Identification and Characterization of Two Isoenzymes of Methionine γ-Lyase from Entamoeba histolytica
2003; Elsevier BV; Volume: 278; Issue: 43 Linguagem: Inglês
10.1074/jbc.m212414200
ISSN1083-351X
AutoresMasaharu Tokoro, Takashi Asai, Seiki Kobayashi, Tsutomu Takeuchi, Tomoyoshi Nozaki,
Tópico(s)Amoebic Infections and Treatments
ResumoTo 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 γ-lyase (EC 4.4.1.11, 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 α-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. 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 γ-lyase (EC 4.4.1.11, 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 α-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. Entamoeba histolytica is a causative agent of amebiasis, which annually affects an estimated 48 million people and results in 70,000 deaths (1Behbehani K. Bull World Health Organ. 1998; 76: 64-67PubMed Google Scholar). 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 (2Ravdin, J. I. (2000) AMEBIASIS Series on Tropical Medicine and Practice, Vol. 2, pp. 1-45 Imperial College Press, Covent Garden, London, UKGoogle Scholar). However, a recent demonstration of genes encoding mitochondrial proteins, i.e. cpn60 and pyridine nucleotide transhydrogenase (3Clark C.G. Roger A.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6518-6521Crossref PubMed Scopus (236) Google Scholar), together with electron micrographic demonstration of the rough endoplasmic reticulum and the Golgi apparatus (4Mazzuco A. Benchimol M. De Souza W. Micron. 1997; 28: 241-247Crossref PubMed Scopus (45) Google Scholar), suggested the presence of a residual organelle of mitochondria (called crypton or mitosome) (5Mai Z. Ghosh S. Frisardi M. Rosenthal B. Rogers R. Samuelson J. Mol. Cell. Biol. 1999; 19: 2198-2205Crossref PubMed Scopus (153) Google Scholar, 54Tovar J. Fischer A. Clark C.G. Mol. Microbiol. 1999; 32: 1013-1021Crossref PubMed Scopus (293) Google Scholar) and also indicated that this group of parasitic protists possess a unique organelle organization. This parasite also reveals numerous unusual aspects in its metabolism (6Müller M. Biosystems. 1992; 28: 33-40Crossref PubMed Scopus (43) Google Scholar), highlighted by the lack of the tricarboxylic acid cycle (7Reeves R.E. Adv. Parasitol. 1984; 23: 105-142Crossref PubMed Scopus (163) Google Scholar) and glutathione metabolism (8Fahey R.C. Newton G.L. Arrick B. Overdank-Bogart T. Aley S.B. Science. 1984; 224: 70-72Crossref PubMed Scopus (162) Google Scholar). In addition, recent studies suggesting the horizontal transfer of genes encoding a variety of fermentation enzymes from bacteria (9Rosenthal B. Mai Z. Caplivski D. Ghosh S. de la Vega H. Graf T. Samuelson J. J. Bacteriol. 1997; 179: 3736-3745Crossref PubMed Scopus (94) Google Scholar), and genes encoding malic enzyme and acetyl-CoA synthase from the Archaea (10Field J. Rosenthal B. Samuelson J. Mol. Microbiol. 2000; 38: 446-455Crossref PubMed Scopus (72) Google Scholar) 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 (11Diamond L.S. Harlow D.R. Cunnick C.C. Trans. R. Soc. Trop. Med. Hyg. 1978; 72: 431-432Abstract Full Text PDF PubMed Scopus (1565) Google Scholar, 12Gillin F.D. Diamond L.S. J. Protozool. 1980; 27: 474-478Crossref PubMed Scopus (31) Google Scholar). Sulfur-containing amino acid metabolism varies among organisms (Fig. 1, also reviewed in Ref. 13Walker J. Barrett J. Int. J. Parasitol. 1997; 27: 883-897Crossref PubMed Scopus (34) Google Scholar). 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) 1The abbreviations used are: OAS, O-acetylserine; TFMET, trifluoromethionine; TIGR, The Institute for Genomic Research; SAT, serine acetyltransferase; CS, cysteine synthase; MGL, methionine γ-lyase; PPG, dl-propargylglycine; PLP, pyridoxal 5′-phosphate; CGL, cystathionine γ-lyase; CGS, cystathionine γ-synthase; CBL, cystathionine β-lyase; CBS, cystathionine β-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.1The abbreviations used are: OAS, O-acetylserine; TFMET, trifluoromethionine; TIGR, The Institute for Genomic Research; SAT, serine acetyltransferase; CS, cysteine synthase; MGL, methionine γ-lyase; PPG, dl-propargylglycine; PLP, pyridoxal 5′-phosphate; CGL, cystathionine γ-lyase; CGS, cystathionine γ-synthase; CBL, cystathionine β-lyase; CBS, cystathionine β-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. 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 (14Nozaki T. Asai T. Kobayashi S. Ikegami F. Noji M. Saito K. Takeuchi T. Mol. Biochem. Parasitol. 1998; 97: 33-44Crossref PubMed Scopus (82) Google Scholar, 15Nozaki T. Asai T. Sanchez L.B. Kobayashi S. Nakazawa M. Takeuchi T. J. Biol. Chem. 1999; 274: 32445-32452Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). We have also demonstrated (15Nozaki T. Asai T. Sanchez L.B. Kobayashi S. Nakazawa M. Takeuchi T. J. Biol. Chem. 1999; 274: 32445-32452Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) 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 (8Fahey R.C. Newton G.L. Arrick B. Overdank-Bogart T. Aley S.B. Science. 1984; 224: 70-72Crossref PubMed Scopus (162) Google Scholar). 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 γ-lyase (MGL; EC 4.4.1.11) 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 (10Field J. Rosenthal B. Samuelson J. Mol. Microbiol. 2000; 38: 446-455Crossref PubMed Scopus (72) Google Scholar). 