Crystal structures of Toxoplasma gondii uracil phosphoribosyltransferase reveal the atomic basis of pyrimidine discrimination and prodrug binding.
Uracil phosphoribosyltransferase (UPRTase) catalyzes the transfer of a ribosyl phosphate group from alpha-D-5-phosphoribosyl-1-pyrophosphate to the N1 nitrogen of uracil. The UPRTase from the opportunistic pathogen Toxoplasma gondii is a rational target for antiparasitic drug design. To aid in structure-based drug design studies against toxoplasmosis, the crystal structures of the T.gondii apo UPRTase (1.93 A resolution), the UPRTase bound to its substrate, uracil (2.2 A resolution), its product, UMP (2.5 A resolution), and the prodrug, 5-fluorouracil (2.3 A resolution), have been determined. These structures reveal that UPRTase recognizes uracil through polypeptide backbone hydrogen bonds to the uracil exocyclic O2 and endocyclic N3 atoms and a backbone-water-exocyclic O4 oxygen hydrogen bond. This stereochemical arrangement and the architecture of the uracil-binding pocket reveal why cytosine and pyrimidines with exocyclic substituents at ring position 5 larger than fluorine, including thymine, cannot bind to the enzyme. Strikingly, the T. gondii UPRTase contains a 22 residue insertion within the conserved PRTase fold that forms an extended antiparallel beta-arm. Leu92, at the tip of this arm, functions to cap the active site of its dimer mate, thereby inhibiting the escape of the substrate-binding water molecule.
Schumacher
M A
MA
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, OR 97201-3098, USA.
Carter
D
D
Scott
D M
DM
Roos
D S
DS
Ullman
B
B
Brennan
R G
RG
D000595:Amino Acid Sequence; D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D019281:Dimerization; D015195:Drug Design; D005472:Fluorouracil / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010430:Pentosyltransferases / Q000737:chemistry*; D011355:Prodrugs / Q000378:metabolism; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000737:chemistry*; D011743:Pyrimidines / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014122:Toxoplasma / Q000201:enzymology*; D014542:Uridine Monophosphate / Q000378:metabolism
D000963:Antimetabolites; D011355:Prodrugs; D015800:Protozoan Proteins; D011743:Pyrimidines; D014542:Uridine Monophosphate; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase; C030986:pyrimidine; D005472:Fluorouracil