Gene 209 (1998) 219���228 Cloning and heterologous expression of Entamoeba histolytica adenylate kinase and uridylate/cytidylate kinase Lidya B. Sanchez �� *, Miklos �� Muller �� The Rockefeller University, New York, NY 10021, USA Received 25 November 1997 accepted 5 January 1998 Received by W. Martin Abstract We have isolated two cDNA clones encoding Entamoeba histolytica nucleotide kinases, EhAK and EhUK, expressed them in E. coli and performed functional studies of the recombinant enzymes. Nucleotide sequence analysis showed that EhAK and EhUK genes exhibited the features characteristic of E. histolytica genes, such as transcripts with relatively short 5��� and 3��� untranslated flanking regions containing the conserved E. histolytica transcription promoter elements located 5��� to the initiation codon and a polyadenylation signal in the 3��� UTR, a distinctive codon usage bias for A or T in the third position and an AT bias greater than 75% in the flanking regions of the transcripts. At the protein level, both enzymes belong to the short variant nucleoside monophosphate (NMP) kinases, which lack a 29 amino acid LID region present in the long variant isoenzymes. EhAK was 30���38% identical to the members of the adenylate kinase (AK) family while EhUK was more similar (48���49% identity) to UMP/CMP kinases. Both enzymes used ATP as preferred phosphate-group donor but each one exhibited strict specificity for the acceptor NMP, EhAK for AMP and EhUK for the pyrimidine nucleoside monophosphates UMP and CMP. Biochemical characterization of the enzymes and phylogenetic reconstruction showed that EhUK is an authentic and well conserved member of the UMP/CMP kinase group while EhAK is the most divergent member known of the AK1 isoenzymes. �� 1998 Elsevier Science B.V. Keywords: Amitochondriate protist NMP kinase recombinant enzyme 1. Introduction nucleoside triphosphates to nucleoside monophosphates. Of these, adenylate kinase, AK, has been most extens- Studies on the energy metabolism of Entamoeba histo- ively studied in view of its role in maintaining homeosta- lytica, an amitochondriate parasite that resides in the sis of adenosine phosphates. Several isoforms of NMP human intestine, have shown distinctive metabolic fea- kinases have been identified, which diVer in size, sub- tures not observed in most eukaryotes. Its glycolysis has strate specificity and subcellular location. Based on their been studied in some detail (Reeves, 1984 Muller, �� molecular size these enzymes can be divided into short 1988), however, the mechanisms this organism uses to and long variant isoforms. These diVer in the absence maintain nucleotide pools within the cell are virtually or presence of a 30 amino acid stretch in the central unknown. part of the molecule, called the ������LID region������ (Fukami- Nucleoside monophosphate (NMP) kinases (EC Kobayashi et al., 1996 Diederichs and Schulz, 1991). 2.7.4.-) represent a family of ubiquitous enzymes that Short variant type enzymes comprise the adenylate catalyse the reversible phosphate group transfer from kinases AK1 (EC 2.7.4.3), which utilize preferentially AMP as phosphoryl-acceptor and are present in the * Corresponding author: Rockefeller University, 1230 York Avenue, cytosol of vertebrates and the uridylate/cytidylate New York, NY 10021, USA. Tel.: +1 212 327 8144 Fax: +1 212 327 (UMP/CMP) kinases (EC 2.3.4.14), with higher speci- 7974 e-mail: sanchel@rockvax.rockefeller.edu ficity for UMP and CMP. Long variant isoforms include Abbreviations: AK, adenylate kinase Ap 5 A, P1P5-di(adenosine-5���) the adenylate kinases AK2 from the mitochondrial pentaphosphate bp, base pair(s) DTNB, 5,5���-dithiobis(2-nitro- intermembrane space and cytosol, and the adenylate benzoic acid) kDa, kilodalton b-ME, b-mercaptoethanol NMP, kinases AK3 from the mitochondrial matrix. The latter nucleotide monophosphate ORF, open reading frame PCR, polymer- ase chain reaction UK, uridylate kinase. utilize GTP more eYciently than ATP (Nakazawa et al., 0378-1119/98/$19.00 �� 1998 Elsevier Science B.V. All rights reserved. PII S0378-1119 ( 98 ) 00053-5

