Figure 7
From: Inhibiting mycobacterial tryptophan synthase by targeting the inter-subunit interface
Insights into inhibitor mode of action using gel filtration of tryptophan synthase. Gel filtration was used to analyze the TrpA and TrpB subunits and conditions required for complex formation to give insights into the impact of the TrpB-F188L mutation and the mode of action of the inhibitors. (a) Complex formation requirements of tryptophan synthase. TrpA and TrpB eluted as individual peaks in the presence or absence of compound 2. PLP induced complex formation, which was enhanced in the presence of the inhibitor. Samples were incubated for 1 h and analyzed with absorbance at 210 nm. (b) l-Tryptophan synthesis by TrpB and TrpB-F188L in the presence and absence of compound 3. Compound 3 inhibited l-tryptophan synthesis by TrpB, but not TrpB-F188L. Samples were incubated for 1 h and 2 h for TrpB and TrpB-F188L containing reactions respectively and analyzed with absorbance at 280 nm (to observe l-tryptophan). (c) Mutant tryptophan synthase (TrpB-F188L) complex formation is driven by compound 3. As observed with WT TrpB, PLP induced complex formation and this was further enhanced by the inhibitor. Samples were incubated for 1 h and analyzed with absorbance at 280 nm. The peaks are labeled accordingly. The samples are colored as follows: TrpA, brown; TrpB/TrpB-F188L, green; TrpA and TrpB/TrpB-F188L, orange; TrpA, TrpB/TrpB-F188L and inhibitor (light blue); TrpA, TrpB/TrpB-F188L and PLP, purple; TrpA, TrpB/TrpB-F188L, PLP and inhibitor, grey; TrpA, TrpB/TrpB-F188L, PLP, indole and l-serine, light green; TrpA, TrpB/TrpB-F188L, PLP, indole, l-serine and inhibitor, dark blue.
