Page 26 - Demo

30 PROCEEDINGS OF INTERNATIONAL SCIENTIFIC CONFERENCE ON APPLIED BIOTECHNOLOGYbacteria, it is an essential gene. ClpC1 associates with and supports ATP-dependent protein degradation by ClpP, a compartmentalized protease complex found in many bacteria, mitochondria, and chloroplasts[9]. In this process, ClpC1 binds certain cell proteins and unfolds and translocates them into ClpP for degradation. To determine whether ClpC1 is indeed the target of ecumicin in M. tuberculosis and to clarify its mode of action, we tested if ecumicin directly affected the ATPase activity of M. tuberculosis ClpC1, expressed and isolated as described recently [10]. Surprisingly, ecumicin did not inhibit this activity but instead stimulated the hydrolysis of ATP by several fold (Fig. 6A).In mycobacteria, ClpC1 functions with ClpP1P2, a novel member of the ClpP family found only in mycobacteria, where it is essential for viability[10]. Therefore, ClpC1%u2019s ability to stimulate ATP-dependent degradation of the model substrate, FITC-casein, by ClpP1P2 was also analyzed in the presence of ecumicin. Surprisingly, even though ecumicin stimulated ATP hydrolysis, it caused a strong inhibition of the ClpC1-dependent degradation of casein (Fig. 6B). Thus, the antibiotic uncoupled ATP hydrolysis from protein degradation.