Leishmania donovani phosphofructokinaseGene characterization, biochemical properties and structure-modelling studies

Claudia Lopez1,2, Nathalie Chevalier2, Veronique Hannaert2, Daniel J. Rigden3, Paul A. M. Michels2

and Jose Luis Ramirez1,4

1Instituto de Biologıa Experimental, Universidad Central de Venezuela, Caracas, Venezuela; 2Research Unit for Tropical Diseases,

Christian de Duve Institute of Cellular Pathology and Laboratory of Biochemistry, Universite Catholique de Louvain, Brussels,

Belgium; 3CENARGEN/EMBRAPA, Brasılia, Brazil; 4Instituto de Estudios Avanzados-Ministerio de Ciencia y Tecnologıa,

Caracas, Venezuela

The characterization of the gene encoding Leishmaniadonovani phosphofructokinase (PFK) and the biochemicalproperties of the expressed enzyme are reported.L. donovanihas a singlePFKgene copyper haploid genome that encodesa polypeptide with a deduced molecular mass of 53 988 anda pI of 9.26. The predicted amino acid sequence contains aC-terminal tripeptide that conforms to an established signalfor glycosome targeting. L. donovani PFK showed mostsequence similarity to inorganic pyrophosphate (PPi)-dependent PFKs, despite being ATP-dependent. It therebyresembles PFKs from other Kinetoplastida such asTrypanosoma brucei, Trypanoplasma borreli (characterizedin this study), and a PFK found in Entamoeba histolytica. Itexhibited hyperbolic kinetics with respect to ATP whereas

the binding of the other substrate, fructose 6-phosphate,showed slight positive cooperativity. PPi, even at high con-centrations, did not have any effect. AMP acted as an acti-vator of PFK, shifting its kinetics for fructose 6-phosphatefrom slightly sigmoid to hyperbolic, and increasing consid-erably the affinity for this substrate, whereas GDP did nothave any effect. Modelling studies and site-directed muta-genesis were employed to shed light on the structural basisfor the AMP effector specificity and on ATP/PPi specificityamong PFKs.

Keywords: phosphofructokinase; Kinetoplastida; allostericregulation; 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 glycolyticenzymes have been remarkably conserved during evolution,considerable sequence variability is found among PFKs ofdifferent taxonomic groups [1].

In Kinetoplastida, a taxonomic order of protozoanorganisms that includes important pathogens (species ofTrypanosoma, Leishmania, Phytomonas) to man, animalsand plants, the first seven enzymes of the glycolytic pathwayare confined to anorganelle called the glycosome [2,3]. PFKs

can be divided into ATP-dependent and PPi-dependentenzymes; the formeruseATPasphosphodonor in a reactionthat is essentially irreversible under physiological conditions,whereas the latter use PPi in a reversible reaction that can benear equilibrium in vivo [4].Wehavepreviously reported thatTrypanosoma brucei PFK, despite being an ATP-dependentenzyme, has an amino-acid sequence typical of PPi-PFKs [5].Furthermore, the activity of the T. brucei enzyme appearsonly regulated to a limited extent: effectors that modulatePFK activity in other organisms (vertebrates, yeast) such asATP, citrate, fructose 2,6-bisphosphate, ADP and Pi, haveno effect on the Trypanosoma enzyme [6,7]. Only activationby AMP was observed.

We hypothesized that an ancestor of the trypanosomesmust have possessed a PPi-dependent PFK that changed itsspecificity for phospho donor from PPi to ATP duringevolution [5]. The many structural differences between theactive site of the two classes of PFKs, and the strikingdifferences in ligand-binding properties between the humanand parasite enzymes suggest great potential for structure-based design of drugs [5,8].

For comparative purposes we decided to study thePFK of Leishmania donovani another kinetoplastidorganism that, contrary to the bloodstream-dwellingT. 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 thePFK gene of the fish parasite Trypanoplasma borreli (arepresentative of the Bodonina, a different sub-order ofthe Kinetoplastida).

Correspondence to J. R. Ramirez, Instituto de Biologıa Experimental –

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; PPi-PFK, PPi-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) � FEBS 2002 doi:10.1046/j.1432-1033.2002.03086.x

M A T E R I A L S A N D M E T H O D S

Cloning and characterization of the L. donovaniand T. borreli PFK genes

A genomic library of L. donovani (kindly donated byT. deVos, Seattle Biomedical Research Institute, WA,USA) constructed in Supercos7 vector was screened witha 500 bp fragment of the PFK gene of T. brucei as a probe[5]. One positive colony was chosen and the bacteria weregrown for purification of the recombinant cosmid DNA.

Southern analysis using the T. brucei probe showedhybridization with a 500 bp Sau3AI fragment and a 12 kbNotI fragment. The Sau3AI fragment was cloned inpBluescript II KS– (Stratagene) and subsequentlysequenced. The DNA sequence obtained had 55% identitywith the corresponding portion of the T. brucei PFK gene.This sequence was used as a probe, and to design primersfor further experiments. TheNotI fragment was gel purifiedand digested with EcoRI, and a 6.5 kb EcoRI fragmentrecognized 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 fragmentcontaining the entire L. donovani PFK gene (Fig. 1) wassubcloned in pBluescript II KS– (PFK2.2-PBSKS) andsequenced.

DNA sequences of both strands of recombinant plasmidinserts were determined by using the T7 DNA polymerasekit (Amersham Pharmacia Biotech) and [35S]dATP (NENLife Science Products), or with the Thermo Sequenasefluorescent labelled primer cycle sequencing kit (AmershamPharmacia Biotech), and LI-COR automated DNAsequence equipment.

A genomic library of T. borreli constructed in kGEM11(Promega) [9] was screened with a probe consisting of theentire T. brucei PFK gene [5]. The probe was hybridizedunder moderately stringent conditions: 3 · NaCl/Cit/0.1%SDS, in the presence of 10% dextran sulphate, at 60 �C(1 · NaCl/Cit ¼ 150 mM NaCl/15 mM sodium citrate,pH 7.0). Post-hybridization washes were carried out at60 �C for 2 h with 5 · NaCl/Cit/0.1% SDS, followed for1 h with 3 · NaCl/Cit/0.1% SDS. Ten positive recom-binants were purified and rescreened. High-titre phagelysates were prepared and DNA was purified fromphages as described previously [10]. From one recombi-nant phage a 4 kb EcoRI fragment recognized by theT. brucei probe was subcloned in plasmid pZErO-2(Invitrogen) and sequenced.

A multiple alignment of the amino-acid sequences ofATP-PFKs and PPi-PFKs was made as describedpreviously [5].

Structural analysis

MODELLER [11] was used to construct a model ofL. donovani PFK based on the known structure ofEscherichia coli PFK (PDB code 4pfk [12]). This wasthe most suitable template, given our interest in bothcatalytic and regulatory sites, among the various availablestructures of E. coli and Bacillus stearothermophilus PFK,as this 4pfk structure contains fructose 6-phosphate andADP in the active site, and ADP in the effector site. TheE. coli and L. donovani enzymes share 23% sequenceidentity overall, although functional considerations haveled to much greater conservation of the catalytic site andeffector site. Without these sites, the degree of sequenceidentity of � 20% leads to uncertainty in alignment ofsome regions. However, the conservation of the catalyticand effector sites allows reliable alignment and corres-pondingly accurate modelling of the Leishmania enzyme inthese regions of particular importance. The program O

[13] was used for visualizing structures and for its libraryof commonly observed, rotameric side-chain conforma-tions [14]. PROCHECK [15] was used for stereochemicalanalysis of models and for identifying the most likelyposition of an important one residue insertion, in theL. donovani enzyme relative to that from E. coli, at theeffector site.

Expression and purification of recombinantL. donovani PFK

The following specific primers were synthesized to amplifythe gene by PCR: (1) 5¢-CGAATCTCCATATGGAGACTCG-3¢, containing a NdeI site (underlined) adjacent tothe 5¢ end of the coding region; (2) 5¢-TAGGATCCTTACACCTTAGACGCCAG-3¢, comple-mentary to a 3¢ noncoding region followed by a BamHIsite (underlined). The total volume of the amplificationmixture was 100 lL containing 20 ng of DNA, 0.4 lM ofeach primer, 4 mM MgSO4, 200 lM each of the fourdeoxynucleotides, and 1 unit of Vent DNApolymerase withthe corresponding 1 · ThermoPol Reaction Buffer (NewEngland Biolabs). PCR was performed in a HybaidThermal Reactor (Hybaid, UK) using the followingprogram: 5 min 95 �C; 30 cycles consisting of 1 mindenaturation at 95 �C, 45 s annealing at 65 �C and1.5 min extension at 72 �C; and a final 5 min incubationat 72 �C. The amplified gene was purified and ligated to thepCR2.1-TOPO vector (Invitrogen). The resulting recombin-ant plasmid (PFKLd-TOPO) was used to transform E. colistrain XL-1 Blue, and the sequence of the insert waschecked.

