ELSEVIER Molecular and Biochemical Parasitology 74 (1995) 99- 103

MOLECULAR

ECHEMICAL PARASITOLOGY

Short Communication

Cloning and expression of the adenine phosphoribosyltransferase gene from Leishmania donovani

Thomas Allen, Ho-Yon Hwang, Keith Wilson, Sheri Hanson, Armando Jardim, Buddy Ullman*

Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, OR 97201-3098, USA

Received I9 July 1995; revision received ; accepted 20 July 1995

Keywords: Adenine phosphoribosyltransferase; Leishmania; Purine; Drug development; Molecular cloning; Expression

As a consequence of their purine auxotrophy, each genus of protozoan parasite expresses a uni- que complement of purine salvage enzymes that permits them to scavenge purines from their host milieu [l]. Leishmuniu express three biochemically distinguishable phosphoribosyltransferase (PRT) enzymes that are capable of converting purine bases to the nucleotide level, an adenine PRT (APRT), a hypoxanthine-guanine PRT (HGPRT), and a xanthine PRT [2]. The metabolic capacity of PRT enzymes to trap membrane permeable purine nucleobase analogs intracellularly as phosphory- lated nucleotide products has stimulated con-

Abbreviations; PRT phosphoribosyltransferase; APRT. adenine phosphoribosyltransferase; aprt, adenine phos- phoribosyltransferase gene; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; PRPP, phosphoribosylpyrophos- phate; SDS, sodium dodecylsulfate; TMD. Tris-MgCI,- dithiothreitol; PCR. polymerase chain reaction.

Note: Nucleotide sequence information reported in this paper has been submitted to the GenBankTM database with the accession number L25411.

*Corresponding author, Tel.: +I 503 4948437; Fax: +I 503 4948393; Internet: ullmanb@ohsu.edu.

siderable therapeutic interest with regard to a spectrum of parasitic diseases, since analogs of naturally occurring purine bases, termed subver- sive substrates, can function as prodrugs and enter the nucleotide pool with lethal consequences. This interest in PRT enzymes is bolstered by the ex- istence of a plethora of purine base analogs that have been developed by the pharmaceutical in- dustry and the facility by which novel purine base analogs can be chemically synthesized.

Although a number of HGPRT proteins from protozoan parasites have been characterized in detail [3], essentially no information is available on parasite APRT enzymes. The L. donovani APRT has been purified from promastigotes but in such limiting quantities to preclude subsequent analysis [4]. In order to generate large quantities of APRT for biochemical and structural investiga- tions and for evaluation of the enzyme as a poten- tial therapeutic target, the L. donovani uprt was isolated from a cosmid library by cross hybridiza- tion to a 242-bp fragment of the uprt that was amplified from reversed transcribed RNA using the polymerase chain reaction. Translation of the

01666851/95/%09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0 166-685 I (95)02475-S

100

L. donovani D. melanoraster E. A. thaliana s. cerwisiae limeal Hamster Mouse

L. donovani D. melanoraster E coli ti A. thaliana S. cerwisias E-

Bawter Mouse

L. donovani D. melanoaaster E. coli A. thallana S. cerwisiae H- Hamster Mouse

T. Allen et al. /Molecular and Biochemical Parasitology 74 (1995) 99-103

MPFKEvsPNSFLLDDsEALsQLLKKsYRwrsFJFs PRRVPRFADVssITESPETLKAIRD FLvQrwuksPAP--TEILQ 78 tB-PSLSAEDKL--------DYVKSKIGEY-PRF-PKRGILFRDIFGALTD WLLVDEIRES-A-PIIVG 67 MI-ATa---L-- ------EYLKNSTKSI-QDY-PKFGILFRDVTSLLKLlPKAYALSIDLLVRRYKRA----GI~ 62 MA-TEDVQDPRI---- ----AKIASSIRVI-PDF-PKFGIMFQDITTIUDTEAFKDTIALFVDRYKDK----GIsVVAG 1 MS----ISESYA--------KEIKTAFReQF-TDF-PIRGEQFEDFLPII~VIiT~AKRKKTBLEEICPAI(EICIDPIAO ~______~S~________QLyEcpuRsp-pDF_pT PGwFRD1SPVLKDPAsFRAA1 GLURBLWIBO-GRIDYIAG ~______SS~___-____QLV~~_~S_~ XVLFRDISPLLKDPASFRASIRL.LA~SLK~TRG-GKIDYIAG MS----_-SPU--------KIRVP-PDF_PIPGVL IsPLLKDPDSFRASIRLL.ASBTRS-GKIDYIAG * l ** . .

