From the Department of Parasitology, National
Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo
162-8640, Japan and the ¶ Division of Population Sciences, Fox
Chase Cancer Center, Philadelphia, Pennsylvania 19111
Received for publication, October 26, 2000, and in revised form, November 29, 2000
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ABSTRACT |
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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 Various sulfur compounds, especially cysteine, methionine, and
S-adenosylmethionine, are essential for the growth and
activities of all cells (1, 2). Methionine initiates the synthesis of
proteins, whereas cysteine plays a critical role in the structure, stability, and catalytic function of many proteins.
S-Adenosylmethionine plays a crucial role in methyl group
transfer and in polyamine biosynthesis. Cysteine is also involved in
the synthesis of the major antioxidant glutathione.
In the filamentous fungi, Aspergillus nidulans and
Neurospora crassa, the major route for the synthesis of
cysteine is the condensation of O-acetylserine
(OAS)1 with sulfide,
catalyzed by cysteine synthase (CS, OAS sulfhydrase) (3, 4). This
pathway has been shown to be present in prokaryotes, plants, and
enteric protozoan Entamoebae, and is generally called assimilatory cysteine biosynthetic pathway since this process involves
reduction and fixation of inorganic sulfate to organic amino acids.
Cysteine can also be synthesized by an alternative pathway: the
sulfurylation of O-acetylhomoserine to give homocysteine, which then can be converted to cysteine via cystathionine by the transsulfuration pathway. In vertebrates, cysteine is synthesized from
methionine via cystathionine by the transsulfuration pathway. This
pathway is believed to be the sole route for cysteine synthesis in
vertebrates with cystathionine We have shown previously that the enteric protist parasite
Entamoeba histolytica, a causative agent of amebic colitis
and extraintestinal abscesses (8), and a related parasite
Entamoeba dispar possess sulfur assimilatory cysteine
biosynthetic pathway by cloning and characterization of genes encoding
three key enzymes: CS, serine acetyltransferase (SAT), and ATP
sulfurylase (9-12). Since Entamoebae apparently lack
enzymes necessary to produce cysteine from methionine in
transsulfuration pathway, assimilatory cysteine biosynthesis appears to
be the sole biosynthetic pathway of this amino acid. To better
understand sulfur-containing amino acid metabolism and cysteine
biosynthesis in parasitic protists, we have identified and
characterized transsulfuration and assimilatory cysteine biosynthetic
pathways from the parasitic hemoflagellate Trypanosoma
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
(N1,N8-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.
Microorganisms and Cultivation--
Epimastigotes (the insect
form) of T. cruzi clone YNIH (18) and Tulahuen
strain (19) were grown at 26 °C in liver infusion tryptose liquid
medium, supplemented with 20 µg/ml hemin, 10% heat-inactivated fetal
calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin.
Tissue culture-derived amastigotes and trypomastigotes were obtained
from COS7 cultures. COS7 cell cultures were grown and infected with
trypomastigotes of Tulahuen strain as described previously (20). The
amastigotes and trypomastigotes (the mammalian forms) were harvested
5-7 days after inoculation (50-95% amastigotes).
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
Isolation of CBS cDNA--
Isolation of poly(A)+
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
(
We also cloned two additional full-length CBS cDNA clones based on
possible partial CBS sequences deposited in the T. cruzi data base (GenBankTM/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 5'- and 3'-rapid amplification of cDNA end
(RACE). A 1100-bp SAT cDNA, which lacked only three amino acids at
the carboxyl terminus, was obtained by 5'-RACE using a set of the T3
primer and the SAT-specific antisense primer
(5'-gtgtggtgctctcagatatc-3'), 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 3'-RACE essentially as described for 5'-RACE
except that the SAT-specific sense primer (5'-ggtactggaattgtgattgg-3') and the T7 primer were used. Sequencing of the 5'- and 3'-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 SAT-deficient E. coli strain JM39/5
(F+, 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. coli-produced 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 1010
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 µg/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 × g for 15 min at 4 °C and filtrated with 0.45-µm
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-TOYOPEARL 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 end-trimmed 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 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 5'-untranslated region (UTR),
the protein coding region, and 3'-UTR, respectively. Each cDNAs
contained an identical length (191 bases) of 3'-UTR. Two splice
acceptor sites, resulting in heterogeneity of the length of 5'-UTR (14 or 16 bases), were identified; 5'-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 (Val14 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 head-to-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 3'-UTR
of TcCBS4, the 1.2-kb intergenic region, and the 160-bp
amino-terminal 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 >15 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 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 biochemical 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 ± 1.21 units/mg of protein (Fig.