220 L.B. Sanchez, �� M. Muller �� / Gene 209 (1998) 219���228 1990). Adenylate kinases from eubacteria and from Zea contained 1 mM MgCl 2 , 10 mM glucose, 0.5 mM NADP+, 3 U ml-1 hexokinase and 1 U ml-1 glucose- mays chloroplasts also belong to the long type. Although NMP kinases are among the most studied enzymes, 6-phosphate dehydrogenase in 100 mM Tris���HCl, pH 7.5. For kinetic studies varying concentrations of surprisingly few studies were performed on protists. The hydrogenosomal long type AK of the parabasalids ADP or its Mg complex (0.02, 0.04, 0.1, 0.2 and 0.5 mM) were used at several fixed concentrations of Trichomonas vaginalis and Tritrichomonas foetus have been purified (Declerck and Muller, �� 1987 Dinbergs and MgADP or ADP (0.05, 0.1 or 0.2 mM), respectively. In the reverse reaction, formation of ADP was measured Lindmark, 1990) and the sequence of the former estab- lished (Lange �� et al., 1994). An AK gene has been by a coupled assay using pyruvate kinase and lactate dehydrogenase. The standard mixture contained 0.5 mM sequenced from the diplomonad Giardia lamblia (Rozario and Muller, �� 1995) but the enzyme has not NMP, 0.5 mM NTP, 1 mM MgCl 2 , 0.5 mM phospho- enolpyruvate, 0.16 mM NADH, 10 U ml-1 pyruvate been characterized, although the lack of compartmenta- tion of the energy metabolism of the latter species kinase and 10 U ml-1 lactate dehydrogenase in 50 mM Tris���HCl, pH 7.5. For kinetic studies varying concen- suggests a cytosolic location of this enzyme, which belongs to the long type of mononucleotide kinases. trations (0.02, 0.04, 0.2 and 0.2 mM) of one nucleotide phosphate (ATP or NMP) were used while the second Kinetoplastids contain AK in the glycosome (McLaughlin, 1985) and likely also in other subcellular nucleotide phosphate (NMP or ATP, respectively) was kept constant (at 0.05, 0.1 or 0.2 mM). Concentrations compartments. A molecular characterization of these is still outstanding. of free nucleoside mono-, di- and triphosphates and of the corrresponding Mg-complexes were calculated using In the present study we have isolated and sequenced two E. histolytica genes encoding for an adenylate kinase the following stability constants: K Mg���AMP =70 M-1 K Mg���ADP =4000 M-1, K Mg���ATP =73 000 M-1 and and an uridylate/cytidylate kinase. Recombinant pro- teins were expressed in E. coli and characterized bio- K Mg���UMP =178 M-1. Reactions were initiated by the addition of the protein extract and the change in absor- chemically. We also analysed their phylogenetic relationship to other members of this protein family. bance at 340 nm was followed. One unit of activity was defined as mmole of product (NAD+ or NADPH) formed per min per mg of protein. Protein content was estimated by the method of 2. Materials and methods Lowry with bovine serum albumin as the standard. 2.1. Organisms 2.4. Cloning and sequence procedures Entamoeba histolytica, strain HM-1:IMSS (ATCC 30459), was provided by Dr D. Eichinger (New York Degenerate oligonucleotide primers, corresponding to two highly conserved regions of the adenylate kinase University, New York, NY ). Trophozoites were grown axenically at 37��C in TYI medium (Keister, 1983). molecule (sense: 5���CCNGGNNNNGGNAARGGNA- CNCA3��� coding for amino acid residues 15���22 and Escherichia coli strains M15 [pREP4] and XL1-Blue were maintained in LB medium. antisense: 5���GGNCGNGGRAANCCRTC3���, comple- mentary to sequence coding residues 90���95 in Fig. 2) were used in PCR with E. histolytica genomic DNA as 2.2. Cell homogenates the template. Reactions were performed for 30 cycles of 1 min denaturation at 94��C, 1 min annealing at 50��C Log-phase cultures were chilled on ice for 10 min, then centrifuged at 700g for 7 min. The sedimented cells and 2 min extension at 72��C. The amplified product, about 300 bp long, was purified by electrophoresis in were washed with phosphate-buVered saline (PBS) and resuspended in 250 mM sucrose containing 5 mg ml-1 low-melting agarose followed by gelase digestion (Epicentre Technologies) and random priming radiola- leupeptin and lysed by three cycles of freeze���thawing, followed by two 10 s sonication pulses on ice using a beled (Gibco BRL, Life Technologies System). This product was used to screen an E. histolytica cDNA Branson sonifier. Lysates were clarified by centrifugation at 100 000g for 60 min. High speed supernatants were library in lZAPII (provided by Dr E. Tannich, Bernhard Nocht Institute for Tropical Medicine, Hamburg, stored at -20��C or used immediately for protein purifi- cation or determination of enzymatic activity. Germany). The cDNA clones obtained contained either of two related but diVerent sequences. The complete genes were cloned with the use of the two distinct cDNA 2.3. Nucleotide kinase assays clones as probes, from an E. histolytica lZAPII genomic library in lZAPII (provided by Dr J. Samuelson, In the forward reaction, formation of ATP was fol- lowed by coupling the reaction to hexokinase and glu- Harvard School of Public Health, Boston, MA). Nucleotide sequences of the cDNA and the gDNA cose-6-phosphate dehydrogenase. The standard mixture