Báo cáo khoa học: Leishmania donovani phosphofructokinase Gene characterization, biochemical properties and structure-modelling studies doc

Leishmania donovani phosphofructokinase Gene characterization, biochemical properties and structure-modelling studies Claudia Lo ´ pez 1,2 , Nathalie Chevalier 2 ,Ve ´ ronique Hannaert 2 , Daniel J. Rigden 3 , Paul A. M. Michels 2 and Jose Luis Ramirez 1,4 1 Instituto de Biologı ´ a Experimental, Universidad Central de Venezuela, Caracas, Venezuela; 2 Research Unit for Tropical Diseases, Christian de Duve Institute of Cellular Pathology and Laboratory of Biochemistry, Universite ´ Catholique de Louvain, Brussels, Belgium; 3 CENARGEN/EMBRAPA, Brası ´ lia, Brazil; 4 Instituto de Estudios Avanzados-Ministerio de Ciencia y Tecnologı ´ a, Caracas, Venezuela The characterization of the gene encoding Leishmania donovani phosphofructokinase (PFK) and the biochemical properties of the expressed enzyme are reported. L. donovani has a single PFK gene copy per haploid genome that encodes a polypeptide with a deduced molecular mass of 53 988 and a pI of 9.26. The predicted amino acid sequence contains a C-terminal tripeptide that conforms to an established signal for glycosome targeting. L. donovani PFK showed most sequence similarity to inorganic pyrophosphate (PP i )- dependent PFKs, despite being ATP-dependent. It thereby resembles PFKs from other Kinetoplastida such as Trypanosoma brucei, Trypanoplasma borreli (characterized in this study), and a PFK found in Entamoeba histolytica.It exhibited hyperbolic kinetics with respect to ATP whereas the binding of the other substrate, fructose 6-phosphate, showed slight positive cooperativity. PP i ,evenathighcon- centrations, did not have any effect. AMP acted as an acti- vator of PFK, shifting its kinetics for fructose 6-phosphate from slightly sigmoid to hyperbolic, and increasing consid- erably the affinity for this substrate, whereas GDP did not have any effect. Modelling studies and site-directed muta- genesis were employed to shed light on the structural basis for the AMP effector specificity and on ATP/PP i specificity among PFKs. Keywords: phosphofructokinase; Kinetoplastida; allosteric regulation; mutagenesis; structure modelling. Glycolysis is a central metabolic pathway in all organisms. A key enzyme of this pathway is 6-phospho-1-fructokinase (PFK) or ATP: D -fructose-6-phosphate 1-phosphotransfer- ase. The activity of this enzyme is, in almost all organisms, regulated by multiple mechanisms. Whereas most glycolytic enzymes have been remarkably conserved during evolution, considerable sequence variability is found among PFKs of different taxonomic groups [1]. In Kinetoplastida, a taxonomic order of protozoan organisms that includes important pathogens (species of Trypanosoma, Leishmania, Phytomonas)toman,animals and plants, the first seven enzymes of the glycolytic pathway are confined to an organelle called the glycosome [2,3]. PFKs can be divided into ATP-dependent and PP i -dependent enzymes; the former use ATP as phospho donor in areaction that is essentially irreversible under physiological conditions, whereas the latter use PP i in a reversible reaction that can be near equilibrium in vivo [4]. We have previously reported that Trypanosoma brucei PFK, despite being an ATP-dependent enzyme, has an amino-acid sequence typical of PP i -PFKs [5]. Furthermore, the activity of the T. brucei enzyme appears only regulated to a limited extent: effectors that modulate PFK activity in other organisms (vertebrates, yeast) such as ATP, citrate, fructose 2,6-bisphosphate, ADP and P i , have no effect on the Trypanosoma enzyme [6,7]. Only activation by AMP was observed. We hypothesized that an ancestor of the trypanosomes must have possessed a PP i -dependent PFK that changed its specificity for phospho donor from PP i to ATP during evolution [5]. The many structural differences between the active site of the two classes of PFKs, and the striking differences in ligand-binding properties between the human and parasite enzymes suggest great potential for structure- based design of drugs [5,8]. For comparative purposes we decided to study the PFK of Leishmania donovani another kinetoplastid organism that, contrary to the bloodstream-dwelling T.brucei, lives as an intracellular parasite in humans. The results of this work are presented in this paper, together with a report of the cloning and analysis of the PFK gene of the fish parasite Trypanoplasma borreli (a representative of the Bodonina, a different sub-order of the Kinetoplastida). Correspondence to J. R. Ramirez, Instituto de Biologı ´ aExperimental– UCV, Calle Suapure, Colinas de Bello Monte, Caracas, Venezuela, Caracas 1041-A, Venezuela. Fax: + 58 221 962 1120, Tel.: + 58 221 751 0111, E-mail: jramirez@reacciun.ve Abbreviations: PFK, 6-phosphofructokinase; ATP- PFK, ATP-dependent phosphofructokinase; PP i -PFK, PP i -dependent phosphofructokinase; PTS, peroxisome-targeting signal. Enzymes: 6-phosphofructokinase (EC 2.7.1.11); pyrophosphate– fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90). Note: The novel nucleotide sequences reported in this paper have been deposited in the EMBL, GenBank and DDBJ databases and are available under the accession numbers AY029213 (L. donovani PFK) and AJ310928 (T. borreli PFK). (Received 11 June 2002, accepted 1 July 2002) Eur. J. Biochem. 