The Structural Mechanism of GTP Stabilized Oligomerization and Catalytic Activation of the Toxoplasma gondii Uracil Phosphoribosyltransferase

• Published in: Proceedings of the National Academy of Sciences - PNAS 2002-01, Vol.99 (1), p.78-83
• Main Authors: Schumacher, Maria A., Bashor, Caleb J., Song, Minsun Hong, Otsu, Kanao, Zhu, Shuren, Parry, Ronald J., Ullman, Buddy, Brennan, Richard G.
• Format: Article
• Language: English
• Subjects: Active sites; Animals; Atoms; Biochemistry; Biological Sciences; Biology; Catalysis; Chemistry; Dimerization; Dimers; Enzymes; GTP; Guanosine Triphosphate - chemistry; Guanosine Triphosphate - metabolism; Kinetics; Ligands; Light; Models, Chemical; Models, Molecular; Molecular structure; Molecules; Mutagenesis, Site-Directed; Parasites; Pentosyltransferases - chemistry; Phosphates; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Scattering, Radiation; Substrate Specificity; Toxoplasma - enzymology; Toxoplasma gondii; Uracil - chemistry
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.012399599

Uracil phosphoribosyltransferase (UPRT) is a member of a large family of salvage and biosynthetic enzymes, the phosphoribosyltransferases, and catalyzes the transfer of ribose 5-phosphate from α-D-5-phosphoribosyl-1-pyrophosphate (PRPP) to the N1 nitrogen of uracil. The UPRT from the opportunistic pathogen Toxoplasma gondii represents a promising target for rational drug design, because it can create intracellular, lethal nucleotides from subversive substrates. However, the development of such compounds requires a detailed understanding of the catalytic mechanism. Toward this end we determined the crystal structure of the T. gondii UPRT bound to uracil and cPRPP, a nonhydrolyzable PRPP analogue, to 2.5-Å resolution. The structure suggests that the catalytic mechanism is substrate-assisted, and a tetramer would be the more active oligomeric form of the enzyme. Subsequent biochemical studies revealed that GTP binding, which has been suggested to play a role in catalysis by other UPRTs, causes a 6-fold activation of the T. gondii enzyme and strikingly stabilizes the tetramer form. The basis for stabilization was revealed in the 2.45-Å resolution structure of the UPRT-GTP complex, whereby residues from three subunits contributed to GTP binding. Thus, our studies reveal an allosteric mechanism involving nucleotide stabilization of a more active, higher order oligomer. Such regulation of UPRT could play a role in the balance of purine and pyrimidine nucleotide pools in the cell.