Gene 424 (2008) 25–32

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A biochemical and genetic study of Leishmania donovani pyruvate kinase☆

Will Sandoval a, Raúl Isea b, Evelyn Rodriguez a, Jose Luis Ramirez a,c,⁎a Centro de Biotecnologia, Instituto de Estudios Avanzados, Ministerio de Ciencia y Tecnologia de Venezuela, Carretera Nacional Hoyo de la Puerta, Caracas 1080 A, Venezuelab CeCalCULA Parque Tecnológico de Mérida-Edif Masini, Piso 3, Ofic. B32, Mérida 5101, Venezuelac United Nations University Programme UNU-BIOLAC, Venezuela

Abbreviations: CHEF, Contour Clamped HexagonInversion Gel Electrophoresis; F1,6BP, fructose 1,6 bipbiphosphate; MD, Molecular Dynamics; MEM, MinimPyruvate Kinase; RMSD, relative displacements; S0.5, sumaximum velocity; VL, Visceral Leishmaniasis; WT, Wil☆ The DNA and protein sequence of Leishmania doGenBank with accession number EU024521.⁎ Corresponding author. Centro de Biotecnologia, Ins

Ministerio de Ciencia y Tecnologia de Venezuela, CarreteCaracas 1080 A, Venezuela. Tel.: +58 212 903 5084; fax:

E-mail address: ramjoseluis@gmail.com (J.L. Ramirez

0378-1119/$ – see front matter © 2008 Elsevier B.V. Alldoi:10.1016/j.gene.2008.07.034

a b s t r a c t

a r t i c l e i n f o

Article history:

Here we present a biochem Received 24 January 2008Received in revised form 20 June 2008Accepted 29 July 2008Available online 3 August 2008

Keywords:Visceral leishmaniasisGlycolysisIn situ mutagenesisStructural studies

ical and molecular biology study of the enzyme pyruvate kinase (PYK) from theparasitic protozoa Leishmania donovani. The PYK gene was cloned, mutagenised and over expressed and itskinetic parameters determined. Like in other kinetoplastids, L. donovani PYK is allosterically stimulated bythe effector fructose 2,6 biphosphate and not by fructose 1,6 biphosphate. When the putative effector bindingsite of L. donovani PYK was mutagenised, we obtained two mutants with extreme kinetic behavior:Lys453Leu, which retained a sigmoidal kinetics and was little affected by the effector; and His480Gln, whichdeployed a hyperbolic kinetics that was not changed by the addition of the effector. Molecular Dynamics(MD) studies revealed that the mutations not only altered the effector binding site of L. donovani PYK but alsochanged the folding of its domain C.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Leishmania donovani is the etiological agent of Old World Visceralleishmaniasis (VL) or Kalazaar, a deadly disease affecting millions ofpeople in Africa and India. Despite the recent introduction ofmiltefosine, an apparent more effective and little toxic drug againstVL, there are early warnings that drug resistant strains can be easilyproduced (Croft et al., 2006), therefore there is a continuous need todevelop newer, cheaper, and more effective drugs against VL.

The trypanosomatid Leishmania belongs to a group of protozoathat when living in a mammalian host relies heavily on the glycolyticpathway for the production of ATP (Tielens et al., 1998; Cazzulo, 1992).Trypanosomatids glycolysis is particularly suited as a target for highaffinity, non-competitive or irreversible enzyme inhibitors. Moreover,most of the enzymes involved in the conversion of glucose intopyruvate are located in an organelle called the glycosome (Opperdoesand Borst, 1977; Michels et al., 2000) One consequence of thisorganization is that the glycolytic flux through this organelle seemsunregulated (Nwagwu and Opperdoes, 1982). In trypanosomatidshexokinase (EC 2.7.1.1) and phosphofructokinase (EC 2.7.1.11), keyregulatory enzymes of glycolysis (van Schaftingen et al., 1985), are not

al Electric Field; FIGE, Fieldhosphate; F2,6BP, fructose 2,6um Essential Medium; PYK,bstrate concentration at half-d type.novani has been deposited at

tituto de Estudios Avanzados,ra Nacional Hoyo de la Puerta,+58 212 903 5086.).

rights reserved.