A bacteriophage T7 RNA polymerase system [16] wasused to express L. donovani PFK in E. coli. The PFKgene was excised from the PFKLd-TOPO recombinantplasmid and spliced into expression vector pET28b usingthe NdeI and BamHI sites. The new recombinantplasmid named pLdPFK directs, under the control ofthe T7 promoter, the production of a fusion proteinbearing 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–Bertanimedium plus 1 M sorbitol and 2.5 mM 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.

� FEBS 2002 Leishmania donovani phosphofructokinase (Eur. J. Biochem. 269) 3979

supplemented with 30 lgÆmL)1 kanamycin and25 lgÆmL)1 chloramphenicol. Expression was induced atan A600 of 0.6–0.8 by the addition of 1 mM isopropyl thio-b-D-galactoside and growth was continued overnight. Cellswere collected by centrifugation (12 000 g, 10 min at4 �C). The cell pellet was resuspended in 20 mL of lysisbuffer (50 mM triethanolamine/HCl, pH 8.0, 300 mM

NaCl, 200 mM KCl, 1 mM KH2PO4, 5 mM MgCl2, 10%glycerol, 0.1 mM fructose 6-phosphate, 0.3 mM glucose6-phosphate and the protease inhibitors E64, leupeptinand pepstatin, each at a concentration of 1 lM). Cellswere lysed by two passages through a SML-AmincoFrench pressure cell (SML Instruments, USA) at 90 MPa.Nucleic acids were eliminated first by incubation with 500units Benzonase (Merck, Germany) for 15 min at 37 �C,and then with 10 mg of protamine sulphate for 15 min atroom temperature. The lysate was centrifuged (20 000 g15 min at 4 �C) and the supernatant used to further purifythe expressed enzyme using immobilized metal affinitychromatography (TALON resin, Clontech, USA) exploit-ing the (His)6-tag at the N-terminus of the PFK. Thecharged resin was washed with lysis buffer plus 10 mM

imidazole. The enzyme was then eluted with 100 mM

imidazole in lysis buffer. One millilter fractions werecollected to measure enzyme activity and to determine theprotein profile by SDS/PAGE.

Site-directed mutagenesis of the L. donovani PFK genewas performed by PCR techniques as described byMikaelian&Sergeant [18] andusingVentDNApolymerase.TheLeishmaniaPFKLys224 codonAAGwas changed intothe Gly codon GGG. The mutated protein was expressedand purified according to the same protocols as the wild-type enzyme.

Enzyme assays and kinetic analysis

The activity of PFK was determined by measuring thedecrease of NADH absorbance at 340 nm using a BeckmanDU7 spectrophotometer. To follow PFK during purifica-tion, a standard enzymatic assay was performed at 25 �C ina 1 mL reaction mixture containing: 100 mM triethanol-amine/HCl buffer, pH 8.0, 2.5 mM MgSO4, 10 mM KCl,2 mM fructose 6-phosphate, 0.5 mM ATP, 2.2 mM PEP,1.6 mMAMP, 0.42 mMNADH, 2 U lactate dehydrogenaseand 2 U pyruvate kinase. One activity unit is defined as theconversion of 1 lmol substrate per min under thesestandard conditions.

For kinetic analyses an assay was used in which thePFK activity was not coupled to a NAD-dependentreaction through its product ADP, as in the standardassay, but through its product fructose 1,6-bisphosphate.The assay was performed at 25 �C in a 1 mL reac-tion mixture containing 100 mM triethanolamine/HCl,pH 8.0, 2.5 mM MgCl2, 0.42 mM NADH, 0.4 U aldo-lase, 0.8 U glycerol-3-phosphate dehydrogenase and20 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 mM and dithiothreitol 0.5 mM). The effect ofthe fructose 6-phosphate concentration was determinedby fixing the ATP concentration at 1 mM, in thepresence and absence of AMP (1.5 mM) and GDP(1.0 mM).

R E S U L T S A N D D I S C U S S I O N

Analysis of kinetoplastid PFK genes

In the 30 kb insert of the cosmid obtained by screening aL. donovani genomic library, only a single gene copy ofPFK was detected. Figure 1 shows a restriction map of theinsert of recombinant plasmid PFK6.5-pBKS subclonedfrom that cosmid. The coincidence between the restrictionpattern of this cosmid and that obtained by Southernanalysis of whole Leishmania DNA, and the signal inten-sities of the bands (not shown), were consistent with thepresence of one gene copy per haploid genome. When blotsof 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 correspondto chromosomes 27b, 28 or 29 [19]. In L. amazonensis,included for comparative purposes, the probe hybridizedweakly to a band of approximately 1.7 Mb (Fig. 2, lanes 3and 4), the size of the proposed fusion product ofchromosomes 8 and 29 in the L. mexicana group [20].

