Plasmodium falciparum: Isolation and Characterisation of a Gene Encoding Protozoan GMP Synthase

Experimental Parasitology 94, 23–32 (2000) doi:10.1006/expr.1999.4467, available online at http://www.idealibrary.com on

Plasmodium falciparum: Isolation and Characterisation of a Gene Encoding Protozoan GMP Synthase1

Glenn A. McConkey Department of Biology, University of Leeds, Miall Building, Clarendon Way, Leeds LS2 9JT, United Kingdom

McConkey, G. 2000. Plasmodium falciparum: Isolation and characterisation of a gene encoding protozoan GMP synthase. Experimental Parasitology 94, 23–32. The final step in guanylate nucleotide biosynthesis is catalysed by GMP synthase. This paper presents the first isolation of a gene encoding a protozoan GMP synthase. The deduced amino acid sequence from Plasmodium falciparum shares 40% identity with yeast GMP synthase and contains motifs conserved in catalysis. Expression of the gene is regulated through the parasite’s development in human red blood cells with maximal expression during the point of DNA replication. Psicofuranine, which inhibits GMP synthase, interrupts parasite growth, supporting the role of this enzyme. These findings will aid development of inhibitors of purine salvage in malaria parasites. q 2000 Academic Press Index Descriptors and Abbreviations: protozoa, purine, glutamine amidotransferase, Plasmodium falciparum (P. falciparum), GMP synthase (EC 6.3.4.1), GMP (guanosine 58-monophosphate), XMP (xanthosine 58-monophosphate), IMP (inosine 58-monophosphate), hypoxanthine–guanine phosphoribosyltransferase (HGPRT), RACE (random amplification of cDNA ends), IMP dehydrogenase (IMPDH).

and xanthine in human erythrocytes and parasite growth is inhibited by degradation of exogenous hypoxanthine with xanthine oxidase (Berman et al. 1991, Reyes et al. 1982). GMP (guanosine 58-monophosphate) is synthesised from hypoxanthine by a series of enzymes. The enzyme with the highest activity in P. falciparum extracts was hypoxanthine– guanine phosphoribosyltransferase (HGPRT), which metabolises hypoxanthine to IMP (inosine 58-monophosphate) for further metabolism to AMP and GMP. The crystal structure has been solved for HGPRT from the protozoa P. falciparum, Toxoplasma gondii, and Tritrichomonas foetus (Schumacher et al. 1996, Shi et al. 1999, Somoza et al. 1996). P. falciparum HGPRT is also active in metabolising xanthine and guanine to XMP and GMP, respectively (Shahabuddin and Scaife 1990; Queen et al. 1988). GMP is synthesised via IMP dehydrogenase (IMPDH), which is responsible for metabolism of IMP to XMP. Genes encoding IMPDH have been isolated from several protozoa including trypansomatids and T. foetus and the crystal structure of T. foetus IMPDH has been solved (Whitby et al. 1997). The terminal step in GMP synthesis, GMP synthase (xanthosine 58-monophosphate:glutamine amido-ligase, GMP synthetase) activity, has not yet been described in P. falciparum extracts but presumeably XMP is metabolised to GMP by this enzyme. GMP synthase catalyses the transfer of an amido group from glutamine to the C2 carbon of XMP via an adenylyl-XMP intermediate (Fig. 1 (Zalkin 1993)). Based on the assumption that P. falciparum utilises this enzyme, I sought to identify a gene encoding GMP synthase. The dependence of parasitic protozoa on salvage of preformed purines has been considered an attractive target for

INTRODUCTION

All parasitic protozoa studied rely on salvage of preformed purines for RNA and DNA synthesis and cellular metabolism (Ullman and Carter 1995). Hypoxanthine is believed to be the principal source of purines in Plasmodium falciparum because there are limited amounts of guanosine, guanine, 1

The sequence data reported herein have been submitted to GenBank under the Accession Number GenBank AF152349.

0014-4894/00 $35.00 Copyright q 2000 by Academic Press All rights of reproduction in any form reserved.