affected by the typical effectors described for other cells. On the otherhand, trypanosomatid PYK (EC 2.7.1.40) is a cytosolic enzymeseparated from the rest of the glycolytic enzymes. Contrary tomammalian PYKs, which are allosterically regulated by F1,6BP,trypanosomatids PYKs are activated by submicromolar concentrationsof fructose 2,6–biphosphate (F2,6BP) a typical effector of mammalianPFK and are relatively unaffected by fructose 1,6-biphosphate (F1,6BP)(van Schaftingen et al.,1985; Ponte-Sucre et al., 1993; Ernest et al.,1994a,b, 1998). PYK plays an important role in regulating glycolyticflux from F1,6BP to pyruvate and catalyzes the irreversible final step ofthis pathway by which the phosphor group of phosphoenolpyruvate(PEP) is transferred to ADP to form pyruvate and ATP. Like in any otherorganism, trypanosomatids (Trypanosoma brucei, L. mexicana andTrypanoplasma borelli) (van Schaftingen et al., 1985; Ponte-Sucre et al.,1993; Ernest et al., 1994a,b, 1998) PYKs are homotetrameric with asubunit molecular mass of approximately 54,500 and share 47–51%amino acids sequence identity with other eukaryotic PYKs (Ernest etal., 1994b, 1998; Rigden et al., 1999). The crystal structure of L.mexicana PYK has been reported (Rigden et al., 1999). L. mexicana PYKshares the same overall secondary structure with Escherichia coli,rabbit muscle and yeast. Each subunit contains four domains: N-terminal, A, B, and C. The active site lies in a pocket found betweendomains A and B; and in L. mexicana domain C contains binding sitefor the allosteric effector.

In Leishmania, PYK represents an important metabolic crossroadlinking intermediaries of the incipient trypanosomatid's Krebs cycle,lipid synthesis and the glycolytic pathway. Although in a recent reportRNAi gene inactivation seems to be possible in L. braziliensis (Peacock etal., 2007), in general, studies to assess the metabolic importance ofLeishmania glycolytic enzymes have been hampered by the difficultyin implementing in vivo gene inactivating techniques. In T. bruceiwhere

26 W. Sandoval et al. / Gene 424 (2008) 25–32

RNAi inactivating experiments are feasible, the essentiality of PYK onparasite survival has been firmly established (Coustou et al., 2003).

In the present work we have studied biochemically and geneticallyL. donovani PYK, the gene has been cloned, over expressed andmutated. When L. donovani PYK was mutated in critical amino acids ofthe putative binding site for the allosteric effector F2,6BP, mutatedenzymes lacked the normal allosteric transition of the enzymeexhibiting two extreme kinetic behaviors.

2. Materials and methods

2.1. Parasite cells

L. donovani MHOM/SD/00/Khartoum (LSB-52-1) and L. donovaniMHOM/SD/00/Khartoum (LSB-52-1) clone 4.1.3 were kindly donatedby Dr. Kenneth Stuart from Seattle Biomedical Research Institute(SBRI), Seattle, Washington, U.S.A. Leishmania promastigotes werecultured at 24 °C in RPMI 1640 (Gibco-BRL) medium supplementedwith inactivated fetal bovine serum at 10% (vv−1) (Gibco-BRL),essential amino acids at 5% in minimum essential medium (vv−1)(MEM Gibco-BRL) without L-glutamine, sodium pyruvate in MEM,non-essential amino acids in MEM and 0.2% glucose (wv−1).

2.2. Genomic DNA isolation

Leishmania epimatigotes cells in log phase were collected bycentrifugation and washed twice with saline solution. Cell pellet wasresuspended in 0.5mL of saline phosphate buffer (NaPO4 pH 8; 65mMNaCl, 1% Glucose (wv−1)). Parasite disruption was promoted by theaddition of an equal volume of lysis buffer (10 mM Tris–HCl pH 7.5,100 mM NaCl, 0.5% SDS (wv−1), 1 mM EDTA) and incubated overnightat 37 °Cwith proteinase K at 10 μgml−1. The lysatewas extracted twicewith an equal volume of a mixture of chloroform: phenol (1:1 vv−1),and once with a mixture of chloroform: isoamylic alcohol (24:1 vv−1).The aqueous phase was transferred to a new tube and the DNAprecipitated by the addition of 2.5 volumes of ethanol at 99% (vv−1)and storage at −20 °C overnight. The DNA precipitated was collectedby centrifugation at 10,000 ×g for 15 min, vacuum dried andresuspended in sterile bi-distillated water.

2.3. Preparation of high molecular weight DNA

Leishmania chromosome DNA was isolated from early log epimas-tigotes cells following the protocol described by (Galindo and Ramirez,1989). Cells were collected by centrifugation at 4000 ×g for 12 min at4 °C, and the pellet was resuspended (4×108 cells mL−1) in salinephosphate buffer. Cell suspension was mixed with an equal volume oflow melting point agarose 2%, prepared in saline phosphate buffer.Agarose blocks containing Leishmania chromosomal DNAwere lysatedin buffer (0.5 M EDTA pH 9.0, 1% sodium N-lauryl sarcosinate, andproteinase K 2 mg mL−1) for 12 h at 37 °C. Blocks were stored in 0.5 MEDTA pH 9.0 solution at 4 °C.

