25º Congresso Brasileiro de Microbiologia

25º Congresso Brasileiro de Microbiologia

ResumoID:1851-1

Área: Genética e Biologia Molecular ( Divisão N )

THE ENZYME PURINE NUCLEOSIDASE FROM MYCOBACTERIUM TUBERCULOSIS (EC 3.2.2.-) AS A TARGET FOR THE DEVELOPMENT OF NEW ANTIMYCOBACTERIAL&NBSP;DRUGS AND/OR VACCINES

Priscila Lamb Wink (INCT-TB PUCRS); Cristopher Schneider (INCT-TB PUCRS); Luiz Augusto Basso (INCT-TB PUCRS); Diógenes Santiago Santos (INCT-TB PUCRS)

Resumo

Mycobacterium tuberculosis is a pathogenic bacterium which infects one-third of the world population causing tuberculosis (TB), an important infectious disease. Since currently available anti-TB drugs has became ineffective, new drugs and vaccines are needed to treat and prevent TB, respectively. Purine nucleosidase (IunH) is an enzyme involved in the purine salvage pathway which catalyzes the hydrolysis of all the common occurring purine and pyrimidine nucleotides into ribose and its the associated base. This enzyme was characterized in other organisms, for example, Bacillus thuringiensis, Ochrobactrum anthropi and Trypanosoma vivax. Interestingly, to date no report using the sequence of IunH from M. tuberculosis is available. Moreover, no experimental evidences about the probable role of this enzyme in the purine salvage pathway in the latency mechanism of the bacillus. Therefore, this work represents an important step in the production of IunH for use as an anti-TB target. For this, the objectives of this work are amplification, cloning, expression, purification, characterization and site-directed mutagenesis of iunH from M. tuberculosis H37Rv. The iunH (Rv3393) gene was amplified from M. tuberculosis H37Rv genomic DNA. This product (927 bp) was cloned into pCR-Blunt vector and subcloned into pET-23a(+) expression vector and expression assays of the IunH were performed using different strains of E. coli and some different experimental conditions. The overexpression of the protein in C41(DE3) E. coli strains was observed (~32.9 KDa) in the soluble fraction with IPTG induction, at 30 ºC using TB medium. The optimization of protein expression of IunH is necessary to implement high-yield purification protocols. Recombinant homogeneous protein will provide sufficient material for biological activity determination of the protein. Our future goals are to determine if in fact there is a preference of the IunH enzyme for substrates such as uridine and inosine, and determine its basic enzymological features (K_m, K_cat, K_i, pH dependency, thermal stability). Enzyme kinetics, site-directed mutagenesis and structural studies will provide a framework on which can be based the rational design of new inhibitors against IunH from M. tuberculosis. In addition, these data can help us in identifying an attenuated sample that can be used as a vaccine against TB.

Palavras-chave: Mycobacterium tuberculosis, Purine nucleosidase, Purine salvage pathway

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