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Review

TRENDS in Parasitology

Vol.23 No.4

Metabolism of Leishmania: proven and predicted
Fred R. Opperdoes1 and Graham H. Coombs2
1

Research Unit for Tropical Diseases and Laboratory of Biochemistry, Christian de Duve Institute of Cellular Pathology and Catholic University of Louvain, Avenue Hippocrate 74–75, B-1200 Brussels, Belgium 2 Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, The John Arbuthnott Building, Glasgow G4 0NR, UK

The complete analysis of the genomes of three major trypanosomatid parasites has facilitated comparison of the metabolic capabilities of each, as predicted from gene sequences. Not surprisingly, there are differences but is it possible to correlate these with the lives of the parasites themselves and make further predictions of the meaning and physiological importance of the apparently parasite-specific metabolism? In this article, we relate gene predictions with the results from experimental studies. We also speculate on the key metabolic adaptations of Leishmania and reasons why it differs from Trypanosoma brucei and Trypanosoma cruzi. Examining the differences Four hundred genes that code for the enzymes of the major metabolic pathways in Leishmania major have been identified (see Table S1 in the supplementary material online: http://www.icp.be/opperd/leishmania_table1.htm). For 30 (8%) of them, orthologues have not been detected in either Trypanosoma brucei or Trypanosoma cruzi (see Table S2 in the supplementary material online: http:// www.icp.be/opperd/leishmania_table2.htm). By contrast, Leishmania lacks a few genes that encode metabolic enzymes in T. brucei, T. cruzi or both. For example, sedoheptulose-1,7-bisphosphatase and threonine dehydrogenase are absent from Leishmania but occur in both trypanosomes. Three enzymes involved in histidine metabolism are found only in T. cruzi. Overall, however, L. major has the most extensive metabolic machinery, whereas that of T. brucei is the most restricted. The differences between Leishmania and trypanosomes are found mainly at the level of carbohydrate and amino acid metabolism. So, what is the importance of this? One limitation in making meaningful predictions about the metabolic make up of parasites is not knowing which proteins have distinct stage specificity. The amazing variation in energy metabolism exhibited by T. brucei during its life cycle [1,2] has been well documented but this might not be the typical scenario. Unfortunately, there is little information about this for Leishmania (and T. cruzi). Thus, predictions have to involve a certain amount of ‘educated guessing’.
Corresponding author: Coombs, G.H. (graham.coombs@strath.ac.uk). Available online 22 February 2007.
www.sciencedirect.com

Why is Leishmania metabolically different from other trypanosomes? Leishmania and trypanosomes evolved from a common ancestor and share many features. Thus, it was expected that many of the genes of different trypanosomatids would be orthologues, as has now been proven [3]. However, there are some substantial biological differences between trypanosomatids, and these can be illustrated by considering their life history strategies. Notably, Leishmania thrives inside a parasitophorous vacuole within mononuclear cells in a mammalian host; thus, it is adapted for reaching this niche and is metabolically appropriate for it. It is thought that the metabolic capabilities of the amastigote evolved to ensure that this stage of the parasite is fully adapted for its intracellular existence. When in its sand fly host, Leishmania exists as promastigote forms. These are adapted for life in different parts of the intestinal tract of the fly but, unlike trypanosomes, not in the hindgut (except for the braziliensis complex of Leishmania) or the salivary glands. Moreover, sand flies differ from the vectors of trypanosomes in their biology (e.g. their food source) and this has implications for the Leishmania parasites within them. There is now extensive information about the genes of Leishmania and this should help researchers to deduce the adaptations of Leishmania for each of the stages of its life cycle. However, it is not yet known when in the life cycle many of the genes are functionally important. Presumably, such analyses will be completed soon, although it is doubtful that global transcriptomic and proteomic analyses [4] will provide all of the answers, and detailed study of gene groups will be required for many years. Moreover, knowledge of the environments in which the various parasite forms reside, such as the parasitophorous vacuole, remains poor, which limits researchers’ ability to mimic these conditions experimentally. One known variation is pH – the amastigote resides in an acidic environment, whereas the habitat of promastigotes is believed to be closer to neutral. This has implications for nutrient acquisition [5], although the internal pH of both parasite forms is thought to be around neutral. Another aspect that might be important in determining metabolism is the availability of oxygen and carbon dioxide. Amastigotes and promastigotes respond differently, in metabolic terms, to variation in gas availability [6,7] and there are almost certainly differences in the metabolism of the two parasite stages because of the differences between the environments in which they reside.