The amino-acid sequences encoded by the ORFs foundin the L. donovani and T. borreli recombinants are shownin Fig. 3. The ORF in L. donovani is 1461 bp, coding for apolypeptide of 486 amino acids (excluding the initiatormethionine) with a molecular mass of 53 988 and acalculated isoelectric point (pI) of 9.26. The C-terminus hasthe tripeptide -SKV, a type 1 peroxisome-targeting signal(PTS-1) with an acceptable degeneracy of the canonicalmotif -SKL [21,22]. The same tripeptide was previouslyfound in another glycosomal enzyme of this organism,namely hypoxanthine-guanidine phosphoribosyltransferase

Fig. 2. Chromosomal assignment of the PFK genes in L. donovani and

L. amazonensis. 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. The posi-

tions of yeast chromosomes that were used as molecular size markers

are indicated at the right-hand side.

3980 C. Lopez et al. (Eur. J. Biochem. 269) � FEBS 2002

[23]. The sequence predicted from the T. borreliORF codesfor a polypeptide of 489 amino acids (excluding the firstmethionine) with a molecular mass of 53 211 and a pI 8.9.The typical PTS-1 motif -SKL was found as the C-terminaltripeptide. An excess of positively charged residues andresulting high pI are features often associated withglycosomal enzymes, particularly in T. brucei [24,25].

Figure 3 also shows the alignment of L. donovani andT. borreli PFK sequences with those of some otherorganisms. Except for two N-terminal insertions inT. borreli PFK, the new sequences share the characteristicsof T. brucei PFK as described previously [5]. Thepercentage identity in a pairwise comparison (Table 1)of the amino acid sequences of L. donovani and T. bruceiis high (70%), whereas the value obtained by comparingT. borreli PFK with the L. donovani and T. bruceienzymes is 54% in both cases. The extent of sequencesimilarity among the Kinetoplastida PFKs correspondswith 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 kinetoplastidPFKs with those from all other major taxonomic groupsare in the range 15–30%. As already previously observedfor the T. brucei PFK [5], the L. donovani and T. borrelienzymes show signatures typical of PPi-PFKs (see Fig. 3)and, in a phylogenetic analysis, they appear firmly placedwithin the cluster of the PPi-dependent enzymes, wellseparated from the nonkinetoplastid ATP-PFKs (notshown). Interestingly, the kinetoplastid PFKs showed thehighest percentage identity (37–38%) with the minor48 kDa PFK from another protist, Entamoeba histolytica.Despite the higher overall similarity and its phylogeneticrelationship with the subset of PPi-PFKs [5,29,30], it wasrecently reported that this E. histolytica PFK uses ATP asphospho donor (in contrast to the very different 60 kDaPPi-dependent PFK of this organism, see Fig. 3) [31]similar to the observation previously reported for theT. brucei enzyme [5]. The PFK activity in Leishmaniaspecies [32–34] and in T. borreli (J. Van Roy, F. Opper-does, N. Chevalier & P. A. M. Michels, unpublishedresults) is also known to be ATP-dependent.

Kinetics of Leishmania PFK

The L. donovani PFK was expressed in E. coli with anN-terminal His-tag and purified for kinetic analysis. Theactivity of the enzyme was ATP dependent. No activity (lessthan 1%) was observed when PPi (at concentrations up to5 mM) was used as alternative phospho donor. As reportedpreviously for PFK of T. brucei [7] and other Leishmaniaspecies [32], the activity of the enzyme depends hyperbol-ically on the concentration of ATP. The kinetic behaviourof the expressed PFK was determined as a function offructose 6-phosphate at fixed, saturated ATP concentration(1.0 mM), and in the presence or absence of AMP andGDP(Fig. 4). AMP, ADP and GDP are well-known effectors ofbacterial PFKs; in assays, GDP rather than ADP is oftenused as effector, because ADP, being a reaction product,may act as a competitive inhibitor with respect to ATP. Inthe absence of AMP and at low fructose 6-phosphateconcentrations (less than approximately 0.2 mM) theenzyme showed slightly cooperative binding of thesubstrate, with a Hill coefficient of 1.41. At higher substrate

concentrations, the enzyme displayed hyperbolic kinetics;the S0.5 ¼ 3.60 ± 0.48 mM. In the presence of AMP, thecurve is hyperbolic over the entire range of substrateconcentrations; AMP has a clear stimulatory effect byincreasing the affinity for fructose 6-phosphate till aKm ¼ 0.157 ± 0.028 mM. In contrast, GDP has no effectwhatsoever on the activity of the enzyme.

