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Originally published In Press as doi:10.1074/jbc.M212414200 on August 14, 2003

J. Biol. Chem., Vol. 278, Issue 43, 42717-42727, October 24, 2003
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Identification and Characterization of Two Isoenzymes of Methionine {gamma}-Lyase from Entamoeba histolytica

A KEY ENZYME OF SULFUR-AMINO ACID DEGRADATION IN AN ANAEROBIC PARASITIC PROTIST THAT LACKS FORWARD AND REVERSE TRANS-SULFURATION PATHWAYS*

Masaharu Tokoro{ddagger}, Takashi Asai{ddagger}, Seiki Kobayashi{ddagger}, Tsutomu Takeuchi{ddagger}, and Tomoyoshi Nozaki§||

From the {ddagger}Department of Tropical Medicine and Parasitology, Keio University School of Medicine, Tokyo 160-8582, Japan, the §Department of Parasitology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan, and the Precursory Research for Embryonic Science and Technology, Japan Science and Technology Corporation, Tokyo 190-0012, Japan

Received for publication, December 5, 2002 , and in revised form, August 11, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
To better understand the metabolism of sulfur-containing amino acids, which likely plays a key role in a variety of cell functions, in Entamoeba histolytica, we searched the genome data base for genes encoding putative orthologs of enzymes known to be involved in the metabolism. The search revealed that E. histolytica possesses only incomplete cysteine-methionine conversion pathways in both directions. Instead, this parasite possesses genes encoding two isoenzymes of methionine {gamma}-lyase (EC 4.4.1.11 [EC] , EhMGL1/2), which has been implicated in the degradation of sulfur-containing amino acids. The two amebic MGL isoenzymes, showing 69% identity to each other, encode 389- and 392-amino acid polypeptides with predicted molecular masses of 42.3 and 42.7 kDa and pIs of 6.01 and 6.63, respectively. Amino acid comparison and phylogenetic analysis suggested that these amebic MGLs are likely to have been horizontally transferred from the Archaea, whereas an MGL from another anaerobic protist Trichomonas vaginalis has MGL isotypes that share a common ancestor with bacteria. Enzymological and immunoblot analyses of the partially purified native amebic MGL confirmed that both of the MGL isotypes are expressed in a comparable amount predominantly in the cytosol and form a homotetramer. Recombinant EhMGL1 and 2 proteins catalyzed degradation of L-methionine, DL-homocysteine, L-cysteine, and O-acetyl-L-serine to form {alpha}-keto acid, ammonia, and hydrogen sulfide or methanethiol, whereas activity toward cystathionine was negligible. These two isoenzymes showed notable differences in substrate specificity and pH optimum. In addition, we showed that EhMGL is an ideal target for the development of new chemotherapeutic agents against amebiasis by demonstrating an amebicidal effect of the methionine analog trifluoromethionine on trophozoites in culture (IC50 18 µM) and that this effect of trifluoromethionine was completely abolished by the addition of the MGL-specific inhibitor DL-propargylglycine.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Entamoeba histolytica is a causative agent of amebiasis, which annually affects an estimated 48 million people and results in 70,000 deaths (1). The most common clinical presentation of amebiasis is amebic dysentery and colitis; extraintestinal abscesses, i.e. hepatic, pulmonary, and cerebral, however, are also common and often lethal. This microaerophilic anaerobe has been considered to be a unique eukaryotic organism because it apparently lacks organelles typical of eukaryotic organisms such as mitochondria, the rough endoplasmic reticulum, and the Golgi apparatus (2). However, a recent demonstration of genes encoding mitochondrial proteins, i.e. cpn60 and pyridine nucleotide transhydrogenase (3), together with electron micrographic demonstration of the rough endoplasmic reticulum and the Golgi apparatus (4), suggested the presence of a residual organelle of mitochondria (called crypton or mitosome) (5, 54) and also indicated that this group of parasitic protists possess a unique organelle organization. This parasite also reveals numerous unusual aspects in its metabolism (6), highlighted by the lack of the tricarboxylic acid cycle (7) and glutathione metabolism (8). In addition, recent studies suggesting the horizontal transfer of genes encoding a variety of fermentation enzymes from bacteria (9), and genes encoding malic enzyme and acetyl-CoA synthase from the Archaea (10) have placed this protozoan organism at a unique position in eukaryotic evolution.

One of these unique metabolic pathways found in this parasite is the biosynthetic and degradative pathway of sulfur-containing amino acids, especially cysteine, which has been demonstrated to be essential for the growth and various cellular activities of amoebae (11, 12). Sulfur-containing amino acid metabolism varies among organisms (Fig. 1, also reviewed in Ref. 13). In mammals, cysteine is produced solely from incorporated methionine and serine via S-adenosylmethionine, homocysteine, and cystathionine in a pathway called the reverse trans-sulfuration pathway. In contrast, plants, fungi, and some bacteria have a so-called sulfur assimilation pathway to fix inorganic sulfur onto a serine derivative (O-acetylserine, OAS)1 to synthesize cysteine. These organisms are also capable of converting cysteine into methionine via a trans-sulfuration sequence in the opposite orientation (also called the methionine biosynthetic pathway). We previously demonstrated that E. histolytica possesses the sulfur assimilatory cysteine biosynthetic pathway, and is capable of producing cysteine de novo (14, 15). We have also demonstrated (15) that major enzymes in this pathway, serine acetyltransferase (SAT) and cysteine synthase (CS), play a central role in the control of the intracellular cysteine concentrations, and in the antioxidative stress defense mechanism of this gluthathione-lacking parasite (8).



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  		FIG. 1.
A schematic representation of sulfur-containing amino acid metabolism in general (A) and in Entamoeba (B). Only enzymes that belong to the {gamma}-subfamily of PLP-dependent enzymes are shown (in bold), together with CS and CBS, which belong to the {beta}-family, and SAT. Biochemical steps involved in both forward and reverse trans-sulfuration reactions are indicated by filled and open arrows, respectively. Genes encoding the first three steps of the reverse trans-sulfuration pathway have been identified in the E. histolytica genome data base (data not shown). Gray arrows indicate reactions catalyzed by MGLs and hatched arrows indicate sulfur assimilatory steps we previously reported (14, 15).

 

One important question remaining about the sulfur-containing amino acid metabolism in this parasite, and also in anaerobic protists in general, is how these parasites degrade toxic sulfur-containing amino acids since they possess apparently incomplete trans-sulfuration pathways in both the forward and reverse orientation (data not shown, see the present study). Thus, in order to better understand the metabolism, particularly degradation, of these sulfur-containing amino acids in E. histolytica, we attempted to isolate other essential genes encoding proteins involved in sulfur amino acid metabolism.

We identified and characterized two isotypes of the unique enzyme, methionine {gamma}-lyase (MGL; EC 4.4.1.11 [EC] ) and their encoding genes, which, we propose, function in the degradation of sulfur amino acids in this parasite. We show a line of evidence suggesting that the MGL genes and their proteins were likely derived from the Archaea by horizontal transfer as shown for other metabolic enzymes in this parasite (10). In addition, we also demonstrate that the methionine analog trifluoromethionine (TFMET) has a cytotoxic effect on amebic trophozoites that is abolished by a specific inhibitor of MGL, indicating that MGL is exploitable as an attractive target for the development of new amebicidal compounds.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Chemicals and Reagents—L-methionine, L-cysteine, DL-homocysteine, OAS, O-succinyl-L-homocysteine, O-acetyl-L-homoserine, DL-propargylglycine (PPG), 3-methyl-2-benzothiazolinone hydrazon hydrochloride, trichloroacetic acid, pyridoxal 5'-phosphate (PLP), and other chemicals were commercial products of the highest purity available unless otherwise stated. TFMET was a gift from Dr. Cyrus J. Bacchi (Haskins Laboratories and Department of Biology, Pace University, New York).

Microorganisms and Cultivation—Trophozoites of E. histolytica strain HM-1:IMSS cl-6 (16) were maintained axenically in Diamond's BI-S-33 medium (11) at 35.5 °C. Trophozoites were harvested at the late-logarithmic growth phase 2-3 days after inoculation of one-twelfth to one-sixth of a total culture volume. After the cultures were chilled on ice for 5 min, trophozoites were collected by centrifugation at 500 x g for 10 min at 4 °C and washed twice with ice-cold phosphate-buffered saline, pH 7.4. Cell pellets were stored at -80 °C until use.

Search of the Genome Data Base of E. histolytica—The E. histolytica genome data base at the Institute for Genomic Research (TIGR, //www.tigr.org/tdb/) was searched using the TBLASTN algorithm with protein sequences corresponding to the PLP-attachment site of cysteine- and methionine-metabolizing enzymes (PROSITE access number PS00868). This motif is conserved among the {gamma}-subfamily ({alpha}-family) of PLP enzymes (for the classification of PLP enzymes used in this study, see Ref. 17), i.e. cystathionine {gamma}-lyase (CGL), cystathionine {gamma}-synthase (CGS), and cystathionine {beta}-lyase (CBL) from a variety of organisms. We also searched for amebic orthologs that belong to the {beta}-family of PLP enzymes using the PLP-attachment site from CS of E. histolytica and cystathionine {beta}-synthase (CBS) from yeast and mammals.

Cloning of E. histolytica MGL1 and MGL2 and Production of their Recombinant Proteins—Based on nucleotide sequences of the protein-encoding region of the two putative amebic MGL genes (EhMGL1 and EhMGL2), two sets of primers, shown below, were designed to amplify the open reading frames (ORF) of EhMGL1 and to construct plasmids to produce glutathione S-transferase (GST)-EhMGL fusion proteins. The two sense and two antisense primers contained the SmaI restriction site (underlined) either prior to the translation initiation site or next to the stop codon (bold), respectively. The primers used are: EhMGL1 (sense), 5'-CATCCCGGGGATGACTGCTCAAGATATTACTACTACT-3' (37-mer); EhMGL1 (antisense), 5'-TAGCCCGGGATTACCAAAGCTCTAATGCTTGTTTTAA-3' (37-mer); EhMGL2 (sense), 5'-CATCCCGGGTATGTCTCAATTGAAGGATTTACAAACA-3' (37-mer); EhMGL2 (antisense), 5'-TAGCCCGGGATTAGCATTGTTCAAGAGCTTGTTTTAA-3' (37-mer).

The cDNA library of E. histolytica trophozoites constructed in a lambda phage (14) was used as the template for polymerase chain reaction (PCR) using the following parameters. An initial step for denaturation and rTaq (Takara Bio Inc., Shiga, Japan) activation at 94 °C for 15 min was followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 45 °C for 30 s, and extension at 72 °C for 1 min. A final step at 72 °C for 10 min was used to complete the extension. Approximately 1.1-kb PCR fragments were obtained and cloned into the SmaI site of a pGEX-6P-1 expression vector (Amersham Biosciences K.K., Tokyo, Japan). The final constructs were designated as pGEX6P1/MGL1 and pGEX6P1/MGL2, respectively.

Nucleotide sequences were confirmed by using appropriate synthetic sequencing primers, a BigDye Terminator Cycle Sequencing Ready Reaction Kit, and an ABI PRISM 310 genetic analyzer (Applied Biosystems Japan Ltd., Tokyo, Japan), according to the manufacturer's protocol. To express the recombinant proteins in Escherichia coli, pGEX6P1/MGL1 and pGEX6P1/MGL2 were introduced into BL21 (DE3) (Novagen Inc., Madison, WI) host cells. Expression of the GST-MGL1 and GST-MGL2 fusion proteins was induced with 1 mM isopropyl-{beta}-thiogalactoside at 18 °C for 20 h. The fusion proteins were purified using a glutathione-Sepharose 4B column (Amersham Biosciences) according to the manufacturer's instructions. The recombinant EhMGL1/2 (rEhMGL1/2) were obtained by digestion of these fusion proteins with PreScission Protease (Amersham Biosciences) in the column, followed by elution from the column and dialysis at 4 °C with 100 mM sodium phosphate buffer, pH 6.8, containing 0.02 mM PLP.

The final purified recombinant EhMGL (rEhMGL) proteins were presumed to contain 10 additional amino acids (GPLGSPEFPG) at the N terminus. The purified enzymes were stored at -80 °C with 30-50% dimethyl sulfoxide until use. No decrease in enzyme activity was observed under these conditions for at least 3 months. Protein concentrations were determined by Coomassie Brilliant Blue assay (Nacalai Tesque, Inc., Kyoto, Japan) with bovine serum albumin as the standard.