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150 Review TRENDS in Parasitology Vol. The situation in Leishmania promastigotes requires similar reanalysis. so the functionality of the ascorbate biosynthetic pathway in Leishmania is uncertain.20]. cruzi and T. acetate and small amounts of pyruvate and D-lactate. brucei procyclic forms have prompted reinterpretation of the extent to which the TCA cycle is involved (or not) in energy generation [11]. it is the amastigote glycosome that seems to contain the greater number of enzymes www. Recent extensive experimental analyses of T. This indicates the presence of ascorbic acid in trypanosomatids and the possibility that it is used in their defence against oxidative stress (e. Leishmania (as with T. However. at least for some species and with the caveat that the axenic forms could differ from natural amastigotes. of the metabolism of Leishmania provides a framework on which to attempt to interpret the extensive information that is now available about the gene content of Leishmania. Studies using Leishmania mexicana amastigotes isolated from in vivo lesions revealed that. CO2 production results from the pathways that produce acetate and succinate. because analyses of glycosomes that are purified from amastigotes are yet to be conducted. which is considered to be a potential energy reserve. whether these in vitro forms metabolize in the same manner as promastigotes that occur naturally in sand flies is not known. cruzi have an ascorbate-dependent peroxidase. studies have been rather limited so far and the results largely agree with the findings with purified amastigotes. which depend on their respiratory chain and rapidly die if it is nonfunctional because of the absence of oxygen. In addition. which indicates that the glycosome. Knowledge of the energy metabolism of Leishmania amastigotes is considerably more fragmentary than that of promastigotes. in addition to glucose. The application of metabolomics to this parasite stage [17] could revolutionize the understanding of its metabolic capabilities. it is probable that. amino acids (notably proline) and glucose can be used as energy sources. which comprises an active electron-transport chain. Predictions of how Leishmania differs metabolically from trypanosomes Sugar use Leishmania has genes that encode both an amylase and a sucrase-like protein. A thioldependent enzyme that might be involved in the reduction of dehydroascorbic acid has been reported [21] but it could . This correlates well with published findings [18] and.16].23 No. primarily because amastigotes have been less available for study.be/opperd/leishmania_table3. However. Interestingly. by Leishmania in the host macrophage). brucei has many copies of an iron–ascorbate oxidoreductase. so some of the enzymes could also be involved in catabolizing this polysaccharide. It is also thought that other sugars. although considerably fewer than promastigotes [13. However. Results from such studies indicated that the metabolism of promastigotes has similarities to that of the vector stages of both T.H. similar to when promastigotes are starved.14]. amastigotes have an increased b-oxidation of fatty acids and a reduced need for proline and glucose consumption [12]. Amastigotes contain glycosomes.g. The advent of methods for culturing Leishmania amastigotes in the absence of host cells in vitro provided an opportunity for further analyses of the energy metabolism of amastigotes. There are enzymes of the tricarboxylic acid (TCA) cycle present.g. and energy generation seems to involve both glycolysis (and glycosomes) and mitochondrial metabolism. the substantial stage variation in mitochondrial activity that occurs with T. Leishmania has been reported to contain mannan [19. The bacterial nature of these enzymes indicates that they have probably been acquired from a bacterium through horizontal transfer and facilitate the digestion of a range of sugars that is present in the nectar upon which sand flies feed. This knowledge.htm). unpublished). although current evidence does not enable judgment on its effectiveness and other roles cannot be ruled out. Nevertheless. This is in contrast to procyclic stages of African trypanosomes. Denton and G. however.4 Energy metabolism of Leishmania Biochemical analyses of Leishmania promastigotes have almost invariably involved the parasite stages grown in vitro in nutrient-rich medium. For example. the enzymes are used by promastigotes for the digestion of plant starch and disaccharides taken in by sand flies that feed on nectar. might be used.icp. cruzi) also has genes that encode several sugar kinases such as ribulokinase and xylulokinase. This substrate use is facilitated by an apparently fully functional TCA cycle (but this needs reanalysis) and a linked respiratory chain. brucei – although. mixed populations of promastigotes were used (e. Therefore. major and T. ammonia and urea are also released. Alanine. in addition to being the site of glucose metabolism. It has been postulated that this ability of Leishmania promastigotes to undergo metabolic arrest might be an adaptation for survival during life in the sand fly [9]. at least in some studies. is also central to the degradation of other sugars. brucei does not occur with Leishmania. usually with air as the gas phase. whereas T. the current view of the metabolism of promastigotes is now supported by the finding of all the expected genes [3]. All these sugar kinases carry targeting signals for import into the glycosomes. metacyclic promastigotes in addition to multiplicative forms). inhibition of the electron transport chain results in reversible metabolic arrest [9]. Genome mining and experimental evidence indicate that a fungal type of biosynthesis might be functional in the trypanosomatids (Box 1 and see Table S3 in the supplementary material online: http://www. compared with promastigotes. The end products identified include succinate. brucei. Caution is required. although experimental evidence indicates that the cycle is not active [8]. Coombs.com such as phosphoenolpyruvate carboxykinase and malate dehydrogenase [15. The presence of a glyoxylate cycle was reported [10] but the genome analysis shows this to be unlikely. albeit limited. with Leishmania.sciencedirect. presumably. including gene deletions to determine the requirement for the various pathways. Ascorbate metabolism L. attempts to detect ascorbate or erythroascorbate in Leishmania proved unsuccessful (H. There is some evidence for stage variation in the metabolism of these organelles – as exists with glycosomes of T.

y. Thus.4-lactone as a substrate.4-lactone to gulono-1. The genes present include glucose-6-phosphate dehydrogenase.sciencedirect. gulono-1. oxidized glutathione. uridine diphosphate. major but present in Leishmania braziliensis. Footnotes: *. major. www. Interestingly. was characterized [38]. It is not yet clear. which catalyses the formation of D-arabinono-1. z. Trypanosoma brucei and Trypanosoma cruzi. Animal. the operation of such a pathway cannot be excluded but three out of the nine of the genes that code for the respective enzymes of a plant-type pathway could not be identified in the trypanosomatid genomes. where the substrate for the pathway (D-arabinose) comes from. so far. however. Thus.4 151 Box 1. 6-phosphogluconolactonase. brucei and T. and the trypanosomatid genomes contain a gene that encodes an NADP-dependent arabinose dehydrogenase. brucei. The Leishmania enzyme also has a potential PTS1. cruzi has all the genes for the enzymes of the animal-type pathway. the 6-phosphogluconate dehydrogenase required . not present in L. reduced glutathione. The animal-type pathway.com cruzi and likely to be involved in a modified pentose phosphate pathway. brucei). The importance of its absence is unknown because candidate genes encoding most of the enzymes of the conventional pentose phosphate pathway are present and the parasites are reported to contain a functional pathway [22. where identified. which indicates that ascorbate might be generated within glycosomes as antioxidants.4-lactone oxidase has an intermediate activity with Lgalactono-1. Pentose phosphate pathway Leishmania seems to lack sedoheptulose bisphosphatase. however. plants and fungi.23]. Abbreviations: GSH. with arabinolactone as the intermediate and erythroascorbate as end product. Leishmania major seems to lack two of them and Leishmania braziliensis lacks one (Figure I). is shown on the left hand side. It is an extremely rare sugar. UDP. The protein contains a flavine adenine dinucleotide (FAD)-binding motif and a type-1 peroxisome targeting signal (PTS1). is boxed. The trypanosome enzyme was shown to prefer D-arabino-1. however. However. are indicated for L. Ascorbate metabolism The genes that encode enzymes of an animal-type ascorbate biosynthetic pathway have been tentatively identified in the Trypanosoma cruzi genome. enzymes present in glycosomes. Recently. is operational in this trypanosomatid. which is known only in polysaccharides of plants and. with the retained enzymes being active in other ways. In all three trypanosomatids the genes for the enzymes of the yeast-type pathway were identified. only T. there are no obvious candidate enzymes in trypanosomatids that are able to form D-arabinose from other C5 sugars. biochemically identified in T. This enzyme is homologous to the enzymes that catalyse the last step in the formation of (erythro)ascorbate in animals. The yeast-like pathway results in the formation of erythroascorbate from arabinose. Accession codes. The trypanosomal gulono-1. current evidence indicates that a yeast-like pathway.4-lactone [38].23 No. major has recently been reported to incorporate Darabinose into lipophosphoglycan side chains [39].Review TRENDS in Parasitology Vol. Figure I. with gulonate and gulonolactone as intermediates and ascorbate as the end product. Whereas ascorbate biosynthesis seems still to exist in T. consistent with its demonstrated location inside glycosomes [38]. The yeast-type pathway. 4-lactone from D-arabinose.and yeast-type pathways of ascorbate biosynthesis in Leishmania major. GSSG. an enzyme that is predicted to be present in T. cruzi. have other activities in the parasite. cruzi.4lactone oxidase. rather than an animal-type pathway. L. it might have been lost from Leishmania (and T. the substrate for the formation of ascorbate through a plant-type pathway. the last enzyme of this pathway in T.

