Equilibrium binding and kinetic characterization of putative tetracycline repressor family transcription regulator Fad35R from Mycobacterium tuberculosis

Abstract

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 ) 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 palmitylCoA, 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. Structured digital abstractl Fad35R binds to palindromic sequence shown by surface plasmon resonance l Fad35R binds to tetracycline and activated fatty acids as shown by fluorescence spectroscopy

The DNA-binding network of Mycobacterium tuberculosi s

“…The average number of binding sites for TFs with a motif was 246 (range 14–859), compared with an average of 28 peaks (range 3–437) for those TFs where a significant consensus motif could not be identified. For TFs with previously characterized DNA-binding motifs, this analysis corresponded well with previous reports (for example, Rv2506 (refs 37 , 38 ), Rv2359 (ref. 39 ), DosR (ref.…”

Structural and Functional Characterization of a Ketosteroid Transcriptional Regulator of Mycobacterium tuberculosis

“…Comparison of sequence topology maps of 48 structures of TFRs identified by Yu et al (18) indicate that 13 contain at least two of the three conserved basic residues in orientations permissive to CoA binding. Of note, Fad35R of M. tuberculosis (PDB code 4G12) regulates the expression of fad35, which encodes an acetyl-CoA synthetase involved in fatty acid degradation (41). Although Fad35R binds tetracycline, other evidence suggested that a fatty acyl-CoA could be the physiological effector (41).…”

“…Of note, Fad35R of M. tuberculosis (PDB code 4G12) regulates the expression of fad35, which encodes an acetyl-CoA synthetase involved in fatty acid degradation (41). Although Fad35R binds tetracycline, other evidence suggested that a fatty acyl-CoA could be the physiological effector (41). The presence of Lys-184 on the ␣8-␣9 loop, Arg-166 on the apical end of helix ␣8, and His-170 in the middle of helix ␣8 supports this hypothesis.…”

bkaRis a TetR-type repressor that controls an operon associated with branched-chain keto-acid metabolism inMycobacteria

“…While writing this manuscript, a paper was published describing a single motif in the intergenic region of bkaR_fadD35 in M. tuberculosis. Binding was demonstrated to this region together with affinity measurements, and it was shown that both tetracycline and palmityl-coA could interfere with binding (Anand et al, 2012). These authors suggested that bkaR (which they call fad35R and consider to be a homologue of E. coli FadR) controls the expression of fadD35 in M. tuberculosis.…”