Identification and Characterization of Two Isoenzymes ofMethionine �-Lyase from Entamoeba histolyticaA KEY ENZYME OF SULFUR-AMINO ACID DEGRADATION IN AN ANAEROBIC PARASITIC PROTIST THATLACKS FORWARD AND REVERSE TRANS-SULFURATION PATHWAYS*

Received for publication, December 5, 2002, and in revised form, August 11, 2003Published, JBC Papers in Press, August 14, 2003, DOI 10.1074/jbc.M212414200

Masaharu Tokoro‡, Takashi Asai‡, Seiki Kobayashi‡, Tsutomu Takeuchi‡,and Tomoyoshi Nozaki§¶�

From the ‡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

To better understand the metabolism of sulfur-con-taining amino acids, which likely plays a key role in avariety of cell functions, in Entamoeba histolytica, wesearched the genome data base for genes encoding pu-tative orthologs of enzymes known to be involved in themetabolism. The search revealed that E. histolytica pos-sesses only incomplete cysteine-methionine conversionpathways in both directions. Instead, this parasite pos-sesses genes encoding two isoenzymes of methionine�-lyase (EC 4.4.1.11, EhMGL1/2), which has been impli-cated in the degradation of sulfur-containing amino ac-ids. The two amebic MGL isoenzymes, showing 69% iden-tity to each other, encode 389- and 392-amino acidpolypeptides with predicted molecular masses of 42.3and 42.7 kDa and pIs of 6.01 and 6.63, respectively.Amino acid comparison and phylogenetic analysis sug-gested that these amebic MGLs are likely to have beenhorizontally transferred from the Archaea, whereas anMGL from another anaerobic protist Trichomonas vagi-nalis has MGL isotypes that share a common ancestorwith bacteria. Enzymological and immunoblot analysesof the partially purified native amebic MGL confirmedthat both of the MGL isotypes are expressed in a com-parable amount predominantly in the cytosol and form ahomotetramer. Recombinant EhMGL1 and 2 proteinscatalyzed degradation of L-methionine, DL-homocys-teine, L-cysteine, and O-acetyl-L-serine to form �-ketoacid, ammonia, and hydrogen sulfide or methanethiol,whereas activity toward cystathionine was negligible.These two isoenzymes showed notable differences insubstrate specificity and pH optimum. In addition, weshowed that EhMGL is an ideal target for the develop-

ment of new chemotherapeutic agents against amebia-sis by demonstrating an amebicidal effect of the methi-onine analog trifluoromethionine on trophozoites inculture (IC50 18 �M) and that this effect of trifluorome-thionine was completely abolished by the addition of theMGL-specific inhibitor DL-propargylglycine.

Entamoeba histolytica is a causative agent of amebiasis,which annually affects an estimated 48 million people andresults in 70,000 deaths (1). The most common clinical presen-tation of amebiasis is amebic dysentery and colitis; extraintes-tinal abscesses, i.e. hepatic, pulmonary, and cerebral, however,are also common and often lethal. This microaerophilic anaer-obe has been considered to be a unique eukaryotic organismbecause it apparently lacks organelles typical of eukaryoticorganisms such as mitochondria, the rough endoplasmic retic-ulum, and the Golgi apparatus (2). However, a recent demon-stration of genes encoding mitochondrial proteins, i.e. cpn60and pyridine nucleotide transhydrogenase (3), together withelectron micrographic demonstration of the rough endoplasmicreticulum and the Golgi apparatus (4), suggested the presenceof a residual organelle of mitochondria (called crypton or mito-some) (5, 54) and also indicated that this group of parasiticprotists possess a unique organelle organization. This parasitealso reveals numerous unusual aspects in its metabolism (6),highlighted by the lack of the tricarboxylic acid cycle (7) andglutathione metabolism (8). In addition, recent studies suggest-ing the horizontal transfer of genes encoding a variety of fer-mentation enzymes from bacteria (9), and genes encoding malicenzyme and acetyl-CoA synthase from the Archaea (10) haveplaced this protozoan organism at a unique position in eukary-otic evolution.

One of these unique metabolic pathways found in this para-site is the biosynthetic and degradative pathway of sulfur-containing amino acids, especially cysteine, which has beendemonstrated to be essential for the growth and various cellu-lar activities of amoebae (11, 12). Sulfur-containing amino acidmetabolism varies among organisms (Fig. 1, also reviewed inRef. 13). In mammals, cysteine is produced solely from incor-porated methionine and serine via S-adenosylmethionine,homocysteine, and cystathionine in a pathway called the re-verse trans-sulfuration pathway. In contrast, plants, fungi, andsome bacteria have a so-called sulfur assimilation pathway tofix inorganic sulfur onto a serine derivative (O-acetylserine,

* This work was supported by a grant for Precursory Research forEmbryonic Science and Technology (PRESTO), Japan Science andTechnology Corporation, Grants-in-aid for Scientific Research on Pri-ority Areas from the Ministry of Education, Culture, Sports, Scienceand Technology of Japan (15019120, 15590378), a grant for Research onEmerging and Re-emerging Infectious Diseases from Ministry ofHealth, Labor, and Welfare of Japan, and a grant from the Project toPromote Development of Anti-AIDS Pharmaceuticals from JapanHealth Sciences Foundation (SA 14706) (to T. N.). The costs of publi-cation of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the DDBJ/GenBankTM/EBI Data Bank with accession number(s)AB094499 and AB094500.

� 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@nih.go.jp.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 43, Issue of October 24, pp. 42717–42727, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 42717

by guest on July 16, 2020http://w

ww

.jbc.org/D

ownloaded from

OAS)1 to synthesize cysteine. These organisms are also capableof converting cysteine into methionine via a trans-sulfurationsequence in the opposite orientation (also called the methioninebiosynthetic pathway). We previously demonstrated thatE. histolytica possesses the sulfur assimilatory cysteine biosyn-thetic pathway, and is capable of producing cysteine de novo(14, 15). We have also demonstrated (15) that major enzymes inthis pathway, serine acetyltransferase (SAT) and cysteine syn-thase (CS), play a central role in the control of the intracellularcysteine concentrations, and in the antioxidative stress defensemechanism of this gluthathione-lacking parasite (8).

One important question remaining about the sulfur-contain-ing amino acid metabolism in this parasite, and also in anaer-obic protists in general, is how these parasites degrade toxicsulfur-containing amino acids since they possess apparentlyincomplete trans-sulfuration pathways in both the forward andreverse orientation (data not shown, see the present study).Thus, in order to better understand the metabolism, particu-larly degradation, of these sulfur-containing amino acids inE. histolytica, we attempted to isolate other essential genesencoding proteins involved in sulfur amino acid metabolism.

We identified and characterized two isotypes of the uniqueenzyme, methionine �-lyase (MGL; EC 4.4.1.11) and their en-coding genes, which, we propose, function in the degradation ofsulfur amino acids in this parasite. We show a line of evidencesuggesting that the MGL genes and their proteins were likelyderived from the Archaea by horizontal transfer as shown forother metabolic enzymes in this parasite (10). In addition, wealso demonstrate that the methionine analog trifluoromethi-onine (TFMET) has a cytotoxic effect on amebic trophozoitesthat is abolished by a specific inhibitor of MGL, indicating thatMGL is exploitable as an attractive target for the developmentof new amebicidal compounds.

EXPERIMENTAL PROCEDURES

Chemicals and Reagents—L-methionine, L-cysteine, DL-homocys-teine, OAS, O-succinyl-L-homocysteine, O-acetyl-L-homoserine, DL-propargylglycine (PPG), 3-methyl-2-benzothiazolinone hydrazon hydro-chloride, trichloroacetic acid, pyridoxal 5�-phosphate (PLP), and otherchemicals were commercial products of the highest purity availableunless 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. histolyticastrain HM-1:IMSS cl-6 (16) were maintained axenically in Diamond’sBI-S-33 medium (11) at 35.5 °C. Trophozoites were harvested at thelate-logarithmic growth phase 2–3 days after inoculation of one-twelfthto one-sixth of a total culture volume. After the cultures were chilled onice for 5 min, trophozoites were collected by centrifugation at 500 � gfor 10 min at 4 °C and washed twice with ice-cold phosphate-bufferedsaline, pH 7.4. Cell pellets were stored at �80 °C until use.

Search of the Genome Data Base of E. histolytica—The E. histolyticagenome data base at the Institute for Genomic Research (TIGR, //ww-w.tigr.org/tdb/) was searched using the TBLASTN algorithm with pro-tein sequences corresponding to the PLP-attachment site of cysteine-and methionine-metabolizing enzymes (PROSITE access numberPS00868). This motif is conserved among the ��subfamily (�-family) ofPLP enzymes (for the classification of PLP enzymes used in this study,see Ref. 17), i.e. cystathionine �-lyase (CGL), cystathionine �-synthase(CGS), and cystathionine �-lyase (CBL) from a variety of organisms. Wealso searched for amebic orthologs that belong to the �-family of PLPenzymes using the PLP-attachment site from CS of E. histolytica andcystathionine �-synthase (CBS) from yeast and mammals.

Cloning of E. histolytica MGL1 and MGL2 and Production of theirRecombinant Proteins—Based on nucleotide sequences of the protein-encoding region of the two putative amebic MGL genes (EhMGL1 andEhMGL2), two sets of primers, shown below, were designed to amplifythe open reading frames (ORF) of EhMGL1 and to construct plasmids toproduce glutathione S-transferase (GST)-EhMGL fusion proteins. Thetwo sense and two antisense primers contained the SmaI restrictionsite (underlined) either prior to the translation initiation site or next tothe stop codon (bold), respectively. The primers used are: EhMGL1(sense), 5�-CATCCCGGGGATGACTGCTCAAGATATTACTACTAC-T-3� (37-mer); EhMGL1 (antisense), 5�-TAGCCCGGGATTACCAAAG-CTCTAATGCTTGTTTTAA-3� (37-mer); EhMGL2 (sense), 5�-CATCCC-GGGTATGTCTCAATTGAAGGATTTACAAACA-3� (37-mer); EhMGL2(antisense), 5�-TAGCCCGGGATTAGCATTGTTCAAGAGCTTGTTTT-AA-3� (37-mer).

The cDNA library of E. histolytica trophozoites constructed in alambda phage (14) was used as the template for polymerase chainreaction (PCR) using the following parameters. An initial step fordenaturation and rTaq (Takara Bio Inc., Shiga, Japan) activation at94 °C for 15 min was followed by 35 cycles of denaturation at 94 °C for30 s, annealing at 45 °C for 30 s, and extension at 72 °C for 1 min. Afinal step at 72 °C for 10 min was used to complete the extension.Approximately 1.1-kb PCR fragments were obtained and cloned into theSmaI site of a pGEX-6P-1 expression vector (Amersham BiosciencesK.K., Tokyo, Japan). The final constructs were designated as pGEX6P1/MGL1 and pGEX6P1/MGL2, respectively.

Nucleotide sequences were confirmed by using appropriate syntheticsequencing primers, a BigDye Terminator Cycle Sequencing ReadyReaction Kit, and an ABI PRISM 310 genetic analyzer (Applied Biosys-tems Japan Ltd., Tokyo, Japan), according to the manufacturer’s pro-tocol. 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 isopro-

1 The abbreviations used are: OAS, O-acetylserine; TFMET, triflu-oromethionine; TIGR, The Institute for Genomic Research; SAT, serineacetyltransferase; CS, cysteine synthase; MGL, methionine �-lyase;PPG, DL-propargylglycine; PLP, pyridoxal 5�-phosphate; CGL, cystathi-onine �-lyase; CGS, cystathionine �-synthase; CBL, cystathionine�-lyase; CBS, cystathionine �-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-morpholineethane-sulfonic acid.

FIG. 1. A schematic representation of sulfur-containing aminoacid metabolism in general (A) and in Entamoeba (B). Only en-zymes that belong to the �-subfamily of PLP-dependent enzymes areshown (in bold), together with CS and CBS, which belong to the �-fam-ily, and SAT. Biochemical steps involved in both forward and reversetrans-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 genomedata base (data not shown). Gray arrows indicate reactions catalyzed byMGLs and hatched arrows indicate sulfur assimilatory steps we previ-ously reported (14, 15).

MGL from E. histolytica42718

by guest on July 16, 2020http://w

ww

.jbc.org/D

ownloaded from

pyl-�-thiogalactoside at 18 °C for 20 h. The fusion proteins were puri-fied using a glutathione-Sepharose 4B column (Amersham Biosciences)according to the manufacturer’s instructions. The recombinant Eh-MGL1/2 (rEhMGL1/2) were obtained by digestion of these fusion pro-teins 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 theN terminus. The purified enzymes were stored at �80 °C with 30–50%dimethyl sulfoxide until use. No decrease in enzyme activity was ob-served under these conditions for at least 3 months. Protein concentra-tions were determined by Coomassie Brilliant Blue assay (NacalaiTesque, Inc., Kyoto, Japan) with bovine serum albumin as the standard.

Amino Acid Alignments and Phylogenetic Analyses—The sequencesof MGL and other members of the �-subfamily of PLP enzymes showingsimilarities to the amino acid sequences of EhMGL were obtained fromthe National Center for Biotechnology Information (NCBI, //www.ncbi.nih.gov/) by using the BLASTP algorithm. The alignment andphylogenetic analyses were performed with ClustalW version 1.81 (18)using the Neighbor-Joining (NJ) method with the Blosum matrix. Anunrooted NJ tree composed of the amino acid sequences of 13 MGLs and10 other members of the �-subfamily of PLP enzymes from variousorganisms with two EhCSs (�-family of PLP enzymes) as the outgroupwas drawn by Tree View ver.1.6.0 (19). Branch lengths and bootstrapvalues (1000 replicates) were derived from the NJ analysis. Phyloge-netic analyses by the maximum parsimony method (MP) and maximumlikelihood 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 �106 E. histolytica trophozoites was prepared by two cycles of freezingand thawing in 1 ml of cell lysis buffer: 100 mM sodium phosphatebuffer, pH 7.0, containing 1 mM EDTA, 0.02 mM PLP, 1 mM dithiothre-itol, and a protease inhibitor mixture (Complete Mini EDTA-free, RocheApplied Science, Tokyo, Japan), and 1 �g/ml of N-(3-carboxyoxirane-2-carbonyl)-leucyl-amino(4-guanido)butane (E-64, Sigma). The whole ly-sate was then centrifuged at 14,000 � g in a microcentrifuge tube for 20min at 4 °C to separate the supernatant (soluble cytosolic fraction) andthe pellet (debris, membrane, and nuclear fraction).