Amino Acid Alignments and Phylogenetic Analyses—The sequences of MGL and other members of the {gamma}-subfamily of PLP enzymes showing similarities to the amino acid sequences of EhMGL were obtained from the National Center for Biotechnology Information (NCBI, //www.ncbi.nih.gov/) by using the BLASTP algorithm. The alignment and phylogenetic analyses were performed with ClustalW version 1.81 (18) using the Neighbor-Joining (NJ) method with the Blosum matrix. An unrooted NJ tree composed of the amino acid sequences of 13 MGLs and 10 other members of the {gamma}-subfamily of PLP enzymes from various organisms with two EhCSs ({beta}-family of PLP enzymes) as the outgroup was drawn by Tree View ver.1.6.0 (19). Branch lengths and bootstrap values (1000 replicates) were derived from the NJ analysis. Phylogenetic analyses by the maximum parsimony method (MP) and maximum likelihood method (ML) were also conducted using PROTPARS (PHYLIP version 3.57c, Ref. 20) and ProtML (MOLPHY version 2.3, Ref. 21), respectively.

Subcellular Fractionation of the Crude Extract—The lysate of ~3 x 106 E. histolytica trophozoites was prepared by two cycles of freezing and thawing in 1 ml of cell lysis buffer: 100 mM sodium phosphate buffer, pH 7.0, containing 1 mM EDTA, 0.02 mM PLP, 1 mM dithiothreitol, and a protease inhibitor mixture (Complete Mini EDTA-free, Roche Applied Science, Tokyo, Japan), and 1 µg/ml of N-(3-carboxyoxirane-2-carbonyl)-leucyl-amino(4-guanido)butane (E-64, Sigma). The whole lysate was then centrifuged at 14,000 x g in a microcentrifuge tube for 20 min at 4 °C to separate the supernatant (soluble cytosolic fraction) and the pellet (debris, membrane, and nuclear fraction).

Anion Exchange Chromatography of the Native Form MGLs—A supernatant fraction obtained from 2 g (wet weight) of the trophozoite pellet, as described above, was filtered with a 0.45-µm-pore mixed cellulose membrane (Millex-HA, Millipore Corporation, Bedford, MA). The sample buffer was exchanged with buffer A (20 mM Tris-HCl, pH 8.0, 0.02 mM PLP, 1 mM dithiothreitol, 1 mM EDTA, and 0.1 µg/ml of E-64) by using a Centricon Plus-20 (Millipore). A 20-ml sample containing ~100 mg of total protein was loaded on a DEAE-Toyopearl HW-650S column (7.5 x 1.6 cm, 15-ml bed volume, Tosoh, Tokyo, Japan) that was previously equilibrated with buffer A. The column was further washed with ~50 ml of buffer A until the A280 dropped below 0.1. The bound proteins were then eluted with a 50-ml linear potassium chloride gradient (0-0.5 M) in buffer A at a flow rate of 0.8 ml/min. All 0.8-ml fractions were concentrated to 0.2 ml with a Centricon YM-10 (Millipore). All procedures were performed at 4 °C. The amount of MGL in each fraction was assessed using the hydrogen sulfide assay and immunoblotting as described below.

Size Exclusion Chromatography of Recombinant and Native EhMGLs—To estimate the molecular mass of the recombinant and native EhMGLs, gel filtration chromatography was performed. Approximately 500 µg of recombinant and 100 µg of partially purified native EhMGL were dialyzed against buffer B (20 mM Tris-HCl, pH 8.0, 0.02 mM PLP, and 0.2 M KCl) and concentrated to 1 ml with the Centricon Plus-20. The concentrated samples were then applied to a column of Toyopearl HW-65S (70 x 1.6 cm, 140-ml bed volume, Tosoh) preequilibrated with buffer B. The recombinant and native MGLs were eluted with buffer B at a flow rate of 0.8 ml/min. The peaks were detected by measuring absorbance at A280 (recombinant MGLs) and immunoblotting (native MGLs). The same column was calibrated with blue dextran (2000 kDa), ferritin (440 kDa), catalase (232 kDa), and aldolase (158 kDa) (Amersham Biosciences).

Immunoblot Anaylsis—Polyclonal antisera against recombinant EhMGL1 and 2 were raised in rabbits by Sigma-Genosys (Hokkaido, Japan). Immunoblot analysis was carried out using a polyvinylidene difluoride (PVDF) membrane as described in (22). The blot membrane was visualized by using alkaline phosphatase conjugate-coupled secondary antibody with NBT/BCIP solution (Roche Applied Science) according to the manufacturer's protocol.

Two-dimensional Polyacrylamide Gel Electrophoresis—First-dimensional electrofocusing of two-dimensional PAGE was performed using Immobiline Drystrip, pH 3-10 NL, 7 cm and IPG Buffer pH 3-10 NL (Amersham Biosciences) according to the manufacturer's protocol. Second dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was performed on 12% SDS-PAGE gel using prestained SDS-PAGE standards, Broad Range (Bio-Rad Laboratories, Inc., Tokyo, Japan), as a molecular marker.

Enzyme Assays and Kinetic Calculations—The enzymatic activitiy of MGL was monitored by measuring the production of {alpha}-keto acid, ammonia, and hydrogen sulfide or methanethiol. The standard MGL reaction was performed in 200 µl of 100 mM sodium phosphate buffer, pH 6.8, a reaction mixture containing 0.02 mM PLP, and 0.1-10 mM of each substrate with appropriate amounts of each enzyme.

The {alpha}-keto acid assay was performed as described (23). The MGL reaction was terminated by adding 25 µl of 50% trichloroacetic acid. After the proteins were precipitated by centrifugation at 14,000 x g for 5 min at 4 °C, 100 µl of the supernatant was mixed with 200 µl of 0.5 M sodium acetate buffer, pH 5.0, and 80 µl of 0.1% of 3-methyl-2-benzothiazolinone hydrazon hydrochloride, and then incubated at 50 °C for 30 min. After the mixture had cooled to room temperature, absorbance at A320 was measured. Pyruvic acid and 2-{alpha}-butyric acid were used as standards.

For the detection of ammonia, the nitrogen assay (24) was used. A 50-µl sample of the supernatant, as for the {alpha}-keto acid assay, was mixed with 50 µl of Nessler's reagent (Nakalai) and 75 µl of 2 N sodium hydroxide, and then incubated at 25 °C for 15 min. Absorbance at A440 was measured. Ammonium sulfate was used as a standard.

The hydrogen sulfide assay was performed as described (25-27). Briefly, the MGL reaction was terminated by adding 20 µl of 20 mM N,N-dimethyl-p-phenylenediamine sulfate in 7.2 N HCl and 20 µl of 30 mM FeCl3 in 1.2 N HCl. After further incubation in the dark for 20 min, the proteins were precipitated by centrifugation at 14,000 x g for 5 min at 4 °C, and then the absorption at OD650 of the supernatant was measured to quantitate the formed methylene blue. Na2S was used as a standard.

