Metabolism of Leishmania: proven and predicted

Metabolism of Leishmania: proven andpredictedFred 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, Belgium2 Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, The John Arbuthnott Building, Glasgow G4

0NR, UK

Review TRENDS in Parasitology Vol.23 No.4

The complete analysis of the genomes of three majortrypanosomatid parasites has facilitated comparison ofthe metabolic capabilities of each, as predicted fromgene sequences. Not surprisingly, there are differencesbut is it possible to correlate these with the lives of theparasites themselves and make further predictions ofthe meaning and physiological importance of theapparently parasite-specific metabolism? In this article,we relate gene predictions with the results from exper-imental studies. We also speculate on the key meta-bolic adaptations of Leishmania and reasons why itdiffers from Trypanosoma brucei and Trypanosomacruzi.

Examining the differencesFour hundred genes that code for the enzymes of the majormetabolic pathways in Leishmania major have been ident-ified (see Table S1 in the supplementary material online:http://www.icp.be/�opperd/leishmania_table1.htm). For30 (8%) of them, orthologues have not been detected ineither Trypanosoma brucei or Trypanosoma cruzi (seeTable S2 in the supplementary material online: http://www.icp.be/�opperd/leishmania_table2.htm). By contrast,Leishmania lacks a few genes that encode metabolicenzymes in T. brucei, T. cruzi or both. For example, sedo-heptulose-1,7-bisphosphatase and threonine dehydrogen-ase are absent from Leishmania but occur in bothtrypanosomes. Three enzymes involved in histidinemetab-olism are found only in T. cruzi. Overall, however, L. majorhas the most extensive metabolic machinery, whereas thatof T. brucei is the most restricted. The differences betweenLeishmania and trypanosomes are found mainly at thelevel of carbohydrate and amino acid metabolism. So, whatis the importance of this?

One limitation in making meaningful predictions aboutthe metabolic make up of parasites is not knowing whichproteins have distinct stage specificity. The amazing vari-ation in energy metabolism exhibited by T. brucei duringits life cycle [1,2] has been well documented but this mightnot be the typical scenario. Unfortunately, there is littleinformation about this for Leishmania (andT. cruzi). Thus,predictions have to involve a certain amount of ‘educatedguessing’.

Corresponding author: Coombs, G.H. ([email protected]).Available online 22 February 2007.

www.sciencedirect.com 1471-4922/$ – see front matter � 2007 Elsevier Ltd. All rights reserve

Why is Leishmania metabolically different from othertrypanosomes?Leishmania and trypanosomes evolved from a commonancestor and share many features. Thus, it was expectedthat many of the genes of different trypanosomatids wouldbe orthologues, as has now been proven [3]. However, thereare some substantial biological differences between trypa-nosomatids, and these can be illustrated by consideringtheir life history strategies. Notably, Leishmania thrivesinside a parasitophorous vacuole within mononuclearcells in a mammalian host; thus, it is adapted for reachingthis niche and is metabolically appropriate for it. It isthought that the metabolic capabilities of the amastigoteevolved to ensure that this stage of the parasite is fullyadapted for its intracellular existence. When in its sand flyhost, Leishmania exists as promastigote forms. These areadapted for life in different parts of the intestinal tract of thefly but, unlike trypanosomes, not in the hindgut (except forthe braziliensis complex of Leishmania) or the salivaryglands. Moreover, sand flies differ from the vectors of try-panosomes in their biology (e.g. their food source) and thishas implications for the Leishmania parasites within them.

There is now extensive information about the genes ofLeishmania and this should help researchers to deducethe adaptations of Leishmania for each of the stages of itslife cycle. However, it is not yet known when in the life cyclemany of the genes are functionally important. Presumably,suchanalyseswill be completed soon, although it is doubtfulthat global transcriptomic and proteomic analyses [4] willprovide all of the answers, and detailed study of gene groupswill be required formany years.Moreover, knowledge of theenvironments in which the various parasite forms reside,such as the parasitophorous vacuole, remains poor, whichlimits researchers’ ability to mimic these conditions exper-imentally. One known variation is pH – the amastigoteresides in an acidic environment, whereas the habitat ofpromastigotes is believed to be closer to neutral. This hasimplications for nutrient acquisition [5], although theinternal pH of both parasite forms is thought to be aroundneutral. Another aspect that might be important in deter-mining metabolism is the availability of oxygen and carbondioxide. Amastigotes and promastigotes respond differ-ently, in metabolic terms, to variation in gas availability[6,7] and thereare almost certainly differences in themetab-olism of the two parasite stages because of the differencesbetween the environments in which they reside.

