Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis

Anne Drumond Villela, Rodrigo G. 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 Citations (Scopus)

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
Externally publishedYes

Fingerprint

uracil phosphoribosyltransferase
Phosphoribosyl Pyrophosphate
Uridine Monophosphate
uracil
Uracil
Mycobacterium tuberculosis
pyrophosphates
Catalysis
uridine
Pyrimidine Nucleotides
Cytidine Triphosphate
Gene Knockout Techniques
Calorimetry
Uridine Triphosphate
Uridine Diphosphate
Ultracentrifugation
Protein Sequence Analysis
Guanosine Triphosphate
catalytic activity
Salvaging

ASJC Scopus subject areas

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

Cite this

Villela, A. D., Ducati, R. G., Rosado, L. A., Bloch, C. J., Prates, M. V., Gonçalves, D. C., ... 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

Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis. / Villela, Anne Drumond; Ducati, Rodrigo G.; Rosado, Leonardo Astolfi; Bloch, Carlos Junior; Prates, Maura Vianna; Gonçalves, Danieli Cristina; Ramos, Carlos Henrique Inacio; Basso, Luiz Augusto; Santos, Diogenes Santiago.

In: PLoS One, Vol. 8, No. 2, e56445, 12.02.2013.

Research output: Contribution to journalArticle

Villela, AD, Ducati, RG, Rosado, LA, Bloch, CJ, Prates, MV, Gonçalves, DC, Ramos, CHI, Basso, LA & Santos, DS 2013, 'Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis', PLoS One, vol. 8, no. 2, e56445. https://doi.org/10.1371/journal.pone.0056445
Villela AD, Ducati RG, Rosado LA, Bloch CJ, Prates MV, Gonçalves DC et al. Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis. PLoS One. 2013 Feb 12;8(2). e56445. https://doi.org/10.1371/journal.pone.0056445
Villela, Anne Drumond ; Ducati, Rodrigo G. ; Rosado, Leonardo Astolfi ; Bloch, Carlos Junior ; Prates, Maura Vianna ; Gonçalves, Danieli Cristina ; Ramos, Carlos Henrique Inacio ; Basso, Luiz Augusto ; Santos, Diogenes Santiago. / Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis. In: PLoS One. 2013 ; Vol. 8, No. 2.
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