Glycerol kinase of Trypanosoma brucei

Eur. J. Biochem. 267, 2323±2333 (2000) q FEBS 2000

Glycerol kinase of Trypanosoma bruceiCloning, molecular characterization and mutagenesis

Ivica KraÂ lovaÂ 1*, Daniel J. Rigden2, Fred R. Opperdoes1 and Paul A. M. Michels1

1Research Unit for Tropical Diseases, Christian de Duve Institute of Cellular Pathology and Laboratory of Biochemistry,

UniversiteÂ Catholique de Louvain, Brussels, Belgium; 2CENARGEN/EMBRAPA, Brasilia, Brazil

Trypanosoma brucei contains two tandemly arranged genes for glycerol kinase. The downstream gene was

analysed in detail. It contains an ORF for a polypeptide of 512 amino acids. The polypeptide has a calculated

molecular mass of 56 363 Da and a pI of 8.6. Comparison of the T. brucei glycerol kinase amino-acid sequence

with the glycerol kinase sequences available in databases revealed positional identities of 39.0±50.4%. The

T. brucei glycerol kinase gene was overexpressed in Escherichia coli cells and the recombinant protein obtained

was purified and characterized biochemically. Its kinetic properties with regard to both the forward and reverse

reaction were measured. The values corresponded to those determined previously for the natural glycerol kinase

purified from the parasite, and confirmed that the apparent Km values of the trypanosome enzyme for its

substrates are relatively high compared with those of other glycerol kinases. Alignment of the amino-acid

sequences of T. brucei glycerol kinase and other eukaryotic and prokaryotic glycerol kinases, as well as

inspection of the available three-dimensional structure of E. coli glycerol kinase showed that most residues of the

magnesium-, glycerol- and ADP-binding sites are well conserved in T. brucei glycerol kinase. However, a

number of remarkable substitutions was identified, which could be responsible for the low affinity for the

substrates. Most striking is amino-acid Ala137 in T. brucei glycerol kinase; in all other organisms a serine is

present at the corresponding position. We mutated Ala137 of T. brucei glycerol kinase into a serine and this

mutant glycerol kinase was over-expressed and purified. The affinity of the mutant enzyme for its substrates

glycerol and glycerol 3-phosphate appeared to be 3.1-fold to 3.6-fold higher than in the wild-type enzyme. Part of

the glycerol kinase gene comprising this residue 137 was amplified in eight different kinetoplastid species and

sequenced. Interestingly, an alanine occurs not only in T. brucei, but also in other trypanosomatids which can

convert glucose into equimolar amounts of glycerol and pyruvate: T. gambiense, T. equiperdum and T. evansi. In

trypanosomatids with no or only a limited capacity to produce glycerol, a hydroxy group-containing residue is

found as in all other organisms: T. vivax and T. congolense possess a serine while Phytomonas sp., Leishmania

brasiliensis and L. mexicana have a threonine.

Keywords: cloning; glycerol kinase; kinetics; site-directed mutagenesis; trypanosomes.

In Trypanosoma brucei, as in all Kinetoplastida studied to date,the first seven enzymes of glycolysis and two enzymes involvedin glycerol metabolism are present in peroxisome-like organ-elles called glycosomes [1±3]. Such compartmentation of theglycolytic pathway is unique; in other eukaryotes, glycolyticactivity is usually found only in the cytosol [4].

Glycolysis is the sole ATP-yielding metabolic pathway inlong-slender bloodstream-form trypanosomes and glucose istheir most important nutrient. Trypanosomes do not containlactate dehydrogenase and the NADH formed in the glyco-somes during the reaction catalysed by glyceraldehyde-3-phosphate dehydrogenase is oxidized indirectly by O2 via aglycerol 3-phosphate/dihydroxyacetone phosphate shuttle,involving a NAD-linked glycerol-3-phosphate dehydrogenase,which is localized in the glycosome, and a mitochondrialglycerol-3-phosphate oxidase (Fig. 1A). The compartmentationof the enzymes is such that ATP consumption and productionare balanced inside the glycosome; net glycolytic ATPsynthesis occurs only in the cytosol, in the reaction catalysedby pyruvate kinase. Thus, under aerobic conditions, glucose ismetabolized almost completely to pyruvate, about twomolecules of pyruvate being produced per molecule of glucoseconsumed [5], with the concomitant synthesis of two moleculesof ATP.

Correspondence to: P. A. M. Michels, ICP-TROP 74.39, Avenue

Hippocrate 74, B-1200, Brussels, Belgium. Fax: 1 32 276 26853,

Tel.: 1 32 276 47473, E-mail: [email protected]

Abbreviation: GK, glycerol kinase.

Enzyme: glycerol kinase (EC 2.7.1.30).

Note: the novel nucleotide sequence data published here have been

submitted to the EMBL sequence data bank and are available under

accession numbers AF132295 (Trypanosoma brucei), AF153346 (T. vivax),

AF153347 (T. congolense), AF153842 (T. gambiense, strain MA60),

AF153843 (T. gambiense, strain SA), AF153839 (T. equiperdum),

AF153840 (T. evansi, strain ILRAD2374), AF153841 (T. evansi, strain

KE), AF153348 (Phytomonas sp.), AF153349 (Leishmania mexicana),

AF153838 (L. brasiliensis).

Note: throughout this paper, the amino acids in glycerol kinase sequences

are numbered according to the Escherichia coli enzyme, the crystal

structure of which is known.

*Present address: Institute of Parasitology, Slovak Academy of Sciences,

Hlinkova 3, 04001 Kosice, Slovakia.