d. doi:10.1016/j.pt.2007.02.004

http://www.icp.be/~opperd/leishmania_table1.htm





mailto:[email protected]

http://dx.doi.org/10.1016/j.pt.2007.02.004

150 Review TRENDS in Parasitology Vol.23 No.4

Energy metabolism of Leishmania

Biochemical analyses of Leishmania promastigotes havealmost invariably involved the parasite stages grown invitro in nutrient-rich medium, usually with air as the gasphase. However, whether these in vitro forms metabolizein the same manner as promastigotes that occur naturallyin sand flies is not known. In addition, it is probable that, atleast in some studies, mixed populations of promastigoteswere used (e.g. metacyclic promastigotes in addition tomultiplicative forms). Results from such studies indicatedthat the metabolism of promastigotes has similarities tothat of the vector stages of both T. cruzi and T. brucei. Forexample, amino acids (notably proline) and glucose can beused as energy sources, and energy generation seems toinvolve both glycolysis (and glycosomes) andmitochondrialmetabolism, which comprises an active electron-transportchain. There are enzymes of the tricarboxylic acid (TCA)cycle present, although experimental evidence indicatesthat the cycle is not active [8]. Interestingly, inhibition ofthe electron transport chain results in reversible metabolicarrest [9], similar to when promastigotes are starved. Thisis in contrast to procyclic stages of African trypanosomes,which depend on their respiratory chain and rapidly die ifit is nonfunctional because of the absence of oxygen. It hasbeen postulated that this ability of Leishmania promasti-gotes to undergo metabolic arrest might be an adaptationfor survival during life in the sand fly [9]. It is also thoughtthat other sugars, in addition to glucose, might be used.The presence of a glyoxylate cycle was reported [10] but thegenome analysis shows this to be unlikely. The end pro-ducts identified include succinate, acetate and smallamounts of pyruvate and D-lactate. CO2 production resultsfrom the pathways that produce acetate and succinate.Alanine, ammonia and urea are also released. Recentextensive experimental analyses of T. brucei procyclicforms have prompted reinterpretation of the extent towhich the TCA cycle is involved (or not) in energy gener-ation [11]. The situation in Leishmania promastigotesrequires similar reanalysis, including gene deletions todetermine the requirement for the various pathways.Nevertheless, the current view of the metabolism of pro-mastigotes is now supported by the finding of all theexpected genes [3].

Knowledge of the energy metabolism of Leishmaniaamastigotes is considerably more fragmentary than thatof promastigotes, primarily because amastigotes have beenless available for study. Studies using Leishmania mex-icana amastigotes isolated from in vivo lesions revealedthat, compared with promastigotes, amastigotes have anincreased b-oxidation of fatty acids and a reduced need forproline and glucose consumption [12]. This substrate use isfacilitated by an apparently fully functional TCA cycle (butthis needs reanalysis) and a linked respiratory chain.Therefore, the substantial stage variation inmitochondrialactivity that occurs with T. brucei does not occur withLeishmania. Amastigotes contain glycosomes, althoughconsiderably fewer than promastigotes [13,14]. There issome evidence for stage variation in the metabolism ofthese organelles – as exists with glycosomes of T. brucei –although, with Leishmania, it is the amastigote glycosomethat seems to contain the greater number of enzymes

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such as phosphoenolpyruvate carboxykinase and malatedehydrogenase [15,16]. Caution is required, however,because analyses of glycosomes that are purified fromamastigotes are yet to be conducted. The advent ofmethods for culturing Leishmania amastigotes in theabsence of host cells in vitro provided an opportunity forfurther analyses of the energy metabolism of amastigotes,at least for some species and with the caveat that theaxenic forms could differ from natural amastigotes. How-ever, studies have been rather limited so far and theresults largely agree with the findings with purified amas-tigotes. The application of metabolomics to this parasitestage [17] could revolutionize the understanding of itsmetabolic capabilities.

