6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase in Trypanosomatidae Molecular characterization, database searches, modelling studies and evolutionary analysis Nathalie Chevalier1,*, Luc Bertrand2,3,*, Mark H Rider2, Fred R Opperdoes1, Daniel J Rigden4 and Paul A M Michels1 ´ Research Unit for Tropical Diseases, Christian de Duve Institute of Cellular Pathology and Laboratory of Biochemistry, Universite catholique de Louvain, Brussels, Belgium ´ Hormone and Metabolic Research Unit, Christian de Duve Institute of Cellular Pathology and Laboratory of Biochemistry, Universite catholique de Louvain, Brussels, Belgium ´ Division of Cardiology, Universite catholique de Louvain, Brussels, Belgium School of Biological Sciences, University of Liverpool, UK Keywords ankyrin-repeat motif; fructose 2,6-bisphosphate; glycolysis regulation; 6-phosphofructo-2-kinase ⁄ fructose-2,6bisphosphatase; trypanosome Correspondence P A M Michels, ICP-TROP 74.39, Avenue Hippocrate 74, B-1200 Brussels, Belgium Fax: +32 27 62 68 53 Tel: +32 27 64 74 73 E-mail: michels@bchm.ucl.ac.be *These authors contributed equally to this work Database Nucleotide sequence data are available in the DDJB ⁄ EMBL ⁄ GenBank databases under accession numbers AY571277 (Tb4), AY571278 (Tb1) and AY999068 (Tb2) (Received April 2005, revised 10 May 2005, accepted 16 May 2005) doi:10.1111/j.1742-4658.2005.04774.x Fructose 2,6-bisphosphate is a potent allosteric activator of trypanosomatid pyruvate kinase and thus represents an important regulator of energy metabolism in these protozoan parasites A 6-phosphofructo-2-kinase, responsible for the synthesis of this regulator, was highly purified from the bloodstream form of Trypanosoma brucei and kinetically characterized By searching trypanosomatid genome databases, four genes encoding proteins homologous to the mammalian bifunctional enzyme 6-phosphofructo-2kinase ⁄ fructose-2,6-bisphosphatase (PFK-2 ⁄ FBPase-2) were found for both T brucei and the related parasite Leishmania major and four pairs in Trypanosoma cruzi These genes were predicted to each encode a protein in which, at most, only a single domain would be active Two of the T brucei proteins showed most conservation in the PFK-2 domain, although one of them was predicted to be inactive due to substitution of residues responsible for ligating the catalytically essential divalent metal cation; the two other proteins were most conserved in the FBPase-2 domain The two PFK-2-like proteins were expressed in Escherichia coli Indeed, the first displayed PFK-2 activity with similar kinetic properties to that of the enzyme purified from T brucei, whereas no activity was found for the second Interestingly, several of the predicted trypanosomatid PFK-2 ⁄ FBPase-2 proteins have long N-terminal extensions The N-terminal domains of the two polypeptides with most similarity to mammalian PFK-2s contain a series of tandem repeat ankyrin motifs In other proteins such motifs are known to mediate protein–protein interactions Phylogenetic analysis suggests that the four different PFK-2 ⁄ FBPase-2 isoenzymes found in Trypanosoma and Leishmania evolved from a single ancestral bifunctional enzyme within the trypanosomatid lineage A possible explanation for the evolution of multiple monofunctional enzymes and for the presence of the ankyrin-motif repeats in the PFK-2 isoenzymes is presented Abbreviations CDD, conserved domain databases; Fru2,6-P2, fructose 2,6-bisphosphate; FBPase-2, fructose-2,6-bisphosphatase; PEP, phosphoenolpyruvate; PFK-1, 6-phosphofructo-1-kinase; PFK-2, 6-phosphofructo-2-kinase; PKA, protein kinase A; PKC, protein kinase C; TbFBPase-2, Trypanosoma brucei fructose-2,6-bisphosphatase; TbPFK-2, Trypanosoma brucei 6-phosphofructo-2-kinase 3542 FEBS Journal 272 (2005) 3542–3560 ª 2005 FEBS N Chevalier et al PFK-2 ⁄ FBPase-2 of Trypanosomatidae Fig Diagrammatic representation of the regulation of carbohydrate metabolism by fructose 2,6-bisphosphate in mammalian cells (A) and Trypanosomatidae (B) Glycolysis in mammalian cells occurs in the cytosol In Trypanosomatidae, the glycolytic enzymes responsible for the conversion of glucose into 3-phosphoglycerate, and the gluconeogenic enzyme fructose-1,6-bisphosphatase are present in glycosomes Abbreviations: FBPase-1, fructose-1,6-bisphosphatase; PYK, pyruvate kinase Fructose 2,6-bisphosphate (Fru2,6-P2) is a key regulator of glycolysis in almost all eukaryotes, but it is absent from prokaryotes In animals, plants and fungi, this sugar phosphate stimulates glycolysis via allosteric stimulation of 6-phosphofructo-1-kinase (PFK-1) and inhibits gluconeogenesis by acting as a negative effector of fructose-1,6-bisphosphatase [1,2] (Fig 1A) In contrast, in protozoan organisms belonging to the Kinetoplastida (comprising pathogenic organisms such as Trypanosoma and Leishmania) Fru2,6-P2 is not a stimulator of PFK-1, but rather acts on pyruvate kinase [3–9] This latter enzyme is stimulated at submicromolar concentrations, 2000-fold lower than by fructose 1,6-bisphosphate, the usual regulator of pyruvate kinase activity in other organisms Similarly, trypanosomatid fructose-1,6-bisphosphatase is insensitive to Fru2,6-P2 This different enzyme specificity is most likely related to the unique metabolic regulation in Kinetoplastida (Fig 1B) The majority of glycolytic enzymes responsible for the conversion of glucose into 3-phosphoglycerate are compartmentalized in peroxisome-like organelles called glycosomes [10–12] Only the last three enzymes, phosphoglycerate mutase, enolase and pyruvate kinase are present in the cytosol The gluconeogenic enzyme fructose-1,6-bisphosphatase also has a glycosomal localization [12] Strikingly, glycosomal enzymes such as hexokinase and PFK-1 lack the regulatory mechanisms found in most other organisms, namely product inhibition and control by metabolites further downstream in the pathway or by effectors [13] In Kinetoplastida, such mechanisms seem to be redundant as a result of the sequestering of the enzymes within a separate compartment bounded by a membrane with low permeability to many metabolites [12,14,15] Interestingly, this compartmentation FEBS Journal 272 (2005) 3542–3560 ª 2005 FEBS seems to have resulted in a kind of ‘re-routing’ of regulatory mechanisms (Fig 1) and cytosolic pyruvate kinase appears the most important regulated enzyme [5,6,11,12,14] In mammalian tissues, the bifunctional enzyme 6-phosphofructo-2-kinase (PFK-2; EC 2.7.1.105) ⁄ fructose-2,6-bisphosphatase (FBPase-2; EC 3.1.3.46) catalyses both the synthesis and degradation of Fru2,6-P2 [16,17] PFK-2 and FBPase-2 activities have also been detected in Kinetoplastida In line with the activity regulation of cytosolic pyruvate kinase by Fru2,6-P2, the PFK-2 and FBPase-2 activities are localized in the cytosol of the kinetoplastid Trypanosoma brucei [4] However, these activities could be separated by partial protein purification, indicating that they reside in distinct enzymes [4] In higher animals, different tissue-specific PFK-2 ⁄ FBPase-2 bifunctional isoenzymes exist with kinetic properties and regulatory mechanisms related to metabolism (glycolysis vs gluconeogenesis) [1,2,16,17] The isoenzymes are homodimers, typically with subunit masses of 50–60 kDa They have a common structure, with the PFK-2 domain comprising most of the N-terminal half of the enzyme subunit and the FBPase-2 domain in the C-terminal half This central core contains the two catalytic activities and is well conserved Both extremities often contain regulatory phosphorylation sites, involved in the fine tuning of enzyme activity In plants (Arabidopsis, spinach, potato), the bifunctional enzyme sequence possesses a large N-terminal extension that provides a regulatory domain [18] In Saccharomyces cerevisiae, three isoforms of PFK-2 ⁄ FBPase-2 are present, but each displays only a single activity [19–21] Nevertheless, they are clearly homologous to the mammalian bifunctional enzyme 3543 PFK-2 ⁄ FBPase-2 of Trypanosomatidae In two isoforms (called PFK26 and PFK27), FBPase-2 activity appears to have been lost during evolution as the result of a crucial substitution or major deletions, respectively In the third form (FBP26), the PFK-2 domain has undergone multiple substitutions rendering it inactive For mammalian PFK-2 ⁄ FBPase-2 isoenzymes, it has been shown that the functional, active form is a dimer