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

LARA, JULIA; DIACOVICH, LAUTARO; GAGO, GABRIELA AND GRAMAJO, HUGO

Rosario

Congreso; IX Congreso de Microbiologia General; 2013

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 in the regulatory network ofs fatty acids biosynthesis. MabR that controls the expression of fasII operon genes by binding to the fasII promoter region, and FasR which specifically binds to fas promoter region and controlls the de novo fatty acid biosynthesis. To deeply characterize the molecular bases of MabR and FasR interaction with their corresponding DNA targets and effector molecules, we set up obtain the crystal structures of both transcriptional regulators. For this we have cloned, expressed and purified MabR and FasR from M. tuberculosis as His-tagged recombinant proteins. In order to find better conditions to analyze MabR and FasR through crystallographic studies we performed a solubility screening test.Both regulatory proteins were screened based on sitting drop vapor diffusion. çAt first, laminar crystals of His-MabR were obtained, although not suitable for structure determination. We then explored new crystallization conditions with the native protein (without hexa-histidine (His6)-tag) and found rod-like crystals in the presence of the DNA interacting probe.. Additionally, we recently found conditions where small crystals of FasR were obtained. Currently, we are optimizing the crystallization conditions for both proteins in order to obtain diffraction data. The structural characterization of these novel transcriptional regulators will allowed us to gain new insights into the transcriptional regulation fo the fatty acid and mycolic acid biosynthesis pathways in M. tuberculosis. Furthermore, these essential proteins could represent attractive targets for the development of conceptually new antibuberculosis drugs.