CONICET | Buscador de Institutos y Recursos Humanos

Mycobacteria have two fatty acid synthases, FAS I and FAS II, which work in concert to synthesize fatty acids and mycolic acids.We identified a transcriptional regulator essential for mycobacterial viability: FasR, which specifically binds to fas promoter regionand controls the de novo fatty acid biosynthesis. The main purpose of our studies is to understand at the molecular level howmycobacteria exert a fine control over the biosynthesis of their membrane. The effector molecule that modulates the affinity of FasRfor its DNA target was studied using EMSA, SPR and in vitro transcription. In order to deeply understand the molecular bases ofFasR activity; we performed experiments, based on sitting drop vapor diffusion, to obtain the crystal structures of FasR, FasR-DNAand FasR-effector. To identify crystallization conditions, full length protein was initially screened using commercial crystallizationkits and FasR resulted in small crystals, although not suitable for structural determination. In consequence, sequences alignment andsecondary structure prediction tools were used to determine physicochemical parameters and generate alternative constructs to obtaintruncated versions of FasR from Mycobacterium tuberculosis (FasRD-), minimizing unstructured regions that would be causingsolubility problems and/or hindering crystallization. Crystallization screenings using FasRD- provided two different sets of conditionsproducing crystals of FasRD- alone and in complex with acyl C20-CoA. We obtained X-ray diffraction data of both conditions. Finally,we have recently been able to solve both crystal structures, refinement and validation is in progress. In this work, we show thatlong-chain acyl-CoAs are key effector molecules that regulate the expression of FAS I system, by directly binding to FasR. Futureefforts will be concentrated in obtaining crystals of FasR in complex with its cognate DNA oligonucleotide. A better understanding ofthis complex regulatory process of lipid homeostasis in mycobacteria together with the structural characterization of this noveltranscriptional regulator will greatly contribute to the development of new strategies to control this disease.