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 ␣-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.

The competence gene, comF, from Synechocystis sp. strain PCC 6803 is involved in natural transformation, phototactic motility and piliation

“…We propose to designate this gene comF despite only moderate sequence identity to the ComF proteins of B. subtilis, H. influenzae, P. stutzeri and S. pneumoniae, used in its identification. The ComF proteins, including Slr0388, harbour a PRT domain, which is a highly conserved region in the PRT family of enzymes involved in the biosynthesis and salvage pathways of purines, pyrimidines and pyridines in bacteria and lower eukaryotes Schumacher et al, 2002). This domain is unique to this family of proteins, and is not found in other PRTases of the tryptophan, histidine and nicotinamide synthetic and salvage pathways (InterPro identifier IPR000836).…”

“…adenine) Craig & Eakin, 2000). Apart from this conserved region, the PRT family of enzymes have low sequence similarity, ranging from 20 to 45 % (Schumacher et al, 2002). However, they also possess other regions that confer substrate selectivity and catalytic stability, which may correspond to the conserved region between residues 127 and 147 of the ComF group of proteins ( Fig.…”

Substrate Inhibition of Uracil Phosphoribosyltransferase by Uracil Can Account for the Uracil Growth Sensitivity of Leishmania donovani Pyrimidine Auxotrophs

“…Kinetic parameters to the naturally occurring substrates, as well as to several important uracil analogs, were determined, and the L. donovani enzyme, unlike its T. gondii counterpart (17), was shown to form a stable tetramer in the absence of GTP. Furthermore, profound substrate inhibition of LdUPRT to nucleobase substrates was demonstrated, providing a mechanism by which L. donovani, T. gondii, T. cruzi, or T. brucei, genetically deficient in pyrimidine biosynthesis, would exhibit a dramatic growth sensitivity to exogenous uracil.…”

Identification and characterization of human uracil phosphoribosyltransferase (UPRTase)

“…The UPRTases from different organisms differ in their regulatory behavior. UPRTase is activated by GTP in several organisms, for example E. coli (Jensen and Mygind 1996), G. intestinalis (Martinussen and Hammer 1994), T. gondii (Schumacher et al 2002), S. shibatae (Linde and Jensen 1996), and S. solfataricus (Jensen et al 2005), whereas its activity seems unregulated in other organisms, for example yeast (Natalini et al 1979) and Bacillus caldolyticus (Kadziola et al 2002). It has been shown that CTP allosterically inhibits S. shibatae (Linde and Jensen 1996), S. solfataricus (Jensen et al 2005), and yeast UPRTase activity whereas UMP, dUMP, dCMP, UDP, TTP, and dCTP allosterically inhibit yeast UPRTase activity (Natalini et al 1979).…”

“…By studying the mechanism of activation of UPRTase activity by GTP it was found that UPRTase has two forms, dimeric and tetrameric. The tetrameric form is stabilized by PRPP and/or GTP, both of which contact two dimers of the tetramer; for this reason the stimulatory effect of GTP is seen at subsaturating concentrations of PRPP only (Schumacher et al 2002).…”