major and T. This is also the major route of threonine degradation in mammals but it is not operational in Leishmania because of the absence of the mitochondrial TDH. Second. brucei is the mitochondrion-located terminal oxidase that is involved in the reoxidation of NADH using a dihydroxyacetone-phosphate–glycerol-3-phosphate shuttle. import these metabolites from an exogenous source [26. which indicates that the entire pentose phosphate pathway might occur inside glycosomes. protoporphyrinogen oxidase and ferrochelatase. Interestingly. so the pathway probably functions in both compartments. By contrast. This terminal oxidase reduces molecular oxygen to water and transfers electrons directly from ubiquinol to oxygen. Consistent www. other enzymes of the haem biosynthesis pathway. are juxtaposed as a separate transcription unit on one end of chromosome six and. which is then oxidized to succinyl CoA (Figure 2). which might function in Leishmania when glycerol is used [25]. However. the genes that encode the first two enzymes in the haem biosynthetic pathway. transketolase. which correlates well with the fact that. major. in L. SHMT is also able to cleave the Ca–Cb bond of threonine. Does this mean that Leishmania has acquired the ability to synthesize haem from precursors available in the sand fly midgut? Alternatively. Proline oxidation is confirmed at the gene level for all three trypanosomatids and is mediated by proline oxidase and d-1-pyrroline-5-carboxylate dehydrogenase.29]. In the first. However. a partial biosynthetic pathway is needed by amastigotes to obtain the haem that they require? Lack of an alternative oxidase An unusual and key feature of the energy metabolism of bloodstream T. however. which is produced by the TCA cycle. major lacks the next enzyme (HMGCoA lyase) of the classical oxidative pathway and.27]. The resulting glycine can be converted through serine to pyruvate by the THF-dependent glycine cleavage system (GCS) followed by a serine/threonine dehydratase (STD). therefore. together with a pteridine transporter gene.icp. There seem to be two reasons for this. brucei is the alternative oxidase functional.33]. First. the same dehydratase might convert threonine to 2-ketobutyrate.htm). HMGCoA is incorporated directly into sterols by the isoprenoid synthetic pathway. major genome. in T. one with a glycosomal targeting sequence and one without. therefore. cruzi. the enzymatic reduction of folate to become active as tetrahydrofolate (THF). were previously known to occur only in cyanobacteria and in plant plastids [30]. Interestingly. it can convert threonine through a THF-dependent pathway to glycine using the enzyme serine hydroxymethyltransferase (SHMT) (also known as threonine aldolase).sciencedirect. ribulose-5-phosphate 3-epimerase and ribose-5-phosphate isomerase. folate and biopterin) and must. Leishmania has two pathways to metabolize threonine (Figure 2).4 to generate NADPH and ribose 5-phosphate for other biosynthetic pathways. brucei lacks the dehydratase and SHMT. [32. which are also present in trypanosomes. some of the enzymes are mainly cytosolic in promastigotes [23. a coenzyme required for one-carbon (C1) transfer reactions (Box 2 and see Table S4 in the supplementary material online: http://www. Alternatively. T. thus. Only in T. whereas. hence. differs from trypanosomes in that methionine can be oxidized completely. there are two isoenzymes for ribulose-5-phosphate 3-epimerase. for which genes that encode most of the enzymes have been detected and which was experimentally demonstrated by Ginger et al.27]. major or T. transaldolase. The two oxidase genes. Synthesis of haem Haem is required for the synthesis of iron-containing proteins such as cytochromes and is normally synthesized from succinyl coenzyme A (CoA).23 No. no drugs that target the folate pathway have been found to be effective against Leishmania infections [26. such as bacteria and Plasmodium. L. . and seems to metabolize threonine using the alternative aminoacetone pathway. such as coproporphyrinogen III oxidase.be/opperd/leishmania_ table4. However. have not been detected in the trypanosomatids – which correlates well with the requirement of exogenous haem for the growth of these organisms. together with the ferrochelatase gene. Folate metabolism In many other microorganisms. cruzi. can be catalysed by both bifunctional dihydrofolate reductase–thymidylate synthase (DHFR–TS) [31] and pteridine reductase 1 (PTR1) (Box 2). respiration is not sensitive to inhibition by salicylhydroxamic acid. in the two trypanosomes. PTR1 is overexpressed if DHFR–TS is inhibited. No functional oxidase gene was detected in either L. Leishmania. seem to be of bacterial origin.com with this. Leishmania cannot synthesize folates (e. which involves a mitochondrial threonine dehydrogenase (TDH) and an aminoacetone synthase (2-amino-3-ketobutyrate CoA ligase). brucei and T. All three organisms contain a flavine-dependent glycerol-3-phosphate dehydrogenase in the mitochondrion. whereas there are as many as 12 genes that encode a novel class of membrane transport protein that is responsible for folate transport [28. All but one (transaldolase) have a glycosomal targeting signal. Leucine is transaminated in the cytosol to 2-ketoisocaproate by a cytosolic aminotransferase and can be further oxidized in the mitochondrion by a shortbranched chain acyl-CoA dehydrogenase.152 Review TRENDS in Parasitology Vol. perhaps the haem of host origin is partially degraded within the parasitophorous vacuole and. aminolevulinate synthase and aminolevulinate dehydratase. a carboxylase and a hydratase to hydroxymethylglutaryl CoA (HMGCoA). Such transporters. cruzi. it might be necessary to block both DHFR–TS and PTR1 simultaneously for effective interference with folate metabolism. Amino acid oxidation There are multiple differences among the three trypanosomatids in the area of amino acid metabolism (Figure 1).g. the folate pathway provides a valuable target for drug intervention. no genes that encode enzymes of folate biosynthesis are present in the L.24]. its degradation halts at the level of 2-ketobutyrate because the mitochondrial enzymes (methylmalonyl-CoA epimerase and methylmalonyl-CoA mutase) for the oxidation of 2-ketobutyrate to succinyl CoA seem to be absent. are encoded by L. In addition to the demethylation of serine.