269, 3978–3989 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03086.x MATERIALS AND METHODS Cloning and characterization of the L. donovani and T. borreli PFK genes A genomic library of L. donovani (kindly donated by T. deVos, Seattle Biomedical Research Institute, WA, USA) constructed in Supercos7 vector was screened with a 500 bp fragment of the PFK gene of T. brucei as a probe [5]. One positive colony was chosen and the bacteria were grown for purification of the recombinant cosmid DNA. Southern analysis using the T. brucei probe showed hybridization with a 500 bp Sau3AI fragment and a 12 kb NotI fragment. The Sau3AI fragment was cloned in pBluescript II KS – (Stratagene) and subsequently sequenced. The DNA sequence obtained had 55% identity with the corresponding portion of the T.bruceiPFK gene. This sequence was used as a probe, and to design primers for further experiments. The NotI fragment was gel purified anddigestedwithEcoRI, and a 6.5 kb EcoRI fragment recognized by the Sau3AI probe was subcloned in pBlue- script II KS – (PFK6.5-PBSKS). Finally, from the recom- binant PFK6.5-PBSKS, a 2.2 kb EcoRI–PvuII fragment containing the entire L. donovani PFK gene (Fig. 1) was subcloned in pBluescript II KS – (PFK2.2-PBSKS) and sequenced. DNA sequences of both strands of recombinant plasmid inserts were determined by using the T7 DNA polymerase kit (Amersham Pharmacia Biotech) and [ 35 S]dATP (NEN Life Science Products), or with the Thermo Sequenase fluorescent labelled primer cycle sequencing kit (Amersham Pharmacia Biotech), and LI-COR automated DNA sequence equipment. A genomic library of T. borreli constructed in kGEM11 (Promega) [9] was screened with a probe consisting of the entire T. brucei PFK gene [5]. The probe was hybridized under moderately stringent conditions: 3 · NaCl/Cit/0.1% SDS, in the presence of 10% dextran sulphate, at 60 °C (1 · NaCl/Cit ¼ 150 m M NaCl/15 m M sodium citrate, pH 7.0). Post-hybridization washes were carried out at 60 °Cfor2hwith5· NaCl/Cit/0.1% SDS, followed for 1h with 3· NaCl/Cit/0.1% SDS. Ten positive recom- binants were purified and rescreened. High-titre phage lysates were prepared and DNA was purified from phages as described previously [10]. From one recombi- nant phage a 4 kb EcoRI fragment recognized by the T. brucei probe was subcloned in plasmid pZErO-2 (Invitrogen) and sequenced. A multiple alignment of the amino-acid sequences of ATP-PFKs and PP i -PFKs was made as described previously [5]. Structural analysis MODELLER [11] was used to construct a model of L. donovani PFK based on the known structure of Escherichia coli PFK (PDB code 4pfk [12]). This was the most suitable template, given our interest in both catalytic and regulatory sites, among the various available structures of E. coli and Bacillus stearothermophilus PFK, as this 4pfk structure contains fructose 6-phosphate and ADP in the active site, and ADP in the effector site. The E. coli and L. donovani enzymes share 23% sequence identity overall, although functional considerations have led to much greater conservation of the catalytic site and effector site. Without these sites, the degree of sequence identity of  20% leads to uncertainty in alignment of some regions. However, the conservation of the catalytic and effector sites allows reliable alignment and corres- pondingly accurate modelling of the Leishmania enzyme in these regions of particular importance. The program O [13] was used for visualizing structures and for its library of commonly observed, rotameric side-chain conforma- tions [14]. PROCHECK [15] was used for stereochemical analysis of models and for identifying the most likely position of an important one residue insertion, in the L. donovani enzyme relative to that from E. coli,atthe effector site. Expression and purification of recombinant L. donovani PFK The following specific primers were synthesized to amplify the gene by PCR: (1) 5¢-CGAATCTC CATATGGAGA CTCG-3¢, containing a NdeI site (underlined) adjacent to the 5¢ end of the coding region; (2) 5¢- TA GGATCCTTACACCTTAGACGCCAG-3¢, comple- mentarytoa3¢ noncoding region followed by a BamHI site (underlined). The total volume of the amplification mixture was 100 lL containing 20 ng of DNA, 0.4 l M of each primer, 4 m M MgSO 4 , 200 l M each of the four deoxynucleotides, and 1 unit of Vent DNA polymerase with the corresponding 1 · ThermoPol Reaction Buffer (New England Biolabs). PCR was performed in a Hybaid Thermal Reactor (Hybaid, UK) using the following program: 5 min 95 °C; 30 cycles consisting of 1 min denaturation at 95 °C, 45 s annealing at 65 °Cand 1.5 min extension at 72 °C; and a final 5 min incubation at 72 °C. The amplified gene was purified and ligated to the pCR2.1-TOPO vector (Invitrogen). The resulting recombin- ant plasmid (PFKLd-TOPO) was used to transform E. coli strain XL-1 Blue, and the sequence of the insert was checked. A bacteriophage T7 RNA polymerase system [16] was used to express L. donovani PFK in E. coli.ThePFK gene was excised from the PFKLd-TOPO recombinant plasmid and spliced into expression vector pET28b using the NdeIandBamHI sites. The new recombinant plasmid named pLdPFK directs, under the control of the T7 promoter, the production of a fusion protein bearing a N-terminal extension of 20 residues including a (His) 6 -tag. E. coli strain BL21(DE3)pLysS transformed with pLdPFK was grown at 30 °C in 50 mL Luria–Bertani medium plus 1 M sorbitol and 2.5 m M betaine [17] Fig. 1. Restriction map of recombinant plasmid PFK6.5-pBSKS. The hashed box marks the open-reading frame of the L. donovani PFK gene. The ATG of the initiator methionine is indicated. T7 and T3 indicate the orientation of the insert with respect to the promoter sequences of the pBSKS vector. The 2.2 kb EcoRI–PvuII fragment was used as hybridization probe and for sequence analysis. Ó FEBS 2002 Leishmania donovani phosphofructokinase (Eur. J. Biochem. 269) 3979 supplemented with 30 lgÆmL )1 kanamycin and 25 lgÆmL )1 chloramphenicol. Expression was induced at an A 600 of 0.6–0.8 by the addition of 1 m M isopropyl thio- b- D -galactoside and growth was continued overnight. Cells were collected by centrifugation (12 000 g,10minat 4 °C). The cell pellet was resuspended in 20 mL of lysis buffer (50 m M triethanolamine/HCl, pH 8.0, 300 m M NaCl, 200 m M KCl, 1 m M KH 2 PO 4 ,5m M MgCl 2 , 10% glycerol, 0.1 m M fructose 6-phosphate, 0.3 m M glucose 6-phosphate and the protease inhibitors E64, leupeptin and pepstatin, each at a concentration of 1 l M ). Cells were lysed by two passages through a SML-Aminco French pressure cell (SML Instruments, USA) at 90 MPa. Nucleic acids were eliminated first by incubation with 500 units Benzonase (Merck, Germany) for 15 min at 37 °C, and then with 10 mg of protamine sulphate for 15 min at room temperature. The lysate was centrifuged (20 000 g 15 min at 4 °C) and the supernatant used to further purify the expressed enzyme using immobilized metal affinity chromatography (TALON resin, Clontech, USA) exploit- ing the (His) 6 -tag at the N-terminus of the PFK. The charged resin was washed with lysis buffer plus 10 m M imidazole. The enzyme was then eluted with 100 m M imidazole in lysis buffer. One millilter fractions were collected to measure enzyme activity and to determine the protein profile by SDS/PAGE. Site-directed mutagenesis of the L. donovani PFK gene was performed by PCR techniques as described by Mikaelian & Sergeant [18] and using Vent DNA polymerase. The Leishmania PFK Lys224 codon AAG was changed into the Gly codon GGG. The mutated protein was expressed and purified according to the same protocols as the wild- type enzyme. Enzyme assays and kinetic analysis The activity of PFK was determined by measuring the decrease of NADH absorbance at 340 nm using a Beckman DU7 spectrophotometer. To follow PFK during purifica- tion, a standard enzymatic assay was performed at 25 °Cin a 1 mL reaction mixture containing: 100 m M triethanol- amine/HCl buffer, pH 8.0, 2.5 m M MgSO 4 ,10m M KCl, 2m M fructose 6-phosphate, 0.5 m M ATP, 2.2 m M PEP, 1.6 m M AMP, 0.42 m M NADH, 2 U lactate dehydrogenase and 2 U pyruvate kinase. One activity unit is defined as the conversion of 1 lmol substrate per min under these standard conditions. For kinetic analyses an assay was used in which the PFK activity was not coupled to a NAD-dependent reaction through its product ADP, as in the standard assay, but through its product fructose 1,6-bisphosphate. The assay was performed at 25 °C in a 1 mL reac- tion mixture containing 100 m M triethanolamine/HCl, pH 8.0, 2.5 m M MgCl 2 ,0.42m M NADH, 0.4 U aldo- lase, 0.8 U glycerol-3-phosphate dehydrogenase and 20 U triosephosphate isomerase. The reaction was initi- ated by the addition of 5 lL of enzyme diluted in buffer (0.1 M triethanolamine/HCl, pH 7.4, BSA 0.1 mgÆmL )1 , EDTA 0.2 m M and dithiothreitol 0.5 m M ). The effect of the fructose 6-phosphate concentration was determined by fixing the ATP concentration at 1 m M ,inthe presence and absence of AMP (1.5 m M ) and GDP (1.0 m M ). RESULTS AND DISCUSSION Analysis of kinetoplastid PFK genes In the 30 kb insert of the cosmid obtained by screening a L. donovani genomic library, only a single gene copy of PFK was detected. Figure 1 shows a restriction map of the insert of recombinant plasmid PFK6.5-pBKS subcloned from that cosmid. The coincidence between the restriction pattern of this cosmid and that obtained by Southern analysis of whole Leishmania DNA, and the signal inten- sities of the bands (not shown), were consistent with the presence of one gene copy per haploid genome. When blots of L. donovani chromosomal bands separated by pulsed- field gel electrophoresis were hybridized with an EcoRI– PvuII fragment from recombinant PFK6.5-pBSKS (Fig. 1), a unique hybridization signal of 1.3 Mb was observed (Fig. 2, lanes 1 and 2). This 1.3 Mb band may correspond to chromosomes 27b, 28 or 29 [19]. In L. amazonensis, included for comparative purposes, the probe hybridized weakly to a band of approximately 1.7 Mb (Fig. 2, lanes 3 and 4), the size of the proposed fusion product of chromosomes8and29intheL. mexicana group [20]. The amino-acid sequences encoded by the ORFs found in the L. donovani and T. borreli recombinants are shown in Fig. 3. The ORF in L. donovani is 1461 bp, coding for a polypeptide of 486 amino acids (excluding the initiator methionine) with a molecular mass of 53 988 and a calculated isoelectric point (pI) of 9.26. The C-terminus has the tripeptide -SKV, a type 1 peroxisome-targeting signal (PTS-1) with an acceptable degeneracy of the canonical motif -SKL [21,22]. The same tripeptide was previously found in another glycosomal enzyme of this organism, namely hypoxanthine-guanidine phosphoribosyltransferase Fig. 2. Chromosomal assignment of the PFK genes in L. donovani and L. a mazon ens is. Southern blot of Leishmania chromosomal bands separated by pulsed-field gel electrophoresis after hybridization with a probe consisting of a radioactively labelled EcoRI–PvuII restriction fragment (see Fig. 1) containing the whole L. donovani PFK gene. Lanes 1 and 2, L. donovani, lanes 3 and 4, L. amazonensis.Theposi- tions of yeast chromosomes that were used as molecular size markers are indicated at the right-hand side. 3980 C. Lo ´ pez et al. (Eur. J. Biochem. 