Our data thus showed that the expressed L. donovaniPFK binds its substrate fructose 6-phosphate in a cooper-ative manner, similar to many other PFKs, such as theenzymes frommammals [35] and bacteria (reviewed in [36]).This behaviour, and the increased affinity for the substratein the presence of AMP, have also been reported for theenzymes partially purified from cultured L. donovani andL. braziliensis [32] and forT. brucei PFK [6,7]. The observedcooperative substrate binding and allosteric activation byAMP suggest amultimeric structure for the enzyme. Indeed,T. brucei PFK was shown to be tetrameric [25], like theATP-PFKs ofmost other organisms [1]. Hyperbolic kineticshave been reported for the Trypanosoma cruzi enzyme, butthe relevance of this finding may be questioned, because theauthors described an enzyme with a 17 kDa subunit mass[37]. Despite the relatively high sequence identity ofKinetoplastida PFK and PPi-dependent enzymes, AMPstimulation has only been described for one PPi-dependentPFK, that fromNaegleria fowleri [38]. In this case the AMPeffect was attributed to promoting a more active enzymeaggregate.

Active site of kinetoplastid PFKs

Table 2 presents a summary of the active-site residues ofE. coli PFK involved in the binding of ADP and fructose6-phosphate as observed in its crystal structure [12], and thecorresponding residues in the PFKs of human, T. brucei,L. donovani, T. borreli and in the minor 48 kDa enzyme ofE. histolytica. A comparison of the kinetoplastid andhuman PFKs shows four differences in the residues involvedin nucleotide binding and three differences in the residuesinvolved in the binding of fructose 6-phosphate. The same

Fig. 3. Alignment of L. donovani and T. borreli PFK amino acid

sequenceswithotherATPandPPi 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, and the PPi-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. Symbols are: black arrows, 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. histolyticaATP-PFKs and the set of typical

ATP-PFKs, or the kinetoplastid andE. histolyticaATP-PFKs and the

set of PPi-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].

� FEBS 2002 Leishmania donovani phosphofructokinase (Eur. J. Biochem. 269) 3981

substitutionsoccur in the48 kDaATP-PFKofE. histolytica(Fig. 3) [39], as well as in one of the PFKs of the prokaryoticSpirochaetes Treponema pallidum and Borrelia burgdorferi(data not shown; GenBank accession numbers AE001195and AE001172). Indeed, the identity of active-site residues

in all these organisms is in agreement with the branchingorder in a phylogenetic analysis based on full-length PFKsequences [5,29,30]. The PFKs of these organisms form awell-supported monophyletic cluster within the PPi-PFKsubset, well separated from the typical ATP-PFKs [5,29,30].

3982 C. Lopez et al. (Eur. J. Biochem. 269) � FEBS 2002

The identity of the phospho donor of the PFKs fromT. pallidum and B. burgdorferi has not been establishedyet.

The E. coli residues involved in fructose 6-phosphatebinding that have been substituted in the kinetoplastid andrelated PFKs are Arg162, Arg243 and His249. The

Fig. 3. (Continued.)

� FEBS 2002 Leishmania donovani phosphofructokinase (Eur. J. Biochem. 269) 3983

corresponding residues found in all these PFKs are Gly, Lysand Tyr, respectively. Whereas Arg162 seems conserved inall typical ATP-PFKs, a positively charged residue at thecorresponding position seems not essential for substratebinding in the PPi-PFKs and the atypical ATP-PFKs. TheArg243 of ATP-PFKs is replaced by Lys in all PFKs of thesubset comprising the Kinetoplastida; apparently, apositively charged residue at this position is essential in allATP-PFKs. It is possibly equivalent to Lys315 of thePPi-dependent PFK of Propionibacterium freundenreichii(E. coli position 241 in Fig. 3), because Xu et al. [40] haveshown that an alteration of this residue by site-directedmutagenesis causes a 389-fold increase of the Km forfructose 6-phosphate. The substitution of His249 (E. colinumbering) by Tyr in the Kinetoplastida is also found insome PPi-PFKs such as the enzymes ofP. freudenreichii andE. histolytica (Fig. 3), and is possibly without much effect.