This might also reflect the use by amastigotes of amino acids from the vacuole. PGA. major and T. cruzi (see key). This indicates that arginine. citrulline. brucei (but not T. Leishmania is similar to T. which could be incompatible with release of ammonia in quantity because it could affect the pH. dihydrofolate. although a fully functional urea cycle is missing from L. B. HMGCoA follows the classical pathway of leucine oxidation and is cleaved into acetyl CoA and acetoacetate. hydroxymethylglutaryl CoA. with permission. oxaloacetic acid. is present in all three organisms but argininosuccinate synthase and arginase occur only in L. Amino acid metabolism in Leishmania major compared with Trypanosoma brucei and Trypanosoma cruzi. acetoacetate. tetrahydrofolate. major.sciencedirect. THF. unlike trypanosomes. [37]. AdoMet. quinoid form of dihydrobiopterin. brucei or T. The first enzyme.com produce ammonia [34]. biopterin. cruzi and explains an old observation that some Leishmania excrete urea. cruzi) in that it contains ornithine decarboxylase. the glyoxylate cycle and the urea cycle [which are present in most other eukaryotes (grey)] and the differences between L. brucei and T. . from Ref. Orn.4 153 Figure 1. Abbreviations: AcAc. Cit. qH2B. The lack of efficacy of this drug against Leishmania seems to relate to both the transporters on the parasite and the environment of the parasite. This might relate to life in an acidic parasitophorous vacuole. the target of the antitrypanosomal drug difluoromethylornithine. The figure shows the pathways that are shared by all three trypanosomatids (black).Review TRENDS in Parasitology Vol. Question marks represent enzyme-catalysed steps for which no unambiguous gene identification could be made. OAA.23 No. HMGCoA. ornithine. ornithine and urea can be formed only in Leishmania and not in T. DHF. major. the absence of many enzymes that are involved in amino acid metabolism. T. phosphoglyceric acid. adenosylmethionine. Leishmania contains several enzymes of the urea cycle. Adapted. carbamoyl-phosphate synthetase. rather than a lack of the target enzyme [35]. brucei and T. whereas trypanosomes www. cruzi.

TS.sciencedirect. Interestingly. Activated C1 units are used in the synthesis of thymidylate by the thymidylate-synthase half of DHFR–TS and for the formation of methionine from cysteine by the enzyme methionine synthase. DHF. MTFC. cobalamin-independent methionine synthase.4 Synthesis of amino acids The capacity for the synthesis of amino acids by L. this loss does not mean that trypanosomes now obtain methionine from an external source. By contrast. QDPR. is present in L. Abbreviations (enzymes are in italics in figure): BT. Most genes that code for enzymes that are Box 2. MTR.23 No. This pathway seems to occur only in Leishmania because the upstream enzyme methylenyltetrahydrofolate reductase. which indicates independent methionine synthase. PCD. glycine-cleavage system. However. dihydrofolate. However. dihydrofolate reductase. D-3-phosphoglycerate dehydrogenase. Folate metabolism The activation of C1 units into the THF pool takes place through the formation of N5. biopterin transporter. cruzi as a separate monofunctional ligase and a bifunctional cyclohydrolase–dehydrogenase enzyme (Figure II). TK. FTHS. methyl tRNA formyltransferase. cobalamin-dependent methionine synthase. methylenyl tetrahydrofolate reductase. HMT. serine hydroxymethyltransferase. pteridine reductase 1. PTR1. major is limited to the nonessential ones plus threonine and methionine. T. tetrahydrofolate. homocysteine S-methyltransferase. nucleoside transporter. all three organisms have the genes that encode the GCS. methylene tetrahydrofolate dehydrogenase. one cytosolic and one mitochondrial [40]. DHFR.N10-methylene-THF by either SHMT or the GCS (see Figures 1 and 2 in the main text). quinonoid dihydropteridin reductase. The trypanosomatid FTHS. brucei lacks the gene for the ligase. whereas T.com . However. NT.154 Review TRENDS in Parasitology Vol. fMet is essential for the initiation of mitochondrial protein synthesis. MTFD. all trypanosomatids seem to be able to synthesize this amino acid by an alternative route using homocysteine S-methyltransferase (HMT). methylene-THF-cyclohydrolase and methyleneTHF dehydrogenase reactions. Trypanosoma brucei and Trypanosoma cruzi.0010). Leishmania has two isofunctional methionine synthases for this purpose: a cobalamin-dependent one (LmjF07. Formyl-C1 units are also used for the formation of formyl-methionine (fMet) by methionyl-tRNA formyl transferase (Figure I). phenylalanine hydroxylase. Biopterin and folate metabolism in Leishmania major. LmjF31. cruzi lacks both the cofactor-dependent and the cofactor-independent enzymes. thymidine kinase.0090) and a cobalamin-independent one (LmjF31.0010 seems to be the result of a lateral transfer event from a bacterium. The inactivity of this pathway by the loss of the reductase from an ancestral trypanosome probably explains why T. pterin-4-a-carbinolamine dehydratase. folate transporter. MTFC and MTFD enzymes differ in structure from their mammalian counterparts (Figure II). the formate-THF ligase. which have been combined in humans into a trifunctional enzyme called one-carbon-tetrahydrofolate synthase (C1-THF). Leishmania has two SHMT isoenzymes. formate tetrahydrofolate ligase. Trypanosoma cruzi has only the cytosolic isoenzyme and Trypanosoma brucei seems to lack both. brucei has only the cofactor- involved in the synthesis of these amino acids are clearly present. GCS. major but is absent from the two trypanosomes. indeed. is not present in the two trypanosomes. dMS. are present in Leishmania and T. www. PAH. Methotrexate functions as an inhibitor of DHFR. THF. iMS. which is necessary for the formation of methyl THF. the first enzyme in the synthesis of serine. methylene tetrahydrofolate cyclohydrolase. MtRFT. Figure I. other THF-dependent formyl transferases such as those involved in the biosynthesis of either purines or histidine are all absent and this correlates with the absence from trypanosomatids of the biosynthetic pathways for these molecules. SHMT. thymidylate synthase. FT.