4) with a Km of
1.0 ± 0.2 mM for L-serine and a
Km of 0.9 ± 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 ± 0.85 units/mg of
protein with a Km of 1.1 ± 0.2 mM
for L-serine and a Km of 3.1 ± 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 ± 1.74 units/mg of protein) and
a Km of 4.9 mM for OAS and a
Km of 4.1 mM for sodium sulfide. rTcCBS
also catalyzed the production of hydrogen sulfide from L-cysteine and 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 epimastigote
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 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
TcCBS--
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 ± 25 kDa, and
the rest of the activity was eluted at molecular mass of 65 ± 7 and 30 ± 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 ± 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 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 stage-specific 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 ~50% 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 stage-specific regulation of transsulfuration pathway are likely
associated with the complex life cycle of T. cruzi. Within the cytoplasm of mammalian cells, where amastigotes 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
sulfur-containing 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 3' end of spliced
leader sequence, a 17-bp 5'-UTR, a 1047-bp ORF, and a 86-bp 3'-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 Enzymatic Characterization of the Recombinant TcSAT--
We
produced a recombinant GST-TcSAT fusion protein (rGST-TcSAT) 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 Km of 0.24 ± 0.1 mM for L-serine (in the presence of 0.1 mM acetyl-CoA) and 0.92 ± 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
(Ki was 12 ± 4 µM for
L-serine, and 0.14 ± 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 ± 7% inhibition. An oxidized
form of L-cysteine (L-cystine) also showed
70 ± 10% reduction Only slight or moderate inhibition was
observed at 0.2 mM with structurally similar amino acids,
L-homocysteine (11 ± 3%), L-homoserine
(13 ± 5%), and N-acetyl-L-cysteine
(7 ± 4%). Surprisingly, the activity of rTcSAT was also
inhibited in the presence of 0.2 mM glutathione by 36 ± 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.
-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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-synthase (CBS) acting as the flux-controlling enzyme (1). In mammals, this pathway also functions as
catabolic pathway of methionine and its toxic intermediates including
homocysteine. Genetically determined impairment of the activities of
CBS and/or cystathionine
-lyase results in serious clinical
disorders (1, 5, 6). In contrast to vertebrates, prokaryotes, fungi,
and plants synthesize methionine from cysteine via the reverse
transsulfuration pathway employing the complementary enzymes
cystathionine
-synthase and cystathionine
-lyase (7).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-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
(A232) 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 µmol of
thioester bond of acetyl-CoA cleaved or 1 µmol of
L-cysteine, L-cystathionine, or hydrogen
sulfide synthesized at 25 °C for 1 min.
tryE5 leu-6 thi hsdR hsdM+ 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 ×104 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.
(MATa leu2 ura3-52 ade2 trp1 cys4::LEU2) (31), and selected on SC (
Ura) 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of
microheterogeneity of TcCBS cDNA sequences.
Upper panel represents the TcCBS1
nucleotide sequence. Note that only the nucleotides that vary among the
TcCBS cDNA isotypes are shown. An arrow and
thick horizontal lines represent ORF and untranslated
regions, respectively. Lower panel shows the
heterogeneous nucleotides in TcCBS2-TcCBS6 that are
distinct from those in TcCBS1. A nucleotide sequence
corresponding to spliced leader sequence (59) is present in all of the
obtained cDNA clones. 5'-UTR of TcCBS3 is two base
shorter than that of the others. "A" of the translation
initiation codon corresponds to +1. Nucleotide substitutions that cause
amino acid substitutions in TcCBS6 are highlighted by
gray boxes.
Ala and
Tyr261
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.
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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.
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Fig. 3.
Southern blot analysis of TcCBS
genes. Ten µg of the total DNA from T. cruzi
epimastigotes was digested with a variety of restriction endonucleases,
separated by agarose gel electrophoresis, transferred onto a nylon
membrane, and then hybridized with the [32P]dCTP-labeled
TcCBS cDNA probe, corresponding to an entire open
reading frame, as described (27). Restriction endonucleases used were
EcoRI (lane 1), BamHI
(lane 2), HindIII (lane
3), ClaI (lane 4),
XbaI (lane 5), and XhoI
(lane 6).
-mercaptoethanol with a
Km of 2.4 ± 0.5 mM for
L-cysteine and a Km of 26 ± 9 mM for
-mercaptoethanol. This hydrogen sulfide-forming
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, rTcCBS 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.
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Fig. 4.
CBS, SS, and CS activities in the lysate of
T. cruzi epimastigotes and tissue culture-derived
amastigotes/trypomastigotes, and of the recombinant TcCBS.
Open bars, gray bars, and filled bars
represent CBS, SS, and CS activities, respectively. All activities are
expressed in units/mg of protein from three to five separate triplicate
experiments. Error bars represent standard error
of mean.
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Fig. 5.
A, elution profile of the native CBS,
SS, and CS activities from the T. cruzi epimastigote lysate
by DEAE anion exchange chromatography. CBS, SS, and CS activities in
the parasite lysate were separated on a DEAE-TOYOPEARL 650S column. The
trypanosomal lysate was eluted with Buffer A containing a linear
gradient of 0-0.5 mM KCl. Circles and a
thick solid line represent CBS activities.