269) Ó FEBS 2002 [23]. The sequence predicted from the T. borreli ORF codes for a polypeptide of 489 amino acids (excluding the first methionine) with a molecular mass of 53 211 and a pI 8.9. The typical PTS-1 motif -SKL was found as the C-terminal tripeptide. An excess of positively charged residues and resulting high pI are features often associated with glycosomal enzymes, particularly in T. brucei [24,25]. Figure 3 also shows the alignment of L. donovani and T. borreli PFK sequences with those of some other organisms. Except for two N-terminal insertions in T. borreli PFK, the new sequences share the characteristics of T.brucei PFK as described previously [5]. The percentage identity in a pairwise comparison (Table 1) of the amino acid sequences of L. donovani and T.brucei is high (70%), whereas the value obtained by comparing T. borreli PFK with the L. donovani and T.brucei enzymes is 54% in both cases. The extent of sequence similarity among the Kinetoplastida PFKs corresponds with the values found for some other glycolytic enzymes [9,26] and with the proposed phylogeny of this order [27,28]. The percentage identities of the kinetoplastid PFKs with those from all other major taxonomic groups are in the range 15–30%. As already previously observed for the T. brucei PFK [5], the L. donovani and T. borreli enzymes show signatures typical of PP i -PFKs (see Fig. 3) and, in a phylogenetic analysis, they appear firmly placed within the cluster of the PP i -dependent enzymes, well separated from the nonkinetoplastid ATP-PFKs (not shown). Interestingly, the kinetoplastid PFKs showed the highest percentage identity (37–38%) with the minor 48 kDa PFK from another protist, Entamoeba histolytica. Despite the higher overall similarity and its phylogenetic relationship with the subset of PP i -PFKs [5,29,30], it was recently reported that this E. histolytica PFK uses ATP as phospho donor (in contrast to the very different 60 kDa PP i -dependent PFK of this organism, see Fig. 3) [31] similar to the observation previously reported for the T. brucei enzyme [5]. The PFK activity in Leishmania species [32–34] and in T. borreli (J.VanRoy,F.Opper- does, N. Chevalier & P. A. M. Michels, unpublished results) is also known to be ATP-dependent. Kinetics of Leishmania PFK The L. donovani PFK was expressed in E. coli with an N-terminal His-tag and purified for kinetic analysis. The activity of the enzyme was ATP dependent. No activity (less than 1%) was observed when PP i (at concentrations up to 5m M ) was used as alternative phospho donor. As reported previously for PFK of T. brucei [7] and other Leishmania species [32], the activity of the enzyme depends hyperbol- ically on the concentration of ATP. The kinetic behaviour of the expressed PFK was determined as a function of fructose 6-phosphate at fixed, saturated ATP concentration (1.0 m M ), and in the presence or absence of AMP and GDP (Fig. 4). AMP, ADP and GDP are well-known effectors of bacterial PFKs; in assays, GDP rather than ADP is often used as effector, because ADP, being a reaction product, may act as a competitive inhibitor with respect to ATP. In the absence of AMP and at low fructose 6-phosphate concentrations (less than approximately 0.2 m M )the enzyme showed slightly cooperative binding of the substrate, with a Hill coefficient of 1.41. At higher substrate concentrations, the enzyme displayed hyperbolic kinetics; the S 0.5 ¼ 3.60 ± 0.48 m M . In the presence of AMP, the curve is hyperbolic over the entire range of substrate concentrations; AMP has a clear stimulatory effect by increasing the affinity for fructose 6-phosphate till a K m ¼ 0.157 ± 0.028 m M . In contrast, GDP has no effect whatsoever on the activity of the enzyme. Our data thus showed that the expressed L. donovani PFK binds its substrate fructose 6-phosphate in a cooper- ative manner, similar to many other PFKs, such as the enzymes from mammals [35] and bacteria (reviewed in [36]). This behaviour, and the increased affinity for the substrate in the presence of AMP, have also been reported for the enzymes partially purified from cultured L. donovani and L. braziliensis [32] and for T.bruceiPFK [6,7]. The observed cooperative substrate binding and allosteric activation by AMP suggest a multimeric structure for the enzyme. Indeed, T. brucei PFKwasshowntobetetrameric[25],likethe ATP-PFKs of most other organisms [1]. Hyperbolic kinetics have been reported for the Trypanosoma cruzi enzyme, but the relevance of this finding may be questioned, because the authors described an enzyme with a 17 kDa subunit mass [37]. Despite the relatively high sequence identity of Kinetoplastida PFK and PP i -dependent enzymes, AMP stimulation has only been described for one PP i -dependent PFK, that from Naegleria fowleri [38]. In this case the AMP effect was attributed to promoting a more active enzyme aggregate. Active site of kinetoplastid PFKs Table 2 presents a summary of the active-site residues of E. coli PFK involved in the binding of ADP and fructose 6-phosphate as observed in its crystal structure [12], and the corresponding residues in the PFKs of human, T.brucei, L. donovani, T. borreli and in the minor 48 kDa enzyme of E. histolytica. A comparison of