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. 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 (16Diamond L.S. Mattern C.F. Bartgis I.L. J. Virol. 1972; 9: 326-341Crossref PubMed Google Scholar) were maintained axenically in Diamond's BI-S-33 medium (11Diamond L.S. Harlow D.R. Cunnick C.C. Trans. R. Soc. Trop. Med. Hyg. 1978; 72: 431-432Abstract Full Text PDF PubMed Scopus (1565) Google Scholar) 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 × 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 γ-subfamily (α-family) of PLP enzymes (for the classification of PLP enzymes used in this study, see Ref. 17Mehta P.K. Christen P. Adv. Enzymol. Relat. Areas. Mol. Biol. 2000; 74: 129-184PubMed Google Scholar), i.e. cystathionine γ-lyase (CGL), cystathionine γ-synthase (CGS), and cystathionine β-lyase (CBL) from a variety of organisms. We also searched for amebic orthologs that belong to the β-family of PLP enzymes using the PLP-attachment site from CS of E. histolytica and cystathionine β-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 (14Nozaki T. Asai T. Kobayashi S. Ikegami F. Noji M. Saito K. Takeuchi T. Mol. Biochem. Parasitol. 1998; 97: 33-44Crossref PubMed Scopus (82) Google Scholar) 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-β-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 γ-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 (18Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55409) Google Scholar) 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 γ-subfamily of PLP enzymes from various organisms with two EhCSs (β-family of PLP enzymes) as the outgroup was drawn by Tree View ver.1.6.0 (19Page R.D. Comput. Appl. Biosci. 1996; 12: 357-358PubMed Google Scholar). 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. 20Kuhner M.K. Felsenstein J. Mol. Biol. Evol. 1994; 11: 459-468PubMed Google Scholar) and ProtML (MOLPHY version 2.3, Ref. 21Adachi J. Hasegawa M. Computer Science Monographs, Institute of Statistical Mathematics. 28. Tokyo, Japan1996: 1-150Google Scholar), respectively. Subcellular Fractionation of the Crude Extract—The lysate of ∼3 × 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 × 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 × 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 A 280 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 × 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 A 280 (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 (22Sambrook J. Russell D.W. Molecular Cloning: a Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001: 18.62-18.74Google Scholar). 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 α-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 α-keto acid assay was performed as described (23Soda K. Agr. Biol. Chem. 1967; 31: 1054-1060Crossref Scopus (58) Google Scholar). The MGL reaction was terminated by adding 25 μl of 50% trichloroacetic acid. After the proteins were precipitated by centrifugation at 14,000 × 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 A 320 was measured. Pyruvic acid and 2-α-butyric acid were used as standards. For the detection of ammonia, the nitrogen assay (24Thompson J.F. Morrison G.R. Anal. Chem. 1951; 23: 1153-1157Crossref Scopus (101) Google Scholar) was used. A 50-μl sample of the supernatant, as for the α-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 A 440 was measured. Ammonium sulfate was used as a standard. The hydrogen sulfide assay was performed as described (25Siegel L.M. Anal. Biochem. 1965; 11: 126-132Crossref PubMed Scopus (370) Google Scholar, 26Schneider D. Jaschkowitz K. Seidler A. Rogner M. Indian J. Biochem. Biophys. 2000; 37: 441-446PubMed Google Scholar, 27Jaschkowitz K. Seidler A. Biochemistry. 2000; 39: 3416-3423Crossref PubMed Scopus (40) Google Scholar). 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 × 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 (28Laakso S. Nurmikko V. Anal. Biochem. 1976; 72: 600-605Crossref PubMed Scopus (23) Google Scholar) 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 A 412 was measured. l-cysteine was used as a standard. The cysteine and cystathionine assay was performed as described (29Kashiwamata S. Greenberg D.M. Biochim. Biophys. Acta. 1970; 212: 488-500Crossref PubMed Scopus (104) Google Scholar). 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 A 455 was measured. Cysteine concentrations were determined using the same protocol except for the measurement of absorbance at A 560. 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 α-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 × 104 cells per well (1 × 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 A 445 was measured with that at A 595 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 × 103 to 2.0 × 105 cells/ml). 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 γ-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 β-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 (14Nozaki T. Asai T. Kobayashi S. Ikegami F. Noji M. Saito K. Takeuchi T. Mol. Biochem. Parasitol. 1998; 97: 33-44Crossref PubMed Scopus (82) Google Scholar), 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 γ-subfamily of PLP enzymes, and lacks the β-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 s
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