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FIG. 1. Scheme for the reaction catalysed by GMP synthase.

antiprotozoal drugs for over 2 decades, although no current antimalarial drugs target this pathway (Craig and Eakin 1997). Substrate analogues for HGPRT and IMPDH inhibitors mycophenolic acid (MPA) and bredinin inhibit Plasmodium growth but lack specificity (Queen et al. 1988, 1990; Webster and Whaun 1982). The enzyme activities present in the parasite suggest that a combination of inhibitors may be required to effectively inhibit parasite growth. High levels of phosphribosyltransferase, adenosine deaminase, and purine nucleoside phosphorylase activities and lack of nucleoside kinase activity for inosine, xanthosine, or guanosine indicate that blocking two different branches of the pathway may provide an effective chemotherapy (Reyes et al. 1982). Isolation of the P. falciparum gene encoding GMP synthase will contribute to the development of inhibitors of protozoan purine salvage. This paper describes the isolation of the gene encoding GMP synthase, its expression, and the potential of GMP synthase as a drug target.

MATERIALS AND METHODS In vitro cultivation of P. falciparum. P. falciparum clonal strain 3D7 was cultured in human red blood cells by the method of Trager as previously described (McConkey et al. 1994). Briefly, culture medium consisted of RPMI 1640 (Life Technologies Inc., Gaithersburg, MD) containing 0.2% NaHCO3, 20–40 mM Hepes, 40 mg/L gentamycin, and 10% (V/V) human serum (Blood Transfusion Service, Seacroft Hospital, Seacroft, West Yorkshire) at pH 7.1. Parasites were grown in a 3% CO2, 2% O2, 95% N2 gas phase. Cloning a gene homologous to GMP synthases. P. falciparum genomic DNA (5 mg), purified as described previously, was partially

digested with Sau3A and inserted into BamH1-digested lZAP (Stratagene, Inc., La Jolla, CA) (Rogers et al. 1995). The lZAP library (average insert of 1–2 kb) was screened with a degenerate oligonucleotide (58-TT(C/T)GGIGTICA(A/G)T(A/T)(C/T)CA(C/T)CCIGA-38; I5inosine) based on a conserved sequence in the glutamine binding domain. The library was hybridised with radiolabeled oligonucleotide and washed in low stringency conditions (458C in 2 3 SSC, 0.2% SDS) according to the manufacturer’s suggested protocol. Plaques hybridising to the oligonucleotide were confirmed by secondary screening. A plasmid clone was excised from lZAP according to the manufacturer’s procedures. The plasmid insert ('1 kb) was sequenced with processive oligonucleotides by the Sanger dideoxynucleotide method using a dye-terminator and an automated sequencer (Applied Biosystems, Perkin–Elmer Inc., Norwalk, CT). A BLASTX search (at http:// www.ebi2.ac.uk) of the SwissProt protein database with six possible reading frames of the identified sequence showed similarity to GMP synthases from other organisms. To isolate the remaining 58 end of the gene, a PCR-based ligation library procedure was developed. pBluescript II (Stratagene) was digested with several of the enzymes which cleave at single sites in the multiple cloning site. P. falciparum DNA was digested with the same enzyme set and ligated to the respective restriction-enzyme-digested pBluescript II. An aliquot of the ligation mixture was initially subjected to repeated primer extensions with an antisense oligonucleotide specific for the gene (58-ATGGTGTTCAATATCATCCAGAGGTG-38) in a standard PCR reaction with AmpliTaq (Perkin–Elmer). The reactions were incubated in an Omnigene thermal cycler (Hybaid Inc., Ashford, Middlesex): 948C, 40 s; 508C, 1 min; 688C, 2 min repeated for 10 cycles. An universal T7 promoter primer (Amersham Ltd., Little Chalfont, Buckinghamshire) was then added to each reaction and the reaction was continued for 40 incubation cycles. The products were resolved by agarose gel electrophoresis. Amplification of the EcoR1 and HindIIIdigested DNA yielded the largest fragments (1.2 and 1 kb, respectively). These products were purified, treated with Pfu DNA polymerase, and ligated to pBluescript II in the presence of SmaI restriction enzyme, as previously described (Li et al. 1994). Several clones from each restriction enzyme digestion were purified and sequenced. The 38 end of the gene was cloned by 38 RACE. Total P. falciparum 3D7 RNA was isolated (RNagents kit, ProMega Inc., Madison, WI)