the kinetoplastid and human PFKs shows four differences in the residues involved in nucleotide binding and three differences in the residues involved in the binding of fructose 6-phosphate. The same Fig. 3. Alignment of L. donovani and T. borreli PFK amino acid sequences with other ATP and PP i dependent enzymes. Sequences include the ATP-dependent enzymes of T.brucei, E. coli, B. stearothermo- philus, E. histolytica, the N-catalytic domain of the human muscle enzyme and the catalytic subunit of S. cerevisiae,andthePP i -depen- dent enzymes of E. histolytica, A. methanolica and P. freundenreichii. Sequences of yeast and human expanding beyond the N- or C-termini of Kinetoplastida sequences are not shown. The E. coli and L. dono- vani enzymes are numbered above and below the alignment, respec- tively.Symbolsare:blackarrows,b strands; black cylinders, ahelices; open circles, substrate ATP-binding residues; black circles, fructose 6-phosphate binding residues; black triangles, effector-binding resi- dues. Boxes mark regions sharing identical residues between either the set of kinetoplastid and E. histolytica ATP-PFKs and the set of typical ATP-PFKs, or the kinetoplastid and E. histolytica ATP-PFKs and the setofPP i -PFKs. Residues common to all sequences are in bold + italic font; bold only is used where there is one disagreement among the entire sequence set. Residue 224 of L. donovani PFK that was studied by mutagenesis is indicated by a black triangle underneath the align- ment. The figure was made using ALSCRIPT [53]. Ó FEBS 2002 Leishmania donovani phosphofructokinase (Eur. J. Biochem. 269) 3981 substitutions occur in the 48 kDa ATP-PFK of E. histolytica (Fig. 3) [39], as well as in one of the PFKs of the prokaryotic Spirochaetes Treponema pallidum and Borrelia burgdorferi (data not shown; GenBank accession numbers AE001195 and AE001172). Indeed, the identity of active-site residues in all these organisms is in agreement with the branching order in a phylogenetic analysis based on full-length PFK sequences [5,29,30]. The PFKs of these organisms form a well-supported monophyletic cluster within the PP i -PFK subset, well separated from the typical ATP-PFKs [5,29,30]. 3982 C. Lo ´ pez et al. (Eur. J. Biochem. 269) Ó FEBS 2002 The identity of the phospho donor of the PFKs from T. pallidum and B. burgdorferi has not been established yet. The E. coli residues involved in fructose 6-phosphate binding that have been substituted in the kinetoplastid and related PFKs are Arg162, Arg243 and His249. The Fig. 3. (Continued.) Ó FEBS 2002 Leishmania donovani phosphofructokinase (Eur. J. Biochem. 269) 3983 corresponding residues found in all these PFKs are Gly, Lys and Tyr, respectively. Whereas Arg162 seems conserved in all typical ATP-PFKs, a positively charged residue at the corresponding position seems not essential for substrate binding in the PP i -PFKs and the atypical ATP-PFKs. The Arg243 of ATP-PFKs is replaced by Lys in all PFKs of the subset comprising the Kinetoplastida; apparently, a positively charged residue at this position is essential in all ATP-PFKs. It is possibly equivalent to Lys315 of the PP i -dependent PFK of Propionibacterium freundenreichii (E. coli position 241 in Fig. 3), because Xu et al. [40] have shown that an alteration of this residue by site-directed mutagenesis causes a 389-fold increase of the K m for fructose 6-phosphate. The substitution of His249 (E. coli numbering) by Tyr in the Kinetoplastida is also found in some PP i -PFKs such as the enzymes of P. freudenreichii and E. histolytica (Fig. 3), and is possibly without much effect. Out of 10 residues involved in ADP binding in E. coli PFK, four are not conserved in Kinetoplastida (Table 2). Strikingly, the ATP-dependent E. histolytica PFK and the putative ATP-PFKs of two Spirochaetes discussed above have the same residues as the ATP-dependent kinetoplastid enzymes [39,41]. Therefore, these residues may be important determinants for the phospho-donor specificity if, in future research, the isoenzymes of the Spirochaetes turn out to be ATP-dependent as well. In addition to the substrate-binding residues in Table 2, another active-site residue is of particular interest: the residue corresponding to E. coli Gly124 is a Lys in the kinetoplastid ATP-PFKs, in the ATP-dependent isoenzyme of E. histolytica (see Fig. 3) and in the PFKs of the two Spirochaetes (not shown). A Gly is typical of ATP-PFKs, Table 1. Percentage identity of the PFK amino-acid sequences given in Fig. 3. B. stearo. Human muscle-N S. cere N T. bruce i L. donovani T. borreli E. histo. ATP E. histo. PP i A. meth. B. stearothermophilus 54 Human muscle-N 39 42 S. cerevisiae-N 34 37 46 T. brucei 24 30 17 18 L. donovani 23 29 19 18 70 T. borreli 25 25 18 19 54 54 E. histolytica ATP 25 27 19 21 38 37 38 E. histolytica PP i 21 25 15 12 16 15 16 18 A. methanolica 37 43 32 33 30 29 26 30 21 P. freudenreichii 23 22 18 18 20 20 20 22 17 22 Fig. 4. Kinetics of recombinant L. donovani PFK with respect to fruc- tose 6-phosphate. Activity was measured at a fixed ATP concentration of 1.0 m M . Symbols are: j, no additions; d,+1.5m M AMP; m,+1.0m M GDP. Values of kinetic parameters (see text) were cal- culated after optimal curve fitting of the experimentally determined data using the SIGMAPLOT program. The values given in the text are ± SD for the fit. Table 2. Amino acid residues involved in binding fructose 6-phosphate and ADP in E. coli PFK, and the corresponding residues in the organisms L. donovani, T. bruc ei, T. borreli, E. histolytica-ATP, T. pallidum and B. burgdorferi. Differences are highlighted in bold. E. coli Other organisms Fructose 6-phosphate Thr125 Thr Asp127 Asp Asp129 Asp Arg162 Gly Met169 Met Gly170 Gly Arg171 Arg Glu222 Glu Arg243 Lys His249 Tyr Arg252 Arg ADP Gly11 Gly Tyr41 Tyr Arg72 Arg Phe73 Gly Arg77 (gap) Asp103 Asp Gly104 Gly Ser105 Thr Met107 Arg Gly109 Gly 3984 C. Lo ´ pez et al. (Eur. J. Biochem. 269) Ó FEBS 2002 whereasaLysispresentinallPP i -dependent enzymes. Xu et al. [40] have shown that a change of this lysine into a methionine in P. freudenreichii PFK caused a 132-fold increase in the K m for PP i and a 490-fold decrease in k cat , providing strong indication for a direct involvement of this Lys residue in PP i binding. From a phylogenetic analysis, we previously concluded that the kinetoplastid PFKs must have been derived from a PP i -dependent ancestral PFK, which changed its phospho-donor specificity early in the evolution of the lineage [5]. However, the Lys is no longer involved in PP i binding, so why has it been retained in the present-day kinetoplastid PFKs which are all ATP dependent, and in the ATP-dependent isoenzyme of E. histolytica? Has it obtained a function in ATP binding, implying that the mode of nucleotide binding is different in kinetoplastid PFKs from that in other ATP-PFKs? Or has it been retained for structural reasons? Relevant to these questions is the observation that the Lys is also present in the ATP-dependent enzyme from the actinomycete Strep- tomyces coelicolor that, in a phylogenetic analysis, also clusters with the PP i -dependent PFKs and that must have had a common ancestor with the PP i -dependent PFKs of other Actinomycetes such as Amycolatopsis methanolica [42]. We therefore investigated whether substituting the Lys would have any effect on the kinetoplastid enzyme. To this end, we replaced it by Gly in L. donovani PFK. The resulting LdPFK Lys224fiGly mutant did not display any activity. In contrast, the corresponding LysfiGly mutant of T. brucei (TbPFK Lys226fiGly) appeared to be active [43]. Strikingly, this T. brucei mutant showed only a slight decrease in its affinity for ATP, but the mutation had a major effect on the enzyme’s behaviour with respect to fructose 6-phosphate. In the absence of AMP, the S 0.5 for fructose 6-phosphate was  5m M compared to 0.59 m M in the wild-type enzyme. However, the higher substrate affinity can still be induced by the addition of AMP: K m for fructose 6-phosphate ¼ 0.82 m M compared to 0.15 m M in the wild- type PFK [43]. It seems that the mutation leads to a local disruption of the active site with accompanying lowering of fructose 6-phosphate affinity. This is independent of the allosteric changes in fructose 6-phosphate affinity as AMP is capable of similar enhancements of fructose 6-phosphate affinity in both wild-type and mutant enzymes. In the case of the L. donovani enzyme, a greater degree of disruption leads to abolition of fructose 6-phosphate binding, either through local or global effects. As discussed previously, the comparative analysis of PFK sequences suggests that only subtle changes may be required for a change of phospho donor specificity [5]. This notion was reinforced by the recent publication [44] describing mutations in the ATP-PFK of E. coli and the PP i -PFK of E. histolytica, similar to those described above for trypan- osomatid PFKs. The Gly124fiLys substitution in E. coli effectively eliminated activity with ATP as a substrate, but no PP i -dependent activity was observed. However, the reverse Lys201fiGly mutation in the PP i -dependent, major PFK of E. histolytica reduced the k cat with PP i as the phospho donor by four orders of magnitude, while having only a limited effect on the apparent PP i affinity of the residual enzyme activity. Importantly, the performance of the enzyme with ATP as a phospho donor increased about eightfold (although this is still 10 5 times less than the performance of the wild-type enzyme with PP i ) essentially by an increase in k cat . Understanding of the role of Lys224 in L. donovani PFK, and corresponding residues in other PFKs, is hampered by the lack of a crystal structure with a lysine at this position. Modelling of the Gly to Lys mutation shows that, without significant local structural changes, the lysine side chain becomes entirely buried in the protein interior, with no possibility of electrostatic interaction with an acidic residue, a situation essentially unknown in protein structure. One hypothesis is that, in enzymes containing a lysine at this position, the peptide bond with the preceding proline adopts a cis configuration [45]. It is suggestive that proline is entirely conserved at position 123 (E. coli numbering) when a lysine is present at position 124, while valine is also tolerated in other PFKs. Modelling of possibilities for a L. donovani model containing such a cis peptide bond yields structures in which the lysine side chain is solvent-exposed such as that illustrated in Fig. 5. In this structure the lysine side chain is placed at the heart of the catalytic site. A direct interaction with substrate ATP seems unlikely from the kinetic results presented here, although it is unfortunate that structural inferences may only be drawn from a product- bound PFK structure (Fig. 5). However, a limited descrip- tion of a PFK-AMPPNP-fructose 6-phosphate substrate analogue complex [46] (coordinates