2.4. Plasmid DNA isolation

Recombinant plasmidic DNA was isolated from bacteria accordingto the protocol of Birnboim and Doly, 1979. For DNA sequencing,plasmids were isolated with the kit Wizard® Plus SV Minipreps DNAPurification System, following (Promega) indications.

2.5. DNA gel electrophoresis

DNA gel electrophoresis was done on agarose gels of variousconcentrations in a horizontal chamber using either l× TBE buffer(80 mM Tris–HCl pH 7.6, 90 mM boric acid, 2 mM EDTA) or 1× TAEbuffer (40 mM Tris–acetate pH 8, 0.5 M EDTA). Samples were loaded

on gels after adjusting them to l× loading buffer (0.042% bromophenolblue (wv−1), 0.042% xylene-cyanol (wv−1), 5% (vv−1) glycerol).Electrophoretical runs were done with voltage gradient of 5 V. cm−1.After runs, gels were stained with ethidium bromide at 0.5 μg mL−1

and DNA bands were observed at 302 nm in a UV mini-transillumi-nator Gel Doc 1000® (BioRad).

2.6. Pulse field electrophoresis

2.6.1. Field Inversion Gel Electrophoresis (FIGE)This systemwas used to purify cosmid 11A7. Samples were loaded

on 8% agarose gels in 0.5× TBE in an electrophoresis chamberBRL model H3. Electrophoresis was done with a voltage gradient of4 V cm−1 for 10 h at 16 °C. Field inversion pulse times were controlledwith the system PPI-200 (MJ Research, Inc.) using the followingprogram: initial pulse ramp 0:0.01 s with 0.05 s increments on eachstep. Steps were repeated 20 times.

2.6.2. Contour clamped hexagonal electric field (CHEF) electrophoresisLeishmania chromosomal size DNA was separated in 1% agarose

gels in 0.5× TBE buffer using a CHEF-DR (BioRad) apparatus (Chu et al.,1986). The electrophoresis run was done at 14 °C with a voltagegradient of 4 V cm−1, and the following pulse program: initial time60 s, final time 120 s, run length 20 h, electric field angle 120°.

2.7. Isolation of DNA fragments from agarose gels

DNA bands resolved in low melting point agarose (Gibco-BRL)were sliced out with a razor blade and the agarose blocks werecovered with solution of 3 M sodium acetate and 1× TE pH 5.2 andmelted by incubation at 65 °C for 10 min. DNA was extracted twicewith 500 μL of saturated phenol in Tris–HCl pH 7.5 at 65 °C. Toprecipitate the DNA from the aqueous phase, two volumes of 99%(vv−1) ethanol were added and incubated overnight at −20 °C. DNAwas collected by centrifugation at 10,000 ×g for 15 min, and thepellet was washed with 500 μL of 70% (vv−1) ethanol and air dried.

2.8. Southern transfers

DNA was vacuum transferred into nylon Hybond-N+filters (Amer-

sham) using a LKB 2016 VacuGene instrument with a pressure of50 cm Hg. Gel was equilibrated with 10X SSC (3 M NaCl, 0.3 M sodiumcitrate) for 10 min. Then the gel was washed in 0.25 N HCl during7 min. After this incubation, this solution was removed and replacedby alkaline solution (0.5 N NaOH,1.5 M NaCl). The gel was equilibratedin a buffer solution (0.5 N Tris–HCl pH 7.5, 1.5 M NaCl) for 10 min.Finally, the transference was done running 20× SSC solution throughthe gel during 45 min. The filter was removed, and the DNAwas fixedby heating for 2 h at 80 °C.

2.9. Probe labeling

Radioactive labeling: Probes were labelled with [α-32P] dCTP(3000 Ci mmol−1) using the Random Primer kit Megaprime®(Amersham) and following Manufacturer's protocol. Non-radioactivelabeling was done by adding dUTP-digoxigenine tails to the oligosusing terminal transferase (Boehringer Mannheim).

2.10. Cloning L. donovani PYK

A genomic library of L. donovani (kindly donated for Dr. Theo DeVos of the Seattle Biomedical Research Institute) constructed inSupercos 7 vector was screened with a probe of L. mexicana wholegene kindly donated by Dr. Paul Michels, Institute of CellularPathology, Université Catholique de Louvaine, Brussels, Belgium. Onepositive colony was grown for cosmid DNA purification.

Fig. 1. Gene organization of pyruvate kinase genes in the genus Leishmania. Leishmania donovani pyruvate kinase restriction map is presented, and for comparative purposes theorganization of other Leishmania PYK genes and pseudogenes are included. Map distances at the bottom are approximated. The source of information for these genes was obtainedfrom GeneDB database (www.geneDB.org). TATE is retrotransposon described in L. braziliensis.

