Chalcone, acyl hydrazide, and related amides kill cultured Trypanosoma brucei brucei

BACKGROUND: Protozoan parasites of the genus Trypanosoma cause disease in a wide range of mammalian hosts. Trypanosoma brucei brucei, transmitted by tsetse fly to cattle, causes a disease (Nagana) of great economic importance in parts of Africa. T. b. brucei also serves as a model for related Trypanosoma species, which cause human sleeping sickness. MATERIALS AND METHODS: Chalcone and acyl hydrazide derivatives are known to retard the growth of Plasmodium falciparum in vitro and inhibit the malarial cysteine proteinase, falcipain. We tested the effects of these compounds on the growth of bloodstream forms of T. b. brucei in cell culture and in a murine trypanosomiasis model, and investigated their ability to inhibit trypanopain-Tb, the major cysteine proteinase of T. b. brucei. RESULTS: Several related chalcones, acyl hydrazides, and amides killed cultured bloodstream forms of T. b. brucei, with the most effective compound reducing parasite numbers by 50% relative to control populations at a concentration of 240 nM. The most effective inhibitors protected mice from an otherwise lethal T. b. brucei infection in an in vivo model of acute parasite infection. Many of the compounds also inhibited trypanopain-Tb, with the most effective inhibitor having a Ki value of 27 nM. Ki values for trypanopain-Tb inhibition were up to 50- to 100-fold lower than for inhibition of mammalian cathepsin L, suggesting the possibility of selective inhibition of the parasite enzyme. CONCLUSIONS: Chalcones, acyl hydrazides, and amides show promise as antitrypanosomal chemotherapeutic agents, with trypanopain-Tb possibly being one of their in vivo targets.     

The population genetics of Trypanosoma brucei and the origin of human infectivity

The African trypanosome, Trypanosoma brucei, is a zoonotic parasite transmitted by tsetse flies. Two of the three subspecies, T. brucei gambiense and T.b. rhodesiense, cause sleeping sickness in humans whereas the third subspecies, T.b. brucei, is not infective to humans. We propose that the key to understanding genetic relationships within this species is the analysis of gene flow to determine the importance of genetic exchange within populations and the relatedness of populations. T.brucei parasites undergo genetic exchange when present in infections of mixed genotypes in tsetse flies in the laboratory, although this is not an obligatory process. Infections of mixed genotype are surprisingly common in field isolates from tsetse flies such that there is opportunity for genetic exchange to occur. Population genetic analyses, taking into account geographical and host species of origin, show that genetic exchange occurs sufficiently frequently in the field to be an important determinant of genetic diversity, except where particular clones have acquired the ability to infect humans. Thus, T. brucei populations have an 'epidemic' genetic structure, but the better-characterized human-infective populations have a 'clonal' structure. Remarkably, the ability to infect humans appears to have arisen on multiple occasions in different geographical locations in sub-Saharan Africa. Our data indicate that the classical subspecies terminology for T. brucei is genetically inappropriate. It is an implicit assumption in most infectious disease biology that when a zoonotic pathogen acquires the capability to infect humans, it does so once and then spreads through the human population from that single-source event. For at least one major pathogen in tropical medicine, T. brucei, this assumption is invalid.     

A multiplex PCR that discriminates between Trypanosoma brucei brucei and zoonotic T. b. rhodesiense

Two subspecies of Trypanosoma brucei s.l. co-exist within the animal populations of Eastern Africa; T. b. brucei a parasite which only infects livestock and wildlife and T. b. rhodesiense a zoonotic parasite which infects domestic livestock, wildlife, and which in humans, results in the disease known as Human African Trypanosomiasis (HAT) or sleeping sickness. In order to assess the risk posed to humans from HAT it is necessary to identify animals harbouring potentially human infective parasites. The multiplex PCR method described here permits differentiation of human and non-human infective parasites T. b. rhodesiense and T. b. brucei based on the presence or absence of the SRA gene (specific for East African T. b. rhodesiense), inclusion of GPI-PLC as an internal control indicates whether sufficient genomic material is present for detection of a single copy T. brucei gene in the PCR reaction.     

