Structural and kinetic characterization of quinolinate phosphoribosyltransferase (hQPRTase) from homo sapiens.

Human quinolinate phosphoribosyltransferase (EC 2.4.2.19) (hQPRTase) is a member of the type II phosphoribosyltransferase family involved in the catabolism of quinolinic acid (QA). It catalyses the formation of nicotinic acid mononucleotide from quinolinic acid, which involves a phosphoribosyl transfer reaction followed by decarboxylation. hQPRTase has been implicated in a number of neurological conditions and in order to study it further, we have carried out structural and kinetic studies on recombinant hQPRTase. The structure of the fully active enzyme overexpressed in Escherichia coli was solved using multiwavelength methods to a resolution of 2.0 A. hQPRTase has a alpha/beta barrel fold sharing a similar overall structure with the bacterial QPRTases. The active site of hQPRTase is located at an alpha/beta open sandwich structure that serves as a cup for the alpha/beta barrel of the adjacent subunit with a QA binding site consisting of three arginine residues (R102, R138 and R161) and two lysine residues (K139 and K171). Mutation of these residues affected substrate binding or abolished the enzymatic activity. The kinetics of the human enzyme are different to the bacterial enzymes studied, hQPRTase is inhibited competitively and non-competitively by one of its substrates, 5-phosphoribosylpyrophosphate (PRPP). The human enzyme adopts a hexameric arrangement, which places the active sites in close proximity to each other.

[1]  S. Eom,et al.  Crystal structure of quinolinic acid phosphoribosyltransferase from Helicobacter pylori , 2006, Proteins.

[2]  S. Doublié.  [29] Preparation of selenomethionyl proteins for phase determination. , 1997, Methods in enzymology.

[3]  Hughes,et al.  The Salmonella typhimurium nadC gene: sequence determination by use of Mud-P22 and purification of quinolinate phosphoribosyltransferase , 1993, Journal of bacteriology.

[4]  A. Rougier,et al.  Quinolinic‐Phosphoribosyl Transferase Activity is Decreased in Epileptic Human Brain Tissue , 1988, Epilepsia.

[5]  David C. Richardson,et al.  MOLPROBITY: structure validation and all-atom contact analysis for nucleic acids and their complexes , 2004, Nucleic Acids Res..

[6]  R. Schwarcz,et al.  Characterization of Quinolinic Acid Phosphoribosyltransferase in Human Blood and Observations in Huntington's Disease , 1985, Journal of neurochemistry.

[7]  C. Grubmeyer,et al.  Quinolinate phosphoribosyltransferase: kinetic mechanism for a type II PRTase. , 2002, Biochemistry.

[8]  M. Hørder,et al.  An improved PCR-based method for site directed mutagenesis using megaprimers. , 1998, Molecular and cellular probes.

[9]  J C Sacchettini,et al.  A new function for a common fold: the crystal structure of quinolinic acid phosphoribosyltransferase. , 1997, Structure.

[10]  K. Iwai,et al.  Distribution of quinolinate phosphoribosyl-transferase in animals, plants and microorganisms. , 1973, Journal of nutritional science and vitaminology.

[11]  K. Calvo,et al.  The sequencing expression, purification, and steady-state kinetic analysis of quinolinate phosphoribosyl transferase from Escherichia coli. , 1996, Archives of biochemistry and biophysics.

[12]  K. Iwai,et al.  Purification and crystallization of quinolinate phosphoribosyltransferase from hog liver. , 1974, Biochemical and biophysical research communications.

[13]  K. Shibata,et al.  Characterization and functional expression of the cDNA encoding human brain quinolinate phosphoribosyltransferase. , 1998, Biochimica et biophysica acta.

[14]  R. Schwarcz,et al.  Quinolinic acid metabolism in the rat brain. Immunohistochemical identification of 3-hydroxyanthranilic acid oxygenase and quinolinic acid phosphoribosyltransferase in the hippocampal region , 1988, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[15]  James C. Sacchettini,et al.  The crystal structure of human hypoxanthine-guanine phosphoribosyltransferase with bound GMP , 1994, Cell.

[16]  R. Schwarcz,et al.  Quinolinic acid phosphoribosyltransferase in human and rat brain: Activity in Huntington's disease and in quinolinate-lesioned rat striatum , 1985, Brain Research.

[17]  Kim Henrick,et al.  Detection of Protein Assemblies in Crystals , 2005, CompLife.

[18]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[19]  K. Calvo,et al.  Inhibition of quinolinate phosphoribosyl transferase by pyridine analogs of quinolinic acid. , 1988, Biochemical and biophysical research communications.

[20]  S. Larsen,et al.  Structural basis for the function of Bacillus subtilis phosphoribosyl-pyrophosphate synthetase , 2000, Nature Structural Biology.

[21]  L. DeLucas,et al.  Crystallization and preliminary crystallographic investigation of porcine quinolinate phosphoribosyltransferase. , 1998, Acta crystallographica. Section D, Biological crystallography.

[22]  D. Roos,et al.  Crystal structures of Toxoplasma gondii uracil phosphoribosyltransferase reveal the atomic basis of pyrimidine discrimination and prodrug binding , 1998, The EMBO journal.

[23]  J. L. Smith,et al.  Glutamine PRPP amidotransferase: snapshots of an enzyme in action. , 1998, Current opinion in structural biology.

[24]  R. Schwarcz,et al.  Quinolinic acid phosphoribosyltransferase: purification and partial characterization from human liver and brain. , 1988, Journal of biochemistry.

[25]  Slawomir K. Grzechnik,et al.  Crystal structure of a type II quinolic acid phosphoribosyltransferase (TM1645) from Thermotoga maritima at 2.50 Å resolution , 2004, Proteins.

[26]  S. J. Sinclair,et al.  Molecular characterization of quinolinate phosphoribosyltransferase (QPRTase) in Nicotiana , 2000, Plant Molecular Biology.

[27]  C. Grubmeyer,et al.  Transition state structure of Salmonella typhimurium orotate phosphoribosyltransferase. , 1996, Biochemistry.

[28]  R. Schwarcz,et al.  Quinolinic Acid Phosphoribosyltransferase in Rat Brain , 1985, Journal of neurochemistry.

[29]  K. Iwai,et al.  Crystallization and properties of quinolinate phosphoribosyltransferase from hog liver. , 1980, Methods in enzymology.

[30]  George M Sheldrick,et al.  Substructure solution with SHELXD. , 2002, Acta crystallographica. Section D, Biological crystallography.

[31]  Janet L. Smith.  Forming and inhibiting PRT active sites , 1999, Nature Structural Biology.

[32]  C. Grubmeyer,et al.  Crystal structure of orotate phosphoribosyltransferase. , 1994, Biochemistry.

[33]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[34]  P. Loll,et al.  The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1 , 1994, Nature.

[35]  J C Sacchettini,et al.  Crystal structure of quinolinic acid phosphoribosyltransferase from Mmycobacterium tuberculosis: a potential TB drug target. , 1998, Structure.

[36]  R. Schwarcz,et al.  Purification of quinolinic acid phosphoribosyltransferase from rat liver and brain. , 1985, Biochimica et biophysica acta.

[37]  R. Schwarcz,et al.  Increased quinolinic acid metabolism following neuronal degeneration in the rat hippocampus , 1987, Brain Research.