Out of 10 residues involved in ADP binding in E. coliPFK, four are not conserved in Kinetoplastida (Table 2).Strikingly, the ATP-dependent E. histolytica PFK and theputative ATP-PFKs of two Spirochaetes discussed abovehave the same residues as the ATP-dependent kinetoplastidenzymes [39,41]. Therefore, these residues may be importantdeterminants for the phospho-donor specificity if, in futureresearch, the isoenzymes of the Spirochaetes turn out to beATP-dependent as well.

In addition to the substrate-binding residues in Table 2,another active-site residue is of particular interest: theresidue corresponding to E. coli Gly124 is a Lys in thekinetoplastid ATP-PFKs, in the ATP-dependent isoenzymeof E. histolytica (see Fig. 3) and in the PFKs of the twoSpirochaetes (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.

PPi 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 PPi 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 mM. Symbols are: j, no additions; d, + 1.5 mM AMP;

m, + 1.0 mM 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. brucei, 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. Lopez et al. (Eur. J. Biochem. 269) � FEBS 2002

whereas a Lys is present in all PPi-dependent enzymes. Xuet al. [40] have shown that a change of this lysine into amethionine in P. freudenreichii PFK caused a 132-foldincrease in the Km for PPi and a 490-fold decrease in kcat,providing strong indication for a direct involvement of thisLys residue in PPi binding. From a phylogenetic analysis, wepreviously concluded that the kinetoplastid PFKs musthave been derived from a PPi-dependent ancestral PFK,which changed its phospho-donor specificity early in theevolution of the lineage [5]. However, the Lys is no longerinvolved in PPi binding, so why has it been retained inthe present-day kinetoplastid PFKs which are all ATPdependent, and in the ATP-dependent isoenzyme ofE. histolytica? Has it obtained a function in ATP binding,implying that the mode of nucleotide binding is different inkinetoplastid PFKs from that in otherATP-PFKs?Or has itbeen retained for structural reasons? Relevant to thesequestions is the observation that the Lys is also present inthe ATP-dependent enzyme from the actinomycete Strep-tomyces coelicolor that, in a phylogenetic analysis, alsoclusters with the PPi-dependent PFKs and that must havehad a common ancestor with the PPi-dependent PFKs ofother Actinomycetes such as Amycolatopsis methanolica[42].

We therefore investigated whether substituting the Lyswould have any effect on the kinetoplastid enzyme. To thisend, we replaced it by Gly in L. donovani PFK. Theresulting LdPFK Lys224fiGly mutant did not display anyactivity. In contrast, the corresponding LysfiGlymutant ofT. brucei (TbPFK Lys226fiGly) appeared to be active [43].Strikingly, this T. brucei mutant showed only a slightdecrease in its affinity for ATP, but the mutation had amajor effect on the enzyme’s behaviour with respect tofructose 6-phosphate. In the absence of AMP, the S0.5 forfructose 6-phosphate was � 5 mM compared to 0.59 mM inthe wild-type enzyme. However, the higher substrate affinitycan still be induced by the addition of AMP:Km for fructose6-phosphate ¼ 0.82 mM compared to 0.15 mM in the wild-type PFK [43]. It seems that the mutation leads to a localdisruption of the active site with accompanying lowering offructose 6-phosphate affinity. This is independent of theallosteric changes in fructose 6-phosphate affinity asAMP iscapable of similar enhancements of fructose 6-phosphateaffinity in both wild-type and mutant enzymes. In the caseof the L. donovani enzyme, a greater degree of disruptionleads to abolition of fructose 6-phosphate binding, eitherthrough local or global effects.

As discussed previously, the comparative analysis of PFKsequences suggests that only subtle changes may be requiredfor a change of phospho donor specificity [5]. This notionwas reinforced by the recent publication [44] describingmutations in the ATP-PFK of E. coli and the PPi-PFK ofE. histolytica, similar to those described above for trypan-osomatid PFKs. The Gly124fiLys substitution in E. colieffectively eliminated activity with ATP as a substrate, butno PPi-dependent activity was observed. However, thereverse Lys201fiGly mutation in the PPi-dependent, majorPFK of E. histolytica reduced the kcat with PPi as thephospho donor by four orders of magnitude, while havingonly a limited effect on the apparent PPi affinity of theresidual enzyme activity. Importantly, the performance ofthe enzyme with ATP as a phospho donor increased abouteightfold (although this is still 105 times less than the

performance of the wild-type enzyme with PPi) essentiallyby an increase in kcat.