The flagellum of Trypanosoma brucei: new tricks from an old dog

African trypanosomes, i.e. Trypanosoma brucei and related sub-species, are devastating human and animal pathogens that cause significant human mortality and limit sustained economic development in sub-Saharan Africa. T. brucei is a highly motile protozoan parasite and coordinated motility is central to both disease pathogenesis in the mammalian host and parasite development in the tsetse fly vector. Therefore, understanding unique aspects of the T. brucei flagellum may uncover novel targets for therapeutic intervention in African sleeping sickness. Moreover, studies of conserved features of the T. brucei flagellum are directly relevant to understanding fundamental aspects of flagellum and cilium function in other eukaryotes, making T. brucei an important model system. The T. brucei flagellum contains a canonical 9+2 axoneme, together with additional features that are unique to kinetoplastids and a few closely-related organisms. Until recently, much of our knowledge of the structure and function of the trypanosome flagellum was based on analogy and inference from other organisms. There has been an explosion in functional studies in T. brucei in recent years, revealing conserved as well as novel and unexpected structural and functional features of the flagellum. Most notably, the flagellum has been found to be an essential organelle, with critical roles in parasite motility, morphogenesis, cell division and immune evasion. This review highlights recent discoveries on the T. brucei flagellum.     

Characterization of di‐, tri‐ and tetranucleotide microsatellite markers with perfect repeats for Trypanosoma brucei and related species

Trypanosoma brucei, a unicellular parasite causing human sleeping sickness and animal nagana, has a great impact on the socioeconomic environment of sub‐Saharan Africa. The dynamics of the parasite are still poorly understood. We have characterized 14 polymorphic di‐, tri‐ and tetranucleotide microsatellite loci with perfect repeats (only one motif) exhibiting between five and 16 alleles in T. brucei isolates from all over Africa and from all described subspecies. The microsatellites will be useful in addressing population genetic questions in T. brucei to better understand the population structure and spread of this important parasite.     

Evidence for Multiple Origins of Human Infectivity in Trypanosoma brucei Revealed by Minisatellite Variant Repeat Mapping

In recent years a wide variety of biochemical and molecular typing systems has been employed in the study of parasite diversity aimed at investigating the level of genetic diversity and delineating the relationship between different species and subspecies. However, such methods have failed to differentiate between two of the classically defined subspecies of the protozoan parasite Trypanosoma brucei: the human infective, T. b. rhodesiense, which causes African sleeping sickness, and the non-human infective T. b. brucei. This has led to the hypothesis that T. b. rhodesiense is a host range variant of T. b. brucei. In this paper we test this hypothesis by examining highly polymorphic tandemly repeated regions of the trypanosome genome, i.e., minisatellite loci. We have employed the technique of minisatellite variant repeat mapping by PCR (MVR-PCR), which determines the distribution of variant repeat units along the tandem array of one minisatellite, MS42. The maps generated by this technique not only allow unequivocal allele identification but also contain within them cladistic information which we used to determine the possible genetic relationship between the different subspecies of T. brucei. Our findings revealed that human infective (T. b. rhodesiense) isolates from Uganda are more closely related to the local non-human infective isolates (T. b. brucei) than they are to other human infective stocks from different regions, suggesting that human infectivity has originated independently in these different geographical regions. This would infer that the separate classification of all human infective stocks from East Africa into the subspecies T. b. rhodesiense is genetically inappropriate and it would be better to consider geographically separate populations as host range variants of T. brucei brucei or perhaps as a series of different subspecies. Based on these data, it is clear that MVR mapping is a very useful tool for the analysis of zoonotic eukaryotic pathogens where delineation of the origins of outbreaks of disease and definition of human infective strains are key questions.     

What happens when Trypanosoma brucei leaves Africa

Julius Lukes and co-workers evaluated the evolutionary origin of Trypanosoma equiperdum and Trypanosoma evansi, parasites that cause horse and camel diseases. Although similar to T. brucei, the sleeping-sickness parasite, these trypanosomes do not cycle through the tsetse fly and have been able to spread beyond Africa. Transmission occurs sexually, or via blood-sucking flies or vampire bats. They concluded that these parasites, which resemble yeast petite mutants, are T. brucei sub-species, which have evolved recently through changes in mitochondrial DNA.     