Anion Exchange Chromatography of the Native Form MGLs—A su-pernatant fraction obtained from 2 g (wet weight) of the trophozoitepellet, as described above, was filtered with a 0.45-�m-pore mixedcellulose membrane (Millex-HA, Millipore Corporation, Bedford, MA).The sample buffer was exchanged with buffer A (20 mM Tris-HCl, pH8.0, 0.02 mM PLP, 1 mM dithiothreitol, 1 mM EDTA, and 0.1 �g/ml ofE-64) by using a Centricon Plus-20 (Millipore). A 20-ml sample contain-ing �100 mg of total protein was loaded on a DEAE-Toyopearl HW-650S column (7.5 � 1.6 cm, 15-ml bed volume, Tosoh, Tokyo, Japan)that was previously equilibrated with buffer A. The column was furtherwashed with �50 ml of buffer A until the A280 dropped below 0.1. Thebound proteins were then eluted with a 50-ml linear potassium chloridegradient (0–0.5 M) in buffer A at a flow rate of 0.8 ml/min. All 0.8-mlfractions were concentrated to 0.2 ml with a Centricon YM-10 (Milli-pore). All procedures were performed at 4 °C. The amount of MGL ineach fraction was assessed using the hydrogen sulfide assay and im-munoblotting as described below.

Size Exclusion Chromatography of Recombinant and Native Eh-MGLs—To estimate the molecular mass of the recombinant and nativeEhMGLs, gel filtration chromatography was performed. Approximately500 �g of recombinant and 100 �g of partially purified native EhMGLwere 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 ToyopearlHW-65S (70 � 1.6 cm, 140-ml bed volume, Tosoh) preequilibrated withbuffer B. The recombinant and native MGLs were eluted with buffer Bat a flow rate of 0.8 ml/min. The peaks were detected by measuringabsorbance at A280 (recombinant MGLs) and immunoblotting (nativeMGLs). The same column was calibrated with blue dextran (2000 kDa),ferritin (440 kDa), catalase (232 kDa), and aldolase (158 kDa) (Amer-sham Biosciences).

Immunoblot Anaylsis—Polyclonal antisera against recombinant Eh-MGL1 and 2 were raised in rabbits by Sigma-Genosys (Hokkaido,Japan). Immunoblot analysis was carried out using a polyvinylidenedifluoride (PVDF) membrane as described in (22). The blot membranewas visualized by using alkaline phosphatase conjugate-coupled sec-ondary antibody with NBT/BCIP solution (Roche Applied Science) ac-cording to the manufacturer’s protocol.

Two-dimensional Polyacrylamide Gel Electrophoresis—First-dimen-sional electrofocusing of two-dimensional PAGE was performed usingImmobiline Drystrip, pH 3–10 NL, 7 cm and IPG Buffer pH 3–10 NL(Amersham Biosciences) according to the manufacturer’s protocol. Sec-ond dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel elec-trophoresis was performed on 12% SDS-PAGE gel using prestainedSDS-PAGE standards, Broad Range (Bio-Rad Laboratories, Inc., Tokyo,Japan), as a molecular marker.

Enzyme Assays and Kinetic Calculations—The enzymatic activitiy ofMGL was monitored by measuring the production of �-keto acid, am-monia, and hydrogen sulfide or methanethiol. The standard MGL re-action was performed in 200 �l of 100 mM sodium phosphate buffer, pH6.8, a reaction mixture containing 0.02 mM PLP, and 0.1–10 mM of eachsubstrate with appropriate amounts of each enzyme.

The �-keto acid assay was performed as described (23). The MGLreaction was terminated by adding 25 �l of 50% trichloroacetic acid.After the proteins were precipitated by centrifugation at 14,000 � g for5 min at 4 °C, 100 �l of the supernatant was mixed with 200 �l of 0.5 M

sodium acetate buffer, pH 5.0, and 80 �l of 0.1% of 3-methyl-2-benzo-thiazolinone hydrazon hydrochloride, and then incubated at 50 °C for30 min. After the mixture had cooled to room temperature, absorbanceat A320 was measured. Pyruvic acid and 2-�-butyric acid were used asstandards.

For the detection of ammonia, the nitrogen assay (24) was used. A50-�l sample of the supernatant, as for the �-keto acid assay, was mixedwith 50 �l of Nessler’s reagent (Nakalai) and 75 �l of 2 N sodiumhydroxide, 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 �l of 20 mM

N,N-dimethyl-p-phenylenediamine sulfate in 7.2 N HCl and 20 �l of 30mM FeCl3 in 1.2 N HCl. After further incubation in the dark for 20 min,the proteins were precipitated by centrifugation at 14,000 � g for 5 minat 4 °C, and then the absorption at OD650 of the supernatant wasmeasured to quantitate the formed methylene blue. Na2S was used asa standard.

The methanethiol assay was performed as described (28) using 5,5�-dithio-bis-(2-nitrobenzoic acid). One-hundred microliters of the samplesupernatant were mixed with 1 �l of 100 mM 5,5�-dithio-bis-(2-nitro-benzoic acid) in ethanol, and after 2 min incubation at room tempera-ture, absorbance at A412 was measured. L-cysteine was used as astandard.

The cysteine and cystathionine assay was performed as described(29). The ninhydrin reagent was prepared by dissolving 1 g of ninhydrinin 100 ml of glacial acetic acid and adding 33 ml of glacial phosphoricacid. For the determination of cystathionine, 0.2 ml of cystathionine-containing solution was mixed with 0.33 ml of the ninhydrin reagentand boiled at 100 °C for 5 min. The solution was then cooled on ice for2 min and at room temperature for a further 10 min. Absorbance at A455

was measured. Cysteine concentrations were determined using thesame protocol except for the measurement of absorbance at A560.

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

Assay of the Inhibition of rEhMGL by DL-Propargylglycine—An�-keto acid assay (described above) with L-methionine as the substratewas performed to evaluate the inhibitory effects of DL-propargylglycine(PPG) on the activity of rEhMGLs. rEhMGL (5 �g) was preincubatedwith various concentrations of PPG in the standard MGL reactionmixtures (described above) in the absence of L-methionine at 36 °C. Thepreincubation time was 5 min for kinetic analyses and 1 to 60 min tocharacterize the slow binding of this inhibitor. After preincubation, thereaction was initiated by adding an appropriate amount of L-methionineto the reaction mixture.

