Characterization of transsulfuration and cysteine biosynthetic pathways in the protozoan hemoflagellate, Trypanosoma cruzi. Isolation and molecular characterization of cystathionine beta-synthase and serine acetyltransferase from Trypanosoma.

Sulfur-containing amino acids play an important role in a variety of cellular functions such as protein synthesis, methylation, and polyamine and glutathione synthesis. We cloned and characterized cDNA encoding cystathionine beta-synthase (CBS), which is a key enzyme of transsulfuration pathway, from a hemoflagellate protozoan parasite Trypanosoma cruzi. T. cruzi CBS, unlike mammalian CBS, lacks the regulatory carboxyl terminus, does not contain heme, and is not activated by S-adenosylmethionine. T. cruzi CBS mRNA is expressed as at least six independent isotypes with sequence microheterogeneity from tandemly linked multicopy genes. The enzyme forms a homotetramer and, in addition to CBS activity, the enzyme has serine sulfhydrylase and cysteine synthase (CS) activities in vitro. Expression of the T. cruzi CBS in Saccharomyces cerevisiae and Escherichia coli demonstrates that the CBS and CS activities are functional in vivo. Enzymatic studies on T. cruzi extracts indicate that there is an additional CS enzyme and stage-specific control of CBS and CS expression. We also cloned and characterized cDNA encoding serine acetyltransferase (SAT), a key enzyme in the sulfate assimilatory cysteine biosynthetic pathway. Dissimilar to bacterial and plant SAT, a recombinant T. cruzi SAT showed allosteric inhibition by l-cysteine, l-cystine, and, to a lesser extent, glutathione. Together, these studies demonstrate the T. cruzi is a unique protist in possessing both transsulfuration and sulfur assimilatory pathways.

soma cruzi, a causative agent of Chagas' disease that affects more than 15 million people in Central and South America (13). Members of the family Trypanosomatidae are characterized by complex life cycles involving both vertebrate and invertebrate hosts (14). During its developmental transitions, T. cruzi encounters numerous nutritional and environmental changes both in the alimentary tract of insect hosts and in the cytoplasm of mammalian cells. In addition, in trypanosomes and their related organisms, the majority of glutathione is conjugated with spermidine to form a kinetoplastid-specific antioxidant, trypanothione (N 1 ,N 8 -bis(glutathionyl)spermidine), which constitutes about 70% of total glutathione (15). Trypanothione and trypanothione-dependent enzymes seem to replace glutathione and glutathione-dependent enzymes present in a variety of prokaryotic and eukaryotic cells. Trypanothione system plays a vital role in cellular metabolism, especially maintaining the intracellular redox balance and protecting the cells from oxidative damage of free radicals and peroxides in trypanosomatids (16,17). Therefore, understanding sulfur amino acid metabolism in trypanosomes may allow us to further exploit trypanothione metabolism to develop new drugs against trypanosomiasis. In this work, we show, by cloning and molecular characterization of the genes encoding the major enzymes associated with cysteine biosynthesis, that T. cruzi is capable of producing cysteine by two independent pathways: sulfur assimilatory cysteine biosynthesis and transsulfuration from methionine via cystathionine. In the literature, this is the first demonstration of a unicellular protist possessing both of these two pathways.
Enzyme Assays-The enzymatic activity of CBS was assayed by measuring cystathionine production as described (21). The enzymatic activity of CS was assayed by measuring cysteine production (22). The standard mixture of CS assay was identical to the above mentioned except that serine and homocysteine were replaced with OAS and sodium sulfide. The enzymatic activity of serine sulfhydrylase (SS) was measured in the same way as described for CS assay except that serine was used instead of OAS. The rate of cysteine desulfhydration in ␤-replacement reaction was monitored by measuring hydrogen sulfide production (23) in the mixture of 50 mM Tris-HCl, pH 8.0, 10 mM L-cysteine, 20 mM ␤-mercaptoethanol, 0.1 mM pyridoxal phosphate, 0.075 mM lead acetate. For all assays using a recombinant CBS, bovine serum albumin was included at 1 mg/ml. The enzymatic activity of SAT was measured by monitoring of the decrease in absorbance at 232 nm (A 232 ) due to the thioester bond of acetyl-coenzyme A (CoA) (24). The standard mixture for SAT assay contained 50 mM Tris-HCl, pH 8.0, 0.1 mM acetyl-CoA, 0.2 mM L-serine, and enzyme solution. One unit of the enzymatic activity was defined as 1 mol of thioester bond of acetyl-CoA cleaved or 1 mol of L-cysteine, L-cystathionine, or hydrogen sulfide synthesized at 25°C for 1 min.
Isolation of CBS cDNA-Isolation of poly(A) ϩ RNA and construction of phage and phagemid cDNA library from epimastigotes were described previously (25). To obtain CBS/CS cDNA encoding functional enzymes, we took advantage of functional rescue of the cysteine-auxotrophic Escherichia coli NK3 strain (⌬tryE5 leu-6 thi hsdR hsdMϩ cysK cysM) (26), a gift by Masaaki Noji and Kazuki Saito (Chiba University), which is defective of the two CS genes, with putative CBS/CS cDNA from T. cruzi. Procedures for functional rescue were described previously (9). Ten colonies out of 5 ϫ10 4 grew in the absence of cysteine. After screening twice, all these cDNA clones were confirmed to complement cysteine auxotrophy of NK3 cells. Six clones were randomly chosen, and fully sequenced.
We also cloned two additional full-length CBS cDNA clones based on possible partial CBS sequences deposited in the T. cruzi data base (GenBank/EMBL/DDBJ accession nos. AQ445547 and AQ445365). We obtained a 0.5-kb fragment by polymerase chain reaction (PCR) using CBS/CS-specific sense and antisense primers, a lysate of the T. cruzi phage cDNA library as template, and Taq polymerase with the following cycling parameters: 1) denaturation at 94°C for 1 min; 2) annealing at 50°C for 1 min; 3) elongation at 72°C for 2 min; 4) 30 cycles. To obtain a full-length CBS cDNA, the T. cruzi cDNA library was screened by hybridization with the partial CBS cDNA fragment as described (27). Two CBS cDNA clones were randomly chosen and sequenced.
Isolation of SAT cDNA-Based on possible SAT sequences deposited in the T. cruzi data base (AQ445417 and AQ445365), we amplified a partial SAT cDNA fragment by PCR. Since screening of the cDNA library with this partial SAT cDNA probe was unsuccessful presumably due to a low representation of SAT mRNA, we cloned SAT cDNA by 5Јand 3Ј-rapid amplification of cDNA end (RACE). A 1100-bp SAT cDNA, which lacked only three amino acids at the carboxyl terminus, was obtained by 5Ј-RACE using a set of the T3 primer and the SAT-specific antisense primer (5Ј-gtgtggtgctctcagatatc-3Ј), a lysate of the T. cruzi phage cDNA library as template, and Taq polymerase under the condition described above. A 400-bp COOH-terminal portion of the SAT cDNA was obtained by 3Ј-RACE essentially as described for 5Ј-RACE except that the SAT-specific sense primer (5Ј-ggtactggaattgtgattgg-3Ј) and the T7 primer were used. Sequencing of the 5Ј-and 3Ј-RACE cDNA clones revealed that the overlapped region of these sequences were completely identical, suggesting that they were derived from a single gene.
Bacterial Expression and Purification of Recombinant Trypanosomal CBS-A plasmid was constructed to produce a glutathione S-transferase (GST)-T. cruzi CBS fusion protein (GST-TcCBS). An open reading frame (ORF) encoding TcCBS was amplified by PCR using a plasmid containing TcCBS1 cDNA (see "RESULTS AND DISCUSSION") as template and appropriate oligonucleotide primers including SmaI site at the end. PCR was performed as described except that Pfu polymerase was used instead of Taq polymerase. The 1.2-kb PCR fragment was digested with SmaI and cloned into SmaI-digested pGEX-2T in the same orientation as the tac promoter to produce pGEX-TcCBS. E. coli NK3 strain was transformed with pGEX-TcCBS, and cultivated in the presence of 1 mM isopropyl-D-thiogalactoside at 37°C for 2 h. The GST-TcCBS fusion protein was purified using glutathione-Sepharose 4B column (Amersham Pharmacia Biotech, Tokyo, Japan). The purified GST-TcCBS was dialyzed against phosphate-buffered saline at 4°C for 10 h, re-applied onto the glutathione-Sepharose 4B column, and digested in the column with thrombin at room temperature for 24 h. The recombinant TcCBS, which contained TcCBS and extra four amino acids (GSPG) at the amino terminus, was eluted from the column and used for further analyses.
