Ancestral Sequence\nReconstruction Reveals Determinants\nof Regioselectivity in C(sp<sup>3</sup>)‑H Oxyfunctionalization\nReactions by CYP505Es

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