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J. Biol. Chem., Vol. 278, Issue 43, 42717-42727, October 24, 2003

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Identification and Characterization of Two Isoenzymes of Methionine -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, 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-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 -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 -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.

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

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

^* 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.

^|| 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.

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