(Received 13 December 1999, revised 17 February 2000, accepted

21 February 2000)

2324 I. KraÂlovaÂ et al. (Eur. J. Biochem. 267) q FEBS 2000

Under anaerobic conditions or in the presence of an inhibitorof glycerol-3-phosphate oxidase, such as salicylhydroxamicacid, glycerol 3-phosphate cannot be oxidized via the mito-chondrion and accumulates inside the glycosome [6,7]. Thislarge increase in glycerol 3-phosphate concentration leads to areversal of the glycosomal glycerol kinase (GK) reaction. Thisreversed reaction is unfavourable thermodynamically but seemspossible in trypanosomes because of the unique form ofmetabolic compartmentation [8,9]. In contrast, in trypanosomesgrowing under aerobic conditions, and generally in otherorganisms, GK serves to phosphorylate glycerol at theexpense of ATP. Glycerol could also be used as acarbohydrate source by trypanosomes [10]. Dephosphorylationof glycerol 3-phosphate is usually catalysed by glycerol-3-phosphatase. However, bloodstream form trypanosomes do notcontain any significant activity of this latter enzyme [1]. Thereversal of the GK reaction thus also permits the regeneration ofglycosomal NAD1 by glycerol-3-phosphate dehydrogenase inthe absence of glycerol-3-phosphate oxidase activity [8,9,11](Fig. 1B). Moreover, it ensures that the ATP/ADP balanceinside the glycosome is also maintained under anaerobicconditions, thus permitting the continuation of the glycolyticprocess [1,12]. Glucose is then metabolized to equimolarquantities of glycerol and pyruvate with a net gain of only oneATP molecule per glucose. The exceedingly high GK activityfound in glycosomes of bloodstream form trypanosomes hasbeen invoked as a prerequisite for the formation of glycerol andATP [1]. Indeed, high GK activity has been found in organismsbelonging to the Trypanosoma species that produce glycerolanaerobically, but only low activities are present in T. cruzi,T. lewisi and Crithidia fasciculata, organisms that do notproduce glycerol [8].

GK has been purified previously from glycosomes ofbloodstream form T. brucei, and used for a preliminarybiochemical analysis [13] and kinetic studies [14]. The kineticsof the forward [8,14] and reverse [8] reactions have beendetermined. However, because the reverse reaction proceedsonly very poorly in vitro, a high priority has been to identify,clone and express the T. brucei gene in large amounts forfurther kinetic studies [14]. Here, we report the cloning andsequence determination of the GK gene. Furthermore, GK wasexpressed as a recombinant enzyme, purified and used forpreliminary characterization. Using sequence comparisons,

structure modelling and site-directed mutagenesis we searchedfor structural features which could be responsible for somespecial kinetic properties of the trypanosome enzyme. Wediscuss how these properties may be an adaptation to theparasite's life in the mammalian bloodstream.

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

Parasites

The following kinetoplastid organisms were used in thisstudy: T. brucei (strain 427), T. vivax (strain ILRAD1392),T. congolense (strain MRC33 KIBOKO), T. gambiense(strains MA60 and SA), T. equiperdum (strain Berenice),T. evansi (strains ILRAD2374 and KE), Phytomonas sp.(isolated from Euphorbia characias [15]), Leishmania brasi-liensis (strain MHOM/BR/75/M2903) and L. mexicana (strainMHOM/BZ/84/BEL46). Purified genomic DNA samples ofT. equiperdum, T. gambiense SA and T. evansi KE were kindlydonated by Dr F. Bringaud (UniversiteÂ de Bordeaux) and thoseof T. vivax, T. congolense, T. gambiense MA60 and T. evansiILRAD2374 by Dr E. Pays (UniversiteÂ Libre de Bruxelles).

Cloning and sequencing of the glycerol kinase gene ofT. brucei

A search in the EST subset of the GenBank nucleic aciddatabase revealed a 377-bp T. brucei rhodesiense sequence(accession number N45857; cDNA clone no. T1532 [16]);coding for an amino-acid sequence, 55 residues of whichshowed 47% identity with the N-terminal part of human GK.This cDNA clone (kindly provided by N. El-Sayed andJ. Donelson, University of Iowa, Iowa City, USA) was usedas a hybridization probe to screen a genomic T. bruceilibrary in the phage vector lGEM11 (Promega, USA) [17].Hybridization was performed under stringent conditions withpost-hybridization washes as described previously [18].Positive recombinant phages were rescreened. High-titrephage lysates were prepared and phage DNA was purified asdescribed previously [19]. The GK genes were further studiedin DNA from one selected positive phage by Southern blotanalysis of restriction fragments. Fragments containing GKgene segments were subcloned into the pBluescript IIKS(±)

Fig. 1. Simplified, stoichiometric scheme of

glucose metabolism in bloodstream form

T. brucei, under aerobic (A) and

anaerobic (B) conditions. (1) Glycerol kinase,

(2) glycerol-3-phosphate dehydrogenase,

(3) glycerol-3-phosphate oxidase.

q FEBS 2000 Trypanosoma brucei glycerol kinase (Eur. J. Biochem. 267) 2325

(Stratagene, USA) and pZErO-2 (Invitrogen, USA) phagemidvectors and sequenced from both strands using [35]S-dATP,the T7 DNA polymerase system of Amersham PharmaciaBiotech (Sweden) and 11 custom-synthesized oligonucleotides(GIBCO-BRL, UK).

PCR amplification, cloning and sequencing of a glycerolkinase gene fragment from different Kinetoplastida

For PCR amplification of a GK gene fragment from differentKinetoplastida, two highly conserved regions were selectedfrom an alignment of the T. brucei GK sequence with 18 GKsof various prokaryotes and eukaryotes. These regions arein the T. brucei sequence: IGITNQRET (amino acids atpositions 77±85 in Fig. 2, and KNTYGTGCF (262±270).Based on these motifs a forward primer (5 0-ATTGGAATCAC-GAACCAACGTGAGACA-3 0) and a reverse primer (5 0-GAA-GCAGCCGGTGCCGTATGTGTTCTT-3 0) were designed. Thetwo primers were used to amplify the corresponding GKregion in the following Kinetoplastida: T. vivax, T. congolense,T. gambiense, T. equiperdum, T. evansi, Phytomonas sp.,L. brasiliensis and L. mexicana.

PCR was performed in a total volume of 100 mLcontaining 300±400 ng of genomic DNA (for T. gambiense,T. equiperdum and T. evansi the experiments were alsoperformed with < 4 ng), 0.25 mm of each primer, 250 mm eachof the four deoxynucleotides and 2 U of Taq polymerase (fromQiagen, Germany or TaKaRa Shuzo Co, Japan) with thecorresponding reaction buffer. PCR was carried out using thefollowing programme: first 3 min at 95 8C; 35 cycles:denaturation 30 s, 95 8C; annealing 1 min, 55 8C; extension2 min, 72 8C; and a final incubation for 8 min at 72 8C.Amplified PCR products with a length of < 600 bp wereligated into the pCR2.1-TOPO phagemid vector designed forTA cloning (Invitrogen) and used to transform Escherichia coliTOP10 competent cells (Invitrogen). The cloned fragmentswere sequenced using the Thermo Sequence fluorescent-labelled primer cycle sequencing kit (Amersham PharmaciaBiotech, UK) and the LI-COR automated sequence equipment,based on a fluorescent detection system.

Computer analysis of the glycerol kinase sequences

Pairwise distances between the sequences were calculated afterelimination of gaps using the program clustal w [20]. Amultiple alignment of the amino-acid sequences of T. bruceiGK and 18 GKs available in databases was initially made byusing the pileup program of the Genetics Computer Group(GCG) package. Subsequently, the alignment was adjustedmanually taking into account elements of secondary structureas observed in the crystal structure of E. coli GK [21] using thedssp program [22] and formatted and displayed graphicallyusing the alscript program [23].

Overexpression of T. brucei glycerol kinase in E. coli

The complete GK gene was amplified via PCR using twocustom-synthesized oligonucleotides: 5 0-GGCGCCGCATAT-GAAGTACGTCGG-3 0, containing an NdeI site (underlined)adjacent to the 5 0-end of the GK gene coding region, and5 0-GGGAGATCTCTCAGAATACTACAA-3 0, complementaryto the 3 0-terminal coding region of GK, followed by a BglIIrestriction site (underlined). The total volume of the amplifi-cation mixture was 100 mL containing 10 ng of genomicDNA, 0.25 mm of each primer, 250 mm each of the four

deoxynucleotides and 2 U of Vent DNA polymerase with thecorresponding reaction buffer (New England Biolabs). PCRwas performed according to the programme: first 3 min at95 8C; 35 cycles: denaturation 30 s, 95 8C; annealing 1 min,60 8C; extension 2 min, 72 8C; and a final incubation for10 min at 72 8C. The amplified fragment was purified andligated to the blunt EcoRV ends of linearized phagemidpZErO-2. The GK gene was checked by automated sequencing,liberated from the recombinant plasmid by digestion with NdeIand Bgl II and ligated in the expression vector pET15b(Novagen) in such a way that an ORF was created for a fusionprotein of GK with a N-terminal extension of 20 aminoacids including a stretch of six histidine residues. E. coliBL21(DE3)pLysS [24] was used for expression of this protein.Cells were grown at 37 8C in 50 mL of Luria±Bertanimedium with 100 mg´mL21 ampicilin and 10 mg´mL21

chloramphenicol to a D600 of < 0.6. Isopropyl thio-b-d-galactoside was then added to a final concentration of 1 mmto induce the expression of the protein. Growth was continuedfor 4 h after addition of the inducer. The same conditionswithout any modifications were used for the over-expression ofan Ala137Ser GK mutant of T. brucei.

Site-directed mutagenesis

To produce a mutated T. brucei GK with an Ala137Sersubstitution, a short gene fragment containing the corres-ponding codon was amplified and simultaneously mutated. As aforward PCR primer the same oligonucleotide was used as forthe amplification of the entire GK gene of T. brucei (seeabove). The reverse primer was designed so that it introducedthe Ala137!Ser mutation and in addition included the uniqueXhoI restriction site of the GK gene. The amino-acid tripletcoding for Ala137 is GCT (nucleotide positions 421±423). Thistriplet is close to the unique XhoI restriction site (CTCGAG,positions 445±450). The reverse primer 5 0-CATTCTCGAGCA-TCCACCGCATCTTGAACGCAGAGAAATACGTGC-3 0 coversthe gene segment from position 410 to 454; the unique XhoIrestriction site is underlined and the mutated codon (TCT forSer ± in reverse AGA) is in bold. PCR was performed with10 ng of T. brucei genomic DNA, under conditions essentiallyidentical to those described above for the preparation of theexpression construct. The 460-bp GK fragment thus amplifiedwas incubated with 250 mm dATP, 1 U of TaKaRa Taqpolymerase and the corresponding buffer at 72 8C for 20 minto produce a 3 0-overhanging A. Subsequently, it was ligated inthe pCR2.1-TOPO vector and used to transform E. coli TOP10competent cells. After checking its sequence by automatedsequencing the mutated gene fragment was liberated from thevector by NdeI and XhoI digestion and inserted into theexpression construct from which the corresponding segment ofthe wild-type gene had been removed.

Purification of T. brucei glycerol kinase from E. coli cells

E. coli cells, expressing either wild-type trypanosome GK orthe Ala137Ser mutated protein, were harvested by centrifu-gation and resuspended in 10 mL of lysis buffer (100 mmtriethanolamine/HCl pH 7.6, 200 mm NaCl) containing aprotease inhibitor cocktail (Roche Molecular Biochemicals,Germany). The cells were disrupted by two passages through aSLM-Aminco French pressure cell (SLM Instruments, USA) at90 MPa. The nucleic acids were removed by treatment with250 U of benzonase (15 min at 37 8C; Merck, Germany) andprotamine sulfate (0.5 mg´mL21; 15 min at room temperature)


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followed by centrifugation for 10 min at 12 000 g. GK wasprecipitated with 40% ammonium sulfate and dissolved in10 mL of lysis buffer. Next, 1 mL of washed metal affinityresin (Talon resin; Clontech) was added to the sample and thesuspension was mixed for 20 min. The resin with bound proteinwas washed three times with lysis buffer (with centrifugation at700 g for 5 min), transferred to a gravity column and washedanother twice with 4 mL of lysis buffer. Finally, protein waseluted with 4 mL of lysis buffer supplemented with 100 mmimidazole. This protocol was used to purify both wild-type andmutant GK. In order to remove 17 amino acids of the 20-residue long N-terminal extension including the stretch of sixhistidine residues, the recombinant GK was treated withthrombin (Novagen). The GK was first washed on the Econo-Pac 10DG Column (Bio-Rad) with the thrombin cleavagebuffer (20 mm Tris/HCl pH 8.4, 150 mm NaCl, 2.5 mm CaCl2)and subsequently incubated with 1 U´mg21 thrombin for 20 hat 30 8C.

Protein measurements, SDS/PAGE and isoelectric focusing

Protein concentrations were determined using the Bio-Radprotein assay, based on Coomassie Brilliant Blue [25]. BSA wasused as a standard. PAGE in the presence of 0.1% SDS wascarried out in Laemmli's buffer system following standardtechniques [26]. Isoelectric focusing was performed using anAmpholine PAGplate (Pharmacia, Sweden), with a pH range of3.5±9.5, following the manufacturer's instructions. The pHgradient was measured using a Broad pI isoelectric pointcalibration kit (pI 3.5±9.3) (Amersham Pharmacia Biotech).

Enzyme assays and kinetic studies

The GK activity was measured by following the decrease inNADH absorbance at 340 nm using a Beckman DU7 spectro-photometer. The assay was performed at 25 8C in 1 mLreaction mixture containing 945 mL of 0.1 m triethanolamine/HCl buffer pH 8.0, 50 mL of the 20 � concentrated GK assaymixture (stored at 220 8C) and 5 mL of 1/10 diluted enzymesample, i.e. < 100 ng of enzyme per assay. The finalconcentrations of the reactants in the GK assay were asfollows: 2.5 mm MgSO4, 10 mm KCl, 5 mm glycerol, 2.5 mmATP (neutralized with equimolar quantities of NaHCO3),2.2 mm phosphoenolpyruvate and 0.42 mm NADH. Pyruvatekinase and lactate dehydrogenase were used as auxiliaryenzymes at final activities of 5 and 3 U, respectively. ATP,phosphoenolpyruvate, NADH, pyruvate kinase and lactatedehydrogenase were purchased from Roche Molecular Bio-chemicals. One activity unit is defined as the conversion of1 mmol substrate´min21 under these standard conditions.

The Michaelis±Menten constants for the substrates of theforward reaction were determined under the above-mentionedreaction conditions, with the same quantity of enzyme and withthe following substrate concentrations: the apparent Km for ATPwas determined at 5 mm glycerol, whereas the ATP concen-tration was varied between 0.05 and 2.5 mm. The apparent Km

for glycerol was measured at 2.5 mm ATP and with glycerolconcentrations varying between 0.002 and 2.0 mm.

The assay for the reverse reaction contained 0.1 m Mopsbuffer pH 6.8, 1 mm EDTA, 5 mm MgSO4, 0.4 mm NADP,55 mm glucose, 2 mm ADP and 10 mm l-glycerol 3-phosphate.Hexokinase and glucose-6-phosphate dehydrogenase wereused as auxiliary enzymes, both at final activities of 1 U.NADP, ADP, l-glycerol 3-phosphate, hexokinase andglucose-6-phosphate dehydrogenase were purchased from

Roche Molecular Biochemicals. The apparent Km for ADPwas determined at 10 mm l-glycerol 3-phosphate and ADPconcentrations varying between 0.1 and 1.0 mm. Theapparent Km for l-glycerol 3-phosphate was measured at2.0 mm ADP, while the l-glycerol 3-phosphate concentrationwas varied between 1.0 and 10.0 mm. For this determination ofthe kinetic parameters of the reverse reaction, 15 mL ofundiluted enzyme, i.e. < 3 mg of protein was used. The kineticparameters (Km , Vmax) given are the data as calculated fromHanes plots.

To determine the optimal pH for the forward reaction oftrypanosomal GK, the following buffer compounds, kept ata constant ionic strength of 0.1 m, were used: Mes (Sigma)pH between 4.9 and 7.2, triethanolamine (Merck) pH 5.2±8.4,2-[N-cyclohexylamino]ethanesulfonic acid (Sigma) pH 8.4±8.9,and Caps (Serva) pH 9.6±10.