This knowledge, albeit limited, of the metabolism ofLeishmania provides a framework on which to attemptto interpret the extensive information that is now availableabout the gene content of Leishmania.

Predictions of how Leishmania differs metabolicallyfrom trypanosomesSugar use

Leishmania has genes that encode both an amylase and asucrase-like protein. This correlates well with publishedfindings [18] and, presumably, the enzymes are used bypromastigotes for the digestion of plant starch and dis-accharides taken in by sand flies that feed on nectar.Leishmania has been reported to contain mannan[19,20], which is considered to be a potential energyreserve, although current evidence does not enable judg-ment on its effectiveness and other roles cannot be ruledout, so some of the enzymes could also be involved incatabolizing this polysaccharide. Leishmania (as with T.cruzi) also has genes that encode several sugar kinasessuch as ribulokinase and xylulokinase. The bacterialnature of these enzymes indicates that they have probablybeen acquired from a bacterium through horizontal trans-fer and facilitate the digestion of a range of sugars that ispresent in the nectar upon which sand flies feed. All thesesugar kinases carry targeting signals for import into theglycosomes, which indicates that the glycosome, inaddition to being the site of glucose metabolism, is alsocentral to the degradation of other sugars.

Ascorbate metabolism

L. major and T. cruzi have an ascorbate-dependentperoxidase, whereas T. brucei has many copies of aniron–ascorbate oxidoreductase. This indicates the presenceof ascorbic acid in trypanosomatids and the possibility thatit is used in their defence against oxidative stress (e.g. byLeishmania in the host macrophage). Genome mining andexperimental evidence indicate that a fungal type of bio-synthesis might be functional in the trypanosomatids (Box1 and see Table S3 in the supplementary material online:http://www.icp.be/�opperd/leishmania_table3.htm). How-ever, attempts to detect ascorbate or erythroascorbate inLeishmania proved unsuccessful (H. Denton and G.H.Coombs, unpublished), so the functionality of the ascorbatebiosynthetic pathway in Leishmania is uncertain. A thiol-dependent enzyme that might be involved in the reductionof dehydroascorbic acid has been reported [21] but it could



Box 1. Ascorbate metabolism

The genes that encode enzymes of an animal-type ascorbate biosyn-

thetic pathway have been tentatively identified in the Trypanosoma

cruzi genome. However, Leishmania major seems to lack two of them

and Leishmania braziliensis lacks one (Figure I). Recently, gulono-1,4-

lactone oxidase, the last enzyme of this pathway in T. cruzi, was

characterized [38]. This enzyme is homologous to the enzymes that

catalyse the last step in the formation of (erythro)ascorbate in animals,

plants and fungi. The protein contains a flavine adenine dinucleotide

(FAD)-binding motif and a type-1 peroxisome targeting signal (PTS1),

consistent with its demonstrated location inside glycosomes [38]. The

trypanosome enzyme was shown to prefer D-arabino-1,4-lactone to

gulono-1,4-lactone as a substrate. Thus, current evidence indicates that

a yeast-like pathway, rather than an animal-type pathway, is opera-

tional in this trypanosomatid. The yeast-like pathway results in the

formation of erythroascorbate from arabinose, and the trypanosomatid

genomes contain a gene that encodes an NADP-dependent arabinose

dehydrogenase, which catalyses the formation of D-arabinono-1,

4-lactone from D-arabinose. The Leishmania enzyme also has a

potential PTS1, which indicates that ascorbate might be generated

within glycosomes as antioxidants. It is not yet clear, however, where

the substrate for the pathway (D-arabinose) comes from. It is an

extremely rare sugar, which is known only in polysaccharides of plants

and, so far, there are no obvious candidate enzymes in trypanosoma-

tids that are able to form D-arabinose from other C5 sugars. Interest-

ingly, however, L. major has recently been reported to incorporate D-

arabinose into lipophosphoglycan side chains [39]. The trypanosomal

gulono-1,4-lactone oxidase has an intermediate activity with L-

galactono-1,4-lactone [38], the substrate for the formation of ascorbate

through a plant-type pathway. Thus, 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.