Biochemical Characterization of TcCBS-Heme concentration was determined by the pyridine hemochrome assay as described previously (28). The absorption spectrum of rTcCBS was recorded using visible/UV spectrometer Beckman model DU530 thermostatted at 25°C.
Bacterial Expression and Purification of Recombinant Trypanosomal SAT-A plasmid was constructed to produce a GST-T. cruzi SAT fusion protein. An ORF encoding SAT from T. cruzi (TcSAT) was amplified, as described above, by PCR using a sense and an antisense primer containing BamHI and SmaI site, respectively. The 1.1-kb PCR fragment was double-digested with BamHI and SmaI and cloned into BamHI-SmaI-double-digested pGEX-4T-3 to produce pGEX-TcSAT. The SATdeficient E. coli strain JM39/5 (F ϩ , cysE51, recA56) (29), a gift by Masaaki Noji and Kazuki Saito, was transformed with pGEX-TcSAT and cultivated as described above. The GST-TcSAT was produced and purified as described for the GST-TcCBS. The purified rGST-TcSAT was free from bacterial contamination, as judged by SDS-polyacrylamide gel electrophoresis. The purified GST-TcSAT was further analyzed as a fusion protein for the following reasons: 1) the E. coliproduced GST-TcSAT was mostly insoluble, and a very small amount of soluble GST-TcSAT was obtained despite of several efforts to increase solubility, including use of a variety of culture temperatures, induction protocols, and detergents during sonication; 2) TcSAT became inactive during thrombin digestion for unknown reasons.
Chromatographic Characterization of CBS and CS from T. cruzi Epimastigote Lysate and Recombinant TcCBS-After 10 10 epimastigotes were washed twice with ice-cold phosphate-buffered saline, the cell pellet was resuspended in 10 ml of 50 mM Tris-HCl, pH 7.5, containing 0.1 mM sodium ethylenediaminetetracetate, 1 mM each of dithiothreitol, phenylmethylsulfonyl fluoride, and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), and 10 g/ml each of antipain, aprotinin, leupeptin, and pepstatin (Buffer A). The suspension was then subjected to three cycles of freezing and thawing. The cell lysate was centrifuged at 15,000 ϫ g for 15 min at 4°C and filtrated with 0.45-m cellulose acetate membrane. For gel filtration chromatography, the lysate was applied to a column of TOYOPEARL HW 55S (Tosoh Corp., Tokyo, Japan) that had been equilibrated with Buffer A. Elution was carried out with the same solution and each fraction was subjected to CBS, CS, and SS assays. For anion exchange chromatography, DEAE-TOYOPE-ARL 650S was pre-equilibrated with Buffer A. The pre-equilibrated DEAE column was loaded with the parasite lysate, washed extensively with Buffer A, and eluted with a 300-ml linear gradient of 0 -0.5 M KCl in Buffer A.
Functional Rescue of the Yeast CBS-deficient Mutant Strain with Expression of TcCBS-The ORF of TcCBS1 was PCR-amplified as described above, end-trimmed, and cloned into EcoRI-digested and endtrimmed pKT10 (25) to produce the plasmid construct pyTcCBS to express the trypanosomal CBS under the control of the TDH3 promoter in yeast. Each of the three plasmid constructs: pyTcCBS, pKT10, and WB68, which contained yeast CBS ORF at BglII site of pAB23BX (30), was introduced into WK63yCBS⌬ (MATa leu2 ura3-52 ade2 trp1 cys4::LEU2) (31), and selected on SC (ϪUra) plates. The transformants were incubated on SD plates in either the presence or absence of cysteine at 26°C. Standard yeast genetic methods were performed essentially as described (32,33).