not deposited) supports the notion of a close resemblance between substrate- and product-bound protein structures. Why then, in contrast, should PP i binding be dramatically affected when this lysine is mutated in E. histolytica and P. freudenreichii PFKs [40,44]? The explanation may lie in another residue, clearly implicated in substrate specificity [44]. Position 104 (E. coli numbering) is always a Gly in ATP-PFKs and an Asp in PP i -PFKs. Structural examination (Fig. 5) shows that the presence of any non-Gly residue leads to steric clashes with the bound nucleotide in its crystallographically observed Fig. 5. Positions of phospho-donor specificity-determining residues rel- ative to the catalytic site of E. coli PFK bound to products. Numbering is according to the E. coli enzyme. The figure was produced using MOLSCRIPT [54]. Ó FEBS 2002 Leishmania donovani phosphofructokinase (Eur. J. Biochem. 269) 3985 position. In the structure of a PP i -dependent PFK bound to substrates, it would be reasonable to expect that the PP i substrate binds in the corresponding position as the b-and c-phospho groups of bound ATP in ATP-dependent enzymes. However, analysis shows that any rotameric conformation of an Asp104 side chain leads to positioning of its negative charge near to the phospho group occupying the Ôa positionÕ, also negatively charged. Minimum oxygen- oxygen interatomic distances range from 1.1 to 3.9 A ˚ , depending on Asp104 rotamer. This electrostatic repulsion may therefore force the PP i into a slightly different conformation, further from Asp104 and hence nearer to Lys124. This hypothesis allows an explanation of the apparent involvement of this lysine in PP i binding [40,44] but not in ATP binding. A more prosaic explanation may underlie the almost complete lack of PP i - or ATP-dependent PFK activity seen for the Gly124fiLys E. coli mutant [44]. Without a preceding cis peptide bond only side chain conformations that unfavourably bury the positive charge of the new Lys are attainable and the protein would therefore be destabilized. The lack of confirmation of native fold for the mutant, by CD experiments for example, suggests that the mutant may have undergone gross structural changes resulting in loss of activity. The modelled lysine in the L. donovani modeliswellpackedandnot apparently well positioned to interact directly with fructose 6-phosphate. These considerations support the previously advanced explanation of the effects of the Lys224fiGly mutation in terms of destabilizing local structural changes. Effector-binding site of trypanosomid PFKs Table 3 presents a comparison of the residues in B. stearo- thermophilus and E. coli PFKs involved in the binding of the allosteric activator ADP with the corresponding residues in the L. donovani and T.bruceienzymes (according to the alignment in Fig. 3). A structural comparison of the residues binding the activator ADP in B. stearothermophilus PFK and a putative AMP binding mode for the L. donovani, suggested by modelling, is shown in Fig. 6. This comparison suggests that the kinetoplastid enzymes may employ the same region for binding their allosteric activator AMP. The b-phospho group binding residues of the bacterial enzymes show the most changes, with the most striking substitution being the replacement of the Mg 2 +-ligating Glu187 with Asn. The loss of Mg 2+ and the replacements of Arg25 and Arg154, both of which electrostatically interact with the b-phospho group of ADP (Fig. 6A), effectively eliminate the b-phospho-binding pocket in the trypanosomatid enzymes. The residues at positions 211 and 213 of the B. stearothermophilus enzyme that bind the a-phospho Fig. 6. Comparison of (A) the crystallographically observed effector site of E. coli PFK with bound ADP and (B) the modelled structure of L. donovani PFK effector site bound to AMP. Ligand and protein are shown in ball-and-stick representation with the exception of the protein backbone, in the vicinity of the one residue insertion, which is drawn as a tube. Possible hydrogen bonds are shown with dotted lines. The figure was produced using MOLSCRIPT [54]. Table 3. Amino acid residues involved in binding the allosteric activator ADP in B. stearothermophilus and E. coli PFK, and corresponding residues in T. brucei and L. donovani PFKs. Differences are highlighted in bold. B. stearothermophilus E.coli T. brucei L. donovani Arg21 Arg Arg Arg Arg25 Arg Leu Leu Val54 Arg Arg Arg Gly58 Ser Thr Arg Arg154 Arg Tyr Tyr Glu187 Glu Asn Asn Arg211 Lys His Gln Lys213 Lys Arg Arg 3986 C. Lo ´ pez et al. (Eur. J. Biochem. 269) Ó FEBS 2002 group of ADP (Fig. 6A) are better conserved and may be involved in binding the phospho group of AMP (Fig. 6B). The modelling reveals that, in addition to the residue differences identified by sequence comparisons, other significant structural differences may exist. The L. donovani enzyme, along with others from kinetoplastids and the E. histolytica ATP-dependent PFK, has a one residue insertion, relative to bacterial enzymes, at around position 287 (L. donovani enzyme numbering). Two possible posi- tions for this insertion were analysed and one, as shown in Fig. 6B, found to be favoured for avoiding the positioning of model residues in unusual areas of the Ramachandran plot. The altered main chain conformation in the vicinity causes the Gln287 side chain to intrude into the area corresponding to the b-phospho-binding site of the bacterial enzymes. Furthermore, it may form