Hypoxanthine-guanine phosphoribosyltransferase from Mycobacterium tuberculosis H37Rv: cloning, expression, and biochemical characterization.
Human tuberculosis (TB) is a major cause of morbidity and mortality worldwide, especially in poor and developing countries. Moreover, the emergence of Mycobacterium tuberculosis strains resistant to first- and second-line anti-TB drugs raises the prospect of virtually incurable TB. Enzymes of the purine phosphoribosyltransferase (PRTase) family are components of purine salvage pathway and have been proposed as drug targets for the development of chemotherapeutic agents against infective and parasitic diseases. The PRTase-catalyzed chemical reaction involves the ribophosphorylation in one step of purine bases (adenine, guanine, hypoxanthine, or xanthine) and their analogues to the respective nucleoside 5'-monophosphate and pyrophosphate. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8) is a purine salvage pathway enzyme that specifically recycles hypoxanthine and guanine from the medium, which are in turn converted to, respectively, IMP and GMP. Here we report cloning, DNA sequencing, expression in Escherichia coli BL21 (DE3) cells, purification to homogeneity, N-terminal amino acid sequencing, mass spectrometry analysis, and determination of apparent steady-state kinetic parameters for an in silico predicted M. tuberculosis HGPRT enzyme. These data represent an initial step towards future functional and structural studies, and provide a solid foundation on which to base M. tuberculosis HGPRT-encoding gene manipulation experiments to demonstrate its role in the biology of the bacillus.
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, Brazil.
Gisele
G
Biazus
Cristopher Z
CZ
Schneider
Mario S
MS
Palma
Luiz A
LA
Basso
Diógenes S
DS
Santos
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003198:Computer Simulation; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007041:Hypoxanthine Phosphoribosyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D007041:Hypoxanthine Phosphoribosyltransferase