RESULTS AND DISCUSSION
Isolation of Six Isotypes of TcCBS cDNA-We attempted to obtain functional CS cDNA from T. cruzi epimastigotes by using the strategy of functional rescue of the CS-deficient E. coli strain. Ten cDNAs obtained by this strategy apparently encoded a protein with high sequence similarity to CBS from a variety of species. This suggests that trypanosomal CBS functions as CS in E. coli in vivo. Although the native and recombinant yeast and mammalian CBS showed CS and CBS activities in vitro (23,34), this is, in the literature, the first demonstration that a eukaryotic CBS physiologically functions as CS in prokaryotes. Sequencing of six randomly chosen cDNA clones obtained by rescue, and two additional CBS cDNA clones obtained by hybridization revealed that all of these clones contained an insert with a 1155-bp ORF, encoding a protein of 384 amino acids with a calculated molecular mass of 42.0 kDa. These cDNA sequences were heterogeneous; most of these individual cDNAs were independent. Based on nucleotide sequences, the eight cDNAs encoding CBS from T. cruzi (TcCBS) were grouped into six isotypes, TcCBS1-TcCBS6 (Fig. 1). Nucleotide variations among the cDNA isotypes were found at one, eight, and one nucleotide positions in 5Ј-untranslated region (UTR), the protein coding region, and 3Ј-UTR, respectively. Each cDNAs contained an identical length (191 bases) of 3Ј-UTR. Two splice acceptor sites, resulting in heterogeneity of the length of 5Ј-UTR (14 or 16 bases), were identified; 5Ј-UTR of TcCBS3 was two bases shorter than the rest of the isotypes.
Features of the Deduced Amino Acid Sequences of TcCBS-Five of the six cDNA isotypes encoded identical proteins, while one (TcCBS6) contained two nucleotide substitutions that resulted in amino acid alterations (Val 14 3 Ala and Tyr 261 3 Cys) (Fig. 1). TcCBS revealed significant identities to CBS and CS from a variety of organisms. Both TcCBS isotypes show higher identities to CBS from eukaryotic organisms (47-66%) than to CS from bacteria and plants (32-39%). Specifically, TcCBS shows a 66% identity to CBS from Leishmania tarentolae, a 54% identity to CBS from Dictyostelium discoideum, and a 50% identity to Rat CBS. Sequence alignment (Fig. 2) shows that TcCBS lacks the 90 -120-amino acid COOH-terminal extension present in CBS from all other organisms except with Leishmania. Thus, trypanosomal CBS is one of the shortest CBS enzymes reported to date. The consensus sequence for the putative pyridoxal phosphate-binding domain and three of four lysine residues shown to be important for the catalytic activity of spinach CS-A by site-directed mutagenesis (35) are very well conserved in TcCBS.
Genomic Organization and Significance of Heterogeneity of TcCBS-Digestion of T. cruzi genomic DNA with restriction enzymes that cut once within TcCBS ORF gave 2.5-kb ladders, suggesting that TcCBS genes are tandemly linked in a headto-tail fashion (Fig. 3). The hybridization patterns of different enzymes were not identical, suggesting that there is heterogeneity of these enzyme recognition sites in the different TcCBS isotypes. However, these differences must be within the intergenic region, as all three enzymes tested, BamHI, EcoRI, and HindIII, cut once in each of the isotype cDNAs. Differences in the ladder patterns were not due to incomplete digestion since hybridization of the identical blot with a single-copy gene, e.g. gp72 (36), gave single bands (data not shown). The premise that TcCBS genes are tandemly linked in a head-to-tail fashion was also supported by the fact that a genomic fragment containing the 10-bp carboxyl end of ORF and the 191-bp 3Ј-UTR of TcCBS4, the 1.2-kb intergenic region, and the 160-bp aminoterminal end of TcCBS2 or TcCBS3 or TcCBS4, was obtained by PCR (data not shown). Assuming that each hybridizing band of the ladder in the BamHI-digest represents an identical number of molecules, TcCBS genes appeared to be present as Ͼ15 copies per haploid. The fact that XhoI and XbaI digest gave large hybridizing bands suggests that TcCBS genes likely clustered at a single or two loci.
