title: The structural mechanism of GTP stabilized oligomerization and catalytic activation of the Toxoplasma gondii uracil phosphoribosyltransferase.
abstract: 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 alpha-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-A 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-A 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health and Science University, 3181 Southwest Sam Jackson Park Road, Portland, OR 97201-3098, USA.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Schumacher
authors.forename: Caleb J
authors.initials: CJ
authors.lastname: Bashor
authors.forename: Minsun Hong
authors.initials: MH
authors.lastname: Song
authors.forename: Kanao
authors.initials: K
authors.lastname: Otsu
authors.forename: Shuren
authors.initials: S
authors.lastname: Zhu
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Parry
authors.forename: Buddy
authors.initials: B
authors.lastname: Ullman
authors.forename: Richard G
authors.initials: RG
authors.lastname: Brennan
mesh_terms: D000818:Animals; D002384:Catalysis; D019281:Dimerization; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008027:Light; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010430:Pentosyltransferases / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D013379:Substrate Specificity; D014122:Toxoplasma / Q000201:enzymology*; D014498:Uracil / Q000737:chemistry
chemical_list: D008024:Ligands; D014498:Uracil; D006160:Guanosine Triphosphate; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase