title: Biochemical characterization of uracil phosphoribosyltransferase from Mycobacterium tuberculosis.
abstract: Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5'-monophosphate (UMP) and pyrophosphate (PP(i)). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PP(i) product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis.
authors: Centro de Pesquisas em Biologia Molecular e Funcional-CPBMF, Instituto Nacional de Ciência e Tecnologia em Tuberculose-INCT-TB, Pontifícia Universidade Católica do Rio Grande do Sul-PUCRS, Porto Alegre, Rio Grande do Sul, Brazil.
authors: Anne Drumond
authors: AD
authors: Villela
authors: Rodrigo Gay
authors: RG
authors: Ducati
authors: Leonardo Astolfi
authors: LA
authors: Rosado
authors: Carlos Junior
authors: CJ
authors: Bloch
authors: Maura Vianna
authors: MV
authors: Prates
authors: Danieli Cristina
authors: DC
authors: Gonçalves
authors: Carlos Henrique Inacio
authors: CH
authors: Ramos
authors: Luiz Augusto
authors: LA
authors: Basso
authors: Diogenes Santiago
authors: DS
authors: Santos
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009711:Nucleotides / Q000378:metabolism; D010430:Pentosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017421:Sequence Analysis; D013379:Substrate Specificity
chemical_list: D009711:Nucleotides; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase