Abstract
Crystal structures of substrate-free and XMP-soaked hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) of the opportunistic pathogen Toxoplasma gondii have been determined to 2.4 and 2.9 Å resolution, respectively. HGXPRTase displays the conserved PRTase fold. In the structure of the enzyme bound to its product, a long flexible loop (residues 115–126) is located away from the active site. Comparison to the substrate-free structure reveals a striking relocation of the loop, which is poised to cover the catalytic pocket, thus providing a mechanism by which the HG(X)PRTases shield their oxocarbonium transition states from nucleophilic attack by the bulk solvent. The conserved Ser 117-Tyr 118 dipeptide within the loop is brought to the active site, completing the ensemble of catalytic residues.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Frenkel, J.K. in The Cocddia (ed. D.M. Hammond) 343–410 (Baltimore University Press, 1973).
McAuley, J. et al. Early and longitudinal evaluations of treated infants and children and untreated historical patients with congenital toxoplasmosis: the Chicago Collaborative Treatment Trial. Clinical Infectious Diseases 19, 38–72 (1994).
Daffos, F. et al. Toxoplasma in pregnancy. Lancet 344, 54–61 (1994).
Luft, B.J. & Remington, J.S. Toxoplasmic encephalitis in AIDS. Clinical Infectious Diseases 15, 211–222 (1992).
Mariuz, P., Bosler, E.M. & Luft, B.J. Toxoplasmosis in individuals with AIDS. Infectious Disease Clinics of North America 8, 365–381 (1994).
Leport, C. et al. Treatment of central nervous system toxoplasmosis with pyrimethamine/sulfadiazine combination in 35 patients with the Acquired Immunodeficiency Syndrome. Amer. J. Med. 84, 94–100 (1987).
Fernandez-Martin, J. et al. Pyrimethamine-clarithromycin combination for therapy of acute toxoplasma encephalitis in patients with AIDS. Antimicrobial Agents and Chemotherapy 35, 2049–2052 (1991).
Tenant-Flowers, M. Sulfadiazine desensitization in patients with AIDS and cerebral toxoplasmosis. AIDS 5, 311–315 (1991).
Laughon, B.E. et al. Summary of the workshop on future directions in discovery and development of therapeutic agents for opportunistic infections associated with AIDS. J. Infect. Dis. 164, 244–251 (1991).
Dannemann, B. et al. Treatment of toxoplasmic encephalitis in patients with AIDS. A randomized trial comparing pyrimethamine plus clindamycin to pyrimethamine and sulfadiazine. Annals of Internal Medicine 116, 33–43 (1992).
Ullman, B. & Allen, T.E. Hypoxanthine-guanine phosphoribosyl-transferase in trypanosomatids: a rational target for antiparasitic chemotherapy. Molecular Approaches to Parasitology. (Ed. John Boothroyd) 123–141 (Wiley-Liss Division, 1995).
Musick, D.L. Structural features of the phosphoribosyltransferases and their relationship to the human deficiency disorders of purine and pyrimidine metabolism. CRC Crit. Rev. Biochem. 11, 1–33 (1981).
Argos, P., Hanei, M., Wilson, J.M. & Kelley, W.N. A possible nucleotide-binding domain in the tertiary fold of phosphoribosyltransferases. J. Biol. Chem. 258, 6450–6457 (1983).
Smith, J.L. Enzymes of nucleotide synthesis. Curr. Opin. Struct. Biol. 5, 752–757 (1995).
Donald, R.G.K., Carter, D., Ullman, B. & Roos, D.S. Insertional tagging, cloning and expression of the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase gene: characterization of a new genetic marker for stable transformation. J. Biol. Chem. 271, 14010–14019 (1996).
Giacomello, A. & Salerno, C. Human hypoxanthine-guanine phosphoribosyltransferase. J. Biol. Chem. 253, 6038–6044 (1978).
Yuan, L. Craig III, S.P., McKerrow, J.H. & Wang, C.C. Steady-state kinetics of the schistosomal hypoxanthine-guanine phosphoribosyltransferase. Biochemistry 31, 806–810 (1992).
Goitein, R.K., Chelsky, D. & Parsons, S.M. Primary 14C and α secondary 3H substrate kinetic isotope effects for some phosphoribosyl-transferases. J. Biol. Chem. 253, 2963–2971 (1978).
Tao, W., Grubmeyer, C. & Blanchard, J.S. Transition state structure of Salmonella typhimurium orotate phosphoribosyltransferase. Biochemistry 35, 14–21 (1996).
