Fig. 4

AncCAR tolerance to loop-dependent environmental factors. a AncCAR and ExCAR activity on 5 mM (E)-3-phenylprop-2-enoic acid was assessed in aprotic and protic solvents by solvent titration from 25% (v/v). For all proteins on all solvents besides DMSO (A, 88.9%; PA, 86.5%; PF 92.4%) considerable activity is lost at 10% solvent concentration for AncCARs, while ExCARs also lose activity in DMSO (MpCAR, 74%; NiCAR, 67%). As DMSO had the smallest inhibitory effect and lowest variance between enzymes, 10% DMSO was chosen for further kinetic analyses. Graphs represent relative activity of each ancestral enzyme at increasing concentrations of solvent compared to 0% solvent. Data for all solvents can be found in Supplementary Table 2. b To assess the resistance of AncCARs folding to pH, AncCARs and ExCARs were incubated for 30 min in 0.5 pH increments between pH 3 and 11, before being assayed for their turnover of NADPH in the presence of 5 mM (E)-3-phenylprop-2-enoic acid relative to turnover at pH 7.5 (100%). Data were analyzed in GraphPad Prism 7.0. pK₁ and pK₂ values were calculated respectively as: AncCAR-A—4.96 ± 0.06 and 10.83 ± 0.06; AncCAR-PA—5.12 ± 0.05 and 11.11 ± 0.07; AncCAR-PF—5.011 ± 0.06 and 11.56 ± 0.11. NiCAR—4.55 ± 0.09 and 9.70 ± 0.09; MpCAR—4.3 ± 0.1 and 11.8 ± 0.3. Error bars represent standard error, calculated from three experimental replicates.