27W. Sandoval et al. / Gene 424 (2008) 25–32

2.11. Subcloning of L. donovani PYK

A restriction analysis of cosmid was done with restriction enzymesEco RI, Not I, Pst I, Sau 3AI, Xba I, Cla I, Xho I y Hind III and L. mexicanaPYK DNA as probe. Pst I restriction fragments (Fig. 1) positive for theprobe were isolated and cloned in plasmid pBlueScript® II KS (+)(STRATAGENE).

2.12. Expression and purification of recombinant PYK of L. donovani

L. donovani recombinant PYK was over-expressed in E. coli using abacteriophage T7 RNA polymerase system (Studier et al., 1990). Thegene of PYK of L. donovani was sliced out of PYK recombinant andspliced into the Nde I site of expression vector pET28b. The newrecombinant named pLdPYK directs, under the control of the T7promoter, the production of a fusion protein bearing an N-terminal(His)6 tag.

E. coli strain BL21 (DE3) pLysS transformed with the pLdPYK wasgrown at 30 °C in LB medium supplemented with 30 μg ml−1

kanamycine and 25 μg ml−1 choramphenicol. Expression wasinduced at an OD600 nm of 0.6–0.8 by the addition of 1 mMisopropyl-β-D-thiogalactopyranoside (IPTG) for 3 h, and growth was

Fig. 2. Strategy for in situ mutagenesis of L. donovani PYK gene. Plasmid pBKS/LDPYK contarepresented by arrows in the figure, rhombs on the arrowsmark the points where nucleotideThe proximity of codon 453 to aNco I restriction site allowed the introduction of the nucleotidfour different oligonucleotides. Mutated amplified PCR fragments were digested with the re

continued overnight. Cells were collected by centrifugation(12,000 ×g, 10 min at 4 °C). The cell pellet was resuspended in10 ml of buffer A (50 mM Triethanolamine–HCl pH 7.6, 200 mM KCl,1 mM KH2PO4, 5 mM MgCl2, 20% glycerol, 0.1 mM fructose 6-phosphate, 0.3 mM glucose 6 phosphate, protease inhibitors E64,leupeptine and pepstatine 1 μM of each). Cells were lysed by twopassages through a SML-Aminco Frech pressure cell (SML Instru-ments, USA) one at 13,000 psi, and the second pass at 8000 psi.Nucleic acids were eliminated first by incubation with 750 unitsBenzonase (Merck, Germany) for 30 min at 37 °C, and then with0.1 mg of protamine sulfate for 15 min at room temperature. Thelysate was spun down (12,000 ×g 15 min at 20 °C), and thesupernatant used to further purify (His)6-PYK. Recombinant enzymewas purified using immobilized metal affinity resin in batch (TALONresin, Clontech, USA) exploiting the (His)6 tag at the N-terminus ofthe protein. The resin was first incubated with 5 mL of buffer A for20 min. The 10 mL of lysate was added and incubated for another20 min. Then two washes with 10 mL of buffer A were done, and theresin was loaded into a 5 mL column and equilibrated with buffer A.Then, the column was washed with 5 mL of buffer B+10 mMImidazol, and finally washed with 5 mL of buffer A plus 100 mMImidazole, and 1 mL fractions were collected.

ining the entire ORF for L. donovani PYK was mutagenised using the oligonucleotideschangeswere introduced. The sequences of the oligonucleotides are presented in Table 1.e change in one PCR step,whereas for site 480 itwas necessary a two steps approachwithstriction enzymes indicated in the figure to be reinserted in pBKS/LdPYK.

Table 1List of oligonucleotides

Oligonucleotides Sequence 5′-3′ Tm (°C)

For PCR amplificationPK870F CTC ATT CTA CCA TAT GTC GCA GTT GG 65PK670R GTT ATT CTT CAT ATG CGG GAA GCC CC 66PK14-5 TTG TCT AGA CCC GGG TGC CGT TTT TGA ACT CAT TT 59PK15-3 GCA GAT ATC GGA TTC CGT CGT CGA TGT AGA TGT AG 62PK16-5 ATG TCG CAG TTG GCT CAC AAC CTG ACG CTC 61

In situ mutagenesisM1-453 TGT GTG TGA CGA CGC GTC TGC AGA CGT GCC GC 76.2M2-453 CAC CCA TGG CCA CGC GCT GCT CCA GGC CCT CAT 78.3M1-480 GCG CGT GGC CAT GGG TGT TG 68.6M2-480 TGT ATC ATT GGG TAA ACA CAT G 57.1M3-480 GTT GTG ATT CAG GCT GAT ACA A 60.8M4-480 GAA CTA GTG GAT CCC CCG GGC T 68.3