GMP SYNTHASE OF MALARIA PARASITES

and treated with DNase I (amplification grade, Life Technologies) as per the manufacturer’s instructions. A generic 38 RACE oligonucleotide (58-GGCCACGCGAAGCTTCAGCT18-38) was annealed to P. falciparum RNA (2 mg) and extended with Superscript RT (Life Technologies). The cDNA from reverse transcription was used to amplify the 38 end of the gene in a PCR reaction containing an amplification primer (58-GGCCACGCGAAGCTTCAGC-38) and a gene-specific primer (58-CTACAAGGAGTTACAGATCCC-38). The reactions were incubated 948C, 30 s; 558C, 45 s; 688C, 2 min for 40 cycles. The PCR product was purified and cloned into pCR-Script using the manufacturer’s kit (Stratagene). Transcript analysis of the P. falciparum GMP synthase gene. Parasites were synchronised by two rounds of sorbitol treatment (McConkey et al. 1994). Briefly, asynchronous parasites (5% parasitemia) were pelleted at 1000g for 5 min and suspended in 10 ml 5% sorbitol; after 10 min incubation at 378C, the culture was pelleted as above. The parasites were suspended in complete medium and grown under normal conditions. After 40 h, the sorbitol treatment was repeated. Greater than 98% of the parasitised red blood cells were ring-stage trophozoites. The culture (1.5 3 108 parasites) was suspended in complete medium and aliquots (5 ml) were removed at 12, 24, 36, and 48 h for RNA. The aliquots were centrifuged as above to pellet the cells. A thin smear of the cells was Giemsa-stained for microscopic examination of the stages. The cells were lysed with 1.5 ml guanidine isothiocyanate solution (RNagents, ProMega). The lysates were processed for RNA as described above. Equal amounts of total RNA (10 mg) were denatured and bound onto a nylon membrane by filtration through a dot blot manifold as described (Ausubel et al. 1997). The DNA fragment containing the central region of the P. falciparum GMP synthase gene (1 kb) was radiolabeled using random primers (Life Technologies) and hybridised to the dot blot with 5X SSC/0.5% SDS at 658C overnight. Excess probe was removed by rinsing the blot several times with 4X SSC/0.5% SDS at 378C for 15 min. The final stringent wash was 0.1X SSC/0.1% SDS at 658C for 30 min. Dots were excised and quantitated by Cerenkov counting on a MicroBeta counter (Wallac, EG&G Ltd., Crownhill, Milton Keynes). Growth inhibition assay. Psicofuranine (a kind gift of Upjohn Pharmaceuticals, Inc., Kalamazoo, MI) was dissolved at 100 mM in dimethyl sulfoxide. The growth inhibition assay was conducted as described (McConkey et al. 1994). Briefly, the parasite culture was adjusted to 0.25% parasitemia, 2.5% hematocrit, and 180-ml aliquots placed in wells of a microtitre dish. Dilutions of psicofuranine (20 ml) were added to the cultures in the microtitre plate, in triplicate. The medium was replaced with fresh dilutions of inhibitor at 48 h. After a further 48 h, thin smears were taken, Giemsa-stained, and counted microscopically. The experiment was repeat thrice. Each point is an average of triplicates. Data analysis. The DNA sequence and deduced amino acid sequence were compared to the GenBank database using the BLASTX and BLASTP algorithms at European Molecular Biology Laboratory (http://www.ebi2.ac.uk). Multiple alignment of GMP synthases and the glutamine amidotransferase domains was performed with CLUSTALV in the DNAStar package (Lasergene Software, Madison, WI).

RESULTS Isolation of a gene encoding P. falciparum GMP synthase. A degenerate oligonucleotide based on a conserved

25 region in GMP synthases from bacteria, archaebacteria, fungi, and mammals was used to screen a P. falciparum library. A plasmid clone was isolated containing an insert whose open-reading frame exhibited identity to GMP synthases to other organisms. Also, the sequence of the oligonucleotide used to screen the library was included in the openreading frame (Fig. 2). The remainder of the gene was isolated utilising the sequence information from the cloned fragment. The 38 end of the gene (349 nucleotides) is also confirmed by the presence of an identical sequence in the raw sequence data produced by the P. falciparum Sequencing Group at the Sanger Centre and can be obtained from http:// www.sanger.ac.uk/cgi-bin/Protozoa/Blast Server#M3I4f6. r1t (accessed Nov. 8, 1999). An ORF of 555 amino acids was deduced from the sequence. There is no evidence for introns in this gene based on PCR and RT-PCR from total RNA of fragments throughout the gene (data not shown). The sequence aligns well with GMP synthases from other organisms (Fig. 2). A BLASTX search of translated GenBank and SwissProt databanks with the P. falciparum sequence found the 10 most similar sequences to be GMP synthases with the highest probability scores for bacterial and yeast GMP synthases (P values of 2.6 3 102165 to 2.1 3 102150). The P. falciparum sequence shares 35–40% identity with bacterial and yeast GMP synthases and 22– 25% identity with Caenorhabditus elegans and Dictyostelium discoideum GMP synthase sequences based on multiple alignment of sequences (data not shown). The least identity of the comparisons between bacterial, yeast, and eukaryotic GMP synthase sequences is between P. falciparum and the human sequence (19%). GMP synthase domains encoded in the P. falciparum gene. GMP synthases have a modular design containing separate domains for glutamine-binding and synthase activity (Zalkin and Smith 1998). The deduced P. falciparum GMP synthase was examined to determine whether the primary structure and conserved motifs are conserved in this protozoan GMP synthase. The conserved glutamine-binding module is responsible for the transfer of an amido group from glutamine to the substrate. The second module, “synthase domain,” present in GMP synthases, was identified by structural studies and includes a dinucleotide-binding motif conserved in a family of ATP pyrophosphatases that catalyses changes in nucleotides through a dinucleotide intermediate (Tesmer et al. 1996). The glutamine-binding module in the amino-terminal fragment of the sequence encompasses three conserved regions of 8 to 12 amino acids separated by nonconserved regions. The P. falciparum GMP synthase sequence was