Genetic analysis of the human infective trypanosome <Emphasis Type="Italic">Trypanosoma brucei gambiense</Emphasis>: chromosomal segregation,crossing over,and the construction of a genetic map

Background  

Trypanosoma brucei is the causative agent of human sleeping sickness and animal trypanosomiasis in sub-Saharan Africa, and it has been subdivided into three subspecies: Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, which cause sleeping sickness in humans, and the nonhuman infective Trypanosoma brucei brucei. T. b. gambiense is the most clinically relevant subspecies, being responsible for more than 90% of all trypanosomal disease in humans. The genome sequence is now available, and a Mendelian genetic system has been demonstrated in T. brucei, facilitating genetic analysis in this diploid protozoan parasite. As an essential step toward identifying loci that determine important traits in the human-infective subspecies, we report the construction of a high-resolution genetic map of the STIB 386 strain of T. b. gambiense.     

The de novo synthesis of GDP-fucose is essential for flagellar adhesion and cell growth in Trypanosoma brucei

The protozoan parasite Trypanosoma brucei causes human African sleeping sickness in sub-Saharan Africa. The parasite makes several essential glycoproteins, which has led to the investigation of the sugar nucleotides and glycosyltransferases required to synthesize these structures. Fucose is a common sugar in glycoconjugates from many organisms; however, the sugar nucleotide donor GDP-fucose was only recently detected in T. brucei, and the importance of fucose metabolism in this organism is not known. In this paper, we identified the genes encoding functional GDP-fucose biosynthesis enzymes in T. brucei and created conditional null mutants of TbGMD, the gene encoding the first enzyme in the pathway from GDP-mannose to GDP-fucose, in both bloodstream form and procyclic form parasites. Under nonpermissive conditions, both life cycle forms of the parasite became depleted in GDP-fucose and suffered growth arrest, demonstrating that fucose metabolism is essential to both life cycle stages. In procyclic form parasites, flagellar detachment from the cell body was also observed under nonpermissive conditions, suggesting that fucose plays a significant role in flagellar adhesion. Fluorescence microscopy of epitope-tagged TbGMD revealed that this enzyme is localized in glycosomes, despite the absence of PTS-1 or PTS-2 target sequences.     

Differential effect of 1,10-phenanthroline on mammalian, yeast, and parasite glycosylphosphatidylinositol anchor synthesis.

Glycosylphosphatidylinositol (GPI) anchoring of proteins to the plasma membrane is a common mechanism utilized by all eukaryotes including mammals, yeast, and the Trypanosoma brucei parasite. We have previously shown that in mammals phenanthroline (PNT) blocks the attachment of phosphoethanolamine (P-EthN) groups to mannose residues in GPI anchor intermediates, thus preventing the synthesis of mammalian GPI anchors. Therefore, PNT is likely to inhibit GPI-phosphoethanolamine transferases (GPI-PETs). Here we report that in yeast, PNT also inhibits the synthesis of the GPI anchor as well as GPI-anchored proteins. Interestingly, the mechanism of PNT inhibition of GPI synthesis is different from that of YW3548, another putative GPI-PET inhibitor. In contrast to mammals and yeast, the synthesis of GPIs in T. brucei is not affected by PNT. Our results indicate that the T. brucei GPI-PET could be a potential target for antiparasitic drugs.     