DNA sequencingT3 TAA CCC TCA CTA AAG GGA 57T7 TAA TAC GAC TCA CTA TAG GG 58M13 Forward CCC AGT CAC GAC GTT GTA AAA CG 66M13 Reverse AGC GGA TAA CAA TTT CAC ACA GG 64PK506F GTA CAT GGC GCG CAT CTG CC 67PK670R TTG CCC TCA TCG TGG CCG AG 67

The different oligonucleotide primers of this work are grouped according to the assayswhere they were used, their Tm are also included. Restriction sites in oligonucleotidesused for in situmutagenesis are in bold letters:M1-53Mlu I,M2-453Nco I,M1-480Nco Iand M4-480 Bam HI. Sites where the mutations were introduced are underlined.

28 W. Sandoval et al. / Gene 424 (2008) 25–32

2.13. Expression and purification of recombinant mutated PYK ofL. donovani

The expression and purification of recombinant mutated PYK wasmade as described for of the wild-type enzyme.

2.14. Site-directed mutagenesis of L. donovani PYK

Site directed mutagenesis of the PYK gene was performed onplasmid pBKS/LdPYK with PCR techniques (Mikaelian and Sergeant,1992) and using the proof reading Pfu DNA polymerase for amplifica-tion. The following substitutions were made (Fig. 2): Lys453 AAGcodonwas change into the Leu codon CTG, whereas the His480 codonCAC was changed into the Gln codon CAG. The amplified fragmentswere purified and ligated into the Eco RV site of plasmid pBlueScript IIKS (+) to form the plasmids pBKS/Lys453Leu and pBKS/His480Gln. Themutagenised fragment Lys453Leu was then excised from the plasmidspBKS/Lys453Leu by digestion with Mlu I and Nco I and the fragmentHis480Gln from the plasmid pBKS/His481Gln with Nco I and Bam HI.Both fragments were used to replace the corresponding segment inthe original plasmid containing the wild type gene (pLdPYK). Therecombinants thus obtained were named pLdPYKmutLys453Leu andpLdPYKmutHis480Gln.We also placed bothmutations together on thesame PYK gene. The mutagenised fragment Lys453Leu was excisedfrom the plasmid pBKS/Lys453Leu by digestion with Mlu I and Nco Iand used to replace the corresponding segment mutagenised plasmidpLdPYKmut480. This recombinant was named pLdPYKmut453/480.Correctly mutagenised plasmids were confirmed by DNA sequencing.The mutated proteins were expressed and purified according to thesame protocols as thewild type enzyme. Primers used in this work arelisted in Table 1.

2.15. DNA sequencing and analysis

DNA sequencing was performed with a fluorescent dideoxyterminator technique using an Applied Biosystems ABI Prism™ 310apparatus.

All sequence analysis and oligonucleotide designs were made withDNAMAN® (Lynnon Biosoft) and DNASTAR software.

2.16. Enzyme assays and kinetic analysis

The activity of PYK was determined by measuring the decrease ofNADH absorbance at 340 nm using a Lambda 35 UV/VIS spectro-photometer (Perkin-Elmer). To follow PYK activity during purification,a standard enzymatic assay was performed at 25 °C in a 1 mL reactionmixture containing: 50 mM triethanolamine–HCl buffer, pH 7.2, 6 mMMgSO4, 50 mM KCl, 2 mM ADP, 2.5 mM PEP, 0.42 mM NADH, 1.6 unitsof beef heart lactate dehydrogenase (Roche, Germany). The reactionwas initiated by the addition of 5 μL of diluted enzyme. Dilutionbuffer: 50 mM triethanolamine–HCl, pH 7.2, 0.2 mM EDTA, 0.1 mg/mLBSA, 25% glycerol, 1 mM DTT and 0.5 mM KH2PO4. Under thesestandard conditions an activity unit is defined as the conversion of1 μmol substrate per min. The behavior of purified PYK with regard toits two substrates PEP and ADP was measured by fixing one substrateat time at a concentration of 2 mM, while varying the other. In thekinetic experiments here presented the allosteric effector F2,6BPconcentration was adjusted to at 10 μM, ADP was adjusted to aconcentration of 2 mM, whereas the PEP concentration variedbetween 0 and 18 mM. Kinetics parameters were calculated from

Fig. 3. Protein sequence alignment of L. donovani pyruvate kinase with enzymes of other or(lower numbers) proteins, black arrows indicate β-chain regions; white cylinders indicateα-and E effector binding residues. Italicized residues are identical in all organisms. Gaps represeresidues at some Leishmania PYK pseudogenes.

Michaelis–Menten and Hill plots after optimal curve fitting ofexperimentally determined data, using the SigmaPlot® program.

2.17. 3D structural studies through molecular dynamics simulations

Studies comparing native and mutated L. donovani PYK enzymeswere done simulating the tridimensional structure of native L.donovani PYK. This structure was generated by homology-based-modeling using L. mexicana pyruvate kinase structure PDB code 1PKL(sequences similarity 96%), and the software Modeller version 8 (Saliand Blundell 1993).

All calculationswere carried out with Charmmversion 27 (Brooks etal.,1983) using the potential provided by theCharmmpackage. The forcefield was modified to consider the interaction of the hydrogen atoms.The calculationswere conductedwith a cut-off distance for non-bondedVan derWalls interaction at 14 Å. To simplify the simulation time, only asolvatation layer of water molecules (∼5 Å in thickness) was explicitlymodeled in the system. All of the water molecules were generatedaccording to their minor energy conformation. Constraints and/orrestraints were imposed on the structure during the calculations. Thestarting structurewasfirstminimizedby the conjugate gradientmethodto a rms. grd.b0.05 kcal. mol−1 Å−2followed by 1.5 ps (1500 steps)molecular dynamics and minimized, again. The calculations werefollowed by 50 ps MD simulations. The simulation was carried out atemperature of 300 K. In order to explore the configuration spaceavailable to the molecule, ten successive annealing cycles wereperformed. The systemwas heated and equilibrated from 300 to 700 Kin 10 K increments with velocities assigned every 0.001 ps, and thengradually cooled to 300 K. For the conformations obtained, those oflowest energy were chosen. All calculations were performed in periodicboundary conditions. Using as starting point the structure of native L.donovani PYK, we generated two other conformations corresponding tothe mutated PYKs. For each mutant structure we only calculated theenergyminimization as beforewith a 0.05 kcalmol−1 error in the energygradient. For eachmutant, theminimizationwasdone,without the need

ganisms. Numbering of residues was based in E. coli (upper numbers) and L. mexicanahelix regions. Thick cursive lettersmark: A ATP binding residues; P PEP binding residues;nt absent residues. The thick black line underlining L. major sequence marks themissing

29W. Sandoval et al. / Gene 424 (2008) 25–32

Table 3Kinetic parameters of L. donovani PYKs

Ligand Effector Parameter WT Lys453Leu His480Gln Double

PEP – S0.5 (mM) 1.16±0.12 1.01±0.07 0.7±0.06 0.76±0.02q – n 1.56±0.17 1.67±0.15 1.23±0.09 2.13±0.11q F2,6BP S0.5 (mM) 0.08±0.01 0.97±0.08 0.49±0.05 0.97±0.06q q n 0.95±0.10 1.60±0.17 1.24±0.08 1.56±0.12

S0.5 substrate concentration at half-maximum velocity at a fix 2 mM ADP concentration,with and without the addition of 10 μM F2,6BP; n is the Hill's coefficient. WT is wildtype PYK (recombinant), Lys453Leu is Mutant 1, His480Gln is Mutant 2, and double isthe double mutagenised Lys453Leu-His480Gln PYK. Results represent the average ofthree independent experiments.

30 W. Sandoval et al. / Gene 424 (2008) 25–32

to minimize the whole structure, in an eleven amino acids segmentcomprising thefive aminoacidspreceding themutatedone, themutatedamino acids, and the five amino acids following this site.

3. Results

3.1. Leishmania PYK gene organization

Restriction analysis sequence and cloning resulted in the mappresented in Fig. 1, for comparative purposes the approximated mapsfor other Leishmania were included. Similar to L. mexicana, there aretwo gene copies of PYK per genome and at 1.2 Kbp downstream fromthe gene there is a PYK pseudogene (Ernest et al., 1994a). In L. majorthe pseudogene was located at about the same distance but upstreamof the gene, whereas in L. infantum there were two pseudogenesflanking the gene at variable distances, and in L. braziliensis, none ofthe four PYK sequences so far reported in the database (www.geneDB.org) are whole genes, and one of these copies has an invertedorientation after a retrotransposon element (Fig. 1). When wecompared different Leishmania PYK pseudogenes, the most commonones lacked the first 43 amino acids that included the N-terminalregion (Nα1) and the structures Aβ1 and Aα1 from Domain A. Thisregion has been underlined for L. major PYK in Fig. 3. In the case of L.donovani where we only have sequenced 50% of the pseudogene, theonly difference was the lack of the methionine initiation codon, andfew amino acids changed (not shown). In Fig. 3 we also presented analignment of L. donovani PYK protein sequence with other enzymesfrom bacteria, trypanosomatids and other eukaryotes. This alignmentwas based on the sequence and secondary structure of L. mexicanaPYK as described by Rigden et al., 1999.

