Fig. 5

Test of mutants from PcaV to Van2 and the Van mutants a. Point mutants of the 3 amino acid positions ranging from PcaV (I110, M113, N114) to Van2 (V110, S113, A114) were generated and all systems were induced with increasing concentrations of vanillin, PCA and vanillic acid. PcaV I110V kept the PCA detection performance and ligand specificity pattern. PcaV N114A, PcaV M113S and PcaV I110V N114A led to reduced PCA detection without ligand specificity change. PcaV I110V M113S led to reduced PCA detection and ligand specificity change with detection of vanillin and vanillic acid. PcaV M113S N114A showed complete ligand specificity change being responsive to vanillin, partially responsive to vanillic acid, and unresponsive to PCA, however the system had a high basal expression. b. Homology modelling and docking of Van2 and the double mutant PcaV M113S N114A. Docking of the models for Van2 and PcaV M113S N114A with vanillin showed, in the 2nd best docked conformation, an interacting with S113 of Van2 via a hydrogen bond between the aldehyde functional group of vanillin and the hydroxyl group of the serine. This conformation was only observed for Van2 and PcaV M113S N114A, mutants that displayed vanillin detection in vivo. c. Further mutants of Van2, to explore additional amino acids substitutions at positions 110 and 113, were induced with vanillin, PCA and vanillic acid at 2.5 mM. Presence of apolar small, medium and bulky amino acids at 110 on Van3, Van4 and Van5 respectively, as well as a bulky amino acid at 113 on Van6 kept a high basal expression of the systems leading to low max/min. The fluorescent gene expression normalised to cell density (RFU/OD₆₀₀) is shown. Each value represents the mean and standard deviation of 3 biological triplicates