Trypanosoma brucei ornithine decarboxylase: enzyme purification, characterization, and expression in Escherichia coli

Ornithine decarboxylase from the African trypanosome is an important target for antitrypanosomal chemotherapy. Despite this, the enzyme had not been previously purified or extensively characterized as it is a very low level protein. In this paper we describe the purification of Trypanosoma brucei brucei ornithine decarboxylase from bloodstream form trypomastigotes by 107,000-fold to a specific activity of 2.7 x 10(6) nmol CO2/h/mg of protein in the parasite. T. brucei ornithine decarboxylase had a native molecular weight of 90,000 and a subunit molecular weight of 45,000. The isoelectric point of the protein was 5.0. The Km for ornithine was 280 microM and the Ki for the irreversible inhibitor alpha-difluoromethylornithine (DFMO) was 220 microM with a half-time of inactivation at saturating DFMO concentration of 2.7 min. T. brucei ornithine decarboxylase appears similar to mouse ornithine decarboxylase, further supporting our previous suggestion that the selective toxicity of DFMO to the parasite is not due to catalytic differences between the two proteins. Although a small quantity of T. brucei ornithine decarboxylase was purified from T. brucei, extensive structural and kinetic studies will require a more ample source of the enzyme. We therefore expressed our previously cloned T. brucei ornithine decarboxylase gene in Escherichia coli using a vector that contains an inducible lambda promoter. T. brucei ornithine decarboxylase activity was induced in E. coli to levels that were 50 to 200 fold of that present in the long-slender bloodstream form of T. brucei. Ornithine decarboxylase activity in the crude E. coli lysate was 1500-6000 nmol of CO2/h/mg of protein and represented 0.05-0.2% of the total cell protein. The recombinant T. brucei ornithine decarboxylase was purified to apparent homogeneity from the transformed E. coli. The purified recombinant enzyme had kinetic and physical properties essentially identical to those of the native enzyme.     

Molecular cloning and characterization of Trypanosoma vivax alternative oxidase (AOX) gene, a target of the trypanocide ascofuranone

Trypanosoma vivax causes nagana disease in cattle. Since T. vivax is transmitted not only by tsetse flies but also by other biting flies (non-cyclic transmission), the parasite has been distributed to and has had a significant economic impact on wide geographical areas, including Africa and South America. Our previous study on Trypanosoma brucei brucei showed that the trypanosome alternative oxidase (TAO, TbAOX) is a promising target of chemotherapy. For this reason, we also have cloned the T vivax AOX (TvAOX) gene and characterized the recombinant enzyme. The deduced amino acid sequence (328 a.a.) of TvAOX shares 76% identity with TbAOX and contains the diiron-coordination motifs (-E-, -EXXH-) that are conserved among AOXs. The Km of recombinant TvAOX (rTvAOX) expressed in Escherichia coli for ubiquinol (87.0 +/- 0.54 microM) was significantly lower than the value for recombinant TbAOX (rTbAOX) (714 +/- 4.5 microM). Ascofuranone, the most potent inhibitor of TbAOX, was a competitive inhibitor of rTvAOX with a Ki value (0.40 +/- 0.00 nM) significantly lower than that for rTbAOX (1.29 +/- 0.00 nM). The non-cyclic transmission ability of T. vivax and the in vivo chemotherapeutic efficacy of ascofuranone against T. vivax and T. b. brucei infection are discussed in terms of these Km and Ki values.     

RNA interference of Trypanosoma brucei cathepsin B and L affects disease progression in a mouse model

We investigated the roles played by the cysteine proteases cathepsin B and cathepsin L (brucipain) in the pathogenesis of Trypansoma brucei brucei in both an in vivo mouse model and an in vitro model of the blood-brain barrier. Doxycycline induction of RNAi targeting cathepsin B led to parasite clearance from the bloodstream and prevent a lethal infection in the mice. In contrast, all mice infected with T. brucei containing the uninduced Trypanosoma brucei cathepsin B (TbCatB) RNA construct died by day 13. Induction of RNAi against brucipain did not cure mice from infection; however, 50% of these mice survived 60 days longer than uninduced controls. The ability of T. b. brucei to cross an in vitro model of the human blood-brain barrier was also reduced by brucipain RNAi induction. Taken together, the data suggest that while TbCatB is the more likely target for the development of new chemotherapy, a possible role for brucipain is in facilitating parasite entry into the brain.     