There is, to our knowledge, no precedent for an example of heterogeneous CBS expressed in a single organism although expression of alternatively spliced CBS mRNA isotypes from a single gene was reported (37). Whether a single cell expresses  (59) is present in all of the obtained cDNA clones. 5Ј-UTR of TcCBS3 is two base shorter than that of the others. "A" of the translation initiation codon corresponds to ϩ1. Nucleotide substitutions that cause amino acid substitutions in TcCBS6 are highlighted by gray boxes.
multiple TcCBS genes or whether different cells in the population express different TcCBS isotypes has not been determined. However, the tandemly linked gene organization of TcCBS and polycistronic transcription of trypanosomal genes (e.g. Refs. 38 and 39) suggest that the former is likely the case. What is the biological significance of TcCBS forming tandemly linked multicopy genes with microheterogeneity? One possibility is that an increase in gene copy numbers may be one of the primary strategies of trypanosomes to augment protein expression, as suggested for other genes (e.g. Refs. 40 -42). Although heterogeneity of TcCBS at the protein level was not extensive, we could not rule out a possibility that isotype-dependent bio-chemical differences including substrate preference exist. It is also conceivable to speculate that some, if not all, of these TcCBS isotypes are expressed in a stage-specific fashion.
Biochemical Characterization of Recombinant TcCBS-Purified recombinant TcCBS (rTcCBS) showed both CBS and CS activities. rTcCBS showed CBS activity of 7.51 Ϯ 1.21 units/mg of protein (Fig. 4) with a K m of 1.0 Ϯ 0.2 mM for L-serine and a K m of 0.9 Ϯ 0.3 mM for L-homocysteine. Similar to yeast and mammalian CBS (23,34), rTcCBS also showed SS activity, which forms cysteine from serine and sodium sulfide. The SS activity was 4.58 Ϯ 0.85 units/mg of protein with a K m of 1.1 Ϯ 0.2 mM for L-serine and a K m of 3.1 Ϯ 0.2 mM for sodium sulfide. In agreement with the functional rescue of the CS-deficient E. coli strain, rTcCBS showed remarkably high CS activity (4.45 Ϯ 1.74 units/mg of protein) and a K m of 4.9 mM for OAS and a K m of 4.1 mM for sodium sulfide. rTcCBS also catalyzed the production of hydrogen sulfide from L-cysteine and ␤-mercaptoethanol with a K m of 2.4 Ϯ 0.5 mM for L-cysteine and a K m of 26 Ϯ 9 mM for ␤-mercaptoethanol. This hydrogen sulfideforming activity was relatively insensitive to 10 mM aminooxyacetic acid (only 50% inhibition) and 1 mM hydroxylamine (only 20% inhibition), which are known to inhibit mammalian CBS (55-100% inhibition at 0.1 mM) (23). Unlike human CBS, rTc-CBS was not activated by the presence of 1 mM S-adenosylmethionine and did not contain detectable amounts of heme (data not shown). The absorption spectrum of rTcCBS exhibited major peaks at 280 and 410 nm in a ratio of 1:0.16, typical of a pyridoxal phosphate-containing enzyme (43). However, no peak was visible around 428 nm, which was indicative of heme in case of rat and human CBS (43). Removal of the pyridoxal phosphate resulted in an apoenzyme having no absorbance in the visible range (data not shown). The absence of heme in trypanosomal CBS was not due to expression in E. coli; human CBS expressed in E. coli does contain heme. In addition, supplementation of the bacterial medium with ␦-aminolevulinate did not change the absence of heme in rTcCBS, although this precursor was shown to increase the heme content of the recombinant human CBS (44). These data indicate that physicochemical properties of TcCBS substantially differ from the yeast and mammalian CBS.
Chromatographic Separation of the Native and Recombinant CBS Activities-To examine how the activities observed with rTcCBS relate to the native proteins in the T. cruzi epimastig-FIG. 2. Multiple alignment of deduced amino acid sequences of CBS from T. cruzi and other representative organisms. Sequences are as follows. Tc, T. cruzi TcCBS1 (this study); Lt, Leishmania tarentolae (accession no. AF256080); Dd, Dictyostelium discoideum (U27536); Ra, Rattus norvegicus (D01098); Sc, Saccharomyces cerevisiae (X72922). These sequences were aligned using the Clustal W program (60). Asterisks indicate identical amino acids. Dots and colons indicate conserved amino acid substitutions. Box indicates the consensus sequence for the putative pyridoxal phosphate-binding domain (PXXSVKDR). Dotted rectangles indicate the amino acids conserved among CBS described above and CS from Streptococcus suis, Mycobacterium tuberculosis, E. histolytica, Synechocystis sp., Helicobacter pylori, Clostoridium perfringens, Bacillus subtilis, and Arabidopsis thaliana. Three of four lysine residues shown to be important for the catalytic activity of spinach CS-A by site-directed mutagenesis are marked with triangles. ote lysate, a soluble parasite lysate was fractionated by DEAE anion exchange chromatography, and each fraction was assayed for CBS, SS, and CS activities (Fig. 5A). CBS and SS activities were eluted as a symmetrical overlapped peak (fractions [43][44]. To confirm that this peak was actually due to TcCBS, rTcCBS was loaded on the same column and was shown to elute with the same fractions that contained CBS, SS, and CS activities. Altogether, these data have extended the previous findings suggesting the presence of the transsulfuration pathway in trypanosomes (45,46). The presence of an intact transsulfuration pathway in T. cruzi is further bolstered by our finding that another important enzyme in transsulfuration pathway, cystathionine ␥-lyase, which forms cysteine, ammonia, and 2-oxobutyric acid from cystathionine, was eluted at fractions 39 -40 on DEAE anion exchange chromatography (data not shown, Fig. 5A).