Eads, J.C., Scapin, G., Xu, Y., Grubmeyer, C. & Sacchettini, J.C. The crystal structure of human hypoxanthine-guanine phosphoribosyl-transferase with bound GMP. Cell 78, 325–334 (1994).
Somoza, J.R., Chin, M.S., Focia, P.J., Wang, C.C. & Fletterick, R.J. Crystal structure of the hypoxanthine-guanine-xanthine phosphoribosyltransferase from the protozoan parasite Tritrichomonas foetus . Biochemistry 35, 7032–7040 (1996).
Tronrud, D.E., Ten Eyck, L.F. & Matthews, B.W. An efficient general purpose least-squares refinement program for macromolecular structures. Acta crystallogr. A43, 489–501 (1985).
Laskowski, R.A., MacArthur, M.W. & Thorton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993).
Johnson, G.G., Eisenberg, L.R. & Migeon, B.R. Human and mouse hypoxanthine-guanine phosphoribosyltransferase: dinners and tetramers. Science 203, 174–176 (1979).
Ullman, B. & Carter, D. Hypoxanthine-guanine phosphoribosyl-transferase as a therapeutic target in protozoal infections. Infectious Agents and Disease 4, 29–40 (1995).
Wilson, J.M. et al. A molecular survey of hypoxanthine-guanine phosphoribosyltransferase deficiency in man. J. Clin. Invest. 77, 188–195 (1986).
Henriksen, A., Aghajari, N., Jensen, K.F. & Gajhede, M. A flexible loop at the dimer interface is a part of the active site of the adjacent monomer of Escherichia coli orotate phosphoribosyltransferase. Biochemistry 35, 3803–3809 (1996).
Parry, R.J. & Haridas, K. Synthesis of 1-α-pyrophosphoryl-2α,3α- dihydroxy-4-β-cyclopentanemethanol-5-phosphate. Tetrahedron Lett. 34, 7013–7016 (1993).
Kim, J.H. et al. (1995). A stable carbocyclic analog of 5-phosphoribosyl-1-pyrophosphate to probe the mechanism of catalysis and regulation of glutamine phosphoribosylpyrophosphate amidotransferase. J. Biol. Chem. 270, 17394–17399 (1995).
Smith, J.L. et al. Structure of the allosteric regulatory enzyme of purine biosynthesis. Science 264, 1427–1433 (1994).
Scapin, G., Ozturk, D.H., Grubmeyer, C. & Sacchettini, J.C. The crystal structure of the orotate phosphoribosyltransferase complexed with orotate and α-D-phosphoribosyl-1-pyrophosphate. Biochemistry 34, 10744–10754 (1995).
Cohen, F.C., Blaney, J.M., Humblet, C., Gund, P. & Barry, D.C. Molecular software and methods for medicinal chemistry. J. Med. Chem. 33, 883–894 (1995).
Roos, D.S., Donald, R.G.K., Morrissette, N.S. & Moulton, A.L.C. Molecular tools for genetic dissection of the protozoan parasite Toxoplasma gondii . Meths. Cell Biol. 45, 27–63 (1994).
Xuong, N.H., Nielsen, C., Hamlin, R. & Anderson, D.J. Strategy for data collection from protein crystals using a multiwire counter area detector diffractometer. J. Appl. Crystallogr. 18, 342–350 (1985).
LeMaster, D.M. & Richards, F.M. 1H-15N heteronuclear NMR studies of Escherichia coli thioredoxin in samples isotopically labeled by residue type. Biochemistry 24, 7263–7268 (1985).
Terwilliger, T.C. & Eisenberg, D. Unbiased three-dimensional refinement of heavy atom parameters by correlation of origin-removed patterson functions. Acta Crystallogr. A39, 813–817 (1983).
Jones, T.A. Interactive computer program graphics: FRODO. Meths. Enzymol. B115, 157–171 (1985).
Kraulis, P.J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Cryst 24, 946–950 (1991).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Schumacher, M., Carter, D., Roos, D. et al. Crystal structures of Toxoplasma gondii HGXPRTase reveal the catalytic role of a long flexible loop. Nat Struct Mol Biol 3, 881–887 (1996). https://doi.org/10.1038/nsb1096-881
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/nsb1096-881
This article is cited by
-
The biosynthetic pathway of 2-azahypoxanthine in fairy-ring forming fungus
Scientific Reports (2016)
-
The importance of recycling
Nature Structural & Molecular Biology (1996)