Although the FBPase-2 domain does not seem to be involved in dimerization, its presence, whether active or not, seems to be essential because a mammalian PFK-2 domain expressed alone in bacteria forms inactive aggregates [22] By contrast, the bacterially expressed mammalian FBPase-2 domain is active as a monomer [23] The crystal structures of the rat testis [24] and rat liver bifunctional enzymes [25] have been solved The subunits of the dimer are arranged in a head-to-head fashion with the PFK-2 domains of the two subunits being intimately associated in both testis and liver structures The FBPase-2 domains are independent of each other in the testis enzyme but form contacts in the liver isozyme The fact that the PFK-2 domain is structurally related to the adenylate kinase family, whereas the FBPase-2 domain is similar to the phosphoglycerate mutase family [24,26], seems to reveal that the bifunctional enzyme resulted from the fusion of two ancestral genes We studied some properties of PFK-2 (TbPFK-2) purified from T brucei as well as bacterially expressed forms of TbPFK-2 and investigated whether the T brucei PFK-2 and FBPase-2 enzymes are homologous to their counterparts in higher eukaryotes Results and Discussion Purification and characterization of T brucei PFK-2 PFK-2 was purified from pooled cytosol fractions from the bloodstream form of T brucei stock 427 using ionexchange chromatography and specific elution from Blue Sepharose with buffer containing PFK-2 substrates The purification was 9000-fold compared with the activity in the initial extract and the purified enzyme had a specific activity of 11 mUnitsỈmg)1 of protein, which is comparable with the specific activity of PFK-2 in preparations purified from mammalian tissues [27] Nevertheless, several bands were visible in Coomassie Brilliant Blue-stained gels On gel filtration, PFK-2 activity eluted from a Superose 12 column as a single symmetrical peak with a Mr of 76 400 (not shown) As yeast PFK-2 has been shown to be phosphorylated by protein kinase A (PKA) [28,29] and 3544 N Chevalier et al Table Kinetic properties of PFK-2 purified from bloodstream-form T brucei and effect of phosphorylation by protein kinases Trypanosome PFK-2 (30 lgỈmL)1) was incubated for 15 at 30 °C with or without protein kinases (0.6 unitỈmL)1), purified and assayed as described previously [87] Aliquots were taken for PFK-2 activity measurements For the fructose 6-phosphate (F6P) saturation curves, concentrations were varied up to 30 mM For inhibition by PEP, the concentrations of fructose 6-phosphate and MgATP were mM The results are the means ± SEM of three separate determinations, otherwise individual values are given ND, not determined Enzyme Km F6P (mM) Km ATP (mM) Vmax [mUnits (mg of protein))1] Untreated PKA-treated PKC-treated 5.8 ± 1.2 39 5.8 0.88 ND ND 7.1 ± 3.2 7.1 6.7 protein kinase C (PKC) [30] with accompanying changes in PFK-2 activity, we tested the effect of phosphorylation by these protein kinases on T brucei PFK-2 activity (Table 1) Treatment with PKA led to PFK-2 inactivation via a sevenfold increase in Km for fructose 6-phosphate with no change in Vmax, whereas treatment with PKC was without effect This contrasts with yeast PFK-2, in which PKA treatment led to PFK-2 activation by increasing the Vmax and lowering the Km for fructose 6-phosphate [28,29] and PKC treatment led to PFK-2 inactivation [30] Purified T brucei PFK-2 had a pH optimum around (not shown), was inhibited by phosphoenolpyruvate (PEP; K0.5 ¼ 0.7 mm) and citrate (60% inhibition at mm) but like heart PFK-2 [31], was rather insensitive to inhibition by glycerol 3-phosphate (20% inhibition at mm) Database searches and sequence analysis tblastn searches were performed in the databases of the three trypanosomatid genome projects (T brucei, T cruzi and Leishmania major) using a query of mammalian and yeast bifunctional PFK-2 ⁄ FBPase-2 sequences Analysis of the various T brucei and L major databases surprisingly revealed four homologous sequences The T brucei sequences and their close homologues in L major were named Tb1 ⁄ Lm1, Tb2 ⁄ Lm2, Tb3 ⁄ Lm3 and Tb4 ⁄ Lm4 and have the database codes shown in Table A diagrammatic representation of the four T brucei sequences is presented in Fig For each of these four isoforms, two corresponding sequences could be found in the T cruzi genome database (Table 2), presumably reflecting the known hybrid genotype of the strain chosen for genome sequencing, as a result of genetic exchange FEBS Journal 272 (2005) 3542–3560 ª 2005 FEBS PFK-2 ⁄ FBPase-2 of Trypanosomatidae N Chevalier et al Table Properties of predicted PFK-2 ⁄ FBPase-2 isoenzymes of trypanosomatids Conservation PFK2-domain Accession no Lm Lm Lm Lm Tb1 Tb2 Tb3 Tb4 Tc1–1 Tc1–2 Tc2–1 Tc2–2 Tc3–1 Tc3–2 Tc4–1 Tc4–2 Chromosome LmjF3.0800 LmjF26.0310 LmjF36.0150 LmjF07.0760 Tb03.48O8.70 Tb07.27M11.980 Tb10.70.2700 Tb08.29O4.60 Tc00.1047053508153.950 Tc00.1047053508181.20 Tc00.1047053508207.230 Tc00.1047053509509.30 Tc00.1047053510963.50 Tc00.1047053508625.50 Tc00.1047053508569.130 Tc00.1047053503733.20 26 36 7 10 FBPase-2 domain Overallc (%) Residuesb [key catalytic (total 5); other binding (total 8)] Overallc (%) Presumed activity 24–40 17–29 9–15 13–25 24–37 13–25 10–16 13–26 23–41 23–41 14–29 14–28 10–15 10–15 13–28 13–27 0; 3; 5; 4; 0; 3; 5; 4; 0; 0; 3; 3; 5; 5; 5; 5; 20–23 24–32 27–36 26–40 18–23 23–29 25–34 30–37 19–25 10–24 24–30 24–29 28–36 28–37 28–40 28–40 PFK-2 PFK-2 FBPase-2 ? PFK-2 ? FBPase-2 ? PFK-2 PFK-2 ? ? FBPase-2 FBPase-2 FBPase-2 FBPase-2 No residues Mass (kDa)a Residuesb [key catalytic (total 4); other binding (total 13)] 2422 667 485 1245 1023 648 478 702 1021 1023 705 702 481 481 749 749 251 74 55 132 111 72 54 79 112 113 79 79 55 54 84 84 4; 4; 1; 3; 4; 2; 0; 2; 4; 4; 3; 3; 0; 0; 2; 2; 13 10 13 13 13 10 10 3 6 2 2 2 2 8 7 a Molecular mass calculated from ORF b Conservation of residues in the predicted trypanosomatid enzymes compared to functional mammalian and S cerevisiae PFK-2 and FBPase-2 domains (and corresponding to boxed residues in Fig 3) c Overall percentage of amino acid sequence identity between the predicted trypanosomatid enzymes and functional PFK-2 and FBPase-2 domains from other eukaryotes between two distantly related lineages [32] Each pair, for example Tc1–1 and Tc1–2 corresponding to Tb1, share 95–98% sequence identity overall Between the three trypanosomatids, isoenzyme (Tb1, Lm1, Tc1–1 and Tc1–2) representatives share 35–43% sequence identity The corresponding figures for isoenzymes 2, and are 43–49, 37–52 and 33–40% (Table 2) Tb1 Tb2 Tb3 Tb4 500 residues Fig Diagrammatic representation of the domain structure of the various PFK-2 ⁄ FBPase-2 isoenzymes of T brucei The domain structure of the enzymes was inferred from the amino acid sequences predicted from the ORFs Light grey, FBPase-2 domain; dark grey, PFK-2 domain; white, insertions ⁄ extensions compared with mammalian PFK-2 ⁄ FBPase-2 sequences FEBS Journal 272 (2005) 3542–3560 ª 2005 FEBS Mammalian and yeast enzymes usually have molecular masses of 50–60 kDa By comparison, most of the trypanosomatidal homologues are atypically large (Table 2) Most notable are the isoenzymes 1, which range in size from 1021 residues (112 kDa) for Tc1–1 to 2422 residues (251 kDa) for Lm1 The L major representative of the isoenzymes is also particularly large (1241 residues; 132 kDa) compared with the corresponding trypanosomal sequences at around 700–750 residues The bulk of known homologues of the bifunctional enzymes possess both kinase and phosphatase activities Nevertheless, there is a precedent for homologues having retained only a single activity in the three yeast members of the family In order to predict likely activities for the trypanosomatidal sequences, an analysis was made of the conservation (or lack of conservation) of catalytic and substrate binding-site residues in each domain For this purpose, we defined sets of key catalytic residues for each of the PFK-2 and FBPase-2 activities (boxed and highlighted in Fig 3): nonconservative replacement of any of these residues would be expected to abolish activity For PFK-2 activity key catalytic residues were Lys51, Thr52, Asp128 and Lys172 Site-directed mutation of these residues 3545 PFK-2 ⁄ FBPase-2 of Trypanosomatidae N Chevalier et al A B Fig Sequence alignments of the kinase domain (A) and bisphosphatase