The methanethiol assay was performed as described (28) using 5,5'-dithio-bis-(2-nitrobenzoic acid). One-hundred microliters of the sample supernatant were mixed with 1 µl of 100 mM 5,5'-dithio-bis-(2-nitrobenzoic acid) in ethanol, and after 2 min incubation at room temperature, absorbance at A412 was measured. L-cysteine was used as a standard.

The cysteine and cystathionine assay was performed as described (29). The ninhydrin reagent was prepared by dissolving 1 g of ninhydrin in 100 ml of glacial acetic acid and adding 33 ml of glacial phosphoric acid. For the determination of cystathionine, 0.2 ml of cystathionine-containing solution was mixed with 0.33 ml of the ninhydrin reagent and boiled at 100 °C for 5 min. The solution was then cooled on ice for 2 min and at room temperature for a further 10 min. Absorbance at A455 was measured. Cysteine concentrations were determined using the same protocol except for the measurement of absorbance at A560.

Kinetic parameters were estimated with Lineweaver-Burk plots using Sigma Plot 2000 software (SPSS Inc., Chicago, IL) with the Enzyme Kinetics module (version 6.0, Hulinks, Inc., Tokyo, Japan).

Assay of the Inhibition of rEhMGL by DL-Propargylglycine—An {alpha}-keto acid assay (described above) with L-methionine as the substrate was performed to evaluate the inhibitory effects of DL-propargylglycine (PPG) on the activity of rEhMGLs. rEhMGL (5 µg) was preincubated with various concentrations of PPG in the standard MGL reaction mixtures (described above) in the absence of L-methionine at 36 °C. The preincubation time was 5 min for kinetic analyses and 1 to 60 min to characterize the slow binding of this inhibitor. After preincubation, the reaction was initiated by adding an appropriate amount of L-methionine to the reaction mixture.

In Vitro Assessment of Amebicidal Reagents—To assess the amebicidal effect of TFMET, the trophozoites were cultured in the BI-S-33 medium containing various concentrations of TFMET or metronidazole, the therapeutic compound commonly used for amebiasis, as a control. After cultivation at 35.5 °C for 18 h, cell survival was assessed with the cell proliferation reagent WST-1 (Roche Applied Science). Briefly, the trophozoites were seeded on 96-well microtiter plates in 200 µl of BI-S-33 medium at a density of 2 x 104 cells per well (1 x 105 cells/ml), and the lid was completely sealed with a sterilized adhesive silicon sheet (Corning, New York). After these plates were further incubated at 35.5 °C for 18 h, 20 µl of WST-1 reagent was added to each well and the incubation was continued for 2 more hours. The optical density at A445 was measured with that at A595 as a reference using a microplate reader (Model 550, Bio-Rad, Tokyo, Japan). The initial density and incubation period of the cultures were chosen to maintain the control trophozoites in the late-logarithmic growth phase throughout the experiment, and also to allow the measurement of optical density in the linear portion of the curves (between 4 x 103 to 2.0 x 105 cells/ml).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Data Base Search of the PLP-dependent Enzymes—In an attempt to obtain genes encoding the PLP-dependent enzymes involved in the metabolism of sulfur-containing amino acids, we searched the genome data base for putative proteins that possessed a conserved PLP-binding domain as described under "Experimental Procedures." Two independent contigs were found in the genome data base. These contigs (Contig 315785 and 316820, TIGR) contained two similar but not identical ORFs that encode proteins possessing a region containing the PLP-binding motif of the {gamma}-subfamily of PLP enzymes. Non-coding flanking regions within these contigs also showed significant variations (data not shown). All other contigs or singletons showing significant identity to these two contigs perfectly overlapped them, which is consistent with the notion that these fragments are present as a single copy in the genome.

We also searched for genes containing the PLP-binding site of the {beta}-family of PLP enzymes using both the amebic CS and the yeast and mammalian CBS. However, after eliminating contigs and singletons that contain genes encoding the two CS isotypes described previously (14), no contig or singleton was found to contain this motif. This suggests that E. histolytica possesses two uncharacterized genes encoding proteins that belong to the {gamma}-subfamily of PLP enzymes, and lacks the {beta}-family of PLP enzymes (i.e. CBS) known to be involved in the reverse trans-sulfuration pathway in other organisms. We also tentatively concluded that the trans-sulfuration sequences in both the forward and reverse orientation are incomplete since the amebic genome lacks putative genes for CBL, CGS, and CGL (also see below). In addition, the major pathways for cysteine degradation of sulfur amino acids present in mammals, i.e. the cysteine sulfinic acid (cysteine dioxygenase as a key enzyme), 4'-phosphopanthetheine (leading to synthesis of coenzyme A and cysteamine), and mitochondrial mercaptopyruvate pathways, are apparently absent in this organism (data not shown). Therefore, E. histolytica must have alternative enzymes involved in the degradation of toxic sulfur-containing amino acids, e.g. homocysteine, which is implicated in the well characterized human genetic disease homocysteinuria and its cytotoxicity (30).

Identification and Features of EhMGL Genes and Their Proteins—The nucleotide sequences of the 1170- and 1179-bp ORFs recognized in the contigs described above were homologous to those of the {gamma}-subfamily of PLP-dependent enzymes, including MGL, CBL, CGS, and CGL. These putative genes encode 389- and 392-amino acid polypeptides with predicted molecular masses of 42.3 and 42.7 kDa and predicted pIs of 6.01 and 6.63, respectively. We designated these genes EhMGL1 and EhMGL2 since their predicted proteins showed highest similarity to methionine {gamma}-lyase (MGL; EC 4.4.1.11 [EC] ) from the Archaea. The deduced amino acid sequences of EhMGL1 and 2 are 69% identical to each other. The EhMGL1 and 2 proteins showed 44-49/44-48% identity to MGL from three archael species, i.e. Methanosarcina mazei (MmMGL)2 Methanosarcina acetivorans (MaMGL), and Methanosarcina barkeri (MbMGL), respectively. EhMGL1 and 2 also showed 39-46/40-43% identity to MGL from bacteria, i.e. Fusobacterium nucleatum (FnMGL, 46/43%) and Pseudomonas putida (PpMGL1, 39/40%), and 45/43% identity to MGL from another eukaryotic protozoan parasite, i.e. T. vaginalis (TvMGL1). In addition, EhMGL1 and 2 showed 39/41% identity to CGS from Helicobacter pylori (HpCGS), 37/36% identity to human CGL (HsCGL), and 26/28% identity to E. coli CBL (EcCBL).

