Solving the expression of ADP-glucose pyrophosphorylase from Mycobacterium tuberculosis.

GUERRERO, SERGIO ADRIÁN; DIEZ MATIAS, ASENCIÓN; DEMONTE ANA,; IGLESIAS ALBERTO,

Rosario

Congreso; 42th Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2006

SAIB

Solving the expression of recombinant ADP-glucose pyrophosphorylase from Mycobacterium tuberculosis. Matias D. Asención Diez, Ana M. Demonte, Sergio A. Guerrero, Alberto A. Iglesias UNL-CONICET, Lab. de Enzimología Mol. y de Bioq. Microbiana, FBCB. Santa Fe ademonte@fbcb.unl.edu.ar   In Gram negative bacteria, the biosynthesis of glycogen have been extensively characterized, specially the key enzyme ADP-glucose pyrophosphorylase (ADPGlcPPase, EC 2.7.7.27). M. tuberculosis is a human pathogen of very slow growing velocity and consequently the isolation of the enzyme from the microorganism is a time consuming practice. Expression of the gene encoding the M. tuberculosis ADPGlcPPase in Escherichia coli resulted recalcitrant to obtain the soluble protein. We solved this problem through the expression of the gene in M. smegmatis. Thus, the M. tuberculosis glgC gene was cloned into the shuttle pMIP12 vector. Competent M. smegmatis mc2155 cells were transformed with the recombinant construct (pMIP12/MtglgC), the protein was expressed with a C-term HisTag and purified chromatographically. Eluted fractions were tested by Western blotting and enzymatic activity. Functional characterization was performed in both ADPGlc synthesis and pyrophosphorolysis. Values for Km of  0.53 mM (ATP) and 0.52mM  (ADPGlc) were determined. The enzyme was sensitive to allosteric effectors, different to that found for Bacillus stearothermophilus, the unique studied recombinant enzyme of Gram positive bacterium. The developed recombinant system is being useful not only for the functional characterization of the enzyme, but also for generation of glgC null mutants.