Updated project metadata. The anaerobic degradation of cholesterol fundamentally differs from the cytochrome P450 monooxygenase dependent activation of the isoprenoid side chain at C26 in aerobes. In the denitrifying β-proteobacterium Sterolibacterium denitrificans, activation of primary C26 proceeds via a three-step cascade involving (i) a water-dependent hydroxylation at tertiary C25, (ii) an ATP-dependent dehydration to an alkene, and (iii) a water-dependent hydroxylation at primary C26 to an allylic alcohol. Here, we enriched a 25-hydroxy-steroid kinase (25-HSK) from Sterolibacterium denitrificans that catalyses the ATP-dependent transformation of 25-OH-cholest-1,4-diene-3-one (25-OH-CDO) to 25-phospho-CDO.