As other species of the subgenus Leishmania (www.geneDB.org), L.donovani PYK gene was located at chromosome 35 by pulse fieldelectrophoresis and DNA hybridisation (not shown).

3.2. L. donovani PYK properties

L. donovani PYK gene has 1500 bp (including the initiation andtermination codons) coding for a 498 amino acids protein with acalculated molecular mass of 54,400 and net charge and pI of −3.56and 6.5 respectively.

3.3. Enzyme activity

Table 2 shows the recombinant PYKenzyme activity in the differentbacterial strains used.When comparedwith the non-mutated enzyme,themutated enzymes specific activity suffered a slight decrease of 12%in mutant 1, 62% for mutant 2, and 45% for the double mutant.

3.4. Kinetic studies

The Km for PEP and ADP of purified recombinant L. donovani PYKwas first determined. In each case the concentration of one substratewas kept constant whereas the second one was varied. The assay wasrepeatedwith orwithout the addition of 10 μMF2,6BP. The kinetics for

Table 2L. donovani PYKs activities

Enzyme Protein μg Total activity U Specific activity U μg−1

WT 620 110.14 178.1Lys453Leu 450 70.6 156.9His480Gln 525 35.8 68.2Double mutant 390 38.2 98

Total and specific activities for purified enzymes:WTwild type (recombinant); Mutant1Lys453Leu, Mutant 2 His480Gln, and the double mutant. One enzymatic activity unit isdefined as 1 μmol of PEP converted to lactate per minute.

PEP at a fix concentration of 2 mM ADP and in the presence of theeffector F2,6BF displayed, as expected, a hyperbolic saturation curve,whereas in the absence of F2,6BF the curve was sigmoid. The PEPconcentration for S0.5 with F2,6BP was 0.08 mM, and 1.16 mMwithoutit, with a concomitant increase in Vmax from 492.1 (without) to506.99 U mg−1 (with). When ADP concentration was varied in thepresence of a fix concentration of 2 mM PEP, a hyperbolic curve wasobserved and a Km of 0.32 mMwas calculated (not shown). All kineticfigures represent an average of three independent experiments.

3.5. Mutagenised PYKs

As in the previous case each one of the different PYK recombinantswas purified and assayed. Fig. 4 showed that when mutant 1(Lys453Leu) without F2,6BP was assayed for PEP, similar to the wildtype enzyme, there was a sigmoid kinetics, however when F2,6BP wasadded, no kinetic change was detected. For mutant 2 (M2)(His480Gln) in the absence of F2,6BF the kinetics was hyperbolicwith a S0.5 of 0.49 mM. When the effector was added, the kinetics wasstill hyperbolic but with a S0.5of 0.7 mM (Fig. 4) (Table 3). The doublemutated enzyme was more hyperbolic, and in the presence of F2,6BPthe S0.5 went from 0.76 to 0.97 mM indicating that the two residuesare important for the binding of the effector (Fig. 4) (Table 3).

3.6. Molecular dynamics studies on mutated L. donovani PYK

In order to reveal possible structural changes introduced by thetwo mutants, molecular dynamics simulation was done with bothprotein sequences, the relative displacements (RMSD) vs residuenumber inwild-type (WT) and the twomutants. In M1 although smallchanges were observed for the α-helixes of domain A (not shown),most changes occurred around the amino acids conforming domain C(Fig. 5).

On the other hand using MD calculations we have also determinedthe mobility on domain C with respect to the rest of the protein. Thismethod describes the largest atomic displacement that can beexpected for the protein under study. In our calculations we describeddomain C mobility through the angle formed by amino acids Ser33,Pro87 and Asp145. Mutant 1 favored an open conformation for thisangle, which included a higher mobility for domain C, whereas in theamino acids residues of otherdomains littlemobilitywas detected. Thedefined angle for M1 ranged from 66.15° to 80.22°, whereas the anglefor theWT ranged from 67.6° to 70.77°. InM2 a close conformationwasfavored with an angle variation from 55.86° to 64.63°.

4. Discussion

L. donovani pyruvate kinase gene as in the case of L. mexicana andother Leishmania presented a pseudogene at approximately 1.2 kbpfrom its C-terminal sequence (Fig. 1).

An examination of the first half of the pseudogene DNA sequencerevealed a lack of the initiation codon, andminor amino acids changes,whereas in other Leishmania the typical pseudogene lacked the first

Fig. 4. L. donovani pyruvate kinases kinetics for PEP. Enzyme assays were done at a fix 2mMADP concentration, while increasing PEP concentration. In all cases black dots are kineticswithout the effector F2,6BP, and black squares in the presence of this effector (10 μM). (A) wild type PYK; (B) Mutant 1 Lys453Leu; (C) Mutant 2 His480Gln; and (D) double mutant. InTable 3 the kinetic parameters derived from three independent experiments are shown with their respective standard deviations.