Comparison of the deduced amino acid sequences of these MGLs as well as 18 other PLP enzymes (Fig. 2A, only MGLs are shown) revealed conserved amino acids as well as residues unique to MGLs. Phe44, Met84, Cys110, and Val331 (amino acid numbers are based on EhMGL1) were conserved among all MGLs and absent in all other PLP enzymes. Conservation of Cys110 was previously reported for MGL from P. putida (31) and T. vaginalis (32). Amino acid residues implicated in substrate binding and catalysis from the crystal structure of MGL from P. putida (Tyr108, Asp180, Lys205, Arg367) (33) were also conserved in EhMGLs, although these residues were also shared by other PLP-dependent enzymes.




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  		FIG. 2.
Amino acid comparison of MGLs and phylogenetic analysis of {gamma}-subfamily of PLP enzymes. A, comparison of deduced amino acid sequences of MGL from Entamoeba and other organisms. Pair-wise alignment was performed as described under "Experimental Procedures". Computer-generated gaps are indicated by hyphens. Residues that are conserved among all MGL sequences as well as other members of the {gamma}-subfamily of PLP enzymes are shown on a gray background, and "MGL signature-like residues" conserved in MGLs, but not in other members of the {gamma}-subfamily of PLP enzymes, are shown as reversed letters on a black background and marked with sharps. The two amebic and two archaeal MGLs are labeled in bold, and their conserved residues are shown on a gray background marked with asterisks. A region corresponding to the PLP attachment site in cysteine/methionine-metabolizing enzymes (PROSITE accession number: PS00868) is marked with an open rectangle. Active-site residues depicted from a crystal structure of PpMGL (33) are indicated with arrowheads. Protein accession numbers are shown in Fig. 2B. B, phylogenetic reconstruction of MGLs and other related PLP enzymes belonging to the {gamma}-subfamily. An unrooted NJ tree, which is representative of the results of three independent methods for phylogenetic reconstruction, is shown. The root is arbitrarily placed using EhCSs ({beta}-family of PLP enzyme) as the outgroupc, and numbers beside the nodes indicate bootstrap values from 1000 replicates. The horizontal length of each branch is proportional to the estimated number of amino acid substitutions. EhMGLa accession numbers are shown in the DDBJ footnote, and other accession numbers are shown in parentheses (NCBI protein data base except bSwiss-Prot accession number). Abbreviations are defined in Footnote 2.

 

Considering these MGL-specific residues, the MmMGL and MbMGL genes, which were initially deposited in the data base as CGS (NP_635109 [GenBank] ) and a hypothetical protein (NZ_AAAR01001136), respectively, likely encode MGL since all the important residues that were shown to be unique to and shared by biochemically characterized MGLs from other organisms, including the amebic MGL, were completely conserved in these 2 sequences. In addition, amino acid residues of Phe188, Leu200, Cys248, Gly302, and Asp337, and a deletion (at position 232-233) were uniquely conserved among the two EhMGL isozymes and all the archaeal MGLs.

Phylogenetic Analyses of EhMGLs—Phylogenetic reconstruction of the 23 protein sequences that belong to the {gamma}-subfamily of PLP enzymes from a variety of organisms, together with two EhCS isotypes ({beta}-family of PLP enzymes) as the outgroup, was performed with the NJ, MP, and ML methods as described under "Experimental Procedures." These analyses (only the result of the NJ method is shown in Fig. 2B) revealed that PLP enzymes involved in sulfur amino acid metabolism were clearly divided into four distinct groups, i.e. MGL, CGL, CGS, and CBL, which was supported by high bootstrap proportions (98.9-100%). In the MGL clade, a monophyletic relationship among the MGLs from E. histolytica and three Archaea, i.e. M. acetivorans, M. mazei, and M. barkeri, was confirmed (bootstrap proportion 82.4%), while bacterial MGLs and TvMGLs formed an independent clade (93.0%). Therefore, MGL appeared to be subdivided into the Entamoeba-Archaea and the Trichomonas-Bacteria groups. TvMGLs and EhMGLs did not form a statistically supported clade with any of the three independent analytical methods (data not shown). That is, despite a predicted close relationship between these two protozoan organisms based on several biological and biochemical characteristics (e.g. anaerobic metabolism and a lack of mitochondria and the glutathione system), they do not likely share a common ancestor for their MGLs.

Evolutionary Distribution of MGL—The presence of an MGL gene or its encoded protein has been demonstrated in only a fraction of bacteria, including Clostridium sporogenes (34), P. putida (= ovalis) (35), Pseudomonas taetrolenz (36), Bacillus halodurans (37), Aeromonas sp. (38), Citrobacter intermedius (39), and Brevibacterium linenese (40), and only two eukaryotic organisms, T. vaginalis (41) and E. histolytica (this study). Coombs et al. (41, 42) also reported that MGL activity was not detected in crude extracts from the other anaerobic protozoan parasites Entamoeba invadens, Trichomonas fetus, Trichomitus batrachorum, and Giardia lamblia, suggesting that the presence of MGL is not directly associated with anaerobic metabolism. In addition, our search for a putative MGL gene in 23 archaeal genome databases available at NCBI revealed that only 3 Archaea, M. mazei, M. acetivorans, and M. barkeri, possess orthologous genes. We were also unable to find a MGL ortholog in other eukaryotes including yeasts, fungi, slime mold, and higher eukaryotes. This unique distribution of MGL strongly supports the premise that two distinct MGL subgroups have been horizontally transferred, i.e. from a subgroup of Archaea to E. histolytica and from a subgroup of bacteria to T. vaginalis.

Molecular and Structural Characterization of Recombinant EhMGL Isoenzymes—In order to understand the biochemical properties of the two EhMGL isoenzymes, recombinant proteins were produced. The recombinant proteins (rEhMGL1 and 2) were assessed to be >95% pure with Coomassie-stained SDS-PAGE gel (data not shown). The apparent molecular masses of rEhMGL1 and 2 (Fig. 4A, immunoblots using antibodies against rEhMGL1 and 2 are shown) agreed well with the predicted values (43.3 and 43.7 kDa, respectively, with 10 extra amino acids attached at the N terminus). Two-dimensional PAGE (Fig. 4C, upper two panels; also see below) showed that these rEhMGL isoenzymes had pIs consistent with those calculated (5.9 and 6.5, respectively). Gel filtration chromatography showed that the molecular mass of the native forms of rEhMGL1 and 2 was 171-177 kDa (Fig. 3A), indicating that both rEhMGL proteins form a homotetramer.



