The structural mechanism of GTP stabilized oligomerization and catalytic activation of the Toxoplasma gondii uracil phosphoribosyltransferase.
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.
Schumacher
Maria A
MA
Department of Biochemistry and Molecular Biology, Oregon Health and Science University, 3181 Southwest Sam Jackson Park Road, Portland, OR 97201-3098, USA.
Bashor
Caleb J
CJ
Song
Minsun Hong
MH
Otsu
Kanao
K
Zhu
Shuren
S
Parry
Ronald J
RJ
Ullman
Buddy
B
Brennan
Richard G
RG
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
D008024:Ligands; D014498:Uracil; D006160:Guanosine Triphosphate; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase