Journal of Biological Chemistry

Sterols are ubiquitous membrane constituents that persist to a large extent in the environment due to their water insolubility and chemical inertness. Recently, an oxygenase-independent sterol degradation pathway was discovered in a cholesterol-grown denitrifying bacterium Sterolibacterium (S.) denitrificans. It achieves hydroxylation of the unactivated primary C26 of the isoprenoid side chain to an allylic alcohol via a phosphorylated intermediate in a four-step ATP-dependent enzyme cascade. However, this pathway is incompatible with the degradation of widely distributed steroids containing a double bond at C22 in the isoprenoid side chain such as the plant sterol stigmasterol. Here, we have enriched a prototypical delta-24 desaturase from S. denitrificans, which catalyzes the electron acceptor-dependent oxidation of the intermediate stigmast-1,4-diene-3-one to a conjugated (22,24)-diene. We suggest an α4β4 architecture of the 440 kDa enzyme, with each subunit covalently binding an flavin mononucleotide cofactor to a histidyl residue. As isolated, both flavins are present as red semiquinone radicals, which can be reduced by stigmast-1,4-diene-3-one but cannot be oxidized even with strong oxidizing agents. We propose a mechanism involving an allylic radical intermediate in which two flavin semiquinones each abstract one hydrogen atom from the substrate. The conjugated delta-22,24 moiety formed allows for the subsequent hydroxylation of the terminal C26 with water by a heterologously produced molybdenum-dependent steroid C26 dehydrogenase 2. In conclusion, the pathway elucidated for delta-22 steroids achieves oxygen-independent hydroxylation of the isoprenoid side chain by bypassing the ATP-dependent formation of a phosphorylated intermediate.

steroid degradation

anaerobic metabolism

stigmasterol

flavin radical enzyme

desaturase

molybdenum enzyme

(24E)-26-hydroxy-stigmasta-1,4,24-triene-3-one

cholest-1,4-diene-3-one

2,6-dichlorophenolindophenol

desmost-1,4-diene-3-one

electron paramagnetic resonance

2-hydroxypropyl-β-cyclodextrin

3-(n-morpholino)propanesulfonic acid

steroid C25 dehydrogenase

steroid C26 dehydrogenase

stigmast-1,4-diene-3-one

stigmasta-1,4,24-triene-3-one

ultra-performance liquid chromatography

© 2024 The Authors. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology.