Crystal structures of Toxoplasma gondii uracil phosphoribosyltransferase reveal the atomic basis of pyrimidine discrimination and prodrug binding

Abstract

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.

Clade-Specific Flucytosine Resistance Is Due to a Single Nucleotide Change in the FUR1 Gene of Candida albicans

“…It has been shown that 5FC-resistant C. albicans isolates have undetectable UPRTase activity (25). Crystallography studies of the T. gondii UPRTase enzyme have shown that the Arg126 residue plays a role in the interactions at the interface between FUR1 monomers in the formation of the dimers necessary for the function of the enzyme (23). The Arg101-to-Cys101 mutation found in FUR R of C. albicans corresponds to Arg126 of T. gondii (Fig.…”

Identification and characterization of human uracil phosphoribosyltransferase (UPRTase)

“…1). Four short regions (I-IV) of the UPRTase family encircled the active site in T. gondii UPRTase (Schumacher et al 1998) and were important to substrate recognition, catalysis, or the stability of the protein. All UPRTases contain a conserved thirteen-residue PRPP-binding motif region, which typically comprised four hydrophobic residues then two acidic residues, two hydrophobic residues, and four small residues, for example glycine (Argos et al 1983).…”

“…5). The result showed that UPRTase selectivity for its pyrimidine substrate was provided solely by backbone contacts and a water-mediated hydrogen bond; one proline and two glycines were critical for the conformation of this region and for uracil binding (Schumacher et al 1998;Islam et al (2006). Although in the UPRTases of human and other higher eukaryotes Pro298 is highly conserved, two glycines are absent from the uracil-binding region (Fig.…”

“…1). Met166 in T. gondii UPRTase and B. subtilis PyrR are also conserved and are important in stacking interactions for PRPP binding (Schumacher et al 1998), whereas the corresponding site was Ile or Thr in the UPRTases of higher eukaryotes. It was also found that seven amino acid residues (region V, Fig.…”

“…Several crystal structures of UPRTases have been determined, for example those of UPRTases from B. caldolyticus (Kadziola et al 2002), T. gondii (Schumacher et al 1998), and Thermus thermophilus (Kukimoto-Niino et al 2005). All UPRTases contain a conserved thirteenresidue fingerprint region, with a core five-stranded parallel b-sheet that binds the 5-phosphoribosyl moiety of PRPP and the nucleotide product; this is termed the ''PRPPbinding motif'' and is characteristic for the UPRTases (Argos et al 1983;Lundegaard and Jensen (1999).…”