TY - JOUR
T1 - The structural mechanism of GTP stabilized oligomerization and catalytic activation of the Toxoplasma gondii uracil phosphoribosyltransferase
AU - Schumacher, Maria A.
AU - Bashor, Caleb J.
AU - Minsun, Hong Song
AU - Otsu, Kanao
AU - Zhu, Shuren
AU - Parry, Ronald J.
AU - Ullman, Buddy
AU - Brennan, Richard G.
PY - 2002/1/8
Y1 - 2002/1/8
N2 - 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 Toxoplasrna 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.
AB - 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 Toxoplasrna 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.
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U2 - 10.1073/pnas.012399599
DO - 10.1073/pnas.012399599
M3 - Article
C2 - 11773618
AN - SCOPUS:0037039462
SN - 0027-8424
VL - 99
SP - 78
EP - 83
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 1
ER -