Squares and a thin solid line represent SS
activities. Triangles and a thick dashed line
represent CS activities. A thin dotted line represents
absorbance at 280 nm. B, elution profile of the recombinant
TcCBS and the native CBS and CS from the T. cruzi
epimastigote by gel filtration chromatography. The recombinant rTcCBS
and a mixture (2 ml) of fractions 43-44, corresponding to the native
CBS, and a mixture of fractions 61-62, corresponding to the native CS,
from DEAE anion exchange chromatography, were separated on TOYOPEAL HW
55S gel filtration chromatography. Each fraction was assayed for CS
activity. Circles and a dashed line
represent an elution profile of the recombinant rTcCBS.
Triangles and a solid line represent that of the
native CBS. Squares and a dotted line represent
that of the native CS. Note that the peaks for rTcCBS and native TcCBS
were completely overlapped. Molecular standards shown above, purchased
from Bio-Rad (Tokyo, Japan), are aldolase (158 kDa), bovine serum
albumin (68 kDa), ovalbumin (45 kDa), and chymotrypsin (25 kDa).
-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).
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 S-adenosylmethionine. This might contribute, at least
in part, to the fact that the trypanosomal CBS functions as CS in a
prokaryotic organism.
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Fig. 6.
Functional rescue of a Saccharomyces
cerevisiae CBS mutant with the expression of TcCBS.
WK63yCBS was transformed with pyTcCBS (containing TcCBS; labeled as
TcCBS), WB68 (containing yeast CBS; ScCBS), and
pKT10 (control). The transformants were incubated on SD (
Ura) plates
supplemented with (top) or without cysteine
(bottom).
-helix (51), found
in a variety of acetyl- and 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).
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Fig. 7.
Multiple alignment of the deduced amino acid
sequences of SAT from T. cruzi and a variety of
organisms. Av, Azotobacter vinelandii
(accession no. AF010139); Mt, M. tuberculosis;
S, Synechococcus sp. (L41665); Bs,
B. subtilis (Z99104); Ec, E. coli
(P05796); At, A. thaliana cytosolic form (L42212,
L34076, Z34888); SrpH, plasmid-borne isoform from
Synechococcus sp. (U23436); Eh, E. histolytica (AB023954); Tc, T. cruzi (this
study). Asterisks indicate identical amino acids.
Dots and colons indicate conserved amino acid
substitutions. Structural motifs composed of hexapeptides (coils 1-4),
parallel -strands, and turns, predicted based on homology to LpxA,
are depicted above the sequences as filled boxes (C1-C4),
arrows, and open bars, respectively. An insertion
unique for Synechococcus SrpH, E. histolytica,
and T. cruzi is also boxed.
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Fig. 8.
Double-reciprocal plots of the recombinant
TcSAT showing competitive inhibition of the enzyme
with L-cysteine. Kinetic studies were performed by
monitoring the decrease in A232 with a fixed
concentration of 0.1 mM acetyl-CoA (left) and 10 mM L-serine (right) in the absence
(circles and solid lines) and presence of
10 µM (squares and dashed line), 30 µM (triangles and dotted line), and
100 µM (diamonds and dotted/dashed
line) of L-cysteine.
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ACKNOWLEDGEMENTS |
---|
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 assistance for sequencing; and to Masaaki Noji and Kazuki Saito (Laboratory of Molecular Biology and Biotechnology in Research Center of Medicinal Resources, Chiba University, Chiba, Japan) for helpful discussions and for providing E. coli NK3 and JM39/5 strains.
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FOOTNOTES |
---|
* This work was supported by Grant-in-aid 11770132 from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by grants for research on emerging and re-emerging infectious diseases from the Ministry of Health, Labour and Welfare of Japan, and by Grant K-1037 from Japan Health Sciences Foundation for research on health sciences focusing on drug innovation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession numbers AF296842-AF296848.
§ To whom correspondence should be addressed. Tel.: 81-3-5285-1111 (ext. 2733); Fax: 81-3-5285-1173;E-mail: nozaki@nih.go.jp.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M009774200
2 T. Nozaki, Y. Shigeta, Y. Saito-Nakano, and M. Imada, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
OAS, O-acetylserine;
CS, cysteine synthase;
CBS, cystathionine
-synthase;
SAT, serine acetyltransferase;
SS, serine sulfhydrylase;
PCR, polymerase chain reaction;
RACE, rapid amplification of cDNA
ends;
GST, glutathione S-transferase;
ORF, open reading
frame;
TcCBS, cystathionine
-synthase from T. cruzi;
TcCBS, a gene encoding TcCBS;
TcSAT, serine
acetyltransferase from T. cruzi;
TcSAT, a gene
encoding TcSAT;
UTR, untranslated region;
kb, kilobase pair(s);
bp, base pair(s).
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