Tuberculosis is currently a major cause of mortality in both developing and industrialized countries with eight million people developing active tuberculosis and with two million dying from the disease every year. Difficulties to treat tuberculosis are mainly due to M. tuberculosis multidrug-resistant strains and the limited number of antitubercular agents . In light of the dependence of M. tuberculosis on heme-containing enzymes , we have identified glutamyl-tRNA synthetase (GluRS) and glutamyl-tRNA reductase (GluTR) as potential targets for new drug design. GluRS is not only an essential enzyme because it provides Glu-tRNA for protein biosynthesis, but also because it forms with GluTR and glutamate 1-semialdehyde aminomutase the path leading to the synthesis of δ-aminolevulinic acid (ALA), the first common precursor of tetrapyrrole biosynthesis [3, 4]. The M. tuberculosis gltX gene encoding Mt-GluRS has been cloned, sequenced, and used to construct a plasmid for its overproduction in E. coli BL21(DE3) cells. Soluble recombinant protein was obtained in large amounts and purified to homogeneity. The catalytic properties of Mt-GluRS are being investigated using the well characterized E. coli GluRS as a reference, in order to highlight peculiar properties of the M. tuberculosis enzyme. The purified native protein and its seleno-methionine derivative have also been subjected to extensive crystallization trials. Crystals that diffracted to greater than 2.5 Ǻ were obtained. However, a twinning phenomenon and the small size of single crystals did not allow the resolution of Mt-GluRS structure. In order to gain insight on the enzyme structure and conformational flexibility, which may hamper crystallization, we are using a combination of dynamic light scattering, limited proteolysis, analytical gel filtration as well as small-angle X-ray scattering. Investigation of Mt-GluTR is hampered by the low levels of soluble protein that can be produced in E. coli cells transformed with plasmids containing the putative hemA gene (Rv0509), as found for GluTR from various sources. Indeed, up to now, only a few GluTR have been characterized [5-7]. However, the red phenotype of E. coli cells indicates that Rv0509 is indeed the hemA gene. Recently, significant amounts of soluble Mt-GluTR have been obtained by producing chimeric proteins resulting from the fusion of Mt-GluTR and either a GyrA intein-Chitin Binding Domain fragment or the Maltose Binding Protein. In these cases, the coexpression of E. coli chaperon proteins DnaK, DnaJ and GrpE and low temperature greatly enhanced the amount of soluble protein we could obtain. On the basis of these results attempts to obtain stable and catalytically active preparations of the enzyme are being carried out.
|Titolo:||Mycobacterium tuberculosis glutamyl tRNA synthetase and glutamyl-tRNA reductase|
|Autori interni:||VANONI, MARIA ANTONIETTA (Ultimo)|
PARAVISI, STEFANO (Primo)
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Data di pubblicazione:||2006|
|Enti collegati al convegno:||Societa' Italiana di Biochimica e Biologia Molecolare|
|Appare nelle tipologie:||01 - Articolo su periodico|