Biochemical characterization of uracil phosphoribosyltransferase from Mycobacterium tuberculosis.
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...
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2013-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3570474?pdf=render |
| _version_ | 1828927240757313536 |
|---|---|
| author | 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 |
| author_facet | 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 |
| author_sort | Anne Drumond Villela |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-13T23:46:20Z |
| format | Article |
| id | doaj.art-1016413b77c342b193a7a517ac53c811 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-13T23:46:20Z |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-1016413b77c342b193a7a517ac53c8112022-12-21T23:26:56ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0182e5644510.1371/journal.pone.0056445Biochemical characterization of uracil phosphoribosyltransferase from Mycobacterium tuberculosis.Anne Drumond VillelaRodrigo Gay DucatiLeonardo Astolfi RosadoCarlos Junior BlochMaura Vianna PratesDanieli Cristina GonçalvesCarlos Henrique Inacio RamosLuiz Augusto BassoDiogenes Santiago SantosUracil 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.http://europepmc.org/articles/PMC3570474?pdf=render |
| spellingShingle | 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 Biochemical characterization of uracil phosphoribosyltransferase from Mycobacterium tuberculosis. PLoS ONE |
| title | Biochemical characterization of uracil phosphoribosyltransferase from Mycobacterium tuberculosis. |
| title_full | Biochemical characterization of uracil phosphoribosyltransferase from Mycobacterium tuberculosis. |
| title_fullStr | Biochemical characterization of uracil phosphoribosyltransferase from Mycobacterium tuberculosis. |
| title_full_unstemmed | Biochemical characterization of uracil phosphoribosyltransferase from Mycobacterium tuberculosis. |
| title_short | Biochemical characterization of uracil phosphoribosyltransferase from Mycobacterium tuberculosis. |
| title_sort | biochemical characterization of uracil phosphoribosyltransferase from mycobacterium tuberculosis |
| url | http://europepmc.org/articles/PMC3570474?pdf=render |
| work_keys_str_mv | AT annedrumondvillela biochemicalcharacterizationofuracilphosphoribosyltransferasefrommycobacteriumtuberculosis AT rodrigogayducati biochemicalcharacterizationofuracilphosphoribosyltransferasefrommycobacteriumtuberculosis AT leonardoastolfirosado biochemicalcharacterizationofuracilphosphoribosyltransferasefrommycobacteriumtuberculosis AT carlosjuniorbloch biochemicalcharacterizationofuracilphosphoribosyltransferasefrommycobacteriumtuberculosis AT mauraviannaprates biochemicalcharacterizationofuracilphosphoribosyltransferasefrommycobacteriumtuberculosis AT danielicristinagoncalves biochemicalcharacterizationofuracilphosphoribosyltransferasefrommycobacteriumtuberculosis AT carloshenriqueinacioramos biochemicalcharacterizationofuracilphosphoribosyltransferasefrommycobacteriumtuberculosis AT luizaugustobasso biochemicalcharacterizationofuracilphosphoribosyltransferasefrommycobacteriumtuberculosis AT diogenessantiagosantos biochemicalcharacterizationofuracilphosphoribosyltransferasefrommycobacteriumtuberculosis |