Selective Oxidation of Tertiary C–H Bonds in Steroids Catalyzed by the Heterodimeric Fe(II)/α-Ketoglutarate-Dependent Oxygenase TlxI–J

Selective oxidation of tertiary carbons in steroids remains challenging due to their steric congestion and electronic similarity. Here, we identify the heterodimeric Fe(II)/α-ketoglutarate-dependent oxygenase TlxI-J as a highly versatile oxidative biocatalyst capable of targeting multiple sterically hindered positions across diverse scaffolds. TlxI-J accepts several steroid hormones and catalyzes distinct oxidations, including C-8β, C-9α, and C-14α hydroxylations and C-9/C-10 epoxidation. The enzyme also catalyzes the C-2/C-3 desaturation of flavanones, indicating broad substrate promiscuity beyond steroid frameworks. Docking studies suggest that the flexible lid-like loop and hydrophobic residues cooperate to position distinct scaffolds for selective oxidation. These results provide a foundation for engineering oxidative biocatalysts for the late-stage diversification of steroids and other complex molecules, and highlight the potential of TlxI-J for the chemoenzymatic synthesis of high-value steroid derivatives and related bioactive compounds.