Des T mutants Upon viewing of the 3D structure of DesT, it is clear that specific residues are key mediators in coordinating

the shape of the ligand to the shape of the DNA binding domain. To ascertain information on the key residues, site directed mutagenesis was performed. In vivo analysis of the desCB activity was used to confirm the results. Expression of the wild type DesT showed repressive activity, due to the presence of endogenous ligands which brought about an equilibrium between the relaxed and tense state of DesT. In the presence of an external unsaturated fatty acid ligand, desCB activity remained repressed as the unsaturated fatty acid stabilised the relaxed state. However, in the presence of a saturated fatty acid ligand, the tense state was stabilised, allowing transcription to occur. DesT Y115A In this mutant, the tyrosine at position 155, found at the C terminus of 6, is mutated to an alanine. As a result, the DesT mutant binds DNA regardless of the structure of the bound acylCoA. This shows that Tyr115 is important in the tense state to fix the orientation of the helix at the dimer interface. The affinity of the DesT mutant to bind saturated acyl-CoA is similar to that of the affinity of the DesT mutant to bind unsaturated acyl-CoA. This indicates that the inability to bind DNA did not occur simply due to the inability to bind saturated acyl chains. DesT Y155A is permanebtly locked into the relaxed state, and the repression of desCB activity indicates this. DesT F166A Phenylalanine can be found at residue 166, directly below 6. Its positioning allows the detection of the presence of a cis double bond at the 9 position of the acyl chain. DesT F166A is able to bind either the saturated or unsaturated acyl chains, but is not able to bind DNA, under any conditions; the protein is locked into the tense state. Due to the lack of aromatic ring in the mutant, transmission of the ligand shape to 6 does not occur. Phe166 is able to stabilise DesT in the relaxed state. As the mutant is locjked into the tense state, desCB transcription is not repressed, no matter the ligand present. Upon further analysis, the levels of desCB mRNA present in a cell with the DesT F166A mutant are equivalent to that of cells containing no DesT gene. This shows that the F166A mutant is unable to repress desCB in vivo. DesT F71A The side chain of phenylalanine at position 71 is able to contact the ligand and introduce a bend into the 4 helix in the tense state. As a result, the mutant binds DNA constitutively. DesT F96A and L169A Leucine at position 169 and phenylalanine at position 96 have side chains that stabilise the hydrophobic core of the tense state. Both mutants are able to bind saturated and unsaturated ligands with similar affinities, therefore bind DNA constitutively. It can also be noted that the F71A, F96A AND L169A mutants leave a void within the hydrophobic core. Saturated acyl chains can enter this, which contributes to the failure of the formation of the tense state, even in the presence of a saturated CoA.

Introduction

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