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J. Biol. Chem., Vol. 282, Issue 42, 30804-30816, October 19, 2007

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The Farnesyl-diphosphate/Geranylgeranyl-diphosphate Synthase of Toxoplasma gondii Is a Bifunctional Enzyme and a Molecular Target of Bisphosphonates^*

Yan Ling, Zhu-Hong Li, Kildare Miranda, Eric Oldfield^¶, and Silvia N. J. Moreno¹

From the Department of Pathobiology and ^¶Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 and Department of Cellular Biology and Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia 30602

Farnesyl-diphosphate synthase (FPPS) catalyzes the synthesis of farnesyl diphosphate, an important precursor of sterols, dolichols, ubiquinones, and prenylated proteins. We report the cloning and characterization of two Toxoplasma gondii farnesyl-diphosphate synthase (TgFPPS) homologs. A single genetic locus produces two transcripts, TgFPPS and TgFPPSi, by alternative splicing. Both isoforms were heterologously expressed in Escherichia coli, but only TgFPPS was active. The protein products predicted from the nucleotide sequences have 646 and 605 amino acids and apparent molecular masses of 69.5 and 64.5 kDa, respectively. Several conserved sequence motifs found in other prenyl-diphosphate synthases are present in both TgFPPSs. TgFPPS was also expressed in the baculovirus system and was biochemically characterized. In contrast to the FPPS of other eukaryotic organisms, TgFPPS is bifunctional, catalyzing the formation of both farnesyl diphosphate and geranylgeranyl diphosphate. TgFPPS localizes to the mitochondria, as determined by the co-localisation of the affinity-purified antibodies against the protein with MitoTracker, and in accord with the presence of an N-terminal mitochondria-targeting signal in the protein. This enzyme is an attractive target for drug development, because the order of inhibition of the enzyme by a number of bisphosphonates is the same as that for inhibition of parasite growth. In summary, we report the first bifunctional farnesyl-diphosphate/geranylgeranyl-diphosphate synthase identified in eukaryotes, which, together with previous results, establishes this enzyme as a valid target for the chemotherapy of toxoplasmosis.

Received for publication, April 16, 2007 , and in revised form, July 27, 2007.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY196327 and DQ630749.

^* This work was supported by a Burroughs Welcome New Investigator award (to S. N. J. M.) and National Institutes of Health Grants AI68467 (to S. N. J. M.) and GM65307 (to E. O.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ To whom correspondence should be addressed: Center for Tropical and Emerging Global Diseases and Dept. of Cellular Biology, 500 DW Brooks Dr., Paul D. Coverdell Center for Biomedical and Health Sciences, University of Georgia, Athens, GA 30602. Tel.: 706-542-4736; Fax: 706-583-0181; E-mail: smoreno{at}cb.uga.edu.

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