Figure I. Animal- and yeast-type pathways of ascorbate biosynthesis in Leishmania major, Trypanosoma brucei and Trypanosoma cruzi. The animal-type pathway, with

gulonate and gulonolactone as intermediates and ascorbate as the end product, is shown on the left hand side. The yeast-type pathway, with arabinolactone as the

intermediate and erythroascorbate as end product, is boxed. In all three trypanosomatids the genes for the enzymes of the yeast-type pathway were identified; however,

only T. cruzi has all the genes for the enzymes of the animal-type pathway. Accession codes, where identified, are indicated for L. major. Abbreviations: GSH, reduced

glutathione; GSSG, oxidized glutathione; UDP, uridine diphosphate. Footnotes: *, not present in L. major but present in Leishmania braziliensis; y, biochemically

identified in T. brucei; z, enzymes present in glycosomes.

Review TRENDS in Parasitology Vol.23 No.4 151

have other activities in the parasite. Whereas ascorbatebiosynthesis seems still to exist in T. cruzi, it mighthave been lost from Leishmania (and T. brucei), with theretained enzymes being active in other ways.

Pentose phosphate pathway

Leishmania seems to lack sedoheptulose bisphosphatase,an enzyme that is predicted to be present inT. brucei andT.


cruzi and likely to be involved in a modified pentosephosphate pathway. The importance of its absence isunknown because candidate genes encoding most of theenzymes of the conventional pentose phosphate pathwayare present and the parasites are reported to contain afunctional pathway [22,23]. The genes present includeglucose-6-phosphate dehydrogenase, 6-phosphogluconolac-tonase, the 6-phosphogluconate dehydrogenase required


to generate NADPH and ribose 5-phosphate for otherbiosynthetic pathways, transaldolase, transketolase, ribu-lose-5-phosphate 3-epimerase and ribose-5-phosphate iso-merase. All but one (transaldolase) have a glycosomaltargeting signal, which indicates that the entire pentosephosphate pathway might occur inside glycosomes. How-ever, some of the enzymes are mainly cytosolic in promas-tigotes [23,24], so the pathway probably functions in bothcompartments. Interestingly, there are two isoenzymes forribulose-5-phosphate 3-epimerase, one with a glycosomaltargeting sequence and one without.

Synthesis of haem

Haem is required for the synthesis of iron-containingproteins such as cytochromes and is normally synthesizedfrom succinyl coenzyme A (CoA), which is produced by theTCA cycle. However, the genes that encode the first twoenzymes in the haem biosynthetic pathway, aminolevuli-nate synthase and aminolevulinate dehydratase, have notbeen detected in the trypanosomatids – which correlateswell with the requirement of exogenous haem for thegrowth of these organisms. Interestingly, other enzymesof the haem biosynthesis pathway, such as coproporphyr-inogen III oxidase, protoporphyrinogen oxidase and ferro-chelatase, are encoded by L. major. The two oxidase genes,together with a pteridine transporter gene, are juxtaposedas a separate transcription unit on one end of chromosomesix and, together with the ferrochelatase gene, seem to beof bacterial origin. Does this mean that Leishmaniahas acquired the ability to synthesize haem from precur-sors available in the sand fly midgut? Alternatively,perhaps the haem of host origin is partially degradedwithin the parasitophorous vacuole and, hence, a partialbiosynthetic pathway is needed by amastigotes to obtainthe haem that they require?

Lack of an alternative oxidase

An unusual and key feature of the energy metabolism ofbloodstreamT. brucei is themitochondrion-located terminaloxidase that is involved in the reoxidation of NADH using adihydroxyacetone-phosphate–glycerol-3-phosphateshuttle.This terminal oxidase reduces molecular oxygen to waterand transfers electrons directly from ubiquinol to oxygen.Only in T. brucei is the alternative oxidase functional. Nofunctional oxidase genewas detected in eitherL.major orT.cruzi, which correlates well with the fact that, in L. majorand T. cruzi, respiration is not sensitive to inhibition bysalicylhydroxamic acid. All three organisms contain a fla-vine-dependent glycerol-3-phosphate dehydrogenase in themitochondrion, which might function in Leishmania whenglycerol is used [25].

