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      Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis

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          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 p K values of 5.7 and 8.1 are important for catalysis, and a group with a p K 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.

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          Genetic requirements for mycobacterial survival during infection.

          Despite the importance of tuberculosis as a public health problem, we know relatively little about the molecular mechanisms used by the causative organism, Mycobacterium tuberculosis, to persist in the host. To define these mechanisms, we have mutated virtually every nonessential gene of M. tuberculosis and determined the effect disrupting each gene on the growth rate of this pathogen during infection. A total of 194 genes that are specifically required for mycobacterial growth in vivo were identified. The behavior of these mutants provides a detailed view of the changing environment that the bacterium encounters as infection proceeds. A surprisingly large fraction of these genes are unique to mycobacteria and closely related species, indicating that many of the strategies used by this unusual group of organisms are fundamentally different from other pathogens
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            Mycobacterium tuberculosis and the macrophage: maintaining a balance.

            Mycobacterium tuberculosis is a highly efficient pathogen, killing millions of infected people annually. The capacity of M. tuberculosis to survive and cause disease is strongly correlated to their ability to escape immune defense mechanisms. In particular, M. tuberculosis has the remarkable capacity to survive within the hostile environment of the macrophage. Understanding M. tuberculosis virulence strategies will not only define novel targets for drug development but will also help to uncover previously unknown signaling pathways related to the host's response to M. tuberculosis infection.
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              Tuberculosis: a problem with persistence.

              Mycobacterium tuberculosis is one of most successful pathogens of mankind, infecting one-third of the global population and claiming two million lives every year. The ability of the bacteria to persist in the form of a long-term asymptomatic infection, referred to as latent tuberculosis, is central to the biology of the disease. The persistence of bacteria in superficially normal tissue was recognized soon after the discovery of the tubercle bacillus, and much of our knowledge about persistent populations of M. tuberculosis dates back to the first half of the last century. Recent advances in microbial genetics and host immunity provide an opportunity for renewed investigation of this persistent threat to human health.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                12 February 2013
                : 8
                : 2
                : e56445
                Affiliations
                [1 ]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
                [2 ]Programa de Pós-Graduação em Biologia Celular e Molecular, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, Rio Grande do Sul, Brazil
                [3 ]Programa de Pós-Graduação em Medicina e Ciências da Saúde, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, Rio Grande do Sul, Brazil
                [4 ]Laboratório de Espectrometria de Massa, Empresa Brasileira de Pesquisa Agropecuária - Recursos Genéticos e Biotecnologia, Estação Parque Biológico, Brasília, Federal District, Brazil
                [5 ]Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, São Paulo, Brazil
                [6 ]Instituto de Química, Universidade Estadual de Campinas (UNICAMP), Campinas, São Paulo, Brazil
                Institute of Enzymology of the Hungarian Academy of Science, Hungary
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: ADV LAB DSS CB CHIR. Performed the experiments: ADV RGD LAR DCG MVP. Analyzed the data: ADV RGD LAR DCG MVP. Contributed reagents/materials/analysis tools: LAB DSS CB CHIR. Wrote the paper: ADV LAB DSS.

                Article
                PONE-D-12-11837
                10.1371/journal.pone.0056445
                3570474
                23424660
                3bb0c43e-1720-4f88-883a-49f17a727d04
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 25 April 2012
                : 14 January 2013
                Page count
                Pages: 14
                Funding
                This work was supported by funds of Decit/SCTIE/MS-MCT-CNPq-FNDCT-CAPES to National Institute of Science and Technology on Tuberculosis (INCT-TB) to DSS and LAB. LAB and DSS also acknowledge financial support awarded by FAPERGS-CNPq-PRONEX-2009 (Fundacao de Amparoa Pesquisa do estado do Rio Grand do Sul). CB acknowledges financial support from “Embrapa Recursos Genéticos e Biotecnologia”, Brazil. CHIR acknowledges financial support from FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo). LAB (CNPq, 520182/99-5), DSS (CNPq, 304051/1975-06), CBJ (304034/2008-8), and CHIR are Research Career Awardees of the National Research Council of Brazil (CNPq). RGD was a post-doctoral fellow of CNPq (The National Council for Scientific and Technological Development). ADV and LAR acknowledge scholarships awarded by CNPq (The National Council for Scientific and Technological Development). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Biochemistry
                Enzymes
                Enzyme Kinetics
                Proteins
                Protein Chemistry
                Recombinant Proteins
                Drug Discovery
                Biophysics
                Biomacromolecule-Ligand Interactions
                Microbiology
                Bacterial Pathogens
                Chemistry
                Chemical Reactions
                Catalysis
                Chromatography
                Liquid Chromatography
                Physical Chemistry
                Thermodynamics

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                Uncategorized

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