Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
Expression of M. tuberculosis Fatty Acid Synthase I in M. smegmatis
We have previously shown that an analog of antitubercular agent pyrazinamide (PZA), 5-chloropyrazinamide (5-Cl-PZA) inhibits fatty acid synthase I (FASI) in Mycobacterium tuberculosis (Mtb). FASI has been purified from Mycobacterium smegmatis mc2 2700, a recombinant strain where the native fas1 gene has been deleted and replaced with Mtb fas1 gene. To further prove that 5-Cl-PZA and PZA bind to FASI, we used saturation transfer difference (STD) NMR experiment. NMR shows that PZA and 5ClPZA not only bind to FASI but they also compete for the same binding sites of FASI. Based on STD competition titration, 5-Cl-PZA binds to FASI with dissociation binding constant KD of 90 μM, which is significantly lower than the PZA binding constant KI of 2.5 mM. However, FASI isolated from mc2 2700 yields natural expression levels that make further testing by (STD) NMR not viable. To overcome this problem FASI was successfully overexpressed in E.coli but yielded inactive protein. Since the expression of FASI in E. coli results in inactive enzyme, we are currently working to move fas1 into an E.coli ? Mycobacterial shuttle vector, pVV16 which has been used to overexpress mycobacterial protein in M. smegmatis. Expressing FASI in M. smegmatis should allow overexpression of the protein and minimize any protein folding issues that may have occurred in E. coli.
1Dept of Chemistry