In Vitro Assessment of Amebicidal Reagents—To assess the amebi-cidal effect of TFMET, the trophozoites were cultured in the BI-S-33medium 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 thecell proliferation reagent WST-1 (Roche Applied Science). Briefly, thetrophozoites were seeded on 96-well microtiter plates in 200 �l ofBI-S-33 medium at a density of 2 � 104 cells per well (1 � 105 cells/ml),and the lid was completely sealed with a sterilized adhesive siliconsheet (Corning, New York). After these plates were further incubated at35.5 °C for 18 h, 20 �l of WST-1 reagent was added to each well and theincubation 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

MGL from E. histolytica 42719

by guest on July 16, 2020http://w

ww

.jbc.org/D

ownloaded from

FIG. 2. Amino acid comparison of MGLs and phylogenetic analysis of �-subfamily of PLP enzymes. A, comparison of deduced amino acidsequences 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 �-subfamilyof PLP enzymes are shown on a gray background, and “MGL signature-like residues” conserved in MGLs, but not in other members of the �-subfamilyof PLP enzymes, are shown as reversed letters on a black background and marked with sharps. The two amebic and two archaeal MGLs are labeledin bold, and their conserved residues are shown on a gray background marked with asterisks. A region corresponding to the PLP attachment site incysteine/methionine-metabolizing enzymes (PROSITE accession number: PS00868) is marked with an open rectangle. Active-site residues depictedfrom a crystal structure of PpMGL (33) are indicated with arrowheads. Protein accession numbers are shown in Fig. 2B. B, phylogenetic reconstructionof MGLs and other related PLP enzymes belonging to the �-subfamily. An unrooted NJ tree, which is representative of the results of three independentmethods for phylogenetic reconstruction, is shown. The root is arbitrarily placed using EhCSs (�-family of PLP enzyme) as the outgroupc, and numbersbeside the nodes indicate bootstrap values from 1000 replicates. The horizontal length of each branch is proportional to the estimated number of aminoacid substitutions. EhMGLa accession numbers are shown in the DDBJ footnote, and other accession numbers are shown in parentheses (NCBI proteindata base except bSwiss-Prot accession number). Abbreviations are defined in Footnote 2.

MGL from E. histolytica42720

by guest on July 16, 2020http://w

ww

.jbc.org/D

ownloaded from

period of the cultures were chosen to maintain the control trophozoitesin the late-logarithmic growth phase throughout the experiment, andalso to allow the measurement of optical density in the linear portion ofthe curves (between 4 � 103 to 2.0 � 105 cells/ml).

RESULTS AND DISCUSSION

Data Base Search of the PLP-dependent Enzymes—In anattempt to obtain genes encoding the PLP-dependent enzymesinvolved in the metabolism of sulfur-containing amino acids,we searched the genome data base for putative proteins thatpossessed a conserved PLP-binding domain as described under“Experimental Procedures.” Two independent contigs werefound in the genome data base. These contigs (Contig 315785and 316820, TIGR) contained two similar but not identicalORFs that encode proteins possessing a region containing thePLP-binding motif of the �-subfamily of PLP enzymes. Non-

coding flanking regions within these contigs also showed sig-nificant variations (data not shown). All other contigs or sin-gletons showing significant identity to these two contigsperfectly overlapped them, which is consistent with the notionthat these fragments are present as a single copy in thegenome.

We also searched for genes containing the PLP-binding siteof the �-family of PLP enzymes using both the amebic CS andthe yeast and mammalian CBS. However, after eliminatingcontigs and singletons that contain genes encoding the two CSisotypes described previously (14), no contig or singleton wasfound to contain this motif. This suggests that E. histolyticapossesses two uncharacterized genes encoding proteins thatbelong to the �-subfamily of PLP enzymes, and lacks the �-fam-ily of PLP enzymes (i.e. CBS) known to be involved in the

FIG. 2—continued

MGL from E. histolytica 42721

by guest on July 16, 2020http://w

ww

.jbc.org/D

ownloaded from

reverse trans-sulfuration pathway in other organisms. We alsotentatively concluded that the trans-sulfuration sequences inboth the forward and reverse orientation are incomplete sincethe amebic genome lacks putative genes for CBL, CGS, andCGL (also see below). In addition, the major pathways for

cysteine degradation of sulfur amino acids present in mam-mals, i.e. the cysteine sulfinic acid (cysteine dioxygenase as akey enzyme), 4�-phosphopanthetheine (leading to synthesis ofcoenzyme A and cysteamine), and mitochondrial mercaptopy-ruvate pathways, are apparently absent in this organism (data

FIG. 3. Enzymological characteristics of the recombinant EhMGL isotypes. A, Size exclusion chromatography of the recombinantEhMGLs. rEhMGL1 (�) and 2 (f)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 voidvolume). 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 (E) and 2 (F) were monitored by hydrogen sulfide assay for 5 min using 5 mM

DL-homocysteine and 1 �g of each rEhMGL in a 200 �l 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 (F), 10 (‚), or 20 �M (�) PPG in a reaction mixture without L-methionine at 36 °C for 5 min. Reactionswere initiated by adding appropriate concentrations of L-methionine, and pyruvate production was assayed after 5 min. D, slow binding inhibitionof rEhMGL2 by PPG. rEhMGL2 was preincubated with 0 (●), 10 (E), or 20 �M (Œ) PPG in a reaction mixture without L-methionine at 36 °C foreach period indicated. Reactions were initiated by adding L-methionine and carried out for 5 min. Percentages of MGL activity relative to theuntreated control are shown.

MGL from E. histolytica42722

by guest on July 16, 2020http://w

ww

.jbc.org/D

ownloaded from

not shown). Therefore, E. histolytica must have alternativeenzymes involved in the degradation of toxic sulfur-containingamino acids, e.g. homocysteine, which is implicated in the wellcharacterized human genetic disease homocysteinuria and itscytotoxicity (30).

Identification and Features of EhMGL Genes and Their Pro-teins—The nucleotide sequences of the 1170- and 1179-bpORFs recognized in the contigs described above were homolo-gous to those of the �-subfamily of PLP-dependent enzymes,including MGL, CBL, CGS, and CGL. These putative genesencode 389- and 392-amino acid polypeptides with predictedmolecular masses of 42.3 and 42.7 kDa and predicted pIs of6.01 and 6.63, respectively. We designated these genes Eh-MGL1 and EhMGL2 since their predicted proteins showedhighest similarity to methionine �-lyase (MGL; EC 4.4.1.11)from the Archaea. The deduced amino acid sequences of Eh-MGL1 and 2 are 69% identical to each other. The EhMGL1 and2 proteins showed 44–49/44–48% identity to MGL from threearchael species, i.e. Methanosarcina mazei (MmMGL)2 Meth-anosarcina acetivorans (MaMGL), and Methanosarcina barkeri(MbMGL), respectively. EhMGL1 and 2 also showed 39–46/40–43% identity to MGL from bacteria, i.e. Fusobacteriumnucleatum (FnMGL, 46/43%) and Pseudomonas putida (Pp-MGL1, 39/40%), and 45/43% identity to MGL from anothereukaryotic protozoan parasite, i.e. T. vaginalis (TvMGL1). Inaddition, EhMGL1 and 2 showed 39/41% identity to CGS fromHelicobacter 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 theseMGLs as well as 18 other PLP enzymes (Fig. 2A, only MGLsare shown) revealed conserved amino acids as well as residuesunique to MGLs. Phe44, Met84, Cys110, and Val331 (amino acidnumbers are based on EhMGL1) were conserved among allMGLs and absent in all other PLP enzymes. Conservation ofCys110 was previously reported for MGL from P. putida (31)and T. vaginalis (32). Amino acid residues implicated in sub-

strate binding and catalysis from the crystal structure of MGLfrom P. putida (Tyr108, Asp180, Lys205, Arg367) (33) were alsoconserved in EhMGLs, although these residues were alsoshared by other PLP-dependent enzymes.

Considering these MGL-specific residues, the MmMGL andMbMGL genes, which were initially deposited in the data baseas CGS (NP_635109) and a hypothetical protein (NZ_AAAR-01001136), respectively, likely encode MGL since all the impor-tant residues that were shown to be unique to and shared bybiochemically characterized MGLs from other organisms, in-cluding the amebic MGL, were completely conserved in these 2sequences. 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 andall the archaeal MGLs.