31W. Sandoval et al. / Gene 424 (2008) 25–32

43 amino acids of the protein (Fig. 3). Considering that PYKpseudogenes are present in all Leishmania species, and that thereare not pseudogenes neighboring the rest of glycolytic enzymes genes,we wondered whether this highly conserved feature has anyfunctional meaning in regulating the PYK gene, or it is the result ofa genetic drift without selection. Interestingly, we did not find PYKpseudogenes in Trypanosoma brucei or Trypanosoma cruzi.

Sequence alignment (Fig. 3) and comparison of L. donovani PYKswith L. mexicana PYK and other organisms confirmed the four welldefined domains: a highly variable (in length) N-terminal domain, A,

Fig. 5. RMS Displacements versus residue number in L. donovani pyruvate kinasedomain C. Wild type, WT; M1, Lys453Leu; and M2, His480Gln.

B, and C. The active site is formed by domains A and B, whereas Charbors all the sites for the effector binding sites. The N-terminaldomain is typical of all eukaryotes, and in Leishmania it runs fromamino acid 1 to 17 and has a simple helix, whereas it is much larger inhuman skeletal muscle. A is the larger and best preserved domainharboring 11 α-helixes and 8 β-folded sheets, and it is formed by twoseparate polypeptides: from residue 18 to 88 the first, and from aminoacid 89 to 186 the second. Domain B ranges from residues 187 to 357and has 8 β-sheets and a small α-helix. Domain C located betweenresidues 358 and 498 is the less conserved domain and contains acenter of β-sheets flanked byα-helixes. The alignment in Fig. 3 revealsa stretch of 121 residues that are well conserved in all PYKs.

Structural comparisons between Saccharomyces cerevisiae pyru-vate kinase crystallized in R-state, and L. mexicana in T-state, havesuggested that residues Lysine 453 and Histidine 480 bind the 2-phospho group of the effector F2,6BP. To test the validity of thisassumption Haenert et al., 2002 introduced the following mutationsLys453Glu, His480Leu and His480Gln. Of all these mutants Lys453Glushowed a dramatic reduction of affinity for the effector that wasconsistent with a charge repulsion for the incoming ligand. In theexperiments here described when Lysine 453 residue was replaced bythe hydrophobic residue Leucine, the total activity and specific wentfrom 0.76 to 0.97 mM.

Activity of themutated enzymewere smaller than theWTenzyme,and the enzyme displayed a sigmoid kinetics for PEP that was notaffected by the presence of F2,6BP. Therefore despite no existingcharge repulsion, there was a total abrogation of the allostericbehavior, a loss of affinity for the effector, and a reduction ofenzymatic activity. This result suggested that the role of this residue

32 W. Sandoval et al. / Gene 424 (2008) 25–32

is not only explained by its positive charge but that additionalstructural factors must be considered (see below). In the case ofHis480Gln, similar to Haennert et al., 2002, we observed a moredramatic reduction in total and specific activity, but the mutatedenzyme retained some affinity for the effector. However different fromthese Authors, the kinetics for PEP was hyperbolic, and remainedhyperbolic even after the addition of the effector, with an increase inS0.5 from 0.49 mM to 0.7 mM. In the case of the double mutant thereduction in activity was similar to the one observed for mutant 2,however the kinetics was more hyperbolic, and in the presence ofF2,6BP the S0.5 increased from 0.76 to 0.97. In summary the mutatedenzymes seemed to be trapped in one case in an inactivated state forM1 and in an activated state for M2.

In a search for additional structural changes that might explain thekinetic behavior of the mutated enzymes, we did MD simulations forboth proteins. As is shown in Fig. 5, MD calculations indicated that themobility between helixes Aa6, Aa7, Aa8 is identical for M1 and M2,therefore in the observed phase (state) displacements, domain A doesnot appear to be involved. On the other hand the main changes inmobility were observed in the angle defined by amino acids Ser33,Pro87 and Asp145 in domain C, where an open conformation for thisangle is favored in M1, whereas a close conformation was favored inM2. Since domain C togetherwith domain A harbors the contact zoneswith the other PYK sub-units, the mutated amino amino acids herestudied are likely affecting the allosteric transition from R to T state. L.mexicana pyruvate kinase T-state structure has beenwell studied, andthe allosteric transition to the R state has been suggested throughcomparative studies with yeast R-state crystallized enzyme. If ourassumptions are correct, then crystallized M2 protein could providean insight into Leishmania pyruvate kinase R state and help thedevelopment of compounds to target this important enzyme.

Acknowledgments

This work received support from the EU grant ICA4-2000-10084.Thanks to Mrs. Sharon Sumpter for reviewing the English version.

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