Folate metabolism

In many other microorganisms, such as bacteria andPlasmodium, the folate pathway provides a valuable targetfor drug intervention. However, no drugs that target thefolate pathway have been found to be effective againstLeishmania infections [26,27]. There seem to be two reasonsfor this. First, Leishmania cannot synthesize folates (e.g.folate and biopterin) and must, therefore, import thesemetabolites from an exogenous source [26,27]. Consistent


with this, no genes that encode enzymes of folatebiosynthesis are present in the L. major genome, whereasthere are as many as 12 genes that encode a novel class ofmembrane transport protein that is responsible for folatetransport [28,29]. Such transporters, which are also presentin trypanosomes, were previously known to occur only incyanobacteria and in plant plastids [30]. Second, the enzy-matic reduction of folate to become active as tetrahydrofo-late (THF), a coenzymerequired for one-carbon (C1) transferreactions (Box 2 and see Table S4 in the supplementarymaterial online: http://www.icp.be/�opperd/leishmania_table4.htm), can be catalysed by both bifunctional dihydro-folate reductase–thymidylate synthase (DHFR–TS) [31]and pteridine reductase 1 (PTR1) (Box 2). PTR1 is over-expressed if DHFR–TS is inhibited; therefore, it might benecessary to block both DHFR–TS and PTR1 simul-taneously for effective interference with folate metabolism.

Amino acid oxidation

There are multiple differences among the threetrypanosomatids in the area of amino acid metabolism(Figure 1). Proline oxidation is confirmed at the gene levelfor all three trypanosomatids and is mediated by prolineoxidase and d-1-pyrroline-5-carboxylate dehydrogenase.Leishmania, however, differs from trypanosomes in thatmethionine can be oxidized completely, whereas, in the twotrypanosomes, its degradation halts at the level of 2-keto-butyrate because the mitochondrial enzymes (methylma-lonyl-CoA epimerase and methylmalonyl-CoA mutase) forthe oxidation of 2-ketobutyrate to succinyl CoA seem to beabsent.

Leishmania has two pathways to metabolize threonine(Figure 2). In the first, it can convert threonine through aTHF-dependent pathway to glycine using the enzymeserine hydroxymethyltransferase (SHMT) (also known asthreonine aldolase). In addition to the demethylation ofserine, SHMT is also able to cleave the Ca–Cb bond ofthreonine. The resulting glycine can be converted throughserine to pyruvate by the THF-dependent glycine cleavagesystem (GCS) followed by a serine/threonine dehydratase(STD). Alternatively, the same dehydratase might convertthreonine to 2-ketobutyrate, which is then oxidized tosuccinyl CoA (Figure 2). T. brucei lacks the dehydrataseand SHMT, and seems to metabolize threonine using thealternative aminoacetone pathway, which involves a mito-chondrial threonine dehydrogenase (TDH) and an aminoa-cetone synthase (2-amino-3-ketobutyrate CoA ligase). Thisis also the major route of threonine degradation in mam-mals but it is not operational in Leishmania because of theabsence of the mitochondrial TDH.

Leucine is transaminated in the cytosol to2-ketoisocaproate by a cytosolic aminotransferase and canbe further oxidized in the mitochondrion by a short-branched chain acyl-CoA dehydrogenase, a carboxylaseand a hydratase to hydroxymethylglutaryl CoA (HMGCoA).L. major lacks the next enzyme (HMGCoA lyase) of theclassical oxidative pathway and, thus, HMGCoA is incorp-orated directly into sterols by the isoprenoid synthetic path-way, for which genes that encode most of the enzymes havebeen detected and which was experimentally demonstratedbyGinger et al. [32,33]. By contrast, inT. brucei andT. cruzi,




Figure 1. Amino acid metabolism in Leishmania major compared with Trypanosoma brucei and Trypanosoma cruzi. The figure shows the pathways that are shared by all

three trypanosomatids (black), the absence of many enzymes that are involved in amino acid metabolism, the glyoxylate cycle and the urea cycle [which are present in most

other eukaryotes (grey)] and the differences between L. major and T. brucei and T. cruzi (see key). Question marks represent enzyme-catalysed steps for which no

unambiguous gene identification could be made. Abbreviations: AcAc, acetoacetate; AdoMet, adenosylmethionine; B, biopterin; Cit, citrulline; DHF, dihydrofolate; HMG-

CoA, hydroxymethylglutaryl CoA; OAA, oxaloacetic acid; Orn, ornithine; PGA, phosphoglyceric acid; qH2B, quinoid form of dihydrobiopterin; THF, tetrahydrofolate.