Phylogenetic Analyses of EhMGLs—Phylogenetic reconstruc-tion of the 23 protein sequences that belong to the �-subfamilyof PLP enzymes from a variety of organisms, together with twoEhCS isotypes (�-family of PLP enzymes) as the outgroup, wasperformed with the NJ, MP, and ML methods as describedunder “Experimental Procedures.” These analyses (only theresult of the NJ method is shown in Fig. 2B) revealed that PLPenzymes involved in sulfur amino acid metabolism were clearlydivided into four distinct groups, i.e. MGL, CGL, CGS, andCBL, which was supported by high bootstrap proportions(98.9–100%). In the MGL clade, a monophyletic relationshipamong the MGLs from E. histolytica and three Archaea, i.e.M. acetivorans, M. mazei, and M. barkeri, was confirmed (boot-strap proportion 82.4%), while bacterial MGLs and TvMGLsformed an independent clade (93.0%). Therefore, MGL ap-peared to be subdivided into the Entamoeba-Archaea and theTrichomonas-Bacteria groups. TvMGLs and EhMGLs did notform a statistically supported clade with any of the three inde-pendent analytical methods (data not shown). That is, despitea predicted close relationship between these two protozoanorganisms based on several biological and biochemical charac-teristics (e.g. anaerobic metabolism and a lack of mitochondriaand the glutathione system), they do not likely share a commonancestor for their MGLs.

Evolutionary Distribution of MGL—The presence of an MGLgene or its encoded protein has been demonstrated in only afraction of bacteria, including Clostridium sporogenes (34),P. putida (� ovalis) (35), Pseudomonas taetrolenz (36), Bacillushalodurans (37), Aeromonas sp. (38), Citrobacter intermedius(39), and Brevibacterium linenese (40), and only two eukaryoticorganisms, T. vaginalis (41) and E. histolytica (this study).Coombs et al. (41, 42) also reported that MGL activity was notdetected in crude extracts from the other anaerobic protozoanparasites Entamoeba invadens, Trichomonas fetus, Trichomi-tus batrachorum, and Giardia lamblia, suggesting that thepresence of MGL is not directly associated with anaerobic me-tabolism. In addition, our search for a putative MGL gene in 23archaeal genome databases available at NCBI revealed thatonly 3 Archaea, M. mazei, M. acetivorans, and M. barkeri,possess orthologous genes. We were also unable to find a MGLortholog in other eukaryotes including yeasts, fungi, slimemold, and higher eukaryotes. This unique distribution of MGLstrongly supports the premise that two distinct MGL sub-groups have been horizontally transferred, i.e. from a subgroupof Archaea to E. histolytica and from a subgroup of bacteria toT. vaginalis.

Molecular and Structural Characterization of RecombinantEhMGL Isoenzymes—In order to understand the biochemicalproperties of the two EhMGL isoenzymes, recombinant pro-teins were produced. The recombinant proteins (rEhMGL1 and2) were assessed to be �95% pure with Coomassie-stained

2 Names of organisms: Eh, Entamoeba histolytica; Mm, Methanosar-cina mazei Goe1; Tv, Trichomonas vaginalis; Fn, Fusobacterium nu-cleatum subsp. C2A nucleatum ATCC 25586; Ma, Methanosarcina ace-tivorans; Mb, Methanosarcina barkeri; Pp, Pseudomonas putida; Hs,Homo sapiens; Ec, Escherichia coli K12; Bh, Bacillus halodurans; Cc,Caulobacter crescentus CB15; Oi, Oceanobacillus iheyensis; Rn, Rattusnorvegicus; 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.

TABLE IProducts of L-methionine degradation by the amebic crude extract and

recombinant EhMGLsRates 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 describedunder “Experimental Procedures.” Reactions were carried out with 10mM L-methionine at 37 °C for 30 min with 200 �g of crude extract or for10 min with 3 �g of recombinant enzyme in a 200 �l of reaction mixture.Crude extracts from trophozoites cultured for 48 hrs in BI-S-33 mediumsupplemented with or without 20 �M PPG were also used. Methanethioland nitrogen could not be measured when the crude extract was used asa source of MGL because the backgrounds due to endogenous organicand inorganic thiols and nitrogen were not negligible.

Product

Rate of production [nmol/min/mg]

Crude extract Recombinant enzyme

BI-S-33 BI-S-33 � 20�M PPG rEhMGL1 rEhMGL2

Methanethiol n.d.a n.d. 524.7 738.7�-Buryric acid 0.157 0.004 288.7 468.7Nitrogen n.d. n.d. 385.3 793.3

a n.d., not determined.

MGL from E. histolytica 42723

by guest on July 16, 2020http://w

ww

.jbc.org/D

ownloaded from

SDS-PAGE gel (data not shown). The apparent molecularmasses of rEhMGL1 and 2 (Fig. 4A, immunoblots using anti-bodies against rEhMGL1 and 2 are shown) agreed well withthe predicted values (43.3 and 43.7 kDa, respectively, with 10extra amino acids attached at the N terminus). Two-dimen-sional PAGE (Fig. 4C, upper two panels; also see below) showedthat these rEhMGL isoenzymes had pIs consistent with thosecalculated (5.9 and 6.5, respectively). Gel filtration chromatog-raphy showed that the molecular mass of the native forms ofrEhMGL1 and 2 was 171–177 kDa (Fig. 3A), indicating thatboth rEhMGL proteins form a homotetramer.

Enzymological Characterization of EhMGL Isoenzymes—Both rEhMGL1 and 2 catalyzed �-, �- or �-, �-elimination ofL-methionine, DL-homocysteine, L-cysteine, and OAS, but notL-cystathionine, to form �-keto acid, ammonia, and hydrogensulfide (from cysteine), methanethiol (from methionine) or ac-etate (from OAS) (Tables I and II). The specific activity ofrEhMGL1 and 2 (e.g. 0.36 and 0.44 �mol/min/mg toward L-methionine, respectively) was significantly lower than that ofrecombinant MGLs from other organisms (e.g. rPpMGL, 45.3�mol/min/mg (43) and rTvMGL1/2, 10.4 � 0.31/0.67 � 0.05�mol/min/mg (32), while the Kms for substrates were compara-ble (0.9–3.4 mM) (Table II). Although EhMGL1 and 2 showedmoderate homology in their amino acid sequences to EcCBL,HsCGL and HpCGS (shown above), L-cystathionine degrada-tion by either rEhMGL1 or rEhMGL2 was negligible.

Marked differences in substrate specificity and specific ac-tivity exist between EhMGL isotypes. rEhMGL1 preferentiallydegraded L-methionine and DL-homocysteine and showed lessactivity toward cysteine and OAS, whereas rEhMGL2 cata-lyzed the degradation of these four amino acids with a compa-rable efficiency. That is, the ratio of rEhMGL1 activity towardL-cysteine to that toward L-methionine was 0.20 whereas thatof rEhMGL2 was 0.88. In addition, rEhMGL2 generally showed1.8- to 8-fold higher level of specific activity than rEhMGL1,independent of substrates (Table II).