the first two reactions are normally carried out by a bifunctional enzyme called pyrroline-5-carboxylate synthetase.23 No. The corresponding gene is found in L. In addition.com . Phylogenetic reconstruction Figure 2. Cysteine can be produced by cysteine synthase. cysteine can be generated from homocysteine by the transsulfuration pathway. Enzyme names are in italics.sciencedirect.Review TRENDS in Parasitology Vol. Both of these enzymes have been found in L.org) and Uniprot/SwissProt (http://www. and the enzymes are g-glutamyl kinase. which are present in all three organisms. cruzi should be able to synthesize proline from glutamate. cruzi). Leishmania and T.org) databases. cruzi but not in T. The synthesis of L-proline from glutamate occurs through g-glutamyl-phosphate and d-1-pyrroline-5-carboxylate. as detailed earlier. the conversion of asparagine to aspartate does not seem to occur in T. Threonine metabolism in Leishmania major compared with Trypanosoma brucei and Trypanosoma cruzi. brucei should not. The third activity is catalysed by a separate enzyme and its gene is present in all three trypanosomatids.4 155 Figure II. cruzi or T.expasy. g-glutamyl phosphate reductase and d-pyrroline-5-carboxylate reductase. which is present in all three organisms. brucei. whereas T. major. major (and T. www. which comprises formate tetrahydrofolate ligase (FTHS). It also shows the absence from T. is absent from trypanosomatids and has been replaced by a monofunctional FTHS and a bifunctional enzyme with MTFC and MTFD activities. However. major [36] and are predicted to be present in T. The abilities of trypanosomatids to synthesize proline differ substantially. glycine and serine can be interconverted by the action of SHMT. that they differ in their ability to synthesize this amino acid. In prokaryotes. methylene THF cyclohydrolase (MTFC) and methylene THF dehydrogenase (MTFD) activities. major (and in T. A mammalian-type trifunctional C1-THF synthase. brucei and T. Labelling of sequences is exactly as in the GeneDB (http:// www. brucei. Alanine is formed from pyruvate by a cytosolic alanine aminotransferase. This scheme shows the presence of the aminoacetone pathway in T. major but not in T. Interestingly. Thus. cruzi. brucei of enzymes required for the metabolism of threonine (violet). in eukaryotes. genes encoding a separate g-glutamyl kinase and g-glutamyl-phosphate reductase are also present in L.genedb. cruzi (orange) as opposed to the presence of the ketobutanoate pathway (green) in L. In L. with the possible involvement of 3-mercaptopyruvate sulfurtransferase. brucei). whereas. Schematic alignment of C1-THF sequences and their homologues. See also Figure I in Box 2. and aspartate and asparagine can be formed from oxaloacetate by a mitochondrial aspartate aminotransferase and an asparagine synthase. these activities are encoded by separate genes.