R E S U LT S

Cloning and characterization of the gene for glycerol kinaseof T. brucei

A genomic library of T. brucei stock 427 was screened using asa hybridization probe a T. brucei rhodesiense cDNA clonecoding for a stretch of amino acids with a significant degree ofsimilarity to the N-terminal part of human GK [16]. Sixhybridizing clones were obtained. Two tandemly arrangedgenes were identified in the T. brucei genome by Southern blotanalysis of restricted total genomic DNA and variousrecombinant phage clones (data not shown). Subcloned DNAfragments from one recombinant phage were sequenced in bothdirections. One entire gene and also 435 bp of the 3 0-end of thesecond (upstream) gene were so analysed; the translated amino-acid sequence (145 residues) of the latter gene appeared to be100% identical to the corresponding part of the fully sequenceddownstream GK gene.

The ORF identified for the downstream gene codes for apolypeptide of 512 amino acids (excluding the initiatormethionine) with a calculated molecular mass of 56 363 Da.This value corresponds reasonably well with the subunit massof 51±52.5 kDa determined for the enzyme purified fromglycosomes of T. brucei [13,14]. The calculated net chargeand pI of the polypeptide are 16 and 8.6, respectively.The C-terminal tripeptide of the GK has the sequence -AKL,conforming to a type-1 peroxisome-targeting signal.

Comparison of the T. brucei glycerol kinase sequence withthe sequences of the corresponding protein in otherorganisms

The T. brucei GK amino-acid sequence has been aligned withall 18 other GK sequences that could be retrieved from variousprotein sequence databases: five sequences of eukaryotes, 12 ofeubacteria and one of an archaebacterium. An alignment of fiveselected sequences, representative of different taxonomicgroups (Homo sapiens, mammals; Caenorhabditis elegans,invertebrates; Escherichia coli and Bacillus subtilis, Gram-negative and Gram-positive eubacteria; Sulfolobus solfataricus,archaebacteria) plus that of T. brucei GK, is shown in Fig. 2.Fifty-nine residues are fully conserved among all 18 other GKsequences, and 55 are also shared by the T. brucei enzyme.From the comparison it is apparent that the residues con-stituting the binding sites of Mg21 and glycerol, as known fromthe structure of ligated E. coli GK [21], are generally wellconserved (including the T. brucei enzyme). Most of the


residues involved in ADP binding are also conserved, but someare more variant in the different GKs. There are 10 positions atwhich the T. brucei sequence has a residue which differs fromthat of all the others, or from 17 of the 18 other sequences(vertical arrowheads in Fig. 2). From the E. coli crystalstructure it could be inferred that none of these residues playsa role in substrate binding.

The percentage identity between all aligned GK sequenceswas calculated; the T. brucei sequence has 39.0±50.4% identitywith the others. It is more related to other eukaryotic GKs(49.9±50.4% identity with mammals, 45.5% with C. elegans,albeit only 40.4% identity with yeast) than to prokaryotic GKs(39.0±47.9%). The cyanobacterial Synechocystis sp. GK is themost different from the T. brucei enzyme (39.0%), and ingeneral, the yeast sequence appears more dissimilar from allother GKs (35.6±47.2%).

Over-expression of T. brucei glycerol kinase in E. coli andpurification of the enzyme

Recombinant GK was expressed in E. coli BL21(DE3)pLysScells and purified as described in Materials and methods. Acapture step based on precipitation with 40% ammoniumsulfate was introduced in order to eliminate some bacterialproteins prior to affinity purification using the His-tag ofGK (Fig. 3). Essentially, all impurities were eliminated bymetal affinity resin chromatography and elution with buffercontaining 100 mm imidazole. Highly similar data were

obtained in five different, independently performed purifi-cations. This procedure yields, on average, < 2 mg of purifiedGK from a 50-mL bacterial culture, with a specific activity indifferent batches ranging from 133 to 495 U´mg21. An accuratedetermination of the overall purification factor could not bemade because T. brucei GK activity appears to be inhibited byan endogenous factor in the crude bacterial lysate.

In order to test any possible effect of the 20 amino-acidstretch at the N-terminus of protein on its activity, 17 of themwere removed by thrombin cleavage. Enzymatic activity for theprotein thus obtained was measured and compared with thehistidine-tagged protein. The values obtained were highlysimilar (not shown) indicating that the histidine tag has nosignificant influence on the enzyme activity.

Biochemical characterization of the recombinant T. bruceiglycerol kinase

The purified GK migrates on SDS/PAGE as a polypeptide of55 kDa; this apparent molecular mass is somewhat larger thanthe value of 52.5±53 kDa determined previously for the proteinpurified from bloodstream form T. brucei [13,14] and is inagreement with the presence of the N-terminal extension of 20residues in the recombinant GK (calculated molecular mass58 kDa). The pI of T. brucei GK as calculated from its amino-acid sequence is 8.6, the calculated pI value of the GK with theN-terminal extension is 8.7. By isoelectrofocusing, wedetermined experimentally a pI value of 9 for the recombinantprotein in exact agreement with the value determined for thenatural trypanosome GK by Misset et al. [13].

