Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis

Anne Drumond Villela, Rodrigo Gay Ducati, Leonardo Astolfi Rosado, Carlos Junior Bloch, Maura Vianna Prates, Danieli Cristina Gonçalves, Carlos Henrique Inacio Ramos, Luiz Augusto Basso, Diogenes Santiago Santos

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5′-monophosphate (UMP) and pyrophosphate (PPi). 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 PPi 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.

Original languageEnglish (US)
Article numbere56445
JournalPloS one
Volume8
Issue number2
DOIs
StatePublished - Feb 12 2013

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

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    Villela, A. D., Ducati, R. G., Rosado, L. A., Bloch, C. J., Prates, M. V., Gonçalves, D. C., Ramos, C. H. I., Basso, L. A., & Santos, D. S. (2013). Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis. PloS one, 8(2), [e56445]. https://doi.org/10.1371/journal.pone.0056445