Identification of the Putative CS-CS activity was eluted on the DEAE column as two distinct peaks (fractions 43-44 and 60 -62). The first CS peak overlapped those of CBS and SS, suggesting that this activity was likely attributed to TcCBS. However, the fractions from the second peak solely showed CS activity; neither CBS nor SS activity was detected in these fractions (see below). The presence of this putative CS activity, lacking both CBS and SS activities, together with that of SAT (see below), strongly indicates the presence of the bacteria/ plant-like assimilatory cysteine biosynthetic pathway in trypanosomes. It is possible that, although TcCBS can function as a CS in E. coli, this other putative CS enzyme is the key player in assimilatory cysteine biosynthesis in vivo in T. cruzi. The fact that we did not obtain cDNA encoding this putative CS by functional rescue of the CS-deficient E. coli strain suggests that either T. cruzi CS does not function as CS in a prokaryote, or that CS mRNA was not abundant in epimastigotes, comparing to CBS mRNA. The former possibility indicates the presence of a novel CS in trypanosomes.
Multimerization State of the Native and Recombinant Tc-CBS-Multimerization state of TcCBS was examined by isolating the first peak from the DEAE chromatography, containing CBS/SS/CS activities, then loading and eluting on the gel filtration column. Fractions were then assessed for CS activity (Fig. 5B). More than 70% of total CS activity was eluted at 155 Ϯ 25 kDa, and the rest of the activity was eluted at molecular mass of 65 Ϯ 7 and 30 Ϯ 5 kDa. These data are consistent with the majority of rTcCBS being a homotetramer, and a small proportion of the protein existing as a homodimer and monomer. In contrast to rTcCBS, the native TcCBS was eluted as a single peak at 155 Ϯ 35 kDa, suggesting that the native TcCBS also forms a homotetramer. Formation of homotetramer of the native and recombinant TcCBS implies that the COOH-terminal region, present in yeast and mammalian CBSs and absent in trypanosomal CBS, is not exclusively responsible for tetrameric formation as suggested for the mammalian enzyme (47).

Functional Rescue of the Yeast CBS-deficient Mutant Strain with TcCBS and In Vivo Function of TcCBS-To see if TcCBS
also functions as a CBS enzyme in vivo, we attempted to rescue the growth defect of the CBS-deficient yeast strain, WB63yCBS⌬ by the expression of TcCBS (Fig. 6). As predicted, introduction of a plasmid containing either yeast or trypanosomal CBS gene suppressed growth defect of WB63yCBS⌬ in either the absence or presence of cysteine, whereas the transformant harboring the vector control grew only in the presence of cysteine. Together with the fact that TcCBS functions as CS in E. coli, this result suggests that trypanosomal CBS may be a bifunctional enzyme involved in the two cysteine-synthesizing pathways in vivo. As mentioned above, TcCBS is one of the shortest "prototype" CBS, based on the primary protein sequence, no interaction with heme, and no activation with Sadenosylmethionine. This might contribute, at least in part, to the fact that the trypanosomal CBS functions as CS in a prokaryotic organism.
