Having explored the range in regioselectivity in the ancestral CYP116B enzymes, we compared their sequences overall and within their active sites (Fig. ) to identify determinants of selectivity. Ancestral sequence identities (Supplementary Table ) ranged from 59 (earlier ancestors) to 77% (more recent ancestors) compared to the P450-TT holoenzyme. Most of the non-conserved residues were found in the regions that are predicted to be surface-exposed (Fig. ), with more conservation in the core regions of each domain. The only changes in the active site were two consecutive amino acids (Ala205Thr and Phe206Trp), which increased the active site polarity and were present in all ancestors apart from the two closest to P450-TT (N115 and N106). This suggested a more polar active site may favour sub-terminal hydroxylation in the CYP116B family. Only 17 of the 467 initial extant sequences in the alignment (Fig. ) lacked the Thr205-Trp206 motif present in most of the characterised ancestral sequences, instead having either Ala-Phe, Ile-Trp or Gln-Phe residues, suggesting these could be key active site positions governing regioselectivity towards fatty acid hydroxylation.