Inter-isotype differences in substrate specificity were previ-ously reported for two MGL isozymes from T. vaginalis (32).rTvMGL1 was shown to possess a broader substrate rangethan rTvMGL2; rTvMGL1 prefers methionine whereasrTvMGL2 is able to utilize methionine, homocysteine, OAS,and cysteine at comparable levels (e.g. the ratio of activitytoward L-cysteine to that toward L-methionine was 0.58 forrTvMGL1 whereas for rTvMGL2 it was 1.58). The most strik-ing difference between the amebic and trichomonal MGLs wasthat the latter have a strong preference toward homocysteine(more than 30-fold higher activity for homocysteine than bothmethionine and cysteine). Reactivity toward L-cystathioninewas absent in both the amebic and trichomonal MGLs. This is

in good contrast to a recombinant Pseudomonas MGL (43). Itshould be noted that the recombinant TvMGLs and a nativeform P. putida MGL (44) also lacked reactivity for L-cystathi-onine, which may suggest that reactivity for L-cystathionine iseasily lost during purification or in case of ectopic expressionusing the bacterial system.

The pH optima for the two EhMGL isoenzymes were alsosignificantly different (Fig. 3B). Such isotype-dependent differ-ences in the optimum pH have not previously been describedfor MGLs in other organisms. This, together with the fact thatthe two EhMGL isotypes show only 69% identity and havedistinct pI values, indicates that they may interact with differ-ent proteins and also may be localized in distinct subcellularcompartments. Immunolocalization of each MGL isotype inamebic transformants expressing epitope-tagged EhMGL1 and2, which is now underway, should help to further clarify thesepossibilities.

Next, in order to verify the stoichiometry of the reactionscatalyzed by rEhMGLs, individual products of L-methioninecatabolism, i.e. methanethiol, �-butyric acid, and ammonia,were measured. Approximately equal amounts (within a rangeof 2-fold) of these compounds were detected (Table I), verifyingthat rEhMGLs possess comparable �- and �-lyase activitiesagainst methionine.

We also examined whether EhMGL catalyzed the formationof cystathionine from cysteine and, as a source of the homocys-teine moiety, O-acetyl-L-homoserine or O-succinyl-L-homo-serine (45), in a reaction known to be catalyzed by cystathi-onine �-synthase. However, neither rEhMGL1 nor 2 catalyzedthese reactions (data not shown); instead, both enzymes usedL-cysteine as substrate for �-, �-elimination. Thus, given ourfailure to find CBS, CGL, CGS, and CBL homologs in thegenome data base, we concluded that, unlike other organisms,E. histolytica lacks several key enzymes and their genes in-volved in the forward and reverse trans-sulfuration reactions.

Inhibition of MGL by PPG—To better understand the bio-chemical characteristics of EhMGL, we evaluated effects ofDL-propargylglycine (PPG), a potent inhibitor of the �-subfam-ily of PLP-dependent enzymes (46, 47), on the recombinantEhMGLs. Activity of both rEhMGL1 and 2 was inhibited byPPG in an irreversible and slow-binding manner (Fig. 3, C andD) with an apparent Ki of 35 �M (for rEhMGL2) with 5 min ofpreincubation, which agreed well with a previous report onhuman CGL (47).

We further assessed the effect of PPG on the parasite’s MGLin cultures (Table I). The MGL activity of trophozoites wasalmost completely inhibited (97.5%) when they were cultivatedin the BI-S-33 medium supplemented with 20 �M PPG, whilecontrol CS activity was not affected by PPG; control CS activity

TABLE IIRelative activities and kinetic properties of rEhMGLs

Relative activities and kinetic parameters were determined by using �-keto acid or nitrogen assays. Reactions were performed for 10 min at 36°Cwith 5 mM (for relative activities) or 0.2-10 mM (for kinetics) of each substrate and an appropriate amount of enzyme. Kinetic parameters weredetermined by �-keto acid assay for all the substrates except DL-homocysteine and O-acetyl-L-serine, which were monitored by nitrogen assay. ForL-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 calculateparameters using Sigma Plot 2000 software (SPSS Inc., Chicago, IL) with the Enzyme Kinetics module (version 6.0, Hulinks, Inc., Tokyo, Japan).

SubstrateRelative activity Km Vmax

rEhMGL1 rEhMGL2 rEhMGL1 rEhMGL2 rEhMGL1 rEhMGL2

% mM �mol/min/mg

L-Methionine 100.0 181.6 0.94 � 0.16 1.90 � 0.15 0.36 � 0.20 0.44 � 0.30DL-Homocysteine 112.2 294.7 3.40 � 0.67 1.87 � 0.77 0.98 � 0.11 1.31 � 0.24L-Cystathionine 10 10 NDa ND ND NDL-Cysteine 19.7 160.1 n.d.b 2.30 � 0.10 n.d. 1.26 � 0.81O-Acetyl L-serine 11.1 33.8 n.d. 0.89 � 0.23 n.d. 0.085 � 0.05

a ND, not detectable.b n.d., not determined.

MGL from E. histolytica42724

by guest on July 16, 2020http://w

ww

.jbc.org/D

ownloaded from

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 negligibleunder these conditions (data not shown). Further, the growthinhibition was less than 5% even at higher PPG concentrations(up to 0.5 mM) (Fig. 5B).

These results suggest that MGL may not be essential for theamebae cultured in vitro using rich media. Considering the factthat this parasite possesses only incomplete methionine-cys-teine conversion (i.e. trans-sulfuration) pathways and alsolacks the cysteine degradation pathways present in other or-ganisms, e.g. mammals, as described above, it is not understoodhow toxic sulfur-containing amino acids are degraded in theabsence of MGL. A trace amount of MGL, together with otherunidentified enzymes, may compensate for the decrease ofMGL activity. It is also conceivable that the production of

�-keto acids, i.e. pyruvate and butyrate, by MGL may not beessential in amebae in a nutrient-rich environment despite thefact that these products are used to form acetyl-CoA and��propionic acid in a reaction catalyzed by pyruvate:ferre-doxin oxidoreductase, and thus play a critical role in energyproduction in anaerobic protozoa (48). Furthermore, the otherproducts of MGL, i.e. methanethiol and hydrogen sulfide,which have been implicated in the pathogenesis of oral micro-organisms (49, 50), may not be required for in vitro growth ofthe amebic trophozoites. We are currently testing whetherMGL shows more detrimental effects on amebae in nutrient-limited xenic cultures and also in animal intestine and livermodels.

Characterization of Native Form EhMGL Isotypes in E. his-tolytica Trophozoites—Immunoblot analysis of the fractionatedtrophozoite lysate using specific antibody against rEhMGL1

FIG. 4. Immunoblot analyses of thenative and recombinant EhMGL. A,immunoblot analysis showing relativeabundance and subcellular localization oftwo isotypes of EhMGLs. RecombinantEhMGL1 (lanes 1 and 6, 5 ng), recombi-nant EhMGL2 (lanes 2 and 7, 5 ng), awhole trophozoite lysate (lanes 3 and 8, 20�g), a soluble fraction (lanes 4 and 9, 20�g), and a pellet fraction (lanes 5 and 1,20 �g) were electrophoresed on 10% SDS-PAGE gel, and subjected to immunoblotanalyses with either anti-EhMGL1 (lanes1–5) or anti-EhMGL2 (lanes 6–10) anti-body as described under “ExperimentalProcedures.” B, elution profile of the na-tive EhMGL1 and EhMGL2 obtained byDEAE anion exchange chromatography.Upper panel shows MGL activities andA280 of individual fractions. Arrows indi-cate fractions in which EhMGLs were rec-ognized with the antisera. Middle andlower panels show immunoblots of eachfraction with anti-EhMGL1 (middle) andanti-EhMGL2 (lower) antibodies. C, two-dimensional PAGE analyses of the nativeand recombinant EhMGLs. Upper panels,one-hundred nanograms of rEhMGL1 orrEhMGL2 was subjected to two-dimen-sional PAGE and immunoblot analysis.Lower panels, two major MGL-containingpeak fractions that were eluted from theDEAE column (frs. 5 and 10) were sub-jected to two-dimensional PAGE, followedby immunoblot analyses with the anti-EhMGL1 (fr. 10) or anti-EhMGL2 (fr. 5)antibody.