31. enolase. Abbreviations: Fru. phosphoglycerate kinase. phosphoglyceric acid. hexokinase. 23. pyruvate dehydrogenase. 16. GDP-mannose pyrophosphorylase. amylase-like protein. 40. 3. The pathways of core metabolism in Leishmania major. www.sciencedirect. glucose. that are involved in carbohydrate metabolism. fumarate hydratase. Pathways in blue are thought to be more important in promastigotes and pathways in red are thought to be more important in the amastigote. GAP. glyceraldehyde-3-phosphate dehydrogenase. Glc. 8. 14. pyruvate phosphate dikinase. 2. glucosamine-6-phosphate deaminase. alanine aminotransferase. 2-ketoglutarate dehydrogenase. 10. 24. proline oxidation pathway. clearly indicates that these individual enzymes are of bacterial origin. citrate lyase.23 No. 21. threonine oxidation pathway. phosphoenolpyruvate. 35. succinate dehydrogenase. 34. 33. 12. 28.4 Figure 3. 38. xylulokinase. malic enzyme. 32. phosphoglucose isomerase. mannose-6-phosphate isomerase. NADH-dependent fumarate reductase. 39. ribulokinase. Figure shows reactions taking place in the glycosome. 15. H-5-P. malate dehydrogenase. fructose. Man. 7. pentose-phosphate pathway. It is unclear why L. 5. hexose 5-phosphate. PPP. aspartate aminotransferase. 4. 22. Boxed metabolites are substrates (grey) or end products (black) of metabolism. Thick arrows represent major metabolite fluxes. 25. sucrase-like protein. PGA. pyruvate kinase. glycerol kinase. glycerol-3-phosphate dehydrogenase. 13. Enzymes: 1. glyceraldehyde 3 phosphate. PEP. 37. phosphomannomutase. 36. 27.com Of the so-called essential amino acids. citrate synthase. phosphoenolpyruvate carboxykinase. 6. acetyl-CoA synthetase. adenylate kinase. fructosebisphosphate aldolase. and in the mitochondrion. the pathways for the synthesis of lysine.156 Review TRENDS in Parasitology Vol. 11. ribokinase. 20. 17. phosphoglycerate mutase. 30. threonine and methionine from aspartic acid seem to be either absent or incomplete in all three trypanosomatids. triosephosphate isomerase. whereas the bifunctional pyrroline-5-carboxylate synthetase is of eukaryotic origin. 26. acetate–succinate CoA transferase. mannose. major has these additional proline synthesis enzymes. phosphofructokinase. 29. and the flux of metabolites between these two organelles. 9. Genes that encode aspartokinase . 18. 19. succinyl-CoA ligase. with its tricarboxylic acid cycle.