domain (B) In each case, the trypanosomatid sequences are compared with domains of confirmed activity, rat testis bifunctional enzyme (PDB code 2BIF) [24] in both cases, and the respective monofunctional S cerevisiae enzymes [SWISSPROT codes 6P21_YEAST in (A) and F26_YEAST in (B)] Numbers substitute large insertions to the rat testis enzyme For clarity, only one of each pair of T cruzi homologues is shown Rat testis enzyme numbering is shown beneath the alignment Key catalytic residues and additional binding residues are boxed, with the former also shown emboldened and italicized Within each box shading is used for functional conservation of the particular residue, i.e as an indication that the residue present would have the same capacity for electrostatic interaction, hydrogen bonding or hydrophobic interaction, as the residue present in rat testis enzyme The figures were produced with ALSCRIPT [88] 3546 FEBS Journal 272 (2005) 3542–3560 ª 2005 FEBS N Chevalier et al drastically reduces activity [33–35] Similarly, at the FBPase-2 site, Arg255, His256, Arg305, Glu325 and His390 were considered to be key residues [36–38] We also considered other residues involved in substrate binding (boxed in Fig 3) based mainly on X-ray crystal structures The binding of ATP analogues in the PFK-2 active site and of fructose 6-phosphate and inorganic phosphate ions to the FBPase-2 active site have been visualized crystallographically [24,25,39] The binding site for fructose 6-phosphate in the PFK-2 catalytic site has yet to be directly visualized, but conserved residues in the vicinity have been the subject of several docking and site-directed mutagenesis studies enabling modelling of fructose 6-phosphate binding [26,35,40–42] Striking differences in the patterns of conservation of these residues were immediately apparent For isoenzyme 1, all key kinase catalytic residues were conserved and additional substrate-binding residues were also well conserved (Fig 3A; Table 2) In sharp contrast, no FBPase-2 key catalytic residues were conserved (Fig 3B; Table 2) For example, His256, which is transiently phosphorylated during the catalytic cycle of FBPase-2, is replaced by proline in all isoenzyme sequences Comparisons of whole domains tell a similar tale – the isoenzyme kinase domains are 23–41% identical to presumed active PFK-2 domains, whereas the corresponding range for the bisphosphatase domains is 18–25% Similarly, the isoenzyme sequences are better conserved in their N-terminal domains (59–68% sequence identity, excluding the comparison of Tc1–1 and Tc1–2) than in their C-terminal domains (40–59%) These data strongly suggest that Tb1, Lm1 and Tc1 homologues are monofunctional PFK-2s Lm2 also conserves the key catalytic kinase residues and none of the changes in other substrate binding residues seems incompatible with kinase activity, although Asn63 and Arg193, predicted to hydrogen bond to fructose 6-phosphate, are replaced by Phe and Val, respectively Surprisingly, the trypanosomal representatives of isoenzyme have nonconservative replacements in the key kinase residues; Tb2 has Met and Ala for Thr52 and Asp128, whereas both T cruzi sequences replace Asp128 with Asn (Fig 3A; Table 2) Given the key role of Asp128 in coordination to the catalytically essential divalent metal cation [24], these substitutions appear to rule out kinase activity for Tb2, Tc1–1 and Tc1–2 Indeed, additional substrate binding residues are less well conserved in these sequences At the FBPase-2 site, both key catalytic residues and additional substrate-binding residues are poorly conserved (Fig 3B; Table 2) In particular, the replacements of His256 and Glu325 rule out FBPase-2 FEBS Journal 272 (2005) 3542–3560 ª 2005 FEBS PFK-2 ⁄ FBPase-2 of Trypanosomatidae activity for isoenzyme Comparisons of whole domain conservation are uninformative: the kinase domains of the isoenzymes are 13–29% identical to active kinase domains, whereas their bisphosphatase domains are 22–32% identical to other bisphosphatase domains Surprisingly, given the patterns of residue conservation, the C-terminal domain is slightly better conserved between isoenzyme sequences (58–65%, again excluding the comparison of Tc2–1 and Tc2–2) than the N-terminal domain (47–58%) Taken together, the data suggest that Lm2 probably has monofunctional kinase activity but that experimental characterization would be needed to confirm the prediction Tb2, Tc1–1 and Tc1–2 are predicted, surprisingly, to have neither kinase nor FBPase-2 activity For isoenzyme 3, clear-cut predictions may once again be made In the kinase domain, key catalytic residues are absent from all sequences and other substrate-binding residues are not conserved (Fig 3A) At the FBPase-2 catalytic site, all residues are very highly conserved in all four sequences Similarly, the kinase domains of isoenzyme sequences are just 9–15% identical with active domain sequences, whereas the bisphosphatase domain is well conserved at 25–36% identity Also, the C-terminal domain is much better conserved in an intertrypanosomatid comparison (62–73%) than the N-terminal domain (16–34%) Thus Lm3, Tb3, Tc3–1 and Tc3–2 would clearly be monofunctional FBPase-2 enzymes In the kinase domain of isoenzyme 4, the replacement of Asp128 with Asn in all four sequences, as well as various substitutions of Lys172, rules out kinase activity Additional substrate-binding residues are also poorly conserved (Fig 3A; Table 2) Isoenzyme sequences are also much better conserved, compared with active domain homologues, in the bisphosphatase domain than in the kinase domain Conservation in the bisphosphatase domain is in the range 26–40% compared with just 13–28% in the kinase domain The corresponding figures for the intertrypanosomatid isoenzyme comparison are 47–59% for the bisphosphatase domain and 34–53% identity in the kinase domain The T cruzi sequences have all the required FBPase-2 key catalytic residues and well-conserved additional substrate-binding residues The other two isoenzyme sequences have nonconservative replacements of key catalytic residues; the substitution of Arg255 by Leu in Lm4 and the replacement of His256 by Asn in Tb4 These argue against their having FBPase-2 activity, but in each case mutations elsewhere in the catalytic site make it difficult to completely rule out activity The loss of Arg255, a residue that binds the 2-phospho group of substrate and the 3547 PFK-2 ⁄ FBPase-2 of Trypanosomatidae phosphohistidine intermediate [37,43], could be partially compensated by the presence of neighboring His416 (replacing Leu in rat testis) which could form an ionic interaction with the 2-phosphate group Equally, the lack of phosphorylable His256 in Tb4 would typically be thought to be sufficient to abolish bisphosphatase activity However, experiments on FBPase-2 and relatives show that caution should be exercised Most importantly, when this His was replaced by Ala in the rat testis enzyme, a surprising 17% of catalytic activity was maintained [44], probably due to water taking over the nucleophilic role [39] It is also relevant to note the surprising variations in key catalytic residues in members of the related phosphoglycerate mutase superfamily [45] In the case of Tb4, it is also intriguing to note the replacement of Asn262 (in rat testis enzyme) with an additional His at position 262 The new His is well placed to interact with the 2-phosphate group of the incoming substrate However, it seems unlikely that this new His may take over the role of the missing His256 to form the phospho-enzyme intermediate, as it is not suitably positioned for in-line attack on the 2-phosphate group of the substrate In summary, although Tc4–1 and Tc4–2 are probably monofunctional bisphosphatases, experimental data would be required to test the possibility of Lm4 and Tb4 sharing the same function Heterologous expression, purification and characterization of T brucei PFK-2/FBPase-2 We set out to test experimentally the results of the bioinformatics analysis of sequences retrieved from the databases To that end, PCR amplification was first performed for Tb1 and Tb4 fragments, using as template genomic DNA from our laboratory strain, T brucei stock 427 The fragments thus obtained were used to screen an available genomic library DNA fragments were subcloned in plasmids and sequenced All clones obtained contained either of the two distinct genes The Tb1 and Tb4 sequences in the database of T brucei stock TREU927 ⁄ have the same length as the proteins encoded by the genes analysed by us for T brucei stock 427 (Table 2), but differ by a number of substitutions For Tb1, substitutions were found at five positions (Thr18Met, Pro51Leu, His384Arg, Ala422Gly and Lys630Glu) Only the latter substitution, corresponding to position 138 in the rat testis enzyme [24], is within the PFK-2 domain, but the residue is not part of the active site The other four positions lie in the N-terminal extension The Tb4 amino acid sequences of stocks TREU927 ⁄ and 427 differ at 12 positions by single amino acid changes; two 3548 N Chevalier et al substitutions (Ser–Asn at position 536 of the fulllength predicted protein and His–Gln at position 589) are within the PFK-2 ⁄ FBPase-2-specific region, but are not expected to have any consequence for enzyme activity The first of these positions is on an insertion relative to the rat testis enzyme and the second corresponds to position 347 These differences should, most likely, be attributed to polymorphisms between the T brucei strains used by us and in the genome-sequencing project For Tb2, the full-length gene of stock 427 was amplified and sequenced No differences were found between the Tb2 nucleotide sequences of the two stocks To prove the identity of Tb1 as an active PFK-2, and to confirm that the Tb2 is inactive as predicted, we expressed the proteins in a heterologous system However, it was anticipated that the large Tb1 polypeptide as predicted from the full-length open reading frame (ORF), would be difficult to express as a soluble active protein Therefore, a shorter part comprising the region homologous to the bifunctional PFK2 ⁄ FBPase-2 enzymes of higher eukaryotes was chosen for expression A protein starting at codon ATG 505 (giving the N-terminal sequence MSSSYTTVSDAVSL-) corresponds quite well with the beginning of the structurally resolved part of the rat testis bifunctional enzyme and from where good alignment is possible (the multiple alignment in Fig 3A starts with the underlined last three residues) Moreover, this protein still contains the region corresponding to the N-terminal part of other PFK-2s that is involved in dimerization The shorter ORF codes for a polypeptide of 519 amino acids (including the initiator methionine), with a calculated molecular mass of 57 078 Da and a pI value of 9.29 When expressed with a His-tag, as described in Experimental procedures, a protein of 547 residues with a predicted molecular mass of 60 306 Da and a pI of 9.29 is produced Under rather specific growth conditions, adapted from Oza et al [46], low amounts of soluble enzyme could be obtained that indeed displayed PFK-2 activity The protein was partially purified On SDS ⁄ PAGE several bands were visible, but the identity of a polypeptide of Mr  60 000 was confirmed as Tb1 after western blotting and immunodetection with anti-(poly His) sera (not shown) Expression of larger constructs was also attempted, both using Escherichia coli cells and in vitro, in a coupled transcription–translation system (Rapid Translation System, Roche Molecular Biochemicals), using different vector systems, differently placed tags for affinity purification, and a variety of conditions for bacterial growth and induction of FEBS Journal 272 (2005) 3542–3560 ª 2005 FEBS N Chevalier et al protein expression However, in most cases very poor expression of an inactive, unstable protein was obtained, or the protein was expressed as insoluble inactive enzyme The active 60 000 Mr Tb1 was subjected to a preliminary kinetic analysis The K mapp for fructose 6-phosphate varied between 1.9 ± 0.12 and 4.6 ± 0.8 mm, whereas the K mapp for ATP was between 1.6 ± 0.27 and 2.0 ± 0.30 mm in four different preparations of enzyme These values are similar to those for the enzyme partially purified from bloodstream-form trypanosomes (see above) and to values reported previously [4] With regard to their PFK-2 activity, the various mammalian isoenzymes display much lower Km values: 15–150 times for fructose 6-phosphate and 3–20 times for ATP [4,16] The relatively good conservation of the ATP-binding site residues between mammalian and Tb1 proteins suggests that the explanation for the lower ATP affinity of the latter must lie with the three significantly different positions, residue 220 (Val in mammalian enzymes, Lm1 and Tc1, but Ala in Tb1), residue 246 (Val or Ile in mammalian enzymes, Pro in the trypanosomatid proteins) and position 427 (Tyr in mammalian enzymes, Gly, Glu and Asp in Lm1, Tc1 and Tb1, respectively) The branched side chains of residues 220 and 246 form the side of the adenine-binding pocket further away from the catalytic site and each make multiple hydrophobic interactions with the heterocyclic ring Their replacement with nonbranched Ala and Pro would reduce the steric complementarity of adenine and its pocket, thereby reducing the strength of the interaction Also, the hydrogen bond from Tyr427 to the a-phosphate of ATP is not present in Tb1 Instead, the replacement Asp could, assuming local correctness of the sequence alignment, lead to electrostatic repulsion of the negatively charged phosphate groups of ATP Bacterially expressed Tb1 was analysed by gel filtration over a Superdex 200 HR 10 ⁄ 30 column, to determine its oligomeric state However, under all conditions tested (various buffers, variable ionic strength, presence of reducing agents – Experimental procedures) all Tb1, as detected by western blotting using an antiserum specific for the His6-tag, eluted as an entity of high mass (> 600 kDa) with a low PFK-2 activity In addition, a second protein peak with higher PFK-2 activity eluted with a relative molecular mass of  140 kDa, suggesting it was a dimer Purified rat liver PFK-2 ⁄ FBPase-2, used as a control, eluted as a 110 kDa dimer as detected by both PFK-2 activity and western blotting using a homologous antiserum These results suggest that the bacterially produced Tb1 FEBS Journal 272 (2005) 3542–3560 ª 2005 FEBS PFK-2 ⁄ FBPase-2 of Trypanosomatidae enzyme is an active dimer that has a strong tendency to aggregate In contrast to Tb1, no activity was found for bacterially expressed, soluble Tb2, in agreement with the predictions of the sequence analysis Surprising in this respect is that the Tb2 gene sequences found in the two different T brucei stocks were identical and that an Expressed Sequence Tag corresponding to Tb2 has been found in procyclic T brucei rhodesiense libraries (GenBank accession no AA689209.1) This suggests that this protein, despite its lack of PFK-2 activity, may play a role in these trypanosomes The fact that only Tb1 displays activity, and that no other sequences with typical PFK-2 features could be detected in any of the trypanosomatid databases, strongly suggests that the 76.4 kDa protein purified from trypanosomes is a Tb1 form, despite the fact that the complete ORF of Tb1 gene codes for a 110 kDa polypeptide We hypothesize, therefore, that the purified protein represents a processed form of the protein (see also below) Future studies will also include a detailed experimental analysis of the two T brucei proteins with likely and possible FBPase-2 activity, Tb3 and Tb4, respectively A Tb4 construct of stock 427 trypanosomes has already been expressed in E coli, although so far in mainly an insoluble form, and Tb3 will be expressed in the future FBPase-2 assays were not performed for Tb1 and Tb2, as it is inconceivable that these proteins would possess any FBPase-2 activity, as explained above (also Table 2) Evolution of PFK-2/FBPase-2 The amino acid sequences of the bifunctional PFK-2 ⁄ FBPase-2 enzymes from many organisms were retrieved from the swissprot database, aligned with those of the various trypanosomatid PFK-2 ⁄ FBPase-2 homologues and used for evolutionary analysis All the PFK-2 ⁄ FBPase-2 sequences can be conveniently divided into an N-terminal PFK-2 domain and a C-terminal FBPase-2 domain In the yeast PFK27 sequence, the FBPase-2 domain is difficult to recognize Presumably its sequence diverged considerably and was truncated during evolution The PFK-2 domain is related to a family of nucleotide-binding proteins including adenylate kinase, p21 ras, EF-Tu, the mitochondrial ATPase b-subunit and myosin ATPase, all of which have a similar fold and contain the Walker A and B motifs The FBPase-2 domain also belongs to a protein family comprising the cofactor-dependent phosphoglycerate mutases and acid phosphatases The bifunctional PFK-2 ⁄ FBPase-2 must have originated by fusion of representatives of these two families in a common 3549 PFK-2 ⁄ FBPase-2 of Trypanosomatidae ancestor of all eukaryotic organisms studied here (trypanosomatids, yeasts and fungi, plants and animals) The PFK-2 and FBPase-2 domains can be flanked by extensions of variable lengths In plants, Neurospora crassa and yeast PFK26 the N-terminal extensions can be long and increase the molecular mass from  55 to 90 kDa From the ORFs, we infer that in some trypanosomatid isoenzymes, the N-terminal extensions can be even longer The extremities of mammalian isoenzymes serve as regulatory domains often containing phosphorylation sites Moreover, it has been shown that the N-terminal domain of the Arabidopsis enzyme is important both for subunit assembly and for defining the kinetic properties of the enzyme [47] In each of the yeast and trypanosomatid isoenzymes, one of the catalytic cores seems to have been inactivated, rendering the bifunctional enzyme monofunctional The sequences of PFK-2 ⁄ FBPase-2 in animals and plants form distinct clusters in phylogenetic trees made separately for the PFK-2 and FBPase-2 domains, with adenylate kinase and phosphoglycerate mutase as outgroups, respectively (Fig 4) In plants, only a single gene of the bifunctional enzyme was detected [18,48], N Chevalier et al whereas animals have different bifunctional isoenzymes, represented by four subtrees (corresponding to the liver ⁄ muscle, heart ⁄ kidney, testis and brain ⁄ placenta groups, respectively) The evolution of the PFK-2 ⁄ FBPase-2 in the lineages of yeasts ⁄ fungi and Trypanosomatidae is more difficult to deduce from the phylogenetic trees This is due to: (a) the relatively low level of conservation of these sequences, and the longer evolutionary distances when these organisms are considered; and (b) the fact that the inactivated domains of these enzymes have possibly been subject to a very high evolution rate For the same reason, the highly aberrant C-terminal domain of S cerevisiae PFK27 was omitted from the FBPase-2 tree Nevertheless, our preliminary analysis suggests that most isoenzymes of the fungi result from gene duplications within this group Furthermore, in the FBPase-2 domain tree, all the Trypanosomatidae sequences are together in one cluster separated from the sequences of all other organisms (but containing the outgroup) The isoenzymes form individual groups Isoenzymes and 2, containing all the putative PFK-2s (Lm1, Tb1, Tc1–1, Tc1–2 and Lm2), cluster together, as isoenzymes Fig Phylogenetic trees of the PFK-2 and FBPase-2 domains of both the bifunctional and monofunctional proteins All PFK-2 and ⁄ or FBPase-2 containing sequences from animals, invertebrates, fungi and protists, as obtained from the SWISSPROT ⁄ TREMBL databases (Experimental procedures) were aligned with each other using CLUSTALX [80] From this alignment subalignments were created containing either the PFK-2 domain or the FBPase-2 domain Each of the subalignments was used for the creation of a neighbour-joining tree from a matrix of uncorrected pair-wise distances using the tree option within CLUSTALX Regions with insertions or deletions were excluded from the analyses Horizontal bars represent 10 substitutions per 100 residues The trees were rooted using either an Arabidopsis thaliana chloroplast adenylate kinase or E coli cofactor-dependent phosphoglycerate mutase as an outgroup 3550 FEBS Journal 272 (2005) 3542–3560 ª 2005 FEBS N Chevalier et al and 4, containing all the likely FBPase-2s (Lm3, Tb3, Tc3–1, Tc3–2, Tc4–1 and Tc4–2) The situation for the trypanosomatids in the PFK-2 tree is very similar, except also for the presence of the S cerevisiae PFK27 in the cluster Furthermore, the phylogenetic analysis also showed that the formation of the four isoenzymes in the trypanosomatids has occurred already in the common ancestor of the genera Trypanosoma and Leishmania The presence of multiple isoforms of PFK-2 ⁄ FBPase-2 in mammals can be understood as a need for distinct enzymes each with different kinetic properties and regulatory mechanisms optimized in regulating glycolysis and ⁄ or gluconeogenesis in the different tissues [1,16] With regard to yeast, the two isoenzymes with PFK-2 activity differ in that only PFK26 is activated by protein phosphorylation [29], whereas the synthesis of PFK27 is only induced by fermentable carbon sources [21] However, the growth rates and glycolytic flux of both the PFK26 and PFK27 deletion mutants of S cerevisiae are similar to that of wild-type cells [19,21], and did not reveal an essential role of Fru2,6-P2 in the regulation of carbon fluxes in this organism [49] Nor could different roles for the two PFK-2s be demonstrated by metabolome analysis of the mutants [50] Our data not permit us to draw any conclusions as to the reason why trypanosomatids have four isoenzymes Sequence inspection and structure modelling suggested that some of them (Tb1, Lm1, Tc1–1, Tc1–2 and Lm2) are monofunctional PFK-2s, whereas others (Tb3, Lm3, Tc3–1, Tc3–2, Tc4–1 and Tc4–2) most likely only have FBPase-2 activity The sequence analysis suggested that these proteins are all soluble It did not reveal obvious topogenic signals indicative for functioning of isoenzymes in different cell compartments Only Tb2, the inactive PFK-2 of T brucei, contains a potential peroxisome-targeting signal at its C-terminus (-NKL) [51], but a similar tripeptide was not found on the corresponding sequences of the other trypanosomatids It could be imagined that isoenzymes with different properties are necessary at different stages of the life cycle of these organisms Many trypanosomatid species are pathogenic organisms with a highly complicated life cycle T brucei cycles between the mammalian bloodstream, the tsetse fly midgut and the insect’s salivary gland These are radically different environments where the parasite encounters different nutrients and has to adapt its metabolism accordingly Leishmania species undergo similar transitions between flies and mammals where they predominantly live intracellularly in the phagolysosomes of macrophages FEBS Journal 272 (2005) 3542–3560 ª 2005 FEBS PFK-2 ⁄ FBPase-2 of Trypanosomatidae Why the bifunctional PFK-2 ⁄ FBPase-2 evolved into different monofunctional enzymes in the yeasts and, most likely, also in trypanosomatids is not clear Possibly, it represents an as yet not understood adaptation to the specific requirements of glucose metabolism in these unicellular organisms, different from the requirements in the ancestral eukaryote where the fusion of the PFK-2 and FBPase-2 domains in a single enzyme occurred and different from that in extant animals and plants with their bifunctional enzymes Usually, opposite metabolic reactions are catalysed by separate enzymes; PFK-2 ⁄ FBPase-2 is an exception Moreover, bifunctionality extends to its substrate ⁄ product, Fru2,6-P2, which in higher eukaryotes (but not in trypanosomatids) has two targets, PFK-1 and FBPase-1 The advantages of the association of opposite reactions may be: (a) simplicity in short-term control, such as regulation at a single site by an allosteric effector or phosphorylation; and (b) simplicity of long-term regulation (one gene, one mRNA) One possible reason why several microorganisms have, at a later stage of evolution, again uncoupled the PFK-2 and FBPase-2 activities is that it may have endowed an increased flexibility to adapt to different growth conditions: different combinations of monofunctional PFK-2 and FBPase-2 enzymes may be expressed in cells growing in different environments However, this remains to be studied by following the expression of the different enzymes throughout the life cycle of the trypanosomatids That each of the monofunctional enzymes retained (part of) the inactivated domain is in line with the notion that both domains may be required for proper folding or oligomerization The inferred activities of some trypanosomatid isoenzymes remain to be confirmed Enzymes with similar activities may differ in kinetic and regulatory properties We have demonstrated that Tb1 has PFK-2 activity, whereas no activity could be found for