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FIG. 2. Comparison of P. falciparum GMP synthase with human, yeast, and bacterial GMP synthases. The deduced amino acid sequence of the P. falciparum gene is aligned with Homo sapiens, the yeast Saccharomyces cerevisiae, and Escherichia coli. The residues matching the P. falciparum sequence are denoted by a dot.

examined to determine whether it contained the conserved motifs. The glutamine amidotransferase domain (as defined by Van Lookeren Campagne et al. 1991) from yeast, human, slime mold, bacteria, and archaebacteria GMP synthase was aligned with P. falciparum (Fig. 3). The P. falciparum enzyme contains 16 of the 17 amino acids absolutely conserved in GMP synthases (denoted by * in Fig. 3). This includes the consensus sequences SGGP and P(I/V)(L/F)G(I/V)C(Y/L)G(M/H)Q (with X as any amino acid). The nonconserved amino acid is a tyrosine substituting for phenylalanine in the consensus sequence GXQFHPEV. The spacing between the first two consensus regions is conserved among P. falciparum and other GMP synthases (11–18 amino acids). In contrast, the spacing between the second and third consensus region is significantly greater for P. falciparum (105 amino acids) than other organisms (range 5 73–85 amino acids). This is due to an insertion in the coding sequence of 20 amino acids (Fig. 2).

GMP synthase is a member of Class I glutamine amidotransferases. Other glutamine amidotransferases include carbamoyl phosphate synthases, CTP synthases, anthranilate synthases, para-aminobenzoate synthases, formyl-glycinamide ribonucleotide synthases, and imidazole glycerol phosphate synthases (Zalkin 1993). The glutamine amidotransferase domain of 15 representative glutamine amidotransferases was aligned with P. falciparum GMP synthase (Fig. 3), including sequences in the databanks for carbamoyl phosphate synthetase, CTP synthetase, anthranilate synthase, and para-aminobenzoate synthase derived from bacteria, protozoa, yeast, and humans. Included are two P. falciparum published glutamine amidotransferases; carbamoyl phosphate synthetase and CTP synthetase. The only other published protozoal glutamine amidotransferase is trypanosome carbamoyl phosphate synthase. P. falciparum GMP synthase contains the nine amino acids conserved among 59 Class I glutamine amidotransferases (Tesmer et al.) and underlined

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FIG. 3. Comparison of the glutamine amidotransferase domain of P. falciparum GMP synthase with other glutamine amidotransferases. The glutamine amidotransferase domain from Class I glutamine amidotransferases of Saccharomyces cerevisiae, Home sapiens, Dictyostelium discoideum, Escherichia coli, Methanococcus jannaschii, and Trypanosoma cruzi are aligned with P. falciparum. The amino acids conserved among GMP synthases are denoted by * above the sequences. The consensus sequence for the glutamine amidotransferases is shown below the sequences with the absolutely conserved residues underlined.

residues in the consensus sequence in Fig. 3). The tyrosine substitution found in the P. falciparum GMP synthase sequence is also found in human and yeast CTP synthetase. The spacing between regions is also conserved among glutamine amidotransferases. Glutamine amidotransferases have a similar spacing between the first two regions (with the exception of yeast para-aminobenzoate synthase) whereas the spacing between the second and third regions appears to correlate with enzyme type. Of interest, the two exceptions are P. falciparum GMP synthase and P. falciparum CTP synthetase which contain insertions in this part of the molecule. The synthase module of GMP synthases contains a signature nucleotide-binding motif, dimerisation segment, and phosphate-binding segment. The P. falciparum GMP synthase sequence contains the signature dinucleotide-binding motif (SGGVDS), or P-loop, conserved in ATP pyrophosphatases (amino acid residue 262 in Fig. 2). The valine is not strictly conserved in P-loops and in P. falciparum is substituted with an isoleucine. The peptide for dimerisation

(PFPGPG) is also conserved in the P. falciparum GMP synthase sequence. It is located at the junction of the glutaminebinding and synthase modules (residues 432–437 in Fig. 2) as found in other organisms. Dimerisation is essential for activity of prokaryotic GMP synthase, although its function in eukaryotes is unknown. Finally the carboxy-terminal amino acids (including adjacent proline residues that are conserved among GMP synthases and are likely to bind the phosphate of the substrate) are conserved in P. falciparum. Hence, the P. falciparum sequence contains the motifs conserved in GMP synthases. The overall structure is likely to be similar to other GMP synthases which may promote stuctural alignments of P. falciparum with the crystal structure of the bacterial homologue (Tesmer et al. 1996), although amino acid differences exist throughout the sequence. Developmental expression of the gene encoding GMP synthase. As GMP is necessary for DNA and RNA synthesis, the levels of expression may correlate with the amount of DNA and RNA synthesis at different points in the life

28 cycle, although GMP is also involved in other cellular processes (e.g., signal transduction and amino acid chain elongation) in other organisms (Van Lookeren Campagne et al. 1991). The expression of the gene encoding GMP synthase was analysed through a cycle of P. falciparum development in red blood cells. Following invasion of the red blood cell, ring-stage parasites synthesise low levels of RNA. As the parasite increases in size and progresses through the cycle during the trophozoite stage, the levels of RNA synthesis increase with levels peaking in the late trophozoite stage. The point of DNA synthesis (S phase) is the mature trophozoite stage (Gritzmacher and Reese 1984). Levels of P. falciparum mRNA encoding GMP synthase were quantified through a complete cycle of asexual development in red blood cells. Synchronised parasites were examined at 12-h time points through the 48-h cycle. The stages of the parasites and the level of GMP synthase mRNA relative to total RNA were assessed. The levels of transcripts encoding GMP synthase are greater in the time points with a major proportion of mature trophozoites (t) compared to mostly ring-stage trophozoites (r) (Fig. 4). The peak of

GLENN A. McCONKEY

expression coincides with the majority of parasites in the mature trophozoite stage. Levels are lower in the time points with the principal proportion of parasites in schizogony (s), the parasite’s M phase, and the lowest detected levels of transcripts in the time point containing mostly mature schizonts of newly segregated parasites and ring-stage trophozoites. Expression of the gene encoding GMP synthase coincides with the points in the life cycle with increased DNA and RNA synthesis. The levels of transcripts encoding GMP synthase increase during the trophozoite stage and peak during the late trophozoite stage. The lowest levels were detected in the time points enriched in schizonts and ring stages, correlating with observations of DNA and RNA synthesis. The level of expression of GMP synthase correlates well with its likely role in the generation of precursors for DNA and RNA synthesis in P. falciparum. Inhibition of parasite growth with a GMP synthase inhibitor. The identification of a gene containing an ORF homologous to GMP synthase and its expression suggest that P. falciparum is synthesising GMP from hypoxanthine via

FIG. 4. Developmental expression of P. falciparum GMP synthase. The relative levels of GMP synthase mRNA in synchronised cultures is plotted versus time points. The distribution of stages of parasites at the time points is plotted as bars (r, ring; t, trophozoite; s, schizont).

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XMP. This is also supported by the sensitivity of P. falciparum to bredinin and mycophenolic acid, which inhibit the enzyme preceding GMP synthase in the salvage pathway (Webster and Whaun 1982). Psicofuranine specifically inhibits bacterial GMP synthase as demonstrated by isolation of psicofuranine-resistant Escherichia coli mutants with mutations in the gene encoding GMP synthase and inhibition of bacterial growth (Fukuyama 1966; Udaka and Moyed 1963). The sensitivity of P. falciparum to psicofuranine inhibition of GMP synthase was assessed. P. falciparum was treated with different doses of psicofuranine. Psicofuranine exhibited a dose-dependent inhibition of P. falciparum growth with an IC50 of 3 3 1024 M (Fig. 5). The effect of 6-thioguanine on growth is included as a control. The psicofuranine inhibitory concentration is similar to that of E. coli (Fukuyama 1966). The inhibition by psicofuranine was antagonised by the addition of exogenous guanine (99.9%) supporting the specificity of psicofuranine for GMP synthase (data not shown). Hence, GMP appears to be synthesised via GMP synthase in P. falciparum. These

results also support the hypothesis that GMP synthase is a viable target for development of antimalarial chemotherapy.

