STRUCTURAL CHARACTERIZATION OF MabR AND FasR THE TRANSCRIPTIONAL REGULATORS OF MYCOLIC ACID BIOSYNTHESIS IN Mycobacterium tuberculosis

LARA J, DIACOVICH L, GAGO G, GRAMAJO H.

Rosario

Congreso; IX Congreso Nacional de Microbiología General (SAMIGE 2013); 2013

Sociedad Argentina de Microbiología General (SAMIGE)

Mycolic acids, the dominant feature of the Mycobacterium tuberculosis outer membrane, are essential for the survival, virulence, and antibiotic exclusion of this human pathogen. Mycobacteria, unlike most bacteria, have two fatty acid synthases (FAS-I and FAS-II). Both enzymes are necessary for the biosynthesis of mycolic acids, however, how these pathways were regulated at the transcriptional level was unknown until very recently. Our research group identified two transcriptional regulators involved on this fatty acids biosynthesis. MabR controls the expression of fasII operon genes, by binding to the fasII promoter region and FasR a regulatory protein that specifically binds fasI promoter region to regulate the de novo fatty acid biosynthesis. To deeply characterize the roles of MabR and FasR in fatty acid and mycolic acid biosynthesis, we have cloned, expressed and purified MabR and FasR as His-Tagged recombinant proteins from M. tuberculosis. We were enabled to find more appropriate conditions to analyze MabR and FasR through crystallographic studies, using a solubility screening test.The proteins were screened based on sitting drop vapor diffusion where laminar crystals of MabR were obtained, although not suitable for structure determination. Therefore, we explored new crystallization conditions with native proteins constructions without the Hexa-histidine (His6)-tag. In adittion we tested new crystallization conditions for MabR with the additional presence of DNA interacting probe.. We recently found conditions where small crystals of FasR and rod-like crystals of MabR-DNA complex appeared. Currently, we are optimizing this condition to obtain diffraction data. The characterization of these novel transcriptional regulators open the possibility to further studies relating to fatty acid and mycolic acid biosynthesis in M. tuberculosis and represents an attractive new drug target to be explored.