Lysis of Trypanosoma brucei by a toxic subspecies of human high density lipoprotein

Trypanosoma brucei brucei is an important pathogen of domestic cattle in sub-Saharan Africa and is closely related to the human sleeping sickness parasites, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. However, T. b. brucei is non-infectious to humans. The restriction of the host range of T. b. brucei results from the sensitivity of the parasite to lysis by toxic human high density lipoproteins (HDL) (Rifkin, M. R. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3450-3454). We show in this report that trypanosome lytic activity is not a universal feature of all human HDL particles but rather that it is associated with a minor subclass of HDL. We have purified the lytic activity about 8,000-fold and have identified and characterized the subspecies of HDL responsible for trypanosome lysis. This class of HDL has a relative molecular weight of 490,000, a buoyant density of 1.21-1.24 g/ml, and a particle diameter of 150-210 A. It contains apolipoproteins AI, AII, CI, CII, and CIII, and monoclonal antibodies against apo-AI and apo-AII inhibit trypanocidal activity. In addition to these common apolipoproteins, the particles also contain at least three unique proteins, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Treatment of the particles with dithiothreitol resulted in the disappearance of two of the proteins and abolished trypanocidal activity. Two-dimensional gel electrophoresis showed that these proteins were a disulfide-linked trimer of 45,000, 36,000, and 13,500-Da polypeptides and dimers of the 36,000- and 13,500-Da polypeptides or of 65,000- and 8,500-Da polypeptides. Studies on the lysis of T. b. brucei by the purified particle suggest that the lytic pathway may involve the uptake of the trypanocidal subspecies of HDL by endocytosis.     

Identification and functional characterization of thioredoxin from Trypanosoma brucei brucei

Trypanosomes and Leishmania, the causative agents of several tropical diseases, lack the glutathione/glutathione reductase system but have trypanothione/trypanothione reductase instead. The uniqueness of this thiol metabolism and the failure to detect thioredoxin reductases in these parasites have led to the suggestion that these protozoa lack a thioredoxin system. As presented here, this is not the case. A gene encoding thioredoxin has been cloned from Trypanosoma brucei, the causative agent of African sleeping sickness. The single copy gene, which encodes a protein of 107 amino acid residues, is expressed in all developmental stages of the parasite. The deduced protein sequence is 56% identical with a putative thioredoxin revealed by the genome project of Leishmania major. The relationship to other thioredoxins is low. T. brucei thioredoxin is unusual in having a calculated pI value of 8.5. The gene has been overexpressed in Escherichia coli. The recombinant protein is a substrate of human thioredoxin reductase with a K(m) value of 6 microM but is not reduced by trypanothione reductase. T. brucei thioredoxin catalyzes the reduction of insulin by dithioerythritol, and functions as an electron donor for T. brucei ribonucleotide reductase. The parasite protein is the first classical thioredoxin of the order Kinetoplastida characterized so far.     

Further studies on difluoromethylornithine in African trypanosomes

DL-alpha-Difluoromethylornithine (DFMO), a specific enzyme-activated irreversible inhibitor of ornithine decarboxylase (ODC) was previously shown to cure mice infected with Trypanosoma brucei brucei, a parasite of game and cattle in Africa and Trypanosoma brucei rhodesiense, a human African Sleeping Sickness pathogen. Our studies now indicate that DFMO blocks ornithine decarboxylase and lowers trypanosome polyamine levels in vivo. Polyamine uptake in T.b. brucei also resembles that previously described for mammalian cells. The therapeutic potential of DFMO can now also be extended to another human pathogen, Trypanosoma brucei gambiense. Finally, DFMO acts synergistically with another drug, bleomycin, to cure acute trypanosome infections, and furthermore, this same drug combination provides a new approach to the treatment of trypanosomal infections of the central nervous system.     