DISCUSSION

This paper presents the first demonstration of a GMP synthase in Plasmodium based on isolation of a gene whose deduced amino acid sequence has significant identity with GMP synthases from other organisms (22–40%), expression of the gene during points in the life cycle of nucleic acid synthesis, and inhibition of parasite growth with a GMP synthase inhibitor. These findings support this point of purine salvage for antimalarial chemotherapy development. The sequence information may be exploited for development of specific inhibitors and for identification of motifs in enzyme families to search the sequences arising in the Malaria Genome Project.

FIG. 5. Effect of a GMP synthase inhibitor on growth of P. falciparum. The percentage inhibition of growth of psicofuranine-treated cultures, ●, (expressed as a percentage of untreated cultures) is plotted vs the concentration of inhibitor. 6-Thioguanine treatment is included as a control, n.

30 The role of P. falciparum GMP synthase in supplying GMP remains of interest. In this study, increased expression of the gene encoding GMP synthase correlated with the points in the life cycle with high levels of DNA and RNA synthesis probably because nucleic acid synthesis represents the principal need for GMP. If this is the sole point of GMP requirement, the treatment of P. falciparum with a GMP synthase inhibitor might inhibit growth at a specific stage, analogous to treatment with aphidicolin (Inselburg and Banyal 1984). Treated parasites were arrested at all stages in development (unpublished observations), suggesting that GMP synthesis is required in several stages in the life cycle. Indeed, transcripts of the GMP synthase gene were observed in all samples from points through the life cycle (Fig. 4). GMP may be required for signal transduction or other cellular processes (e.g., translational elongation) as in other cells. Increased levels of GMP synthase and IMPDH have been observed in several human cancer cell lines (Weber 1983). Inhibitors of GMP synthesis such as tiazofurin and mycophenolic acid induce tumor cell differentiation (Hatse et al. 1999). A similar increase in differentiated forms (gametocytes) of P. falciparum was not observed in our experiments possibly due to the lack of de novo purine synthesis as a source of purines (data not shown). A decrease in GMP synthesis has been correlated with differentiation of the slime mold D. discoideum. The level of GMP synthase mRNA decreases with differentiation of D. discoideum (van Lookeren Campagne et al. 1991). A similar diminishing level of GMP synthase mRNA was observed with P. falciparum in differentiating parasites (i.e., gametocytes) (preliminary data not shown). The role of GMP synthesis via GMP synthase versus salvage of guanine via HGPRT in P. falciparum during infection will be of great interest in development of chemotherapy. In P. falciparum, GMP can be supplied by guanine salvage utilising HGPRT or synthesis from hypoxanthine, xanthine, inosine, or adenosine via a series of enzymes terminating with GMP synthase. P. falciparum lacks nucleoside kinase for salvage of guanosine (Reyes et al. 1982). Therefore GMP is only synthesised through GMP synthase or salvage of guanine by HGPRT. The significance of guanine salvage is questionable due to the scarcity of guanine in erythrocytes (Reyes et al. 1982). In humans, cells contain enzymes for de novo purine synthesis and hypoxanthine–guanine– phosphoribosyltransferase (HPRT) for guanine salvage, yet HPRT deficiency has pathologic consequences (i.e. Lesch– Nyhan syndrome, gout) as observed in HPRT genetic disorders (Hershfield and Seegmiller 1976). Therefore any

