania DHFR-TS Sequence with Other DHFR and TS Sequences. The amino acid sequence of the DHFR-TS enzyme was derived from the nucleotide sequence and compared with published DHFR and TS sequences (Fig. 3, Table 1). It is evident that the amino-terminal region of the Leishmania DHFR-TS exhibits significant sequence homology (up to 37%) with DHFRs of diverse origin, whereas the carboxyl-terminal region of the leishmanial protein exhibits homology (up to 63%) with TS sequences. The DHFR-TS also exhibits a hydrophilic 22amino acid extension prior to the DHFR domain. The homologies with both DHFR and TS sequences include amino acids known to be important in ligand binding and/or located at the catalytic site (discussed below). We, therefore, infer that the leishmanial protein is encoded as a fused polypeptide chain of two separate domains, an amino-terminal DHFR domain followed by a carboxyl-terminal TS domain. Our genomic DNA sequence data indicate the Leishmania DHFR gene does not contain intervening sequences, an observation confirmed by S1 nuclease analysis oftheDHFR-coding region ofthe Leishmania gene (data not shown).

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There is a nonrandom utilization of the four nucleotide bases at degenerate codon positions of the DHFR-TS. Within the coding region the first codon position is 62% G+C, the second position 41% G+C, and the third position 86% G+C. Neither the 3'- nor the 5'-untranslated regions show a positional bias. In contrast, genes of the intracellular parasite Plasmodiumfalciparum exhibit an extreme A+T bias at third positions as well as within the genome as a whole (35). Conservation of Amino Acid Sequence. The amino acid sequence alignment presented in Fig. 3 indicates that certain residues are highly conserved among DHFR and TS sequences. In the DHFR domain these residues include many of those implicated in the interactions of DHFR with substrates, cofactors, and/or inhibitors (31). Of the 18 residues shared in all eukaryotic DHFRs, the Leishmania DHFR domain shares 15 (positions 32, 39, 40, 46, 47, 56, 60, 80, 83, 94, 97, 102, 157, 158, and 180). For TS two separate regions have been identified in ligand binding studies: a 5-FdUMP binding site, located at the conserved tripeptide Pro-Cys-His of the TS in many species (5) (residues 399-401 in Fig. 3), and a folylpolyglutamate binding site in Lactobacillus casei (36) (residues 279-292). Both of these sites are conserved in the Leishmania TS domain. There are additionally several other regions within the TS domain that are highly conserved among all species and that presumably represent functionally important residues. The Leishmania TS domain shares an insertion of 12 amino acids with the human sequence (residues 322-333), the site of which has been shown to be a target for protease inactivation (21). Relationships of the Bifunctional DHFR-TS Enzyme to Other Species. Table 1 shows that that Leishmania TS and DHFR are both more closely related to vertebrate enzymes than to the prokaryotic or phage T4 enzymes. The DHFR domain of the Leishmania DHFR-TS exhibits 37% amino acid sequence homology with vertebrate DHFRs, 29% homology with prokaryotes, and 18% homology with phage T4. Similarly, the TS domain of the DHFR-TS exhibits 63% homology with vertebrates, 48% homology with prokaryotes, and 45% homology with phage T4. These findings are in accordance with the current view of the evolutionary relationships among metazoa, protists, and prokaryotes (37). Examination of the evolutionary insertions of amino acid sequence within both the TS and DHFR genes confirms that the Leishmania DHFR-TS is more closely related to the vertebrate genes than to either prokaryotic or phage T4 genes. Within the TS genes there are six introduced gaps among the five sequences. Two of these are specific for phage T4 (positions 306 and 498) and two are specific for Leishmania (positions 275 and 411). The remaining two (positions 322 and 349) are complex, in that the insertion boundaries are variable among species. Examination of these insertions reveals that the Leishmania and human TS sequences are precisely the same length and homologous at the amino acid sequence level, in contrast to the comparisons involving the other prokaryotic and phage sequences. Similar findings are evident in the insertions found within the DHFR sequence alignments; however, as the degree of sequence homology among these genes is considerably less than for TS, there is less certainty about the exact placement of the sites of insertion.