MGL from E. histolytica 42725

by guest on July 16, 2020http://w

ww

.jbc.org/D

ownloaded from

or rEhMGL2 showed that both of the native EhMGL iso-types were predominantly present in the cytosol fraction of theE. histolytica trophozoites (Fig. 4A). A quantitative estimateusing densitometric measurements of native EhMGL in thetrophozoite lysate with recombinant EhMGLs as controls re-vealed that EhMGL1 and 2 constitute �0.05–0.1% of the totalsoluble protein of the cell (Fig. 4A, data for estimation notshown), which agreed well with the activity in the crude extract(Table I).

Fractionation of the whole trophozoite lysate by anion ex-change chromatography, followed by the measurement of MGLactivity in each fraction, revealed two major peaks possessingMGL activity (Fig. 4A, fractions (frs.) 4–7 and 9–11). Immuno-blot analyses using anti-EhMGL1 and 2 antibodies identifiedthe first and second peaks as EhMGL2 and EhMGL1, respec-tively. To correlate the recombinant and native form EhMGLsfurther, we subjected concentrated samples corresponding tothe first and second DEAE peaks (frs. 5 and 10) to two-dimen-sional gel electrophoresis and immunoblotting. We identified asingle spot for each native EhMGL isotype on a two-dimen-sional gel with measured pIs that agreed well with theoreticalpIs (6.01 for rEhMGL1, 6.66 for rEhMGL2). We also examined,

by size exclusion chromatography, the subunit structure ofthese native EhMGL isotypes obtained from the DEAE col-umns. The apparent molecular mass of the native EhMGL1and 2 was determined to be �170 kDa (data not shown). Theseresults suggest, based on the size of the EhMGL monomersobserved on SDS-PAGE and two-dimensional gels, that thenative EhMGL1 and EhMGL2, similar to the recombinant pro-teins, also form a homotetramer. This contrasts well to a nativeMGL from T. vaginalis, which was suggested to form a het-erotetramer (i.e. 2 molecules each of MGL1 and MGL2) (32).

Toxic Effects of TFMET on the Amebic Trophozoites—Toexploit MGL as a potential target to develop a new therapeuticagainst the ameba, we tested if the methinonine analog triflu-oromethionine (TFMET) shows an inhibitory effect on tropho-zoite growth. TFMET, a fluorine substitution-containing ana-log of methionine, is presumed to be catabolized by MGL toform �-keto butyrate, ammonia, and trifluoromethanethiol.Trifluoromethanethiol is non-enzymatically converted to car-bonothionic difluoride (CSF2), a potent cross-linker of primaryamine groups (51).

TFMET caused significant growth inhibition in the tropho-zoites at concentrations as low as 20 �M. In addition, TFMETshowed not only a growth inhibitory effect, but also a notablecytolytic effect on the trophozoites under the same condition;e.g. trophozoites cultivated in the presence of 20 �M of TFMETwere completely lysed within 72 h. The IC50 of TFMET (for thegrowth inhibition) was determined to be 18 �M, which isslightly higher than that of the most commonly used anti-amebic drug metronidazole [1-(2-hydroxyethyl)-2-methyl-5-ni-troimidazole], which showed an IC50 of 7 �M under the sameconditions (Fig. 5A). This amebicidal effect of TFMET wascompletely abolished when the trophozoites were co-incubatedwith 0.5 mM of PPG, an inhibitor of EhMGL (Fig. 5B). Underthe same conditions, PPG did not abolish the growth inhibitionby metronidazole (data not shown). These results strongly sup-port the premise that catabolism of TFMET by MGL is a partof the cytotoxic mechanism of TFMET in the trophozoites. Weshould also note that the activity of CS, which is the majorPLP-dependent enzyme of this parasite, is not inhibited by upto 100 �M PPG (data not shown), further supporting the pre-mise that MGL is a major target of TFMET. The cytotoxic effectof TFMET was also reported for T. vaginalis (52).

Finally, the lack of MGL in higher eukaryotes includinghumans also highlights this enzyme as a suitable and attrac-tive target for the development of a novel chemotherapeuticagent against amebiasis. Combining conventional metronida-zole and a new potent drug, e.g. TFMET, for the chemotherapyof amebiasis patients should prevent the rise of metronidazoleresistance (53).

Acknowledgments—We thank Masanobu Tanabe, Keio University,for technical assistance on two-dimensional gel electrophoresis andCyrus J. Bacchi, Pace University, for generously donating TFMET. Thedata base search was conducted with a 7� E. histolytica genome database available at The Institute for Genomic Research (TIGR) andSanger Institute with financial support from National Institute of Al-lergy and Infectious Diseases and The Wellcome Trust.

REFERENCES

1. Behbehani, K. (1998) Bull World Health Organ. 76, Suppl. 2, 64–672. Ravdin, J. I. (2000) AMEBIASIS Series on Tropical Medicine and Practice, Vol.

2, pp. 1–45 Imperial College Press, Covent Garden, London, UK3. Clark, C. G., and Roger, A. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92,

6518–65214. Mazzuco, A., Benchimol, M., and De Souza, W. (1997) Micron. 28, 241–2475. Mai, Z., Ghosh, S., Frisardi, M., Rosenthal, B., Rogers, R., and Samuelson, J.

(1999) Mol. Cell. Biol. 19, 2198–22056. Muller, M. (1992) Biosystems 28, 33–407. Reeves, R. E. (1984) Adv. Parasitol. 23, 105–1428. Fahey, R. C., Newton, G. L., Arrick, B., Overdank-Bogart, T., and Aley, S. B.

(1984) Science 224, 70–729. Rosenthal, B., Mai, Z., Caplivski, D., Ghosh, S., de la Vega, H., Graf, T., and

FIG. 5. Effects of TFMET on the E. histolytica trophozoites. A,cytotoxic effects of TFMET and metronidazole on the E. histolyticatrophozoites. Trophozoites (1 � 105 cells/ml) were cultured with variousconcentrations of TFMET (●) and metronidazole (E) in BI-S-33 for 18 h.Numbers of live cells were assessed as described under “ExperimentalProcedures.” Data shown are means � S.D. of four independent exper-iments. B, amebicidal effect of TFMET was abolished by PPG. Thetrophozoites were cultivated with various concentrations of TFMETwith (E) or without (●) 0.5 m M PPG. Percentages of the trophozoitesrelative to the untreated control are shown. Data shown are means �S.D. derived from four independent experiments.