Tb2, in agreement with sequence analysis predictions The apparent Km values of Tb1 for fructose 6-phosphate and ATP are similar to those of the enzyme partially purified from the bloodstream-form trypanosomes (Table 1) [4] The lack of FBPase-2 activity in this enzyme is highly likely It should be noted, however, that we not know if the absence of N-terminal domains may have affected the activity of the bacterially expressed enzyme It is interesting to note that Expressed Sequence Tags corresponding to Tb2 (GenBank accession no AA689209.1; unpublished data) and Tb3 (T26149) [52] have been obtained from procyclic T brucei rhodesiense libraries Our unpublished data (N Chevalier and P A M Michels, unpublished) showing the presence 3551 PFK-2 ⁄ FBPase-2 of Trypanosomatidae of Tb1 protein during the same procyclic stage, suggest that proteins with opposite catalytic activities (Tb1 is a PFK-2; Tb3 is strongly predicted to have FBPase-2 activity) are simultaneously present during the trypanosomatid life cycle, reinforcing the notion that the trypanosomatid enzymes should be subject to regulation Indeed, we have shown that the activity of PFK-2 purified from trypanosomes can be regulated by phosphorylation Conserved motifs in the N-terminal regions of trypanosomatid PFK-2s The ORFs of trypanosomatid isoenzymes 1, and extend well upstream of the region corresponding to the mammalian bifunctional enzymes (Fig 2) The predicted amino acid sequences contain normal amounts of predicted regular secondary structure Except for a small region (see below), no obvious homology with other sequences in databases was observed In the gene sequences, no trypanosomatid-specific motifs indicative of RNA trans-splicing [53,54] could be found Cissplicing is very rare in these organisms and only one example has been reported to date [55] The possibility of splicing was further explored To that end, PCR experiments were performed with different sets of primers, covering the various parts of the Tb1 (TbPFK-2) ORF, using as a template cDNA prepared with oligo-dT as primer on total RNA from the cultured bloodstream form of T brucei The results confirmed that the entire ORF was present as a single mRNA (not shown) Among the trypanosomatid sequences, the upstream regions are not conserved either, except for an approximately 135-residue stretch that could be detected in the putative PFK-2s – isoenzymes and Searches in primary and secondary structure databases revealed the presence of two ankyrin repeats within this region For example, an e-value of 1e)16 was obtained for entry cd00204, comprising four ankyrin repeats, when searching in the Conserved Domain Databases (CDD) with the Lm1 N-terminal extension sequence Only 60% of the cd00204 entry was matched to the Lm1 sequence and searches with the other trypanosomal homologues in secondary sequence databases also gave hits for just two ankyrin repeats Because small numbers of consecutive ankyrin repeats are rare, apparently for stability reasons [56], we carried out more sensitive fold recognition experiments With the prekinase domain portions of isoenzymes and 2, more ankyrin repeats were revealed, both within the conserved region shared by the four sequences and in the remaining portions not apparently homologous 3552 N Chevalier et al between the sequences of isoenzymes and Although the fold-recognition scores were highly significant for various ankyrin repeat structures in the PDB, differences in the extent of matches, resulting in different predicted numbers of ankyrin repeats, were evident in the results of different fold-recognition methods However, these uncertainties were confined to the apparently nonhomologous parts of the prekinase domain regions In the conserved region, there was an excellent match between predicted secondary structure and actual secondary structure of a designed artificial ankyrin repeat (PDB code 1MJ0) [57], for example (Fig 5) It should be remembered that the characteristic b-turns of the ankyrin repeat structure (shown as pairs of arrows in Fig 5) are not predicted by 3-state secondary structure prediction programs With the exception of T cruzi isoenzyme sequences, the conserved region clearly contains four ankyrin repeats In Tc2–1 and Tc2–2, a deletion compared with the other trypanosomatid sequences is evident, corresponding exactly to a whole ankyrin repeat The b-turn prior to the first helix pair is not present, but this is often the case for ankyrin repeat structures including the designed ankyrin protein included in the alignment The pairwise sequence identity between Lm1, Tb1 and Tc1–1 in this region is between 60 and 65%, whereas Lm2, Tb and Tc1–2 share 27–43% identity The two groups share 23–29% identity between them, whereas the trypanosomal sequences share 26–33% sequence identity with the designed ankyrin repeat protein [57] Outside the portions shown in Fig 5, additional ankyrin repeats are present in each of the four homologues (Fig 6) Although the sequences seem to diverge more from the standard ankyrin repeat consensus, fold recognition alignments and secondary structure predictions of characteristic pairs of helices (as in Fig 5) provide strong evidence for extra repeats In Lm1, up to seven repeats may be present immediately following the conserved region in Fig However, this still leaves around 500 residues after the extra repeats before the start of the kinase domain, for which no structure may be proposed In the cases of Tc1–1, Tc1–2 and Tb1, there is evidence for three ankyrin repeats before the conserved region and the same number following For Lm2 and Tb2, there appears to be a single additional ankyrin repeat before the conserved region and another immediately following (Fig 6) Tc2–1 and Tc2–2 have a single clear ankyrin repeat following those shown in Fig These different numbers of ankyrin repeats most likely reflect the results of domain duplication and ⁄ or deletion The results (not shown) of the radar repeat detection program [58] for Tc1–1, Tb1 and Lm2 suggest that the third and FEBS Journal 272 (2005) 3542–3560 ª 2005 FEBS PFK-2 ⁄ FBPase-2 of Trypanosomatidae N Chevalier et al Fig Ankyrin motifs in trypanosomatid PFK-2 ⁄ FBPase-2 sequences Alignment of the four ankyrin repeats in the conserved upstream region of the putative trypanosomal PFK-2 sequences with the designed ankyrin repeat protein (1MJO) of Kohl et al [57] For clarity, only one of each pair of T cruzi homologues is shown Regular secondary structure predicted by PSIPRED [79] is shown above the alignment for the L major sequences and the true secondary structure of 1MJO below the alignment Cylinders represent a helices, whereas pairs of arrows are used for the b turns Shading marks positions where the residue of the trypanosomal homologue is identical to that of the crystal structure and positions well conserved among the homologues are emboldened The figure was produced with ALSCRIPT [88] fourth repeats in Fig arose through a duplication Similarly, the two repeats predicted to immediately precede the conserved region in Tb1 and Tc1 have similarity detectable by radar The same program suggests that the region beginning with the clear ankyrin repeats shown in Fig and extending to the start of the kinase domain in Lm1 may consist largely of five highly diverged duplicated sequences The first of these copies likely contains six ankyrin repeats, the last three of the four shown in Fig along with three more Although the clear independent evidence for ankyrin repeats in the second copy supports the idea Tb1 Lm1 Tc1_1 Tb2 Lm2 Tc2_1 500 residues Fig Diagrammatic representation of ankyrin-motif repeats in Tb1, Lm1, Tc1–1, Tb2, Lm2 and Tc2–1 PFK-2 and FBPase-2 domains in the C-terminal halves of the proteins are indicated as in Fig In the N-terminal halves, the ankyrin motifs with a primary structure having high similarity to the consensus motifs are indicated in darker grey than the motifs showing less conservation of the primary structure, but having a repeat length and predicted secondary structure that conform to the consensus FEBS Journal 272 (2005) 3542–3560 ª 2005 FEBS of the duplication, the alignment of the five copies reveals only poor sequence conservation overall so that a definitive conclusion is impossible The ankyrin repeat is a protein sequence motif that is widespread in nature It is found in proteins from