Fig. 3

AncCARs are thermostable enzymes. a AncCARs and the CAR from Mycobacterium phlei (MpCAR) were incubated in 50 mM HEPES at temperatures from 30 to 70 °C for 30 min. Each point represents the rate of NADPH oxidation in 5 mM (E)-3-phenylprop-2-enoic acid at temperature relative to the rate of NADPH oxidation in 5 mM (E)-3-phenylprop-2-enoic acid at 30 °C. Black dotted horizontal line represents 50% activity. Colored vertical dotted lines represent temperature at which A₅₀ is reached. b AncCARs have environment-dependent temperature resistance. AncCARs were incubated in in vivo-like ionic concentrations that model the internal environment of a S. cerevisiae⁵⁶ cell at temperatures from 30 to 70 °C for 30 min. Data were determined as in panel a. Black dotted horizontal line represents 50% activity. Colored vertical dotted lines represent temperature at which A₅₀ is reached. c Differential scanning fluorimetry. AncCARs were incubated in HEPES and analyzed from 25 to 100 °C. Thermal shift curves were drawn from raw DSF data in GraphPad. d To assess half-life at 37 °C, AncCARs and MpCAR were incubated at this temperature over a period of 10 days. Relative activity versus a zero-time point was assessed by activity on 5 mM (E)-3-phenylprop-2-enoic acid. Black dotted horizontal line represents 50% activity. Colored vertical dotted lines represent time taken to reach 50% enzyme activity. Error bars represent standard error, in all cases calculated from three experimental replicates.