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  		FIG. 4.
Immunoblot analyses of the native and recombinant EhMGL. A, immunoblot analysis showing relative abundance and subcellular localization of two isotypes of EhMGLs. Recombinant EhMGL1 (lanes 1 and 6, 5 ng), recombinant EhMGL2 (lanes 2 and 7, 5 ng), a whole trophozoite lysate (lanes 3 and 8, 20 µg), a soluble fraction (lanes 4 and 9, 20 µg), and a pellet fraction (lanes 5 and 1, 20 µg) were electrophoresed on 10% SDS-PAGE gel, and subjected to immunoblot analyses with either anti-EhMGL1 (lanes 1-5) or anti-EhMGL2 (lanes 6-10) antibody as described under "Experimental Procedures." B, elution profile of the native EhMGL1 and EhMGL2 obtained by DEAE anion exchange chromatography. Upper panel shows MGL activities and A280 of individual fractions. Arrows indicate fractions in which EhMGLs were recognized with the antisera. Middle and lower panels show immunoblots of each fraction with anti-EhMGL1 (middle) and anti-EhMGL2 (lower) antibodies. C, two-dimensional PAGE analyses of the native and recombinant EhMGLs. Upper panels, one-hundred nanograms of rEhMGL1 or rEhMGL2 was subjected to two-dimensional PAGE and immunoblot analysis. Lower panels, two major MGL-containing peak fractions that were eluted from the DEAE column (frs. 5 and 10) were subjected to two-dimensional PAGE, followed by immunoblot analyses with the anti-EhMGL1 (fr. 10) or anti-EhMGL2 (fr. 5) antibody.

 


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  		FIG. 3.
Enzymological characteristics of the recombinant EhMGL isotypes. A, Size exclusion chromatography of the recombinant EhMGLs. rEhMGL1 ({square}) and 2 ({blacksquare})were applied to a column of Toyopearl HW-65S and detected as described under "Experimental Procedures." Standard calibration of this column was performed with blue dextran (2000 kDa), ferritin (440 kDa), catalase (232 kDa), and aldolase (158 kDa). The Kav of each protein was calculated using the equation Kav = (Ve - V0)/(Vt - V0) (Ve, elution volume; Vt, total bed volume; V0, column void volume). B, pH optimum of rEhMGLs. The optimum pH was determined at 37 °C with 50 mM MES buffer (pH 5.6-6.2), MOPS buffer (pH 6.4-7.4), and HEPES buffer (pH 7.4-8.2). Activities of rEhMGL1 ({circ}) and 2 (•) were monitored by hydrogen sulfide assay for 5 min using 5 mM DL-homocysteine and 1 µg of each rEhMGL in a 200 µl reaction mixture as described under "Experimental Procedures." Data shown are means ± S.D. of triplicates. C, Lineweaver-Burk double reciprocal plot of MGL showing non-competitive inhibition of rEhMGL2 by DL-propargylglycine (PPG). rEhMGL2 was preincubated with 0 (•), 10 ({triangleup}), or 20 µM ({square}) PPG in a reaction mixture without L-methionine at 36 °C for 5 min. Reactions were initiated by adding appropriate concentrations of L-methionine, and pyruvate production was assayed after 5 min. D, slow binding inhibition of rEhMGL2 by PPG. rEhMGL2 was preincubated with 0 (•), 10 ({circ}), or 20 µM ({blacktriangleup}) PPG in a reaction mixture without L-methionine at 36 °C for each period indicated. Reactions were initiated by adding L-methionine and carried out for 5 min. Percentages of MGL activity relative to the untreated control are shown.

 

Enzymological Characterization of EhMGL Isoenzymes—Both rEhMGL1 and 2 catalyzed {alpha}-, {gamma}- or {alpha}-, {beta}-elimination of L-methionine, DL-homocysteine, L-cysteine, and OAS, but not L-cystathionine, to form {alpha}-keto acid, ammonia, and hydrogen sulfide (from cysteine), methanethiol (from methionine) or acetate (from OAS) (Tables I and II). The specific activity of rEhMGL1 and 2 (e.g. 0.36 and 0.44 µmol/min/mg toward L-methionine, respectively) was significantly lower than that of recombinant MGLs from other organisms (e.g. rPpMGL, 45.3 µmol/min/mg (43) and rTvMGL1/2, 10.4 ± 0.31/0.67 ± 0.05 µmol/min/mg (32), while the Kms for substrates were comparable (0.9-3.4 mM) (Table II). Although EhMGL1 and 2 showed moderate homology in their amino acid sequences to EcCBL, HsCGL and HpCGS (shown above), L-cystathionine degradation by either rEhMGL1 or rEhMGL2 was negligible.


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  		TABLE I
Products of L-methionine degradation by the amebic crude extract and recombinant EhMGLs

Rates of product formation were determined from three independent experiments, and results of one representative experiment are shown. Assays to measure individual products were performed as described under "Experimental Procedures." Reactions were carried out with 10 mM L-methionine at 37 °C for 30 min with 200 µg of crude extract or for 10 min with 3 µg of recombinant enzyme in a 200 µl of reaction mixture. Crude extracts from trophozoites cultured for 48 hrs in BI-S-33 medium supplemented with or without 20 µM PPG were also used. Methanethiol and nitrogen could not be measured when the crude extract was used as a source of MGL because the backgrounds due to endogenous organic and inorganic thiols and nitrogen were not negligible.

 

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  		TABLE II
Relative activities and kinetic properties of rEhMGLs

Relative activities and kinetic parameters were determined by using {alpha}-keto acid or nitrogen assays. Reactions were performed for 10 min at 36°C with 5 mM (for relative activities) or 0.2-10 mM (for kinetics) of each substrate and an appropriate amount of enzyme. Kinetic parameters were determined by {alpha}-keto acid assay for all the substrates except DL-homocysteine and O-acetyl-L-serine, which were monitored by nitrogen assay. For L-methionine, both assays were used. Experiments were repeated three times, and results of one representative experiment are shown (means ± S.D.). To determine kinetic constants, at least 7 different substrate concentrations were used. Lineweaver-Burk plots were obtained to calculate parameters using Sigma Plot 2000 software (SPSS Inc., Chicago, IL) with the Enzyme Kinetics module (version 6.0, Hulinks, Inc., Tokyo, Japan).

