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Alanine mutants of the tyrosine residues were also created by another group member and were used to carry out gel shift assays. It appears that despite the NH2 group being present in alanine, the change the shape of the binding pocket by the smaller amino acid was enough to significantly alter MMF binding in the Y144A mutant. This therefore gives further evidence to tyrosine 144 being key to ligand binding as well as the tyrosine in position 85.

4.4.5

Discussion of MmfR/MMF Binding Data

Figure 4.19 summarises the data found on MmfR/MMF binding using the luciferase reporter gene assay, combining both the analyses done on the binding affinities of the five MMFs to the wild type MmfR as well as the MmfR ligand binding pocket tyrosine mutants. Unless otherwise stated, this figure refers to the wild type mmfR strains.

Figure 4.19. Bar chart showing a comparison of the binding potentials of MMF1-5 for WT MmfR, as shown by the levels of luxCDABE expression when under the control of mmfLp and WT MmfR, compared to the binding potential for MMF4 with the Y85F or Y144F MmfR mutants

Binding potential = Bmax/Kd Strains used: L1+WTmmfR – luxCDABE under the control of mmfLp and wild type mmfR under the control of ermEp* (pKMS01), L1+mmfR Y85F – luxCDABE under the control of mmfLp and mmfR with a mutation to tyrosine 85 under the control of ermEp* (pKMS85), L1+mmfR Y144F – luxCDABE under the control of

mmfLp and mmfR with a mutation to tyrosine 144 under the control of ermEp* (pKMS144). Unless otherwise stated, L1+WTmmfR is used for all data points.

Results from the luciferase assay revealed that there are detectable changes in lux gene expression with concentrations of the MMFs as low as 5 µM, with the Kd values ranging between 18 and 70 µM for the five molecules. Saturation of MmfR appears to occur sometime after around 200 µM, and varies between the particular ligands. The binding potentials varied between the different MMFs, with the branched alkyl chains providing the best efficacy. Four out of the five MMFs had a calculated Bmax bioluminescence reading greater than the maximal reading for the positive control (L1+pCC4) indicating that they may have more of a dose effect that just releasing MmfR and may work as some kind of activator. This is something which would be exciting to investigate further, potentially with a more high throughput assay than the luciferase one used here.

An in silico analysis of MmfR and its homologues indicated that there are two key residues

involved in ligand binding in MmfR, that of tyrosines in amino acid positions 85 and 144. This was indicated to be consistent in binding across all five MMFs and mutants created for these residues provided an interesting set of data.

MMF1 MMF2 MMF3 MMF4 MMF5 Y85F MMF4 Y144F MMF4 0.0 0.2 0.4 0.6 0.8 Bi n d n g p o te n tia l ( Bma x /Kd )

coelicolor

To summarise the findings from Section 4.4, the following binding potentials were calculated for all of the samples tested (see Figure 4.19);

MMF1 > Y144F MMF4 > MMF3 > MMF5 > MMF4 > MMF2 > Y85F MMF4

The binding potential of the Y85F MmfR mutant to MMF4 is much lower than the wild type binding to the same ligand, or indeed any of the other furans. The Y144F MmfR mutant on the other hand appears to have increased release from the MARE operator in the presence of MMF4 when compared to the wild type. This mutant also appears to have greater binding potential to MMF4 than the wild type strain does to either MMF2, 3 or 5.

Although the Y144F mutant appears to be more sensitive to the MMFs, it was not deemed suitable for later use in the novel inducible expression system due to its decreased repressive activities. These decreased repressive activities were also seen for the Y85F mutant, possibly as a result of the ligand binding residues selected for mutation being close to the dimer interface of MmfR and therefore are potentially having an effect on MmfR conformation and consequently, DNA-binding ability.

4.5

Outlook for Further Investigations

In this chapter MmfR has been shown to cause repression at the three known methylenomycin gene cluster MARE operators as well as being released upon the binding of all five MMFs, in line with the hypotheses for this investigation. When stating these hypothesis, it was explained that the role of the MmfR paralogue, MmyR was much less understood. Knockouts of this protein, a potential pseudo MMF receptor, produce the phenotype of methylenomycin overproduction.(71) It is therefore clear that it has a repressive role. The DNA binding sequences and ligands of this second type of TFR are ambiguous however. The next stage of this investigation into methylenomycin cluster repressor/ligand interactions is to use the luciferase assay to study the role of paralogue, MmyR. Many of the methods used in Chapter 4 were also used for this investigation. For example, an investigation into strength of MARE operator binding could be done in the same way with this alternate repressor.