DNA metabolism and genetic diversity in Trypanosomes

Trypanosomes are protozoan parasites that cause major diseases in humans and other animals. Trypanosoma brucei and Trypanosoma cruzi are the etiologic agents of African and American Trypanosomiasis, respectively. In spite of large amounts of information regarding various aspects of their biology, including the essentially complete sequences of their genomes, studies directed towards an understanding of mechanisms related to DNA metabolism have been very limited. Recent reports, however, describing genes involved with DNA recombination and repair in T. brucei and T. cruzi, indicated the importance of these processes in the generation of genetic variability, which is crucial to the success of these parasites. Here, we review these data and discuss how the DNA repair and recombination machineries may contribute to strikingly different strategies evolved by the two Trypanosomes to create genetic variability that is needed for survival in their hosts. In T. brucei, two genetic components are critical to the success of antigenic variation, a strategy that allows the parasite to evade the host immune system by periodically changing the expression of a group of variant surface glycoproteins (VSGs). One component is a mechanism that provides for the exclusive expression of a single VSG at any one time, and the second is a large repository of antigenically distinct VSGs. Work from various groups showing the importance of recombination reactions in T. brucei, primarily to move a silent VSG into an active VSG expression site, is discussed. T. cruzi does not use the strategy of antigenic variation for host immune evasion but counts on the extreme heterogeneity of their population for parasite adaptation to different hosts. We discuss recent evidence indicating the existence of major differences in the levels of genomic heterogeneity among T. cruzi strains, and suggest that metabolic changes in the mismatch repair pathway could be an important source of antigenic diversity found within the T. cruzi population.     

Characterization of Trypanosoma brucei dihydroorotate dehydrogenase as a possible drug target; structural, kinetic and RNAi studies

Nucleotide biosynthesis pathways have been reported to be essential in some protozoan pathogens. Hence, we evaluated the essentiality of one enzyme in the pyrimidine biosynthetic pathway, dihydroorotate dehydrogenase (DHODH) from the eukaryotic parasite Trypanosoma brucei through gene knockdown studies. RNAi knockdown of DHODH expression in bloodstream form T. brucei did not inhibit growth in normal medium, but profoundly retarded growth in pyrimidine-depleted media or in the presence of the known pyrimidine uptake antagonist 5-fluorouracil (5-FU). These results have significant implications for the development of therapeutics to combat T. brucei infection. Specifically, a combination therapy including a T. brucei -specific DHODH inhibitor plus 5-FU may prove to be an effective therapeutic strategy. We also show that this trypanosomal enzyme is inhibited by known inhibitors of bacterial Class 1A DHODH, in distinction to the sensitivity of DHODH from human and other higher eukaryotes. This selectivity is supported by the crystal structure of the T. brucei enzyme, which is reported here at a resolution of 1.95 Å. Additional research, guided by the crystal structure described herein, is needed to identify potent inhibitors of T. brucei DHODH.     

Trypanosoma brucei brucei: antigenic stability of its LDL receptor and immunological cross-reactivity with the LDL receptor of the mammalian host.

The rapid growth of Trypanosoma brucei brucei in the blood and tissue fluids of vertebrates requires the receptor-mediated endocytosis of LDL from the host (Coppens et al. 1987; Gillett and Owen 1987) and is slowed by a monospecific rabbit antiserum against the purified LDL receptor of the parasite. We have used this antiserum in combination with several well-characterized antigenic variants (originating from stock 427: MITat 1.1a, 1.3a, 1.4a, 1.5a, 1.5d, 1.8b) to examine whether the LDL receptor of T. b. brucei is a stable surface antigen, common to all parasite variants despite antigenic variation of the major surface glycoprotein, and whether it is immunologically distinct from the LDL receptor of the host. At low concentrations, binding at 4 degrees C of rat LDL to several variants of T. b. brucei and to isolated rat hepatocytes was inhibited to a similar extent by the antiserum. In double immunodiffusion, a single precipitation line was observed, showing continuity between the extracts of all variants as well as between that of trypanosomes and of mammalian tissues. In Western blots of trypanosome extracts, the LDL receptor was strongly labeled as a single band of Mr 145,000, whereas with a rat liver extract, a single band of similar electrophoretic mobility was weakly labeled at a high concentration of the antiserum. In conclusion, the LDL receptor occurred in all variants of T. b. brucei, was a stable surface antigen despite variation of the major surface glycoprotein, and displayed biochemical and immunological similarities with the LDL receptor of the rat host.     

Glycerol kinase of Trypanosoma brucei. Cloning, molecular characterization and mutagenesis.

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.