Relative Rates of Evolution of Bifunctional vs. Monofunctional DHFRs and TS. It is possible that new properties of the bifunctional DHFR-TS relative to monofunctional DHFR and TS may have given rise to new evolutionary pressures, resulting in a change in the rate of evolution of the DHFR-TS genes. We compared the Leishmania and human genes separately with the more distantly related prokaryote and phage genes (Table 2). Alterations in evolutionary rate would be revealed by differences in the distance between the outside reference group and the Leishmania or human

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-1

-120

TGGCGGACGAAACTCGCACACAGGCACGCCGCCTCCTTTCACCCGTCATAGArAGTTGAArrAGACGCCCTCCTCCTCCCTCATCATCGCCGTCGTCATCCGGGTCCGAGCACTACGAAG 120

PvuII

1

AiTGTCCAGGGCAGCTGCGAGGTTTAAGATTCCGATGCCGGAGACGCAACGWAGACTTTCTTTCCCCTCCCTGCGCGCCTTCTCCATCGTCGTCGCCTCGrATATGCAGCACGCCATCGOC

MetSerArgAloAlaAlaArgPheLysI leProMetPro0luThrLySAlaAspPheAl1PheProSerLeuArgAlaPheSerI1eValValAlILeuAspMet~lnHisGlyIleGly 240

GACGGCGAGTCGATCCCGTGGCGGGTGCCCCGAGGACATGACG'TTC'rTC AAGAACCA GACGACCCGCTGTGWAACAAGAAGCCGCCGACGCACA AGA AGCGCAACGCCGTCCTGATGGGC AspGlyGluSerI leProTrpArgVal1ProGl1uAspMetTh rP hePheLysAsnGlnThrThrL~euLeuArgAsnLY3LysProP roThrC 1uLysLysA rgAsnAl aVal1V alMet~ly 360

CGCAAGACTTOGGAGAGCGTCCCGGTAA AGTTCCGACCACTCAACCGGACWT;GCAACATCGTGTTATCCTCGAAGGCCACCGTC(;AGGAWCTTCTGCGCCGCI'GlCCGGA~tibGAC ACC(;C ArgLysThrTrpGl uSerVa1ProVa Lys PheA rgProLeu LyslyArgLeu AsnIleVaLeuSerSerLysAaThrVa1CuGluLeuLeuA1aProLeuProGlu1yG1nArg 480

GCGGCGGCGGCGCAGGATGTGGTGGrGGTGA ACGGCGGTC TGCGCCACGCGCTCCGCCTC CTCGCACGCCC GCTGCTACTGCAGTC CA TCGAGCAtC~sGT ATTWcGTC GGTG( T ¢GCWA ArgProLeuTyrCysSerSerIleC1uThrAlaTyrCysVal~ly~lyA!aGin IVylAsnGlyGlyyLeuAlaluAlaLeuArgLeuLeuA1 AleAlsAaArlaGlnAspVlValVa bOO

GSTTTACGCGGACGCCATGCTGTCGCCGTGCATCGAGAAACTGCAGGAAGTGTACCTCACCCGCATCTACCGCCAUCGtCCCTGCGCTCTACGCGCTTCTTTCCCTTTCCGCCCCGAG^AACGtCG TyrLeuThrArgl leTyrAlaThrAlaProAlaCysThrArgPhePheProPheProProGluAsnAla ValTyrAlaAspAlaIetIeUSerProCY3IleGluLY3LeuGflnAOI

7zo

BglII

CCAGTACC rTGAGCTG G(CCACGG;CGTGGGAWCTGGCGTCGTCTCAGGGACGCCWAAGAWCGAGGCGGAGGGCCCTCGAGTTCGAGATCTCCAAG'TAC CTGCCGCGC AACCAC;AC(;GAGC u tlaThrAlsTrpAspLeuAlaSerSerGlnGlyArgArtLysSerGluAlaOluGl yLeuGluPheGCuIl eCysLysTyrVal ProArgAsnHisul s;uArgGlnryrLeuGluLeu

840

ATTGACCGCATCATGACACGGGGATCGTGAAGGAGGACCGCACCGGC GTGCGCACCATCAGCCTCTTCCG;CWCCAGATcGCWT'rCTC( CTA CGCGACA ACcCUt ,tI'CCXuTGCTGiACG 960

PavII

A^CGAA^GCGrGTCTTCTGGCGCGGCGTGTGCGAGGACCGCTCXTCC~jTTCCTUCeCC;GGC#^AAC GAGTUCCCAGCTGCTGC;CAG;AC AAGCAACATTCAC ATCTGGGACWCAACCGTTCGCGC

ThrLysArgValPheTrp~r5GlyValCysluGl uLeu LeuTrpPheLeuA rgGl yl uThrSerA1al nLeu LeuA laAspLysAspI eHisI leTrpAspulyAsnblySerArg 1080

GAGTTTCTCGA^CAGCCGCGC;CTTGAsCAGA^GAATAAGGAGATGGUACCTC CGCCCTGTCTAC GGCTTCC A C rvCcCCACTTCUGGiGCA CA rT ACA A GCGGG1TTGA AGC GAA(;TACGACGOC GluPheLuspSerAr&GlyLevThrGluAsnLysGlUeApLeuGly ProVa lTyr~ly PheGl n'r rp~rlis Phe~lyA1AspTy rLysGl y PheG1uA1aAsnTyrAspGly 1200

G^AAGOGT~GGACCAGAkTCAAGCTbCATCGTGGAGACCATCAAGACGAACCCAA^CGACCGCCGCCTC'T AtiTC ACTGCCTGC;AAC CCG rGCGCGCT(;CAA AAG;ATG;CGCTUCCCCCCCTCC G1uGlyValAspGlnIleLysLeuIleValGluThrIleLysThrAfsnProAsnAspArgArgLeuLeuVai rhrAiaTrpAsnProCysAlaLeuo;l!nLysMetAlaLeuProProCys 1320

CACCTTGCTTCCAGTTCTACGTGAACACAGACACGAGCGAGCTATCCTGCATGTTGTACCAGCGCT'CGTGTGACATGGGTC'rTGGCGTCCCCTTCAACATTGCCTCCTACGCGCTGCTC HisL~LeuAl&Gl nPheTyrValAsnThrA pThrbrGlu Leu~rCys tLeuTyrGl nA rrCysAs pMe tGl yLeuGl yVal ProPheAsn I leAlaSerTyrAlaLeuLeu 1440

ACCATCCTCATT~OCAAGGCGACGGCTCTGCGOCTGGTGAGCTTGTGCACACCCTCGGCGACGCCCACGTCTACCGCAACCACGTTGATGCCCTCAAGGCGCA GCTCGAGCGAGTCCCG ThlrIleLouIle~llLysAloThrly L uArgPr~o~lyGl uLeuValH lsThrL u~l y~spAlaHilsV lTyrArgAsnHi sVa 1AspA 1 aLeuLysA laGl nLeuC luA rgV a lPro 1560

CWCCCGfTCCCGACCCTCATCrTCAAGCGAGGGCGGCCGTACCTCGAGGACTACGAGTTGACGGACATGGAGCTGATCGACTACGTTCCACACCCCGGCGATCAAGATGGAGATGGCCGTA Nl&AloPbe~roThrLe~ll PheL~ys~luGl uArgGlnTyrLeuGlu~spTyr~l uL uThrAsp~et~luVel I eAs pTyrVa 1Pr~oHl ProAl1a Ile LysMe tGl uMetA laV al ~~~~~~~~~~~~~~~~~~~~~~~1680

. . *

TAGAGAGAGGGAGGGTGTCATGTCCGsTTGTATGCATGCAGCCACCGCCGC'GACGCTGCC;CTCACCTGliCTCCCACCTCCTCGTCGCACGACGACCGCCCCTTGCGCAGACTCGTTGGT

1800

AACCATGAGCGGCGCGCGAGGGTACGCGCCT&CGTTTCTCGCACATGGCTGCGGCTGATATCCCGCCCGCACCCGCGCGCGCTCAGGCCGCGrGTCG;TCG;TWTCTTTCCATTTrTTTTT 1920

GATTTGG^GCTGCTCTCCGTTGTGTGCTGGGGACCCTCCrTCCC TCGATCTCCTC GTGCGGGGTCTCCGACWCGCAGACGCGGTGCGTGCGAGCGTGCACGTGCTGT

PvuII

2040

CCCMMVOCTOTAOTTGCGAGCGaGAGGAGAGAGAGAGGGAGGGGGGAGGGCAGAGGGCAGAGGACATC;CGGGTGGGAACGTGCACCGGCCTCGTCTCACGCAGCTGGAGCCCACGAAT 2160

