Sterols are abundant hydrophobic organic compounds that persist in the environment due to their water insolubility and chemical inertness. Recently, a sterol degradation pathway was discovered in a cholesterol-grown denitrifying bacterium Sterolibacterium (S.) denitrificans under anoxic conditions. The initial activation of primary C26 of the isoprenoid side chain of cholesterol is catalyzed by a four-step ATP-dependent enzyme cascade involving hydroxylation to an allylic alcohol via a phosphorylated intermediate.¹ However, this degradation mechanism is not applicable to steroids with a double bond at C-22 in the isoprenoid side chain, such as the plant sterol stigmasterol. Here, we have enriched delta-24 desaturase (Δ24‑SD) from S. denitrificans, which catalyzes the oxidation of the intermediate Δ22‑en to a conjugated Δ22,Δ24-diene in the presence of an electron acceptor. Biochemical analysis suggests a α₄β₄ architecture of 440 kDa for Δ24-SD, in which each subunit covalently binds an FMN cofactor to a histidyl residue. UV/vis and EPR analysis revealed the presence of both flavins as red semiquinone radicals, indicating a radical-based mechanism involving an allylic radical intermediate. Our proposed model involves a homodimeric catalytic subcomplex in which two flavin semiquinones each abstract a hydrogen atom from the substrate. Subsequently, the formed C24 double bond enables the hydroxylation of the terminal C26 with water by a heterologously produced molybdenum-dependent steroid C26 dehydrogenase 2 (S26DH₂). In conclusion, Δ24-SD allows for direct hydroxylation of the unactivated primary C26 of Δ22-steroids by circumventing the ATP-dependent formation of a phosphorylated intermediate.²

¹Jacoby, C. et al., Nat. Commun. 11, 3906 (2020)

²Zhan, T. et al., (2024) [submitted for publication]