Adapted, with permission, from Ref. [37].


HMGCoA follows the classical pathway of leucine oxidationand is cleaved into acetyl CoA and acetoacetate.

Leishmania contains several enzymes of the urea cycle,unlike trypanosomes, although a fully functional urea cycleis missing from L. major, T. brucei and T. cruzi. The firstenzyme, carbamoyl-phosphate synthetase, is present in allthree organisms but argininosuccinate synthase and argi-nase occur only in L. major. This indicates that arginine,ornithine and urea can be formed only in Leishmania andnot in T. brucei or T. cruzi and explains an old observationthat some Leishmania excrete urea, whereas trypanosomes


produce ammonia [34]. This might relate to life in an acidicparasitophorous vacuole, which could be incompatible withrelease of ammonia in quantity because it could affect thepH. This might also reflect the use by amastigotes of aminoacids from the vacuole. Leishmania is similar to T. brucei(but notT. cruzi) in that it contains ornithine decarboxylase,the target of the antitrypanosomal drug difluoromethylor-nithine. The lack of efficacy of this drug againstLeishmaniaseems to relate to both the transporters on the parasite andthe environment of the parasite, rather than a lack of thetarget enzyme [35].


Synthesis of amino acids

The capacity for the synthesis of amino acids by L. majoris limited to the nonessential ones plus threonine andmethionine. Most genes that code for enzymes that are

Box 2. Folate metabolism

The activation of C1 units into the THF pool takes place through the

formation of N5,N10-methylene-THF by either SHMT or the GCS (see

Figures 1 and 2 in the main text). Leishmania has two SHMT

isoenzymes, one cytosolic and one mitochondrial [40]. By contrast,

Trypanosoma cruzi has only the cytosolic isoenzyme and Trypanosoma

brucei seems to lack both. However, all three organisms have the genes

that encode the GCS. 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. Leishmania has two isofunctional methionine synthases for

this purpose: a cobalamin-dependent one (LmjF07.0090) and a

cobalamin-independent one (LmjF31.0010). LmjF31.0010 seems to be

the result of a lateral transfer event from a bacterium. This pathway

seems to occur only in Leishmania because the upstream enzyme

methylenyltetrahydrofolate reductase, which is necessary for the for-

mation of methyl THF, is not present in the two trypanosomes. The

inactivity of this pathway by the loss of the reductase from an ancestral

trypanosome probably explains why T. brucei has only the cofactor-

Figure I. Biopterin and folate metabolism in Leishmania major, Trypanosoma brucei an

transporter; DHF, dihydrofolate; DHFR, dihydrofolate reductase; dMS, cobalamin-depen

ligase; GCS, glycine-cleavage system; HMT, homocysteine S-methyltransferase; iMS,

cyclohydrolase; MTFD, methylene tetrahydrofolate dehydrogenase; MTR, methylenyl t

transporter; PAH, phenylalanine hydroxylase; PCD, pterin-4-a-carbinolamine dehydratas

serine hydroxymethyltransferase; THF, tetrahydrofolate; TK, thymidine kinase; TS, thym

structure from their mammalian counterparts (Figure II). Methotrexate functions as an


involved in the synthesis of these amino acids are clearlypresent. D-3-phosphoglycerate dehydrogenase, the firstenzyme in the synthesis of serine, is present in L. majorbut is absent from the two trypanosomes, which indicates

independent methionine synthase, whereas T. cruzi lacks both the

cofactor-dependent and the cofactor-independent enzymes. However,

this loss does not mean that trypanosomes now obtain methionine

from an external source; indeed, all trypanosomatids seem to be able to

synthesize this amino acid by an alternative route using homocysteine

S-methyltransferase (HMT). Formyl-C1 units are also used for the

formation of formyl-methionine (fMet) by methionyl-tRNA formyl

transferase (Figure I). fMet is essential for the initiation of mitochondrial

protein synthesis. However, 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 trypanosoma-

tids of the biosynthetic pathways for these molecules. Interestingly, the

formate-THF ligase, methylene-THF-cyclohydrolase and methylene-

THF dehydrogenase reactions, which have been combined in humans

into a trifunctional enzyme called one-carbon-tetrahydrofolate synthase

(C1-THF), are present in Leishmania and T. cruzi as a separate

monofunctional ligase and a bifunctional cyclohydrolase–dehydrogen-

ase enzyme (Figure II). T. brucei lacks the gene for the ligase.

d Trypanosoma cruzi. Abbreviations (enzymes are in italics in figure): BT, biopterin

dent methionine synthase; FT, folate transporter; FTHS, formate tetrahydrofolate

cobalamin-independent methionine synthase; MTFC, methylene tetrahydrofolate

etrahydrofolate reductase; MtRFT, methyl tRNA formyltransferase; NT, nucleoside

e; PTR1, pteridine reductase 1; QDPR, quinonoid dihydropteridin reductase; SHMT,

idylate synthase. The trypanosomatid FTHS, MTFC and MTFD enzymes differ in

inhibitor of DHFR.

Figure II. Schematic alignment of C1-THF sequences and their homologues. A mammalian-type trifunctional C1-THF synthase, which comprises formate

tetrahydrofolate ligase (FTHS), methylene THF cyclohydrolase (MTFC) and methylene THF dehydrogenase (MTFD) activities, is absent from trypanosomatids and

has been replaced by a monofunctional FTHS and a bifunctional enzyme with MTFC and MTFD activities. Labelling of sequences is exactly as in the GeneDB (http://

www.genedb.org) and Uniprot/SwissProt (http://www.expasy.org) databases.


that they differ in their ability to synthesize this aminoacid.

Cysteine can be produced by cysteine synthase, with thepossible involvement of 3-mercaptopyruvate sulfurtrans-ferase. Both of these enzymes have been found in L. major[36] and are predicted to be present in T. cruzi. In addition,cysteine can be generated from homocysteine by the trans-sulfuration pathway, which is present in all three organ-isms. In L. major (and T. cruzi), glycine and serine can beinterconverted by the action of SHMT, as detailed earlier.Alanine is formed from pyruvate by a cytosolic alanineaminotransferase, and aspartate and asparagine canbe formed from oxaloacetate by a mitochondrial aspartateaminotransferase and an asparagine synthase, whichare present in all three organisms. However, the conver-sion of asparagine to aspartate does not seem to occur in T.brucei.

Figure 2. Threonine metabolism in Leishmania major compared with Trypanosoma bru

pathway in T. brucei and T. cruzi (orange) as opposed to the presence of the ketobutanoa

required for the metabolism of threonine (violet). See also Figure I in Box 2. Enzyme n


The abilities of trypanosomatids to synthesize prolinediffer substantially. The synthesis of L-proline from gluta-mate occurs through g-glutamyl-phosphate and d-1-pyrro-line-5-carboxylate, and the enzymes are g-glutamyl kinase,g-glutamyl phosphate reductase and d-pyrroline-5-carbox-ylate reductase. In prokaryotes, these activities are encodedby separate genes, whereas, in eukaryotes, the first tworeactions are normally carried out by a bifunctional enzymecalled pyrroline-5-carboxylate synthetase. The correspond-ing gene is found in L. major (and in T. cruzi but not in T.brucei). The third activity is catalysed by a separate enzymeand its gene is present in all three trypanosomatids. Thus,LeishmaniaandT. cruzi shouldbeable to synthesize prolinefromglutamate,whereasT.brucei shouldnot. Interestingly,genes encoding a separate g-glutamyl kinase and g-gluta-myl-phosphate reductase are also present in L. major butnot in T. cruzi or T. brucei. Phylogenetic reconstruction

cei and Trypanosoma cruzi. This scheme shows the presence of the aminoacetone

te pathway (green) in L. major. It also shows the absence from T. brucei of enzymes

ames are in italics.

http://www.genedb.org/

http://www.genedb.org/

http://www.expasy.org/

Figure 3. The pathways of core metabolism in Leishmania major. Figure shows reactions taking place in the glycosome, that are involved in carbohydrate metabolism, and

in the mitochondrion, with its tricarboxylic acid cycle, and the flux of metabolites between these two organelles. Boxed metabolites are substrates (grey) or end products