 

Marked differences in substrate specificity and specific activity exist between EhMGL isotypes. rEhMGL1 preferentially degraded L-methionine and DL-homocysteine and showed less activity toward cysteine and OAS, whereas rEhMGL2 catalyzed the degradation of these four amino acids with a comparable efficiency. That is, the ratio of rEhMGL1 activity toward L-cysteine to that toward L-methionine was 0.20 whereas that of rEhMGL2 was 0.88. In addition, rEhMGL2 generally showed 1.8- to 8-fold higher level of specific activity than rEhMGL1, independent of substrates (Table II).

Inter-isotype differences in substrate specificity were previously reported for two MGL isozymes from T. vaginalis (32). rTvMGL1 was shown to possess a broader substrate range than rTvMGL2; rTvMGL1 prefers methionine whereas rTvMGL2 is able to utilize methionine, homocysteine, OAS, and cysteine at comparable levels (e.g. the ratio of activity toward L-cysteine to that toward L-methionine was 0.58 for rTvMGL1 whereas for rTvMGL2 it was 1.58). The most striking difference between the amebic and trichomonal MGLs was that the latter have a strong preference toward homocysteine (more than 30-fold higher activity for homocysteine than both methionine and cysteine). Reactivity toward L-cystathionine was absent in both the amebic and trichomonal MGLs. This is in good contrast to a recombinant Pseudomonas MGL (43). It should be noted that the recombinant TvMGLs and a native form P. putida MGL (44) also lacked reactivity for L-cystathionine, which may suggest that reactivity for L-cystathionine is easily lost during purification or in case of ectopic expression using the bacterial system.

The pH optima for the two EhMGL isoenzymes were also significantly different (Fig. 3B). Such isotype-dependent differences in the optimum pH have not previously been described for MGLs in other organisms. This, together with the fact that the two EhMGL isotypes show only 69% identity and have distinct pI values, indicates that they may interact with different proteins and also may be localized in distinct subcellular compartments. Immunolocalization of each MGL isotype in amebic transformants expressing epitope-tagged EhMGL1 and 2, which is now underway, should help to further clarify these possibilities.

Next, in order to verify the stoichiometry of the reactions catalyzed by rEhMGLs, individual products of L-methionine catabolism, i.e. methanethiol, {alpha}-butyric acid, and ammonia, were measured. Approximately equal amounts (within a range of 2-fold) of these compounds were detected (Table I), verifying that rEhMGLs possess comparable {alpha}- and {gamma}-lyase activities against methionine.

We also examined whether EhMGL catalyzed the formation of cystathionine from cysteine and, as a source of the homocysteine moiety, O-acetyl-L-homoserine or O-succinyl-L-homoserine (45), in a reaction known to be catalyzed by cystathionine {gamma}-synthase. However, neither rEhMGL1 nor 2 catalyzed these reactions (data not shown); instead, both enzymes used L-cysteine as substrate for {alpha}-, {beta}-elimination. Thus, given our failure to find CBS, CGL, CGS, and CBL homologs in the genome data base, we concluded that, unlike other organisms, E. histolytica lacks several key enzymes and their genes involved in the forward and reverse trans-sulfuration reactions.

Inhibition of MGL by PPG—To better understand the biochemical characteristics of EhMGL, we evaluated effects of DL-propargylglycine (PPG), a potent inhibitor of the {gamma}-subfamily of PLP-dependent enzymes (46, 47), on the recombinant EhMGLs. Activity of both rEhMGL1 and 2 was inhibited by PPG in an irreversible and slow-binding manner (Fig. 3, C and D) with an apparent Ki of 35 µM (for rEhMGL2) with 5 min of preincubation, which agreed well with a previous report on human CGL (47).

We further assessed the effect of PPG on the parasite's MGL in cultures (Table I). The MGL activity of trophozoites was almost completely inhibited (97.5%) when they were cultivated in the BI-S-33 medium supplemented with 20 µM PPG, while control CS activity was not affected by PPG; control CS activity from the trophozoites cultured without PPG (72.8 nmol/min/mg) was comparable to that with PPG (69.4 nmol/min/mg). Interestingly, growth inhibition of trophozoites was negligible under these conditions (data not shown). Further, the growth inhibition was less than 5% even at higher PPG concentrations (up to 0.5 mM) (Fig. 5B).



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  		FIG. 5.
Effects of TFMET on the E. histolytica trophozoites. A, cytotoxic effects of TFMET and metronidazole on the E. histolytica trophozoites. Trophozoites (1 x 105 cells/ml) were cultured with various concentrations of TFMET (•) and metronidazole ({circ}) in BI-S-33 for 18 h. Numbers of live cells were assessed as described under "Experimental Procedures." Data shown are means ± S.D. of four independent experiments. B, amebicidal effect of TFMET was abolished by PPG. The trophozoites were cultivated with various concentrations of TFMET with ({circ}) or without (•) 0.5 mM PPG. Percentages of the trophozoites relative to the untreated control are shown. Data shown are means ± S.D. derived from four independent experiments.

 

These results suggest that MGL may not be essential for the amebae cultured in vitro using rich media. Considering the fact that this parasite possesses only incomplete methionine-cysteine conversion (i.e. trans-sulfuration) pathways and also lacks the cysteine degradation pathways present in other organisms, e.g. mammals, as described above, it is not understood how toxic sulfur-containing amino acids are degraded in the absence of MGL. A trace amount of MGL, together with other unidentified enzymes, may compensate for the decrease of MGL activity. It is also conceivable that the production of {alpha}-keto acids, i.e. pyruvate and butyrate, by MGL may not be essential in amebae in a nutrient-rich environment despite the fact that these products are used to form acetyl-CoA and {alpha}-propionic acid in a reaction catalyzed by pyruvate:ferredoxin oxidoreductase, and thus play a critical role in energy production in anaerobic protozoa (48). Furthermore, the other products of MGL, i.e. methanethiol and hydrogen sulfide, which have been implicated in the pathogenesis of oral microorganisms (49, 50), may not be required for in vitro growth of the amebic trophozoites. We are currently testing whether MGL shows more detrimental effects on amebae in nutrient-limited xenic cultures and also in animal intestine and liver models.

