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