WCCACCACCACACCCTcTsTCCCCCCCCCTCCCTCTTCTTCCCACGGCGGCGACGACGACGGGGGCTCAGCTCACGCTTCTGTAGGGTATTATATTAAAGCACATGTGCC;TGCTGTGCTT 2280

CCMC~rCTCTTMaGGcGTTcGCrTTCAAGTCCACGACTCCTCCCGTTGCTCACCGCCGGGCTGTCTTCTTTCGCCCCTCCCTCGGTGCCTCTTCCGCCGCCGCACOCGTGCGCTGAT 2400

CACO=tCTTOCGTGATGTGCCTGTC;TGTATACACACCGTGCACGGAGAGAGCGAGCGAGCGAGAGAGAGAGAGGCCGAGAGGAAGAATGWCGGTGCCTCGCGTGGGCAAGCGTGCGCG 2520

OmGrffOTGTCGTOCOCCGCGTG^GATCGTCGCCAGCOCAAGACCCCCGTGCGCCTGAAACC;TGAAGGGAGG;TCGAGAirGCGTGCCGCA'TGAGGCCTTAAGGCAGGlkA'AGTGAAAAGAG 3st I

,

,

2640

CTCGACG~OTGACCAGCACGCGCGACATCG^AcACGACGGAATAGACACTCAOCCCCTTCCCCTTTCAAAAACTGAATGGACGGACATCGTAACGCGCTCTCTCCCCTCCACTCCCCCT CDNA:.. CCCCTTTCAAAAACTGAAAAAAn

lineages. It is evident that there are no significant or consistent differences between the human and Leishmania DHFR or TS domains in these tests, indicating that the rate of evolution of DHFR and TS has not been detectably altered upon formation of the bifunctional enzyme. Furthermore, each protein domain has maintained its characteristic rate of evolution, with DHFR evolving more rapidly than TS. DISCUSSION In earlier work we described the selection and properties of the methotrexate-resistant R1000 line of Leishmania. This line exhibits unstable amplification of two regions of DNA, and overproduction of the bifunctional DHFR-TS enzyme. As enzyme overproduction and R region amplification were invariably associated (6, 10), we proposed that the R region encoded the structural gene for DHFR-TS. We have now confirmed by nucleotide sequencing and evolutionary comparisons with other DHFR and TS genes that the R region does, in fact, encode the structural gene for the DHFR-TS of Leishmania.

FIG. 2. Sequence of the DHFR-TS gene of Leishmania major. The nucleotide sequence is numbered with position

1 as the first base of the initiation codon. The underlined peptide corresponds to that determined from partial protein sequencing reported for the DHFR-TS from a 5,8-dideaza-lO-propargyl folateresistant line by Garvey and Santi (21). The terminal portion of the cDNA sequence is shown at position 2598; the sequence to the 5' end of this clone is identical to the genomic sequence shown.

DHFR and TS form two successive steps of the metabolic cycle responsible for de novo synthesis of thymidylate in Leishmania (6). Significant interactions of DHFR and TS occur within the bifunctional enzyme complex of Leishmania, as Meek et al. (9) have shown that the bifunctional DHFR-TS exhibits channeling of dihydrofolate, a product of the TS-catalyzed reaction and the substrate of DHFR. Furthermore, the bifunctional DHFR-TS of Leishmania and Crithidia exhibit disparate binding of the usually stoichiometric ligand methotrexate, with 0.5 mol bound per mol of DHFR-TS subunit (8, 9). The disparate binding may be eliminated by limited proteolysis (21). In contrast, it has also been shown that the DHFR and TS domains may be separately inhibited by agents selective for one activity or the other, suggesting that the two domains retain a measure of independence (8, 9). Correspondingly, we can find no evidence indicating that DHFR and TS sequences have become intermixed within the bifunctional DHFR (Fig. 3). Furthermore, evolutionary comparisons indicate that bifunctionality has not grossly altered the rate of evolution of the Leishmania

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