(black) of metabolism. Thick arrows represent major metabolite fluxes. Pathways in blue are thought to be more important in promastigotes and pathways in red are

thought to be more important in the amastigote. Abbreviations: Fru, fructose; GAP, glyceraldehyde 3 phosphate; Glc, glucose; H-5-P, hexose 5-phosphate; Man, mannose;

PEP, phosphoenolpyruvate; PGA, phosphoglyceric acid; PPP, pentose-phosphate pathway. Enzymes: 1, hexokinase; 2, phosphoglucose isomerase; 3, phosphofructokinase;

4, fructosebisphosphate aldolase; 5, triosephosphate isomerase; 6, glyceraldehyde-3-phosphate dehydrogenase; 7, phosphoglycerate kinase; 8, glycerol-3-phosphate

dehydrogenase; 9, glycerol kinase; 10, adenylate kinase; 11, glucosamine-6-phosphate deaminase; 12, mannose-6-phosphate isomerase; 13, phosphomannomutase; 14,

GDP-mannose pyrophosphorylase; 15, phosphoglycerate mutase; 16, enolase; 17, pyruvate kinase; 18, phosphoenolpyruvate carboxykinase; 19, malate dehydrogenase; 20,

fumarate hydratase; 21, NADH-dependent fumarate reductase; 22, malic enzyme; 23, alanine aminotransferase; 24, aspartate aminotransferase; 25, pyruvate phosphate

dikinase; 26, citrate synthase; 27, 2-ketoglutarate dehydrogenase; 28, succinyl-CoA ligase; 29, succinate dehydrogenase; 30, acetate–succinate CoA transferase; 31, pyruvate

dehydrogenase; 32, citrate lyase; 33, acetyl-CoA synthetase; 34, proline oxidation pathway; 35, threonine oxidation pathway; 36, ribulokinase; 37, ribokinase; 38,

xylulokinase; 39, amylase-like protein; 40, sucrase-like protein.


clearly indicates that these individual enzymes are ofbacterial origin, whereas the bifunctional pyrroline-5-car-boxylate synthetase is of eukaryotic origin. It is unclearwhyL. major has these additional proline synthesis enzymes.


Of the so-called essential amino acids, the pathways forthe synthesis of lysine, threonine and methionine fromaspartic acid seem to be either absent or incomplete in allthree trypanosomatids. Genes that encode aspartokinase


and aspartate semialdehyde dehydrogenase could not bedetected. Therefore, the de novo synthesis from aspartate ofthese three amino acids seems unlikely. L. major should beable to synthesize both methionine and threonine startingfrom aspartate semialdehyde, whereas this conversionseems to be absent from the two trypanosomes. However,aromatic amino acids (phenylalanine, tyrosine or trypto-phan), the branched amino acids (leucine, isoleucine andvaline)and lysineandhistidine cannotbesynthesizedby thetrypanosomatids.

Concluding remarksThe importance of some of the detected differences in genecontent between trypanosomatids can be readily hypothes-ized: for instance, for the use of sugars that are available inthe sandflybutnot thevectors of trypanosomes, andalso thenecessity for different end-product production in the para-sitophorous vacuole. Some of the other differences detected,such as in amino acid metabolism, are hard to interpret.Moreover, some of the knownmetabolic differences betweentrypanosomatids are not fully explained by the presence orabsence of genes that encode metabolic enzymes. Clearly,the situation will be affected by the control of gene expres-sion and by enzyme stability at different stages of the lifecycle. The current ‘best guess’ on themetabolism that occursin Leishmania amastigotes and promastigotes is shown inFigure 3. Many detailed studies are now required, and genemining has indicated some sensible approaches to pursueand experiments that should be done.

AcknowledgementsWe thank Valerio Losasso (University of Modena) for communicating herinitial analyses of Leishmania folate metabolism, and the Wellcome TrustSanger Institute for providing sequence information from the Leishmaniagenomes to the community. F.R.O. was supported by IUAP grant 5–29from the Belgian Government. G.H.C. acknowledges support from the UKMedical Research Council and the Wellcome Trust.

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