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Proc Natl Acad Sci U S A. 2002 Jan 8;99(1):78-83. Epub 2002 Jan 2.

The structural mechanism of GTP stabilized oligomerization and catalytic activation of the Toxoplasma gondii uracil phosphoribosyltransferase.

Author information

1
Department of Biochemistry and Molecular Biology, Oregon Health and Science University, 3181 Southwest Sam Jackson Park Road, Portland, OR 97201-3098, USA.

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.

PMID:
11773618
PMCID:
PMC117517
DOI:
10.1073/pnas.012399599
[Indexed for MEDLINE]
Free PMC Article

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