65 66 63 63 63

FDARGFLFGFMIAVELKI ~KRAGLLIRsEPIRYG~I~VVLIDDVLATWTAL~G LDsRGFLnuI~- sVIWUY---GSD~DLUTGGSLVAA TI-SKTYDLKY---GTLXJLRIRVD AXFGDK%WDDLLATGGTIKAT WMRGFIFGPPIALMG PC;I(VI-SEEYSLHY---~TI~~#iEIUIIlllDtIATOOILAM IEARGLLPGPSW~~~-SIrr LDsRGPLn;PsUqELGU;Ca~~~-~YSLE UsRopLFGpsLAQ ~~xRGKI.PGPTV-~ASWLEY---~IWMLE~DLLA~ LDsRGFLFGPs~IRKQGKL~Prv-SASYSLEY ---GKAKLEIQKDALR# ..**..* .* . .*. *. . . . . . . . . l ******** .

LQLVF.ASDAWVE-MVSILSIPFLKMMI HSTANSRYKDIKFiSLLSDDAL~PRVLSCGDVLAEHPS TF.LI ~p_G_______-L~

VKLIRRLGOEVADA-AFIISLFDLNK@UXW ------ITSYSLVPFFGS IRLLERVGVKnEC-ACVIELBTS------L--FVLVKsAA GDLIRQXARILEY-DFVLVLDSLEGRKKL---S------APIFSILES CEUQUAAEVLEC-VSLVELTSLIWEKLA-- P------VPFFSLLJJYE CEUGQLQAFWEC-VSLVELTSW _s______vppp-YE CDLLBELwvvu;r-VSLVELTSLKfXERLG--P------IPFFSLLEYLl *. . . . . . *. . . . .

150 143 136 141 142 139 139 139

237 163 183 183 181 180 180 180

Fig. 1. Alignment of the L. donovani, D. melanogaster, E. coli, A thaliana, S. cerevisiae, human, hamster and mouse APRTs. The amino acid sequences of the L. donovani, D. melanogaster, E. coli, A thaliana, S. cerevisiae, human, hamster and mouse APRTs were aligned with the CLUSTAL V program multisequence alignment program [I 11. Amino acids identities among the 8 APRT sequences are denoted with an asterisk beneath the aligned sequences, whereas like amino acids with similarity scores > 10, as calculated by the log-odds amino acid similarity matrix of Dayhoff (121, are indicated with a dot under the aligned sequences. The nine amino acids of the native protein that were obtained by amino acid sequencing are indicated by the box. Amino acid positions are designated on the right. The primary structure of the L. donovani APRT was inferred from the aprt nucleotide sequence. To isolate a homologous probe for the aprt, a 242-bp fragment corresponding to the 5’ terminus of the aprt mRNA was amplified by PCR (standard PCR reaction conditions: IO cycles at 94°C - 60 s, 50°C - 30 s, 72°C - 30 s; followed by 20 cycles at 94°C - 30 s, 50% - 30 s, 72°C - 30 s) using reverse transcribed RNA as a template, a nondegenerate sense primer, S’-CTCGGGATCCCAACGCTATATA- AGTATCAGTMCTGTACITTATTG-3 ‘, that encoded the L. donovani mini-exon [13], and a degenerate antisense primer, 5’- TcTGAA~CGT(G/C)AGGAA(G/C)(G/C)(T/A)G~(G/C)~G/C)(G/CM~ T)(G/C)A CCTC constructed to the NH*-terminal peptide sequence obtained from the native APRT that had been purified as described [4]. Positive clones were isolated and purified from a cosmid library using the 242-bp PCR fragment as a probe, and a 5.4-kb BamHI-XbaI fragment encompassing the entire coding portion of aprt was ligated into pBluescript KS+ and sequenced by the dideoxynucleotide sequencing method [141.

aprt gDNA sequence revealed an open reading than its counterpart in other organisms. Much of frame of 711 nucleotides (GenBank Accession No. the extra length of the protein can be attributed to L25411) encoding a polypeptide of 237 amino a COOH-terminal extension that exhibits no sig- acids with a molecular mass of 26.2 kDa and a nificant homology to any other protein in the pro- calculated p1 = 6.0 (Fig. 1). The predicted primary tein databases. The size of the L. donovani APRT structure of the L. donovani APRT protein encom- inferred from translation of the uprt sequence is passed the NHrterminal peptide sequence that supported by the electrophoretic migration of both was obtained from the native protein the native [4] and recombinant (Fig. 2) enzymes in (EVSPNSFLL) and is 54-57 amino acids longer SDS gels.

T. Allen et al. /Molecular and Biochemical Parasitology 74 (1995) 99-103 lOI

21.5 -

A B C

Fig. 2 Expression of aprt in E. coli and purification of the recombinant APRT. The L. abnovani aprt in the pBAce vector was transformed into S&446 E. coli [IO] and induced in low phosphate medium as described 1151. S&I46 cells transformed with pBAce alone served as a negative control. Recombinant L. abnovani APRT was purified in a single step on an AMP- agarose affinity column. Proteins were separated on SDS gels. Lane A: 10 pg of cell extract of .%&I46 E. coli transformed with pBAce alone; lane B; 10 pg of cell extract from S&l46 cells transformed with the aprt-pBAce vector; lane C; purified APRT. Molecular size markers are indicated.

Alignment of the L. donovani APRT with other APRT proteins revealed little conservation of pri- mary structure among the eight aligned APRT proteins from phylogenetically diverse organisms with only two regions of conspicuous homology (Fig. 1). The most conserved region of amino acid homology corresponds to amino acids 143- 154 of the L. donovuni APRT in which there are found six identical and six highly conserved residues. This stretch of 12 amino acids encompasses the bulk of the PRPP binding domain that is conserved

among most PRT family members [5]. A shorter region of homology, equivalent to amino acids 80-86 of the L. donovuni protein, is also present on all eight aligned APRTs. The function of this con- served motif is unclear. There are only nine other amino acids, none of which are contiguous within the primary structure, that are common to all APRT proteins indicating that primary structure conservation is not crucial for catalytic activity. Particularly noteworthy among these nine con- served residues is the Asp at position 44 of the L. donovuni APRT. This Asp could theoretically ac- count for the narrow nucleobase substrate specificity of the APRT enzymes by permitting the formation of hydrogen bond contacts between the enzyme and the exocyclic 6-NH,-group of the purine ring. Finally, it should be noted that the APRT protein, unlike the HGPRT proteins from T. brucei, T. cruzi and L. donovani [3], does not possess a COOH-terminal tripeptide that is consis- tent with the acceptable degeneracy of the topogenic signal that mediates protein transfer into the glycosome [6,7].

Southern blot analysis indicated that the uprt structural locus was not organized as a tandemly repeated array and was likely present as a single copy within the L. donovuni genome (data not shown). Northern blot analysis revealed a single 2.2-kb uprt transcript that was expressed to the same extent in wild type cells and previously isola- ted mutant strains of L. donovuni that were either 50 or 100% deficient in APRT activity [8].

The L. donovuni uprt was ligated into the pBAce bacterial expression vector [9] and transformed into APRT-deficient St&t46 E. coli [IO]. After uprt induction, APRT was the most abundant soluble protein in the S&446 cells transformed with the pBAce-uprt construct and was not found in bacte- ria transformed with the pBAce vector alone (Fig. 2). Induction of uprt expression also enabled the pBAce-uprr S@46 transformants to grow in semi- solid medium containing adenine as a source of adenylate nucleotides, while cells transformed with pBAce alone could not grow on the adenine plates (data not shown).

The recombinant L. donovuni APRT was purified to virtual homogeneity in a single step by

102 T. Allen et al. /Molecular and Biochemical Parasitology 74 (1995) 99-103

affinity chromatography with an AMP-agarose matrix and elution in buffer containing 1.0 mM PRPP (Fig. 2). The purified recombinant APRT, as expected, catalyzed the phosphoribosylation of adenine but did not recognize hypoxanthine, guanine or xanthine as substrates. A K,,, value of 3.9 f 2.3 PM was caluclated for adenine from a double reciprocal plot. Size exclusion high perfor- mance liquid chromatography using a silica-based gel filtration column indicated that the pure L. donovuni APRT protein migrated as a monomer in TMD buffer in the absence or presence of PRPP.

The capacity to overexpress the L. donovani aprt

in a genetic background lacking a functional bac- terial counterpart and to purify large and replenishable quantities of the enzyme to homogeneity is crucial for a thorough molecular, biochemical and structural dissection of the enzyme and a prerequisite for the authentication of APRT as a therapeutic target. The ability of the L. donovani aprt to complement the bacterial aprt

deficiency is particularly noteworthy, because site- directed mutagenesis can be implemented to estab- lish active site determinants on the protein that function in catalysis or govern substrate specilici- ty. Thus, one can genetically alter any residue within the protein by site-directed mutagenesis and select directly for functional alterations by com- plementation or by selection in cytotoxic sub- strate. Moreover, since APRT can function as a negative selectable marker, E. coli expressing aprt can be manipulated as a rapid and safe screen for potential antiparasitic agents that require APRT for conversion to the nucleotide level and cytotoxi- city. Finally, the abundant and replenishable amount of recombinant L. donovuni APRT avail- able provides the requisite reagent for a structure- based approach for the design or discovery of novel APRT substrate analogs or possibly in- hibitors that can serve as potential drugs for the treatment of leishmaniasis.

Acknowledgements

This work was supported by funds from grant AI-23682 from the National Institute of Allergy and Infectious Disease. Thomas Allen was a recip- ient of an N.L. Tartar Trust Fellowship from the

Medical Research Foundation of Oregon. Buddy Ullman is a Burroughs Wellcome Fund Scholar in Molecular Parasitology, and this work was sup- ported in part by a grant from The Burroughs Wellcome Fund.

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