Understanding of the role of Lys224 inL. donovani PFK,and corresponding residues in other PFKs, is hampered bythe lack of a crystal structure with a lysine at this position.Modelling of the Gly to Lys mutation shows that, withoutsignificant local structural changes, the lysine side chainbecomes entirely buried in the protein interior, with nopossibility of electrostatic interaction with an acidic residue,a situation essentially unknown in protein structure. Onehypothesis is that, in enzymes containing a lysine at thisposition, the peptide bondwith the preceding proline adoptsa cis configuration [45]. It is suggestive that proline isentirely conserved at position 123 (E. coli numbering) whena lysine is present at position 124, while valine is alsotolerated in other PFKs. Modelling of possibilities for aL. donovanimodel containing such a cis peptide bond yieldsstructures in which the lysine side chain is solvent-exposedsuch as that illustrated in Fig. 5. In this structure the lysineside chain is placed at the heart of the catalytic site. A directinteraction with substrate ATP seems unlikely from thekinetic results presented here, although it is unfortunate thatstructural 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 substrateanalogue complex [46] (coordinates not deposited) supportsthe notion of a close resemblance between substrate- andproduct-bound protein structures. Why then, in contrast,should PPi binding be dramatically affected when this lysineis mutated in E. histolytica and P. freudenreichii PFKs[40,44]? The explanation may lie in another residue, clearlyimplicated in substrate specificity [44]. Position 104 (E. colinumbering) is always a Gly in ATP-PFKs and an Asp inPPi-PFKs. Structural examination (Fig. 5) shows that thepresence of any non-Gly residue leads to steric clashes withthe bound nucleotide in its crystallographically observed

Fig. 5. Positions of phospho-donor specificity-determining residues rel-

ative to the catalytic site ofE. coliPFK bound to products.Numbering is

according to the E. coli enzyme. The figure was produced using

MOLSCRIPT [54].

� FEBS 2002 Leishmania donovani phosphofructokinase (Eur. J. Biochem. 269) 3985

position. In the structure of a PPi-dependent PFK bound tosubstrates, it would be reasonable to expect that the PPisubstrate binds in the corresponding position as the b- andc-phospho groups of bound ATP in ATP-dependentenzymes. However, analysis shows that any rotamericconformation of an Asp104 side chain leads to positioningof its negative charge near to the phospho group occupyingthe �a position�, also negatively charged. Minimum oxygen-oxygen interatomic distances range from 1.1 to 3.9 A,depending on Asp104 rotamer. This electrostatic repulsionmay therefore force the PPi into a slightly differentconformation, further from Asp104 and hence nearer toLys124. This hypothesis allows an explanation of theapparent involvement of this lysine in PPi binding [40,44]but not in ATP binding. A more prosaic explanation mayunderlie the almost complete lack of PPi- or ATP-dependentPFK activity seen for the Gly124fiLys E. coli mutant [44].Without a preceding cis peptide bond only side chainconformations that unfavourably bury the positive chargeof the new Lys are attainable and the protein wouldtherefore be destabilized. The lack of confirmation of nativefold for the mutant, by CD experiments for example,suggests that the mutant may have undergone grossstructural changes resulting in loss of activity. The modelledlysine in the L. donovani model is well packed and notapparently well positioned to interact directly with fructose6-phosphate. These considerations support the previouslyadvanced explanation of the effects of the Lys224fiGlymutation 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 andE. coliPFKs involved in the binding of the

allosteric activator ADP with the corresponding residues inthe L. donovani and T. brucei enzymes (according to thealignment in Fig. 3). A structural comparison of theresidues binding the activator ADP inB. stearothermophilusPFKandaputativeAMPbindingmode for theL. donovani,suggested bymodelling, is shown in Fig. 6. This comparisonsuggests that the kinetoplastid enzymes may employ thesame region for binding their allosteric activator AMP. Theb-phospho group binding residues of the bacterial enzymesshow the most changes, with the most striking substitutionbeing the replacement of the Mg2+-ligating Glu187 withAsn. The loss of Mg2+ and the replacements of Arg25 andArg154, both of which electrostatically interact with theb-phospho group of ADP (Fig. 6A), effectively eliminatethe b-phospho-binding pocket in the trypanosomatidenzymes. The residues at positions 211 and 213 of theB. stearothermophilus enzyme that bind the a-phospho