Significance of the Two Independent Pathways for Cysteine Production in T. cruzi-T. cruzi is the first protist, in the literature, shown to possess two independent pathways for cysteine production: sulfur-assimilation sequence from sulfate and transsulfuration sequence from methionine via cystathionine. Extracellular L-cysteine, but not L-cystine or any reducing agents, has been shown to be essential for growth of Trypanosoma brucei bloodstream forms (48). Since trypanosomes require cysteine not only for protein biosynthesis, but also for formation of glutathione and trypanothione, which are present in high amounts (15), trypanosomes may require multiple cysteine-acquiring pathways to ensure there is cysteine available to fulfill these needs. The presence of two independent cysteine-synthetic pathways in a single protist may be unique for T. cruzi or trypanosomatids, since the other parasitic protozoans Entamoebae contain only the sulfur assimilation pathway and not the transsulfuration pathway (9 -11). 2 Stage-specific Expression of Transsulfuration and Assimilatory Cysteine Biosynthetic Pathways-We next examined whether expression of CBS, SS, and CS activities is stagespecific using the lysate of the insect-form (epimastigotes) and the mammalian form parasites (tissue culture-derived amastigotes and trypomastigotes) (Fig. 4). Both CBS and SS activities were 8 -9 times higher in the lysate of epimastigotes than those in the lysate of amastigotes and trypomastigotes. The ratio of CBS to SS activity in the lysates was 1.3-1.5, which was comparable to that of rTcCBS (1.6), indicating that a majority of CBS activity in the parasite lysate is attributable to TcCBS. In contrast, significant CS activity was detected in both epimastiogotes and amastigotes/trypomastigotes. CS activity in amastigotes and trypomastigotes was about half of that in epimastigotes. Based on the fact that rTcCBS showed comparable SS and CS activities (1:1.0), we attribute ϳ50% of CS activity in the epimastigote lysate to TcCBS and the other half of CS activity to the second putative CS, whereas we attribute about 90% of CS activity in the amastigote/trypomastigote lysate to the putative CS. Thus, the putative CS activity is expressed in both epimastigotes and amastigotes/trypomastigotes at a comparable level.
The actively replicate, sulfur-containing amino acids and their intermediates are readily available, whereas these compounds are scarce in the insect alimentary tract, where epimastigotes and metacyclic trypomastigotes reside. Thus, abundance of sulfur-containing amino acids may be a major cause of the down-regulation of TcCBS expression in the mammalian forms. It was shown that CBS activity was up-regulated 3-fold when yeast cells were cultivated under aerobic condition (49). However, cultivation of amastigotes/trypomastigotes in liver infusion tryptose medium, which presumably provides a less aerobic condition than the condition for amastigote/trypomastigote cultivation, resulted in an 4 -6-fold increase of CBS activity in the parasite lysates (data not shown). The contradiction between the yeast and the T. cruzi data could be explained if the anaerobic up-regulation was due to factors other than oxygen tension, e.g. temperature, nutrients, or a lack of nutrients.
In contrast to the down-regulation of CBS activity in the mammalian forms of T. cruzi, CBS and other activities involved in transsulfuration pathway were detected in the mammalian forms (bloodstream trypomastigotes) of T. brucei (45,46). This indicates that substantial differences in metabolism of sulfurcontaining amino acids exist between T. cruzi and T. brucei. Entamoeba, which involves only mammalian hosts, solely possesses sulfate assimilatory pathway (9 -11), but not transsulfuration pathway. This may suggest that the complexity of life cycles of parasitic protozoa and the redundancy of cysteine biosynthetic pathways are causally connected.
Cloning of TcSAT cDNA and Features of the Deduced Amino Acid Sequence of TcSAT-Sequencing of a putative SAT cDNA obtained by RACE revealed that this cDNA contained a 20-bp 3Ј end of spliced leader sequence, a 17-bp 5Ј-UTR, a 1047-bp ORF, and a 86-bp 3Ј-UTR, followed by poly(A) tail. The ORF of TcSAT cDNA encoded a protein of 348 amino acids with a calculated molecular mass of 38.4 kDa. The deduced protein sequence of TcSAT showed 8 -34% identities to SAT from a variety of organisms. Among them TcSAT revealed highest identities to a plasmid-encoded SAT in Synechococcus (SrpH (Ref. 50); 34%) and SAT from enteric protozoan parasites E. histolytica and E. dispar (Refs. 10 and 11; 31%). Sequence alignment (Fig. 7) showed several important aspects of TcSAT. First, TcSAT contains a conserved structural domain, called a left-handed parallel ␤-helix (51), found in a variety of acetyland acyltransferases (see references in Ref. 51). Second, TcSAT contains an amino-terminal extension that is absent in cytosolic isoforms of SAT from other species. Third, the COOH-terminus of TcSAT is 20 -70 amino acids shorter than SAT from other species. Finally, TcSAT contains a unique 10-amino acid insertion (amino acids 264 -273) that is only found in Entamoeba SATs and Synechococcus SrpH. These data reinforce the premise that protozoan and cyanococcal plasmid-borne SAT shared a common ancestor as suggested previously (10).