MGL from E. histolytica42726

by guest on July 16, 2020http://w

ww

.jbc.org/D

ownloaded from

Samuelson, J. (1997) J. Bacteriol. 179, 3736–374510. Field, J., Rosenthal, B., and Samuelson, J. (2000) Mol. Microbiol. 38, 446–45511. Diamond, L. S., Harlow, D. R., and Cunnick, C. C. (1978) Trans. R. Soc. Trop.

Med. Hyg. 72, 431–43212. Gillin, F. D., and Diamond, L. S. (1980) J. Protozool. 27, 474–47813. Walker, J., and Barrett, J. (1997) Int. J. Parasitol. 27, 883–89714. Nozaki, T., Asai, T., Kobayashi, S., Ikegami, F., Noji, M., Saito, K., and

Takeuchi, T. (1998) Mol. Biochem. Parasitol. 97, 33–4415. Nozaki, T., Asai, T., Sanchez, L. B., Kobayashi, S., Nakazawa, M., and Takeu-

chi, T. (1999) J. Biol. Chem. 274, 32445–3245216. Diamond, L. S., Mattern, C. F., and Bartgis, I. L. (1972) J. Virol. 9, 326–34117. Mehta, P. K., and Christen, P. (2000) Adv. Enzymol. Relat. Areas. Mol. Biol.

74, 129–18418. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic Acids Res. 22,

4673–468019. Page, R. D. (1996) Comput. Appl. Biosci. 12, 357–35820. Kuhner, M. K., and Felsenstein, J. (1994) Mol. Biol. Evol. 11, 459–46821. Adachi, J., and Hasegawa, M. (1996) Computer Science Monographs, Institute

of Statistical Mathematics, Tokyo, Japan 28, 1–15022. Sambrook, J., and Russell, D. W. (2001) Molecular Cloning: a Laboratory

Manual, 3rd Ed., pp. 18.62–18.74, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, NY

23. Soda, K. (1967) Agr. Biol. Chem. 31, 1054–106024. Thompson, J. F., and Morrison, G. R. (1951) Anal. Chem. 23, 1153–115725. Siegel, L. M. (1965) Anal. Biochem. 11, 126–13226. Schneider, D., Jaschkowitz, K., Seidler, A., and Rogner, M. (2000) Indian

J. Biochem. Biophys 37, 441–44627. Jaschkowitz, K., and Seidler, A. (2000) Biochemistry 39, 3416–342328. Laakso, S., and Nurmikko, V. (1976) Anal. Biochem. 72, 600–60529. Kashiwamata, S., and Greenberg, D. M. (1970) Biochim. Biophys. Acta. 212,

488–50030. Starkebaum, G., and Harlan, J. M. (1986) J. Clin. Investig. 77, 1370–137631. Nakayama, T., Esaki, N., Tanaka, H., and Soda, K. (1988) Biochemistry 27,

1587–159132. McKie, A. E., Edlind, T., Walker, J., Mottram, J. C., and Coombs, G. H. (1998)

J. Biol. Chem. 273, 5549–555633. Motoshima, H., Inagaki, K., Kumasaka, T., Furuichi, M., Inoue, H., Tamura,

T., Esaki, N., Soda, K., Tanaka, N., Yamamoto, M., and Tanaka, H. (2000)

J. Biochem. (Tokyo) 128, 349–35434. Kreis, W., and Hession, C. (1973) Cancer Res. 33, 1862–186535. Ito, S., Nakamura, T., and Eguchi, Y. (1976) J. Biochem. (Tokyo) 80,

1327–133436. Zanin, V. A., Lukina, V. I., and Berezov, T. T. (1989) Vopr. Med. Khim. 35,

84–8937. Cuhel, R. L., Taylor, C. D., and Jannasch, H. W. (1981) J. Bacteriol 147,

340–34938. Nakayama, T., Esaki, N., Sugie, K., Beresov, T. T., Tanaka, H., and Soda, K.

(1984) Anal. Biochem. 138, 421–42439. Faleev, N. G., Troitskaya, M. V., Paskonova, E. A., Saporovskaya, M. B., and

Belikov, V. M. (1996) Enzyme and Microbial Technology 19, 590–59340. Dias, B., and Weimer, B. (1998) Appl. Environ Microbiol 64, 3327–333141. Thong, K. W., Coombs, G. H., and Sanderson, B. E. (1987) Mol. Biochem.

Parasitol 23, 223–23142. Lockwood, B. C., and Coombs, G. H. (1991) Biochem. J. 279, 675–68243. Hori, H., Takabayashi, K., Orvis, L., Carson, D. A., and Nobori, T. (1996)

Cancer Res. 56, 2116–212244. Esaki, N., and Soda, K. (1987) Methods Enzymol. 143, 459–46545. Shimizu, H., Yamagata, S., Masui, R., Inoue, Y., Shibata, T., Yokoyama, S.,

Kuramitsu, S., and Iwama, T. (2001) Biochim Biophys Acta. 1549, 61–7246. Johnston, M., Jankowski, D., Marcotte, P., Tanaka, H., Esaki, N., Soda, K.,

and Walsh, C. (1979) Biochemistry 18, 4690–470147. Steegborn, C., Clausen, T., Sondermann, P., Jacob, U., Worbs, M., Marinkovic,

S., Huber, R., and Wahl, M. C. (1999) J. Biol. Chem. 274, 12675–1268448. Upcroft, J. A., and Upcroft, P. (1999) J. Eukaryot. Microbiol 46, 447–44949. Lancero, H., Niu, J., and Johnson, P. W. (1996) J. Dent. Res. 75, 1994–200250. Kapatral, V., Anderson, I., Ivanova, N., Reznik, G., Los, T., Lykidis, A., Bhat-

tacharyya, A., Bartman, A., Gardner, W., Grechkin, G., Zhu, L., Vasieva, O.,Chu, L., Kogan, Y., Chaga, O., Goltsman, E., Bernal, A., Larsen, N.,D’Souza, M., Walunas, T., Pusch, G., Haselkorn, R., Fonstein, M., Kyrpides,N., and Overbeek, R. (2002) J. Bacteriol. 184, 2005–2018

51. Alston, T. A., and Bright, H. J. (1983) Biochem. Pharmacol. 32, 947–95052. Coombs, G. H., and Mottram, J. C. (2001) Antimicrob. Agents Chemother. 45,

1743–174553. Wassmann, C., Hellberg, A., Tannich, E., and Bruchhaus, I. (1999) J. Biol.

Chem. 274, 26051–2605654. Tovar, J., Fischer, A., and Clark, C. G. (1999) Mol. Microbiol. 32, 1013–1021

MGL from E. histolytica 42727

by guest on July 16, 2020http://w

ww

.jbc.org/D

ownloaded from

Masaharu Tokoro, Takashi Asai, Seiki Kobayashi, Tsutomu Takeuchi and Tomoyoshi NozakiFORWARD AND REVERSE TRANS-SULFURATION PATHWAYS

DEGRADATION IN AN ANAEROBIC PARASITIC PROTIST THAT LACKS : A KEY ENZYME OF SULFUR-AMINO ACIDEntamoeba histolytica

-Lyase from γIdentification and Characterization of Two Isoenzymes of Methionine

doi: 10.1074/jbc.M212414200 originally published online August 14, 20032003, 278:42717-42727.J. Biol. Chem. 

  10.1074/jbc.M212414200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/278/43/42717.full.html#ref-list-1

This article cites 49 references, 15 of which can be accessed free at

by guest on July 16, 2020http://w

ww

.jbc.org/D

ownloaded from