Fig. 6. Comparison of (A) the crystallographically observed effector site of E. coliPFKwith 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. Lopez et al. (Eur. J. Biochem. 269) � FEBS 2002

group of ADP (Fig. 6A) are better conserved and may beinvolved in binding the phospho group of AMP (Fig. 6B).The modelling reveals that, in addition to the residuedifferences identified by sequence comparisons, othersignificant structural differences may exist. The L. donovanienzyme, along with others from kinetoplastids and theE. histolytica ATP-dependent PFK, has a one residueinsertion, relative to bacterial enzymes, at around position287 (L. donovani enzyme numbering). Two possible posi-tions for this insertion were analysed and one, as shown inFig. 6B, found to be favoured for avoiding the positioningof model residues in unusual areas of the Ramachandranplot. The altered main chain conformation in the vicinitycauses the Gln287 side chain to intrude into the areacorresponding to the b-phospho-binding site of the bacterialenzymes. Furthermore, it may form a hydrogen bond withthe phospho group of effector AMP (Fig. 6B). Anotherinteresting structural difference revealed by modellingrelates to the side chain position of Arg157 in theL. donovani enzyme which replaces a Ser or Gly in thebacterial enzymes. Adopting a rotameric conformation, thisresidue may fill the space caused by the replacements ofB. stearothermophilus Arg211 with Gln and Arg25 withLeu. In this position it may hydrogen bond to the phosphoof effector AMP in the L. donovani model structure andcontribute to the positive electrostatic potential of theeffector binding pocket.

C O N C L U S I O N S

The PFK genes of L. donovani and T. borreli have beencloned and sequenced. The encoded enzymes show mostsimilarity to the subset of PPi-PFKs, as did the previouslyanalysed T. brucei PFK. Nevertheless, ATP is the phos-pho substrate of all these kinetoplastid PFKs. It ispossible that a common ancestral organism changed itsphospho donor specificity during evolution. The currentlyavailable data do not allow us to draw any conclusion asto how and why the Kinetoplastida and other protistssuch as Entamoeba obtained their ATP-dependent, PPi-like PFKs. Did they evolve from a PPi-PFK in bothlineages independently, or did they originate in a commonancestor of these protists? Were they acquired fromSpirochaetes by lateral gene transfer? In this respect, itmay be relevant that phylogenetic studies based onsequences of other glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase and enolase, showed groupingof Kinetoplastida (and/or the related Euglenoida) andSpirochaetes [47–49].

Strikingly, all kinetoplastid PFKs, as well as theEntamoeba PFK contain a Lys on position 124 (E. colinumbering), whereas all other ATP-PFKs contain a Gly.Previous mutagenesis studies have provided strong evidencethat this Lys residue is involved in PPi binding. Structuremodelling suggests that the Lys may have been retained inthe kinetoplastid PFKs to maintain the stability of theactive-site structure. These results are supported by muta-genesis studies. No active L. donovani Lys224fiGly mutantcould be obtained, whereas the kinetic properties of acorresponding Lys226fiGly mutant of T. brucei PFKcould be interpreted in terms of a destabilized active site.

The L. donovani PFK shows slightly cooperative bindingof fructose 6-phosphate at low concentrations of this

substrate. The enzyme was allosterically activated byAMP by a significant increase in the affinity for thesubstrate. However, trypanosomatid PFKs are not activa-ted by ADP, in contrast to their counterparts in the bacteriaE. coli and B. stearothermophilus. Modelling studies haveprovided a possible structural basis for the AMP specificity.

We have provided evidence for significant structuraldifferences between trypanosomatid PFK and other ATP-PFKs including the human enzyme. Such differences werefound in both the active site and the region of the enzymepresumably involved in effector binding. Indeed, thedifferences in the effector-binding site tally with theapparently low level of activity regulation of trypanosoma-tid PFK as compared to that of the human enzyme. Thislimited regulation of trypanosomatid PFK seems physio-logically relevant in view of the intraglycosomal localizationof the enzyme and the low permeability of the organelle’smembrane for many metabolic intermediates that in othercells act as PFK effectors [3,50]. The structural differencesobserved offer great potential for the design or selection ofdrugs. Although our computer analysis using a kineticmodel of glycolysis suggested that PFK in bloodstream-form T. brucei is present in excess [51], we have arguedelsewhere [8,52] that this does not necessarily exclude theenzyme as a target for selective inhibitors that bind withhigh affinity, particularly irreversibly binding inhibitors.The most important aspects to consider in drug targetselection are that an enzyme should have an essential (or atleast very important) metabolic role and that its structureshould be sufficiently different from that of the correspond-ing host enzyme. Moreover, metabolism in bloodstream-form T. brucei is highly specialized, and in many respectsnot representative for the infective stages of other trypan-osomatid parasites such as the trypomastigotes andamastigotes of Leishmania species and T. cruzi [3].Therefore, we consider the trypanosomatid PFK as a highlypromising drug target.

A C K N O W L E D G E M E N T S

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 Fundacion Gran

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.

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