Thermostability profiles. Enhanced thermostability has been repeatedly observed in reconstructed ancestral proteins, including other P450s, compared to the extant forms from which they are inferred, where sequences have been derived from mesophilic organisms. However, to the best of our knowledge, no reconstructions have been performed to trace the evolution of stability in protein families from thermophiles. As the CYP116B family contains proteins from both mesophiles and thermophiles (Supplementary Fig. ), we were interested in whether thermostability in CYP116B family enzymes is a recent development and a specific adaptation in CYP116B sequences from thermophilic organisms. We assessed the thermostability of the CYP116B ancestors by incubating the proteins at a range of temperatures (25-80 °C) and assessing the loss of the P450 holoenzyme by changes to the characteristic P450 spectrum. Two incubation times were used (15 and 60 minutes), which allowed us to determine both the 15 T50 and 60 T50 stabilities for each ancestor (Table ; Supplementary Fig. and). A wide range of thermostabilities ( 15 T50 38-54 °C and 60 T50 <30-55 °C) was observed among different ancestors across the CYP116B tree. As expected, the 60 T50 values were generally lower than the 15 T50 values, indicating a lower tolerance for prolonged heating, particularly in the ancestors and wild-type enzymes with lower stability e.g., T50 <45 °C (Fig. , Table ).