Enzymatic Characterization of the Recombinant TcSAT-We produced a recombinant GST-TcSAT fusion protein (rGST-Tc-SAT) for enzymatic studies since identification and biochemical characterization of TcSAT is the second piece of evidence supporting the significance of sulfate assimilatory cysteine biosynthesis in T. cruzi. The enzymatic reaction of rGST-TcSAT followed the Michaelis-Menten kinetics and showed an apparent K m of 0.24 Ϯ 0.1 mM for L-serine (in the presence of 0.1 mM acetyl-CoA) and 0.92 Ϯ 0.3 mM for acetyl-CoA (10 mM L-serine). Enzymatic activity of TcSAT was inhibited by L-cysteine in a competitive manner with both L-serine and acetyl-CoA (K i was 12 Ϯ 4 M for L-serine, and 0.14 Ϯ 0.03 mM for acetyl-CoA) (Fig.  8). This inhibitory effect was specific for stereoisomeric structure of L-cysteine, but not specific for the redox state of this amino acid. In the presence of 0.02 mM acetyl-CoA and 0.2 mM L-serine, 0.2 mM D-cysteine showed no inhibition whereas 0.2 mM L-cysteine showed 85 Ϯ 7% inhibition. An oxidized form of L-cysteine (L-cystine) also showed 70 Ϯ 10% reduction Only slight or moderate inhibition was observed at 0.2 mM with structurally similar amino acids, L-homocysteine (11 Ϯ 3%), L-homoserine (13 Ϯ 5%), and N-acetyl-L-cysteine (7 Ϯ 4%). Surprisingly, the activity of rTcSAT was also inhibited in the presence of 0.2 mM glutathione by 36 Ϯ 8%. Allosteric inhibition by glutathione has not been shown for SAT (e.g. Refs. 52 and 53). Therefore, trypanosomal SAT may play an important role in the monitoring of total thiol concentration and perhaps the cellular redox state. A comparable level of inhibition by L-cysteine and L-cystine was also observed for SAT from E. histolytica (10). Therefore, this biochemical property may be a common feature shared by the protozoan SATs.
Possible in Vivo Function of TcSAT-Why do trypanosomes need SAT when they possess CBS, which can form cysteine from serine and hydrogen sulfide (SS activity), and therefore, compensates the reactions catalyzed by SAT and CS? In yeasts, where cysteine is exclusively synthesized through transsulfuration pathway (54), OAS solely serves as a coinducer of the sulfate assimilation pathway (55). In contrast, OAS serves as both a metabolic intermediate and a regulatory element in enteric bacteria (56). Taken together, we propose that the trypanosomal SAT plays a major role to regulate sulfate incorporation, activation, and assimilation pathway, by controlling OAS abundance, as a part of a complex mechanism to balance thiol concentration. The fact that the putative CS activity and TcSAT mRNA are constitutively expressed in both the insect and mammalian stages of T. cruzi (Fig. 4, data not shown) also indicates that this pathway plays a housekeeping role. A biological role of individual enzymes in the assimilatory cysteine biosynthetic pathway in trypanosomes shall be unequivocally demonstrated by gene replacement or disruption (e.g. Refs. 57 and 58) of TcSAT, which is a single copy gene (data not shown), and the putative CS gene.
Acknowledgments-We express our appreciation to Masaharu Tokoro, Seiki Kobayashi, Takashi Asai, Miki Nakazawa, and Tsutomu Takeuchi (Department of Tropical Medicine and Parasitology, School of Medicine, Keio University, Tokyo, Japan) for technical assistance and fruitful discussions; to Shin-ichiro Kawazu and Shigeyuki Kano (International Medical Center of Japan, Tokyo, Japan) for technical assist-