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Antimicrobial Agents and Chemotherapy, June 2005, p. 2172-2179, Vol. 49, No. 6
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.6.2172-2179.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

IMP Dehydrogenase from the Protozoan Parasite Toxoplasma gondii

Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202,¹ Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, GA 30602²

Received 17 November 2004/ Returned for modification 26 January 2005/ Accepted 8 February 2005

The opportunistic apicomplexan parasite Toxoplasma gondii damages fetuses in utero and threatens immunocompromised individuals. The toxicity associated with standard antitoxoplasmal therapies, which target the folate pathway, underscores the importance of examining alternative pharmacological strategies. Parasitic protozoa cannot synthesize purines de novo; consequently, targeting purine salvage enzymes is a plausible pharmacological strategy. Several enzymes critical to purine metabolism have been studied in T. gondii, but IMP dehydrogenase (IMPDH), which catalyzes the conversion of IMP to XMP, has yet to be characterized. Thus, we have cloned the gene encoding this enzyme in T. gondii. Northern blot analysis shows that two IMPDH transcripts are present in T. gondii tachyzoites. The larger transcript contains an open reading frame of 1,656 nucleotides whose deduced protein sequence consists of 551 amino acids (TgIMPDH). The shorter transcript is an alternative splice product that generates a 371-amino-acid protein lacking the active-site flap (TgIMPDH-S). When TgIMPDH is expressed as a recombinant protein fused to a FLAG tag, the fusion protein localizes to the parasite cytoplasm. Immunoprecipitation with anti-FLAG was employed to purify recombinant TgIMPDH, which converts IMP to XMP as expected. Mycophenolic acid is an uncompetitive inhibitor relative to NAD⁺, with a intercept inhibition constant (K_ii) of 0.03 ± 0.004 µM. Tiazofurin and its seleno analog were not inhibitory to the purified enzyme, but adenine dinucleotide analogs such as TAD and the nonhydrolyzable ß-methylene derivatives of TAD or SAD were inhibitory, with K_ii values 13- to 60-fold higher than that of mycophenolic acid.