Characterization of Native Form EhMGL Isotypes in E. histolytica Trophozoites—Immunoblot analysis of the fractionated trophozoite lysate using specific antibody against rEhMGL1 or rEhMGL2 showed that both of the native EhMGL isotypes were predominantly present in the cytosol fraction of the E. histolytica trophozoites (Fig. 4A). A quantitative estimate using densitometric measurements of native EhMGL in the trophozoite lysate with recombinant EhMGLs as controls revealed that EhMGL1 and 2 constitute ~0.05-0.1% of the total soluble protein of the cell (Fig. 4A, data for estimation not shown), which agreed well with the activity in the crude extract (Table I).

Fractionation of the whole trophozoite lysate by anion exchange chromatography, followed by the measurement of MGL activity in each fraction, revealed two major peaks possessing MGL activity (Fig. 4A, fractions (frs.) 4-7 and 9-11). Immunoblot analyses using anti-EhMGL1 and 2 antibodies identified the first and second peaks as EhMGL2 and EhMGL1, respectively. To correlate the recombinant and native form EhMGLs further, we subjected concentrated samples corresponding to the first and second DEAE peaks (frs. 5 and 10) to two-dimensional gel electrophoresis and immunoblotting. We identified a single spot for each native EhMGL isotype on a two-dimensional gel with measured pIs that agreed well with theoretical pIs (6.01 for rEhMGL1, 6.66 for rEhMGL2). We also examined, by size exclusion chromatography, the subunit structure of these native EhMGL isotypes obtained from the DEAE columns. The apparent molecular mass of the native EhMGL1 and 2 was determined to be ~170 kDa (data not shown). These results suggest, based on the size of the EhMGL monomers observed on SDS-PAGE and two-dimensional gels, that the native EhMGL1 and EhMGL2, similar to the recombinant proteins, also form a homotetramer. This contrasts well to a native MGL from T. vaginalis, which was suggested to form a heterotetramer (i.e. 2 molecules each of MGL1 and MGL2) (32).

Toxic Effects of TFMET on the Amebic Trophozoites—To exploit MGL as a potential target to develop a new therapeutic against the ameba, we tested if the methinonine analog trifluoromethionine (TFMET) shows an inhibitory effect on trophozoite growth. TFMET, a fluorine substitution-containing analog of methionine, is presumed to be catabolized by MGL to form {alpha}-keto butyrate, ammonia, and trifluoromethanethiol. Trifluoromethanethiol is non-enzymatically converted to carbonothionic difluoride (CSF2), a potent cross-linker of primary amine groups (51).

TFMET caused significant growth inhibition in the trophozoites at concentrations as low as 20 µM. In addition, TFMET showed not only a growth inhibitory effect, but also a notable cytolytic effect on the trophozoites under the same condition; e.g. trophozoites cultivated in the presence of 20 µM of TFMET were completely lysed within 72 h. The IC50 of TFMET (for the growth inhibition) was determined to be 18 µM, which is slightly higher than that of the most commonly used anti-amebic drug metronidazole [1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole], which showed an IC50 of 7 µM under the same conditions (Fig. 5A). This amebicidal effect of TFMET was completely abolished when the trophozoites were co-incubated with 0.5 mM of PPG, an inhibitor of EhMGL (Fig. 5B). Under the same conditions, PPG did not abolish the growth inhibition by metronidazole (data not shown). These results strongly support the premise that catabolism of TFMET by MGL is a part of the cytotoxic mechanism of TFMET in the trophozoites. We should also note that the activity of CS, which is the major PLP-dependent enzyme of this parasite, is not inhibited by up to 100 µM PPG (data not shown), further supporting the premise that MGL is a major target of TFMET. The cytotoxic effect of TFMET was also reported for T. vaginalis (52).

Finally, the lack of MGL in higher eukaryotes including humans also highlights this enzyme as a suitable and attractive target for the development of a novel chemotherapeutic agent against amebiasis. Combining conventional metronidazole and a new potent drug, e.g. TFMET, for the chemotherapy of amebiasis patients should prevent the rise of metronidazole resistance (53).


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB094499 [GenBank] and AB094500 [GenBank] .

* This work was supported by a grant for Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Corporation, Grants-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan (15019120, 15590378), a grant for Research on Emerging and Re-emerging Infectious Diseases from Ministry of Health, Labor, and Welfare of Japan, and a grant from the Project to Promote Development of Anti-AIDS Pharmaceuticals from Japan Health Sciences Foundation (SA 14706) (to T. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: 1-23-1 Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan. Tel.: 81-3-5285-1111 (ext. 2733); Fax: 81-3-5285-1173; E-mail: nozaki{at}nih.go.jp.

1 The abbreviations used are: OAS, O-acetylserine; TFMET, trifluoromethionine; TIGR, The Institute for Genomic Research; SAT, serine acetyltransferase; CS, cysteine synthase; MGL, methionine {gamma}-lyase; PPG, DL-propargylglycine; PLP, pyridoxal 5'-phosphate; CGL, cystathionine {gamma}-lyase; CGS, cystathionine {gamma}-synthase; CBL, cystathionine {beta}-lyase; CBS, cystathionine {beta}-synthase; ORF, open reading frame; GST, glutathione S-transferase; rEhMGL, recombinant EhMGL; NJ, Neighbor-Joining; MP, maximum parsimony; ML, maximum likelihood; MOPS, 4-morpholinepropanesulfonic acid; MES, 4-morpholineethanesulfonic acid. Back

2 Names of organisms: Eh, Entamoeba histolytica; Mm, Methanosarcina mazei Goe1; Tv, Trichomonas vaginalis; Fn, Fusobacterium nucleatum subsp. C2A nucleatum ATCC 25586; Ma, Methanosarcina acetivorans; Mb, Methanosarcina barkeri; Pp, Pseudomonas putida; Hs, Homo sapiens; Ec, Escherichia coli K12; Bh, Bacillus halodurans; Cc, Caulobacter crescentus CB15; Oi, Oceanobacillus iheyensis; Rn, Rattus norvegicus; Ce, Caenorhabditis elegans; Cp, Clostridium perfringens; Hp, Helicobacter pylori; Sa, Staphylococcus aureus subsp. aureus N315; Hi, Haemophilus influenzae Rd; St, Salmonella typhimurium LT2; Yp, Yersinia pestis. Back


   ACKNOWLEDGMENTS
 
We thank Masanobu Tanabe, Keio University, for technical assistance on two-dimensional gel electrophoresis and Cyrus J. Bacchi, Pace University, for generously donating TFMET. The data base search was conducted with a 7x E. histolytica genome data base available at The Institute for Genomic Research (TIGR) and Sanger Institute with financial support from National Institute of Allergy and Infectious Diseases and The Wellcome Trust.



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 RESULTS AND DISCUSSION
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