Kinetic analysis of recombinant T. brucei glycerol kinase

Values of kinetic parameters of the enzyme for its forward andreverse reactions were determined by separately varying theconcentration of one of its substrates, ATP and glycerol forthe forward reaction, ADP and l-glycerol 3-phosphate for thereverse reaction, while maintaining the other substrate at(almost) the saturating level. With glycerol as the substrate aVmax of 226 ^ 56 U´mg21 was measured; with ATP as thesubstrate a Vmax of 183 ^ 20 U´mg21 was found. For thereverse reaction, with l-glycerol 3-phosphate as the substrate aVmax of 3.3 ^ 1.8 U´mg21 was determined; with ADP as thesubstrate a Vmax of 3.42 ^ 0.06 U´mg21 was found. Theapparent Km values of recombinant GK for all four substratesare given in Table 1. These values correspond reasonably wellwith those determined previously for the natural GK purifiedfrom T. brucei glycosomes [8,14].

To estimate the optimal pH for the forward reaction of GK,various buffers (see Materials and methods) were used atdifferent pH values, while maintaining a constant ionic strengthof 0.1 m. A bell-shaped relationship between pH and activitywas found (data not shown) with maximal activity at pH 8.0.

Fig. 3. Electrophoretic analysis of the subsequent steps in the purifica-

tion of T. brucei recombinant GK. Lane M, molecular mass marker

(Pharmacia); lane 1, bacterial lysate after benzonase and protamine sulfate

treatment; lane 2, supernatant after 40% ammonium sulfate precipitation;

lane 3, pellet after 40% ammonium sulfate precipitation; lanes 4±8,

successive washings of the affinity column; lane 9, eluate with 100 mm

imidazole. The proteins were visualized using Coomassie Brilliant Blue

staining.

Table 1. Km values of the wild-type and the Ala137Ser mutant glycerol kinase of T. brucei. Km values were calculated from Hanes plots of

experimentally determined data. The values given are expressed as mean ^SD, as determined in four (l-glycerol 3 phosphate) or five (glycerol) experiments.

Km (mm)

Type of enzyme ATP glycerol ADP l-glycerol 3 phosphate

Wild-type 0.24 �^ 0.09 0.44 �^ 0.09 0.56 �^ 0.02 3.83 �^ 1.15

Mutant 0.14 �^ 0.03 1.06 �^ 0.35


Outside the pH range 7.5±8.5 the activity decreased steadilywith 50% activity remaining at pH 7.0 and < 30% at pH 9.5.

Structure modelling of T. brucei glycerol kinase

The apparent Km values of trypanosome GK for its substratesare relatively high compared with those of other GKs. Inparticular the value of 0.44 mm for glycerol is high, values of0.01±0.02 mm have been reported for E. coli, Candidamycoderma and mammalian GKs. The apparent Km of< 0.24 mm for MgATP is also relatively high, but is closer tothe values reported for other GKs, although more variationseems to occur in the affinity for the nucleotide±mammalian GKs, 0.01±0.17 mm; C. mycoderma, 0.009 mm;E. coli, 0.08±0.5 mm [27±30].

Could we relate the low substrate affinity of the T. brucei GKto the presently available information of its structure? To thisend, the possible implications of unique substitutions in thetrypanosome's enzyme were evaluated in the three-dimensionalstructure of the E. coli enzyme, the only GK for which a crystalstructure has been reported. Coordinates are available for theenzyme with and without bound glycerol and ADP [21,32].Glycerol binding residues are also all well conserved inT. brucei GK. Interestingly, E. coli Ser137 makes a hydrogenbond with the backbone carbonyl of Glu84 that holds inplace the glycerol substrate (Fig. 4). In T. brucei GK, thecorresponding position harbours a unique substitution,Ser137!Ala. This substitution does not cause any constraints,but the hydrogen bond would be lost. This may have an indirectinfluence on the binding of the substrate and thus explain thelower affinity of the T. brucei enzyme for glycerol and glycerol3-phosphate. This hypothesis was tested using site-directedmutagenesis.

Site-directed mutagenesis of T. brucei glycerol kinase

The codon for Ala137 was replaced by a Ser triplet in thewild-type GK gene and the mutated GK was expressed inE. coli BL21(DE3)pLysS. Overexpression and purificationprotocols were carried out as described for the wild-typeenzyme. From the 50-mL culture < 1 mg of mutant protein wasobtained with a specific activity in different batches rangingfrom 200 to 340 U´mg21. The kinetic parameters (apparent Km

and Vmax for glycerol and l-glycerol 3-phosphate) of the mutantGK were determined in parallel and under conditions identicalto those for the wild-type enzyme. With l-glycerol 3-phosphateas the substrate a Vmax of 3.8 ^ 0.4 U´mg21 was found;with glycerol as the substrate the Vmax value was163 ^ 77 U´mg21. The apparent Km values of mutant GKfor glycerol and l-glycerol 3-phosphate are given in Table 1.They are compared with the data for recombinant wild-typeGK; the ratio of the Michaelis±Menten constants for glycerolof the mutant and wild-type enzyme, Km(mutant) /Km(wild-type) is3.1; the ratio Km(mutant) /Km(wild-type) for l-glycerol 3-phosphateis 3.6. These data, which appeared to be reproducible usingdifferent batches of enzyme, support the hypothesis that thenature of residue 137 affects the affinity of GK for glycerol andl-glycerol 3-phosphate; the Ala contributes to the relatively lowaffinity of the wild-type T. brucei GK for these substrates.