a hydrogen bond with the phospho group of effector AMP (Fig. 6B). Another interesting structural difference revealed by modelling relates to the side chain position of Arg157 in the L. donovani enzyme which replaces a Ser or Gly in the bacterial enzymes. Adopting a rotameric conformation, this residue may fill the space caused by the replacements of B. stearothermophilus Arg211 with Gln and Arg25 with Leu. In this position it may hydrogen bond to the phospho of effector AMP in the L. donovani model structure and contribute to the positive electrostatic potential of the effector binding pocket. CONCLUSIONS The PFK genes of L. donovani and T. borreli have been cloned and sequenced. The encoded enzymes show most similarity to the subset of PP i -PFKs, as did the previously analysed T.brucei PFK. Nevertheless, ATP is the phos- pho substrate of all these kinetoplastid PFKs. It is possible that a common ancestral organism changed its phospho donor specificity during evolution. The currently available data do not allow us to draw any conclusion as to how and why the Kinetoplastida and other protists such as Entamoeba obtained their ATP-dependent, PP i - like PFKs. Did they evolve from a PP i -PFK in both lineages independently, or did they originate in a common ancestor of these protists? Were they acquired from Spirochaetes by lateral gene transfer? In this respect, it may be relevant that phylogenetic studies based on sequences of other glycolytic enzymes, glyceraldehyde-3- phosphate dehydrogenase and enolase, showed grouping of Kinetoplastida (and/or the related Euglenoida) and Spirochaetes [47–49]. Strikingly, all kinetoplastid PFKs, as well as the Entamoeba PFK contain a Lys on position 124 (E. coli numbering), whereas all other ATP-PFKs contain a Gly. Previous mutagenesis studies have provided strong evidence that this Lys residue is involved in PP i binding. Structure modelling suggests that the Lys may have been retained in the kinetoplastid PFKs to maintain the stability of the active-site structure. These results are supported by muta- genesis studies. No active L. donovani Lys224fiGly mutant could be obtained, whereas the kinetic properties of a corresponding Lys226fiGly mutant of T. brucei PFK could be interpreted in terms of a destabilized active site. The L. donovani PFK shows slightly cooperative binding of fructose 6-phosphate at low concentrations of this substrate. The enzyme was allosterically activated by AMP by a significant increase in the affinity for the substrate. However, trypanosomatid PFKs are not activa- ted by ADP, in contrast to their counterparts in the bacteria E. coli and B. stearothermophilus. Modelling studies have provided a possible structural basis for the AMP specificity. We have provided evidence for significant structural differences between trypanosomatid PFK and other ATP- PFKs including the human enzyme. Such differences were found in both the active site and the region of the enzyme presumably involved in effector binding. Indeed, the differences in the effector-binding site tally with the apparently low level of activity regulation of trypanosoma- tid PFK as compared to that of the human enzyme. This limited regulation of trypanosomatid PFK seems physio- logically relevant in view of the intraglycosomal localization of the enzyme and the low permeability of the organelle’s membrane for many metabolic intermediates that in other cells act as PFK effectors [3,50]. The structural differences observed offer great potential for the design or selection of drugs. Although our computer analysis using a kinetic model of glycolysis suggested that PFK in bloodstream- form T. brucei is present in excess [51], we have argued elsewhere [8,52] that this does not necessarily exclude the enzyme as a target for selective inhibitors that bind with high affinity, particularly irreversibly binding inhibitors. The most important aspects to consider in drug target selection are that an enzyme should have an essential (or at least very important) metabolic role and that its structure should be sufficiently different from that of the correspond- ing host enzyme. Moreover, metabolism in bloodstream- form T. brucei is highly specialized, and in many respects not representative for the infective stages of other trypan- osomatid parasites such as the trypomastigotes and amastigotes of Leishmania species and T.cruzi [3]. Therefore, we consider the trypanosomatid PFK as a highly promising drug target. ACKNOWLEDGEMENTS This research was financially supported by the European Commission (programmes STD3 and INCO-DC). Financial support for C. L. for a 1 year stay at the ICP in Brussels was provided by the Fundacio ´ nGran Mariscal de Ayacucho and CONICIT Venezuela (grant S1-9500524). We are grateful to Dr Theo deVos (SBBI, Seattle) for providing the genomic L. donovani library, and to Drs Linda Fothergill-Gilmore (University of Edinburgh) and Fred Opperdoes (ICP, Brussels) for critical reading of the manuscript. REFERENCES 1. Fothergill-Gilmore, L.A. & Michels, P.A.M. (1993) Evolution of glycolysis. Prog. Biophys. Mol. Biol. 59, 105–235. 2. 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Leishmania donovani phosphofructokinase Gene characterization, biochemical properties and structure-modelling studies Claudia Lo ´ pez 1,2 ,. characterization of the gene encoding Leishmania donovani phosphofructokinase (PFK) and the biochemical properties of the expressed enzyme are reported. L. donovani has