Ancestral Sequence Reconstruction Reveals Determinants of Regioselectivity in C(sp³)-H Oxyfunctionalization Reactions by CYP505Es

Regioselective C–H functionalization of fatty acids and alcohols is a challenging reaction, especially in-chain/midchain hydroxylation. These hydroxy fatty acids or diols offer a synthetic route to valuable δ- and γ-lactones. Although terminal and subterminal hydroxylation of fatty acids and alcohols by cytochrome P450 monooxygenases have been extensively explored, the molecular determinants of in-chain hydroxylation are unknown. Here we performed ancestral sequence reconstruction (ASR) of the subfamily of CYP505Es, able to perform in-chain hydroxylation, together with their closest related subterminal hydroxylases. Three ancestors were resurrected, which represented the in-chain and subterminal hydroxylases, as well as their common ancestor, which displayed little regioselectivity. Mutations were introduced to investigate the divergence in regioselectivity observed in the natural evolution. Whereas subterminal hydroxylation appears to be through multiple additive mutations in the active site, in-chain hydroxylation was greatly affected by the BC-loop. ASR provides not only insight for directed evolution studies but also more promiscuous ancestors as templates for the starting point for laboratory evolution.