351–360 9 van Hellemond. (2006) Energy metabolism of trypanosomatids: adaptation to available carbon sources. Parasitol. Microbiol. 83–90 15 Mottram. Therefore. such as in amino acid metabolism. (1981) The effects of carbon dioxide and oxygen upon the growth and in vitro transformation of Leishmania mexicana mexicana. 397– 409 13 Coombs. K.H. N. the de novo synthesis from aspartate of these three amino acids seems unlikely. Mol.N. (2001) Targeting polyamines of parasitic protozoa in chemotherapy. Bioinformatics 22. (1986) Primary structure of the gene encoding the bifunctional dihydrofolate reductase–thymidylate synthase of Leishmania major.sciencedirect. M. Mol.J. 151–160 16 Mottram. for the use of sugars that are available in the sand fly but not the vectors of trypanosomes. and Blum. F.T. Parasitology 114. H. J. et al. L. A. D. and Tielens. 135–138 10 Mukkada. Microbiol.M. J. J. 117–127 7 Darling. Kinetoplastid Biol.H. 34. aromatic amino acids (phenylalanine.H. Biochem.J. 228–231 19 Keegan. 277.P. and Blum. Trends Parasitol. A. G. 191–202 8 van Hellemond. 32.L. J. 2584–2588 32 Ginger. References 1 Bringaud.H. Acad.A. 13–23 14 Tetley. C. Dis.P. 265–274 17 De Souza. et al. Bacteriol. (2006) Progressive peak clustering in GC–MS Metabolomic experiments applied to Leishmania parasites. Parasitol.G. et al. TDR1. M. R. et al. M. 11674–11682 34 Yoshida. Acknowledgements We thank Valerio Losasso (University of Modena) for communicating her initial analyses of Leishmania folate metabolism. J. Biol. 1–9 2 Hannaert.R. 3567–3581 5 Burchmore. et al. Chem. 113. (2005) New functions for parts of the Krebs cycle in procyclic Trypanosoma brucei. 17. Eukaryot. 167–175 www. J. et al. S. (2003) Evolution of energy metabolism and its compartmentation in Kinetoplastida.H. 382. Some of the other differences detected. Biochem. (2002) Pterin transport and metabolism in Leishmania and related trypanosomatid parasites. FEBS Lett. et al. 405–412 22 Cronin. (2001) Life in vacuoles – nutrient acquisition by Leishmania amastigotes. (1978) Ureotelism and ammonotelism in trypanosomatids. (2003) Pentose phosphate metabolism in Leishmania mexicana. Parasitol.com 11 van Weelden. F. 33.4 157 and aspartate semialdehyde dehydrogenase could not be detected. 136. Parasitol.M. are hard to interpret.R.M. J. Chem. Eur.23 No. 385–398 28 Richard. Acta Trop. and Barrett. 381. 1391–1396 18 Blum.J. 26–30 23 Maugeri.L. Chem. acknowledges support from the UK Medical Research Council and the Wellcome Trust. Biochem. et al. and Coombs. (1985) Enzyme activities of amastigotes and promastigotes and their inhibition by antimonials and arsenicals. N. Endotrypanum monterogeii and Trypanosoma cruzi. Mol. et al.T. D. et al. Mol. J. Proteomics 6. (1991) The glycosomes of trypanosomes: number and distribution as revealed by electron spectroscopic imaging and 3D reconstruction. and the Wellcome Trust Sanger Institute for providing sequence information from the Leishmania genomes to the community. E. (1992) Utilization of a carbohydrate reserve comprised primarily of mannose by Leishmania donovani. (2006) The mitochondrial FAD-dependent glycerol3-phosphate dehydrogenase of Trypanosomatidae and the glycosomal redox balance of insect stages of Trypanosoma brucei and Leishmania spp. 29460– 29467 29 Cunningham. Biochem.J. (1985) Leishmania mexicana: subcellular distribution of enzymes in amastigotes and promastigotes. 59. 40. was supported by IUAP grant 5–29 from the Belgian Government.). Biochem. However. et al. Mol. Science 309. 59. (2001) The biosynthetic incorporation of the intact leucine skeleton into sterol by the trypanosomatid Leishmania mexicana.P.J. Parasitol. (2006) A combined proteomic and transcriptomic approach to the study of stage differentiation in Leishmania infantum. Biochem.M. J. Parasitol. Parasitol. F. 267. D. 149. 2555–2566 33 Ginger. some of the known metabolic differences between trypanosomatids are not fully explained by the presence or absence of genes that encode metabolic enzymes.H. 149. Clearly. 38457–38463 31 Beverley.N. Mol. 2. G. Microsc. J. Proc. F. Sci. 117–125 24 Veitch. et al. 130.C. 12451–12460 12 Hart. et al. U. the situation will be affected by the control of gene expression and by enzyme stability at different stages of the life cycle. 244. et al. a cycle not operating as a cycle. Concluding remarks The importance of some of the detected differences in gene content between trypanosomatids can be readily hypothesized: for instance.L. and Coombs. Biochem. (1994) Secretion of sucrase by Leishmania donovani.G. Exp. et al. Exp. (1989) The enzymes of the classical pentose phosphate pathway display differential activities in procyclic and bloodstream forms of Trypanosoma brucei.C. Exp. 4. Biochem. and Coombs. Parasitol. et al. Biochem. Biol. G. 242–249 . D. F. Parasitol.J. 83. Int. Parasitology 114 (Suppl. S. B. J. J. and Camargo. (1993) Incorporation of label from acetate and laurate into the mannan of Leishmania donovani via the glyoxylate cycle. et al. J. Mol. isoleucine and valine) and lysine and histidine cannot be synthesized by the trypanosomatids. Parasitol. (1989) Carbon dioxide abolishes the reverse Pasteur effect in Leishmania major promastigotes. et al. et al. S.J. Parasitol. (1986) Three dimensional structure of the Leishmania amastigote as revealed by computer-aided reconstruction from serial sections. Mol. N. 31. G. 53. G. A. S. (2000) Utilization of leucine and acetate as carbon sources for sterol and fatty acid biosynthesis by Old and New World Leishmania species. 155–169 26 Nare. J. 193–200 20 Keegan. M. F. the branched amino acids (leucine.C. (1977) Tricarboxylic acid and glyoxylate cycles in the Leishmaniae. Int.J. Parasitology 92. J. G. Chem. Natl. D. V. 85. (2004) Transketolase from Leishmania mexicana has a dual subcellular localization. J. 276. Many detailed studies are now required. 280.P. Biochem. (1997) New approaches to Leishmania chemotherapy: pteridine reductase 1 (PTR1) as a target and modulator of antifolate sensitivity.P. J. M. (1997) Leishmania infantum promastigotes have a poor capacity for anaerobic functioning and depend mainly upon respiration for their energy generation. and Vickerman. T. 759–767 25 Guerra. 730–732 21 Denton. Parasitol. (2005) Comparative genomics of trypanosomatid parasitic protozoa. (1997) Inhibition of the respiratory chain results in a reversible metabolic arrest in Leishmania promastigotes. and Beverley.Review TRENDS in Parasitology Vol. (2002) A new type of high affinity folic acid transporter in the protozoan parasite Leishmania and deletion of its gene in methotrexate-resistant cells. Biol. 162. and also the necessity for different end-product production in the parasitophorous vacuole.W. D. tyrosine or tryptophan). J. et al. Parasitol. L. (2001) Pteridine salvage throughout the Leishmania infectious cycle: implications for antifolate chemotherapy. et al. major should be able to synthesize both methionine and threonine starting from aspartate semialdehyde. and gene mining has indicated some sensible approaches to pursue and experiments that should be done. and Coombs. 1311– 1320 6 Hart. (1982) Leishmania mexicana: energy metabolism of amastigotes and promastigotes. (2004) Reduction of anti-leishmanial pentavalent antimonial drugs by a parasite-specific thiol-dependent reductase. and Opperdoes. S. Biochem. whereas this conversion seems to be absent from the two trypanosomes. 54. Moreover.O. S. J. Eukaryot. 404–409 4 McNicoll. 280. J. Biol. 11 3 El-Sayed. S101–S110 27 Ouellette. The current ‘best guess’ on the metabolism that occurs in Leishmania amastigotes and promastigotes is shown in Figure 3. 41. 199–213 30 Klaus. et al. 1184–1186 35 Muller. (2005) Higher plant plastids and cyanobacteria have folate carriers related to those of trypanosomatids.

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