Analysis of partial glycerol kinase sequences in differentKinetoplastida

The low affinity of T. brucei GK for glycerol and glycerol3-phosphate may be relevant for the peculiar form ofmetabolism of this parasite. Therefore, we decided to analysethe distribution of the Ser137!Ala substitution in differentkinetoplastid organisms: parasites which are entirely dependenton glycolysis when living in the mammalian bloodstream, andcapable of converting glucose into equimolar amounts ofglycerol and pyruvate under anaerobic conditions [33]; andparasites with a more elaborate energy and carbohydratemetabolism, and with no or only a limited capacity to formglycerol. The first category was represented by T. gambiense,T. equiperdum and T. evansi and the second by T. vivax,T. congolense, L. mexicana, L. brasiliensis and Phytomonas sp.Based on highly conserved regions within various GK amino-acid sequences (Fig. 2) two primers were specified to amplify afragment of < 600 bp in the various kinetoplastids. Aftercloning and DNA sequence determination of the amplifiedfragments, amino-acid sequences were obtained whichappeared highly similar to the corresponding region ofthe T. brucei GK sequence. The alignment of the partialGK sequences of six kinetoplastids is shown in Fig. 5.T. gambiense, T. equiperdum and T. evansi are not includedin the alignment because their sequence is 100% identical to theT. brucei sequence at both the amino-acid and the DNA level.In order to exclude the possibility that this identity was due toPCR artefacts, DNA preparations from two different strains ofT. gambiense and T. evansi were used; two significantlydifferent DNA concentrations were also used (see Materials andmethods). Indeed the near-identity of the genomes of theseparasites, despite the differences in hosts and pathogenicpatterns they provoke, was shown recently by Stevens et al.[34] who noticed that the < 2 kb small subunit RNAs of thesefour Trypanosoma species differ at not more than two positions.With regard to position 137 of GK, an Ala is found inT. gambiense, T. equiperdum and T. evansi, as in T. brucei. Thetwo other Trypanosoma species, T. vivax and T. congolense,

Fig. 4. View of residues involved in the binding of glycerol (drawn with

grey bonds) to E. coli GK. Other residues referred to in the text are also

shown. The predicted position of the tyrosine usually found at position 352

in eukaryotic GKs is drawn using thin black lines. Hydrogen bonds are

shown as dotted lines. With the exception of Glu84, only the side chain and

Ca atoms are shown. The coordinates of the E. coli GK structure used for

the analysis were taken from the Brookhaven Protein Data Bank, entry 1glb.

The figure was drawn using the program molscript [41].


contain a Ser, whereas Phytomonas sp., L. mexicana andL. brasiliensis possess a Thr which, like Ser, has a hydroxylgroup that should also be able to form a hydrogen bond with theactive-site Glu. These results show that Ala appears to beunique to trypanosomes that are able to produce large amountsof glycerol under anaerobic conditions [33].

D I S C U S S I O N

In this paper we presented the cloning of T. brucei GK and apreliminary characterization of the recombinant enzyme. Theenzyme expressed in E. coli has kinetic properties similar tothose of the natural enzyme purified from trypanosomes. Itcould thus be considered to be a good model for further studiesof the enzyme's catalytic mechanism and structure. Inparticular, the availability of the recombinant enzyme willallow us to perform a detailed study of the kinetics of thereverse reaction. As in other organisms, the trypanosomeGK-catalysed reverse reaction proceeds at a very low rate. Instudies with C. mycoderma GK, having relative velocities of100 : 1.5 for the forward and reverse reaction, respectively,Janson & Cleland [27] had to use 50 mg of purified enzyme perassay to monitor the reverse reaction. In our studies withrecombinant GK from T. brucei, < 3 mg of recombinantenzyme was required in order to assay the reverse reaction,30 times more than the amount needed for the forward reaction(100 ng). The ratio of the relative rates of the forward andreverse reactions (Vf /Vr), as determined in our studies, is 61.For natural trypanosomal GK, Vf /Vr values ranging from 21 to170, depending on the method of preparation, were reported byHammond & Bowman [8].

The apparent Km values of trypanosome GK for its substratesare relatively high compared with those of other GKs. We haveshown that the high Km for glycerol and glycerol 3-phosphatecould be attributed, at least in part, to the Ala at position 137.Mutagenesis of the Ala into Ser resulted in a 3.1-fold to3.6-fold increase in the affinity. Although this increase ismodest, it appeared to be statistically significant. Furtherinspection of the modelled structure provided us with anotherpossible reason for the low affinity for glycerol. InvariantTyr135 makes a H-bond with the glycerol (Fig. 4). It is packedby various hydrophobic residues including invariant Trp356 andTrp103. In the E. coli structure it is also packed by Phe307. A

Phe is found at this position in all prokaryotes analysed, but anAla or Ser is found in eukaryotes. The resulting gap ineukaryotic GKs is filled by the large side chain of Tyr352,where prokaryotes have the small Gly. However, trypanosomespossess the smaller Leu352. It seems possible that this Leudoes not pack as well against the glycerol binding Tyr135,thus allowing more movement/displacement away from idealH-bonding distance/angle.

The nature of residue 137 in GK from different Trypano-somatidae tallies with the known phylogenetic relationships ofthese organisms [34] and their metabolic pattern [33]. Allmembers of the subgenus Trypanozoon (T. brucei, T. gam-biense, T. evansi and T. equiperdum), which have a highlyrepressed mitochondrion in the bloodstream stage and theability to produce large amounts of glycerol, contain an Ala. Incontrast, T. vivax and T. congolense, which in their blood-stream form possess an active mitochondrial metabolism andwhich have only a limited capacity to produce glycerol, have ahydroxy group-containing residue, as is also found in the othertrypanosomatid genera and in all other organisms studied.Therefore, this aspect of the GK structure seems to reflect anexample of divergent evolution, possibly correlated with theselective pressure exerted on the organism.

