Characterization of protein geranylgeranyltransferase I from the enteric protist Entamoeba histolytica

Entamoeba histolytica is a unique protozoan parasite possessing both protein farnesyltransferase and geranylgeranyltrasferase I (GGT-I) for isoprenylation of small GTPases. In this study, we demonstrated unique enzymological properties of the amebic GGT-I (EhGGT-I), including substrate specificity and insensitivity to known mammalian inhibitors. Some of important residues of the catalytic β subunit implicated in the specificity for GTPase acceptors and prenyl donors are substituted in EhGGT-I. Recombinant α and β subunits of EhGGT-I, co-expressed in Escherichia coli, showed activity to transfer geranylgeranyl to both human wild-type (CVLS) and mutant (CVLL) H-Ras, while the mammalian GGT-I geranylgeranylated, but not farnesylated, only mutant H-Ras. All the representative amebic Ras and Rho/Rac small GTPases with phenylalanine, leucine, methionine, or alanine terminus were preferentially geranylgeranylated by EhGGT-I. This indicates that the acceptor specificity of the amebic GGT-I is remarkably broader than that of its mammalian counterpart. In contrast to EhFT, which farnesylates but not geranylgeranlylates solely EhRas4-CVVA, EhGGT-I also showed significant farnesyltransferase activity against Ras GTPase acceptors. EhGGT-I showed remarkable resistance to peptidomimetics known to inhibit mammalian GGT-I. Together with our previous observation that this parasite does not appear to depend on farnesylation for a majority of Ras and Rho/Rac, these data indicate that biological and biochemical advantages leading to the evolutional selection of this isoprenyl modification must exist uniquely in this parasitic protist. Finally, remarkable biochemical differences in binding to substrates and inhibitors between amebic and mammalian GGT-I highlight this enzyme as an attractive target for the development of new chemotherapeutics against amebiasis.