TY  - JOUR
N1  - Copyright of this article belongs to John Wiley and sons.
ID  - open1307
UR  - http://onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2012.08707.x/abstract
IS  - 17
A1  - Anand, Sushma
A1  - Singh, Vijay
A1  - Singh, Appu Kumar
A1  - Mittal, Monica
A1  - Datt, Manish
A1  - Subramani, Bala
A1  - Kumaran, Sangaralingam
Y1  - 2012/09//
N2  - Fatty acids play critical role in the survival and virulence of Mycobacterium tuberculosis (Mtb). Activation of fatty acids by acyl-CoA synthetases (Fad) into fatty acyl-CoA is the first and one of the crucial steps in fatty acid metabolism. Mtb possesses 36 fatty acyl-CoA synthetases, unlike Escherichia coli, which has single enzyme. However, the mechanisms by which the expression of these multiple Fad genes is regulated remain uncharacterized. We characterized the DNA- and ligand-binding properties of a putative tetracycline repressor family regulator, named Fad35R, located upstream of the Fad35 gene and ScoA-citE operon. We identified a palindromic regulatory motif upstream of Fad35 and characterized the binding of Fad35R to this motif. Equilibrium binding studies show that Fad35R binds to this motif with high affinity (K(d) ? 0.033 ?m) and the specificity of binding was confirmed by an electromobility gel shift assay. Kinetic studies indicate that faster association (k(a,avg) ? 5.4 × 10(4) m(-1) · s(-1)) and slower dissociation rates (k(d,avg) ? 5.84 × 10(-4) s(-1)) confer higher affinity. The affinity for the promoter is maximum at 300 mm NaCl but decreases rapidly beyond this range. Ligand-binding studies indicate that Fad35R binds specifically to tetracycline and also binds to fatty acid derivatives. The promoter-binding affinity is decreased significantly in the presence of palmityl-CoA, suggesting that Fad35R can sense the levels of activated fatty acids and alter its DNA-binding activity. Our results suggest that Fad35R may be the functional homologue of FadR and controls the expression of genes in a metabolite-dependent manner.
PB  - John and Wiley
JF  - The FEBS journal
VL  - 279
KW  -  fatty acid;protein?DNA interactions; surface plasmon resonance; TetR family; transcriptional regulation
SN  - 1742-4658
TI  - Equilibrium binding and kinetic characterization of putative tetracycline repressor family transcription regulator Fad35R from Mycobacterium tuberculosis.
SP  - 3214
EP  - 28
ER  -