GLENN A. McCONKEY

antimalarial chemotherapy directed towards phosphoribosyltransferases must be very selective for the parasite homologue. In contrast, interruption of GMP synthesis through IMPDH and GMP synthase is considered a valid target for development of cancer chemotherapy (Hatse et al. 1999). Hence, combination therapy consisting of a potent GMP synthase inhibitor and a highly selective HGPRT inhibitor may prove to be the most effective for antimalarial chemotherapy. Conserved elements in glutamine amidotransferases and ATP pyrophosphatases identified in the P. falciparum GMP synthase may allow identification of other members of these enzyme classes in P. falciparum as the genome is sequenced. These include enzymes in biochemical pathways not yet described in Plasmodium. The conserved motifs in the three P. falciparum glutamine amidotransferases can be used to screen sequence data from the P. falciparum genome project GMP (http://www.sanger.ac.uk/Projects/P falciparum/). synthase is the first member of the ATP pyrophosphatase family which has been identified in P. falciparum. Other members of the ATP pyrophosphatase enzyme family, such as asparagine synthetase and NAD synthetase, may also be identified based on conserved motifs. This approach may identify enzymes involved in purine, pyrimidine, tryptophan, arginine, histidine, NAD, and folic acid biosynthesis. This is particularly relevant as several of these pathways (i.e., tryptophan, arginine, and histidine synthesis) have not been defined in Plasmodium. The conservation of the GMP synthase motifs in the deduced P. falciparum sequence permits the determination of sites for potential inhibitor design. The primary structure exhibits the conserved glutamine, NTP, and phosphate binding sites; organised in an amino to carboxy order (Figs. 2 and 3). The high level of conservation of motifs allows the P. falciparum GMP synthase sequence to be superimposed on the published crystal structure from E. coli (Tesmer et al. 1996). Based on the alignment of GMP synthases (Fig. 2), the conserved residues were mapped to their position on the E. coli GMP synthase structure (Fig. 6). If we examine the residues conserved between E. coli and P. falciparum but differing from human in Fig. 2, then several differences on the model of the structure can be observed. For example, residues Ser475, Gln476, and Ile552 (P. falciparum sequence numbering) are conserved among E. coli and P. falciparum but differ from human. Their positions in the structure map to the edge of the pocket that binds the phosphate of the substrate XMP (Fig. 6). Additionally, there is an insertion in the human GMP synthase between residues Ser475 and Gln476 in the alignment. Development of inhibitors interacting with these residues may prove specific for the parasite

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FIG. 6. The conserved residues between P. falciparum and E. coli GMP synthases superimposed on the E. coli crystal structure. A ribbon drawing of the crystal structure of E. coli GMP synthase is shown (Tesmer et al. 1996). Identical amino acid residues, based on the alignment of GMP synthases in Fig. 2, are gray; differences are black. The three residues labeled are described in the text (with E. coli numbering). The drawing was performed using a computer algorithm written by S. Green (School of Chemistry, University of Leeds) and RasMol 2.6 (R. Sayle).

enzyme. The similarity between the bacterial and P. falciparum GMP synthase combined with the low overall similarity between the human and P. falciparum homologues (19%) is encouraging for development of compounds which specifically bind the parasite GMP synthase. With the description of the sequence and function of GMP synthase in P. falciparum, future characterisation of its structure should identify unique features for rational design of inhibitors to complement inhibitors developed to other enzymes in purine salvage.

ACKNOWLEDGMENTS I thank Martin Looker and Helen Cannon for technical support and S. Green for modelling the structure. Preliminary experiments were performed with T. F. McCutchan at Laboratory of Parasitic Diseases, N.I.H., U.S.A.

REFERENCES Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. 1997. Analysis of RNA by Northern

and slot blot hybridization. In “Current Protocols in Molecular Biology”, p. 4.9. Wiley, Boston. Berman, P. A., Human, L. and Freese, J. A. 1991. Xanthine oxidase inhibits growth of Plasmodium falciparum in human erythrocytes in vitro. J. Clin. Invest. 88, 1848–1855. Craig, S. P., and Eakin, A. E. 1997. Purine salvage enzymes of parasites as targets for structure-based inhibitor design. Parasitol. Today 13, 238–241. Fukuyama, T. T. 1966. Formation of an adenylyl xanthosine monophosphate intermediate by xanthosine 58-phosphate aminase and its inhibition by psicofuranine. J. Biol. Chem. 241, 4745–4749. Gritzmacher, C. A., and Reese, R. T. 1984. Protein and nucleic acid synthesis during synchronized growth of Plasmodium falciparum. J. Bacteriol. 160, 1165–1167. Hatse, S., De Clercq, E., and Balzarini, J. 1999. Role of antimetabolites of purine and pyrimidine nucleotide metabolism in tumor cell differentiation. Biochem. Pharmacol. 58, 539–555. Hershfield, M. S., and Seegmiller, J. E. 1976. Gout and the regulation of purine biosynthesis. Horiz. Biochem. Biophys. 2, 134–62. Inselburg, J. and Banyal, H. S. 1984. Plasmodium falciparum: Synchronization of asexual development with aphidicolin, a DNA synthesis inhibitor. Exp. Parasitol. 57, 48–54. Li, J., Wirtz, R. A., McConkey, G. A., Sattabongkot, J., and McCutchan, T. F. 1994. Transition of Plasmodium vivax ribosome types corresponds to sporozoite differentiation in the mosquito. Mol. Biochem. Parasitol. 65, 283–289. McConkey, G. A., Ittarat, I., Meshnick, S. R., and McCutchan,

32 T. F. 1994. Auxotrophs of Plasmodium falciparum dependent on p-aminobenzoic acid for growth. Proc. Natl. Acad. Sci. USA 91, 4244–4248. Queen, S. A., Jagt, D. L., and Reyes, P. 1990. In vitro susceptibilities of Plasmodium falciparum to compounds which inhibit nucleotide metabolism. Antimicrob. Agents. Chemother. 34, 1393–1398. Queen, S. A., Vander Jagt, D. and Reyes, P. 1988. Properties and substrate specificity of a purine phosphoribosyltransferase from the human malaria parasite, Plasmodium falciparum. Mol. Biochem. Parasitol. 30, 123–133. Reyes, P., Rathod, P. K., Sanchez, D. J., Mrema, J. E., Rieckmann, K. H. and Heidrich, H. G. 1982. Enzymes of purine and pyrimidine metabolism from the human malaria parasite, Plasmodium falciparum. Mol. Biochem. Parasitol. 5, 275–290. Rogers, M. J., McConkey, G. A., Li, J. and McCutchan, T. F. 1995. The ribosomal DNA loci in Plasmodium falciparum accumulate mutations independently. J. Mol. Biol. 254, 881–891. Schumacher, M. A., Carter, D., Ross D. S., Ullman, B., and Brennan, R. G. 1996. Crystal structures of Toxoplasma gondii HGXPRTase reveal the catalytic role of a long flexible loop. Nat. Struct. Biol. 3, 881–887. Shahabuddin, M., and Scaife, J. 1990. The gene for hypoxanthine phosphoribosyl transferase of Plasmodium falciparum complements a bacterial HPT mutation. Mol Biochem. Parasitol. 41, 281–288. Shi, W., Li, C. M., Tyler, P. C., Furneaux, R. H., Cahill, S. M., Girvin, M. E., Grubmeyer, C., Schramm, V. L., and Almo, S. C. 1999. The 2.0 A structure of malarial purine phosphoribosyltransferase in complex with a transition-state analogue inhibitor Biochemistry 38, 9872–80. Somoza, J. R., Chin, M. S., Focia, P. J., Wang, C. C., and Fletterick, R. J. 1996. Crystal structure of the hypoxanthine–guanine–xanthine

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phosphoribosyltransferase from the protozoan parasite Tritrichomonas foetus. Biochemistry 35, 7032–7040. Tesmer, J. J., Klem, T. J., Deras, M. L., Davisson, V. J., and Smith, J. L. 1996. The crystal structure of GMP synthetase reveals a novel catalytic triad and is a structural paradigm for two enzyme families. Nat. Struct. Biol. 3, 74–86. Udaka, S., and Moyed, H. S. 1963. Inhibition of parental and mutant xanthosine 58-phosphate aminases by psicofuranine. J. Biol. Chem. 238, 2797–2803. Ullman, B., and Carter, D. 1995. Hypoxanthine–guanine phosphoribosyltransferase as a therapeutic target in protozoal infections. Infect Agents Dis. 4, 29–40. Van Lookeren Campagne, M. M., Franke, J., and Kessin, R. H. 1991. Functional cloning of a Dictyostelium discoideum cDNA encoding GMP synthetase. J. Biol. Chem. 266, 16448–16452. Weber, G. 1983. Enzymes of purine metabolism in cancer. Clin. Biochem. 16, 57–63. Webster, H. K. & Whaun, J. M. 1982. Antimalarial properties of bredinin. Prediction based on identification of differences in human host–parasite purine metabolism. J. Clin. Invest. 70, 461–469. Whitby, F. G., Luecke, H., Kuhn, P., Somoza, J. R., Huete-Perez, J. A., Phillips, J. D., Hill, C. P., Fletterick, R. J., and Wang, C. C. 1997. Crystal structure of Tritrichomonas foetus inosine-58-monophosphate dehydrogenase and the enzyme–product complex. Biochemistry 36, 10666–10674. Zalkin, H. 1993. The amidotransferases. Adv. Enzymol. Relat. Areas Mol. Biol. 66, 203–309. Zalkin, H. & Smith, J. L. 1998. Enzymes utilizing glutamine as an amide donor. Adv. Enzymol. Relat. Areas Mol. Biol. 72, 87–144. Received 2 June 1999; accepted with revision 24 September, 1999