What could be the reason for the relatively high apparentKm values of the trypanosome GK? Krakow & Wang [14]postulated that they may reflect the relatively high concen-trations of substrates within the glycosome [7]. However, highKm values are not generally found for the glycosomal enzymes.Because of the high glycosomal enzyme concentrations [13],one of the assumptions of Michaelis±Menten kinetics does nothold in the glycosome: the concentrations of metabolites boundto the active sites cannot be neglected compared with the freemetabolite concentrations. The high glycosomal metaboliteconcentrations calculated previously [13] most likely reflectglycolytic intermediates that are sequestered by the relativelyhigh concentration of active sites of the glycosomal proteins,while the actual free metabolite concentrations may not beelevated [35].

An alternative explanation may be the following. The GKactivity in African trypanosomes capable of producing glycerolunder anaerobic conditions is exceptionally high [1±3 U´(mg ofprotein)21] and much higher than the activity required to satisfythe trypanosome's need for glycerol in the formation of

Fig. 5. Comparison of the partial glycerol kinase amino-acid sequences from various trypanosomatids, as predicted from amplified DNA fragments.

Amino acids were aligned to obtain maximal positional identity, using the program clustal w. The T. brucei sequence given corresponds with the peptide

77±270 in Fig. 2. The partial GK sequences of T. gambiense, T. evansi and T. equiperdum appeared identical to that of T. brucei and are therefore not

included in this alignment. The peptide sequences used for primer selection are given in bold characters. The position of Ala137 (T. brucei ) is indicated by a

black dot above the alignment, those of Lys94 and Tyr171 (Trypanosoma specific substitutions nos 3 and 5 in Fig. 2) are indicated by vertical arrowheads.

Asterisks underneath the alignment indicate conserved residues, small dots indicate conservative substitutions in one or more sequences.


triglycerides. Moreover, the other Trypanosomatidae, which arenot capable of producing glycerol anaerobically, have a GKactivity that is one to two orders of magnitude less than thatfound in T. brucei [8]. It has been proposed that this highGK activity contributes to T. brucei's capacity to catalysethe reverse reaction under anaerobic conditions and thusproduce pyruvate and glycerol in equimolar quantities [1].Glycerol 3-phosphate and glycerol accumulate inside theglycosome under these conditions until concentrations wellabove the respective Km values of the enzyme are reached.Therefore, a change in Km as explained by the Ser±Alasubstitution is not expected to have any significant effect on theefficiency of the reverse reaction and the rate of glycerolproduction. However, under aerobic conditions in blood wherea concentration of glycerol of < 50 mm prevails [36], thepresence of a high-affinity GK would lead to an excessive rateof glycerol consumption and this would be at the expense of theconsumption of glucose, because both glycerol and glucosecompete for the same glycosomal ATP pool. Glycerol (at 5 mm)is converted to pyruvate at a rate that is 50% higher than that ofglucose (F. Opperdoes & A. Fairlamb, unpublished observa-tions). However, when glycerol alone is used as the energysubstrate this would result in a net yield of 1 mole of ATP permol of glycerol consumed compared with 2 moles of ATP permol when glucose serves as the energy substrate. Thus, anefficient competition by glycerol for glycosomal ATP wouldresult in a reduction in the trypanosome's ATP production of50%. There are two possible ways by which the trypanosomewould be able to cope with such a situation. This could beachieved either by a permanent reduction in the affinity of GKfor glycerol or by a temporary inhibition of GK activity underaerobic conditions. Our data strongly suggest that the trypano-some has opted for the first possibility, modifying the affinityby specific amino-acid substitutions. To date, no evidence hasbeen found for the regulation of GK activity in T. brucei.

Some data suggest that the activity of T. brucei GK may beregulated in an as yet unknown manner. Previously, it has beenclaimed that glucose uptake by bloodstream form trypanosomesis not decreased by the presence of glycerol [37], but recent,more detailed, experiments have shown that the presence ofglycerol does lead to inhibition of glucose uptake that was mostpronounced at higher concentrations of the sugar [38]. Indeed,in a trypanosome lysate competition for ATP betweenhexokinase and GK was found to occur [11]. Using a detailedkinetic model of trypanosome glycolysis [12] this competitioncould be confirmed. However, the modelled glycolysis wasmore sensitive to glycerol than real glycolysis [38], suggestingthat the actual GK activity is regulated by special effectorswhich control this competition. However, such regulationremains to be established. Similarly, it remains to bedetermined whether effectors are involved in affecting GK'sactivity differently under aerobic and anaerobic conditions. Orare the direction and rate of the GK-catalysed reaction merelydetermined by kinetic properties and thermodynamics? In thisrespect, it may be relevant that the above-mentioned kineticmodel also reproduced the observed inhibition of anaerobicglycolysis by glycerol [39], but the concentration of glycerolneeded to inhibit glycolysis completely was significantly lowerthan that determined experimentally [12,40].

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

I.K. received postdoctoral fellowships from the ICP and the S & T

Cooperation Belgium ± Central and Eastern Europe. This research was

supported financially by the `Interuniversity Poles of Attraction Programme

± Belgium State, Prime Minister's Office ± Federal Office for Sciencific,

Technical and Cultural Affairs'. The authors would like to thank Dr Luise

Kalbe for help in the library screening, Mr Joris Van Roy for assistance in

performing kinetic analyses, Drs John Donelson and Najib El-Sayed

(University of Iowa) for kindly providing us with the T. b. rhodesience

cDNA clone, Drs Etienne Pays (UniversiteÂ Libre de Bruxelles) and FreÂdeÂric

Bringaud (UniversiteÂ de Bordeaux) for kindly providing genomic DNA

samples of different trypanosomatids, Dr Barbara Bakker for helpful

discussions, and Dr VeÂronique Hannaert for critical reading of the

manuscript.

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