bacteria, fungi, plants and animals, and is present in proteins at different locations of the cell as well as in secreted proteins Its function is to mediate protein– protein interactions Within a protein, the motif can form distinct units of 2–20 consecutive stacking repeats with a hydrophobic core The units thus form scaffolds with a stability depending on the number of repeats and displaying a variable surface, each optimized to interact specifically with one out of a wide range of other, unrelated nonankyrin repeat-containing macromolecules [56,57,59] The trypanosomatid PFK-2 molecules seem to be produced as long molecules with a variable number of authentic ankyrin-motif repeats in their N-terminal extensions It also seems likely that in these trypanosomatid proteins, the ankyrin repeat structures are involved in establishing interactions between proteins However, it remains to be determined with which proteins Known interaction partners of homologues from other species include other enzymes of sugar metabolism – glucokinase [60] and PFK-1 [61] – but also 14-3-3s proteins whose binding to cardiac [62] and plant [63] enzymes regulates their activity Other attractive candidate partners for the trypanoso3553 PFK-2 ⁄ FBPase-2 of Trypanosomatidae N Chevalier et al matid enzymes are the (putative) FBPase-2s, the isoenzyme sequences, which have a long N-terminal extension that may contain a binding site for the PFK-2 ankyrin repeats However, there is no detectable similarity between the N-terminal extensions of Lm4 and Tb4 The notion of a noncovalent interaction between distinct PFK-2 isoenzymes and a FBPase-2 would combine the advantages of both the flexibility offered by different combinations of multiple isoenzymes and the formation of a bifunctional (in this case heteromeric) enzyme, as discussed above However, alternative functions for the ankyrin repeat motif cannot be excluded For example: (a) mediating the interaction of PFK-2 with other proteins in the cell, such as cytoskeleton constituents or membrane proteins; or (b) targeting the PFK-2 precursor protein to a possible macromolecular complex for processing Further research is required to unravel the complex picture of the PFK-2 ⁄ FBPase-2 isoenzymes in the Trypanosomatidae cose 6-phosphate before loading a DEAE–Sepharose column (5 · 6.5 cm) After extensive washing with 20 mm Hepes pH 7.5, 50 mm KCl, 2.5 mm MgCl2, 0.5 mm EDTA, 0.25 mm EGTA, mm benzamidine ⁄ HCl, 20% (v ⁄ v) glycerol, 15 mm 2-mercaptoethanol, mm potassium phosphate, 0.2 mm phenylmethanesulfonyl fluoride, lgỈmL)1 antipain, 0.1 mm fructose 6-phosphate and 0.3 mm glucose 6-phosphate (buffer A), the column was eluted in a linear salt gradient (0–0.75 m KCl) in 500 mL buffer A Fractions (10 mL) were pooled, diluted with an equal volume of buffer A and applied to a column of Blue Sepharose (1.5 · cm) equilibrated with buffer A After washing overnight with 500 mL buffer A supplemented with 0.2 m KCl, the enzyme was eluted in buffer A supplemented with 0.18 m KCl and mm MgATP Fractions containing activity were pooled, concentrated by ultrafiltration, dialysed overnight against 500 mL of buffer A and stored in aliquots at )80 °C Conclusions Encouraged by the near-complete status of various trypanosomatid genome projects, we carried out searches in trypanosomatid genome databases These employed the blast [65] facilities of the GeneDB resource (http://www.genedb org) [66] which unites nucleic acid and protein sequence information obtained from the T brucei and L major genome projects and a separate resource (http://www TcruziDB.org/) [67] for T cruzi Sequences of confirmed PFK-2 ⁄ FBPase-2 homologues were obtained from the enzyme database [68] and supplemented with others seen in blast searches of the nr database [69] These homologues are all presumed to be bifunctional with the exception of the three yeast enzymes [19–21] Searches in secondary sequence databases were carried out at the CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) [70] Fold recognition experiments employed metaserver (http://www.bioinfo.pl/Meta/) [71] and their results were interpreted in the light of the Livebench benchmarking effort (http://www.bioinfo.pl/LiveBench/) [72] Sequences were aligned using t-coffee [73] or muscle [74] and the resulting alignments hand-edited and manipulated using jalview [75] modeller [76] was used to calculate percentage identities between sequences, using the formula number of identities divided by the length of the shorter sequence [77] The likely structural and functional effects of sequence differences between the trypanosomal and mammalian homologues were envisaged using the available crystal structures and the program o [78] Protein secondary structure predictions were made with psipred [79] For a phylogenetic analysis, sequences for most of the organisms were taken from the SWISSPROT database (6P21_YEAST, 6P22_YEAST, F261_BOVIN, F261_ HUMAN, F261_RAT, F262_ARATH, F262_BOVIN, F262_HUMAN, F262_MOUSE, F262_RAT, F262_ Unexpectedly, trypanosomatids have been shown to contain four genes for PFK-2 ⁄ FBPase-2 isoenzymes, or four pairs in the case of T cruzi Mirroring the situation in S cerevisiae, each of the four trypanosomal homologues, which seem to have arisen through trypanosomatid lineage-specific duplications, is predicted to be, at most, monofunctional It seems likely that expression of multiple monofunctional enzymes offers additional flexibility of metabolic regulation in the trypanosomes whose life cycles involve dramatic environmental shifts One T brucei homologue was demonstrated to possess PFK-2 activity when expressed in E coli Its kinetic parameters match those of PFK-2 purified from bloodstream-form T brucei, which is regulated by phosphorylation as seen in other species Most surprisingly, all predicted kinases contain ankyrin repeats in long, variably sized N-terminal extensions, which presumably interact with protein targets yet to be identified Experimental procedures Purification of PFK-2 from T brucei Bloodstream-form trypomastigotes of T brucei stock 427 were grown in rats and purified, and lysates prepared as described previously [64] To a high-speed supernatant fraction (1500 mL) from  200 g wet weight of trypanosomes was added 15 mm 2-mercaptoethanol, mm potassium phosphate, 0.2 mm phenylmethanesulfonyl fluoride, lgỈmL)1 antipain, 0.1 mm fructose 6-phosphate and 0.3 mm glu- 3554 Database searches, sequence alignment, structure modelling and phylogenetic analysis FEBS Journal 272 (2005) 3542–3560 ª 2005 FEBS N Chevalier et al SOLTU, F262_SPIOL, F263_BOVIN, F263_HUMAN, F263_RAT, F264_HUMAN, F264_RAT, F26L_CHICK, F26 °CAEEL, F26_YEAST) and for Schizosaccharomyces pombe (Q8TFH0 and T50154) and Neurospora crassa (Q9P522) from the TREMBL database All sequences were aligned together with the trypanosomatid sequences using clustalx [80] Adenylate kinase (KADC_ARATH) and cofactor-dependent phosphoglycerate mutase (GPMB_ECOLI) were added as outgroups for the PFK-2 and FBPase-2 domains, respectively The two separate domains were used for the creation of phylogenetic trees using the tree option of clustalx after exclusion of positions with gaps Owing to the low degree of some of the pairwise identities between sequences, no correction for multiple substitution could be applied Therefore, branch lengths represent observed distance percentages between sequences rather than evolutionary distances Construction of expression clones, production and purification of recombinant T brucei PFK-2 Fragments of two potential PFK-2 ⁄ FBPase-2 sequences (denoted Tb1 and Tb4, see Results and Discussion section), as recognized in the databases of the T brucei (stock TREU927 ⁄ 4) genome project, were amplified on T brucei stock 427 genomic DNA and used as a radioactively labelled hybridization probe to screen a genomic library of this strain prepared in E coli with the phage vector kGEM11 (Promega, Madison, WI) [81] Plaques of positive clones were processed, the DNA was purified and appropriate restriction fragments containing the PFK-2 and FBPase-2 genes were subcloned in plasmid vectors Each part of the genes was sequenced at least once in both directions, using a Beckman CEQ 2000 sequencer (Beckman Instruments, Fullerton, CA, USA) A third gene, coding for the protein Tb2, was amplified over its full length, cloned and sequenced In order to express Tb1 in E coli, different parts of the ORF, each with a different potential start codon but the same stop codon (Results and Discussion) were amplified by PCR, using a sense oligonucleotide containing a NdeI restriction site just upstream of the chosen start codon and an antisense oligonucleotide with an XhoI site immediately before the PFK-2 stop codon The PCR products of the expected size were purified and ligated into the pGEM-T Easy vector (Promega) After checking their sequence, the amplified fragments were excised from the recombinant plasmid by digestion with NdeI and XhoI and ligated in the expression plasmid pET28a (Novagen, Inc., Madison, WI, USA), digested with the same enzymes E coli BL21(DE3) cells were transfected with these constructs Each plasmid directs the synthesis of a Tb1 with a 20-amino acid N-terminal extension including six adjacent His residues (‘Histag’) and an eight-residue C-terminal extension, also having a His6-tag To develop a Tb2 expression construct, a gene- FEBS Journal 272 (2005) 3542–3560 ª 2005 FEBS PFK-2 ⁄ FBPase-2 of Trypanosomatidae internal NdeI site was mutated first by introducing a silent, single-nucleotide substitution (using the QuickChange protocol of Stratagene) Subsequently, the amplified full-length gene, with NdeI and BamHI sites at the start and stop codon positions, was ligated in the corresponding sites of pET28 Cells harbouring a recombinant plasmid Tb1 or Tb2 were grown at 37 °C and 250 r.p.m in 500 mL of Terrific Broth medium [82] supplemented with 30 lgỈmL)1 kanamycin When the culture reached an D600 of  1.4, the temperature was reduced to 30 °C, agitation was reduced to 100 r.p.m and isopropyl thio-b-d-galactoside was added to a final concentration of 0.5 mm to induce the expression of the protein and growth was continued for about 16 h Cells were collected by centrifugation (3000 g, 15 min, °C) and resuspended in 30 mL of cell lysis buffer containing 20 mm Tris ⁄ HCl, pH 8, 0.5 m NaCl, mm MgCl2, 0.03% (w ⁄ v) Brij35, 0.1 mm fructose 6-phosphate, 0.3 mm glucose 6-phosphate and a protease inhibitor mixture (Roche Molecular Biochemicals, Mannheim, Germany) Cells were lysed by two passages through a SLM-Aminco French pressure cell (SLM Instruments, Inc., Urbana, IL, USA) at 90 MPa Nucleic acids were degraded by treatment with 125 units of Benzonase (Merck, Germany) for 10 at 37 °C The lysate was centrifuged (12 000 g, 10 min, °C) and TbPFK-2 was purified from the soluble cell fraction by metal-affinity chromatography (TALON resin, BD Biosciences-Clontech, Franklin lakes, NJ, USA) Briefly, mL of resin was added to the suspension and mixed for 20 at room temperature The resin with bound protein was washed twice for 10 with 20 mL lysis buffer (with centrifugation at 700 g, min), transferred to a column and washed twice again with mL lysis buffer supplemented with and 10 mm imidazole, respectively Finally, protein was eluted with 10 mL lysis buffer containing 50 mm imidazole and 1-mL fractions were collected EDTA and dithiothreitol were immediately added to each fraction at concentrations of 2.5 and mm, respectively Protein measurements, SDS/PAGE, western blotting, gel filtration Protein concentrations were determined using the Bio-Rad (Hercules, CA) protein assay, based on the Bradford Coomassie Brilliant Blue-binding procedure [83], using bovine serum albumin as a standard SDS ⁄ PAGE was carried out by the Laemmli method [84] After electrophoresis, gels were either stained with Coomassie Brilliant Blue, or used for immunoblotting according to the method of Towbin [85] The membranes (polyvinylidene difluoride membrane, Roche Molecular Biochemicals) were blocked by incubation in phosphatebuffered saline (NaCl ⁄ Pi) containing 0.1% Tween 20 and 5% (w ⁄ v) low-fat milk powder For detection of the protein, the primary antibody (monoclonal anti-His serum, 3555 PFK-2 ⁄ FBPase-2 of Trypanosomatidae Amersham Biosciences, Amersham, UK) was diluted (1 : 15,000–25 000) in NaCl ⁄ Pi containing 0.5% milk powder The secondary antibody, anti-(mouse IgG) conjugated to horseradish peroxidase (Rockland Immunochemicals, Inc., Gilbertsville, PA, USA), was diluted : 40 000 and visualized with the ECL Western Blotting System, a luminol-based system (Amersham Biosciences) The native molecular mass of TbPFK-2 was determined by gel filtration Protein purified from parasites (0.15 mL) was loaded on a Superose 12 column (Amersham Biosciences) equilibrated in 50 mm Hepes pH 7.5, 100 mm KCl, 0.1 mm EDTA, mm dithiothreitol, mm potassium phosphate, 0.1 mm fructose 6-phosphate and 0.3 mm glucose 6-phosphate Fractions (0.2 mL) were collected at a flow rate of 0.3 mLỈmin)1 and assayed for PFK-2 activity The native molecular mass of the bacterially expressed form of TbPFK-2 ⁄ FBPase-2 with PFK-2 activity (Tb1) was determined using a Superdex 200 HR 10 ⁄ 30 column (Amersham Biosciences) equilibrated in a buffer specified below A Tb1 preparation (2 mL), eluted from the TALON resin and concentrated to 0.25 mL, was loaded onto the column and eluted at a flow rate of 0.4 mLỈmin)1 using the equilibration buffer Fractions of 0.4 mL were collected and the presence of Tb1 was determined by enzymatic PFK-2 assays and western blotting The columns were calibrated with gel filtration standards from Bio-Rad ranging from 1350 to 670 000 Da, and purified rat liver PFK-2 ⁄ FBPase-2 For the recombinant enzyme, gel-filtration experiments were performed with various protein batches prepared in different ways When the protein had been purified under the standard conditions, using a Tris ⁄ HCl buffer, 0.5 m NaCl and various additions as described above, the equilibration buffer used contained 20 mm Tris ⁄ HCl, pH 8, 0.5 m NaCl, mm MgCl2, 0.03% (w ⁄ v) Brij35, 10% (v ⁄ v) glycerol, 0.1 mm fructose 6-phosphate, 0.3 mm glucose 6-phosphate, a protease inhibitor mixture, with or without 15 mm 2-mercaptoethanol Other batches of protein had been prepared specifically for the gel-filtration experiments with a buffer containing 50 mm Hepes ⁄ KOH, pH 7.5, 250 mm KCl, 0.03% (w ⁄ v) Brij35, 0.1 mm fructose 6-phosphate, 0.3 mm glucose 6-phosphate, 50 mm imidazole, 2.5 mm EDTA, mm dithiothreitol and a protease inhibitor mixture In these cases, gel filtration was performed with 50 mm Hepes ⁄ KOH, pH 7.5, 100 mm KCl, 0.03% (w ⁄ v) Brij35, 10% (v ⁄ v) glycerol, mm EDTA, 0.1 mm fructose 6-phosphate, 0.3 mm glucose 6-phosphate, 15 mm 2-mercaptoethanol and a protease inhibitor mixture Enzyme assay and kinetic studies PFK-2 activity was assayed in buffer containing 50 mm Tris ⁄ HCl at pH 7.1, mm potassium phosphate, mm dithiothreitol, 100 mm KCl, 20 mm KF, mgỈmL)1 of albumin and appropriate concentrations of substrates as 3556 N Chevalier et al previously described [86] For Km measurements, the concentrations of substrate were up to 10 times the Km value For fructose 6-phosphate saturation curves, the concentration of MgATP was 10 mm For MgATP saturation curves, the concentration of fructose 6-phosphate was 10 mm Kinetic constants were calculated by fitting the data to a hyperbola by nonlinear least-squares regression using the sigmaplot computer package Auxiliary enzymes (aldolase, triosephosphate isomerase and glycerol-3-phosphate dehydrogenase) and cofactors (ATP, NADH) were from Roche Molecular Biochemicals Acknowledgements This research was supported by grants from the European Commission through its INCO-DEV programme (contract ICA4-CT-2001–10075) to PM and DR, ´ the Belgian ‘Fonds de la Recherche Scientifique Medicale’ (FRSM) to PM, and the Belgian Interuniversity Attraction Poles – Federal Office for Scientific, Technical and Cultural Affairs to FO LB was supported by ´ the ‘Actions de Recherche Concertees’ 98 ⁄ 03-216 from the French Community of Belgium and is currently a Research Associate of the ‘Fonds National de la Recherche Scientifique’ (Belgium) We thank Prof Louis Hue (ICP, Brussels) for many stimulating discussions and his 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Tb2, Lm2 and Tc2–1 PFK-2 and FBPase-2 domains in the C-terminal halves of the proteins are indicated as in Fig In the N-terminal halves, the ankyrin motifs with a primary structure having high... for ankyrin repeat structures including the designed ankyrin protein included in the alignment The pairwise sequence identity between Lm1, Tb1 and Tc1–1 in this region is between 60 and 65%,